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Mechanism of extracellular Ca2+ receptor‐stimulated hormone release from sheep thyroid parafollicular cells
The Journal of Physiology
Abstract
Expression of receptors to extracellular calcium enables parafollicular cells of the thyroid gland (PF cells) to release calcitonin (CT) and serotonin (5‐HT) in response to increased external Ca ²⁺ . Recently, a calcium‐sensing receptor (CaR), similar to the G protein‐coupled receptor for external Ca ²⁺ cloned from parathyroid gland, was shown to be expressed in PF cells. Using a highly purified preparation of sheep PF cells, we have examined the electrical and biochemical processes coupling CaR activation to hormone release.
Whole‐cell recordings in the permeabilized‐patch configuration show that elevated extracellular Ca ²⁺ concentration ([Ca ²⁺ ] o ) depolarizes these cells and induces oscillations in membrane potential. In voltage clamp, high [Ca ²⁺ ] o activates a cation conductance that underlies the depolarization. This conductance is cation selective, with a reversal potential near −25 mV indicating poor ion selectivity.
The CaR expressed in these cells is activated by other multivalent cations with a rank order potency of Gd ³⁺ > Ba ²⁺ > Ca ²⁺ ≫ Mg ²⁺ . The insensitivity of these cells to high external Mg ²⁺ contrasts with the reported sensitivity of the cloned CaR from parathyroid.
Elevation of [Ca ²⁺ ] o also stimulates increases in intracellular Ca ²⁺ concentration ([Ca ²⁺ ] o ) and this effect is largely inhibited by the Ca ²⁺ channel blocker nimodipine, indicating that L‐type voltage‐gated Ca ²⁺ channels contribute to the response to elevated [Ca ²⁺ ] o .
Elevated [Ca ²⁺ ] o induces an inward current under conditions where the only permeant external cation is Ca ²⁺ , indicating that influx via the cation conductance is another source of the increases in [Ca ²⁺ ] i .
Extracellular Ca ²⁺ stimulates 5‐HT release with an EC 50 of 1.5 m m . Nimodipine blocks 90% of the Ca ²⁺ ‐induced 5‐HT release, while other inhibitors of voltage‐gated calcium channels had no effect. These data support an important role for L‐type Ca ²⁺ channels in CaR‐induced hormone secretion. Although earlier studies indicate that high [Ca ²⁺ ] o induces release of Ca ²⁺ from intracellular stores, thapsigargin‐induced depletion of these stores did not affect secretion from these cells, indicating that Ca ²⁺ influx is necessary and sufficient for the Ca ²⁺ ‐induced 5‐HT secretion.
Inhibition of protein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Ca ²⁺ ‐induced 5‐HT release by 50% while phorbol ester‐induced 5‐HT secretion was completely inhibited. Thus, PKC is an important component of the pathway linking CaR activation to hormone release. However, another as yet unknown second messenger also contributes to this pathway.
We tested the contribution of two different phospholipases to the CaR responses to determine the source of the PKC activator diacylglycerol (DAG). Selective inhibition of phosphatidylinositol‐specific phospholipase C (PI‐PLC) with U73122 had no effect on the response to elevated [Ca ²⁺ ] o . However, pretreatment with D609, a selective inhibitor of phosphatidylcholine‐specific phospholipase C (PC‐PLC), inhibited Ca ²⁺ ‐induced 5‐HT release to 50% of control indicating that phosphatidylcholine is a likely source of DAG in the response of PF cells to elevated [Ca ²⁺ ] o .
... Sr is also known to be a modulator and agonist of calcium-sensing receptors (CaRs). 42,43 These receptors are expressed throughout osteoblast development, aiding the proliferation, differentiation, and mineralization processes of osteogenesis. 44 This study established that the ALP activities of CaP-Mg and Sr-CaP-Mg were higher than that of PM after 14 days of cell culture [ Fig. 5(c)]. ...
This study investigated the corrosion resistance and biocompatibility of magnesium coated with strontium-doped calcium phosphate (Sr-CaP) for dental and orthopedic applications. Sr-CaP was coated on biodegradable magnesium using a chemical dipping method. Magnesium coated with Sr-CaP exhibited better corrosion resistance than pure magnesium. Sr-CaP-coated magnesium showed excellent cell proliferation and differentiation. Additionally, new bone formation was confirmed in vivo. Therefore, Sr-CaP-coated magnesium with reduced degradation and improved biocompatibility can be used for orthopedic and dental implant applications.
... The extracellular calcium-sensing receptor (CaSR) is a member of the G-protein-coupled receptor C family and is expressed at multiple sites including the parathyroid, thyroid, kidney, bone, and gastrointestinal tract (Brown et al., 1993;Zaidi et al., 1993;McGehee et al., 1997;Dufner et al., 2005;Theman and Collins, 2009;Riccardi and Brown, 2010). CaSR in cardiomyocytes was first discovered in 2003 (Wang et al., 2003). ...
As a common complication of many cardiovascular diseases, cardiac hypertrophy is characterized by increased cardiac cell volume, reorganization of the cytoskeleton, and the reactivation of fetal genes such as cardiac natriuretic peptide and β-myosin heavy chain. Cardiac hypertrophy is a distinguishing feature of some cardiovascular diseases. Our previous study showed that sodium ferulate (SF) alleviates myocardial hypertrophy induced by coarctation of the abdominal aorta, and these protective effects may be related to the inhibition of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signaling pathways. This study investigated the inhibitory effect and mechanism of SF on myocardial hypertrophy in spontaneously hypertensive rats (SHRs). The effects of SF on cardiac hypertrophy were evaluated using echocardiographic measurement, pathological analysis, and detection of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) expression. To investigate the mechanisms underlying the anti-hypertrophic effects of SF, the calcium-sensing receptor (CaSR), calcineurin (CaN), nuclear factor of activated T cells 3 (NFAT3), zinc finger transcription factor 4 (GATA4), protein kinase C beta (PKC-β), Raf-1, extracellular signal-regulated kinase 1/2 (ERK 1/2), and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by molecular biology techniques. Treatment with SF ameliorated myocardial hypertrophy in 26-week-old SHRs. In addition, it downregulated the levels of ANP, β-MHC, CaSR, CaN, NFAT3, phosphorylated GATA4 (p-GATA4), PKC-β, Raf-1, and p-ERK 1/2; and upregulated the levels of p-NFAT3 and MKP-1. These results suggest that the effects of SF on cardiac hypertrophy are related to regulation of the CaSR-mediated signaling pathway.
... The CaSR also increases Ca 2+ i via influx through L-type voltage-gated and transient receptor potential ion channels on the plasma membrane, 19−21 in part via a PKC-dependent mechanism. 20 In addition to canonical G protein coupling, the CaSR stimulates mitogen activated protein kinases (MAPK) downstream from G q/11 , G i/o , and β arrestin. 22 −25 In some cell types, the CaSR couples to G 12/13 , 26 although the (patho)physiological relevance is unknown. ...
The CaSR is a class C G protein-coupled receptor (GPCR) that acts as a multimodal chemosensor to maintain diverse homeostatic functions. The CaSR is a clinical therapeutic target in hyperparathyroidism and has emerged as a putative target in several other diseases. These include hyper- and hypocalcaemia caused either by mutations in the CASR gene or in genes that regulate CaSR signaling and expression, and more recently in asthma. The development of CaSR-targeting drugs is complicated by the fact that the CaSR possesses many different binding sites for endogenous and exogenous agonists and allosteric modulators. Binding sites for endogenous and exogenous ligands are located throughout the large CaSR protein and are interconnected in ways that we do not yet fully understand. This review summarizes our current understanding of CaSR physiology, signaling, and structure and how the many different binding sites of the CaSR may be targeted to treat disease.
... Unlike the effect of rising CaCC to inhibit the secretion of PTH, a rise of extracellular CaCC acts through the CaS-R of thyroidal parafollicular C-cells to stimulate the secretion of calcitonin (Garrett et al. 1995, McGehee et al. 1997. It may not, however, cause hyperplasia of C-cells (Conte-Devolx et al. 2010). ...
Five syndromes share predominantly hyperplastic glands with primary excess of hormone. 1) NEONATAL SEVERE PRIMARY HYPERPARATHYROIDISM, from homozygous mutated CASR, begins severely in utero. 2) CONGENITAL NON-AUTOIMMUNE THYROTOXICOSIS, from mutated TSHR, varies from severe with fetal onset to mild with adult onset. 3) FAMILIAL MALE-LIMITED PRECOCIOUS PUBERTY, from mutated LHR, expresses testosterone over-secretion in young boys. 4) HEREDITARY OVARIAN HYPERSTIMULATION SYNDROME, from mutated FSHR, expresses symptomatic vascular permeabilities during pregnancy. 5) FAMILIAL HYPERALDOSTERONISM TYPE IIIA, from mutated KCNJ5, presents in young children with hypertension and hypokalemia. Grouping of these 5 syndromes highlights predominant hyperplasia as a stable tissue endpoint, and as their tissue stage for all the hormone excess. Comparisons were made among this and 2 other groups of syndromes, forming a continuum of tissue staging [A-B-C]. A) Predominant oversecretions express little or no hyperplasia. B) Predominant hyperplasias express little or no neoplasia. C) Predominant neoplasias express nodules, adenomas, or cancers. Hyperplasias may progress (5 of 5) to neoplastic stages while predominant oversecrertions rarely do (1 of 6; frequencies differ P<0.02). Hyperplasias do not show tumor multiplicity (0 of 5) unlike neoplasias that do (14 of 20; P<.02). Hyperplasias express mutation of a plasma membrane-bound sensor (5 of 5) while neoplasias rarely do (RET) (1 of 18; P<.0002). In conclusion, the multiple distinguishing themes within the hyperplasias establish a robust pathophysiology. It has the shared and novel feature of mutant sensors in the plasma membrane, suggesting that these are a major contributor to hyperplasia.
... This hypothesis was based upon results demonstrating that CaSR agonists (gentamicin, neomycin, spermine, and LaCl 3 ) elevated the [Ca 2+ ]i and that the CaSR antagonist NPS2390 inhibited nifedipine-induced [Ca 2+ ]i elevation [5]. It has been reported that signals from CaSR stimulation activate NSCCs [6] and Ca 2+ release from Ca 2+ stores [7]. ...
Biallelic loss of function mutations in the CLDN16 gene cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), and chronic kidney disease. Here we report two cases of FHHNC with diverse clinical presentations and hypercalcemia in one as a novel finding. Pt#1 initially presented with urinary tract infection and failure to thrive at 5.5 months of age to another center. Bilateral nephrocalcinosis, hypercalcemia (Ca: 12.2 mg/dl), elevated parathyroid hormone (PTH) level, and hypercalciuria were detected. Persistently elevated PTH with high/normal Ca levels led to subtotal-parathyroidectomy at the age of 2.5. However, PTH levels remained elevated with progressive deterioration in renal function. At 9-year-old, she was referred to us for evaluation of hyperparathyroidism and, hypomagnesemia together with hypercalciuria, elevated PTH with normal Ca levels, and medullary nephrocalcinosis were detected. Compound heterozygosity of CLDN16 variants (c.715G>A, p.G239R; and novel c.360C>A, p.C120*) confirmed the diagnosis. Pt#2 was a 10-month-old boy, admitted with irritability and urinary crystals. Hypocalcemia, hypophosphatemia, elevated PTH and ALP, low 25(OH)D levels, and radiographic findings of rickets were detected. However, additional findings of hypercalciuria and bilateral nephrocalcinosis were inconsistent with the nutritional rickets. Low/normal serum Mg levels suggested the diagnosis of FHHNC which was confirmed genetically as a homozygous missense (c.602G > A; p.G201E) variant in CLDN16. Yet, hypocalcemia and hypomagnesemia persisted in spite of treatment. In conclusion, FHHNC may present with diverse clinical features with mild hypomagnesemia leading to secondary hyperparathyroidism with changing Ca levels from low to high. Early and accurate clinical and molecular genetic diagnosis is important for proper management.
