Signal Transduction of Physiological Concentrations of Vasopressin in A7r5 Vascular Smooth Muscle Cells

Abstract

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 nonreceptor tyrosine kinase, PYK2, in A7r5 cells treated with 4␤-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 M) or chelerythrine chloride (1-20 M). In fura-2-loaded A7r5 cells, the stimulation of Ca 2؉ spiking by 100 pM AVP or 1 nM 4␤-phorbol 12-myristate 13-acetate was completely blocked by PP2 (10 M, a Src family kinase inhibitor). Salicylate (20 mM, recently identified as a PYK2 inhibitor) and the tyrosine kinase inhibitor, tyrphostin A47 (50 M), 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 M) 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. It remains to be elucidated how activation of PKC ultimately produces Ca 2ϩ spiking. One possibility is that PKC activation leads to membrane depolarization and consequently to activation of L-type voltage-sensitive Ca 2ϩ channels. We have preliminary data that suggest that inhibition of delayed rectifier K ϩ channels (K v channels) may provide the trigger for L-type Ca 2ϩ channel activation (16). The present study examines the possibility that one or more tyrosine kinases may serve as intermediary links in this signaling cascade. In particular, we focus on the non-receptor tyrosine kinase PYK2 (proline-rich tyrosine kinase 2, also known as RAFTK or CADTK), a member of the focal adhesion kinase (p125 FAK ) family, which is acti-

Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells

“…Treatment of A7r5 cells with 100 pM AVP leads to increased tyrosine phosphorylation of Kv1.2 delayed rectifier K + channels (3). We previously speculated (3) that tyrosine phosphorylation of Kv1.2 channels inhibits their function and thereby induces the membrane depolarization that is required for L-type Ca 2+ channel activation.The signal transduction pathway leading to Kv1.2 phosphorylation and Ca 2+ spiking requires activation of protein kinase C (PKC) (3,14). The present study provides the first electrophysiological evidence that activation of PKC at physiological concentrations of AVP leads to inhibition of an outward voltage-sensitive K + current, depolarizes the membrane, and induces action potential generation in vascular smooth muscle cells.…”

“…The stimulation of Ca 2+ -dependent action potentials, which underlie AVP-stimulated Ca 2+ spiking, involves activation of a novel signaling pathway (5). Treatment of A7r5 cells with 100 pM AVP leads to increased tyrosine phosphorylation of Kv1.2 delayed rectifier K + channels (3). We previously speculated (3) that tyrosine phosphorylation of Kv1.2 channels inhibits their function and thereby induces the membrane depolarization that is required for L-type Ca 2+ channel activation.…”

“…We have previously presented evidence that PKC activation is a necessary step in the stimulation of Ca 2+ spiking by AVP (14). Direct activation of PKC with PMA was shown to be effective in stimulating Ca 2+ spiking (3,14). To test the hypothesis that inhibition of K v currents may be a consequence of PKC activation, we examined the effects of PMA on I Kv .…”

Long Lasting Inhibition of Adenylyl Cyclase Selectively Mediated by Inositol 1,4,5-Trisphosphate-evoked Calcium Release

“…In A7r5 cells, Arg 8 -vasopressin (AVP), via V 1A receptors (37), stimulates phospholipase C and thereby both release of Ca 2ϩ from intracellular stores and Ca 2ϩ entry across the plasma membrane. The latter involves a complex interplay between different Ca 2ϩ channels: AVP promotes opening of L-type voltage-gated Ca 2ϩ channels (38), and it can reciprocally regulate CCE and a noncapacitative Ca 2ϩ entry (NCCE) pathway (39,40). Given the importance of Ca 2ϩ -cAMP interactions in vascular smooth muscle, the evidence that not all Ca 2ϩ signals are equally effective in regulating AC (7), and evidence that AVP inhibits AC in A7r5 cells (33), we decided to examine whether inhibition of AC in these cells was regulated selectively by distinct Ca 2ϩ entry pathways.…”

Arginine-Vasopressin Activates the JAK-STAT Pathway in Vascular Smooth Muscle Cells

“…Src activation was previously demonstrated upon AVP stimulation of various cell types (34,35). Considering these studies and the results presented in this study, we propose that AVP binding to its GPCR activates both c-Src and JAK2, thereby inducing the formation of a c-Src-JAK2 complex.…”