Slow ATP loss and the defense of ion homeostasis in the anoxic frog brain
Department of Biological Sciences, Florida Atlantic University, Boca Raton, FL 33431, USA
*Author for correspondence (e-mail: lutz{at}fau.edu)
Accepted July 18, 2001
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Summary |
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Key words: brain anoxia, adenosine, ATP, frog, Rana pipiens, channel arrest, depolarization.
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Introduction |
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There are, however, a few truly anoxia-tolerant exceptions, such as the freshwater turtle Trachemys scripta and the crucian carp Carassius carassius, which have brains that are able to survive days of anoxia at room temperature (Lutz and Nilsson, 1997). The most important compensation in the turtle brain for surviving anoxia is, almost uniquely, lowering its energy consumption to such a degree (by 7080 %) that brain energy needs can be fully met by anaerobic glycolysis (Lutz et al., 1984; Doll et al., 1993). As a result, the turtle brain is able to maintain ATP levels and ionic gradients during anoxia and thus avoid the fatal consequences of energy failure (Lutz, 1992). A reduction in membrane ion leakage (channel arrest) may provide important energy savings for the anoxia-tolerant brain (Lutz et al., 1985; Hochachka, 1986; Hand and Hardewig, 1996), and there is evidence that the rate of K+ flux is significantly lower in the anoxic turtle brain than in the normoxic brain (Chih et al., 1989; Jiang and Haddad, 1991; Pek and Lutz, 1997).
Some frog species (Rana temporaria, Rana pipiens) appear to be intermediate in anoxia-tolerance, having brains that can tolerate approximately 3 h without oxygen at room temperature (Lutz and Reiners, 1997; Wegener and Krause, 1993) and at least 30 h of anoxia at 5°C (Hermes-Lima and Storey, 1996). The peculiarity is that, unlike anoxia-tolerant species, these frogs do not defend brain ATP levels during anoxia but, in contrast to anoxia-vulnerable species, ATP depletion takes hours instead of minutes (Okada and McDougal, 1971; Lutz and Reiners, 1997; Wegener and Krause, 1993). After approximately 100 min of anoxia, there is a release of adenosine, but an excitotoxic release of neurotransmitters does not occur until after 3 h of anoxia (Lutz and Reiners, 1997). However, it is not known whether ionic gradients are maintained during the hours of anoxia survival in the frog brain, a critical factor in understanding this phenomenon.
The release of adenosine may be a factor in anoxia-tolerance in the frog brain (Lutz and Reiners, 1997). Extracellular adenosine plays an important protective role in the turtle (Lutz and Nilsson, 1997) and mammalian (Sweeney, 1996) brain and appears to be involved in the activation of channel arrest at the onset of anoxia in the turtle brain (Pék-Scott and Lutz, 1998). Whether it has such a role in the frog brain is not known.
The frog brain therefore offers a unique model in which to examine the relationship between ATP depletion and the failure of ionic-gradient-maintaining pumps. It is also of interest to investigate the mechanisms that slow ATP loss in the anoxic frog brain.
The aims of this study were (i) to determine whether extracellular [K+] is maintained in the anoxic frog brain over the known period of survival, (ii) to identify a critical ATP level at which ionic homeostasis is lost, (iii) to determine whether a decrease in the rate of K+ efflux is an energy-saving strategy in the anoxic frog brain and (iv) to investigate whether adenosine plays a role in anoxia survival in the frog brain.
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Materials and methods |
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Animal preparation
The frogs were placed in a 500 ml sealed polyethylene box containing saline in which was dissolved the anesthetic tricaine methanesulfonate, MS222 (250300 g l1), buffered to pH 7.4. Until surgery, this chamber was continuously aerated with 100 % O2 to ensure that the anesthetized frogs remained fully aerobic. After a surgical plane had been achieved (as indicated by the lack of an ocular reflex), an area of the skull (4 mmx5 mm) directly over the optic lobes was thinned and removed with jewellers forceps, exposing the optic tectum. In all experiments, the brain was superfused with frog ACSF (116 mmol l1 NaCl, 2 mmol l1 KCl, 26 mmol l1 NaHCO3, 1.25 mmol l1 NaH2PO4, 2 mmol l1 CaCl2, 2 mmol l1 MgSO4, 2 mmol l1 glucose, buffered to pH 7.4) at a flow rate of approximately 1 ml h1. The chamber was sealed with a tight-fitting lid during the entire experiment, except for a small hole above the brain for the placement of the microelectrode. The water temperature in the sealed aerated chamber was 16°C. Anoxic conditions were achieved by aerating the chamber with 100 % N2 (County Welding/Air Products).
Using a micromanipulator, a double-barrelled K+-selective glass microelectrode (Sick and Kreisman, 1981) was inserted 500700 µm below the dorsal surface of the optic lobe. Calibration of electrical activities with K+ was performed before and after experimentation using different concentrations of KCl, with NaCl maintaining a constant ionic strength of 200 mosmol l1. Electrodes were used only if they showed stability and sensitivity to changing K+ concentrations (a voltage reading change of at least 70 mV in response to a change from a 1 mmol l1 to a 50 mmol l1 solution) during the calibration.
Experimental groups
To investigate the levels of extracellular K+ during anoxia and their relationship to brain ATP levels, experiments were performed on four groups of five frogs. Brain extracellular [K+] was monitored in all frogs, and at the end of each experiment the brain was dissected out and frozen in liquid nitrogen in less than 1 min. Tissues were then stored at 80°C until used.
