Effect of FeCl3 on ion transport in isolated frog skin

Summary The effect of addition of FeCl₃ to the media bathing the isolated skin ofRana pipiens was studied by measuring short-circuit current, transepithelial potential, and resistance, and by determining the influx and efflux of sodium (J ₁₃ ^Na andJ ₃₁ ^Na , respectively) and the influx and efflux of chloride (J ₁₃ ^Cl andJ ₃₁ ^Cl , respectively) across the epithelium. With normal Ringer's solution on both sides of the skin, addition of 10^–3 m FeCl₃ to the external medium resulted in nearly complete inhibition of active Na transport (J ₁₃ ^Na decreased from 1.30±0.14 to 0.10±0.04 eq/cm² hr (N=8)) and in appearance of active chloride transport in outward direction due to an 80% increase inJ ₃₁ ^Cl . Average (J ₃₁ ^Cl –J ₁₃ ^Cl ) obtained from means of 8 skins in 6 consecutive control and last 3 experimental periods was –0.17±0.04 and 0.38±0.05 eq/cm² hr, respectively. FeCl₃ added to external medium also induced substantial net chloride movement in outward direction when external medium contained Na-free choline chloride Ringer's or low ionic strength solution. Under the latter condition net Na movement was virtually eliminated by external FeCl₃. After addition of FeCl₃ to serosal medium there was delayed inhibition ofJ ₁₃ ^Na but no change in chloride fluxes. Immediate and profound changes in Na and Cl transport systems seen after external application of FeCl₃ indicate charge effects of Fe³⁺ on surface of apical cell membranes, possibly close to or in ion channels.     

Benzodiazepines stimulate sodium ion transport in frog skin epithelium

Benzodiazepine binding sites are present in a variety of non-neuronal tissues including the kidney where they are localized to distal nephron segments. It is postulated that renal binding sites are involved in modulating ion transport. This study examined the effects of two benzodiazepines on sodium transport in frog skin epithelium, a model system for sodium transport in renal collecting duct. Treatment of short-circuited frog skin with diazepam (a non-selective benzodiazepine agonist) stimulated amiloride-sensitive short-circuit current, reflecting stimulation of active sodium transport. The diazepam response was equally effective with either serosal or mucosal application of the drug. Maximal stimulation of the current (42 +/- 8%) was achieved with 10 microM diazepam (serosal). Short-circuit current was similarly augmented by serosal or mucosal addition of Ro5-4864, a benzodiazepine agonist with selective activity at peripheral (non-neuronal) receptors. The natriferic response to diazepam was additive to that of vasopressin or cyclic AMP suggesting that the mode of action of benzodiazepines is probably distinct from the cyclic AMP pathway. Thus, frog skin appears to be a useful model to examine the epithelial effects of benzodiazepines. Whether stimulation of sodium transport, however, involves peripheral-type benzodiazepine receptors in this tissue requires further studies.     

Transepithelial transport of sodium and chloride ions in isolated skin of the frog,Rana esculenta L

Isolated frog skin, mounted in a Ussing apparatus, was investigated electrophysiologically. Application of amiloride, an inhibitor of sodium ion transport, and bumetanide, known to block the transport of chloride ions, revealed the effect of these ions on PD, both under control conditions and following mechanical stimulation. Under control conditions, mechanical stimulation of the skin caused hyperpolarization, i.e. a transient increase in the electrical potential difference. Preincubation in the presence of amiloride, or amiloride plus bumetanide, brought about both a decrease in electrical potential and an inhibition of the reaction upon stimulation. On the other hand, incubation with bumetanide resulted in a decrease in electrical potential, but did not affect the skin reaction after mechanical stimulation. The above results indicate that hyperpolarization of the frog skin following mechanical stimulation is caused by enhanced transepithelial transport of sodium ions which, in turn, is induced by stimulation of sensory receptors.     

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.     

