Vasopressin Stimulates Ca 2+ Spiking Activity in A7r5 Vascular Smooth Muscle Cells via Activation of Phospholipase A 2

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Abstract
[Arg8]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line (Kd ≈2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) by releasing intracellular Ca2+ stores and increasing Ca2+ influx; the EC50 for these effects is ≈5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 ≈0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca2+ spiking activity in fura 2–loaded A7r5 cells. Frequency of Ca2+ spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca2+]i changes ascribed to the release of Ca2+ stores and increased Ca2+ entry. The effects of both low and high concentrations of AVP were inhibited by [1-(β-mercapto-β,β,-pentamethylenepropionic acid),2-O-methyltyrosine]arginine vasopressin, a selective V1a vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca2+ channels, abolished the Ca2+-spiking activity without inhibiting a maximal [Ca2+]i response to AVP (1 μmol/L). AVP-stimulated Ca2+ spiking, but not release of intracellular Ca2+ stores, was also abolished by ONO-RS-082 (1 μmol/L), an inhibitor of phospholipase A2. These results suggest that occupation of a small fraction of V1a vasopressin receptors by AVP results in stimulation of phospholipase A2 and leads to increased Ca2+-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis.