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Effect of temperature on mitochondrial respiration in a hibernator (Myotis Austroriparius) and a non-hibernator (Rattus rattus)
Comp. Biochem. Physiol., 1968, Vol. 24, pp. 385 to 394. Pergamon Press. Printed in Great Britain
E F F E C T OF T E M P E R A T U R E ON M I T O C H O N D R I A L R E S P I R A T I O N IN A H I B E R N A T O R (MYOTIS A USTRORIPARIUS) A N D A N O N - H I B E R N A T O R
(RA TTUS RA TTUS)* B. A. HORWITZt and L. NELSON{ Department of Physiology, Emory University, Atlanta, Georgia 30322 (Received 4 July 1967)
A b s t r a c t - - 1 . Respiratory rates of washed and unwashed liver mitochondria from torpid and aroused bats as well as control and cold-acclimated rats were measured at five incubation temperatures. 2. The effects of washing suggest the influence of at least three extramitochondrial factors on succinate oxidation. 3. Although species differences occur at the higher incubation temperatures (37, 29°C), the data do not indicate any differential effects of cold exposure on the metabolic capacity of the mitochondria per se. Rather, the differences observed seem to reflect the influence of extramitochondrial substances which themselves may have been affected by the animal's trophie response to cold. INTRODUCTION THE ABILITY of cells f r o m hibernating m a m m a l s to function at lower temperatures t h a n do those f r o m non-hibernators (Chatfield et al., 1948) raises the possibility that phylogenefic adaptations to t e m p e r a t u r e have developed or " o c c u r " in the cellular c o m p o n e n t s themselves. Increases in the activity of liver mitochondrial oxidative enzymes (measured at 37°C) f r o m hibernating hamsters and chipmunks c o m p a r e d to those f r o m control and cold-adapted rats have been reported (Chaffee et al., 1951; F r e h n & Anthony, 1962), although at 7°C, the liver mitochondrial succinoxidase activity of hibernating hamsters was less than that of the euthermic animals (Chaffee, 1962). T h e s e results m i g h t be interpreted as indicative of the existence of inherent differences in the t e m p e r a t u r e response of the liver mitochondria f r o m hibernating and non-hibernating species. * This study is part of a thesis submitted to the Graduate School of Emory University in partial fulfillment of the requirements for the Doctor of Philosophy degree. This research was supported in part by Training Grant USPHS 5T1-GM-206, Research Grant U.S. Army Medical Research and Development Command, contract number DA-49-193MD-2432, USPHS Research Grants GM-06815 and GM-09652, and NASA Research Grant NGR-11-001-009. Present address : Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, California 95616. ++Recipient of Career Development Award 2K3-GM-15, 193-06 from the National Institute of General Medical Sciences. Present address : Department of Physiology, Medical College of Ohio at Toledo, Toledo, Ohio 43614. 385
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B. A. HORWlTZ AND L. NELSON
T h e p r e s e n t s t u d y was u n d e r t a k e n in a n a t t e m p t to d e t e r m i n e t h e e x t e n t to w h i c h s u c h d i f f e r e n c e s m a y o c c u r . T h e effect of t h e a c t i v i t y state o f t h e h i b e r n a t o r s - - a c t i v e o r t o r p i d - - - o n t h e rates o f m i t o c h o n d r i a l o x y g e n c o n s u m p t i o n was e x a m i n e d at five i n c u b a t i o n t e m p e r a t u r e s . T h e t e m p e r a t u r e r e s p o n s e p a t t e r n s o f t h e s e m e t a b o l i c p a r a m e t e r s w e r e c o m p a r e d to s i m i l a r m e a s u r e m e n t s o b t a i n e d w i t h l i v e r m i t o c h o n d r i a f r o m c o n t r o l a n d c o l d - a d a p t e d a l b i n o rats. T h i s c o m p a r i s o n w a s m a d e to e v a l u a t e t h e d i r e c t effect o f t e m p e r a t u r e o n t h e m e t a b o l i c b e h a v i o r of m i t o c h o n d r i a i s o l a t e d f r o m a n i m a l s w h i c h r e s p o n d d i f f e r e n t l y to c o l d e x p o s u r e . MATERIALS AND METHODS Adult southeastern brown bats, Myotis a. austroriparius (Rhoads), were maintained at a temperature of 3 + 2°C. No selection was made for sex, age or body weight (4.9-6"3 g). Bats sacrificed immediately after removal from the cold are designated as torpid, and those allowed to rewarm for 3 hr at room temperature as aroused. T h e male and female albino rats (Rattus rattus) were 3-4 months of age and weighed between 270 and 325 g. T h e normal rats were housed at 26°C, while the cold-adapted animals were continuously exposed to 5°C for 4-5 weeks. T h e bats were sacrificed by decapitation and the rats exsanguinated after being stunned by a blow to the skull. T h e liver was homogenized in 0-25 M sucrose (4°C) and the homogenate centrifuged at 980 g for 10 min. T h e resulting supernatant was used to isolate two mitoehondrial fractions : the unwashed fraction (Mx) was sedimented by centrifugation at 25,000 g for 10 min, and the washed fraction (M2) was obtained after resuspension of the unwashed pellet in 0"25 M sucrose and recentrifugation at 25,000g for 10 rain. T h e mitoehondrial fractions were resuspended in 0-015 M phosphate buffer (pH 7"35) containing 0"25 M sucrose and 10 -3 M MgC12. All preparatory procedures were conducted at 4°C. A rotating platinum electrode, polarized at - 0 " 8 V, was used to measure the oxygen consumption (Horwitz et al., 1967). Samples of mitochondrial suspensions were removed from the ice bath and added to the reaction cell containing air-saturated phosphate buffer (0"015 M), p H 7"35, 10 -3 M MgC1B, 10 -5 M cytochrome-c, and 0"25 M sucrose. Sodium succinate (50/~moles) was added to the vessel after the mitochondria had utilized all their endogenous substrate, and 50--70 sec later, 0"4-0'5/zmoles of A D P was added. T h e rates of oxygen consumption with substrate alone (Rs) as well as those with substrate and exogenous A D P ( R ~ p ) were measured at five incubation temperatures (5, 13, 21, 29 and 37°C), the ratios of the respiratory rates before and after A D P addition were calculated. Total nitrogen was determined by the micro-Kjeldahl technique (Ma & Zuazaga, 1942). Although the mitochondrial concentration used varied considerably over the temperature range, there was no apparent correlation between these concentrations and the measured respiratory rates. Analyses of variance were performed on all the data and 0.05 was selected as the probability level for significance. RESULTS
