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Seasonal changes in the cardiorespiratory responses to hypercarbia and temperature in the bullfrog, Rana catesbeiana
Comparative Biochemistry and Physiology Part A 124 (1999) 221 – 229 www.elsevier.com/locate/cbpa
Seasonal changes in the cardiorespiratory responses to hypercarbia and temperature in the bullfrog, Rana catesbeiana Keˆnia C. Bı´cego-Nahas, Luiz G.S. Branco * Departamento of Fisiologia, Faculdade de Medicina de Ribeira˜o Preto e Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, 14040 -904. Ribeira˜o Preto, Sa˜o Paulo, Brazil Received 17 March 1999; received in revised form 20 July 1999; accepted 19 August 1999
Abstract We assessed the seasonal variations in the effects of hypercarbia (3 or 5% inspired CO2) on cardiorespiratory responses in the bullfrog Rana catesbeiana at different temperatures (10, 20 and 30°C). We measured breathing frequency, blood gases, acid–base status, hematocrit, heart rate, blood pressure and oxygen consumption. At 20 and 30°C, the rate of oxygen consumption had a tendency to be lowest during winter and highest during summer. Hypercarbia-induced changes in breathing frequency were proportional to body temperature during summer and spring, but not during winter (20 and 30°C). Moreover, during winter, the effects of CO2 on breathing frequency at 30°C were smaller than during summer and spring. These facts indicate a decreased ventilatory sensitivity during winter. PaO2 and pHa showed no significant change during the year, but PaCO2 was almost twice as high during winter than in summer and spring, indicating increased plasma bicarbonate levels. The hematocrit values showed no significant changes induced by temperature, hypercarbia or season, indicating that the oxygen carrying capacity of blood is kept constant throughout the year. Decreased body temperature was accompanied by a reduction in heart rate during all four seasons, and a reduction in blood pressure during summer and spring. Blood pressure was higher during winter than during any other seasons whereas no seasonal change was observed in heart rate. This may indicate that peripheral resistance and/or stroke volume may be elevated during this season. Taken together, these results suggest that the decreased ventilatory sensitivity to hypercarbia during winter occurs while cardiovascular parameters are kept constant. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Breathing frequency; Arterial pH; Blood gases; Hematocrit; Blood pressure; Heart rate; Bullfrog; Hypercarbia; Seasonal variations; Temperature
1. Introduction Amphibians have a remarkable tolerance to diverse environmental conditions and they range widely over the globe. Much of this extensive environmental range is due to the ability of amphibians to withstand long periods of unfavorable conditions (starvation, cold and/ or drought) and to condense their life cycle into brief active periods when conditions are less stringent [38]. However, nearly all studies of cardiorespiratory physiology of amphibians have been performed only on active animals. * Corresponding author. Tel.: +55-16-602-4049; fax: + 55-16-6330999. E-mail address: [email protected] (L.G.S. Branco)
The bullfrog, Rana catesbeiana, family Ranidae, is an aquatic amphibian native to the temperate Northern USA, which has been found overwintering under 5 cm of leaf litter (cf. [38]) or underwater ([7], cf. [38,45]). While burrowed, amphibians may be exposed to hypoxic and hypercapnic conditions [5]. Seasonal variations in the hypoxia- (but not hypercarbia) induced changes in the cardiorespiratory responses of the bullfrog have been studied recently [40]. Thus, the present study was designed to determine if during the overwintering period the known metabolic rate reduction (cf. [38]) of amphibians is accompanied by alterations of the respiratory and cardiovascular responses to hypercarbia. In order to determine such seasonal variations, we measured the effects of hypercarbia on breathing frequency, hematocrit, heart rate
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and blood pressure, at 10, 20 and 30°C, and on blood gases and acid–base status at 20 and 30°C, in R. catesbeiana.
Oxygen consumption was measured under normocarbia conditions using a Krogh respirometer [29] and the values were corrected to STPD.
2.4. Analysis of blood gases and hematocrit 2. Materials and methods
2.1. Animals Four groups (one for each season) of adult bullfrogs (R. catesbeiana) of either sex, weighing 133 – 226 g in summer (N =24), 129 – 233 g in autumn (N= 27), 142– 200 g in winter (N = 30) and 146 – 226 g in spring (N = 31) were obtained from a local frog farm. At the farm, they were held at ambient temperature with free access to running tap water and were fed mealworms throughout the year. Upon arrival at the laboratory, the animals were kept indoors in aquaria with free access to tap water and basking areas. All animals were fed cow liver once a week until at least 5 days before surgery. Experiments were performed throughout the year avoiding transition weeks between seasons (summer experiments were conducted during February and March, autumn experiments during May and June, winter experiments during July, August and September, and spring experiments during October and November). The mean ambient temperatures throughout the year were (mean9 S.E.M.) 24.8 9 0.2°C for summer, 21.5 9 0.3°C for autumn, 22.49 0.3°C for winter and 249 0.3°C for spring.
2.2. Surgery The bullfrogs were anesthetized in an aqueous solution of ethyl-m-amino benzoate (MS-222; 0.3%) (Sigma). Arterial cannulae (PE-50) filled with heparinized Ringer’s solution were occlusively inserted into the femoral artery. A second catheter (PE-100), inserted into the frog’s buccal cavity via a tight-fitting hole made in the tympanic membrane, was used to measure breathing frequency. All animals recovered promptly from anesthesia. After surgery, the animals were left undisturbed for at least 24 h at 23 – 25°C, in containers with free access to tap water and basking areas.
2.3. Measurements of breathing frequency, blood pressure, heart rate and O2 consumption Breathing frequency was recorded using a differential air-pressure transducer (model MP45-14-871; Validyne, Northridge, CA, USA) connected to the buccal catheter. Arterial blood pressure was measured by connecting the arterial catheter to a Narco pressure transducer (model P-1000B; Austin, TX, USA). Signals from transducers were recorded on paper (Narcotrace 80).
Arterial blood samples were analyzed for oxygen pressure (PO2) (model 204A; FAC Instruments, Sa˜o Carlos, Brazil) and pH (model 654; Metrohm, Switzerland) immediately after withdrawal. The O2 electrode (FAC Instruments) was calibrated with pure N2 and atmospheric air. The pH electrode (Metrohm) was adjusted using Radiometer (Copenhagen, Denmark) precision buffer solutions (S1510 and S1500). Electrodes were kept at the temperature of the experimental animal (20 or 30°C) using a constant-temperature circulator (model 1160A; VWR Scientific, Niles, IL, USA). Technical limitations of our PO2 electrode prevented us from measuring PaO2 at 10°C. Blood PCO2 was estimated by the Astrup technique [2] at ambient temperature (25°C) to assess the possible seasonal variations of acid–base status of the animals. Hematocrit was determined in 10 ml blood samples using a microhematocrit centrifuge (model 211; FANEN, Sa˜o Paulo, Brazil).
2.5. Experimental procedure Experiments were performed on conscious, unrestrained and undisturbed frogs. During the experiments, the frogs were housed in an acrylic chamber kept at the experimental temperature of 10, 20 or 30°C using a constant-temperature circulator (model 1160A; VWR Scientific, Niles). The animals were acclimated to each temperature for approximately 10 h (34 h after surgery). Cloacal temperature probes confirmed that there was no difference between animal temperature and environmental chamber temperature. The animal chamber was continuously flushed with humidified room air at a rate of 1.5 l min − 1. Once conditions were stable in the normocarbic gas, buccal and arterial blood pressures were recorded for approximately 15 min, and arterial blood was sampled for analysis of blood gases, pH and hematocrit. Hypercapnic gas mixtures (3 or 5% inspired CO2; AGA, Serta˜ozinho, SP, Brazil) were then applied in random order for 30 min each. Buccal and arterial blood pressures were recorded, and 1 ml arterial blood samples were withdrawn at the end of each experimental condition. Approximately 80% of this volume was reinfused into the animal’s circulation after blood gas measurements.
