Sodium-transporting Epithelia Research Articles

Hypertension Editors' Picks: Hyperaldosteronism.

Hypertension Editors' Picks: Hyperaldosteronism.

Role of the 11β-hydroxysteroid dehydrogenase type 2 in blood pressure regulation

The renal 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme inactivates 11-hydroxy steroids in the kidney, thus protecting the nonselective mineralocorticoid receptor (MR) from occupation by glucocorticoids. The gene is highly expressed in all sodium-transporting epithelia, but also in human placenta, pancreas, and thyroid. Mutations in the HSD11B2 gene cause a rare monogenic juvenile hypertensive syndrome called apparent mineralocorticoid excess (AME). In AME, compromised 11betaHSD2 enzyme activity results in overstimulation of the MR by cortisol, causing sodium retention, hypokalemia, and salt-dependent hypertension. Recent evidence suggests a role of the 11betaHSD2 in essential hypertension. We found hypertension with no other characteristic signs of AME in the heterozygous father of a child with AME and in a girl with a homozygous gene mutation resulting in a mild deficiency of 11betaHSD2. Moreover, some studies in patients with essential hypertension showed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites, suggesting a deficient 11betaHSD2 activity. These abnormalities may be genetically determined. A genetic association of a microsatellite flanking the HSD11B2 gene and hypertension in black patients with end-stage renal disease has been reported. We recently analyzed a CA-repeat allele polymorphism in unselected patients with essential hypertension, but did not find any correlation between this marker and blood pressure. However, we did find an association between this polymorphic CA microsatellite marker and salt sensitivity. Moreover, the activity of the 11betaHSD2, as shown by elevated mean ratios of urinary cortisol to cortisone metabolites, was decreased in salt-sensitive compared with salt-resistant subjects. These findings indicate that variants of the HSD11B2 gene contribute to the enhanced blood pressure response to salt in humans.

Transgenic mouse models to study human mineralocorticoid receptor function in vivo

The mineralocorticoid receptor (MR) is a transcription factor that mediates aldosterone action. MR is expressed in a wide variety of tissues, most notably in sodium-transporting epithelia, but also in nonepithelial cells of the cardiovascular and central nervous systems. However, molecular mechanisms underlying mineralocorticoid signaling and the primary mineralocorticoid-regulated genes are not fully identified. We recently showed that the human MR (hMR) gene possesses two first 5'-untranslated exons 1alpha and 1beta, and demonstrated that the 5'-flanking regions of these exons, named P1 and P2, respectively, are functional promoters that differ by their basal and corticosteroid-regulated transcriptional activities. To gain insight into the tissue-specific expression and function of MR, we have established transgenic mouse models using both targeted oncogenesis and receptor overexpression strategies. P1 and P2 promoters were used to direct expression of the large T antigen (TAg) of SV40 in constitutively MR-expressing cells. P1.TAg mice developed lethal hibernomas, while P2.TAg animals died from cerebral neuroectodermal tumors and leiomyosarcomas. Quantification of TAg messenger RNA levels revealed that P1 and P2 were differentially utilized. P1 promoter was transcriptionally active in all MR-expressing tissues and importantly directed an appropriate transgene expression in the distal nephron. Conversely, P2 activity was weak and spatially restricted. Several immortalized cell lines were established, thus constituting valuable models to investigate on aldosterone-regulated proteins. We also used P1 and P2 to target overexpression of hMR cDNA in mice. Phenotypic characterization of these mice is currently under investigation. Some transgenic lines should represent useful systems to further explore multiple functions of MR in vivo.

