Multiple signaling pathways of V1-vascular vasopressin receptors of A7r5 cells.
We explored the nature and time course of the multiple signal transduction pathways for V1-vascular vasopressin (AVP) receptors of A7r5 aortic smooth muscle cells in culture by using radioligand binding techniques, intracellular calcium monitoring, and polyphosphoinositide and phospholipid analyses. V1-vascular AVP receptors of A7r5 cells were characterized by the agonist radioligand [3H]AVP and the antagonist radioligand [3H]d(CH2)5Tyr(Me)AVP. Affinity and capacity of agonist but not antagonist binding were modulated by MgCl2 and aluminum fluoride, suggesting that the receptors are coupled to a guanine nucleotide regulatory protein. In fura-2-loaded A7r5 cells, AVP induced within seconds a dose-dependent increase of free intracellular Ca++ ([Ca++]i) consisting of a rapid transient spike and a sustained increase lasting for 3-5 min. The baseline [Ca++]i was 136 +/- 18 nM, the maximum [Ca++]i response to AVP was 1,582 +/- 297 nM, and AVP ED50 was 1.87 +/- 0.15 nM. Diverse experiments performed with EGTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester, Mn++, ionomycin, terbutylbenzo hydroquinone, and nicardipine suggested that the initial spike resulted from both intracellular Ca++ release from the endoplasmic reticulum and extracellular Ca++ influx, whereas the sustained phase depended on dihydropyridine-insensitive extracellular Ca++ influx. Experiments done with indomethacin and arachidonic acid indicated that AVP-induced extracellular Ca++ influx was in part dependent on phospholipase A2 activation. In [3H]myoinositol and [3H]arachidonate-labeled A7r5 cells, AVP stimulated inositol 1,4,5 trisphosphate and 1,2 diacylglycerol production via activation of phospholipase C. Also, AVP stimulated a transphosphatidylation reaction through activation of phospholipase D in A7r5 cells labeled with [3H]1-O-alkyl lysoglycerophosphocholine. Thus, the stimulation of V1-vascular AVP receptors of A7r5 cells triggers several signaling pathways. The immediate and transient [Ca++]i rise due to mobilization of intracellular and extracellular Ca++ is associated with the activation of phospholipases A2 and C, and the sustained activation of phospholipase D.
Thibonnier
M
M
Department of Medicine, University Hospitals of Cleveland, Ohio.
Bayer
A L
AL
Simonson
M S
MS
Kester
M
M
D000818:Animals; D001011:Aorta; D001127:Arginine Vasopressin / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis; D004622:Embryo, Mammalian; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010713:Phosphatidylcholines / Q000378:metabolism; D051381:Rats; D011945:Receptors, Angiotensin / Q000378:metabolism*; D017483:Receptors, Vasopressin; D015398:Signal Transduction
C011439:1,2-diacylglycerol; D015220:Calcium Channels; D004075:Diglycerides; D010713:Phosphatidylcholines; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D015636:Magnesium Chloride; D001127:Arginine Vasopressin; D008345:Manganese; D019204:GTP-Binding Proteins; D002118:Calcium