Intracellular signalling pathway involved in ATP release from isolated smooth muscle cells.

在最近的研究中，人们知道ATP通过激活P2X和P2Y-嘌呤受体而表现出各种各样的细胞反应，因此似乎扮演着将信号传递到“细胞到细胞”的中介的角色。在我们的一系列研究中，我们的一系列研究发现，去甲肾上腺素和ACh刺激递质受体可以引起平滑肌节段释放ATP。在该项目的第一年(1995年)，用荧光素酶方法测定了豚鼠回肠和结肠带的培养的平滑肌细胞向超融合液中释放的ATP。在α，β-亚甲基三磷酸腺苷、苯二酚和血管紧张素Ⅱ等多肽的存在下，三磷酸腺苷的释放增加，增加的释放可被各自的拮抗剂减弱。因此，研究结果提示，ATP的释放来自于平滑肌细胞。1996年，该研究项目集中在有无α，β-亚甲基ATP存在和不存在的情况下，测定回肠纵肌匀浆中肌醇1，4，5-三磷酸(IP3)的积累。IP3的测定采用放射受体测定法，使用IP3分析试剂盒(amersham，TRK 1000)。P2激动剂促进了IP3在匀浆中的积累。新霉素和Li~+分别阻断磷脂酶C和磷脂酰肌醇的周转可抑制这种增强积累。α，β-亚甲基三磷酸腺苷诱导的三磷酸腺苷的释放对百日咳毒素不敏感，可被新霉素、精胺和Li^+拮抗，提示促进三磷酸腺苷的积累可能参与了三磷酸腺苷的释放。

In recent investigations, ATP is known to exhibit a wide-variety of cell responses mediated by activation of P2X and P2Y-purinoceptors and, thus, seems to play roles of mediators to transfer signals to "cell to cell".In a series of our studies, it has been persented findings that stimulation of transmitters' receptors with noradrenaline and ACh elicits ATP release from smooth muscle segments.During the first year (1995) of the project, ATP released to superfusate from cultured smooth muscle cells from ileum and taenia coli of guinea-pig was measured by the luciferase method. The release of ATP was increased in the presence of alpha, beta-methylene ATP,bethanechol and peptides such as angiotensin II.The increased release was attenuated by respective antagonists. Accordingly, the findings suggest that ATP release comes from the smooth muscle cells.In 1996, the research project was focused on determination of inositol 1,4,5-trisphosphate (IP3) accumulation from the homogenate of ileal longitudinal muscles in the persence and absence of alpha, beta-methylene ATP.IP3 was measured by a radio-receptor assay using a IP3 assay kit (Amersham, TRK 1000). The P2-agonist enhanced the accumulation of IP3 in the homogenate. The enhanced accumulation was inhibited by blockade of phospholipase C and phosphoinositide turnover with neomycin and Li^+, respectively. The release of ATP evoked by alpha, beta-methylene ATP was insensitive to pertussis toxin and was counteracted by neomycin and spermine and by Li^+.Taken together, it is concluded that the enhancement of IP3 accumulation may be involved in the evoked release of ATP.