The role of the 11β-hydroxysteroid dehydrogenase type 2 in human hypertension

The 11 beta-hydroxysteroid dehydrogenase type 2 (11 PHSD2) enzyme inactivates 11 betahydroxy steroids in sodium-transporting epithelia such as the kidney, thus protecting the non-selective mineralocorticoid receptor (MR) from occupation by cortisol in humans. Inhibition by xenobiotics such as liquorice or mutations in the HSD11 B2 gene, as occur in the rare monogenic hypertensive syndrome of apparent mineralocorticoid excess (AME), result in a compromised 11 betaHSD2 enzyme activity, which in turn leads to overstimulation of the MR by cortisol, sodium retention, hypokalaemia, low plasma renin and aldosterone concentrations, and hypertension. Whereas the first patients described with AME had a severe form of hypertension and metabolic derangements, with an increased urinary ratio of cortisol (THF+5alphaTHF) to cortisone (THE) metabolites, more subtle effects of mild 11 beta HSD2 deficiency on blood pressure have recently been observed. Hypertension with no other characteristic signs of AME was found in the heterozygous father of a child with AME, and we described a girl with a homozygous gene mutation resulting in only a slightly reduced 11 beta HSD2 activity causing 'essential' hypertension. Thus, depending on the degree of loss of enzyme activity, 11 beta HSD2 mutations can cause a spectrum of phenotypes ranging from severe, life-threatening hypertension in infancy to a milder form of the disease in adults. Patients with essential hypertension usually do not have overt signs of mineralocorticoid excess, but nevertheless show a positive correlation between blood pressure and serum sodium levels, or a negative correlation with potassium concentrations, suggesting a mineralocorticoid influence. Recent studies revealed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites in some patients with essential hypertension. These abnormalities may be genetically determined. A genetic association of a HSD11 B2 flanking microsatellite and hypertension in black patients with end-stage renal disease has been reported. A recent analysis of a CA-repeat allele polymorphism in unselected patients with essential hypertension did not find a correlation between this marker and blood pressure. Since steroid hormones with mineralocorticoid action modulate renal sodium retention, one might hypothesize that genetic impairment of 11 beta HSD2 activity would be more prevalent in salt-sensitive as compared with salt-resistant subjects. Accordingly, we found a significant association between the polymorphic CA-microsatellite marker and salt-sensitivity. Moreover, the mean ratio of urinary cortisol to cortisone metabolites, as a measure for 11betaHSD2 activity, was markedly elevated in salt-sensitive subjects. These findings suggest that variants of the HSD11 B2 gene may contribute to the enhanced blood pressure response to salt in some humans.

Modulation of human mineralocorticoid receptor function by protein kinase A.

The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.

Modulation of Human Mineralocorticoid Receptor Function by Protein Kinase A

The mineralocorticoid receptor (MR) acts as a ligand-dependent transcription factor modulating specific gene expression in sodium-transporting epithelia. Physiological evidence suggest a cross-talk between the cAMP- and aldosterone-signaling pathways. We provide evidence that protein kinase A (PKA), a major mediator of signal transduction pathways, modulates transcriptional activity of the human MR (hMR). Using transient transfection assays in HepG2 cells, we show that 8-bromo-cAMP, a protein kinase A activator, stimulates glucocorticoid response element (GRE)-containing promoters in a ligand-independent manner. This effect was strictly MR dependent since no activation of the reporter gene was observed in the absence of cotransfected hMR expression plasmid. Furthermore, a synergistic activation was achieved when cells were treated with both aldosterone and cAMP. This synergistic effect was also observed in the CV1 and the stable hMR-expressing M cells but was dependent on the promoter used. In particular, synergism was less pronounced in promoters containing several GREs. We show that (protein kinase-inhibiting peptide (PKI), the peptide inhibitor of PKA, prevented both cAMP and aldosterone induction, which indicates that a functional cAMP pathway is required for stimulation of transcription by aldosterone. Using MR-enriched baculovirus extracts in gel shift assays, we have shown that the binding of the MR to a GRE-containing oligonucleotide was enhanced by PKA. Increased DNA binding of hMR is likely to reflect an increase in the number of active receptors, as measured by Scatchard analysis. Using a truncated MR, we show that the N-terminal domain is required for the effect. Finally, the N-terminal truncated MR was not directly phosphorylated by PKA in vitro. We conclude that PKA acts indirectly, probably by relieving the effect of an MR repressor.