2.6. Calculations and statistic analysis Breathing frequency was obtained by counting the number of large amplitude buccal movements (lung breaths), distinguished from small amplitude buccal
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3. Results
Fig. 1. Seasonal comparisons of the relationship between rates of oxygen consumption and body temperature under normocarbic conditions. Data are reported as means 9S.E.M. (n =5–6 during summer and autumn and n = 4–5 during winter and spring). †† and †††, Significant effects (PB 0.01 and PB 0.001, respectively) of temperature within a season (relative to values at 10°C).
oscillations [27]. Mean arterial blood pressure was estimated from the pressure pulse using the following formula: mean pressure= diastolic pressure plus 1/ 3(systolic pressure minus diastolic pressure) [31]. All values are reported as means9S.E.M. The effects of season, temperature and hypercarbia on breathing frequency, blood pressure, heart rate, PaO2, pHa and hematocrit were analyzed by three-way multivariate analysis of variance (MANOVA) on log-transformed data (factors: season, temperature and percentage inspired CO2) and by Duncan’s multiple range test. For the oxygen consumption experiments, two-way MANOVA and Duncan’s multiple range test were performed (factors: season and temperature), and PaCO2 were analyzed by one-way ANOVA and the Tukey test. Values of PB0.05 were considered significant.
The effects of temperature and season on oxygen consumption are shown in Fig. 1. At 20 and 30°C, the rate of oxygen consumption tended to be higher during summer and lower during winter, but differences were not significant. Regardless of season, breathing frequency was reduced at lower temperatures under normocarbic conditions (PB0.01; Table 1). Fig. 2 shows the effects of hypercarbia on breathing frequency at different temperatures during the four seasons. A seasonal difference in the effect of hypercarbia was observed, i.e. during winter, breathing frequency at 30°C was lower than during summer (with 3 and 5% inspired CO2; P B 0.001) and spring (5% inspired CO2; PB 0.001). Moreover, in contrast to summer and spring, the ventilatory responses to hypercarbia during winter were similar at 30 and 20°C. Table 2 shows the effects of hypercarbia on blood gases of frogs equilibrated at different temperatures during the four seasons. PaO2 and pHa were similar during summer, autumn, winter and spring. However, PaCO2 values were significantly higher (PB 0.01) in winter than in spring and summer. The effect of hypercarbia on PaO2 and pHa did not change among seasons. The hematocrit values showed no significant changes induced by temperature, hypercarbia or season (Table 3). Fig. 3 shows heart rate of the frogs at different temperatures and under 0, 3 and 5% of inspired CO2 during the four seasons. Under normocarbia, heart rate decreased significantly (PB0.001; Table 1) with reduced temperature. The decrease was similar in all seasons.
Table 1 Breathing frequency, heart rate and mean blood pressure of R. catesbeiana under normocarbia during all four seasonsa Temperature (°C)
Summer
Autumn
Winter
Spring
Breathing frequency (breaths min−1)
10 20 30
0.56 9 0.13 8.05 91.46†† 14.62 91.70††
0.59 90.14 5.88 9 0.90†† 12.36 92.69††
1.85 90.91 5.00 9 1.24 8.78 9 1.13†
Heart rate (beats min−1)
10 20 30
11.81 9 0.60 25.29 91.46†† 48.60 93.32††
10.94 90.43 27.29 91.30†† 50.17 94.60††
12.70 9 1.30 28.63 9 2.24†† 53.83 91.08††
13.35 90.88 23.44 9 0.95†† 50.25 92.60††
Mean blood pressure (mmHg)
10 20 30
22.18 90.58‡‡‡ 25.45 92.00 30.10 91.30†
21.45 91.30‡‡‡ 25.06 90.90 24.32 91.80‡‡
29.62 91.45 30.38 9 2.82 32.52 93.13
18.55 9 0.74‡‡‡ 21.3 9 0.79‡‡ 26.46 91.01†
0.68 90.23 3.06 90.64††,‡ 7.589 1.09††
Values are means 9 S.E.M. (n= 5–10 during summer, n =5–9 during autumn, n = 5–9 during winter and n = 5–10 during spring). Significant (PB0.01) difference from the value at 10°C. †† Significant (PB0.001) difference from the value at 10°C. ‡ Significant (PB0.05) difference from the value during summer, at the same temperature. ‡‡ Significant (PB0.05) difference from the values during winter, at the same temperature. ‡‡‡ Significant (PB0.001) difference from the values during winter, at the same temperature. a †
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blood pressure during any season or at any experimental temperature (Fig. 4). 4. Discussion The present study provides data about the effect of temperature on cardiorespiratory responses to hypercarbia not only during the active period of R. catesbeiana, but also during winter, when adult frogs in the wild become quieter, but are not torpid. In the field, overwintering bullfrogs have been found under leaf litter in Michigan (USA) in March [38] and submerged on the bottoms of ponds in Ohio (USA) from December to February [45]. Our experiments were performed under artificial laboratory conditions, which differs considerably from the field. We cannot assure that our frogs were indeed hibernating/estivating, but both oxygen consumption measurements (Fig. 1) and behavioral observations (Rocha and Branco, unpublished) indicate that they were quieter and tended to burrow if provided with soil, during winter.
4.1. Metabolic rate
Fig. 2. Seasonal comparisons of the effect of hypercarbia on breathing frequency at different temperatures. Data are reported as means9S.E.M. (n=5 – 10 during summer, n= 5–9 during autumn and winter, and n =5 – 7 during spring). †† and †††, Significant (PB 0.01 and P B0.001, respectively) effect of temperature within a season (relative to values at 10°C). *, ** and ***, Significant (PB 0.05, P B 0.01 and P B0.001, respectively) effect of hypercarbia relative to normocarbic control value at the same temperature.
Reduced body temperature was accompanied by decreased (PB 0.01) mean blood pressure during summer and spring (Table 1). Season also altered blood pressure. At 10°C, blood pressure was significantly higher (PB 0.001) during winter than during the other seasons and, at 20 and 30°C, it was higher (P B0.05) during winter than during spring and autumn, respectively (Table 1). Hypercarbia caused no significant changes in
During winter, the rate of oxygen consumption by the bullfrogs was approximately 21 and 17% lower than during summer at 20 and 30°C, respectively (Fig. 1). Although we did not observe significant differences, these data are consistent with a recent study on the same species [40]. Previous studies have reported that the resting rate of oxygen consumption is reduced during estivation in Scaphiopus hammondii, Schaphiopus couchii [41], Pyxicephalus adspersus [32], Lepdobranchus llaninsis [35], Physalaemus fuscomaculatus, Leptodactylus fuscus [1] and Neobatrachus kunapalari [16], and Bufo paracnemis [19]. Thus, metabolic depression during adverse periods seems to be a widespread strategy in amphibians from various families. This hypometabolic state may extend survival, while animals rely on stored fuel supplies during unfavorable periods [22]. In the present study, O2 consumption was determined under normocarbia only, to confirm that our frogs would follow the known tendency of hypometabolism previously described for the species [40]. Further studies are needed to assess the seasonal effects of CO2 on metabolic rate.
