The alpha-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5'-flanking region of the gene.

Aldosterone stimulates Na(+) reabsorption in the collecting ducts by increasing the activity of the epithelial sodium channel, ENaC. Systemic administration of aldosterone increases alpha ENaC mRNA expression in mammalian kidney, suggesting that the alpha ENaC gene is a target for aldosterone action in the distal nephron. To determine whether aldosterone increases alpha ENaC gene transcription, a portion of the alpha ENaC 5'- flanking region coupled to luciferase was transfected into MDCK-C7 cells, a collecting duct cell line with aldosterone-stimulated Na(+) transport. Both dexamethasone and aldosterone stimulated alpha ENaC-coupled reporter gene activity via the glucocorticoid receptor (GR), and this response correlated with the effect of these hormones on endogenous alpha ENaC expression. The aldosterone-stimulated alpha ENaC expression was blocked by actinomycin D, and aldosterone had no effect on alpha ENaC mRNA decay, confirming a transcriptional effect. In HT-29 cells, a GR/mineralocorticoid receptor (MR)-deficient colonic cell line with constitutive alpha ENaC expression, cotransfection with GR or MR restored aldosterone-stimulated alpha ENaC gene transcription, although aldosterone had a functional preference for MR. Analysis of deletion constructs confirmed that a single imperfect glucocorticoid response element (GRE) is necessary and sufficient to confer the aldosterone responsiveness to the alpha ENaC gene promoter in MDCK-C7 and HT-29 cells. These results confirm that alpha ENaC is an aldosterone-induced transcript in the collecting duct and delineates the molecular mechanism for this effect.

Kinase regulation of hENaC mediated through a region in the COOH-terminal portion of the alpha-subunit.

In an effort to gain insight into how kinases might regulate epithelial Na(+) channel (ENaC) activity, we expressed human ENaC (hENaC) in Xenopus oocytes and examined the effect of agents that modulate the activity of some kinases. Activation of protein kinase C (PKC) by phorbol ester increased the activity of ENaC, but only in oocytes with a baseline current of <2,000 nA. Inhibitors of protein kinases produced varying effects. Chelerythrine, an inhibitor of PKC, produced a significant inhibition of ENaC current, but calphostin C, another PKC inhibitor, had no effect. The PKA/protein kinase G inhibitor H-8 had no effect, whereas the p38 mitogen-activated protein kinase inhibitor, SB-203580 had a significant inhibitory effect. Staurosporine, a nonspecific kinase inhibitor, was the most potent tested. It inhibited ENaC currents in both oocytes and in M-1 cells, a model for the collecting duct. Site-directed mutagenesis revealed that the staurosporine effect did not require an intact COOH terminus of either the beta- or gamma-hENaC subunit. However, an intact COOH terminus of the alpha-subunit was required for this effect. These results suggest that an integrated kinase network regulates ENaC activity through an action that requires a portion of the alpha-subunit.

Mechanisms of inactivation of the action of aldosterone on collecting duct by TGF-beta.

The purpose of these experiments was to investigate the mechanisms whereby transforming growth factor-beta (TGF-beta) antagonizes the action of adrenocorticoid hormones on Na(+) transport by the rat inner medullary collecting duct in primary culture. Steroid hormones 1) increased Na(+) transport by three- to fourfold, 2) increased the maximum capacity of the Na(+)-K(+) pump by 30-50%, 3) increased the steady-state levels of the alpha(1)-subunit of the Na(+)-K(+)-ATPase by approximately 30%, and 4) increased the steady-state levels of the alpha-subunit of the rat epithelial Na(+) channel (alpha-rENaC) by nearly fourfold. TGF-beta blocked the effects of steroids on the increase in Na(+) transport and the stimulation of the Na(+)-K(+)-ATPase and pump capacity. However, there was no effect of TGF-beta on the steroid-induced increase in mRNA levels of alpha-rENaC. The effects of TGF-beta were not secondary to the decrease in Na(+) transport per se, inasmuch as benzamil inhibited the increase in Na(+) transport but did not block the increase in pump capacity or Na(+)-K(+)-ATPase mRNA. The results indicate that TGF-beta does not inactivate the steroid receptor or its translocation to the nucleus. Rather, they indicate complex pathways involving interruption of the enhancement of pump activity and activation/inactivation of pathways distal to the steroid-induced increase in the transcription of alpha-rENaC.

