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CICR from an intracellular store, here directly characterized as the ER, usually refers to net [Ca.sup.2+] release that amplifies evoked elevations in cytosolic free calcium ([[[Ca.sup.2+]].sub.i]). However, the companion paper (Albrecht, M.A., S.L. Colegrove, J. Hongpaisan, N.B. Pivovarova, S.B. Andrews, and D.D. Friel. 2001. J. Gen. Physiol. 118:83-100) shows that in sympathetic neurons, small [[[Ca.sup.2+]].sub.i] elevations evoked by weak depolarization stimulate ER Ca accumulation, but at a rate attenuated by activation of a ryanodine-sensitive CICR pathway. Here, we have measured depolarization-evoked changes in total ER Ca concentration ([[Ca].sub.ER]) as a function of [[[Ca.sup.2+]].sub.i], and found that progressively larger [[[Ca.sup.2+]].sub.i] elevations cause a graded transition from ER Ca accumulation to net release, consistent with the expression of multiple modes of CICR. [[Ca].sub.ER] is relatively high at rest (12.8 [+ or -] 0.9 mmol/kg dry weight, mean [+ or -] SEM) and is reduced by thapsigargin or ryanodine (5.5 [+ or -] 0.7 and 4.7 [+ or -] 1.1 mmol/ kg, respectively). [[Ca].sub.ER] rises during weak depolarization (to 17.0 [+ or -] 1.6 mmol/kg over 120s, [[[Ca.sup.2+]].sub.i] less than ~350 nM), changes little in response to stronger depolarization (12.1 [+ or -] 1.1 mmol/kg, [[[Ca.sup.2+]].sub.i] ~700 nM), and declines (to 6.5 [+ or -] 1.0 mmol/kg) with larger [[[Ca.sup.2+]].sub.i] elevations ([is greater than] 1 [micro]M) evoked by the same depolarization when mitochondrial [Ca.sup.2+] uptake is inhibited (FCCP). Thus, net ER [Ca.sup.2+] transport exhibits a biphasic dependence on [[[Ca.sup.2+]].sub.i]. With mitochondrial [Ca.sup.2+] uptake enabled, [[Ca].sub.ER] rises after repolarization (to 16.6 [+ or -] 1.8 mmol/kg at 15 min) as [[[Ca.sup.2+]].sub.i] falls within the permissive range for ER Ca accumulation over a period lengthened by mitochondrial [Ca.sup.2+] release. Finally, although spatially averaged [[Ca].sub.ER] is unchanged during strong depolarization, net ER [Ca.sup.2+] release still occurs, but only in the outermost ~5-[micro]m cytoplasmic shell where [[[Ca.sup.2+]].sub.i] should reach its highest levels. Since mitochondrial Ca accumulation occurs preferentially in peripheral cytoplasm, as demonstrated here by electron energy loss Ca maps, the Ca content of ER and mitochondria exhibit reciprocal dependencies on proximity to sites of [Ca.sup.2+] entry, possibly reflecting indirect mitochondrial regulation of ER [Ca.sup.2+] transport. KEY WORDS: calcium signaling * mitochondria * ryanodine * electron probe X-ray microanalysis * electron energy loss spectrum imaging

