AMPA receptors undergo channel arrest in the anoxic turtle cortex

Without oxygen, all mammals suffer neuronal injury and excitotoxic cell death mediated by overactivation of the glutamatergic N-methyl-d-aspartate receptor (NMDAR). The western painted turtle can survive anoxia for months, and downregulation of NMDAR activity is thought to be neuroprotective during anoxia. NMDAR activity is related to the activity of another glutamate receptor, the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR). AMPAR blockade is neuroprotective against anoxic insult in mammals, but the role of AMPARs in the turtle's anoxia tolerance has not been investigated. To determine whether AMPAR activity changes during hypoxia or anoxia in the turtle cortex, whole cell AMPAR currents, AMPAR-mediated excitatory postsynaptic potentials (EPSPs), and excitatory postsynaptic currents (EPSCs) were measured. The effect of AMPAR blockade on normoxic and anoxic NMDAR currents was also examined. During 60 min of normoxia, evoked peak AMPAR currents and the frequencies and amplitudes of EPSPs and EPSCs did not change. During anoxic perfusion, evoked AMPAR peak currents decreased 59.2 ± 5.5 and 60.2 ± 3.5% at 20 and 40 min, respectively. EPSP frequency (EPSPƒ) and amplitude decreased 28.7 ± 6.4% and 13.2 ± 1.7%, respectively, and EPSCƒ and amplitude decreased 50.7 ± 5.1% and 51.3 ± 4.7%, respectively. In contrast, hypoxic (Po2 = 5%) AMPAR peak currents were potentiated 56.6 ± 20.5 and 54.6 ± 15.8% at 20 and 40 min, respectively. All changes were reversed by reoxygenation. AMPAR currents and EPSPs were abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In neurons pretreated with CNQX, anoxic NMDAR currents were reversibly depressed by 49.8 ± 7.9%. These data suggest that AMPARs may undergo channel arrest in the anoxic turtle cortex.

ATP-sensitive K+ channel activation provides transient protection to the anoxic turtle brain

There is wide speculation that ATP-sensitive K+(KATP) channels serve a protective function in the mammalian brain, being activated during periods of energy failure. The aim of the present study was to determine if KATP channels also have a protective role in the anoxia-tolerant turtle brain. After ouabain administration, rates of change in extracellular K+ were measured in the telencephalon of normoxic and anoxic turtles ( Trachemys scripta). The rate of K+ efflux was reduced by 50% within 1 h of anoxia and by 70% at 2 h of anoxia, and no further decrease was seen at 4 h of anoxia. The addition of the KATP channel blocker glibenclamide or 2,3-butanedione monoxime prevented the anoxia-induced decrease in K+ efflux during the first hour of anoxia, but the effect of these blockers was diminished at 2 h of anoxia and was not seen after 4 h of anoxia. This pattern of change in KATP channel blocker sensitivity can be related to a previously established temporary fall and subsequent recovery of tissue ATP during early anoxia. We suggest that activated KATP channels are involved in the downregulation of membrane ion permeability (channel arrest) during the initial energy crisis period but are switched off when the full anoxic state is established and tissue ATP levels have been restored. We also found that, in contrast to those in mammals, KATP channels are not a major route for K+ efflux in the energy-depleted turtle brain.

Adaptations of vertebrate neurons to hypoxia and anoxia: maintaining critical Ca2+ concentrations.

Down-regulation of ion channel activity ('channel arrest'), which aids in preserving critical ion gradients in concert with greatly diminished energy production, is one important strategy by which anoxia-tolerant neurons adapt to O2 shortage. Channel arrest results in the elimination of action potentials and neurotransmission and also decreases the need for ion transport, which normally requires a large energy expenditure. Important targets of this down-regulation may be channels in which activity would otherwise result in the toxic increases in intracellular [Ca2+] characteristic of anoxia-sensitive mammalian neurons. In turtles, Na+ channels and the Ca2+-permeable ion channel of the N-methyl-d-aspartate (NMDA)-type glutamate receptor undergo down-regulation during anoxia. Inactivation of NMDA receptors during hypoxia occurs by a variety of mechanisms, including alterations in the phosphorylation state of ion channel subunits, Ca2+-dependent second messenger activation, changes in Ca2+-dependent polymerization/depolymerization of actin to postsynaptic receptors and activation of other G-protein-coupled receptors. Release of inhibitory neurotransmitters (e.g. gamma-aminobutyrate) and neuromodulators (e.g. adenosine) into the brain extracellular fluids may play an important role in the down-regulation of these and other types of ion channels.