Glucocorticoid induction of epithelial sodium channel expression in lung and renal epithelia occurs via trans-activation of a hormone response element in the 5'-flanking region of the human epithelial sodium channel alpha subunit gene.

In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na+ transport is associated with increased expression of the epithelial Na+ channel alpha subunit (alphaENaC). In H441 lung cells, 100 nM dexamethasone increases amiloride-sensitive short-circuit current (3.3 microA/cm2 to 7.5 microA/cm2), correlating with a 5-fold increase in alphaENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of alphaENaC, the human alphaENaC 5'-flanking region was cloned and tested in H441 cells. By deletion analysis, a approximately 150-base pair region 5' to the upstream promoter was identified that, when stimulated with 100 nM dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na+ transport, malphaENaC expression and luciferase expression from alphaENaC genomic fragments were also increased by 100 nM dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated alphaENaC gene transcription. We conclude that glucocorticoids stimulate alphaENaC expression in kidney and lung via activation of a hormone response element in the 5'-flanking region of halphaENaC and this response, in part, is the likely basis for the up-regulation of Na+ transport in these sites.

Concerted actions of IL-1beta inhibit Na+ absorption and stimulate anion secretion by IMCD cells.

Increasing evidence indicates that factors other than adrenocorticoid hormones can influence long-term regulation of Na+ transport by inner medullary collecting duct (IMCD) cells. We now report that, of 14 interleukins tested, only interleukin-1alpha (IL-1alpha) and IL-1beta inhibited Na+ transport by primary cultures of rat IMCD. IL-1beta reduced both basal and mineralocorticoid (MC)-stimulated Na+ transport by 50-70%; its effect on glucocorticoid (GC)-stimulated Na+ transport was significantly less. IL-1beta continued to blunt MC stimulation of Na+ transport even after it had been removed from the medium for 24 h. The onset of action to inhibit Na+ transport was within 20 min. The acute effect from the basolateral surface was greater than that from the apical surface, but the effect from each surface was additive. In addition to its inhibitory effect on Na+ transport, chronic IL-1beta exposure increased both basal and cAMP-stimulated anion secretion rates. IL-1beta had no acute effect on anion secretion. Monolayers chronically treated with IL-1beta had an increased capacity to secrete fluid, as predicted from its effects on ion transport. Inhibitors of cyclooxygenase did not blunt the actions of IL-1beta. Furthermore, IL-1beta did not produce a rise in intracellular Ca2+. These results suggest novel signaling pathways induced by IL-1beta regulating Na+ and Cl- transport by the IMCD.

Candidate genes in the regulation of Na+ transport by inner medullary collecting duct cells from Dahl rats.

Recently, we reported that primary cultures of inner medullary collecting duct cells from Dahl salt-sensitive (S) rats absorb more Na+ than do cells cultured from Dahl salt-resistant (R) rats. To begin to evaluate the molecular basis for this difference, we selected four candidate gene products that on the basis of their physiology and genetics could participate in regulation of Na+ transport by these cells. During 24-hour exposure, inhibitors of the cytochrome P450 enzymes had no effect on Na+ transport by either S or R monolayers. Twenty-four-hour exposure to NG-monomethyl-L-arginine (0.5 mmol/L), a nonspecific inhibitor of NO synthase, also had no effect on Na+ transport by either S or R monolayers. Neither atrial natriuretic peptide 1-28 (100 nmol/L) nor 8-Br-cyclic GMP (100 micromol/L) had any short-term effect on Na+ transport by either S or R monolayers. 18-Hydroxy-11-deoxycorticosterone (100 nmol/L), an adrenocorticoid hormone that is produced in greater amounts in S rats, stimulated Na+ transport by both S and R monolayers via the mineralocorticoid receptor; however, its effect was less potent than aldosterone. Congenic rats in which the R isoform of the 11beta-hydroxylase gene was bred onto the S background had monolayers that transported Na+ at a rate similar to the S rats. These results demonstrate that neither cytochrome P450 genes, NO synthase genes, the atrial natriuretic peptide receptor gene, nor the 11beta-hydroxylase gene is a likely candidate to explain the difference in Na+ transport between S and R inner medullary collecting duct monolayers in primary culture.