The objectives were to determine the effects of dietary cation-anion difference (DCAD) fed to pregnant cows during the last 22 d of gestation on offspring acid-base balance, metabolism, growth, and health preweaning. A total of 132 nulliparous Holstein cows were enrolled at 250 (248 to 253) d of gestation in a randomized block design. Cows were blocked by genomic merit of energy-corrected milk yield and assigned randomly to diets varying in DCAD: +200 (P200, n = 43), −50 (N50, n = 45), or −150 (N150, n = 44) mEq/kg of dry matter (DM). Newborn calves (15 males and 28 females in P200, 22 males and 23 females in N50, and 18 males and 26 females in N150) were followed for the first 7 or 56 d of age if males or females, respectively. Measures of acid-base balance and concentrations of minerals in blood were measured in all calves on d 0 before colostrum feeding, and on d 1, 3, and 7. Each calf was fed 3.78 L of colostrum from the respective treatment, and apparent efficiency of IgG absorption was determined. All calves were weighed at birth, and females were weighed again at 21, 42, and 56 d of age. Concentrations in serum of total calcium (tCa), total magnesium (tMg), and total phosphorus (tP) were measured up to 56 d of age; intakes of milk and starter grain DM were measured daily from 21 to 56 d of age; and incidence of disease was recorded for the first 56 d of age in females. Treatment did not affect acid-base balance measured in all calves. Calves were born with metabolic and respiratory acidosis, which reversed by 1 d of age. In the first 24 h after birth, blood pH increased from 7.215 to 7.421 and bicarbonate from 26.2 to 31.7 mM, whereas partial pressure of CO2 decreased from 64.1 to 48.7 mm of Hg in all treatments. Maternal DCAD did not affect colostrum IgG content fed to calves (P200 = 95.0 vs. N50 = 91.0 vs. N150 = 97.1 ± 4.1 g/L) or apparent efficiency of IgG absorption (P200 = 33.1 vs. N50 = 33.1 vs. N150 = 34.2 ± 1.9%). Males were born heavier than females, but maternal DCAD did not affect birth weight of all calves (P200 = 37.7 vs. N50 = 37.3 vs. N150 = 37.8 ± 0.7 kg) or daily weight gain in females in the first 56 d of life (P200 = 0.80 vs. N50 = 0.81 vs. N150 = 0.77 ± 0.03 kg/d). Treatment did not affect intake of milk (P200 = 1.11 vs. N50 = 1.04 vs. N150 = 1.19 ± 0.06 kg/d) or starter grain DM (P200 = 0.27 vs. N50 = 0.27 vs. N150 = 0.21 ± 0.06 kg/d), or measures of feed efficiency. Treatment did not affect concentrations of minerals in serum, morbidity, or age at morbidity. Manipulating the DCAD of pregnant nulliparous dams during late gestation did not affect offspring performance in the first 2 mo of age.
Despite the fact that there can be argued that no single cell in the human body can be devoid of molecular tools that fit into the broad definition of an endocrine function, some organs are primarily dedicated to hormone secretion and are therefore designated endocrine glands. Under regulation by pituitary gland (reviewed on the previous chapter), three peripheral organs are exclusively devoted to endocrine functions: the thyroid, the parathyroid and the adrenal glands. This Chapter on endocrine system will cover the signaling pathways implied in these three organs, with identification of their particular and shared features.
The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor that responds to multiple endogenous agonists and allosteric modulators, including divalent and trivalent cations, L-amino acids, γ-glutamyl peptides, polyamines, polycationic peptides, and protons. The CaSR plays a critical role in extracellular calcium (Ca2+o) homeostasis, as demonstrated by the many naturally occurring mutations in the CaSR or its signaling partners that cause Ca2+o homeostasis disorders. However, CaSR tissue expression in mammals is broad and includes tissues unrelated to Ca2+o homeostasis, in which it, for example, regulates the secretion of digestive hormones, airway constriction, cardiovascular effects, cellular differentiation, and proliferation. Thus, although the CaSR is targeted clinically by the positive allosteric modulators (PAMs) cinacalcet, evocalcet, and etelcalcetide in hyperparathyroidism, it is also a putative therapeutic target in diabetes, asthma, cardiovascular disease, and cancer. The CaSR is somewhat unique in possessing multiple ligand binding sites, including at least five putative sites for the "orthosteric" agonist Ca2+o, an allosteric site for endogenous L-amino acids, two further allosteric sites for small molecules and the peptide PAM, etelcalcetide, and additional sites for other cations and anions. The CaSR is promiscuous in its G protein-coupling preferences, and signals via Gq/11, Gi/o, potentially G12/13, and even Gs in some cell types. Not surprisingly, the CaSR is subject to biased agonism, in which distinct ligands preferentially stimulate a subset of the CaSR's possible signaling responses, to the exclusion of others. The CaSR thus serves as a model receptor to study natural bias and allostery. SIGNIFICANCE STATEMENT: The calcium-sensing receptor (CaSR) is a complex G protein-coupled receptor that possesses multiple orthosteric and allosteric binding sites, is subject to biased signaling via several different G proteins, and has numerous (patho)physiological roles. Understanding the complexities of CaSR structure, function, and biology will aid future drug discovery efforts seeking to target this receptor for a diversity of diseases. This review summarizes what is known to date regarding key structural, pharmacological, and physiological features of the CaSR.
Our blood serum Ca²⁺ levels are maintained within a narrow range (Ca²⁺ homeostasis) through a complex feedback system. However, local bone marrow Ca²⁺ levels can reach high concentrations, at least transiently, due to bone resorption, which is one of the notable features of the bone marrow stroma. Bone homeostasis is maintained by both the balance between osteoblastic bone formation and osteoclastic bone resorption and the balance of mesenchymal stem cell differentiation into osteoblasts and adipocytes. It has been reported that under culture conditions of infrequent adipocyte differentiation (no treatment with insulin or dexamethasone), high extracellular Ca²⁺ enhances osteoblast but not adipocyte accumulation in bone marrow stromal cells. In contrast, under culture conditions of predominant adipocyte differentiation (treatment with insulin and dexamethasone), high extracellular Ca²⁺ enhances adipocyte but not osteoblast accumulation in bone marrow stromal cells. Thus, the increased extracellular Ca²⁺ caused by bone resorption might enhance osteoblast development to reform missing bone under conditions of infrequent adipocyte differentiation (such as the normal physiological state) and might accelerate adipocyte accumulation instead of osteoblastic bone formation under conditions of predominant adipocyte differentiation (such as aging, obesity, use of glucocorticoids, and postmenopause). Moreover, increased adipocyte accumulation in bone marrow suppresses lymphohematopoiesis and contributes to a dysfunction of osteogenesis.
Human calcitonin release is promoted by elevated extracellular Ca²⁺ (Ca²⁺o) concentration acting, at least in part, via the calcium-sensing receptor (CaSR). The CaSR is positively modulated by L-amino acids including the aromatic amino acids L-Phe and L-Trp. To investigate the effect of L-amino acids on human calcitonin secretion we selected thyroid TT cells and exposed them to various Ca²⁺o concentrations in the absence or presence of L-Phe, plasma-like mixtures of L-amino acids, or the clinically effective positive modulator (calcimimetic), cinacalcet. In the presence of L-Phe or plasma-like mixtures of amino acids, TT cells exhibited enhanced Ca²⁺o sensitivity in assays of calcitonin release and intracellular Ca²⁺ (Ca²⁺i) mobilization. Furthermore, the effect of elevated Ca²⁺o and L-Phe on calcitonin release was markedly suppressed by the calcilytic NPS-2143. These effects were dependent upon CaSR-mediated activation of Gq/11 as revealed by the specific inhibitor YM-254890. The findings support the hypothesis that calcitonin release is stimulated by increases in plasma L-amino acid levels as well as elevated extracellular Ca²⁺. They also demonstrate that stimulated calcitonin release as well as basal levels of calcitonin secretion are mediated by a CaSR:Gq/11 signaling mechanism.
Divalent and trivalent salts exhibit a complex taste profile. They are perceived as being astringent/drying, sour, bitter, and metallic. We hypothesized that human bitter-taste receptors may mediate some taste attributes of these salts. Using a cell-based functional assay, we found that TAS2R7 responds to a broad range of divalent and trivalent salts, including zinc, calcium, magnesium, copper, manganese, and aluminum, but not to potassium, suggesting TAS2R7 may act as a metal cation receptor mediating bitterness of divalent and trivalent salts. Molecular modeling and mutagenesis analysis identified 2 residues, H943.37 and E2647.32, in TAS2R7 that appear to be responsible for the interaction of TAS2R7 with metallic ions. Taste receptors are found in both oral and extraoral tissues. The responsiveness of TAS2R7 to various mineral salts suggests it may act as a broad sensor, similar to the calcium-sensing receptor, for biologically relevant metal cations in both oral and extraoral tissues.
This chapter describes the properties and functions of the calcium‐sensing receptor (CaSR), a G‐protein coupled receptor (GPCR) that plays a central role in Ca²⁺o homeostasis by virtue of its ability to sense Ca²⁺o. Knowledge of CaSR structure and activation was based upon extracellular domain (ECD) crystal structures of the related metabolic glutamate receptors (mGluRs) in active and inactive conformations. Protein sequence alignment and phylogenetic analysis provide evidence of a functional association of the CaSR, cloned initially from parathyroid gland, with the vertebrate skeleton that has an ancient origin. The single copy CASR gene maps to chromosome 3q in humans. The CaSR is subject to biased signaling, the phenomenon by which distinct ligands stabilize the receptor conformation such that it activates a preferred downstream signaling pathway. There is the potential for CaSR allosteric modulators that cause biased signaling and promote selective activity in different tissues to be useful for specific disease states.
This chapter describes the properties and functions of the Ca2+o-sensing receptor (CaSR), a G-protein coupled receptor (GPCR) that plays a central role in Ca2+o homeostasis by virtue of its capacity to sense extracellular ionized calcium Ca2+o. The CaSR was originally cloned from bovine parathyroid gland and then from several tissues of a variety of other species, including humans. The chapter describes the CaSR gene properties, biogenesis and structure of the CaSR. It explains the binding and activation of CaSR-mediated signaling. In addition to the roles that it plays in tissues participating in Ca2+o homeostasis, the CaSR is expressed in and modulates the functions of numerous other cells uninvolved in mineral ion homeostasis.
Bacterial infection are serious complications for biomedical implants in the orthopedic and dental fields, and the ideal implants should combine good antibacterial ability and bioactivity. In this paper, we have fabricated the strontium/copper substituted hydroxyapatite (SrCuHA) coating on the commercially pure titanium (CP-Ti) and studied their effect on antibacterial and in vitro cytocompatible properties. Cu was incorporated into HA in order to improve its antimicrobial properties. Sr was added as a second binary element to improve the biocompatibility. The structural and morphological characteristics of the SrCuHA coatings were investigated using various analytical techniques. The presence of Sr2+ and Cu2+ in solution led to reduced roughness of the coating and finer nucleus size formed. The results highlight that Sr2+ and Cu2+ were homogenously incorporated into HA lattice to form SrCuHA coatings. Inductively coupled plasma mass spectrometry (ICP-MS) was used for the leach out analysis of the samples. A low contact angle value revealed the hydrophilic nature. In vitro electrochemical corrosion studies indicated that the SrCuHA coating sustain in the stimulated body-fluid (SBF), exhibiting superior corrosion resistance with a lower corrosion penetration rate than the bare CP-Ti substrate. The SrCuHA coatings can kill Escherichia coli to a certain extent during the first few days, which might be due to the Cu substitution in the coating. An enhancement of in vitro osteoblast adhesion, proliferation, and alkaline phosphatase activity was observed, which could lead to the optimistic orthopedic and dental applications.
The neuromodulatory inward current (IMI) generated by crab Cancer borealis stomatogastric ganglion neurons is an inward current whose voltage dependence has been shown to be crucial in the activation of oscillatory activity of the pyloric network of this system. It has been previously shown that IMI loses its voltage dependence in conditions of low extracellular calcium, but that this effect appears to be regulated by intracellular calmodulin. Voltage dependence is only rarely regulated by intracellular signaling mechanisms. Here we address the hypothesis that the voltage dependence of IMI is mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce IMI voltage dependence in normal Ca²⁺, but that, in conditions of low Ca²⁺, calmodulin activators do not restore IMI voltage dependence. Further, we show evidence that CaMKII alters IMI voltage dependence. These results suggest that calmodulin is necessary but not sufficient for IMI voltage dependence. We therefore hypothesize that the Ca²⁺/calmodulin requirement for IMI voltage dependence is due to an active sensing of extracellular calcium by a GPCR family calcium-sensing receptor (CaSR) and that the reduction in IMI voltage dependence by a calmodulin inhibitor is due to CaSR endocytosis. Supporting this, preincubation with an endocytosis inhibitor prevented W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride)-induced loss of IMI voltage dependence, and a CaSR antagonist reduced IMI voltage dependence. Additionally, myosin light chain kinase, which is known to act downstream of the CaSR, seems to play a role in regulating IMI voltage dependence. Finally, a Gβγ-subunit inhibitor also affects IMI voltage dependence, in support of the hypothesis that this process is regulated by a G-protein-coupled CaSR.
