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Anaerobic glycolysis in cardiac tissue from a hibernator and non-hibernator as effected by temperature and hypoxia
Comp. Biochem. Physiol., 1966, Vol. 17, pp. 183 to 189. Pergamon Press Ltd. Printed in Great Britain
ANAEROBIC GLYCOLYSIS IN CARDIAC TISSUE FROM A HIBERNATOR AND NON-HIBERNATOR AS EFFECTED BY TEMPERATURE AND HYPOXIA* ROY F. B U R L I N G T O N t and JACOB E. WIEBERS Department of Biological Sciences, Purdue University, Lafayette, Indiana (Received 31 M a y 1965) A b s t r a c t - - 1 . Rates of anaerobic glycolysis were measured in cardiac tissue from hypoxic ground squirrels (Citellus tridecemlineatus) and albino rats.
2. After hypoxia glycolysis increased significantly in both species at 38°C. At temperatures from 5 to 38°C glycolyticrates were significantlyhigher in tissue from control active (summer) or hibernating (winter) ground squirrels as compared to control rats. Glycolysis increased significantly during hibernation. 3. At low temperatures energies of activation (Ea) for glycolysisin rat tissue were higher than those in ground squirrel tissue. 4. Interspecific differences in glycolyticrates support the hypothesis that the hibernator is better adapted to hypoxia and hypothermia than the homeotherm. INTRODUCTION HIBEI~ATING mammals maintain physiological integrity in deep hypothermia for extended periods (Lyman & Chatfield, 1955) and they survive severe anoxic hypoxia longer than adult homeotherms (Hiestand et al., 1950). The body temperature of the hibernator Often approaches 3 ° to 6°C, whereas in homeothermic mammals neural and cardiac function are impaired at temperatures ranging from 10 ° to 20°C (Hegnauer et al., 1950; South, 1961). South (1958) postulated that cardiac function in hibernating mammals is maintained at low temperatures by a greater energy-producing capacity than that in the homeotherm heart. The mechanisms responsible for increased hypoxic resistance in the hibernator are not well documented although Kayser & Malan (1963) and Andjus et al. (1964) have implicated an increased capacity for cerebral anaerobic glycolysis. On the basis of the above observations, it is reasonable to propose that cellular energy-transforming mechanisms in the heart of the hibernator are modified for function in low temperatures and/or hypoxia. The present investigation was undertaken to clarify the role of anaerobic glycolysis as a factor contributing to hypoxic and hypothermic tolerance in the hibernator, Citellus tridecemlineatus. * Supported by a Public Health Service Fellowship (IFI GM-13 210-01A1) from the National Institute of General Medical Sciences, Public Health Service. t Present address: U.S. Army Medical Research and Nutrition Laboratory, Fitzsimons General Hospital, Denver, Colorado. 183
184
RoY F. BURLINGTONAND JACOB E. WIEBERS
MATERIALS AND METHODS Active (summer) or hibernating (winter) ground squirrels and HarvardWistar albino rats were placed in a Plexiglass chamber and exposed to continuously flowing gas mixtures of 95% N2-5 % 0 2 and 91% N2-9 % 03, respectively. The gas flow was maintained at a rate of 2 l./min for 2 hr at N.A.P. Rats and active ground squirrels were exposed at an ambient temperature of 25 _+I°C, whereas the hibernating ground squirrels were exposed at 6+ I°C. After exposure, the animals were killed by cervical dislocation. The heart was immediately removed, placed in ice cold Krebs-Ringer-bicarbonate solution and sliced to a thickness of 0"5 mm with a Stadie-Riggs microtome. Anaerobic glycolysis (/xl CO2 