11 beta-Hydroxysteroid dehydrogenase type II in the human endometrium: localization and activity during the menstrual cycle.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) is a potent inactivator of glucocorticoids and is present in high amounts in the placental syncytiotrophoblast and sodium-transporting epithelia. Placental 11 beta HSD2 is thought to protect the fetus from high circulating levels of maternal glucocorticoids, whereas the renal enzyme is important in conferring aldosterone specificity on the mineralocorticoid receptor. An isoform of 11 beta HSD (11 beta HSD1) is also present in a wide range of tissues, but usually acts as an oxoreductase, converting the biologically inactive cortisone to cortisol. In the present study we have used an immunopurified antibody to the carboxy-terminus of human 11 beta HSD2 (HUH23) to demonstrate localization of the enzyme in luminal and glandular epithelia of human endometrium. In some specimens staining was uniformly distributed, but in others there was clear evidence of heterogeneity both between and within epithelia. Although 11 beta HSD2 was found mainly in the cytoplasm, some cells showed evidence of nuclear staining only. Western blot analysis showed a band at 41 kDa in endometrium and myometrium, confirming the presence of 11 beta HSD2. Measurement of activity throughout the menstrual cycle showed that mean levels (+/- SEM) of activity were 156 +/- 17 and 6.1 +/- 1.1 pmol product/min.g homogenate protein for 11 beta HSD2 and 11 beta HSD1, respectively. Patients taking combined estrogen/progesterone contraceptives had significantly lower activities of both enzymes (76 +/- 19 and 1.9 +/- 0.4; both P < 0.01) compared with the control group. 11 beta HSD2 activity was significantly higher in the secretory than in the proliferative phase of the cycle in controls (193 +/- 22 vs. 120 +/- 23; P < 0.05). All groups contained outliers with elevated enzyme activities, with some patients displaying 11 beta HSD2 levels comparable to those observed in human kidney (> 1000 pmol/min.g). Further analysis showed that there was a statistically significant correlation (r = 0.43; P < 0.001) between the levels of 11 beta HSD1 and 11 beta HSD2. There was no detectable mineralocorticoid receptor binding in endometrial cytosols prepared from patients with a range of 11 beta HSD2 activities. It remains to be determined whether elevated or suppressed levels of either isoform are associated with fertility or endometrial pathology.

Colocalization of 11 beta-hydroxysteroid dehydrogenase type II and mineralocorticoid receptor in human epithelia.

The enzyme 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2) has been shown to confer specificity on mineralocorticoid receptors (MR) by inactivating glucocorticoids. In the present study we examined the colocalization of 11 beta HSD2 and MR in various exocrine and secretory glands by immunostaining of serial mirror tissue sections with subsequent computerized image analysis. Both 11 beta HSD2 and MR proteins were expressed in the same cells in the distal convoluted tubules, Henle's loop, and collecting tubules of the kidney and the absorptive epithelia of duodenum, jejunum, ileum, colon, and excretory ducts of anal and esophageal glands. Significantly, 11 beta HSD2 and MR immunoreactivity also colocalized in the respiratory tract, in collecting ducts of the tracheal and bronchial glands, ciliated bronchial epithelial cells, and type II alveolar epithelial cells, suggesting important and unexpected roles for mineralocorticoids in the lung. In the skin, 11 beta HSD2 and MR were present only in excretory ducts of eccrine sweat glands, but not in sebaceous or apocrine glands. In eccrine glands, MR immunoreactivity was present in the basal cells of excretory ducts, while 11 beta HSD2 immunoreactivity was localized in the luminal cells. Neither 11 beta HSD2 nor MR proteins were expressed in the lacrimal gland, prostate, bile ducts, gall bladder, urinary bladder, urethra, or ureter. These results indicate that 11 beta HSD2 protein colocalizes with MR protein in the great majority of sodium-transporting epithelia involved in serous secretion and supports the proposal that 11 beta HSD2 is a pivotal determinant of mineralocorticoid receptor occupancy in man. Furthermore, our demonstration of colocalization in discrete areas of the lung suggests that mineralocorticoid agonists or antagonists, and/or inhibitors of 11 beta HSD2, may have unexpected applications in respiratory disease.

