External Ni2 + and ENaC in A6 cells: Na+ current stimulation by competition at a binding site for amiloride and Na+.

In cultured A6 monolayers from distal Xenopus kidney, external Ni2+ stimulated active Na+ uptake via the epithelial Na+ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni2+ effect. Na+ current noise analysis was performed using the neutral Na(+) -channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni2+ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni2+ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni2+ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na(+)--known to compete with amiloride and so producing the "self-inhibition" phenomenon--and Ni2+ exerted mutually exclusive analogous effects on amiloride kinetics. Na+ current kinetics revealed that Ni2+ prevents ENaC to be downregulated by self-inhibition. Co2+ behaved similarly to Ni2+, whereas Zn2+ did not. Attempts to disclose the chemical nature of the site reacting with Ni2+ suggested cysteine but not histidine as reaction partner.

Changes in element composition of A6 cells following hypotonic stress.

Cellular element concentrations and dry weight contents were determined in A6 epithelia using electron microprobe analysis. This was done to assess the quantitative contributions of Na, K and Cl to the regulatory volume decrease (RVD) and isovolumetric regulation (IVR) after decreasing the basolateral osmolality from 260 to 140 mosmol/kg in a stepwise or gradual way. Two minutes after inducing acute hypotonic stress the cells behaved almost like ideal osmometers, as indicated by a pronounced increase in cell height and decreases in the cellular dry weight and concentrations of all measured elements by about the same degree. Sixty minutes after inducing acute hypotonic stress the dry weight and concentrations of the impermeant elements P, Mg and Ca had returned approximately to control values, indicating normalized cell volume. Na, K and Cl concentrations, however, remained greatly reduced. The cellular amounts of Na, K and Cl diminished during RVD by approximately 31%, 24% and 46%, respectively. The dry weights and element concentrations measured 60 min after inducing acute hypotonic stress were similar to those obtained after a continuous reduction of basolateral osmolality. The cellular loss of Na and K following hypotonic stress exceeded that of Cl by about 40 mmol/kg wet wt., suggesting the exit of an other anion and/or the titration of fixed negative charges. The contribution of Na, K and Cl to total cellular osmolality increased from about 75% under control conditions to about 85% during RVD and IVR. Since only approximately 70% of the loss of cellular osmolytes necessary for the observed RVD and IVR is accounted for by the cellular exit of Na, K and Cl, other osmolytes, possibly amino acids, must leave the cells following hypotonic stress.

Functional integrity of the vesicle transporting machinery is required for complete activation of cFTR expressed in xenopus laevis oocytes.

We expressed the human cystic fibrosis transmembrane conductance regulator (CFTR) in oocytes of the South African clawed frog Xenopus laevis. We performed simultaneous and continuous recording of membrane current (Im), conductance (Gm) and capacitance (Cm), the latter being a direct measure of membrane surface area. A cAMP-cocktail containing cAMP and isobutylmethylxanthine (IBMX) increased all parameters, demonstrating that CFTR activation was partly achieved by exocytotic delivery and insertion of preformed CFTR molecules into the plasma membrane. CFTR currents after cAMP-cocktail were correlated with the capacitance of the oocytes: oocytes with larger Cm exhibited larger currents. Expression of CFTR itself did not change the Cm of the oocytes. However, activation of CFTR with cAMP-cocktail increased Im and Gm 15- and 20-fold, respectively while membrane surface area increased by about 7%, indicating the functional insertion of preformed CFTR into the plasma membrane. While cAMP-cocktail yielded maximal CFTR stimulation, IBMX alone, but not caffeine or theophylline, was sufficient to stimulate more than half of the increases in Im and Gm as observed with cAMP-cocktail. Since Cm was not significantly stimulated by IBMX, we conclude that IBMX alone activated the CFTR channels already present in the oocyte membrane. CFTR stimulation by cAMP-cocktail was independent of external Ca2+ and ATP had no additional activating potency. The role of protein trafficking in the activation of CFTR evoked by increases of cytoplasmic cAMP was assessed by measuring the effects of brefeldin A (BFA), nocodazole and primaquine on the bioelectric parameters and membrane surface area. All these compounds that interfere with the protein trafficking machinery at different stages prevented the translocation of CFTR from intracellular pools to the plasma membrane. These data confirm and extend our previous observations that CFTR expressed in Xenopus laevis oocytes is activated via dual pathways including direct activation of CFTR already present in the membrane and exocytotic insertion of preformed CFTR channels into the membrane. Furthermore, we show that complete activation of CFTR requires an intact protein trafficking machinery.

