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.     

Membrane transport parameters in frog corneal epithelium measured using impedance analysis techniques

Summary Active Cl^– transport in bullfrog corneal epithelium was studied using transepithelial impendance analysis methods, and direct-current (DC) measurements of membrane voltages and resistance ratios. The technique allows the estimation of the apical and basolateral membrane conductances, and the paracellular conductance, and does not rely on the use of membrane conductance-altering agents to obtain these measurements as was requisite in earlier DC equivalent-circuit analysis studies. In addition, the analysis results in estimates of the apical and basolateral membrane capacitances, and allows resolution of the paracellular conductance into properties of the tight junctions and lateral spaces. Membrane capacitances (proportional to areas) were used to estimate the specific conductances of the apical and basolateral membranes, as well as to evaluate coupling between the cell layers. We confirm results obtained from earlier studies: (1) apical membrane conductance is proportional to the rate of active Cl^– transport and is, highly Cl^– selective; (2) intracellular Cl^– activity is above electrochemical equilibrium, thereby providing a net driving force for apical membrane Cl^– exit; (3) the paracellular conductance is comparable to the transcellular conductance. We also found that: (1) the paracellular conductance is composed of the series combination of the junctional conductance and a nonnegligible lateral space resistance; (2) a small K⁺ conductance reported in the apical membrane may result from Cl^– channels possessing a finite permeability to K⁺; (3) the basolateral membrane areas is 36 times greater than the apical membrane area which is consistent with the notion of electrical coupling between the five to six cell layers of the epithelium; (4) the specific conductance of the basolateral membrane is many times lower than that of the apical membrane; (5) the net transport of Cl^– is modulated primarily by changes in the conductance of the apical membrane and not by changes in the net electrochemical gradient resulting from opposite changes in the electrical and chemical gradients; (6) the conductance of the basolateral membrane does not change with transport which implies that the net driving force for K⁺ exit increases with transport, possibly due to an increase in the intracellular K⁺ activity.     

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.     

Interrelations/cross talk between transcellular transport function and paracellular tight junctional properties in lung epithelial and endothelial barriers

In this synopsis of a symposium at EB2007, we start with an overview of noise and impedance analyses that have been applied to various epithelial barriers. Noise analysis yields specific information about ion channels and their regulation in epithelial and endothelial barriers. Impedance analysis can yield information about apical and basolateral membrane conductances and paracellular conductance of both epithelial and endothelial barriers. Using a morphologically based model, impedance analysis has been used to assess changes in apical and basolateral membrane surface areas and dimensions of the lateral intercellular space. Impedance analysis of an in vitro airway epithelial barrier under normal, nucleotide-stimulated, and cigarette smoke-exposed conditions yielded information on how activation and inhibition of secretion occur in airway epithelial cells. Similarly, impedance analysis of primary rat alveolar epithelial cell monolayer model under control and EGTA exposure conditions indicate that EGTA causes decreases in resistances of tight junctional routes as well as apical and basolateral cell membranes without causing much change in cell capacitances. In a stretch-caused injury model of alveolar epithelium, transcellular ion transport function and paracellular permeability of solute transport appear to be differentially regulated. Finally, inhibition of caveolae-mediated transcytosis in lung endothelium led to disruption of paracellular routes, increasing the physical dimension and permeability of tight junctional region. These data together demonstrate the cross talk between transcellular and paracellular transport (function and routes) of lung epithelial and endothelial barriers. Mechanistic (e.g., signaling cascades) information on such cross talk remain to be determined.     

NaCl flux between apical and basolateral side recruits claudin-1 to tight junction strands and regulates paracellular transport

In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side.     

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.     

Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport.     

Intracellular K+ activity and its relation to basolateral membrane ion transport in Necturus gallbladder epithelium

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.     

Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium.

Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.     