Diphenylamine-2-carboxylate stimulates sodium ion transport in frog skin epithelium

1. Diphenylamine-2-carboxylate (DPC), added to the mucosal side of the frog skin, increased reversibly the short-circuit current (I0), even in SO2-(4) Ringer. Amiloride blocked this effect. 2. The maximal stimulation was 140% of the control value and the EC50 was 0.26 mM DPC. 3. The stimulatory effect of DPC was additive to that of oxytocin. 4. The dose-response curves for amiloride determined in the absence and in the presence of 1 mM DPC showed an IC50 of 1.0 microM and 0.8 microM amiloride, respectively. 5. Thus DPC, a blocker of Cl- channels in various Cl-transporting epithelia, exerts a stimulatory effect on the amiloride-sensitive Na+ transport in frog skin.     

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.     

Effect of acetazolamide on sodium transport through the isolated frog skin at low and high external sodium concentrations.

The action of acetazolamine on sodium transport in $\text{Rana esculenta}$ skin was studied with the external face bathed in dilute (2mMM) or concentrated (Ringer) solutions of sodium chloride.The absorption of Na⁺ from a dilute solution is inhibited at an acetazolamide concentration of 10⁻⁵M. This is due to an inhibition of the influx: the efflux remains unchanged. Acetazolamide has no effect, however, on transport from Ringer solution.The graphic determination of the Na⁺ transport pool at the 2 mM NaCl concentration showed that acetazolamide diminished the pool without affecting the $\text{t}\text{1}\text{2}$. The inhibitor had no effect on the pool at the higher (Ringer) concentration.These results indicate that acetazolamide acts on the external barrier of the sodium transport compartment without affecting the active pump of this ion when it is being transported from a dilute sodium chloride solution.     

Reversed potentials in isolated frog skin. II. Active transport of chloride

Net inward transport of Cl in the absence of an electrochemical potential difference was demonstrated in the skin of two species of frog, R. pipiens and R. esculenta under conditions of low (2 mM) Cl concentration in the bathing solutions. The electrical potential profile of skins of R. pipiens was examined with microelectrodes under conditions in which the inside solution was negative relative to the outside solution. This reversal of the normal potential difference was found to arise as a result of potential changes across the outward facing electrical barriers in the skin. The reversed potential difference appears to arise, at least in part, as a result of the inward Cl transport. The effect may be due either to electrogenic Cl transfer or to variations in internal composition of the epithelial cells arising as a result of Cl transport.     

Effect of thallium on enzyme activity and unidirectional sodium transport in frog skin

The cytophotometric analysis of enzyme activity in the frog skin epithelium has shown that Tl+ accumulated by cells at millimolar concentrations causes a 70-80% inhibition of both succinate and alpha-ketoglutarate dehydrogenases, while the activity of Na, K-ATPase decreased only slightly. The opposite situation was true for the ouabain treatment. The accumulation of Tl+ by frog skin caused a substantial swelling of mitochondria. It is suggested that the earlier observed inhibition of the unidirectional Na+ transport by Tl+ might be resulted from a blocking of oxidative metabolism. The same cells poisoned by Tl+ were able to maintain their ion composition presumably at the expense of glycolysis.     

Effect of sodium on amiloride- and triamterene-induced current fluctuations in isolated frog skin

The apparent association constants of two agents, amiloride and triamterene, that block the Na-selective channel of apical membrane of frog skin are shown to decrease as the Na concentration is increased in the apical bathing solution in isolated skin of the frog, Rana temporaria, Rana esculenta, and Rana pipiens. These results were obtained in "normally polarized" skins. These effects were independent of the anion used (chloride or methylsulfate) or the cation used as the Na substitute (Tris, DDA, or K ion). When NaCl was replaced with mannitol, the Na effect on the amiloride association rate constant persisted, which shows that ionic strength was not critically involved. The amiloride corner frequency was unaffected when the clamp potential was altered from +100 to -60 mV. The Na dependence was greatly attenuated or absent when the serosal surface was bathed in 120 mM K Ringer's, an effect that appears to be attributable to some pharmacological effect of high serosal K. A previously described three-state model is used to analyze the inhibitory effect of Na on the blocker association rate constant.