In group A (normoxic control), individual animals were aerated with O2 for 30 min. Group B frogs were aerated with N2 for 1 h. Only animals that showed no increase in extracellular [K+] from either group were used. Group C animals were left in anoxic conditions until extracellular [K+] was seen to increase (phase 1 of anoxic depolarization) (Hansen, 1977). Group D animals were left in anoxic conditions until complete anoxic depolarization (see Fig. 1).
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To investigate the effects of anoxia on K+ efflux, the frogs were allocated to four groups. At fixed points, 10 mmol l1 ouabain was superfused onto the brain to inhibit Na+/K+-ATPase (Chih et al., 1989; Pék-Scott and Lutz, 1998) and in some experiments theophylline was added to inhibit adenosine receptors (Pék and Lutz, 1997). Group 1 was normoxic for 45 min before ouabain was added. Group 2 was held in anoxia for 2 h, and the brain was then superfused with ouabain. In normoxic group 3, the brain was superfused with ACSF containing 100 µmol l1 theophylline for 1 h before ouabain was added to ensure sufficient blocking of adenosine receptors (Pék and Lutz, 1997). A similar procedure (theophylline then ouabain) was adopted for the fourth group after 2 h of N2 aeration.
All statistical analyses were performed using analysis of variance (ANOVA) and Dunnetts tests. All data are expressed as means ± S.E.M.
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Results |
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The normoxic ATP concentration in frog brain was 0.95±0.06 mmol kg1, at the low end of the range previously measured for this species (1.11.56 mmol kg1) (McDougal et al., 1968). Brain ATP had fallen within the first hour and was almost fully depleted in 4 h (Fig. 2). Previous researcher have similarly found that frog brain ATP levels are not defended during anoxia (Wegener and Krause, 1993; Lutz and Reiners, 1997).
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Compared with air-breathing animals, the time taken for extracellular [K+] to increase by 1 mmol l1 over resting levels had more than doubled after 2 h of anoxia (+251 %), indicating a corresponding reduction in the rate of K+ leakage (Fig. 5). However, the value of the inflection point, after which [K+] increased rapidly, did not change (4.80±0.87 mmol l1 in normoxia, 4.77±0.84 mmol l1 in anoxia). As a consequence, the time to reach the critical extracellular [K+], i.e. the duration of phase 1, also increased in anoxia (normoxic 11.9±2.1 min versus anoxic 21.5±1.8 min, P<0.05). During phase 2, [K+] increased rapidly in both groups, and there was no significant difference in the total duration of phase 2 between normoxic (2.84±0.39 min) and anoxic (2.24±0.33 min) animals.
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Discussion |
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Simultaneous measurements of the relatively slow changes in K+ and ATP levels in the anoxic turtle brain allowed a determination of the critical ATP levels at which ionic homeostasis starts to be lost. In the rat, there is a precipitous efflux of K+ when ATP levels fall below 50 % of normoxic values (Erecinska and Silver, 1998; Katsura et al., 1994), and this is followed by a rapid increase in extracellular glutamate and GABA levels as ATP continues to be depleted (Shimizu et al., 1993). But, in the mammal, the precise critical level of ATP at which the uncontrolled efflux of K+ occurs is difficult to determine because the anoxia-driven changes are so rapid and because pathological events and defense mechanisms are inextricably mixed. The critical ATP level determined in the present study was 0.33±0.06 mmol l1, which is 35 % of normoxic levels. These values are thought to represent an energy minimum for ion homeostasis to be maintained. In the mammal, the opening of KATP channels following ATP depletion is thought to be a major route of K+ efflux (Jiang et al., 1992).
The 50 % reduction in the rate of K+ leakage in anoxic the frog brain is one factor contributing to the delayed time to the onset of depolarization. It would lower the cost of ion pumping (and consequently the rate of depletion of ATP) and the rate of increase in extracellular [K+] (phase 1) (and consequently extend the time to reach the extracellular [K+] threshold value). A similar but greater reduction (70 %) in the rate of K+ efflux is seen in the anoxic turtle brain (Pék and Lutz, 1997). Interestingly, in sharp contrast to the brain, the frog R. esculanta defends skeletal muscle ATP levels during anoxia (Wegener and Krause, 1993) and there is a 75 % decrease in the rate of K+ leakage in severely hypoxic muscle (Donohoe et al., 2000).
Although adenosine is released during anoxia in the frog brain, unlike in the turtle, it did not appear to play a role in slowing the K+ efflux (Pék and Lutz, 1997). In the turtle and rat, adenosine is released early in the energy crisis and acts as a retaliatory metabolite to restore energy balance (Sweeney, 1996; Lutz and Nilsson, 1997), but in the frog adenosine is not released until after ATP has been almost fully depleted (Lutz and Reiners, 1997). The late release of adenosine in the frog may be a pathological event associated with energy failure. It is possible that, in the frog, the reduction in the rate of K+ efflux is a consequence of the suppression of action potentials and synaptic transmission, spike arrest (Sick et al., 1993), rather than channel arrest. Transmembrane ionic flux would be lower in a quiescent neuron. Indeed, there is evidence of a significant reduction in neuronal activity in the frog brain during early anoxia: in the frog optic tectum, signal transmission disappeared within 30 min of anoxia (Wegener and Krause, 1993) and there was a complete (but reversible) suppression of action potentials in the isolated sciatic nerve within 60 min of anoxia (Okada et al., 1971).
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Acknowledgments |
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References |
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