Respiration Physiology 118 (1999) 49 – 59
www.elsevier.com/locate/resphysiol
Constant set points for pH and PCO2 in cold-submerged
skin-breathing frogs
Glenn J. Tattersall *, Robert G. Boutilier
Department of Zoology, Uni6ersity of Cambridge, Cambridge CB2 3EJ, UK
Accepted 21 July 1999
Abstract
The low temperatures encountered by overwintering frogs result in a large downregulation of metabolism and
behaviour. However, little is known about acid–base regulation in the extreme cold, especially when frogs become
exclusive skin-breathers during their winter submergence. Blood and muscle tissue acid – base parameters (pH, PCO2,
bicarbonate and lactic acid concentrations) were determined in submerged frogs exposed to a range of low
temperatures (0.2–7°C). At overwintering temperatures between T = 0.2 and 4°C plasma pH and PCO2 were
maintained constant, whereas intracellular pH regulation resulted in larger pH-temperature slopes occurring in the
presumably more active heart muscle (DpH/DT= − 0.0313) than in the gastrocnemius muscle (DpH/DT= −
0.00799). Although blood pH was not significantly affected by submergence between 0.2 and 4°C (pH =8.220 – 8.253),
it declined in the 7°C frogs (pH = 8.086), a decrease not linked to the recruitment of anaerobiosis. Plasma PCO2 and
pH in the cold appear to be regulated at constant levels, implying that cutaneous CO2 conductance in submerged
frogs is adjusted within the range of overwintering temperatures. This is likely geared toward facilitating the uptake
of oxygen under conditions of greater metabolic demand, however there remains the possibility that acid – base
balance itself is maintained at a constant set point at the frog’s natural overwintering temperatures. © 1999 Elsevier
Science B.V. All rights reserved.
Keywords: Acid – base, overwintering frog; Amphibians, frog; Gas exchange, skin; Overwintering, metabolism; Temperature,
overwintering
1. Introduction
Aquatic overwintering frogs encounter nearfreezing temperatures in the winter, which can
* Corresponding author. Present address: Department of
Physiology, Northeastern Ohio Universities College of
Medicine, OH 44272-0095, USA; Tel.: + 1-330-3256441; fax:
+1-330-3255912.
E-mail address: [email protected] (G.J. Tattersall)
bring about drastic reductions in activity and
metabolism (Pinder et al., 1992). Although these
low temperatures produce large metabolic savings, both in terms of prolonging metabolic fuel
stores during the winter (Donohoe et al., 1998),
and in the behavioural adjustments to reducing
stress (Tattersall and Boutilier, 1997), the actual
homeostatic adjustments to changes within this
range of cold temperatures are not well known.
0034-5687/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 3 4 - 5 6 8 7 ( 9 9 ) 0 0 0 7 3 - 0
50
G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
Nearly all the previous work on acid – base balance in ectotherms has only examined pH adjustments above 5°C, ignoring the overwintering
temperatures of many ectotherms, such as the
common frog, Rana temporaria. Thus, making
predictions concerning acid – base balance at the
extreme cold end of non-freezing temperatures is
difficult and complicated by behavioural and
metabolic factors, both of which will be subject to
the high thermal sensitivities commonly encountered at low temperatures. Moreover, frogs can
spend months submerged underwater in the winter, during which time CO2 and oxygen homeostasis can be met only through cutaneous respiration.
Since the skin has often been thought to be capable of only passive regulation of gas exchange
(Gottlieb and Jackson, 1976; Mackenzie and
Jackson, 1978, although see Burggren and Moalli,
1984; Feder and Burggren, 1985; Pinder, 1987)
and thereby of acid – base balance (Moalli et al.,
1981; Johnson et al., 1993), examining the pH –
temperature relationships of cold-submerged frogs
may prove to be a useful approach in understanding the extent to which cutaneous respiration can
regulate acid–base balance.
