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Cerebral resistance to anoxia in the marine turtle
Respiration Physiology (1980), 41, 241-251 © Elsevier/North-Holland Biomedical Press
CEREBRAL RESISTANCE TO ANOXIA IN THE MARINE TURTLE*
P. L, L U T Z j, J . C . L a M A N N A 2, M. R. A D A M S 2 and M, R O S E N T H A L 2 l Rosenstiel School o[' Mar#w and Atmospheric Seienee, Universi O, (~/Miami. Miami, Florida 33149 and 2 Department ~71"Neurology, Universi O' (~/"Miami Medical Sehool, Miami, Florida 33101. U.S.A.
Abstract. The extraordinary ability of the turtle to withstand prolonged anoxia was examined in cerebral cortex m situ by recording changes in the reduction/oxidation ratio of cytochrome a,a 3 by reflection spectrophotometry. Inspiration of 100"i, oxygen increased the oxidation of cytochrome a,a 3 beyond that of the air breathing control, suggesting that cytochrome a,a3 is not fully oxidized under normoxic conditions in turtle brain. A similar response was seen also in the cerebral cortex of the rat. The significance of the cytochrome a,a 3 reduction in these intact tissues is discussed. Both severe hypoxia (100",i N 2) and asphyxia produced increasing levels of reduced cytochrome a,a~ in turtle and rat brains. The rate of change produced by N 2 inspiration was greater than that produced by asphyxia in both species. This is interpreted as demonstrating an open pulmonary blood circulation during anoxia. In turtles, levels of reduced cytochrome a,a 3 were maintained for over 3 h of continual N 2 inspiration. Subsequent inspiration of room air resulted in a full restoration of turtle brain cytochrome a,a 3 redox state within 30 s e c In the rat, continued N 2 inspiration resulted in a rapid reduction of cytochrome a,a~ to a plateau (3 rain) which became irreversible within a short period, An extended tolerance of N 2 inspiration found in rats cooled to temperatures approximating that of the turtle was inadequate to account for the wide species difference. We suggest that special adaptations, not related to the redox state of cytochrome oxidase under normoxic conditions, are responsible for maintaining the functional integrity and the capacity for cytochrome oxidase re-oxidation of turtle brain mitochondria under prolonged anoxia.
Anoxia Asphyxia Brain Cerebral cortex
Cytochrome a,a 3 Diving turtle Mitochondria Rat
Turtles may be the most remarkable of airbreathing vertebrates in their tolerance of total anoxia (Belkin, 1968; Bennett and Dawson, 1976). For example, Robin et al. (1964) reported that Pseudemys scripta can endure at least 48 hr in a 100°/,, N, Accepted~or publication 19 Mc(v 1980
* Supported by NSF Grant PCM78-09281 and NIH Grants NS-14319 and NS-14325. 241
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P.L. LUTZ et al,
environment and can survive anaerobic dives of up to two weeks at 16-18°C. Berkson (1966) recorded that a green turtle (Chelonia mydas) sustained a voluntary six hour dive, the last five hours of which were without measurable oxygen in the trachea or carotid artery. Folk (1974) demonstrated that Chrvsemys tolerated 93 hours in oxygen free water while Felger et al. (1976) found that marine green turtles may bury themselves for 1-3 months in mud on the ocean floor. Much effort has gone to characterizing the respiratory physiology of diving turtles and to explain different vulnerabilities to anoxia (Bennett and Dawson, 1976). In this report, we consider whether turtle anaerobic capacity is linked to a distinctive property of turtle mitochondria in vivo. The impetus of this work comes from the premise that the anaerobic capacity of turtles results from specific adaptations in various tissues. For example, Reeves (1963) found that the isolated turtle heart maintained its mechanical integrity over 15 hours in an oxygen free medium. Bing et al. (1972) found that isolated turtle ventricular strips were markedly more tolerant of hypoxia than those of rat, and McDougal et al. (1968) reported that decapitated turtle heads exhibited periodic movements at long as 3 1/2 h after beheading. However, it is not likely that the anaerobic ability of turtles is due, in any significant way, to unique reaction pathways to energy conservation. As in other vertebrates, anaerobic energy is primarily supplied from glycolysis (Millen et al., 1964) and turtles have particularly high capacities in this respect (Clark and Miller, 1973: Penney, 1974; Robin et al., 1979). Nevertheless, during nitrogen anoxia, glycogen levels in the heart and liver of P. scripta are extensively depleted (Clark and Miller, 1973: Penney, I974). Some supplement, however, may be supplied by more unorthodox mechanisms (Altman and Robin, 1969), and in the green turtle, at least, amino acid catabolism to succinate and alanine appears to have some role (Hochachka et al., 1975). The most sensitive tissue to oxygen lack, however, is commonly considered to be the central nervous system, where energy must continuously be expended particularly for ion transport functioning required for the maintenance of transmembrane potentials (e.g., Robin et al., 1964). Since the adult mammalian brain is highly vulnerable to anoxia, the extraordinary tolerance of turtle brain to anoxia suggests that some particularly interesting adaptations may occur. For example, lactate dehydrogenase (LDH) activity in the brain of the fresh water turtle P. elega~ls is twice that of the rat brain and has a high proportion of M - L D H , the isozyme which is less limited by anoxia (Miller and Hale, 1968). However, compared with other turtle tissues, brain glycogen is low and apparently is depleted very quickly (McDougal et al., 1968) or may not be utilized as a substrate t~r glycolysis (Clark and Miller, 1973), There is nevertheless, a large increase in brain tissue lactic acid levels during prolonged anoxia, and both creatine phosphate and ATP are utilized as energy sources (McDougal et al., 1968: Clark and Miller, 1973). A fall in brain cytoplasmic and mitochondrial N A D + / N A D H ratios has also been noted following anoxia (Lai and Miller, 1973). At the same temperature (21 °C), the rates of oxygen consumption and aerobic ATP production are similar in rat and turtle (P. scripta)
CEREBRAL RESISTANCE TO ANOXIA IN THE MARINE TURTLE
243
brain slices (Robin et al., 1979). However the glycolytic capacity is much greater in the turtle brain and turtle brain pyruvate kinase activity is enhanced (Robin et al., 1979). Direct information on cell oxidative metabolism can be gathered from the reduction/oxidation ratios of the mitochondrial respiratory enzymes. The recent development of dual wavelength reflection spectrophotometry (J6bsis et al., 1977) allows continuous, on-line, recording of changes in redox ratio of cytochromes, particularly cytochrome a,a, (cytochrome c oxidase) from tissue in situ. Cytochrome a,a3 is the terminal oxidase which reacts directly with oxygen and its redox level is, therefore, a particularly sensitive index of mitochondrial oxidative activity. We report here that turtle brain cytochrome a,a 3 is partially reduced under 'resting' conditions and responds to changes in oxygen availability. These findings are in agreement with results from mammalian brain (Rosenthal et al., 1976), including the human neocortex (Austin et al., 1977). This is in contrast, however, to the fully oxidized state of cytochrome oxidase in mitochondria isolated in vitro (Chance and Williams, 1956). A preliminary report of this work has been presented (Lutz et al., 1978).