4.2. Pulmonary 6entilation During summer, breathing frequency was directly related to temperature, and hypercarbic-induced tachypnoea was greater at higher temperatures. In most amphibians [10,30] and reptiles [8,15,17,37], pulmonary ventilation increases with rising body temperature. As to the effect of hypercarbia, Branco et al. [10] reported
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Table 2 Effect of hypercarbia and body temperature on PaO2, pHa, and PaCO2 values of R. catesbeiana during all four seasonsa
PaO2 (mmHg)
Temperature (°C)
Inspired CO2 (%)
Summer
Autumn
Winter
Spring
20
0 3 5 0 3 5
54.30 93.16 61.80 92.75 78.22 94.30** 77.40 93.53† 80.50 9 4.04 82.02 9 5.50
51.77 9 6.30 58.80 9 6.21 78.57 9 2.03* 71.57 96.70† 76.93 9 1.60 88.90 9 4.50
51.14 9 4.90 58.82 9 6.12 77.86 9 6.54* 76.32 9 4.85† 80.90 9 5.74 89.60 9 4.72
52.98 9 4.14 62.76 9 4.30 75.70 9 4.50** 75.86 94.08† 79.14 9 3.30 83.88 9 4.38
30
pHa
20
30
PaCO2 (mmHg)
7.85 9 0.03 7.69 90.02 7.40 9 0.05** 7.84 90.05 7.51 90.05** 7.45 90.05***
0 3 5 0 3 5
18.50 91.80
0
25
7.87 9 0.05 7.64 9 0.03* 7.43 90.06** 7.79 9 0.02 7.56 9 0.05** 7.44 90.04** 26.20 9 4.60
7.86 90.04 7.62 90.04** 7.41 90.03*** 7.81 90.03 7.58 90.04** 7.43 9 0.04*** 34.80 9 2.40‡‡
7.84 9 0.02 7.68 9 0.04* 7.41 9 0.05*** 7.80 9 0.06 7.55 9 0.05* 7.48 9 0.05** 18.80 9 0.90
Values are means 9 S.E.M. for five animals in each group (except during autumn, when n =3); * Significant (PB0.05) effect of hypercarbia relative to normocarbic control values, at the same temperature. ** Significant (PB 0.01) effect of hypercarbia relative to normocarbic control values, at the same temperature. *** Significant (PB0.001) effect of hypercarbia relative to normocarbic control values, at the same temperature. † Significant (PB0.001) difference from the value at 20°C (normocarbic conditions). ‡‡ Significant (PB0.01) difference from the value during summer. a
that hypercarbia-induced hyperventilation in the toad B. paracnemis is augmented in response to increased body temperature. Similar data were obtained for the alligator Alligator mississipiensis [8,14] and the turtle Chrysemys picta [18]. The present study provides the first evidence that the effect of hypercarbia on breathing frequency depends on the season (P B 0.001). During winter, hypercarbiainduced tachypnoea was similar at 20 and 30°C. Moreover, during this season, at 30°C, hypercarbia-induced tachypnoea was lower than during summer and spring (Fig. 2). This indicates that there may be a reduction in the sensitivity for the hypercarbic ventilatory response during winter. No significant increase in breathing frequency was observed during autumn when the animals inspired 5%
of CO2 at 20°C, perhaps because these responses varied considerably between individuals (Fig. 2). In this study, frogs were exposed to hypercarbia for 30 min at all experimental temperatures. Perhaps this period of time was not enough for a full cardiovascular and respiratory response at 10°C. However, this seems unlikely because pilot studies revealed that a 30min period is sufficient for steady-state measurements. Additionally, we assessed breathing frequency by measurements of buccal pressure, which is a widely used [27,43,52] and extremely useful method for studies on ventilatory control. However, it does not provide a measure of the actual volumes of ventilated air and, therefore, does not allow calculation of parameters such as air convection requirement (V/VO2) (cf. [50]).
Table 3 Values of hematocrit (%) of R. catesbeiana during all four seasonsa Temperature (°C)
Inspired CO2 (%)
Summer
Autumn
Winter
Spring
10
0 3 5
16.71 91.05 18.00 91.00 19.29 91.00
15.67 9 1.20 16.44 9 1.50 15.22 9 1.00
16.47 9 1.66 18.00 91.58 15.53 9 1.89
17.88 9 0.70 17.94 90.87 18.00 9 0.73
20
0 3 5
16.79 9 1.04 17.07 9 0.99 19.479 1.71
13.78 9 1.00 14.39 91.10 14.94 9 1.00
16.95 9 1.39 20.40 91.39 19.00 91.60
17.44 90.75 17.78 9 0.97 18.74 90.98
30
0 3 5
15.70 9 1.62 15.60 91.20 16.66 92.04
15.07 9 1.60 15.29 9 2.20 17.21 9 2.00
17.69 9 0.96 18.56 91.02 19.25 9 1.45
17.22 9 0.85 16.94 90.78 16.37 9 0.69
a
Values are means 9 S.E.M. (n= 5–7 during summer, n= 7–9 during autumn, n = 8–10 during winter and n = 8–9 during spring).
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reduced and breathing frequency unaltered during winter. Thus, the mechanism responsible for the twofold elevation of PaCO2 may involve a reduction in cutaneous CO2 elimination. Actually, it is well known that amphibians excrete excess CO2 passively across the skin [23,28] and R. catesbeiana, specifically, liberates approximately 80% of the total CO2 produced across cutaneous surface [12]. Moreover, Burggren and Moalli [11] suggest that a partial inhibition of CO2 excretion is linked with a decrease in the extent and pattern of blood flow through the skin. These blood flow changes may be related to the increased mean blood pressure during winter (Table 1).
Fig. 3. Seasonal comparisons of the effect of temperature on heart rate. Data are reported as means 9 S.E.M. (n = 5–8 during summer and winter, n=6 – 8 during autumn and n = 8–10 during spring). †††, Significant (P B 0.001) effect of temperature under normocarbia conditions within a season (relative to values at 10°C).
4.3. Blood gas le6els and acid– base status PaO2, pHa and PaCO2 values obtained here during summer (Table 2) were similar to those reported earlier for R. catesbeiana [27], and the temperature- and hypercarbia-induced alterations in PaO2 and pHa during summer agreed with previous studies on anuran amphibians [3,6,10,30,40,47,53]. It is interesting to note that no seasonal variation in PaO2 or pHa occurred, but winter PaCO2 values were twice as high as summer and spring values. This increased PaCO2 is unexpected since O2 consumption was
Fig. 4. Seasonal comparisons of the effect of temperature on blood pressure. Data are reported as means 9 S.E.M. (n =5 – 8 during summer, n =6 – 8 during autumn, n =7 – 8 during winter and n=7–9 during spring). ††, Significant (P B0.01) effect of temperature under normocarbia conditions within a season (relative to values at 10°C).
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According to the Henderson – Hasselbalch equation, increased PaCO2 should cause a drop in pH unless a compensatory increase in bicarbonate concentrations occurs. This suggests an increase in plasma bicarbonate levels during winter, which could not be calculated in the present study because measurements were performed at different temperatures. Perhaps the increased PaCO2 is caused by a net bicarbonate reabsorption through the urinary bladder wall and/or mobilization of internal calcareous deposits [46]. Boutilier et al. [5] observed a similar increase of PaCO2 in B. marinus. Under laboratory conditions, B. marinus burrows, hypoventilates and reduces its cutaneous respiration, with a resulting twofold increase in arterial blood PCO2 despite a reduction in metabolism. This rise in PaCO2 leads to the development of marked respiratory acidosis, which is completely compensated for a rise in plasma bicarbonate levels in the animals after 3 days of burrowing [5]. As pointed out by Rocha and Branco [40], this suggests that short-term [5] and long-term (present study) acid– base compensation might be similar among anuran amphibians. During active periods, such high PaCO2 values would induce hyperventilation [3,4,9,10,38] but, during winter, ventilation under normocarbia is unaltered in R. catesbeiana (Table 1). This indicates reduced sensitivity of chemoreceptors, alteration in the central respiratory control system response and/or reduced effector response during this season.
