![Real time RT-PCR to detect transcripts of ␣ - ( top ),  - ( middle ), and ␥ - ( bottom ) epithelial Na channel (ENaC) in different brain areas [organum vasculosum of lamina terminalis (OVLT); subfornical organ (SFO); supraoptic nucleus (SON); paraventricular nucleus (PVN); choroid plexis (CP); hippocampus (Hippo); and median preoptic nucleus (MnPo)]. Total RNA was isolated from the kidney (Kid) and different brain areas of Wistar rats. cDNAs were synthesized and subjected to real-time PCR. The 429-, 220-, and 301-bp PCR products were amplified for ␣ -,  -, and ␥ -ENaC, respectively, by using ENaC-specific primers. Kidney served as positive control, and the RT reaction with absence of reverse transcriptase served as negative control (Neg).](https://www.researchgate.net/profile/Frans-Leenen/publication/7620618/figure/fig1/AS:318884235235328@1453039334131/Real-time-RT-PCR-to-detect-transcripts-of-top-middle-and-bottom_Q320.jpg)
Content uploaded by Frans Leenen
Author content
All content in this area was uploaded by Frans Leenen on Jan 17, 2016
Content may be subject to copyright.
A preview of the PDF is not available
Distribution of epithelial sodium channels and mineralocorticoid receptors in cardiovascular regulatory centers in rat brain
Abstract and Figures
Epithelial sodium channels (ENaC) are important for regulating sodium transport across epithelia. Functional studies indicate that neural mechanisms acting through mineralocorticoid receptors (MR) and sodium channels (presumably ENaC) are crucial to the development of sympathoexcitation and hypertension in experimental models of salt-sensitive hypertension. However, expression and localization of the ENaC in cardiovascular regulatory centers of the brain have not yet been studied. RT-PCR and immunohistochemistry were performed to study ENaC and MR expression at the mRNA and protein levels, respectively. Both mRNA and protein for alpha-, beta-, and gamma-ENaC subunits and MR were found to be expressed in the rat brain. All three ENaC subunits and MR were present in the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels, suggesting the presence of multimeric channels and possible regulation by mineralocorticoids. In most cortical areas, thalamus, amygdala, and suprachiasmatic nucleus, notable expression of gamma-ENaC was undetectable, whereas alpha- and beta-ENaC were abundantly expressed pointing to the possibility of a heterogeneous population of channels. The findings suggest that stoichiometrically different populations of ENaC may be present in both epithelial and neural components in the brain, which may contribute to regulation of cerebrospinal fluid and interstitial Na+ concentration as well as neuronal excitation.
Figures - uploaded by Frans Leenen
Author content
All figure content in this area was uploaded by Frans Leenen
Content may be subject to copyright.
... These regions then complete the homeostatic loop regulating water and electrolyte balance by affecting motivation for water drinking and salt appetite. The role ENaCs play in cardiovascular mechanisms of water and electrolyte homeostasis also suggests ENaCs may be components of the molecular machinery underlying the homeostatic mechanisms in the brain regions listed above (Voisin and Bourque 2002;Amin et al. 2005). ...
... Moreover, these few tdTomato-labeled cells did not co-label with anti-NeuN and were therefore unlikely to be neurons (Fig. 8a-c). These observations differ significantly from previous reports (Voisin and Bourque 2002;Amin et al. 2005;Teruyama et al. 2012). ...
