Ion transport across posterior gills of hyperosmoregulating shore crabs (Carcinus maenas): amiloride blocks the cuticular Na(+) conductance and induces current-noise.

Abstract

Split gill lamellae and gill cuticles of shore crabs (Carcinus maenas) adapted to 10 per thousand salinity were mounted in a modified Ussing-type chamber. With NaCl saline on both sides, split gill lamellae generated a short-circuit current (I(sc)) of -301+/-16 microA cm(-2) at a conductance (G(te)) of 40+/-2 mS cm(-2). The net influxes of Na(+) and Cl(-) were 8.3+/-2.6 and 18.2+/-2.7 micromol cm(-2) h(-1), respectively. External amiloride (100 micromol l(-1)) reduced G(te) to approximately 50 % of the original value at unchanged I(sc); Cl(-) fluxes remained unaffected, whereas Na(+) fluxes were markedly reduced by 70-80 %. The I(sc) in the presence of external amiloride was almost completely inhibited by internal ouabain. At a clamp voltage of 50 mV (outside-positive), a positive current was measured at unchanged G(te). Under these conditions, amiloride reduced the current and conductance at half-maximal concentrations of 3.6 and 2.0 micromol l(-1), respectively. At outside-positive voltages, but not under short-circuit conditions, external amiloride induced Lorentzian components in the power density spectra. The amiloride-dependent changes in the corner frequency (linear) and of the low-frequency plateau ('bell-shaped') were as expected for channel blockade by amiloride with pseudo-first-order kinetics. With an outside-positive clamp voltage of 50 mV across isolated cuticles, a positive cuticular current (I(cut)) of 25 188+/-3791 microA cm(-2) and a cuticular conductance (G(cut)) of 547+/-76 mS cm(-2) were measured. External amiloride reduced I(cut) and G(cut) at half-maximal concentrations of 0.7 and 0.6 micromol l(-1), respectively. Amiloride-induced current-noise analysis gave similar results to those observed with split gill lamellae. Ion-substitution experiments with isolated cuticles further support inhibition by external amiloride of the cuticular Na(+) conductance of shore crab gills and not amiloride-sensitive transporters (Na(+) channels or Na(+)/H(+) antiports) in the apical membrane.