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Underwater Physiology: Proceedings of the Fourth Symposium on Underwater Physiology
Underwater Physiology: Proceedings of the Fourth Symposium on Underwater Physiology
Underwater Physiology: Proceedings of the Fourth Symposium on Underwater Physiology
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Underwater Physiology: Proceedings of the Fourth Symposium on Underwater Physiology

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Underwater Physiology is a collection of papers that deals with the physiologically limiting effects of undersea, high pressure exposure ranging from fundamental biological reactions, through integration of physiological stresses, and to limits actually experienced in deep diving. Papers discuss oxygen, the mechanisms of toxicity, and the effects of oxygen on cells and systems such as its pathological and physiological influences in the neurosensory ocular tissue. Other papers discuss the physical effects of pressure and gases on cellular function, protein structure, and the possibility of alleviating symptoms through the administration of drugs. Tests in mice show that various gases exhibit qualitative and semi-quantitative differences in the characteristics of sickness, reactions to hypoxia, and the time before the onset of symptoms. A computer, programmed for nonlinear gas transfer and other variables, running in real time can compute directly from the breathing mixture and provide a real time solution to decompression sickness under various conditions. A combined therapeutic approach, recompression and dextran (an effective lipemic clearing agent) should be capable of treating decompression sickness in humans. Other papers investigate the influence of inert gases and pressure on the central nervous system, as well as, situations in undersea and manned chamber operations. This collection can prove valuable for physiologists, biochemists, cellular biologists, and researchers involved in deep sea diving.
LanguageEnglish
Release dateOct 22, 2013
ISBN9781483272559
Underwater Physiology: Proceedings of the Fourth Symposium on Underwater Physiology

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    Underwater Physiology - C. J. Lambertsen

    efforts.

    Part I

    OXYGEN. MECHANISMS OF TOXICITY

    Outline

    Chapter 1: THE SCOPE OF CHEMICAL OXYGEN POISONING

    Chapter 2: OXYGEN TOXICITY IN NEURONAL ELEMENTS

    Chapter 3: THE INTRACELLULAR OXIDATION–REDUCTION STATE AT HIGH AND LOW OXYGEN CONCENTRATIONS

    Chapter 4: NATURAL RESISTANCE TO OXYGEN POISONING

    Chapter 5: CHEMICAL PROTECTION AGAINST OXYGEN TOXICITY

    Chapter 6: EFFECTS OF OXYGEN ON BLOOD FORMATION AND DESTRUCTION

    THE SCOPE OF CHEMICAL OXYGEN POISONING

    Niels Haugaard

    Publisher Summary

    This chapter discusses the scope of chemical oxygen poisoning. Oxygen poisoning is not a phenomenon confined to animals with a central nervous system but can be elicited in lower animal organisms and plants as well. Studies with isolated enzymes and tissue preparations in vitro have shown that many enzymic reactions are resistant to prolonged exposure to high pressures of oxygen, but these enzymes are also quite easily inactivated by O2. In the search for a biochemical site of O2 toxicity, very little emphasis has probably been placed on localization of particular physiological functions that may be interfered with, inhibited, or possibly stimulated in the organism exposed to an elevated pressure of O2. Oxygen poisoning must involve many fundamental biochemical reactions such as those concerned with energy production, transport of substances across membranes, and the oxidation and synthesis of vital tissue constituents.

    The work of Paul Bert (3) in the last century established without doubt that breathing oxygen at pressures above a certain value is incompatible with normal life. He observed that increased pressures of oxygen produced a characteristic syndrome in mammals that he called oxygen poisoning. The most marked and dramatic manifestation of oxygen toxicity was the opisthotonus and clonic–tonic convulsions induced in mammals exposed to hyperbaric oxygen. Paul Bert also understood and demonstrated that oxygen poisoning was not a phenomenon confined to animals with a central nervous system, but could be elicited in lower animal organisms and plants as well. At a time when the existence of enzymes had not been firmly established, he recognized that ferments, substances that were capable of degradative reactions such as digestion of meat, could be inactivated by oxygen.

    The history of the discovery of O2 poisoning and the early work in this field has been treated in several review articles (1, 5, 6, 8, 14) and will not be discussed in detail here.

