RESUMEN

The cardiovascular system is now recognized as an important mineralocorticoid target. All -components required for specific and selective aldosterone effects are present in the cardiovascular system. Mineralocorticoid receptors (MR) are expressed in the heart and large blood vessels together with the 11 B-hydroxysteroid dehydrogenase type II, which ensures the enzymatic protection of MR against glucocorticoids. The recent description of local vascular and cardiac aldosterone biosynthesis strongly supports an autocrine/paracrine hormonal action. Establishment of transgenic mice models of targeted overexpression of the mineralocorticoid receptor should facilitate new insights into the molecular and cellular mechanisms of aldo-sterone actions in the cardiovascular system.

Asunto(s)

RESUMEN

The classical short-term effect (within minutes) of arginine vasopressin (AVP) consists in increasing sodium, chloride and water transport in kidney cells. More recently, long-term actions (several hours) of the hormone have been evidenced on water and sodium fluxes, due to transcriptional enhancement in the expression of their transporters. The present study demonstrates that AVP is also responsible for a long-term increase in net chloride secretion. In the RCCD(1) rat cortical collecting duct cell line, 10(-8) M AVP induced, after several hours, an increase in net (36)Cl(-) secretion. This delayed effect of AVP was inhibited by basal addition of 10(-4) M bumetanide and apical addition of 10(-4) M glibenclamide, suggesting chloride entry at the basal membrane through a Na(+)/K(+)/2Cl(-) and apical secretion through a chloride conductance. An original acute cell permeabilization method was developed to allow for entry of antibodies directed against the regulatory region (R) of the cystic fibrosis transmembrane regulator (CFTR) into the cells. This procedure led to a complete and specific blocking of the long-term net chloride secretion induced by AVP. Finally, it was observed that CFTR transcripts steady-state level was significantly increased by AVP treatment. Besides the well-documented short-term effect of AVP on chloride transport, these results provide evidence that in RCCD(1) cells, AVP induces a delayed increase in transepithelial net chloride secretion that is mediated by a Na(+)/K(+)/2Cl(-) co-transporter and CFTR.

Asunto(s)

RESUMEN

Several K+ conductances have been identified in the kidney, with specific properties and localization in distinct cell types and membrane domains. On the other hand, several K+ channels have been characterized at the molecular level. By immunolocalization, we show that a new inward rectifying K+ channel, TWIK-1, is specifically expressed in distinct tubular segments and cell types of the rat kidney. In the proximal tubule, TWIK-1 prevails in the initial portions (convoluted part), where it is restricted to the apical (brush-border) membrane. In the collecting duct, immunofluorescence was intracellular or confined to the apical membrane and restricted to intercalated cells, i.e., in cells lacking aquaporin-2, as shown by double immunofluorescence. TWIK was also expressed in medullary and cortical parts of the thick limb of the loop of Henle, identified with an anti-Tamm-Horsfall protein antibody (double immunofluorescence). The intensity of TWIK-1 immunolabeling was unchanged in rats fed a low-Na+ or a low-K+ diet. Because TWIK-1 shares common properties with the low-conductance apical K+ channel of the collecting duct, we propose that it could play a role in K+ secretion, complementary to ROMK, another recently characterized K+ channel located in principal cells of the cortical collecting duct and in the loop of Henle.

Asunto(s)

RESUMEN

The main mechanisms involved in the regulation of sodium transport by steroid hormones are briefly reviewed. The respective roles of the apical epithelial sodium channel, which is likely to be the limitant step of steroid-regulated transepithelial sodium transport, and Na,K-ATPase are described. Regulation of these ion transporting proteins by aldosterone and glucocorticoid hormones, probably via a two step mechanism (rapid activation of channels or pumps by unknown regulators, and modulation of the transcription/translation rate of these transporters), is discussed. The mechanisms of mineralocorticoid selectivity, that is, the integrated process allowing a specific action of aldosterone, in spite of high concentrations of glucocorticoids that crossbind with aldosterone to the mineralocorticoid receptor (MR), are explained, as is the role of the enzyme 11 beta-hydroxysteroid dehydrogenase and the differential interactions of MR with steroid ligands and hormone responsive elements of DNA. Finally, synergism between aldosterone and antidiuretic hormone for the stimulation of sodium transport is evoked.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

