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1 Centre for Respiratory Adaptation, Institute of Biology, University of Southern Denmark, Main Campus: Odense University, DK-5230 Odense M, Denmark; and 2 School of Biological Sciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Massive feeding in ectothermic
vertebrates causes changes in metabolism and acid-base and respiratory
parameters. Most investigations have focused on only one aspect of
these complex changes, and different species have been used, making
comparison among studies difficult. The purpose of the present study
was, therefore, to provide an integrative study of the multiple
physiological changes taking place after feeding. Bullfrogs (Rana
catesbeiana) partly submerged in water were fed meals (mice or
rats) amounting to ~
of their body weight. Oxygen
consumption increased and peaked at a value three times the
predigestive level 72-96 h after feeding. Arterial
PO2 decreased slightly during
digestion, whereas hemoglobin-bound oxygen saturation was unaffected.
Yet, arterial blood oxygen content was pronouncedly elevated because of
a 60% increase in hematocrit, which appeared mediated via release of
red blood cells from the spleen. Gastric acid secretion was associated
with a 60% increase in plasma HCO
3 concentration
([HCO3]) 48 h
after feeding. Arterial pH only increased from 7.86 to 7.94, because
the metabolic alkalosis was countered by an increase in
PCO2 from 10.8 to 13.7 mmHg. Feeding
also induced a small intracellular alkalosis in the sartorius muscle.
Arterial pH and HCO3 returned to
control values 96-120 h after feeding. There was no sign of
anaerobic energy production during digestion as plasma and tissue
lactate levels remained low and intracellular ATP concentration stayed
high. However, phosphocreatine was reduced in the sartorius muscle and
ventricle 48 h after feeding.
specific dynamic action; O2 transport; alkaline tide; metabolites; high-energy phosphates
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ECTOTHERMIC CARNIVOROUS VERTEBRATES habitually ingest
very large meals that induce several-fold increases in oxygen uptake (
O2). This response, often
referred to as specific dynamic action (SDA), varies among species and
depends on the size of the meal. In snakes,
O2 increases more than
10-fold after feeding (1, 28), and the peak value exceeds that attained
during muscular exercise (1, 27). Endothermic vertebrates, in contrast,
have high standard metabolic rates and typically feed often and on relatively small meals, limiting the magnitude of their SDA. Many physiological and biochemical factors contribute to SDA. Preabsorptive factors include mechanical processing of the meal, secretion of digestive fluids into the gastrointestinal tract, upregulation of
digestive enzyme and nutrient transporter levels, and, in some cases,
extensive intestinal hypertrophy (17, 30). Postabsorptive factors
include transmembrane transfer of nutrient into target organs and
protein synthesis.
The elevated metabolism after feeding places increased demands on the
gas transport by the cardiopulmonary system, but respiratory physiology
during digestion remains little studied. In toads, SDA has been
elicited by peptone injection into the stomach (36), which leads to an
increased heart rate (10) and an elevation of blood hemoglobin
concentration, whereas arterial PO2 (PaO2) remains high. In contrast, it has
been reported that PO2 decreases
drastically during digestion in pythons. Feeding has also been reported
to increase plasma [HCO3] and pH ("alkaline tide") as a result of acid secretion into the stomach in reptiles (8, 27). However, in these studies, blood was
sampled by cardiac puncture from conscious animals, which is likely to
affect blood pH due to struggling proceeding sampling. Furthermore, as
cardiac puncture yields an uncertain mixture of venous and arterial
blood, the reported PO2 and
PCO2 measurements are unlikely to
reflect arterial blood gases of undisturbed animals. Thus, at present,
there are very limited data on blood gases and acid-base parameters in
undisturbed animals after feeding, and, in some cases, the findings are
conflicting. A major goal of the present study was, therefore, to
determine acid-base status and respiratory parameters of arterial blood
in the frog Rana catesbeiana after feeding. Because of the
intracellular presence of pH-sensitive enzymes, intracellular
acid-base status may be regulated at the expense of the
extracellular space. However, there are no reports on intracellular
acid-base status during digestion. Accordingly, to provide an
integrated picture of whole animal acid-base regulation, intracellular
acid-base status was also determined.
