Microelectrode studies in toad urinary bladder epithelium. effects of Na concentration changes in the mucosal solution on equivalent electromotive forces

Microelectrode techniques were employed to measure membrane potentials, the electrical resistance of the cell membranes, and the shunt pathway, and to compute the equivalent electromotive forces (EMF) at both cell borders in toad urinary bladder epithelium before and after reductions in mucosal sodium concentration. Basal electrical parameters were not significantly different from those obtained with impalements from the serosal side, indicating that mucosal impalements do not produce significant leaks in the apical membrane. A decrease in mucosal Na concentration caused the cellular resistance to increase and both apical and basolateral EMF to depolarize. When Na was reduced from 112 to 2.4 mM in bladders with spontaneously different baseline values of transepithelial potential difference (Vms), a direct relationship was found between the change in Vms brought about by the Na reduction and the base-line Vms before the change. A direct relationship was also found by plotting the change in EMF at the apical or basolateral border caused by a mucosal Na reduction with the corresponding base-line EMF before the change. These results indicate that resting apical membrane EMF (and, therefore, resting apical membrane potential) is determined by the Na selectivity of the apical membrane, whereas basolateral EMF is at least in part the result of rheogenic Na transport. These results are consistent with data of others that suggested a link between the activity of the basolateral Na pump and apical Na conductance.     

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.     

Electrical properties of the cellular transepithelial pathway in Necturus gallbladder. II. Ionic permeability of the apical cell membrane.

Microelectrode techniques were employed to study the ionic permeability of the apical cell membrane of Necturus gallbladder epithelium. Results obtained from continuous records in single cells, and from several cellular impalements shortly after a change in solution, were similar and indicate that both the apical membrane equivalent electromotive force (Va) and electrical resistance (Ra) strongly depend on external [K]. Cl substitutions produced smaller effects, while the effects of Na substitutions with N-methyl-D-glucamine on both Va and Ra were minimal. These results indicate that the permeability sequence of the apical membrane is PKgreater thanPClgreater than PNa. From the calculated absolute value of PNa it is possible to estimate the diffusional Na flux from the mucosal solution into the cells (from the cell potential and an assumed intracellular Na concentration). The calculated flux is roughly three orders of magnitude smaller than the measured net transepithelial flux in this tissue and in gallbladders of other species. Thus, only a minimal portion of Na entry can be attributed to independent diffusion. From estimations of the electrochemical potential gradient across the apical membrane, Cl transport at that site must be active. At the serosal cell membrane, Na transport takes place against both chemical and electrical potentials, while a significant portion of the Cl flux can be passive, if this membrane has a significant Cl conductance. The changes in shunt electromotive force and in transepithelial potential after mucosal substitutions were very similar, indicating that transepithelial bi-ionic potentials yield appropriate results on the properties of shunt pathway.     

Electrophysiology of flounder intestinal mucosa. II. Relation of the electrical potential profile to coupled NaCl absorption

We characterized the hyperpolarization of the electrical potential profile of flounder intestinal cells that accompanies inhibition of NaCl cotransport. Several observations indicate that hyperpolarization of psi a and psi b (delta psi a,b) results from inhibition of NaCl entry across the apical membrane: (a) the response was elicited by replacement of mucosal solution Cl or Na by nontransported ions, and (b) mucosal bumetanide or serosal cGMP, inhibitors of NaCl influx, elicited delta psi a,b and decreased the transepithelial potential (psi t) in parallel. Regardless of initial values, psi a and psi b approached the equilibrium potential for K (EK) so that in the steady state following inhibition of NaCl entry, psi a approximately equal to psi b approximately equal to ECl approximately equal to EK. Bumetanide decreased cell Cl activity (aClc) toward equilibrium levels. Bumetanide and cGMP decreased the fractional apical membrane resistance (fRa), increased the slope of the relation of psi a to [K]m, and decreased cellular conductance (Gc) by approximately 85%, which indicates a marked increase in basolateral membrane conductance (Gb). Since the basolateral membrane normally shows a high conductance to Cl, a direct relation between apical salt entry and GClb is suggested by these findings. As judged by the response to bumetanide or ion replacement in the presence of mucosal Ba, inhibition of Na/K/Cl co-transport alone is not sufficient to elicit delta psi a,b. This suggests the presence of a parallel NaCl co-transport mechanism that may be activated when Na/K/Cl co-transport is compromised. The delta psi a,b response to reduced apical NaCl entry would assist in maintaining the driving force for Na-coupled amino acid uptake across the apical membrane as luminal [NaCl] falls during absorption.     