Effects of extracellular Mg2+ on transepithelial capacitance and Na+ transport in A6 cells under different osmotic conditions.

The electrophysiological characteristics of monolayers of cultured renal epithelial A6 cells were studied under short-circuit conditions. Replacing basolateral isosmotic (260 mOsm/kg H2O) media by hyposmotic (140 mOsm/kg H2O) solutions transiently increased the transepithelial capacitance (C(T)) by 57.3+/-2.3% after 16 min. The transepithelial Na+ current (I(Na)) increased concomitantly from 4.2+/-0.7 to 26.1+/-2.6 microA/cm2 with a time course that was noticeably slower, reaching its maximum after 60 min of hypotonicity. The transepithelial conductance (G(T)) increased synchronously with I(Na). Analysis of blocker-induced noise in I(Na), using the amiloride analogue 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), showed that the hypotonic shock increased Na+ channel density (N(T)) at the apical border. The presence of 10 mM Mg2+ on both sides of the epithelium suppressed the hypotonicity-induced C(T) increase to 14.3+/-0.5%, whereas the I(Na) increase was even larger than without Mg2+. Both effects of Mg2+ were located at an extracellular, basolateral site, because apical administration was without effect, whereas the acute basolateral addition of Mg2+ at the moment of the hypotonic shock was sufficient. Interaction between Mg2+ and Ca2+ influenced the behaviour of C(T). At constant osmolality (200 mOsm/kg H2O) 10 mM Mg2+ increased I(Na), leaving C(T) unaffected, whereas 10 mM Ca2+ stimulated both I(Na) and CT. In the presence of 1 mM Mg2+, however, the Ca(2+)-induced CT increase was abolished. The failure of CT to increase during stimulation of I(Na) by Mg2+ suggests that the divalent cation activates pre-existing channels in the apical membrane. Noise analysis showed that the natriferic effects of Mg2+ were also mediated by an increase in NT. The moderate initial increase in CT in the presence of Mg2+ under hypotonic conditions, occurring in parallel with increases in GT and I(Na), reflects most likely Na+ channel insertion induced by the hypotonic treatment. However, the large, transient, Mg(2+)-sensitive increase in CT, not correlated with increases in GT and I(Na), seems to be unrelated to Na+ channel recruitment.

Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia.

A sine wave method was used to measure transepithelial capacitance (CT) at 4.1 kHz (CHFT ). Model calculations show that CHFT reflects the equivalent capacitance of the series arrangement of apical and basolateral membrane capacitance. Cell swelling induced by reducing the basolateral osmolality from 260 to 140 mosmol/kg H2O (NaCl or sucrose removal) transiently decreased CHFT. The decrease in CHFT (DeltaCHFT ) reached its maximum 30 s after the onset of cell swelling and a complete recovery of CHFT was attained within 3-4 min. DeltaCHFT could be diminished by manoeuvres that reduced the rate or amplitude of cell swelling, i.e. lowering the temperature or treatment with low concentrations of glutaraldehyde (0.025%). DeltaCHFT increased with the magnitude of the osmotic perturbation but saturated at large volume expansions. DeltaCHFT increased with culture time. Electron micrographs showed a clear correlation between time course of CHFT changes and the closure of the lateral interspace (LIS). A striking correlation between the occurrence of CHFT recovery and the ability of the cells to develop a regulatory volume decrease (RVD) was found: Gd3+ (0.5 mM) inhibited both phenomena. The frequency dependence of CT was obtained from impedance spectra recorded over the range of 4 Hz to 22 kHz. These data agree with model calculations in which the contribution of the access resistance to the lateral membrane was included. All observations are consistent with the idea that DeltaCHFT originates from the closure of the LIS during cell swelling. The latter phenomenon increases the access resistance to the lateral membrane, which results in a marked reduction of the basolateral membrane area detected at high frequencies with capacitance measurements.

Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations.

