DISTRIBUTION OF ADENYLATE-CYCLASE AND GTP-BINDING PROTEINS IN HEPATICPLASMA-MEMBRANES

Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolater al (sinusoidal) but not the apical (canalicular) plasma membrane. Afte r bile duct ligation (BDL) for 12 or 24 h, there was an increase in FS K-stimulated adenylate cyclase activity in the apical membrane (54.2 /- 3.9 pmol . mg-1 . min-1). The mechanism for this increase was explo red further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol . mg-1 . min-1); however, total adenosinetriphosphatase (ATPase) acti vity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increa se in adenylate cyclase activity (30.6 +/- 3.6 pmol . mg-1. min-1; det ergent C12E8) This suggested that bile duct ligation was acting via th e detergent-like action of bile acids to uncover latent adenylate cycl ase activity on apical membranes. Further studies demonstrated that bo th BDL and detergent extraction also enhanced toxin-directed ADP-ribos ylation of G(s)alpha (cholera toxin) and G(i)alpha (pertussis toxin) i n the apical but not the basolateral membrane. After BDL, G(i)alpha wa s found to be twofold greater in the apical membrane than the basolate ral membrane. Immunoblotting using specific G protein antibodies furth er confirmed that apical membranes from control rats had a higher conc entration of G(i1), 2alpha and beta and slightly elevated levels of G( i3)alpha and G(s)alpha compared with the basolateral membrane. The res ults demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical mem brane vesicles and is limited by the presence of high ATPase activity.