Acid-base status in the toad Bufo viridis in vivo

Respiration Physiology (1980) 41, 105-115 © Elsevier/North-Holland Biomedical Press

A C I D - B A S E S T A T U S IN T H E T O A D BUFO VIRIDIS IN VIVO

U. K A T Z * Department of Zoology, The Hebrew University of Jerusalem, Israel

Abstract. The acid-base status in the toad Bufo viridis was assessed on blood samples taken through a chronical cannula from unrestrained animals. Blood gases and the p H were very constant in animals which were kept either under control conditions (free access to tap water) or immersed in shallow water, p H was 7.646 +_0.032 (mean + SEM) in 14 control toads at 26°C. Pco2 was 13.1 +_ 1.2 m m Hg and [HCO~-] was 17.0 +_ 0.7 mmol/1 at the same temperature, p H a n d P¢o2 were independent of the hematocrit and the haemoglobin in the blood. Diamox induced a characteristic metabolic acidosis which is apparently the result of the inhibition of carbonic anhydrase both in the kidney and in the skin. The results are discussed in relation to the role of the kidney and the skin in regulating the acid-base balance in the toad. Acetazolamid Acid-base Blood gases

Bufo viridis Hematocrit Metabolic acidosis

Acid-base balance in amphibians has been studied mostly as part of extensive studies on poikilotherms' acid-base balance carried out in the last decade. These studies were concerned mainly with the effect of temperature on the acid-base status (see Reeves, 1977, for recent review). Other studies were concerned with evolutionary developments (Lenfant and Johansen, 1967; Rahn and Howell, 1976), or the effects of hypoxia and diving (Boutilier and Toews, 1977; Lillo, 1978, among others). The general picture of the acid-base status in amphibians is related to that of higher vertebrates with a dependence on temperature similar to other poikilotherms (Howell et al., 1970). Its regulation, however, is certainly different from that in mammals (Yoshimura et al., 1961), or in fish (Cameron, 1978), and is known only poorly. Accepted for publication 22 February 1980 * On leave at the The Weizmann Institute of Science, Rehovot. With the technical assistance of Mrs. Y. Shimoni. 105

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An arterial cannula, chronically implanted into the dorsal artery was used in this study to collect blood, for the assessment of the acid-base status in the toad Bufo viridis under normal conditions.

Materials and methods

Toads (Bufo viridis), of both sexes were collected in Jerusalem (Israel) and were kept outdoors with free access to tap water. They were fed twice a week with maggots. During the experiments they were kept in the laboratory (19-25 °C) without food. To collect blood, toads were cannulated chronically through the dorsal artery. After anesthetization (MS-222 given in the mouth), a small incision was made in the skin on the back, near the right leg. The cannula (thinned PE 10 connected through a PE 50 to PE 160 clay-Adams polyethylene tubes), was inserted into the dorsal artery through the external iliac artery after puncturing it with a No. 30 needle. The open end of the cannula was left free in the artery, permitting the flow of blood to the hind muscles. The cannula was tightened with silk threads to the muscle behind, near the exit point, and then outside on the back to the skin. Care was taken not to occlude the knot. Sometimes infections developed, but healthy toads (up to 5-6 weeks after capture) usually did not show any ill effects and were used over 3 weeks in the experiments. Animals were used 2-3 days after the operation, and only after checking the cannula. About 80-100 ~tl of blood was collected anaerobically from unrestrained toads into heparinized pyrex capillaries. After collection of the blood, the cannula was flushed back with saline containing heparin, and was stoppered with a small stainless steel rod. pH and gases (CO 2 and 02) were measured with a Radiometer BMS 3 MK2 blood microsystem attached to a PHM73 pH/blood gas monitor. Blood was sucked into the pH electrode filling the cuvette of the Pco,-Po~ electrodes. ]'he pH electrode was calibrated with a Radiometer type S1510 buffer, corrected to the temperature of measurement. The Pco2 electrode (Radiometer E-5037) was calibrated with two concentrations of CO_~ supplied through a Radiometer GMA2 precision gas supply, and the Po~ electrode (Radiometer E-5047) with air saturated water; all at the temperature of the instrument, which matched the temperature of the animals. TABLE 1 Comparison of the composition of arterial (cannula) and ventricular blood of the toad Bufo viridis (1978). t = 26 °C. Mean _+ SEM from 4 toads Blood from

pH

PCO2 (mm Hg)