produced/hr/mg dry tissue) was measured in tissues from control and hypoxic animals in a Warburg apparatus at 5 °, 16 °, 27 ° and 38°C (Umbreit et al., 1957). The incubation medium consisted of KrebsRinger-bicarbonate solution with 200 mg % glucose, a pH of 7"4 and a gas phase of 950/0 N2-5 % COs. The difference between average values obtained from tissues of control and hypoxic animals at each temperature were subjected to Student's t test. The same test was used to compare interspecific and intraspecific differences between mean rates of anaerobic glycolysis in tissues from control animals. Arrhenius plots were utilized to depict the effect of temperature on glycolytic rates. Critical thermal increments (apparent energy of activation, E.4) were calculated for each temperature segment (5-16 °, 16-27 °, and 27-38°C) as k2_Ea(1 1) lnk I R Tll-~O2 " RESULTS Comparative rates of anaerobic glycolysis in heart tissue and t values for differences between glycolytic rates in control and hypoxic animals are presented in Table 1. Table 2 is constructed to elucidate interspecific differences between mean glycolytic rates in control animals as well as intraspecific differences between control active and hibernating ground squirrels. At 38 ° glycolytic rates were significantly higher in tissues from both species after hypoxic exposure, but similar results were not observed at lower temperatures (Table 1). The mean values for anaerobic glycolysis in tissue from exposed rats were consistently lower than those in hypoxic active or hibernating ground squirrels. At all incubation temperatures glycolysis was significantly higher in cardiac tissue from control ground squirrels as compared to control rats (Table 2). A comparison of glycolytic rates in tissue from active and hibernating ground squirrels revealed consistently higher values in the latter animals. Fig. 1 and 2 suggest that inherent differences exist between the effect of temperature on rates of anaerobic glycolysis in the hibernator and homeotherm. The rate of anaerobic glycolysis in rat cardiac tissue decreased sharply between 16 ° and 5°C. In this temperature
185
ANAEROBIC GLYCOLYSIS IN CARDIAC TISSUE T A B L E 1 - - A N A E R O B I C GLYCOLYSIS IN HEART FROM CONTROL AND HYPOXIC ANIMALS
Tempera- Observature tion (°C) ~' QCOI
Hibernating ground squirrel Control
Active ground squirrel
Exposed
Control
Exposed
Rat Control
Exposed
38
Average* Range S.D. t
1.42 2"50 0"83-2"57 2.00-3"64 0"50 0"46 8.44+
0"87 1 '30 0"50-1"29 0"52-1"97 0"23 0"35 3-36,+
0"59 1"04 0"40-0-92 0"53-1"61 0"13 0-28 3-56+
27
Average Range S.D. t
1"58 1"23 0"34-2"73 0"82-1"92 0-74 0"34 2'36t
1"04 1"17 0"46-1-69 0"68-1"71 0"38 0-33 0-88
0"22 0-57 0"09-0"44 0"34-0"96 0"09 0"21 2"36t
16
Average Range S.D. t
0'83 0"65 0"48-1'19 0'33-0"87 0"22 0.14 2"50t
0"48 0"60 0-20-0"83 0'17-1"18 0"15 0"34 1"67
0"13 0-0"29 0"05
Average Range S.D. t
0-21 0"18 0.06-0.41 0.08-0.30 0"11 0.07 1.43
0"07 0"09 0.02-0-17 0-03-0.18 0.05 0.05 0.95
0 0 0
0.19 0"03-0"45 0"13 0"83 0 0 0 0
* n = 15 for each value. t Significant at the 0"05 level: to.o5 (28) = 2.048. ++Significant at the 0"01 level: to.ol (28) = 2-763.
TABLE 2 - - t
VALUES : DIFFERENCES BETWEEN MEANS FOR ANAEROBIC GLYCOLYSIS IN CONTROL ANIMALS
Temperature (°C) 38 27 16 5
Rat and hibernating ground squirrel 6"48* 9"19" 9"72* 10"00"
t Rat and active ground squirrel
Active ground squirrel and hibernating ground squirrel
2"19t 5'54* 4"86* 3'33 *
4'30* 3"65* 4"86* 6'54*
* Significant at the 0.01 level: t0.01 (28) = 2"763. t Significant at the 0"05 level: t0.05 (28) = 2.048.