A significant contribution from anaerobic metabolism to energy production during digestion is unlikely, considering the intensity and duration (several days) of the SDA response and the low efficiency of anaerobic pathways. This does not, however, exclude a significant effect of anaerobic metabolism on acid-base status in the extracellular space or in selected organs. Peripheral tissues (e.g., skeletal muscles) and central aerobic organs (e.g., liver, kidney, and ventricle) could experience oxygen delivery limitations because of tissue-dependent changes in blood flow and/or oxygen demand during digestion. A final goal was, therefore, to investigate key metabolites (lactate and high-energy phosphates) in selected body compartments to evaluate metabolism in the postprandial period.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental Animals
Adult bullfrogs (Rana catesbeiana) weighing 358-588 g (mean ± SE: 421 ± 14 g, n = 20) were obtained from North Carolina Biological Supply or Charles D. Sullivan several months before experimentation and kept in large plastic cages with free access to tap water at 20 ± 2°C. A light bulb provided a basking site for thermoregulation and a 12:12-h light-dark rhythm. Frogs were force-fed liver or heart (~10% of body wt) once a week, but starved for 2 wk before experimentation to ensure that digestion was completed. For experimentation, frogs were fed mice or rats equivalent to 10% of their body weight (see Experimental Protocol). Most reports on massive feeding in anurans are anecdotal, but several sit-and-wait foraging species, including Rana catesbeiana, often feed on large prey items such as mammals, reptiles, and other anurans (9). Accordingly, a meal size amounting to 10% of the body mass seems realistic. To our knowledge, there are no data for clearance rates of food after massive feeding in amphibians. However, as defecation did not start until after at least 5 days, digestion of big meals is a prolonged process, and 2 wk of starvation before experimentation appeared realistic.Experimental Protocol
Series I: Oxygen consumption and carbon dioxide excretion.O2 and carbon dioxide
production (CO2) were
determined on uncatheterized frogs (445 ± 44 g, n = 5) with
closed-system respirometry using 2.6-liter chambers supplied with
flowing water (covering the bottom) and air during nonmeasurement
periods. The whole system was thermostated at 22 ± 1°C. Frogs
were placed in the chambers and allowed to acclimate for ~24 h before
measurements were started. During measurements, the chambers were
sealed for 20-60 min. Air samples were drawn at the beginning and
the end of this period and analyzed for O2 and
CO2 with a Datex (Helsinki, Finland) Normocap 200 gas
analyzer. Gas exchange between animal and the small water volume
(~100 ml) remaining in the sealed chamber was considered
insignificant. After measurement of standard metabolic rate
(O2 and
CO2), frogs were fed mice
or rats (44 ± 4 g). The frogs did not eat voluntarily but did swallow
the meal whenever it was placed in the mouth. Measurements were then
repeated with 24-h intervals until 144 h after feeding. The respiratory
gas exchange ratio was calculated as
CO2/O2.
Series II: Blood and transepithelial flux measurements. For experiments involving blood and tissue sampling, frogs were anesthetized by immersion in a 1-g/l solution of MS-222 (3-aminobenzoic acid ethyl ester; Sigma) and occlusively cannulated in the right femoral artery (4). Catheters were filled with heparinized saline and flushed daily.
After 24 h of recovery from anesthesia, catheterized frogs (424 ± 10 g, n = 8) were transferred to an experimental chamber with a
volume of 16 liters. The chamber was fitted with a transparent lid, but
the sides were covered with black plastic to minimize visual
disturbance. The bottom was covered with 2.5 liters of aerated water at
22 ± 1°C. Tetracyclinhydrochloride (12.5 mg/l) was
added to the water to prevent any bacterial growth. To determine transepithelial fluxes (skin and/or renal system) of acid-base equivalents and blood acid-base and respiratory parameters, blood and
water samples were taken according to the following protocol. Frogs
were allowed a 24-h acclimation period in the experimental chamber,
whereupon the first water sample (beginning of the control flux period)
was taken. After 24 h, a second water sample was taken. A 1-ml control
blood sample was subsequently drawn (defined as time 0) and
analyzed for acid-base and respiratory parameters. Remaining blood was
centrifuged (3,000 rpm for 2 min) to separate plasma and red blood
cells (RBCs). The plasma was stored at 
80°C, and the RBCs
were resuspended in amphibian Ringer and reinjected into the animals.
