TEMPERATURE ACCLIMATION IN THE PANCAKE TORTOISE, MALACOCHERSUS TORNIERI:

METABOLIC RATE, BLOOD pH, OXYGEN AFFINITY AND RED CELL ORGANIC PHOSPHATES

STEPHEN C. WOOD,* GUNNAR LYKKEBOE, KJEL~ JOHANSEN, ROY E. WEBER AND G. M. 0. MALOIY?

Department of Zoophysiology. University of Aarhus, DK-8000 Aarhus C., Denmark

(Reeei~e~ 29 farce 1977)

Abstract-I. The effect of temperature acclimation (20 and 35°C) on blood gases, pH and the oxyhe- moglobin dissociation curve was studied in the pancake tortoise.

2. Cold-acclimated tortoises had blood with a lower 0, aflinity (Fig. la) and lower Bohr effect (Fig. 1 b) than warm-a~limated tortoises‘

3. The concentration of ATP in red cells was s~gni~~ntly higher in old-a~ii~ted tortoises, provid- ing a mechanism for the temperature-induced a@inity change of whole blood (Table 1; Fig. la and b).

4. The cofactor-free hemoglobins from warm- and cold-acclimated tortoises showed no significant differences in oxygen-binding properties or in charge heterogeneity (Figs. 3 and 4).

5. Two major hemoglobin components isolated show slight differences in oxygen affinities but both are sensitive to ATP (Fig. 5).

6. The effect of temperature on blood pH. Pco,. HCQ; and ventilation/Ot uptake was similar to that reported for turtles (Jackson, 1971; Jackson et al., 1974).

7. Cold-a~li~t~ tortoises had a signi~cantly higher O2 uptake at 20°C and a lower Qio than warm-acclimated tortoises (Fig. 2).

8. The lower 0, affinity of blood after cold acclimation may be adaptive to the increased requirement for oxygen delivery to the tissues.

INTRODUCJ’ION

The oxygen affinity of most respiratory pigments is temperature dependent. In the case of hemoglobin, the apparent heat of oxygenation (AH) ranges from -10 to - 13 kcal/mol for most species (Rossi-Fanelli et at., 1964). The effect of temperature on oxygen affinity can be calculated from the van’t Hoff equa- tion :

A log P,, = AH AT/4574 T, Tz,

where P,, is the Po, of 50% saturated hem~lob~n, and AT is the difference between Ts and T2, two abso- lute temperatures (Sullivan & Riggs, 1971 b). For most hemoglobins, A log P5JAT is approximately constant at 0.024 between 14 and 38°C (~veringhau~ 1966).

This effect of temperature, moderately irn~r~nt in homeotherms, can have extreme effects on respira- tory function in poikilothermic animals. For exampie, consider a reptile with blood having a PsO of 30mm Hg at pH 7.8 and 20°C. if body temperature increased to 35”C, the P5e would increase to 69 mm Hg at pH 7.8 (if 4 log P&AT= 0.024). However, tempera- ture also has an indirect effect on hemoglobin func- tion in poikilotherms, i.e. increased body temperature

* Present address: Department of Physiology, School of Medicine, Universitv of New Mexico, Albuaueraue. NM 87131 U.S.A. -

. 1

t Present address: Department of Animal Physiology, University of Nairobi, Nairobi, Kenya.

results in lowered blood pH. Assuming a ApH/AT of -0.017 (cf. Howell & Rahn, 1976), blood pH would decrease from 7.8 to 7.55 as temperature in- creases from 20 to 35°C. Using the average Bohr fac- tor for reptile blood (A log PSO/ApH = -0.54; Wood & Lenfant, 19763, the P5e would then be 94mm Hg at 35°C and pH 7.55. Obviously, these dual effects of temperature may have effects on oxygen delivery which are disproportionate to the temperature- induced changes in oxygen demand.

Two patterns of compensation for this effect of tem- perature are found. Some euryther~~ species of fish and reptiles have hemoglobin (or components of mul- tiple hemoglobins) with low values of AH (Rossi- Fanelii & Antonini, 1960; Hashimoto et al.. 1960; Pough, 1969). This prevents large shifts of the oxygen dissociation curve (ODC) during rapid changes of ambient temperature or, in the case of tuna, due to large regional differences in body temperature (Carey & Teal, 1966). The second type of compensation in- volves acclimation of the function of hemoglobin (with normal AH values) to prolonged, or seasonal, temperature changes. This has been described for the frog, Rana esculenta (Kirberger, 1953; Straub, 1957; Gahienbeck & Bartels, 1968) and a fish, fctalurus nebulous (Grigg, 1969). fn all these studies, the ODC of animals acclimated to the cold was shifted to the right of that for warm-acclimated animals. Many potential mechanisms were examined in these studies, but none provided an unequivocal expl~ation for the