4.4. Hematocrit The hematocrit values showed no significant changes induced by temperature, hypercarbia or season (Table 3), indicating that bullfrogs kept a constant oxygen-carrying capacity of blood during the year. Similarly, no change in hemoglobin concentration or hematocrit occurs in naturally estivating S. couchii and S. hammondii [42] or in Xenopus lae6is estivating in the laboratory (cf. [38]). In contrast, in Rana pipiens, hematocrit is higher during winter [26] than during summer [25]. These discrepancies may be due to interspecific differences among amphibians.
4.5. Cardio6ascular system In agreement with previous studies [20,21,36], falling temperatures were accompanied by decreasing heart rate (Fig. 3), which may be due to a direct effect on pacemaker cells [13]. As to seasonal variations of cardiac function in anuran amphibians, Jones [24] evaluated driving bradycardia of Bufo bufo and Rana temporaria during prehibernation, breeding season and summer. The author found that, although statistically significant, the seasonal variations in the response of heart rate to submer-
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gence observed in R. temporaria and B. bufo are not so marked as to make a clear seasonal characterization of animals possible. In agreement, in the present study, no seasonal variations were observed in relation to cardiovascular responses to hypercarbia (Figs. 3 and 4). The relationship between heart rate and body temperature in the bullfrog was not influenced by season (Table 1), as previously observed [40]. In contrast, the frogs Rana pipiens [36] and R. temporaria [20] and the toads, Bufo fowleri [44] and Bufo paracnemis [19] showed seasonal variation of the effect of temperature on heart rate during summer and winter. These contrasting responses may be indicating interspecific differences among amphibians. We observed seasonal variation in the effect of temperature on blood pressure (Table 1). Our results are in agreement with those of Weathers [51] and Rocha and Branco [40], who observed that blood pressures in the bullfrog were slightly higher in winter than in summer. Higher arterial blood pressure, without any change in heart rate, indicates an increase in peripheral resistance and/or in the stroke volume in the frogs during winter.
4.6. Conclusion We have shown that R. catesbeiana has an endogenous annual rhythm of cardiorespiratory function. The present study suggests that there is a relationship between oxygen-consumption variability and cardiorespiratory system function in bullfrogs, which is temperature independent. The mechanisms involved in such seasonal rhythm are not known, and will require further research. Perhaps, plasma concentration of some peptides such as arginine-vasotocin (AVT), a non-mammalian arginine vasopressin (AVP) analog, is elevated during winter. This would be consistent with the notion that, during seasonal periods of drought (winter in south eastern Brazil), amphibians would have an increased plasma osmolarity which is known to cause behavioral hypothermia [34], and increased blood pressure [33]. Moreover, ventilation is stimulated after AVP V1 receptor blockage in normocapnia [48] and hypercapnia [49] in dogs. Finally, winter frogs have an increased PaCO2 (Table 2) and AVP is known to be released during hypercapnia in rats (cf. [54]) and dogs [39]. Although the exact mechanisms remain to be determined, R. catesbeiana is an interesting model to access this question.
Acknowledgements This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). K.C.B.-N. was the recipient of a
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FAPESP postgraduate fellowship. We thank Mauro F Silva for technical assistance. References [1] Abe AS, Garcia LS. Response to temperature in the oxygen uptake of awake and dormant frogs (Amphibia, Leptodactylidae). Studies Neotrop Fauna Environ 1991;26:135–41. [2] Astrup P. A simple electrometric technique for the determination of carbon dioxide tension in blood and plasma, total content of carbon dioxide in plasma and bicarbonate content in ‘separate’ plasma at a fixed carbon dioxide tension (40 mmHg). Scand J Clin Invest 1956;8:33–43. [3] Boutilier RG, Heisler N. Acid–base regulation and blood gases in the anuran amphibian, Bufo marinus, during environmental hypercapnia. J Exp Biol 1988;134:79–98. [4] Boutilier RG, Randall DJ, Shelton G, Toews DP. Acid – base relationships in the blood of the toad, Bufo marinus. I. The effects of environmental CO2. J Exp Biol 1979;82:331–44. [5] Boutilier RG, Randall DJ, Shelton G, Toews DP. Acid – base relationships in the blood of the toad, Bufo marinus. III. The effects of burrowing. J Exp Biol 1979;82:357–65. [6] Boutilier RG, Glass ML, Heisler N. Blood gases and extracellular/intracellular acid –base status as a function of temperature in the anuran amphibians Xenopus lae6is and Bufo marinus. J Exp Biol 1987;130:13 – 25. [7] Boutilier RG, Donohoe PH, Tattersal GJ, West TG. Hypometabolic homeostasis in overwintering aquatic amphibians. J Exp Biol 1997;200:387–400. [8] Branco LGS, Wood SC. Effects of temperature on central chemical control of ventilation in the alligator Alligator mississippiensis. J Exp Biol 1993;179:261–72. [9] Branco LGS, Glass ML, Hoffmann A. Central chemoreceptor drive to breathing in unanesthetized toads, Bufo paracnemis. Respir Physiol 1992;87:195–204. [10] Branco LGS, Glass ML, Wang T, Hoffmann A. Temperature and central chemoreceptor drive to ventilation in toad (Bufo paracnemis). Respir Physiol 1993;93:337–46. [11] Burggren WW, Moalli R. ’Active’ regulation of cutaneous gas exchange by capillary recritment in amphibians: experimental evidence and a revised model for skin respiration. Respir Physiol 1984;55(3):379– 92. [12] Burggren WW, West NH. Changing respiratory importance of gills, lungs and skin during metamorphosis in the bullfrog Rana catesbeiana. Respir Physiol 1982;47(2):151–64. [13] Clark AJ. The effect of alterations of temperature upon the functions of isolated heart. J Physiol Lond 1920;54:275– 86. [14] Davies DG, Thomas JL, Smith EN. Effect of body temperature on ventilatory control in the alligator. J Appl Physiol 1982;52(1):114– 8. [15] De Vera Porcell L, Gonzalez Gonzalez J. Effect of body temperature on the ventilatory responses in the lizard Gallotia galloti. Respir Physiol 1986;65(1):29–37. [16] Flannigan JE, Guppy M. Metabolic depression and sodium – potassium ATPase in the aestivating frog, Neobatrachus kunapalari. J Comp Physiol B 1997;167:135–45. [17] Frappell PB, Daniels CB. Temperature effects on ventilation and metabolism in the lizard, Ctenophorus nuchalis. Respir Physiol 1991;86(2):257– 70. [18] Funk GD, Milsom WK. Changes in ventilation and breathing pattern produced by changing body temperature and inspired CO2 concentration in turtles. Respir Physiol 1987;67(1):37 – 51. [19] Glass ML, Fernandes MS, Soncini R, Glass H, Wasser JS. Effects of dry season dormancy on oxygen uptake, heart rate, and blood pressures in the toad, Bufo paracnemis. J Exp Zool 1997;279(4):330– 6.
