EFFECT OF ETHANOL ON INTRACELLULAR VESICULAR TRANSPORT FROM GOLGI TO THE APICAL CELL-MEMBRANE - ROLE OF PHOSPHATIDYLINOSITOL 3-KINASE AND PHOSPHOLIPASE A(2) IN GOLGI TRANSPORT VESICLES ASSOCIATION AND FUSION WITH THE APICAL MEMBRANE

The study of ethanol effects on intracellular transport and membrane b iogenesis in rat hepatocytes revealed that, during synthesis of transp ort vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the ve sicular phosphatidylinositol was phosphorylated to phosphatidylinosito l 3-phosphate. Association of the enzyme with Golgi transport vesicles and the transport to the apical portion of the cell membrane was not affected by 0 to 120 mM ethanol, but was dependent on the presence of the p85 subunit of the phosphatidylinositol 3-kinase. In the presence of ATP-enriched cytosol and calcium ions, association of Golgi transpo rt vesicles with the apical membrane was followed by phospholipase A(2 )-specific hydrolysis of phosphatidylinositol 3-phosphate and incorpor ation of the transport vesicle membrane into the apical membrane. Asso ciation of Golgi transport vesicles with apical membranes was not affe cted by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidyl inositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Go(gi transport vesicles with the apical membrane and generation of lysophosphatidylinositol 3 -phosphate and arachidonate was inhibited with EGTA or after depletion of ATP from cytosol. Results of these studies provide evidence that p hosphatidylinositol 3-kinase and phospholipase A,activities are crucia l for the final step of exocytotic transport. The process consists of two stages. First, the p85 subunit of phosphatidylinositol 3-kinase is involved in the specific association of the vesicle with membrane rec eptor, and that is followed by phospholipase A(2)-specific lysophospho lipid generation, perturbation of the membranes, and fusion of the tra nsport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transpo rt vesicles, but had no effect on their association with apical membra ne or fusion with the membrane.