"Active' water transport?
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Biological Transport↗
Biomedical subjects
L Reuss
Publications and source records attributed to L Reuss.
A new method to measure alveolar-epithelium permeability in intact lung.
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Animals↗
P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody.
The relationships between P-glycoprotein (PGP) expression and plasma membrane ion currents activated by cell swelling were studied in several cell lines by use of the whole cell configuration of the patch-clamp technique. Swelling-activated Cl- currents (ICls) had similar characteristics independently of whether PGP was expressed. Addition of the anti-PGP monoclonal antibody C219 or its Fab fragment to the pipette solution prevented ICls in cells expressing functional PGP (assessed by immunoblots, immunofluorescence, and transport of rhodamine 123) but not in cells lacking PGP expression. A peptide analogue of the C219 epitope abolished the effect of C219. Other anti-PGP antibodies and mouse immunoglobulin G were ineffective. C219 did not alter swelling-activated cation currents. Inasmuch as ICls is present in cells that do not express PGP and C219 has no effect on ICls in these cells, we conclude that PGP is not required for the ICls phenotype. However, when expressed in the plasma membrane, PGP is involved, directly or indirectly, in ICls but not in swelling-activated K+ currents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Preservation of structural and functional polarity in isolated epithelial cells.
We describe a method to isolate epithelial cells from gallbladders of Necturus maculosus with preserved structural and functional polarity. Isolation was carried out with a mixture of collagenase and protease, with only a brief exposure to a divalent-cation-free medium. About 40% of the isolated epithelial cells had a "figure-eight" shape and retained metabolic and cell membrane integrity. Figure-eight cells display features consistent with preserved polarity for several hours, including the following: 1) the "apical" and "basolateral" membrane domains were differentially labeled by a hydrophobic fluorescent dye; 2) freeze fracture electron microscopy verified two plasma membrane domains differing in the presence of microvilli and folds and separated by tight junctions; 3) proteins such as ZO-1, NHE3, and Na(+)-K(+)-ATPase remained localized in the junctional, apical, and basolateral regions, respectively; 4) after apical surface exposure to wheat germ agglutinin, the label remained in the apical membrane after cell isolation; and 5) patch-clamp experiments demonstrated polarized expression of K+ channels. Polarity was rapidly lost after removal of extracellular Ca2+, exposure to trypsin, or ATP depletion. Therefore, this preparation allows for structural and functional studies of epithelial transport in single cells retaining the essential features present in the assembled epithelium.
Animals↗
Polarized expression of cAMP-activated chloride channels in isolated epithelial cells.
We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.
Animals↗
Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane.
P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is an ATPase thought to actively export cytotoxic drugs. It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium ("vacuum cleaner" hypothesis). A possible mechanism for this model is that the Pgp is a flippase--i.e., it catalyzes the translocation of hydrophobic substrates from the inner to the outer leaflet of the cell membrane. Two immediate predictions of the vacuum cleaner and flippase hypotheses are that the apparent unidirectional influx of substrate should be less in Pgp-expressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp. We used Chinese hamster fibroblasts with different levels of Pgp expression to measure true unidirectional fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye that accumulates in mitochondria (hence, its cytosolic concentration remains low at short times after external addition). The unidirectional efflux of R123 was proportional to the level of Pgp expression and was reduced by Pgp inhibitors. The unidirectional influx of R123 was the same in sensitive and resistant cells--i.e., independent of the level of Pgp expression and insensitive to inhibitors of R123 efflux. From these results, we rule out the vacuum cleaner and flippase hypotheses and conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Relationships between rhodamine 123 transport, cell volume, and ion-channel function of P-glycoprotein.