K+ secretion across frog skin. Induction by removal of basolateral Cl-

We examined the development of K+ secretion after removing Cl- from the basolateral surface of isolated skins of Rana temporaria using noise analysis. K+ secretion was defined by the appearance of a Lorentzian component in the power density spectrum (PDS) when Ba2+ was present in the apical bath (0.5 mM). No Lorentzians were observed when tissues were bathed in control, NaCl Ringer solution. Replacement of basolateral Cl- by gluconate, nitrate, or SO4- (0-Clb) yielded Lorentzians with corner frequencies near 25 Hz, and plateau values (So) that were used to estimate the magnitude of K+ secretion through channels in the apical cell membranes of the principal cells. The response was reversible and reproducible. In contrast, removing apical Cl- did not alter the PDS. Reduction of basolateral Cl- to 11.5 mM induced Lorentzians, but with lower values of So. Inhibition of Na+ transport with amiloride or by omitting apical Na+ depressed K+ secretion but did not prevent its appearance in response to 0-Clb. Using microelectrodes, we observed depolarization of the intracellular voltage concomitant with increased resistance of the basolateral membrane after 0-Clb. Basolateral application of Ba2+ to depolarize cells also induced K+ secretion. Because apical conductance and channel density are unchanged after 0-Clb, we conclude that K+ secretion is "induced" simply by an increase of the electrical driving force for K+ exit across this membrane. Repolarization of the apical membrane after 0-Clb eliminated K+ secretion, while further depolarization increased the magnitude of the secretory current. The cell depolarization after 0-Clb is most likely caused directly by a decrease of the basolateral membrane K+ conductance. Ba2(+)-induced Lorentzians also were elicited by basolateral hypertonic solutions but with lower values of So, indicating that cell shrinkage per se could not entirely account for the response to 0-Clb and that the effects of 0-Clb may be partly related to a fall of intracellular Cl-.     

Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane potential (Vbl) was observed between tubules and ducts in response to ion substitutions, inhibitor and agonist applications. Cl- substitution experiments indicated a small Cl- conductance in the basolateral membrane. The apical membrane did not have a significant Cl- conductance. Luminal [Na+] steps and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na+] in the bath, demonstrating the presence of a Na+-coupled anion transporter. [HCO3-] steps revealed that this transporter is not a Na+-HCO3- cotransporter. Together, our results indicate that a major task of the collecting duct system in B. bufo is not conductive NaCl transport but rather K+ secretion, as shown by our previous studies. Moreover, our results indicate the presence of a novel basolateral Na+-coupled anion transporter, the identity of which remains to be elucidated.     

Characterization of the basolateral membrane conductance of Necturus urinary bladder

Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that permitted rapid changes in the ion composition of the serosal solution. The transepithelial electrical properties exhibited a marked seasonal variation that could be attributed to variations in the conductance of the shunt pathway, apical membrane selectivity, and basolateral Na+ transport. In contrast, the passive electrical properties of the basolateral membrane remained constant throughout the year. The apparent transference numbers (Ti) of the basolateral membrane for K+ and Cl- were determined from the effect on the basolateral membrane equivalent electromotive force of a sudden increase in the serosal K+ concentration from 2.5 to 50 mM/liter or a decrease in the Cl- concentration from 101 to 10 mM/liter. TK and TCl were 0.71 +/- 0.05 and 0.04 +/- 0.01, respectively. The basolateral K+ conductance could be blocked by Ba2+ (0.5 mM), Cs+ (10 mM), or Rb+ (10 mM), but was unaffected by 3,4-diaminopyridine (100 microM), decamethonium (100 microM), or tetraethylammonium (10 mM). We conclude that a highly selective K+ conductance dominates the electrical properties of the basolateral membrane and that this conductance is different from those found in nerve and muscle membranes.     

Anthracene-9-carboxylic acid inhibits an apical membrane,chloride conductance in canine tracheal epithelium

Summary Canine tracheal epithelium secretes Cl from the submucosal to the mucosal surface via an electrogenic transport process that appears to apply to a wide variety of secretory epithelia. Cl exit across the apical membrane is thought to be a passive, electrically conductive process. To examine the cellular mechanism of Cl secretion we studied the effect of anthracene-9-carboxylic acid (9-AC), an agent known to inhibit the Cl conductance of muscle membrane. When added to the mucosal solution, 9-AC rapidly and reversibly decreases short-circuit current and transepithelial conductance, reflecting a reduction in electrogenic Cl secretion. The inhibition is concentration-dependent and 9-AC does not appear to compete with Cl for the transport process. The decrease in current and conductance results from a decrease in the net and both unidirectional transepithelial Cl fluxes without substantial alterations of Na fluxes. Furthermore, 9-AC specifically inhibits a Cl conductance: tissues bathed in Cl-free solutions showed no response to 9-AC. Likewise, when the rate of secretion and Cl conductance were minimized with indomethacin, addition of 9-AC did not alter transepithelial conductance. In contrast, neither removal of Na from the media nor blockade of the apical Na conductance with amiloride prevented a 9-AC-induced decrease in transepithelial conductance. We also found that the effect of 9-AC is independent of transepithelial transport: 9-AC decreases transepithelial conductance despite inhibition of Cl secretion with ouabain or furosemide. Intracellular electrophysiologic techniques were used to localize the effect of 9-AC to a reduction of the electrical conductance of the apical cell membrane: 9-AC hyperpolarizes the electrical potential difference across the apical membrane and decreases its relative conductance. 9-AC also prevents the characteristic changes in the cellular electrical potential profile, transepithelial conductance, and the ratio of membrane conductances produced by a reduction in mucosal bathing solution Cl concentration. These results indicate that 9-AC inhibits Cl secretion in tracheal epithelium by blocking an electrically conductive Cl exit step in the apical cell membrane. Thus, they support a cellular model of Cl secretion in which Cl leaves the cell across a Cl permeable apical membrane driven by its electrochemical gradient.     

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder

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.     

The sodium and chloride dependence of chloride secretion by the opercular epithelium

The effects of ion substitutions on the Cl- secretion rate and tissue conductance of isolated short-circuited opercular epithelia from sea-water-adapted Fundulus heteroclitus were investigated. Serosal Na+ substitution had the same effect on the Cl- secretion rate that serosal Cl- substitution had on the active component of the Cl- efflux. This similarity indicated a 1:1 Na-Cl requirement for active Cl- secretion across this epithelium, which supports the proposal of a coupled NaCl uptake mechanism at the serosal membrane of Cl- secretory epithelia. Mucosal Na+ and Cl- substitutions appeared to inhibit completely the active Cl- secretory flux. The reductions in the tissue conductance with mucosal ion substitutions suggested that this effect can be attributed to a blocking of the apical membrane Cl- conductance. These mucosal ion effects suggested a possible direct regulatory influence of the external salinity on the Cl- secretion rate and tissue conductance, which provide alternative explanations for observations with the teleost gill epithelium.     

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.     

Model of ion transport regulation in chloride-secreting airway epithelial cells. Integrated description of electrical, chemical, and fluorescence measurements.