Epithelial sodium channel (ENaC) is responsible for regulating Na⁺ homeostasis. While its physiological functions have been investigated extensively in peripheral tissues, far fewer studies have explored its functions in the brain. Since our limited knowledge of ENaC’s distribution in the brain impedes our understanding of its functions there, we decided to explore the whole-brain expression pattern of the Scnn1a gene, which encodes the core ENaC complex component ENaCα. To visualize Scnn1a expression in the brain, we crossed Scnn1a-Cre mice with Rosa26-lsl-tdTomato mice. Brain sections were subjected to immunofluorescence staining using antibodies against NeuN or Myelin Binding Protein (MBP), followed by the acquisition of confocal images. We observed robust tdTomato fluorescence not only in the soma of cortical layer 4, the thalamus, and a subset of amygdalar nuclei, but also in axonal projections in the hippocampus and striatum. We also observed expression in specific hypothalamic nuclei. Contrary to previous reports, however, we did not detect significant expression in the circumventricular organs, which are known for their role in regulating Na⁺ balance. Finally, we detected fluorescence in cells lining the ventricles and in the perivascular cells of the median eminence. Our comprehensive mapping of Scnn1a-expressing cells in the brain will provide a solid foundation for further investigations of the physiological roles ENaC plays within the central nervous system.
... These Na channels are non-voltagedependent, and conduct Na D ions across the apical membrane of cells in salt-reabsorbing epithelia, such as in the distal nephron, 35) in which they play a master role in regulating the volume of extracellular fluid. ENaCs are also expressed in astrocytes, endothelial cells, and the choroid plexus, 36) which may contribute to the maintenance of physiological [Na D ] in CSF. Besides central neurons, 36),37) ENaCs function as Na detectors of the salt taste receptors in the tongue. ...
... ENaCs are also expressed in astrocytes, endothelial cells, and the choroid plexus, 36) which may contribute to the maintenance of physiological [Na D ] in CSF. Besides central neurons, 36),37) ENaCs function as Na detectors of the salt taste receptors in the tongue. 38),39) Loewy's group reported that ENaCpositive neurons are highly concentrated in sCVOs. ...
Extracellular fluids, including blood, lymphatic fluid, and cerebrospinal fluid, are collectively called body fluids. The Na+ concentration ([Na+]) in body fluids is maintained at 135-145 mM and is broadly conserved among terrestrial animals. Homeostatic osmoregulation by Na+ is vital for life because severe hyper- or hypotonicity elicits irreversible organ damage and lethal neurological trauma. To achieve "body fluid homeostasis" or "Na homeostasis", the brain continuously monitors [Na+] in body fluids and controls water/salt intake and water/salt excretion by the kidneys. These physiological functions are primarily regulated based on information on [Na+] and relevant circulating hormones, such as angiotensin II, aldosterone, and vasopressin. In this review, we discuss sensing mechanisms for [Na+] and hormones in the brain that control water/salt intake behaviors, together with the responsible sensors (receptors) and relevant neural pathways. We also describe mechanisms in the brain by which [Na+] increases in body fluids activate the sympathetic neural activity leading to hypertension.
... To illustrate, sustained intracerebroventricular (ICV) administration of aldosterone induces hypertension (Gomez-Sanchez, 1986). Moreover, mineralocorticoid receptors and epithelial sodium channels have been identified throughout the lamina terminalis of the brain (Amin et al., 2005;Miller et al., 2013), and blocking central ...
We have previously reported that the subfornical organ (SFO) does not contribute to the chronic hypertensive response to DOCA‐salt in rats, and yet the organum vasculosum of the lamina terminalis (OVLT) plays a significant role in the development of deoxycorticosterone acetate (DOCA)‐salt hypertension. Since efferent fibers of the OVLT project to and through the median preoptic nucleus (MnPO), the present study was designed to test the hypothesis that the MnPO is necessary for DOCA‐salt hypertension in the rat. Male Sprague‐Dawley rats underwent SHAM (MnPOsham; n = 5) or electrolytic lesion of the MnPO (MnPOx; n = 7) followed by subsequent unilateral nephrectomy and telemetry instrumentation. After recovery and during the experimental protocol, rats consumed a 0.1% NaCl diet and 0.9% NaCl drinking solution. Mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). The chronic pressor response to DOCA was attenuated in MnPOx rats by Day 11 of treatment and continued such that MAP increased 25 ± 3 mmHg in MnPOsham rats by Day 21 of DOCA compared to 14 ± 3 mmHg in MnPOx rats. These results support the hypothesis that the MnPO is an important brain site of action and necessary for the full development of DOCA‐salt hypertension in the rat.
