Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.
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Abstract:
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.Keywords:
Necturus
Hyperpolarization
Amiloride
Tetraethylammonium
Tetrodotoxin
Apical membrane
Epithelial polarity
Tetramethylammonium
A study of the mechanisms of the effects of amphotericin B and ouabain on cell membrane and transepithelial potentials and intracellular K activity (alpha Ki) of Necturus gallbladder epithelium was undertaken with conventional and K-selective intracellular microelectrode techniques. Amphotericin B produced a mucosa-negative change of transepithelial potential (Vms) and depolarization of both apical and basolateral membranes. Rapid fall of alpha Ki was also observed, with the consequent reduction of the K equilibrium potential (EK) across both the apical and the basolateral membrane. It was also shown that, unless the mucosal bathing medium is rapidly exchanged, K accumulates in the unstirred fluid layers near the luminal membrane generating a paracellular K diffusion potential, which contributes to the Vms change. Exposure to ouabain resulted in a slow decrease of alpha Ki and slow depolarization of both cell membranes. Cell membrane potentials and alpha Ki could be partially restored by a brief (3-4 min) mucosal substitution of K for Na. Under all experimental conditions (control, amphotericin B, and ouabain), EK at the basolateral membrane was larger than the basolateral membrane equivalent emf (Eb). Therefore, the K chemical potential difference appears to account for Eb and the magnitude of the cell membrane potentials, without the need to postulate an electrogenic Na pump. Comparison of the rate of Na transport across the tissue with the electrodiffusional K flux across the basolateral membrane indicates that maintenance of a steady-state alpha Ki cannot be explained by a simple Na,K pump-K leak model. It is suggested that either a NaCl pump operates in parallel with the Na,K pump, or that a KCl downhill neutral extrusion mechanism exists in addition to the electrodiffusional K pathway.
Necturus
Paracellular transport
Epithelial polarity
Apical membrane
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The crystal structure of [(C 2 H 5 ) 4 N][(CH 3 ) 4 N][ZnCl 4 ] [abbreviated to (TEA)(TMA)ZnCl 4 ] was investigated by means of X-ray diffraction at room temperature. The crystal structure can be regarded as layered by repeating the sequence (TMA) + /(ZnCl 4 ) 2− /(TEA) + /(TEA) + / (ZnCl 4 ) 2− . In the same layer, the (TMA) + tetrahedra may be ordered or disordered according to their position in the layer
Tetramethylammonium
Tetraethylammonium
Tetramethylammonium hydroxide
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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.
Epithelial polarity
Necturus
Apical membrane
Tetraethylammonium
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Eight cations were reversibly and isosmotically substituted for sodium (45 m<i>M</i>) in peritubular circulation of the proximal tubule of the doubly perfused Necturus kidney. Li, choline and tetramethylammonium produced small shifts in peritubular potential difference with no change in conductance. Tris- and tetraethylammonium depolarized the membrane by about 10 mV without affecting its conductance. Rb and Cs produced a decrease of membrane permeability to potassium. Potassium for sodium substitution depolarized the membrane (3.1 mV) and increased its conductance. It is concluded that none among the cations tested may be considered as readily less permeant than sodium across the peritubular membrane.
Tetramethylammonium
Necturus
Tetraethylammonium
Peritubular capillaries
Choline
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Tetramethylammonium
Tetraethylammonium
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Necturus
Apical membrane
Epithelial polarity
Cell membrane
Hyperpolarization
Intracellular pH
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Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.
Necturus
Epithelial polarity
Apical membrane
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Tetraethylammonium
Tetramethylammonium
Residue (chemistry)
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Tetramethylammonium
Tetraethylammonium
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Voltage‐sensitive glass micro‐electrodes were used to determine the electrical characteristics of Necturus proximal duodenal epithelium. Some comparative experiments with amiloride were performed with gastric antrum. The apical and the basolateral cell membrane potential differences in duodenum averaged ‐32 mV and ‐34 mV (cell negative) respectively. The transepithelial potential difference was ‐2 mV (lumen negative). The EMF across the apical cell membrane was ‐29 mV and that across the basolateral cell membrane ‐39 mV. The transepithelial resistance (R 1 ) of 63 Ω cm 2 and the paracellular pathway resistance (R s ) of 8 o Ω cm 2 are of magnitudes similar to that previously reported for more distal amphibian small intestine. The apical and basolateral cell membrane resistances, however, were lower than those reported for distal small intestine. Ion permeabilities for Na + , K + and Cl ‐ across the apical cell membrane were calculated from ion substitution experiments. The permeability sequence across the apical cell membrane was P K :P cl :P Na 3.02:1.31:1.00. Luminal amiloride (10 ‐4 M) was without significant effect, further indicating a low duodenal membrane conductance for Na + . The low conductances for K + , Na + and Cl ‐ suggest that the major ion transport modes across the apical duodenal cell membrane are electroneutral in nature. In contrast, amiloride caused a marked increase in the transmembrane potentials in the antrum.
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