Effect of intracellular pH on cytosolic free [Ca 2+] in human epidermoid A-431 cells

Abstract

This study characterizes the correlation between intracellular pH (pH i) and the cytosolic free Ca 2+ concentration ([Ca 2+] i) in suspended and adherent human epidermoid A-431 cells. Using the fluorescent dyes 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester (BCECF) and fura-2, the resting pH i and [Ca 2+] i in suspended cells were 7.23 ± 0.03 and 209 ± 30 nM; those in adherent cells were 7.28 ± 0.02 and 87 ± 5 nM. Removal of external Ca 2+ did not change the resting pH i but reduced the resting [Ca 2+] i, indicating the resting level of [Ca 2+] i is in part maintained by an influx of Ca 2+ from the external medium. When both suspended and adherent cells were acidified or alkalinized, resting [Ca 2+] i was altered. An intracellular acidification induced a fall in [Ca 2+] i, and a rise in pH i induced a rise in [Ca 2+] i. These changes in [Ca 2+] i were correlated with an uptake of 45Ca 2+ from the external medium, whereas no Ca 2+ efflux occurred. The alteration in [Ca 2+] i induced by modification of pH i was abolished in the absence of external Ca 2+ or by adding 2 mM CoCl 2, LaCl 3, and attenuated by the addition of 2 mM MnCl 2 to the bathing medium. It was insensitive to the voltage-gated Ca 2+ channel blockers nifedipine or verapamil (1 mM). CoCl 2, LaCl 3, and MnCl 2 each induced changes in pH i and [Ca 2+] i but verapamil and nifedipine did not. Because CoCl 2, LaCl 3, and MnCl 2 are also known to block Na +/Ca 2+ exchange, intracellular Na + ([Na +] i) was measured by flame photometry in acidified or alkalinized cells. In either case no change in [Na +] i was observed. Furthermore, treatment with amiloride (100 μM), a blocker of the Na +/Ca 2+ exchanger, did not inhibit the pH-induced changes in [Ca 2+] i. 1,2-bis(o-Aminophenoxy)ethane-N,N,N′,N′-te-traacetic acid (BAPTA) (100 μM), a Ca 2+ chelator, induced a decrease in pH i as well as a reduction of [Ca 2+] i, also supporting the direct relation between pH i and [Ca 2+] i. 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)octyl ester HCl (TMB-8) (100 μM), a known blocker of intracellular Ca 2+ mobilization, did not change the resting pH i and [Ca 2+] i in normal cells or cells acidified or alkalinized. This observation, taken together with data from cells incubated in the absence of external Ca 2+, suggests intracellular Ca 2+ pools are not involved in changes in [Ca 2+] i that result from a modification of pH i. Resting pH i and [Ca 2+] i in cells treated with either 8-bromo-dibutyryl cAMP (1 mM) or forskolin (150 μM) are not changed. These results suggest a positive correlation between pH i and [Ca 2+] i in human A-431 cells that is primarily due to Ca 2+ influx and not related to voltage, second messenger-operated Ca 2+ channels, or the Na +/Ca 2+ exchange.