Exogenous fructose 1,6-bisphosphate reduces K+ permeability in isolated rat hepatocytes

The relationship between the protective effect of fructose 1,6-bisphosphate (F-1,6-P2) against cell injury and the modifications produced in the metabolic fluxes and in the membrane permeability to K+ was studied in isolated rat hepatocytes. Incubation of these cells in the presence of F-1,6-P2 reduced metabolic activity without affecting the ATP content, which suggests a downregulation of the ATP turnover. Using 86Rb+ as a tracer, we analyzed the relationship between these metabolic changes and alterations in K+ fluxes. In the presence of F-1,6-P2 the passive and the active K+ fluxes in hepatocytes decreased. However, the Na(+)-K+ pump from semipurified membranes was not directly affected by F-1,6-P2, which suggests a secondarily induced reduction of Na(+)-K+ pump activity. Moreover, galactosamine-treated cells showed a marked increase in permeability to K+ that was abolished by the presence of F-1,6-P2. This protective effect may be related to the prevention of K+ efflux. The results reported here strongly suggest the induction of channel arrest, and the associated metabolic downregulation, as the primary protective effect of F-1,6-P2, as has been shown in the prevention of galactosamine-induced hepatotoxicity.

Role for adenosine in channel arrest in the anoxic turtle brain.

The remarkable ability of the turtle brain to survive anoxia is based on its ability to match energy demand flexibly to energy production. Earlier studies indicate that reduced ion leakage is an important mechanism for energy conservation during anoxia. We tested the hypothesis that extracellular adenosine plays a role in the reduction of K+ flux (channel arrest) that occurs in the anoxic turtle brain. Changes in extracellular K+ concentration ([K+]o in the in situ brain of the turtle Trachemys scripta were monitored following inhibition of Na+/K(+)-ATPase with ouabain. The time to reach full depolarization ([K+]o plateau) was three times longer in the anoxic brain than in normoxic controls and the initial rate of K+ leakage was reduced by approximately 70%. Superfusing the brain before the during anoxia with the general adenosine receptor blocker theophylline, or the specific adenosine A1 receptor blocker 8-cyclopentyltheophylline, significantly shortened the time to full depolarization in the ouabain-challenged anoxic brain and increased the rate of K+ efflux. The results suggest that adenosine A1 receptors are involved in the expression of anoxia-induced ion channel arrest in the turtle brain.

Contrasting strategies for anoxic brain survival--glycolysis up or down.

Anoxia-tolerant turtles and carp (Carassius) exhibit contrasting strategies for anoxic brain survival. In the turtle brain, the energy consumption is deeply depressed to the extent of producing a comatose-like state. Brain metabolic depression is brought about by activating channel arrest to reduce ion flux and through the release of inhibitory gamma-aminobutyric acid (GABA) and the upregulation of GABAA receptors. Key glycolytic enzymes are down-regulated during prolonged anoxia. The result is a suppression of neurotransmission and a substantial depression in brain electrical activity. By contrast, Carassius remain active during anoxia, though at a reduced level. As in the turtle, there is an adenosine-mediated increase in brain blood flow but, in contrast to the turtle, this increase is sustained throughout the anoxic period. Key glycolytic enzymes are up-regulated and anaerobic glycolysis is enhanced. There is no evidence of channel arrest in Carassius brain. The probable result is that electrical activity in the brain is not suppressed but instead maintained at a level sufficient to regulate and control the locomotory and sensory activities of the anoxic carp. The key adaptations permitting the continued high level of glycolysis in Carassius are the production and excretion of ethanol as the glycolytic end-product, which avoids self-pollution by lactate produced during glycolysis that occurs in other vertebrates.

Roles of energy status, KATP channels and channel arrest in fish brain K+ gradient dissipation during anoxia

The crucian carp (Carassius carassius L.) is one of the most anoxia-tolerant vertebrates known, being able to maintain ion homeostasis in its brain for many hours of anoxia. This study aims to clarify the importance of glycolysis during anoxia and also to investigate whether the extreme tolerance to anoxia could be due to down-regulation of K+ permeability ('channel arrest') and/or activation of ATP-sensitive K+ (KATP) channels. The latter was also tested in rainbow trout (Oncorhynchus mykiss). The results suggest that, during anoxia, the crucian carp brain is completely dependent on glycolysis, since blocking glycolysis with iodoacetic acid (IAA) rapidly caused an increase in [K+]o that coincided with a drastic drop in ATP level and energy charge. Testing the channel arrest hypothesis by measuring the K+ efflux rate after Na+/K+-ATPase had been blocked by ouabain revealed no change in K+ permeability in crucian carp brain in response to anoxia. Furthermore, superfusing the brain of anoxic crucian carp with the KATP channel blocker glibenclamide did not alter the efflux rate of K+ after glycolysis had been inhibited with IAA. Glibenclamide had no effect on K+ efflux rate in rainbow trout brain during anoxia.