scholarly journals Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD)

2012 ◽  
Vol 302 (1) ◽  
pp. F205-F215 ◽  
Author(s):  
Wen Liu ◽  
Núria M. Pastor-Soler ◽  
Carlos Schreck ◽  
Beth Zavilowitz ◽  
Thomas R. Kleyman ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Axial Flow ◽  
Bk Channel ◽  
Cortical Collecting Duct ◽  
Dolichos Biflorus ◽  
Intracellular Ca ◽  
Acid Load ◽  
Luminal Flow ◽  
Principal And Intercalated Cells

Epithelial Na+ channel (ENaC)-mediated Na+ absorption and BK channel-mediated K+ secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca2+ concentration ([Ca2+]i), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO3− reabsorption in the proximal tubule due to changes in both luminal Na+/H+ exchanger 3 and H+-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289–F296, 2006), we sought to test the hypothesis that flow also regulates H+-ATPase activity in the CCD. H+-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH4Cl prepulse technique). H+-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pHi) recovery in the absence of luminal K+, bilateral Na+, and CO2/HCO3−, from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min−1·mm−1 stimulated H+-ATPase activity, 2) flow-stimulated H+ pumping was Ca2+ dependent and required microtubule integrity, and 3) basal and flow-stimulated pHi recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H+-ATPase activity in the rabbit CCD and that H+-ATPases therein are present in both principal and intercalated cells.

scholarly journals Potassium depletion increases proton pump (H+-ATPase) activity in intercalated cells of cortical collecting duct

2000 ◽  
Vol 279 (1) ◽  
pp. F195-F202 ◽  
Author(s):  
Randi B. Silver ◽  
Sylvie Breton ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Collecting Duct ◽  
Proton Pumps ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Collecting Ducts ◽  
Acid Load ◽  
Collecting Tubules ◽  
Atpase Staining

Intercalated cells (ICs) from kidney collecting ducts contain proton-transporting ATPases (H+-ATPases) whose plasma membrane expression is regulated under a variety of conditions. It has been shown that net proton secretion occurs in the distal nephron from chronically K+-depleted rats and that upregulation of tubular H+- ATPase is involved in this process. However, regulation of this protein at the level of individual cells has not so far been examined. In the present study, H+-ATPase activity was determined in individually identified ICs from control and chronically K+-depleted rats (9–14 days on a low-K+ diet) by monitoring K+- and Na+-independent H+ extrusion rates after an acute acid load. Split-open rat cortical collecting tubules were loaded with the intracellular pH (pHi) indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHiwas determined by using ratiometric fluorescence imaging. The rate of pHi recovery in ICs in response to an acute acid load, a measure of plasma membrane H+-ATPase activity, was increased after K+ depletion to almost three times that of controls. Furthermore, the lag time before the start of pHirecovery after the cells were maximally acidified fell from 93.5 ± 13.7 s in controls to 24.5 ± 2.1 s in K+-depleted rats. In all ICs tested, Na+- and K+-independent pHi recovery was abolished in the presence of bafilomycin (100 nM), an inhibitor of the H+-ATPase. Analysis of the cell-to-cell variability in the rate of pHi recovery reveals a change in the distribution of membrane-bound proton pumps in the IC population of cortical collecting duct from K+-depleted rats. Immunocytochemical analysis of collecting ducts from control and K+-depleted rats showed that K+-depletion increased the number of ICs with tight apical H+ATPase staining and decreased the number of cells with diffuse or basolateral H+-ATPase staining. Taken together, these data indicate that chronic K+ depletion induces a marked increase in plasma membrane H+ATPase activity in individual ICs.

Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct

2007 ◽  
Vol 293 (1) ◽  
pp. F227-F235 ◽  
Author(s):  
Wen Liu ◽  
Tetsuji Morimoto ◽  
Craig Woda ◽  
Thomas R. Kleyman ◽  
Lisa M. Satlin
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Brefeldin A ◽  
Bk Channels ◽  
Bk Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Intracellular Ca ◽  
K Secretion ◽  
High Conductance

Apical low-conductance SK and high-conductance Ca2+-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion ( JK) in the CCD is 1) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and 2) associated with an increase in [Ca2+]i, leading us to conclude that BK channels mediate flow-stimulated JK. To examine the Ca2+ dependence and sources of Ca2+ contributing to flow-stimulated JK, JK and net Na absorption ( JNa) were measured at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in the absence of luminal Ca2+ or after pretreatment with BAPTA-AM to chelate intracellular Ca2+, 2-aminoethoxydiphenyl borate (2-APB), to inhibit the inositol 1,4,5-trisphosphate (IP3) receptor or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated JNa (Morimoto et al. Am J Physiol Renal Physiol 291: F663–F669, 2006), inhibited flow-stimulated JK. Increases in [Ca2+]i stimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 μM colchicine (COL) to disrupt microtubule function or 5 μg/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated JK but not JNa (Morimoto et al. Am J Physiol Renal Physiol 291: F663–F669, 2006), although COL but not BFA also blocked the flow-induced [Ca2+]i transient. We thus speculate that BK channel-mediated, flow-stimulated JK requires an increase in [Ca2+]i due, in part, to luminal Ca2+ entry and ER Ca2+ release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane.