Rates of oxygen consumption without A D P (R~) T h e effect on R s o f c o l d e x p o s u r e o n t h e r a t m i t o c h o n d r i a is m o r e p r o n o u n c e d at t h e h i g h e r i n c u b a t i o n t e m p e r a t u r e s ( T a b l e 1). T h e c o l d - a d a p t e d rats h a v e i n c r e a s e d m i t o c h o n d r i a l r e s p i r a t o r y rates at all t e m p e r a t u r e s e x c e p t 21°C, b u t t h e d i f f e r e n c e s b e t w e e n t h e n o r m a l a n d c o l d - e x p o s e d a n i m a l s b e c o m e s m a l l e r at t h e l o w e r i n c u b a t i o n t e m p e r a t u r e s . T h e diffezences b e t w e e n t h e rates o f m i t o c h o n d r i a l o x y g e n c o n s u m p t i o n in t h e t o r p i d a n d a r o u s e d b a t s are n o t as large as t h o s e
387
M I T O C H O N D R I A L RESPIRATION I N HIBERNATOR AND N O N - H I B E R N A T O R
between the rats, and the effect of the incubation temperature on the rates differs greatly between the two species (Table 1). The torpid bats exhibit increased respiratory rates at 37 and 29°C, but at 21°C and below, these rates are considerably lower than those of the aroused bats. Furthermore, in both groups of bats, the maximum rate of oxygen consumption is seen at 29°C. A comparison of the cold-adapted rats and the torpid bats indicates that the rates of oxygen uptake of the mitochondria are significantly higher in the former at all incubation temperatures except 29 and 13°C. TABI.E 1--EFFECT OF TEMPERATUREAND ANIMAL GROUP ON THE RATE OF RESPIRATION (/~moles O~/mg N/rain) WITHOUT ADP (R,)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat: Aroused Torpid M i n i m u m differences for significance (Harter, 1960) Standard error
5% 1%
37
29
21
13
5
0"2370 0"4031
0"1275 0-2164
0"1227 0"1292
0"0353 0"0511
0"0054 0-0083
0"1674 0"2427
0"2392 0"2823
0"1223 0"0663
0"0592 0"0502
0"0149 0-0099
0"0737 0"0967
0"0331 0-0433
0"01740"0228
0"0075 0"0098
0.0019 0"0025
0"0120
0"0054
0"0028
0"0012
0"0003
* Each mean is the average of twenty measurements.
The effect of washing on the mitochondrial R 8 (Table 2) is quite evident at 37 and 29°C, with the rates of the washed mitochondria being greater than those of the unwashed in all animal groups. This trend continues over the lower temperature range in the rats. However, the respiratory rates of the washed and unwashed fractions in both the aroused and torpid bats show no significant differences from 21 to 5°C with one exception; at 5°C, the rate of the M1 oxygen consumption of the aroused bat is statistically higher than that of the M~. The rates of the washed mitochondrial preparations from the cold-adapted rats are not significantly different from those of the aroused or torpid bats at 13 or 5°C.
Rates of oxygen consumption with exogenous ADP
(RADp)
Although the mitochondrial rates of oxygen consumption after ADP addition are greater in the cold-exposed rats than in the normal rats at the higher incubation temperatures, these differences decrease at 13°C and disappear at 5°C (Table 3). In the bats, however, the rates of mitochondrial respiration in the torpid animals are significantly different from those of the aroused bats at 29, 21 and 5°C, and at the two latter temperatures, the mean RAn r of the aroused animals is greater than
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B.A. HORWITZANDL. NELSON
that of the torpid bats. At 29°C, the rates of the bat mitochondrialoxygenuptake are again greater than those at 37°C, although these rates do decrease with temperatures below 29°C.
TABLE 2--EFFECT OF TEMPERATURE,ANIMAL GROUP AND MITOCHONDRIALWASHING ON THE RATE OF RESPIRATION (/~moles Oz/mg N/rain) WITHOUT A D P (Rs)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat: Aroused Torpid M i n i m u m differences for significance (Hatter, 1960) Standard error
37
29
21
13
5
M1 Ms M1 Ms
0"2020 0"2719 0"2664 0"5399
0"1044 0"1505 0"1878 0-2451
0.1021 0-1434 0"0969 0.1614
0"0267 0"0438 0"0489 0"0533
0"0040 0'0069 0"0054 0"0111
M1 Ms MI Ms
0"0805 0"2543 0"1695 0"3160
0"1607 0"3178 0"2047 0"3600
0-1586 0"1586 0"0663 0"0663
0"0629 0'0556 0"0530 0"0474
0"0174 0"0124 0"0086 0'0113
5% 1%
0"1080 0"1402
0"0498 0"6646
0"0258 0'0336
0"0112 0"0146
0"0028 0"0037
0-0120
0"0054
0"0028
0"0012
0"0003
* Each mean is the average of ten measurements.
TABLE 3--EFFECT OF TEMPERATUREAND ANIMAL GROUP ON THE RATE OF RESPIRATION (/~moles Os/mg N/min) WITH A D P (RAPe)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat: Aroused Torpid M i n i m u m differences for significance (Harter, 1960) Standard error
5% 1%
37
29
21
13
5
0-3121 0"4736
0'1554 0"2614
0"1418 0"2046
0-0748 0"0905
0.0081 0'0100
0-2014 0'2838
0-2780 0-3381
0.1452 0"0852
0'0701 0-0575
0-0175 0"0129
0'0868 0"1139
0-0415 0.0545
0-0268 0"0352
0'0147 0"0193
0"0021 0"0028
0"0142
0'0068
0.0044
0-0024
0"0004
* Each mean is the average of twenty measurements.