    It should be emphasized that, although O2 is necessary for the production of energy and survival of all aerobic cells, it is also a universal cellular poison. It is only because cells in the course of evolution have developed special defense mechanisms against the toxic effects of O2 that life as we know it has been able to flourish. In a sense, the study of O2 toxicity is the study of the ways in which organisms manage to protect themselves against the oxidizing potential of molecular O2. Viewed in this way, O2 poisoning is not a special phenomenon seen only under unusual circumstances such as in deep-sea diving or when animals or men are exposed to elevated pressures of oxygen in high-pressure chambers. Toxicity of O2 is a fact that all organisms on earth have to contend with. The difference between the biological effects of O2 at 0.2 atm and at 1, 2, 3, or 10 atm is only one of degree. Physiological or biochemical studies of animals or man exposed to elevated pressures of O2 can therefore also be expected to lead to important information about the role of O2 in regulating cellular activity at the tension of O2 normally present at sea level.

    Another important consideration in this connection is that the concentration of O2 that produces a toxic effect on cellular metabolism is not necessarily that present in the gaseous atmosphere surrounding the organism. In a mammalian cell the tension of O2 is a function of the blood supply, the diffusion of O2 from the blood vessel to the cell, and finally the rate of O2 uptake per unit weight of tissue. In rapidly respiring tissues, such as brain cells, the O2 tension is therefore far less than that of the arterial blood (11). Therefore, when convulsions occur in animals or man exposed, for example, to inspired O2 tensions of 3 atm, the Po2 that produces the toxic effects in the CNS ranges from nearly 3 atm at some enzymic locations to considerably below 1 atm in other intracellular sites (12).

    The mechanism by which O2 exerts its toxic effect on cells has intrigued many investigators and is a problem that is still unsolved. One reason for this is that there is not one but probably a great many sites at which O2 exerts effects on metabolic reactions or on specific cellular functions.

    Studies with isolated enzymes and tissue preparations in vitro have shown that many enzymic reactions are resistant to prolonged exposure to high pressures of oxygen (HPO), but also that many enzymes are quite easily inactivated by O2 (1, 5, 8, 14). Among these are the so-called sulfhydryl (SH) enzymes, which contain an SH group which is necessary for their activity. Among these are several hydrolytic enzymes, many dehydrogenases (including a key enzyme in glycolysis), glyceraldehyde phosphate dehydrogenase (GAPD), and certain enzymes concerned with the respiratory chain and oxidative phosphorylation.

    Inhibition of SH enzymes by O2 theoretically can occur by one of several mechanisms. These are illustrated in Fig. 1. If two SH groups are present close to each other on the surface of the protein enzyme, oxidation can occur within the molecule to form a disulfide (S-S) linkage. This may be the mechanism of O2 effect upon GAPD, which has two SH groups in cysteine moieties separated by only three amino acids. If an enzyme protein contains only one SH group or SH groups widely separated, oxidation may occur between two enzyme molecules. Such a bimolecular reaction between enzymes is not likely to occur to any great extent and inactivation of an SH enzyme will probably take place mostly by mechanism 3 illustrated in Fig. 1. This mechanism represents a prior oxidation of a nonprotein, cellular SH compound such as glutathione followed by mixed disulfide formation. The last reaction is reversible and is also the reaction by which oxidized SH enzymes can be reactivated by a substance such as reduced glutathione (GSH).

    FIG. 1 Possible mechanisms involved in the inactivation of SH enzymes by O2.

    The schema demonstrates the dual role that gluthathione, a universal constituent of cells, can play. By being oxidized it can react with SH groups of enzymes to inactivate them. In its reduced state it can regenerate enzyme thiol groups and in this way reverse the toxic effects of O2.

    Considerable thought has been given to the manner in which the oxidation of SH groups by O2 is brought about. Two possibilities are illustrated in Table I. In one, an increased concentration of O2 may by mass action drive a reaction such as the oxidation of GSH toward the right (2GSH + ½O2 → GSSG + H2O). On the other hand, free radicals such as RS· and HO2· could be formed during hyperbaric oxygenation. HO2· subsequently can oxidize a second thiol group to produce the free radical RS· and hydrogen peroxide. Two moieties of RS· can then combine to form a disulfide. The hydrogen peroxide can be expected to be degraded by the ubiquitous enzyme catalase. Note that the net result of the two processes is the same. Which mechanism is the predominant one is not known; however, the fact that certain trace metals, such as cupric and ferrous ions, are essential for the oxidation of SH groups by O2 favors the view that free radicals are involved in this mechanism of O2 toxicity.

    TABLE I

    MOLECULAR MECHANISMS OF OXYGEN TOXICITYa

    aFor a discussion of free radical mechanisms in O2 toxicity, see Gilbert (7).

