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ABSTRACT: Restoration of the functional potency of pancreatic islets, either through enhanced proliferation (hyperplasia) or increase in size (hypertrophy) of beta cells is a major objective for intervention in diabetes. We have obtained experimental evidence that global knockout of a small, single-span regulatory subunit of Na,K-ATPase, FXYD2, alters glucose control. Adult Fxyd2-/- mice showed significantly lower blood glucose level, no signs of peripheral insulin resistance, and improved glucose tolerance compared to their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the Fxyd2-/- mice compared to the wild type littermates, compatible with an observed increase in the level of circulating insulin. No changes were seen in the exocrine compartment of the pancreas, and the mice had only a mild, well-adapted renal phenotype. Morphometric analysis revealed an increase in beta cell mass in KO compared to WT mice. This appears to explain a phenotype of hyperinsulinemia. By RT-PCR, Western blot and immunocytochemistry we showed the FXYD2b splice variant in pancreatic beta cells from wild type mice. Phosphorylation of Akt kinase was significantly higher under basal conditions in freshly isolated islets from Fxyd2-/- mice compared to their WT littermates. Inducible expression of FXYD2 in INS 832/13 cells produced a reduction in the phosphorylation level of Akt, and phosphorylation was restored in parallel with degradation of FXYD2. Thus we suggest that in pancreatic beta cells FXYD2 plays a role in Akt signaling pathways associated with cell growth and proliferation.
Journal of Biological Chemistry 01/2013; · 4.77 Impact Factor
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ABSTRACT: Oculodentodigital dysplasia (ODDD) is a human disorder caused by mutations in the gap junction alpha 1 (GJA1) gene encoding the connexin43 (Cx43) gap junction protein. Causal links between GJA1 mutations and glaucoma are not understood. The purpose in this study was to examine the ocular phenotype for Gja1(Jrt/+) mice harboring a Cx43 G60S mutation. METHODS; In young Gja1(Jrt/+) mice, Cx43 abundance was assessed with a Western blot, and Cx43 localization was visualized using immunohistochemistry and confocal microscopy. Intraocular pressure (IOP) was measured by rebound tonometry, and eye anatomy was imaged using ocular coherence tomography (OCT). Hematoxylin and eosin (H&E)-stained eye sections were examined for ocular histopathology related to the development of glaucoma.
Decreased Cx43 protein levels were evident in whole eyes from Gja1(Jrt/+) mice compared with those of wild-type mice at postnatal day 1 (P = 0.005). Cx43 immunofluorescence in ciliary bodies of Gja1(Jrt/+) mice was diffuse and intracellular, unlike the gap junction plaques prevalent in wild-type mice. IOP in Gja1(Jrt/+) mice changed during postnatal development, with significantly lower IOP at 21 weeks of age in comparison to the IOP of wild-type eyes. Microphthalmia, enophthalmia, anterior angle closure, and reduced pupil diameter were observed in Gja1(Jrt/+) mice at all ages examined. Ocular histology showed prominent separations between the pigmented and nonpigmented ciliary epithelium of Gja1(Jrt/+) mice, split irides, and alterations in the number and distribution of nuclei in the retina.
Detailed phenotyping of Gja1(Jrt/+) eyes offers a framework for elucidating human ODDD ocular disease mechanisms and evaluating new treatments designed to protect ocular synaptic network integrity.
Investigative ophthalmology & visual science 01/2011; 52(6):3539-47. · 3.43 Impact Factor
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ABSTRACT: WNTs are secreted extracellular signaling molecules that transduce their signals by binding to G protein-coupled receptors of the frizzled (FZD) family. They control diverse developmental processes, such as cell fate specification, cell proliferation, cell differentiation, and apoptosis. Although WNT signaling has been shown to be essential for development of the ovary, its mechanistic role in folliculogenesis within the adult ovary has not been studied extensively. Therefore, the objective of this study was to investigate the regulation and function of WNT2 signaling in mouse granulosa cells. Immunostaining identified WNT2 as being expressed in granulosa cells throughout folliculogenesis, but with varying signal strength: in sequential sections, WNT2 immunoreactivity was strongest in healthy antral follicles but weak in atretic follicles. Knockdown of WNT2 expression using transfected short interfering RNA decreased DNA synthesis in granulosa cells, whereas WNT2 overexpression using a recombinant viral vector enhanced it. WNT2 knockdown led to accumulation of glycogen synthase kinase-3beta (GSK3B) in the cytoplasm but reduced the expression of beta-catenin. Conversely, WNT2 overexpression reduced the expression of GSK3B in the cytoplasm and induced beta-catenin translocation from the membrane into the nucleus. Beta-catenin knockdown also inhibited DNA synthesis in granulosa cells and neutralized the effect of WNT2 overexpression. WNT2/beta-catenin signaling had a slight effect on the apoptosis of granulosa cells. Taken together, the data indicate that WNT2 regulates beta-catenin localization in granulosa cells, and WNT2/beta-catenin signaling contributes to regulating their proliferation.