Arginine vasopressin (AVP) and corticosteroid hormones are involved in sodium reabsorption regulation in the renal collecting duct. Synergy between AVP and aldosterone has been well documented, although its mechanism remains unclear. Both aldosterone and glucocorticoid hormones bind to the mineralocorticoid receptor (MR), and mineralocorticoid selectivity depends on the MR-protecting enzyme 11 beta hydroxysteroid deshydrogenase (11-HSD), which metabolizes glucocorticoids into derivatives with low affinity for MR. We have investigated whether the activity of 11-HSD could be influenced by AVP and corticosteroid hormones. This study shows that in isolated rat renal collecting ducts, AVP increases 11-HSD catalytic activity. This effect is maximal at 10(-8) M AVP (a concentration clearly above the normal physiological range of AVP concentrations) and involves the V2 receptor pathway, while activation of protein kinase C or changes in intracellular calcium are ineffective. The stimulatory effect of AVP on 11-HSD is largely reduced after adrenalectomy, and is selectively restored by infusion of aldosterone, not glucocorticoids. We conclude that this synergy between AVP and aldosterone in controlling the activity of 11-HSD is likely to play a pivotal role in resetting mineralocorticoid selectivity, and hence sodium reabsorption capacities of the renal collecting duct.

Asunto(s)

RESUMEN

Expression of the mineralocorticoid receptor (MR) is restricted to some sodium-transporting epithelia and a few nonepithelial target tissues. Determination of the genomic structure of the human MR (hMR) revealed two different untranslated exons (1alpha and 1beta), which splice alternatively into the common exon 2, giving rise to two hMR mRNA isoforms (hMR alpha and hMR beta). We have investigated expression of hMR transcripts in renal, cardiac, skin, and colonic tissue samples by in situ hybridization with exon 1alpha and 1beta specific riboprobes, using an exon 2 probe as internal control. Specific signals for either exon 1alpha- and 1beta-containing mRNAs were detected in typically hMR-expressing cells in all tissues analyzed. hMR alpha and hMR beta were present in distal tubules of the kidney, in cardiomyocytes, in enterocytes of the colonic mucosa, and in keratinocytes and sweat glands. Interestingly, although both isoforms appear to be expressed at approximately the same level, the relative abundance of each message compared with that of exon 2-containing mRNA strikingly differs among aldosterone target tissues, suggesting the possibility of other tissue-specific transcripts originating from alternative splicing. Finally, functional hypermineralocorticism was associated with reduced expression of hMR beta in sweat glands of two patients affected by Conn's and Liddle's syndrome, whereas normal levels of hMR isoforms were found in one case of pseudohypoaldosteronism. Altogether, our results indicate a differential, tissue-specific expression of hMR mRNA isoforms, hMR beta being down-regulated in situations of positive sodium balance, independently of aldosterone levels.

Asunto(s)

RESUMEN

Distal colon and renal cortical collecting ducts are major effectors of aldosterone-dependent Na homeostasis. Na is absorbed by entry through an apical amiloride-sensitive Na channel and extruded by Na-K-ATPase at the basolateral membrane. Using a ribonuclease protection assay, we studied, in vivo, aldosterone regulation of alpha-, beta-, gamma-subunits of the rat epithelial Na channel (rENaC) and alpha 1- and beta 1-subunits of Na-K-ATPase. In the kidney, Na-K-ATPase mRNAs were also assayed over discrete tubular segments by in situ hybridization. In rat colon, all three rENaC mRNAs were decreased by adrenalectomy, with a major effect on beta- and gamma-subunits, and were restored with 7 days, but not 2 days, of aldosterone treatment; in the kidney, however, only alpha-transcripts varied. Na-K-ATPase alpha 1- and beta 1-subunit mRNAs in both organs were not (in the case of the beta 1-subunit) or were mildly (in the case of the alpha 1-subunit) affected after adrenalectomy. Our conclusions are as follows: 1) Transcripts of rENaC and Na-K-ATPase subunits are not coordinately regulated by aldosterone in vivo; i.e., modulation involves mainly the Na channel, not Na-K-ATPase; the effect is not of comparable magnitude on each subunit mRNA and differs between tissues. 2) The delay of the aldosterone effect on transcripts is much longer than that required to restore normal Na transport in adrenalectomized rats, indicating that rENaC and Na-K-ATPase subunit transcript levels may depend on unidentified early aldosterone-induced proteins.