PO2 (mm Hg)

Hb saturation

Osmolality (mOsm)

[CI ] (mmol/l)

Ht (%)

312-+2 312_+2

125+5 121_+6

18.1_+1.3 19.9+1.1

(%) 1. Cannula 2. Ventricle

7.698_+0.068 7,550_+0.037

11.8-+2.1 16.6_+2.3

100.0+12.9 44.9_+ 4.2

80-+3 20+6

ACID-BASE STATUS IN A TOAD

107

After each measurement all electrodes were recalibrated, and readings were corrected accordingly. Haemoglobin was determined with a Radiometer OSM2 Hemoximeter at 37°C on whole blood. H C O f was calculated according to the HendersonHasselbach equation from the pH and Pco, determinations, and the values of pK' and CO2 solubility taken from Severinghaus et al. (1956). At the end, the blood from the electrodes' cuvette was sucked back and centrifuged. After determination of the hematocrit, the plasma was separated and analyzed. Osmolality was determined on a Knauer, Berlin semimicro osmometer, and chloride on a Radiometer CMTI0 chloride titrator. Comparison of hematocrit, osmolality and the concentration of chloride of fresh samples with samples taken from the cuvette, gave similar results. Diamox was from Lab. Theraplix Paris, and was dissolved in saline (0.75 NaC1), which was used also for control. Student 't'-test was used for statistical analyses.

Results

Table 1 compares the composition of arterial blood (taken through the cannula) with blood from the same toad taken from the ventricle of the heart; the latter was taken immediately after killing the toad by double pitting and exposure of the heart. It can be seen that the arterial blood had similar composition in respect to the hematocrit, concentration of chloride and osmolality, pH, Pco2 and Po2, differed however, as could be expected since the blood taken from the ventricle represents a mixture of both arterial and venous blood (Tazawa et al., 1979). Figure 1 shows the mean values of the pH, Pco.,, Po2, osmolality and hematocrit of arterial blood in a group of 6 toads, sampled 6 times successively during 10 days. The toads were kept first under control conditions (with free access to tap water), then on the fifth day they were transferred, and kept in 1-2 cm deep tap water throughout. It can be seen (see also table 2) that the pH, Pco, and Po2 were hardly affected by the experimental conditions (successive blood sampling, and control to tap water transfer). The osmolality and the concentration of chloride decreased, however, by an average of 15%, under the tap water conditions. Table 3 is a summary of another experiment which further checks this point. 4 toads weighing an average of 54 + 9 g (mean + SEM) were kept for 2 days under control conditions (they appear quite dry under these conditions, since they go into the water only seldomly). Arterial blood was analyzed and the toads were then immersed in tap water up to their heads for 24 h, permitting them to breathe freely; then, the toads were left for another 24 h in 1-2 cm deep tap water. It can be seen that the Pco~ and Po2 were unaffected by the experimental treatments. The pH however changed a little, significantly only in the third condition. Attention should be drawn to the systematic and characteristic changes in the osmolality of the blood and the concentration of chloride upon water immersion, which are shown in the table. Comparison of the 2 sets of experiments in table 2, which were carried out at

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7.6

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Time (days) Fig. 1. Mean values (+ SEM) of the pH, P c o , , Po,, hematocrit (Ht), and osmolality (mOsm) of 6 toads. The toads were cannulated and sampled as described in the methods. They were kept first with free acess to tap water; on the 5th day they were transferred to 2-3 cm deep tap water, tl~a) = 21-23°C. Determinations were made at 26 °C. Average weight of the toads was 33,0 + 1.7 g (1978).

