Regulation of apical K and Na channels and Na/K pumps in rat cortical collecting tubule by dietary K

The patch-clamp technique was used to study the properties and the density of conducting K and Na channels in the apical membrane of rat cortical collecting tubule. The predominant K channel observed in cell- attached patches (SK channels) had an outward single-channel conductance (with LiCl in the pipette) of 10 pS. The inward conductance (with KCl in the pipette) was 42 pS. The channel had a high open probability that increased with depolarization. Kinetic analysis indicated the presence of a single open state and two closed states. Increasing K intake by maintaining animals on a high K diet for 12-16 d increased the number of SK channels per patch by threefold (0.7- 2.0/patch) over control levels. In addition, conducting Na-selective channels, which were not observed in control animals, were seen at low density (0.5/patch). These channels had properties similar to those observed when the animals were on a low Na diet, except that the mean open probability (0.84) was higher. In other experiments, the whole- cell patch clamp technique was used to measure Na channel activity (as amiloride-sensitive current, INa) and Na pump activity (as ouabain- sensitive current, Ipump). In animals on a high K diet, INa was greater than in controls but much less than in rats on a low Na diet. Ipump was greater after K loading than in controls or Na-depleted animals. These K diet-dependent effects were not accompanied by a significant increase in plasma aldosterone concentrations. To further investigate the relationship between K channel activity and mineralocorticoids, rats were maintained on a low Na diet to increase endogenous aldosterone secretion. Under these conditions, no increase in SK channel density was observed, although there was a large increase in the number of Na channels (to 2.7/patch). Aldosterone was also administered exogenously through osmotic minipumps. As with the low Na diet, there was no change in the density of conducting SK channels, although Na channel activity was induced. These results suggest that SK channels, Na channels and Na/K pumps are regulated during changes in K intake by factors other than aldosterone.     

Protein-tyrosine phosphatase reduces the number of apical small conductance K+ channels in the rat cortical collecting duct

Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K(+) (SK) channels in the cortical collecting duct (CCD) from rats on a K(+)-deficient diet (). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K(+) diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 microm phenylarsine oxide (PAO) or 1 mm benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP(o) reversibly from control value 3.20 to 0.40. Addition of 1 microm herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K(+) diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.     

Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct

We used the patch-clamp technique to study the effects of ATP on the small-conductance potassium channel in the apical membrane of rat cortical collecting duct (CCD). This channel has a high open probability (0.96) in the cell-attached mode but activity frequently disappeared progressively within 1-10 min after channel excision (channel "run-down"). Two effects of ATP were observed. Using inside-out patches, low concentrations of ATP (0.05-0.1 mM) restored channel activity in the presence of cAMP-dependent protein kinase A (PKA). In contrast, high concentrations (1 mM) of adenosine triphosphate (ATP) reduced the open probability (Po) of the channel in inside-out patches from 0.96 to 0. 1.2 mM adenosine diphosphate (ADP) also blocked channel activity completely, but 2 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a nonhydrolyzable ATP analogue, reduced Po only from 0.96 to 0.87. The half-maximal inhibition (Ki) of ATP and ADP was 0.5 and 0.6 mM, respectively, and the Hill coefficient of both ATP and ADP was close to 3. Addition of 0.2 or 0.4 mM ADP shifted the Ki of ATP to 1.0 and 2.0 mM, respectively. ADP did not alter the Hill coefficient. Reduction of the bath pH from 7.4 to 7.2 reduced the Ki of ATP to 0.3 mM. In contrast, a decrease of the free Mg2+ concentration from 1.6 mM to 20 microM increased the Ki of ATP to 1.6 mM without changing the Hill coefficient; ADP was still able to relieve the ATP-induced inhibition of channel activity over this low range of free Mg2+ concentrations. The blocking effect of ATP on channel activity in inside-out patches could be attenuated by adding exogenous PKA catalytic subunit to the bath. The dual effects of ATP on the potassium channel can be explained by assuming that (a) ATP is a substrate for PKA that phosphorylates the potassium channel to maintain normal function. (b) High concentrations of ATP inhibit the channel activity; we propose that the ATP-induced blockade results from inhibition of PKA-induced channel phosphorylation.     

