Vasopressin-stimulated Ca2+ spiking in vascular smooth muscle cells involves phospholipase D.
Physiological concentrations of [Arg(8)]vasopressin (AVP; 10-500 pM) stimulate oscillations of cytosolic free Ca2+ concentration (Ca2+ spikes) in A7r5 vascular smooth muscle cells. We previously reported that this effect of AVP was blocked by a putative phospholipase A2 (PLA2) inhibitor, ONO-RS-082 (5 microM). In the present study, the products of PLA2, arachidonic acid (AA), and lysophospholipids were found to be ineffective in stimulating Ca2+ spiking, and inhibitors of AA metabolism did not prevent AVP-stimulated Ca2+ spiking. Thin layer chromatography was used to monitor the release of AA and phosphatidic acid (PA), which are the products of PLA2 and phospholipase D (PLD), respectively. AVP (100 pM) stimulated both AA and PA formation, but only PA formation was inhibited by ONO-RS-082 (5 microM). Exogenous PLD (type VII; 2.5 U/ml) stimulated Ca2+ spiking equivalent to the effect of 100 pM AVP. AVP stimulated transphosphatidylation of 1-butanol (a PLD-catalyzed reaction) but not 2-butanol, and 1-butanol (but not 2-butanol) completely prevented AVP-stimulated Ca2+ spiking. Protein kinase C (PKC) inhibition, which completely prevents AVP-stimulated Ca2+ spiking, did not inhibit AVP-stimulated phosphatidylbutanol formation. These results suggest that AVP-stimulated Ca2+ spiking depends on activation of PLD rather than PLA2 and that PKC activation may be downstream of PLD in the signaling cascade.
Li
Y
Y
Department of Physiology, Loyola University, Chicago, Illinois 60626, USA.
Shiels
A J
AJ
Maszak
G
G
Byron
K L
KL
D020001:1-Butanol / Q000494:pharmacology; D062365:Aminobenzoates / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002723:Chlorobenzoates; D002934:Cinnamates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008246:Lysophospholipids / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014667:Vasopressins / Q000378:metabolism* / Q000494:pharmacology; D062367:ortho-Aminobenzoates
D062365:Aminobenzoates; D002723:Chlorobenzoates; D002934:Cinnamates; D004791:Enzyme Inhibitors; D008246:Lysophospholipids; D062367:ortho-Aminobenzoates; D014667:Vasopressins; D016718:Arachidonic Acid; D020001:1-Butanol; C060048:2-(4-amylcinnamoyl)amino-4-chlorobenzoic acid; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate