Exchange diffusion,electrodiffusion and rectification in the chloride transport pathway of frog skin

Measurements of chloride flux ratios across frog skin at different clamping voltages showed that chloride transport at clamping voltages from 0 mV to and beyond the spontaneous potential is probably electrodiffusion. At reversed potentials a significant fraction of chloride transport could be described formally as exchange diffusion. Chloride conductance was found to be highly voltage dependent, being largest at hyperpolarizing clamping voltages. The transition from the less conducting state to the more conducting one was studied by recording the time course of the current after a step change in clamping voltage from 0 mV to hyperpolarizing voltages. The shape of the curve is sigmoidal, and the relative rate of change of current increases with increasing hyperpolarization. It is proposed that the change in conductance is governed by the same mechanism as in the toad skin, namely a change in chloride permeability due to voltage gating of chloride channels. The time course of transepithelial conductance after addition of amiloride to the outside solution indicates that a fraction of the decrease in conductance is due to closure of chloride channels caused by the change in intracellular potential due to the inhibition of the sodium channels.     

Electrical parameters of the toad skin: effects of forskolin.

Forskolin stimulated short-circuit current (SCC) and transepitelial electrical conductance (G) in the isolated skin of the toad Bufo arenarum in a concentration-dependent manner, between 1.0 x 10(-6) and 2.4 x 10(-5) M. At the latter concentration, glandular secretion appeared to be stimulated also. The increase in G was considerably greater in skins bathed in Ringer solution than in solutions containing no chloride. The increased SCC was abolished by amiloride, a specific blocker of sodium transport in amphibian membranes, irrespective of the anion present in the solution bathing the skin. G was also decreased by amiloride to control values in skins bathed in solutions without chloride, but remained elevated in the presence of Cl-. The increase in SCC following exposure to forskolin, 4.4 x 10(-6) M, was not altered when furosemide, a specific blocker of chloride transport, was present in the Ringer solution bathing the dermal side of the skin. The response to forskolin, 2.4 x 10(-5) M, however, was significantly decreased by dermal furosemide; the inhibitor was ineffective in the absence of chloride. The data indicate that forskolin acts on at least two sites: stratum granulosum cells (the main pathway for sodium transport, and an alternate site, responsible for the increase in permeability to chloride. In addition, at high concentration of the agent, glandular secretion is also stimulated. The data suggest that the adenylate cyclase-cyclic AMP system is involved in the regulation of the permeability of the toad skin to sodium and chloride, probably by separate cell types.     

Interactive effects of ethanol and silver on sodium transport across toad skin

Both ethanol and silver ions have been shown to affect ion transport across various epithelia. This investigation was principally undertaken to further define mechanisms of silver ions and ethanol, and their possible interactions, on sodium transport across toad skin. Isolated toad skin, mounted between identical oxygenated amphibian bicarbonate Ringer solutions, maintained stable transepithelial potential differences (serosa positive) and short-circuit currents for several hours at 25 degrees C. It was observed that (1) ethanol inhibited the active transcellular component of sodium absorption and this effect was reversible; (2) inhibition of sodium transport by ethanol was directly proportional to the applied concentration; (3) pretreatment with silver ions prevented any ethanol effects; and (4) pretreatment with ethanol prevented any silver ion effects. It was concluded from these results that ethanol induced its inhibitory effects on membrane phospholipids thereby perturbing the function of a sulfhydryl ligand, while silver ion or silver chloride complex binding to this ligand would maintain its function in sodium transport despite the presence of ethanol.     

Differences in c-AMP levels in epithelial cells from pelvic and pectoral regions of the toad skin

Arginine vasopressin (AVP) stimulated active Na+ transport (JNa+) and osmotic water flow (JH2O) across the pelvic skin but only JNa+ across the pectoral skin of the toad, Bufo woodhouseii. Isolated epithelial cells from the pelvic skin had a maximal c-AMP level of 11.16 pmoles/mg protein after 5 min of AVP treatment while that of pectoral skin was 3.64 pmoles/mg protein. The c-AMP level of both skin areas fell to unstimulated values after 20 min of AVP treatment; however, JH2O (pelvic skin) and JNa+ (pelvic and pectoral skin) remained elevated during 3 hr of treatment. Dibutyryl c-AMP and theophylline stimulated JH2O across the pelvic but not the pectoral skin. Maintaining toads in water for 12-24 hr resulted in a substantial lowering of JH2O across the pectoral skin which was not reversible by treatment with c-AMP and theophylline.     