TABLE 2 Summary of blood data obtained from Bufo viridis under 'control' (free access to tap water) conditions, and tap water (1-2 cm in tap water)conditions (1978). Mean + SEM pH

PCO, (ram Hg)

[HCO~] (mmo!-/l)

Pox (mm Hg)

Osmolality (mOsm)

1. 6 toads at 26 °C (the group shown in fig. l) (number of determinations in parentheses) Control* 7.646_+0.032 I3.1_+1.2 17.0_+0.7 96_+3 359-+11 (3.5-7.5) (14) (14) (14) (13) (10) Tap water* 7.661_+0.041 12.1_+0.8 16.2_+ 1.1 102_+5 312_+8 (8.5 12.5) (11) (11) (11) (11) (6) 11. Toads at 18-22 °C** (1 determination from each toad) Control 7.721_+0.019 11.9_+0.8 21.2_+0.9 (20 toads) Tap water 7.810-+0.018 ll.4_+0.4 24.1_+1.7 (3 toads)

[CI ] Ht (mmol/l ) C/o)

140_+2 (12) 129±4 (7)

16.8_+1.2 (13) 14.7 + 1.4 (6)

91.6+_2.7

354_+7

130_+4

21.3+_1.7

95.3+_9.0

297_+3

109+8

23.4t 5.0

* Summary of the 3 successivemeasurements, corresponding to the times in fig. 1. ** Summary of control values of toads used in other experiments.

A C I D - B A S E S T A T U S IN A T O A D

109

TABLE 3 The effect of 'control' conditions, water immersion (up to the head) and tap water (1-2 cm deep) on arterial blood data in Bufo viridis, t = 23 °C. Mean + SEM of 4 toads

1. Control (36 h) 2. Water immersion (24 h) 3. Shallow water (24 h)

pH

PCO_~ (ram Hg)

PO_, (mm Hg)

Osmolality (mOsm)

[C1 ] (rnmol/l- 1)

Ht (%)

7.694+_0.058 7.800+_0.038

11.0_+0.7 10.7+_0.6

91.0+11.3 97.7+_ 5.4

371+_33 329+_26

148+_18 130+_14

20.1+_2.0 18.2+_3.6

7.839+0.047

10.7+-0.4

94.3+- 7.0

360+-15

145_+13

17.0+_3.6

different temperatures, gives an average dpH/dt value of 0.015 pH/1 °C, which is similar to the value (0.016 pH/1 °C) given by Howell et al. (1970) for three other poikilotherms including a toad (B. marinus). The differences in Pco2 and H C O ; concentration between the two groups in table 2 may be accounted for by the difference in the temperatures. The relation of the concentration of H C O 3 in the blood to the pH was calculated from the in vivo measurements. A linear regression analysis from 16 toads under control conditions gave pH = 0.013 [HCO3] + 7.455; the correlation coefficient was r a t h e r poor (r=0.597). (Average pH was 7.719_+0.029, and HCO 3 20.9 _+ 1.4 mM/l; mean _+ SEM). In fig. 2 the pH, Pco2 and Po~ values of arterial blood from control toads is drawn on the ordinate, with the hematocrit values of each sample on the abcissa. Linear regression analyses gave the following equations (table 4). These three parameters do not correlate then, with the hematocrit. It should be noted here, that the arterial Po2 is not expected to be dependent on the concentration of the haemoglobin in the blood (Roughton, 1964). It is quite surprising however, how little the pH and Pco2 depend on the concentration of haemoglobin in the blood. On two extreme occasions the hematocrit fell to nearly zero, and no visible erythrocytes could be seen in the blood. These toads were killed later and central blood (from the ventricle) was examined. Chloride and osmolality were found to be the same as in the arterial (cannula) blood, but there were no visible erythrocytes either. Table 5 compares the pH, Pco2 and Po2 of arterial blood in one of these two toads, to 5 other toads having a mean hematocrit value of 16.5~. The only difference which is significant is found in the Po: value, while the pH and Pco2 are practically the same. The extent of saturation of the haemoglobin (in percent, as measured in the Hemoximeter) was quite fairly correlated with the Po2 of the arterial blood. The regression line for 16 toads under control conditions gave ~o SAT(Hb)= 0.399 Po: + 41.5 (r = 0.689). Table 6 summarizes experiments in which Diamox was used. The drug which inhibits the carbonic anhydrase catalyzed hydration of C02 and dehydration of HCO3, was injected intra-peritoneally. It can be seen that after 3 h,

