Effect of buffer systems and pH_i on the measurement of [Ca²⁺]_i with fura 2

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca²⁺]_i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO₂/HCO₃^-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca²⁺]_i when HEPES vs. CO₂/HCO₃^- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca²⁺]_i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO₂/HCO₃^- buffer, agonist addition led to an identical transient increase in [Ca²⁺]_i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca²⁺]_i in the different buffers was examined in relationship to known differences in intracellular pH (pH_i). It was found that measurements of [Ca²⁺]_i with fura 2 were influenced by shifts in pH_i that occur when cells are incubated in either HEPES-buffered or CO₂/HCO₃^- media of differing pH_○ values. However, at any given value of pH_i, the apparent [Ca²⁺]_i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO₂/HCO₃^- buffered media.