MITOCHONDRIAL RESPIRATION I N HIBERNATOR AND NON-HIBERNATOR
389
T h e effect of mitochondrial washing on R.~Dp (Table 4) is seen at the higher incubation temperatures in the bats wherein the respiratory rates of M s are greater than those of M 1. At 21°C, however, the RADp of M s from the aroused TABLE 4
EFFECT OF TEMPERATURE~ ANIMAL GROUP AND MITOCHONDRIAL WASHING ON THE RATE OF RESPIRATION
(/zmoles O,/mg N/min) WITH ADP (R~Dp)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat : Aroused Torpid Minimum differences for significance (Harter, 1960) Standard error
37
29
21
13
5
M1 Me M1 M2
0"2792 0"3500 0"3277 0"6196
0"1262 0"1846 0"2243 0'2985
0"1184 0"1652 0"2080 0"2012
0"0586 0'0912 0-1074 0"0736
0"0068 0"0093 0"0072 0"0135
l~ir1
Me M1 Me
0-0989 0-3040 0"1987 0"3639
0-1845 0-3715 0"2390 0"4372
0"1817 0"1088 0"0904 0"0851
0.0787 0"0616 0"0628 0"0522
0-0206 0"0144 0"0108 0"0140
5% 1°';
0'1309 0"1700
0"0625 0"0812
0"0404 0"0527
0"0219 0'0285
0"0033 0"0042
0"0142
0"0068
0"0044
0"0024
0'0004
* Each value is the average of ten measurements. bats falls significantly below that of the M1, but this does not occur in the torpid bat. T h e RAn v of these two fractions in both groups of bats are also similar at 13°C, but at 5°C, the rates of M 1 f r o m the aroused bats are significantly greater than those of the M 2 fractions. I n turn, the M s fractions f r o m the aroused bats are similar to those of the torpid bats at all temperatures. T h e R~_Dp of the normal rat mitochondria is affected b y washing only at 21°C and at 13°C where the rate of M 2 is greater than that of M v T h e rates of oxygen consumption of the washed mitochondria from the cold-exposed rats, however, are higher than those of the unwashed (M1) fractions at all temperatures except 21 and 13°C. T h e rates of M 2 from the cold-adapted rats are also similar to those of both aroused and torpid bats at 13 and 5°C.
Ratios of RADp/R s T h e effect of temperature, animal group and mitochondrial washing on the means of the ratios of respiration with and without exogenous A D P are shown in T a b l e 5. No differences between the respiratory ratios of the two groups of bats are seen at any of the incubation temperatures. Neither washing nor t e m p e r a t u r e influences
390
B.A. HORWITZANDL. NELSON
the magnitude of these ratios in the bat preparations. Decreasing the incubation temperature, however, does increase the respiratory ratios in the rats. This increase occurs at 13°C in the normal rat fractions and at 21°C as well as 13°C in the coldexposed rat mitochondria. In the latter group, washing abolishes the increase. At 5°C, the ratios of the mitochondrial rates of oxygen consumption from both groups of rats are not significantly different from those at 37°C. TABLE 5 - - E F F E C T OF TEMPERATURE,
ANIMAL GROUP AND MITOCHONDRIAL WASHING ON
RADp/R,* Temperature (°C) Animfl group Rat: Normal Cold-exposed Bat: Aroused Torpid
37
29
21
13
5
M1 Me Mt Me
1"38 1"33 1"22 1"18
1"23 1"22 1"19 1"21
1"15 1"14 2'05 1"17
2"52 2"23 2"15 1"37
1"51 1"35 1"32 1-25
M1 Me Mt Me
1"21 1"19 1"21 1"15
1"17 1"20 1"18 1"20
1"14 1-26 1"26 1"31
1"26 1"11 1"18 1"10
1-19 1"18 1"29 1"26
* Minimum differences for significance (Hatter, 1960): 5% = 0.24, 1% = 0-32. Standard error -- 0"01. Each mean is the average of ten measurements. DISCUSSION Since electron microscopic examination of mitochondria isolated in the manner described above indicated that neither mitochondrial washing nor cold exposure increases the extent of ultrastructural modification (Horwitz et al., 1967), the changes seen after washing cannot be attributed to fine structure disorganization. Although the addition of ADP elevates the respiratory rates of the mitochondria from the control rats, the temperature-response patterns of these rates are not markedly influenced. In both the presence and absence of exogenous ADP, the rates appear to level off between incubation temperatures of 21 and 29°C. This plateau region is also evident in the respiratory rates of the cold-adapted rat mitochondria, but only after ADP addition. Thus, in this animal group ADP affects the interaction of succinate oxidation and incubation temperature. The observation that washing abolishes this effect of ADP in the fractions from the cold-exposed rats suggests that the plateau effect of the nucleotide is not exerted directly on the mitochondria but through some substance which can be removed by mitochondrial washing. Furthermore, the absence of such a plateau unless ADP is added to the incubation medium may indicate that the endogenous amounts of this nucleotide fall below a certain critical concentration necessary to interact with the intermediate substance in the preparations from the cold-acclimated rats.
M I T O C H O N D R I A L R E S P I R A T I O N I N H I B E R N A T O R AND N O N - H I B E R N A T O R
391
The significance of the plateau region is not quite clear. In a related experiment, respiration of control rat liver slices displayed a similar temperature response between 23 and 30°C (Horwitz, 1964). These results, for which glucose was used as a substrate with no exogenous ADP present, indicate that this plateau region is not an artefact due to the mitochondrial isolation procedure and further, that the presence of extramitochondrial components of the surviving cells do not eliminate the plateau. Both RxDp and Rs of the washed mitochondria from the cold-adapted rats tend to be higher than those of the unwashed fractions except at 21 and 13°C. In the normal rat preparations, however, significant differences occurred only at the lower temperatures (21, 13 and 5°C). The fact that washing did not significantly increase the specific activities of the respiratory rates at all incubation temperatures suggests that this effect results not from the removal of "non-metabolizing" nitrogenous compounds, but rather from the removal of some soluble or extramitochondrial factor which normally exerts an inhibitory control on the respiratory rate. Loss of this factor appears to have a more profound effect on the coldexposed rat fractions than on those of the control animals. The temperature-response patterns of the bat mitochondrial rates of oxygen uptake are considerably different from those of the rats at the higher incubation temperatures. In all measurements of the torpid and aroused bat mitochondrial fractions, the respiratory rates reach a maximum near 29°C and decrease at 37°C. This pattern is similar to an enzyme-temperature optimum curve, and one might infer that exposure to temperatures much higher than 29°C inactivates the enzymes associated with succinate oxidation in the bat liver mitochondria. It is also possible, however, that a heat-labile activator, rather than an enzyme itself, is involved. Such an in vitro effect at this temperature range would appear unlikely, since the body temperatures of these bats often reach 39°C when they are active (Burbank & Young, 1934). However, the fact that no such temperature lability of the respiratory rates appeared when bat liver slices were exposed to the same incubation temperatures (Horwitz, 1964) might imply that a protective effect can be exerted by extramitochondrial components of the cell. It is also possible that the apparent lack of temperature sensitivity in the tissue slices is due to the presence of the cellular machinery necessary for the continuous synthesis of sufficient quantities of the enzymes to compensate for any inactivation, as seems to be the situation for some of the thermophile bacteria (Allen, 1950, 1953). On the other hand, if the absence of such a temperature lability in the intact liver cells were interpreted as support for the artefactual nature of the mitochondrial response, it would become necessary to explain the lack of a similar lability in the rat mitochondrial fractions by a differential effect of the isolation procedure on the preparations from the two species. A plateau region between 21 and 29°C appears in the respiratory rates of the aroused bat mitochondria and, as in the cold-adapted rat extracts, is abolished by washing. Unlike the response in the cold-exposed rat fractions, however, this effect is independent of the presence of exogenous ADP. It thus appears that the