The basis of higher Na+ transport by inner medullary collecting duct cells from Dahl salt-sensitive rats: implicating the apical membrane Na+ channel.

The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant (R) rats. Previous reports have suggested that there is a difference in primary sequence in the alpha 1 subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion in these two strains which in turn could influence the systemic blood pressure. Using "back-door" phosphorylation of pumps isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers cultured from S rats transport about twice as much Na+ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of the affinities for ouabain, Na+, and K+, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na+ transport rates. The analysis of the steady-state Na+ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of Na+ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na+. They suggest that the epithelial Na+ channel is intrinsically different or differently regulated in S and R rats.

IMCD cells cultured from Dahl S rats absorb more Na+ than Dahl R rats.

Dahl salt-sensitive (S) rats develop hypertension in response to a high-salt diet, whereas Dahl salt-resistant (R) rats do not. There is good evidence that the Dahl S kidneys have diminished natriuretic capacity. We studied the rate of Na+ transport by primary cultures of the inner medullary collecting duct from these two strains to determine whether there were intrinsic differences. Monolayers obtained from prehypertensive S rats transported Na+ at twice the rate as monolayers from age-matched R rats. Mineralocorticoid and glucocorticoid hormones increased Na+ transport from both strains; the S rat monolayers always displayed higher transport rates than R rat monolayers with the same treatment. The Na+ entry pathway in both S and R rat monolayers was via an Na+ channel. The difference in Na+ transport was not explained by a difference in the metabolism of corticosterone, ATP content, citrate synthase activity, ultrastructural appearance, or rate of maturation. Monolayers from S rats tended to have higher protein and DNA content, but these differences could not account for the difference in Na+ transport. Anion secretion in response to adenosine 3',5'-cyclic monophosphate agonists was similar. These results demonstrate intrinsic differences in renal tubular cells that may play an important role in the pathogenesis of salt-sensitive hypertension.

Cl- current in IMCD cells activated by hypotonicity: time course, ATP dependence, and inhibitors.

The hypertonic environment of the renal medulla can change rapidly according to the state of hydration of the animal. We used primary cultures of rat inner medullary collecting duct (IMCD) cells to investigate the characteristics of Cl- currents activated by an acute reduction in osmolarity (ICl(osm)). Using the whole cell patch-clamp technique, we identified an outwardly rectifying current that decayed slowly at strongly depolarizing voltages. The onset of ICl(osm) began 6.7 min after the fall in bath osmolarity, a delay longer than reported in other cell types. Hypotonicity did not induce an increase in intracellular Ca2+ concentration, and activation of ICl(osm) did not require the presence of Ca2+. Intracellular ATP was needed to evoke ICl(osm) when the hypotonic stimulus was modest (50 mosmol/l or less) but was not necessary when the stimulus was stronger (100 mosmol/ l). ICl(osm) was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by tamoxifen or glibenclamide. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid produced a voltage-dependent block. Acute reduction in osmolarity using cells grown on filters did not induce a Cl- secretory current. The ICl(osm) of IMCD cells appears to be on the basolateral membrane and displays some unique features.

Separate regulation of Na+ and anion transport by IMCD: location, aldosterone, hypertonicity, TGF-beta 1, and cAMP.

