| Budget Amount *help |
¥3,760,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥360,000)
Fiscal Year 2007: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Preventing and treating atherosclerosis is an important clinical issue since its development is the primary cause of cardiovascular diseases. The aberrant proliferation of vascular smooth muscle cells (SMCs) has been accepted as a key event in the pathophysiology of atherosclerosis. To search for new molecular target(s) of anti-atherosclerotic drugs, we intended to regulate SMC growth by controlling intracellular calcium signaling and nitric oxide (NO)-mediated signaling. Calcium channel blockers (CCBs) are useful for this purpose, because some of these drugs exhibit anti-atherosclerotic action via mechanism(s) other than L type Ca^<2+> channel blockade. Human epidermoid carcinoma A431 cell line which lacks the L type Ca^<2+> channel was used as a target cell line. Analysis of cell growth demonstrated that dihydropyridine CCBs such as amlodipine inhibit the growth of A431 cells. Antiproliferative CCBs specifically attenuated the capacitative Ca^<2+> entry evoked by Ca^<2+> store deplet
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ion and phospholipase C-coupled receptor stimulation. Reverse transcription-polymerase chain reaction analysis showed that A431 cells express mRNAs of canonical transient receptor potential 1 and 5 (TRPC1, TRPC5), molecular candidates for store-operated and receptor-activated cation channels. Cell cycle analysis demonstrated that amlodipine induced Gi cell cycle arrest in A431 cells. Under this condition, amlodipine decreased the phosphorylation of retinoblastoma protein, a regulator of G1 to S phase transition, and kinase activities associated with cell cycle regulators such as cyclin Dl and cyclin-dependent kinase 4 (CDK4), whereas increased CDK inhibitor p21^<waf1/Cip1> protein expression. These results demonstrated that amlodipine caused GI cell cycle arrest and growth inhibition in A431 cells through induction of p21^<waf1/Cip1> (Yoshida J., et al., Biochem. Pharmacol., 73: 943-953, 2007). Therefore, p21^<waf1/Cip1> might be a key molecule for the inhibition of SMC growth, because its negative role in the progression of atherosclerosis is suggested. In this study, we established a method for quantifying the nanomolar levels of nitrite (NO_2), a metabolite of nitric oxide (NO), in biological samples (Ishibashi T., et al., Tohoku J. Exp. Med., 215:2008, in press). This sensitive method is applicable for exploring mechanism(s) underlying the NO-releasing effect of amlodipine and SMC growth inhibition by NO. Less
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