Biology of Reproduction 05/2010; 82(5):865-75. · 4.01 Impact Factor
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ABSTRACT: Female fertility is determined to a large extent by the quality (developmental competence) of the oocyte as reflected in its ability to undergo meiosis, be fertilized, and give rise to a healthy embryo. Growth of the mammalian oocyte is coordinated with that of the follicle that encloses it by the actions of signals that pass in both directions between the germline and somatic components. This review summarizes what is known about the roles played by 2 different modes of intrafollicular signalling in oogenesis: paracrine factors activating receptors on the opposite cell type, and direct sharing of small molecules throughout the follicle via gap junction channels. Recent evidence indicates that these 2 modes of signalling interact to regulate oocyte growth and granulosa cell proliferation and that defects in either can contribute to female infertility.
Canadian Journal of Physiology and Pharmacology 04/2010; 88(4):399-413. · 1.95 Impact Factor
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ABSTRACT: We have previously shown that increased nitric oxide (NO) production in sepsis impairs arteriolar-conducted vasoconstriction cGMP independently and that the gap junction protein connexin (Cx) 37 is required for this conducted response. In the present study, we hypothesized that NO impairs interendothelial electrical coupling in sepsis by targeting Cx37. We examined the effect of exogenous NO on coupling in monolayers of cultured microvascular endothelial cells derived from the hindlimb skeletal muscle of wild-type (WT), Cx37 null, Cx40 null, and Cx43(G60S) (nonfunctional mutant) mice. To assess coupling, we measured the spread of electrical current injected in the monolayer and calculated the monolayer intercellular resistance (inverse measure of coupling). The NO donor 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (DETA) rapidly and reversibly reduced coupling in cells from WT mice, cGMP independently. NO scavenger HbO(2) did not affect baseline coupling, but it eliminated DETA-induced reduction in coupling. Reduced coupling in response to DETA was also seen in cells from Cx40 null and Cx43(G60S) mice, but not in cells from Cx37 null mice. DETA did not alter the expression of Cx37, Cx40, and Cx43 in WT cells analyzed by immunoblotting and immunofluorescence. Furthermore, neither the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III), superoxide scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, nor preloading of WT cells with the antioxidant ascorbate affected this reduction. We conclude that NO-induced reduction of electrical coupling between microvascular endothelial cells depends on Cx37 and propose that NO in sepsis impairs arteriolar-conducted vasoconstriction by targeting Cx37 within the arteriolar wall.
AJP Heart and Circulatory Physiology 06/2009; 297(1):H93-H101. · 3.71 Impact Factor
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ABSTRACT: Gap junctional coupling among cumulus cells is important for oogenesis since its deficiency in mice leads to impaired folliculogenesis. Multiple connexins (Cx), the subunits of gap junction channels, have been found within ovarian follicles in several species but little is known about the connexins in human follicles. The aim of this study was to determine which connexins contribute to gap junctions in human cumulus cells and to explore the possible relationship between connexin expression and pregnancy outcome from in vitro fertilization (IVF). Cumulus cells were obtained from IVF patients undergoing intracytoplasmic sperm injection (ICSI). Connexin expression was examined by RT-PCR and confocal microscopy. Cx43 was quantified by immunoblotting and gap junctional coupling was measured by patch-clamp electrophysiology. All but five of 20 connexin mRNAs were detected. Of the connexin proteins detected, Cx43 forms numerous gap junction-like plaques but Cx26, Cx30, Cx30.3, Cx32, and Cx40 appeared to be restricted to the cytoplasm. The strength of gap junctional conductance varied between patients and was significantly and positively correlated with Cx43 level, but neither was correlated with patient age. Interestingly, Cx43 level and intercellular conductance were positively correlated with embryo quality as judged by cleavage rate and morphology, and were significantly higher in patients who became pregnant than in those who did not. Thus, despite the presence of multiple connexins, Cx43 is a major contributor to gap junctions in human cumulus cells and its expression level may influence pregnancy outcome after ICSI.