We have investigated some of the factors known or suspected to influence ion transport by the rat inner medullary collecting duct and have analyzed their actions on active Na+ absorption and active anion secretion by primary cultures. Cells from the terminal 1-2 mm (tip) of the papilla had a lower basal rate of Na+ absorption (2.0 microA/cm2) than cells from the more proximal portions (6.5 microA/cm2). Aldosterone increased Na+ transport approximately sevenfold in the tip cells and approximately threefold in the proximal cells. The magnitude of anion secretion in response to adenosine 3',5'-cyclic monophosphate (cAMP) agonists was similar in the two regions and was unaffected by aldosterone. The morphology of monolayers from both regions was also similar. In monolayers cultured from the entire inner medulla, hypertonic (100 mosM) urea, NaCl, or sucrose reduced Na+ transport but had no significant effect on anion secretion. Transforming growth factor-beta 1, known to blunt the effect of steroids on Na+ transport, had no effect on anion secretion. Finally, cAMP had no effect on Na+ transport, a result that contrasts with its effect on Na+ transport by other epithelial cells demonstrating steroid-responsive, electrogenic Na+ transport. These results demonstrate some potential differences in the magnitude of Na+ transport by position along the inner medulla. They further demonstrate separate regulation of Na+ and anion transport.

Effect of cAMP agonists on cell pH and anion transport by cultured rat inner medullary collecting duct cells.

The rat inner medullary collecting duct is capable of secreting anions. We previously showed that adenosine 3',5'-cyclic monophosphate (cAMP) stimulates anion secretion; the apical membrane anion exit pathway activated by cAMP appears to be the cystic fibrosis transmembrane conductance regulator Cl- channel. The present experiments were designed to test the hypothesis that the entry pathway across the basolateral membrane is a Cl-/HCO3- exchanger operating in parallel with an Na+/H+ exchanger. We investigated the mechanism by measuring cell Cl-, cell pH, and short-circuit current under a variety of conditions designed to uncover these pathways. cAMP agonists caused little change in cell Cl-, but they produced a consistent intracellular acidification. This acidification was dependent on HCO3-, but not on Cl-, and was not inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The presence of the basolateral Cl-/HCO3- exchanger was demonstrated by several maneuvers, and its activity was inhibited by DIDS. Applied to the basolateral solution, DIDS did not inhibit the cAMP-dependent anion current but actually stimulated it. We conclude that cAMP-stimulated anion secretion does not require activation of the basolateral Cl-/HCO3- exchanger. The transporter responsible for Cl- entry across the basolateral membrane remains unknown and is not inhibited by a variety of anion transport inhibitors, including DIDS, bumetanide, and hydrochlorothiazide. The cell acidification induced by cAMP appears to be independent of acid secretion and is the result of activation of one or more HCO3- exit pathways that are resistant to DIDS but are inhibited by a nonspecific anion transport inhibitor, 5-nitro-2-(3-phenylpro-pylamino) benzoic acid. We present a revised model for anion transport by the rat inner medullary collecting duct.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

Induction of resistance to mineralocorticoid hormone in cultured inner medullary collecting duct cells by TGF-beta 1.

The renal collecting duct is a major target for the mineralocorticoid hormone aldosterone which acts to enhance electrogenic Na+ absorption. The cortical portion of the collecting duct displays a vigorous response to mineralocorticoids administered in vivo. The terminal, or inner medullary portion, does not usually display such a vigorous response; the reason for this difference is unknown. To explore one possible mechanism for this lack of response, we varied the conditions of culturing these cells and determined that serum inhibited the ability of aldosterone to enhance Na+ transport. By screening 11 peptides, we found that transforming growth factor (TGF)-beta 1 produced a concentration-dependent inhibition of the action of aldosterone. The action of TGF-beta 1 required at least several hours of incubation. Resistance to the action of aldosterone could be produced by preincubating the monolayers with TGF-beta 1 for a few hours; subsequent exposure to aldosterone for up to 48 h failed to stimulate Na+ transport. TGF-beta 1 did not produce a change in cell morphology or the content of DNA, ATP, or ADP; there was a small reduction in protein content. Pretreatment with cycloheximide failed to reproduce the TGF-beta 1 effect. The induction of resistance to mineralocorticoid hormone may play an important role in modulating the effects of aldosterone on Na+ homeostasis.

Functional and molecular evidence for Shaker-like K+ channels in rabbit renal papillary epithelial cell line.

