Vasopressin-stimulated Ca²⁺spiking in vascular smooth muscle cells involves phospholipase D

Abstract

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.

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+‐stimulated phospholipase A2

“…Both BEL and ONO-RS-082 inhibited Ca spiking, and ONO-RS-082 was shown also to inhibit PLD, but not the Ca 2+ signals evoked by higher concentrations of AVP [17]. The latter is consistent with our results (Fig.…”

“…At low concentrations (61 nM), AVP stimulates Ca 2+ spiking mediated by L-type Ca 2+ channels: the likely steps in this sequence have been identified by Byron and colleagues and are shown in Fig. 4 [17,18]. Phosphatidylcholine (PC) probably provides the major substrate for the PLD that initiates this sequence [42], but the links between the V 1A receptors and stimulation of PLD remain to be identified [17].…”

“…4 [17,18]. Phosphatidylcholine (PC) probably provides the major substrate for the PLD that initiates this sequence [42], but the links between the V 1A receptors and stimulation of PLD remain to be identified [17]. Higher concentrations of AVP ( P 1 nM, Fig.…”

“…The V 1A -AVP receptors of A7r5 vascular smooth muscle cells stimulate an initial activation of PLC and a more delayed activation of PLD and PLA 2 [14,17] but see [24]. The relationships between these events are not entirely clear: Gq links the V 1A receptor to activation of PLC, but the role of these initial events in the activation of PLD and PLA 2 , each of which can lead to release of AA, is unresolved.…”

“…The relationships between these events are not entirely clear: Gq links the V 1A receptor to activation of PLC, but the role of these initial events in the activation of PLD and PLA 2 , each of which can lead to release of AA, is unresolved. Indeed, in both A7r5 cells and primary aortic smooth muscle [17], persuasive evidence suggests that AVP can activate PLA 2 [25][26][27] and PLD [17] independent of its effects on PLC. The present study was undertaken to measure directly the effects of AVP on AA production in A7r5 cells.…”