An electrokinetic model was developed to calculate the time course of electrical parameters, ion fluxes, and intracellular ion activities for experiments performed in airway epithelial cells. Model variables included cell [Na], [K], [Cl], volume, and membrane potentials. The model contained apical membrane Cl, Na, and K conductances, basolateral membrane K conductance, Na/K/2 Cl and Na/Cl symport, and 3 Na/2 K ATPase, and a paracellular conductance. Transporter permeabilities and ion saturabilities were determined from reported ion flux data and membrane potentials in intact canine trachea. Without additional assumptions, the model predicted accurately the measured short-circuit current (Isc), cellular conductances, voltage-divider ratios, open-circuit potentials, and the time course of cell ion composition in ion substitution experiments. The model was used to examine quantitatively: (a) the effect of transport inhibitors on Isc and membrane potentials, (b) the dual role of apical Cl and basolateral K conductance in cell secretion, (c) whether the basolateral symporter requires K, and (d) the regulation of apical Cl conductance by cAMP and Ca-dependent signaling pathways. Model predictions gave improved understanding of the interrelations among transporting systems and in many cases gave surprising predictions that were not obvious without a detailed model. The model developed here has direct application to secretory or absorptive epithelial cells in the kidney thick ascending limb, cornea, sweat duct, and intestine in normal and pathophysiological states such as cystic fibrosis and cholera.     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.     

The cellular mechanism of active chloride secretion in vertebrate epithelia: studies in intestine and trachea

The cellular mechanism of active chloride secretion, as it is manifested in the intestine and trachea, appears to possess the following elements: (1)NaCl cl-transport across the basolateral membrane; (2) Cl- accumulation in the cell above electrochemical equilibrium due to the Na+ gradient; (3) a basolateral Na+-K+ pump that maintains the Na+ gradient; (4) a hormone-regulated Cl- permeability in the apical membrane; (5) passive Na/ secretion through a paracellular route, driven by the transepithelial potential difference; and (6) an increase in basolateral membrane K+ permeability occurring in conjunction with an increase in Na+-K+ pump rate. Electrophysiological studies in canine trachea support this model. Adrenalin, a potent secretory stimulus in that tissue, increases apical membrane conductance through a selective increase in Cl- permeability. Adrenalin also appears to increase basolateral membrane K+ permeability. Whether or not adrenalin also increases paracellular Na+ permeability is unclear. Some of the testable implications of the above secretion model are discussed.     

Cyclic AMP-induced chloride permeability in the apical membrane of necturus gallbladder epithelium

The effects of theophylline, 8-Br-cAMP, and cAMP on necturus gallbladder epithelium were investigated using microelectrode techniques. Each of these substances depolarized the cell membranes by approximately 15 mV and decreased the apparent ratio of apical to basolateral membrane resistances to a value not significantly different from zero. Examination of the ionic selectivity of the apical membrane by ion substitutions in the mucosal bathing medium revealed a large increase in Cl permeability with no apparent changes in K and Na permeabilities. Intracellular Cl activity ((a)CL(i)) was measured using Cl- sensitive liquid ion-exchanger microelectrodes. Under control conditions, (a)Cl(i) was approximately 20 mM, 2.5 times higher than the value expected for equilibrium distribution ((a)Cl(i/eq). After addition of 8-Br-cAMP, (a)Cl(i) decreased within less than 60 s to approximately 13 mM, a value not significantly different from ((a)Cl(i/eq)). Virtually identical results were obtained with theophylline. Under control conditions, luminal Cl removal caused (a)Cl(i) to fall at an initial rate of 1.8 mM/min, whereas in tissues exposed to 8-Br- cAMP or theophylline a rate of 11.6 mM/min was observed. The apical membrane Cl transference number was estimated from the change of (a)Cl(i) upon exposure to 8-Br-cAMP as well as from the changes in apical membrane potential during variation of the luminal Cl concentration. The results, 0.91 and 0.88, respectively, are indicative of a high Cl permeability of the apical membrane during cAMP. This effect may explain, at least in part, the complete inhibition of fluid absorption produced by theophylline in this tissue. Moreover, enhancement of apical membrane Cl permeability may account for a variety of cAMP effects in epithelial tissues.     