Tissue-specific expression of alpha and beta messenger ribonucleic acid isoforms of the human mineralocorticoid receptor in normal and pathological states.

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.

Immunohistochemical and molecular characterization of the rat 11 beta-hydroxysteroid dehydrogenase type II enzyme.

Mineralocorticoid action is facilitated by 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2), which metabolizes glucocorticoids and allows aldosterone to bind to the nonselective mineralocorticoid receptor. We have recently demonstrated the presence of the 11 beta HSD2 protein in a wide range of human epithelia, suggesting that it is the sole isoform endowing specificity in man. In the present study we have used an immunopurified polyclonal antibody (RAH23) raised against a C-terminal peptide derived from the cloned rat 11 beta HSD2 protein to perform immunohistochemical and molecular analysis in rat tissues. In frozen sections of rat kidney, strong staining was seen with the RAH23 antibody in the distal tubule; weaker staining was observed in the thick ascending loop of Henle and the medullary and papillary collecting ducts. Punctate cortical staining was observed in the fetus at 20 days gestation and in 8-day-old rats, with a noticeable increase in the staining pattern at 16 days of age. The kidney did not attain the adult pattern of staining until 28 days of age. Epithelia of ileum and colon also stained with RAH23, as did excretory ducts of the submandibular gland. Intrahepatic and excretory bile ducts displayed strong immunoreactivity in the epithelial lining. Rat adrenal glands showed evidence of the 11 beta HSD2 antigen in the zona fasciculata and zona reticularis, but not in the zona glomerulosa or medulla. Western blot analysis with the RAH23 antibody revealed strong bands in the kidney, colon, adrenal gland, and submandibular gland at 40 kDa, colinear with the migration of the cloned 11 beta HSD2 enzyme. A band of medium intensity was also seen at this size in the pancreas, whereas a band of moderate intensity was seen in the bile duct, and weaker bands were noticed in the stomach, small intestine, and liver, with a diffuse band at 36-42 kDa in the prostate. Strong bands were seen in the pancreas and prostate at 78 kDa, with weaker signals in the colon, adrenal, stomach, and bile duct. A number of tissues also displayed multiple bands at about 30 kDa. Enzymatic assays on tissue homogenates showed extensive conversion of corticosterone to its 11-dehydro product in an NAD-dependent manner in the submandibular gland, adrenal gland, and kidney, but not in the pancreas or prostate. This study confirms the ubiquitous presence of 11 beta HSD2 in sodium-transporting epithelia, demonstrates the high level of 11 beta HSD2 protein and enzyme activity in the rat adrenal, and suggests a possible role for the enzyme in the biliary system. Further studies are required to determine the relevance of the various molecular species to the activity, latency, and processing of the enzyme.

Localization of 11 beta-hydroxysteroid dehydrogenase type II in human epithelial tissues.

