Mechanism of desensitization of the cloned vasopressin V1a receptor expressed in Xenopus oocytes.
The vasopressin V1a receptor exerts its effects by G protein-mediated increases in cytosolic Ca2+ (Cai2+) and activation of protein kinase C. The V1a receptor also undergoes autologous desensitization. To clarify the mechanism of this desensitization, we expressed the cloned receptor in Xenopus oocytes, and vasopressin-induced Cai2+ waves were examined as an index of V1a activation using confocal microscopy. Pretreatment of oocytes with a minimal concentration of vasopressin inhibited further generation of Cai2+ waves upon maximal stimulation. Such pretreatment did not abolish Cai2+ waves induced by subsequent microinjection of inositol trisphosphate, suggesting that this phenomenon represents receptor desensitization rather than depletion of inositol trisphosphate-sensitive Cai2+ stores. Pretreatment with phorbol dibutyrate, ionomycin, or 8-bromoadenosine 3',5'-cyclic monophosphate had no effect on vasopressin-induced Cai2+ 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 Kd for the V1a receptor. These findings indicate that 1) expression of the cloned V1a receptor in Xenopus oocytes, coupled with subcellular Cai2+ imaging, provides a useful system to examine mechanisms of V1a desensitization, 2) the V1a receptor undergoes autologous desensitization in this experimental system, and 3) protein kinase C, Cai2+, 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.
Nathanson
M H
MH
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Burgstahler
A D
AD
Orloff
J J
JJ
Mani
A
A
Moyer
M S
MS
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D015759:Ionomycin / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D017483:Receptors, Vasopressin / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014981:Xenopus
D011943:Receptors, Adrenergic, beta; D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin; D010656:Phenylephrine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D015240:Phorbol 12,13-Dibutyrate; D015759:Ionomycin; D000242:Cyclic AMP; D011493:Protein Kinase C; D002118:Calcium