When overwintering under the ice of frozen
lakes and ponds, frogs have the opportunity to
select temperatures within a narrow range (0 –
4°C). Indeed, frogs use behavioural thermoregulation and respond to these low temperatures,
resulting in large changes in metabolic rate due to
the strong Q10 effect (Tattersall and Boutilier,
1999). Depending on latitude or the severity of
winter, Rana temporaria overwinter either underwater in frozen lakes or in the moist soil on land
(Pasanen and Sorjonen, 1994). This choice of
habitat may affect the acid – base balance of overwintering frogs due to the shift in the primary
respiratory organ between these different environments. In fact, at overwintering temperatures,
frogs should be considered facultative airbreathers, since their actual metabolic rate (M: O2)
is much less than the total oxygen uptake which
the skin can accommodate. Therefore, provided
the water does not fall to severely low levels of
oxygen (e.g. B 30 mmHg), frogs will remain entirely aerobic throughout the winter, and maintain
tissue energetic status (e.g. constant ATP) without
the need to recruit a potentially lethal and substrate-inefficient anaerobiosis (Donohoe and
Boutilier, 1998). Since the solubility and capacitance of oxygen in water is much lower than that
of CO2, gas exchange in an exclusive skinbreather is more likely to be geared toward oxygen acquisition rather than CO2 excretion (M: CO2).
Thus, increasing cutaneous conductance in order
to facilitate oxygen uptake will result in submerged frogs retaining less CO2 than air breathing
ones, and possibly lead to differences in their
capacity for acid–base regulation.
The simplified model of cutaneous CO2 excretion, M: CO2 = GCO2 × DPCO2 (where GCO2 is the
cutaneous CO2 conductance, and DPCO2 is the
transcutaneous partial pressure gradient for CO2)
suggests that the regulation of gas exchange can
be accomplished either by increasing the conductance for that gas or by increasing the driving
gradient for diffusion. However, if cutaneous
GCO2 in amphibians is indeed as poorly controlled
as is thought (Mackenzie and Jackson, 1978),
then CO2 elimination must result passively from
the increased cutaneous driving gradient due to
increases in metabolic CO2 production (Mackenzie and Jackson, 1978; Moalli et al., 1981). However, these studies have all been performed on
bimodally breathing amphibians at higher temperatures, where further increases in cutaneous blood
flow may conflict with other regulatory processes,
such as the control of cutaneous water loss. It is
possible that submergence itself is a cue which
allows for some degree of control of cutaneous
GCO2 through changes in capillary recruitment
(Feder and Burggren, 1985), and thus simultaneously manipulate both CO2 excretion and acid–
base balance (Gottlieb and Jackson, 1976). In
other words, are the PaCO2 levels in the frog
passively ‘set’ by the requirements for oxygen
uptake or is GCO2 changed in order to regulate
PaCO2?
Acid–base regulation in response to temperature has been well-documented in amphibians
(Toews and Boutilier, 1986). Numerous body
compartments have been observed to show pH
changes with temperature (DpH/DT) which vary
between species, tissue, and even temperature
range. This regulation of pH preserves protein
G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
function and is thought to occur through either
the maintenance of a constant charge balance on
the imidazole moieties of histidine residues in
proteins through ventilatory adjustments in PCO2
(e.g. alphastat regulation; Reeves, 1972), or simply through the maintenance of a constant relative alkalinity (Rahn and Baumgardner, 1972).
However, experiments on salamanders have not
helped to resolve the issue of whether skinbreathers are fully capable of regulating their
tissue (pHi) and blood pH (pHe). Moalli et al.
(1981) demonstrated that the large, primarily
skin-breathing salamander, Cryptobranchus alleganiensis is able to regulate its blood pH in a
fashion consistent with alphastat regulation,
whereas the small, lungless salamander, Plethodon
cinereus is apparently incapable of tissue pH regulation due to oxygen delivery problems at increasing temperatures (Johnson et al., 1993). The
suggestion from both of these studies was that
even if pH-temperature regulation occurs in skinbreathers, it can be explained by entirely passive
mechanisms, and becomes potentially limiting at
the higher temperatures naturally encountered by
amphibians.
However, these adjustments in pH have been
measured exclusively above 5 – 10°C, outside the
range of most overwintering temperatures. At low
temperatures, pHe values (8.0 – 8.4) are high
enough that temperature-induced changes in pH
(e.g. − DpH/DT) are larger than those predicted
at higher temperatures, simply due to the logarithmic nature of the pH scale. Moreover, numerous
physiological (e.g. ion exchange, blood flow and
metabolism) and behavioural processes (e.g. activity) interact with potentially different Q10 values,
thus complicating any attempt to maintain extracellular or intracellular acid – base balance. Although pHi changes with temperature in a similar
fashion to pHe, it does not always parallel
changes observed in the blood (Boutilier et al.,
1987; Donohoe, 1997). Particularly relevant to the
acid –base status of overwintering frogs is pHi
regulation in active and inactive tissues. Since
activity is likely to be minimal in the cold, the
inactive muscle tissue might show a different pattern of pHi regulation than a continuously active
organ like the heart.