Methods
Experiments were performed on 7 turtles (loggerhead hatchlings, Caretta caretm) approximately six months old, weighing about 200 g. Pentobarbital sodium (Nembutal) was administered i.p. in doses of around l0 mg/kg. Since pentobarbital is known to depress respiration in reptiles, the drug was injected at below the expected anesthetic dose initially and increments were added until adequate surgical anesthesia was attained, as suggested by McDonald (1976). A cannula was placed in the trachea and the animals were either allowed to respire room air or were respired artificially with a positive pressure respirator. Respirator volumes were kept below the magnitude of extensively visible thoracic expansion (1.5-2.5 ml/ stroke) and stroke rate was set at approximately 30 cycles/rain. Changes in inspired gases were produced by altering the pump supply. For turtles breathing independently through the tracheal cannula, input lines to the cannula were held within large (4 L) respiratory bags that acted as a reservoir for the ambient gas mixture (21 i'ii 02, 79°J,i N,) and then open ends of the cannula were placed in a bag that acted as a reservoir for the required test gas mixture. Bags with different gas mixtures were substituted as necessary. No significant differences were apparent in the two methods of providing respiratory gases. In all experiments, turtles were maintained at ambient temperature (25 °C). Eight rats of the Sprague-Dawley strain (approx. 250--300 g) were anesthetized with pentobarbital sodium (Nembutal) i.p. in doses of 50 mg/kg. The trachea was provided with a cannula, the animals were paralyzed with tubocurarine and respired on a positive pressure respirator. Rats were normally respired with 30?, O~/
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70% N~ to maintain blood gases at physiological values. Rats were maintained at 37°C with a heating pad and rectal thermoprobe. When required, rats were cooled by placing bags of ice in close proximity. In both turtles and rats, optical signals were recorded through the intact dura after removal of a portion of the bone covering the brain. The spectrophotometer procedure had been described in detail (J6bsis et al., 1977). In brief, light at the absorption peak of cytochrome a,a~ (605 nm) and at a reference wavelength (590 nm) alternately illuminated a 3-ram diameter area of cortical surface. The reference wavelength was chosen so that changes in absorption due to changes in blood volume, or to changes in the ratio of oxygenated/deoxygenated hemoglobin were equal at the reference and at the 605 nm wavelength. Light reflected back from the cortex was monitored by a photomultiplier tube housed in the barrel of a microscope. The difference signal (605 590 nm) has been shown to accurately indicate changes in the redox ratio of cytochrome a,a., even when maximal changes in blood volume or hemoglobin oxygenation occur (J6bsis et al., 1977). The signal at 590 nm has been shown to be indicative of changes in blood volume (J6bsis et al., 1977). This technique is based on the fact that reduced cytochrome a,a, absorbs more light than does the oxidized form. The difference (or cytochrome) signal is calibrated as a percent of the full scale (F.S.) light level with zero being the level when the cortex is illuminated fully by reference (590 nm) light but without light at 605 nm. The 100'}~, value is based upon the light reflected when equal signals are recorded from 605 nm and 590 nm with the cortex in a 'resting' state previous to experimental manipulation.
Results
When turtles were switched from respiration of 21')~i O, in 79?'o N~ to 30~, O, in 70% N~_, cytochrome a,a~. became more oxidized (fig. 1). Raising the oxygen content of the respiratory gas to 100~i resulted in a greater cytochrome oxidation. This response is similar to that found for the normothermic rat on switching from 30°% to 100°o oxygen inspiration (fig. 1, bottom trace). When respiratory bags were used as gas reservoirs for turtles freely breathing without a respiratory pump, transitions to higher Flo. values were also accompanied by cytochrome a,a~ oxidation, indicating that the apparent high level of reduction of cytochrome a,a~ observed at 'rest', is not an artifact of positive pressure respiration. When either turtle or rat was respired with 100% N 2, cytochrome a,a3 became reduced. The.effects of a short period of N 2 respiration in turtle (approximately 3 rain) are compared with the reduction of cytochrome a,a3 in a rat respired for approximately 20 sec with N, in fig. 2. Consistently, short periods of N2 hypoxia in turtles were followed by reoxidation of cytochrome a,a~ on air back only to baseline levels. The oxidative overshoot, characteristic of the mammalian recovery period following N~, did not become apparent in turtles unless N~ was presented
245
C E R E B R A L RESISTANCE TO A N O X I A IN THE M A R I N E T U R T L E
TURTLE
AIR
I
I
30% 02
I
AIR
I ~12%
TURTLE AIR
I
100% 02
I
F.S.
AIR 3 MIN
I
RAT
30% 02 , 70%N2
I
I
I
I
30% 02 70% N 2
100% O --
2
I'~-
5% F.$.
1/~
1 MIN
Fig. 1. Responses of cytochrome a , a 3 to increased inspired oxygen in the turtle and rat. Upper trace shows the transition from air inspiration to 30% O~/70% N, in turtles. The second trace shows a transition to 100% O , . The bottom trace demonstrates a response to 100% O, in the rat. In this and subsequent figures, an increase in the level of oxidized cytochrome a , ( l ) is signalled as a downward deflection. Optical traces are calibrated to full scale (F.S.) values as described in the text.
for longer (approximately 15-rain) periods. In mammalian brains, the overshoot following N2 respiration has been attributed to hyperemia (increased blood volume) that occurs during the hypoxic period and remains during the recovery period (Rosenthal et al., 1976). Such an increase in blood volume was not consistently observed during short hypoxic periods in the turtle.
CYT
A,A 3
(605 - 590 NM) AIR
N
I
AIR
TURTLE
I 1% F.S.
2 MIN RAT 30% 02 70% N2
IN~//'~, N2
30% 02 70 % N2
~ - I J 10% F.S. 20 SEC
Fig. 2. Responses of cytochrome a,a~ to short periods of N 2 respiration in the turtle and rat. Top trace shows the reduction of cytochrome a,a 3 with 3 min of N_~ breathing in the turtle. Very short N 2 respiratory periods (approximately 13 sec and 20 sec) are shown in the bottom traces taken from the rat.
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P. L, L U T Z et al. CYTOCHROME A,A 3 (605
- 59o
NM)
TURTLE AIR
PUMP OFF
I
AIR
_
AIR
N2
I
_~-'~
AIR
[ 5% FS
Fig. 3. Changes in cytochrome a,a~ redox ratio produced in the turtle by asphyxia or prolonged N 2 respiration. In the upper trace, the first dashed vertical line represents the point at which the respiratory p u m p was stopped. The subsequent vertical dashed line represents the point at which the p u m p was restarted. In the lower trace, the first dashed vertical line represents onset of N 2 respiration while the second line represents a return to air inspiration.
Figure 3 shows the response of cytochrome a , a 3 in the turtle when the respiratory pump was stopped (upper trace) and the response when N, was presented for approximately 20 min (lower trace). Consistently, the reduction of turtle cytochrome a,a~ in asphyxia was much slower than that produced by N_, respiration. As seen in fig. 3, the amplitude of reduction produced by asphyxia in 20 min, however, was similar to that which occurred following 10 rain of N~. Also, the oxidative overshoot following asphyxia was decreased compared to the overshoot following N2 respiration and the post-asphyxia oxidation was less prolonged. This is consistent with the observations that the oxidative overshoot amplitude and duration depended upon two factors: (a) the amplitude of cytochrome reduction, and (b) the duration of cytochrome reduction. For comparison, fig. 4 shows responses to asphyxia and to N~ respiration in the normothermic rat cerebrum. Note that the time scale is an order of magnitude less. As in the turtle, the response to asphyxia was consistently slower but hypoxia produced by asphyxia or by N2 produced much more rapid reduction responses of cytochrome a,a3 in mammals than in turtles. The longest continuous provision of N~ in the respiratory gas of turtles was 210 min. During this experiment (c¢i fig. 5) cytochrome a , a 3 became reduced with time course similar to that shown in fig. 3 and reached a plateau value within approximately 40 rain. In preliminary studies it appears that turtle brain electrical
247
C E R E B R A L R E S I S T A N C E TO A N O X I A IN T H E M A R I N E T U R T L E
CYTOCHROME
A,A 3
(605 - 590 NM)
RAT I PUMP O F F
3o,~ 0 2
,o..,
,
I
so,~ 0 2
-
~ l _ j /
,o..,
/
]~
10% F,8.