The enzyme 11 beta-hydroxysteroid dehydrogenase type II (11 beta HSD2) confers specificity on the renal mineralocorticoid receptor by inactivating glucocorticoids. Mutations in this gene give rise to the syndrome of apparent mineralocorticoid excess, a congenital condition characterized by sodium retention, severe hypertension, and often by growth retardation. It is not known whether 11 beta HSD2 or another enzyme confers specificity in nonrenal sodium transporting epithelia, such as those in the sweat gland, salivary gland, and gastrointestinal tract. We previously have used the HUH23 antibody to localize 11 beta HSD2 in the human kidney, vascular smooth muscle cells, and placenta. In the present study, we have examined a range of human epithelia for the presence of 11 beta HSD2. In the skin, staining was seen in eccrine sweat glands and arterioles, whereas weak HUH23 immunostaining was observed in the epidermis. Staining was absent from sebaceous glands and hair follicles. In the parotid gland, the 11 beta HSD2 enzyme was present in striated and excretory ducts, whereas in the submandibular gland, it was found in striated and interlobular ducts. Acini, adipocytes, and associated tumor tissue did not stain with the HUH23 antibody. In the gastrointestinal tract, HUH23 stained ileal enterocytes, colonic absorptive cells, and epithelial goblet cells, whereas the rectum contained areas of staining and nonstaining absorptive cells. Gastrointestinal structures, such as the lamina propria, Peyer's patch, and goblet cells within the crypts of Lieberkuhn did not stain with the antibody. This study demonstrates the presence of 11 beta HSD2 in nonrenal sodium-transporting epithelia and describes a range of tissues affected in the syndrome of apparent mineralocorticoid excess.

Aldosterone interaction on sodium transport and chloride permeability: influence of epithelial structure

The effects of aldosterone on sodium transport and chloride permeability were investigated by electrophysiology in two structurally distinct epithelia used as models for the distal renal tubule: the A6 cell monolayer as compared with the amphibian skin epithelium (ASE). Short-circuit current ( I sc) and transepithelial conductance ( G t) were measured in A6 monolayers incubated overnight with(out) aldosterone. Cell and shunt conductances ( G cell and G sh) were also determined, as well as the conductive nature of the chloride pathway. These parameters were correlated with sodium and chloride fluxes in A6 cells ( J Na and J Cl) and compared with the data recorded across ASE ( Bufo marinus). The existence of a cAMP-dependent chloride secretory pathway in A6 cells was also investigated upon exposition to arginine vasopressin (AVP) or oxytocin. When A6 monolayers were incubated with aldosterone, G t significantly increased with respect to control preparations; this increase resulted solely from an increase in G cell, and was reflected by a 3-fold increase in I sc. There was a significant relationship between I sc and G cell, as well as between I sc and J Na in both control and aldosterone-stimulated preparations. The A6 clone used was devoid of cAMP-dependent chloride secretory activity and was unresponsive to AVP or oxytocin. Thus, comparison between ASE and A6 preparations revealed two major differences: unlike ASE, (i) aldosterone has no effect on G sh and (ii) no conductive reabsorptive chloride pathway is operative in A6 monolayers tested. In addition, cobalt had no effect on electrical parameters of A6 monolayers. These observations show that difference in epithelial structure is reflected in terms of electrophysiological response to aldosterone, which suggests that cell heterogeneity could be a prerequisite for observing a conductive reabsorptive chloride pathway in aldosterone-responsive, sodium-transporting epithelia.

Isolation of a rat amiloride-binding protein cDNA clone: tissue distribution and regulation of expression

Sodium transport across high-resistance epithelia involves both an apical amiloride-sensitive sodium channel and the basal Na(+)-K(+)-ATPase pump. Aldosterone regulates sodium transport by increasing the sodium permeability of the sodium channels. To study further the regulation of gene expression in sodium-transporting epithelia by corticosteroids, we have cloned an amiloride-binding protein (ABP) cDNA from rat descending colon and kidney. Identical 311 nucleotide cDNAs were amplified from both rat descending colon and kidney, and the predicted amino acid sequence exhibited 83% homology to the equivalent region of the human peptide sequence. Use of this cDNA as a probe resulted in detection of a transcript in both the small and large bowel, thymus, and seminal vesicle. The latter tissue exhibited the highest level of rat ABP expression. Low to undetectable levels of rat ABP were expressed in the descending colon and kidney. No regulation of rat ABP by either class of corticosteroids was observed. Levels of ABP were low at birth and increased gradually to adult levels just before weaning in the bowel. The distribution of rat ABP is not as would be predicted for an aldosterone-induced gene and is thus unlikely to be a component of the amiloride-sensitive electrogenic sodium channel.

