Potentiation by m-cresol on transepithelial sodium transport across frog skin induced by insulin

1. In this study we found that insulin mixed with m-cresol, normally used as pharmaceutical preparation, shows an earlier and larger stimulating effect on transepithelial sodium transport than insulin alone.2. The action displayed by the m-cresol seems to be specific for insulin because m-cresol mixed with ADH, a hormone known to stimulate sodium transport, failed to show the potentiation seen for insulin.3. It is proposed that m-cresol could facilitate the interaction with its receptor by modifying the insulin molecule.4. This fincling could be of biological and pharmacological importance.     

Does intracellular sodium regulate sodium transport across the mucosal surface of frog skin?

A method has been devised to functionally remove the serosal membrane of frog skin. Skins treated in this way have no spontaneous potential. However, if sodium gradients are placed across the tissues diffusion potentials and hence short-circuit currents of either sign, depending on the direction of the gradient, could be recorded. These short-circuit currents were completely imhibited by amiloride only from the mucosal face. However, the concentration of amiloride causing 50% inhibition of the short-circuit curent (Km) in treated skins was 2.3 . 10(-3)M, when a sodium gradient was applied from serosa to mucosa, whereas both in untreated skins without a sodium gradient and in treated skins with a mucosal to serosal sodium gradient, the Km of amiloride was 2 . 10(-7)-4 . 10(-7)M. The mechanism by which amiloride is able to inhibit the short-circuit currents of either sign is discussed.     

Direct measurement of uptake of sodium at the outer surface of the frog skin

A combination of the methods described by Schultz et al. (6) and by Ussing and Zerahn (9) was used to measure directly the unidirectional uptake of sodium from the outside solution into the frog skin, under short-circuit conditions. The sodium uptake was determined at six sodium concentrations ranging from 3.4 to 114 mM. NaCl was replaced by choline chloride in the solutions bathing both sides of the skin. Sodium uptake is not a linear function of sodium concentration but appears to be composed of two components, a saturating one and one that varies linearly with concentration. The sodium uptake is inhibited by the addition of lithium to the outside solution. The effect appears to be primarily on the saturating component and has the characteristics of competitive inhibition. In addition, lithium uptake by the skin is inhibited by sodium. The effects of lithium cannot be ascribed to changes in electrical potential difference. Measurements with microelectrodes indicate that under short-circuit condition there is no change in the intracellular potential when lithium chloride is added to the outside solution.     

Ouabain on active transepithelial sodium transport in frog skin: studies with microelectrodes

Studies were done with isolated frog skin to determine the effects of 10(-4) M ouabain on the electrophysiological parameters of outer and inner barriers of the Na-transporting cells. Microelectrodes were used to impale the skins from the outer surface to determine the intracellular voltages (Vsco) under conditions of short-circuiting and under conditions where a voltage clamp was used to vary the transepithelial voltage, VT. From this, the electrical resistances of outer (Rfo) and inner (RI) barriers were estimated. In addition, the driving force for active transepithelial Na transport (ENa = E'1) was estimated from the values of VT when the Vo = 0 mV (Helman and Fisher. 1977. J. Gen. Physiol. 69: 571-604). Studies were done with skins bathed with the usual 2.4 meq/liter [K]i in the inner solution as well as with reduced [K]i of 0.5 and 0 meq/liter. Characteristically, the responses to ouabain could be described by an initial rapid phase (5-10 min) during which time the Ri was increased markedly and the E'1 was decreased from control values. Thereafter, during the slow phases of the response, the resistances of both outer and inner barriers increased continuously and markedly with time leading ultimately to essentially complete inhibition of the short-circuit current. Similar studies were done with skins exposed to 10(-4) M amiloride in the outer solution. Although estimates of Ri could not be obtained under these conditions, the effects on the Vsco and E'1 were similar to those observed for the Na-transporting skins. However, the magnitudes of the effects were less and relatively slower than observed for the Na-transporting skins. The results of these studies were analyzed within the context of a proposed electrical model that takes into account the observation that the magnitude of the voltage at the inner barrier appears to exceed the equilibrium potential for K especially when transepithelial Na transport is inhibited at the apical barrier of the cells.     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Calcium modulates transepithelial potassium secretion across isolated frog skin

1. Transepithelial K+ movements across isolated frog skin consist of four components: (i) a passive component; (ii) an active inward transport of K+ which occurs via the epithelial cells; (iii + iv) two active outward-directed components, one via the skin glands, the other via the epithelial cells. 2. Incubation of frog skin in gluconate Ringer's solution activates the K+ secretion via the epithelial cells. 3. A decrease in the Ca2+ activity of the epithelial cells increases the K+ permeability of the apical membrane and reduces the K+ permeability of the basolateral membrane.     

Effect of Mercurascan on sodium transport in frog skin and bladder

The authors studied the effect of Mercurascan (MSC) (a hydroxy- mercury derivative of fluorescein) on electrical parameters, namely potential difference (P.D.) and short circuit current (S.C.C.) of frog skin and on the ability of frog bladder tissue to accumulate sodium ions in experiments in vitro. It was found that MSC, in 10(-4) mol/l concentration, reduced the S.C.C., after a brief initial increase, to 5% of the original value and that the P.D. fell steadily right from the outset. In 10(-5) mol/l concentration it raised the S.C.C. by 60% and the increase lasted several hours. The P.D. was unaffected. In 10(-7) and 10(-6) mol/l concentration MSC had no effect on the NA+ content of a nonpolarized frog bladder tissue preparation, but a 10(-5) nol/l concentration sharply reduced it. The effect of MSC on membrane Na+--K+ ATPase, i.e. on the energy metabolism of cellular tissue, is discussed with reference to these results.     