51
The objectives of this study were to characterise
blood and tissue acid–base balance of submerged
frogs subjected to temperatures typical of their
overwintering environment. The null hypothesis
was that pHe and pHi of submerged frogs would
be regulated at low temperatures in a fashion
similar to other ectotherms at higher temperatures
(e.g. alphastat or constant relative alkalinity).
This was tested by subjecting submerged frogs to
waters spanning a narrow range of winter temperatures (0.2, 1.5, 4, and 7°C), and measuring blood
pH, plasma bicarbonate and PCO2, tissue bicarbonate, PCO2 and pHi, along with lactate and
glucose concentrations in order to ensure that
metabolic homeostasis was not disturbed by the
acclimation period. These three temperatures were
chosen since 0.2°C was as close to 0°C as possible
without the risk of freezing, 4°C was representative of many overwintering environments, and
7°C was considered an upper limit estimate of the
aerobic threshold of submerged frogs, although
outside the normal overwintering temperature
range.
2. Materials and methods
2.1. Maintenance of experimental animals
Frogs (Rana temporaria) were purchased in December 1996 from Blades Biological who had
caught them from wild populations originating in
Ireland. Before arriving in Cambridge, the frogs
were housed with access to air at 10°C. Upon
arrival in the laboratory, they were transferred to
aerated tanks (Living Stream, Frigid Units,
Toledo, OH) at 3–4°C for at least 2 months
before transfer to final experimental temperatures
(0.2, 1.5, 4 and 7°C) in February, 1997. During
this pre-acclimation period all frogs had access to
air, however since they were housed in relatively
deep water (40 cm) it was unlikely that they chose
to breathe air during this period.
2.2. Acid–base and metabolite experiments
Following the pre-acclimation period, groups of
frogs (n= 6 each group) were transferred to hold-
52
G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
ing tanks and submerged completely. On three
separate occasions, frogs were housed in these
tanks at 0.2, 4, and 7°C for a period of 7 days.
One week was deemed an upper limit estimate of
a suitable acclimation period for acid – base adjustments; 3–7 days being a common acclimation
period in studies of acid – base regulation during
temperature fluctuations (Rahn and Baumgardner, 1972; Reeves, 1972; Moalli et al., 1981). In
some cases (blood and gastrocnemius muscle),
acid –base data was available for frogs kept at 1.5
and 7°C (corresponding to preferred temperatures
in hypoxia and normoxia; Tattersall and
Boutilier, 1997) for 1 – 2 days, and these data were
included in the results section. These shorter term
acclimation periods were not deemed to be a
problem for assessing pH regulation, as the larger
passive proportion of pH adjustment occurs relatively quickly (Pörtner et al., 1997), and often
within 2–8 h. As well, long-term acclimation to
overwintering temperatures has not shown any
significant changes in the acid – base properties of
the blood or tissues of cold-submerged frogs
(Donohoe et al., 1998).
2.3. Acid–base measurements
Upon completion of temperature acclimation,
frogs were completely anaesthetised in aerated
0.3% (w/v) MS-222 (buffered with 0.4% NaHCO3)
for 10–15 min at the acclimation temperature.
Hearts were surgically exposed and mixed arteriovenous blood ( 300 ml) drawn into heparinised
capillary tubes from the left aortic arch, sealed
and put on ice for a maximum of 5 min. Blood
pH (pHe) was subsequently determined on 100 ml
of blood using a PHM84 (Radiometer, Copenhagen) pH meter and glass microelectrode (G297/
G2,
Radiometer)
water-jacketed
to
the
acclimation temperature and calibrated with Radiometer S1500 and S1510 precision buffers at the
correct value for the given acclimation temperature. The remainder of the blood was centrifuged
(15 800×g) for less than 10 sec and the plasma
total CO2 determined on 50 ml of plasma with a
Corning 965 CO2 Analyser. Plasma bicarbonate
and PCO2 were calculated from TCO2 as described
by Boutilier et al. (1993), using aCO2 and apparent
pK values derived from empirical formulae described by Heisler (1989). Following the blood
isolation, which took up to 3 min, gastrocnemius
muscles from both legs and the heart (ventricle)
muscle (rinsed briefly in 300 mOsmol L − 1 sucrose
to remove excess blood and minimising extracellular contamination) were isolated, freeze-clamped
between aluminium plates at −196°C and immediately stored at − 80°C for up to one month
before metabolite determination (metabolites
measured only in gastrocnemius since all ventricular tissue was used to measure pHi). pHi determinations for gastrocnemius and heart tissue were
performed within one month of the experiments,
using the method described by Pörtner et al.
