Inhibition of A Chloride Pump By Acetazolamide In The Intestine of Aplysia Californica

Abstract

ABSTRACT The isolated intestine of Aplysia californica, bathed in a substrate-free NaCl seawater bathing medium, generates a spontaneous transepithelial potential difference such that the serosal surface is negative relative to the mucosal surface (Gerencser, 1978b). The short-circuit current (Isc) is accounted for by active absorptive mechanisms for both Na+ and Cl−, the Cl− transport mechanism being more vigorous than that for Na+ (Gerencser, 1978a). However, Cl− transport appeared to be independent of Na+ transport, for the Isc measured in an Na+-free seawater bathing medium was shown to be identical to a net active absorptive flux of Cl− (Gerencser, 1984a). It was hypothesized that active Cl− absorption in Aplysia enterocytes was mediated by a primary active transport process, because it had been demonstrated that the intracellular Cl− electrochemical potential was less than that measured in the extracellular medium (Gerencser & White, 1980), even in the absence of extracellular Na+ (Gerencser, 1983). Lending strength to this hypothesis, Gerencser & Lee (1983, 1985a) demonstrated the existence of a CU-stimulated ATPase activity in Aplysia enterocyte plasma membranes, suggesting a cause-and-effect relationship between ATPase activity and Cl− transport. The ATPase activity stimulated by Cl− was strongly inhibited by acetazolamide. In addition, Gerencser (1984b) and Gerencser & Lee (1985b) have demonstrated an ATP-dependent Cl− uptake in Aplysia inside-out enterocyte plasma membrane vesicles (EPMV). Therefore, the present study was undertaken to assess the effect of acetazolamide on the ATP-driven Cl− uptake mechanism in EPMV.. Seahares (Aplysia californica) were obtained from Marinus Inc. (Westchester, CA) and were maintained at 25 °C in circulating filtered seawater. Adult Aplysia (600-1000 g) were used in these experiments. The plasma membrane vesicles were prepared from Aplysia intestinal enterocytes by homogenization and differential and discontinuous sucrose density-gradient centrifugation techniques as described previously (Gerencser & Lee, 1985a). Vesicle transport experiments were also performed as previously described (Gerencser & Lee, 1985b).