| Budget Amount *help |
¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
The small G protein, rho, has been shown to inhibit myosin light chain phosphatase (MLCP) through the phosphorylation of this enzyme by rho kinase. It was thus obvious that MLCP may be the key protein for the rho-mediated signal transduction to induce modulation of calcium sensitivity of the vascular smooth muscle. MLCP is composed of three subunits, namely, catalytic subunit, small regulatory subunit and large regulatory subunit. We focused on the function of two regulatory subunits of MLCP on the smooth muscle contractile apparatus in the present research program. The major findings were as follows.
1. The effects of the small regulatory subunit of myosin light chain phosphatase (MLCPsr) on the Ca^<2+>-induced contraction of smooth muscle were investigated in the Triton X-100-permeabilized porcine renal artery. The full-length recombinant chicken MLCPsr obtained by the bacterial expression system induced an additional contraction at a constant [Ca^<2+>] i and shifted the [Ca^<2+>] i-f
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orce relation curve to the left. A deletion mutant containing the N-terminal 78 amino acids of MLCPsr retained the full action, compared with the full-length MLCPsr, while the deletion of this region completely abolished its effect. The process of relaxation was also delayed by the fragment containing the N-terminal 78 amino acids. These results indicated that MLCPsr increases the Ca^<2+> sensitivity of the contractile apparatus while the N-terminal 78 amino acids are responsible for this effect in vascular smooth muscle.
2. The effects of the NH2-terminal fragments of the large regulatory subunit of myosin light chain phosphatase (MLCPlr) on the Ca^<2+>-induced contraction of smooth muscle were investigated in the Triton X-100-permeabilized porcine renal artery. MLCPlr 1-374, MLCPlr 304-511 and MLCPlr 297-374 induced leftward shifts of the [Ca^<2+>] i-force relationship. Deletion of residues 304-374 from the most potent construct, MLCPlr 1-374, abolished the Ca^<2+>-sensitizing effect. M1301-374 slowed the rate of relaxation in Ca^<2+>-free medium. The levels of myosin light chain phosphorylation (22.4%) and force (34.5%) obtained with 300 nM Ca^<2+> were increased by 3 microMMLCPlr 1-374 to 35.7 and 92.2%, respectively.These results indicated that the NH2-terminal MLCPlr fragments containing residues 304-374 inhibited myosin phosphatase, increased myosin light chain phosphorylation and increased the Ca^<2+> sensitivity of the contractile apparatus in permeabilized porcine renal artery.
3. We have isolated cDNAs for heart-specific small regulatory subunit of myosin light chain phosphatase (HS-MLCPsr) that were transcribed from a heart-specific promoter in intron 13 of the MYPT2 gene. Functional analysis in permeabilized rat cardiac myocytes and porcine renal artery revealed that HS-MLCPsr enhanced Ca^<2+>-induced contraction, and the main functional domain was mapped in the N-terminal half of HS-MLCPsr. In addition, HS-MLCPsr bound to C-terminal one-third of MYPT1, and the binding domain was also mapped in the N-terminal half of HS-MLCPsr. These results indicated that HS-MLCPsr plays a role in regulation of the cardiac muscle contraction via binding to MYPT1. Less
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