|Budget Amount *help
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1990: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1989: ¥5,400,000 (Direct Cost: ¥5,400,000)
The regulatory protein of small molecular GTP-binding protein(smgP25A), GDI(GDP dissociation inhibitor), is a 50 KG single peptide which inhibits smg mediated physiological processes. GDI is introduced into chromaffin cells of rats through patch-clamp glass pipettes and increases in membrane capacitance was monitored. Activation of Ca^<2+> channels with a step-voltage depolarization consistently induced an increase in membrane capacitance. In several such trials with GDI of 200 nM(prepared from bovine brain by Prof. Takai, Y.), the increases in membrane capacitance were reduced during first 20 min of cell dialysis. However, the efficacy of GDI was quite variable, and in some batches of cells using a GDI preparation, no effects were observed. The purity of GDI was about 90%, so we could not rule oul the possibility that contaminating phospholipid reduced Ca^<2+>-dependent exocytosis or increases in membrane capacitance in these cells. We now try to improve the purity of GDI.
GDS is a another regulatory protein of smgP21B(GDS : GDP dissociation stimulator). It is possible that GDS synergistically acts- with A-kinase to activate smgP21B. In pancreatic acinar cells, VIP(vasointestinal polypeptide)is thought to induce enzyme secretion via cAMP-dependent process. Thus, it is expected that smg and/or GDS play roles in VIP mediated exocytosis. We tried to stimulate pancreatic acinar cells using patch-clamp internal dialysis with CAMP and GTPYS containing solution. With this stimulation, some cells showed increases in membrane capacitance but some did not. Probably we are missing an another cofactor since external application of VIP(10 nM)consistently induced an increase in membrane capacitance. The missing factor probably relates the formation of arachidonic acid(AA), and we are now studying the effects of a inhibitor of AA, 4-BPB, on the acinar cell response to internal CAMP and GTPyS.