[20] Harri MNE, Talo A. Effect of season and temperature acclimation on the heart rate – temperature relationship in the frog, Rana temporaria. Comp Biochem Physiol 1975;50A:469 – 72. [21] Herman CA, Robleto DO, Mata PL, Heller RS. Cardiovascular responses to catecholamines at 12 degrees C in the american bullfrog (Rana catesbeiana). J Exp Zool 1986;240(1):17–23. [22] Hochachka PW, Dunn JF. Metabolic arrest: the most effective means of protecting tissues against hypoxia. Progr Clin Biol Res 1983;136:297 – 309. [23] Hutchison VH, Whitford WG, Kohl M. Relation of body size and surface area to gas exchange in anurans. Physiol Zool 1968;41:65 – 85. [24] Jones DR. Specific and seasonal variations in development of diving bradycardia in anuran amphibia. Comp Biochem Physiol 1968;25:821 – 34. [25] Jungreis AM. The effects of long-term starvation and acclimation temperature on glucose regulation and nitrogen anabolism in the frog, Rana pipiens – II. Summer animals. Comp Biochem Physiol 1970;32:433 – 44. [26] Jungreis AM, Hooper AB. The effects of long-term starvation and acclimation temperature on glucose regulation and nitrogen anabolism in the frog, Rana pipiens — I. Winter animals. Comp Biochem Physiol 1970;32:417 – 32. [27] Kinkead R, Milsom WK. Chemoreceptors and control of episodic breathing in the bullfrog (Rana catesbeiana). Respir Physiol 1994;95:81 – 98. [28] Krogh A. Some experiments on the cutaneous respiration of vertebrate animals. Scand Arch Physiol 1904:348 – 357. [29] Krogh A. The quantitative relation between temperature and standard metabolism in animals. Int Z Phys Chem Biol 1914;1:491 – 508. [30] Kruhøffer M, Glass ML, Abe AS, Johansen K. Control of breathing in an amphibian Bufo paracnemis: effects of temperature and hypoxia. Respir Physiol 1987;69:267 – 75. [31] Lillo RS. Heart rate and blood pressure in bullfrogs during prolonged maintenance in water at low temperature. Comp Biochem Physiol 1979;65A:251 – 3. [32] Loveridge JP, Withers PC. Metabolism and water balance of active and cocooned african bullfrog Pyxicephalus adspersus. Physiol Zool 1981;54:203 – 14. [33] Malvin GM. Vascular effects of arginine vasotocin in toad skin. Am J Physiol 1993;265:R426– 32. [34] Malvin GM, Wood SC. Behavioral thermoregulation in the toad Bufo marinus: effects of air humidity. J Exp Zool 1991;258:322– 6. [35] McClanahan LL, Ruibal R, Shoemaker VH. Rate of cocoon formation and its physiological correlates in a ceratophryd frog. Physiol Zool 1983;56:430 – 5. [36] Miller LC, Mizell S. Seasonal variation in heart rate response to core temperature changes. Comp Biochem Physiol 1972;42A: 773 – 9. [37] Munns SL, Frappell PB, Evans BK. The effects of environmental temperature, hypoxia, and hypercapnia on the breathing pattern of saltwater crocodiles (Crocodylus porosus). Physiol Zool 1998;71(3):267– 73. [38] Pinder AW, Storey KB, Ultsch GR. Estivation and hibernation. In: Feder ME, Burgreen WW, editors. Environmental Physiology of the Amphibians. Chicago, IL: University of Chicago Press, 1992:250 – 74. [39] Raff H, Shinsako J, Keil LC, Dallman MF. Vasopressin, ACTH, and corticosteroids during hypercapnia and graded hypoxia in dogs. Am J Physiol 1983;244:E453– 8. [40] Rocha PL, Branco LGS. Seasonal changes of cardiovascular, respiratory and metabolic responses to temperature and hypoxia in the bullfrog Rana catesbeiana. J Exp Biol 1998;201:761–8. [41] Seymour RS. Energy metabolism of dormant spadefoot toads (Scaphiopus). Copeia 1973:435 – 45.
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Seasonal changes in the cardiorespiratory responses to hypercarbia and temperature in the bullfrog, Rana catesbeiana Keˆnia C. Bı´cego-Nahas, Luiz G.S. Branco * Departamento of Fisiologia, Faculdade de Medicina de Ribeira˜o Preto e Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeira˜o Preto, Uni6ersidade de Sa˜o Paulo, 14040 -904. Ribeira˜o Preto, Sa˜o Paulo, Brazil Received 17 March 1999; received in revised form 20 July 1999; accepted 19 August 1999
Abstract We assessed the seasonal variations in the effects of hypercarbia (3 or 5% inspired CO2) on cardiorespiratory responses in the bullfrog Rana catesbeiana at different temperatures (10, 20 and 30°C). We measured breathing frequency, blood gases, acid–base status, hematocrit, heart rate, blood pressure and oxygen consumption. At 20 and 30°C, the rate of oxygen consumption had a tendency to be lowest during winter and highest during summer. Hypercarbia-induced changes in breathing frequency were proportional to body temperature during summer and spring, but not during winter (20 and 30°C). Moreover, during winter, the effects of CO2 on breathing frequency at 30°C were smaller than during summer and spring. These facts indicate a decreased ventilatory sensitivity during winter. PaO2 and pHa showed no significant change during the year, but PaCO2 was almost twice as high during winter than in summer and spring, indicating increased plasma bicarbonate levels. The hematocrit values showed no significant changes induced by temperature, hypercarbia or season, indicating that the oxygen carrying capacity of blood is kept constant throughout the year. Decreased body temperature was accompanied by a reduction in heart rate during all four seasons, and a reduction in blood pressure during summer and spring. Blood pressure was higher during winter than during any other seasons whereas no seasonal change was observed in heart rate. This may indicate that peripheral resistance and/or stroke volume may be elevated during this season. Taken together, these results suggest that the decreased ventilatory sensitivity to hypercarbia during winter occurs while cardiovascular parameters are kept constant. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Breathing frequency; Arterial pH; Blood gases; Hematocrit; Blood pressure; Heart rate; Bullfrog; Hypercarbia; Seasonal variations; Temperature
1. Introduction Amphibians have a remarkable tolerance to diverse environmental conditions and they range widely over the globe. Much of this extensive environmental range is due to the ability of amphibians to withstand long periods of unfavorable conditions (starvation, cold and/ or drought) and to condense their life cycle into brief active periods when conditions are less stringent [38]. However, nearly all studies of cardiorespiratory physiology of amphibians have been performed only on active animals. * Corresponding author. Tel.: +55-16-602-4049; fax: + 55-16-6330999. E-mail address: [email protected] (L.G.S. Branco)
The bullfrog, Rana catesbeiana, family Ranidae, is an aquatic amphibian native to the temperate Northern USA, which has been found overwintering under 5 cm of leaf litter (cf. [38]) or underwater ([7], cf. [38,45]). While burrowed, amphibians may be exposed to hypoxic and hypercapnic conditions [5]. Seasonal variations in the hypoxia- (but not hypercarbia) induced changes in the cardiorespiratory responses of the bullfrog have been studied recently [40]. Thus, the present study was designed to determine if during the overwintering period the known metabolic rate reduction (cf. [38]) of amphibians is accompanied by alterations of the respiratory and cardiovascular responses to hypercarbia. In order to determine such seasonal variations, we measured the effects of hypercarbia on breathing frequency, hematocrit, heart rate
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and blood pressure, at 10, 20 and 30°C, and on blood gases and acid–base status at 20 and 30°C, in R. catesbeiana.
