N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) does not modify the angiotensin II-stimulated calcium signal in cultured bovine glomerulosa cells

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.