... 73 Although ENaCs are mainly localized in epithelial tissues, alpha and beta ENaC subunits are abundantly co-expressed in many brain regions. 74 Regarding its in vivo function, our results have shown that del-4 mutants are insensitive to solution at pH 4.5 indicating that DEL-4 could be directly involved in acid sensation. Additionally, we have also shown that DEL-4 may play a more modulatory role during neurotransmission. ...
The nervous system participates in the initiation and modulation of systemic stress. Ionstasis is of utmost importance for neuronal function. Imbalance in neuronal sodium homeostasis is associated with pathologies of the nervous system. However, the effects of stress on neuronal Na+ homeostasis, excitability, and survival remain unclear. We report that the DEG/ENaC family member DEL-4 assembles into a proton-inactivated sodium channel. DEL-4 operates at the neuronal membrane and synapse to modulate Caenorhabditis elegans locomotion. Heat stress and starvation alter DEL-4 expression, which in turn alters the expression and activity of key stress-response transcription factors and triggers appropriate motor adaptations. Similar to heat stress and starvation, DEL-4 deficiency causes hyperpolarization of dopaminergic neurons and affects neurotransmission. Using humanized models of neurodegenerative diseases in C. elegans, we showed that DEL-4 promotes neuronal survival. Our findings provide insights into the molecular mechanisms by which sodium channels promote neuronal function and adaptation under stress.
... Another possible mechanism is the expression of both mineralocorticoid responsive epithelial sodium channel receptors on the basolateral membrane of the CSF producing epithelial cells of the choroid plexus as well as the expression of 11-beta hydroxysteroid dehydrogenase type 1 enzyme, which is a bidirectional enzyme that mainly functions to convert the inactive cortisone to active cortisol. These mechanisms play a role in maintaining the balance between CSF production and absorption [13,14]. ...
The association between empty sella turcica (EST) syndrome and Cushing's disease has been rarely reported. It is plausible to hypothesize that EST syndrome in association with Cushing's disease can be attributed to intracranial hypertension. In this case report, we present a 47-year-old male patient who presented with weight loss, fatigue, easy bruising, acanthosis nigricans, and skin creases hyperpigmentation. Investigations revealed hypokalemia and confirmed the diagnosis of Cushing's disease. Magnetic resonance imaging (MRI) brain showed a partial EST syndrome and a new pituitary nodule as compared with previous brain imaging. Transsphenoidal surgery was pursued and was complicated by cerebrospinal fluid leakage. This case reflects the rare association of EST syndrome and Cushing's disease, suggesting the increased risk of postoperative complications in this setting and the diagnostic challenge that EST syndrome imposes. We review the literature for a possible mechanism of this association.
... 339 Several ENaCs are present in the central nervous system, being expressed in neurones, ependymocytes, choroid plexus cells and in astrocytes. 340 Fibrous astrocytes at the borders of circumventricular organs specifically express homotrimeric gsubunit assembled (SCNN1G) ENaCs, possibly involved in Na þ sensing. 341 Astrocytes also show immunoreactivity for ASIC1, ASIC2a and ASIC3 channels, which are localised intracellularly. ...
... Non-voltage dependent channels, particularly the epithelial Na + channel (ENaC), were identified in the SON by immunocytochemistry (Amin et al., 2005) and their functionality was ascertained by Teruyama et al. (2012), by associating pharmacology and electrophysiological recordings in brain slice preparations. They demonstrated that ENaC has a significant influence on the resting membrane potential of MNCs, since its blockage, by amiloride or benzamil, resulted in hyperpolarization of the membrane potential and cessation of action potential firing . ...
