Mechanism of the effect of cyanide on cell membrane potentials in Necturus gall‐bladder epithelium.

The Journal of General Physiology

1985

Self Cite

106

The effects of elevating intracellular cAMP levels on Na' transport across the apical membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques . Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 pM). During elevation of intracellular cAMP, intracellular Na' activity (aNai) and intracellular pH (pHi) decreased significantly . In addition, acidification of the mucosal solution, which contained either 100 or 10 mM Na', was inhibited by^-50%. The inhibition was independent of the presence of Cl -in the bathing media. The rates of change of aNai upon rapid alterations of mucosal [Na'] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate of pHi recovery upon acid loading was also reduced by elevated cAMP levels . Inhibition was^-50% for all of these processes. These results indicate that cAMP inhibits apical membrane Na'/H' exchange . The results of measurements of pH i recovery at 10 and 100 mM mucosal [Na'] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in apical membrane electrodiffusional Cl -permeability, produced by cAMP, which causes a decrease in net Cl -entry (Petersen, K.U., and L. Reuss, 1983, J: Gen. Physiol., 81 :705

Section: Discussionmentioning

confidence: 99%

Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium.

The Journal of General Physiology

1985

Self Cite

106

“…In Necturus gallbladder epithelium, estimates of the cell membrane resistances have been obtained utilizing AC-impedance analysis (Kottra & Fr6mter, 1984a,b), flat-sheet intracellular cable analysis (Fr6mter, 1972;Reuss & Finn, 1975a), or variations of these techniques including squarewave pulse analysis (Suzuki et al, 1982) and twopoint intracellular cable analysis (Bello- Reuss, Grady & Reuss, 1981;Petersen & Reuss, 1985;and see Materials and Methods). Regardless of the approach used, voltage-dependent and/or time-dependent alterations in cell membrane resistances during the measurement or during experimental perturbations can mask expected changes in resistance and give misleading results.…”

Section: Introductionmentioning

confidence: 99%

Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Stoddard

1988

J. Membrain Biol.

The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances of Necturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (Vmc) results in a marked decrease in apical membrane fractional resistance (fRa), whereas hyperpolarization of Vmc results in either no change in fRa or a small increase, and b) the voltage-dependent changes in fRa are essentially complete within 500 msec. Exposure of the tissue to 5 mM TEA+ on the mucosal side caused no significant change in baseline Vmc (-69 +/- 2 mV) and yet virtually abolished the voltage dependence of fRa. A possible interpretation of these results is that two types of K+ channels exist in the apical membrane, with different voltage dependencies and TEA+ sensitivities. Acidification or Ba2+ addition to the mucosal solution also reduced the voltage-dependent changes in fRa. The time courses of the changes in fRa and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (+/- 200 microA/cm2). No secondary change in fRa was observed with mucosa-to-serosa currents, but a slow TEA+-sensitive decrease in fRa (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (less than 500 msec) voltage-dependent decrease in fRa is due to a fall in apical membrane resistance. The later decrease in fRa is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimating fRa than the current-induced effects on the lateral intercellular spaces. In principle, TEA+ can be used to prevent the nonlinear behavior of Ra during measurements of the voltage divider or membrane resistance ratio.

“…Previous results indicate that there are Ca2+-activated K ÷ channels at the apical and basolateral membranes (Bello- Reuss et al, 1981;Garcia-Diaz et al, 1983;Reuss, 1988b, 1989b;Reuss, 1989;Segal and Reuss, 1990a). Here we study the mechanisms of the electrophysiological effects of lowering [Na+]~, in particular the possibility that an elevation in intracellular free [Ca 2+] hyperpolarizes Vc~ by increasing gK +.…”

Section: Introductionmentioning

confidence: 84%

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.

Altenberg

Stoddard

The Journal of General Physiology

1992

Self Cite

In Necturus gallbladder epithelium, lowering serosal [Na ÷] ([Na+]3 reversibly hyperpolarized the basolateral cell membrane voltage (Vc,) and reduced the fractional resistance of the apical membrane (fR~). Previous results have suggested that there is no sizable basolateral Na + conductance and that there are apical Ca~+-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 [Ca 2+] hyperpolarizes V,, by increasing gK ÷. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), V,~ hyperpolarized from -68 -+ 2 to a peak value of -82 -2 mV (P < 0.001), and fR a decreased from 0.84 +-0.02 to 0.62 +-0.02 (P < 0.001). Addition of 5 mM tetraethylammonium (TEA +) to the mucosal solution reduced both the hyperpolarization of Vc~ and the change in fR~, whereas serosal addition of TEA + had no effect. Ouabain (10 -4 M, serosal side) produced a small depolarization of Vc, and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fR a. The effects of mucosal TEA + and serosal ouabain were additive. Neither amiloride (10 -5 or 10 -s M) nor tetrodotoxin (10 -~ M) had any effects on Vcs or fR a or on their responses to lowering [Na+]~, suggesting that basolateral Na + channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]5. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vc~ upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral C1-conductance (gC1-) 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 fR~ by lowering [Na÷]~ is mainly caused by an increase in intraceUular free [Ca2÷], which activates TEA+-sensitive maxi K ÷ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vc, is due to increases in: (a) apical membrane gK ÷, (b) the contribution of the Na + pump to Vc~, (c) basolateral membrane K ÷ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.