The P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is thought to be both an ATPase that actively exports cytotoxic drugs and a Cl- channel activated by cell swelling. The partial reversal of multidrug resistance by Cl- transport blockers suggests a possible role for Cl- in Pgp-mediated drug transport. We used multidrug-resistant Chinese hamster fibroblasts and human breast cancer cells expressing Pgp to study the roles of Cl- (and also Na+ and HCO3-/CO2) on Pgp-mediated efflux of the fluorescent dye rhodamine 123 (R123). In Pgp-expressing Chinese hamster fibroblasts, exposed to isosmotic solutions, the unidirectional efflux of R123 was not measurably changed by a approximately 60-min removal of Cl- (or by exposure to Na(+)-free, or nominally HCO3-/CO2-free medium); short term (2-3 min) ion substitutions were also ineffective. In human breast cancer cells transfected with human mdr1 cDNA, hyposmotic solutions activated a Cl- current but had no effect on the Pgp-mediated unidirectional efflux of R123. Additionally, in human breast cancer cells, the intracellular presence of R123 did not prevent activation of the Cl- current by hyposmotic solution. The lack of detectable effect of removal of Cl-, Na+, or HCO3- on Pgp-mediated R123 transport rules out direct coupling between substrate transport and transport of either of these ions by Pgp. The persistence of Pgp-mediated R123 efflux in osmotically swollen cells indicates that activation of the Pgp-associated Cl- current does not hinder the Pgp pump function. The lack of effect of R123 on swelling-activated Cl- current denotes that Pgp-mediated transport of organic substrates and Pgp-associated Cl- currents can occur at the same time in a single cell. These results underscore the dissociation between Pgp-mediated active drug transport and electrodiffusive Cl- transport.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
P-glycoprotein-associated Cl- currents are activated by cell swelling but do not contribute to cell volume regulation.
P-glycoprotein-associated Cl- conductance is activated by cell swelling; the ensuing Cl- efflux is thought to contribute to cell volume regulation. We tested this hypothesis in human breast cancer cells transfected with human mdr1 complementary DNA, which display P-glycoprotein-associated, swelling-activated Cl- currents. The Cl- electrochemical driving force favors Cl- efflux, but there was no appreciable Cl- loss or regulatory volume decrease (both assessed with fluorescent dyes) during the exposure to hyposmotic solution. Calculations indicate that the swelling-activated Cl- current is insufficient to cause a significant Cl- efflux. Hence, regulatory volume decrease is not a function of P-glycoprotein.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium.
Regulation of the cAMP-activated apical membrane Cl- conductance (GaCl) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. GaCl was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca2+] using ionomycin had effects on GaCl or interfered with activation of GaCl by forskolin. N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the GaCl response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated GaCl but had no effects on intracellular [cAMP]. GaCl was unaffected by 4 alpha-phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed the activation of GaCl by 8-Br-cAMP, hastened the return of GaCl to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the GaCl changes elicited by theophylline. We conclude that: (a) NGB GaCl can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) GaCl can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of GaCl; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects GaCl activation, and (d) the apical membrane of NGB epithelium lacks a Ca(2+)-activated Cl- conductance.
Animals↗
Muscarinic stimulation of gallbladder epithelium. III. Antagonism of cAMP-mediated effects.
Elevation of adenosine 3',5'-cyclic monophosphate (cAMP) levels in Necturus gallbladder (NGB) epithelium activates an apical membrane Cl- conductance and decreases transepithelial fluid transport (Jv). Acetylcholine (ACh), which had no effects on Jv by itself, antagonized the electrophysiological effects of forskolin (FSK) and theophylline and the decrease in Jv produced by FSK. By itself, ACh had no effects on basal cAMP levels but antagonized the increases in cAMP induced by FSK and theophylline. ACh had no effect on phosphodiesterase activity and prevented both the electrophysiological response and the elevation in cAMP by theophylline. In conclusion, the effect of ACh is mediated by inhibition of adenylate cyclase. A pertussis toxin (PTX)-sensitive G protein may mediate inhibition of adenylate cyclase because pretreatment with PTX prevented the reversal of the electrophysiological effects of FSK by ACh, and PTX catalyzed the ribosylation of cell membranes from NGB epithelium. ACh could have a physiological role in modulating the effects of secretagogues that act via elevation of cAMP levels.
Acetylcholine↗
Transmembrane ion movements elicited by sodium pump inhibition in Helix aspersa neurons.