Effects of basolateral monovalent cation replacements (Na+ by Li+, K+, Cs+, methylammonium, and guanidinium) on permeability to 86Rb of volume-sensitive cation channels (VSCC) in the basolateral membrane and on regulatory volume decrease (RVD), elicited by a hyposmotic shock, were studied in A6 epithelia in the absence of apical Na+ uptake. A complete and quick RVD occurred only when the cells were perfused with Na+ or Li+ saline. With both cations, hypotonicity increased basolateral 86Rb release (RblRb), which reached a maximum after 15 min and declined back to control level. When the major cation was K+, Cs+, methylammonium, or guanidinium, the RVD was abolished. Methylammonium induced a biphasic time course of cell thickness (Tc), with an initial decline of Tc followed by a gradual increase. With K+, Cs+, or guanidinium, Tc increased monotonously after the rapid initial rise evoked by the hypotonic challenge. In the presence of K+, Cs+, or methylammonium, RblRb remained high during most of the hypotonic period, whereas with guanidinium blockage of RblRb was initiated after 6 min of hypotonicity, suggesting an intracellular location of the site of action. With all cations, 0.5 mM basolateral Gd3+ completely blocked RVD and fully abolished the RblRb increase induced by the hypotonic shock. The lanthanide also blocked the additional volume increase induced by Cs+, K+, guanidinium, or methylammonium. When pH was lowered from 7. 4 to 6.0, RVD and RblRb were markedly inhibited. This study demonstrates that the VSCCs in the basolateral membrane of A6 cells are permeable to K+, Rb+, Cs+, methylammonium, and guanidinium, whereas a marked inhibitory effect is exerted by Gd3+, protons, and possibly intracellular guanidinium.

Cu2+ reveals different binding sites of amiloride and CDPC on the apical Na channel of frog skin.

The effect of Cu2+ ions, present in the mucosal bathing solution, on the transepithelial short-circuit current (Isc) and conductance (Gt) and on the blocker-induced noise of apical Na channels, was studied on the isolated ventral skin of the frog Rana temporaria. Cu2+ effects were concentration-dependent, the full effect being reached at 50 micromol/l. Cu2+ increased Isc and Gt; this effect was eliminated by high concentrations of amiloride (30 micromol/l) and of CDPC (150 micromol/l). Cu2+ markedly reduced the corner frequency (fc) of the Na channel noise, while having virtually no effect on the fc of CDPC-induced noise. Cu2+ reduces the association rate constant of amiloride to the Na channel to one third; this effect is interpreted as indicating competition between Cu2+ and amiloride for the same (negatively charged) binding site on the channel, while CDPC appears to bind on a different site.

Regulatory volume decrease in a renal distal tubular cell line (A6). II. Effect of Na+ transport rate.

A6 epithelia, a cell line originating from the distal tubular part of the kidney of Xenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (Tc), short-circuit current (Isc) and transepithelial conductance (Gt) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na+ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease of Tc described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na+ uptake by replacing apical Na+ by N-methyl-d-glucamine (NMDG+) did not affect cell volume under isotonic conditions. An increase of Tc by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na+ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na+uptake or Na+ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K+ channels by 5 mM Ba2+ elicited a significant increase in Tc of 16.3% above control. Quinine, a potent blocker of volume-activated K+ channels, did not change Tc significantly. Basolateral hypotonicity elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na+ uptake as well as after stimulating apical Na+ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K+ efflux at the basolateral side with Ba2+ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K+, whereas the effects of Ba2+ and quinine are most likely due to the blockage of the basolateral K+ pathway.

Regulatory volume decrease in a renal distal tubular cell line (A6). I. Role of K+ and Cl-.

Changes in volume of A6 epithelial cells were monitored by recording cell thickness (Tc). The response of Tc to a reduction of the basolateral osmolality from 260 to 140 mosmol/kg was recorded while transepithelial Na+ transport was inhibited by 20 microM amiloride. With Cl--containing bathing media, this osmotic challenge elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). Substitution of SO4(2-) or gluconate for Cl- markedly reduced the RVD, whereas cells completely maintained their ability to regulate their volume after replacing Cl- by NO3(-). A conductive pathway for Cl- excretion is suggested, which is insensitive to NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid], an inhibitor of some types of Cl- channels. Ba2+ (5 or 20 mM) reduced the RVD. A more pronounced inhibition of the RVD was obtained with 500 microM quinine, a potent blocker of volume-activated K+ channels. K+-induced depolarization of the basolateral membranes of tissues incubated with SO4(2-)-containing solutions completely abolished the RVD. Noise analysis in the presence of Ba2+ showed the activation of an apical K+ conductive pathway. These results demonstrate that cell volume regulation is controlled by processes involving Cl- and K+ excretion through conductive pathways.