Bacterial infection are serious complications for biomedical implants in the orthopedic and dental fields, and the ideal implants should combine good antibacterial ability and bioactivity. In this paper, we have fabricated the strontium/copper substituted hydroxyapatite (SrCuHA) coating on the commercially pure titanium (CP-Ti) and studied their effect on antibacterial and in vitro cytocompatible properties. Cu was incorporated into HA in order to improve its antimicrobial properties. Sr was added as a second binary element to improve the biocompatibility. The structural and morphological characteristics of the SrCuHA coatings were investigated using various analytical techniques. The presence of Sr2+ and Cu2+ in solution led to reduced roughness of the coating and finer nucleus size formed. The results highlight that Sr2+ and Cu2+ were homogenously incorporated into HA lattice to form SrCuHA coatings. Inductively coupled plasma mass spectrometry (ICP-MS) was used for the leach out analysis of the samples. A low contact angle value revealed the hydrophilic nature. In vitro electrochemical corrosion studies indicated that the SrCuHA coating sustain in the stimulated body-fluid (SBF), exhibiting superior corrosion resistance with a lower corrosion penetration rate than the bare CP-Ti substrate. The SrCuHA coatings can kill Escherichia coli to a certain extent during the first few days, which might be due to the Cu substitution in the coating. An enhancement of in vitro osteoblast adhesion, proliferation, and alkaline phosphatase activity was observed, which could lead to the optimistic orthopedic and dental applications.
The metastases of breast cancer cells to bone result in osteolysis and release of Ca2+. Ca2+ as a primary signal transducer can regulate the expression patterns of cell signaling systems. The extracellular calcium ion concentration sensing receptor CaR is a 123 kDa G-protein coupled membrane protein that resides within caveolin-rich regions as a dimer. CaR is involved in regulating several cellular processes such as proliferation, differentiation, secretion, and apoptosis. Calbindin-D-28k is a 28 kDa high affinity calcium-binding protein and it is involved in regulating the intracellular calcium ion concentration, [Ca2+](i), and thus influences signal transduction. The role of CaR in sensing and responding to extracellular calcium ion concentration, [Ca2+](o), and neomycin sulfate, and spatial interactions of CaR with calbindin-D-28k in MCF-7 human breast cancer cells were studied. Fura-2 loaded MCF-7 cells were exposed to increasing concentrations of CaCl2, or neomycin sulfate and the [Ca2+], was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 or neomycin sulfate caused an increase in [Ca2+](i). The normalized dose response curves fitting yielded Hill coefficient values of 4.32+/-0.63 and 1.49+/-0.14 for Ca2+ and neomycin sulfate respectively, thus indicating highly cooperative, 4-5 binding sites for Ca2+ and 1-2 binding site(s) for neomycin sulfate on CaR. The EC50 values were 21+/-1.6 mM and 43+/-3.5 muM for CaCl2, and neomycin sulfate respectively. The confocal microscopy data, obtained by using a highly sensitive tyramide signal amplification technology for immunofluorescence detection, showed CaR and calbindin-D-28k were co-localized when cells were exposed to 200 muM neomycin sulfate, whereas in control cells there was no co-localization of these two proteins. We hypothesize that sensing and responses to increasing [Ca2+], that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D-28k. towards CaR.
The key property in the preparation of a biomaterial is to facilitate the regeneration and/or replacement of injured organs and tissues. To obtain such a biomaterial, strontium and manganese co-substituted hydroxyapatite (SrMnHA) ceramic coatings on commercially pure titanium (CP-Ti) were successfully prepared by cathodic electrodeposition method. The formation of SrMnHA coating was investigated using Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, energy dispersive X-ray analysis and inductively coupled plasma. Co-doping of Sr2 + and Mn2 + ions decreased hydroxyapatite unit cell volume and grain sizes. Obtained coatings were crack-free and dense, which led to the decrease in the corrosion current densities of CP-Ti in physiological solutions. The dissolution tests performed on SrMnHA resulted in the leaching of Sr2 + and Mn2 + at low levels. SrMnHAs have moderately hydrophilic surfaces with a contact angle of 23.8°. Additionally, the MC3T3-E1 cells show better cell morphology, adhesion, spreading, proliferation and expression of alkaline phosphatase on SrMnHA than on HA. The excellent biocompatibility of SrMnHA is mainly attributed to a probable effect of a combination of good surface wettability and ion release (Sr2 + and Mn2 +). Results suggest that SrMnHA composite-coated CP-Ti can be a potential candidate for orthopaedic applications.
Cell surface sensors for extracellular Ca 2+ and Mg 2+ 51 provide an important mechanism for the regulation of diverse physiological processes by extracellular divalent mineral ions. [53] ; [190] ; [402] These ion sensors function as “calciostats” for Ca 2+ and/or Mg 2+ that not only regulate divalent mineral metabolism at the level of the whole organism but also control a variety of other cellular processes (e.g., salt and water handing in various epithelia and cell proliferation-differentiation) in terrestrial and aquatic animals, as well as in plants. This chapter will focus on the role of the extracellular calcium-sensing receptor (CaSR) in the mammalian parathyroid, kidney, and other tissues participating in divalent mineral ion homeostasis. The unique properties of the mammalian CaSR include: (1) Having extracellular Ca 2+ and Mg 2+ as its primary physiological ligands, establishing that ions can function as first messengers. (2) Responding with a millimolar EC 50 , close to the normal plasma ionized Ca 2+ concentration, but several orders of magnitude higher than that for ligands of other G protein–coupled receptors. (3) Possessing a remarkable ability to detect small deviations from the normal ionized calcium concentration of 1.1–1.3 mM, making it an ideal sensor for Ca 2+ , functioning as a “calciostat.”
Calcium and vitamin D have been inextricably linked for decades by their combined roles in promoting mineral ion homeostasis and the growth and remodeling of the skeleton. This chapter briefly reviews the structure and functional properties of the calcium sensing receptor (CaSR) and describes how it regulates, in concert with 1,25(OH)2D3, the various cells types expressing both the CaSR and vitamin D receptor (VDR) that participate in the maintenance of Ca2+ homeostasis as well as bone growth and development. An important advance in this area has been the identification and characterization of the calcium sensing receptor CaSR. As a result, it has become possible to understand the role of extracellular calcium ions as not just serving as a passive participant in processes such as bone development and renal calcium excretion, but also acting as an extracellular first messenger. As such, calcium can actively regulate the tissues that make up the systems governing mineral ion and bone metabolism. Finally, this chapter presents selected examples of the roles of the CaSR and VDR when they are coexpressed in selected cell types uninvolved in Ca2+ homeostasis.
The calcium sensing receptor (CaSR) was cloned for the first time from a bovine parathyroid cDNA library by Brown, Hebert, and colleagues and reported in 1993 [1]. This event paved the way for far deeper understandings of the regulatory mechanisms that underpin the control of whole body calcium and bone metabolism, particularly in the parathyroid and kidney, along with various other tissues including gut and bone [2].
In this study calcium phosphate coatings with different amounts of strontium (Sr) were prepared using a biomineralization method. The incorporation of Sr changed the composition and morphology of coatings from plate-like to sphere-like morphology. Dissolution testing indicated that the solubility of the coatings increased with increased Sr concentration. Evaluation of extracts (with Sr concentrations ranging from 0 to 2.37μg/mL) from the HA, 0.06Sr, 0.6Sr, and 1.2Sr coatings during in vitro cell cultures showed that Sr incorporation into coatings significantly enhanced the ALP activity in comparison to cells treated with control and HA eluted media. These findings show that calcium phosphate coatings could promote osteogenic differentiation even in a low amount of strontium.
Copyright © 2015. Published by Elsevier B.V.
Background and purpose:
Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies.
Experimental approach:
We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca(2+) i ) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression.
Key results:
Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca(2+) i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G(670) E).
Conclusions and implications:
The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses.
The endocrine activity of the thyroid gland is accomplished by its follicular and parafollicular cells. In these cells, numerous G proteins-dependent pathways are active and potentially could be regulated by a 33-kDa cytoplasmic protein phosducin, which interacts with the Gβ subunit and may compete with Gα or Gβγ dimer effectors. Significant expression of phosducin has been shown in the retina, pineal gland, and some neurons. Here, we studied postoperative thyroid tissue samples collected from patients with nodular goiter and 2 thyroid-derived cell lines for the presence of phosducin. Using reverse transcription PCR with product sequencing and highly sensitive immunodetection we identified phosducin mRNA and protein in the thyroid gland and parafollicular C TT cells, but not in the follicular Nthy-ori 3-1 cell line. We also observed that siRNA-mediated silencing of phosducin gene expression decreased Ca(2+)-stimulated secretion of calcitonin and serotonin by TT cells.
Dietary inorganic phosphate (Pi) is the most important factor in the regulation of renal Pi excretion. Recent studies suggest the presence of an enteric-renal signaling axis for dietary Pi as well as the existence of a mechanism by which the intestine detects changes in luminal Pi concentrations. The mechanisms of intestinal Pi sensing, however, are unknown. In the present study, we focused on Pi depletion signals and investigated the effects of dietary components on intestinal Pi sensing. After feeding rats experimental diets for 3 days, we investigated urinary Pi excretion and plasma biochemical parameters. Renal Pi excretion was suppressed in rats fed a low-Pi diet (0.02% Pi). Elimination of dietary calcium (Ca) completely blocked the suppression of Pi excretion, suggesting that the presence of Ca is essential for the Pi depletion signal. Furthermore, a minimum Ca content of more than 0.02% was necessary for the Pi depletion signal. Magnesium, lanthanum, and strontium, which are agonists of calcium sensing receptor, instead of Ca, reduced Pi excretion. Therefore, dietary Ca appears to be important for the Pi depletion-sensing mechanism in the gastrointestinal tract. In addition, the calcium sensing receptor may be involved in the Pi depletion signal. J. Med. Invest. 61: 162-170, February, 2014.
Regulation of the synthesis and/or secretion of hypocalcemic and hypercalcemic hormones by calcium-sensing receptor (CaSR) is believed to be a major pathway for maintaining Ca(2+) homeostasis in vertebrates, based primarily on findings in mammals. However, understanding the evolution of this physiological process requires that it be described in non-mammalian species. Here, we describe the use of zebrafish as a model to investigate whether CaSR contributes to body fluid Ca(2+) homeostasis by regulating synthesis of hypercalcemic (PTH1 and PTH2) and hypocalcemic hormones (stanniocalcin (STC-1)). We report that PTH1, but not PTH2, increases Ca(2+) uptake through stimulating the expression of the gene encoding epithelial Ca(2+) channel (ecac). Furthermore, we demonstrate that CaSR, as a Ca(2+) sensor, may differently affect stc-1 and pth1 expressions, thereby suppressing ecac expression and Ca(2+) uptake. Finally, we show that CaSR knockdown has time-dependent effects on STC-1 and PTH1 expression, and these two hormones have mutual effects on the expressions, thus forming a possible counterbalance. These findings enhance our understanding of CaSR-PTH-STC control of Ca(2+) homeostasis in vertebrates.
The human calcium‐sensing receptor ( CaSR ) is widely expressed in the body, where its activity is regulated by multiple orthosteric and endogenous allosteric ligands. Each ligand stabilizes a unique subset of conformational states, which enables the CaSR to couple to distinct intracellular signalling pathways depending on the extracellular milieu in which it is bathed. Differential signalling arising from distinct receptor conformations favoured by each ligand is referred to as biased signalling. The outcome of CaSR activation also depends on the cell type in which it is expressed. Thus, the same ligand may activate diverse pathways in distinct cell types. Given that the CaSR is implicated in numerous physiological and pathophysiological processes, it is an ideal target for biased ligands that could be rationally designed to selectively regulate desired signalling pathways in preferred cell types.
Linked Articles
This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue‐5
The cloning of a G protein-coupled, extracellular Ca2+ (Ca
o
2+
)-sensing receptor (CaR) has afforded a molecular basis for a number of the known effects of Ca
o
2+
on tissues involved in maintaining systemic calcium homeostasis, especially parathyroid gland and kidney. In addition to providing molecular tools for showing that CaR messenger RNA and protein are present within these tissues, the cloned CaR has permitted documentation that several human diseases are the result of inactivating or activating mutations of this receptor as well as generation of mice that have targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in these patients as well as in the CaR “knockout” mice have elucidated considerably the CaR’s physiological roles in mineral ion homeostasis. Nevertheless, a great deal remains to be learned about how this receptor regulates the functioning of other tissues involved in Ca
o
2+
metabolism, such as bone and intestine. Further study of these human diseases and of the mouse models will doubtless be useful in gaining additional understanding of the CaR’s roles in these latter tissues. Furthermore, we understand little of the CaR’s functions in tissues that are not directly involved in systemic mineral ion metabolism, where the receptor probably serves diverse other roles. Some of these functions may be related to the control of intra- and local extracellular concentrations of Ca2+, while others may be unrelated to either systemic or local ionic homeostasis. In any case, the CaR and conceivably additional receptors/sensors for Ca2+ or other extracellular ions represent versatile regulators of a wide variety of cellular functions and represent important targets for novel classes of therapeutics.