Synergistic action of vasopressin and aldosterone on basolateral Na+-K+-ATPase in the cortical collecting duct

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 40% increase in the number of pumps. This was accompanied by a 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.     

Regulation of Na channels of the rat cortical collecting tubule by aldosterone

The activity of apical membrane Na channels in the rat cortical collecting tubule was studied during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet or the diuretic furosemide. Tubules were isolated and split open to expose the luminal membrane surface. Induction of Na channel activity was studied in cell- attached patches of the split tubules. No activity was observed with control animals on a normal diet. Channel activity could be induced by putting the animals on the low-Na diet for at least 48 h. The mean number of open channels per patch (NPo) was maximal after 1 wk on low Na. Channels were also induced within 3 h after injection of furosemide (20 mg/kg body wt per d). NPo was maximal 48 h after the first injection. In both cases, increases in NPo were primarily due to increases in the number of channels per patch (N) at a constant open probability (Po). With salt depletion or furosemide injection NPo is a saturable function of aldosterone concentration with half-maximal activity at approximately 8 nM. When animals were salt repleted after 1- 2 wk of salt depletion, both plasma aldosterone and NPo fell markedly within 6 h. NPo continued to decrease over the next 14 h, while plasma aldosterone rebounded partially. Channel activity may be dissociated from aldosterone concentrations under conditions of salt repletion.     

Regulation by GTP of a Ca2+-activated K+ channel in the apical membrane of rabbit cortical collecting duct cells

Ca²⁺-activated K⁺ channels play an important role in Ca²⁺ signal transduction and may be regulated by mechanisms other than a direct effect of Ca²⁺. Inside-out patches of the apical membrane of confluent transformed rabbit cortical collecting duct cells cultured on collagen were subjected to patch clamp analysis. Two types of K⁺ channel, of medium and high conductance, were observed. The latter channel was characterized by a K⁺/Na⁺ permeability ratio of 10, an inwardly rectified current, a conductance of 80 pS at 0 mV, and an open probability dependent on both voltage and Ca²⁺. Guanosine 5-triphosphate (GTP) but not a guanosine 5-diphosphate (GDP) analogue, adenosine 5-triphosphate (ATP), cytidine 5-triphosphate (CTP), or inosine 5-triphosphate (ITP), inhibited the activity of this Ca²⁺-activated K⁺ channel. The inhibitory effect of GTP was dose dependent, with a 50% inhibitory concentration of 10^–5 m in the absence of Mg²⁺. In the presence of Mg²⁺ (1 mm), which is required for the binding of GTP to G proteins, the 50% inhibitory concentration decreased to 3×10^–12 m. Pertussis toxin or cholera toxin (each at 10 ng/ml) did not prevent the inhibitory effect of GTP. After removal of GTP from the medium bathing an inhibited channel, subsequent application of Ca²⁺ failed to activate the channel. Ca²⁺-activated K⁺ channels of smooth muscle cells and proximal tubule cells did not respond to GTP. Thus, the Ca²⁺-activated K⁺ channel in the apical membrane of collecting duct cells is inhibited by GTP, which appears to exert its effect via a G protein that is insensitive to both cholera and pertussis toxins.     

Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.     

Biphasic effect of protein kinase C on rat renal cortical Na+, K+-ATPase.