(1990). Tissue PCO2 and bicarbonate were calculated, as opposed to intracellular values, since
estimates of fractional tissue water were either
unavailable or found not to be reliable at low
temperatures. Relative alkalinity ([OH−]/[H+])
was also assessed in blood and tissue samples as
the ratio of hydroxyl ions to hydrogen ions based
on pKw estimates:
[OH-] 10pH − pKw
=
[H+]
10 − pH
(1)
Overall, this sampling method was deemed to
be preferable to cannulation, since these animals
were acclimated to low temperatures and tissue
and blood samples were collected quickly enough
to minimise possible biases (errors in body temperature estimation, non-resting state of animals
and the grab and stab blood collection). This is
reflected by the low, temperature-independent
plasma lactate concentrations which are not substantially different from values obtained by cannulated or pithed frogs in the cold (Pinder, 1987;
Donohoe, 1997), indicating that anaesthesia at
low temperatures is an effective and consistently
low-stress means of collecting blood and tissue
samples from frogs.
2.4. Metabolite measurements
Frozen plasma and gastrocnemius (pulverised
under liquid nitrogen) samples were first extracted
in 7% perchloric acid, and the supernatants, after
centrifugation at 15 800× g for 10 min, were neu-
G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
tralised to pH 7 with 2 mol L − 1 KOH/0.4 mol
L − 1 sodium imidazole. These neutralised samples
were subsequently analysed for lactate using a
standard enzymatic assay (lactate dehydrogenase
coupled to glutamic pyruvic transaminase; Passoneau and Lowry, 1993) and an ELX 800 UV
Biotek Instruments Plate Reader following the
appearance of NADH at 340 nm. In addition to
plasma lactate, glucose was also determined on
the extracted plasma samples using an enzymatic
method coupling glucose phosphorylation with
hexokinase to glucose-6-phosphate oxidation with
glucose-6-phosphate dehydrogenase (Passoneau
and Lowry, 1993). All concentrations are expressed on the basis of total plasma or tissue
water (mmol·L − 1).
2.5. Statistical analysis
All acid–base and metabolite data were
analysed using One-Way ANOVA, unless indicated otherwise, with temperature as the effect.
When ANOVA revealed significant effects, post
hoc analysis was performed using the multiple
comparison Student Newman – Keul’s test (Sigmastat). Temperature – pH slopes were calculated
using least squares linear regression and included
all temperature points, except in the case of
blood, where a serious deviation from a linear fit
prompted analysis at only the normal overwintering temperatures (i.e. below 5°C). In all cases,
53
PB 0.05 was chosen as a significant effect. All
data are reported as mean9 S.E.M.
3. Results
3.1. Acid–base and metabolite experiments
Relative alkalinity and metabolite measurements for the submerged frogs at 0.2, 4, and 7°C
are summarised in Table 1. The effect of temperature on extracellular acid–base balance was only
pronounced at 7°C (Table 1; Fig. 1), however, the
high variability obscured any significant effects.
pHe and plasma PCO2 and bicarbonate were not
significantly affected by temperature, especially at
and below 4°C where all the parameters remained
nearly constant. pHe at and below 4°C did not
appear to follow alphastat regulation (Fig. 1;
Table 1), changing only slightly between 0.2 and
4°C (pH =8.253, 8.226 and 8.220 at 0.2, 1.5 and
4°C; DpH/DT = −0.00779; P= 0.21). Relative alkalinity was not significantly affected by temperature (Table 1), although it did exhibit high
variability, ranging from 28.9–46.2. Other blood
parameters, such as haematocrit (30.9–34.1%)
and plasma lactate concentrations (0.73–1.31
mmol L − 1) were unaffected by temperature, however, plasma glucose was found to be significantly
lower at 0.2°C (0.909 0.11 mmol L − 1) than at 4
and 7°C (1.51 90.15 and 1.58 9 0.08 mmol L − 1).