Intracellular ionic activities and transmembrane electrochemical potential differences in gallbladder epithelium

Summary Intracellular ion activities inNecturus gallbladder epithelium were measured with liquid ion-exchanger microelectrodes. Mean values for K, Cl and Na activities were 87, 35 and 22mm, respectively. The intracellular activities of both K and Cl are above their respective equilibrium values, whereas the Na activity is far below. This indicates that K and Cl are transported uphill toward the cell interior, whereas Na is extruded against its electrochemical gradient. The epithelium transports NaCl from mucosa to serosa. From the data presented and the known Na and Cl conductances of the cell membranes, we conclude that neutral transport driven by the Na electrochemical potential difference can account for NaCl entry at the apical membrane. At the basolateral membrane, Na is actively transported. Because of the low Cl conductance of the membrane, only a small fraction of Cl transport can be explained by diffusion. These data suggest that Cl transport across the basolateral membrane is a coupled process which involves a neutral NaCl pump, downhill KCl transport, or a Cl-anion exchange system.     

Sodium chloride transport by rabbit gallbladder. Direct evidence for a coupled NaCl influx process

The results of the present study that NaCl transport by in vitro rabbit gallbladder must be a consequence of a neutral coupled carrier-mediated mechanism that ultimately results in the active absorption of both ions; pure electrical coupling between the movements of Na and Cl can be excluded on the grounds of electrphysiologic considerations. Studies on the unidirectional influxes of Na and Cl have localized the site of this coupled mechanism to the mucosal membranes. Studies on the intracellular ion concentrations and the intracellular electrical potential are consistent with the notion that (a) the coupled NaCl influx process results in the movement of Cl from the mucosal solution into the cell against an apparent electrochemical potential difference; (b) the energy for the uphill movement of Cl is derived from the Na gradient across the mucosal membrane which is maintained by an active Na extrusion mechanism located at the basolateral membranes; and (c) Cl exit from the cell across the basolateral membranes is directed down an electrochemical potential gradient and may be diffusional. Finally, as for the case of rabbit ileum, the coupled NaCl influx process is inhibited by elevated intracellular levels of cyclic 3',5'-adenosine monophosphate. A working model for transcellular and paracellular NaCl transport by in vitro rabbit gallbladder is proposed.     

Inactivation of a volume-sensitive basolateral potassium conductance in turtle colon: effect of metabolic inhibitors.

Previous work has shown that the basolateral membrane of turtle colon epithelium contains a quinidine-sensitive potassium conductance which can be activated by osmotic cell swelling. In this work and in the present study, potassium flow across the basolateral membrane was measured as a short-circuit current across intact pieces of epithelial tissue in which amphotericin B was used to permeabilize the apical membrane. Quinidine-sensitive currents were generated when the mucosal bath contained chloride, a permeant anion. Replacement of chloride by sulfate or addition of sucrose to the bathing solutions abolished 75-90% of the current and caused the quinidine-inhibitable fraction of the current to go from over 90% to around 6%--suggesting that decreases in cell volume had brought about inactivation of the quinidine-sensitive conductance. When metabolic inhibitors were present, inactivation of the conductance by these maneuvers was prevented. Activation of the conductance by replacement of mucosal SO4 by Cl, however, was not affected.     

A TSH/dibutyryl cAMP activated Cl-/I- channel in FRTL-5 cells.

An iodide (I) and chloride (Cl) channel has been identified in the continuously cultured FRTL-5 thyroid cell line using a cell attached patch clamp technique. The channel is activated by TSH and dibutyryladenosine cyclic monophosphate (Bt2-cAMP) but not by phorbol 12-myristate 13-acetate (TPA). Gluconate can not replace chloride or iodide and the channel is impermeable to Na+,K+ and tetraethylammonium ions. The current-voltage relationship demonstrates that the single channel current is a linear function of the clamp voltage. Single channel currents reversed at a pipette potential close to 0 mV. The mean single channel conductance was 60 pS for Cl- and 50 pS for I-. From the I-V relationship there was a strong outward rectification with Cl-, and a complete block with I-, in the single channel current above +40 mV. The feature of the channel is manifested in the single channel records by four distinct, equally spaced conductance levels. We suggest the channel is important for the transport of I and Cl ions across the apical membrane into the colloid space and is important for hormone synthesis and follicle formation.