186
RoY F. BURLINGTON AND JACOBE. WIEBERS
range, the energies of activation (Ea) for glycolysis in the rat tissue far exceed those in the active or hibernating ground squirrel tissue. This phenomenon appeared consistently in tissue from control and hypoxic animals. DISCUSSION Entrance into hibernation and the physiological responses to hypoxia are quite similar in the ground squirrel, i.e. a rapid metabolic depression and body cooling occurs after hypoxia (Bullard, 1960). Although the metabolic needs of the hibernator are substantially lowered in hypothermia and/or hypoxia, the heart and central nervous system must be supplied with energy to insure survival. The capacity for aerobic metabolism is reduced in cardiac tissue from the hibernating animal (Covino & Harmon, 1959; Hannon et al., 1961) ; consequently, anaerobic metabolism could be an increasingly important energy source during hibernation or hypoxia. Studies of glycolytic enzyme activities in heart tissue indicate that the potential for glucose utilization may be increased during hibernation (Vaughan &Hannon, 1960). During hibernation ATP levels decrease in the myocardium (Zimny, 1956). Anaerobic glycolysis is inversely dependent on ATP concentration (Kaplan, 1951). Thus the above data are in agreement with the increased anaerobic glycolysis reported here. Energy transformed via the Embden-Meyerhof pathway is minimal when compared with the oxidative processes of the Krebs cycle; however, a higher rate of anaerobic glycolysis may provide sufficient energy for increased survival of severe hypoxia. Thus higher rates of glycolysis in cardiac tissue from the ground squirrel as compared to the rat are indicative of a cellular adaptation to hypoxia, especially at lower temperatures. A high capacity for anaerobic glycolysis is associated with increased anoxic resistance in infant mammals (Himwich, 1951). Maturation in the homeotherm coincides with a decrease in the initial predominance of anaerobic energy sources and a subsequent loss of hypoxic tolerance. The high capacity for anaerobic glycolysis in the adult hibernator resembles that in the infant homeotherm (Burlington & Wiebers, 1965). The lack of linearity shown in Figs. 1 and 2 reveals that the Arrhenius equation is not wholly adequate for describing the effects of temperature on the complex series of reactions in the Embden-Meyerhof pathway. Nevertheless, assuming that a lower energy of activation (E~) reflects greater enzyme activity, it may be postulated that the high E~ values for rat tissue between 16° and 5°C are due to alteration of the enzymes associated with glycolysis. Theoretically E A is that energy required by chemical reactants to reach an intermediate complex in order that a reaction can proceed to completion. The E~ for a series of biochemical reactions, i.e. anaerobic glycolysis, reflects the slowest or pacemaker reaction in the series (Sizer, 1943). Enzymes reduce the E A needed for reactions in biological systems to proceed at temperatures compatible to life (Glasstone et al., 1941). Temperatures nearing the lower extreme of the biologically tolerable range apparently inactivate enzymes by altering their
0.!
0.5-
i
38C
.....
i
i
27 C i
RAT ACTIVE G.S. HIBERNATING G.S.
i
IBC
I0, 190 3,480 13,716
i
17,307 8,945 8,047
0.0033 0.0034 0.0035 Reciprocol of Absolute Temperature, / T
-~
\
16°C
I
0-0036
~5C
\
42,978 27,693 19,139
5-
FIc. 1. Relationship between heart Q~e6, and temperature in control rats, active ground squirrels and hibernating ground squirrels.
0.0032
0.01
0.05
o
zu
~0
-
1.0-
5~.
I0,0
EA(cal) 27-38 ° 16-27"
0,1
58C
o.o, ,.ob3~
0.05-
~n o
-J
~o ~ u
- - -
~
o.ob3~
0.0534
I
\, /T
o ohms I
16C
"-
\
16,712 8,283 3,021 12,177 1,810 10,133
16-2"/"
EA(cal)
Reciprocal of Absolute Temperature
i
27C
RAT ACTIVE G. S. HIBERNATING GIS
-~3 °
~
\ •
5C
o.oo35
N\
57,445 28,102 20,073
5 - 16*C
FIG. 2. Relationship between heart Qco, ~' and temperature in hypoxic rats, active ground squirrels and hibernating ground squirrels.
z
0'5
I-O
5"0,
lifO-
27
~o
188
RoY F. BURLINGTON AND JACOR E. WIEBERS
molecular configuration (Kavanau, 1950). This p h e n o m e n o n may account for the high E l values which appear at low temperatures in rat tissue. Higher rates of anaerobic glycolysis in the hibernator represent a cellular adaptation to adverse environmental conditions; however, the mechanisms responsible for this p h e n o m e n o n remain unclear and must await further investigation.