110

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(ram Hg}

70

50 19 17 15

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Pco2 13 (mm Hg) 11

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•

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9 7

7.9 7.8

pH

7.7 7.6 7.5 I

i

J

10

I

20

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30

40 H t (%)

Fig. 2. The relation of pH, Pco_~ and Po, to the hematocrit value in determinations on arterial blood taken from 27 toads under control conditions, t = 22 °C. See also table 4 (1978).

the pH decreased markedly with apparently no effect on the Pco~. There was some 35% decrease in the concentration of HCO[, which was, however, somewhat low in these experiments in the control period. TABLE 4 Linear regression (y = ax + b) analysis of the dependence o f p H , Pco 2, Po2 and haemoglobin concentration on the hematocrit (x = hematocrit in percent). Data from 21 control toads. Average hematocrit was 21.2 _+ 7.5% Related parameter

Mean + SEM

Y (related parameter)

r

pH Pco2 (mm Hg) Po2(mmHg) Hb (g%)

7.719_+0.023 11.9 _+0.6 90.4 _ + 3 . 5 7.3 _+0.5

- 0.004x + 7.811 0.142x+ 8.945 -1.153x+115.063 0.307x+ 0.780

0.305 0.384 0.547 0.956

111

A C I D - B A S E S T A T U S IN A T O A D

TABLE 5 Comparison of the p H Pco2 a n d Po2 in artei'ial blood of a haemoglobin-'less' toad with 5 other toads with mean hematocrit of 16.5%. t = 26 °C. Mean + SEM

1. Normal toads* 2. Hb-less toad**

pH

PCO2 (mm Hg)

[HCOf] (mmol/l)

P02 (mm Hg)

Osmolality (mOsm)

Ht (%)

7.638+0.018 7.654+_0.040

13.0+0.8 12.6+0.5

16.6 16.7

100.1+_1.8 84.5+_4.7

360+_7 348+_15

16.5+0.9 1.7+1.0

* 20 measurements from 5 toads. ** 5 measurements from 1 toad.

TABLE 6 The effect of acetazolamide (Diamox) 10 -3 M on blood data analyses in the toad Bufo viridis. Average weight of the toads was 54 + 6 g and the hematocrit 15 +6%. t = 23 °C (1979). Mean _+SEM. N u m b e r of toads in parentheses

Control (5) Diamox* (5) Control group ** (6)

pH

Pcoz ( m m Hg)

[HCO3] (mmol/l)

Po., ( m m Hg)

7.606 + 0.037 7.343 _+0.060 7.626 +_0.014

11.0 + 1.2 12.8 +0.7 10.4 +0.6

13.6 + 1.0 8.3 +0.8 13.5 -+0.8

89 + 12 92 + 15 97 + 12

* After a control sample, ihe toads were injected with Diamox, and the next sample (Diamox) was taken 3 h later. ** A group of toads injected with 0.8% NaCI, and analyzed in parallel to the experimental group.

No correlation could be established between the concentrations of chloride and HCO? in the plasma of the arterial blood under ontrol conditions (HCO3 = 0.03 [C1 ] + 25.04; r = 0.097 for 20 toads).

Discussion The results presented in this paper give an acount of the acid-base status in the toad Bufo viridis under normal resting conditions. The blood delivered through the cannula proved to represent arterial blood. The importance of such methodology is emphasized in table 1, where the arterial blood is compared to the blood taken from the heart; the latter is considerably different in respect to acid-base, and blood gases, but not in respect to the osmolality and the concentration of chloride. The values which were obtained in this study (table 2) compare fairly well with values of others (Campbell, 1926; Howell et al., 1970; Just et al., 1973; Emilio, 1974; Tazawa et al., 1979) in other amphibians. The hematocrit value with an average of 20-25% was quite low. Weissberg (1973) found a higher value (36 +29/0;