scholarly journals Dynamic coupling between TRPV4 and Ca2+-activated SK1/3 and IK1 K+ channels plays a critical role in regulating the K+-secretory BK channel in kidney collecting duct cells

2017 ◽  
Vol 312 (6) ◽  
pp. F1081-F1089 ◽  
Author(s):  
Yue Li ◽  
Hongxiang Hu ◽  
Jin-Bin Tian ◽  
Michael X. Zhu ◽  
Roger G. O’Neil
Keyword(s):  
Membrane Potential ◽  
Transient Receptor Potential ◽  
Collecting Duct ◽  
Critical Role ◽  
Receptor Potential ◽  
Bk Channel ◽  
Transient Receptor Potential Vanilloid ◽  
Cortical Collecting Duct ◽  
Intracellular Ca ◽  
Transient Receptor

The large-conductance Ca2+-activated K+ channel, BK (KCNMA1), is expressed along the connecting tubule (CNT) and cortical collecting duct (CCD) where it underlies flow- and Ca2+-dependent K+ secretion. Its activity is partially under the control of the mechanosensitive transient receptor potential vanilloid type 4 (TRPV4) Ca2+-permeable channel. Recently, we identified three small-/intermediate-conductance Ca2+-activated K+ channels, SK1 (KCNN1), SK3 (KCNN3), and IK1 (KCNN4), with notably high Ca2+-binding affinities, that are expressed in CNT/CCD and may be regulated by TRPV4-mediated Ca2+ influx. The K+-secreting CCD mCCDcl1 cells, which express these channels, were used to determine whether SK1/3 and IK1 are activated on TRPV4 stimulation and whether they contribute to Ca2+ influx and activation of BK. Activation of TRPV4 (GSK1016790A) modestly depolarized the membrane potential and robustly increased intracellular Ca2+, [Ca2+]i. Inhibition of both SK1/3 and IK1 by application of apamin and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), respectively, further depolarized the membrane potential and markedly suppressed the TRPV4-mediated rise in [Ca2+]i. Application of BK inhibitor iberiotoxin after activation of TRPV4 without apamin/TRAM-34 also reduced [Ca2+]i and further intensified membrane depolarization, demonstrating BK involvement. However, the BK-dependent effects on [Ca2+]i and membrane potential were largely abolished by pretreatment with apamin and TRAM-34, identical to that observed by separately suppressing TRPV4-mediated Ca2+ influx, demonstrating that SK1/3-IK1 channels potently contribute to TRPV4-mediated BK activation. Our data indicate a direct correlation between TRPV4-mediated Ca2+ signal and BK activation but where early activation of SK1/3 and IK1 channels are critical to sufficiently enhanced Ca2+ entry and [Ca2+]i levels required for activation of BK.

High-conductance K channels in intercalated cells of the rat distal nephron

2007 ◽  
Vol 292 (3) ◽  
pp. F966-F973 ◽  
Author(s):  
Lawrence G. Palmer ◽  
Gustavo Frindt
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Reversal Potential ◽  
Bk Channel ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
High K ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
High Conductance

High-conductance (BK or maxi) K+ channels were observed in cell-attached patches of the apical membrane of the isolated split-open rat connecting tubule (CNT). These channels were quite rare in cells identified visually as principal cells (PCs; 5/162 patches) but common in intercalated cells (ICs; 24/26 patches). The BK-expressing intercalated cells in the CNT and cortical collecting duct (CCD) were characterized by a low membrane potential (−36 mV) under short-circuit conditions, measured from the reversal potential of the channel currents with similar K+ concentrations on both sides of the membrane. Under whole-cell clamp conditions with low intracellular Ca2+, ICs had a very low K+ conductance. When cell Ca2+ was increased to 200 nM, a voltage-dependent, tetraethylammonium (TEA)-sensitive outward conductance was activated with a limiting value of 90 and 140 nS/cell in the CNT and CCD, respectively. Feeding animals a high-K diet for 1 wk did not increase these currents. TEA-sensitive currents were much smaller in PCs and usually below detection limits. To examine the possibility that the ICs participate in transepithelial K+ secretion, we measured Na/K pump activity as a ouabain-sensitive current. Although these currents were easily observed in PCs, averaging 79 ± 14 and 250 ± 50 pA/cell in the CCD and CNT, respectively, they were below the level of detection in the ICs. We conclude that ICs have BK channel densities that are sufficient to support renal secretion of K+ if cell Ca2+ is elevated. However. a pathway for K+ entry into these cells has not been identified.