392
B.A. HORWITZAND L. NELSON
endogenous concentrations of ADP in the aroused bat preparations may be sufficient to interact with the postulated intermediate although they appear to be insufficient in the cold-adapted rat fractions. No plateau region is observed in the torpid bat mitochondria, suggesting that the activity state of the animal (or its body temperature) influences the succinate metabolic pathway. This influence may be a direct effect on the enzymes or may be exerted indirectly on the substance responsible for the plateau region observed in the unwashed fractions from the aroused bat. The effect of washing upon the respiratory rates of the bat mitochondrial preparations differs from that seen in the rat fractions. Washing increases the bat mitochondrial rates only at the higher temperatures, whereas it affects the respiratory rates of the normal rat mitochondria only at the lower incubation temperatures. Furthermore, after washing, the mitochondrial respiration of the torpid and aroused bats is similar in the presence of exogenous ADP over the entire range of incubation temperatures, suggesting that the activity state of the bats has no effect on the capacity of the mitochondria to respond to temperature. The ratios of the respiratory rates with and without exogenous ADP in the control rat mitochondrial fractions are similar except at 13°C where they are considerably elevated. In the cold-adapted rat fractions, this increase, which is seen at 21°C as well as at 13°C, is eliminated by washing. Consideration of this ratio in terms of mitochondrial respiratory control leads to the conclusion that such "control" is not only temperature sensitive but is more easily affected by washing in cold-acclimated rats. Upon examination of the temperature-response patterns of R s and RADp, however, it appears that the elevated ratios reflect a greater temperature sensitivity of the respiratory rates without exogenous ADP. That is, exogenous ADP seems to lower the magnitude of the temperature sensitivities of the respiratory enzymes. The elimination of this increased ratio in the washed mitochondrial preparations from the cold-adapted rats again suggests the loss of a substance, the presence of which seems to result in an increase of the temperature sensitivities. The absence of this effect at 37 and 29°C may indicate that the regulatory mechanism by which the effect of this postulated factor is expressed is itself affected by the incubation temperature. The fact that the respiratory ratios show no such increase at 5°C reflects a greater temperature sensitivity of RXDP from 13 to 5°C than is observed from 21 to 13°C; i.e. at 5°C, addition of ADP does not lower the temperature sensitivities of the respiratory enzymes. This apparent lack of "protective effect" of the nucleotide may be ascribed to limitations on the rates of the enzyme activities imposed by the low temperatures. Thus, the effect of ADP may not be detectable at 5°C because the enzyme activities themselves are so low. The respiratory ratios of the bat mitochondrial fractions are not influenced by the incubation temperatures or washing, suggesting that the substance responsible for the elevated ratios in the rat preparations may not be present in the bat material. The fact that exogenous ADP stimulates the rate of oxygen consumption to the same degree in both the torpid and aroused bat fractions may be interpreted
MITOCHONDRIALRESPIRATION
IN
HIBERNATORANDNON-HIBERNATOR
393
as indicating that the activity state of the bat does not affect the levels of endogenous ADP available as a phosphate acceptor during oxidative metabolism. CONCLUSION The data obtained reveal species differences between bats and rats in the temperature-response patterns of rates of oxygen consumption at higher incubation temperatures. These differences reflect the presence of what appears to be a heat-labile activator or enzyme involved in the bat succinate metabolic pathway. The effect of mitochondrial washing suggests the influence of at least three extramitochondrial (or soluble mitochondrial) factors on the enzyme systems concerned with succinate oxidation. One of these factors appears to interact with ADP, resulting in the occurrence of a plateau region between 21 and 29°C. Only the torpid bat fractions did not show this response. A second factor, which seems to be present in all four animal groups, influences the magnitude of the respiratory rates. Removal of this substance by washing results in elevated rates of oxygen consumption. The third factor, present only in the rat fractions, appears to increase the temperature sensitivities of the succinate metabolic pathway unless exogenous ADP is added. This effect is manifested in increased RADI,/R 8 ratios. The fact that the respiratory rates (with and without exogenous ADP) of washed mitochondria from cold-adapted rats are not significantly different from those of torpid or aroused bats at the lower incubation temperatures suggests that there is no differential effect of cold exposure on the metabolic capacity of the mitochondria themselves. That is, the response of the isolated bat mitochondria to incubation temperature does not appear to reflect the capacity of this species to hibernate. The differences in the metabolic behavior of the preparations from the four animal groups, however, appear to be determined by the influence of extramitochondrial factors which themselves have been affected by changes in the internal environment associated with the state of the animal. Acknowledgements--Grateful acknowledgement is made to Dr. Paul L. Pfahler for his assistance with the statistical analyses and to Dr. Gerhard A. Brecher, Vojin P. Popovic, Raymond Shapira and Morton B. Waitzman who have kindly read this paper and offered their criticisms of the content. REFERENCES
ALLENM. B. (1950) The dynamic nature of thermophily. 07. gen. Physiol. 33, 205-214. ALLENM. B. (1953) The thermophilic aerobic sporeforming bacteria. Bacteriol. Rev. 17, 125-173.
BURBANKR. C. & YOUNGJ. Z. (1934) Temperature changes and winter sleep of bats. 07. Physiol. 83, 459-467. CHAFFEE R. R. J. (1962) Mitochondrial changes during the process of awakening from hibernation. Nature, Lond. 196, 789-790. CHAFFEER. R. J., HOCHF. L. & LYMANC. P. (1961) Mitochondrial oxidative enzymes and phosphorylations in cold exposure and hibernation. Am..7. Physiol. 201, 29-32.
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CHATFIELD P. O., BATTISTAA. F., LYMAN C. P. & Garcia J. P. (1948) Effects of cooling on nerve conduction in a hibernator (golden hamster) and non-hibernator (albino rat). Am. 07. Physiol. 1$5, 179-185. FszI-L~ J. L. & ANTHONY A. (1962) Respiration and phosphorylation of liver mitochondria from cold exposed rats and chipmunks. Am.W. Physiol. 203, 821-824. H . ~ T ~ H. L. (1960) Critical values for Duncan's new multiple range test. Biometrics 16, 671-685. HO~WITZ B. A. (1964) Temperature effects on oxygen uptake of liver and kidney tissues of a hibernating and a non-hibernating mammal. Physiol. Zool. 37, 231-239. HoRwITz B. A., NELSON L. & Povovm V. P. (1967) Effect of temperature on oxidative phosphorylation in hibernators and non-hibernators. 07. appl. Physiol. 22, 639-644. M A T . S. & ZUAZACAG. (1942) Micro-Kjeldahl determination of nitrogen. A new indicator and an improved rapid method. Industr. Engng Chem. Analyt. Ed. 14, 280-282.