Immediately after the control sampling of blood, each frog was fed
freshly killed mice or rats (37 ± 2 g). When the meal was swallowed,
1.5 liters of the water in the chamber was changed and shortly
thereafter a water sample was taken. The procedure for blood/water
sampling and water shift was repeated every 24 h until 144 h after
feeding. Occasionally, experiments had to be terminated before 144 h
due to clotting of the catheter. To evaluate if blood chemistry was
influenced by repetitive blood sampling per se, a control group of
frogs (412 ± 11 g, n = 3) was treated as described above,
except that the frogs were not fed.
Series III: Intracellular acid-base parameters, buffer values, and metabolite status. To reduce the number of animals needed for analysis of the influence of feeding on intracellular acid-base parameters and metabolites, catheterized frogs were divided into two groups only: 1) a control group (starved frogs) and 2) a 48-h postprandial group (frogs sampled 48 h after feeding). Sampling 48 h after feeding was chosen because the first two experimental series revealed substantial changes in both metabolic rate and plasma acid-base parameters at this time. Control frogs (395 ± 18, n = 4) were placed in the experimental chamber for at least 96 h, and a blood sample was drawn. Frogs in the other group (438 ± 36, n = 5) were placed in the experimental chamber for 48 h and then fed mice or rats (43 ± 4 g). Forty-eight hours after feeding, a blood sample was drawn. After blood sampling and analysis, frogs in both groups were anesthetized by adding a pH-neutralized solution of MS-222 (final concentration 3 g/l) to the animal chamber. After ~12-15 min, with very limited preceding activity, anesthesia was completed and the sartorius muscle, liver, and ventricle were quickly excised, freeze-clamped, and stored in liquid nitrogen.
Analytic Procedures
Blood measurements. Arterial blood was analyzed for PaO2 and pH using a Radiometer (Copenhagen, Denmark) O2 electrode (E5046-0) and a capillary pH electrode (PS-1 204), respectively. Both electrodes were maintained at 22°C in a BMS Mk3 electrode assembly. Total contents of O2 (CO2) and CO2 (CCO2) in whole blood and plasma were measured as described by Tucker (34) and Cameron (6) and corrected according to Bridges et al. (5). Arterial Pco2 (PaCO2) was calculated from the Henderson-Hasselbalch equation using pK'and CO2 solubility values (
CO2) from
Heisler (13). The plasma HCO3
concentration ([HCO3]) was
calculated as plasma CCO2 CO2×PCO2. Hematocrit (Hct) was determined after 3 min centrifugation at 12,000 rpm in capillary tubes. The apparent RBC bicarbonate
concentration ([HCO3]RBC)
was calculated from the plasma (pl) and whole blood (wb)
CCO2 and Hct using the formula
[HCO3]RBC = { [CCO2]wb [1 (Hct/100)]×[Cco2]pl/(0.01×Hct)} PCO2×CO2. [HCO3]RBC was
expressed per kilogram of RBC water, assuming a fractional water
content of 0.71 as determined for Bufo marinus (19).
