Active absorption of Na+ and Cl- across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion-flux studies

Abstract

Mechanisms of active NaCl uptake across the posterior gills of the shore crab Carcinus maenas were examined using radiochemical and electrophysiological techniques. In order to measure short-circuit current (Isc), transepithelial conductance (Gte) and area-related unidirectional fluxes of Na+ and Cl-, single split gill lamellae (epithelium plus cuticle) of hyperregulating shore crabs were mounted in a modified Ussing chamber. The negative short-circuit current measured with haemolymph-like NaCl saline on both sides of the epithelium could be inhibited by application of basolateral ouabain (ouabain inhibitor constant KOua=56±10 µmol l-1), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; KNPPB=7.5±2.5 mmol l-1) or Cs+ (10 mmol l-1). From the apical side, Isc was nearly completely blocked by Cs+ (10 mmol l-1) or Ba2+ (15 µmol l-1), whereas apical addition of furosemide (1 mmol l-1) resulted in only a small current decrease. Cl- influxes were linearly related to negative Isc. The ratio between net influxes of Cl- and Na+ was found to be approximately 2:1. With a single membrane preparation, achieved by permeabilizing the basolateral membrane with amphotericin B, Cl- influxes which were driven by a concentration gradient were shown to depend on the presence of apical Na+ and K+. On the basis of these observations, we propose that active and electrogenic absorption of NaCl across the gill epithelium of hyperregulating shore crabs proceeds as in the thick ascending limb of Henle's loop in the mammalian nephron. Accordingly, branchial NaCl transport is mediated by apical K+ channels in cooperation with apical Na+/K+/2Cl- cotransporters and by the basolateral Na+/K+-ATPase and basolateral Cl- channels.