目的:为了量化新鲜分离的膀胱尿路上皮细胞释放的ATP量,研究其细胞内和细胞外钙的控制,并确定其释放的途径。
方法:从雄性豚鼠膀胱中分离出尿路上皮细胞,并通过反复移液施加阻力刺激释放ATP。使用萤光素-萤光素酶测定法测量ATP,并研究改变内部和外部钙浓度和潜在释放途径的阻断剂的影响。
结果:新鲜分离的豚鼠尿路上皮细胞以1.9(0.1)pmoles/mm(2)细胞膜的平均(sem)速率释放ATP,相当于约700皮摩尔/克组织,大约一半[49(6)%,n=9)的可用细胞ATP。这种释放减少到平均(sem)0.46(0.08)pmoles/mm(2)(160pmoles/g)与1.8mm外部钙,并通过增加细胞内钙而增加约两倍。伞形细胞的释放与混合的中间和基础细胞群体没有显着差异,这表明三组细胞每单位面积释放相似量的ATP。通过阻断pannexin和连接蛋白半通道的药物,ATP释放减少了约50%。建议其余的可能涉及囊泡释放。
结论:通过施加引起细胞活力最小损失的阻力,从分离的尿路上皮细胞释放显著部分的细胞ATP。这种释放涉及多种释放途径,包括半通道和囊泡释放。
OBJECTIVE: To quantify the amount of ATP released from freshly isolated bladder urothelial cells,
study its control by intracellular and extracellular calcium and identify the pathways responsible for its release.
METHODS: Urothelial cells were isolated from male guinea-pig urinary bladders and stimulated to release ATP by imposition of drag forces by repeated pipetting. ATP was measured using a luciferin-luciferase assay and the effects of modifying internal and external calcium concentration and blockers of potential release pathways studied.
RESULTS: Freshly isolated guinea-pig urothelial cells released ATP at a mean (sem) rate of 1.9 (0.1) pmoles/mm(2) cell membrane, corresponding to about 700 pmoles/g of tissue, and about half [49 (6)%, n = 9) of the available cell ATP. This release was reduced to a mean (sem) of 0.46 (0.08) pmoles/mm(2) (160 pmoles/g) with 1.8 mm external calcium, and was increased about two-fold by increasing intracellular calcium. The release from umbrella cells was not significantly different from a mixed intermediate and basal cell population, suggesting that all three groups of cells release a similar amount of ATP per unit area. ATP release was reduced by ≈ 50% by agents that block pannexin and connexin hemichannels. It is suggested that the remainder may involve vesicular release.
CONCLUSIONS: A significant fraction of cellular ATP is released from isolated urothelial cells by imposing drag forces that cause minimal loss of cell viability. This release involves multiple release pathways, including hemichannels and vesicular release.