Table 1
Relative alkalinity and metabolic properties of blood and tissues from submerged frogs at 0.2, 4, and 7°Ca
0.2°C
4°C
7°C
Significant effect
Plasma
Relative alkalinityb
Haematocrit (%)
Lactate (mmol L−1)
Glucose (mmol L−1)
38.093.1
31.892.2
0.739 0.20
0.90 90.11
46.29 2.7
30.9 92.0
1.319 0.25
1.5190.15
28.9 96.2
34.1 9 2.2
1.17 90.13
1.58 90.08
P= 0.10
P= 0.60
P = 0.13
PB0.001
Gastrocnemius
Relative alkalinity
Lactate (mmol L−1)
1.239 0.15
2.6991.23
1.319 0.13
1.799 0.30
1.50 90.14
1.42 9 0.36
P = 0.48
P= 0.6
Ventricle
Relative alkalinity
0.38990.046
0.337 9 0.035
0.269 9 0.021
P = 0.09
Values are mean 9 S.E.M.
Kruskal–Wallis One-Way ANOVA on Ranks. Note: underscoring denotes statistical similarity of means (PB0.05) based on
Student-Newman–Keul’s method conducted when a significant ANOVA was found.
a
b
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G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
Ventricular muscle, on the other hand, was
more acidic than gastrocnemius muscle (7.040–
7.254 compared to 7.373–7.501) and showed a
considerably larger decline with increasing temperature, which was highly significant (PB 0.001;
DpH/DT = −0.0313; Table 1 and Fig. 2). These
decreases in pHi were accompanied by gradually
increasing PCO2’s (7.56–12.6 mmHg) with increasing temperature, whereas ventricular bicarbonate
concentrations
remained
relatively
constant across all three temperatures (4.59–5.35
mmol L − 1).
Fig. 1. Plot (Mean9 S.E.M.) of blood acid–base parameters
(pHe, plasma PCO2 and bicarbonate). The pH-temperature
slope (shown 995% confidence interval) in the range of overwintering temperatures was not significant (DpH/DT= −
0.00799 versus − 0.016 expected for anuran amphibians).
Plasma PCO2 and bicarbonate were not significantly affected
by temperature (Kruskal–Wallis test).
Intracellular pH in the gastrocnemius muscle
was not significantly affected by temperature, but
did show an overall DpH/DT of −0.00763 (nonsignificant; P= 0.13). This small change in pHi
with temperature was accompanied by slight increases in tissue PCO2 and bicarbonate, culminating in the 0.2°C values being significantly lower
than any measurements at 1.5, 4, or 7°C (Fig. 2).
These tissue adjustments in acid – base parameters
were not accompanied by anaerobiosis in the
gastrocnemius muscle, as lactate concentrations
were not significantly affected by temperature
(Table 1).
Fig. 2. Plot (Mean 9S.E.M.) of gastrocnemius (circles) and
ventricular muscle (triangles) pHi, tissue PCO2 and bicarbonate
concentrations. Gastrocnemius pHi showed no significant correlation with temperature (0.2 – 7°C), although the DpH/DT
was −0.00763 versus the significant ventricular muscle DpH/
Dt of −0.0313 (both shown 9 95% CI). Letters correspond to
statistically similar values for each tissue.
G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
4. Discussion
Many temperate anurans overwinter for several
months in ice-covered ponds when all of their gas
exchange requirements must be satisfied by cutaneous respiration. They achieve this across the
range of overwintering temperatures (0 – 4°C)
without the need to recruit anaerobiosis (Donohoe et al., 1998). However, studies on amphibians
at higher temperatures (15 – 30°C) have repeatedly
suggested that cutaneous respiration is primarily
diffusion limited (see Feder et al., 1988) and, thus
potentially poorly regulated (see Piiper and
Scheid, 1982). At the low overwintering temperatures, M: O2 may be reduced by so much, that
oxygen is no longer limiting and cutaneous gas
exchange is sufficient to accommodate routine
changes in metabolic oxygen demand. However,
at higher temperatures, these animals normally
maintain their temperature-dependent acid – base
set points by pulmonary regulation of PCO2, since
oxygen in air is so readily available. When they
are forced to become skin breathers while overwintering under ice, two things could happen. If
their ability to extract oxygen to meet metabolic
demand is limiting, then PCO2 levels would be set
by the requirements for oxygen uptake. Under
these conditions there would be little control of
PCO2 (as in fish) and ‘skin-ventilation’ (e.g. rocking or bobbing behaviour; Pinder, 1987) would be
primarily oxygen-driven. If, on the other hand,
the capacity for oxygen uptake by the skin far
exceeded that of oxygen demand, there could be
some potential for adjusting PCO2 by changing
cutaneous conductance for the respiratory gases.