We examined the dependence of rat renal Na+, K+-ATPase activity on protein kinase C (PKC) stimulation. Infusion of either phorbol 12, 13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) into rat abdominal aorta resulted in dose-dependent changes of renal cortical Na+, K+-ATPase activity. Low doses of these esters (3 x 10(-11) mol/kg/min) increased activity of Na+, K+-ATPase whereas high doses (3 x 10(-9) mol/kg/min) decreased it. The changes in Na+, K+-ATPase activity induced by PDBu and PMA were prevented by staurosporine, a PKC inhibitor. 4Alpha phorbol didecanoate (4alpha PDD), phorbol ester which does not activate PKC had no effect on cortical Na+, K+-ATPase. PDBu and PMA did not change Na+, K+-ATPase activity in the renal medulla. The stimulatory effect of PDBu (3 x 10(-11) mol/kg/min) was neither mimicked by amphotericin B, a sodium ionophore nor blocked by amiloride, an inhibitor of Na+/H+-exchanger. The inhibitory effect of 3 x 10(-9) mol/kg/min PDBu was not mimicked by amiloride indicating that the observed effects of PKC stimulation are not secondary to alterations in intracellular sodium concentration. The inhibitory effect of PDBu was prevented by infusion of ethoxyresorufin, an inhibitor of cytochrome P450-dependent arachidonate metabolism. These results suggest that the inhibitory effect of PKC on renal cortical Na+, K+-ATPase is mediated by cytochrome P450-dependent arachidonate metabolites.     

Flow-induced prostaglandin E2 release regulates Na and K transport in the collecting duct

Fluid shear stress (FSS) is a critical regulator of cation transport in the collecting duct (CD). High-dietary sodium (Na) consumption increases urine flow, Na excretion, and prostaglandin E(2) (PGE(2)) excretion. We hypothesize that increases in FSS elicited by increasing tubular flow rate induce the release of PGE(2) from renal epithelial cells into the extracellular compartment and regulate ion transport. Media retrieved from CD cells exposed to physiologic levels of FSS reveal several fold higher concentration of PGE(2) compared with static controls. Treatment of CD cells with either cyclooxygenase-1 (COX-1) or COX-2 inhibitors during exposure to FSS limited the increase in PGE(2) concentration to an equal extent, suggesting COX-1 and COX-2 contribute equally to FSS-induced PGE(2) release. Cytosolic phospholipase A2 (cPLA2), the principal enzyme that generates the COX substrate arachidonic acid, is regulated by mitogen-activated protein-kinase-dependent phosphorylation and intracellular Ca(2+) concentration ([Ca(2+)](i)), both signaling processes, of which, are activated by FSS. Inhibition of the ERK and p38 pathways reduced PGE(2) release by 53.3 ± 8.4 and 32.6 ± 11.3%, respectively, while antagonizing the JNK pathway had no effect. In addition, chelation of [Ca(2+)](i) limited the FSS-mediated increase in PGE(2) concentration by 47.5 ± 7.5% of that observed in untreated sheared cells. Sheared cells expressed greater phospho-cPLA2 protein abundance than static cells; however, COX-2 protein expression was unaffected (P = 0.064) by FSS. In microperfused CDs, COX inhibition enhanced flow-stimulated Na reabsorption and abolished flow-stimulated potassium (K) secretion, but did not affect ion transport at a slow flow rate, implicating that high tubular flow activates autocrine/paracrine PGE(2) release and, in turn, regulates flow-stimulated cation transport. In conclusion, FSS activates cPLA2 to generate PGE(2) that regulates flow-mediated Na and K transport in the native CD. We speculate that dietary sodium intake modulates tubular flow rate to regulate paracrine PGE(2) release and cation transport in the CD.     

Subcellular distribution of K+-pNPPase (Na+,K+-ATPase) in the Golgi apparatus of developing rat cortical neurons

The distribution of K+-pNPPase (Na+,K+-ATPase) activity in the compartments of the Golgi apparatus in neurons of the cerebral cortex of young and adult Wistar rats was studied by ultrastructural cytochemistry. In adult rats, mainly the cis-most cisterna was associated with reaction deposits. In 10- and especially in 15-day-old rats, not only the cis-cisternae, but the cis- and trans-Golgi, as well as components of the Golgi stack, also revealed K+-pNPPase activity. The dynamic changes of K+ -pNPPase localization in the compartments of the neuronal Golgi complexes were discussed with respect to the biochemical evidence concerning the building, assembly and processing of Na+,K+-ATPase as plasma membrane glycoprotein. It was suggested that the high activity in the Golgi complexes seen in 15-day-old rats has to be associated with the advancing myelinization in this period and the necessity of Na+,K+-ATPase equipment of nodes of Ranvier.     

Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li

The behavior of individual Na channels in the apical membrane of the rat cortical collecting tubule (CCT) was studied at different concentrations of the permeant ions Na and Li. Tubules were opened to expose their luminal surfaces and bathed in K-gluconate medium to minimize tubule-to-tubule variation in cell membrane potential and intracellular Na concentration. The patch-clamp technique was used to resolve currents through individual channels. The patch-clamp pipette was filled with solutions containing variable concentrations of either NaCl or LiCl. In one series of experiments, the concentrations were changed without substitutions. In another series, the ionic strength and Cl concentration were maintained constant by partial substitution of Li with N-methyl-D-glucamine (NMDG). In cell-attached patches, both the single-channel conductance (g) and the single-channel current (i) saturated as functions of the Na or Li activity in the pipette. Without NMDG, the saturation of i was well described by Michaelis-Menten kinetics with an apparent Km of approximately 20 mM activity for Na and approximately 50 mM activity for Li. Km was independent of voltage for both ions. With substitution for Li by NMDG, the apparent Km value for Li transport through the channels increased. The values of the probability of a channel's being open (Po) varied from patch to patch, but no effect of pipette ion activity on Po could be demonstrated. A weak dependence of Po on membrane voltage was observed, with hyperpolarization increasing Po by an average of 2.3%/mV.     

Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

We examined the effect of angiotensin II (ANG II) on epithelial Na(+) channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NP(o) (a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca(2+)-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H(2)O(2) diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca(2+)-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.     

Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma

Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+-adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity.     

Electrophysiological identification of cell types in cortical collecting duct monolayers.

Double-barrel microelectrodes were used to determine membrane voltages and the intracellular pH (pHi) in primary cultures of cortical collecting duct cells (CCD) grown in the absence of aldosterone. Electrophysiologically, two main cell types were identified. In cell type 1, the apical membrane voltage (Va) was -60 +/- 5 mV. The fractional resistance of the apical membrane (fRa) was 0.40 +/- 0.03, and pHi was 7.21 +/- 0.04. Exposure to 50 mM K+ on the apical side depolarized Va by 21 +/- 4 mV. When Cl- was replaced by cyclamate two types of responses were observed: (a) depolarization of Va by 26 +/- 3 mV while pHi remained unchanged, and (b) no change in Va. In cell type 2, Va was -36 +/- 5 mV, fRa was 0.91 +/- 0.03 and increasing apical [K+] from 5 to 50 mM did not change Va. Two subpopulations were distinguished by the response of pHi to lowering apical [Cl-]. In one of them pHi increased from 6.99 +/- 0.05 to 7.11 +/- 0.07. In the other, pHi was significantly decreased from 7.16 +/- 0.08 to 7.03 +/- 0.07. These results are compatible with the conclusion that about 50% of the impaled cells type 2 have a Cl-/HCO-3 exchanger at the apical membrane. In summary, two different cell types can be identified electrophysiologically in CCD monolayers. Cell type 1 has the electrical characteristics of principal cells. Cell type 2 resembles the intercalated cells. The cell alkalinization observed in approximately 50% of the cells type 2 in response to Cl- removal suggests the presence of an apical Cl-/HCO-3 exchanger. Thus, these cells should be the bicarbonate-secreting cells. The remaining cells should correspond to the acid-secreting cells.     

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

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 Ca(2+) concentration ([Ca(2+)](i)), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO(3)(-) 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 (NH(4)Cl prepulse technique). H(+)-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pH(i)) recovery in the absence of luminal K(+), bilateral Na(+), and CO(2)/HCO(3)(-), 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 Ca(2+) dependent and required microtubule integrity, and 3) basal and flow-stimulated pH(i) 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.