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.