Asunto(s)

RESUMEN

Na+ reabsorption from the epithelial surface of the respiratory tract plays a fundamental role in respiratory physiology. As in the epithelia of the renal collecting tubule and distal colon, Na+ enters across the luminal surface of respiratory epithelial cells via a recently cloned amiloride-sensitive multisubunit (alpha, beta, gamma) epithelial Na+ channel. We have examined the cellular expression at the mRNA level of the alpha-, beta-, and gamma-subunits of rat epithelial Na+ channel (rENaC) in the rat lung and upper airway epithelial cells using in situ hybridization. A large prevalence of alpha- and gamma-rENaC subunit expression (over beta) was found in tracheal epithelium, in a subpopulation of alveolar cells, presumably type II pneumocytes, and in nasal and tracheal gland acini. In contrast, equivalent levels of expression of all three subunits were detected in bronchiolar epithelium and in rat nasal gland ducts. This diversity of expression may reflect cell-specific functions of the amiloride-sensitive Na+ channel along the respiratory tract.

Asunto(s)

RESUMEN

We have examined whether arginine vasopressin (AVP) can induce a long-term modulation of transepithelial ion transport in addition to its well known short-term effect. In the RCCD1 rat cortical collecting duct cell line, an increase in both short-circuit current and 22Na transport was observed after several hours of 10(-8) M AVP treatment (a concentration above the in vivo physiological range). This delayed effect was partially prevented by apical addition of 10(-5) M amiloride and was blocked by 10(-6) M actinomycin D and 2 x 10(-6) M cycloheximide. The amounts of mRNA encoding the alpha1 (not beta1) subunit of Na+/K+-ATPase and the beta and gamma (not alpha) subunits of the amiloride-sensitive epithelial Na+ channel were significantly increased by AVP treatment. The increase in mRNA was blocked by actinomycin D, not by amiloride, suggesting a Na+-independent increase in the rate of transcription of these subunits. The translation rates of the alpha1 subunit of Na+/K+-ATPase and the beta and gamma subunits of the rat epithelial sodium channel increased significantly, whereas the translation rates of the other subunits remained unchanged. Finally, the number of Na+ channels present in the apical membrane of the cells increased, as demonstrated by enhanced specific [3H]phenamil binding.

Asunto(s)

RESUMEN

Channel inducing factor (CHIF) is a novel cDNA recently cloned from a rat distal colon cDNA library of dexamethasone-treated animals. While its expression in Xenopus oocytes evokes a potassium channel activity similar to that induced by Isk (minK), its cellular role is not clear. CHIF exhibits significant homologies with proteins that are putatively regulatory (phospholemman, gamma-subunit of Na(+)-K(+)-ATPase, Mat-8) while it differs from the small-conductance potassium channel Isk. We have studied the tissue specificity of CHIF expression in rat by in situ hybridization. CHIF is selectively present in the distal parts of the nephron (medullary and papillary collecting ducts and end portions of cortical collecting tubule) and in the epithelial cells of the distal colon. No expression of CHIF was found in renal proximal tubule, loop of Henle and distal tubule, proximal colon, small intestine, lung, choroid plexus, salivary glands, or brain. To gain some insight into CHIF function, we have investigated, using in situ hybridization and ribonuclease protection assay, whether CHIF mRNA expression could be altered in some situations. In the distal colon, corticosteroid hormones, sodium restriction, low-potassium diet, and metabolic acidosis significantly increased CHIF mRNA expression. In the kidney, metabolic acidosis was the only condition that showed an increase in CHIF mRNA expression. Some of these treatments also altered the expression of the colonic H(+)-K(+)-ATPase mRNA. In summary, CHIF mRNA is selectively expressed in the medullary collecting duct of the kidney and in the epithelium of the distal colon; its expression varies differently in these two target tissues after alterations in corticosteroid status, potassium depletion, and metabolic acidosis. The precise cell-specific functions of CHIF remain to be established.