155

STf.PHfJU c.

affnity changes. However. a key observation by Grigg (1969) was that the affinity difference pertained only to whole blood and disappeared when purified hemo- globin solutions from cold- and warm-acclimated fish were compared. These adjustments of oxygen afhnity. analogous to temperature acclimation of metabolic rate (Bullock, 1955), have not. to the authors’ knowl- edge. been described for reptiles. This fact. and the still unresolved mechanism of this adaptation of hemoglobin function. prompted us to examine the effects of temperature acclimation on hemoglobin function. red cell organic phosphates. and metabolic rate in the pancake tortoise.

MATERIALS AND METHODS

Pancake tortoises. Malacochrrsus rornicri. were obtained in Kenya. They were air-shipped to Denmark and main- tained at 25 + 2°C. on a diet of mixed vegetables. for I month before use. The tortoises were then divided mto two groups and kept at either 20 or 35’C in constant tem- perature rooms for at least I month. Food was withheld for 3 days prior to experiments. Body weight ranged from 292 to 485g (mean = 385.4; SD. + 67).

Blood was obtained by cardiac puncture. simplified by the thin plastron of this species. from unanesthetized and minimally restrained animals. Samples were immediately analyzed for O2 content and pH. Oxygen saturation (S) was calculated from the ratio of O2 content to O2 capacity. wrth corrections for dissolved OZ. Oxygen capacity was determined from the 0, content of blood equilibrated with 75”” 02. A fuel cell technique (Lexington Inst. Co.. Wal- tham, MA) was used for measurements of 0, content. Blood pH was measured at the appropriate temperature with Radiometer electrodes and meter (BMS-3: PHM-64). IU IVVO Pco, was obtained using the Astrup technique (Sig- gaard-Andersen, 1974). Oxygen dissociation curves were obtained by measurements of 0, content and pH in blood samples equilibrated with a known PO.. The PO, was varied from zero I 155 mm Hg and mixed with either 3 or 6:; CO2 using gas mixing pumps (Wijsthoff. Bochum. Germany). The P,” (PO2 of 50% saturated blood) was obtained from the least squares regression of Hill plots. I.e. log S!lOO-S vs Iota PO.. The Hill coefficient. II. is the slope V of the reg~es&n line. The Bohr factor. A log P,,/ApH. was calculated from ODCs measured at i and 6” CO*.

Red ceil organic phosphates were measured as total nuc- leoside triphosphates (NTP) using an enzymatic method (Siema Chemical Co.. MI). In addition. the fractions of to&l NTP derived from adenosine triphosphate (ATP) and guanosine triphosphate (GTP) were determined using thin layer chromatography (Johansen rr al.. 1976). lnosine pen- taphosphate (IPP). a minor component of the total organic phosphate in turtles (Bartlett. 1976) was not measured.

All measurements on hemoglobm solutions pertain to tortoises which have been acclimated to 20 and 35°C. re- spectively. for at least 2 months. Hemoglobin solutions were prepared by washing the red cells twice in 0.9% NaCI. lysmg the cells in 0.1 M Tris buffer. pH 7.8 containing 5 x 10m4M Tris. and separating the red cell ghosts by centrifugation.

Hemoglobin solutions were stripped of ions by passage through a column of Sephadex G25, using 0.05 M Tris. pH 7.5 containing 0.1 M sodium chloride as elution buffer (Berman rt al.. 1971). Hemoglobin concentration was

measured spectrophotometrlcally usmg the extinctton cocf- fictents for human hemoglobin

The ODCs of hemoglobin solutions were measured by a modtfied diffusion chamber method (Sick & Gersonde. 1969: Mangum et al.. 1975). The major individual hemo- globin components were Isolated by iso-electric focusing of carbon monoxide saturated hemolvsates. Thus was done in I IO ml preparative columns containing LKB ampholinc (0.35”,, pH 7 IO. 0.20”,, pH S- 8 and 0.20°f{, pH 7-9). The pH values of fractrons of the column contents were measured at 5 C usmg a Radrometer micro-electrode (type G297). Hemoglobrn components retrieved from the column were prepared for oxygen equrlibrium studies by dialysis for 2 days against three changes of 0.01 M Tris buffer. pH 7.X. contaming 5 x IV4 M EDTA

Oxygen uptake (I’j,) and CO2 production (i&,,) were measured rn food-deprived (7 days) and resting ammals using an open circuit technique. Animals were placed m a plastic box which was maintained at either 20 or 35°C. The sample probe of a mass spectrometer (Medspect II. Searle, Houston. TX) provrded a constant flow of air through the box and measured the concentration of 0, and COZ in the excurrent air ii,, and (Vco,) were calcu- lated from the flow rate and differences between mspired and expired gas concentrattons. Calculations of Vo, were corrected for values of the respiratory exchange ratio less than I.0 (Hill. 1977).