Journal of Cellular and Molecular Medicine 05/2009; · 4.13 Impact Factor
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ABSTRACT: Expression of GJA1 (commonly known as connexin43 or Cx43), a major myometrial gap junction protein, is upregulated before the onset of delivery, suggesting an essential role for Cx43-mediated gap junctional intercellular communication (GJIC) in normal uterine contraction during parturition. To determine how a disease-linked Cx43 mutation affects myometrial function, we studied a mutant mouse model carrying an autosomal dominant mutation (Gja1(Jrt)) in the gene encoding Cx43 that displays features of the human genetic disease oculodentodigital dysplasia. We found that Cx43 level, specifically the phosphorylated species of the protein, is significantly reduced in the myometrium of the mutant mice (Gja1(Jrt)/+), as revealed by Western blotting and immunostaining. Patch-clamp electrophysiological measurements demonstrated that coupling between myometrial smooth muscle cells is reduced to <15% of wild-type, indicating that the mutant protein acts dominantly on its wild-type counterpart. The phosphorylated species of Cx43 in the mutant myometrium failed to increase prior to parturition as well as in response to exogenous estrogen. Correspondingly, in vitro experiments with uterine strips revealed weaker contraction of the mutant myometrium and reduced responsiveness to oxytocin, providing an explanation for the prolonged gestation and presence of suffocated fetuses in the uteri that were observed in some of the mutant mice. We conclude that the Gja1(Jrt) mutation has a dominant-negative effect on Cx43 function in the myometrium, severely reducing GJIC, leading to impaired parturition.
Biology of Reproduction 01/2009; 80(6):1099-106. · 4.01 Impact Factor
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ABSTRACT: Signaling via the conserved WNT/beta-CATENIN pathway controls diverse developmental processes. To explore its potential role in the ovary, we investigated the expression of WNTs, frizzled (FZD) receptors and other pathway components in human cumulus cells obtained from oocytes collected for in vitro fertilization. Proteins were detected in cultured cells using immunofluorescence microscopy. Protein-protein interactions were analyzed by means of immunoprecipitation. WNT2, FZD2, FZD3 and FZD9 were identified but WNT1, WNT4 and FZD4 were not detected. WNT2 is co-expressed with FZD2, FZD3 and FZD9. Co-immunoprecipitation using WNT2 antibody demonstrated that WNT2 interacts with both FZD3 and FZD9, but only FZD9 antibody precipitated WNT2. We also identified DVL (disheveled), AXIN, GSK-3beta (glycogen synthase kinase-3beta) and beta-CATENIN. beta-CATENIN is concentrated in the plasma membranes. DVL co-localizes with FZD9 and AXIN in the membranes, but GSK-3beta has little co-localization with AXIN and beta-CATENIN. Interestingly, beta-CATENIN is highly co-localized with FZD9 and AXIN. CDH1 (E-cadherin) was also detected in the plasma membranes and cytoplasm, co-localized with beta-CATENIN, and CDH1 antibody precipitated beta-CATENIN. The results suggest that WNT2 could act through its receptor FZD9 to regulate the beta-CATENIN pathway in cumulus cells, recruiting beta-CATENIN into plasma membranes and promoting the formation of adherens junctions involving CDH1.