Oxygen consumption was measured under normocarbia conditions using a Krogh respirometer [29] and the values were corrected to STPD.
2.4. Analysis of blood gases and hematocrit 2. Materials and methods
2.1. Animals Four groups (one for each season) of adult bullfrogs (R. catesbeiana) of either sex, weighing 133 – 226 g in summer (N =24), 129 – 233 g in autumn (N= 27), 142– 200 g in winter (N = 30) and 146 – 226 g in spring (N = 31) were obtained from a local frog farm. At the farm, they were held at ambient temperature with free access to running tap water and were fed mealworms throughout the year. Upon arrival at the laboratory, the animals were kept indoors in aquaria with free access to tap water and basking areas. All animals were fed cow liver once a week until at least 5 days before surgery. Experiments were performed throughout the year avoiding transition weeks between seasons (summer experiments were conducted during February and March, autumn experiments during May and June, winter experiments during July, August and September, and spring experiments during October and November). The mean ambient temperatures throughout the year were (mean9 S.E.M.) 24.8 9 0.2°C for summer, 21.5 9 0.3°C for autumn, 22.49 0.3°C for winter and 249 0.3°C for spring.
2.2. Surgery The bullfrogs were anesthetized in an aqueous solution of ethyl-m-amino benzoate (MS-222; 0.3%) (Sigma). Arterial cannulae (PE-50) filled with heparinized Ringer’s solution were occlusively inserted into the femoral artery. A second catheter (PE-100), inserted into the frog’s buccal cavity via a tight-fitting hole made in the tympanic membrane, was used to measure breathing frequency. All animals recovered promptly from anesthesia. After surgery, the animals were left undisturbed for at least 24 h at 23 – 25°C, in containers with free access to tap water and basking areas.
2.3. Measurements of breathing frequency, blood pressure, heart rate and O2 consumption Breathing frequency was recorded using a differential air-pressure transducer (model MP45-14-871; Validyne, Northridge, CA, USA) connected to the buccal catheter. Arterial blood pressure was measured by connecting the arterial catheter to a Narco pressure transducer (model P-1000B; Austin, TX, USA). Signals from transducers were recorded on paper (Narcotrace 80).
Arterial blood samples were analyzed for oxygen pressure (PO2) (model 204A; FAC Instruments, Sa˜o Carlos, Brazil) and pH (model 654; Metrohm, Switzerland) immediately after withdrawal. The O2 electrode (FAC Instruments) was calibrated with pure N2 and atmospheric air. The pH electrode (Metrohm) was adjusted using Radiometer (Copenhagen, Denmark) precision buffer solutions (S1510 and S1500). Electrodes were kept at the temperature of the experimental animal (20 or 30°C) using a constant-temperature circulator (model 1160A; VWR Scientific, Niles, IL, USA). Technical limitations of our PO2 electrode prevented us from measuring PaO2 at 10°C. Blood PCO2 was estimated by the Astrup technique [2] at ambient temperature (25°C) to assess the possible seasonal variations of acid–base status of the animals. Hematocrit was determined in 10 ml blood samples using a microhematocrit centrifuge (model 211; FANEN, Sa˜o Paulo, Brazil).
2.5. Experimental procedure Experiments were performed on conscious, unrestrained and undisturbed frogs. During the experiments, the frogs were housed in an acrylic chamber kept at the experimental temperature of 10, 20 or 30°C using a constant-temperature circulator (model 1160A; VWR Scientific, Niles). The animals were acclimated to each temperature for approximately 10 h (34 h after surgery). Cloacal temperature probes confirmed that there was no difference between animal temperature and environmental chamber temperature. The animal chamber was continuously flushed with humidified room air at a rate of 1.5 l min − 1. Once conditions were stable in the normocarbic gas, buccal and arterial blood pressures were recorded for approximately 15 min, and arterial blood was sampled for analysis of blood gases, pH and hematocrit. Hypercapnic gas mixtures (3 or 5% inspired CO2; AGA, Serta˜ozinho, SP, Brazil) were then applied in random order for 30 min each. Buccal and arterial blood pressures were recorded, and 1 ml arterial blood samples were withdrawn at the end of each experimental condition. Approximately 80% of this volume was reinfused into the animal’s circulation after blood gas measurements.
2.6. Calculations and statistic analysis Breathing frequency was obtained by counting the number of large amplitude buccal movements (lung breaths), distinguished from small amplitude buccal
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3. Results
Fig. 1. Seasonal comparisons of the relationship between rates of oxygen consumption and body temperature under normocarbic conditions. Data are reported as means 9S.E.M. (n =5–6 during summer and autumn and n = 4–5 during winter and spring). †† and †††, Significant effects (PB 0.01 and PB 0.001, respectively) of temperature within a season (relative to values at 10°C).
oscillations [27]. Mean arterial blood pressure was estimated from the pressure pulse using the following formula: mean pressure= diastolic pressure plus 1/ 3(systolic pressure minus diastolic pressure) [31]. All values are reported as means9S.E.M. The effects of season, temperature and hypercarbia on breathing frequency, blood pressure, heart rate, PaO2, pHa and hematocrit were analyzed by three-way multivariate analysis of variance (MANOVA) on log-transformed data (factors: season, temperature and percentage inspired CO2) and by Duncan’s multiple range test. For the oxygen consumption experiments, two-way MANOVA and Duncan’s multiple range test were performed (factors: season and temperature), and PaCO2 were analyzed by one-way ANOVA and the Tukey test. Values of PB0.05 were considered significant.
The effects of temperature and season on oxygen consumption are shown in Fig. 1. At 20 and 30°C, the rate of oxygen consumption tended to be higher during summer and lower during winter, but differences were not significant. Regardless of season, breathing frequency was reduced at lower temperatures under normocarbic conditions (PB0.01; Table 1). Fig. 2 shows the effects of hypercarbia on breathing frequency at different temperatures during the four seasons. A seasonal difference in the effect of hypercarbia was observed, i.e. during winter, breathing frequency at 30°C was lower than during summer (with 3 and 5% inspired CO2; P B 0.001) and spring (5% inspired CO2; PB 0.001). Moreover, in contrast to summer and spring, the ventilatory responses to hypercarbia during winter were similar at 30 and 20°C. Table 2 shows the effects of hypercarbia on blood gases of frogs equilibrated at different temperatures during the four seasons. PaO2 and pHa were similar during summer, autumn, winter and spring. However, PaCO2 values were significantly higher (PB 0.01) in winter than in spring and summer. The effect of hypercarbia on PaO2 and pHa did not change among seasons. The hematocrit values showed no significant changes induced by temperature, hypercarbia or season (Table 3). Fig. 3 shows heart rate of the frogs at different temperatures and under 0, 3 and 5% of inspired CO2 during the four seasons. Under normocarbia, heart rate decreased significantly (PB0.001; Table 1) with reduced temperature. The decrease was similar in all seasons.
