The cytoplasmic free calcium concentration ([Ca2+](i)) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P-2 purinergic receptors by ATP or UTP always evoked a [Ca2+](i) elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+](i) increase. Following this rebound, [Ca2+](i) did not return to the initial resting level, bur remained for long periods of time (up to 20 min), at a new; higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+](i) level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+](i).Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 mu M) caused a decrease in [Ca2+](i), despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+](i) level, stimulation of P-2U metabotropic purinoreceptors did not induce a [Ca2+](i) response. In addition, application of either thapsigargin (1 mu M) or carbonyl cyanide chlorophenyl hydrazone (10 mu M) failed to affect [Ca2+](i). We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted. (C) 1998 IBRO. Published by Elsevier Science Ltd.