Molecular Human Reproduction 12/2008; 15(1):11-7. · 3.85 Impact Factor
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ABSTRACT: We showed that lipopolysaccharide (LPS) or hypoxia and reoxygenation (H/R) decreases electrical coupling between microvascular endothelial cells by targeting the gap junction protein connexin40 (Cx40), tyrosine kinase-, ERK1/2-, and PKA-dependently. Since LPS can compromise microvascular blood flow, resulting in micro-regional H/R, the concurrent LPS + H/R could reduce coupling to a much greater extent than LPS or H/R alone. We examined this possibility in a model of cultured microvascular endothelial cells (mouse skeletal muscle origin) in terms of electrical coupling and the phosphorylation status of Cx40. To assess coupling, we measured the spread of electrical current injected into the cell monolayer and computed the intercellular resistance as an inversed measure of coupling. In wild type cells, but not in Cx40 null cells, concurrent LPS + H/R synergistically increased resistance by approximately 270%, well above the level observed for LPS or H/R alone. Cx37 and Cx43 protein expression did not differ between Cx40 null and wild type cells. LPS + H/R increased resistance PKA- and PKC-dependently. By immunoprecipitating Cx40, we found that LPS + H/R reduced serine phosphorylation to a much greater degree than that observed for LPS or H/R alone. Further, PKA-specific, but not PKC-specific serine phosphorylation of Cx40 was also significantly reduced following LPS + H/R. This reduction was prevented by tyrosine kinase and MEK1/2 inhibition, by PKA activation, and mimicked in control cells by PKA inhibition. We conclude that LPS + H/R initiates tyrosine kinase- and ERK1/2-sensitive signaling that synergistically reduces inter-endothelial electrical coupling by dephosphorylating PKA-specific serine residues of Cx40.
Journal of Cellular Physiology 11/2008; 217(2):350-9. · 3.87 Impact Factor
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ABSTRACT: Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD.
We expressed human and mouse ODDD-linked Cx43 mutants in MC3T3-E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin.
We showed that ODDD-linked Cx43 mutants are loss-of-function and dominant-negative to co-expressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation.
Expression of human and mouse ODDD-linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43-based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 07/2008; 23(6):928-38. · 6.04 Impact Factor
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ABSTRACT: Mammalian oocytes and surrounding granulosa cells are metabolically coupled via gap junctions. In growing follicles of the mouse, gap junctions between oocytes and granulosa cells are assembled from connexin 37 (Cx37, encoded by Gja4), whereas those between granulosa cells are assembled from connexin 43 (Cx43, encoded by Gja1). This spatial separation, and the different permeability properties of gap junctions composed of Cx37 and Cx43, suggests that Cx37 channels serve a unique function in oogenesis. Female mice lacking Cx37 are sterile because oocytes do not complete their development. To test the hypothesis that the unique properties of Cx37 make it irreplaceable in oocytes, Cx43 was ectopically expressed in growing oocytes lacking Cx37. Transgenic mice were produced in which Gja1 is expressed in oocytes under control of the Zp3 (zona pellucida protein 3) gene promoter. When the transgene was crossed into the Cx37-null mutant line, oocyte-granulosa-cell coupling, oocyte growth and maturation, and fertility were all restored. Thus, despite their different properties, Cx43 is physiologically equivalent to Cx37 in coupling oocytes with granulosa cells.
Journal of Cell Science 01/2008; 120(Pt 23):4117-25. · 6.11 Impact Factor
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ABSTRACT: Connexin43 (Cx43, encoded by Gja1) is required for ovarian follicle development in the mouse. It is strongly expressed in granulosa cells, in which it forms intercellular gap junction channels that couple the cells metabolically. However, recent evidence indicates that undocked gap junction hemichannels can also have physiological roles such as mediating the release of small messenger molecules, including ATP. In this study, the presence of undocked Cx43 hemichannels in granulosa cells was revealed by dye uptake induced either by mechanical stimulation or by the reduction of extracellular divalent cations, both of which are known triggers for hemichannel opening. ATP release was also detected, and could be abolished by connexin-channel blockers. None of these putative hemichannel-mediated activities were detected in Cx43-deficient granulosa cells. Therefore, we hypothesized that hemichannels account for the essential role of Cx43 in folliculogenesis. To test this, a Cx43 mutant lacking the conserved cysteines on the extracellular loops (cys-less Cx43), reported to form hemichannels but not intercellular channels, was retrovirally expressed in Cx43-deficient granulosa cells. The infected cells were then combined with wild-type oocytes to make reaggregated ovaries, which were grafted into host kidneys. Although re-introduction of wild-type Cx43 rescued folliculogenesis, introduction of cys-less Cx43 did not. Therefore, although Cx43 gap junction hemichannels might play a role in ovarian folliculogenesis, their contribution does not supplant the need for intercellular gap junction channels.