A constant extracellular Ca2+ concentration is required for numerous physiological functions at tissue and cellular levels. This suggests that minor changes in Ca2+ will be corrected by appropriate homeostatic systems. The system regulating Ca2+ homeostasis involves several organs and hormones. The former are mainly the kidneys, skeleton, intestine and the parathyroid glands. The latter comprise, amongst others, the parathyroid hormone, vitamin D and calcitonin. Progress has recently been made in the identification and characterisation of Ca2+ transport proteins CaT1 and ECaC and this has provided new insights into the molecular mechanisms of Ca2+ transport in cells. The G-protein coupled calcium-sensing receptor, responsible for the exquisite ability of the parathyroid gland to respond to small changes in serum Ca2+ concentration was discovered about a decade ago. Research has focussed on the molecular mechanisms determining the serum levels of 1,25(OH)2D3, and on the transcriptional activity of the vitamin D receptor. The aim of recent work has been to elucidate the mechanisms and the intracellular signalling pathways by which parathyroid hormone, vitamin D and calcitonin affect Ca2+ homeostasis. This article summarises recent advances in the understanding and the molecular basis of physiological Ca2+ homeostasis.
Cardiac injury is a common pathological change frequently accompanied by diabetes mellitus. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiac tissue. However, the functional role of CaSR in diabetic cardiac injury remains unclear. The present study was designed to investigate the relationship between CaSR activation and diabetes-induced cardiac injury. Diabetic model was successfully established by administration of streptozotocin (STZ) in vivo, and cardiomyocyte injury was simulated by 25.5 mM glucose in vitro. Apoptotic rate, intracellular calcium concentration ([Ca(2+)]i) and the expression of Bcl-2, Bax, extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 were examined. We demonstrated a significant increase in left ventricular end-diastolic pressure (LVEDP) as well as decrease in maximum rate of left ventricular pressure rise and fall (±dp/dtmax), and left ventricular systolic pressure (LVSP), apoptosis of cardiomyocytes was also observed by TUNEL staining. In vitro, 25.5 mM glucose-induced apoptosis was detected by flow cytometry in neonatal rat cardiomyocytes. Further results showed that 25.5 mM glucose significantly increased [Ca(2+)]i, up-regulated the expression of Bax, P-ERK and P-JNK, and suppressed Bcl-2 expression. However, the above deleterious changes were further confirmed when co-treatment with CaSR agonist GdCl3 (300 µM). But the effects of GdCl3 were attenuated by 10 µM NPS-2390, a specific CaSR inhibitor. When CaSR was silence by siRNA transfection, the effects of high glucose were inhibited. These results suggest that CaSR activation could lead to the apoptosis of cardiomyocytes in diabetic cardiac injury through the induction of calcium overload, the activation of the mitochondrial, and mitogen-activated protein kinase pathway.
Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.
Calcium (Ca) and magnesium (Mg) homeostasis are interrelated and share common regulatory hormones, including parathyroid hormone (PTH) and vitamin D. However, the role of the calcium-sensing receptor (CaSR) in Mg homeostasis in vivo is not well-understood. We sought to investigate the interactions between Mg and Ca homeostasis using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR) (double knockout, DKO). Serum Mg is lower in PTH KO and DKO mice than in WT mice on standard chow, while supplemental dietary Ca leads to equivalent Mg levels for all three genotypes. Mg loading increases serum Mg in all genotypes; however, the increase in serum Mg is most pronounced in the DKO mice. Serum Ca is increased with Mg loading in the PTH KO and DKO mice, but not in the WT mice. Here too, the hypercalcemia is much greater in the DKO mice. Serum and urinary phosphate are greatly reduced during Mg loading, likely due to intestinal chelation of phosphate by Mg. Mg loading decreases serum PTH in WT mice and increases serum calcitonin in both WT and PTH KO mice but not DKO mice. Furthermore, Mg loading elevates serum 1,25-dihydroxyvitamin D in all genotypes, with greater effects in PTH KO and DKO mice, possibly due to reduced levels of serum phosphorus and FGF23. These hormonal responses to Mg loading and the CaSR's role in regulating renal function may help to explain changes in serum Mg and Ca found during Mg loading.
Calcium balance is essential for a multitude of physiological processes, ranging from cell signaling to maintenance of bone health. Adequate intestinal absorption of calcium is a major factor for maintaining systemic calcium homeostasis. Recent observations indicate that a reduction of gastric acidity may impair effective calcium uptake through the intestine. This article reviews the physiology of gastric acid secretion, intestinal calcium absorption, and their respective neuroendocrine regulation and explores the physiological basis of a potential link between these individual systems.
Calcium and phosphorus homeostasis are highly interrelated and share common regulatory hormones, including FGF23. However, little is known about calcium's role in the regulation of FGF23. We sought to investigate the regulatory roles of calcium and phosphorus in FGF23 production using genetic mouse models with targeted inactivation of PTH (PTH KO) or both PTH and the calcium-sensing receptor (CaSR) (PTH-CaSR DKO). In wild-type, PTH KO, and PTH-CaSR DKO mice, elevation of either serum calcium or phosphorus by intraperitoneal injection increased serum FGF23 levels. In PTH KO and PTH-CaSR DKO mice, however, increases in serum phosphorus by dietary manipulation were accompanied by severe hypocalcemia, which appeared to blunt stimulation of FGF23 release. Increases in dietary phosphorus in PTH-CaSR DKO mice markedly decreased serum 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) despite no change in FGF23, suggesting direct regulation of 1,25(OH)(2)D(3) synthesis by serum phosphorus. Calcium-mediated increases in serum FGF23 required a threshold level of serum phosphorus of about 5 mg/dL. Analogously, phosphorus-elicited increases in FGF23 were markedly blunted if serum calcium was less than 8 mg/dl. The best correlation between calcium and phosphorus and serum FGF23 was found between FGF23 and the calcium x phosphorus product. Since calcium stimulated FGF23 production in the PTH-CaSR DKO mice, this effect cannot be mediated by the full length CaSR. Thus the regulation of FGF23 by both calcium and phosphorus appears to be fundamentally important in coordinating the serum levels of both mineral ions and ensuring that the calcium x phosphorus product remains within a physiological range.
The calcium-sensing receptor (CaSR)-specific allosteric modulator, cinacalcet, has revolutionized treatment of secondary hyperparathyroidism in patients with chronic kidney disease. However, its application is limited to patients with end-stage renal disease due to hypocalcemic side effects presumably caused by CaSR-mediated calcitonin secretion from thyroid parafollicular C-cells. These hypocalcemic side effects might be dampened by compounds, which bias the signaling of CaSR causing similar therapeutic effects as cinacalcet without stimulating calcitonin secretion. Since biased signaling of CaSR is poorly understood the objective of the present study was to investigate biased signaling of CaSR using rat medullary thyroid carcinoma 6-23 cells as a model of thyroid parafollicular C-cells. By doing concentration-response experiments we focused on the ability of two well-known CaSR agonists, calcium and strontium, to activate six different signaling entities including G(q/11) signaling, G(i/o) signaling, G(s) signaling, extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling, intracellular calcium ([Ca(2+)](i)) mobilization, and calcitonin secretion. The experiments showed that strontium biases CaSR signaling towards G(i/o) and ERK1/2 signaling, and possibly another pathway independent of G(q/11) signaling and [Ca(2+)](i) mobilization. Interestingly, the potency of strontium-stimulated calcitonin secretion was elevated compared to calcium. Combining these results with experiments investigating signaling pathway components involved in calcitonin secretion, we found that the enhanced potency of strontium-mediated calcitonin secretion was caused by a different signaling pattern than produced by calcium. Together, our results suggest that calcitonin secretion can be affected by CaSR-stimulated signaling bias, which may be utilized to develop novel drugs for treatment of secondary hyperparathyroidism.
While most central nervous system (CNS) neurons receive the majority of their input through direct synaptic connections, there is evidence suggesting that they are in fact susceptible to modulation by changes in extracellular ionic composition during both physiological and pathophysiological conditions. In many regions of the CNS, there exists an identified extracellular receptor with the ability to sense levels of cations, most notably calcium. Here we report that activation of this calcium receptor (CaR) in neurons of the subfornical organ (SFO), a forebrain circumventricular structure, results in profound effects on neuronal excitability through metabotropic actions on a non-selective cation channel. Activation of the CaR by NPS R-467, an allosteric agonist of the CaR, evoked depolarizing plateau potentials ranging in duration from 5 to 30 s. Similarly, 5 mm CaCl2 caused depolarization and increased action potential frequency. NPS R-467 was found to activate a non-selective cation channel with a reversal potential of –48 ± 4 mV, and a slope conductance of 2.54 ± 11 nS. This current could also be elicted by spermine, a known agonist of the CaR. CaR-mediated activation of this channel was dependent upon both G proteins and intracellular Ca2+ signalling, as disruption of these pathways through inclusion of guanosine 5′-O-(2-thiodiphosphate) (GDP-β-S) and 1,2-bis(2-aminophenoxy)ethane-N,N,N ′,N ′-tetraacetic acid (BAPTA), respectively, in the recording pipette prevented activation of the current. Microinjection of CaR agonists into the SFO of anaesthetized rats resulted in a significant, site-specific elevation of blood pressure (mean area under curve, 141 ± 50 mmHg.s). Together, these results indicate that the CaR can play an important role in transducing the effects of changes in the extracellular ionic composition, and that these effects have implications for the neural control of fluid balance.
Calcium (Ca 2+) is the most universal carrier of biological signals: it modulates cell life from its origin at fertilization to its end in the apoptotic process. The signaling function of Ca 2+ has had an unusual history. Discovered serendipitously at the end of the nineteenth century, it received almost no attention for decades. After its rediscovery in the 1960s, it grew in popularity and importance at an exponential pace. As progress advanced, it was recognized that the signaling function of Ca 2+ had a number of unique properties. One is the ability of Ca 2+ to act both as a first and as a second messenger. Ca 2+ may recognize a canonical seven-transmembrane domain receptor at the external side of the plasma membrane, initiating an internal signaling cascade that may even involve Ca 2+ itself. Another distinctive property of the Ca 2+ signal is autoregulation. It occurs at the transcriptional and post-translational levels, as the expression and activity of a number of proteins involved in the transport of Ca 2+ and in the processing of its signal are regulated by Ca 2+ itself. Most importantly, the Ca 2+ signal shows ambivalence. Cells need Ca 2+ to correctly carry out most of their important functions. To this aim, they have developed a sophisticated array of means to carefully control its concentration and movements. But damage of various degrees, up to cell death, invariably follows the failure of the cell systems to properly control Ca 2+ .
A hallmark of chronic kidney disease is hyperphosphatemia due to renal phosphate retention. Prolonged parathyroid gland exposure to hyperphosphatemia leads to secondary hyperparathyroidism characterized by hyperplasia of the glands and excessive secretion of parathyroid hormone (PTH), which causes renal osteodystrophy. PTH secretion from the parathyroid glands is controlled by the calcium-sensing receptor (CaSR) that senses extracellular calcium. High extracellular calcium activates the CaSR causing inhibition of PTH secretion through multiple signaling pathways. Cinacalcet is the first drug targeting the CaSR and can be used to effectively control and reduce PTH secretion in PTH-related diseases. Cinacalcet is a positive allosteric modulator of the CaSR and affects PTH secretion from parathyroid glands by shifting the calcium-PTH concentration-response curve to the left. One major disadvantage of cinacalcet is its hypocalcemic side effect, which may be caused by increased CaSR-mediated calcitonin secretion from the thyroid gland. However, multiple studies indicate that PTH and calcitonin secretion are stimulated by different signaling pathways, and therefore it might be possible to develop a CaSR activating drug that selectively activates signaling pathways that inhibit PTH secretion while having no effect on signaling pathways involved in calcitonin secretion. Such a drug would have the same therapeutic value as cinacalcet in lowering PTH secretion while eliminating the side effect of hypocalcemia by virtue of it not affecting calcitonin secretion. The present review will focus on recent advancements in understanding signaling and biased signaling of the CaSR, and how that may be utilized to discover new and smarter drugs targeting the CaSR.
The extracellular calcium ([Formula: see text])-sensing receptor (CaSR) was the first GPCR identified whose principal physiological ligand is an ion, namely extracellular Ca(2+). It maintains the near constancy of [Formula: see text] that complex organisms require to ensure normal cellular function. A wealth of information has accumulated over the past two decades about the CaSR's structure and function, its role in diseases and CaSR-based therapeutics. This review briefly describes the CaSR and key features of its structure and function, then discusses the extracellular signals modulating its activity, provides an overview of the intracellular signaling pathways that it controls, and, finally, briefly describes CaSR signaling both in tissues participating in [Formula: see text] homeostasis as well as those that do not. Factors controlling CaSR signaling include various factors affecting the expression of the CaSR gene as well as modulation of its trafficking to and from the cell surface. The dimeric cell surface CaSR, in turn, links to various heterotrimeric and small molecular weight G proteins to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. CaSR signaling is impacted by its interactions with several binding partners in addition to signaling elements per se (i.e., G proteins), including filamin-A and caveolin-1. These latter two proteins act as scaffolds that bind signaling components and other key cellular elements (e.g., the cytoskeleton). Thus CaSR signaling likely does not take place randomly throughout the cell, but is compartmentalized and organized so as to facilitate the interaction of the receptor with its various signaling pathways.