Resistive properties of the epithelial membranes of the urinary bladder of the toad, Bufo marinus, determined using the fluorescent dye, RH160.

A technique is described for quantitative epifluorescence studies of the apical membrane of the epithelial cells of the urinary bladder of the toad, Bufo marinus, using the lipid-soluble dye, RH160. When the urinary bladder is appropriately mounted, fluorescence signals, in response to a transepithelial voltage pulse, can be recorded from the epithelium immediately after the addition of the dye to the mucosal bath, and for some hours subsequently. The optical signal, recorded as the change in fluorescence in response to a transepithelial voltage pulse, as a fraction of resting fluorescence, was found to be a linear function of the applied voltage over the range +/- 200 mV, and was approximately 3% for a 100 mV change in transepithelial potential. The signal was enhanced by amiloride (10 mumol.l-1), reduced by bretylium (5 mmol.l-1) and abolished in the presence of nystatin (730 U.ml-1). Calculations based on these data permitted estimation of the fractional resistance of the apical membrane, which was found to be 0.85 under control conditions. Apical membrane resistance was 8.6 k omega.microF, and the basolateral membrane resistance was 1.5 k omega.microF. These findings support the conclusion that the apical membrane of toad urinary bladder epithelial cells is of high resistance, thus resembling other sodium-transporting epithelia.

Immunochemical localization of amiloride-sensitive sodium channels in sodium-transporting epithelia

The localization of amiloride-sensitive Na+ channels in Na+-transporting epithelia was examined using antibodies made against amiloride-binding Na+ channel protein purified from bovine kidney. The distribution of the channel protein was determined in thick frozen sections at the light-microscopic level using indirect immunofluorescence, and at the electron-microscopic level using immunogold labelling. In the cells of both the intact bovine collecting tubule and A6 confluent monolayers, only the luminal or apical-facing surface membranes showed staining. Sodium channel protein was characteristically localized on microvillar domains of the apical plasma membrane. Little or no basolateral membrane staining was evident. Channel protein was also absent from subapical vesicles and tight junctions, and was not found in bovine renal proximal tubules, cultured human secretory sweat coils, non-epithelial Chinese hamster ovary (CHO) cells or human skin fibroblasts. Trypsinization of intact A6 monolayers prior to cell fixation abolished specific staining with antibody. Pretreatment with amiloride protected against this loss of staining. Thus, our probes are specific for amiloride-binding Na+ channel protein, and this channel protein is largely or completely confined to the apical membrane of Na+-transporting epithelia. The level and distribution of specific immunostaining in A6 cells was unchanged by aldosterone treatment, although channel activity, as measured by short-circuit current, increased threefold. This result demonstrates that Na+ channel protein is ever present at the cell surface and exists in both an active and an inactive form. We find no evidence that stimulation of Na+ uptake by aldosterone involves recruitment of new channels from a cytoplasmic pool.

Role of topology in bioenergetics of sodium transport in complex epithelia.

There has been some controversy as to whether in sodium-transporting epithelia (e.g., frog skin, toad urinary bladder) the Na-O2 ratio is independent of the net rate of transepithelial sodium transport or remains constant over a wide range of transport rates. This computer simulation study shows that both views are defensible, depending on whether one wishes to exclude or include "static head" energy in the calculations. This energy arises from intramembrane sodium recirculation, as shown here when applying a multicompartment epithelial membrane model. Assuming reasonable kinetic parameters of a transport model whose sodium transport rate is varied over a wide range by a step-by-step simulated amiloride action, the computations have shown the following. When static head energy (O2 consumption) is excluded from the calculations, the Na-O2 ratio is constant over a wide range of transepithelial sodium flux, up to the tested value of 20 neq X cm-2 X min-1, a "normal" value found in frog skin. The Na-O2 ratios were 19.4 and 28.7 in low- and high-sodium models, respectively. If the static head energy is included in the calculations, the Na-O2 ratios increase with increasing transport rate from zero values to values up to 15.7. These data are in good agreement with laboratory results, as are derived data on phenomenological coefficients and thermodynamic coupling coefficients (LNa = 80, 128; LNa,r = 4.4; Lr = 0.27, 0.58; q = 0.50, 0.90, depending on the chosen model parameters).

