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Anaerobic contributions during progressive hypoxia in the toad Bufo marinus
Cc ,,,, p Biochwx
Ph~,roi.
IU71(.
Vd
6OA.
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1 fo IO
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ANAEROBIC CONTRIBUTIONS DURING PROGRESSIVE HYPOXIA IN THE TOAD BUFO MARINUS MICHAEL Comparative
E. D’EoN, ROBERTG. BOUTILIERand DANIEL P. TOEWS
Physiology
Laboratory. Department of Biology, Acadia Nova Scotia. Canada, BOP IX0
University.
Wolfville,
(Received 27 June 1977) Bufo mrinus were exposed to a 90-min time course of progressive hypoxia. 2. Increases of blood glucose and lactic acid concentrations suggest that anaerobic contributions occur when inspired oxygen levels become very low. 3. The results are discussed in light of previous experiments and it is concluded that anaerobiosis accompanied by cardiovascular adjustments could be of importance during oxygen stress situations such as burrowing. Abstract--l.
Corp., Oshkosh. WI. U.S.A.). Animals of either sex were kept in the laboratory at 23 _+ 2°C in large aquaria. After each animal was anaesthetized in a 1.5 g/l solution of MS-222 (tricaine methanesulfonate, Sandoz), a polyethylene cannula (P.E. 60; id. 0.76 mm; o.d. 1.22 mm) was inserted upstream so as to occlude the femoral artery. Cannulae were kept filled with heparinized amphibian Ringer (250 i.u./ml) to prevent clotting. with I.0 ml/kg of the solution being injected immediately following the operation. Animals were then left undisturbed for a 24-hr recovery period before experimentation. Blood samples were collected in all instances from free-moving unanesthetized animals. Each toad was placed in a gas-tight chamber and exposed to a 90-min period of progressive hypoxia. Prior to and following hypoxic exposure the chamber was flushed with air. All gases were water-saturated and temperature adjusted (22’C) before being put into the chamber. The experimental design was identical with that previously reported by Boutilier & Toews (1977). Blood samples were collected prior to hypoxia and at 30 min intervals throughout the hypoxic and post-hypoxic period. Each sample was then immediately analyzed for glucose (Worthington Biochemical Corp., U.S.A.) and lactic acid (Sigma Chemical Co.. U.S.A.) concentrations using commercially prepared kits. Results were analyzed statistically at the 5”,, level or better using Student’s t-test. Mean data in all instances is presented as k the standard error of the mean (k S.E.).
INTRODUCTION
It has been well documented that amphibians show several respiratory, cardiovascular and metabolic adjustments when exposed to 100% N,. During anoxia, hepatic glycogen depletion in Bufo cognntus (Armentrout & Rose, 1971) and cardiac glycogen reductions in Ambystoma tigrinum (Rose rt al., 1971) are coincident with increases in blood glucose and lactic acid concentrations. Rose & Drotman (1967) reported glycogen reductions in the liver, ventricle and gastrocnemius muscle of anoxic Rana p&ens and found that while iodoacetate poisoned frogs survived aerobic conditions, they succumbed in anoxia much more quickly than normal animals. These data give evidence that anoxic amphibians utilize anaerobic processes. Less well known are the anaerobic contributions that occur when amphibians encounter hypoxic conditions such as during periods of diving or burrowing. Analyses of acid-base balance in Rana esculenta during apnoea (Jones, 1972) Rana ridihunda during periods of diving (Emilio, 1974) and Bufo marinus under conditions of progressive hypoxia (Boutilier & Toews. 