Fig. 4. A comparison of cytochrome a,a 3 redox changes in the rat during asphyxia (upper trace) and N 2 respiration (lower trace).
activity, while changed to a pattern of slower waves, is not suppressed during the prolonged anoxic periods. When 100~ N 2 was replaced with a mixture of 21~Jo O~ in 79 ~°~,;N2, there was again a rapid reoxidation, back to, and then slightly beyond the baseline demonstrating that the mitochondrial capability to pass reducing equivalents to oxygen was not significantly altered by the prolonged anoxic period. There is no reason to believe, however, that an endurance limit had been reached. Since these studies involve a comparison between mammalian (37 °C) and turtle (25°C) reactions to anoxia, it was important to determine whether the lower RAT 10~ F.8
C Y T A,A3 -
-
~
I
I
ECOG
100 UV
I---4 1MIN BP
--
_ - - L -
. . . . . . ~
,,,
i ~ -
~ - - -
i,.o _.,
,
I
TURTLE
I tO0~ N 2
~
/j
I
AIR
I
12~ F.$. I I 3 MIN
Fig. 5. Comparison of cytochrome a , a 3 redox changes in the rat and turtle to prolonged anoxia under approximately similar systemic temperatures. Upper traces show the rat's response when cooled to 22 °C. Lower traces show the effect on the turtle of 210 rain of N 2 and subsequent re-ventilation with air. In this trace, approximately 190 rain are deleted during the period incubated.
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P.L. LUTZ et al.
temperature p e r se of the turtle accounts in any significant way for the differences found. Rats were tested for characteristic responses to transitions from normoxia to 100°/0 02 and to N2 and then were cooled by ice bags placed around their bodies. Rectal temperature was recorded throughout as was the redox state of cytochrome a,a~,. In preliminary studies, it appeared that the cytochrome redox ratio was either unchanged or it became slightly more oxidized during the transition from systemic normothermia to hypothermia (approx. 22°C). The oxidation response of cytochrome a,a~, to hyperoxia was unaltered. The brain temperature, recorded with a microprobe inserted 1 mm below the dura, was approximately 3 °C higher than the rectal temperature. Figure 5 shows the effects of 30 rains of N~ respiration when a rat was cooled systemically to 22:'C. Cytochrome a,a, became reduced slightly more slowly than it did when normothermic conditions although statistical analysis was not done under these conditions. As in normothermia, the cytochrome a,a~ redox state reached peak reduction and remained there throughout the anoxic period. Cortical electrographic activity (ECoG) became suppressed within a short period but at a slower rate than found for normothermia. No reoxidation was seen, however, when the rat was subsequently ventilated with 100°/~; 02. In these animals, systemic blood pressure fell after approximately 15 rains and no recovery appeared possible within a short time afterward.
Discussion The finding that, in the turtle, an increase in inspired oxygen produced a change in the cerebral cytochrome a,a, redox state in the direction of oxidation indicates that turtle cytochrome oxidase is not fully oxidized under normoxic conditions. This is similar to the situation found in mammals (Rosenthal et al., 1976) and suggests that in vivo, high ratios of reduced cerebral cytochrome a,a, are a general vertebrate phenomenon. It contrasts with the in vitro state of fully oxidized cytochrome oxidase (Chance and Williams, 1956). The sensitivity of brain cytochrome a,a, redox state to Po~ changes around normoxia is of significance to respiratory physiology. If, for example, cell metabolic processes are modified around much higher levels of Po~ than has generally been considered (Lubbers and Kessler, 1968), then the blood oxygen dissociation curve, both in shape and affinity, assumes a particular importance for normal tissue respiration, particularly in the tissue normoxic range, i.e., 5-40 Torr O_~. This contrasts with the conventional role given to the dissociation curve of simply providing an oxygen gradient to supply mitochrondria working maximally at incipient anoxia (i.e., 'critical' Po: values less than 0.01 Torr, J6bsis, 1972). Although brain cytochrome a,a) responses to hyperoxia and hypoxia were similar in direction, there were marked quantitative differences between the turtle and mammal when inspiratory gases were changed. In the normothermic rat, peak reduction of cytochrome a,a~ occurred within 3 5 rain of N: respiration and
CEREBRAL RESISTANCE TO ANOXIA IN THE MARINE TURTLE
249
a transition to 30°~i 02/70°o N2 inspiration was not accompanied by reoxidation of cerebral cytochrome a,a,,. Even when rats were cooled to temperatures approximating that of turtles, the recovery capability was extended in time only a short amount and was less than 30 min, However, the reduced cytochrome a.a~ in the turtle was capable of being reoxidized following 210 rain of N2 respiration. This ability of turtle brain mitochondria to survive prolonged anoxia and apparently retain functional integrity (seen as the reversibility of cytochrome a,a,, reduction) suggested that some special structural resiliance may be involved. This is in contrast to mammals where morphological changes take place in mitochondria after only 10-15 rain of anoxia or ischemia and alterations in the reactions of oxidative phosphorylation have been shown to occur after ischemic insults of only 1 min duration (Rosenthal et al., 1976). The data presented answer two additional basic questions about brain metabolism when O: inspiration is decreased. These are: (1) Is there pulmonary storage of O~ in the turtle'?; and (2) Does this pulmonary 02 storage account for prolonged brain viability'? In both rats and turtles. N2 respiration produced more rapid increases in reduced cytochrome a,a~ compared to asphyxia. We interpret this as indicating that oxygen is being purged from the respiratory and circulatory systems when N, is breathed. In fresh water P. scripta, diving is accompanied by a marked increase in pulmonary resistance (White, 1976) and N2 inhalation appears to result in a complete bypass of the lungs (Millen et al., 1964). In the loggerhead it appears. however, that some pulmonary circulation must still be in effect on 100°;, N, inhalation, since the rate of reduction of cytochrome oxidase was more rapid than that following asphyxia. If the asphyxic response of turtle simulates that of the dive, then the transport of oxygen from stores has some initial contribution to normal dive endurance of this animal. This appears to occur in the green turtle CDdo#7.ki m r d a s as oxygen is depleted from the lung during forced dives (Berkson, 1966), though it may not be true for some other diving reptiles (Glass and Johansen, 1979). It is important to note, however, that pulmonary oxygen storage seems not to account for the prolonged viability of the turtle brain on experimental anoxia. We base this interpretation upon the fact that reduction of cytochrome oxidase reached a plateau during N: respiration and also during asphyxia, that these plateau levels were equal in both cases and that long periods of continued N. respiration or asphyxia could be followed by recovery when O, was subsequently made available. In fact, compared to the duration of the anoxic endurance, the transition to anaerobiosis both with asphyxia and N~ respiration occurred within a similar period. It would appear that the extraordinary ability of the turtle to withstand prolonged anoxia is not reflected in differences in the direction of the responses of cytochrome a,a., in turtle and mammalian brain mitochondria to hyperoxia or anoxia. There are, however, marked differences in the time scales of these responses, which could be due to several factors including temperature differences, systemic factors, brain metabolic rate, glycolytic capacity and possible differences in the functional use of
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energy. Temperature effects appear to be involved only to a limited extent because the survival of the hypothermic rats, while prolonged, still does not approach that o f the turtle under similar temperature conditions. This observation is borne out by Qt, measurements of rat brain evoked potential induced metabolic responses (LaManna et al., 1980) and K ~ clearances (Lewis and Schuette, 1975). These are in the range of 2 3 which is much less than the Q], that is necessary to render the rat and turtle responses equivalent with respect to temperature. While systemic factors may make some contribution to the extraordinary anoxic tolerance of the turtle brain the reports of major differences in energy metabolism of rat and turtle brain slices at 26°C (Robin et al., 1979), and our preliminary finding that ECoG activity is not fully suppressed in turtles alter an hour of anoxia, points to an intrinsic anoxic capability in the turtle brain. These observations lead to speculation that the answer to the turtle's viability in anoxia will require studies of brain metabolic and functional activities in vivo.
