Na+-Ca2+ exchanger mediates Ca2+ influx during anoxia in mammalian central nervous system white matter (original) (raw)
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The Journal of Neuroscience, 1992
White matter of the mammalian CNS suffers irreversible injury when subjected to anoxia/ischemia. However, the mechanisms of anoxic injury in central myelinated tracts are not well understood. Although white matter injury depends on the presence of extracellular Ca2+, the mode of entry of Ca2+ into cells has not been fully characterized. We studied the mechanisms of anoxic injury using the in vitro rat optic nerve, a representative central white matter tract. Functional integrity of the nerves was monitored electrophysiologically by quantitatively measuring the area under the compound action potential, which recovered to 33.5 +/- 9.3% of control after a standard 60 min anoxic insult. Reducing Na+ influx through voltage-gated Na+ channels during anoxia by applying Na+ channel blockers (TTX, saxitoxin) substantially improved recovery; TTX was protective even at concentrations that had little effect on the control compound action potential. Conversely, increasing Na+ channel permeabilit...
Annals of the New York Academy of Sciences, 1991
Central white matter (WM) tracts in the mammalian central nervous system (CNS), such as subcortical pathways and spinal cord tracts that are critical to the functional integrity of the CNS, suffer irreversible injury after anoxia/ischemia. Anoxia-induced cell death appears to be caused by sustained increases in intracellular CaZf. In gray matter, Ca2+ influx into the cytoplasm during anoxia is thought to occur via NMDAreceptor-gated The mechanisms of anoxic injury in CNS WM are less well understood; specifically, the critical step by which extracellular Ca2+ enters the cytoplasmic compartment is not known. We have studied this question using the in vitro rat optic nerve, a representative CNS WM tract. Our results indicate that a large part of the damaging CaZ+ influx that occurs during anoxia in WM is mediated by the Na+-Ca2+ exchanger, forced to operate in the reverse mode due to Na+ influx via voltage-gated Naf channels. METHODS AND RESULTS The methods have been described in detail el~ewhere.~.~ Briefly, optic nerves were dissected from adult Long Evans rats and perfused in an interface brain slice chamber with artificial cerebrospinal fluid (CSF) containing, in mM: NaCl 126, KCl3.0, MgSO, 2.0, NaHCO, 26, NaH,PO, 1.25, CaCl, 2.0, dextrose 10 (pH 7.45). Temperature was maintained at 37°C. The tissue was aerated with a 95% 0 , / 5 % C O , gas mixture. The functional integrity of the nerves was monitored electrophysiologcally as the area
Axonal L-Type Ca2+ Channels and Anoxic Injury in Rat CNS White Matter
Journal of Neurophysiology, 2001
We studied the magnitude and route(s) of Ca2+ flux from extra- to intracellular compartments during anoxia in adult rat optic nerve (RON), a central white matter tract, using Ca2+-sensitive microelectrodes to monitor extracellular [Ca2+] ([Ca2+]o). One hour of anoxia caused a rapid loss of the stimulus-evoked compound action potential (CAP), which partially recovered following re-oxygenation, indicating that irreversible injury had occurred. After an initial increase caused by extracellular space shrinkage, anoxia produced a sustained decrease of 0.42 mM (29%) in [Ca2+]o. We quantified the [Ca2+]o decrease as the area below baseline [Ca2+]o during anoxia and used this as a qualitative index of suspected Ca2+ influx. The degree of RON injury was predicted by the amount of Ca2+ leaving the extracellular space. Bepridil, 0 Na+ artificial cerebrospinal fluid or tetrodotoxin reduced suspected Ca2+ influx during anoxia implicating reversal of the Na+-Ca2+ exchanger as a route of Ca2+ infl...
Voltage-gated calcium channels in CNS white matter: role in anoxic injury
Journal of neurophysiology, 1995
1. The effect of Ca2+ channel antagonists on the extent of anoxia-induced white matter injury was studied in the rat optic nerve, a white matter tract. Compound action potentials (CAPs) were recorded before and after a standard 60-min anoxic period to assess the extent of anoxic injury. 2. The L-type Ca2+ channel antagonists verapamil (90 microM), diltiazem (50 microM), and nifedepine (2.5 microM) significantly protected the rat optic nerve from anoxic injury. Mean recovery of CAP area was 51.3 +/- 3.0% (mean +/- SE, n = 8, P < 0.01), 65.6 +/- 5.3% (n = 8, P < 0.01), and 54.3 +/- 6.1% (n = 8, P < 0.01), respectively. Mean CAP recovery under control conditions was 35.2 +/- 0.3 (n = 33). 3. Simultaneous block of L-type and N-type Ca2+ channels by coapplication of 50 microM diltiazem and 1 microM SNX-124 [synthetic omega-conotoxin (CgTx) GVIA], resulted in postanoxic CAP recovery of 73.6 +/- 6.0% (n = 12), significantly larger than CAP recovery in diltiazem alone (P < 0.001...