The aim of this study was to explore the association of parathyroid hormone (PTH) gene Bst BI polymorphism, calciotropic hormone levels, and dental fluorosis of children. A case-control study was conducted in two counties (Kaifeng and Tongxu) in Henan Province, China in 2005-2006. Two hundred and twenty-five children were recruited and divided into three groups including dental fluorosis group (DFG), non-dental fluorosis group (NDFG) from high fluoride areas, and control group (CG). Urine fluoride content was determined using fluoride ion selective electrode; PTH Bst BI were genotyped using PCR-RFLP; osteocalcin (OC) and calcitonin (CT) levels in serum were detected using radioimmunoassay. Genotype distributions were BB 85.3% (58/68), Bb 14.7% (10/68) for DFG; BB 77.6% (52/67), Bb 22.4% (15/67) for NDFG; and BB 73.3% (66/90), Bb 27.7% (24/90) for CG. No significant difference of Bst BI genotypes was observed among three groups (P > 0.05). Serum OC and urine fluoride of children were both significantly higher in DFG and NDFG than in CG (P < 0.05, respectively), while a similar situation was not observed between DFG and NDFG in high fluoride areas (P > 0.05). Serum OC level of children with BB genotype was significantly higher compared to those with Bb genotype in high fluoride areas (P < 0.05). However, no significant difference of serum CT or calcium (Ca) was observed. In conclusion, there is no correlation between dental fluorosis and PTH Bst BI polymorphism. Serum OC might be a more sensitive biomarker for detecting early stages of dental fluorosis, and further studies are needed.
After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off" conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G(q/11) protein, G(i/o) protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G(q/11) and G(i/o) signaling, ERK1/2 is activated through recruitment of β-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.
The purpose of this study was to test the effects of a series of strontium-substituted HA (Sr-HA) ceramics (0, 1, 5, and 10 mol% Sr substitution) on osteoblasts, thereby demonstrating whether strontium incorporation with HA would favor osteoblast metabolism. Rat primary osteoblasts were cultured with culture media containing ions released from the Sr-HA ceramics as they dissolved. MTT test, alkaline phosphatase activity, osteoblast transcription factor gene (cbfa1) expression and Alizarin Red staining were conducted at different time-points. There is no significant difference in cell proliferation between groups. However, compared with HA group, Sr-HA groups presented significant enhancement with regard to ALP activity, cbfa1 mRNA expression, and mineralization nodules. Among Sr-HA groups, 5 and 10% groups showed much better performances in ALP activity, cbfa1 mRNA expression, and mineralization nodules than 1% group, however, no significant difference was found between 5 and 10% groups. This study has demonstrated that Sr incorporation in HA ceramic enhanced osteoblastic cell differentiation and mineralization. However, further detailed studies are needed to understand the mechanistic effects of this Sr incorporation on osteoblastic cells and the optimal percentage of calcium should be substituted with strontium in HA.
The endocrine system is integral for normal growth, development, and reproduction. Endocrinopathies can vary from insignificant to lethal and clinical signs, diagnoses, pathology, and treatment can be straightforward or complicated and convoluted. This chapter presents recent data, literary reviews, and available case reports regarding the endocrine systems of sheep and goats to provide an overview of endocrine function and pathology to aid the clinician in diagnosing and treating endocrine abnormalities.
The calcium-sensing receptors (CaSRs) exist in a variety of tissues and cells. In 2001, Canaff et al. first identified its expression in liver tissue and primary cultured hepatocytes, and demonstrated that GdCl(3) (a specific agonist of CaSR) can cause an increase in intracellular calcium and bile flow. However, authors did not elucidate its mechanisms. Therefore, this study sought to detect CaSR expression in BRL cell line, which is derived from buffalo rat liver, and to reveal the cellular signal transduction pathway by which the CaSR activation results in increased intracellular calcium by BRL cells. In this study, the expression and distribution of CaSR were detected by RT-PCR, Western blotting, and immunofluorescence, and the intracellular calcium concentration [Ca(2+)](i) was measured using LCSM. The results showed that CaSR mRNA and protein were expressed in BRL cells and mainly distributed in cell membrane and cytoplasm. Increased extracellular calcium or GdCl(3) could increase intracellular calcium concentration and CaSR expression. Moreover, this increase of [Ca(2+)](i) could be inhibited or even abolished by U73122 (a specific inhibitor of PLC), 2-APB (an inhibitor of IP(3) receptor), and thapsigargin (an inhibitor of endoplasmic reticulum calcium pump). In conclusion, CaSR is functionally expressed in BRL cells, and activation of CaSR involves in increased intracellular calcium through Gq-PLC-IP(3) pathway.
The calcium-sensing receptor (CaSR) controls parathyroid hormone (PTH) secretion, which, in turn, via direct and indirect actions on kidney, bone, and intestine, maintains a normal extracellular ionized calcium concentration (Ca(2+)(o)). There is less understanding of the CaSR's homeostatic importance outside of the parathyroid gland. We have employed single and double knockout mouse models, namely mice lacking PTH alone (CaSR(+/+) PTH(-/-), referred to as C(+)P(-)), lacking both CaSR and PTH (CaSR(-/-) PTH(-/-), C(-)P(-)) or wild-type (CaSR(+/+) PTH(+/+), C(+)P(+)) mice to study CaSR-specific functions without confounding CaSR-mediated changes in PTH. The mice received three hypercalcemic challenges: an oral Ca(2+) load, injection or constant infusion of PTH via osmotic pump, or a phosphate-deficient diet. C(-)P(-) mice show increased susceptibility to developing hypercalcemia with all three challenges compared with the other two genotypes, whereas C(+)P(-) mice defend against hypercalcemia similarly to C(+)P(+) mice. Reduced renal Ca(2+) clearance contributes to the intolerance of the C(-)P(-) mice to Ca(2+) loads, as they excrete less Ca(2+) at any given Ca(2+)(o) than the other two genotypes, confirming the CaSR's direct role in regulating renal Ca(2+) handling. In addition, C(+)P(+) and C(+)P(-), but not C(-)P(-), mice showed increases in serum calcitonin (CT) levels during hypercalcemia. The level of 1,25(OH)(2)D(3) in C(-)P(-) mice, in contrast, was similar to those in C(+)P(-) and C(+)P(+) mice during an oral Ca(2+) load, indicating that increased 1,25(OH)(2)D(3) production cannot account for the oral Ca(2+)-induced hypercalcemia in the C(-)P(-) mice. Thus, CaSR-stimulated PTH release serves as a "floor" to defend against hypocalcemia. In contrast, high-Ca(2+)(o)-induced inhibition of PTH is not required for a robust defense against hypercalcemia, at least in mice, whereas high-Ca(2+)(o)-stimulated, CaSR-mediated CT secretion and renal Ca(2+) excretion, and perhaps other factors, serve as a "ceiling" to limit hypercalcemia resulting from various types of hypercalcemic challenges.
The calcium-sensing receptor (CaR) belongs to the G protein-coupled receptor superfamily, with a characteristic structure consisting of seven transmembrane helices, an intracellular C-terminal and an extracellular N terminal domain. The primary physiological function of the CaR is the maintenance of constant blood Ca2+ levels, as a result of its ability to sense very small changes in extracellular Ca2+ (Ca2+(o)). Nevertheless, in addition to being expressed in tissues involved in Ca2+(o) homeostasis, the CaR is also expressed in tissues not involved in mineral homeostasis, suggestive of additional physiological functions. Numerous agonists and modulators of the CaR are now known in addition to Ca2+(o), including various divalent and trivalent cations, aromatic l-amino acids, polyamines, and aminoglycoside antibiotics. The signaling of the CaR is also regulated by extracellular pH and ionic strength. The activated CaR couples mainly to the phospholipase Cbeta and extracellular signal-regulated kinase 1/2 signaling pathways, and it decreases intracellular cAMP levels, leading to various physiological effects. The recent identification of synthetic allosteric modulators of the CaR has opened up a new field of research possibilities. Calcimimetics and calcilytics, which increase and decrease agonist signaling via the CaR, respectively, may facilitate the manipulation of the CaR and thus aid in further investigations of its precise signaling. These allosteric modulators, as well as strontium, have been demonstrated to have therapeutic potential for the treatment of disorders involving the CaR. This review discusses the various agonists and modulators of the CaR, differences in their binding and signaling, and their roles as therapeutics in various diseases.
The intracellular Ca2+ pump inhibitor, thapsigargin, added to DDT1MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca2+ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 microM thapsigargin followed by extensive washing, the total releasable InsP3-sensitive Ca2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca(2+)-pumping activity were all similar with the EC50 values for thapsigargin in each case being close to 10 nM. Upon application to DDT1MF-2 cells, thapsigargin transiently increased resting cytosolic Ca2+ (0.15 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca2+ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca2+, indicating that the internal Ca2+ pool remained completely empty. These results suggest that emptying of Ca2+ from intracellular thapsigargin-sensitive Ca(2+)-pumping pools induces profound alteration of cell proliferation.
Application of bradykinin to neonatal rat dorsal root ganglion neurons caused a depolarization associated with an inward current and an increase in membrane conductance that was probably due to the opening of sodium channels. No hyperpolarization or outward current was detected. In addition, bradykinin increased the rate of 45Ca uptake into the neurons by a mechanism that was blocked by the dihydropyridine calcium channel antagonist nifedipine. Direct activation of protein kinase C (PKC) with phorbol esters mimicked the ability of bradykinin to depolarize the neurons and to increase the rate of 45Ca uptake. Down- regulation of PKC by prolonged treatment with phorbol esters and treatment of the cells with staurosporine, which inhibits PKC, blocked both bradykinin- and phorbol ester-induced 45Ca influx, and substantially reduced the proportion of cells that gave electrophysiological responses to either agent. Bradykinin also activated polyphosphoinositidase C in the dorsal root ganglion neurons, elevating levels of inositol(1,4,5)-trisphosphate and 1,2- diacylglycerol, an endogenous activator of PKC. It is suggested, therefore, that PKC may mediate some of the effects of bradykinin in sensory neurons.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
Specific antisera were produced to peptides representing the carboxyl termini of three subtypes of phosphatidylinositol-specific phospholipase C (PIPLC) beta which have been identified by isolation of cDNAs (Kriz, R., Lin, L., Sultzman, L., Ellis, C., Heldin, C., Pawson, T., and Knopf, J. (1990) Ciba Found. Symp. 150, 112-127). Screening with the antisera indicates that PIPLC beta3 is present in a variety of cell lines and rat tissues, whereas the distribution of PIPLC beta1 and beta2 is more restricted. A combination of conventional and immunoaffinity chromatographic techniques was used to purify PIPLC beta1 and beta3 from rat brain membranes. PIPLC beta2 was purified from cytosol of HL60 cells. All three subtypes were activated by purified G protein alpha(q/11) subunits with the following relative efficacies: PIPLC beta3 greater-than-or-equal-to PIPLC beta1 >> PIPLC beta2. All three PIPLC subtypes were also activated by G protein betagamma subunits with varying efficacies. The presence of betagamma subunits depressed the ability of alpha(q/11) to activate PIPLC beta1 and beta3 at low Mg2+ concentrations (1 mM). At higher concentrations of Mg2+ (2 mM or greater), activation of PIPLC beta3, but not PIPLC beta1, by betagamma and alpha(q/11) became additive. PIPLC beta3 was activated by alpha(q/11) even in the presence of a saturating concentration of betagamma subunits. This indicates that there are separate sites for interaction of PIPLCs with G protein subunits and that this interaction differs depending on the enzyme subtype and the concentration of Mg2+.