1977) indicate that metabolic acids are added to the blood as a result of anaerobic processes. In addition, Jones & Mustafa (1973) found that anaerobic contributions did indeed occur in apnoeic Rana pipiens when the oxygen metabolism was reduced to levels not unlike those previously reported for anurans during submergence (Jones, 1967). The present experiments were conducted in order to measure blood glucose and lactic acid concentrations during normal, hypoxic and post-hypoxic situations in the toad BuJo marinus. Under these conditions, the ventilatory and blood acid-base adjustments previously reported from this laboratory (Boutilier & Toews, 1977) indicated that anaerobic processes may have occurred when oxygen levels became verv low. MATERIALS
AND
RESULTS
Table 1 shows the mean blood glucose and lactic acid concentrations for 10 Bufo rnarinus before. during and after a bout of progressive hypoxia. Inspired oxygen tensions (P,02) progressively decreased over a 90-min period after which they were rapidly restored to pre-exposure levels for a 90-min recovery period The glucose
(Table normal
I). pre-exposure
concentration
of blood
was 14.59 ? 1.23 mg% (Table I). During progressive hypoxia. glucose levels rose sightly but the change was not significant until the 30-min recovery period when the highest blood glucose levels (23.90 + 2.47 mg%) were recorded (Table I, Fig. 1). These levels remained significantly above those of normal and hypoxic toads for the first 60 min of recovery
METHODS
The experiments were performed on IO Bufo marinus collected in Mexico and obtained commercially (The Mogul 7
Progrcssivc Preexposure Glucose (mg,‘lOO ml) Lactic acid
14.59 f
I .zi
7.21 f
(mg/lOO ml) PIOz mmHg
I50
s
30 mtn
- I5.Y.? + 0.Xx’
1.22 -
6.16 kO.Y6*
I.55
S
hypo\la
60 min
S
Post-e\po\,l,-~
YO “1111
s
30 mln
s
- 1631 + 1.54” ~ 16.75 * 1.77* +2i.Y0_+‘47t ~
60 70
9.73 & ‘.?4*
+24.20+6.51~
70 75
-2771
0~ IO
60 mill
s
YO mln
20.46 + 2 57t + 16.34*
+6.4Yi
-21.06
150 Ii5
5 2YOt -
I Shf
lY.48 + 1.YXt I 5w
150 155
IS5
P,OL ranges arc glvcn 30 min Interval. S indlcatcs whether the dilkrencc between adjacent means was significant at the 5”,, level of Student’s I-test: + Indicates slgnlficance: ~ Indicates no \Igntficancc Temperature = 22 C. * Not significantly dltl’crent from normal mean. t Significantly diffcrcnt from normal mean
although
as P,02
after the 30 min recovery
to
Fig. I shows that levels began lo decrease period. After 90 min rccovcry, blood glucose lcvcls returned to normal (Table I, Fig. I). The normal preexposure lactic acid concentration was 7.21 + 1.23 mg”,, (Table 1). From P,O, levels of 155 to 20 mmHg there were no significant deviations (Table I ). however. when PI02 tensions declined below 20 mmHg. blood lactate Icvels rose dramatically (Fig. 2) and remained significantly above those previously observed. A further increase after 30 min recovery (Fig. 2) was not significant (Table I). Although lactate levels remained significantly elevated throughout the recovery period. they appeared to progressively decline following the first 30 min of recovery. Comparing both levels together. blood glucose and lactic acid concentrations did not significantly change
levels progressively decreased from I5& 155 The initial significant increase in blood lactate (after 90 min exposure) was unaccompanied by an increase in blood glucose. however, the highest levels of both parameters were recorded after 30 min recovery (Table I ). During the remainder of the recovery period blood glucose and lactic acid levels decreased. Blood lactate showed much greater increases than did glucose. At the highest concentrations (30 min recovery) glucose levels were approximately 6@65”;, higher than preexposure values. On the other hand. lactate levels at the 90-min exposure period and 30 min recovery were approximately 3 -4 times higher than the pre-exposure levels. While 2(& 75 mmHg.
