| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Stretch-induced myosin light chain (MLC) phosphorylation in canine basilar artery was investigated. In the presence of tetraethylammonium (5 mM), slow stretch at a rate of 1 mm/sec up to 1.5 times initial muscle length during a stimulus period of 15 min produced an increase in multiple phosphorylated MLC species (mono-, di- and tri-phosphorylated species). Although this multiple phosphorylation of MLC was partly inhibited by ML-9, an inhibitor of myosin light chain kinase, and calphostin C, a protein kinase C (PKC) inhibitor, it was abolished by ML-9 plus calphostin C. Y-27632, a Rho-kinase inhibitor, almost completely inhibited MLC phosphorylation. Stretch increased the phosphorylation of 130 kDa regulatory subunit of myosin phosphatase (type 1, MBS), resulting the inhibition of myosin phosphatase. Y-27632 inhibited the phosphorylation of MBS. Of 4 PKC isoforms (PKC_α, δ,ζand η), only PKC_α was translocated from the cytosol to the membrane fraction by 15-min stretch. Multiple phosphorylation of MLC was inhibited by Go6976, an inhibitor of classical PKC, but not 5 μM rottlerin, a specific inhibitor of PKCδ. On the other hand, 3μM okadaic acid (OA), which inhibited phosphatase 2A activity at this concentration, attenuated 80 mM KCl-induced contraction of canine basilar artery. This attenuation was countered by Go6976, but not by rotterin. OA produced the multiple phosphorylated MLC species and phosphorylation pattern was similar to that produced by stretch. OA had no effect on the phosphorylation pattern produced by stretch. Actomyosin ATPase activity was inhibited by 15-min stretch and OA. These results suggest than MLCK and PKCα activities unmasked by the inhibition of phosphatases type 1 and type 2A which mediated by Rho/Rho-kinase pathway may be involved in stretch-induced multiple phosphorylation of MLC in the canine basilar artery.
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