The present data suggest that at 4°C, PaCO2 has
nearly reached the theoretical minimum for an
exclusively aquatic vertebrate (Rahn and Baumgardner, 1972). Indeed, the PaCO2 levels of 3
mmHg seen in cold-submerged frogs (Fig. 1) are
not that unlike those observed in cold, waterbreathing fish (reviewed in Heisler, 1984; Ultsch
and Jackson, 1996), whose main respiratory drive
is towards oxygen uptake. Thus, if the demands
for cutaneous oxygen uptake at 4°C dictate a
minimum PaCO2 of 3 mmHg, there would be little
scope to decrease PaCO2 further through respiratory adjustments in order to raise pHe. The result
55
is that blood is maintained at a constant PCO2 and
pHe at temperatures down to 0.2°C. At temperatures above the overwintering range (7°C), pHe
decreases as a consequence of an increase in
PaCO2. Given the increased metabolism and decrease in dissolved CO2 that comes with increasing temperature, it is likely that PaCO2 rises due to
a partial diffusion-limitation for CO2 excretion. In
other words, the capacity for cutaneous oxygen
uptake at 7°C is probably just sufficient to meet
the overall oxygen demand of the animal, and so
the maximal oxygen extraction effectively governs
the PaCO2.
4.1. Acid–base regulation in the cold
Exclusively skin-breathing amphibians are
thought to be unable to regulate pH through the
respiratory control of PCO2, and thus are not
expected to show true alphastat regulation with
changes in temperature (Johnson et al., 1993).
Indeed, lungless salamanders are incapable of the
same degree of intracellular pH regulation as
other air breathing amphibians; i.e. the DpH/DT
values of the skin-breathers far exceeded those
required for alphastat regulation (−0.041 compared to −0.081 in the tissue of a comparatively
sized lunged salamander, Johnson et al., 1993). A
DpH/DT response consistent with alphastat pH
regulation was achieved, however, by providing
the animals with 100% O2, outlining the increasing importance of oxygen delivery in maintaining
tissue acid–base balance, especially at higher temperature ranges. Similar oxygen delivery problems
do not appear to manifest in cold-submerged
frogs, since, at least in the inactive gastrocnemius
muscle, pHi and tissue lactate were unaffected by
temperature (Fig. 2; Table 1). The gastrocnemius
maintains its relative alkalinity by increasing PCO2
and bicarbonate concentrations relative to 0.2°C
values (e.g. an apparent respiratory acidosis) with
increasing temperatures (as would be expected for
a ternary-buffer system where phosphate represents a significant buffer, Reeves and Malan,
1976), suggesting an increased CO2 production as
a result of increased temperature, rather than
regulating a constant intracellular bicarbonate
concentration.
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G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
Fig. 3. Plot of cutaneous CO2 conductance in submerged frogs
(solid circles) as a function of temperature. Conductance was
estimated from the whole animal metabolic rate (mmol g − 1
h − 1; Donohoe, 1997) divided by the PCO2 gradient (DPCO2)
between the plasma and environmental PCO2 (assuming ambient PCO2 =1 mmHg and Q10 for metabolic rate =4.8). The
equation, nopen/ntotal × DCO2 (diffusing capacity of the skin)
refers to the regulation of GCO2 through the recruitment of
under-perfused cutaneous capillaries. High variability in the
7°C group reflects the variable estimates for PaCO2. Recalculated (on a per gram basis) bullfrog (open circles) and Cryptobranchus (solid triangles) cutaneous GCO2 are shown for
comparison to demonstrate the constant cutaneous GCO2 at
higher temperatures (Mackenzie and Jackson, 1978; Moalli et
al., 1981). Typical overwintering and non-overwintering temperatures are indicated for reference.
Ventricular pHi, on the other hand, showed a
greater decrease with increasing temperature
(DpH/DT = − 0.0313, significantly different from
alphastat value of − 0.017, P B0.01; Fig. 2). This
larger pH-temperature slope for ventricular pHi in
cold-submerged frogs likely represents an increased production of metabolic protons and CO2
due to the need of the frog’s heart to work harder
and increase cardiac output at the higher temperatures. That the heart is an active tissue in an
otherwise inactive animal is suggested by the apparent combined respiratory and metabolic
acidosis observed in the higher temperatures (e.g.
increased PCO2 and constant bicarbonate concen-
tration), supported by the fact that ventricular
pHi is actually relatively acidotic compared to
neutral water, rather than the relatively alkaline
environment of the circulating blood (Table 1).