Hemoglobin-bound oxygen (HbO2) was calculated as
CO2 O2×PaO2,
where O2
is the solubility of oxygen in human blood at 22°C (7). Hb
concentration was measured spectrophotometrically after conversion to
cyanmethemoglobin and application of a millimolar extinction
coefficient of 11 (39). Methemoglobin (Hbmet) was measured
according to the method of Benesch et al. (3). The fractional
Hb oxygen saturation (HbO2-sat) was calculated as
HbO2 relative to the O2 capacity of the
functional Hb (i.e., total Hb Hbmet). Plasma
lactate and total ammonium concentrations were measured using standard
enzymatic tests. Plasma chloride was measured by coulometric titration
(Radiometer CMT 10), and sodium and potassium were measured by flame
photometry (Instrumentation Laboratory 243). Plasma osmolality was
measured by a cryoscopic osmometer (Gonotec Osmomat 030), and plasma
protein was measured with the Lowry method (18).
Intracellular acid-base parameters and metabolite measurements.
Intracellular pH and total CO2 in the sartorius muscle
were determined in tissue homogenates as described by Pörtner et
al. (23). In short, frozen tissue samples were ground under liquid nitrogen and metabolism was arrested using 130 mM potassium fluoride (KF) and 5 mM nitrilotriacetic acid (NTA). pH and total CO2
of the supernatant were measured as described above. Total
CO2 was corrected for contamination with extracellular
CO2 (23). Fractional tissue water content and extracellular
water content of sartorius muscle were approximated by values for the
triceps femorius and gracilis complex (38). Finally, intracellular
[HCO3] and
PCO2 were calculated as described for
plasma, using pK'and CO2 solubility values from
Heisler (13). Intracellular metabolites (lactate, total ammonium,
creatine phosphate, creatine, and ATP) were analyzed in sartorius,
ventricle, and liver by standard enzymatic tests after tissue
extraction in 8% HClO4 and neutralization with 3 M
K2CO3 and 0.5 M
HOCH2CH3.
Intracellular nonbicarbonate buffer values. The nonbicarbonate
buffer value for sartorius muscle was determined as described by
Pörtner (22). Approximately 1 g of tissue powder was added to
four times the tissue volume of ice-cold metabolic inhibitor solution
(in mM: 540 KF, 10 NTA, and 5 KHCO3) and briefly vortexed. The tissue homogenate was equilibrated with 0.5, 3, and 7% humidified CO2 (delivered by cascaded Wösthoff gas mixing pumps)
in Eschweiler (Kiel, Germany) tonometers, and the supernatant was
analyzed for pH and total CO2 as described above.
CO2 was
calculated according to Heisler (13), using an osmolality of 850 mM
(osmolality of the supernatant).
Transepithelial transfer of acid-base equivalents. Water
samples (10 ml) were equilibrated to 1% humidified CO2 in
a gas-tight titration beaker thermostated at 30°C. To determine
changes in strong ion difference (
[SID]), water samples
collected at the end of each flux period were titrated back to the pH
of the water samples collected at the beginning of each flux period by
adding either HCl or NaHCO3 from 120 mM standards. pH was
measured with a Radiometer combined pH electrode (GK2401C). Finally,
water samples were analyzed for total ammonia
([NH3]+[NH+4]), with the phenol-hypochlorite method (32) to determine the total transfer of H+ equivalents
[([NH3]+[NH+4]) [SID]] between animal and environmental
water. The total transfer of H+ equivalents was expressed
as a flux rate considering the water volume of the chamber and the body
mass of the frogs.
Statistical Analysis
For the metabolic rate, blood, and transepithelial flux measurements, statistical differences were tested by one-way ANOVA for repeated measurements followed by a Student-Newman-Keuls test. For the intracellular measurements, an unpaired t-test or (if the normality and/or equal variance test failed) the Mann-Whitney rank sum test was used. Differences were accepted to be significant at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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O2 and
CO2
O2 of fasting animals at
rest was 27 ml
O2 · kg1
· h1 (Fig.
1A), which is equivalent to a
standard metabolic rate of 0.54 kJ · kg1 · h1.
After feeding, O2 increased
significantly, peaking at a value approximately three times higher than
the predigestive value 96 h after feeding (Fig. 1A).
O2 remained significantly
elevated above the time zero fasting value throughout the
experimental period (Fig. 1A).