References Altman, M. and E.D. Robin (1969). Survival during prolonged anaerobiosis as a function of an unusual adaptation involving lactate dehydrogenase subunits. Comp. Biochem. Phv,siol. 30: l lT~J 1187. Austin, J., ,I. Hougan and J.C. L a M a n n a (19771. Cortical oxidative metabolism following micromastomosis for brain ischemia, In: Oxygen and Physiological Function, edited by F.F. J6bsis. Dallas, Texas, Professional Inl\)rlnation Library, pp. 531 544. Belkin, D . A . (1968). Anaerobic brain function. Effects of slagnan! and anoxic anoxia on persistence of breathing ill reptile. Science 162:1017 1018. Bennen, A . F . and W. R. Dawson (1976). Metabolism. In: Biology of the Reptilia. Vol. 5. Physiology, edited by C. G a n s and W. R. Dawson. London, New York, Academic Press. Bing, O. H. L., W . W . Brooks. A . N . Inamdar and 1. V. Messer (1972). Tolerance of isolated heart muscle to hypoxia: turtle v.s. rat. Am. J. Physiol. 223:1481 1485. Berkson, H. (1966). Physiological adjustments to prolonged diving in the Pacific green turtle ~Chehmia a eassizii). Comp. Biochem. Physiol. 18 : I01 I 19. Chance, B. and G . R . Williams (1956). The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17 : 65 134. Clark, V.M. and A.T. Miller. Jr. (1973}. Studies on anaerobic inetabolism in the freshwater turtle (Pseudem3's ,s-crt~ota elelzans). Comp, Biochem. Physiol. 44A: 55 62. Felger, R.S.. K. Clifton and P.J. Regal (1976). Winter dormancy in sea turtles: independent discovery and exploitation in t h e G u l f o l C a l i f o r n i a by two localculturcs. Science 191:283 285. Folk. G. E. (1974). Textbook of Environmental Physiology. 2rid edn. Leak and Febiger. Glass, M. and K. Johansen (1979). Periodic breathing in the crocodile, Crocodvlu.s nilotl'cu~: Consequences l\)r the gas exchange ratio control of breathing. J. Evp. Zoo/. 2(18:319 354. Hochachka, P.W., T . G . Owen, J.F. Allen and G . C . Whitton (1975). Multiple end products of anaerobiosis in diving vertebrates. Comp. Biochem. Physiol. 50B: 17 22. J6bsis, F.F.(1972). Oxidative metabolism at Io,~ PO~. Fed. Proc. 31:1404 1413. J6bsis, F.F., J. Keizer, .I.C. L a M a n n a and M. Rosenthal (1977). Rellectance spectrophotometry of the intact cerebral cortex. 1. Dual wavelength technique. J. Appl. Physiol. 4 3 : 8 5 8 g72. Lai, F . M . H . and A.T. Miller. Jr. (1973). Effect o f h y p o x i a on brain and liver N A D I N A D H ~ ratios in the fl'eshwater turtle f P.s'ettdemys scril~ta cl<~ans). ('omp. Biochem. Physiol. 44B: 307 312.
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L a M a n n a , J.C., M. Rosenthal, R. Novack, D , F . Moffctt and F . F . J6bsis (1980). Temperature coefficients for the oxidative metabolic responses to electrical stimulation in cerebral cortex. J. Neurochem. 34: 203-209, Lewis, D. V. and W. H. Schuette (1975). Temperature dependence of potassium clearance in the central nervous system, Braitt Res. 9 9 : 1 7 5 178. Lubbers, D . W . and M. Kessler (1968). Oxygen supply and rate ot" tissue respiration. In: Oxygen Transport in Blood and Tissue, edited by D . W . Lubbers, U.C. kuft, G. Thews and E. Witzleb. Stuttgart, Georg Thieme Verlag. Lutz. P. k., J.C. L a M a n n a and M. Rosenthal (1978). Cerebral resistance to anoxia o1" marine turtles. Am. Zool. 18: 655. McDonald, H.S. (1976). Methods for the physiological study of reptiles. In: Biology of the Reptilia. Vol. 5. Physiology, edited by C. G a n s and W . R . Dawson. London, New York, Academic Press. McDougal, D. B., Jr., J. Holowach, M. C. Howe, E. M. Jones and C. A. T h o m a s (1968), The effects of anoxia upon energy sources and selected metabolic intermediates in the brains of fish, frog and turtle. J. N~,urochen~,. 15 : 577 588. Millen, J.E.. H.V. Murdaugh, Jr., C.B. Bayer and E.D. Robin (1964). Circulatory adaptations to diving in the freshwater turtle. Science 145:591 -593. Miller. A . T . , Jr., and D . M . Hale (1968). C o m p a r i s o n s of lactic dehydrogeuase in rat and turtle organs. Comp. Biochem. Ph.v,',iol. 2 7 : 5 9 7 601. Penncy, D . G . (1974). Effects of prolonged diving anoxia on the turtle, Ps~,udenl3".s ,SUI'I])ItI ~'[~'~,TtlIM. C'o,'Jlp. Biochum. Phr,siol. 47A: 933-941. Reeves, R. B. (1963). Energy cost of work in aerobic and anaerobic turtle heart muscle. Am. J. Physiol. 205 : 17 -22. Robin, E, D., J.W. Vester, H.V. Murdaugh, Jr.. and ]. E. Millcn (1964). Prolonged anaerobiosis in a vertebrate: Anaerobic metabolism in the freshwater turtle. J. Ue/I. Comp. Phv.vio/. 6 3 : 2 8 7 291. Robin, E.D., N. Lewiston, A. Newman, L. M. Simon and J. Theodore (1979). Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial A T P generation. Proc. NaIL 4cad. Sci. U.S.A. 76:3922 3926. Rosenlhal, M., J. C. LaManna, F. F. J6bsis, J.E. Levasseur, H.A. Kontos and J. L. Patterson (1976). Effects of respiratory gases on cytochrome a m intact cerebral cortex: Is there a critical PO~? Brah~ Rus. 108:143 145. Rosenthal, M., D. L. Martel and J. C. LaManna (1976}. Effects of incomplete and complete ischemia on mitochondrial functioning measured in intact cerebral cortex of cats, Exp. Neurol., 52: 433-446. White, F. N. (1976). Circulation. In: Biology of the Reptilia. Vol. 5. Physiology, edited by C. Gaus and W. R. Dawson. London. New York, Academic Press.