Journal of Cell Science 12/2007; 120(Pt 22):4016-24. · 6.11 Impact Factor
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ABSTRACT: To understand the role of connexin43 (Cx43) in epidermal differentiation, we reduced Cx43 levels by RNA-mediated interference knockdown and impaired its functional status by overexpressing loss-of-function Cx43 mutants associated with the human disease oculodentodigital dysplasia (ODDD) in rat epidermal keratinocytes. When Cx43 expression was knocked down by 50-75%, there was a coordinate 55-65% reduction in Cx26 level, gap junction-based dye coupling was reduced by 60%, and transepithelial resistance decreased. Importantly, the overall growth and differentiation of Cx43 knockdown organotypic epidermis was severely impaired as revealed by alterations in the levels of the differentiation markers loricrin and involucrin and by reductions in vital and cornified layer thicknesses. Conversely, although the expression of Cx43 mutants reduced the coupling status of rat epidermal keratinocytes by approximately 80% without altering the levels of endogenous Cx43 or Cx26, their ability to differentiate was not altered. In addition, we used a mouse model of ODDD and found that newborn mice harboring the loss-of-function Cx43(G60S) mutant had slightly reduced Cx43 levels, whereas Cx26 levels, epidermis differentiation, and barrier function remained unaltered. This properly differentiated epidermis was maintained even when Cx43 and Cx26 levels decreased by more than 70% in 3-week-old mutant mice. Our studies indicate that Cx43 and Cx26 collectively co-regulate epidermal differentiation from basal keratinocytes but play a more minimal role in the maintenance of established epidermis. Altogether, these studies provide an explanation as to why the vast majority of ODDD patients, where Cx43 function is highly compromised, do not suffer from skin disease.
Journal of Biological Chemistry 11/2007; 282(41):30171-80. · 4.77 Impact Factor
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ABSTRACT: Electrical coupling along the endothelium is central in the arteriolar conducted response and in control of vascular resistance. It has been shown that exposure of endothelium to lipopolysaccharide (LPS, an initiating factor in sepsis) reduces intercellular communication in vitro and in vivo. The molecular basis for this reduction is not known. We examined the effect of LPS on electrical coupling in monolayers of cultured mouse microvascular endothelial cells (MMEC) derived from the mouse hindlimb skeletal muscle. To assess coupling, we measured the spread of electrical current injected into the monolayer and computed the monolayer intercellular resistance (inverse measure of coupling). LPS (10 microg/ml, 1 h) reduced coupling (i.e., increased resistance) in MMEC isolated from wild-type, connexin37 (Cx37) null and Cx43(G60S) (nonfunctional mutant) mice, but not in MMEC derived from Cx40 null mice. LPS also activated JNK1/2, p38 and ERK1/2 MAP kinases. Pretreatment of WT monolayers with ERK1/2 inhibitor U0126 (20 microM, 1 h) prevented the LPS-induced decrease in coupling, while inhibition of JNK1/2 with SP600125 (20 microM, 1 h) and p38 with a p38 inhibitor (10 nM, 1 h) had no effect. Furthermore, inhibition of tyrosine kinases with PP-2 (10 nM, 1 h), activation of PKA by 8-bromo-cAMP (1 mM, 5 min), and activation of PKC by bryostatin-2 (10 nM, 1 h) also prevented the reduction in coupling. We propose that LPS reduces inter-endothelial electrical coupling via tyrosine-, ERK1/2-, PKA-, and PKC-dependent signaling that targets Cx40. We suggest that this mechanism contributes to compromised arteriolar function following LPS exposure.