    Among the possible biochemical sites of O2 toxicity are: (1) SH enzymes; (2) thiol-containing coenzymes, lipoic acid, coenzyme A, and GSH; (3) flavoprotein enzymes, particularly those containing nonheme iron in addition to SH groups; (4) enzymes requiring pyridoxal phosphate as a coenzyme—of particular interest here is glutamic acid decarboxylase (GAD), the enzyme responsible for the formation of γ-aminobutyric acid (GABA) in the nervous system; and finally, (5) lipid peroxidation should be considered. Lipids containing double bonds are essential constituents of cell membranes and many enzyme aggregates. These lipid substances form peroxides in the presence of a high concentration of O2 and can be destroyed during this process. Lipid peroxidation is catalyzed by several trace metals, especially iron, and may play an important role in the development of O2 toxicity.

    Figure 2 illustrates the steps in carbohydrate metabolism that are susceptible to the toxic action of O2. In glycolysis the enzyme GAPD is quite easily inactivated (10). This has been demonstrated with several in vitro systems. After inactivation by O2, the enzyme can be reactivated by incubation with an SH reagent. The next step in glucose oxidation that has been found to be inactivated by O2 is the oxidation of pyruvate and this may involve the oxidation of either lipoic acid or coenzyme A to the oxidized form. In the TCA cycle itself several dehydrogenases contain SH groups and have been demonstrated to be inactivated in in vitro systems by O2. In the respiratory chain there are a number of flavoprotein enzymes that are exceptionally vulnerable to O2 toxicity. Inactivation of one of these may be involved in the inhibition of reverse electron flow (the reduction of NAD by succinate) demonstrated by Chance and his collaborators (4). Finally, we come to oxidative phosphorylation, the formation of ATP linked to the reactions of the respiratory chain. These processes have recently been found to depend on the presence of free SH groups since agents that react with SH groups, such as organic mercurials and various disulfides, can completely inhibit oxidative phosphorylation by mitochondria (9).

    FIG. 2 Enzyme reactions in carbohydrate metabolism and energy production susceptible to O2 toxicity.

    In the search for a biochemical site of O2 toxicity, too little emphasis has probably been placed on localization of particular physiological functions that may be interfered with, inhibited, or possibly stimulated in the organism exposed to an elevated pressure of O2. The characteristic convulsions exhibited by mammals at 3 atm O2 or greater certainly indicate that neuronal pathways in the CNS do not operate normally. The disturbance of the metabolism of glutamate and GABA as a possible cause of the neuronal dysfunction will be discussed later by Dr. Wood.

    Important though they are, derangements of CNS function are not the only signs of O2 toxicity and many other cellular functions are interfered with during exposure of an animal to an elevated tension of O2. Many physiological processes involve the transfer of molecules or ions across the cell membrane; the transport of some of these such as sodium or glucose constitutes what has been called active transport—movement across a membrane of a substance by a process that requires the expenditure of energy by the cell. The sites of such reactions should be considered as potential sites at which O2 could exert a toxic effect.

    Other possible sites of O2 toxicity are synapses in the nervous system (autonomic or central), sites at which neurohumors are released, exert an action on the postsynaptic membrane, and are finally destroyed. It is conceivable that one or more of the reactions of synaptic transmission are influenced by an elevated pressure of O2.

    A third possible site of O2 toxicity involves the mitochondria. These organelles are involved not only in the production of energy by oxidative phosphorylation but also in the uptake and release of Ca and other ions in the cell. Free SH groups have already been mentioned as essential for oxidative phosphorylation. Oxidation of such groups by O2 can be expected to interfere with normal cell function. Since O2 at 1 atm or less has been found to inhibit cell division in tissue cultures, the cell nucleus should also be considered as a possible site of O2 toxicity.

    Bean and Bohr (2) and Riggs (13) showed that O2 at 7 atm caused an immediate and dramatic decrease in tension of a smooth muscle preparation. This effect was completely reversed by returning the preparation to O2 at 1 atm. These experiments raise the possibility that HPO may affect the contractile proteins themselves or possibly the release or uptake of Ca by cellular elements.

    Finally, in a list which is far from complete, is included the possibility that an increased tension of O2 might interfere with the normal synthesis and release of acetylcholine or norepinephrine at nerve terminals. Thus far, no evidence for or against such a hypothesis has been presented.

    Certain of our own experiments concern the effects of O2 on systems studied in vitro. Such studies may not reveal what happens in the intact organism exposed to O2, but they do illustrate some of the specific actions that O2 can exert on metabolic reactions. Williams, in our laboratory, has shown that O2 at 1 atm, as expected from experiments with SH inhibitors, interferes with oxidative phosphorylation and Ca uptake by rat liver mitochondria (15). However, the damage produced was greater than that observed after addition of most SH reagents and it is possible that the effect of O2 involved not only oxidation of SH groups but other reactions (possibly lipid peroxidations) as well.