The rabbit papillary epithelial cell line GRB-PAP1 was used to determine the ion transport characteristics of a model of the distal nephron and terminal collecting duct. When grown on permeable supports, monolayers developed a significant electrical resistance and a benzamil-sensitive short-circuit current, indicating that they had the property of electrogenic Na+ transport. Using the whole cell patch-clamp technique, we found that the dominant current in these cells was a slowly inactivating, time- and voltage-dependent K+ current. This current was activated by voltages more positive than -30 mV. At +30 mV, the peak outward currents were > 300 pA. The magnitude of the outward currents and their reversal potentials depended strongly on the extracellular concentration of K+ and not on the extracellular concentration of Cl-. These currents were inhibited by either tetraethylammonium, 4-aminopyridine, charybdotoxin, or dendrotoxin. These characteristics, together with the kinetics of activation and inactivation, are the general characteristics of delayed rectifier channels seen in many muscle and neuronal cells. Because many of these types of channels share sequence homology with the Shaker family of channels cloned from Drosophila, we sought to identify a molecular correlate. Using reverse transcription followed by polymerase chain reaction to amplify Shaker-like sequences, we cloned and sequenced a single 881-bp fragment. The sequence shared identity with a recently reported rabbit Shaker channel that belongs to the subclass Kv 1.2. These data show that this renal papillary epithelial cell line, which has the capability of electrogenic Na+ transport, expresses functional delayed rectifier channels.

Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors.

The A6 cell line derived from the toad kidney forms polarized, highly differentiated epithelial monolayers in culture and has been utilized as an experimental model for studying regulation of transepithelial Na+ transport by aldosterone. In the present study we evaluated the specific role(s) of glucocorticoid and mineralocorticoid receptors in mediating this enhanced electrogenic Na+ transport, which was measured experimentally as an increase in short-circuit current (Isc). Our data demonstrate that specific glucocorticoid agonists (100 nM), including RU 28362 and RU 26988, elicit "mineralocorticoid-like" increases in Isc that are blocked by the glucocorticoid antagonist RU 38486 but are unaffected by mineralocorticoid antagonists including RU 28318 and RU 26752. The stimulatory effects of aldosterone (100 nM) were also blocked by RU 38486 and not by mineralocorticoid antagonists. These data extend earlier studies suggesting that in this cell line aldosterone mediates its physiological effects via binding with relatively low affinity (dissociation constant Kd congruent to 25-50 nM) to glucocorticoid receptors, despite the presence of apparently normal mineralocorticoid receptors. Our in vitro biochemical studies also demonstrate that A6 glucocorticoid receptor complexes can be thermally activated or transformed to DNA binding forms which exhibit altered elution profiles from anion-exchange resins. Thus, based on several criteria, these amphibian glucocorticoid receptors appear very similar to classical mammalian receptors and are capable of mediating all of the stimulatory effects of aldosterone on net Na+ transport.

Cellular responses to steroids in the enhancement of Na+ transport by rat collecting duct cells in culture. Differences between glucocorticoid and mineralocorticoid hormones.

It has recently been discovered that both mineralocorticoid (MC) and glucocorticoid (GC) hormones can stimulate electrogenic Na+ absorption by mammalian collecting duct cells in culture. In primary cultures of rat inner medullary collecting duct (IMCD) cells, 24-h incubation with either MC or GC agonist stimulates Na+ transport approximately threefold. We have now determined that the effects were not additive, but the time courses were different. As aldosterone is known to stimulate citrate synthase, Na+/K+ ATPase activity, and ouabain binding in cortical collecting duct principal cells, we determined the effects of steroids on these parameters in IMCD cells. MC and GC agonists both produced a small increase in citrate synthase activity. There was no increase in Na+/K+ ATPase activity but specific ouabain binding was increased more than two-fold by either agonist. To determine the role of apical Na+ entry in the steroid-induced effects, the Na+ channel inhibitor, benzamil, was used. Benzamil did not alter the stimulation of citrate synthase activity by either steroid. In contrast, GC stimulation of ouabain binding was prevented by benzamil, whereas MC stimulation was not. We conclude that there are differences in the way that MC and GC hormones produce an increased Na+ transport. Both appear to produce translocation (or activation) of pumps into the basolateral membrane. GC stimulation of pump translocation requires increased Na+ entry whereas MC stimulation does not.