Regulation of protein phosphorylation and sodium transport in toad bladder

It is well established that active sodium-ion transport and water flow across isolated toad bladder are increased by antidiuretic hormone (ADH) and by cAMP. These agents were also observed in previous studies to cause changes in the amount of radioactive phosphate in a specific protein in the toad bladder. This protein, found by SDS-polyacrylamide gel electrophoresis of toad bladder epithelial preparations, had an apparent molecular weight of 49,000 daltons. In the present study, a correlation was found between the ability of a variety of substances to affect the amount of radioactive phosphate in this 40,000-dalton protein and their ability to alter the rate of sodium transport. Thus several agents (ADH, cAMP, theophylline, adenine, prostaglandin E1, and Mn Cl-2) caused a decrease in the amount of radioactive phosphate in the 49,000-dalton protein and also stimulated active sodium transport across the bladder. Conversely, ZnCl-2 produced an increase in the amount of radioactive phosphate in this protein and an inhibition of sodium transport. With each of these agents, the time-course of change in phosphorylation of this protein was, in general, similar to that for sodium transport. A second phosphoprotein, with an apparent molecular weight of about 42,000 daltons, showed changes in parallel with, but less extensive than, those observed in the 49,000 dalton protein. There was no consistent relationship between changes in level of phosphorylation of either in the 49,000- or 42,000- dalton protein and changes in osmotic water permeability. The results are compatible with the possibility that regulation by ADH and by cAMP of sodium transport in the toad bladder epithelium may be mediated through regulation of the amount of phosphate in a specific protein.     

Fine structural changes associated with the onset of calcium,sodium and water transport by the chick chorioallantoic membrane

Summary An attempt is made to correlate structure and transport function in the embryonic chorioallantoic membrane. The fine structure of the endoderm and ectoderm in the membrane was examined with particular attention given to the morphological changes that occur when transport is established,in vivo. Two distinctive cells, a granule-rich cell and a mitochondria-rich cell, appear in the endoderm at the time allantoic fluid sodium, chloride and water reabsorption commences. These are indistinguishable from the cells described in toad bladder epithelium. It is suggested that the granule-rich cell is responsible for bulk water movement and the mitochondria-rich cell is specifically engaged in active sodium transport. In the ectoderm, two distinctive cell types are also found to be associated with the onset of active calcium transport. These are referred to as the capillary-covering cell and the villus-cavity cell. The preponderate capillary-covering cell is most likely responsible for transcellular calcium transport. It is postulated that the function of the villus-cavity cell is to secrete hydrogen ions which are necessary, along with carbonic anhydrase, to mobilize Ca⁺⁺ from the insoluble calcium carbonate of the eggshell.     

Variation in the effects of antidiuretic hormone on the isolated skin of the toad, Scaphiopus couchi.

The rate of active sodium transport as measured by short-circuit current across the isolated skin of the toad, Scaphiopus couchi, was elevated following vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml) treatment of skins from active animals at all times of the year tested. Skins from dormant animals showed no such elevation at any time of the year. The rate of active sodium transport was elevated following treatment with dibutyryl cyclic AMP (2.5mM) plus theophylline (10 mM) in all skins tested. The hydraulic conductivity of isolated skins from both active and dormant animals showed no significant change following treatment with vasopressin (0.2 units/ml) or arginine vasotocin (0.1 units/ml except on the first day following emergence from dormancy in the field. A correlation was, therefore, observed between the occurrence of a hydroosmotic response to antidiuretic hormones and the seasonal exposure of S. couchi to standing water. A small but significant elevation of hydraulic conductivity was observed across the skins of dormant toads following treatment with dibutyryl cyclic AMP (2.5 mM) plus theophylline (10 mM) whereas a substantial elevation was observed with the skins of active animals.     