Asunto(s)

RESUMEN

This study describes the establishment of a rat kidney cortical collecting duct (CCD) clonal cell line (RCCD1 cells) that maintains high transepithelial resistance and specific hormonal sensitivities. Immortalized cells were obtained by infection of primary cultured CCD cells with the wild-type simian virus 40. Grown on Petri dishes, RCCD1 cells are organized as monolayers of cuboid cells separated by tight junctions and form domes. Grown on permeable filters, confluent RCCD1 cells exhibit high transepithelial resistance (Rt: 2390 +/- 140 omega. cm2), transepithelial potential difference (PD) of -10.5 +/- 1.2 mV lumen negative, an associated short-circuit current (Isc) of 4.3 +/- 0.5 microA/cm2, and generated significant Na+, K+, H+ and HCO3- gradients, reflecting Na+ and H+ reabsorption and K+ and HCO3- secretion. RCCD1 cells exhibit features of both principal (PC) and intercalated (IC) cells. Consistent with PC phenotype, about 50% of the cells were positively stained by a PC-specific agglutinin. In situ hybridization studies revealed the presence of alpha, beta and gamma subunit mRNAs of the amiloride-sensitive epithelial Na+ channel and alpha 1 and beta 1 subunits of Na(+)-K(+)-ATPase. Moreover, Na(+)-K(+)-ATPase was immunolocalized at the basolateral side of the cells. Arginine vasopressin (AVP) induced a significant increase in both cellular cAMP content and Isc. Amiloride decreased in a dose-dependent manner Isc from untreated and AVP-treated RCCD1 cells. In addition, a barium-sensitive K+ conductance was evidenced in the apical side of the cells. Consistent with IC phenotype, isoproterenol (ISO) provoked a large increase in cellular cAMP and stimulated Isc. The effect of ISO on Isc was blocked by 5 x 10(-3) M DPC, a chloride channel blocker. Finally, AVP plus ISO had additive effect on Isc. Taken together, these results provide evidence that the RCCD1 cell line has maintained many of the original properties of rat CCD from which they were derived.

Asunto(s)

RESUMEN

Hormones can regulate the expression of their own receptor. We have examined whether adrenalectomy (ADX) and hormone replacement by physiological doses of aldosterone or dexamethasone could modulate the expression of glucocorticoid receptor (GR) or mineralocorticoid receptor (MR) at the mRNA level in the rat kidney, distal colon, and heart. Adult rats were adrenalectomized and received or did not receive an infusion of aldosterone (5 micrograms.100 g-1.day-1) or dexamethasone (10 micrograms.100 g-1.day-1). No significant change in steady-state levels of both MR and GR mRNA was detectable by using ribonuclease (RNase) protection assay (RPA) after either ADX or hormone replacement. Because the kidney is heterogeneous with regard to MR expression, RPA was adapted for measurements on microdissected nephron segments. GR mRNA is expressed at comparable levels all along the nephron, whereas MR mRNA is restricted to the distal nephron. No effect of ADX or GR and MR mRNA levels was detected in any nephron segment that was either aldosterone sensitive or insensitive. In situ hybridization confirmed the absence of corticosteroid-dependent modulation of MR mRNA in all kidney cell types. We conclude that variations of corticosteroid status do not affect MR and GR mRNA steady-state levels in heart, colon, and kidney and thus do not participate to the functional adaptations that are known to depend on hormonal status.