Due to the relatively high permeability to water of the plasma membrane, water tends to equilibrate its chemical potential gradient between the intra and extracellular compartments. Because of this, changes in osmolality of the extracellular fluid are accompanied by changes in the cell volume. Therefore, osmoregulatory mechanisms have evolved to keep the tonicity of the extracellular compartment within strict limits. This review focuses on the following aspects of osmoregulation: 1) the general problems in adjusting the “milieu interieur” to challenges imposed by water imbalance, with emphasis on conceptual aspects of osmosis and cell volume regulation; 2) osmosensation and the hypothalamic supraoptic nucleus (SON), starting with analysis of the electrophysiological responses of the magnocellular neurosecretory cells (MNCs) involved in the osmoreception phenomenon; 3) transcriptomic plasticity of SON during sustained hyperosmolality, to pinpoint the genes coding membrane channels and transporters already shown to participate in the osmosensation and new candidates that may have their role further investigated in this process, with emphasis on those expressed in the MNCs, discussing the relationships of hydration state, gene expression, and MNCs electrical activity; and 4) somatodendritic release of neuropeptides in relation to osmoregulation. Finally, we expect that by stressing the relationship between gene expression and the electrical activity of MNCs, studies about the newly discovered plastic-regulated genes that code channels and transporters in the SON may emerge.
The epithelial Na ⁺ channel (ENaC) resides on the apical surfaces of specific epithelia in vertebrates and plays a critical role in extracellular fluid homeostasis. Evidence that ENaC senses the external environment emerged well before the molecular identity of the channel was reported three decades ago. This article discusses progress toward elucidating the mechanisms through which specific external factors regulate ENaC function, highlighting insights gained from structural studies of ENaC and related family members. It also reviews our understanding of the role of ENaC regulation by the extracellular environment in physiology and disease. After familiarizing the reader with the channel's physiological roles and structure, we describe the central role protein allostery plays in ENaC's sensitivity to the external environment. We then discuss each of the extracellular factors that directly regulate the channel: proteases, cations and anions, shear stress, and other regulators specific to particular extracellular compartments. For each regulator, we discuss the initial observations that led to discovery, studies investigating molecular mechanism, and the physiological and pathophysiological implications of regulation. © 2024 American Physiological Society. Compr Physiol 14:5407‐5447, 2024.
The cerebrospinal fluid (CSF) fills the brain ventricles and the subarachnoid space surrounding the brain and spinal cord. The fluid compartment of the brain ventricles communicates with the interstitial fluid of the brain across the ependyma. In comparison to blood, the CSF contains very little protein to buffer acid–base challenges. Nevertheless, the CSF responds efficiently to changes in systemic pH by mechanisms that are dependent on the CO2/HCO3− buffer system. This is evident from early studies showing that the CSF secretion is sensitive to inhibitors of acid/base transporters and carbonic anhydrase. The CSF is primarily generated by the choroid plexus, which is a well-vascularized structure arising from the pial lining of the brain ventricles. The epithelial cells of the choroid plexus host a range of acid/base transporters, many of which participate in CSF secretion and most likely contribute to the transport of acid/base equivalents into the ventricles. This review describes the current understanding of the molecular mechanisms in choroid plexus acid/base regulation and the possible role in CSF pH regulation.
The choroid plexus is a small but very active epithelial structure located in the brain ventricles. Here, it secretes the majority of the cerebrospinal fluid that covers the brain and spinal cord. The epithelial cells of the choroid plexus have a high rate of fluid secretion and differs from many other secretory epithelia in the organization of the main ion transporters. One striking difference is the apical localization of the Na,K ATPase. In recent years focus has increased on the role of the ion transporters as more studies have indicated implication of increased transport in disorders such as hydrocephalus, idiopathic intracranial hypertension and sequelae following intraventricular hemorrhages. The role of the choroid plexus ion transporters in regulation of composition of the cerebrospinal fluid has also been the focus in research in recent years as a regulator of breathing, blood pressure and heart rate.