Effect of flow and stretch on the [Ca2+]i response of principal and intercalated cells in cortical collecting duct

2003 ◽  
Vol 285 (5) ◽  
pp. F998-F1012 ◽  
Author(s):  
Wen Liu ◽  
Shiyun Xu ◽  
Craig Woda ◽  
Paul Kim ◽  
Sheldon Weinbaum ◽  
...  
Keyword(s):  
Flow Rate ◽  
Collecting Duct ◽  
Cell Types ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Duration Magnitude ◽  
Luminal Flow ◽  
Principal And Intercalated Cells ◽  
Circumferential Stretch

An acute increase in tubular fluid flow rate in the microperfused cortical collecting duct (CCD), associated with a ∼20% increase in tubular diameter, leads to an increase in intracellular Ca2+ concentration ([Ca2+]i)in both principal and intercalated cells (Woda CB, Leite M Jr, Rohatgi R, and Satlin LM. Am J Physiol Renal Physiol 283: F437-F446, 2002). The apical cilium present in principal but not intercalated cells has been proposed to be a flow sensor. To determine whether flow across the cilium and/or epithelial stretch mediates the [Ca2+]i response, CCDs from New Zealand White rabbits were microperfused in vitro, split-open (to isolate the effect of flow across cilia), or occluded (to examine the effect of stretch and duration/magnitude of the flow impulse), and [Ca2+]i was measured using fura 2. In perfused and occluded CCDs, a rapid (<1 s) but not slow (>3 min) increase in luminal flow rate and/or circumferential stretch led to an approximately threefold increase in [Ca2+]i in both principal and intercalated cells within ∼10 s. This response was mediated by external Ca2+ entry and inositol 1,4,5-trisphosphate-mediated release of cell Ca2+ stores. In split-open CCDs, an increase in superfusate flow led to an approximately twofold increase in [Ca2+]i in both cell types within ∼30 s. These experimental findings are interpreted using mathematical models to predict the fluid stress on the apical membranes of the CCD and the forces and torques on and deformation of the cilia. We conclude that rapid increases in luminal flow rate and circumferential stretch, leading to shear or hydrodynamic impulses at the cilium or apical membrane, lead to increases in [Ca2+]i in both principal and intercalated cells.

H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct

1997 ◽  
Vol 272 (2) ◽  
pp. F167-F177 ◽  
Author(s):  
A. Constantinescu ◽  
R. B. Silver ◽  
L. M. Satlin
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Adult Rabbit ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Load ◽  
Ethanesulfonic Acid

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.

Adenosine-stimulated Ca2+reabsorption is mediated by apical A1 receptors in rabbit cortical collecting system

1998 ◽  
Vol 274 (4) ◽  
pp. F736-F743 ◽  
Author(s):  
Joost G. J. Hoenderop ◽  
Anita Hartog ◽  
Peter H. G. M. Willems ◽  
René J. M. Bindels
Keyword(s):  
Collecting Duct ◽  
Cortical Collecting Duct ◽  
Concomitant Increase ◽  
Cgs 21680 ◽  
Kinase C ◽  
Intracellular Ca ◽  
Collecting Duct Cells ◽  
Camp Formation ◽  
Pkc Activation ◽  
Permeable Supports

Confluent monolayers of immunodissected rabbit connecting tubule and cortical collecting duct cells, cultured on permeable supports, were used to study the effect of adenosine on net apical-to-basolateral Ca2+ transport. Apical, but not basolateral, adenosine increased this transport dose dependently from 48 ± 3 to 110 ± 4 nmol ⋅ h−1 ⋅ cm−2. Although a concomitant increase in cAMP formation suggested the involvement of an A2 receptor, the A2 agonist CGS-21680 did not stimulate Ca2+ transport, while readily increasing cAMP. By contrast, the A1 agonist N 6-cyclopentyladenosine (CPA) maximally stimulated Ca2+transport without significantly affecting cAMP. Adenosine-stimulated transport was effectively inhibited by the A1 antagonist 1,3-dipropyl-8-cyclopenthylxanthine but not the A2 antagonist 3,7-dimethyl-1-propargylxanthine, providing additional evidence for the involvement of an A1 receptor. Both abolishment of the adenosine-induced transient increase in intracellular Ca2+ concentration by 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid and downregulation of protein kinase C (PKC) by prolonged phorbol ester treatment were without effect on adenosine-stimulated Ca2+ transport. The data presented suggest that adenosine interacts with an apical A1 receptor to stimulate Ca2+ transport via a hitherto unknown pathway that does not involve cAMP formation, PKC activation, and/or Ca2+ mobilization.