Inhibition of Na transport by 2-chloroadenosine: dissociation from production of cyclic nucleotides.

The adenosine analogue 2-chloroadenosine (2-CA) is often used to determine the biologic effects of adenosine because 2-CA is less susceptible to degradation than adenosine. We studied the effects of 2-CA on primary cultures of rat inner medullary collecting ducts because there is good evidence that adenosine can influence cell function through its effects on second messengers. 2-CA inhibited Na+ transport across the apical membrane and increased cAMP content of the cells. The major adenosine receptors in these cells appear to be the stimulatory (A2) type. Stimulation of cAMP by 2-CA was more potent when applied to the apical membrane than to the basolateral membrane, an effect opposite to that of vasopressin. These results imply that adenosine receptors are more numerous or more effective on the apical membrane than on the basolateral membrane. Inhibition of Na+ transport was probably not mediated by an adenosine receptor as evidenced by (i) a lack of effect of adenosine and other adenosine analogues on Na+ transport; (ii) a lack of effect of nonmetabolizable cyclic nucleotides on Na+ transport; and (iii) a clear discrepancy in the temporal course of 2-CA effects on a second messenger system (cAMP) and 2-CA inhibition of Na+ transport. Dipyridimole, an inhibitor of adenosine transport, also reduced Na+ transport. Taken together, the data suggest that 2-CA inhibits Na+ transport by interfering with adenosine transport or metabolism.

Enhancement of electrogenic Na+ transport across rat inner medullary collecting duct by glucocorticoid and by mineralocorticoid hormones.

We have investigated the effect of steroid hormones on Na+ transport by rat renal inner medullary collecting duct (IMCD) cells. These cells, grown on permeable supports in primary culture, grow to confluence and develop a transmonolayer voltage oriented such that the apical surface is negative with respect to the basal surface. The results of these experiments demonstrate that this voltage is predominantly (or exclusively) the result of electrogenic Na+ absorption. Na+ transport can be stimulated two- to fourfold by exposure to either dexamethasone or aldosterone (100 nM). Experiments using specific antagonists of the glucocorticoid and mineralocorticoid receptors indicate that activation of either receptor stimulates electrogenic Na+ transport; electroneutral Na+ transport is undetectable. Two other features of the IMCD emerge from these studies. (a) These cells appear to have the capacity to metabolize the naturally occurring glucocorticoid hormone corticosterone. (b) The capacity for K+ secretion is minimal and steroid hormones do not induce or stimulate conductive K+ secretion as they do in the cortical collecting duct.

Characteristics of papillary collecting duct cells in primary culture.

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.

Selectivity of basolateral anion exchange in the acidification pathway of turtle bladder.

The turtle urinary bladder in vitro acidifies the solution bathing its luminal surface. Protons are actively extruded across the apical membrane by an H+-ATPase. Bicarbonate ion exits the cell across the basolateral membrane via a stilbene-sensitive, anion exchange for chloride. Chloride then exits the cell via a conductive pathway. The present studies were undertaken to define the specificity of the basolateral anion exchange mechanism for chloride. Turtle bladders were mounted on chambers in vitro, short-circuited, and treated with ouabain. The current remaining after inhibition of sodium transport was used to measure the acidification rate. Ion replacement studies with bromide, isethionate, sulfate, and nitrate indicated that only bromide supported acidification at rates comparable to chloride. In separate experiments, kinetic analysis of anion interaction with the exchanger indicates that maximal acidification rates decrease in the order: Cl greater than Br greater than SO4 greater than methyl sulfate = gluconate. The affinity of the exchanger decreases in the order: Cl greater than SO4 greater than Br greater than HCO3 greater than methylsulfate greater than gluconate. These selectivity sequences indicate "strong" interaction of the anions with the selectivity site. The differences in position of the polyatomic anions in the two sequences indicates that the "binding" site is accessible but that transport is limited by steric factors.