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 (AngII) 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 (125)I-AngII binding to AT1Rs and acute angiotensin-stimulated intracellular calcium mobilization but no decreases in specific (125)I-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 AngII 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.


CONCLUSIONS
DTT inhibits cell signaling to AngII AT1Rs and AVP V1Rs, at least in part through disruption of disulfide linkages, but the pattern of response depends upon whether disulfides of ligand or receptor are targeted.
Ullian
Michael E
ME
Division of Nephrology, Department of Medicine, Ralph H. Johnson Veterans Administration Hospital, Medical University of South Carolina, Charleston, South Carolina, USA. ullianme@musc.edu
Beck
C Nicole
CN
Walker
Linda P
LP
Fitzgibbon
Wayne R
WR
Morinelli
Thomas A
TA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003894:Deamino Arginine Vasopressin / Q000737:chemistry / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004231:Diuresis / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D009994:Osmolar Concentration; D051381:Rats; D017207:Rats, Sprague-Dawley; D044140:Receptor, Angiotensin, Type 1 / Q000187:drug effects / Q000502:physiology*; D017483:Receptors, Vasopressin / Q000187:drug effects / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D014556:Urine / Q000502:physiology
D000975:Antioxidants; D004220:Disulfides; D044140:Receptor, Angiotensin, Type 1; D017483:Receptors, Vasopressin; D048049:Extracellular Signal-Regulated MAP Kinases; D003894:Deamino Arginine Vasopressin; D002118:Calcium; D004229:Dithiothreitol