    Experiments were also carried out with rat brain homogenates (15) which were incubated with α-oxyglutarate (α-OG) as substrate and AMP as phosphate acceptor. Substrate oxidation and net ATP formation were measured. In every experiment the formation of ATP was inhibited to a greater extent and, what is perhaps more important, at an earlier time than the oxidation of the substrate α-OG. The experiments illustrate the great vulnerability of oxidative phosphorylation to O2 toxicity, at least in in vitro systems.

    The experiments reported in Table II are concerned with the effect of trace metals on O2 toxicity on brain metabolism in vitro. In the absence of added trace metals, glucose utilization was inhibited to a smaller extent in 100% O2 than it was in 10% O2. However, ATP accumulation in the brain preparation is markedly depressed. Ferrous ions inhibited both glucose utilization and net ATP formation and accentuated O2 toxicity. Cobalt ions had quite the opposite effect. They completely prevented O2 toxicity and overcame the effect of ferrous ions so that in the presence of both ferrous and cobalt ions the system behaved as if no trace metal ions had been added. The experiments illustrate the remarkable effect that trace metals have on O2 toxicity. Some metal ions accentuate O2 toxicity and others are capable of protecting enzymes against inactivation by O2. The role of metal ions in influencing O2 poisoning deserves further study.

    TABLE II

    THE ACTION OF TRACE METALS ON OXYGEN TOXICITY IN RAT BRAIN HOMOGENATESa,b

    aThe homogenates were incubated at 37° C in the presence of KCl, MgCl2, phosphate buffer, glucose, and AMP as phosphate acceptor. The results are the mean values from 10 or 12 separate incubations.

    bResults are from Ph.D. thesis of C. Williams, University of Pennsylvania, 1969 (15).

    Figure 3 shows the results of experiments with heart homogenates (10). In this system, in contrast to brain homogenates, O2 at 1 atm produces an inhibition of GAPD leading to a marked decrease in the rate of glycolysis. With fructose diphosphate as substrate there is a definite crossover point at the phosphoglyceraldehyde dehydrogenase reaction leading to an accumulation of metabolites, such as triose phosphates, above the metabolic block and a decrease in concentrations of metabolites, such as 3-PGA, below the block. In this system ATP formation from glycolysis is also inhibited by 100% O2 in comparison with 10% O2. In the presence of the thiol compound dithiothreitol (DTT) all effects of O2 are abolished.

    FIG. 3 Oxygen toxicity in heart homogenates. Heart homogenates were incubated for 15 min at 37° C with fructose diphosphate as substrate. Glycolytic intermediates were determined at the end of the experiment. For details, see Horn et al. () 100% O2.

    The in vitro experiments reported here demonstrate the vulnerability of several metabolic reactions to inactivation by O2 and the effects that trace metals have on the toxic action of O2 on metabolism. The experiments illustrate what may, but not necessarily what does, happen in the intact organism when it is exposed to an elevated pressure of O2.

    It is evident from all the foregoing that the scope of oxygen toxicity is broad both in mechanism and effect. I would like to emphasize what appears to me to be the most important characteristics of O2 toxicity.

    1. It can manifest itself in a great variety of ways. Most, if not all, cells are susceptible to O2 toxicity. Toxic effects of O2 have been demonstrated in bacteria, plants, cell cultures, amphibians, and mammals. Effects vary from inhibition of cellular movement and division of cells to impairment of highly specialized functions such as those performed by the lung, retina, or CNS.

    2. Oxygen poisoning must involve many fundamental biochemical reactions such as those concerned with energy production, transport of substances across membranes, and the oxidation and synthesis of vital tissue constituents.

    3. Oxygen toxicity is most likely associated with the oxidation of certain easily oxidizable chemical entities such as SH groups. Other processes may also be involved, particularly peroxidation of lipids.

    4. Oxygen toxicity is not a unique and esoteric phenomenon seen only when organisms are exposed to extreme conditions of high pressures of O2. It occurs in man in the form of interference with pulmonary function after hours of breathing pure O2. On exposure to a pressure of O2 above 1 atm, there undoubtedly occur a great number of cellular changes, most involving metabolic alterations, long before the overt signs of gross O2 poisoning appear. A study of such early changes is of the utmost importance for the eventual understanding of the mechanism of O2 toxicity and of the way in which O2 tension can influence cell metabolism and function.