This chapter focuses on the role of key ion transporters involved in cerebrospinal fluid secretion and cerebrospinal fluid ion composition. The chapter gives an overview of established factors as well as controversies in the area of ion transporters and finally discusses the future perspectives related to targeting treatment of cerebrospinal fluid disorders toward transporters on the choroid plexus epithelium.
Chronic stress has been associated with degenerative changes in the rodent and primate hippocampus, presumably mediated in part via neuronal glucocorticoid receptors (GRs). In the rat brain, GRs are widely distributed and are particularly dense in the hippocampus. The distribution of GRs in the primate brain, however, has not been fully characterized. In this study, we used in situ hybridization histochemistry and immunohisto-chemistry to map the distribution of GR mRNA and GR protein, respectively, in adult rhesus monkeys (Macaca mulatta). In contrast to its well established distribution in the rat brain, GR mRNA was only weakly detected in the dentate gyrus (DG) and Cornu Ammonis (CA) of the macaque hippocampus, whereas it was abundant in the pituitary (PIT), cerebellum (CBL), hypotha-lamic paraventricular nucleus (PVN), and, to a lesser extent, the neocortex. Immunohistochemical staining indicated a very low density of GR-like immunoreactive cells within the macaque hippocampal formation in contrast to the high density observed within the PVN, prefrontal and entorhinal cortices, and cerebel-lar cortex. Relative to the low level of GR, mineralocorticoid receptor (MR) mRNA and protein expression were abundant within the DG and CA of the rhesus monkey hippocampal formation. These results indicate that, in the primate, neocorti-cal and hypothalamic areas may be more important targets for GR-mediated effects of glucocorticoids than the hippocampus. Alternatively, it is also possible that glucocorticoid effects are mediated through the MRs present in the hippocampal formation.
A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.
Caenorhabditis elegans MEC-4 and MEC-10 are subunits of the degenerin/epithelial Na+ channel (DEG/ENaC) ion channel superfamily thought to be associated with MEC-2 (a stomatin-like protein) in a mechanotransducing molecular complex in specialized touch sensory neurons. A key question is whether analogous molecular complexes in higher organisms transduce mechanical signals. To address this question, we selected mechanoreceptors of the rat vibrissal follicle-sinus complex in the mystacial pad and the trigeminal ganglia for an immunocytochemical and molecular biological study. RT-PCR of poly(A+) mRNA of rat trigeminal ganglia indicated that α-, β-, and γ-ENaC and stomatin mRNA are expressed in rat trigeminal ganglia. Using immunocytochemistry, we found that α-, β-, and γ-ENaC subunits and stomatin are localized in the perikarya of the trigeminal neurons and in a minor fraction of their termination site in the vibrissal follicle-sinus complex, where longitudinal lanceolate endings are immunopositive. We conclude that α-, β-, and γ-ENaC subunits as well as the candidate interacting protein stomatin are coexpressed in a mammalian mechanoreceptor, a location consistent with a possible role in mechanotransduction.
The myogenic response is an essential component of renal blood flow autoregulation and is the inherent ability of vascular smooth muscle cells (VSMCs) to contract in response to increases in intraluminal pressure. Although mechanosensitive ion channels are thought to initiate VSMC stretch-induced contraction, their molecular identity is unknown. Recent reports suggest degenerin/epithelial Na(+) channels (DEG/ENaC) may form mechanotransducers in sensory neurons and VSMCs; however, the role of DEG/ENaC proteins in myogenic constriction of mouse renal arteries has not been established. To test the hypothesis that DEG/ENaC proteins are required for myogenic constriction in renal vessels, we first determined expression of ENaC transcripts and proteins in mouse renal VSMCs. Then, we determined pressure- and agonist-induced constriction and changes in vascular smooth muscle cytosolic Ca(2+) and Na(+) in isolated mouse renal interlobar arteries following DEG/ENaC inhibition with amiloride and benzamil. We detect alpha-, beta-, and gamma ENaC transcript and protein expression in cultured mouse renal VSMC. In contrast, we detect only beta- and gamma- but not alpha ENaC protein in freshly dispersed mrVMSC. Selective DEG/ ENaC inhibition, with low doses of amiloride and benzamil, abolishes pressure- induced constriction and increases in cytosolic Ca(2+) and Na(+) without diminishing agonist-induced responses in isolated mouse interlobar arteries. Our findings indicate that DEG/ ENaC proteins are required for myogenic constriction in mouse interlobar arteries and are consistent with our hypothesis that DEG/ ENaC proteins may be components of mechanosensitive ion channel complexes required for myogenic vasoconstriction.