Table 1 Breathing frequency, heart rate and mean blood pressure of R. catesbeiana under normocarbia during all four seasonsa Temperature (°C)
Summer
Autumn
Winter
Spring
Breathing frequency (breaths min−1)
10 20 30
0.56 9 0.13 8.05 91.46†† 14.62 91.70††
0.59 90.14 5.88 9 0.90†† 12.36 92.69††
1.85 90.91 5.00 9 1.24 8.78 9 1.13†
Heart rate (beats min−1)
10 20 30
11.81 9 0.60 25.29 91.46†† 48.60 93.32††
10.94 90.43 27.29 91.30†† 50.17 94.60††
12.70 9 1.30 28.63 9 2.24†† 53.83 91.08††
13.35 90.88 23.44 9 0.95†† 50.25 92.60††
Mean blood pressure (mmHg)
10 20 30
22.18 90.58‡‡‡ 25.45 92.00 30.10 91.30†
21.45 91.30‡‡‡ 25.06 90.90 24.32 91.80‡‡
29.62 91.45 30.38 9 2.82 32.52 93.13
18.55 9 0.74‡‡‡ 21.3 9 0.79‡‡ 26.46 91.01†
0.68 90.23 3.06 90.64††,‡ 7.589 1.09††
Values are means 9 S.E.M. (n= 5–10 during summer, n =5–9 during autumn, n = 5–9 during winter and n = 5–10 during spring). Significant (PB0.01) difference from the value at 10°C. †† Significant (PB0.001) difference from the value at 10°C. ‡ Significant (PB0.05) difference from the value during summer, at the same temperature. ‡‡ Significant (PB0.05) difference from the values during winter, at the same temperature. ‡‡‡ Significant (PB0.001) difference from the values during winter, at the same temperature. a †
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blood pressure during any season or at any experimental temperature (Fig. 4). 4. Discussion The present study provides data about the effect of temperature on cardiorespiratory responses to hypercarbia not only during the active period of R. catesbeiana, but also during winter, when adult frogs in the wild become quieter, but are not torpid. In the field, overwintering bullfrogs have been found under leaf litter in Michigan (USA) in March [38] and submerged on the bottoms of ponds in Ohio (USA) from December to February [45]. Our experiments were performed under artificial laboratory conditions, which differs considerably from the field. We cannot assure that our frogs were indeed hibernating/estivating, but both oxygen consumption measurements (Fig. 1) and behavioral observations (Rocha and Branco, unpublished) indicate that they were quieter and tended to burrow if provided with soil, during winter.
4.1. Metabolic rate
Fig. 2. Seasonal comparisons of the effect of hypercarbia on breathing frequency at different temperatures. Data are reported as means9S.E.M. (n=5 – 10 during summer, n= 5–9 during autumn and winter, and n =5 – 7 during spring). †† and †††, Significant (PB 0.01 and P B0.001, respectively) effect of temperature within a season (relative to values at 10°C). *, ** and ***, Significant (PB 0.05, P B 0.01 and P B0.001, respectively) effect of hypercarbia relative to normocarbic control value at the same temperature.
Reduced body temperature was accompanied by decreased (PB 0.01) mean blood pressure during summer and spring (Table 1). Season also altered blood pressure. At 10°C, blood pressure was significantly higher (PB 0.001) during winter than during the other seasons and, at 20 and 30°C, it was higher (P B0.05) during winter than during spring and autumn, respectively (Table 1). Hypercarbia caused no significant changes in
During winter, the rate of oxygen consumption by the bullfrogs was approximately 21 and 17% lower than during summer at 20 and 30°C, respectively (Fig. 1). Although we did not observe significant differences, these data are consistent with a recent study on the same species [40]. Previous studies have reported that the resting rate of oxygen consumption is reduced during estivation in Scaphiopus hammondii, Schaphiopus couchii [41], Pyxicephalus adspersus [32], Lepdobranchus llaninsis [35], Physalaemus fuscomaculatus, Leptodactylus fuscus [1] and Neobatrachus kunapalari [16], and Bufo paracnemis [19]. Thus, metabolic depression during adverse periods seems to be a widespread strategy in amphibians from various families. This hypometabolic state may extend survival, while animals rely on stored fuel supplies during unfavorable periods [22]. In the present study, O2 consumption was determined under normocarbia only, to confirm that our frogs would follow the known tendency of hypometabolism previously described for the species [40]. Further studies are needed to assess the seasonal effects of CO2 on metabolic rate.
4.2. Pulmonary 6entilation During summer, breathing frequency was directly related to temperature, and hypercarbic-induced tachypnoea was greater at higher temperatures. In most amphibians [10,30] and reptiles [8,15,17,37], pulmonary ventilation increases with rising body temperature. As to the effect of hypercarbia, Branco et al. [10] reported
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Table 2 Effect of hypercarbia and body temperature on PaO2, pHa, and PaCO2 values of R. catesbeiana during all four seasonsa
PaO2 (mmHg)
Temperature (°C)
Inspired CO2 (%)
Summer
Autumn
Winter
Spring
20
0 3 5 0 3 5
54.30 93.16 61.80 92.75 78.22 94.30** 77.40 93.53† 80.50 9 4.04 82.02 9 5.50
51.77 9 6.30 58.80 9 6.21 78.57 9 2.03* 71.57 96.70† 76.93 9 1.60 88.90 9 4.50
51.14 9 4.90 58.82 9 6.12 77.86 9 6.54* 76.32 9 4.85† 80.90 9 5.74 89.60 9 4.72
52.98 9 4.14 62.76 9 4.30 75.70 9 4.50** 75.86 94.08† 79.14 9 3.30 83.88 9 4.38
30
pHa
20
30
PaCO2 (mmHg)
7.85 9 0.03 7.69 90.02 7.40 9 0.05** 7.84 90.05 7.51 90.05** 7.45 90.05***
0 3 5 0 3 5
18.50 91.80
0
25
7.87 9 0.05 7.64 9 0.03* 7.43 90.06** 7.79 9 0.02 7.56 9 0.05** 7.44 90.04** 26.20 9 4.60
7.86 90.04 7.62 90.04** 7.41 90.03*** 7.81 90.03 7.58 90.04** 7.43 9 0.04*** 34.80 9 2.40‡‡
7.84 9 0.02 7.68 9 0.04* 7.41 9 0.05*** 7.80 9 0.06 7.55 9 0.05* 7.48 9 0.05** 18.80 9 0.90
Values are means 9 S.E.M. for five animals in each group (except during autumn, when n =3); * Significant (PB0.05) effect of hypercarbia relative to normocarbic control values, at the same temperature. ** Significant (PB 0.01) effect of hypercarbia relative to normocarbic control values, at the same temperature. *** Significant (PB0.001) effect of hypercarbia relative to normocarbic control values, at the same temperature. † Significant (PB0.001) difference from the value at 20°C (normocarbic conditions). ‡‡ Significant (PB0.01) difference from the value during summer. a
that hypercarbia-induced hyperventilation in the toad B. paracnemis is augmented in response to increased body temperature. Similar data were obtained for the alligator Alligator mississipiensis [8,14] and the turtle Chrysemys picta [18]. The present study provides the first evidence that the effect of hypercarbia on breathing frequency depends on the season (P B 0.001). During winter, hypercarbiainduced tachypnoea was similar at 20 and 30°C. Moreover, during this season, at 30°C, hypercarbia-induced tachypnoea was lower than during summer and spring (Fig. 2). This indicates that there may be a reduction in the sensitivity for the hypercarbic ventilatory response during winter. No significant increase in breathing frequency was observed during autumn when the animals inspired 5%
of CO2 at 20°C, perhaps because these responses varied considerably between individuals (Fig. 2). In this study, frogs were exposed to hypercarbia for 30 min at all experimental temperatures. Perhaps this period of time was not enough for a full cardiovascular and respiratory response at 10°C. However, this seems unlikely because pilot studies revealed that a 30min period is sufficient for steady-state measurements. Additionally, we assessed breathing frequency by measurements of buccal pressure, which is a widely used [27,43,52] and extremely useful method for studies on ventilatory control. However, it does not provide a measure of the actual volumes of ventilated air and, therefore, does not allow calculation of parameters such as air convection requirement (V/VO2) (cf. [50]).
