Effect of intracellular pH on cytosolic free [Ca2+] in human epidermoid A-431 cells

Abstract

This study characterizes the correlation between intracellular pH (pHi) and the cytosolic free Ca2+ concentration ([Ca2+]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 pHi and [Ca2+]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 Ca2+ did not change the resting pHi but reduced the resting [Ca2+]i, indicating the resting level of [Ca2+]i is in part maintained by an influx of Ca2+ from the external medium. When both suspended and adherent cells were acidified or alkalinized, resting [Ca2+]i was altered. An intracellular acidification induced a fall in [Ca2+]i, and a rise in pHi induced a rise in [Ca2+]i. These changes in [Ca2+]i were correlated with an uptake of 45Ca2+ from the external medium, whereas no Ca2+ efflux occurred. The alteration in [Ca2+]i induced by modification of pHi was abolished in the absence of external Ca2+ or by adding 2 mM CoCl2, LaCl3, and attenuated by the addition of 2 mM MnCl2 to the bathing medium. It was insensitive to the voltage-gated Ca2+ channel blockers nifedipine or verapamil (1 mM). CoCl2, LaCl3, and MnCl2 each induced changes in pHi and [Ca2+]i but verapamil and nifedipine did not. Because CoCl2, LaCl3, and MnCl2 are also known to block Na+/Ca2+ 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 microM), a blocker of the Na+/Ca2+ exchanger, did not inhibit the pH-induced changes in [Ca2+]i. 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (100 microM), a Ca2+ chelator, induced a decrease in pHi as well as a reduction of [Ca2+]i, also supporting the direct relation between pHi and [Ca2+]i. 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)ocytl ester HCl (TMB-8) (100 microM), a known blocker of intracellular Ca2+ mobilization, did not change the resting pHi and [Ca2+]i in normal cells or cells acidified or alkalinized. This observation, taken together with data from cells incubated in the absence of external Ca2+, suggests intracellular Ca2+ pools are not involved in changes in [Ca2+]i that result from a modification of pHi. Resting pHi and [Ca2+]i in cells treated with either 8-bromo-dibutyryl cAMP (1 mM) or forskolin (150 microM) are not changed.(ABSTRACT TRUNCATED AT 400 WORDS)