Regulatory volume decrease in cultured kidney cells (A6): role of amino acids.

Volume regulation was studied in A6 epithelia grown on permeable supports by measuring cell thickness (Tc) while simultaneously recording short circuit current (ISC) and transepithelial conductance (Gt). Lowering the tonicity of the basolateral solution (pi b) from 250 or 215 to 140 mOsm/kg elicited a rapid rise in Tc followed by a regulation of the cell volume towards control. This decrease in Tc displays the characteristics of the regulatory volume decrease (RVD). Upon restoring the isoosmotic conditions, Tc decreased rapidly below its control value. A post RVD regulatory volume increase (RVI) as described for other cell types was not observed. The subsequent reduction of the basolateral osmolality increased Tc to the level recorded at the end of the first hypoosmotic pulse. Because cell content was not altered during the isoosmotic period the second hypoosmotic challenge was isotonic with the cell and did therefore not evoke an RVD. However, the cell did not lose its ability to volume regulate since an RVD could be elicited by further reduction of pi b from 140 to 100 mOsm/kg. The possibility of an involvement of amino acids in the RVD was tested. The amount of amino acids in the cell as well as excreted in the bath was determined by amino acid analysis. Millimolar concentrations of threonine, serine, alanine, glutamate, glycine and aspartate were found in the cell extract. The cellular amino acid concentration was 28.8 +/- 0.4 mM. The amounts of glycine, aspartate and glutamate excreted from the cell during the hypotonic treatment were significantly larger than in control conditions. The excretion of these amino acids during hypotonicity decreased the cellular amino acid concentration by 8.4 +/- 0.2 mM. This quantity cannot completely account for the RVD during the first hypotonic challenge. The addition of glycine, aspartate and glutamate to the bathing solutions, although used at concentrations higher than intracellularly, did not reduce RVD. On the contrary, this maneuver increased the amplitude of the RVD following both hypoosmotic pulses. This result suggests a stimulatory role of the amino acids on the processes responsible for the RVD.

Poorly selective cation channels in apical membranes of epithelia.

The apical membrane of frog skin contains two types of pathways which allow the passage of several monovalent cations in the absence of external Ca2+. Differences between the two pathways concern their open-close kinetics, selectivity, and the affinity for several blocking agents. Type S channels open and close relatively slowly, whereas type F channels display fast open-close kinetics. Both channel types allow the passage of Na+, K+, and Rb+ currents which are blocked by divalent cations and La3+ added to the extracellular side. Type F channels are permeable for Cs+ which is, however, excluded from type S channels. Shifts in open-close kinetics induced by Mg2+ occur at concentrations below 5 microM for type F channels, whereas more than a tenfold higher dose is required for the type S pathway. UO2(2+) concentrations up to 100 microM only occlude type S channels while 100 microM tetracaine selectively blocks type F channels. Apical membranes of toad urinary bladder, cultured amphibian renal epithelia (A6), and toad colon contain only type F channels. In toad bladder and A6 cells volume expansion strongly activates this pathway. Macroscopic currents carried by Ba2+ and Ca2+ could be recorded after activation of toad bladders with oxytocin and treatment of the apical surface with nanomolar concentrations of Ag+, which seems to interact with a site located at the channel interior.

Ca(2+)-blockable, poorly selective cation channels in the apical membrane of amphibian epithelia. Tetracaine blocks the UO2(2+)-insensitive pathway.