CO2 more or less paralleled
O2 (Fig. 1A), whereby
feeding only induced a minor and nonsignificant decrease in the
respiratory gas exchange ratio (Fig. 1B).
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Blood Respiratory Parameters
PaO2 displayed a minor and nonsignificant decrease after feeding (Fig. 2A), and HbO2-sat (Fig. 2B) remained virtually unaffected. Arterial O2 content, however, was significantly increased throughout the experimental period (Fig. 2C). This was due to a significant increase in Hct (Fig. 3A) and hemoglobin concentration (Fig. 3B) with the mean cellular hemoglobin concentration staying constant (Fig. 3C). There were no changes in blood respiratory parameters with time in unfed control animals (Table 1).
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Extra- and Intracellular Acid-Base Parameters and Nonbicarbonate Buffer Values
Feeding resulted in a pronounced increase in the plasma bicarbonate concentration from a control value of 24.2 to a maximum value of 37.8 mM at 48 h (Fig. 4B). Feeding also elevated PaCO2 from 10.8 to 13.7 mmHg within 48 h, where after it remained nearly constant throughout the experiment (Fig. 4C). Thus the feeding-induced metabolic alkalosis was partially compensated by a concomitant respiratory acidosis, resulting in an only minor rise in pH of 0.08-0.09 pH units at 24-48 h (Fig. 4A). The pH compensatory effect of the respiratory acidosis is best illustrated in a Davenport diagram, where the pH values expected if only the metabolic alkalosis were present, is included for comparison (Fig. 5). At 96 h, pH was back to the control value, but then decreased to a significantly lower value 144 h after feeding (Fig. 4A). The postprandial increase in plasma [HCO3] was paralleled by
an increase in RBC
[HCO3] (Fig.
4B), but changes in the latter were nonsignificant. Repetitive
blood sampling per se did not cause time-dependent changes in arterial
acid-base parameters (Table 1).
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Feeding also resulted in a small intracellular alkalosis (pH = 0.05)
in the sartorius muscle 48 h after feeding (Table
2). The intracellular nonbicarbonate buffer
value of sartorius muscle was not significantly affected by feeding. It
was 40.6 ± 6.1 mmol · pH
unit1 · kg1
tissue (n = 3) before feeding and 34.5 ± 1.4 mmol · pH
unit1 · kg1
tissue (n = 3) after feeding.
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Transepithelial Fluxes of Acid-Base Equivalents and Ammonia
The total ammonia excretion rate increased strongly from a low value of 9 µmol · kg1 · h1
before feeding to ~400
µmol · kg1 · h1
between 72 and 120 h after feeding (Fig.
6A). The predigestive H+ excretion rate was low and not significantly different
from zero, and the H+ excretion rates after feeding were
not significantly different from the control value (Fig. 6B).
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Plasma Osmolality, Protein Concentration, and Ionic Status
Plasma osmolality (Fig. 7A) and plasma protein concentration (Fig. 7B) did not change significantly after feeding. Plasma [Na+] and [K+] did not change (Fig. 8, A and B, respectively), whereas plasma [Cl] declined
significantly (Fig. 8C) in the postprandial period. Changes in
plasma [Cl] and
[HCO3] were close to
showing an inverse 1:1 molar relationship (Fig.
9). Blood sampling per se had no effect on
plasma osmolality, protein concentration, or ionic status (Table 1).
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Extra- and Intracellular Metabolite Status
The plasma total ammonia content was significantly increased 96 h after feeding (Fig. 10A), but lactate remained low and nearly constant during the whole postprandial period (Fig. 10B). There were no significant changes in these parameters in nonfed control animals (Table 1). The extracellular increase in the ammonia concentration was accompanied by a significant increase in the sartorius muscle ammonia concentration 48 h after feeding (Table 3). Feeding resulted in significant decreases in the phosphocreatine content of sartorius muscle and ventricle (Table 3). The postprandial drop in ventricular phosphocreatine level was more or less mirrored by a concomitant, although nonsignificant, increase in the creatine content, whereas the sartorius muscle creatine content stayed constant. Despite the drop in phosphocreatine levels, ATP remained constant in all tissues after feeding (Table 3).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CO2
O2 in the bullfrogs
was reached 96 h after feeding (Fig. 1), compared with <12 h in fish
(16), ~24 h in a monitor lizard (29), and up to 48 h in snakes (12, 28, 30). The heterogeneous values reported for intensity and duration
of SDA is probably due to differences in meal size and composition,
temperature, extent of hypertrophy of the gut, and standard metabolic rate.