Asunto(s)

RESUMEN

Three subunits (alpha, beta, gamma) of the amiloride-sensitive epithelial sodium channel have been recently characterized. The channel subunits have significant homologies with the Caenorhabditis elegans mec-4, mec-10 and deg-1 genes, which are involved in control of cell volume and mecanotransduction. These subunits are coexpressed at equivalent levels in the renal collecting duct and the distal colon epithelium which are high resistance sodium transporting epithelia. We have investigated whether these subunits were expressed, at the mRNA level, in transporting as well as non transporting epithelial cells of rat skin. In full-thickness abdominal skin only alpha and gamma subunit mRNAs were detected, while all three subunit mRNAs were present in sole skin, as demonstrated by RNase-protection assay. Furthermore, the level of expression of each subunit varied with the epithelial cell type as demonstrated by in situ hybridization: epidermal and follicular keratinocytes express mostly alpha and gamma subunits (while beta was low); a prevalence of beta and gamma was observed in sweat glands. Thus, it appeared that two out of the three subunit mRNAs predominated in each epithelial structure. In addition, mRNAs of the alpha, beta and gamma subunits of the amiloride-sensitive sodium channel were expressed at a higher level in large suprabasal epidermal keratinocytes (which undergo terminal differentiation) than in small proliferative basal keratinocytes.

Asunto(s)

RESUMEN

K+ homeostasis depends on K+ absorption in digestive and renal epithelia. Recently, a cDNA encoding for a putative K(+)-adenosinetriphosphatase (ATPase) alpha-subunit has been characterized. We studied its expression by ribonuclease protection assay and in situ hybridization in the distal colon and the kidney of rats in various physiological states. In the distal colon of control rats, high expression of the colonic putative K(+)-ATPase mRNA was restricted to the surface epithelial cells. A low-K+ diet did not modify this expression, adrenalectomy decreased it, and aldosterone or dexamethasone treatment for 2 days restored normal levels. In the kidney of control rats, levels of K(+)-ATPase mRNA were very low. A low-K+ diet revealed a clear mRNA expression, which is consistent with a recent report [J.A. Kraut, F. Starr, G. Sachs, and M. Reuben. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F581-F587, 1995]. This expression was restricted to the outer medullary collecting duct, presumably in principal cells. Changes in corticosteroid status did not influence the renal expression. Our results, together with previous studies on K+ absorption and K(+)-ATPase activity, suggest that more than a single molecular form of K(+)-ATPase is likely to be responsible for the regulation of K+ absorption in the colon and distal nephron.

Asunto(s)

RESUMEN

It has been suggested that aldosterone exerts direct effects on heart functioning, in particular by inducing cardiac fibrosis. We examined human heart tissue for the expression of aldosterone receptors (mineralocorticoid receptors, MRs) and of the MR-protecting enzyme, 11 beta hydroxysteroid dehydrogenase (11 beta HSD). In situ hybridization using cRNA probes specific for human MRs revealed the presence of mRNA encoding for MRs in cardiomyocytes. Immunohistochemistry with specific antibodies against the MR exhibited the expression of MR protein in cardiomyocytes. In contrast, intramyocardial small blood vessels showed no evidence of immunolabelling. A distinct 11 beta HSD activity, which was nicotinamide-adenine dinucleotide dependent, was also demonstrated in human cardiac tissue. These results illustrate that all the components required for a specific aldosterone effect are present in the human heart.

Asunto(s)