CEREBRAL RESISTANCE TO ANOXIA IN THE MARINE TURTLE*
P. L, L U T Z j, J . C . L a M A N N A 2, M. R. A D A M S 2 and M, R O S E N T H A L 2 l Rosenstiel School o[' Mar#w and Atmospheric Seienee, Universi O, (~/Miami. Miami, Florida 33149 and 2 Department ~71"Neurology, Universi O' (~/"Miami Medical Sehool, Miami, Florida 33101. U.S.A.
Abstract. The extraordinary ability of the turtle to withstand prolonged anoxia was examined in cerebral cortex m situ by recording changes in the reduction/oxidation ratio of cytochrome a,a 3 by reflection spectrophotometry. Inspiration of 100"i, oxygen increased the oxidation of cytochrome a,a 3 beyond that of the air breathing control, suggesting that cytochrome a,a3 is not fully oxidized under normoxic conditions in turtle brain. A similar response was seen also in the cerebral cortex of the rat. The significance of the cytochrome a,a 3 reduction in these intact tissues is discussed. Both severe hypoxia (100",i N 2) and asphyxia produced increasing levels of reduced cytochrome a,a~ in turtle and rat brains. The rate of change produced by N 2 inspiration was greater than that produced by asphyxia in both species. This is interpreted as demonstrating an open pulmonary blood circulation during anoxia. In turtles, levels of reduced cytochrome a,a 3 were maintained for over 3 h of continual N 2 inspiration. Subsequent inspiration of room air resulted in a full restoration of turtle brain cytochrome a,a 3 redox state within 30 s e c In the rat, continued N 2 inspiration resulted in a rapid reduction of cytochrome a,a~ to a plateau (3 rain) which became irreversible within a short period, An extended tolerance of N 2 inspiration found in rats cooled to temperatures approximating that of the turtle was inadequate to account for the wide species difference. We suggest that special adaptations, not related to the redox state of cytochrome oxidase under normoxic conditions, are responsible for maintaining the functional integrity and the capacity for cytochrome oxidase re-oxidation of turtle brain mitochondria under prolonged anoxia.
Anoxia Asphyxia Brain Cerebral cortex
Cytochrome a,a 3 Diving turtle Mitochondria Rat
Turtles may be the most remarkable of airbreathing vertebrates in their tolerance of total anoxia (Belkin, 1968; Bennett and Dawson, 1976). For example, Robin et al. (1964) reported that Pseudemys scripta can endure at least 48 hr in a 100°/,, N, Accepted~or publication 19 Mc(v 1980
* Supported by NSF Grant PCM78-09281 and NIH Grants NS-14319 and NS-14325. 241
242
P.L. LUTZ et al,
environment and can survive anaerobic dives of up to two weeks at 16-18°C. Berkson (1966) recorded that a green turtle (Chelonia mydas) sustained a voluntary six hour dive, the last five hours of which were without measurable oxygen in the trachea or carotid artery. Folk (1974) demonstrated that Chrvsemys tolerated 93 hours in oxygen free water while Felger et al. (1976) found that marine green turtles may bury themselves for 1-3 months in mud on the ocean floor. Much effort has gone to characterizing the respiratory physiology of diving turtles and to explain different vulnerabilities to anoxia (Bennett and Dawson, 1976). In this report, we consider whether turtle anaerobic capacity is linked to a distinctive property of turtle mitochondria in vivo. The impetus of this work comes from the premise that the anaerobic capacity of turtles results from specific adaptations in various tissues. For example, Reeves (1963) found that the isolated turtle heart maintained its mechanical integrity over 15 hours in an oxygen free medium. Bing et al. (1972) found that isolated turtle ventricular strips were markedly more tolerant of hypoxia than those of rat, and McDougal et al. (1968) reported that decapitated turtle heads exhibited periodic movements at long as 3 1/2 h after beheading. However, it is not likely that the anaerobic ability of turtles is due, in any significant way, to unique reaction pathways to energy conservation. As in other vertebrates, anaerobic energy is primarily supplied from glycolysis (Millen et al., 1964) and turtles have particularly high capacities in this respect (Clark and Miller, 1973: Penney, 1974; Robin et al., 1979). Nevertheless, during nitrogen anoxia, glycogen levels in the heart and liver of P. scripta are extensively depleted (Clark and Miller, 1973: Penney, I974). Some supplement, however, may be supplied by more unorthodox mechanisms (Altman and Robin, 1969), and in the green turtle, at least, amino acid catabolism to succinate and alanine appears to have some role (Hochachka et al., 1975). The most sensitive tissue to oxygen lack, however, is commonly considered to be the central nervous system, where energy must continuously be expended particularly for ion transport functioning required for the maintenance of transmembrane potentials (e.g., Robin et al., 1964). Since the adult mammalian brain is highly vulnerable to anoxia, the extraordinary tolerance of turtle brain to anoxia suggests that some particularly interesting adaptations may occur. For example, lactate dehydrogenase (LDH) activity in the brain of the fresh water turtle P. elega~ls is twice that of the rat brain and has a high proportion of M - L D H , the isozyme which is less limited by anoxia (Miller and Hale, 1968). However, compared with other turtle tissues, brain glycogen is low and apparently is depleted very quickly (McDougal et al., 1968) or may not be utilized as a substrate t~r glycolysis (Clark and Miller, 1973), There is nevertheless, a large increase in brain tissue lactic acid levels during prolonged anoxia, and both creatine phosphate and ATP are utilized as energy sources (McDougal et al., 1968: Clark and Miller, 1973). A fall in brain cytoplasmic and mitochondrial N A D + / N A D H ratios has also been noted following anoxia (Lai and Miller, 1973). At the same temperature (21 °C), the rates of oxygen consumption and aerobic ATP production are similar in rat and turtle (P. scripta)
CEREBRAL RESISTANCE TO ANOXIA IN THE MARINE TURTLE
243
brain slices (Robin et al., 1979). However the glycolytic capacity is much greater in the turtle brain and turtle brain pyruvate kinase activity is enhanced (Robin et al., 1979). Direct information on cell oxidative metabolism can be gathered from the reduction/oxidation ratios of the mitochondrial respiratory enzymes. The recent development of dual wavelength reflection spectrophotometry (J6bsis et al., 1977) allows continuous, on-line, recording of changes in redox ratio of cytochromes, particularly cytochrome a,a, (cytochrome c oxidase) from tissue in situ. Cytochrome a,a3 is the terminal oxidase which reacts directly with oxygen and its redox level is, therefore, a particularly sensitive index of mitochondrial oxidative activity. We report here that turtle brain cytochrome a,a 3 is partially reduced under 'resting' conditions and responds to changes in oxygen availability. These findings are in agreement with results from mammalian brain (Rosenthal et al., 1976), including the human neocortex (Austin et al., 1977). This is in contrast, however, to the fully oxidized state of cytochrome oxidase in mitochondria isolated in vitro (Chance and Williams, 1956). A preliminary report of this work has been presented (Lutz et al., 1978).