Acid–base regulation in the blood differed
somewhat from that of the tissues (cf. Figs. 1 and
2; Table 1), demonstrating that tissues independently regulate their acid–base status. At temperatures of 4°C and below, pHe did not differ
significantly with temperature (DpH/DT = −
0.00763; P=0.21) and only changed by approximately one-half of the expected relationship for
anuran amphibians (DpH/DT = − 0.016; Toews
and Boutilier, 1986). However, at 7°C large decreases in pHe were seen in some individuals,
possibly reflecting a greater level of activity at the
higher temperature. Unlike pHi, the temperature
increase between 4 and 7°C was accompanied by
a pHe change not resembling that expected for
alphastat pH regulation in anuran amphibians
(Reeves, 1972), despite an insignificant change in
relative alkalinity (Rahn and Baumgardner, 1972;
Table 1). It is likely that this fall in pH and
increased PCO2 stems from a greater tendency for
some frogs to become more active and retain CO2
at higher temperatures, perhaps due to different
temperature sensitivities of metabolic and perfusive processes above the normal overwintering
temperatures. To date, there is no evidence of
anaerobiosis being induced in normoxic frogs at
this temperature (Table 1), suggesting that the
explanation lies with the diffusion limitations for
gas exchange. However, at their normal overwintering temperatures (0–4°C), submerged frogs appear to be capable of maintaining a constant pH,
despite the absence of true ‘ventilatory’ control of
PCO2.
4.2. Cutaneous CO2 conductance and constant
PaCO2 in the cold
Since submerged frogs are capable of regulating
cutaneous O2 conductance, it was essential to
know whether cutaneous CO2 conductance (GCO2)
could also be regulated by a skin-breather at low
temperatures. Although previous work has suggested that cutaneous GCO2 does not change with
temperature in amphibians (see Fig. 3; Mackenzie
G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
and Jackson, 1978; Moalli et al., 1981), the constant GCO2 prediction from higher temperatures
does not appear to fit the scenario at these low
temperatures in submerged frogs (cf. Fig. 3). Assuming a typical Q10 of 4.8 (G.J. Tattersall and
R.G. Boutilier, unpublished) and whole animal
CO2 excretion values (M: CO2) conducted on the
same cohort of frogs under similar conditions
(Donohoe et al., 1998), CO2 conductance (GCO2)
was estimated (GCO2 =M: CO2/DPCO2), where DPCO2
represents the PCO2 difference between the plasma
and water. These estimates revealed that cutaneous GCO2 increases at higher temperatures in
submerged frogs, with the minimal cutaneous
GCO2 occurring at 0.2°C (see Fig. 3). This is quite
opposite to the conclusion reached by Mackenzie
and Jackson (1978) in air-breathing frogs at
higher temperatures, and provides tentative evidence for a regulation of GCO2 across the range of
overwintering temperatures.
This is likely to be achieved by increased perfusion or the recruitment of blood flow through
otherwise under-perfused capillaries, presenting a
greater functional surface area for the exchange of
CO2 to the environment (Feder and Burggren,
1985; see equations in Fig. 3). This raises two
important questions: the first being why changes
in cutaneous CO2 conductance were not observed
in air-breathing frogs at higher temperatures
(Mackenzie and Jackson, 1978), and the second
being why regulate CO2 conductance in the cold?
The reason cutaneous GCO2 changes with temperature in the present study is probably because the
metabolic demands of the submerged frog must
be met solely through the skin. Air-breathing
frogs at higher temperatures would have sufficient
control of M: CO2 and M: CO2 through both the lungs
and the skin, but the skin itself will be governed
by other external influences, namely that of water
conservation. Since any increase in cutaneous
conductance for respiratory gases is bound to
accelerate the evaporation of water from the skin,
air-breathing amphibians likely keep cutaneous
conductance at a minimum across a wide temperature range through reductions in cutaneous
blood flow (see Feder and Burggren, 1985, for
references), and thus minimise their water loss.
