Rheogenic transport in the renal proximal tubule

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.     

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.     

Effect of hypertonicity on the increase in basolateral conductance of Necturus small intestine in response to Na+-sugar cotransport

Exposure of Necturus small intestine to a galactose-containing perfusate that is 20% hypertonic compared to the galactose-free (control) perfusate results in a rapid depolarization of the electrical potential difference across the apical membrane, psi mc, and a decrease in the ratio of the resistance of the apical membrane to that of the basolateral membrane, (rm/rs); however, the slow repolarization of psi mc and increase in (rm/rs), observed under isotonic conditions, is blocked. These findings are consistent with the notion that the increase in the conductance of the basolateral membrane in response to Na+-coupled sugar (or amino acid) transport across the apical membrane may be a 'volume regulatory response' to cell swelling.     

Coupling of volume and Na+ transport in frog skin epithelium

Whole skins and isolated epithelia were bathed with isotonic media (congruent to 244 mOsm) containing sucrose or glucose. The serosal osmolality was intermittently reduced (congruent to 137 mOsm) by removing the nonelectrolyte. Transepithelial and intracellular electrophysiological parameters were monitored while serosal osmolality was changed. Serosal hypotonicity increased the short-circuit current (ISC) and the basolateral conductance, hyperpolarized the apical membrane (psi mc), and increased the intracellular Na+ concentration. The increases in apical conductance and apical Na+ permeability (measured from Goldman fits of the relationship between amiloride-sensitive current and psi mc) were not statistically significant. To verify that the osmotically induced changes in ISC were mediated primarily at the basolateral membrane, the basolateral membrane potential of the experimental area was clamped close to 0 mV by replacing the serosal Na+ with K+ in Cl--free media. The adjoining control area was exposed to serosal Na+. Serosal hypotonicity produced a sustained stimulation of ISC across the control, but not across the adjoining depolarized tissue area. The current results support the concept that hypotonic cell swelling increases Na+ transport across frog skin epithelium by increasing the basolateral K+ permeability, hyperpolarizing the apical membrane, and increasing the electrical driving force for apical Na+ entry.     

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes ofNecturus small intestine

Intracellular electrical potentials and K activity, (K)c, were determined simultaneously in Necturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (psi mc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of psi mc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine. In the absence of alanine, psi mc averaged -32 mV and (K)c averaged 67 mM. When a steady state was achieved in the presence of alanine these values averaged -24 mV and 50 mM, respectively. The steady-state electrochemical potential differences for K across the basolateral membrane in the absence and presence of alanine did not differ significantly. Inasmuch as the rate of transcellular active Na transport or "pump activity" was increased two- to threefold in the presence of alanine, it follows that, if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease in rs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in "pump activity". This "homocellular" regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and, (ii) repolarize psi mc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.     

Development and polarization of the Na+/H+ antiport system during reorganization of LLC-PK1A cells into an epithelial membrane

Changes in Na+/H+ antiport activity and transepithelial electrical resistance were analyzed in a clone of LLC-PK1 cells as the dispersed cells became organized into an epithelial membrane. The clone designated LLC-PK1A showed a 250% increase in Na+/H+ exchange activity as compared with the parent cell line. Na+ influx induced by an outwardly oriented H+ gradient is almost completely abolished during active cell proliferation or after cell dispersion. The activity of the Na+/H+ antiport system increases after plating the cells at high density. This increase precedes the increase in the transepithelial electrical resistance. The increase in the Na+/H+ antiport activity was not observed when the cells were plated at low density in the presence of an antimitotic agent indicating that close cell contact is an absolute requirement for the development of the system. The increase in Na+ influx correlated with an increase in Vmax, while the Km for Na+ remained essentially unchanged. Unidirectional Na+ influx measured from the apical or basolateral side as the dispersed cells became reorganized into an epithelial membrane indicated that the insertion of the Na+/H+ antiporter proteins occurred directly in the apical membrane of the epithelial cells. This finding is consistent with the hypothesis that the sorting of native proteins occurs intracellularly prior to their insertion in the apical membrane of the epithelial cells. The delay in the increase of transepithelial electrical resistance as compared with the increase in Na+ influx indicates that the settlement of the limits between the apical and basolateral membrane (fence function) precedes the closing of the intercellular space (barrier function) during the development of the occluding junctions. Further, the development of the Na+/H+ antiporter was inhibited by cycloheximide but not by actinomycin D, suggesting that the expression of epithelial cell polarization is a translational or posttranslational event.     

Comparison of the effects of dDAVP and AVP on the sodium transport in the frog skin

The effect of 1-deamino-8-D-arginine-vasopressin, dDAVP, the synthetic analogue of vasopressin, upon the active sodium transport across the frog skin was studied using standard microelectrode technique and compared with the effect of synthetic arginine-vasopressin, AVP. dDAVP applied to the basolateral side of the epithelium stimulated the active sodium transport as reflected by the increase of short-circuit current, Isc, and transepithelial electrical potential difference, Voc. Potential difference across both the apical, Vo, and the basolateral, Vi, cell membranes decreased. The driving force of transepithelial sodium transport, ENa, did not change. The transepithelial electrical resistance, Rt, ohmic resistance of the active sodium transport, RNa, and apical cell membrane resistance, Ro, rapidly decreased, while the resistance of the basolateral cell membrane, Ri, and the resistance of the shunt pathway, Rs, remained unchanged. It is concluded that dDAVP primarily increases sodium permeability of the apical cell membrane which subsequently stimulates sodium pump activity. This action is similar to that of AVP.     

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.     

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.     

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.     

Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder

Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal Cl- concentration ([Cl]m). The effects of reduction in [Cl]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, where iodide replacements increased it. When gluconate or sulfate substitutions were brought about the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a Cl- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity.     

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.     

Electrolyte transport by gallbladders of rabbit and guinea pig: Effect of amphotericin B and evidence of rheogenic Na transport

Summary Ion transport and electrical properties of rabbit and guinea pig gallbladders were investigated to gain further information about the active transport mechanism that mediates fluid absorption. The intracellular and transepithelial electrical potentials were measured simultaneously using the microelectrode technique. Exposure of the mucosal surface to Amphotericin B resulted in the prompt development of a serosa-positive electrical potential difference (PD) which could not be attributed to an alteration in ion diffusion potentials across either the cell membrane or across the tight junction. Because the Amphotericin B-inducedPD was immediately dependent on warm temperatures and O₂, and was independent of NA and K concentration gradients across the cell membrane, it is suggested that active ion transport is directly responsible for thePD. Since thePD was abolished in the absence of Na in the bathing solutions, a rheogenic Na pump is postulated; this pump also appears to be operative in tissue not exposed to Amphotericin B. The specific tissue properties altered by Amphotericin B to produce a serosa-positivePD remain incompletely defined. The results of the present study indicate that ion transport by rabbit gallbladderin vitro is a consequence of a rheogenic active Na transport mechanism at the basolateral membranes which, in conjunction with a coupled NaCl influx process at the mucosal border, ultimately results in absorption of NaCl and water.     

Potassium secretion by the cortical collecting tubule

The isolated perfused rabbit cortical collecting tubule has been shown to actively transport K from bath to lumen. The first step in this process is active uptake of K across the basolateral membrane via and Na:K exchange pump as evidenced by: 1) basolateral localization and Na:K exchange properties of the ouabain-sensitive Na,K-ATPase, 2) ouabain sensitivity of the Na and K fluxes, 3) interdependence of the Na and K fluxes, and 4) ouabain-sensitivity of 42K uptake into the cell across the basolateral membrane. At the luminal border, a significant K permeability of the apical cell membrane has been identified using electrophysiological techniques. This K permeability is insensitive to the diuretic amiloride, and, thus, differs from the pathway for Na entry, which is highly amiloride sensitive. A significant K permeability of the paracellular pathway is not apparent. It is concluded that K secretion by the rabbit cortical collecting tubule occurs via a two-step process: active uptake of K across the basolateral membrane via the Na:K exchange pump, followed by passive efflux of K across the apical membrane via an amiloride-insensitive K conductive pathway.     

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-.     

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.     

Na+ transport and impedance properties of cultured renal (A6 and 2F3) epithelia

Previous impedance analysis studies of intact epithelia have been complicated by the presence of connective tissue or smooth muscle. We now report the first application of this method to cultured epithelial monolayers. Impedance analysis was used as a nondestructive method for deducing quantitative morphometric parameters for epithelia grown from the renal cell line A6, and its subclonal cell line 2F3. The subclonal 2F3 cell line was chosen for comparison to A6 because of its inherently higher Na+ transport rate. In agreement with previous results, 2F3 epithelia showed significantly higher amiloride-sensitive short-circuit currents (Isc) than A6 epithelia (44 +/- 2 and 27 +/- 2 microA/cm2, respectively). However, transepithelial conductances (GT) were similar for the two epithelia (0.62 +/- 0.04 mS/cm2 for 2F3 and 0.57 +/- 0.04 mS/cm2 for A6) because of reciprocal differences in cellular (Gc) and paracellular (Gj) conductances. Significantly lower Gj and higher Gc values were observed for 2F3 epithelia than A6 (Gj = 0.23 +/- 0.02 and 0.33 +/- 0.04 mS/cm2 and Gc = 0.39 +/- 0.16 and 0.26 +/- 0.10 mS/cm2, respectively). Nonetheless, the cellular driving force for Na+ transport (Ec) and the amount of transcellular Na+ current under open-circuit conditions (Ic) were similar for the two epithelia. Three different morphologically-based equivalent circuit models were derived to assess epithelial impedance properties: a distributed model which takes into account the resistance of the lateral intercellular space and two models (the "dual-layer" and "access resistance" models), which corrected for impedance of small fluid-filled projections of the basal membrane into the underlying filter support. Although the data could be fitted by the distributed model, the estimated value for the ratio of apical to basolateral membrane resistances was unreasonably large. In contrast, the other models provided statistically superior fits and reasonable estimates of the membrane resistance ratio. The dual-layer model and access resistance models also provided similar estimates of apical and basolateral membrane conductances and capacitances. In addition, both models provided new information concerning the conductance and area of the basolateral protrusions. Estimates of the apical membrane conductance were significantly higher for 2F3 (0.79 +/- 0.23 mS/cm2) than A6 epithelia (0.37 +/- 0.07 mS/cm2), but no significant difference could be detected for apical membrane capacitances (1.4 +/- 0.04 and 1.2 +/- 0.1 microF/cm2 for 2F3 and A6, respectively) or basolateral membrane conductances (3.48 +/- 1.67 and 2.95 +/- 0.40 mS/cm2). The similar basolateral membrane properties for the two epithelia may be explained by their comparable transcellular Na+ currents under open-circuit conditions.     

Effects of ouabain on fluid transport and electrical properties of Necturus gallbladder. Evidence in favor of a neutral basolateral sodium transport mechanism

Net fluid transport (Jv) and electrical properties of the cell membranes and paracellular pathway of Necturus gallbladder epithelium were studied before and after the addition of ouabain (10(-4) M) to the serosal bathing medium. The glycoside inhibited Jv by 70% in 15 min and by 100% in 30 min. In contrast, the potentials across both cell membranes did not decrease significantly until 20 min of exposure to ouabain. At 30 min, the basolateral membrane potential (Vcs) fell only by ca 7 mV. If basolateral Na transport were electrogenic, with a coupling ratio (Na:K) of 3:2, the reductions of Vcs at 15 and 30 min should be 12--15 and 17--21 mV, respectively. Thus, we conclude that the mechanism of Na transport from the cells to the serosal bathing solution is not electrogenic under normal transport conditions. The slow depolarization observed in ouabain is caused by a fall of intracellular K concentration, and by a decrease in basolateral cell membrane K permeability. Prolonged exposure to ouabain results also in an increase in paracellular K selectivity, with no change of P Na/P Cl.     

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.     

Inhibition of chloride secretion by BaCl2 in the rectal gland of the spiny dogfish, Squalus acanthias

In the rectal gland of the spiny dogfish (Squalus acanthias), chloride enters the cell via a cotransport system together with sodium and potassium in a 2 Cl-: 1 Na+: 1 K+ stoichiometry. The system is energized by the electrochemical potential for sodium directed into the cell. Sodium is extruded from the cell by Na-K-ATPase located on the basolateral cell membrane. Chloride leaks into the lumen following a favorable electrical gradient. Potassium is thought to recirculate across the basolateral cell membrane. Since barium ions inhibit the efflux of potassium from cells we used barium chloride to explore the role of potassium in the process of stimulated secretion of chloride by the gland. The secretion of chloride was stimulated with theophylline 2.5 X 10(-4)M and dibutyryl cyclic AMP 5 X 10(-5)M. Ba++ inhibited the secretion of chloride in a way that was reversible and dose dependent. The reduction in secretion was associated with a parallel fall in transglandular electrical potential. Inhibition was half maximal at a concentration of Ba++ of 10(-3)M. The reduction in efflux of potassium produced by Ba++ presumably decreases the potassium diffusion potential, thus reducing the electronegativity of the cell and dissipating the driving force for chloride across the apical cell membrane. Recirculation of K+ across the basolateral border of the cell would thus be essential for the maintenance of chloride secretion by the gland.