Cytosolic Ca²⁺Changes duringIn VitroIschemia in Rat Hippocampal Slices: Major Roles for Glutamate and Na⁺-Dependent Ca²⁺Release from Mitochondria

Abstract

This work determined Ca2+ transport processes that contribute to the rise in cytosolic Ca2+ during in vitro ischemia (deprivation of oxygen and glucose) in the hippocampus. The CA1 striatum radiatum of rat hippocampal slices was monitored by confocal microscopy of calcium green-1. There was a 50-60% increase in fluorescence during 10 min of ischemia after a 3 min lag period. During the first 5 min of ischemia the major contribution was from Ca2+ entering via NMDA receptors; most of the fluorescence increase was blocked by MK-801. Approximately one-half of the sustained increase in fluorescence during 10 min of ischemia was caused by activation of Ca2+ release from mitochondria via the mitochondrial 2Na+-Ca2+ exchanger. Inhibition of Na+ influx across the plasmalemma using lidocaine, low extracellular Na+, or the AMPA/kainate receptor blocker CNQX reduced the fluorescence increase by 50%. The 2Na+-Ca2+ exchange blocker CGP37157 also blocked the increase, and this effect was not additive with the effects of blocking Na+ influx. When added together, CNQX and lidocaine inhibited the fluorescence increase more than CGP37157 did. Thus, during ischemia, Ca2+ entry via NMDA receptors accounts for the earliest rise in cytosolic Ca2+. Approximately 50% of the sustained rise is attributable to Na+ entry and subsequent Ca2+ release from the mitochondria via the 2Na+-Ca2+ exchanger. Sodium entry is also hypothesized to compromise clearance of cytosolic Ca2+ by routes other than mitochondrial uptake, probably by enhancing ATP depletion, accounting for the large inhibition of the Ca2+ increase by the combination of CNQX and lidocaine.

Na+ and Ca2+ homeostasis pathways, cell death and protection after oxygen–glucose-deprivation in organotypic hippocampal slice cultures

“…Intracellular Ca 2ϩ is abnormally increased in neurons exposed to ischemic conditions. This effect has been described for in vivo conditions (Nakamura et al, 1999;Silver and Erecinska, 1992), during in vitro ischemia in hippocampal slices (Lobner and Lipton, 1993;Zhang and Lipton, 1999) and in neuronal cell cultures exposed to mitochondrial and glycolytic inhibitors (Dubinsky and Rothman, 1991). In these studies, Ca 2ϩ rises during the first few minutes after ischemia and progressively returns close to the resting level after the end of the ischemic conditions.…”

“…Activation of these receptors results in opening of the associated ion channels and leads to the entry of extracellular Ca 2ϩ and Na ϩ into the cells. NMDAglutamate receptors are widely accepted as an important contributor to the [Ca 2ϩ ] i increase induced by ischemia (Kubo et al, 2001;Zhang and Lipton, 1999). Moreover, antagonists of this receptor type have been shown to protect neurons in vitro (Newell et al, 1995) and after transient global and focal ischemia (Foster et al, 1988;Hatfield et al, 1992).…”

“…Moreover, antagonists of this receptor type have been shown to protect neurons in vitro (Newell et al, 1995) and after transient global and focal ischemia (Foster et al, 1988;Hatfield et al, 1992). In contrast, the contribution of AMPA/ kainate receptors to both intracellular Ca 2ϩ and/or Na ϩ accumulation and neuronal cell loss is still controversial (Laake et al, 1999;Pringle et al, 1997;Tasker et al, 1992;Zhang and Lipton, 1999).…”

“…Under ischemic conditions, mitochondria are thought to accumulate Ca 2ϩ , to induce the generation of harmful reactive oxygen species and to release proapoptotic factors contributing to ischemic cell damage (Kristian and Siesjo, 1996). In acute hippocampal slices, mitochondrial Ca 2ϩ release is involved in the ischemic Ca 2ϩ elevation observed in dendrites of CA1 pyramidal neurons (Zhang and Lipton, 1999). In addition, Ca 2ϩ release from the endoplasmic reticulum has been proposed as an important mediator of ischemic cell loss (Paschen, 2003).…”

Prolonged Positive Modulation of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Induces Calpain-Mediated PSD-95/Dlg/ZO-1 Protein Degradation and AMPA Receptor Down-Regulation in Cultured Hippocampal Slices

“…However, AMPArs are Na ϩ channels and are, by and large, impermeable to Ca 2ϩ ions (Lomeli et al, 1994;Jensen et al, 1998;Carlson et al, 2000;Iizuka et al, 2000;Krampfl et al, 2002;Kumar et al, 2002). Interestingly, elevated intracellular Na ϩ levels can increase Ca 2ϩ release from intracellular stores (Hoyt et al, 1998;Zhang and Lipton 1999), thus CX614-induced increases in AMPAr function and the resulting depolarization may contribute to increased intracellular levels of Ca 2ϩ . Pertinent to this, ryanodine receptors, which regulate intracellular Ca 2ϩ release, are also targets for calpain proteolysis (Shoshan-Barmatz et al, 1994).…”