35r
30 -
30 -
t
i
25 -
Q,=
3:z 2 02009 0; OaE z
25 -
%E &O 80 J =. =oH *
15 -
E 15-
10 -
10 -
5-
5-
I
I 0
JO
60
TIME
90
120
150
180
(min)
Fig. 1. Changes of mean (kS.E.) blood glucose conccntrations recorded from IO BI!/o wuriuu.\ during hypoxia and normoxia. Data as in Table I. Arrows at top of ligurc indicate the onset (1) and termination I?) of exposure to hypoxia. Open circles represent mean pre-exposure values. Zero tlmc indicates the onset of hypoxia. Temperature
I 0
30
60
TIME
90
120
150180
I
(min)
Fig. 2. Changes of mean ( i SE.) blood lactic acid concen trations recorded from IO BI& wwi~~u\ during hypoxia and normoxia. Data as In Table I. Arrows at top of figure induzatr the onset (1) and tcrmmatmn (f ) of cxposurc 10 hypoxia. Open circles represent mean pre-exposure values Zero time mdlcates the onset of hypoxia. Temperature
Progressive
hypoxia
blood glucose returned to normal pre-exposure levels after 90 min recovery. lactic acid concentrations (although progressively decreasing) were still elevated significantly above the normal. DISCUSSION The present data indicates that during periods of oxygen deficiency, Bufo marinus utilize anaerobic processes for energy production. This could be of importance during periods of burrowing when arterial oxygen tensions are known to substantially decline for several days (Boutilier & Toews; in preparation). Normal pre-exposure levels of blood glucose were much lower than those values previously reported for Bufo cognatus (Armentrout & Rose, 1971). Amhystoma tigrinum (Rose et al., 1971) and Rana pipiens (Jones & Mustafa, 1973). Similarly, the normal pre-exposure blood lactate measurements (Table I) were generally lower than those previously reported for Bufonids (Leivestad. 1960; Armentrout & Rose, 1971) and Rana pipirns (Jones & Mustafa. 1973). These relatively low levels in normal resting Eufo marinus (Table I), as compared with those of other anurans, may reflect our method of blood collection. Blood samples in previous studies have been taken by methods which pose at least a brief stress (3@60 set) upon the animals [for example. methods such as pithing or decapitation require that the animal be removed from its enclosure and forceably restrained for a brief period of time] while the present blood samples were collected from animals that were undisturbed and free from any direct experimenter-animal interaction. Bennett & Licht (1974) have shown that body lactate content in a representative Bufonid (Bufi horras) increased 2-3 fold over an activity period of only 30 sec. In addition. previous studies on animals such as rainbow trout have shown that short term (seconds) strenuous exercise can substantially alter the blood glucose (Hammond & Hickman, 1966) and lactic acid concentrations (Stevens & Black. 1966). Because the present experiments and those of Boutilier & Toews (1977) were performed under identical situations. the current data should be directly comparable over the same time course with those previously reported. Consequently, the dramatic increase in blood lactate at P102 tensions of O-IOmmHg (Fig. 2) occurred at the same time that Boutilier & Toews (1977) noted a significant decline in arterial pH. Their earlier suggestion that the pH decrease was at least in Dart caused bv an influx of metabolic acid from anaerobic metabolism has thus been confirmed. It would appear that as the hypoxic conditions become progressively stressful, the normal paths of oxygen delivery cannot meet the total tissue demands and energy for selected areas (i.e. the support of muscle work) is supplied by anaerobic glycolysis which reduces large amounts of pyruvate to lactate. For example, a concomitant bradycardia and skeletal muscle vasoconstriction in anoxic Bufo coynatus (Armentrout & Rose, 1971) suggests that the blood circulation in Bufonids may be regulated to favour the more oxygen sensitive areas of the body (heart and brain) at the expense of the more peripheral tissues. notably the skeletal musculature. In Bufo marinus. a further decline in arterial pH after 30 min re-
in the toad Sufb
9
marinus
covery (Boutilier & Toews, 1977) correlates with the additional increase in blood lactate shown in Fig. 2.
If the regulation of peripheral blood supply, as shown in Bufo cognatus (Armentrout & Rose, 1971). holds true for Sufo marinus, it would appear that the further influx of lactic acid into the blood at the 3@min recovery period was caused by a re-establishment of the skeletal muscle circulation upon returning to normal atmosphere. A delay of 6%90min for the complete recovery of arterial blood pH and total CO, in recently surfaced Rana ridihunda (Emilio. 1974) was thought to be related to the slow removal of organic acids from the blood. Although the present blood lactate measurements remained significantly elevated during the 90-min recovery period (Table 1) a gradual decline following 30 min recovery (Fig. 2) does correlate with the 6&90 min delay in recovery of arterial pH in Bufo tnurinus (Boutilier & Toews. 1977). The elevation of blood glucose levels in the present study occurred after the animals were returned to normoxia for 30 min (Table 1, Fig. 1). Hochachka & Somero (1973) state that upon termination of anoxia. although a portion of the muscle lactate is “burned off” by aerobic metabolism, the largest portion is delivered to glucogenic tissues (i.e. liver and kidney) where it is converted to glucose by the gluconeogenic pathway. Therefore we might suggest that the increase in blood glucose occurs as a result of lactate being metabolized upon termination of anaerobic muscle work. The lactate is probably flushed out of the muscle at this time and delivered to the liver for conversion to glucose. Acknowlrdycvnrnts-Financial support was provided by a National Research Council of Canada operating grant (No. A6641) to D. Toews and a NRCC Postgraduate Scholarship to R. Boutilier.
