THE GTPASE RAB3A NEGATIVELY CONTROLS CALCIUM-DEPENDENT EXOCYTOSIS IN NEUROENDOCRINE CELLS

There is accumulating evidence that small GTPases of the rab family re gulate intracellular vesicle traffic along biosynthetic and endocytoti c pathways in eukaryotic cells. It has been suggested that Rab3a, whic h is associated with synaptic vesicles in neurons and with secretory g ranules in adrenal chromaffin cells, might regulate exocytosis. We rep ort here that overexpression in PC12 cells of Rab3a mutant proteins de fective in either GTP hydrolysis or in guanine nucleotide binding inhi bited exocytosis, as measured by a double indirect immunofluorescence assay. Moreover, injection of the purified mutant proteins into bovine adrenal chromaffin cells also inhibited exocytosis, as monitored by m embrane capacitance measurements. Finally, the electrophysiological ap proach showed that bovine chromaffin cells which were intracellularly injected with antisense oligonucleotides targeted to the rab3a messeng er exhibited an increasing potential to respond to repetitive stimulat ions. In contrast, control cells showed a phenomenon of desensitizatio n. These results provide clear evidence that Rab3a is involved in regu lated exocytosis and suggest that Rab3a is a regulatory factor that pr events exocytosis from occurring unless secretion is triggered. Furthe rmore, it is proposed that Rab3a is involved in adaptive processes suc h as response habituation.