The predigestive respiratory gas exchange ratio of ~0.8 (Fig. 1B) suggests oxidation of a mixture of lipid, protein, and carbohydrate substrates. Although nonsignificant, the decrease in the respiratory ratio to ~0.7 throughout the postprandial period may be related to a shift to a nearly exclusively lipid-based ATP production. The increased ammonium excretion (Fig. 6A), however, suggests increased oxidation of amino acids as well.
Blood Respiratory Parameters
PaO2 and HbO2-sat remained high during digestion (Fig. 2, A and B) as previously reported for chronically cannulated Bufo marinus after peptone injection into the stomach (36). Because of central vascular right-to-left shunt, systemic arterial blood in anurans constitutes a mixture of systemic venous blood and pulmonary venous blood (37). Right-to-left shunt decreases systemic arterial O2 levels relative to left atrial blood and most likely explains why HbO2-sat was ~0.9 (Fig. 2B). The constant HbO2-sat throughout the experiment may indicate that the right-to-left shunt was not affected by digestion, but a complete analysis would have required obtaining additional blood samples from the left and right atria. Nevertheless, it can be concluded that blood in the pulmonary circulation attains virtually full HbO2-sat during the postprandial period. In contrast to our study and the study of Wang et al. (36), Secor and Diamond (27) reported that blood PO2 of pythons plunges to ~20 mmHg during digestion. However, because blood samples were obtained by cardiac puncture, this value is unlikely to reflect arterial values in undisturbed animals.The increased Hct (Fig. 3A) and Hb concentration (Fig.
3B) after feeding is in line with the finding of Wang et al.
(36) that hemoglobin concentration increases after injection of amino acids into the stomach of the toad Bufo marinus. To our
knowledge, there are no other reports on changes in hematological
parameters in lower vertebrates after feeding. The increase in Hct may
result from release of RBCs from the spleen or a reduction in plasma volume caused by water shift to intracellular body compartments. Furthermore, a water shift to the gastrointestinal system, driven by
secretion of osmotic active solutes (e.g., chloride) or disarticulation of the prey, is possible. Considering the increased demand for oxygen
transport by the cardiovascular system during digestion, a release of
RBCs seems likely. The increase in Hct observed in many fish species
during hypoxia results, at least partly, from splenic contractions
(14). However, the increase in Hct caused by hypoxic exposure in
cold-submerged Rana catesbeiana was due to decreased plasma
volume rather than release of RBCs from the spleen (20). There was no
indication of RBC swelling in the present study, as the mean cellular
Hb content stayed constant throughout the experiment (Fig. 3C).
Furthermore, there were only small changes in plasma osmolality (Fig.
7A) and protein (Fig. 7B), and intracellular lactate
remained low (Table 3). Thus a reduction of the plasma volume caused by
osmotic water movement to the intracellular space or stomach seems
unlikely. It is suggested that the increase in Hct and Hb concentration
is due to a release of RBCs from the spleen to improve oxygen
transport. In support of this, changes in Hct and Hb concentration and
changes in metabolic rate showed similar time courses (Figs. 1A
and 3, A and B). Furthermore, the frogs with the lowest
Hct values before feeding attained the highest Hct values after feeding
(Fig. 11), which seems difficult to
relate to osmotic fluid shifts. One reason for this correlation could
be that a low predigestive Hct value correlates with depressed metabolic activities (including digestive activities). Thus a higher
postprandial O2 is needed to
prepare those animals for digestion.
