Plasmalemmal and intracellular Ca2+ pumps as main determinants of slow Ca2+ buffering in rat hippocampal neurones

Using the Ca(2+)-sensitive fluorescent indicator dye fura-2, the mechanisms by which cytoplasmic free Ca2+ concentration, [Ca]i, decays to resting levels were studied in neurones cultured from the rat hippocampus. The time-course of [Ca]i restoration after transient elevations due to CaCl2 injections or brief exposures to 50 mM K Cl were biexponential. Application of specific inhibitors of systems participating in Ca2+ removal from cytoplasm changed both basal [Ca]i and the slow phase of recovery, but the fast phase was unaltered by any treatment. Inhibition of the plasmalemmal Ca2+ pump by external alkalinization or intracellular acidification was reversible, whereas calmodulin inhibitors (calmidazolium and triftazine, W-13) acted irreversibly. The net effects of blockers of the intracellular Ca2+ pump, thapsigargin (Tg) and t-BuHQ, were similar. Suppression of mitochondrial Ca2+ uptake or Ca2+ extrusion due to Na+/Ca2+ exchange, reversibly increased [Ca]i but the time-course of [Ca]i clearance was marginally changed. After glutamate application [Ca]i restoration was prolonged which was mediated by concomitant intracellular acidification causing inhibition of plasmalemmal Ca2+ ATPase. It is concluded that Ca2+ homeostasis in rat hippocampal neurones is mainly determined by Ca2+ pumps in both the surface membrane and internal stores, whereas Na+/Ca2+ exchange and mitochondria play a minor role.