Preliminary Study On The Role Of PI3K And PtdIns-3-P In Cell Secretion

Secretion forms the basis for signal transmission between neurons and hormone release from endocrine cells. It must undergo repeated rounds of exocytosis,endocytosis,refilling and mobilization, a phenomenon called recycling. The temporal and spatial coordination of exo–endocytic recycling requires a complex molecular machinery that integrates cell signaling, dynamic changes within membranes, and cytoskeletal rearrangements. Over the past 25 years, many proteins of this machinery have been identified and characterized. However, less is known about the function of lipids. Now the role of lipids, especially PIs, in the physiology of secretion and vesicle traffic is focused. In this study, we applied advanced biophysical techniques, such as patch clamp,membrane capacitance measurement,microfluoremetric technique,flash photolysis,TIRFM,and so on. Inspection on the role of phospoinositide-3 kinase and phospoinositide -3-phosphate in secretion was carried on PC12 and INS-1 cells which represent widely used models for secretion and membrane traffic. Wortmannin, a specific inhibitor of PI3Ks, and EGFP-2xFYVE fusion protein which bind specifically with PtdIns-3-P were used to block the activity of PI3K and PtdIns-3-P separately. The results showed that the block did not change PC12 cell response to flash including its kinetics and calcium dependence. So, it was demonstrated that PI3K and PtdIns-3-P had no effect on PC12 cell secretion itself, but did not exclude the possibility that they enhance cell to response to repetitive strong stimuli by their accelerating effect on vesicle endocytosis which speed the refilling of releasable vesicle. In INS-1 cells, it was found that the block inhibited fast endocytosis, but did not change the amplitude of slow endocytosis. At the same time, it also did not change calcium dependence of fast and slow endocytosis. From this, we draw a conclusion that PI3K and PtdIns-3-P may be involved in the regulation of endocytosis, but more experiments are required for precise elucidation.