We examined the effect of the local anesthetic tetracaine on the Ca(2+)-blockable, poorly selective cation channels in the isolated skin of Rana temporaria and the urinary bladder of Bufo marinus using noise analysis and microelectrode impalements. Experiments with frog skin demonstrated that mucosal concentrations of the compound up to 100 microM did not affect the Na+ current through type S channels (slowly fluctuating, UO2(2+)-blockable channels) and the associated noise. On the other hand, 20 microM mucosal tetracaine already suffices to inhibit approximately 50% of the current carried by Cs+ and Na+ through channel type F (fast fluctuating, UO2(2+)-insensitive channel) and So of the associated Lorentzian component. With 100 microM of the inhibitor the current and So values were reduced by at least 70-80%. The time course of the response to serosal tetracaine was markedly slower and the effects on the current and So were smaller. Possible effects on the basolateral K+ conductance were excluded on the basis of the lack of response of transepithelial K+ movements to 100 microM tetracaine. UO2(2+) and tetracaine together blocked the poorly selective cation pathways almost completely. Moreover, both agents retain their inhibitory effect in the presence of the other. In toad urinary bladder, the Ca(2+)-blockable channel is also tetracaine blockable. The concentration required for half-maximal inhibition is approximately 100 microM in SO4(2-) and approximately 20 microM in Cl-. The data with tetracaine complement those obtained with UO2(2+) and support the idea that the Ca(2+)-blockable current proceeds through two distinct classes of cation channels. Using tetracaine and UO2(2+) as channel-specific compounds, we demonstrated with microelectrode measurements that both channel types are located in the granulosum cells.

Ca(2+)-blockable, poorly selective cation channels in the apical membrane of amphibian epithelia. UO2(2+) reveals two channel types.

This study deals with the effect of mucosal UO2(2+) on the Ca(2+)-blockable, poorly selective cation channels in the apical membrane of frog skin and toad urinary bladder. Our data show that UO2(2+) inhibits the Na+ currents through the amiloride-insensitive cation pathway and confirm a previously described stimulatory effect on the amiloride-blockade Na+ transport. Noise analysis of the Ca(2+)-blockable current demonstrates that the divalent also depresses the low-frequency Lorentzian (fc = 11.7 Hz) in the power density spectrum (PDS) and reveals the presence of high-frequency relaxation noise (fc = 58.5 Hz). The action of UO2(2+) is not reversed upon washout and is not accompanied by noise, typically induced by reversible blockers. The divalent merely depresses the plateau of the low-frequency Lorentzian, demonstrating a decrease in the number of conductive cation channels. Similarly, with mucosal K+ and Rb+, UO2(2+) also unmasks the high-frequency Lorentzian by depressing the noise from the slowly fluctuating cation channels (type S). In all experiments with mucosal Cs+, the PDS contains high-frequency relaxation noise (fc = 75.1 Hz in Rana temporaria, and 65.4 Hz in Rana ridibunda). An effect of UO2(2+) on the Cs+ currents and Lorentzian plateaus could not be demonstrated, suggesting that this monovalent cation does not pass through type S channels. Experiments with the urinary bladder revealed only a UO2(2+)-insensitive pathway permeable for Na+, K+, Rb+, and Cs+. We submit that in frog skin two cation-selective channels occur, distinguished by their spontaneous gating kinetics, their sensitivity to UO2(2+), and their permeability for Cs+. In toad urinary bladder, only one kind of cation-selective channel is observed, which resembles the UO2(2+)-insensitive channel in frog skin, with fast open-closed kinetics (type F).

Amiloride blockage of Na+ channels in amphibian epithelia does not require external Ca2+.

Noise analysis was used to study the influence of external Ca2+ on the blockage of Na+ transport by amiloride. Experiments were done using frog skin (Rana temporaria and Rana catesbeiana), toad urinary bladder (Bufo marinus) and epithelia of A6 cells. In nondepolarized skins and bladders, removal of Ca2+ from the mucosal bath diminished markedly the inhibitory effect of amiloride. Ca2+ depletion also gave rise to the appearance of an additional noise component related to cation movement through the poorly selective cation channel in the apical membrane [Aelvoet I, Erlij D, Van Driessche W (1988) J Physiol (Lond) 398:555-574; Van Driessche W, Desmedt L, Simaels J (1991) Pflügers Arch 418:193-203]. The amplitude of this Ca(2+)-blockable noise component was elevated by amiloride and markedly exceeded the amiloride-induced Lorentzian noise levels as recorded in the presence of Ca2+. On the other hand, in K(+)-depolarized skins and bladders as well as in nondepolarized epithelial of A6 cells, the Ca(2+)-blockable noise was absent or of much smaller amplitude. Depolarization of frog skin and toad urinary bladder apparently inactivated the poorly selective channels, whereas in A6 cells they were not observed. Under these conditions the typical amiloride-induced blocker noise could also be analysed in the absence of Ca2+ and demonstrated that the on and off rates for amiloride binding were not significantly altered by external Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockage of Na+ currents through poorly selective cation channels in the apical membrane of frog skin and toad urinary bladder.

