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).

The transmembrane electrical potential (Δψ) was estimated from the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) between the extra-and intravesicular space by ultrafiltration as described above and by a doublelabelling method as described by Lee & Pritchard (1983). Non-specific binding of TPMP+ to the vesicular membranes (Goldinger, Duffey & Hong, 1983) was assessed by using non-ionic media in the membrane preparative, reaction mixture and ultrafiltration stages of the TPMP+ electrical potential difference assay. When converted into a Δψ, 31·1 mV was then subtracted from the total to give the ATP-dependent Δψ.

As demonstrated in the present study (Table 1), the addition of ATP, in the presence of Mg2+, to EPMV of Aplysia elicited a rapid Cl uptake significantly above that of control. This difference in Cl uptake is the ATP-dependent portion of the total Cl uptake into the EPMV and it is inhibited 50·3 ±6·1% by 1 mmol I−1 acetazolamide. Similarly, in the same preparation of EPMV, the Δψ was inhibited by acetazolamide 90·6 ±2·1%, which is similar quantitatively to the effect of 1 mmol 1−1 acetazolamide on Cl-stimulated ATPase activity in the same preparation (Gerencser & Lee, 1985a). The above values are means ±S.E. for 9-12 different experiments (36-42 animals).

The present finding (Table 1) that acetazolamide inhibited the ATP-dependent Cl uptake and intravesicular negative potential (Δψ) in Aplysia EPMV is Consistent with the following previous findings: (1) acetazolamide inhibition of active Cl absorption and Isc in in vitro Aplysia intestine (Gerencser, 1984a) and (2) acetazolamide inhibition of Cl-stimulated ATPase activity in Aplysia EPMV (Gerencser & Lee, 1985a). Although acetazolamide, at low concentrations, has been shown to be a specific inhibitor of carbonic anhydrase (Maren, 1977), it has also been demonstrated to be a good Cl transport inhibitor (White, 1980). Thus the data further strengthen the idea that the Cl-stimulated ATPase, which is inhibited by acetazolamide, may be involved in Cl transport across the Aplysia intestine.

Additionally, the finding that ATP, in the presence of Cl, can stimulate (increase in intravesicular negativity), as seen in Table 1, also suggests that the mechanism responsible for this phenomenon is electrogenic.

I would like to acknowledge the excellent technical assistance of C. Burgin and F. Robbins. This investigation was supported by DSR Grant no. 122101010.

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