Membrane capacitance and conductance changes parallel mucin secretion in the human airway epithelium

Measurement of the magnitude and kinetics of exocytosis from intact epithelia has historically been difficult. Using well-differentiated cultures of human bronchial epithelial cells, we describe the use of transepithelial impedance analysis to enable the real-time quantification of mucin secretagogue-induced changes in membrane capacitance (surface area) and conductance. ATPgammaS, UTP, ionomycin, and PMA induced robust increases in total cellular capacitance that were demonstrated to be dominated by a specific increase in apical membrane surface area. The UTP-induced increase in capacitance occurred in parallel with goblet cell emptying and the secretion of mucin and was associated with decreases in apical and basolateral membrane resistances. The magnitude and kinetics of the capacitance increases were dependent on the agonist and the sidedness of the stimulation. The peak increase in capacitance induced by UTP was approximately 30 mucin granule fusions per goblet cell. Secretagogue-induced decreases in apical membrane resistance were independent of exocytosis, although each of the secretagogues induced profound reductions in basolateral membrane resistance. Transepithelial impedance analysis offers the potential to study morphological and conductance changes in cultured human bronchial epithelial cells.     

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.     

Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis

The secondary active Cl(-) secretion in seawater (SW) teleost fish gills and elasmobranch rectal gland involves basolateral Na(+),K(+)-ATPase and NKCC, apical membrane CFTR anion channels, and a paracellular Na(+)-selective conductance. In freshwater (FW) teleost gill, the mechanism of NaCl uptake is more controversial and involves apical V-type H(+)-ATPase linked to an apical Na(+) channel, apical Cl(-)-HCO-3 exchange and basolateral Na(+),K(+)-ATPase. Ca(2+) uptake (in FW and SW) is via Ca(2+) channels in the apical membrane and Ca(2+)-ATPase in the basolateral membrane. Mainly this transport occurs in mitochondria rich (MR) chloride cells, but there is a role for the pavement cells also. Future research will likely expand in two major directions, molded by methodology: first in physiological genomics of all the transporters, including their expression, trafficking, operation, and regulation at the molecular level, and second in biotelemetry to examine multivariable components in behavioral physiological ecology, thus widening the integration of physiology from the molecular to the environmental levels while deepening understanding at all levels.     

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.     

Effects of amphotericin B on the electrical properties ofNecturus gallbladder: Intracellular microelectrode studies

Summary Intracellular microelectrode techniques were employed to study the mechanism by which amphotericin B induces a transient mucosa-negative transepithelial potential (V _ms) in the gallbladder ofNecturus. When the tissue was incubated in standard Na-Ringer's solution, the antibiotic reduced the apical membrane potential by about 40 mV, and the basolateral membrane potential by about 35 mV whereas the transepithelial potential increased by about 5 mV. The electrical resistance of the apical membrane fell by 83%, and that of the basolateral membrane by 40%; the paracellular resistance remained unchanged. Circuit analysis indicated that the equivalent electromotive forces of the apical and basolateral membranes fell by 35 and 11 mV, respectively. Changes in potentials and resistances produced by ionic substitutions in the mucosal bathing medium showed that amphotericin B produces a nonselective increase in apical membrane small monovalent cation conductance (K, Na, Li). In the presence of Na-Ringer's on the mucosal side, this resulted in a reduction of the K permselectivity of the membrane, and thus in a fall of its equivalent emf. During short term exposure to amphotericin B,P _Na/P _Cl across the paracellular pathway did not change significantly, whereasP _K/P _Na doubled. These results indicate that V _ms is due to an increase of g_Na across the luminal membranes of the epithelial cells (Cremaschiet al., 1977,J. Membrane Biol. 34:55); the data do not support the alternative hypothesis (Rose & Nahrwold, 1976.J. Membrane Biol. 29:1) that V _ms results from a reduction in shuntP _Na/P _Cl acting in combination with a rheogenic basolateral Na pump.     

Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells

The vacuolar apical compartment (VAC) is an organelle found in Madin-Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo.     

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.     

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.     

Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.     

Regulation of ion conductance in frog skin by isoproterenol

The effect of isoproterenol on apical and basolateral membrane conductance in principal cells of short-circuited frog skin was analyzed using microelectrodes. Isoproterenol (10(-6) mol/l) increased the apical membrane conductance in addition to stimulating Cl- conductive pathways outside the principal cells. The effect on apical Na+ channels explains the increase in amiloride sensitive short-circuit current. Basolateral membrane conductance increased only slightly. Steady-state I/V relationships of the basolateral membrane indicate that the inward rectification of basolateral membrane K+ channels was not altered.