Journal of Cellular Physiology 05/2007; 211(1):159-66. · 3.87 Impact Factor
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ABSTRACT: Connexin43 (Cx43) is the most abundantly expressed member of the connexin (gap junction protein) family and the only one so far identified in mouse Leydig cell gap junctions. Mice lacking Cx43 were used to investigate its role in testicular androgen production and regulation. Testes from term fetuses were grafted under the kidney capsules of castrated adult males. After 3 weeks, serum from host mice was analyzed for androgens. In order to test their response to stimulation, the grafted testes were incubated in vitro with varying concentrations of LH and their androgen end products analyzed. Incubation with radiolabeled progesterone was followed by high performance liquid chromatography to quantify the androgen-intermediate metabolites. Radiolabeled testosterone in the presence of NADPH was used to determine the activity of testosterone-metabolizing enzymes 17beta-hydroxysteroid dehydrogenase (17betaHSD), 5alpha-reductase (5alphaR), and 3alpha-hydroxysteroid dehydrogenase (3alpha HSD). Serum androgen levels did not differ between hosts carrying wild-type versus null mutant grafts although Cx43-deficient testes had more 17betaHSD and 5alphaR activity than wild-type controls. Furthermore, the genotype of grafted testes did not influence LH-stimulated androgen production in vitro. These results indicate that the steroidogenic function of Leydig cells is not compromised by the absence of Cx43, perhaps because other gap junction proteins are present. Dye transfer experiments demonstrated that Cx43-deficient Leydig cells retain intercellular coupling, indicating that Cx43 is not the only protein contributing to their gap junctions. Thus, despite their prominence in Leydig cells, Cx43 gap junctions are not essential for androgen production.
Reproduction (Cambridge, England) 11/2006; 132(4):607-16. · 3.09 Impact Factor
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ABSTRACT: Increased nitric oxide (NO) production in sepsis precipitates microcirculatory dysfunction. We aimed (i) to determine if NO is the key water-soluble factor in the recently discovered sepsis-induced deficit in arteriolar conducted vasoconstriction, (ii) to identify which nitric oxide synthase (NOS) isoforms account for this deficit, and (iii) to examine the potential role of connexin37 (Cx37, a hypothesized signaling target of NO) in arteriolar conduction.
Using intravital microscopy and the cecal ligation and perforation 24-h model of sepsis, arterioles in the cremaster muscle of male C57BL/6 wild-type (WT), iNOS-/-, eNOS-/-, nNOS-/- and Cx37-/- mice were locally stimulated with KCl to initiate conducted vasoconstriction. We used the ratio of conducted constriction (500 microm upstream) to local constriction as an index of conduction (CR500). NOS enzymatic activity and protein expression were determined in control and septic cremaster muscles.
Sepsis reduced CR500 in WT mice [from 0.77 +/- 0.05 to 0.20 +/- 0.02 (means +/- SE) independent of the site of stimulation along the arteriole], in iNOS-/- and eNOS-/- mice, but not in nNOS-/- mice. The nNOS inhibitor 7-nitroindazole or NO scavenger HbO2 restored CR500 in septic WT mice, but blockade of soluble guanylate cyclase had no effect. Sepsis increased cNOS (eNOS + nNOS) activity in WT mice (from 340 +/- 40 to 490 +/- 30 pmol/mg/h) and in eNOS-/-, but not in nNOS-/- mice (iNOS activity was negligible in all mice). Sepsis did not alter nNOS protein expression in WT mice. CR500 in non-septic Cx37-/- mice (0.15 +/- 0.1) was similar to that observed in septic WT mice.
Increased nNOS activity and the resultant increased NO production in the septic mouse cremaster muscle are the key factors responsible for the deficit in conducted vasoconstriction along the arteriole. Deletion of Cx37 results in reduced CR500, which is consistent with the hypothesis that Cx37 in the arteriole could be a target of NO signaling.
Cardiovascular Research 02/2006; 69(1):236-44. · 6.06 Impact Factor
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ABSTRACT: Genetic ablation of connexin37 (Cx37) or connexin43 (Cx43), the two gap junction proteins expressed by mouse ovarian granulosa cells, has been shown to result in impaired follicle development. We used patch-clamp techniques to evaluate quantitatively the contribution of these connexins to gap junctional intercellular communication (GJIC) among granulosa cells. The coupling conductance derived from a voltage step-induced capacitive current transient was used as a measure of GJIC in cultured granulosa cells. Using this method, we determined that the conductance of wild-type (84.1 +/- 28.6 nS; n = 6) and Cx37-deficient granulosa cells (83.7 +/- 6.4 nS; n = 11) does not differ significantly (P = 0.35), suggesting a limited contribution, if any, of Cx37 to granulosa cell coupling. In contrast, the conductance between granulosa cells of Cx43-deficient mice (2.6 +/- 0.8 nS; n = 5) was not significantly different from that of single, isolated wild-type granulosa cells (2.5 +/- 0.7 nS, n = 5; P = 0.83), indicating that Cx43-deficient granulosa cells were not electrically coupled. A direct measurement of transjunctional conductance between isolated granulosa cell pairs using a dual patch-clamp technique confirmed this conclusion. Interestingly, a partial rescue of folliculogenesis was observed when the Cx43-null mutation in C57BL/6 mice was crossed into the CD1 strain, and capacitive current measurement demonstrated that this rescue was not due to reestablishment of GJIC. These results demonstrate that folliculogenesis is impaired in the absence of GJIC between granulosa cells, but they also indicate that the severity is dependent on genetic background, a phenomenon that cannot be attributed to the expression of additional connexins.