57
There are two possible explanations for why
cutaneous GCO2 may have changed in cold-submerged frogs. The first assumes that respiration in
the submerged frog is primarily oxygen-driven. At
higher temperatures, the metabolic oxygen demand will increase, requiring greater systemic
oxygen delivery, a greater efficiency of gas transfer across the skin, and possibly an increase in the
transcutaneous PO2 gradient in order to facilitate
oxygen uptake. This greater oxygen demand is
easily met by adjustments at the skin. However,
any increase in cutaneous conductance for oxygen
(e.g. through capillary recruitment), could result
in accompanying increases in the cutaneous GCO2,
thus leading to enhanced CO2 excretion and the
apparent regulation of a constant PaCO2. The
second possible rationale is that the apparent
increase in GCO2 is in fact geared toward the
regulation of a constant PaCO2 and pHe. At constant temperature and environmental PO2, increased levels of ambient CO2 can induce
increases in the number of perfused skin capillaries or cutaneous blood flow (Poczopko, 1957;
Courtice, 1981), presumably to regulate CO2 excretion. Thus, it is possible that cutaneous GCO2 is
actually being adjusted in normoxic, submerged
frogs in order to regulate a constant PaCO2. However, the exact reasons for these adjustments will
not be discerned fully until these measurements
are made simultaneously on the same frogs, and
caution should be taken in the interpretation of
these data. For example, if one assumed that no
control exists, but that oxygen uptake was not
limiting over the 0.2–4°C temperature range, then
one would expect metabolism to fall and PCO2 to
decline accordingly with decreasing temperature.
The fact that PCO2 remains constant suggests that
regulation is occurring, although how this occurs
is unknown. The net result, however is that
plasma PCO2 is maintained constant across the
range of overwintering temperatures, a response
which has to date only been observed in exclusively water-breathing fish (Heisler, 1984).
The low PaCO2 values observed in these coldsubmerged frogs are akin to those routinely measured in fish, supporting the fact that water
breathers, by having a primarily oxygen-driven
respiratory drive, have low PCO2 values in the
58
G.J. Tattersall, R.G. Boutilier / Respiration Physiology 118 (1999) 49–59
blood simply because the solubility of CO2 in
water is so much greater than that of oxygen. It is
clear that cold-submerged frogs accomplish this
differently than fish as they still maintain bicarbonate levels similar to when they are airbreathers
(22
mmol
L − 1;
personal
observations), which are much higher than those
of water-breathing teleost fish at similar temperatures (6–16 mmol L − 1; reviewed by Ultsch and
Jackson, 1996). These differences are easily accounted for by the major differences in oxygen
uptake and carbon dioxide excretion between gill
and lung/skin breathers. Gill breathers, by possessing a remarkable ability to regulate acid – base
via ionoregulation and constantly perfusing a thin
respiratory organ of high surface area draw bicarbonate concentrations and PCO2 levels lower than
a skin or lung breather can possibly attain. Therefore, the implication of utilising primarily the skin
for CO2 excretion is that at minimal metabolic
rates (e.g. those at low temperatures) PCO2 is not
allowed to fall below a minimal level (see Fig. 1),
ensuring that a large enough PCO2 gradient exists
across the skin in order to drive CO2 diffusion.
Consequently, plasma bicarbonate concentrations
remain high and pH is regulated at a constant,
upper limit of approximately 8.20 – 8.25 (Fig. 1).
We can only speculate that in this skin breather, a
PCO2 of approximately 3 mmHg represents a
lower limit that cannot be further reduced in the
system under study. However, PCO2 could vary, at
least theoretically, at a constant M: O2 and cutaneous DPO2. For example, because CO2 is only
partly diffusion limited and oxygen is almost entirely diffusion limited (Feder and Burggren,
1985), high skin perfusion with high heterogeneity
of flow could result in a relatively low PCO2 but a
high DPO2, whereas a low skin perfusion with
homogeneous flow through all capillaries could
result in a higher PCO2 but a lower DPO2. This
minimal level of PCO2 is only ever surpassed in
hypoxic- and anoxic-submerged frogs which increase cutaneous diffusing capacity by capillary
recruitment as a means of extracting more oxygen
from the environment (Pinder et al., 1992).
Plasma PCO2 and bicarbonate concentrations only
ever decline in these frogs as a result of the
oxygen-limited hypometabolism and the massively
increased cutaneous perfusion (Pinder et al., 1992;
Tattersall and Boutilier, 1997). Thus, it appears
that plasma PCO2 values in cold-submerged frogs
represent a balance between metabolic CO2 production and CO2 excretion. Whether this is indeed
the case, or whether plasma PCO2 is merely a
reflection of acid–base regulation remains to be
shown.
Acknowledgements
This study, which was part of a Ph.D. dissertation at the University of Cambridge, UK, was
supported by a 1967 Centennial NSERC scholarship to GJT. We wish to thank two referees for
their invaluable assistance in the revision of the
manuscript.
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