Activation of Protein Kinases in Canine Basilar Artery in Vasospasm

Abstract

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebra! artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCI or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Rafl, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase C(alpha) (PKC alpha), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of mu-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCI- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg(2+)-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated in vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.

<b>Different effect of serotonin on intracellular calcium ion dynamics in the smooth muscle cells between rat posterior ciliary artery and vorticose </b><b>vein </b>

“…In the posterior ciliary arteries, the 5-HT-induced response of increased [Ca 2+ ] i was effected by both the Ca 2+ influx from extracellular spaces and its release from internal stores, whereas the [Ca 2+ ] i increase in the vorticose veins was much more dependent on the Ca 2+ influx from extracellular spaces. Furthermore, the activation of smooth muscle contraction is thought to occur through increases in the [Ca 2+ ] i , activation of Ca 2+ / calmodulin-dependent MLCK, and increases in MLC phosphorylation, as in the spastic canine basilar artery, where MLC has been shown to be phosphorylated by MLCK (22,38). Our results using a MLCK inhibitor in both arteries and veins were consistent with this model.…”

“…10c). The effect of PKI (14)(15)(16)(17)(18)(19)(20)(21)(22) (2 μM) was the same as that observed in the presence of H-89 (n = 8) (data not shown). SQ22536 (100 μM) also completely blocked this increase (n = 8) (Fig.…”

“…With the exception of the 5-HT 3 receptor, which is a ligand-gated ion chan-intact arteries and veins were selected and examined under the microscope. Specimens were continuously perfused with HR containing the following agonists and/or antagonists: 5-HT (Nacalai Tesque, 10 μM); GdCl 3 (a nonspecific cation channel blocker; Wako, Japan, 100 μM); diltiazem (a L-type Ca 2+ channel blocker, 40 μM); thapsigargin (a sarco-endoplasmic reticulum Ca 2+ -ATPase inhibitor, 2 μM); U73122 (a phospholipase C inhibitor, 5 μM); SQ22536 (an adenylyl cyclase inhibitor, 100 μM); ketanserin (a 5-HT 2 antagonist, 1 μM); 8-Hydroxy-DPAT (a 5-HT 1a agonist, 50 μM), all from Sigma; CP93129 (a 5-HT 1b agonist, 10 μM); ML 9 (a selective myosin light chain kinase inhibitor, 10 μM); PKI (14)(15)(16)(17)(18)(19)(20)(21)(22) (a cell-permiable protein kinase A (PKA) inhibitor, 2 μM), all from TOCRIS (Minneapolis, MN, USA), α-methylserotonin maleate (a 5-HT 2 agonist, 100 μM); 2-methylserotonin maleate (a 5-HT 3 agonist, 100 μM); and H-89 (a PKA antagonist, 20 μM), all from Enzo Life Sciences (Farmingdale, NY, USA).…”

Mechanism of Endothelin-1–Induced Contraction in Rabbit Basilar Artery

“…2,20 MAPK activity was elevated in canine basilar arteries in a double-hemorrhage model of experimental cerebral vasospasm. 27 This study for the first time demonstrated that PTK and MAPK are involved in ET-1-induced contraction in cerebral arteries. Since both hemolysate and ET-1 are extremely important spasmogens for cerebral vasospasm 1,2 and PTK and MAPK are involved in the signal transduction of both hemolysate 2,20 and ET-1 (this study), PTK and MAPK may be important factors in the pathogenesis of cerebral vasospasm.…”

“…No attempt was made to remove endothelial cells. The modified Krebs-Henseleit solution contained the following (mmol/L): NaCl 120, KCl 4.5, MgSO 4 1, NaHCO 3 27, KH 2 PO 4 1, CaCl 2 2.5, and dextrose 10. All procedures were approved by the Animal Care and Use Committee at the University of Mississippi Medical Center.…”