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

Split gill lamellae and gill cuticles of shore crabs (Carcinus maenas) adapted to 10% 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 muA 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 mumol cm(-2) h(-1), respectively. External amiloride (100 mumol 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 mumol 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 25188 +/- 3791 muA 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 mumol 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.