    5. Finally, the progress and severity of O2 poisoning can be influenced by a number of agents. This subject will be discussed later in this session. Let me just say here that many substances and conditions have been shown to influence O2 toxicity under different experimental conditions: trace metals, chelating agents, SH compounds and disulfides, hormones, body temperature, and diet. It is very likely that studies of the influence of exogenously administered substances on O2 toxicity in animals will lead to the discovery of agents that will offer man significant protection against O2 toxicity when he is exposed to pressures of O2 greater than that found in air at sea level.

    It is clear to all who have become familiar with the subject that O2 toxicity is not an obscure area of physiology, but one of the most important and interesting problems in the whole field of biology.

    REFERENCES

    1. Bean, J. W. Effects of oxygen at increased pressure. Physiol. Rev.. 1945; 25:1–147.

    2. Bean, J. W., Bohr, D. F. The response of mammalian smooth muscle to oxygen at high pressure and its possible relationship to oxygen poisoning of respiratory enzyme systems. Amer. J. Physiol.. 1944; 142:379–390.

    3. Bert, P.Barometric Pressure: Researches in Experimental Physiology.. Paris: Masson, 1878. [(translated by M. A. Hitchcock and F. A. Hitchcock, reprinted by College Bk.Co., Columbus, Ohio, 1943).].

    4. Chance, B., Jamieson, D., Coles, H. Energy-linked pyridine nucleotide reduction: Inhibitory effects of hyperbaric oxygen in vitro and in vivo. Nature (London). 1965; 206:257–263.

    5. Davies, H. C., Davies, R. E., Biochemical aspects of oxygen poisoningField, J., eds. Handbook of Physiology; II. Williams & Wilkins, Baltimore, Maryland, 1965:1047–1058. [Am. Physiol. Soc. Sect. 3, Respiration].

    6. Gerschman, R. Biological effects of oxygen. In: Dickens F., Neil E., eds. Oxygen in the Animal Organism. New York: Macmillan; 1964:475–492.

    7. Gilbert, D. L. The role of pro-oxidants and anti-oxidants in oxygen toxicity. Radiat. Res. Suppl.. 1963; 3:44–53.

    8. Haugaard, N. Cellular mechanisms of oxygen toxicity. Physiol. Rev.. 1968; 48:311–373.

    9. Haugaard, N., Lee, N. H., Kostrzewa, R., Horn, R. S., Haugaard, E. S. The role of sulfhydryl groups in oxidative phosphorylation and ion transport by rat liver mitochondria. Biochim. Biophys. Acta. 1969; 172:198–204.

    10. Horn, R. S., Haugaard, E. S., Haugaard, N. The mechanism of the inhibition of glycolysis by oxygen in rat heart homogenate. Biochim. Biophys. Acta. 1965; 99:549–552.

    11. Lambertsen, C. J., Kough, R. H., Cooper, D. Y., Emmel, G. L., Loeschcke, H. H., Schmidt, C. F. Oxygen toxicity. Effects in man of oxygen inhalation at 1 and 3.5 atmospheres upon blood gas transport, cerebral circulation and cerebral metabolism. J. Appl. Physiol.. 1953; 5:471–486.

    12. Lambertsen, C. J., Ewing, J. H., Rough, R. H., Gould, R. A., Stroud, M. W., III., Oxygen toxicity. Arterial and internal jugular blood gas composition in man during inhalation of air, 100% O2 and 2% CO2 in O2 at 3.5 atmospheres ambient pressure. J. Appl. Physiol. 1955; 8:255–263

    13. Riggs, B. C. The effect of exposure to oxygen at high pressure upon the tonus and respiration of pyloric muscle from the rabbit. Amer. J. Physiol.. 1945; 145:211–217.

    14. Stadie, W. C., Riggs, B. C., Haugaard, N. Oxygen poisoning. Amer. J. Med. Sci.. 1944; 207:84–114.

    15. Williams, C. D. (1969). Studies of toxic effects of oxygen at one atmosphere on tissue metabolism in vitro. Ph.D. Dissertation, University of Pennsylvania.

    OXYGEN TOXICITY IN NEURONAL ELEMENTS

    J.D. Wood

    Publisher Summary

    This chapter discusses the effects of O2 upon γ-aminobutyric acid metabolism. The pathway from α-oxoglutarate (α-OG) to succinate involving gamma-aminobutyric acid (GABA) is known as the GABA shunt, and radioisotope studies indicate that about 17% of oxidative metabolism goes via the shunt in comparison with 83% that goes via the full metabolic cycle. There is a considerable difference among animal species in susceptibility to high-pressures-of-oxygen (HPO) convulsions. The GABA-forming enzyme, glutamic acid decarboxylase (GAD), is found primarily in the nerve endings and is probably associated with the synaptic vesicles. Until a technique is available for the accurate determination of extracellular GABA concentrations, the problem of measurement will remain a major obstacle in the complete evaluation of the role of GABA in HPO seizures.