RESULTS AND DISCUSSION

Whole blood. orgunic phosphate and metabolic rate

Figure I(a) shows the effects of temperature accli- mation on the ODCs of whole blood. As in previous studies of fish and amphibtans. the blood of cold- acclimated tortoises had a higher P,O (lower O2 affinity) at both acclimation temperatures. The mech- anism of this shift in the ODC appears to be a change in the intra-erythrocytic concentration of NTP. As summarized in Table 1. the NTP concentration in the cold-acclimated tortoises was significantly higher than that of the warm-acclimated group. The NTP was found to be primarily ATP with only trace amounts of GTP, an important component of NTP in red cells of some species. The relationship between oxygen affinity and pH (Table 1 and Fig. lb) reveals a significantly lower Bohr effect at 20°C (P < 0.05) in the cold-acclimated animals (4 = A logP,e/ApH = -0.37) than in the warm-acclimated animals (4 = -0.57). This seems inconsistent with previous obser- vations that organic phosphates increase the Bohr effect of purified hemoglobin solutions (Benesch er al..

1969). However. it should be noted that the present CO, Bohr effects are calculated for whole blood on the basis of changes in plasma pH (ApH,). but the changes in oxygen affinity depend on changes in in- tracellular pH (ApHi). At one particular pH,. both pHi and ApHi/ApH, will increase with a decrease in the red cell contents of non-diffusable anions (i.e. NTP). In addition. decreased NTP, together with in- creased pH. will enhance the specific effect of COZ on oxygen affinity (Duhm. 1976). Therefore, in ac- cordance with the present data. the CO, Bohr effect as a function of red cell NTP concentration should have its maximum value at a low NTP concentration.

The ODCs of Fig. l(a) were constructed at the in rioo blood pH for each temperature; pH 7.64 at 20°C

1.70

fz h 1.50

2

1.X

, -

) -

Temperature and blood respiratory properties in tortoise blood

0’ 10 30 50 70 90 110

PO, tmmHg)

-I

Malacochorsus tornior!

50

40

9

: 30 -

a:

20

7.1 0 730 7.50 7.70 PH

Fig. l(a) Oxygen equilibrium curves of whole blood interpolated from the data shown in Fig. l(b) and Table I. n equals 1.9 and 2.6 at 20 and 35°C. respectively. Fig. l(b) CO2 Bohr effect (Alog PJApH) in whole blood from cold-acclimated (A. A) and warm-acclimated (V,V) tortoises.

Upper pair at 35”C, lower pair at 20°C.

Table 1. Effects of temperature acclimation on the oxygen affinity and organic phosphates of red cells in the tortoise Malacochrrsus tornieri

157

Equation relating oxygen affinity (P,,) to pH

NTP ATP ATP + GTP n (mM/l cells) (rnM/l cells1 bI+4ll cells)

(N20 20 log 4) P,, r = = 4.18-0.37 -0.96 pH fS.E.M x- 0 I.84 19 028 5.86 5.16 0 24 016 5.58

35 log P,, = 4.59-0.40 pH 2.28 r = -0.98 027

35 20 log P,, = 5.61-0.57 pH 1.89 3.58 3 21 3.64 (N = 4) I = -0.91 0.12 011 0.14 017

35 log P,, = 5.60-0.54 pH 2.71 I = -0.97 0.22

STEPHEN C. Woon or nl.

Table 2. Effects of temperature acclimation on the acid-base balance and oxygen car,-\rng prolx’l II,:\ of blood in the tortoise Malucochrrsus tornieri

and pH 7.47 at 35°C (Table 2). This change in pH with temperature (ApH/AT= -0.011) conforms to the relative alkalinity concept (Rahn. 1967) as does the change in blood Pco, and plasma bicarbonate (Table 2). Unfortunately, in the present study. both deoxygenated systemic venous, O2 rich pulmonary venous. or mixtures of the two were sampled from the heart. Ideally, only systemic arterial blood should have been analyzed. The O2 saturation of the sampled blood ranged from 25 to 77% (mean = 56.3 f 18% SD.). Consequently, the pH and Pco2 data at each temperature only approximate the temperature dependence of acid-base balance in arterial blood.