112

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mean _+ SEM of 10 toads), in this toad. The difference in the methods (the later author collected the blood from animals which were not operated or touched before), was found to be the reason for the reduced hematocrit in the present study. Successive blood sampling may be of considerable effect on the hematopoetic tissues (even that 500-700 gl of whole blood from toads of 50 + 5 g weight in no days, does not seem to be much). In our experience there was a successive decrease in the hematocrit value (from 23 _+2~o, post-operation value, to 17 _+2 ~ in 12 toads during 10-12 days with 5-7 samplings; 21-24°C). Both the operation, and the sampling had an effect, in addition to the low capacity for haemoglobin synthesis and the production of erythrocytes in the toads which is nearly half at room temperature than in mammals and man at 37°C (Coleman et al., 1953). This, however, did not affect the acid-base status, which was practically independent of the hematocrit (fig. 2). De Graaf (1957) and Ewer (1959) have already reported on two anaemic, apparently normal toads (Xenopus laevis), which they noticed in the laboratory. They differed, however, in their interpretations. Ewer (1959) questioned whether this was not a pathologically unusual phenomenon? The results obtained in the present study seem to support the view that resting toads, as well as other amphibians, are only little dependent on the concentration of haemoglobin in the blood for gas transfer and buffering (table 5). It should be emphasized at this point that the continuous change in the hematocrit during the experiments did not affect the blood data (fig. 2); it thus further supports the view on the relative independence of blood gases and acid-base status in the toad, from the concentration of haemoglobin (fig. 2; table 5). Similar observation was made by Haswell and Randall (1978), in a fish (Salmo gairdneri). These authors found the pH and the arterial Pco: to be independent of the hematocrit, in fish which were made anaemic experimentally. These later authors postulated a mechanism for CO2 excretion in the fish, whereby the majority of CO2 which is excreted through the gills is originated from plasma HCO 3. In their hypothesis the erythrocytes are nearly impermeable to HCO3, which is formed at uncatalyzed rate as CO 2 from the tissues enters the blood through the veins. It is in the gills of these fish that HCO 3 is dehydrated rapidly through the action of the gills' carbonic anhydrase. A similar mechanism could possibly be envisaged in amphibians, with the skin which contains carbonic anhydrase (Rosen and Friedly, 1973), analogous to the gills of the fishes. A clarification of this possibility will need the assessment of the difference in HCO3, at the gas exchange sites in the toad, and a more direct study in vitro. It seems that diffusion and the circulation are sufficient in providing for the needs of these animals. Moreover, the absence of an effect of the immersion in water (tables 2 and 3) on the blood gases proves that this is true also under variable conditions. Krogh (1904) had already pointed out that CO2 elimination is accomplished mostly through the skin in Rana esculenta, and this is true to a greater or lesser extent in most amphibians (Dolk and Postma, 1928 ; Charles, 1931 ; Rahn and