SUMMARY An investigation was conducted to assess the effects of hypoxia and hypothermia on rates of anaerobic glycolysis in heart tissue from albino rats and hibernating or active thirteen-lined ground squirrels. At 38°C anaerobic glycolysis increased significantly in tissues from both species after exposure to hypoxia in vivo ; however, similar increases did not occur consistently at lower temperatures. Between 5~ and 16'~C lower energy of activation values ( E , ) for anaerobic glycolysis in ground squirrel tissue reflects a greater enzyme activity than that in rat tissue. In a species comparison at temperatures ranging from 5 ° to 38°C glycolytic rates were consistently higher in ground squirrel tissue. Intraspecifically, anaerobic glycolysis in cardiac tissue from hibernating ground squirrels was significantly higher than that in active s u m m e r animals. F r o m these data it is suggested that hypoxic or hypothermic tolerance is partially attributable to an increased capacity for anaerobic glycolysis. REFERENCES ANDJUS R. K., (~IRKOVI(~T., (_~UPERLOVI(~N., DAVIDOVI(~J., MARKOVI(~-USKOKOVICV. & VELIMIROVICT. (1964) Brain metabolism and resistance of a hibernator (Citellus citelhts) and the rat to different anoxic conditions, including cardiac arrest in deep hypothermia. Ann. Acad. Sci. Fenn. (Set. A.) 4, 11-23. I]ULLARDR. W., DAVID G. & NICHOLS C. T. (1960) The mechanisms of hypoxic tolerance in hibernating and non-hibernating mammals. Bull. Mus. Comp. Zool., t-Iarvard 124, 321-335. BURLINGTONR. F. ~¢ WIEBERSJ. E. (1965) Effect of hypoxia on cerebral anaerobic glycolysis in the hibernator and infant or adult homeotherm. Comp. Biochem. Physiol. 14, 201-203. COVINO B. G. & HANNON J. P. (1959) Myocardial metabolic and electrical properties of rabbits and ground squirrels at low temperature. Amer. ff. Physiol. 197, 494-498. GLASSTONES., LAIDLERK. J. & EYRINGH. (1941) The Theory of Rate Processes. McGrawHill, N.Y. HANNON J. P., VAUCHAND. A. & HOCK R. J. (1961) The endogenous tissue respiration of the Arctic ground squirrel as effected by hibernation and season. J. cell. comp. Physiol. 57, 5-10. HEGNAUERA. H., SHIRBERW. J. & HATERIUSH. O. (1950) Cardiovascular response of the dog to immersion hypothermia. Amer. J. Physiol. 161, 455-465. tIIESTANOW. S., ROCKtaOLDW. T., STEMLERF. W., STULLKEND. E. & WIEBERSJ. E. (1950) The comparative hypoxic resistance of hibernators and non-hibernators. P~,siol. Zool. 23, 264-269. HIMWICH H. E. (1951) Brain Metabolism and Cerebral Disorders. Williams & Wilkins, Baltimore. KAPLA:~ N. O. (1951) Thermodynamics and mechanism of the phosphate bond. In The Enzymes (SI;MNERJ. B. & MYR~ACt~ K., eds.), chap. 45. Academic Press, N.Y.
ANAEROBIC GLYCOLYSIS IN CARDIAC TISSUE
189
KAVANAUJ. L. (1950) Enzyme kinetics and the rate of biological processes, ft. Gen. Physiol. 34, 193-209. KAYSER C. H. & MALAN A. (1963) Central nervous system and hibernation. Experientia 19, 441-451. LYMAN C. P. ~ CHATFIELDP. O. (1955) Physiology of hibernation in mammals. Physiol. Rev. 35, 403-425. SIZER I. W. (1943) Effects of temperature on enzyme kinetics. Advanc. Enzymol. 3, 35-62. SOUTH F. E. (1958) Rates of oxygen consumption and glycolysis of ventricle and brain slices, obtained from hibernating and non-hibernating mammals, as a function of temperature. Physiol. Zool. 31, 6-15. SOUTH F. E. (1961) Phrenic nerve-diaphragm preparations in relation to temperature and hibernation. Amer..7. Physiol. 200(3), 565-571. UMBREIT W. W . , Bum~is R. H. & STAUFFERJ. F. (1957) Manometric Techniques. Burgess Co., Minneapolis. VAUGHAN n . A. & HANNON J. P. (1960) Activity of selected glycolytic enzymes during hibernation. Fed. Proc. 19, 45. ZIMNY M. L. (1956) Metabolism of some carbohydrate and phosphate compounds during hibernation in the ground squirrel. )t. cell. comp. Physiol. 48, 371-391.