Neuroscience Letters, 1990
The effects of anoxic injury on the functional integrity of mammalian central white matter were studied electrophysiologically using the rat optic nerve model. Previous studies on this model have shown that extracellular Ca 2÷ is critical to the production of irreversible anoxic injury, and suggest that during anoxia Ca 2+ crosses the membrane to enter the intracellular compartment. We attempted to elucidate the mechanism by which this damaging Ca :÷ influx occurs. The inorganic Ca 2÷ channel blockers Mn 2÷ (1 mM), Co 2 + (1 mM) or La 3 + (0.1 mM) had no effect on recovery of the area under the compound action potential after a standard 60 min period of anoxia; only Mg 2+ (I0 mM) significantly improved recovery (54.9 + 8.9% vs. 28.7 + 10.1%, P < 0.005). Treatment with organic Ca 2+ channel blockers of the dihydropyridine class, nifedipine (1-10/iM) or nimodipine (1-40 #M), also had no effect on recovery from anoxia. We conclude that Ca 2+ influx during anoxia does not occur via conventional Ca 2÷ channels sensitive to polyvalent cations or dihydropyridines. In the mammalian brain, both gray (GM) and white matter (WM) structures are susceptible to anoxic injury. Extensive study of GM anoxia has implicated an increase in intracellular Ca 2+ concentration ([Ca2+]i) in the production of irreversible injury. This rise in [Ca2+]i is believed to occur in GM primarily through the opening of channels gated by excitotoxins, especially glutamate [3, 6, 13, 19, 20, 31]. In addition, the administration of organic Ca 2+ channel blockers such as nimodipine has improved neurological recovery in certain in vivo stroke models [25, 34], although this may be attributable to a vascular effect. In contrast, relatively little is known about the pathophysiology of anoxia in WM, despite the fact that both GM and WM are critical for the functional integrity of the nervous system. Moreover, the mechanisms ofanoxic damage are likely to be different in the two tissues since WM contains no neuronal cell bodies or synapses and appears to have a different complement of ion channels [4, 17].
Responses to reversible anoxia of intracellular free and bound Ca2+ in rat cortical slices
Resuscitation, 2000
Severe anoxia induces destabilisation of intracellular calcium homeostasis in neurones. The mechanism of this effect, and particularly the interrelationship between changes in intracellular concentration of free Ca 2 + ions and the content of the intracellular Ca 2 + stores, during and after anoxia, is not clear. We used a superfusion system of rat olfactory cortical slices for the fluorimetric estimation of changes in the intracellular concentration of free Ca 2 + ions and in the level of bound Ca 2 + , utilising the fluorescent indicators Fura-2 and chlortetracycline, respectively. It was found that 10-min normoglycaemic anoxia results in simultaneous decrease in bound and increase in free Ca 2 + levels, whereas during 60-min reoxygenation, we detected an increase in both indices. The NMDA receptor antagonists MK-801 and APV attenuated changes in free Ca 2 + level during anoxia and reoxygenation and intensified anoxia-evoked decrease in bound Ca 2 + content, whereas a late post-anoxic increase in bound Ca 2 + was abolished. These data suggest that the influx of extracellular Ca 2 + to neurones via NMDA receptors, plays a critical role in the rise of intracellular free Ca 2 + concentration during and after anoxia. Biphasic changes in bound Ca 2 + content during anoxia and reoxygenation may reflect an anoxia-induced release of Ca 2 + from intracellular stores, followed later by a neuronal calcium overload and refilling of intracellular Ca 2 + binding sites.
