Abstract
Previously we demonstrated that activation of Gq protein-coupled H1 histamine receptors potentiates stimulus-coupled exocytosis in bovine chromaffin cells, despite inhibiting Ca2+ influx through voltage-gated calcium channels (VGCCs). This potentiation is restricted to the immediately releasable pool (IRP) of vesicles coupled to VGCCs, and requires the priming protein Munc13-1. Doc2B is a calcium-binding protein, known to interact with Munc13 to promote exocytosis; whether it plays a role in agonist-regulated exocytosis is unknown. To address this question, we isolated chromaffin cells from Doc2B (+/+), (+/-) and (-/-) mice and examined the effects of PLC-coupled GPCR activation on depolarization-evoked exocytosis.We found that under control stimulus conditions, depolarization-evoked exocytosis, measured as changes in membrane capacitance, was not significantly different between Doc2B (+/+), (+/-) and (-/-) cells; similarly no differences were found between the sizes of the releasable vesicle pools (IRP, RRP, SRP). Equimolar replacement of extracellular Ca2+ by Ba2+ also revealed no significant differences in the synchronous and asynchronous phases of Ba2+-evoked exocytosis in Doc2B (+/+), (+/-) or (-/-) cells. Finally, although more than 80% of cells, independent of their genotype, responded to histamine with a reduction of Ca2+ influx, potentiation of exocytosis was surprisingly modest (2-fold) and, even in Doc2 (+/+) cells rarely observed (5/17 cells, (+/-): 5/24 cells, (-/-): 2/18). This is in stark contrast to bovine cells, where 80% of the cells show a 5-fold potentiation of exocytosis. Importantly, Doc2B (+/+), (+/-) and (-/-) cells responded to the DAG analog PMA with potentiation. Taken together, the results suggest that there are profound species differences in the coupling between GqPCRs and the exocytotic machinery in chromaffin cells and that Doc2B is not required for agonist-dependent potentiation of exocytosis in mice.
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