1. Transmembrane ion movements upon sodium-pump inhibition were studied in identified neurons of the subesophageal ganglia of Helix aspersa. A two-microelectrode, voltage-clamp technique was used to measure transmembrane currents. Changes in intracellular Na+, K+, and Ca2+ concentrations were measured, in unclamped neurons, with Na(+)-sensitive microelectrodes, K(+)-sensitive microelectrodes, and with the fluorescent probe fura-2, respectively. 2. Inhibition of the sodium pump with ouabain (1 mM) elicited an increase in intracellular Na+ concentration, [Na+]i, at an initial rate of 0.42 +/- 0.05 mM/min (mean +/- SE; n = 27), and a membrane depolarization often followed by hyperpolarization. In cells clamped at -50 or -60 mV, ouabain produced an inward shift in membrane-holding current followed by an outward current usually having two components, transient and sustained, respectively. 3. Replacing external Na+ with either N-methyl-D-glucammonium or tetraethylammonium (TEA+) abolished both the ouabain-induced inward membrane current and the rise in [Na+]i, suggesting that Na+ was the charge carrier of the inward current. 4. Cd2+ (400 microM) reduced the rate of rise of the inward current by 60% and the estimated net Na+ flux by 47%. 5. The outward current was abolished by K(+)-channel blockers (10 mM TEA+ and 5 mM 4-aminopyridine or 10 nM apamin). Cd2+ (400 microM), a Ca(2+)-entry blocker, also abolished the outward current. 6. Inhibition of the sodium pump elicited a fall in [K+]i at an initial rate of 1.4 +/- 0.2 mM/min (n = 9 cells). 7. Upon inhibition of the sodium pump in neurons loaded with fura-2, [Ca2+]i increased from an estimated resting level of 147 +/- 37 nM to a maximum of 764 +/- 248 nM (n = 12 cells). 8. The rise in [Ca2+]i in the sustained presence of ouabain was transient, lasting 19.5 +/- 2.8 min, and could be prevented by removal of external Ca2+ before ouabain application or curtailed by removal of external Ca2+ during sustained ouabain exposure. The latter effect was not a consequence of exhaustion of caffeine-sensitive intracellular Ca2+ stores. 9. It is concluded that 1) the rise in [Ca2+]i upon Na(+)-pump inhibition requires the presence of external Ca2+, 2) the outward current observed upon pump inhibition is a Ca(2+)-activated K+ current flowing through apamin-sensitive channels, 3) the resting Na+ permeability involves a Cd(2+)-sensitive component, 4) a large fraction (approximately 30-60%) of the previously described ouabain-induced cell shrinkage may result from Ca(2+)-activated K+ efflux contributing to net solute and water loss.
Adenosine Triphosphate↗
Changes in intra- or extracellular pH do not mediate P-glycoprotein-dependent multidrug resistance.
P-glycoprotein (Pgp)-mediated multidrug resistance (MDR) is thought to result from active extrusion of lipid-soluble, titratable chemotherapeutic agents. Given the lack of demonstration of coupling between ATP hydrolysis and drug transport, the resistance to chemically unrelated compounds, and findings of elevated intracellular pH (pHi), it has been proposed that reduced intracellular accumulation of drugs in MDR is due to changes in the pH difference across the plasma membrane. Elevation of pHi or decrease in local extracellular pH (pHo) could reduce the intracellular accumulation of the protonated chemotherapeutic drugs and account for Pgp-mediated MDR. Alternatively, changes in pHi or pHo could increase drug efflux by other mechanisms, such as coupled transport involving H+ or OH-, or allosteric effects on Pgp or other proteins. Both mechanisms could operate independently of the charge of the substrate. The possibility of a role of pHi in drug efflux is important to test because of the clinical significance of the phenomenon of MDR of tumors. We tested this hypothesis and found that MDR can occur in cells with low, normal, or high pHi. Further, resistant cells exhibited reduced steady-state drug accumulation and increased efflux without changes in local pHo. Finally, acute changes in pHi had no appreciable effect on Pgp-mediated drug efflux. We conclude that Pgp-mediated MDR is not a consequence of changes in pHi or pHo.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Maternal exercise during pregnancy, physical fitness, and fetal growth.
The value of exercise during pregnancy is controversial; both benefits and risks have been hypothesized. As empiric evidence is scant, the issue was investigated in a prospective study that assessed the impact on fetal growth of maternal exercise in each trimester of pregnancy. A cohort of over 800 prenatal patients was recruited from obstetric practices in Pennsylvania and New York between January 1987 and June 1989. Subjects were interviewed at entry into care and recontacted at 28 and 36 weeks of gestation. In women with prior adverse outcomes or a lack of conditioning, the associations between maternal exercise and fetal growth were equivocal. In fit, low-risk, prenatal patients, exercise was positively associated with fetal growth. With low-moderate exercise levels, the adjusted mean birth weights were about 100 g higher than in nonexercisers (117 g; 95% confidence interval 17 to 217 g). With heavier exercise, larger birth weight increments were seen, close to 300 g in those who exercised throughout pregnancy at levels of about 2,000 kcal/week in energy expenditure (276 g; 95% confidence interval 54 to 497 g). These results suggest that the guidelines issued by the American College of Obstetricians and Gynecologists may be too stringent for well-conditioned, low-risk, prenatal patients. Additional research to define safe limits more precisely seems warranted.