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Intra- and Extracellular Acid-Base Status
Repetitive blood sampling per se had no effect on blood acid-base parameters (Table 1). However, it must be noted that the arterial plasma pH and bicarbonate concentration, but not PCO2, differed between control frogs (Table 1) and frogs from the feeding experiments (Fig. 4). This could be a seasonal effect, because the control experiments were performed in August and feeding experiments were from November to January. Seasonal effects on blood acid-base parameters have been previously reported in Rana catesbeiana even after weeks of acclimation at constant temperature (26).Gastric acid secretion into the stomach after feeding induced a
significant alkaline tide in arterial blood (Figs. 4 and 5). The
acid-base status was composed of a large metabolic alkalosis (increase
in bicarbonate), which was counteracted by a respiratory acidosis
(increase in carbon dioxide tension). The increase in plasma
[HCO3] was mirrored by a
decrease in [Cl] (Fig. 9), which
probably is a consequence of active gastric H+ secretion by
the H+-K+-ATPase followed by passive
K+ and Cl diffusion (25). The
respiratory compensation of the metabolic alkalosis was substantial, as
pH would have increased by 0.18 units 48 h after feeding compared with
the observed 0.08 units if PCO2 had
remained constant (Fig. 5). An increase in PaCO2 during digestion was also
suggested in Bufo marinus (36). This response most likely
results from a reduction in ventilation relative to the rate of
CO2 production, although the possibility that cutaneous
CO2 conductance decreased during digestion cannot be
dismissed. A similar response was also observed in pythons (unpublished
observation), and, consistent with a relative hypoventilation, end-tidal PCO2 increases during
digestion in varanid lizards (11). Thus it appears that a respiratory
compensation of the metabolic alkalosis may be a universal response
among air-breathing ectotherms.
The respiratory compensation of the metabolic alkalosis seen in
digesting frogs and pythons may limit inappropriate changes in blood
oxygen affinity. pH in the RBCs was not measured in the present study,
but the relative changes in RBC and plasma
[HCO3] were similar (Fig.
4B), indicating that the plasma metabolic alkalosis was
transferred to the RBCs. The rise in pH will increase the Hb oxygen
affinity via the Bohr effect, which tends to reduce the unloading
PO2 in tissue capillaries, thus
compromising tissue O2 delivery in a situation where
O2 demand is actually increased and where a lowered
O2 affinity would be favorable (15). The respiratory
acid-base compensation can therefore be viewed as a means to limit the
unfavorable rise in oxygen affinity. In contrast to frogs and pythons,
alligators develop a very large postprandial rise in pH (up to 0.3 pH
units; Ref. 8) and apparently no respiratory compensation takes place
(38). However, due to the unique allosteric binding of bicarbonate to
Hb in alligators (2), the alkaline tide is expected to reduce the
oxygen affinity and therefore improve oxygen unloading in the tissues.
The alkaline tide was also present in the intracellular space (Table
2), but the intracellular acid-base changes were small, suggesting that
another reason for the postprandial hypercapnic acidosis could be
protection of pH-sensitive metabolic pathways from the deleterious
effect of a large alkalosis. Finally, the respiratory acidosis might
play a role in gastric acid secretion, because an elevation of
PCO2 is supposed to speed up carbonic
anhydrase catalyzed proton formation in parietal cells (31).
Considering the above discussion, at least one important question
remains. Why is the minor postprandial alkalosis not completely
compensated? One reason could be that a (further?) reduction of the
ventilation relative to the CO2 production might cause a
decrease in lung PO2 that would
compromise O2.
Between 48 and 72 h after feeding, plasma
[HCO3] started to decrease
in the bullfrog (Figs. 4B and 5). The decline in
HCO3 levels may relate to increased base secretion to the intestine and decreased acid secretion to the
stomach as food moves through the gastrointestinal tract.
