MEMBRANE-DELIMITED G-PROTEIN-MEDIATED COUPLING BETWEEN V-1A VASOPRESSIN RECEPTOR AND DIHYDROPYRIDINE BINDING-SITES IN RAT GLOMERULOSA CELLS

In rat glomerulosa cells, vasopressin stimulates intracellular calcium mobilization via at least two distinct mechanisms: the release of cal cium from inositol-1,4,5-P-3-sensitive stores and the activation of tr ansmembrane calcium influx. In this study, we focused on the second me chanism through three experimental approaches. By videomicroscopically examining Fura-2-loaded cells, we demonstrate that vasopressin induce s a dose-dependent and receptor-mediated calcium influx fully inhibite d by either 1 mu M nifedipine or a pertussis toxin pretreatment and po tentiated by 1 mu M BAY K 8644. Patch-clamp experiments also indicate that vasopressin stimulates L-type calcium current by 87% and only wea kly inhibits T-type calcium current. To further characterize the coupl ing between the vasopressin receptor and the dihydropyridine calcium c hannel, we performed binding studies using tritiated nitrendipine. Wit h this technique, we showed that on intact cells, vasopressin is able to increase the specific binding of tritiated nitrendipine in a dose-d ependent manner (K-act=2 nM). Pharmacological studies using a series o f vasopressin analogs revealed that this effect is mediated via a V-1a vasopressin receptor subtype. Furthermore, the vasopressin-stimulated nitrendipine binding was sensitive to pertussis toxin pretreatment, w hich affected only the maximum binding capacity of nitrendipine-bindin g sites. More interestingly, we demonstrate that vasopressin still inc reases nitrendipine binding to plasma membrane preparation and that GT P is absolutely necessary for such a hormonal effect. Altogether, thes e data confirm the existence of a tight and direct coupling between th e V-1a vasopressin receptor and a dihydropyridine calcium channel via a pertussis toxin-sensitive G protein.