The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kappa Da protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca2+/ calmodulin (CaM), which causes the small GTP- binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM- dependnet protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca2+/CaM directly or indirectly through GTP-binding protein(s) and Ca2+/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca2+/CaM may be important for the regulation of transporters and neurosecretion.
Animals ; Calcium/*metabolism ; Calmodulin/*metabolism ; Carrier Proteins/*chemistry/*metabolism ; Guanine Nucleotide Dissociation Inhibitors/metabolism ; Guanosine Triphosphate/metabolism/*pharmacology ; Molecular Weight ; Monomeric GTP-Binding Proteins/metabolism ; Phosphorylation/drug effects ; Rats ; Recombinant Fusion Proteins/*chemistry/*metabolism ; Synaptic Membranes/chemistry/drug effects/*metabolism ; Synaptic Vesicles/chemistry/drug effects/*metabolism

OBJECTIVETo study the possible mechanism of aqueous extract of detoxified cottonseeds (CTN-W).

METHOD AND RESULTCTN-W 0.01, 0.03, 0.10, 0.30 mg.mL-1 was incubated directly with the synaptic membrane extracted from the cerebral cortex in rats, and adenylyl cyclase (AC) activity was detected by using radio-immunoassay.

RESULTShowed that CTN-W could activate AC in a dose-dependend manner. After incubation with PC12 cells in the presence of corticosterone 2 x 10(-4)mol.L-1 for 48 h, CTN-W 0.08, 0.4, 2 mg.mL-1 protected PC12 cells from the lesion induced by corticosterone.

CONCLUSIONAntidepressant and anxiolytic effects of CTN-W are related with the activation of AC-cAMP pathway in signal transduction system, thus protecting neurons from the lesion. These two aspects maybe partly form the mechanism of CTN-W's action.

Adenylyl Cyclases ; metabolism ; Animals ; Anti-Anxiety Agents ; pharmacology ; Antidepressive Agents ; pharmacology ; Corticosterone ; antagonists & inhibitors ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Gossypium ; chemistry ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; drug effects ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Seeds ; chemistry ; Synaptic Membranes ; enzymology