E F F E C T OF T E M P E R A T U R E ON M I T O C H O N D R I A L R E S P I R A T I O N IN A H I B E R N A T O R (MYOTIS A USTRORIPARIUS) A N D A N O N - H I B E R N A T O R
(RA TTUS RA TTUS)* B. A. HORWITZt and L. NELSON{ Department of Physiology, Emory University, Atlanta, Georgia 30322 (Received 4 July 1967)
A b s t r a c t - - 1 . Respiratory rates of washed and unwashed liver mitochondria from torpid and aroused bats as well as control and cold-acclimated rats were measured at five incubation temperatures. 2. The effects of washing suggest the influence of at least three extramitochondrial factors on succinate oxidation. 3. Although species differences occur at the higher incubation temperatures (37, 29°C), the data do not indicate any differential effects of cold exposure on the metabolic capacity of the mitochondria per se. Rather, the differences observed seem to reflect the influence of extramitochondrial substances which themselves may have been affected by the animal's trophie response to cold. INTRODUCTION THE ABILITY of cells f r o m hibernating m a m m a l s to function at lower temperatures t h a n do those f r o m non-hibernators (Chatfield et al., 1948) raises the possibility that phylogenefic adaptations to t e m p e r a t u r e have developed or " o c c u r " in the cellular c o m p o n e n t s themselves. Increases in the activity of liver mitochondrial oxidative enzymes (measured at 37°C) f r o m hibernating hamsters and chipmunks c o m p a r e d to those f r o m control and cold-adapted rats have been reported (Chaffee et al., 1951; F r e h n & Anthony, 1962), although at 7°C, the liver mitochondrial succinoxidase activity of hibernating hamsters was less than that of the euthermic animals (Chaffee, 1962). T h e s e results m i g h t be interpreted as indicative of the existence of inherent differences in the t e m p e r a t u r e response of the liver mitochondria f r o m hibernating and non-hibernating species. * This study is part of a thesis submitted to the Graduate School of Emory University in partial fulfillment of the requirements for the Doctor of Philosophy degree. This research was supported in part by Training Grant USPHS 5T1-GM-206, Research Grant U.S. Army Medical Research and Development Command, contract number DA-49-193MD-2432, USPHS Research Grants GM-06815 and GM-09652, and NASA Research Grant NGR-11-001-009. Present address : Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, California 95616. ++Recipient of Career Development Award 2K3-GM-15, 193-06 from the National Institute of General Medical Sciences. Present address : Department of Physiology, Medical College of Ohio at Toledo, Toledo, Ohio 43614. 385
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T h e p r e s e n t s t u d y was u n d e r t a k e n in a n a t t e m p t to d e t e r m i n e t h e e x t e n t to w h i c h s u c h d i f f e r e n c e s m a y o c c u r . T h e effect of t h e a c t i v i t y state o f t h e h i b e r n a t o r s - - a c t i v e o r t o r p i d - - - o n t h e rates o f m i t o c h o n d r i a l o x y g e n c o n s u m p t i o n was e x a m i n e d at five i n c u b a t i o n t e m p e r a t u r e s . T h e t e m p e r a t u r e r e s p o n s e p a t t e r n s o f t h e s e m e t a b o l i c p a r a m e t e r s w e r e c o m p a r e d to s i m i l a r m e a s u r e m e n t s o b t a i n e d w i t h l i v e r m i t o c h o n d r i a f r o m c o n t r o l a n d c o l d - a d a p t e d a l b i n o rats. T h i s c o m p a r i s o n w a s m a d e to e v a l u a t e t h e d i r e c t effect o f t e m p e r a t u r e o n t h e m e t a b o l i c b e h a v i o r of m i t o c h o n d r i a i s o l a t e d f r o m a n i m a l s w h i c h r e s p o n d d i f f e r e n t l y to c o l d e x p o s u r e . MATERIALS AND METHODS Adult southeastern brown bats, Myotis a. austroriparius (Rhoads), were maintained at a temperature of 3 + 2°C. No selection was made for sex, age or body weight (4.9-6"3 g). Bats sacrificed immediately after removal from the cold are designated as torpid, and those allowed to rewarm for 3 hr at room temperature as aroused. T h e male and female albino rats (Rattus rattus) were 3-4 months of age and weighed between 270 and 325 g. T h e normal rats were housed at 26°C, while the cold-adapted animals were continuously exposed to 5°C for 4-5 weeks. T h e bats were sacrificed by decapitation and the rats exsanguinated after being stunned by a blow to the skull. T h e liver was homogenized in 0-25 M sucrose (4°C) and the homogenate centrifuged at 980 g for 10 min. T h e resulting supernatant was used to isolate two mitoehondrial fractions : the unwashed fraction (Mx) was sedimented by centrifugation at 25,000 g for 10 min, and the washed fraction (M2) was obtained after resuspension of the unwashed pellet in 0"25 M sucrose and recentrifugation at 25,000g for 10 rain. T h e mitoehondrial fractions were resuspended in 0-015 M phosphate buffer (pH 7"35) containing 0"25 M sucrose and 10 -3 M MgC12. All preparatory procedures were conducted at 4°C. A rotating platinum electrode, polarized at - 0 " 8 V, was used to measure the oxygen consumption (Horwitz et al., 1967). Samples of mitochondrial suspensions were removed from the ice bath and added to the reaction cell containing air-saturated phosphate buffer (0"015 M), p H 7"35, 10 -3 M MgC1B, 10 -5 M cytochrome-c, and 0"25 M sucrose. Sodium succinate (50/~moles) was added to the vessel after the mitochondria had utilized all their endogenous substrate, and 50--70 sec later, 0"4-0'5/zmoles of A D P was added. T h e rates of oxygen consumption with substrate alone (Rs) as well as those with substrate and exogenous A D P ( R ~ p ) were measured at five incubation temperatures (5, 13, 21, 29 and 37°C), the ratios of the respiratory rates before and after A D P addition were calculated. Total nitrogen was determined by the micro-Kjeldahl technique (Ma & Zuazaga, 1942). Although the mitochondrial concentration used varied considerably over the temperature range, there was no apparent correlation between these concentrations and the measured respiratory rates. Analyses of variance were performed on all the data and 0.05 was selected as the probability level for significance. RESULTS