The effects of perfusion of the cutaneous vasculature on sodium uptake across isolated frog skin

Perfusion of cutaneous capillaries in isolated frog skin may remove an unstirred layer along the basolateral membrane of the epidermis that may affect the rate of cutaneous Na(+) uptake. To test this hypothesis, the cutaneous artery and vein of a bullfrog were cannulated to allow perfusion of isolated flank skin while the rate of Na(+) influx was determined. Rates of sodium influx with and without perfusion were determined in the same experiment. Na(+) uptake increased by 59+/-4.8% during the 1st 0.5 h of perfusion relative to the control, pre-perfusion period and then remained at 26+/-5.3% above control values. Concomitant with the increase in sodium uptake, the transepithelial potential difference fell by ca. 10% within the 1st 0.5 h of perfusion. The amount of labeled sodium leaving the skin in the venous effluent decreased exponentially in the 1st 0.5 h of perfusion, suggesting a wash out of an unstirred layer within the interstitial fluid. Sodium in the venous outflow accounted for ca. 25% of the sodium uptake during each perfusion period. Perfusion of the cutaneous vasculature thus has a significant effect on Na(+) transport and may potentially play a role in the acute regulation of cutaneous ion transport.     

Procaine effects on the sodium transport in frog skin

A study on the influence of procaine on the sodium transport properties in frog skin was carried out. The application of procaine hydrochloride on either the mucosal or the serosal sides of the isolated frog skin has opposite effects. When added to the mucosal compartment, the procaine (as well as two procaine based drugs: Gerovital H3 and Aslavital) biphasically increase the short-circuit current (Isc) with a noticeable "recline" phenomenon, and decrease the slope resistance, as given by the I-V curves. When applied in the serosal compartment, Isc is decreased and the slope resistance of the epithelium is increased. The procaine effect on the apical membranes shows a pronounced dependence on the external sodium concentration. The shift of the E2 inflection point (which indicates the critical intensity of the electric field at which the epithelial conductance changes), with respect to the transepithelial open-circuit potential, shows a rapid and quasi-exponential increase following the application of 25 mM procaine in addition to the different mucosal Na concentrations.     

Stimulation of the sodium transport across the frog skin by three N-terminally extended arginine-vasopressins

The standard Ussing method was used to electrophysiologically characterize the effects of three analogs of arginine-vasopressin (AVP) on the frog skin, a model Na-transporting epithelium. The analogs tested were N-terminally extended Arg8-vasopressins: Ala-AVP, Ser-Ala-AVP and Thr-Ser-Ala-AVP; synthetic Arg8-AVP was used as the reference agent. The vasopressins were applied to the basolateral side of the frog skin in concentrations ranging between 10(-8) to 10(-5) mol.l-1. All the three analogs increased both the short-circuit current (Isc) and the open-circuit transepithelial potential (Voc), and decreased the transepithelial d.c. resistance (Rt) similarly as did synthetic Arg8-AVP. The results show that N-terminal extension of the Arg8-AVP did not alter the natriferic properties of AVP.     

Effect of 5-hydroxytryptamine on sodium transport across bullfrog skin

Summary 5-hydroxytryptamine, when present in the solution bathing the inside surface of bullfrog skin at concentrations of 0.25–25.0 mM, reduced both electrical potential difference and short-circuit current across the skin. The magnitude of reduction in potential difference and short-circuit current was dependent on 5 HT concentration. Reduction in sodium influx entirely accounted for the reduction in short-circuit current. Preliminary evidence suggested a competition between 5 HT and vasopressin in the production of their effects on sodium transport across the skin, while high Ca⁺⁺ concentrations and 5 HT seemed to act independently of each other.Dr. Henry C. and Bertha H. Buswell Fellow.     

Effect of thallium ions on sodium and potassium transport in frog skin

It has been shown that Tl+ accumulated in the frog skin cells (Rana temporaria) inhibits irreversibly the unidirectional transport of Na+ estimated by the short circuit current (SCC). The inhibiting effect of Tl+ cannot be attributed to a decrease of Na+ penetration through the apical membranes. The influx of 22Na+ from mucosal bathing solution into the skin poisoned with Tl+ was about 50% of that observed in the intact skin, while the SCC was completely inhibited. The activity of the ouabain-sensitive Na+/K+ pumps located in the basolateral cell membranes was estimated by studying the uptake of 86Rb+ as a tracer for K+. This activity was high enough to maintain the ion composition of epithelial cells in spite of their ability to accomplish the undirectional transport of Na+. Tl+ seems to inhibit the production of respiration energy utilized in the undirectional Na+ transport, while the ion homeostasis of epithelial cells may be supported by the Na+/K+ pumps consuming energy of glycolytic reactions.     

Barriers to sodium movement across frog skin

Summary The aim of this paper is to obtain information on the number, nature and location of the barriers to Na movement across the frog skin, and on the size and location of the Na-pool that might be contained between these barriers. On the basis that Na penetrates passively across an outer barrier, and is actively extruded across an inner barrier which is impermeable to passive movements of Na, we expected to detect at least the Na-pool of a single cell layer containing some 10^–8 moles per cm² of epithelium (i.e., in a cell layer 5 thick and with 21mm Na). Yet no Na-pool with these characteristics was found. The method employed could have detected a Na-pool at least an order of magnitude smaller than the one expected. It is concluded that either a Na-pool does not exist (except for the Na bound to the mechanisms operating the translocation), or else that the Na-pool is contained between barriers with different characteristics than the ones assumed above. In the first case, Na transportacross the epithelium would consist of a translocation across a single asymmetrical functional barrier. In the second case, the experimental results would require that ouabain either directly (by inhibiting an active step) or indirectly (through a mediated decrease of the Na permeability of the outer barrier) prevents Na penetration at the outer border.