REFERENCES AKM~NTKWT D. & ROSL F. L. (1971) Some
responses
to anoxia
in the great
natus. Comp. Biochem.
Physiol.
plains
physiological toad. Bufb coy-
39, 447455.
BENNETT A. F. & LIGHT P. (1974) Anaerobic metabolism during activity in amphibians. Camp. Biochwn. Physiol. 48, 319 327.
BOIJIILIER R. G. & Tol:ws D. P. (1977) The effect of progressive hypoxia on respiration in the toad Bufo ntarinus. J. e.Yp. Biol. 68. 99- 107. EMILIO M. G. (1974) Gas exchanges and blood gas concentrations in the frog Rana ridihunda. J. r.~p. Biol. 68. 901-908. HAMMOND B. R. & HICKMAN C. P.. JR. (1966) The effect of physlcal conditioning on the metabolism of lactate. phosphate and glucose in rainbow trout. Solmo yuirdneri. J. Fish. Res. Bd Can. 23. 65-83.
HOCHACHKA P. W. and SOMCRO G. N. (1973) Strarrgirs of Biochrnlical Adaptation. p. 358. Saunders. Toronto. JONES D. R. (1967) Oxygen consumption and heart rate of several species of anuran amphibia during submergence. Camp. Biochem. PhJsiol. 20, 691-707. JONES D. R. (1972) Anaerobiosis and the oxygen debt in an anuran amphibian. RUIIU ~sculenta (L.) J. camp, Physiol. 77. 356-381. JONES D. R. & MUWAFA T. (1973)
The lactacid oxygen after one hour’s apnoea in air. J. camp. Physiol. 85. I5- 24. LFIVI-STAD H. (1960) The effect of prolonged submersion debt in frogs
Ph~,roi.
IU71(.
Vd
6OA.
pp
1 fo IO
Perymo,,
Pw,s
Prrnr,d
I,, Grror
Bnru,,,
ANAEROBIC CONTRIBUTIONS DURING PROGRESSIVE HYPOXIA IN THE TOAD BUFO MARINUS MICHAEL Comparative
E. D’EoN, ROBERTG. BOUTILIERand DANIEL P. TOEWS
Physiology
Laboratory. Department of Biology, Acadia Nova Scotia. Canada, BOP IX0
University.
Wolfville,
(Received 27 June 1977) Bufo mrinus were exposed to a 90-min time course of progressive hypoxia. 2. Increases of blood glucose and lactic acid concentrations suggest that anaerobic contributions occur when inspired oxygen levels become very low. 3. The results are discussed in light of previous experiments and it is concluded that anaerobiosis accompanied by cardiovascular adjustments could be of importance during oxygen stress situations such as burrowing. Abstract--l.
Corp., Oshkosh. WI. U.S.A.). Animals of either sex were kept in the laboratory at 23 _+ 2°C in large aquaria. After each animal was anaesthetized in a 1.5 g/l solution of MS-222 (tricaine methanesulfonate, Sandoz), a polyethylene cannula (P.E. 60; id. 0.76 mm; o.d. 1.22 mm) was inserted upstream so as to occlude the femoral artery. Cannulae were kept filled with heparinized amphibian Ringer (250 i.u./ml) to prevent clotting. with I.0 ml/kg of the solution being injected immediately following the operation. Animals were then left undisturbed for a 24-hr recovery period before experimentation. Blood samples were collected in all instances from free-moving unanesthetized animals. Each toad was placed in a gas-tight chamber and exposed to a 90-min period of progressive hypoxia. Prior to and following hypoxic exposure the chamber was flushed with air. All gases were water-saturated and temperature adjusted (22’C) before being put into the chamber. The experimental design was identical with that previously reported by Boutilier & Toews (1977). Blood samples were collected prior to hypoxia and at 30 min intervals throughout the hypoxic and post-hypoxic period. Each sample was then immediately analyzed for glucose (Worthington Biochemical Corp., U.S.A.) and lactic acid (Sigma Chemical Co.. U.S.A.) concentrations using commercially prepared kits. Results were analyzed statistically at the 5”,, level or better using Student’s t-test. Mean data in all instances is presented as k the standard error of the mean (k S.E.).
