Signal transduction of physiological concentrations of vasopressin in A7r5 vascular smooth muscle cells - A role for PYK2 and tyrosine phosphorylation of K+ channels in the stimulation of Ca2+ spiking

The signal transduction pathway linking physiological concentrations of [Arg(8)]vasopressin (AVP) to an increase in frequency of Ca2+ spiking was examined in confluent cultures of A7r5 vascular smooth muscle cells. Immunoprecipitation/Western blot studies revealed a robust increase in tyrosine phosphorylation of the nonreceptor tyrosine kinase, PYK2, in A7r5 cells treated with 4beta-phorbol 12-myristate 13-acetate or ionomycin. 100 pm AVP also induced PYK2 tyrosine phosphorylation, and this effect was inhibited by protein kinase C inhibitors Ro-31-8220 (1-10 muM) or chelerythrine chloride (1-20 muM). In fura-2-loaded A7r5 cells, the stimulation of Ca2+ spiking by 100 pM AVP or 1 nM 4beta-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 muM, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 muM), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca2+ spiking. PYK2 phosphorylation was inhibited by both PP2 and salicylate, whereas tyrphostin A47 failed to inhibit PYK2 tyrosine phosphorylation. ERK1/2 kinases did not appear to be involved because 1) 100 pm AVP did not appreciably increase ERK1/2 phosphorylation and U-0126 (2.5 muM) did not inhibit AVP-stimulated Ca2+ spiking; and 2) epidermal growth factor (10 nM) robustly stimulated ERK1/2 phosphorylation but did not induce Ca2+ spiking. Delayed rectifier K+ channels may mediate the PYK2 activity because Kv1.2 channel protein co-immunoprecipitated with PYK2 and tyrosine phosphorylation of Kv1.2 was stimulated by AVP and inhibited by Ro-31-8220, PP2, and salicylate but not tyrphostin A47. Our findings are consistent with a role for PYK2 and phosphorylation of K+ channels in the stimulation of Ca2+ spiking by physiological concentrations of AVP.