ANAEROBIC GLYCOLYSIS IN CARDIAC TISSUE FROM A HIBERNATOR AND NON-HIBERNATOR AS EFFECTED BY TEMPERATURE AND HYPOXIA* ROY F. B U R L I N G T O N t and JACOB E. WIEBERS Department of Biological Sciences, Purdue University, Lafayette, Indiana (Received 31 M a y 1965) A b s t r a c t - - 1 . Rates of anaerobic glycolysis were measured in cardiac tissue from hypoxic ground squirrels (Citellus tridecemlineatus) and albino rats.
2. After hypoxia glycolysis increased significantly in both species at 38°C. At temperatures from 5 to 38°C glycolyticrates were significantlyhigher in tissue from control active (summer) or hibernating (winter) ground squirrels as compared to control rats. Glycolysis increased significantly during hibernation. 3. At low temperatures energies of activation (Ea) for glycolysisin rat tissue were higher than those in ground squirrel tissue. 4. Interspecific differences in glycolyticrates support the hypothesis that the hibernator is better adapted to hypoxia and hypothermia than the homeotherm. INTRODUCTION HIBEI~ATING mammals maintain physiological integrity in deep hypothermia for extended periods (Lyman & Chatfield, 1955) and they survive severe anoxic hypoxia longer than adult homeotherms (Hiestand et al., 1950). The body temperature of the hibernator Often approaches 3 ° to 6°C, whereas in homeothermic mammals neural and cardiac function are impaired at temperatures ranging from 10 ° to 20°C (Hegnauer et al., 1950; South, 1961). South (1958) postulated that cardiac function in hibernating mammals is maintained at low temperatures by a greater energy-producing capacity than that in the homeotherm heart. The mechanisms responsible for increased hypoxic resistance in the hibernator are not well documented although Kayser & Malan (1963) and Andjus et al. (1964) have implicated an increased capacity for cerebral anaerobic glycolysis. On the basis of the above observations, it is reasonable to propose that cellular energy-transforming mechanisms in the heart of the hibernator are modified for function in low temperatures and/or hypoxia. The present investigation was undertaken to clarify the role of anaerobic glycolysis as a factor contributing to hypoxic and hypothermic tolerance in the hibernator, Citellus tridecemlineatus. * Supported by a Public Health Service Fellowship (IFI GM-13 210-01A1) from the National Institute of General Medical Sciences, Public Health Service. t Present address: U.S. Army Medical Research and Nutrition Laboratory, Fitzsimons General Hospital, Denver, Colorado. 183
184
RoY F. BURLINGTONAND JACOB E. WIEBERS
MATERIALS AND METHODS Active (summer) or hibernating (winter) ground squirrels and HarvardWistar albino rats were placed in a Plexiglass chamber and exposed to continuously flowing gas mixtures of 95% N2-5 % 0 2 and 91% N2-9 % 03, respectively. The gas flow was maintained at a rate of 2 l./min for 2 hr at N.A.P. Rats and active ground squirrels were exposed at an ambient temperature of 25 _+I°C, whereas the hibernating ground squirrels were exposed at 6+ I°C. After exposure, the animals were killed by cervical dislocation. The heart was immediately removed, placed in ice cold Krebs-Ringer-bicarbonate solution and sliced to a thickness of 0"5 mm with a Stadie-Riggs microtome. Anaerobic glycolysis (/xl CO2 produced/hr/mg dry tissue) was measured in tissues from control and hypoxic animals in a Warburg apparatus at 5 °, 16 °, 27 ° and 38°C (Umbreit et al., 1957). The incubation medium consisted of KrebsRinger-bicarbonate solution with 200 mg % glucose, a pH of 7"4 and a gas phase of 950/0 N2-5 % COs. The difference between average values obtained from tissues of control and hypoxic animals at each temperature were subjected to Student's t test. The same test was used to compare interspecific and intraspecific differences between mean rates of anaerobic glycolysis in tissues from control animals. Arrhenius plots were utilized to depict the effect of temperature on glycolytic rates. Critical thermal increments (apparent energy of activation, E.4) were calculated for each temperature segment (5-16 °, 16-27 °, and 27-38°C) as k2_Ea(1 1) lnk I R Tll-~O2 " RESULTS Comparative rates of anaerobic glycolysis in heart tissue and t values for differences between glycolytic rates in control and hypoxic animals are presented in Table 1. Table 2 is constructed to elucidate interspecific differences between mean glycolytic rates in control animals as well as intraspecific differences between control active and hibernating ground squirrels. At 38 ° glycolytic rates were significantly higher in tissues from both species after hypoxic exposure, but similar results were not observed at lower temperatures (Table 1). The mean values for anaerobic glycolysis in tissue from exposed rats were consistently lower than those in hypoxic active or hibernating ground squirrels. At all incubation temperatures glycolysis was significantly higher in cardiac tissue from control ground squirrels as compared to control rats (Table 2). A comparison of glycolytic rates in tissue from active and hibernating ground squirrels revealed consistently higher values in the latter animals. Fig. 1 and 2 suggest that inherent differences exist between the effect of temperature on rates of anaerobic glycolysis in the hibernator and homeotherm. The rate of anaerobic glycolysis in rat cardiac tissue decreased sharply between 16 ° and 5°C. In this temperature
185
ANAEROBIC GLYCOLYSIS IN CARDIAC TISSUE T A B L E 1 - - A N A E R O B I C GLYCOLYSIS IN HEART FROM CONTROL AND HYPOXIC ANIMALS
Tempera- Observature tion (°C) ~' QCOI
Hibernating ground squirrel Control
Active ground squirrel
Exposed
Control
Exposed
Rat Control
Exposed
38
Average* Range S.D. t
1.42 2"50 0"83-2"57 2.00-3"64 0"50 0"46 8.44+
0"87 1 '30 0"50-1"29 0"52-1"97 0"23 0"35 3-36,+
0"59 1"04 0"40-0-92 0"53-1"61 0"13 0-28 3-56+
27
Average Range S.D. t
1"58 1"23 0"34-2"73 0"82-1"92 0-74 0"34 2'36t
1"04 1"17 0"46-1-69 0"68-1"71 0"38 0-33 0-88
0"22 0-57 0"09-0"44 0"34-0"96 0"09 0"21 2"36t
16
Average Range S.D. t
0'83 0"65 0"48-1'19 0'33-0"87 0"22 0.14 2"50t
0"48 0"60 0-20-0"83 0'17-1"18 0"15 0"34 1"67
0"13 0-0"29 0"05
Average Range S.D. t
0-21 0"18 0.06-0.41 0.08-0.30 0"11 0.07 1.43
0"07 0"09 0.02-0-17 0-03-0.18 0.05 0.05 0.95
0 0 0
0.19 0"03-0"45 0"13 0"83 0 0 0 0
* n = 15 for each value. t Significant at the 0"05 level: to.o5 (28) = 2.048. ++Significant at the 0"01 level: to.ol (28) = 2-763.
TABLE 2 - - t
VALUES : DIFFERENCES BETWEEN MEANS FOR ANAEROBIC GLYCOLYSIS IN CONTROL ANIMALS
Temperature (°C) 38 27 16 5
Rat and hibernating ground squirrel 6"48* 9"19" 9"72* 10"00"
t Rat and active ground squirrel
Active ground squirrel and hibernating ground squirrel
2"19t 5'54* 4"86* 3'33 *
4'30* 3"65* 4"86* 6'54*
* Significant at the 0.01 level: t0.01 (28) = 2"763. t Significant at the 0"05 level: t0.05 (28) = 2.048.