INTRODUCTION
It has been well documented that amphibians show several respiratory, cardiovascular and metabolic adjustments when exposed to 100% N,. During anoxia, hepatic glycogen depletion in Bufo cognntus (Armentrout & Rose, 1971) and cardiac glycogen reductions in Ambystoma tigrinum (Rose rt al., 1971) are coincident with increases in blood glucose and lactic acid concentrations. Rose & Drotman (1967) reported glycogen reductions in the liver, ventricle and gastrocnemius muscle of anoxic Rana p&ens and found that while iodoacetate poisoned frogs survived aerobic conditions, they succumbed in anoxia much more quickly than normal animals. These data give evidence that anoxic amphibians utilize anaerobic processes. Less well known are the anaerobic contributions that occur when amphibians encounter hypoxic conditions such as during periods of diving or burrowing. Analyses of acid-base balance in Rana esculenta during apnoea (Jones, 1972) Rana ridihunda during periods of diving (Emilio, 1974) and Bufo marinus under conditions of progressive hypoxia (Boutilier & Toews. 1977) indicate that metabolic acids are added to the blood as a result of anaerobic processes. In addition, Jones & Mustafa (1973) found that anaerobic contributions did indeed occur in apnoeic Rana pipiens when the oxygen metabolism was reduced to levels not unlike those previously reported for anurans during submergence (Jones, 1967). The present experiments were conducted in order to measure blood glucose and lactic acid concentrations during normal, hypoxic and post-hypoxic situations in the toad BuJo marinus. Under these conditions, the ventilatory and blood acid-base adjustments previously reported from this laboratory (Boutilier & Toews, 1977) indicated that anaerobic processes may have occurred when oxygen levels became verv low. MATERIALS
AND
RESULTS
Table 1 shows the mean blood glucose and lactic acid concentrations for 10 Bufo rnarinus before. during and after a bout of progressive hypoxia. Inspired oxygen tensions (P,02) progressively decreased over a 90-min period after which they were rapidly restored to pre-exposure levels for a 90-min recovery period The glucose
(Table normal
I). pre-exposure
concentration
of blood
was 14.59 ? 1.23 mg% (Table I). During progressive hypoxia. glucose levels rose sightly but the change was not significant until the 30-min recovery period when the highest blood glucose levels (23.90 + 2.47 mg%) were recorded (Table I, Fig. 1). These levels remained significantly above those of normal and hypoxic toads for the first 60 min of recovery
METHODS
The experiments were performed on IO Bufo marinus collected in Mexico and obtained commercially (The Mogul 7
Progrcssivc Preexposure Glucose (mg,‘lOO ml) Lactic acid
14.59 f
I .zi
7.21 f
(mg/lOO ml) PIOz mmHg
I50
s
30 mtn
- I5.Y.? + 0.Xx’
1.22 -
6.16 kO.Y6*
I.55
S
hypo\la
60 min
S
Post-e\po\,l,-~
YO “1111
s
30 mln
s
- 1631 + 1.54” ~ 16.75 * 1.77* +2i.Y0_+‘47t ~
60 70
9.73 & ‘.?4*
+24.20+6.51~
70 75
-2771
0~ IO
60 mill
s
YO mln
20.46 + 2 57t + 16.34*
+6.4Yi
-21.06
150 Ii5
5 2YOt -
I Shf
lY.48 + 1.YXt I 5w
150 155
IS5
P,OL ranges arc glvcn 30 min Interval. S indlcatcs whether the dilkrencc between adjacent means was significant at the 5”,, level of Student’s I-test: + Indicates slgnlficance: ~ Indicates no \Igntficancc Temperature = 22 C. * Not significantly dltl’crent from normal mean. t Significantly diffcrcnt from normal mean
although
as P,02
after the 30 min recovery
to
Fig. I shows that levels began lo decrease period. After 90 min rccovcry, blood glucose lcvcls returned to normal (Table I, Fig. I). The normal preexposure lactic acid concentration was 7.21 + 1.23 mg”,, (Table 1). From P,O, levels of 155 to 20 mmHg there were no significant deviations (Table I ). however. when PI02 tensions declined below 20 mmHg. blood lactate Icvels rose dramatically (Fig. 2) and remained significantly above those previously observed. A further increase after 30 min recovery (Fig. 2) was not significant (Table I). Although lactate levels remained significantly elevated throughout the recovery period. they appeared to progressively decline following the first 30 min of recovery. Comparing both levels together. blood glucose and lactic acid concentrations did not significantly change
levels progressively decreased from I5& 155 The initial significant increase in blood lactate (after 90 min exposure) was unaccompanied by an increase in blood glucose. however, the highest levels of both parameters were recorded after 30 min recovery (Table I ). During the remainder of the recovery period blood glucose and lactic acid levels decreased. Blood lactate showed much greater increases than did glucose. At the highest concentrations (30 min recovery) glucose levels were approximately 6@65”;, higher than preexposure values. On the other hand. lactate levels at the 90-min exposure period and 30 min recovery were approximately 3 -4 times higher than the pre-exposure levels. While 2(& 75 mmHg.