With the above limitations in mind, it is interesting to compare the present data with those of Jackson (1971) (Jackson et al., 1974) for the turtle, Pseudemys s. elegans. They found that arterial pH changed from 7.67 at 20°C to 7.56 at 30°C (ApH/AT= -0.01 I). The present data for Vco, and Pao, also permit calcula- tions of alveolar ventilation using the relationship

V, = RW~,lPaco,.

where R is the gas constant (2.785 I v Hg/K/lsV I) and T is the absolute temperature. Vco, is in ml STP/kg/min and VR is in ml BTPS/kg/min. In the cold-acclimated group. VA was 2.6.3.at 20°C and 20.7 at 35°C while the ratio V,/V,, was 53.9ml BTPS/ml STP at 20°C and 27.6 ml BTPS/ml SIP at 35°C. In Jackson’s (1971) study (where the turtles were also acclimated to 2&22°C). the ratio of total ventilation (c’) to PO2 decreased from 38.1 at 20°C to 10.8 at 35C.

There was no significant effect of temperature accli- mation on the O2 capacity of blood (Table 2). Other studies of O2 capacity following temperature acclima- tion show no change, an increase or a decrease fol- lowing warm acclimation, even in the same species (cf. Johansen & Weber. 1976).

The temperature sensitivity of oxygen affinity within each group can be calculated from the regres- sion equations in Table I. At a constant pH (7.5). the PsO of blood in the cold-adapted group increased from 25.4 to 38.9 mm Hg as temperature increased from 20 to 35°C (A log Pso/AT= 0.0123). The same

temperature increase in the warm-adapted group caused a P,, increase from 21.6 to 35.5 mm Hg (A log P,,/AT= 0.0144). As a result of acclimation, the A log P,,/AT between groups (AP,, = 25435.5) is 0.009.

The oxygen uptake measured at 20°C was 29.3 + 2.0 S.E.M. for the cold-adapted and 14.5 of: 0.8 for the warm-adapted tortoises (units are ml O2 STP/kg/hr). At 35°C the Vo, was 44.9 + I.4 for the cold-adapted and 46.3 f 2.1 for the warm-adapted group. Thus, as shown in Fig. 2, the metabolic rate

IO 10

_pp_-. _~ i -

20 30 40

TEMPERATURE ‘T

Fig. 2. Standard metabolic rate as a function of tempera- ture in cold- and warm-acclimated tortoises.

showed a compensatory pattern of acclimation in which the temperature coefficient of l&1 (Q1 0) is lower in cold-adapted tortoises [Precht’s Type III acclima- tion, cf. Prosser (1973)].

The respiratory exchange ratio (R) was 0.81 + 0.04 S.E.M. for both acclimation groups at 20°C. and 0.93 k 0.04 S.E.M. at 35-C. These values (assumed to be steady state) may indicate a non-fast- ing (R = 0.7) condition despite 3 days of food depri- vation. It is also possible that the higher than expected R values reflect fat synthesis or uric acid as noted by Rebach (1973) for fasting snakes.

In Jackson’s (1971) study of Psrudrm)x $. &YJU~S. PO, increased from 39 ml STP,/kglbr at 2OC to 52.8 ml STP/kg/hr at 35°C (Q, 0 = I .35). These values are similar to those for the cold-acclimated tortoises. It is noteworthy that Jackson’s turtles were also kept at 20-22°C before being studied.

Hemoglobin solutions

As shown in Fig. 3. the purified hemoglobins from 20- and 35”C-acclimated tortoises have very similar oxygen-binding properties (at pH 7.4, Pso - 556mm Hg. and 4 _ -0.24 to --0.31). The slightly higher oxygen affinity observed for the hemolysates from the cold-acclimated specimens is opposite to the afhnity change found in whole blood. Figure 3 also shows that the hemolysates from both acclimation groups are similarly affected by the presence of ATP. which increases both PSO and t7 values. These data indicate that changes in the intrinsic properties of the hemo- globin do not contribute to the thermo-acclimatory responses of the whole blood.

Iso-electric focusing experiments revealed identical heterogeneity patterns in the hemolysates of cold- and warm-acclimated tortoises (Fig. 4). The two major

Temperature and blood respiratory properties in tortoise blood 159

A Malacochersus _ 20 tornieri

<

15 _

r” . ‘0 F : 6E

l -a. &

6. -. .-.-----.

