Activation of protein kinases in canine basilar artery in vasospasm

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cereb ra! artery called vasospasm. To understand the molecular mechanisms in vaso spasm, signal transduction of tyrosine kinase pathway and phosphorylation o f myosin light chain (MLC) and calponin (CaP) in the basilar artery were st udied. Vasospasm was produced in the canine basilar artery by a two-hemorrh age method, and vasocontraction was induced by a local application of KCl o r serotonin to the basilar artery after a transclival exposure. Intracellul ar substrates of tyrosine kinase pathway, including She, Raf1, and extracel lular-regulated kinases in the basilar artery, were activated after SAH, an d the activation of She suggests stimulation of signal transductions from t yrosine kinase receptors, G-coupled receptors, or both. The activation of t yrosine kinase pathway in vasospasm also was supported by dose-dependent di lation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibitio n of tyrosine phosphorylation of intracellular substrates, including She. I n addition, the generation of protein kinase M, catalytic fragment of prote in kinase C alpha (PKC alpha), in vasospasm on days 0 and 7 was inhibited i n response to genistein, indicating an inactivation of mu-calpain. It is su ggested, 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 vasospa sm were declined on day 7 compared with those on day 0 or 2, suggesting tha t the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The an alysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demon strated three MLC bands in vasospasm on days 2 and 7, as well as in KCl- an d 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 phospho rylation state, the current results suggest that MLC in vasospasm is phosph orylated by MLCK but not by PKC. In basilar artery, CaP was significantly d own-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the signific ance of Cap phosphorylations in vivo still is controversial, CaP downregula tion and phosphorylation may attenuate the inhibition of Mg2+-ATPase activi ty by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated in viva by PKC, activate d PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kin ase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.