Chloride conductance in amphibian skin: regulatory control in the skin of Rana pipiens

Chloride conductance across the isolated skin of Rana pipiens shows a voltage-activated component (G(Cl)(V)) which requires the presence of mucosal Cl. G(Cl)(V) is normally low or dormant. It is stimulated by elevated intracellular cAMP, irrespective whether originating from application of ss-adrenergic agonists (isoproterenol), stimulators of the adenylyl-cyclase (forskolin), inhibitors of the phosphodiesterases (isobutyl-methyl-xanthine) or membrane-permeable cAMP analogues (CPT-cAMP). Baseline G(Cl) under inactivating conditions increases also with cAMP dose-dependently. The data indicate that cAMP is a central regulator of the passive, conductive chloride transport across amphibian skin.     

Comparative effects of catecholamines, angiotensin II and antidiuretic hormone on chloride transport in toad skin

In this work we present data which show stimulation of Cl- transport in the isolated toad skin by four agonists: L-isoproterenol, L-adrenalin, angiotensin II and ADH. This response was demonstrated by raising mucosal amiloride concentration to block the sodium transport in the skin. With transepithelial sodium influx almost completely inhibited, it was likely that the response reflected transport events in the glands. Inhibition of the bioelectric parameters by removing chloride from the serosal bathing medium in the amiloride-inhibited preparation eliminated the response to all four agents, indicating that these responses are chloride dependent. The similarity of the bioelectric responses of the amiloride-treated preparation to db cAMP and to the four agents tested in this work add further evidence that this second messenger may account largely for the Cl- transport mechanism in the toad skin glands by increasing the apical membrane permeability to Cl-.     

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.     

Stimulation by aldosterone of a conductive chloride pathway in toad skin

As a rule, chloride movement (JC1-) across amphibian skin is considered to be passive; this is implied in fact for preparations incubated in Ringer's fluid, since short-circuit current (Isc) is the quantitative expression of net, active sodium transport (JNa+). The nature of the Cl- pathway(s) was investigated by incubating amphibian skin (mostly Bufo marinus) with Cl- present on the epithelial side only, and after blocking JNa+ by combined treatment with ouabain and amiloride. In such conditions, JCl- was found to be equal to (reversed) Isc; furthermore, when JCl- was "translated" in terms of conductance, gCl-, the latter accounted almost quantitatively for transepithelial conductance, g1. When residual intratissue (i.e. intracellular) electronegativity was eliminated by replacing Na+ with K+, JCl- was larger but Isc and JCl- were still found to reflect each other, and gCl- again accounted for most, if not all, of g1. JCl- in the opposite direction, as a result of Cl- being present only on the dermal side, was negligible, and g1 was very low. Thus, in the absence of sodium transport, when experimental conditions are such that a net inward JCl- obtains, the anion apparently flows only through (a) conductive pathway(s). Aldosterone is probably involved in the regulation of this pathway, as JCl- was much lower when toads were maintained in dilute saline than in water or on moist peat; so was the fraction of the apical surface corresponding to mitochondria-rich cells.     

Mechanisms for the effects of acetylcholine on sodium transport in frog skin

Summary In frog skin (Rana temporaria) acetylcholine applied to the serosal surface produces either a sustained inhibition or sustained stimulation of short-circuit current (SCC). The former effect is accompanied by a reduction and the latter by an increase in total tissue conductance. Both effects of acetylcholine can be accounted for, within experimental error, by changes in net sodium flux across the tissue. By use of selective agonists and antagonists it is concluded that acetylcholine interacts with muscarinic receptors in the serosal membrane. The effects of cholinoceptor agents are also seen with isolated epithelium.The stimulatory effect of acetylcholine is potentiated by theophylline and blocked by inhibitors of prostaglandin synthetase and by mepacrine. It is suggested that acetylcholine stimulates transport by liberating prostaglandins which may then activate adenylcyclase. The inhibitory effect of acetylcholine is correlated with a reduction in cyclic AMP content of the epithelium. Calcium appears to be an important determinant of the type of response seen with acetylcholine, but the mechanism is not known.     

