Thiol Antioxidants Regulate Angiotensin II AT1 and Arginine Vasopressin V1 Receptor Functions Differently in Vascular Smooth Muscle Cells

BACKGROUND We compared the effects of the sulfhydryl-containing (thiol) antioxidant dithiothreitol (DTT), which disrupts disulfide bonds, on cell signaling through angiotensin II (Angll) Type 1 receptors (AT1Rs) and arginine vasopressin (AVP)V1 receptors (V1Rs). The AT1R contains two extracellular disulfides bonds but its ligand contains none, whereas the V1R contains no extracellular disufides bonds but its ligand contains 1. METHODS We measured radioligand binding, intracellular calcium responses, and extracellular signal-regulated kinase phosphorylation in cultured rat aortic vascular smooth muscle cells and alterations in urine osmolality in intact rats. RESULTS Preincubation of cells with DTT, a maneuver designed to target receptor disulfides, resulted in concentration-dependent decreases in specific I-125-Angll binding to AT1Rs and acute angiotensin-stimulated intracellular calcium mobilization but no decreases in specific I-125-AVP binding to V1Rs or AVP-stimulated intracellular calcium mobilization. In contrast, preincubation of the ligands with DTT followed by acute exposure to the cells, a maneuver designed to target ligand disulfides, blunted calcium mobilization to AVP robustly but to Angll only minimally. In intact rats, the increase in urine osmolality caused by subcutaneous injection with the AVP analogue desmopressin was significantly diminished when the analogue was preincubated with an excess of DTT. CONCLUSION DTT inhibits cell signaling to Angll AT1Rs and AVPV1Rs, at least in part through disruption of disulfide linkages, but the pattern of response depends upon whether disulfides of ligand or receptor are targeted.