Rates of oxygen consumption without A D P (R~) T h e effect on R s o f c o l d e x p o s u r e o n t h e r a t m i t o c h o n d r i a is m o r e p r o n o u n c e d at t h e h i g h e r i n c u b a t i o n t e m p e r a t u r e s ( T a b l e 1). T h e c o l d - a d a p t e d rats h a v e i n c r e a s e d m i t o c h o n d r i a l r e s p i r a t o r y rates at all t e m p e r a t u r e s e x c e p t 21°C, b u t t h e d i f f e r e n c e s b e t w e e n t h e n o r m a l a n d c o l d - e x p o s e d a n i m a l s b e c o m e s m a l l e r at t h e l o w e r i n c u b a t i o n t e m p e r a t u r e s . T h e diffezences b e t w e e n t h e rates o f m i t o c h o n d r i a l o x y g e n c o n s u m p t i o n in t h e t o r p i d a n d a r o u s e d b a t s are n o t as large as t h o s e
387
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between the rats, and the effect of the incubation temperature on the rates differs greatly between the two species (Table 1). The torpid bats exhibit increased respiratory rates at 37 and 29°C, but at 21°C and below, these rates are considerably lower than those of the aroused bats. Furthermore, in both groups of bats, the maximum rate of oxygen consumption is seen at 29°C. A comparison of the cold-adapted rats and the torpid bats indicates that the rates of oxygen uptake of the mitochondria are significantly higher in the former at all incubation temperatures except 29 and 13°C. TABI.E 1--EFFECT OF TEMPERATUREAND ANIMAL GROUP ON THE RATE OF RESPIRATION (/~moles O~/mg N/rain) WITHOUT ADP (R,)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat: Aroused Torpid M i n i m u m differences for significance (Harter, 1960) Standard error
5% 1%
37
29
21
13
5
0"2370 0"4031
0"1275 0-2164
0"1227 0"1292
0"0353 0"0511
0"0054 0-0083
0"1674 0"2427
0"2392 0"2823
0"1223 0"0663
0"0592 0"0502
0"0149 0-0099
0"0737 0"0967
0"0331 0-0433
0"01740"0228
0"0075 0"0098
0.0019 0"0025
0"0120
0"0054
0"0028
0"0012
0"0003
* Each mean is the average of twenty measurements.
The effect of washing on the mitochondrial R 8 (Table 2) is quite evident at 37 and 29°C, with the rates of the washed mitochondria being greater than those of the unwashed in all animal groups. This trend continues over the lower temperature range in the rats. However, the respiratory rates of the washed and unwashed fractions in both the aroused and torpid bats show no significant differences from 21 to 5°C with one exception; at 5°C, the rate of the M1 oxygen consumption of the aroused bat is statistically higher than that of the M~. The rates of the washed mitochondrial preparations from the cold-adapted rats are not significantly different from those of the aroused or torpid bats at 13 or 5°C.
Rates of oxygen consumption with exogenous ADP
(RADp)
Although the mitochondrial rates of oxygen consumption after ADP addition are greater in the cold-exposed rats than in the normal rats at the higher incubation temperatures, these differences decrease at 13°C and disappear at 5°C (Table 3). In the bats, however, the rates of mitochondrial respiration in the torpid animals are significantly different from those of the aroused bats at 29, 21 and 5°C, and at the two latter temperatures, the mean RAn r of the aroused animals is greater than
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that of the torpid bats. At 29°C, the rates of the bat mitochondrialoxygenuptake are again greater than those at 37°C, although these rates do decrease with temperatures below 29°C.
TABLE 2--EFFECT OF TEMPERATURE,ANIMAL GROUP AND MITOCHONDRIALWASHING ON THE RATE OF RESPIRATION (/~moles Oz/mg N/rain) WITHOUT A D P (Rs)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat: Aroused Torpid M i n i m u m differences for significance (Hatter, 1960) Standard error
37
29
21
13
5
M1 Ms M1 Ms
0"2020 0"2719 0"2664 0"5399
0"1044 0"1505 0"1878 0-2451
0.1021 0-1434 0"0969 0.1614
0"0267 0"0438 0"0489 0"0533
0"0040 0'0069 0"0054 0"0111
M1 Ms MI Ms
0"0805 0"2543 0"1695 0"3160
0"1607 0"3178 0"2047 0"3600
0-1586 0"1586 0"0663 0"0663
0"0629 0'0556 0"0530 0"0474
0"0174 0"0124 0"0086 0'0113
5% 1%
0"1080 0"1402
0"0498 0"6646
0"0258 0'0336
0"0112 0"0146
0"0028 0"0037
0-0120
0"0054
0"0028
0"0012
0"0003
* Each mean is the average of ten measurements.
TABLE 3--EFFECT OF TEMPERATUREAND ANIMAL GROUP ON THE RATE OF RESPIRATION (/~moles Os/mg N/min) WITH A D P (RAPe)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat: Aroused Torpid M i n i m u m differences for significance (Harter, 1960) Standard error
5% 1%
37
29
21
13
5
0-3121 0"4736
0'1554 0"2614
0"1418 0"2046
0-0748 0"0905
0.0081 0'0100
0-2014 0'2838
0-2780 0-3381
0.1452 0"0852
0'0701 0-0575
0-0175 0"0129
0'0868 0"1139
0-0415 0.0545
0-0268 0"0352
0'0147 0"0193
0"0021 0"0028
0"0142
0'0068
0.0044
0-0024
0"0004
* Each mean is the average of twenty measurements.
MITOCHONDRIAL RESPIRATION I N HIBERNATOR AND NON-HIBERNATOR
389
T h e effect of mitochondrial washing on R.~Dp (Table 4) is seen at the higher incubation temperatures in the bats wherein the respiratory rates of M s are greater than those of M 1. At 21°C, however, the RADp of M s from the aroused TABLE 4
EFFECT OF TEMPERATURE~ ANIMAL GROUP AND MITOCHONDRIAL WASHING ON THE RATE OF RESPIRATION
(/zmoles O,/mg N/min) WITH ADP (R~Dp)* Temperature (°C) Animal group Rat: Normal Cold-exposed Bat : Aroused Torpid Minimum differences for significance (Harter, 1960) Standard error
37
29
21
13
5
M1 Me M1 M2
0"2792 0"3500 0"3277 0"6196
0"1262 0"1846 0"2243 0'2985
0"1184 0"1652 0"2080 0"2012
0"0586 0'0912 0-1074 0"0736
0"0068 0"0093 0"0072 0"0135
l~ir1
Me M1 Me
0-0989 0-3040 0"1987 0"3639
0-1845 0-3715 0"2390 0"4372
0"1817 0"1088 0"0904 0"0851
0.0787 0"0616 0"0628 0"0522
0-0206 0"0144 0"0108 0"0140
5% 1°';
0'1309 0"1700
0"0625 0"0812
0"0404 0"0527
0"0219 0'0285
0"0033 0"0042
0"0142
0"0068
0"0044
0"0024
0'0004
* Each value is the average of ten measurements. bats falls significantly below that of the M1, but this does not occur in the torpid bat. T h e RAn v of these two fractions in both groups of bats are also similar at 13°C, but at 5°C, the rates of M 1 f r o m the aroused bats are significantly greater than those of the M 2 fractions. I n turn, the M s fractions f r o m the aroused bats are similar to those of the torpid bats at all temperatures. T h e R~_Dp of the normal rat mitochondria is affected b y washing only at 21°C and at 13°C where the rate of M 2 is greater than that of M v T h e rates of oxygen consumption of the washed mitochondria from the cold-exposed rats, however, are higher than those of the unwashed (M1) fractions at all temperatures except 21 and 13°C. T h e rates of M 2 from the cold-adapted rats are also similar to those of both aroused and torpid bats at 13 and 5°C.
