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 kin ase C. The V-1a, receptor also undergoes autologous desensitization. T o clarify the mechanism of this desensitization, we expressed the clon ed receptor in Xenopus oocytes, and vasopressin-induced Ca-i(2+) waves were examined as an index of V-1a, activation using confocal microsco py. Pretreatment of oocytes with a minimal concentration of vasopressi n inhibited further generation of Ca-i(2+) waves upon maximal stimulat ion. Such pretreatment did not abolish Ca-i(2+) waves induced by subse quent microinjection of inositol trisphosphate, suggesting that this p henomenon represents receptor desensitization rather than depletion of inositol trisphosphate-sensitive Ca-i(2+) stores. Pretreatment with p horbol dibutyrate, ionomycin, or 8-bromoadenosine 3',5'-cyclic monopho sphate had no effect on vasopressin-induced Ca-i(2+) waves. Oocytes re covered from desensitization within 1 h, but the microtubule inhibitor -thienylcarbonyl]-1H-benzimiidazol-2-yl)-carbamate (nocodazole) inhib ited this recovery. Receptor binding sites were reduced by over 50% wi thin 10 min of exposure to vasopressin, with no associated change in t he K-d for the V-1a, receptor. These findings indicate that 1) express ion of the cloned V-1a receptor in Xenopus oocytes, coupled with subce llular Ca-i(2+) imaging, provides a useful system to examine mechanism s of V-1a desensitization, 2) the V-1a receptor undergoes autologous d esensitization in this experimental system, and 3) protein kinase C, C a-i(2+), and adenosine 3',5'-cyclic monophosphate do not appear respon sible for this desensitization, but 4) microtubule-dependent recycling of the receptor is preserved in this system and may be important for receptor desensitization.