The blockage of Na+ movements through the poorly selective cation channels in the apical membrane of frog skin (Rana temporaria) and toad urinary bladder (Bufo marinus) was investigated with noise, impedance analysis and microelectrode techniques. Na+ currents through this pathway were studied with NaCl Ringer solutions on both sides. After removal of Ca2+ and other divalent cations from the mucosal compartment, a considerable part of Isc became insensitive to amiloride. In frog skin, the inhibitory effect of amiloride in mucosal Ca(2+)-free solutions was highly variable. In some experiments a complete lack of inhibition was observed. Similarly, in the absence of amiloride, the inhibitory effect of mucosal Ca2+ varied strongly among frogs. In the absence of mucosal Ca2+, analysis of the fluctuation in Isc revealed a Lorentzian component in the power density spectrum. The corner frequency (fc) of this spontaneous Lorentzian was 12.3 Hz in frog skin and 347 Hz in the toad urinary bladder. In frog skin, nanomolar concentrations of mucosal Ca2+ induced an additional Lorentzian noise component. Its corner frequency shifted upwards with increasing mucosal Ca2+ concentration ([Ca2+]m). The relation between 2 pi fc and [Ca2+]m was linear at small [Ca2+]m whereas a parabolic increase of fc was observed at the highest [Ca2+]m. In the bladder, nanomolar concentrations of mucosal Ca2+ did not induce an additional noise component but modified the spontaneous Lorentzian noise by increasing fc proportionally with [Ca2+]m. Microelectrode recordings demonstrated that at least part of the Ca(2+)-blockable current passes through the granulosum cells and confirmed the apical localization of the poorly selective cation channel. The lack of the inhibitory effect of amiloride in Ca(2+)-free solutions seems to originate from the parallel arrangement of the amiloride- and Ca(2+)-blockable pathways and from influences of the blockage of apical channels on the basolateral membrane conductances. The latter cross-talk seems to find its origin in the voltage dependence of the basolateral membrane conductance [Garty H (1984) J Membr Biol 77:213-222; Nagel W (1985) Pflügers Arch 405 [Suppl 1]:S39-S43].

Role of an apical cation-selective channel in function of tight epithelia.

Some features of oxytocin stimulation of a cation-selective channel of the apical membrane of amphibian tight epithelia were examined in an attempt to understand the channel's role in the regulation of epithelial transport. We first examined the ability of the channel to pass alkaline-earth cations. We found that oxytocin can stimulate the movement of alkaline-earth ions through the channel. This stimulation became greatly enhanced by treatment with Ag+. The stimulation of alkaline-earth movements is discussed together with recently reported experiments which suggest that the channel may be involved in K+ secretion. In addition we carried out comparative studies of the effects of oxytocin on the channel in a variety of epithelia obtained from different amphibians to examine whether the stimulation of ionic currents through the channel and the enhancement of hydrosmotic permeability caused by the hormone are linked. The results of our experiments showed that oxytocin activates the channel in the urinary bladders of Bufo marinus, and Rana catesbeiana as well as in the skin of B. marinus. It is well known that in all these tissues the hormone increases water permeability of the apical membrane. On the other hand, in skins of Rana catesbeiana, Rana pipiens, and Rana temporaria, where oxytocin does not have a hydrosmotic effect, the hormone did not increase the currents through the cation-selective channel.

Cation-selective channels in amphibian epithelia: electrophysiological properties and activation.

1. Na+ as well as Li+ move across the apical membrane through amiloride-sensitive ionic channels. 2. K+ movements across the apical membrane occur through Ba2+- and Cs+-sensitive channels which do not allow the passage of Na+ or Li+. 3. A third pathway in the apical membrane is permeable for Na+, K+, Cs+, Rb+, NH+4 and Ti+. The currents carried by these monovalent cations are blocked by Ca2+ and divalent cations as well as La3+. 4. In the urinary bladder, the Ca2+-sensitive currents are stimulated by oxytocin, activators of cytosolic cAMP and cAMP analogues. Also the oxytocin activated currents are blocked by divalent cations and La3+. 5. Nanomolar concentrations of mucosal Ag+ activate the third channel and open the pathway for movements of Ca2+, Ba2+ and Mg2+, which are known to permeate through Ca2+ channels in excitable tissues.