35r
30 -
30 -
t
i
25 -
Q,=
3:z 2 02009 0; OaE z
25 -
%E &O 80 J =. =oH *
15 -
E 15-
10 -
10 -
5-
5-
I
I 0
JO
60
TIME
90
120
150
180
(min)
Fig. 1. Changes of mean (kS.E.) blood glucose conccntrations recorded from IO BI!/o wuriuu.\ during hypoxia and normoxia. Data as in Table I. Arrows at top of ligurc indicate the onset (1) and termination I?) of exposure to hypoxia. Open circles represent mean pre-exposure values. Zero tlmc indicates the onset of hypoxia. Temperature
I 0
30
60
TIME
90
120
150180
I
(min)
Fig. 2. Changes of mean ( i SE.) blood lactic acid concen trations recorded from IO BI& wwi~~u\ during hypoxia and normoxia. Data as In Table I. Arrows at top of figure induzatr the onset (1) and tcrmmatmn (f ) of cxposurc 10 hypoxia. Open circles represent mean pre-exposure values Zero time mdlcates the onset of hypoxia. Temperature
Progressive
hypoxia
blood glucose returned to normal pre-exposure levels after 90 min recovery. lactic acid concentrations (although progressively decreasing) were still elevated significantly above the normal. DISCUSSION The present data indicates that during periods of oxygen deficiency, Bufo marinus utilize anaerobic processes for energy production. This could be of importance during periods of burrowing when arterial oxygen tensions are known to substantially decline for several days (Boutilier & Toews; in preparation). Normal pre-exposure levels of blood glucose were much lower than those values previously reported for Bufo cognatus (Armentrout & Rose, 1971). Amhystoma tigrinum (Rose et al., 1971) and Rana pipiens (Jones & Mustafa, 1973). Similarly, the normal pre-exposure blood lactate measurements (Table I) were generally lower than those previously reported for Bufonids (Leivestad. 1960; Armentrout & Rose, 1971) and Rana pipirns (Jones & Mustafa. 1973). These relatively low levels in normal resting Eufo marinus (Table I), as compared with those of other anurans, may reflect our method of blood collection. Blood samples in previous studies have been taken by methods which pose at least a brief stress (3@60 set) upon the animals [for example. methods such as pithing or decapitation require that the animal be removed from its enclosure and forceably restrained for a brief period of time] while the present blood samples were collected from animals that were undisturbed and free from any direct experimenter-animal interaction. Bennett & Licht (1974) have shown that body lactate content in a representative Bufonid (Bufi horras) increased 2-3 fold over an activity period of only 30 sec. In addition. previous studies on animals such as rainbow trout have shown that short term (seconds) strenuous exercise can substantially alter the blood glucose (Hammond & Hickman, 1966) and lactic acid concentrations (Stevens & Black. 1966). Because the present experiments and those of Boutilier & Toews (1977) were performed under identical situations. the current data should be directly comparable over the same time course with those previously reported. Consequently, the dramatic increase in blood lactate at P102 tensions of O-IOmmHg (Fig. 2) occurred at the same time that Boutilier & Toews (1977) noted a significant decline in arterial pH. Their earlier suggestion that the pH decrease was at least in Dart caused bv an influx of metabolic acid from anaerobic metabolism has thus been confirmed. It would appear that as the hypoxic conditions become progressively stressful, the normal paths of oxygen delivery cannot meet the total tissue demands and energy for selected areas (i.e. the support of muscle work) is supplied by anaerobic glycolysis which reduces large amounts of pyruvate to lactate. For example, a concomitant bradycardia and skeletal muscle vasoconstriction in anoxic Bufo coynatus (Armentrout & Rose, 1971) suggests that the blood circulation in Bufonids may be regulated to favour the more oxygen sensitive areas of the body (heart and brain) at the expense of the more peripheral tissues. notably the skeletal musculature. In Bufo marinus. a further decline in arterial pH after 30 min re-
in the toad Sufb
9
marinus
covery (Boutilier & Toews, 1977) correlates with the additional increase in blood lactate shown in Fig. 2.
