Na+-independent, electrogenic Cl- uptake across the posterior gills of the Chinese crab (Eriocheir sinensis): Voltage-clamp and microelectrode studies

Abstract

Single gill lamellae from posterior gills of Chinese crabs (Eriocheir sinensis) were isolated, separated into halves and mounted in a modified Ussing chamber. Area-related short-circuit current (Isc) and conductance (Gtot) of this preparation were measured. Epithelial cells were impaled with microelectrodes through the basolateral membrane and cellular potentials (Vi under open- and Vsc under short-circuit conditions) as well as the voltage divider ratios (Fi, Fo) were determined. With NaCl salines on both sides an outside positive PDte (22±2 mV) and an Isc (-64±13 μA·cm-2) with a polarity corresponding to an uptake of negative charges (“inward negative”) were obtained. “Trough”-like potential profiles were recorded across the preparation under open- as well as short-circuit conditions (Vo=-101±5 mV, external bath as reference; Vi=-78±2 mV, internal bath as reference; Vsc=-80±2 mV, extracellular space as reference). The voltage divider ratios of the external (apical membrane plus cuticle) and internal (basolateral membrane) barrier were Fo=0.92±0.01 and Fi=0.08±0.01, respectively. To investigate a Cl--related contribution to the above parameters, Na+-free solutions in the external bath (basolateral NaCl-saline) were used. “Inward negative” Isc under these conditions almost completely depended on external Cl-. Elimination of Cl- in the external bath reversed Isc, and Gtot decreased substantially. Concomitantly, Vsc depolarised and Fo increased. Cl--dependent current and conductance showed saturation kinetics with increasing external [Cl-]. Addition of 20 mmol·1-1 thiocyanate to the external bath had similar, although less pronounced, effects as Cl- substitution. Equally, external SITS (1 mmol·1-1) inhibited the current and, concomitantly, Gtot decreased substantially. Addition of 1 mmol·1-1 acetazolamide to, and omission of NaHCO3 from, the basolateral bath resulted in a decrease of Isc while Gtot remained unchanged. The Cl--channel blocker DPC inhibited Isc almost completely when added to the basolateral saline, whereas Gtot decreased moderately; however, Vsc depolarised without significant change of Fi. Ouabain had no influence on Isc and Gtot. Increasing the basolateral [K+] resulted in a decrease in Isc, while Gtot was not affected. At the same time Vsc largely depolarised and Fi decreased. Addition of the K+-channel blocker Ba++ (5 mmol·1-1) to the basolateral solution resulted in a two-step alteration of the transepithelial (Isc, Gtot) and cellular (Vsc, Fi) parameters. The results are discussed with regard to (i) the mechanisms responsible for active transbranchial Cl- uptake, and (ii) the technical improvement of being able to perform transport studies with crab gill preparations in an Ussing chamber.