The sensitivity of apical Na⁺ permeability in frog skin to hypertonic stress

Abstract

• 1. Na⁺ transport across abdominal skins of the frog speciesRana esculenta andRana pipiens was analyzed by recording short-circuit current (I _sc), transepithelial conductance (G _t), and the current noise generated by the random blockage of apical Na⁺ channels by the diuretic, amiloride.

• 2. Specific Na⁺ current (I _Na) and conductance (G _Na), as reflected by the amiloride-sensitive part ofI _sc andG _t, respectively, were markedly depressed after addition of some osmotically active substances, like sugars or alcohols to the mucosal Na⁺-Ringer solution. These hypertonicity-induced reactions were fast and fully reversible, even at mucusal osmolarities of 1 Osmol.

• 3. With muscosal solutions of moderate hyperosmolarity a recovery ofI _Na andG _Na was observed in presence of the osmotic gradient. This “regulatory” current showed to be carried by Na⁺ through the Na⁺-specific apical channels. Contrary to the fast current drop during the intial phase of hyperosmotic shocks, the “osmoregulation” was considerably slower. The recovery ofI _Na was only complete at smaller osmotic gradients but became more and more suppressed at higher osmolarities.

• 4. Steady-state analysis of the kinetics of the Na⁺-specific current revealed that the current depression by osmotic shocks obeys Michaelis-Menten kinetics. This current depression at high osmolaritics, as well as during the initial phase before “osmoregulation” with small osmotic gradients, can be described in terms of a non-competitive inhibition. This was also suggested by Na⁺-concentration jump experiments indicating a reduction of the maximal, apical Na⁺ permeability as mechanism of the hypertonicity-induced drop inI _Na. TheI _Na kinetics after complete “osmoregulation” were, however, indistinguishable from the isotonic control condition.

• 5. In the presence of the Na⁺-channel blocker, amiloride, the relaxation, or Lorentzian, noise component inI _Na was analyzed to yield microscopic parameters like single Na⁺-channel current (i), channel density (M), as well as the association (k ₀₁) and dissociation (k ₁₀) rate constants for the blocker reaction between amiloride and its receptor at the apical Na⁺ channel. With mucosal hypertonicity amiloride blocks less effectively, becausek ₀₁ is markedly reduced whereas changes ink ₁₀ could not be demonstrated. Furthermore, hypertonicity dramatically reducesM andi. While, after “osmoregulation” with small osmotic gradients, the reduction ink ₀₁ is still clearly observed,M andi gain their original values as if they were in isotonic condition.