ACID-BASE STATUS IN A TOAD

113

Howell, 1976). The extensive vascularization of the skin in the toad (Czopek and Czopek, 1959) would serve this need quite efficiently. Recent studies (Jackson and Braun, 1979) have proved, however, that the major role in regulating the Pco2 of the blood is played by the respiratory system, rather than by the skin. On the other hand, there is a considerable variability in the osmolality of the plasma and the concentration of its chloride (300 to 370 mOsm, and 109 to 140 mmol/1 respectively, in this study; see also Katz, 1973). Since the animals were kept under normal conditions, (free access to tap water or shallow water), which do not seem to be far from their natural habitat, one is inclined to conclude that the osmotic and ionic 'milieu interieur' of the toads is not kept constant, or that it is regulated within a wide range (Katz, 1973). The blood gases on the other hand, and the concentration of hydrogen ion and HCO3 which vary only little, are regulated more strictly under these conditions (tables 2 and 3). The concentration of bicarbonate (16-24 mM/l), which was calculated in the blood of the toads in this study seems to be common to most or all terrestrial amphibians (Rahn and Howell, 1976). Pco2 in this study corresponds to a transitional concentration of water-breathers to air-breathers (Dejours, 1978). The injection of Diamox (acetazolamide) (table 6), resulted in a rapid increase in the concentration of H ÷ in the blood (from 24.0 to 45.7 nmol/l) and a decrease in the concentration of HCO 3 by some 30~o. Pco., did not change. The picture is a characteristic of metabolic acidosis (Pitts, 1974). Carbonic anhydrase, the enzyme which is the target for the action of the Diamox is present in the kidneys of amphibians in large amounts (Toews et al., 1978), and also in the skin (Rosen and Friedly, 1973). Since the enzyme is apparently inhibited in both organs, it is difficult to decide of their proportional contribution to the acid-base balance under ,normal conditions. The fact that the acid-base status was independent of the hematocrit value (fig. 2), seems to exclude an important role to inhibition of the enzyme in the erythrocytes. Yoshimura et al. (1961) have described a situation of respiratory acidosis 6-8 h after the injection of Diamox to Rana catesbeiana. They found a great increase in the excretion of sodium and total CO2 (mostly as H C O 3 ) by the kidneys. At the same time there was some 17~o increase in the calculated serum Pco2, significant at P < 0.05. It may be pointed out here that a transient respiratory acidosis may occur as a result of administration of Diamox (Mudge, 1975). Any increase in the arterial Pco2 could have been regulated through the pulmonary respiration, which was found quite efficient in the bullfrog (Jackson and Braun, 1979). On the other hand, Diamox inhibits the secretion of H + through the Na+/H + exchange across frog skin, both in vivo and in vitro (Ehrenfeld and Garcia-Romeu, 1977), and this has also been found in the isolated toad skin (Katz, 1979). It is not possible at present to estimate the quantitative contribution of each of these organs (as well as that of the urinary bladder), in regulating the acid-base balance of the toad. This will need a study of kidney function, and of the in vivo buffering capacity of the toads. In conclusion, the acid-base status and the blood gases have been determined in

114

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the toad Bufo viridis under normal conditions. Quite surprisingly, these parameters were nearly independent of the concentration of haemoglobin in the blood. This point should be studied more directly in vitro. Control (free access to tap water), or tap water conditions did not affect these parameters, which can be regulated quite readily under these conditions. It is not possible at present to assess the relative importance of the osmoregulatory organs in regulating the acid-base balance in the toad.

Acknowledgements I would like to thank A. Bar-Ilan for introducing me to the measuring system, and for helpful discussions. 1 thank Dr. F. Garcia-Romeu from Nice for critical review of the manuscript, and for helpful suggestions. This study has been supported by the Bat-Sheva de Rothschild Fund for the Advancement of Science.

References Boutilier, R.G. and D.R. Toews (1977). The effect of progressive hypoxia on respiration in the toad Bufo marinus. J. Exp. Biol. 68: 99-107. Cameron, J.N. (1978). Regulation of blood pH in teleost fish. Respir. Physiol. 33: 129-144. Campbell, J.A. (1926). The normal CO 2 and 0 2 tension in the tissues of various animals. J. Physiol. (London) 61: 248-254. Charles, E. (1931). Metabolic changes associated with pigmentary effector activity and pituitary removal in Xenopus laevis I. Respiratory exchange. Proc. R. Soc. B 107: 486-503. Coleman, D.H., A.R. Stevens, H.T. Dodge and C.A. Finch (1953). Rate of blood regeneration after blood loss. Arch. Int. Med. 92:341 349. Czopek, G. and J. Czopek (1959). Vascularization of respiratory surfaces in Bufo viridis and BuJb calamita. Bull. Acad. Polon. Scie. Ser. Sci. Biol. 7: 3%45. De Graaf, A. R. (1957). A note on the oxygen requirements of Xenopus laevis. J. Exp. Biol. 34:173 176. De)ours, P. (1978). Carbon dioxide in water- and air-breathers. Respir. Physiol. 33:121 128. Dolk, H. E. and N. Postma (1928). Ueber die Haut und Lungenatmung von Rana termporaria. Z. Vergleich. Physiol. 5: 417~,44. Ehrenfeld, J. and F. Garcia Romeu (1977). Active hydrogen excretion and sodium absorption through isolated frog skin. Am. J. Physiol. 233: F46-F54. Emilio, M. G. (1974). Gas exchanges and blood gas concentration in the frog Rana ridibunda. J. Exp. Biol. 60: 901-908. Ewer, D.W. (1959). A toad (Xenopus laevis) without haemoglobin. Nature 183: 271. Haswell, M.S. and D.J. Randall (1978). The pattern of carbon dioxide excretion in the rainbow trout Salmo gairdneri. J. Exp. Biol. 72:17-24. Howell, B.J., F.W. Baumgardner, K. Bondi and H. Rahn (1970). Acid base balance in cold-blooded vertebrates as a function of body temperature. Am. J. Physiol. 218: 600-606. Jackson, D.C. and B.A. Braun (1979). Respiratory control in Bullfrogs: Cutaneous versus pulmonary response to selective CO 2 exposure. J. Comp. Physiol. 129: 339-342. Just, J.J., R. N. Gatz and E.C. Crawford (1973). Changes in respiratory functions during metamorphosis of the Bullfrog Rana catesbeiana. Respir. Physiol. 17 : 276-282.