    It would be surprising if O2 seizures should prove to be caused solely by one chemical mechanism. Since numerous systems and compounds in the body tissues are sensitive to HPO, it seems more likely that O2 poisoning is due to a variety of factors—some related to one another, some unrelated, and some more important than others. Effects of O2 upon γ-aminobutyric acid metabolism is in the last category, and it is with this subject that this presentation will deal.

    γ-Aminobutyric acid (GABA: NH2CH2CH2CH2COOH) is a simple short-chain acid containing an amino group at one end and a carboxyl group at the other. It is found in vertebrates in significant amounts only in the CNS where it probably plays two roles—that of a modulator or inhibitor of nerve transmission (3), and that of an intermediate in oxidative metabolism (8) (Fig. 1).

    FIG. 1 Metabolic reactions involving GABA and their relationship to the reactions of the TCA cycle. Only the cycle intermediates pertinent to the present discussion are shown.

    The pathway from α-oxoglutarate (α-OG) to succinate involving GABA is known as the GABA shunt, and radioisotope studies (7) indicate that about 17% of oxidative metabolism goes via the shunt in comparison with 83% that goes via the full metabolic cycle. GABA may therefore be a vital link between nerve transmission and oxidative metabolism in the CNS. The effect of HPO on GABA metabolism has seemed worthy of investigation, and the results of such a study follow.

    Effect of HPO on GABA Metabolism

    As Fig. 2 illustrates, HPO was shown in an earlier investigation (13) to cause a significant decrease in brain GABA levels in rats. Of prime importance was the finding that decreased levels were also observed in those animals that had not convulsed. The decrease in GABA could not therefore be attributed to the seizures per se. The GABA levels were also determined 1 hour after the end of the exposure. They had reverted to almost normal levels by that time, indicating that the changes were reversible. These findings are comparable to those of Lambertsen (5), who has described the reversible nature of O2 poisoning. Moreover, the effect of HPO was specific for GABA since no change in the concentration of total α-amino acids was observed (Table I).

    TABLE I

    Concentration of Total α-Amino Acids in Brains of Rats Exposed to 75 psig O2 for 33 Min

    aAll values are the mean ± S.E. for five groups, each containing three brains (13).

    FIG. 2 Brain GABA levels in rats exposed to HPO. (A) 2-min compression to 75 psig O2 followed immediately by 5-min decompression to ambient pressure. (B, C, and D) 33-min exposure to 75 psig O2 with the occurrence, respectively, of no convulsions, mild convulsions, and severe convulsions (13).

    It has been recognized for many years that there is a considerable difference among animal species in susceptibility to HPO convulsions (1), as illustrated in Fig. 3. In the present investigation, the susceptibility to seizures was determined quantitatively by measuring the HPO exposure required to cause convulsions in 50% of the animals tested (expressed as convulsion threshold for 50% of the animals, CT50). This measurement was compared with the decrease in brain GABA for different species exposed to HPO. It is obvious from Fig. 4 that a correlation existed between the CT50 value and the decrease in GABA.

    FIG. 3 Susceptibility of mammalian species to O2 poisoning. The number of animals per group indicated in parentheses. From Wood et al. (17), by permission of the publishers.

    FIG. 4 Decrease in brain GABA level relative to susceptibility to O2 poisoning. CT50 indicates the 75 psig O2 exposure required to convulse 50% of the animals. Decrease in GABA was measured using a 25-min exposure to 75 psig O2. From Wood et al. (17), by permission of the publishers.

    The time to onset of HPO seizures can, of course, be altered by varying the pressure of the O2. Therefore, using pressure as the variable and mice as the experimental animals, the CT50 values were compared with the rate of decrease in GABA. Once again, the correlation was good (Fig. 5). When the rate of decrease in GABA was plotted against the O2 pressure, a most interesting correlation emerged (Fig. 6). A linear relationship between these factors was observed, but, more importantly, the critical pressure causing brain GABA to decrease was 30 psig (3 atm abs). This is the same pressure that has previously been recognized as the one inducing HPO seizures in animals and in man (5, 11).

    FIG. 5 Correlation between rate of decrease in brain GABA concentrations and susceptibility to HPO seizures in mice at different pressures. CT50 indicates the exposure required to convulse 50% of the mice. Numbers in parentheses indicate psig O2 (18).

    FIG. 6 Rate of decrease in brain GABA concentration in mice as a function of O2 pressure. From Wood et al. (18), by permission of the publishers.