Journal of Neurochemistry, 2003
The effect of anoxia on Na + /H + exchange activity was examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H + -sensitive fluorophore, BCECF. Five-minute anoxia imposed under nominally HCO 3 -/CO 2 -free conditions induced a fall in pH i , the magnitude of which was smaller following prolonged exposure to medium in which N-methyl-D-glucamine (NMDG + ) was employed as an extracellular Na + (Na + o ) substitute. Also consistent with the possibility that Na + /H + exchange becomes inhibited soon after the induction of anoxia, rates of Na + o -dependent pH i recovery from internal acid loads imposed during anoxia were slowed, compared to rates of Na + o -dependent pH i recovery observed prior to anoxia. At the time at which rates of pH i recovery were reduced during anoxia, cellular adenosine triphosphate (ATP) levels had fallen to 35% of preanoxic levels, suggesting that ATP depletion might contribute to the observed inhibition of Na + /H + exchange. In support, incubation of neurons with 2-deoxyglucose and antimycin A under normoxic conditions induced a fall in cellular ATP levels that was also associated with reduced Na + o -dependent rates of pH i recovery from imposed acid loads; conversely, pre-treatment with 10 mM creatine attenuated the effects of anoxia to reduce both ATP levels and Na + o -dependent rates of pH i recovery from internal acid loads. Taken together, the results are consistent with the possibility that functional Na + /H + exchange activity in adult rat CA1 neurons declines soon after the onset of anoxia, possibly as a result of anoxia-induced falls in intracellular ATP. Abbreviations used: ATP, adenosine triphosphate; BCECF, 2¢,7¢-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; [Ca 2+ ] i , intracellular free Ca 2+ concentrations; 2-DG, 2-deoxyglucose; NMDG + , N-methyl-D-glucamine; Na + o , extracellular Na + pH i , intracellular pH; NHE1, Na + /H + exchanger isoform 1.
Role of Na+-H+ and Na+-Ca2+ exchange in hypoxia-related acute astrocyte death
Glia, 2005
Cultured astrocytes do not succumb to hypoxia/zero glucose for up to 24 h, yet astrocyte death following injury can occur within 1 h. It was previously demonstrated that astrocyte loss can occur quickly when the gaseous and interstitial ionic changes of transient brain ischemia are simulated: After a 20-40-min exposure to hypoxic, acidic, ion-shifted Ringer (HAIR), most cells died within 30 min after return to normal saline (i.e., "reperfusion"). Astrocyte death required external Ca 2ϩ and was blocked by KB-R7943, an inhibitor of reversed Na ϩ-Ca 2ϩ exchange, suggesting that injury was triggered by a rise in [Ca 2ϩ ] i. In the present study, we confirmed the elevation of [Ca 2ϩ ] i during reperfusion and studied the role of Na ϩ-Ca 2ϩ and Na ϩ-H ϩ exchange in this process. Upon reperfusion, elevation of [Ca 2ϩ ] i was detectable by Fura-2 and was blocked by KB-R7943. The low-affinity Ca 2ϩ indicator Fura-FF indicated a mean [Ca 2ϩ ] i rise to 4.8 Ϯ 0.4 M. Loading astrocytes with Fura-2 provided significant protection from injury, presumably due to the high affinity of the dye for Ca 2ϩ. Injury was prevented by the Na ϩ-H ϩ exchange inhibitors ethyl isopropyl amiloride or HOE-694, and the rise of [Ca 2ϩ ] i at the onset of reperfusion was blocked by HOE-694. Acidic reperfusion media was also protective. These data are consistent with Na ϩ loading via Na ϩ-H ϩ exchange, fostering reversal of Na ϩ-Ca 2ϩ exchange and cytotoxic elevation of [Ca 2ϩ ] i. The results indicate that mechanisms involved in pH regulation may play a role in the fate of astrocytes following acute CNS injuries.
The Journal of Neuroscience, 1993
Although we and others have previously shown that newborn central mammalian neurons are more tolerant to anoxia than their adult counterparts, we do not know whether neonatal nerve cells accumulate free cytosolic calcium (Ca2+i) less than adults in response to O2 deprivation. In order to determine whether anoxia increases Ca2+i in adult and neonatal neurons, we monitored calcium in CA1 hippocampal neurons using the calcium-sensitive probe fluo-3 and confocal microscopy. These neurons were studied in the dissociated state in order to study their inherent response to anoxia without the influence of modulatory factors such as synaptic input and neurotransmitters. Severe anoxia caused a rapid increase in Ca2+i in adult CA1 hippocampal neurons, followed by swelling and bleb formation. In neonatal neurons, the latency of this calcium rise was about five times longer than in the adult. Removal of extracellular calcium and addition of calcium channel blockers (Co2+) greatly attenuated the i...