Methods
Experiments were performed on 7 turtles (loggerhead hatchlings, Caretta caretm) approximately six months old, weighing about 200 g. Pentobarbital sodium (Nembutal) was administered i.p. in doses of around l0 mg/kg. Since pentobarbital is known to depress respiration in reptiles, the drug was injected at below the expected anesthetic dose initially and increments were added until adequate surgical anesthesia was attained, as suggested by McDonald (1976). A cannula was placed in the trachea and the animals were either allowed to respire room air or were respired artificially with a positive pressure respirator. Respirator volumes were kept below the magnitude of extensively visible thoracic expansion (1.5-2.5 ml/ stroke) and stroke rate was set at approximately 30 cycles/rain. Changes in inspired gases were produced by altering the pump supply. For turtles breathing independently through the tracheal cannula, input lines to the cannula were held within large (4 L) respiratory bags that acted as a reservoir for the ambient gas mixture (21 i'ii 02, 79°J,i N,) and then open ends of the cannula were placed in a bag that acted as a reservoir for the required test gas mixture. Bags with different gas mixtures were substituted as necessary. No significant differences were apparent in the two methods of providing respiratory gases. In all experiments, turtles were maintained at ambient temperature (25 °C). Eight rats of the Sprague-Dawley strain (approx. 250--300 g) were anesthetized with pentobarbital sodium (Nembutal) i.p. in doses of 50 mg/kg. The trachea was provided with a cannula, the animals were paralyzed with tubocurarine and respired on a positive pressure respirator. Rats were normally respired with 30?, O~/
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p.L. LUTZ et al.
70% N~ to maintain blood gases at physiological values. Rats were maintained at 37°C with a heating pad and rectal thermoprobe. When required, rats were cooled by placing bags of ice in close proximity. In both turtles and rats, optical signals were recorded through the intact dura after removal of a portion of the bone covering the brain. The spectrophotometer procedure had been described in detail (J6bsis et al., 1977). In brief, light at the absorption peak of cytochrome a,a~ (605 nm) and at a reference wavelength (590 nm) alternately illuminated a 3-ram diameter area of cortical surface. The reference wavelength was chosen so that changes in absorption due to changes in blood volume, or to changes in the ratio of oxygenated/deoxygenated hemoglobin were equal at the reference and at the 605 nm wavelength. Light reflected back from the cortex was monitored by a photomultiplier tube housed in the barrel of a microscope. The difference signal (605 590 nm) has been shown to accurately indicate changes in the redox ratio of cytochrome a,a., even when maximal changes in blood volume or hemoglobin oxygenation occur (J6bsis et al., 1977). The signal at 590 nm has been shown to be indicative of changes in blood volume (J6bsis et al., 1977). This technique is based on the fact that reduced cytochrome a,a, absorbs more light than does the oxidized form. The difference (or cytochrome) signal is calibrated as a percent of the full scale (F.S.) light level with zero being the level when the cortex is illuminated fully by reference (590 nm) light but without light at 605 nm. The 100'}~, value is based upon the light reflected when equal signals are recorded from 605 nm and 590 nm with the cortex in a 'resting' state previous to experimental manipulation.
Results
When turtles were switched from respiration of 21')~i O, in 79?'o N~ to 30~, O, in 70% N~_, cytochrome a,a~. became more oxidized (fig. 1). Raising the oxygen content of the respiratory gas to 100~i resulted in a greater cytochrome oxidation. This response is similar to that found for the normothermic rat on switching from 30°% to 100°o oxygen inspiration (fig. 1, bottom trace). When respiratory bags were used as gas reservoirs for turtles freely breathing without a respiratory pump, transitions to higher Flo. values were also accompanied by cytochrome a,a~ oxidation, indicating that the apparent high level of reduction of cytochrome a,a~ observed at 'rest', is not an artifact of positive pressure respiration. When either turtle or rat was respired with 100% N 2, cytochrome a,a3 became reduced. The.effects of a short period of N 2 respiration in turtle (approximately 3 rain) are compared with the reduction of cytochrome a,a3 in a rat respired for approximately 20 sec with N, in fig. 2. Consistently, short periods of N2 hypoxia in turtles were followed by reoxidation of cytochrome a,a~ on air back only to baseline levels. The oxidative overshoot, characteristic of the mammalian recovery period following N~, did not become apparent in turtles unless N~ was presented
245
C E R E B R A L RESISTANCE TO A N O X I A IN THE M A R I N E T U R T L E
TURTLE
AIR
I
I
30% 02
I
AIR
I ~12%
TURTLE AIR
I
100% 02
I
F.S.
AIR 3 MIN
I
RAT
30% 02 , 70%N2
I
I
I
I
30% 02 70% N 2
100% O --
2
I'~-
5% F.$.
1/~
1 MIN
Fig. 1. Responses of cytochrome a , a 3 to increased inspired oxygen in the turtle and rat. Upper trace shows the transition from air inspiration to 30% O~/70% N, in turtles. The second trace shows a transition to 100% O , . The bottom trace demonstrates a response to 100% O, in the rat. In this and subsequent figures, an increase in the level of oxidized cytochrome a , ( l ) is signalled as a downward deflection. Optical traces are calibrated to full scale (F.S.) values as described in the text.
for longer (approximately 15-rain) periods. In mammalian brains, the overshoot following N2 respiration has been attributed to hyperemia (increased blood volume) that occurs during the hypoxic period and remains during the recovery period (Rosenthal et al., 1976). Such an increase in blood volume was not consistently observed during short hypoxic periods in the turtle.
CYT
A,A 3
(605 - 590 NM) AIR
N
I
AIR
TURTLE
I 1% F.S.
2 MIN RAT 30% 02 70% N2
IN~//'~, N2
30% 02 70 % N2
~ - I J 10% F.S. 20 SEC
Fig. 2. Responses of cytochrome a,a~ to short periods of N 2 respiration in the turtle and rat. Top trace shows the reduction of cytochrome a,a 3 with 3 min of N_~ breathing in the turtle. Very short N 2 respiratory periods (approximately 13 sec and 20 sec) are shown in the bottom traces taken from the rat.
246
P. L, L U T Z et al. CYTOCHROME A,A 3 (605
- 59o
NM)
TURTLE AIR
PUMP OFF
I
AIR
_
AIR
N2
I
_~-'~
AIR
[ 5% FS
Fig. 3. Changes in cytochrome a,a~ redox ratio produced in the turtle by asphyxia or prolonged N 2 respiration. In the upper trace, the first dashed vertical line represents the point at which the respiratory p u m p was stopped. The subsequent vertical dashed line represents the point at which the p u m p was restarted. In the lower trace, the first dashed vertical line represents onset of N 2 respiration while the second line represents a return to air inspiration.
Figure 3 shows the response of cytochrome a , a 3 in the turtle when the respiratory pump was stopped (upper trace) and the response when N, was presented for approximately 20 min (lower trace). Consistently, the reduction of turtle cytochrome a,a~ in asphyxia was much slower than that produced by N_, respiration. As seen in fig. 3, the amplitude of reduction produced by asphyxia in 20 min, however, was similar to that which occurred following 10 rain of N~. Also, the oxidative overshoot following asphyxia was decreased compared to the overshoot following N2 respiration and the post-asphyxia oxidation was less prolonged. This is consistent with the observations that the oxidative overshoot amplitude and duration depended upon two factors: (a) the amplitude of cytochrome reduction, and (b) the duration of cytochrome reduction. For comparison, fig. 4 shows responses to asphyxia and to N~ respiration in the normothermic rat cerebrum. Note that the time scale is an order of magnitude less. As in the turtle, the response to asphyxia was consistently slower but hypoxia produced by asphyxia or by N2 produced much more rapid reduction responses of cytochrome a,a3 in mammals than in turtles. The longest continuous provision of N~ in the respiratory gas of turtles was 210 min. During this experiment (c¢i fig. 5) cytochrome a , a 3 became reduced with time course similar to that shown in fig. 3 and reached a plateau value within approximately 40 rain. In preliminary studies it appears that turtle brain electrical
247
C E R E B R A L R E S I S T A N C E TO A N O X I A IN T H E M A R I N E T U R T L E
CYTOCHROME
A,A 3
(605 - 590 NM)
RAT I PUMP O F F
3o,~ 0 2
,o..,
,
I
so,~ 0 2
-
~ l _ j /
,o..,
/
]~
10% F,8.