Ratios of RADp/R s T h e effect of temperature, animal group and mitochondrial washing on the means of the ratios of respiration with and without exogenous A D P are shown in T a b l e 5. No differences between the respiratory ratios of the two groups of bats are seen at any of the incubation temperatures. Neither washing nor t e m p e r a t u r e influences
390
B.A. HORWITZANDL. NELSON
the magnitude of these ratios in the bat preparations. Decreasing the incubation temperature, however, does increase the respiratory ratios in the rats. This increase occurs at 13°C in the normal rat fractions and at 21°C as well as 13°C in the coldexposed rat mitochondria. In the latter group, washing abolishes the increase. At 5°C, the ratios of the mitochondrial rates of oxygen consumption from both groups of rats are not significantly different from those at 37°C. TABLE 5 - - E F F E C T OF TEMPERATURE,
ANIMAL GROUP AND MITOCHONDRIAL WASHING ON
RADp/R,* Temperature (°C) Animfl group Rat: Normal Cold-exposed Bat: Aroused Torpid
37
29
21
13
5
M1 Me Mt Me
1"38 1"33 1"22 1"18
1"23 1"22 1"19 1"21
1"15 1"14 2'05 1"17
2"52 2"23 2"15 1"37
1"51 1"35 1"32 1-25
M1 Me Mt Me
1"21 1"19 1"21 1"15
1"17 1"20 1"18 1"20
1"14 1-26 1"26 1"31
1"26 1"11 1"18 1"10
1-19 1"18 1"29 1"26
* Minimum differences for significance (Hatter, 1960): 5% = 0.24, 1% = 0-32. Standard error -- 0"01. Each mean is the average of ten measurements. DISCUSSION Since electron microscopic examination of mitochondria isolated in the manner described above indicated that neither mitochondrial washing nor cold exposure increases the extent of ultrastructural modification (Horwitz et al., 1967), the changes seen after washing cannot be attributed to fine structure disorganization. Although the addition of ADP elevates the respiratory rates of the mitochondria from the control rats, the temperature-response patterns of these rates are not markedly influenced. In both the presence and absence of exogenous ADP, the rates appear to level off between incubation temperatures of 21 and 29°C. This plateau region is also evident in the respiratory rates of the cold-adapted rat mitochondria, but only after ADP addition. Thus, in this animal group ADP affects the interaction of succinate oxidation and incubation temperature. The observation that washing abolishes this effect of ADP in the fractions from the cold-exposed rats suggests that the plateau effect of the nucleotide is not exerted directly on the mitochondria but through some substance which can be removed by mitochondrial washing. Furthermore, the absence of such a plateau unless ADP is added to the incubation medium may indicate that the endogenous amounts of this nucleotide fall below a certain critical concentration necessary to interact with the intermediate substance in the preparations from the cold-acclimated rats.
M I T O C H O N D R I A L R E S P I R A T I O N I N H I B E R N A T O R AND N O N - H I B E R N A T O R
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The significance of the plateau region is not quite clear. In a related experiment, respiration of control rat liver slices displayed a similar temperature response between 23 and 30°C (Horwitz, 1964). These results, for which glucose was used as a substrate with no exogenous ADP present, indicate that this plateau region is not an artefact due to the mitochondrial isolation procedure and further, that the presence of extramitochondrial components of the surviving cells do not eliminate the plateau. Both RxDp and Rs of the washed mitochondria from the cold-adapted rats tend to be higher than those of the unwashed fractions except at 21 and 13°C. In the normal rat preparations, however, significant differences occurred only at the lower temperatures (21, 13 and 5°C). The fact that washing did not significantly increase the specific activities of the respiratory rates at all incubation temperatures suggests that this effect results not from the removal of "non-metabolizing" nitrogenous compounds, but rather from the removal of some soluble or extramitochondrial factor which normally exerts an inhibitory control on the respiratory rate. Loss of this factor appears to have a more profound effect on the coldexposed rat fractions than on those of the control animals. The temperature-response patterns of the bat mitochondrial rates of oxygen uptake are considerably different from those of the rats at the higher incubation temperatures. In all measurements of the torpid and aroused bat mitochondrial fractions, the respiratory rates reach a maximum near 29°C and decrease at 37°C. This pattern is similar to an enzyme-temperature optimum curve, and one might infer that exposure to temperatures much higher than 29°C inactivates the enzymes associated with succinate oxidation in the bat liver mitochondria. It is also possible, however, that a heat-labile activator, rather than an enzyme itself, is involved. Such an in vitro effect at this temperature range would appear unlikely, since the body temperatures of these bats often reach 39°C when they are active (Burbank & Young, 1934). However, the fact that no such temperature lability of the respiratory rates appeared when bat liver slices were exposed to the same incubation temperatures (Horwitz, 1964) might imply that a protective effect can be exerted by extramitochondrial components of the cell. It is also possible that the apparent lack of temperature sensitivity in the tissue slices is due to the presence of the cellular machinery necessary for the continuous synthesis of sufficient quantities of the enzymes to compensate for any inactivation, as seems to be the situation for some of the thermophile bacteria (Allen, 1950, 1953). On the other hand, if the absence of such a temperature lability in the intact liver cells were interpreted as support for the artefactual nature of the mitochondrial response, it would become necessary to explain the lack of a similar lability in the rat mitochondrial fractions by a differential effect of the isolation procedure on the preparations from the two species. A plateau region between 21 and 29°C appears in the respiratory rates of the aroused bat mitochondria and, as in the cold-adapted rat extracts, is abolished by washing. Unlike the response in the cold-exposed rat fractions, however, this effect is independent of the presence of exogenous ADP. It thus appears that the
392
B.A. HORWITZAND L. NELSON
