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 Ca(2+) 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 non-receptor 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 microm) or chelerythrine chloride (1-20 microm). In fura-2-loaded A7r5 cells, the stimulation of Ca(2+) spiking by 100 pm AVP or 1 nm 4beta-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 microm, a Src family kinase inhibitor). Salicylate (20 mm, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 microm), but not its inactive analog, tyrphostin A63, also blocked AVP-stimulated Ca(2+) 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 microm) did not inhibit AVP-stimulated Ca(2+) spiking; and 2) epidermal growth factor (10 nm) robustly stimulated ERK1/2 phosphorylation but did not induce Ca(2+) 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 Ca(2+) spiking by physiological concentrations of AVP.
Mesh Terms:
Animals, Binding Sites, Blotting, Western, Calcium, Cell Line, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors, Focal Adhesion Kinase 2, Ionomycin, Ionophores, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases, Models, Biological, Muscle, Smooth, Phosphorylation, Precipitin Tests, Protein Binding, Protein Kinase C, Protein Structure, Tertiary, Protein-Tyrosine Kinases, Rats, Signal Transduction, Tetradecanoylphorbol Acetate, Time Factors, Transcriptional Activation, Tyrosine, Tyrphostins, Vasopressins
Animals, Binding Sites, Blotting, Western, Calcium, Cell Line, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors, Focal Adhesion Kinase 2, Ionomycin, Ionophores, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases, Models, Biological, Muscle, Smooth, Phosphorylation, Precipitin Tests, Protein Binding, Protein Kinase C, Protein Structure, Tertiary, Protein-Tyrosine Kinases, Rats, Signal Transduction, Tetradecanoylphorbol Acetate, Time Factors, Transcriptional Activation, Tyrosine, Tyrphostins, Vasopressins
J. Biol. Chem.
Date: Mar. 01, 2002
PubMed ID: 11739373
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