Fig. 4. A comparison of cytochrome a,a 3 redox changes in the rat during asphyxia (upper trace) and N 2 respiration (lower trace).
activity, while changed to a pattern of slower waves, is not suppressed during the prolonged anoxic periods. When 100~ N 2 was replaced with a mixture of 21~Jo O~ in 79 ~°~,;N2, there was again a rapid reoxidation, back to, and then slightly beyond the baseline demonstrating that the mitochondrial capability to pass reducing equivalents to oxygen was not significantly altered by the prolonged anoxic period. There is no reason to believe, however, that an endurance limit had been reached. Since these studies involve a comparison between mammalian (37 °C) and turtle (25°C) reactions to anoxia, it was important to determine whether the lower RAT 10~ F.8
C Y T A,A3 -
-
~
I
I
ECOG
100 UV
I---4 1MIN BP
--
_ - - L -
. . . . . . ~
,,,
i ~ -
~ - - -
i,.o _.,
,
I
TURTLE
I tO0~ N 2
~
/j
I
AIR
I
12~ F.$. I I 3 MIN
Fig. 5. Comparison of cytochrome a , a 3 redox changes in the rat and turtle to prolonged anoxia under approximately similar systemic temperatures. Upper traces show the rat's response when cooled to 22 °C. Lower traces show the effect on the turtle of 210 rain of N 2 and subsequent re-ventilation with air. In this trace, approximately 190 rain are deleted during the period incubated.
248
P.L. LUTZ et al.
temperature p e r se of the turtle accounts in any significant way for the differences found. Rats were tested for characteristic responses to transitions from normoxia to 100°/0 02 and to N2 and then were cooled by ice bags placed around their bodies. Rectal temperature was recorded throughout as was the redox state of cytochrome a,a~,. In preliminary studies, it appeared that the cytochrome redox ratio was either unchanged or it became slightly more oxidized during the transition from systemic normothermia to hypothermia (approx. 22°C). The oxidation response of cytochrome a,a~, to hyperoxia was unaltered. The brain temperature, recorded with a microprobe inserted 1 mm below the dura, was approximately 3 °C higher than the rectal temperature. Figure 5 shows the effects of 30 rains of N~ respiration when a rat was cooled systemically to 22:'C. Cytochrome a,a, became reduced slightly more slowly than it did when normothermic conditions although statistical analysis was not done under these conditions. As in normothermia, the cytochrome a,a~ redox state reached peak reduction and remained there throughout the anoxic period. Cortical electrographic activity (ECoG) became suppressed within a short period but at a slower rate than found for normothermia. No reoxidation was seen, however, when the rat was subsequently ventilated with 100°/~; 02. In these animals, systemic blood pressure fell after approximately 15 rains and no recovery appeared possible within a short time afterward.
Discussion The finding that, in the turtle, an increase in inspired oxygen produced a change in the cerebral cytochrome a,a, redox state in the direction of oxidation indicates that turtle cytochrome oxidase is not fully oxidized under normoxic conditions. This is similar to the situation found in mammals (Rosenthal et al., 1976) and suggests that in vivo, high ratios of reduced cerebral cytochrome a,a, are a general vertebrate phenomenon. It contrasts with the in vitro state of fully oxidized cytochrome oxidase (Chance and Williams, 1956). The sensitivity of brain cytochrome a,a, redox state to Po~ changes around normoxia is of significance to respiratory physiology. If, for example, cell metabolic processes are modified around much higher levels of Po~ than has generally been considered (Lubbers and Kessler, 1968), then the blood oxygen dissociation curve, both in shape and affinity, assumes a particular importance for normal tissue respiration, particularly in the tissue normoxic range, i.e., 5-40 Torr O_~. This contrasts with the conventional role given to the dissociation curve of simply providing an oxygen gradient to supply mitochrondria working maximally at incipient anoxia (i.e., 'critical' Po: values less than 0.01 Torr, J6bsis, 1972). Although brain cytochrome a,a) responses to hyperoxia and hypoxia were similar in direction, there were marked quantitative differences between the turtle and mammal when inspiratory gases were changed. In the normothermic rat, peak reduction of cytochrome a,a~ occurred within 3 5 rain of N: respiration and
CEREBRAL RESISTANCE TO ANOXIA IN THE MARINE TURTLE
249
a transition to 30°~i 02/70°o N2 inspiration was not accompanied by reoxidation of cerebral cytochrome a,a,,. Even when rats were cooled to temperatures approximating that of turtles, the recovery capability was extended in time only a short amount and was less than 30 min, However, the reduced cytochrome a.a~ in the turtle was capable of being reoxidized following 210 rain of N2 respiration. This ability of turtle brain mitochondria to survive prolonged anoxia and apparently retain functional integrity (seen as the reversibility of cytochrome a,a,, reduction) suggested that some special structural resiliance may be involved. This is in contrast to mammals where morphological changes take place in mitochondria after only 10-15 rain of anoxia or ischemia and alterations in the reactions of oxidative phosphorylation have been shown to occur after ischemic insults of only 1 min duration (Rosenthal et al., 1976). The data presented answer two additional basic questions about brain metabolism when O: inspiration is decreased. These are: (1) Is there pulmonary storage of O~ in the turtle'?; and (2) Does this pulmonary 02 storage account for prolonged brain viability'? In both rats and turtles. N2 respiration produced more rapid increases in reduced cytochrome a,a~ compared to asphyxia. We interpret this as indicating that oxygen is being purged from the respiratory and circulatory systems when N, is breathed. In fresh water P. scripta, diving is accompanied by a marked increase in pulmonary resistance (White, 1976) and N2 inhalation appears to result in a complete bypass of the lungs (Millen et al., 1964). In the loggerhead it appears. however, that some pulmonary circulation must still be in effect on 100°;, N, inhalation, since the rate of reduction of cytochrome oxidase was more rapid than that following asphyxia. If the asphyxic response of turtle simulates that of the dive, then the transport of oxygen from stores has some initial contribution to normal dive endurance of this animal. This appears to occur in the green turtle CDdo#7.ki m r d a s as oxygen is depleted from the lung during forced dives (Berkson, 1966), though it may not be true for some other diving reptiles (Glass and Johansen, 1979). It is important to note, however, that pulmonary oxygen storage seems not to account for the prolonged viability of the turtle brain on experimental anoxia. We base this interpretation upon the fact that reduction of cytochrome oxidase reached a plateau during N: respiration and also during asphyxia, that these plateau levels were equal in both cases and that long periods of continued N. respiration or asphyxia could be followed by recovery when O, was subsequently made available. In fact, compared to the duration of the anoxic endurance, the transition to anaerobiosis both with asphyxia and N~ respiration occurred within a similar period. It would appear that the extraordinary ability of the turtle to withstand prolonged anoxia is not reflected in differences in the direction of the responses of cytochrome a,a., in turtle and mammalian brain mitochondria to hyperoxia or anoxia. There are, however, marked differences in the time scales of these responses, which could be due to several factors including temperature differences, systemic factors, brain metabolic rate, glycolytic capacity and possible differences in the functional use of
250
P.L. L U T Z et al,
energy. Temperature effects appear to be involved only to a limited extent because the survival of the hypothermic rats, while prolonged, still does not approach that o f the turtle under similar temperature conditions. This observation is borne out by Qt, measurements of rat brain evoked potential induced metabolic responses (LaManna et al., 1980) and K ~ clearances (Lewis and Schuette, 1975). These are in the range of 2 3 which is much less than the Q], that is necessary to render the rat and turtle responses equivalent with respect to temperature. While systemic factors may make some contribution to the extraordinary anoxic tolerance of the turtle brain the reports of major differences in energy metabolism of rat and turtle brain slices at 26°C (Robin et al., 1979), and our preliminary finding that ECoG activity is not fully suppressed in turtles alter an hour of anoxia, points to an intrinsic anoxic capability in the turtle brain. These observations lead to speculation that the answer to the turtle's viability in anoxia will require studies of brain metabolic and functional activities in vivo.