The effect of tanning agents on the permeability of the toad bladder to water and solutes

Summary Previous studies with phloretin have shown that the movement of urea and other solutes across the toad bladder can be inhitited with no effect on osmotic water flow, active sodium transport, or the movement of ethanol and ethylene glycol. These findings have suggested that a vasopressin-sensitive carrier is involved in the transport of solutes such as urea across the luminal membrane of the epithelial cell. The present paper describes the effect of two agents other than phloretin: tannic acid and chromate, on water and solute movement across the bladder. The pattern of action of these two agents resembles that of phloretin, and supports our earlier findings of the independence of solute and water movement. The effect of chromate on urea movement is seen only in the presence of vasopressin, and only if chromate is added prior to vasopressin. Chromate also proves to be an irreversible inhibitor of urea movement. The implications of these findings are discussed. In view of the known interactions of both agents with proteins, it is suggested that carrier-mediated transport of urea proceeds across a protein component of the membrane.Presented in part at the 57th annual meeting, Federation of American Societies for Experimental Biology, Atlantic City, April 1973.     

Cl(-)-ATPases: biological active transporters

Five widely documented mechanisms of chloride transport across plasma membranes are: anion-coupled antiport; sodium and hydrogen-coupled symport; Cl- channels; and an electrochemical coupling process. No genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl(-)-stimulated ATPases co-existing, in the same tissue, with uphill chloride transport that could not be accounted for by the five common chloride transport processes. Cl(-)-stimulated ATPase activity is a common property of practically all biological cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl(-)-stimulated ATPase activity. Recent studies of Cl(-)-stimulated ATPase activity and active chloride transport in the same membrane system, including liposomes, suggest a mediation by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl(-)-stimulated ATPases.     

Conductive chloride flux across amphibian skin: inhibition by indacrinone and cobalt ion.

When amphibian skin was incubated under conditions in which transepithelial sodium transport was abolished, a conductive transepithelial Cl- flux arose when Cl- was removed from one of the compartments. This flux was matched by short-circuit current and it accounted entirely for transepithelial conductance. Cl- influx was larger than efflux; it was linearly related to the magnitude of transepithelial Cl- concentration difference. When applied to the epithelial surface of the tissue, divalent metal cations such as Co2+, and the ethacrynic acid derivative, indacrinone, reduced rapidly and reversibly both transepithelial Cl- (in)flux and short-circuit current. Frog skin proved to be more sensitive to these inhibitors than toad skin. Further characterization of transepithelial Cl- pathway(s) should benefit from the fact that Cl- across amphibian skin can easily be monitored by the short-circuit current method, and from the availability of agents which inhibit this passive flux rapidly and reversibly.     

NaCl adaptation in Rana ridibunda and a comparison with the euryhaline toad Bufo viridis.

The physiological adaptation of the frog Rana ridibunda to saline environment was studied. It was found that blood was always hypertonic to the external solution, but at the highest salinity tolerated (i.e. 300 mOsM) the osmotic gradient across the skin was nearly abolished. Water uptake by the living frog remained unchanged, whereas sodium transport across the skin decreased markedly. Neurohypophyseal hormone increased water uptake and sodium transport to levels similar to those in tap water frogs. Water content of the tissues was not affected by saline adaptation, although it varied appreciably under acute conditions. Oxygen consumption increased in dehydrated frogs, but not in adapted ones. The results are discussed and compared to the euryhaline toad Bufo viridis; the importance of high urea levels for high salt adaptation is stressed.     