Adult↗
cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.
Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Artifactual expression of maxi-K+ channels in basolateral membrane of gallbladder epithelial cells.
To patch clamp the basolateral cell membrane, sheets of Necturus gallbladder epithelium were stripped of the subepithelial tissue layers and affixed apical side down on cover slips coated with Cell-Tak [F. Wehner, L. Garretson, K. Dawson, Y. Segal, and L. Reuss. Am. J. Physiol. 258 (Cell Physiol. 27): C1159-C1164, 1990]. In 90% of the patches we observed K+ channels identical to the maxi-K+ channels previously demonstrated in the apical membrane (Y. Segal and L. Reuss. J. Gen. Physiol. 95: 791-818, 1990). To ascertain whether these channels were present in the native tissue, we carried out intracellular-microelectrode studies. We tested for activation of basolateral membrane K+ conductance by depolarization or by elevation of intracellular Ca2+ and for tetraethylammonium sensitivity of the basolateral membrane voltage and fractional resistance. The results were negative, indicating that maxi-K+ channels are not expressed in the basolateral membrane of the "intact" epithelium. Using the same intracellular-microelectrode protocol on the apical membrane, we demonstrated the presence of an apical K+ conductance attributable to maxi-K+ channels. Additional experiments revealed a Ba(2+)-sensitive basolateral K+ conductance in the native epithelium. We conclude that in the stripped preparation there is artifactual expression of maxi-K+ channels. In addition, the native basolateral membrane K+ channels either are not expressed in this preparation or have a low conductance and cannot be discerned from the background noise.
Animals↗
Muscarinic stimulation of gallbladder epithelium. I. Electrophysiology and signaling mechanisms.
To understand the effects of acetylcholine (ACh) on fluid-absorbing epithelia, we carried out experiments on Necturus gallbladder epithelium. Binding studies with 1-quinuclidinyl[phenyl-4(N)-3H]benzilate (QNB) demonstrated that Necturus gallbladder epithelial cells express high-affinity muscarinic receptors. The effects of ACh and carbachol were exerted from the basolateral surface and consisted of a transient hyperpolarization of both cell membranes and a concomitant decrease in the apparent fractional resistance of the apical membrane. Atropine blocked both effects. ACh also elicited transient elevations of inositol 1,4,5-trisphosphate and intracellular free calcium ([Ca2+]i) levels, the latter by both release from intracellular stores and basolateral influx. The phospholipase C antagonist U-73122 inhibited the effects of ACh, whereas inhibition of prostaglandin and guanosine 3',5'-cyclic monophosphate synthesis with indomethacin or methylene blue, respectively, had no effect. In conclusion, Necturus gallbladder epithelium expresses muscarinic receptors in the basolateral membrane. Receptor activation stimulates phospholipase C and elevates cellular levels of inositol 1,4,5-trisphosphate and [Ca2+]i. The elevation in [Ca2+]i activates K+ channels but apparently not Cl- channels.
Acetylcholine↗
Muscarinic stimulation of gallbladder epithelium. II. Fluid transport, cell volume, and ion permeabilities.
Activation of muscarinic receptors in the fluid-absorptive epithelium of the Necturus gallbladder elevates cytosolic Ca2+ concentration, transiently hyperpolarizes the cell membrane voltages, and decreases the apparent fractional resistance of the apical membrane [G. A. Altenberg, M. Subramanyam, J. S. Bergmann, K. M. Johnson, and L. Reuss. Am. J. Physiol. 265 (Cell Physiol. 34): C1604-C1612, 1993]. In these studies, we show that at the peak of the hyperpolarization both apical and basolateral membrane resistances (Ra and Rb, respectively) decreased, but in 2-3 min Ra returned to control values while Rb rose to a level approximately 60% higher than control. The acetylcholine (ACh)-induced decrease in Ra is caused by activation of apical membrane maxi K+ channels secondary to elevation of cytosolic Ca2+ concentration. The increase in Rb is due to decreases in K+ and Cl- conductances. ACh had no effects on cell KCl content or water volume, although K+ conductance transiently increased. These results can be explained by the changes in basolateral membrane conductances. ACh did not alter fluid absorption. In conclusion, ACh has complex time-dependent effects on K+ and Cl- electrodiffusive permeabilities without measurable changes in cell volume or in the rate of transepithelial fluid transport.
Acetylcholine↗
Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.
In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.
Amiloride↗