186
RoY F. BURLINGTON AND JACOBE. WIEBERS
range, the energies of activation (Ea) for glycolysis in the rat tissue far exceed those in the active or hibernating ground squirrel tissue. This phenomenon appeared consistently in tissue from control and hypoxic animals. DISCUSSION Entrance into hibernation and the physiological responses to hypoxia are quite similar in the ground squirrel, i.e. a rapid metabolic depression and body cooling occurs after hypoxia (Bullard, 1960). Although the metabolic needs of the hibernator are substantially lowered in hypothermia and/or hypoxia, the heart and central nervous system must be supplied with energy to insure survival. The capacity for aerobic metabolism is reduced in cardiac tissue from the hibernating animal (Covino & Harmon, 1959; Hannon et al., 1961) ; consequently, anaerobic metabolism could be an increasingly important energy source during hibernation or hypoxia. Studies of glycolytic enzyme activities in heart tissue indicate that the potential for glucose utilization may be increased during hibernation (Vaughan &Hannon, 1960). During hibernation ATP levels decrease in the myocardium (Zimny, 1956). Anaerobic glycolysis is inversely dependent on ATP concentration (Kaplan, 1951). Thus the above data are in agreement with the increased anaerobic glycolysis reported here. Energy transformed via the Embden-Meyerhof pathway is minimal when compared with the oxidative processes of the Krebs cycle; however, a higher rate of anaerobic glycolysis may provide sufficient energy for increased survival of severe hypoxia. Thus higher rates of glycolysis in cardiac tissue from the ground squirrel as compared to the rat are indicative of a cellular adaptation to hypoxia, especially at lower temperatures. A high capacity for anaerobic glycolysis is associated with increased anoxic resistance in infant mammals (Himwich, 1951). Maturation in the homeotherm coincides with a decrease in the initial predominance of anaerobic energy sources and a subsequent loss of hypoxic tolerance. The high capacity for anaerobic glycolysis in the adult hibernator resembles that in the infant homeotherm (Burlington & Wiebers, 1965). The lack of linearity shown in Figs. 1 and 2 reveals that the Arrhenius equation is not wholly adequate for describing the effects of temperature on the complex series of reactions in the Embden-Meyerhof pathway. Nevertheless, assuming that a lower energy of activation (E~) reflects greater enzyme activity, it may be postulated that the high E~ values for rat tissue between 16° and 5°C are due to alteration of the enzymes associated with glycolysis. Theoretically E A is that energy required by chemical reactants to reach an intermediate complex in order that a reaction can proceed to completion. The E~ for a series of biochemical reactions, i.e. anaerobic glycolysis, reflects the slowest or pacemaker reaction in the series (Sizer, 1943). Enzymes reduce the E A needed for reactions in biological systems to proceed at temperatures compatible to life (Glasstone et al., 1941). Temperatures nearing the lower extreme of the biologically tolerable range apparently inactivate enzymes by altering their
0.!
0.5-
i
38C
.....
i
i
27 C i
RAT ACTIVE G.S. HIBERNATING G.S.
i
IBC
I0, 190 3,480 13,716
i
17,307 8,945 8,047
0.0033 0.0034 0.0035 Reciprocol of Absolute Temperature, / T
-~
\
16°C
I
0-0036
~5C
\
42,978 27,693 19,139
5-
FIc. 1. Relationship between heart Q~e6, and temperature in control rats, active ground squirrels and hibernating ground squirrels.
0.0032
0.01
0.05
o
zu
~0
-
1.0-
5~.
I0,0
EA(cal) 27-38 ° 16-27"
0,1
58C
o.o, ,.ob3~
0.05-
~n o
-J
~o ~ u
- - -
~
o.ob3~
0.0534
I
\, /T
o ohms I
16C
"-
\
16,712 8,283 3,021 12,177 1,810 10,133
16-2"/"
EA(cal)
Reciprocal of Absolute Temperature
i
27C
RAT ACTIVE G. S. HIBERNATING GIS
-~3 °
~
\ •
5C
o.oo35
N\
57,445 28,102 20,073
5 - 16*C
FIG. 2. Relationship between heart Qco, ~' and temperature in hypoxic rats, active ground squirrels and hibernating ground squirrels.
z
0'5
I-O
5"0,
lifO-
27
~o
188
RoY F. BURLINGTON AND JACOR E. WIEBERS
molecular configuration (Kavanau, 1950). This p h e n o m e n o n may account for the high E l values which appear at low temperatures in rat tissue. Higher rates of anaerobic glycolysis in the hibernator represent a cellular adaptation to adverse environmental conditions; however, the mechanisms responsible for this p h e n o m e n o n remain unclear and must await further investigation.