AJP Cell Physiology 02/2006; 290(1):C290-7. · 3.54 Impact Factor
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ABSTRACT: The gap junction proteins connexin37 and connexin43 are required for ovarian folliculogenesis in the mouse. To define their respective roles in oogenesis, chimeric ovaries containing either null mutant oocytes and wild-type granulosa cells or the reverse combination were grafted to the renal capsules of immunodeficient female mice. After three weeks, the oocytes were tested for meiotic competence and fertilizability in vitro. Ovaries composed of connexin43-deficient oocytes and wild-type granulosa cells produced antral follicles enclosing oocytes that could develop to at least the two-cell stage, demonstrating that oocytes need not express connexin43 to reach maturity. Conversely, both follicle development and oocyte maturation were impaired in ovaries containing either wild-type oocytes and connexin43-deficient granulosa cells or connexin37-deficient oocytes and wild-type granulosa cells. Thus absence of connexin43 from granulosa cells or connexin37 from oocytes is sufficient to compromise both oocyte and follicle development. Wild-type oocytes paired with connexin37-deficient granulosa cells generated antral follicles containing oocytes that developed to at least the two-cell stage. Therefore, connexin37 absence from granulosa cells need not impair fertility in mice. Dye transfer experiments revealed persistent oocyte-granulosa cell coupling in those follicles, indicating functional compensation by another connexin. The results indicate that mouse oocytes do not need to express connexin43 in order to develop into meiotically competent, fertilizable gametes, but must express connexin37 for communication with granulosa cells, a requirement for oogenesis.
Journal of Cell Science 12/2005; 118(Pt 21):5071-8. · 6.11 Impact Factor
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ABSTRACT: Before implantation into the uterine wall, the mammalian embryo undergoes a period of cell division, cell shape change, and cell differentiation leading to the formation of an outer epithelium, the trophectoderm. The trophectoderm is the part of the embryo that initiates uterine contact and, after transformation to become the trophoblast, uterine invasion. Similar to the kidney nephron, the trophectoderm is a transporting epithelium with distinct apical and basolateral membrane domains; its function is to facilitate transepithelial Na+ and fluid transport for blastocoel formation. That transport is driven by Na,K-adenosine triphosphatase (ATPase) localized in basolateral membranes of the trophectoderm. Preimplantation embryos express multiple alpha and beta subunit isoforms of Na,K-ATPase, potentially constituting multiple isozymes, but the basolaterally located alpha1beta1 isozyme appears to function uniquely to drive fluid transport. Embryos unable to express alpha1 subunits because of targeted deletion of the gene are able to form a blastocoel, but they fail to maintain their integrity and expire during the peri-implantation period. Preimplantation embryos also express the gamma subunit, a modulator of Na,K-ATPase activity, but targeted deletion of that gene did not reveal an essential developmental role. The preimplantation embryo offers a unique model for understanding the roles of Na,K-ATPase subunit isoforms in epithelial development and transepithelial transport.
Seminars in Nephrology 10/2005; 25(5):352-5. · 2.12 Impact Factor
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Ann M Flenniken,
Lucy R Osborne,
Nicole Anderson,
Nadia Ciliberti,
Craig Fleming,
Joanne E I Gittens,
Xiang-Qun Gong,
Lois B Kelsey,
Crystal Lounsbury,
Luisa Moreno, [......],
Marc Grynpas,
Janet E Henderson,
R Mark Henkelman,
Colin McKerlie,
John G Sled,
William L Stanford,
Dale W Laird, Gerald M Kidder,
S Lee Adamson,
Janet Rossant
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ABSTRACT: Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.
Development 10/2005; 132(19):4375-86. · 6.60 Impact Factor