endogenous concentrations of ADP in the aroused bat preparations may be sufficient to interact with the postulated intermediate although they appear to be insufficient in the cold-adapted rat fractions. No plateau region is observed in the torpid bat mitochondria, suggesting that the activity state of the animal (or its body temperature) influences the succinate metabolic pathway. This influence may be a direct effect on the enzymes or may be exerted indirectly on the substance responsible for the plateau region observed in the unwashed fractions from the aroused bat. The effect of washing upon the respiratory rates of the bat mitochondrial preparations differs from that seen in the rat fractions. Washing increases the bat mitochondrial rates only at the higher temperatures, whereas it affects the respiratory rates of the normal rat mitochondria only at the lower incubation temperatures. Furthermore, after washing, the mitochondrial respiration of the torpid and aroused bats is similar in the presence of exogenous ADP over the entire range of incubation temperatures, suggesting that the activity state of the bats has no effect on the capacity of the mitochondria to respond to temperature. The ratios of the respiratory rates with and without exogenous ADP in the control rat mitochondrial fractions are similar except at 13°C where they are considerably elevated. In the cold-adapted rat fractions, this increase, which is seen at 21°C as well as at 13°C, is eliminated by washing. Consideration of this ratio in terms of mitochondrial respiratory control leads to the conclusion that such "control" is not only temperature sensitive but is more easily affected by washing in cold-acclimated rats. Upon examination of the temperature-response patterns of R s and RADp, however, it appears that the elevated ratios reflect a greater temperature sensitivity of the respiratory rates without exogenous ADP. That is, exogenous ADP seems to lower the magnitude of the temperature sensitivities of the respiratory enzymes. The elimination of this increased ratio in the washed mitochondrial preparations from the cold-adapted rats again suggests the loss of a substance, the presence of which seems to result in an increase of the temperature sensitivities. The absence of this effect at 37 and 29°C may indicate that the regulatory mechanism by which the effect of this postulated factor is expressed is itself affected by the incubation temperature. The fact that the respiratory ratios show no such increase at 5°C reflects a greater temperature sensitivity of RXDP from 13 to 5°C than is observed from 21 to 13°C; i.e. at 5°C, addition of ADP does not lower the temperature sensitivities of the respiratory enzymes. This apparent lack of "protective effect" of the nucleotide may be ascribed to limitations on the rates of the enzyme activities imposed by the low temperatures. Thus, the effect of ADP may not be detectable at 5°C because the enzyme activities themselves are so low. The respiratory ratios of the bat mitochondrial fractions are not influenced by the incubation temperatures or washing, suggesting that the substance responsible for the elevated ratios in the rat preparations may not be present in the bat material. The fact that exogenous ADP stimulates the rate of oxygen consumption to the same degree in both the torpid and aroused bat fractions may be interpreted
MITOCHONDRIALRESPIRATION
IN
HIBERNATORANDNON-HIBERNATOR
393
as indicating that the activity state of the bat does not affect the levels of endogenous ADP available as a phosphate acceptor during oxidative metabolism. CONCLUSION The data obtained reveal species differences between bats and rats in the temperature-response patterns of rates of oxygen consumption at higher incubation temperatures. These differences reflect the presence of what appears to be a heat-labile activator or enzyme involved in the bat succinate metabolic pathway. The effect of mitochondrial washing suggests the influence of at least three extramitochondrial (or soluble mitochondrial) factors on the enzyme systems concerned with succinate oxidation. One of these factors appears to interact with ADP, resulting in the occurrence of a plateau region between 21 and 29°C. Only the torpid bat fractions did not show this response. A second factor, which seems to be present in all four animal groups, influences the magnitude of the respiratory rates. Removal of this substance by washing results in elevated rates of oxygen consumption. The third factor, present only in the rat fractions, appears to increase the temperature sensitivities of the succinate metabolic pathway unless exogenous ADP is added. This effect is manifested in increased RADI,/R 8 ratios. The fact that the respiratory rates (with and without exogenous ADP) of washed mitochondria from cold-adapted rats are not significantly different from those of torpid or aroused bats at the lower incubation temperatures suggests that there is no differential effect of cold exposure on the metabolic capacity of the mitochondria themselves. That is, the response of the isolated bat mitochondria to incubation temperature does not appear to reflect the capacity of this species to hibernate. The differences in the metabolic behavior of the preparations from the four animal groups, however, appear to be determined by the influence of extramitochondrial factors which themselves have been affected by changes in the internal environment associated with the state of the animal. Acknowledgements--Grateful acknowledgement is made to Dr. Paul L. Pfahler for his assistance with the statistical analyses and to Dr. Gerhard A. Brecher, Vojin P. Popovic, Raymond Shapira and Morton B. Waitzman who have kindly read this paper and offered their criticisms of the content. REFERENCES
ALLENM. B. (1950) The dynamic nature of thermophily. 07. gen. Physiol. 33, 205-214. ALLENM. B. (1953) The thermophilic aerobic sporeforming bacteria. Bacteriol. Rev. 17, 125-173.
BURBANKR. C. & YOUNGJ. Z. (1934) Temperature changes and winter sleep of bats. 07. Physiol. 83, 459-467. CHAFFEE R. R. J. (1962) Mitochondrial changes during the process of awakening from hibernation. Nature, Lond. 196, 789-790. CHAFFEER. R. J., HOCHF. L. & LYMANC. P. (1961) Mitochondrial oxidative enzymes and phosphorylations in cold exposure and hibernation. Am..7. Physiol. 201, 29-32.
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CHATFIELD P. O., BATTISTAA. F., LYMAN C. P. & Garcia J. P. (1948) Effects of cooling on nerve conduction in a hibernator (golden hamster) and non-hibernator (albino rat). Am. 07. Physiol. 1$5, 179-185. FszI-L~ J. L. & ANTHONY A. (1962) Respiration and phosphorylation of liver mitochondria from cold exposed rats and chipmunks. Am.W. Physiol. 203, 821-824. H . ~ T ~ H. L. (1960) Critical values for Duncan's new multiple range test. Biometrics 16, 671-685. HO~WITZ B. A. (1964) Temperature effects on oxygen uptake of liver and kidney tissues of a hibernating and a non-hibernating mammal. Physiol. Zool. 37, 231-239. HoRwITz B. A., NELSON L. & Povovm V. P. (1967) Effect of temperature on oxidative phosphorylation in hibernators and non-hibernators. 07. appl. Physiol. 22, 639-644. M A T . S. & ZUAZACAG. (1942) Micro-Kjeldahl determination of nitrogen. A new indicator and an improved rapid method. Industr. Engng Chem. Analyt. Ed. 14, 280-282.