0‘. - : L p

-;. .

+ 2

t

E . . .

I . . A. l . . i

Fig. 3. P5,, and !1 values and their pH dependence of stripped hemolysates from tortoises acclimated to 2o’C (a,~) and 35°C (A. A) measured in 0.05 M Tris buffers at 25°C. Closed symbols. stripped hemolysates; open sym- bols, stripped hemolysates in the presences of ATP (ap- proximately five-fold molar excess over hemoglobin).

hemoglobin concentration 0.54.6 mM.

components constitute about 67 and 28% of the total haem. and the carboxy derivatives are iso-electric at pH 8.1 and 7.0, respectively, at 5°C. In addition, minor components were observed to focus at pH values near 9.0, 7.3 and 5.8.

The minor component (II) shows higher oxygen affinity than component I. Under the same experi- mental conditions, the values of PSo, 4 and n of com- ponent I at pH 7.4 are 3.4 mm, -0.23 and 2.1, com- pared to corresponding values of 1.7 mm, -0.40 and 1.9 of component II (Fig. 5). A mixture of the two components has oxygen equilibrium properties that are intermediate to those of the isolated components, indicating a lack of interaction. Assuming that these components have the same concentration effect, and that their relative intracellular concentrations equal

20

10 20 30 LO FRACTIONS COLLECTED

Fig. 4. Separation of hemoglobin components from a 3S”C-acclimated tortoise by isoelectric focusing. 0, optical density at 540 nm ; O. pH at 5°C. Horizontal bars, fractions pooled for oxygen-equilibrium determination (cf. Fig. 5); fraction size, I.8 ml. A virtually identical profile was obtained using hemolysates from a ZO”C-acclimated

= B.P 59 2*--r

tortoise.

66 72 76 60

PH

Fig. 5. P,, and n values and their pH dependence measured at 25°C in 0.05 M Tris buffers, of hemoglobin components I (O.O.U,O) and 11 (A,A,V,V) from tor- toises acclimated to 20°C (0.0.r. A) and 35°C (W. 0. V, V). Solid symbols, stripped hemoglobins; open symbols, stripped hemoglobins in the presence of ATP (ap- proximately five-fold molar excess over hemoglobin),

hemoglobin concentration. approximately 0.2 mM.

those in the hemolysate, it is likely that the in viw, functional differences between these components will be greater than indicated by the present measure- ments which were conducted at similar concen- trations.

The hemolysate data show that in Malacochersus thermoacclimatory responses at the level of the hemo- globin molecule do not contribute to the correspond- ing adaptation observed in whole blood. This is apparent not only from similar oxygenation proper- ties of the cofactor-free hemolysates of warm- and cold-acclimated tortoises, but also from the identical heterogeneity patterns of the hemoglobins, and simi- larity in oxygen affinities of the components and their sensitivities to pH and ATP. Evidently, the adapta- tional response in the whole blood rests wholly with changes in the physico-chemical environment of the hemoglobin molecules in the red cells. These observa- tions align with previous data showing the absence of changes in inherent properties of vertebrate hemo- globins following changes in environmental factors such as oxygen-availability (Weber rt al., 1976; Johansen rt al., 1976). Unlike the tortoise, however, several fish species show temperature-induced changes in relative abundancies of hemoglobin com- ponents, which may represent altered aggregation of pre-existing subunits rather than synthesis of specific components (Houston et al., 1976; Houston & Rupert, 1976; Weber et al., 1976).

Hemoglobin heterogeneity appears to be of com- mon occurrence in reptiles (Gratzer & Allison. 1964; Sullivan & Riggs, 1967), but there is little information on the functional significance of multiple hemo- globins. Several studies suggest that hemoglobin het- erogeneity in fishes could provide the basis for diverse functioning of the composite hemoglobin in blood (cf. Hashimoto et al., 1960; Powers, 1972). Hypoxic-adag tation in blood oxygen affinity in eels appears almost

entirely attributable to the high sensitivity of oxygen affinity of a single hemoglobin component to nucleo- side triphosphates (Weber c’f al.. 1976). However. the two major components of Mulucochersus hemoglobin have only slightly different loading and unloading properties. Also. the present demonstration of similar phosphate and pH sensitivities suggests that both major components are implicated in the thermo-accli- matory responses of the whole blood respiratory properties.

Acknowledgcvnmts -Research support was provided hy the Danish Natural Science Research Council and National Institutes of Health Grant No. HL 18026 (S.C.W.).

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