SUMMARY An investigation was conducted to assess the effects of hypoxia and hypothermia on rates of anaerobic glycolysis in heart tissue from albino rats and hibernating or active thirteen-lined ground squirrels. At 38°C anaerobic glycolysis increased significantly in tissues from both species after exposure to hypoxia in vivo ; however, similar increases did not occur consistently at lower temperatures. Between 5~ and 16'~C lower energy of activation values ( E , ) for anaerobic glycolysis in ground squirrel tissue reflects a greater enzyme activity than that in rat tissue. In a species comparison at temperatures ranging from 5 ° to 38°C glycolytic rates were consistently higher in ground squirrel tissue. Intraspecifically, anaerobic glycolysis in cardiac tissue from hibernating ground squirrels was significantly higher than that in active s u m m e r animals. F r o m these data it is suggested that hypoxic or hypothermic tolerance is partially attributable to an increased capacity for anaerobic glycolysis. REFERENCES ANDJUS R. K., (~IRKOVI(~T., (_~UPERLOVI(~N., DAVIDOVI(~J., MARKOVI(~-USKOKOVICV. & VELIMIROVICT. (1964) Brain metabolism and resistance of a hibernator (Citellus citelhts) and the rat to different anoxic conditions, including cardiac arrest in deep hypothermia. Ann. Acad. Sci. Fenn. (Set. A.) 4, 11-23. I]ULLARDR. W., DAVID G. & NICHOLS C. T. (1960) The mechanisms of hypoxic tolerance in hibernating and non-hibernating mammals. Bull. Mus. Comp. Zool., t-Iarvard 124, 321-335. BURLINGTONR. F. ~¢ WIEBERSJ. E. (1965) Effect of hypoxia on cerebral anaerobic glycolysis in the hibernator and infant or adult homeotherm. Comp. Biochem. Physiol. 14, 201-203. COVINO B. G. & HANNON J. P. (1959) Myocardial metabolic and electrical properties of rabbits and ground squirrels at low temperature. Amer. ff. Physiol. 197, 494-498. GLASSTONES., LAIDLERK. J. & EYRINGH. (1941) The Theory of Rate Processes. McGrawHill, N.Y. HANNON J. P., VAUCHAND. A. & HOCK R. J. (1961) The endogenous tissue respiration of the Arctic ground squirrel as effected by hibernation and season. J. cell. comp. Physiol. 57, 5-10. HEGNAUERA. H., SHIRBERW. J. & HATERIUSH. O. (1950) Cardiovascular response of the dog to immersion hypothermia. Amer. J. Physiol. 161, 455-465. tIIESTANOW. S., ROCKtaOLDW. T., STEMLERF. W., STULLKEND. E. & WIEBERSJ. E. (1950) The comparative hypoxic resistance of hibernators and non-hibernators. P~,siol. Zool. 23, 264-269. HIMWICH H. E. (1951) Brain Metabolism and Cerebral Disorders. Williams & Wilkins, Baltimore. KAPLA:~ N. O. (1951) Thermodynamics and mechanism of the phosphate bond. In The Enzymes (SI;MNERJ. B. & MYR~ACt~ K., eds.), chap. 45. Academic Press, N.Y.
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KAVANAUJ. L. (1950) Enzyme kinetics and the rate of biological processes, ft. Gen. Physiol. 34, 193-209. KAYSER C. H. & MALAN A. (1963) Central nervous system and hibernation. Experientia 19, 441-451. LYMAN C. P. ~ CHATFIELDP. O. (1955) Physiology of hibernation in mammals. Physiol. Rev. 35, 403-425. SIZER I. W. (1943) Effects of temperature on enzyme kinetics. Advanc. Enzymol. 3, 35-62. SOUTH F. E. (1958) Rates of oxygen consumption and glycolysis of ventricle and brain slices, obtained from hibernating and non-hibernating mammals, as a function of temperature. Physiol. Zool. 31, 6-15. SOUTH F. E. (1961) Phrenic nerve-diaphragm preparations in relation to temperature and hibernation. Amer..7. Physiol. 200(3), 565-571. UMBREIT W. W . , Bum~is R. H. & STAUFFERJ. F. (1957) Manometric Techniques. Burgess Co., Minneapolis. VAUGHAN n . A. & HANNON J. P. (1960) Activity of selected glycolytic enzymes during hibernation. Fed. Proc. 19, 45. ZIMNY M. L. (1956) Metabolism of some carbohydrate and phosphate compounds during hibernation in the ground squirrel. )t. cell. comp. Physiol. 48, 371-391.