Table 3 Values of hematocrit (%) of R. catesbeiana during all four seasonsa Temperature (°C)
Inspired CO2 (%)
Summer
Autumn
Winter
Spring
10
0 3 5
16.71 91.05 18.00 91.00 19.29 91.00
15.67 9 1.20 16.44 9 1.50 15.22 9 1.00
16.47 9 1.66 18.00 91.58 15.53 9 1.89
17.88 9 0.70 17.94 90.87 18.00 9 0.73
20
0 3 5
16.79 9 1.04 17.07 9 0.99 19.479 1.71
13.78 9 1.00 14.39 91.10 14.94 9 1.00
16.95 9 1.39 20.40 91.39 19.00 91.60
17.44 90.75 17.78 9 0.97 18.74 90.98
30
0 3 5
15.70 9 1.62 15.60 91.20 16.66 92.04
15.07 9 1.60 15.29 9 2.20 17.21 9 2.00
17.69 9 0.96 18.56 91.02 19.25 9 1.45
17.22 9 0.85 16.94 90.78 16.37 9 0.69
a
Values are means 9 S.E.M. (n= 5–7 during summer, n= 7–9 during autumn, n = 8–10 during winter and n = 8–9 during spring).
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reduced and breathing frequency unaltered during winter. Thus, the mechanism responsible for the twofold elevation of PaCO2 may involve a reduction in cutaneous CO2 elimination. Actually, it is well known that amphibians excrete excess CO2 passively across the skin [23,28] and R. catesbeiana, specifically, liberates approximately 80% of the total CO2 produced across cutaneous surface [12]. Moreover, Burggren and Moalli [11] suggest that a partial inhibition of CO2 excretion is linked with a decrease in the extent and pattern of blood flow through the skin. These blood flow changes may be related to the increased mean blood pressure during winter (Table 1).
Fig. 3. Seasonal comparisons of the effect of temperature on heart rate. Data are reported as means 9 S.E.M. (n = 5–8 during summer and winter, n=6 – 8 during autumn and n = 8–10 during spring). †††, Significant (P B 0.001) effect of temperature under normocarbia conditions within a season (relative to values at 10°C).
4.3. Blood gas le6els and acid– base status PaO2, pHa and PaCO2 values obtained here during summer (Table 2) were similar to those reported earlier for R. catesbeiana [27], and the temperature- and hypercarbia-induced alterations in PaO2 and pHa during summer agreed with previous studies on anuran amphibians [3,6,10,30,40,47,53]. It is interesting to note that no seasonal variation in PaO2 or pHa occurred, but winter PaCO2 values were twice as high as summer and spring values. This increased PaCO2 is unexpected since O2 consumption was
Fig. 4. Seasonal comparisons of the effect of temperature on blood pressure. Data are reported as means 9 S.E.M. (n =5 – 8 during summer, n =6 – 8 during autumn, n =7 – 8 during winter and n=7–9 during spring). ††, Significant (P B0.01) effect of temperature under normocarbia conditions within a season (relative to values at 10°C).
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According to the Henderson – Hasselbalch equation, increased PaCO2 should cause a drop in pH unless a compensatory increase in bicarbonate concentrations occurs. This suggests an increase in plasma bicarbonate levels during winter, which could not be calculated in the present study because measurements were performed at different temperatures. Perhaps the increased PaCO2 is caused by a net bicarbonate reabsorption through the urinary bladder wall and/or mobilization of internal calcareous deposits [46]. Boutilier et al. [5] observed a similar increase of PaCO2 in B. marinus. Under laboratory conditions, B. marinus burrows, hypoventilates and reduces its cutaneous respiration, with a resulting twofold increase in arterial blood PCO2 despite a reduction in metabolism. This rise in PaCO2 leads to the development of marked respiratory acidosis, which is completely compensated for a rise in plasma bicarbonate levels in the animals after 3 days of burrowing [5]. As pointed out by Rocha and Branco [40], this suggests that short-term [5] and long-term (present study) acid– base compensation might be similar among anuran amphibians. During active periods, such high PaCO2 values would induce hyperventilation [3,4,9,10,38] but, during winter, ventilation under normocarbia is unaltered in R. catesbeiana (Table 1). This indicates reduced sensitivity of chemoreceptors, alteration in the central respiratory control system response and/or reduced effector response during this season.
4.4. Hematocrit The hematocrit values showed no significant changes induced by temperature, hypercarbia or season (Table 3), indicating that bullfrogs kept a constant oxygen-carrying capacity of blood during the year. Similarly, no change in hemoglobin concentration or hematocrit occurs in naturally estivating S. couchii and S. hammondii [42] or in Xenopus lae6is estivating in the laboratory (cf. [38]). In contrast, in Rana pipiens, hematocrit is higher during winter [26] than during summer [25]. These discrepancies may be due to interspecific differences among amphibians.
4.5. Cardio6ascular system In agreement with previous studies [20,21,36], falling temperatures were accompanied by decreasing heart rate (Fig. 3), which may be due to a direct effect on pacemaker cells [13]. As to seasonal variations of cardiac function in anuran amphibians, Jones [24] evaluated driving bradycardia of Bufo bufo and Rana temporaria during prehibernation, breeding season and summer. The author found that, although statistically significant, the seasonal variations in the response of heart rate to submer-
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gence observed in R. temporaria and B. bufo are not so marked as to make a clear seasonal characterization of animals possible. In agreement, in the present study, no seasonal variations were observed in relation to cardiovascular responses to hypercarbia (Figs. 3 and 4). The relationship between heart rate and body temperature in the bullfrog was not influenced by season (Table 1), as previously observed [40]. In contrast, the frogs Rana pipiens [36] and R. temporaria [20] and the toads, Bufo fowleri [44] and Bufo paracnemis [19] showed seasonal variation of the effect of temperature on heart rate during summer and winter. These contrasting responses may be indicating interspecific differences among amphibians. We observed seasonal variation in the effect of temperature on blood pressure (Table 1). Our results are in agreement with those of Weathers [51] and Rocha and Branco [40], who observed that blood pressures in the bullfrog were slightly higher in winter than in summer. Higher arterial blood pressure, without any change in heart rate, indicates an increase in peripheral resistance and/or in the stroke volume in the frogs during winter.
4.6. Conclusion We have shown that R. catesbeiana has an endogenous annual rhythm of cardiorespiratory function. The present study suggests that there is a relationship between oxygen-consumption variability and cardiorespiratory system function in bullfrogs, which is temperature independent. The mechanisms involved in such seasonal rhythm are not known, and will require further research. Perhaps, plasma concentration of some peptides such as arginine-vasotocin (AVT), a non-mammalian arginine vasopressin (AVP) analog, is elevated during winter. This would be consistent with the notion that, during seasonal periods of drought (winter in south eastern Brazil), amphibians would have an increased plasma osmolarity which is known to cause behavioral hypothermia [34], and increased blood pressure [33]. Moreover, ventilation is stimulated after AVP V1 receptor blockage in normocapnia [48] and hypercapnia [49] in dogs. Finally, winter frogs have an increased PaCO2 (Table 2) and AVP is known to be released during hypercapnia in rats (cf. [54]) and dogs [39]. Although the exact mechanisms remain to be determined, R. catesbeiana is an interesting model to access this question.
Acknowledgements This work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). K.C.B.-N. was the recipient of a
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