MECHANISMS OF C5A AND C3A COMPLEMENT FRAGMENT-INDUCED [CA2+](I) SIGNALING IN MOUSE MICROGLIA

Microglial cells are activated in response to brain insults; the mecha nisms of this process are not yet understood. One of the important sig naling mechanisms that might be involved in microglia activation is re lated to changes in the intracellular calcium concentration ([Ca2+](i) ). Using fluo-3 microfluorimetry, we have found that external applicat ion of the complement fragment C5a (4-10 nM) induced [Ca2+](i) elevati on in microglial cells in situ in corpus callosum slices. Similarly, a pplication of complement fragments C5a (0.1-10.0 nM) or C3a (100 nM) g enerates biphasic [Ca2+](i) transients composed of an initial peak fol lowed by a plateau in cultured microglia. Incubation of microglial cel ls for 30 min with pertussis toxin (PTX; 1 mu g/ml) inhibited both C5a - and C3a-triggered [Ca2+](i) responses, suggesting the involvement of PTX-sensitive G-proteins in the signal transduction chain. Removal of Ca2+ ions from the extracellular solution eliminated the plateau phas e and limited the response to the initial peak. The restoration of the extracellular Ca2+ concentration within 30-60 sec after the beginning of the complement fragment-induced [Ca2+](i) elevation led to the rec overy of the plateau phase. Inhibition of the endoplasmic reticulum Ca 2+ pumps with 500 nM thapsigargin transiently increased the [Ca2+](i) and blocked the [Ca2+](i) signals in response to subsequent complement fragment application. Our data suggest that complement factors induce [Ca2+](i) responses by Ca2+ release from internal pools and subsequen t activation of Ca2+ entry controlled by the filling state of the intr acellular Ca2+ depots.