Regulation of the sodium permeability of the luminal border of toad bladder by intracellular sodium and calcium: role of sodium-calcium exchange in the basolateral membrane

Sodium movement across the luminal membrane of the toad bladder is the rate-limiting step for active transepithelial transport. Recent studies suggest that changes in intracellular sodium regulate the Na permeability of the luminal border, either directly or indirectly via increases in cell calcium induced by the high intracellular sodium. To test these proposals, we measured Na movement across the luminal membrane (th Na influx) and found that it is reduced when intracellular Na is increased by ouabain or by removal of external potassium. Removal of serosal sodium also reduced the influx, suggesting that the Na gradient across the serosal border rather than the cell Na concentration is the critical factor. Because in tissues such as muscle and nerve a steep transmembrane sodium gradient is necessary to maintain low cytosolic calcium, it is possible that a reduction in the sodium gradient in the toad bladder reduces luminal permeability by increasing the cell calcium activity. We found that the inhibition of the influx by ouabain or low serosal Na was prevented, in part, by removal of serosal calcium. To test for the existence of a sodium- calcium exchanger, we studied calcium transport in isolated basolateral membrane vesicles and found that calcium uptake was proportional to the outward directed sodium gradient. Uptake was not the result of a sodium diffusion potential. Calcium efflux from preloaded vesicles was accelerated by an inward directed sodium gradient. Preliminary kinetic analysis showed that the sodium gradient changes the Vmax but not the Km of calcium transport. These results suggest that the effect of intracellular sodium on the luminal sodium permeability is due to changes in intracellular calcium.     

Trypsin inhibits voltage-activated chloride conductance of toad skin

The effect of trypsin on the voltage-activated chloride conductance (GCl) of toad skin was investigated. Serosal application of > 0.1 mg ml-1 trypsin decreased the voltage-activated GCl without notable delay. The maximal inhibition to 38% of the control values, exerted within 15 min, was in some experiments partly or completely reversible. Chymotrypsin had much lower effect than trypsin. Mucosal application of trypsin did not have any effect. Trypsin did neither interfere with the conductive pathway opened by supramaximal concentrations of cAMP nor with the inhibitory effect of epinephrine on the voltage-activated GCl. The effect of trypsin required influx of Ca2+ from the extracellular space. It is concluded that protease-activated receptors or trypsin-sensitive proteins in the basolateral membrane of toad skin epithelial cells interfere with regulative steps involved in the voltage-activation of GCl. This may be harmful for the segregation of epithelial cells using this enzyme.     

Cl(-)-ATPases: Novel primary active transporters in biology

Five widely documented mechanisms of chloride transport across plasma membranes are anion-coupled antiport, sodium and hydrogen-coupled symport, Cl(-)channels, and an electrochemical coupling process. No genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl(-)-stimulated ATPases co-existing, in the same tissue, with uphill chloride transport that could not be accounted for by the five common chloride transport processes. Cl(-)-stimulated ATPase activity is a common property of practically all biological cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl(-)-stimulated ATPase activity. Recent studies of Cl(-)-stimulated ATPase activity and active chloride transport in the same membrane system, including liposomes, suggest a medication by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl(-)-stimulated ATPases. J. Exp. Zool. 289:215-223, 2001.     

The significance of changes in thermodynamic affinity induced by aldosterone in sodium-transporting epithelia

Summary The energetics of sodium transport were examined in toad (and occasionally frog) skin, with particular emphasis on the effect of aldosterone.Thermodynamic affinity was computed according to Essig and Caplan. Following treatment with antidiuretic hormone or drugs believed to affect only the apical membrane barrier, no change in thermodynamic affinity was observed either acutely (after one to two hours) or chronically (after 18-odd hours).By contrast, following treatment with aldosterone overnight, thermodynamic affinity was considerably increased, whether or not incubation was conducted in the presence of sodium in the outer solution; addition of glucose at the end of incubation, whereby sodium transport was stimulated further, failed to influence affinity as measured. The stoichiometry between sodium transport and oxygen consumption was, however, unchanged by aldosterone treatment in short-circuit conditions, neither was that fraction of aerobic metabolism unrelated to sodium transport influenced.It is concluded that the change observed with aldosterone can be directly ascribed to the hormone, as it is independent of glucose availability and of sodium transport. Aldosterone action, at least following prolonged incubation, therefore does not involve only an increase in apical conductance for sodium.