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.     

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.     

Inhibitory and stimulatory effects of amiloride analogues on sodium transport in frog skin

Summary Effects of amiloride analogues on Na transport were studied in isolated skins of the frogRana ridibunda. The pattern of structure-activity relationship of these compounds showed that both the –NH₂ group at position 5 and Cl at position 6 of the pyrazine ring of the amiloride molecule were important for their biological activity. The paramount role of the groups at position 5 was further demonstrated by the striking properties of an analogue resulting from dimethylation of that –NH₂ group. A stimulation of Na transport, opposite to the effect of amiloride itself, was observed in this instance. The increase in Na transport could already be seen at 10^–6 m and was equivalent to the measured increase in Na influx, reversible, dose-dependent, and additive to the natriferic action of oxytocin. Such characteristics resemble those reported with external agents like propranolol and La³⁺. Furthermore, mutual inhibition was observed between the stimulatory effects of this analogue and those of propranolol or La³⁺. These results suggest that the analogue may be considered as another external agent acting at sites of the external membrane distinct from those activated by cAMP but similar to the Ca sites described by Herrera and Curran (Herrera, F.C., Curran, P.F. 1963.J. Gen. Physiol. 46:999).     

Potassium dependence of sodium transport in frog skin

22Na+ and 42K+ fluxes across the basolateral membrane of the isolated epithelium of frog skin were investigated with regard to dependence on K+ in the basolateral solution. When K+ was removed from the basolateral solution (K+-free Ringer), there was a transient rise in short circuit current (Isc) that could be eliminated by pretreatment with ouabain. Concurrently, the apparent sodium efflux across the basolateral membrane (JNa*13) showed either no change or an immediate (1-2 min) small decrease (approximately equal to 10%) that was followed by a small transient increase. K+ fluxes showed either no change or a small decrease under these conditions. JNa*13 was partially ouabain sensitive during all of the above treatments. Furosemide partially inhibited both sodium and potassium flux after K+-free treatment. The pump, as defined by ouabain sensitivity of Na+ flux, continued to work even after 20 minutes of K+-free treatment. Pump activity may be maintained by potassium leaking from the cells that is recycled by the pump. However, the ouabain-sensitive transient rise in Isc after K+-free treatment cannot readily be explained by changes in either Na+ or K+ flux. A change in pump coupling ratio provides one explanation for these data.     

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.     

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.     

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.     

Calcium role in base-line and ADH-stimulated sodium transport in frog skin

Treatment of ventral frog skin with serosal A23187 calcium ionophore caused an initially transient increase in transepithelial sodium transport. After 60 min of treatment with A23187, a steady-state transport value was reached which was significantly lower than the initial one. Furthermore, it was found that ionophore treatment greatly inhibited the natriferic response to ADH and to 8br-cAMP. A further analysis on the possible ionophore action mechanism was carried out through pretreatment of the skin with indomethacin, very powerful prostaglandin synthesis inhibitor. In the experimental conditions reported, A23187 seems no longer capable of inducing a transient increase in sodium transport, although it does inhibit the natriferic response to ADH.     

Effect of hibernation on sodium and chloride ion transport in isolated frog skin

The aim of the study was to evaluate the effect of hibernation on electrophysiological parameters of isolated frog skin under control incubation (Ringer solution) and after inhibition of Na+ and CI- transepithelial transport by application of amiloride and bumetanide. The transepithelial electrical potential difference (PD in mV) was measured before and after mechanical stimulation of isolated frog skin. The tissues were mounted in a modified Ussing chamber. The results revealed a reduced PD of frog skin during hibernation. In February, as compared with November, PD of frog skin incubated in Ringer solution decreased by about 50%. Hibernation also affected hyperpolarization (dPD) of frog skin after mechanical stimulation. In November and December, dPD was about 50% and 30% lower, respectively, compared with the subsequent two months of the experiment. The incubation of frog skin with amiloride, a sodium ion channel blocker, resulted in reduced values of all measured electrophysiological parameters irrespective of the phase of hibernation. After application of chloride ion transport inhibitor (bumetanide), the PD in November and December decreased compared with the control incubation by about 80% and 75%, while in January and February by about 40% and 25%, respectively. In January and February dPD increased by four times and three times as compared with November and December. Hibernation reduces net ion flow in isolated frog skin. During the initial period of hibernation the sensitivity of the skin to mechanical stimulation also decreases. Towards the end of hibernation, on the other hand, excitation of mechanosensitive ion channels takes place.     

The effect of aldosterone on the energetics of sodium transport in the frog skin

Uncertainty persists as to whether the stimulation of active sodium transport by aldosterone is attributable to effects on permeability or energetic factors. This question has been examined with the aid of a thermodynamic formulation in which the rate of both active sodium transport J_Na and O₂ consumption J_r are assumed to be linear functions of the electrical potential difference Δψ and the affinity A (negative free energy) of metabolic reaction. Previous studies have indicated constancy of a characteristic affinity on perturbation of Δψ, suggesting the possibility of its evaluation. In studies of paired frog skins the admnistration of aldosterone led to a significant increase in the short-circuit current I₀, a suggestive increase in the associated rate of O₂ consumption J_ro, and a significant increase in the ratio

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. If linearity obtains this ratio is equal to A. Depression of active sodium transport and the associated metabolism with amiloride, which depresses permeability, also results in an increase in the apparent affinity

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. The results indicate that aldosterone does not act simply by increasing the permeability or the number of transport units operating in parallel, but suggests that energetic factors are implicated as well.     

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.