The betagamma subunits of guanine nucleotide-binding proteins (G proteins) have been shown to activate unidentified phospholipase C (PLC) isozymes (Camps, M., Hou, C., Sidiropoulos, D., Stock, J. B., Jakobs, K. H., and Gierschik, P. (1992) Eur. J. Biochem. 206, 821-831; Blank, J. L., Brattain, K. A., and Exton, J. H. (1992) J. Biol. Chem. 267, 23069-23075). To identify these target PLC isozymes, we measured the effect of bovine brain G protein betagamma subunits on PLC-beta1, PLC-beta2, PLC-beta3, PLC-gamma1, and PLC-delta1 activity by reconstituting purified protein components with lipid vesicles containing [H-3]phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2). A nearly saturating concentration of betagamma produced 2.5-, 4-, 8.5-, and 2-fold increases in PLC-beta1, PLC-beta2, PLC-beta3, and PLC-delta1 activity, respectively, and no activation of PLC-gamma1, in the presence of 0.2 muM free Ca2+. The betagamma-dependent activation of the PLC-beta isozymes does not appear to be the result of increased affinity of the enzymes for Ca2+. The betagamma-dependent PLC activation could be reversed by addition of the GDP-bound form of the alpha subunit of G(o). The alpha subunits of G(q) class G proteins have been shown to activate PLC-beta isozymes in the order of PLC-beta1 greater-than-or-equal-to PLC-beta3 >> PLC-beta2, which differs from the order of PLC-beta3 > PLC-beta2 > PLC-beta1 for betagamma-dependent activation. Furthermore, the half-maximal concentration of betagamma (25 nM) required to activate PLC-beta3 is much higher than that of G(q) alpha subunits (0.6 nM) required to activate PLC-beta1. These results suggest that the extracellular signals that induce the dissociation of G(o) or G(i), the heterotrimeric G proteins abundant in brain, should enhance the hydrolysis of PtdIns 4,5-P2 in brain primarily through activation of PLC-beta3 (PLC-beta2 is not detectable in brain). However, signals that activate the less abundant G(q) class heterotrimers should result in the activation primarily of PLC-beta1 and PLC-beta3 by the corresponding alpha subunits.
We have stably expressed cDNA for the rat brain Ca-sensing receptor in Chinese hamster ovary cells. Stimulation of phosphatidylinositol hydrolysis and arachidonic acid (AA)
release displayed markedly cooperative responses to Ca with Hill coefficients of 4-5. Both phosphatidylinositol and AA responses were not detected below a threshold of 1.5 mM Ca. Mg behaved as a partial agonist with only half the maximal inositol phosphate and AA responses displayed by Ca and with a more shallow concentration-response slope. The potency of Mg in augmenting inositol phosphate and AA responses, in the presence of 1.5 mM Ca, implies that serum Mg concentrations attained in clinical conditions will influence the Ca-sensing receptor.
Parafollicular (PF) cells of the thyroid gland are neural crest derivatives. These cells remain plastic even in adult animals and can be induced to exhibit neural properties when exposed to NGF in vitro. A human cell line derived from PF cells, medullary thyroid carcinoma (MTC), has previously been shown to synthesize and store 5-HT, a serotonin-binding protein (SBP), and several neuropeptides; moreover, when grown in impoverished media, MTC cells display neural properties. The purpose of the current study was to utilize MTC cells as a neurally relevant model system to investigate factors involved in mediating 5-HT secretion. Electron microscopic immunocytochemistry revealed that secretory vesicles of MTC cells costore immunoreactive 5-HT with SBP and calcitonin. The cAMP derivative, N6-2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate (dibutyryl-cAMP; 1.0 mM) increased the concentration of 5-HT in MTC cells and almost doubled the rate of synthesis of 5-HT from L-tryptophan. Dibutyryl-cAMP also significantly increased the secretion of 5-HT. Cycloheximide (20 micrograms/ml) and anisomycin (20 microM) inhibited the dibutyryl-cAMP-induced increase of 5-HT release, suggesting that this action of dibutyryl-cAMP requires protein synthesis. Cholera toxin (1.0 microgram/ml) and forskolin (0.05 mM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1.0 mM) both increased 5-HT biosynthesis and secretion. Attempts were made to identify a ligand that stimulates cAMP-mediated secretion of 5-HT. Both thyroid-stimulating hormone (TSH: 50 mU/ml) and elevated [Ca2+]e (7.0 mM), each of which acts as a secretogogue for PF cells, stimulated the secretion of 5-HT. The effect of TSH was Ca2(+)-dependent. Immunocytochemistry with monoclonal antibodies to the TSH receptor confirmed that these receptors are present on MTC cells. Neither TSH nor elevated [Ca2+]e elevated cAMP levels. Measurements of Fura-2 fluorescence, however, indicated that both TSH and elevated [Ca2+]e increased cytosolic calcium ([Ca2+]i), as did elevation of [K+]e. It is concluded that exocytosis can be triggered in MTC cells by multiple signal transduction mechanisms. Either cAMP or elevated [Ca2+]i can stimulate secretion; however, a secretogogue that increases cAMP has yet to be identified.
Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.
Application of bradykinin to neonatal rat dorsal root ganglion neurons caused a depolarization associated with an inward current and an increase in membrane conductance that was probably due to the opening of sodium channels. No hyperpolarization or outward current was detected. In addition, bradykinin increased the rate of 45Ca uptake into the neurons by a mechanism that was blocked by the dihydropyridine calcium channel antagonist nifedipine. Direct activation of protein kinase C (PKC) with phorbol esters mimicked the ability of bradykinin to depolarize the neurons and to increase the rate of 45Ca uptake. Down-regulation of PKC by prolonged treatment with phorbol esters and treatment of the cells with staurosporine, which inhibits PKC, blocked both bradykinin- and phorbol ester-induced 45Ca influx, and substantially reduced the proportion of cells that gave electrophysiological responses to either agent. Bradykinin also activated polyphosphoinositidase C in the dorsal root ganglion neurons, elevating levels of inositol(1,4,5)-trisphosphate and 1,2-diacylglycerol, an endogenous activator of PKC. It is suggested, therefore, that PKC may mediate some of the effects of bradykinin in sensory neurons.
An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.
The thyroid parafollicular cell is an endocrine cell derived from the neural crest that stores 5-hydroxytryptamine (5-HT). In common with serotonergic neurons, but in contrast to 5-HT-storing cells that are not neurectodermal derivatives, parafollicular cells also contain a specific 5-HT binding protein. Despite this similarity to serotonergic neurons, parafollicular cells in situ were found to express an endocrine phenotype with few neural characteristics. Thus, the cells costore 5-HT with calcitonin, not calcitonin gene-related peptide (CGRP), which is the product of the calcitonin gene expressed in neurons, and they do not contain neurofilaments. The ability of adult parafollicular cells to respond to microenvironmental perturbations by expressing neuronal characteristics was examined. Sheep thyroid glands were dissociated, and parafollicular cells were purified by affinity chromatography. The purified parafollicular cells were grown in culture on a variety of substrates in the presence or absence of the beta subunit of nerve growth factor (beta-NGF). Parafollicular cells survived in culture for at least a week but retained a roughly spherical shape. Nevertheless, a subset of the cultured parafollicular cells began to display CGRP immunoreactivity. The addition of beta-NGF to the cultured parafollicular cells induced a number of them to extend neurites and increased the proportion of cells in which CGRP immunoreactivity could be found. Neurite-bearing parafollicular cells appeared not to survive for more than 2 d. While their survival was not enhanced when they were grown on collagen, polylysine, laminin, or reconstituted basal lamina, parafollicular cells that had extended neurites in response to beta-NGF survived for at least a week when cocultured with an explant of aneuronal chick hindgut. The effect of the gut was local and only those neurite-bearing parafollicular cells that were growing in direct contact with the explant survived. The thyroid parafollicular cell therefore resembles another crest-derived endocrine cell, the adrenal chromaffin cell, in being able to manifest neural properties in culture. For the parafollicular cell these neural properties include the processing of RNA encoded by the calcitonin gene to express CGRP and neurite outgrowth in response to beta-NGF.
The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.
The subcellular storage of 5-HT was studied in sheep thyroid parafollicular cells. These cells are neural crest derivatives and were investigated as a serotonergic model system. Light and electron microscopic immunocytochemistry was used to examine the distributions of 5-HT, 45 and 56 kDa forms of 5-HT binding protein (SBP), and calcitonin. A single type of parafollicular cell was found that contained calcitonin, 5-HT, and 45 kDa SBP but not 56 kDa SBP. The secretory granules of parafollicular cells all displayed calcitonin immunoreactivity, and many were also immunoreactive for 5-HT and 45 kDa SBP. Granules were isolated, first by size and then by density, on successive metrizamide gradients. These provided a granular fraction that was enriched in calcitonin, endogenous 5-HT, and 45 kDa SBP. Immunoblots confirmed the presence of 45 kDa SBP in the isolated granules and in suspensions of parafollicular cells that were purified by an affinity chromatographic technique. Parafollicular cell granules did not appear to contain substantial stores of ATP. Granules isolated on Percoll gradients were morphologically homogeneous and took up 3H-5-HT. The specificity of this uptake was confirmed by quantitative electron microscopic radioautography. The granular uptake of 3H-5-HT was inhibited by reserpine (10 microM). It is concluded that parafollicular cell granules are different from other amine-storing vesicles that do contain ATP; nevertheless, since parafollicular cell granules store 5-HT and have the same 45 kDa SBP as is found in serotonergic axon terminals, parafollicular cell granules may be analogous to the synaptic vesicles of serotonergic neurons.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10-fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.
Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.
Ryanodine receptors (RyRs) reside in microsomal membranes where they gate Ca2+ release in response to changes in the cytosolic Ca2+ concentration. In the osteoclast, a divalent cation sensor, the Ca2+ receptor (CaR), located within the cell's plasma membrane, monitors changes in the extracellular Ca2+ concentration. Here we show that a RyR-like molecule is a functional component of this receptor. We have demonstrated that [3H] ryanodine specifically binds to freshly isolated rat osteoclasts. The binding was displaced by ryanodine itself, the CaR agonist Ni2+ and the RyR antagonist ruthenium red. The latter also inhibited cytosolic Ca2+ elevations induced by Ni2+. In contrast, the responses to Ni2+ were strongly potentiated by an antiserum Ab129 raised to an epitope located within the channel-forming domain of the type II RyR. The antiserum also stained the surface of intact, unfixed, trypan blue-negative osteoclasts. Serial confocal sections and immunogold scanning electron microscopy confirmed a plasma membrane localization of this staining. Antiserum Ab34 directed to a putatively intracellular RyR epitope expectedly did not stain live osteoclasts nor did it potentiate CaR activation. It did, however, stain fixed, permeabilized cells in a distinctive cytoplasmic pattern. We conclude that an RyR-like molecule resides within the osteoclast plasma membrane and plays in important role in extracellular Ca2+ sensing.
We have investigated whether rat thyroid C-cells can acquire a phenotype similar to serotonergic neurons. C-cells are neural crest derived endocrine cells with some intrinsic neuronal and serotonergic properties. A relatively simple isolation scheme yielded cultures of about 50% initial purity, as measured by fluorescence activated cell sorting. These enriched C-cells could extend neurites up to 550 microns on a laminin-containing substratum in the presence of NGF. The cultured C-cells expressed neurofilaments and this expression was enhanced by NGF treatment. The C-cells also expressed two markers of the sympathoadrenal neural crest lineage, the mammalian achaete scute homolog-1 (MASH-1) transcription factor, and the B2 cell surface antigen. Interestingly, MASH-1 was not detectable after the C-cells were placed in culture, which is consistent with neuronal differentiation, since MASH-1 is only expressed in neuronal progenitors prior to differentiation. We then demonstrated that C-cells possess the fundamental features of serotonergic neurons: synthesis and secretion, uptake, and feedback control. The enriched C-cells, as well as the CA77 C-cell line, showed 5-HT immunostaining, expression of tryptophan hydroxylase mRNA, 5-HT1B autoreceptor mRNA, and 5-HT transporter mRNA and activity. NGF greatly induced 5-HT transporter activity as determined by sensitivity to sertraline, a selective 5-HT reuptake inhibitor. Based on these results, we propose that thyroid C-cells are derived from a vagal sympathoadrenal progenitor, similar to serotonergic enteric neurons, and can undergo neuronal transdifferentiation. Hence, these cells should provide suitable and convenient models for molecular and cellular studies on serotonergic neurons.
We have molecularly cloned a calcium sensing receptor (CaSR) from a rat striatal cDNA library. Rat CaSR displays 92% overall homology to its bovine counterpart with seven putative transmembrane domains characteristic of the superfamily of guanine nucleotide-binding proteins and significant homology with the metabotropic glutamate receptors. Northern blot analysis reveals two transcripts in thyroid, kidney, lung, ileum, and pituitary. In brain highest regional expression of the RNA occurs in the hypothalamus and the corpus striatum. Immunohistochemistry reveals discrete punctate localizations throughout the brain that appear to be associated with nerve terminals. No staining is evident in cell bodies of neurons or glia. Cerebral arteries display an intense network of CaSR immunoreactive fibers associated with vessel innervation. CaSR on nerve terminal membranes may regulate neurotransmitter disposition in response to Ca2+ levels in the synaptic space.
It has been supposed that a cell-surface Ca2+ receptor enables parathyroid cells to detect and respond to extracellular Ca2+. The recent cloning of a G protein-coupled Ca2+ receptor solidifies this view. Ca2+ receptors regulate the activity of various cells and provide novel molecular targets for new pharmaceuticals.