Many cells express ryanodine receptors (RyRs) whose activation is thought to amplify depolarization-evoked elevations in cytoplasmic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) through a process of [Ca.sup.2+]-induced [Ca.sup.2+] release (CICR). In neurons, it is usually assumed that CICR triggers net [Ca.sup.2+] release from an ER [Ca.sup.2+] store. However, since net ER [Ca.sup.2+] transport depends on the relative rates of [Ca.sup.2+] uptake and release via distinct pathways, weak activation of a CICR pathway during periods of ER Ca accumulation would have a totally different effect: attenuation of [Ca.sup.2+] accumulation. Stronger CICR activation at higher [[[Ca.sup.2+]].sub.i] could further attenuate [Ca.sup.2+] accumulation or trigger net [Ca.sup.2+] release, depending on the quantitative properties of the underlying [Ca.sup.2+] transporters. This and the companion study (Hongpaisan, J., N.B. Pivovarova, S.L. Colgrove, R.D. Leapman, and D.D. Friel, and S.B. Andrews. 2001. J. Gen. Physiol. 118:101-112) investigate which of these CICR 'modes' operate during depolarization-induced [Ca.sup.2+] entry in sympathetic neurons. The present study focuses on small [[[Ca.sup.2+]].sup.i] elevations (less than ~350 nM) evoked by weak depolarization. The following two approaches were used: (1) [Ca.sup.2+] fluxes were estimated from simultaneous measurements of [[[Ca.sup.2+]].sub.i] and [I.sub.Ca] in fura-2-loaded cells (perforated patch conditions), and (2) total ER Ca concentrations ([[Ca].sub.ER]) were measured using X-ray microanalysis. Flux analysis revealed triggered net [Ca.sup.2+] release during depolarization in the presence but not the absence of caffeine, and [[[Ca.sup.2+]].sub.i] responses were accelerated by SERCA inhibitors, implicating ER [Ca.sup.2+] accumulation, which was confirmed by direct [[Ca].sub.ER] measurements. Ryanodine abolished caffeine-induced CICR and enhanced depolarization-induced ER [Ca.sup.2+] accumulation, indicating that activation of the CICR pathway normally attenuates ER [Ca.sup.2+] accumulation, which is a novel mechanism for accelerating evoked [[[Ca.sup.2+]].sub.i] responses. Theory shows how such a low gain mode of CICR can operate during weak stimulation and switch to net [Ca.sup.2+] release at high [[[Ca.sup.2+]].sub.i], a transition demonstrated in the companion study. These results emphasize the importance of the relative rates of [Ca.sup.2+] uptake and release in defining ER contributions to depolarization-induced [Ca.sup.2+] signals. KEY WORDS: calcium signaling * endoplasmic reticulum * caffeine * ryanodine * electron probe X-ray microanalysis

Glutamate excitotoxicity, a major component of many neurodegenerative disorders, is characterized by excessive calcium influx selectively through NMDARs. However, there is a substantial uncertainty concerning why other known routes of significant calcium entry, in particular, VGCCs, are not similarly toxic. Here, we report that in the majority of neurons in rat hippocampal and cortical cultures, maximal L-type VGCC activation induces much lower calcium loading than toxic NMDAR activation. Consequently, few depolarizationactivated neurons exhibit calcium deregulation and cell death. Activation of alternative routes of calcium entry induced neuronal death in proportion to the degree of calcium loading. In a small subset of neurons, depolarization evoked stronger calcium elevations, approaching those induced by toxicNMDA. These neurons were characterized by elevated expression of VGCCs and enhanced voltage-gated calcium currents, mitochondrial dysfunction and cell death. Preventing VGCC-dependent mitochondrial calcium loading resulted in stronger cytoplasmic calcium elevations, whereas inhibiting mitochondrial calcium clearance accelerated mitochondrial depolarization. Both observations further implicate mitochondrial dysfunction in VGCC-mediated cell death. Results indicate that neuronal vulnerability tracks the extent of calcium loading but does not appear to depend explicitly on the route of calcium entry. [ABSTRACT FROM AUTHOR]

In neurons, excitotoxic stimulation induces mitochondrial calcium overload and the release of pro-apoptotic proteins, which triggers delayed cell death. The precise mechanisms of apoptogen release, however, remain controversial. To characterize the linkage between mitochondrial calcium load and cell vulnerability, and to test the hypothesis that only a subpopulation of mitochondria damaged by calcium overload releases apoptogens, we have measured directly the concentrations of total Ca (free plus bound) in individual mitochondria and monitored in parallel structural changes and the subcellular localization of pro-apoptotic cytochrome c after NMDA overstimulation in cultured hippocampal neurons. Beyond transient elevation of cytosolic calcium and perturbation of Na+/K+ homeostasis, NMDA stimulation induced dramatic, but mainly reversible, changes in mitochondria, including strong calcium elevation, membrane potential depolarization, and variable swelling. Elevation of matrix Ca in the approximately one-third of mitochondria that were strongly swollen, as well as the absence of swelling when Ca2+ entry was abolished, indicate an essential role for Ca overload. Shortly after NMDA exposure, cytochrome c, normally localized to mitochondria, became diffusely distributed in the cytoplasm, coincident with the appearance of severely swollen mitochondria with ruptured outer membranes; under these conditions, cytochrome c was retained in intact mitochondria, implying that it was released mainly from damaged mitochondria. Consistent with the role of mitochondrial Ca overload, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone decreased Ca accumulation, prevented cytochrome c release, and was neuroprotective. These results support a mechanism in which delayed excitotoxic death involves apoptogen release from a subpopulation of calcium-overloaded mitochondria, whereas other, undamaged mitochondria maintain normal function. [ABSTRACT FROM AUTHOR]