The measured changes in acid-base parameters allow an estimate of the
relative importance of the intra- and extracellular spaces in providing
protons for gastric HCl secretion. The estimate is presented in Table
4 and is based on the assumptions that the
nonbicarbonate buffer value and the change in acid-base parameters in
the sartorius muscle apply to all intracellular compartments. The blood
nonbicarbonate buffer values were calculated from the relationship
between Hct and buffer value provided by McDonald et al. (19) for
Bufo marinus. The tissue water distribution, extracellular
volume, and blood volume were adopted from Thorson (33). The increase
in extra- and intracellular base excess after 48 h of digestion amounts
to 3.4 and 2.1 mmol/kg animal, respectively, suggesting that
both compartments provide protons for gastric acid secretion. Although
nonsignificant, the change from net transepithelial acid excretion
before feeding to a net acid uptake between 24 and 48 h after feeding
(Fig. 6B) indicates that appropriate changes in
extragastrointestinal transepithelial fluxes of acid-base equivalents may also contribute to acid secretion to the stomach. An estimate of
the total proton secretion into the stomach is not, however, possible
from the data in the present study, because proton turnover from
metabolism is likely to change after feeding (21).
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Metabolite Status
Feeding caused a significant reduction in the phosphocreatine content in the sartorius muscle and ventricle, but ATP levels remained sufficiently buffered (Table 3). As phosphocreatine is a very sensitive indicator of metabolic stress, this may indicate that oxygen and/or substrate delivery to some tissues was compromised during digestion. High-energy phosphates displayed similar changes in the ventricle of Bufo marinus during long-term exposure to severe environmental hypoxia (24). In the anuran heart, which lacks a coronary circulation, a lowering of PO2 in systemic venous blood (resulting from a higher tissue oxygen extraction) may compromise luminal oxygen delivery to the ventricle and therefore contribute to limiting the intensity of the SDA response. In accordance with that, blood drawn from the ventricle of the snake Python molurus showed an 80% decrease in PO2 after feeding (27). The decrease in phosphocreatine levels was not mirrored by a significant accumulation in intracellular lactate (Table 3), which could be either due to a low production rate or a continued release to the blood with subsequent dilution, excretion, or metabolism by other organs (Fig. 10B).Perspectives
The present study is the first integrative study of changes in metabolism, respiratory parameters, and acid-base status after feeding in a cannulated ectothermic vertebrate. Feeding was associated with a large increase in Hct, which appeared due to release of RBCs from the spleen. Further experiments (e.g., involving spleen ligation) would be informative in providing a definite conclusion regarding the mechanism of the Hct increase and in evaluating its importance for safeguarding blood O2 transport during digestion. The extracellular metabolic alkalosis caused by gastric acid secretion after feeding was countered by an increase in PaCO2. This response may help limit the oxygen affinity increase associated with a pH increase, which lowers the unloading oxygen pressure in the tissue capillaries. If this idea is correct, then a comparison with alligators is desirable, because their oxygen-linked binding of bicarbonate to Hb reduces the oxygen affinity during an alkaline tide. Metabolic data indicate that the intensity of the SDA response might be limited by the aerobic capacity of the heart. This could be a special feature in anuran amphibians, which lack coronary circulation and therefore depend on luminal oxygen delivery to the ventricle. Experiments on amphibians with and without coronary arteries may shed light on this possibility. It would also be interesting to see ifO2 reaches a plateau when the
meal size is increased beyond what was used in this study.
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ACKNOWLEDGEMENTS |
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This study was supported by a grant to the Danish Centre for Respiratory Adaptation from the Danish Natural Science Research Council.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Busk, Institute of Biology, Odense Univ., Campusvej 55, DK-5230 Odense M, Denmark (E-mail: busk{at}biology.ou.dk).
Received 20 April 1999; accepted in final form 24 August 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) and carbon dioxide release
(
; A) and in
respiratory gas exchange ratio (RE; B) in
Rana catesbeiana after a food intake of ~
). Dashed lines are PCO2
isopleths, and dotted lines are nonbicarbonate buffer lines calculated
from relationship between hematocrit and buffer value provided for
Bufo marinus (