Homocellular regulatory mechanisms in sodium-transporting epithelia: avoidance of extinction by flush-through

Page F579: S. G. Schultz. “Homocellular regulatory mechanisms in sodium-transporting epithelia: avoidance of extinction by flush-through.” Page 588: in footnote 5 the paper by Hoffman and Tosteson ( J. Gen. Physiol. 58: 438–466, 1971) was erroneously cited for finding that differences in cation composition between HK and LK sheep erythrocytes are the results of changes in both the Na-K pump and the K leak and the suggestion that the “…molecular mechanism of pump and leak may be closely related.” The correct citation is Tosteson and Hoffman ( J. Gen. Physiol. 44: 169–194, 1960).

The significance of changes in thermodynamic affinity induced by aldosterone in sodium-transporting epithelia.

The energetics of sodium transport were examined in toad (and occasionally frog) skin, with particular emphasis on the effect of aldosterone. Thermodynamic affinity was computed according to Essig and Caplan. Following treatment with antidiuretic hormone or drugs believed to affect only the apical membrane barrier, no change in thermodynamic affinity was observed either acutely (after one to two hours) or chronically (after 18-odd hours hours). By contrast, following treatment with aldosterone overnight, thermodynamic affinity was considerably increased, whether or not incubation was conducted in the presence of sodium in the outer solution; addition of glucose at the end of incubation, whereby sodium transport was stimulated further, failed to influence affinity as measured. The stoichiometry between sodium transport and oxygen consumption was, however, unchanged by aldosterone treatment in short-circuit conditions, neither was that fraction of aerobic metabolism unrelated to sodium transport influenced. It is concluded that the change observed with aldosterone can be directly ascribed to the hormone, as it is independent of glucose availability and of sodium transport. Aldosterone action, at least following prolonged incubation, therefore does not involve only an increase in apical conductance for sodium.

Homocellular regulatory mechanisms in sodium-transporting epithelia: avoidance of extinction by "flush-through".

In recent years electrophysiologic studies on several sodium-transporting epithelia have uncovered two "homocellular" (intrinsic) regulatory mechanisms that appear to protect the absorptive cells from marked changes in sodium and potassium content in response to rapid and wide-ranging physiologic variations in the rate of transcellular sodium transport. These are: 1) an inverse relation between intracellular sodium activity and the sodium conductance of the apical (mucosal) membrane, and 2) a parallel relation between the rate of sodium extrusion from the cell across the basolateral membrane ("pump activity") and the conductance of that barrier to potassium. The purpose of this review is to document these homocellular regulatory mechanisms, discuss their physiologic significance, and speculate on possible underlying mechanism(s).

Generation of transepithelial potentials by isolated perfused reptilian distal tubules.

Transepithelial potentials were measured in the most distal segments of garter snake (Thamnophis spp.) distal tubles perfused in vitro. The segments generated high lumen-negative potentials when sodium was in the lumen. The size of the potentials was a saturable function of luminal sodium concentrations between 0 and 32 mM. The potentials were stable with time only when sodium concentrations in the lumen were less than 30 mM. Perfusion with high sodium concentrations resulted in transient potentials. Stable potentials changed markedly when the lumen sodium concentration or the bath potassium concentration was altered suddenly, but they were independent of lumen potassium concentrations and bath sodium concentrations. Amiloride stimulated or inhibited the potentials, ouabain partially depressed them, and ethacrynic acid and cyanide inhibited them slowly and often irreversibly. We conclude that distal transepithelial potentials reflect sodium transport from lumen to bath across a tight asymmetric epithelium which differs from other sodium-transporting epithelia in that stable transepithelial potentials are maintained only with luminal sodium concentrations less than 30 mM.