Abstract. The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca 2+ ([Ca2+]~) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37~ with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15~ temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37~ for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15~ demonstrated in spotty cells a marked apparent blunting of Ca 2 § transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]~ was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca 2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]~ measurements. (34) and, more recently, fura-2 (14, 33). The remarkable advantages of the latter dye with respect to its older congener, discussed in detail by Tsien and his associates (14,33,(35)(36)(37), include higher fluorescence and quantum yield, lower affinity for Ca 2 § and favorable spectral properties, including shift of the exci-1. Abbreviations used in this paper: AM, acetoxy-methyl ester; AO, acridine orange; [Ca2+]i, free ionized Ca2+; HSE human skin fibroblasts; KRH, Krebs-Ringer medium buffered with Hepes. tation spectrum induced by Ca 2 § binding, with reciprocal changes of fluorescence across the isos...

Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP-ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 microM) showed a rapid and transient increase of polyADP-ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non-toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate-mediated neurotoxicity, structurally different inhibitors of pADPRP (3-aminobenzamide, benzamide,3-aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60-100%, assessed 24 hr later. Glutamate-induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca(2+)-mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death.

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The effects of glutamate on intracellular free Ca2+, [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i was measured with fura-2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dose-dependent increases in [Ca2+]i, which tended to be transient for glutamate concentrations in a range of 0.01-0.5 microM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was 6 microM. The LD50 for glutamate-induced neurotoxicity was similar, i.e., 10 microM. The effect of glutamate on [Ca2+]i was greatly diminished when external Ca2+ was removed and blocked by Mg2+ or N-methyl-D-aspartate (NMDA)-type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10-20 min at 25 degrees C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment of the cells with the ganglioside GM1 reduced this late increase in [Ca2+]i as well as the neurotoxic effects of glutamate. This indicates that glutamate-induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.

Excitatory amino acids (EAA) are known to induce an increase in the breakdown of polyphosphoinositides (PI) in brain slices and in dispersed cultures of neurons. We have now used astroglia cultured from newborn rat cerebra to demonstrate that glutamate provokes, in [3H]inositol-labeled cells, an accumulation of inositol phosphates in a time- and concentration-dependent manner. The ED50 value for glutamate was 40 microM. Quisqualate, ibotenate, and kainate were also active, with their relative potencies in the order of quisqualate greater than ibotenate much greater than kainate. No effect was detected with N-methyl-D-aspartate and quinolinic acid in the absence of Mg2+. The nonselective glutamate receptor antagonist gamma-D-glutamylglycine fully inhibited glutamate agonist-induced PI breakdown. A brief pretreatment of the astroglial cells with phorbol esters negated these effects of EAA receptor agonists, suggesting a feedback role for protein kinase C in phospholipase C action. Glutamate also elevated cytosolic free Ca2+ in Fura-2-loaded astroglial cells, as assessed by digital fluorescence imaging microscopy. Since a close metabolic partnership is known to exist between neurons and glia, these findings may have important functional consequences for neural cells in vivo.