scholarly journals Role of NKCC in BK channel-mediated net K+ secretion in the CCD

2011 ◽  
Vol 301 (5) ◽  
pp. F1088-F1097 ◽  
Author(s):  
Wen Liu ◽  
Carlos Schreck ◽  
Richard A. Coleman ◽  
James B. Wade ◽  
Yubelka Hernandez ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Bk Channels ◽  
Bk Channel ◽  
Taqman Assay ◽  
Specific Cell ◽  
Cortical Collecting Duct ◽  
Ph Indicator ◽  
Transport Pathway ◽  
K Secretion

Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion ( JK) and Na absorption ( JNa) at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal JK, JNa, or the flow-induced [Ca2+]i transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce JK. Basolateral Cl removal reversibly inhibited FIKS but not basal JK or JNa. Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH4+ at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH4+ addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH4+ entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.

Nifedipine-activated Ca2+ permeability in newborn rat cortical collecting duct cells in primary culture

2001 ◽  
Vol 280 (5) ◽  
pp. C1193-C1203 ◽  
Author(s):  
Laura Valencia ◽  
Michel Bidet ◽  
Sonia Martial ◽  
Elsa Sanchez ◽  
Estela Melendez ◽  
...  
Keyword(s):  
Primary Culture ◽  
Transient Receptor Potential ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Receptor Potential ◽  
Cortical Collecting Duct ◽  
Newborn Rat ◽  
Adult Rat ◽  
Intracellular Ca ◽  
Transient Receptor

To characterize Ca2+ transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca2+concentration ([Ca2+]i) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 μM) produced an increase in [Ca2+]i from 87.6 ± 3.3 nM to 389.9 ± 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca2+]i in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca2+]i. Experiments in the presence of EGTA showed that external Ca2+ was required for the nifedipine effect, while lanthanum (20 μM), gadolinium (100 μM), and diltiazem (20 μM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K+ channels were not involved in the nifedipine-induced [Ca2+]i rise. H2O2also triggered [Ca2+]i rise. However, nifedipine-induced [Ca2+]i increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca2+transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca2+ channel of capacitive type (either transient receptor potential or leak channel).

Minealocorticoid receptors and 11 beta-steroid dehydrogenase activity in renal principal and intercalated cells

1994 ◽  
Vol 266 (1) ◽  
pp. F76-F80 ◽  
Author(s):  
A. Naray-Fejes-Toth ◽  
E. Rusvai ◽  
G. Fejes-Toth
Keyword(s):  
Cell Sorting ◽  
Direct Action ◽  
Collecting Duct ◽  
Cell Types ◽  
Target Cells ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Specific Receptors ◽  
Principal And Intercalated Cells ◽  
Steroid Dehydrogenase

Aldosterone exerts complex effects on the cortical collecting duct (CCD): it increases Na+ and K+ transport, and it also influences H+ and HCO3 transport. Whether these latter effects represent direct action of aldosterone on intercalated cells (ICC) or are secondary to changes in the transport of other electrolytes is unclear. Because the presence of specific receptors is the prerequisite of a direct steroid action, and mineralocorticoid receptors (MR) have not yet been demonstrated in ICC, in this study we determined the density of MR directly in isolated principal cells (PC) and beta-ICC. Purified populations of these two cell types were obtained from rabbit renal cortex by immunodissection and fluorescence-activated cell sorting. We found that both PC and beta-ICC contained a significant number of MR, although receptor density was higher in PC than in beta-ICC (6,704 +/- 912 vs. 2,181 +/- 388 MR sites/cell; P < 0.001). 11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD), an enzyme that is present predominantly in mineralocorticoid target cells, exhibited a distribution similar to that of MR in the two cell types. 11 beta-OHSD activity, determined by measuring the rate of conversion of [3H]corticosterone to 11-dehydrocorticosterone, was 1.08 +/- 0.14 and 0.34 +/- 0.08 fmol.min-1 x 1,000 cells-1 (P < 0.001) in intact PC and beta-ICC, respectively. 11 beta-OHSD in both cell types utilized NAD as cofactor. These results suggest that beta-ICC are potential direct targets of aldosterone and that MR in both PC and beta-ICC are protected by 11 beta-OHSD.

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