RESUMEN

BACKGROUND: It has been proposed that aldosterone exerts direct effects on heart function, most notably on the development of myocardial fibrosis during ventricular hypertrophy in rat. Initial events in aldosterone action entail its binding to mineralocorticoid receptor (MR). Because MR displays similar affinities for aldosterone and glucocorticoids, the in vivo aldosterone selectivity of MR requires the presence of an enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD), which metabolizes glucocorticoids into inactive derivatives. Although evidence exists for the presence of MR in rodent heart, no data are available for humans; moreover, the existence of cardiac 11-HSD is controversial. METHODS AND RESULTS: The heart samples used originated from tissue removed during cardiac surgery in nontransplant patients or from endocavitary biopsies done for the follow-up of heart transplantation. The expression of MR was examined at the mRNA and protein level by in situ hybridization with cRNA probes specific for human MR mRNA and by immunodetection with two specific anti-MR antibodies. 11-HSD catalytic activity was determined by measurement of the metabolic rate of tritiated corticosteroids by cardiac samples. In nontransplanted hearts, an in situ hybridization signal equivalent to that found in the whole kidney was present on cardiomyocytes. Specific immunolabeling of cardiomyocytes with anti-MR antibodies demonstrated the presence of the MR protein. Cardiac 11-HSD activity was detected (243 +/- 26 fmol.30 min-1.mg protein-1) and was dependent on the cofactor NAD, not NADP, suggesting that it corresponds to the form of the enzyme specifically responsible for MR protection. In transplanted hearts that presented severe alterations, MR immunodetection was weaker and irregular, with no specific hybridization signal. CONCLUSIONS: Our results demonstrate that MR is coexpressed with 11-HSD in human heart, which thus possesses the cellular machinery required for direct aldosterone action.

Asunto(s)

RESUMEN

The respective effects of aldosterone and arginine vasopressin (AVP) were examined on the number of active Na(+)-K(+)-ATPase and their pumping activity in nonperfused microdissected mouse cortical collecting tubules (CCD) by measuring specific 3H-ouabain binding and ouabain-sensitive 86Rb uptake. In adrenalectomized (ADX) animals, incubation of CCD with AVP (10(-8) M for 5 min) had no effect on the number of pumps. In contrast, in ADX animals replete with aldosterone, AVP induced a approximately equal to 40% increase in the number of pumps. This was accompanied by a approximately equal to 60-65% increase in ouabain-sensitive Rb uptake. AVP effect was dose-dependent (10(-10)-10(-8) M) and was reproduced by dDAVP, forskolin and 8-Br cAMP, indicating a V2 pathway. It was inhibited by amiloride 10(-5) M, and did not occur in CCD incubated in hyperosmotic solution, suggesting that the signal was transmitted via apical sodium entry and cell swelling. Finally, the AVP-dependent increase in the number of pumps was rapid (within 5 min) and transient (< 25 min). These results demonstrate that, in the CCD, aldosterone and AVP act synergistically to increase not only the apical sodium entry but also the basolateral Na(+)-K(+)-ATPase transport capacity: AVP allows a rapid recruitment and/or activation of an aldosterone-dependent pool of latent Na(+)-K(+)-ATPase.

Asunto(s)

RESUMEN

Enzymatic properties of the enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD), which confers mineralocorticoid selectivity, have been explored in the aldosterone-sensitive collecting duct (CCD) and the aldosterone-insensitive Pars Recta (PR) of the rat kidney. After incubation of freshly isolated tubular segments with [3H]corticosterone (3H-B) or [3H]dehydrocorticosterone (3H-A), the rate of transformation of 3H-B into 3H-A (dehydrogenase activity), or the reverse reaction (reductase activity) were measured by HPLC, Vmax for dehydrogenase activity was found to be 8- to 10-fold higher in CCD than PR. The enzyme functions over a very wide range (0.1-5000 nM) of corticosterone concentration. In CCD, enzyme kinetics suggest either the presence of two 11-HSD forms, differing by their affinity for corticosterone, or complex kinetics. Addition of NAD or NADP to permeabilized tubules revealed that dehydrogenase activity is NAD-dependent in CCD and NADP-dependent in PR. Cofactor addition was ineffective in intact tubules. CCD exhibited an exclusive dehydrogenase activity, whereas in PR dehydrogenase and reductase activity were found. No regulation of dehydrogenase activity could be evidenced in adrenalectomized rats receiving or not aldosterone, corticosterone or dexamethasone, for 2 h, 3 days or 4 days. We conclude that 11-HSD in the CCD and PR differs by its Vmax and cofactor dependence. Corticosteroid hormones do not influence 11-HSD activity.

Asunto(s)