Temperature compensation of sodium transport and ATPase activity in frog skin

Na⁺ transport across frog skin, measured as short-circuit current (SCC) shows perfect temperature compensation in frogs acclimated to 6°, 12°, and 23°C as SCC values observed at the acclimation temperatures are equal (about 13 μA/cm²). Reacclimation experiments show that this is not a starvation effect. While very little temperature compensation is seen in the activity of Na⁺, K⁺-ATPase in epidermal homogenates from frog skins, the activity of Mg²⁺-ATPase shows inverse compensation at assay temperatures from 4^o to 48^oC. This ATPase is apparently activated either by Mg²⁺ or by Ca²⁺ and it probably controls the passive permeability of epidermal cells. It is suggested that the inverse temperature compensation in the activity of this enzyme is the main mechanism by which the observed perfect temperature compensation of Na⁺ transport across frog skin occurs.     

Effect of Amiloride on sodium transport in frog skin

Summary The effect of Amiloride on several parameters of sodium transport was investigated on the isolated frog skin. Amiloride at a concentration of 10^–4 M/l decreased the sodium concentration in the sodium transport pool from 8.9 meq/kg cell water to 3.7 meq/kg cell water. No effect was observed on the intracellular sodium which is exchangeable from the corium side. Short circuit current and unidirectional sodium influx were diminished to the same extent whilst the unidirectional sodium efflux was not affected. In contrast to the short circuit current, which reaches a new steady state value within seconds, the unidirectional sodium influx reaches its new steady state with a half-time of 3.3 min. From the difference in the time courses of the decreases of short circuit current and unidirectional sodium influx, an amount of sodium could be calculated which agreed well with the directly measured fall in the sodium transport pool.The results provide further evidence for the concept that Amiloride inhibits the passive entrance of sodium into the sodium transport compartment, without influencing the transport capacity of the sodium pump.Supported by the Deutsche Forschungsgemeinschaft.     

Procaine effects on sodium and chloride transport in frog skin

Procaine, a tertiary amine, has previously been shown to stimulate reversibly transepithelial Na transport across frog skin after application from the epithelial side. In the present study with intracellular recording from principal, i.e. amiloride-sensitive cells, we demonstrate that the stimulation results from increase in apical membrane Na permeability. A second effect of procaine (10–25 mmol/l) in the outside perfusion solution is a reversible increase of transepithelial conductance which drastically exceeds the predicted response of the transcellular Na pathway. It requires presence of chloride on the epithelial side and depends on the non-ionized molecule of procaine. Abolition of apical membrane Na uptake by amiloride or Na-free mucosal inbubation decreases the magnitude but does not prevent the stimulatory effect of procaine. The origin of this gain in conductance from stimulation of a Cl-specific pathway is demonstrated by a highly significant correlation between the increases in electrically determined tissue conductance and partial Cl conductance, obtained from measurements of influx and efflux of Cl-36. Measurements with microelectrodes indicate that the stimulated Cl-specific pathway is distinct from the principal cells. Since procaine activates a conductive pathway with similar response pattern as spontaneously existing Cl conductance, it might be a valuable tool for investigating mode and way of Cl movement across epithelial tissues.     

Effect of amiloride on sodium transport of frog skin

Summary The effect of Amiloride on intracellular sodium content of the isolated frog skin was investigated. The maximal effect on intracellular exchangeable sodium concentration (–10 meq/kg cell water) was found at concentrations of Amiloride in the incubation solution between 0.3 and 1.0×10^–4 M/l, and at an incubation time of 6–15 min. Since total intracellular sodium concentration is also reduced by approximately 10 meq/kg cell water, it follows that the intracellular non-exchangeable sodium concentration was not affected by Amiloride. Water content, extracellular volume, and intracellular potassium concentration remained constant. The short circuit current reached a new steady state within a few seconds after addition of Amiloride. It is concluded that Amiloride affects a sodium transport pool, which contains about 10% of the exchangeable intracellular sodium.Supported by the Deutsche Forschungsgemeinschaft.     

EP3 receptors inhibit antidiuretic-hormone-dependent sodium transport across frog skin epithelium

 We examined the effect of prostaglandin E₂ (PGE₂) on antidiuretic hormone (ADH)-dependent Na⁺ transport and cAMP production in isolated frog skin epithelium. ADH caused an increase in transepithelial Na⁺ transport and a decrease in cellular potential, indicating an increase in apical Na⁺ permeability. Subsequent addition of PGE₂ decreased Na⁺ transport and repolarised the cells. The PGE₂ receptor EP_1/3-selective analogue sulprostone and the PGE₂ receptor EP_2/3-selective analogue misoprostol were able to mimic the effect of PGE₂. ADH increased cellular cAMP levels, whereas PGE₂, sulprostone and misoprostol were able to reduce the ADH-dependent cAMP production. Measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) revealed that it was unaffected by both PGE₂ and sulprostone. The inhibitory effect of PGE₂ on ADH-dependent Na⁺ transport was also observed in Ca²⁺-depleted epithelia. We conclude that ADH stimulates transepithelial Na⁺ transport by increasing cellular cAMP levels, whereas PGE₂ inhibits ADH-dependent Na⁺ transport by activating EP₃-type receptors, which decrease cellular cAMP levels. We have found no evidence that [Ca²⁺]_i is involved in the regulation of ADH-dependent Na⁺ transport by PGE₂. Received: 27 May 1998 / Received after revision: 18 August 1998 / Accepted: 1 September 1998     

Effects of standard diuretics and ortho-vanadate on sodium transport across isolated frog skin

Frog (Rana temporaria) skins were studied in an Ussing type lucite chamber adapted to diminish tissue edge damage. The transepithelial electrical potential difference, short circuit current and direct current (DC) resistance of skins mounted in this chamber were 56, 20 and 24% higher, respectively, than those of skins mounted in a conventional chamber. Amiloride, triamterene, ouabain and ortho-vanadate inhibited short circuit current and net mucosal to serosal flux of 22Na. Amiloride and triamterene had rapid onsets of action and were effective only when administered to the mucosal (pond) side of the skin. Ouabain and ortho-vanadate had slower onsets of action and were effective only when administered to the serosal side of the skin. Steady state of effects of these drugs was not reached within the three-hour period of the experiments. The inhibitory effect of ortho-vanadate was blocked by adding a disulfonic stilbene derivative (DIDS) to the serosal side of the skin. Serosal prostaglandin E2 stimulated the short-circuit current and decreased the DC resistance. Thiazides, acetazolamide and loop diuretics had no effects on Na+ transport by frog skin. Thus, frog skin seems to be a useful model only in studies of the mode of action and the structure-activity relationship of diuretic which act by inhibiting sodium entry or Na+-K+-ATPase activity.     

Trifluoperazine stimulated sodium transport through the apical surface of isolated frog skin

When added to the apical solution of isolated frog skin, the calmodulin antagonist trifluoperazine (TFP)* stimulated the short-circuit current (SCC) in a dose-dependent manner. The increase in SCC was due to an enhanced active transepithelial Na transport. After addition of TFP (15 microM) the intracellular voltage depolarized, showing that TFP acts by increasing the sodium (Na) permeability of the apical membrane. The TFP-induced increase in SCC was not accompanied by an increase in prostaglandin E2 release from the skins as observed by basolateral addition of TFP. When the apical Na concentration in fast-flow experiments was changed from 0 to 50 mM, the SCC increased promptly and then reclined. The presence of TFP in the apical solution abolished this recline. The apparent inhibition constant for amiloride changed in the presence of TFP from 1.42 +/- 0.12 microM to 0.38 +/- 0.05 microM (n = II) and TFP abolished the inhibition of SCC caused by high apical Na concentrations. These observations indicate that TFP acts by abolishing the Na self-inhibition of the Na channels. Calmidazolium and chlorpromazine stimulated the SCC to the same degree and in the same concentration range as TFP, suggesting that the effect of TFP was not mediated by the Ca2+-calmodulin complex.     

Chloride transport in glands of frog skin

The effects of beta-adrenergic stimulation on the bidirectional fluxes of Na+ and Cl- across the frog skin glands were determined. Isoproterenol elicited net serosal-to-mucosal fluxes of both Na+ (JNanet) and Cl- (JClnet) equal to 0.19 +/- 0.05 (SE) and 0.57 +/- 0.05 mueq X cm-2 X h-1, respectively. The residual current (JClnet - JNanet) of 0.38 +/- 0.05 mueq X cm-2 X h-1 closely approximates the isoproterenol-induced short-circuit current of 0.30 +/- 0.04 mueq X cm-2 X h-1. Furosemide added to the serosal side prior to isoproterenol inhibited the isoproterenol-induced net fluxes of both Na+ and Cl-. The addition of dibutyryl cAMP and 3-isobutyl-1-methylxanthine to the serosal side mimicked the action of isoproterenol by stimulating glandular short-circuit current. We conclude that an active Cl(-)-transport mechanism resides in the frog skin glands and is 1) stimulated by a beta-adrenergic agonist (its action is mimicked by cAMP) and 2) inhibited by the loop diuretic furosemide.