    Another well-known factor influencing O2 toxicity is the amount of CO2 in the breathing mixture (1, 2, 12). As earlier workers such as Marshall and Lambertsen (6) have observed, 0.5% or 1.0% CO2 in the mixture hastens the onset of seizures in mice breathing O2 at 60 psig, whereas 5% CO2 prevents the seizures (Table II). Most interestingly, a 0.5% or 1.0% CO2 level accelerated the rate of decrease in brain GABA caused by HPO, whereas a 5% CO2 level almost completely prevented any decrease in GABA. These results are therefore in keeping with the correlation previously observed between susceptibility to HPO seizures and decrease in brain GABA concentrations.

    TABLE II

    Effect of CO2 on HPO-Induced Convulsions (60 psig O2 for 20 Min) and Changes in GABA Levels in Brains of Mice (18)

    The correlation between CT50 values and decrease in GABA under all the conditions described above is illustrated in Fig. 7, in which all points lie on or close to the curve of best fit.

    FIG. 7 Correlation between susceptibility to HPO seizures and decrease in brain GABA. CT50 same as ) Various species, 75 psig O2; (•) mice, various pressures; (×) mice, 60 psig O2 + CO2. From Wood et al. (18), by permission of the publishers.

    If a deranged GABA metabolism is involved in the etiology of HPO-induced seizures, then the administration of GABA prior to exposure to HPO might prevent or delay convulsions. That this protection does indeed exist is demonstrated in the results shown in Table III.

    TABLE III

    Protective Action of Different Dosages of GABA against Oxygen Poisoning (45-Min Exposure at 75 psig) in Rats (15)

    The evidence we have gathered concerning the involvement of GABA metabolism in the production of HPO seizures may be thus summarized:

    1. HPO causes a decrease in GABA levels.

    2. The GABA decrease occurs prior to convulsions.

    3. The GABA decrease is reversible.

    4. The decrease is specific for GABA among the amino acids.

    5. Susceptibility to seizures correlates with the rate of decrease in GABA levels for (a) different animal species, (b) different pressures, and (c) different CO2 concentrations.

    6. The same O2 pressure that induces convulsions also brings about decreases in GABA levels.

    7. GABA administered intraperitoneally protects an animal against HPO seizures.

    Causes and Effects of Decreased GABA Levels

    The possible causes and effects of low GABA levels are shown in the schematic diagram of a nerve synapse in Fig. 8. The GABA-forming enzyme, glutamic acid decarboxylase (GAD), is found primarily in the nerve endings (9) and is probably associated with the synaptic vesicles (4, 9). In contrast, the GABA-degrading enzyme system, GABA-α-oxoglutarate transaminase (GABA-T), is found only in the mitochondria (9). Roberts and Eidelberg (8) found that brain GABA levels are normally determined by the GAD activity rather than by the GABA-T activity, despite the much greater potential activity of the latter enzyme (17).

    FIG. 8 Structural and biochemical organization of a synaptic complex. Mit = mitochondria; Ves = synaptic vesicles.

    This finding suggests that a large portion of GABA in the brain tissues does not have ready access to the catabolizing enzyme. In other words, membrane permeability probably plays a major role in the control of GABA levels. Salganicoff and DeRobertis (9) suggest that GABA formed in nerve endings is released into the synaptic cleft either directly or via the release of synaptic vesicles into the cleft. In either event, it appears that this extracellular GABA is the one involved in the inhibition or modulation of nerve transmission (3).

    The HPO-induced reductions in brain GABA could therefore be brought about by any one, or a combination, of three mechanisms: (1) inhibition of GAD, (2) activation of GABA-T, or (3) increased membrane permeability, which would allow GABA more rapid access to GABA-T. There is good evidence from both in vitro and in vivo studies that GAD is inhibited by HPO (10, 14, 16). This inhibition does not, however, seem to be the sole cause of low GABA levels, in view of recent work (17) indicating that an increased catabolism by GABA-T may also occur. There is not yet evidence indicating whether this increased catabolism is due to an actual activation of GABA-T or to the greater permeability of GABA, but I suspect the latter.

    If low GABA levels induce HPO seizures, the cause is probably a low extracellular concentration of the amino acid. If this is so, total brain GABA levels are useful in assessing the role of GABA in HPO seizures only if they accurately reflect the extracellular concentration. We have perhaps been lucky in maintaining a normal intracellular–extracellular ratio throughout our experiments; it is conceivable that certain medical treatments or physiological conditions might very well alter this ratio of GABA in the tissues. Until a technique is available for the accurate determination of extracellular GABA concentrations, the problem of measurement will remain a major obstacle in the complete evaluation of the role of GABA in HPO seizures.

