| Budget Amount *help |
¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
The studies were undertaken to determine what kind of intracellular Ca^<2+> signals is involved in the autocrine/paracrine release of ATP evoked by receptor stimulation from cultured smooth muscle cells (SMCs) from guinea-pigs. Angiotensin II (Ang II)(0.3-1 μM) elicited substantial release of ATP from cultured taenia coli SMCs, but not from a human fibroblast cell line. However, Ang II even at 10 μM failed to cause a leakage of lactate dehydrogenase (LDH) from the SMCs. The release of ATP by Ang II was suppressed by 10 μM SC52458, an AT_1 receptor antagonist, not by 10 μM PD123319, an AT_2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 μM U-73122, a phospholipase C inhibitor, and 0.5 μM thapsigargin, a Ca^<2+>-ATPase inhibitor. Furthermore, the release was hampered by 50 μM BAPTA/AM, an intracellular Ca^<2+> chelator, but not by 0.1 μM nifedipine, a voltage gated Ca^<2+> channel inhibitor. The basal release of ATP was increased by B
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APTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P_3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca^<2+> signals activated via stimulation of Ins(1,4,5)P_3 receptor are involved in the release of ATP evoked by Ang II. In Ca^<2+> free Krebs solution, caffeine at 3 mM instantaneously caused a remarkable release of ATP from cultured vas deferens SMCs, not from a human fibroblast cell line. The evoked release of ATP was markedly abolished by treatment with BAPTA/AM and thapsigargin. Furthermore, the release was completely inhibited by ryanodine and tetracaine, ryanodine receptor antagonists. From the study of [Ca^<2+>]i measurement with fluo 4, caffeine induced an elevation of [Ca^<2+>]i and the effect of caffeine was absolutely antagonized by tetracaine. In RT-PCR studies, the expression of mRNA from RyR-2, but not from RyR-1, was observed in vas deferens SMCs as well as ileal longitudinal SMCs from guinea-pigs. These findings suggest that the release of ATP induced by caffeine is triggered by the intracellular Ca^<2+> signal via ryanodine receptor (RyR-2) stimulation on endoplasmic/sarcoplasmic reticulum. Less
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