Polyclonal antibodies have been raised against the , and subunits of the amiloride-sensitive Na+ channel. The three subunits were detected by immunohistochemistry at the apical membrane of epithelial cells from the distal colon, the lung and the distal segments of the kidney tubules. No significant labelling was detected in lung alveoli, suggesting that it is not a major site of expression of the Na+ channel. Effects of a low Na+ diet or of dexamethasone treatment were measured at the mRNA level and at the protein level by immunohistochemistry. In the colon, steroids controlled Na+ channel activity via the stimulation of the transcription of and subunits. The mRNA was constitutively expressed. However, while neither , nor proteins were detected in the colon of control animals, they were all detected in the colon of steroid-treated animals. In the lung, Na+ channel expression was regulated by glucocorticoids the circulating level of which was sufficiently high to induce a maximal expression of the three subunits, even in control animals. Adrenalectomy drastically reduced expression of the three subunits. A surprising finding was the apparent absence of steroid effects on , and subunit expression in the kidney. Neither the expression of the mRNAs nor the expression of the proteins were significantly altered by aldosterone or by dexamethasone. These results could be due to mixed gluco -and mineralocorticoid regulations in different segments of the kidney tubule, but their interpretation also requires regulations that are apparently not found in the lung or colon.
Previous maps of Type I corticosteroid receptor binding in the rat central nervous system (CNS) revealed a restricted distribution of the receptor in limbic regions, hypothalamus, and circumventricular organs. More recent studies have shown a more widespread expression of the receptor, with high levels of Type I receptor mRNA in limbic, motor, and sensory systems. We have used two antisera against peptide sequences derived from the cDNA of the human Type I corticosteroid receptor to map the regional distribution and corticosteroid regulation of the intracellular location of Type I corticosteroid receptor-like immunoreactivity (Type I-ir) in the rat CNS.
Neurons showing Type I-ir were observed at all levels of the CNS. Highest densities of immunoreactive neurons were observed in limbic regions, isocortex, and some thalamic nuclei. Motor, sensory, and visceral systems often showed moderate densities of immunoreactive neurons. Type I-ir glia were observed in some fiber systems, e.g., corpus callosum, medial lemniscus, cerebral peduncles, spinal trigeminal tract, and funiculi of the spinal cord. In the majority of neurons and in glia, Type I-ir showed a diffusely nuclear and cytoplasmic location, Long-term adrenalectomy reduced immunoreactivity in most neurons and glia. Neuronal Type I-ir was localized mainly in the cytoplasm after long-term adrenalectomy. Nuclear immunoreactivity was retained in some neurons in the globus pallidus, motor trigeminal nucleus, and laminae 8 and 9 of the spinal cord. Acute treatment with corticosterone or aldosterone restored neuronal and glial Type I-ir to densities below that seen in intact rats.
Using a polyclonal antiserum against the hinge region of the recently cloned human mineralocorticoid receptor (MR) and indirect peroxidase immunohistochemistry, we have shown MR-like immunoreactivity (LI) in superficial nephron segments, including distal convoluted tubule, connecting piece and initial cortical collecting duct. The absence of staining in cells tentatively identified as intercalated cells on light microscopy was confirmed by pre-embedding electron microscopy. Though the intracellular distribution of immunostaining varied with the fixative used, the cellular distribution of MR-LI is in good general agreement with earlier micropuncture and autoradiographic studies.