MECHANISM OF DESENSITIZATION OF THE CLONED VASOPRESSIN V-1A RECEPTOR EXPRESSED IN XENOPUS-OOCYTES

The vasopressin V-1a receptor exerts its effects by G protein-mediated increases in cytosolic Ca2+ (Ca-i(2a+)) and activation of protein kinase C. The V-1a, receptor also undergoes autologous desensitization. To clarify the mechanism of this desensitization, we expressed the cloned receptor in Xenopus oocytes, and vasopressin-induced Ca-i(2+) waves were examined as an index of V-1a, activation using confocal microscopy. Pretreatment of oocytes with a minimal concentration of vasopressin inhibited further generation of Ca-i(2+) waves upon maximal stimulation. Such pretreatment did not abolish Ca-i(2+) waves induced by subsequent microinjection of inositol trisphosphate, suggesting that this phenomenon represents receptor desensitization rather than depletion of inositol trisphosphate-sensitive Ca-i(2+) stores. Pretreatment with phorbol dibutyrate, ionomycin, or 8-bromoadenosine 3',5'-cyclic monophosphate had no effect on vasopressin-induced Ca-i(2+) waves. Oocytes recovered from desensitization within 1 h, but the microtubule inhibitor methyl-5-[2-thienylcarbonyl]-1H-benzimiidazol-2-yl)-carbamate (nocodazole) inhibited this recovery. Receptor binding sites were reduced by over 50% within 10 min of exposure to vasopressin, with no associated change in the K-d for the V-1a, receptor. These findings indicate that 1) expression of the cloned V-1a receptor in Xenopus oocytes, coupled with subcellular Ca-i(2+) imaging, provides a useful system to examine mechanisms of V-1a desensitization, 2) the V-1a receptor undergoes autologous desensitization in this experimental system, and 3) protein kinase C, Ca-i(2+), and adenosine 3',5'-cyclic monophosphate do not appear responsible for this desensitization, but 4) microtubule-dependent recycling of the receptor is preserved in this system and may be important for receptor desensitization.