If the regulation of peripheral blood supply, as shown in Bufo cognatus (Armentrout & Rose, 1971). holds true for Sufo marinus, it would appear that the further influx of lactic acid into the blood at the 3@min recovery period was caused by a re-establishment of the skeletal muscle circulation upon returning to normal atmosphere. A delay of 6%90min for the complete recovery of arterial blood pH and total CO, in recently surfaced Rana ridihunda (Emilio. 1974) was thought to be related to the slow removal of organic acids from the blood. Although the present blood lactate measurements remained significantly elevated during the 90-min recovery period (Table 1) a gradual decline following 30 min recovery (Fig. 2) does correlate with the 6&90 min delay in recovery of arterial pH in Bufo tnurinus (Boutilier & Toews. 1977). The elevation of blood glucose levels in the present study occurred after the animals were returned to normoxia for 30 min (Table 1, Fig. 1). Hochachka & Somero (1973) state that upon termination of anoxia. although a portion of the muscle lactate is “burned off” by aerobic metabolism, the largest portion is delivered to glucogenic tissues (i.e. liver and kidney) where it is converted to glucose by the gluconeogenic pathway. Therefore we might suggest that the increase in blood glucose occurs as a result of lactate being metabolized upon termination of anaerobic muscle work. The lactate is probably flushed out of the muscle at this time and delivered to the liver for conversion to glucose. Acknowlrdycvnrnts-Financial support was provided by a National Research Council of Canada operating grant (No. A6641) to D. Toews and a NRCC Postgraduate Scholarship to R. Boutilier.
REFERENCES AKM~NTKWT D. & ROSL F. L. (1971) Some
responses
to anoxia
in the great
natus. Comp. Biochem.
Physiol.
plains
physiological toad. Bufb coy-
39, 447455.
BENNETT A. F. & LIGHT P. (1974) Anaerobic metabolism during activity in amphibians. Camp. Biochwn. Physiol. 48, 319 327.
BOIJIILIER R. G. & Tol:ws D. P. (1977) The effect of progressive hypoxia on respiration in the toad Bufo ntarinus. J. e.Yp. Biol. 68. 99- 107. EMILIO M. G. (1974) Gas exchanges and blood gas concentrations in the frog Rana ridihunda. J. r.~p. Biol. 68. 901-908. HAMMOND B. R. & HICKMAN C. P.. JR. (1966) The effect of physlcal conditioning on the metabolism of lactate. phosphate and glucose in rainbow trout. Solmo yuirdneri. J. Fish. Res. Bd Can. 23. 65-83.
HOCHACHKA P. W. and SOMCRO G. N. (1973) Strarrgirs of Biochrnlical Adaptation. p. 358. Saunders. Toronto. JONES D. R. (1967) Oxygen consumption and heart rate of several species of anuran amphibia during submergence. Camp. Biochem. PhJsiol. 20, 691-707. JONES D. R. (1972) Anaerobiosis and the oxygen debt in an anuran amphibian. RUIIU ~sculenta (L.) J. camp, Physiol. 77. 356-381. JONES D. R. & MUWAFA T. (1973)
The lactacid oxygen after one hour’s apnoea in air. J. camp. Physiol. 85. I5- 24. LFIVI-STAD H. (1960) The effect of prolonged submersion debt in frogs