The influence of calcium transients on intracellular pH in cortical neurons in primary culture.

The objective of this study was to assess the influence of Ca2+ influx on intracellular pH (pHi) of neocortical neurons in primary culture. Neurons were exposed to glutamate (100-500 microM) or KCl (50 mM), and pHi was recorded with microspectrofluorometric techniques. Additional experiments were carried out in which calcium influx was triggered by ionomycin (2 microM) or the calcium ionophore 4-Br-A23187 (2 microM). Glutamate exposure either caused no, or only a small decrease in pHi (delta pH approximately 0.06 units). When a decrease was observed, a rebound rise in pHi above control was observed upon termination of glutamate exposure. In about 20% of the cells, the acidification was more pronounced (delta pH approximately 0.20 units), but all these cells had high control pHi values, and showed gradual acidification. Exposure of cells to 50 mM KCl consistently increased pHi. Since this increase was similar in the presence and nominal absence of HCO3-, it probably did not reflect influx of HCO3- via a Na(+)-HCO3- symporter. Furthermore, since it occurred in the absence of external Ca2+ (or a measurable rise in Cai2+) it seemed independent of Ca2+ influx. It is tentatively concluded that the rise in pHi was due to reduced passive influx of H+ along the electrochemical gradient, which is reduced by depolarization. In Ca(2+)-containing solutions, depolarization led to a rebound increase in pHi above control. This, and the rebound found after glutamate transients, may reflect Ca(2+)-triggered phosphorylation and upregulation of the Na+/H+ antiporter which extrudes H+ from the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of acid-base changes on the intracellular calcium concentration of neurons in primary culture.

The influence of changes in intra- and extracellular pH (pHi and pHe, respectively) on the cytosolic, free calcium concentration ([Ca2+]i) of neocortical neurons was studied by microspectrofluorometric techniques and the fluorophore fura-2. When, at constant pHe, pHi was lowered with the NH4Cl prepulse technique, or by a transient increase in CO2 tension, [Ca2+]i invariably increased, the magnitude of the rise being proportional to delta pHi. Since similar results were obtained in Ca(2+)-free solutions, the results suggest that the rise in [Ca2+]i was due to calcium release from intracellular stores. The initial alkaline transient during NH4Cl exposure was associated with a rise in [Ca2+]i. However, this rise seemed to reflect influx of Ca2+ from the external solution. Thus, in Ca(2+)-free solution NH4Cl exposure led to a decrease in [Ca2+]i. This result and others suggest that, at constant pHe, intracellular alkalosis reduces [Ca2+]i, probably by enhancing sequestration of calcium. When cells were exposed to a CO2 transient at reduced pHe, Ca2+ rose initially but then fell, often below basal values. Similar results were obtained when extracellular HCO3- concentration was reduced at constant CO2 tension. Unexpectedly, such results were obtained only in Ca(2+)-containing solutions. In Ca(2+)-free solutions, acidosis always raised [Ca2+]i. It is suggested that a lowering of pHe stimulates extrusion of Ca2+ by ATP-driven Ca2+/2H+ antiport.