Early metabolic inhibition‐induced intracellular sodium and calcium increase in rat cerebellar granule cells

Abstract

Possible mechanisms responsible for the increases in intracellular calcium ([Ca2+]i) and sodium ([Na+]i) levels seen during metabolic inhibition were investigated by continuous [Ca2+]i and [Na+]i measurement in cultured rat cerebellar granule cells. An initial small mitochondrial Ca2+ release was seen, followed by a large influx of extracellular Ca2+. A large influx of extracellular Na+ was also seen. The large [Ca2+]i increase was not due to opening of voltage‐dependent or voltage‐independent calcium channels, activation of NMDA/non‐NMDA channels, activation of the Na+i‐Ca2+o exchanger, or inability of plasmalemmal Ca2+‐ATPase to extrude, or mitochondria to take up, calcium. The large [Na+]i increase was not due to activation of the TTX‐sensitive Na+ channel, the Na+i‐Ca2+o exchanger, the Na+‐H+ exchanger, or the Na+‐K+‐2Cl− cotransporter, or an inability of Na+‐K+‐ATPase to extrude the intracellular sodium. Phospholipase A2 (PLA2) activation may be involved in the large influx, since both were completely inhibited by PLA2 inhibitors. Moreover, melittin (a PLA2 activator) or lysophosphatidylcholine or arachidonic acid (both PLA2 activation products) caused similar responses. Inhibition of PLA2 activity may help prevent the influx of these ions that may result in serious brain injury and oedema during hypoxia/ischaemia.

Sodium Influx Pathways during and after Anoxia in Rat Hippocampal Neurons

“…Before anoxia, resting [Na ϩ ] i was 11 Ϯ 1 mM (n ϭ 444), a value similar to those reported previously in mammalian central neurons both in culture (Rose and Ransom, 1997;Chen et al, 1999;Diarra et al, 2001) and in slice preparations (Pisani et al, 1998;Calabresi et al, 1999). Five-minute anoxia evoked an increase in [Na ϩ ] i that began ϳ90 sec after the start of anoxia and recovered to resting levels within ϳ6 -10 min of the return to normoxia (Fig.…”

“…As reported previously (Rose and Ransom, 1997) that in turn may contribute to the pathogenesis of injury (Chen et al, 1999;Barros et al, 2001;Aarts et al, 2003). To examine the contribution of Na ϩ influx through NSCCs to the rise in [Na ϩ ] i during anoxia, we applied Gd 3ϩ (Caldwell et al, 1998 (Fig.…”

“…Although Na ϩ entry attributable to the activation of ionotropic glutamate receptors has received some attention, with conflicting results (cf. Pisani et al, 1998;Chen et al, 1999;Müller and Somjen, 2000;LoPachin et al, 2001), glutamate-mediated excitotoxicity cannot fully account for the direct actions of anoxia or ischemia on neurons, and the potential contribution from mechanisms integral to neurons (i.e., independent from glutamate receptor activation and changes in the external microenvironment) to Na ϩ influx during anoxia or ischemia has not been systematically addressed. Furthermore, despite indications that continued Na ϩ influx after reperfusion may be as damaging as Na ϩ entry during anoxia or ischemia (Lipton, 1999), the pathways that mediate Na ϩ influx after anoxia or ischemia have not been well characterized, and it remains unknown whether these pathways might differ from those active during an insult, as reported for Ca 2ϩ Ereciń ska, 1990, 1992).…”

Activation of the transient receptor potential M2 channel and poly(ADP-ribose) polymerase is involved in oxidative stress-induced cardiomyocyte death

“…34 Using confocal microscopy with a thin optical section (0.04 mm), it can be used to simultaneously report changes in the [Na þ ] cyt and [Na þ ] m , since only a small proportion of the probe is found in the cytosolic compartment, 34 the majority being trapped by the negative mitochondrial potential because of the positive charge of the probe. Since the nuclear membrane is no barrier to cytosolic ion movement, 35,36 averaging the signal over a small nuclear optical section (ER-free and mitochondria-free) or the mitochondria (identified by a mitochondrial marker, MTG, last frame in Figures 2a and c Figure S1) or after clamping the [Na þ ] i at 60 mM using Na-ionophore cocktail (5 mM gramicidin D, 40 mM monensin, and 100 mM strophanthidine) in Ca-free medium, 33 Significant chromatin condensation (arrows in Figure 2fi) and DNA fragmentation ( Figure 2fii, green for TUNEL ( þ ) staining) were seen after 4.5 h wash-out of H 2 O 2 or of Naionophore cocktail/Ca-free medium (Figure 2g). Approximately 18% inhibition of condensation was seen in Ca-free medium (Figure 2g), in which the [Ca 2 þ ] m overload should be totally abolished.…”

Relationships between superoxide levels and delayed calcium deregulation in cultured cerebellar granule cells exposed continuously to glutamate

“…Phospholipase A2 inhibitors prevent the increase in superoxide following DCD Ionomycin, metabolic inhibition or glutamate have each been reported to activate phospholipase A2 in CGNs (Lazarewicz et al 1990;Gunasekar et al 1995;Chen et al 1999) and cortical neurons (Tapia-Arancibia et al 1992;Stella et al 1995;Taylor and Hewett 2002) while PLA2 inhibitors decrease cell death measured by lactic dehydrogenase release (Ciani et al 1996). In addition Lafon-Cazal et al (1993) reported that superoxide production from glutamate-exposed CGNs was due in part to arachidonic acid release.…”