References Altman, M. and E.D. Robin (1969). Survival during prolonged anaerobiosis as a function of an unusual adaptation involving lactate dehydrogenase subunits. Comp. Biochem. Phv,siol. 30: l lT~J 1187. Austin, J., ,I. Hougan and J.C. L a M a n n a (19771. Cortical oxidative metabolism following micromastomosis for brain ischemia, In: Oxygen and Physiological Function, edited by F.F. J6bsis. Dallas, Texas, Professional Inl\)rlnation Library, pp. 531 544. Belkin, D . A . (1968). Anaerobic brain function. Effects of slagnan! and anoxic anoxia on persistence of breathing ill reptile. Science 162:1017 1018. Bennen, A . F . and W. R. Dawson (1976). Metabolism. In: Biology of the Reptilia. Vol. 5. Physiology, edited by C. G a n s and W. R. Dawson. London, New York, Academic Press. Bing, O. H. L., W . W . Brooks. A . N . Inamdar and 1. V. Messer (1972). Tolerance of isolated heart muscle to hypoxia: turtle v.s. rat. Am. J. Physiol. 223:1481 1485. Berkson, H. (1966). Physiological adjustments to prolonged diving in the Pacific green turtle ~Chehmia a eassizii). Comp. Biochem. Physiol. 18 : I01 I 19. Chance, B. and G . R . Williams (1956). The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17 : 65 134. Clark, V.M. and A.T. Miller. Jr. (1973}. Studies on anaerobic inetabolism in the freshwater turtle (Pseudem3's ,s-crt~ota elelzans). Comp, Biochem. Physiol. 44A: 55 62. Felger, R.S.. K. Clifton and P.J. Regal (1976). Winter dormancy in sea turtles: independent discovery and exploitation in t h e G u l f o l C a l i f o r n i a by two localculturcs. Science 191:283 285. Folk. G. E. (1974). Textbook of Environmental Physiology. 2rid edn. Leak and Febiger. Glass, M. and K. Johansen (1979). Periodic breathing in the crocodile, Crocodvlu.s nilotl'cu~: Consequences l\)r the gas exchange ratio control of breathing. J. Evp. Zoo/. 2(18:319 354. Hochachka, P.W., T . G . Owen, J.F. Allen and G . C . Whitton (1975). Multiple end products of anaerobiosis in diving vertebrates. Comp. Biochem. Physiol. 50B: 17 22. J6bsis, F.F.(1972). Oxidative metabolism at Io,~ PO~. Fed. Proc. 31:1404 1413. J6bsis, F.F., J. Keizer, .I.C. L a M a n n a and M. Rosenthal (1977). Rellectance spectrophotometry of the intact cerebral cortex. 1. Dual wavelength technique. J. Appl. Physiol. 4 3 : 8 5 8 g72. Lai, F . M . H . and A.T. Miller. Jr. (1973). Effect o f h y p o x i a on brain and liver N A D I N A D H ~ ratios in the fl'eshwater turtle f P.s'ettdemys scril~ta cl<~ans). ('omp. Biochem. Physiol. 44B: 307 312.
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L a M a n n a , J.C., M. Rosenthal, R. Novack, D , F . Moffctt and F . F . J6bsis (1980). Temperature coefficients for the oxidative metabolic responses to electrical stimulation in cerebral cortex. J. Neurochem. 34: 203-209, Lewis, D. V. and W. H. Schuette (1975). Temperature dependence of potassium clearance in the central nervous system, Braitt Res. 9 9 : 1 7 5 178. Lubbers, D . W . and M. Kessler (1968). Oxygen supply and rate ot" tissue respiration. In: Oxygen Transport in Blood and Tissue, edited by D . W . Lubbers, U.C. kuft, G. Thews and E. Witzleb. Stuttgart, Georg Thieme Verlag. Lutz. P. k., J.C. L a M a n n a and M. Rosenthal (1978). Cerebral resistance to anoxia o1" marine turtles. Am. Zool. 18: 655. McDonald, H.S. (1976). Methods for the physiological study of reptiles. In: Biology of the Reptilia. Vol. 5. Physiology, edited by C. G a n s and W . R . Dawson. London, New York, Academic Press. McDougal, D. B., Jr., J. Holowach, M. C. Howe, E. M. Jones and C. A. T h o m a s (1968), The effects of anoxia upon energy sources and selected metabolic intermediates in the brains of fish, frog and turtle. J. N~,urochen~,. 15 : 577 588. Millen, J.E.. H.V. Murdaugh, Jr., C.B. Bayer and E.D. Robin (1964). Circulatory adaptations to diving in the freshwater turtle. Science 145:591 -593. Miller. A . T . , Jr., and D . M . Hale (1968). C o m p a r i s o n s of lactic dehydrogeuase in rat and turtle organs. Comp. Biochem. Ph.v,',iol. 2 7 : 5 9 7 601. Penncy, D . G . (1974). Effects of prolonged diving anoxia on the turtle, Ps~,udenl3".s ,SUI'I])ItI ~'[~'~,TtlIM. C'o,'Jlp. Biochum. Phr,siol. 47A: 933-941. Reeves, R. B. (1963). Energy cost of work in aerobic and anaerobic turtle heart muscle. Am. J. Physiol. 205 : 17 -22. Robin, E, D., J.W. Vester, H.V. Murdaugh, Jr.. and ]. E. Millcn (1964). Prolonged anaerobiosis in a vertebrate: Anaerobic metabolism in the freshwater turtle. J. Ue/I. Comp. Phv.vio/. 6 3 : 2 8 7 291. Robin, E.D., N. Lewiston, A. Newman, L. M. Simon and J. Theodore (1979). Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial A T P generation. Proc. NaIL 4cad. Sci. U.S.A. 76:3922 3926. Rosenlhal, M., J. C. LaManna, F. F. J6bsis, J.E. Levasseur, H.A. Kontos and J. L. Patterson (1976). Effects of respiratory gases on cytochrome a m intact cerebral cortex: Is there a critical PO~? Brah~ Rus. 108:143 145. Rosenthal, M., D. L. Martel and J. C. LaManna (1976}. Effects of incomplete and complete ischemia on mitochondrial functioning measured in intact cerebral cortex of cats, Exp. Neurol., 52: 433-446. White, F. N. (1976). Circulation. In: Biology of the Reptilia. Vol. 5. Physiology, edited by C. Gaus and W. R. Dawson. London. New York, Academic Press.