The Signaling Pathway of SPC-Induced Ca2+-Sensitization Leading to Vasospasm
ROK is regarded as a molecule to mediate Ca2+-sensitization. The active ROK phosphorylated myosin light chain phosphatase target subunit 1 (MYPT1) and inactivated myosin light chain phosphatase (Kimura et al., 1998). In addition, ROK can directly phosphorylate and activate MLC (Kureishi et al., 1997). Therefore, ROK mediates Ca2+-sensitization through the inactivation of myosin light chain phosphatase and activation of MLC. However, the mechanism by which ROK mediated Ca2+-sensitization has not been fully elucidated. We identified SPC as an upstream signaling molecule for the ROK-mediated Ca2+-sensitization (Todoroki- Ikeda et al., 2000). First, SPC-induced Ca2+-sensitization of VSM contraction was inhibited by the specific ROK inhibitor Y27632, suggesting that ROK involved in SPC-induced contraction. Second, both dominant negative ROK (dn-ROK) and ROK inhibiter Y27632 abolished SPC-induced Ca2+-sensitization of VSM contraction in VSM strips permeabilized with b-escin. Conversely, dn-ROK showed no effect on Ca2+-sensitization of VSM contraction induced by phorbol ester, a protein kinase C activator. In addition, SPC induced the translocation of ROK from the cytosol to the cell membrane of VSM cells (Shirao et al., 2002). This translocation was demonstrated to play an important role in the activation of ROK. These results indicated that SPC-ROK pathway mediates Ca2+-sensitization of VSM contraction.
We then found that a member of the Src family protein tyrosine kinases (Src-PTKs), Fyn, was involved in the SPC-ROK pathway mediated Ca2+-sensitization of contraction as an upstream signaling molecule of ROK (Nakao et al., 2002). PP1, a specific inhibitor of Src-PTKs, abolished SPC-induced Ca2+-sensitization of contraction. However, PP3, an inactive analogue of PP1, could not inhibit SPC-induced contraction. In VSM cells stimulated by SPC, Fyn, not Src, translocated from the cytosol to the cell membrane (Nakao et al., 2002). This translocation is related to S-palmitoylation playing an important role in protein activation and signal transduction. 2-Bromopalmitate, a specific inhibitor for S-palmitoylation of Fyn, inhibited SPC-induced Ca2+-sensitization of contraction. Eicosapentaenoic acid (EPA), an inhibitor for Fyn S-palmitoylation, inhibited both SPC-induced contraction and translocation of Fyn (Nakao et al., 2002). Moreover, PP1 and EPA inhibited SPC-induced Ca2+-sensitization of VSM contraction, translocation, and activation of ROK. These results suggested that Fyn as an upstream signaling molecule of ROK is involved in SPC-ROK-mediated Ca2+-sensitization leading to vasospasm.