This book examines what is known about the molecular biology of multiple endocrine neoplasia, type 2 (MEN2). Mutations and alterations in gene expressions and signal transduction are placed in the context of their effects on the biology of the thyroid and adrenal cells which generate cancers in MEN2. Thus, this volume addresses the ret mutations, the effects these mutations may have on signal transduction, and developmental and neurobiological aspects of the cells affected of MEN2, and provides the basic researcher - in genetics, signal transduction, cell transformation, developmental or neurobiology - with a better understanding of how the knowledge generated by each of these fields intercalates within the overall biology of MEN2.
Auniversal aspect of cancer development is its multistage nature. Precancerous stages evolve to more advanced stages of malignancy and metastasis. This has been reviewed extensively by Foulds.1,2 Based on observations of karyotypic progression in hematopoietic and other neoplasms, Nowell3 proposed a clonal evolution model for tumor progression. In this model, variant cell clones which possess some growth advantage overgrow the other cells in the tumor.
We have stably expressed cDNA for the rat brain Ca2+-sensing receptor in Chinese hamster ovary cells. Stimulation of phosphatidylinositol hydrolysis and arachidonic acid (AA) release displayed markedly cooperative responses to Ca2+ with Hill coefficients of 4-5. Both phosphatidylinositol and AA responses were not detected below a threshold of 1.5 mM Ca2+. Mg2+ behaved as a partial agonist with only half the maximal inositol phosphate and AA responses displayed by Ca2+ and with a more shallow concentration-response slope. The potency of Mg2+ in augmenting inositol phosphate and AA responses, in the presence of 1.5 mM Ca2+, implies that serum Mg2+ concentrations attained in clinical conditions will influence the Ca2+-sensing receptor.
Calcitonin (CT) secretion by parafollicular cells of the thyroid (C cells) is regulated by small changes in the concentration of extracellular calcium ([Ca2+]e). Elevation of [Ca2+]e elicits a rise in the C cell cytoplasmic calcium concentration and stimulates CT release. The molecular entity through which C cells detect changes in [Ca2+]e and modulate hormone secretion is unknown. Recently, an extracellular calcium-sensing receptor (CaR) complementary DNA was isolated from bovine parathyroid gland. To assess whether parathyroid cells and C cells use similar mechanisms to detect changes in ambient Ca2+, rat, human, and sheep C cells were examined for expression of the parathyroid CaR or a related receptor isoform. Reverse transcription-polymerase chain reaction analysis identified CaR transcripts in rat and human thyroid gland. Northern blot analysis demonstrated CaR messenger RNA (mRNA) in rat thyroid gland, a human medullary thyroid carcinoma (MTC) isolate, and a highly enriched preparation of sheep C cells. Rat MTC 44-2 cells, a cell line responsive to changes in [Ca2+]e, express abundant levels of CaR mRNA. Human TT cells, a C cell line lacking the extracellular calcium-sensing function, have undetectable levels of CaR mRNA by Northern blot analysis. Western blot analysis, using antiserum specific to the parathyroid CaR, detected CaR protein in rMTC 44-2, but not TT cells. Immunostaining of both dispersed sheep C cells and rat thyroid gland sections identified C cell-specific expression of the CaR protein, and in situ hybridization analysis confirmed the C cell-specific expression of CaR mRNA in the intact rat thyroid. The nucleotide sequence of the coding region of the rMTC 44-2 CaR transcripts was found to encode the same CaR protein as that expressed in the parathyroid and kidney. The results demonstrate that C cells express the same extracellular calcium-sensing receptor that is found in parathyroid and kidney, and the presence of this receptor protein in C cell lines correlates with the extracellular calcium-sensing function. This CaR is likely to represent the primary molecular entity through which C cells detect changes in [Ca2+]e and control CT release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types.
Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.
Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.
Application of the nonapeptide bradykinin (BK) to rat sensory neurons in culture elicits two types of electrical responses in a subpopulation of the cells: (i) an excitatory inward cation current (I(BK)), and (ii) an inhibition of voltage-activated calcium currents (I(Ca)). Ion replacement experiments and current-voltage measurements indicate that I(BK) shows little selectivity for monovalent cations. We investigated the role of GTP-binding proteins in the BK signal transduction pathway. Internal dialysis of neurons with the inhibitory guanine nucleotide analogue, GDPbetaS, decreased the percentage of cells responding and the magnitude of the responses. This finding lead us to propose that a G protein is involved in this transduction pathway. We have attempted to identify the G protein subtype involved using pretreatment with the bacterial toxins pertussis toxin (PTX) and cholera toxin (CTX). PTX potentiated I(BK) slightly, but had no effect on the inhibition of I(Ca) by BK. In contrast, PTX pretreatment blocked the effects of norepinephrine and neuropeptide Y on I(Ca), confirming that the PTX was capable of interfering with appropriate G protein signaling in these neurons. Pretreatment with CTX had no effect on the BK-induced inward current response. Therefore we propose that the BK signal transduction pathway utilizes a G protein that is not part of the G(i), G(o), G(t), or G(s) families.
1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
This chapter discusses the cytophysiology of thyroid parafollicular cells. The thyroid gland of the mammal is a bilobed structure located at the base of the neck on either side of the trachea. The most important function of the thyroid gland is the synthesis, storage, and secretion into the blood of two iodinated amino-acid hormones, L-thyroxine and 3,5,3'-triiodo-L-thyronine. Thyroid hormones are required for normal growth and development and for normal metabolic activity. Thyroid hormones act by accelerating general and specific metabolic processes of the body, leading to an increase in oxygen consumption and heat production. The mammalian thyroid gland is also responsible for the elaboration, storage, and secretion of a second type of hormone, calcitonin, which lowers the calcium concentration of blood.
Parafollicular (PF) cells of the thyroid gland are neural crest derivatives, which costore the neurotransmitter, 5-hydroxytryptamine (5-HT) with calcitonin. PF cells are located adjacent to follicular (F) cells within the basement membrane of thyroid follicles. It has been proposed that 5-HT serves an intercellular signalling function in the thyroid and that F cells are its target. This proposal was tested by using cell lines derived from PF (medullary thyroid carcinoma [MTC]) and F (FRTL-5) cells to study the mechanisms that mediate the secretion and action of 5-HT. Secretion of 5-HT by MTC cells was evoked by thyroid stimulating hormone, thyrotropin (TSH), elevated extracellular calcium (↑[Ca2+]e), or by agents that increase intracellular cAMP (↑[cAMP]i). When protein kinase C (PKC) was down-regulated by prolonged treatment of MTC cells with phorbol 12-myristate 13-acetate (PMA), or PKC was inhibited by staurosporin, the TSH-or PMA-evoked secretion of 5-HT was blocked; however, interference with PKC function did not affect 5-HT secretion evoked by ↑ [Ca2+]e or ↑ [cAMP]i. In the putative targets, FRTL-5 cells, 5-HT increased the turnover of phosphoinositides (PI), cytosolic calcium (↑[Ca2+]i), ↑[cAMP]i, and biphasically modified the effect of TSH on cAMP. All of these 5-HT effects were inhibited by 5-HT2 receptor antagonists (spiperone and ketanserin) and by pertussis toxin (PTx), suggesting that the actions of 5-HT are mediated by 5-HT2 receptors, which are coupled to a G protein. This suggestion was supported by the following additional observations: FRTL-5 membranes bound the 5-HT2 agonist, [125I]2,5-dimethoxy-4-iodophenylisopropylamine ([125I]-DOI), and anti-idiotypic anti-bodies, which recognize 5-HT2 receptors. [125I]-DOI binding was inhibited by guanosine-5′-O-(3-thiotriphosphate) (GTP-γ-S) and the antibodies were displaced by spiperone. Data are consistent with the hypothesis that 5-HT serves as a PF to F cell messenger.
Calcium and BAY K 8644 acutely stimulate calcitonin secretion by influx of extracellular calcium (Ca) through voltage-dependent calcium channels, leading to an increase in cytosolic free Ca. Repetitive exposure to BAY K 8644 (10(-6) M) resulted in an increase in calcitonin (CT) secretion in the rat C-cell line (rMTC 6-23) lasting 9 hours, in comparison to that of 3 mM Ca2+ which lasted 6 hours. Equimolar concentration of nifedipine did not inhibit the stimulatory effect of BAY K 8644 as compared to the nifedipine only group. The decrease in stimulated CT secretion during long-term exposure to BAY K 8644 is due to desensitization of cells which may be attributed to down-regulation of dihydropyridine receptors. After 12 h exposures to 3 mM Ca2+ alone, BAY K 8644 (10(-6) M) alone or in combination with nifedipine (10(-6) M), CT content decreased below the control level, indicating a decrease in synthesis. Overall cellular protein content was not affected by the test agents. Repetitive exposure of C-cells to BAY K 8644 revealed a desensitization of the stimulatory effect on CT secretion and a decrease in CT cell content.
1. Calcitonin secretion is regulated by the external Ca2+ concentration ([Ca2+]o) via a rise in intracellular Ca2+ concentration ([Ca2+]i). The mechanism which couples an increase in [Ca2+]o to an increase in [Ca2+]i was explored in a rat calcitonin-secreting cell line (rMTC 44-2). [Ca2+]i was monitored using Fura-2 AM, and the membrane potential or current was simultaneously measured. 2. Using the conventional whole-cell clamp, tetrodotoxin-sensitive voltage-gated Na+ channels, T- and L-type Ca2+ channels, and three types of K+ channels, the delayed K+ channel, the A-channel and the inward-rectifying channel were observed. 3. Using the nystatin-perforated whole-cell-clamp technique, the resting potential measured under current clamp in standard extracellular medium was -59.0 +/- 5.0 mV (mean +/- S.D., n = 25), and the input resistance was 3.9 +/- 1.9 G omega (n = 10). In 0.5 mM [Ca2+]o most cells (22/25) showed spontaneous action potentials. 4. An increase in [Ca2+]o depolarized the cell membrane and elevated [Ca2+]i even in the presence of 10 microM-tetrodotoxin. The rise in [Ca2+]i was greatly reduced when action potentials were inhibited by applying hyperpolarizing current. The increase in [Ca2+]i saturated with 3-4 mM [Ca2+]o. In 3 mM [Ca2+]o, [Ca2+]i was 188.9 +/- 40.5% (n = 12) of that in 0.5 mM [Ca2+]o. 5. In high [Ca2+]o the duration of action potentials was prolonged, but the action potential frequency did not always increase. In some cases it even decreased in high [Ca2+]o. 6. Two types of action potential were observed in high [Ca2+]o, one with a shorter duration and the other with a longer duration. [Ca2+]i transiently increased in association with the long-duration action potentials. These long-duration action potentials were also accompanied by a larger after-hyperpolarization. 7. Under voltage clamp, high [Ca2+]o caused a membrane conductance increase to Na+ ions. 8. Even when the membrane potential was clamped at a level below the threshold for Ca2+ channel activation, high [Ca2+]o provoked an increase of [Ca2+]i which was composed of an initial transient increase followed by a sustained increase, indicating an involvement of mechanisms other than Ca2+ influx through voltage-gated channels. The sustained increase was more frequently observed than the initial transient increase. The amplitude of the sustained phase was dependent on [Ca2+]o, and in 5 mM [Ca2+]o it was 120.9 +/- 18.9% (103-194%) (n = 58) of that in 0.5 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)
The intracellular Ca2+ pump inhibitor, thapsigargin, added to DDT1MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca2+ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 microM thapsigargin followed by extensive washing, the total releasable InsP3-sensitive Ca2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca(2+)-pumping activity were all similar with the EC50 values for thapsigargin in each case being close to 10 nM. Upon application to DDT1MF-2 cells, thapsigargin transiently increased resting cytosolic Ca2+ (0.15 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca2+ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca2+, indicating that the internal Ca2+ pool remained completely empty. These results suggest that emptying of Ca2+ from intracellular thapsigargin-sensitive Ca(2+)-pumping pools induces profound alteration of cell proliferation.
Few endocrine tissues can detect changes in the extracellular Ca2+ concentration within the physiological range and modify their hormone secretion accordingly. A rat cell line of C-cell origin (rMTC 44-2) secretes calcitonin and neurotensin in response to small increases in external Ca2+. To better understand the mechanism of extracellular Ca2+ sensing in this cell type, we studied single fura-2-loaded rMTC 44-2 cells perfused with increasing concentrations of Ca2+ and K+. In the basal state (Ca2+ = 0.5 mM), cytosolic Ca2+ levels were 53 nM, with 27% of the cells having spikes or oscillations. With elevation of the external Ca2+ to between 0.5 and 4 mM, 84% of the cells showed a rapid (less than 5 s) rise in cytosolic Ca2+ to values 2- to 10-fold higher than basal levels. Most of the responding cells exhibited complex patterns of cytosolic Ca2+ fluctuations, including oscillations with frequencies varying from less than 1/min to as many as 6/min. When averaged over time, the cytosolic Ca2+ of individual cells showed a dose-dependent response with changes in external Ca2+, resembling the relationship between extracellular Ca2+ and calcitonin secretion. With continued or repeated stimulation, the spike amplitude often declined. These cytosolic Ca2+ responses were attenuated in the presence of the Ca(2+)-channel blockers cadmium and nifedipine. Cytosolic Ca2+ responses to perfusion with elevated K+ (20 mM) were similar in waveform to those seen with Ca2+ stimulation. Most cells displayed cytosolic Ca2+ changes in response to both ionic secretagogues when stimulated with external Ca2+ or K+.(ABSTRACT TRUNCATED AT 250 WORDS)
We present experimental procedures describing the creation of perforated patches by use of amphotericin B. In 13 different cellular preparations, access resistances below 10 M omega were achieved and with blunt electrode tips, access resistances of 3-4 M omega were possible. In addition to using the techniques to measure whole cell currents, we have used them to measure single channel currents in a new "outside-out patch" preparation and we have utilized them to measure the resting voltage of epithelial monolayers. We conclude that these new approaches can provide a substantial increase in versatility and quality for many kinds of electrophysiological measurements.
The two dihydropyridine enantiomers, (+)202-791 and (-)202-791, that act as voltage-sensitive Ca2+ channel agonist and antagonist, respectively, were examined for effects on cytosolic Ca2+ concentrations ([Ca2+]i) and on hormones secretion in dispersed bovine parathyroid cells and a rat medullary thyroid carcinoma (rMTC) cell line. In both cell types, small increases in the concentration of extracellular Ca2+ evoked transient followed by sustained increases in [Ca2+]i, as measured with fura-2. Increases in [Ca2+]i obtained by raised extracellular Ca2+ were associated with a stimulation of secretion of calcitonin (CT) and calcitonin gene-related peptide (CGRP) in rMTC cells, but an inhibition of secretion of parathyroid hormone (PTH) in parathyroid cells. The Ca2+ channel agonist (+)202-791 stimulated whereas the antagonist (-)202-791 inhibited both transient and sustained increases in [Ca2+]i induced by extracellular Ca2+ in rMTC cells. Secretion of CT and CGRP was correspondingly enhanced and depressed by (+)202-791 and (-)202-791, respectively. In contrast, neither the agonist nor the antagonist affected [Ca2+]i and PTH secretion in parathyroid cells. Depolarizing concentrations of extracellular K+ increased [Ca2+]i and hormone secretion in rMTC cells and both these responses were potentiated or inhibited by the Ca2+ channel agonist or antagonist, respectively. The results suggest a major role of voltage-sensitive Ca2+ influx in the regulation of cytosolic Ca2+ and hormones secretion in rMTC cells. Parathyroid cells, on the other hand, appear to lack voltage-sensitive Ca2+ influx pathways and regulate PTH secretion by some alternative mechanism.
The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.
This chapter contains a summary of previous work, as well as some new data concerning the roles of potassium and calcium in electrically and chemically induced seizures. During tonic-clonic seizure discharges, the extracellular concentration of potassium, [K+]o, increases from its resting level of 3.0 to 3.5 mM to between 8.0 and 12.0 mM. The time course of the [K+]o increase is such that it cannot play a part in causing either the onset or termination of paroxysmal firing, but its magnitude is in the range where K+ ions have a profound influence on the functions of excitable membranes and synapses. During nonparoxysmal activation of central nervous system (CNS) tissue, [Ca2+]o may decrease, increase, or remain unchanged. When the same stimulus train is repeated every few seconds, in time the [Ca2+]o response may change polarity even if the experimental conditions have not deliberately been altered. Changes in cerebral pH can cause small changes in the level of free Ca2+ ions in the CNS interstitium, possibly contributing to the variability of its response. At the site of origin of seizure discharges, however, [Ca2+]o does decrease in most or all cases. Paroxysmal firing provoked in hippocampal formation by repetitive stimulation of an afferent pathway and recorded with extracellular microelectrodes in a cell-body layer consists of "giant" population spikes riding on a sustained negative shift of the baseline potential. The paroxysmal sustained potential (SP) shift appears to be generated by intense and sustained depolarization of the cell bodies of dentate granule cells, and of hippocampal pyramidal cells. This is different from spinal cord and cerebral neocortex, where paroxysmal SP shifts are generated mainly by depolarization of neuroglial cells. The giant population spikes are probably the result of lockstep firing of granule cells and of pyramidal cells.
Three hormones were demonstrated in ultrathin sections of the rat thyroid using immunocytochemical methods with either a PAP complex or a protein A-gold complex as the label. In control rats, calcitonin was found to be present in all parafollicular cells and somatostatin in occasional cells. In rats pretreated with 5-hydroxytryptophan, serotonin was detected in all parafollicular cells as well. In serial ultrathin sections, the three hormones were seen to be localized in the same secretory granules.
The importance of intracellular calcium in regulating cell function is well recognized. No less important, but less well understood (and probably appreciated), is the fundamental role played by extracellular calcium, Ca2+o, in the modulation of cell function. The recent cloning of Ca2+o-sensing, G-protein-coupled receptors from bovine (and human) parathyroid and rat kidney (and brain) has clearly demonstrated that Ca2+o can function as a traditional 'first messenger'. The identification of 'inactivating' and 'activating' mutations in this Ca2+o-sensing receptor in two hypercalcemic disorders and in an autosomal dominant form of hypocalcemia, respectively, has underscored the physiological relevance of this receptor in Ca2+ homeostasis in man. These advances have significantly enhanced our understanding of the molecular mechanisms involved in extracellular calcium sensing in parathyroid and kidney. Moreover, the localization of the Ca2+o-sensing receptor in tissues previously not known to have Ca2+o-sensing capability has suggested novel and potentially quite important roles for Ca2+o in regulating the function of cells not apparently directly involved in Ca2+ homeostasis.
Thyroid parafollicular (PF) cells are neural crest-derived endocrine cells that secrete serotonin and calcitonin. The secretory vesicles of PF cells acidify when secretion is induced by increased extracellular Ca2+ or TSH. We tested the hypothesis that acidification is regulated by secretogogue-gated Cl- channels in vesicular membranes. Cl- channel (p64) immunoreactivity was enriched in purified PF vesicles. X-Ray microanalysis showed a change in chlorine level in PF vesicles in response to secretogogue-stimulation of isolated cells. Secretogogue stimulation also altered the degree of p64 channel phosphorylation. Protein kinase and phosphatase inhibitors antagonized secretogogue-induced vesicle acidification and secretion; however, secretion could occur even when acidification was blocked. We conclude that acidification of PF vesicles is regulated by a gatable Cl- channel in vesicle membranes and that protein phosphorylation and dephosphorylation are involved in channel activation. Acidification of vesicles is not required for exocytosis.
The RET proto-oncogene encodes a protein receptor tyrosine kinase. RET mutations are associated with the dominantly inherited
cancer syndromes multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC). In MEN
2A, MEN 2B, and FMTC, direct detection of RET mutations can be used to identify disease allele carriers prior to the development
of clinically evident neoplasms. RET mutations are also associated with sporadic thyroid carcinomas. The effects of RET mutation
on protein function have been investigated both in vivo and in vitro, and the study of RET has served to provide insights
into the mechanisms of tumorigenesis in general. [J Natl Cancer Inst 1995; 87: 1515–23]
1. The floor plate is a ventral mid-line structure that plays a pivotal role in the organization of the developing vertebrate central nervous system. Previous studies have demonstrated that the floor plate may provide signals that induce neuronal differentiation and guide axons; however, it is not known whether the floor plate can itself respond to signals that derive from surrounding tissue. 2. The peptide substance P is one of the first transmitters to be expressed in the developing spinal cord. To determine whether the floor plate may respond to substance P we have examined the expression of the principal substance P receptor (the tachykinin NK1 receptor) by floor plate cells of the rat embryonic spinal cord using immunocytochemistry, in situ hybridization and fura-2 calcium imaging. 3. Immunocytochemistry demonstrated selective expression of the NK1 receptor by cells at the ventral mid-line of the spinal cord. Double immunofluorescence labelling with the specific floor plate marker FP3 indicated that NK1 receptor expression is confined to cells in the lateral region of the floor plate. 4. In order to confirm the specificity of the NK1 receptor immunoreactivity we performed in situ hybridization histochemistry using antisense cRNA probes directed against the NK1 receptor. In situ hybridization demonstrated selective expression of NK1 receptor mRNA by floor plate cells. 5. The ontogeny of NK1 receptor protein and mRNA expression in the floor plate was defined. NK1 receptor expression occurred in a rostrocaudal progression that begins at embryonic day 10-11 (E10-E11) and is complete by E12-E14. The restriction of NK1 receptor expression to the lateral part of the floor plate was conserved throughout embryonic development. 6. NK1 receptor signalling was assessed by monitoring substance P-evoked changes in the intracellular concentration of calcium ions ([Ca2+]i) of acutely dissociated cells from the floor plate region. Application of substance P (5 nM) elevated [Ca2+]i in 10% of cells examined. 7. Selective neurokinin agonists were used to identify the receptor subtype involved in the substance P-evoked elevation of [Ca2+]i. Acetyl-[Arg6,Sar9,Met(O2)11]-substance P(6-11) (5 nM) and [Sar9,Met(O2)11]-substance P (5 nM), two highly selective NK1 receptor agonists, both elevated [Ca2+]i in floor plate cells that responded to substance P. [beta-Ala8]-neurokinin A(4-10) (50 nM) and senktide (50 nM), selective agonists respectively of NK2 and NK3 receptors, had no effect on [Ca2+]i.
Calcitonin (CT) secretion by parafollicular cells of the thyroid (C cells) is regulated by small changes in the concentration of extracellular calcium ([Ca2+]e). Elevation of [Ca2+]e elicits a rise in the C cell cytoplasmic calcium concentration and stimulates CT release. The molecular entity through which C cells detect changes in [Ca2+]e and modulate hormone secretion is unknown. Recently, an extracellular calcium-sensing receptor (CaR) complementary DNA was isolated from bovine parathyroid gland. To assess whether parathyroid cells and C cells use similar mechanisms to detect changes in ambient Ca2+, rat, human, and sheep C cells were examined for expression of the parathyroid CaR or a related receptor isoform. Reverse transcription-polymerase chain reaction analysis identified CaR transcripts in rat and human thyroid gland. Northern blot analysis demonstrated CaR messenger RNA (mRNA) in rat thyroid gland, a human medullary thyroid carcinoma (MTC) isolate, and a highly enriched preparation of sheep C cells. Rat MTC 44-2 cells, a cell line responsive to changes in [Ca2+]e, express abundant levels of CaR mRNA. Human TT cells, a C cell line lacking the extracellular calcium-sensing function, have undetectable levels of CaR mRNA by Northern blot analysis. Western blot analysis, using antiserum specific to the parathyroid CaR, detected CaR protein in rMTC 44-2, but not TT cells. Immunostaining of both dispersed sheep C cells and rat thyroid gland sections identified C cell-specific expression of the CaR protein, and in situ hybridization analysis confirmed the C cell-specific expression of CaR mRNA in the intact rat thyroid. The nucleotide sequence of the coding region of the rMTC 44-2 CaR transcripts was found to encode the same CaR protein as that expressed in the parathyroid and kidney. The results demonstrate that C cells express the same extracellular calcium-sensing receptor that is found in parathyroid and kidney, and the presence of this receptor protein in C cell lines correlates with the extracellular calcium-sensing function. This CaR is likely to represent the primary molecular entity through which C cells detect changes in [Ca2+]e and control CT release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types.
The role of protein kinase C (PKC) in regulating cytosolic Ca2+ concentrations ([Ca2+]i) and parathyroid hormone (PTH) secretion was studied in bovine parathyroid cells rendered deficient in PKC activity by incubation with phorbol 12-myristate 13-acetate (PMA). Pretreatment with PMA caused a time- and concentration-dependent loss of functional PKC activity as assessed by the failure of [Ca2+]i and PTH secretion to respond to the subsequent addition of PKC activators or the inhibitor staurosporine. Pretreatment for 24 h with 1 microM PMA caused a loss of 85% of the total and 98% of the cytosolic PKC activity. Cells so pretreated were considered to be "PKC downregulated." Increasing the concentration of extracellular Ca2+ or Mg2+ caused corresponding increases in [Ca2+]i that were similar in control and in PKC-downregulated cells. PTH secretion regulated by extracellular Ca2+ or Mg2+ was likewise similar in control and PKC-downregulated cells. Stimulus-secretion coupling is thus unimpaired in parathyroid cells deficient in PKC activity. Cytosolic Ca2+ responses remained depressed in cells incubated for 24 h with low concentrations of PMA (30 or 100 nM). However, under these conditions, extracellular Ca2+ still suppressed PTH secretion similarly to control cells. These results reveal a dissociation between cytosolic Ca2+ and PTH secretion and suggest that signals other than cytosolic Ca2+ are involved in the regulation of PTH secretion.