Effect of Ethanol on Intracellular Vesicular Transport from Golgi to the Apical Cell Membrane: Role of Phosphatidylinositol 3‐Kinase and Phospholipase A₂ in Golgi Transport Vesicles Association and Fusion with the Apical Membrane

Abstract

The study of ethanol effects on intracellular transport and membrane biogenesis in rat hepatocytes revealed that, during synthesis of transport vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the vesicular phosphatidylinositol was phosphorylated to phosphatidylinositol 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 transport vesicles with the apical membrane was followed by phospholipase A2-specific hydrolysis of phosphatidylinositol 3-phosphate and incorporation of the transport vesicle membrane into the apical membrane. Association of Golgi transport vesicles with apical membranes was not affected by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidylinositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Golgi 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 phosphatidylinositol 3-kinase and phospholipase A2 activities are crucial 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 receptor, and that is followed by phospholipase A2-specific lysophospholipid generation, perturbation of the membranes, and fusion of the transport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transport vesicles, but had no effect on their association with apical membrane or fusion with the membrane.

Intracellular Calcium Signals Regulating Cytosolic Phospholipase A2 Translocation to Internal Membranes

“…Interestingly, PLA 2 activity has been implicated in Golgi formation (18,29) and tubulation (29), in membrane remodeling (46), and in membrane trafficking events including recycling between Golgi and ER (28,32,47), endocytosis (48), exocytosis (49), and trafficking of plasma membrane and secretory proteins (18,50). It is not clear from most reports whether cPLA 2 or other PLA 2 s (or multiple PLA 2 species) are involved in the described processes.…”

Association of cPLA2-α and COX-1 with the Golgi apparatus of A549 human lung epithelial cells

“…These include studies on Golgi formation (Choukroun et al, 2000), tubulation (de Figueiredo et al, 1999) and membrane remodelling (Schmidt et al, 1999). In addition, several studies using PLA2 inhibitors have highlighted the importance of PLA2 activity in trafficking between the ER and the Golgi (de Figueiredo et al, 2000;Drecktrah and Brown, 1999;Slomiany et al, 1992), endocytosis (Mayorga et al, 1993), exocytosis (Slomiany et al, 1998) and the intracellular trafficking of secretory proteins (Choukroun et al, 2000;Tagaya et al, 1993). Since most of the inhibitors used in such studies were broad-range inhibitors, the precise species of PLA2 involved in these processes is unclear.…”

Activation of apoptotic caspase‐3 and nitric oxide synthase‐2 in buccal mucosa with chronic alcohol ingestion

“…BIOCHEMISTRYend MOLECULAR BIOLOGY INTERNATIONAL perturbations in cell membrane architecture and disruption of the gated processes responsible for the preservation of intracellular electroosmotic homeostasis (29,30). Our findings indicate that these disturbances may well be responsible for the activation of proapoptotic caspase-3, induction of NOS-2, and the enhanced buccal epithelial cells apoptosis with chronic alcohol ingestion.…”