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Katz, U. (1973). Studies on the adaptation of the toad Bufo viridis to high salinities: oxygen consumption, plasma concentration and water content of the tissues. J. Exp. Biol. 58 : 785-796. Katz, U. (1979). Role of the skin in osmoregulation and in the acid-base status of toads (Bufo viridis) under adaptation to high salinity. INSERM, Symp. Paris. 85: 351-358. Krogh, A. (1904). On the cutaneous and pulmonary respiration of the frog. Skand. Arch. Physiol. 15: 328~,19. Lenfant, C. and K. Johansen (1967). Respiratory adaptation in selected amphibians. Respir. Physiol. 2: 247-260. Lillo, R.S. (1978), The effect of arterial-blood Po2, P¢o2 and pH on diving bradycardia in the Bullfrog Rana catesbeiana. Physiol. Zool. 51 : 340-346. Mudge, G.H. (1975). In: Pharmacological Basis of Therapeutics. Ch. 39, edited by L.A. Goodman and A. Gilman, 5th edition. New York, Macmillan. Pitts, R.F. (1974). Physiology of the Kidney and Body Fluids. Chicago, Year Book Medical Publications. Rahn, H. and B.J. Howell (1976). Bimodal gas exchange. In: Respiration of Amphibious Vertebrates, edited by Hughes. Academic Press, pp. 271-285. Reeves, R.B. (1977). The interaction of body temperature and acid-base balance in ectothermic vertebrates. Ann. Rev. Physiol. 38: 559-586. Rosen, S. and N. Friedly (1973). Carbonic anhydrase activity in Rana pipiens skin: biochemical and histochemical analysis. Histochemie 36 : 14. Roughton, F.J.W. (1964). Transport of oxygen and carbon dioxide. In: Handbook of Physiology. Respiration. Vol. I, edited by W.O. Fenn and H. Rahn. Washington, D.C., Am. Physiol. Soc., pp. 767-825. Severinghaus, J.W., M. Stupfel and A.F. Bradley (1956). Variations of serum carbonic ackl pK with pH and temperature. J. AppL Physiol. 9: 197-200. Tazawa, H., M. Mochizuki and J. Piiper (1979). Respiratory gas transport by the incompletely separated double circulation in Bullfrog Rana Catesbeiana. Respir. Physiol. 36:77 95. Toews, D., R. Boutilier, L. Todd and N. Fuller (1978). Carbonic anhydrase in the amphibia. Comp. Biochem. Physiol. 59A: 211-213. Weissberg, J. (1973). The effect of high salt adaptation of the toad Bufo viridis on the salt balance in the erythrocytes. M. Sc. thesis. The Hebrew University of Jerusalem. (In Hebrew). Yoshimura, H., M. Yata, M. Yursa and R.A. Wolbach (1961). Renal regulation of acid-base balance in the Bullfrog. Am. J. Physiol. 201 : 980-986.