Kramer R E
Department of Pharmacology, University of Tennessee, Memphis 38163.
Life Sci. 1991;48(1):27-35. doi: 10.1016/0024-3205(91)90422-8.
Studies were performed to determine if the sustained elevation in [Ca2+]c noted previously in glomerulosa cells in response to Ang II resulted from the presence of HEPES in the experimental medium. At confluence, primary cultures of bovine glomerulosa cells were maintained for 24-30 h in the presence of either 14 mM NaHCO3/5% CO2 or 25 mM HEPES/4 mM NaHCO3/air. During subsequent experimental periods, cells were incubated in the presence of the corresponding or reciprocal buffer, and the effects of Ang II on [Ca2+]c were monitored by fura 2 fluorescence. Increases in [Ca2+]c produced by Ang II in cells continuously maintained in either HCO3(-) - or HEPES-buffered media were similar, and with the same monolayer the nature of the Ang II-stimulated Ca2+ signal was independent of the buffer employed. Moreover, the Ang II-stimulated Ca2+ signal was not significantly affected by the removal of HCO3- from the superfusate. These results indicate that the sustained increase in [Ca2+]c is not an artifact introduced by the use of HEPES as an experimental buffer, but rather a normal component of the Ang II-stimulated Ca2+ signal.
开展了多项研究，以确定先前在球状带细胞中观察到的、对血管紧张素II产生反应的细胞内钙离子浓度（[Ca2+]c）持续升高是否源于实验培养基中存在HEPES。汇合后，将原代培养的牛球状带细胞在含有14 mM碳酸氢钠/5%二氧化碳或25 mM HEPES/4 mM碳酸氢钠/空气的条件下维持培养24 - 30小时。在随后的实验阶段，将细胞在相应或相反的缓冲液存在下孵育，并通过fura 2荧光监测血管紧张素II对[Ca2+]c的影响。在持续维持于碳酸氢根（HCO3(-)）缓冲或HEPES缓冲培养基中的细胞中，血管紧张素II所产生的[Ca2+]c升高情况相似，并且对于同一细胞单层，血管紧张素II刺激的钙离子信号性质与所使用的缓冲液无关。此外，从灌流液中去除HCO3-对血管紧张素II刺激的钙离子信号没有显著影响。这些结果表明，[Ca2+]c的持续升高并非使用HEPES作为实验缓冲液所引入的假象，而是血管紧张素II刺激的钙离子信号的正常组成部分。
