Subcellular Carbonic Anhydrase Profile in Aplysia Gut

The isolated gut of Aplysia californica (seahare), bathed in a substrate-and Na⁺-free seawater bathing medium, generates a serosa-negative transepithelial potential difference and the concomitant short-circuit current (I_sc), which is inhibitable by acetazolamide, was shown to be identical to a net active absorptive flux of Cl⁻ (Gerencser, 1984). Additionally, Gerencser and Lee (1983, 1985) demonstrated the existence of a Cl⁻-stimulated ATPase activity in Aplysia gut absorptive cell basolateral membranes (BLM), which was also strongly inhibited by acetazolamide. Furthermore, Gerencser (1986) demonstrated acetazolamide inhibition of ATP-dependent Cl⁻ uptake and ATP-dependent vesicular membrane potential change in Aplysia inside-out absorptive cell BLM vesicles. A recent study demonstrating reconstitution of Cl⁻-stimulated ATPase activity into liposomes with subsequent Cl” accumulation in the presence of ATP provided strong evidence for the existence of a primary active transport mechanism for Cl⁻ (Gerencser, 1990) and this, coupled with a previous observation (Gerencser, 1988) that FCCP (a protonophore) had no inhibitory effect on ATP-driven Cl⁻ accumulation in the BLM vesicles, strongly suggested that eukaryotic H⁺-ATPases could not express Cl⁻ pump activity. In view of acetazolamide being a specific inhibitor of carbonic anhydrase (Maren, 1977), the present study was undertaken to determine whether carbonic anhydrase activity was an intermediate in the inhibition of the primary active Cl⁻ transport process in Aplysia gut.

Seahares (Aplysia californica) were obtained from Marinus Inc. (Westchester, CA) and were maintained at 25 °C in circulating filtered sea water. Adult Aplysia (600–1200g) were used in these experiments. The various subcellular membrane fractions were prepared from Aplysia gut epithelial absorptive cells by homogenization and differential and discontinuous sucrose density-gradient centrifugation techniques as described previously (Gerencser and Lee, 1985). Carbonic anhydrase activity was measured by the micromethod developed by Silverman and Gerster (1973), which based enzyme activity on enzyme-catalyzed ¹⁸O exchange between bicarbonate and water. Alkaline phosphatase (brush border membrane marker), Na⁺/K⁺-ATPase (basolateral membrane marker), cytochrome c oxidase (mitochondrial marker) and CD-stimulated ATPase were assayed as previously described (Gerencser and Lee, 1985), as was the ATP-dependent Cl⁻ transport in the BLM vesicles (Gerencser, 1986, 1988). All data are reported as means±S.E. Differences between means were analyzed statistically using Student’s t-test with P<0.05 used as the statistical significant difference criterion.

As demonstrated in the present study (Table 1), carbonic anhydrase activity occurs in both the brush border membrane and the mitochondrial fractions of the Aplysia gut absorptive cells. It is apparent that the bulk of carbonic anhydrase activity resides in the mitochondria rather than the brush border because of the greater enrichment of this enzyme in that fraction. However, no detectable carbonic anhydrase activity resided in the basolateral membrane fraction (Table 1).

As seen in Table 2, Cl⁻-stimulated ATPase is significantly (P<0.05) more active than Mg²⁺-stimulated ATPase activity in the BLM vesicle population. Acetazolamide (0.1 mmol l⁻¹) inhibited this CD-stimulated ATPase activity. There is a significant ATP-dependent Cl” uptake into these BLM vesicles compared with control vesicles in the absence of ATP (P<0.05) and this ATP-dependent Cl⁻ uptake is also inhibited by 0.1 mmol l⁻¹ acetazolamide (Table 2). All these values are means±S.E. for 3–5 different experiments (12–20 animals).

The previous findings, combined with the present findings that acetazolamide inhibits (i) active Cl⁻ absorption and in Aplysia gut in vitro (Gerencser, 1984); (ii) Cl⁻-stimulated ATPase activity in Aplysia gut absorptive cell BLM (Gerencser and Lee, 1985, and Table 2); and (iii) the ATP-dependent Cl⁻ uptake (Table 2) and ATP-dependent intravesicular negative potential change in Aplysia gut absorptive cell BLM vesicles (Gerencser, 1986, 1988; Gerencser et al. 1988), are consistent with the possibility that carbonic anhydrase activity is an intermediate, directly or indirectly, in active Cl⁻ transport in the Aplysia gut because acetazolamide, at low concentrations, has been shown to be a specific inhibitor of carbonic anhydrase (Maren, 1977). However, since no carbonic anhydrase activity was detectable in the BLM of Aplysia gut (Table 1) and acetazolamide inhibited both Cl “-stimulated ATPase activity and ATP-dependent Cl” transport in the BLM (Table 2), this hypothesis is not tenable. Lee (1982) has demonstrated acetazolamide inhibition of blue crab gill HCO3⁻-ATPase, while White (1980) has shown acetazolamide to be a Cl⁻ transport inhibitor, supporting a precedent for acetazolamide inhibition of both anion-ATPase activity and anion transport. Thus, the present data further strengthen the idea that the BLM-localized Cl⁻ pump, which is inhibitable by acetazolamide, is independent of carbonic anhydrase activity.