Demonstration of processing and recycling of biologically active V1 vasopressin receptors in vascular smooth muscle.
The present study examines the binding and postbinding cellular processing and recycling of the V1 arginine vasopressin (AVP) receptor in cultured vascular smooth muscle cells (VSMCs). The surface binding of AVP to VSMCs was temperature dependent and reached equilibrium within 60 min at 4 degrees C. Displacement studies with unlabeled AVP or a specific V1 AVP antagonist revealed a single class of V1 receptors (Bmax, 1.99 pmol [corrected] per mg of protein; Kd, 2.15 nM). Incubation of VSMCs with unlabeled 10 nM AVP to promote receptor internalization resulted in a time- and temperature-dependent loss of AVP surface binding. At 37 degrees C, maximum loss of binding sites (65%) occurred within 20 min. Recovery of AVP binding occurred rapidly (t1/2, 15-20 min at room temperature) and was uninfluenced by inhibiting protein synthesis with cycloheximide. Pretreating VSMCs with chloroquine prevented AVP receptor recycling, indicating that the AVP-receptor complex requires endosomal processing. The biological competence of the recycled AVP receptor was shown by AVP-induced Ca2+ uptake. The results of these studies therefore indicate that, after surface binding, the AVP-receptor complex internalizes and dissociates in an endosomal compartment. It is demonstrated that in VSMCs biologically active V1 AVP receptors recycle back to the cell surface, thus attenuating the loss of AVP surface binding sites.
Department of Medicine, University of Colorado School of Medicine, Denver 80262.
V A
VA
Briner
B
B
Williams
P
P
Tsai
R W
RW
Schrier
D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002451:Cell Compartmentation; D002478:Cells, Cultured; D015536:Down-Regulation; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D051381:Rats; D011945:Receptors, Angiotensin / Q000378:metabolism*; D017483:Receptors, Vasopressin; D015398:Signal Transduction; D013997:Time Factors
D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D001127:Arginine Vasopressin; D002118:Calcium