    REFERENCES

    1. Bean, J. W. Effects of oxygen at increased pressure. Physiol. Rev.. 1945; 25:1–147.

    2. Chapin, J. L. Anticonvulsant threshold of CO2 in oxygen under high pressure. Proc. Soc. Exp. Biol. Med.. 1955; 90:663–664.

    3. Krnjevic, K., Schwartz, S. Is gamma-aminobutyric acid an inhibitory transmitter? Nature (London). 1966; 211:1372–1374.

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    THE INTRACELLULAR OXIDATION–REDUCTION STATE AT HIGH AND LOW OXYGEN CONCENTRATIONS

    Britton Chance

    Publisher Summary

    This chapter discusses intracellular oxidation. Convulsions are the first external sign of hyperbaric toxicity in small animals and man because their primary consequences occur at the cellular level in the same time range of a few minutes. The various cellular oxidase systems that must be considered in the O2 metabolism show different affinities for O2. The microsomal system that is responsible for the detoxification reactions and steroid metabolism of the liver, and the peroxisomal system that may operate in the pathway of glyoxylate metabolism in certain tissues, have considerably higher O2 requirements than mitochondria. The mitochondrial response to hyperbaric O2 represents one of its most rapid and sensitively localized biochemical effects. As further technological advances are made, other incisive comparisons of the intracellular redox state with gross physiological manifestations of hyperbaric toxicity may become possible.

    The necessity of O2 for intracellular bioenergetic reactions, on the one hand, and the damage to sensitive enzymes by higher pressures of O2, on the other, suggest the need to elaborate in some detail the quantitative nature of dangerously low and dangerously high O2 tensions. The cells of mammalian organs—liver, kidney, heart, and brain—afford experimental data that are relevant to human responses to HPO. Our attention will be focused mainly upon primary rather than secondary responses, i.e., those occurring in the first few minutes of anoxia or hyperbaric conditions. Convulsions are the first external sign of hyperbaric toxicity in small animals and man; the primary consequences occurring at the cellular level in the same time range of a few minutes are the topic of this paper. The more involved effects which occur in a quarter of an hour or several hours will not be discussed here, nor will the somewhat different responses of the organs of amphibia, for example, the toad bladder (J. Allen and H. Rasmussen, communication at this symposium).

    In Fig. 1 I have attempted to present in a single diagram the range of O2 concentrations approaching tissue anoxia at the left-hand end of the scale, and tissue damage at the right-hand end, for the different cellular oxidase systems (4, 5). The abscissa represents the logarithm of the O2 concentration in micromolar units, which are approximately equal to Po2 in mmHg at these levels. The biochemical responses at the two ends of the scale are evaluated by direct recording of the fluorescence of intracellular reduced pyridine nucleotide (PN). At the left, this component is too far reduced for appropriate energy metabolism, while on the right it has become too highly oxidized as a consequence of primary events in hyperbaric toxicity affecting mitochondrial reactions.

    FIG. 1 Schematic diagram of O2 affinity of respiratory systems.

    The various cellular oxidase systems which must be considered in the total picture of O2 metabolism show different affinities for O2 (6). Thus, the ordinates of Fig. 1 differ for the various curves. The curve labeled mitochondria illustrates the increasing activity of the mitochondrial respiratory chain as the Po2 increases from left to right, and its effectiveness in causing increasing oxidation of reduced PN in the mitochondria. Intracellular O2 concentrations of as little as 0.1 μM satisfy the needs of the mitochondrial system, and provide satisfactory oxidation of the pool of reduced PN and activation of the energy resources of the mitochondria. Tissue gradients may cause the capillary O2 tension to be somewhat higher than 0.1 μM; our experiments suggest that small mammals breathing 4 to 5% O2 have reached the critical intracellular Po2 as indicated by direct measurement of tissue fluorescence (5). On the other hand, when the O2 pressure reaches approximately 5 atm, as shown at the right-hand side of the diagram, the PN oxidation proceeds to a much higher level and the toxic effects of HPO become evident.

    The microsomal system which is responsible for the detoxification reactions and steroid metabolism of the liver, and the peroxisomal system which may operate in the pathway of glyoxylate metabolism in certain tissues, have considerably higher O2 requirements than do the mitochondria (R. W. Estabrook, C. De Duve, personal communication). It is probable that temporary anoxias which inactivate these systems by reducing the intracellular O2 tension to 0.1 μM do not have any immediate biochemical or physiological repercussions, because this O2 tension is not low enough to impair the operation of the cytochrome system of the

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