INTRODUCTION

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ON THE BASIS OF permeation coefficients alone, CO₂ should be more diffusible in tissue than O₂ (39). However, in teleost fish, O₂ uptake appears to be perfusion limited, whereas CO₂ excretion behaves as a diffusion-limited system (7, 8, 25, 27, 28, 33). This apparent discrepancy likely reflects chemical equilibrium limitations on CO₂ excretion. The majority of CO₂ that is excreted is transported in the plasma as bicarbonate ions owing to the low solubility of plasma for CO₂ (4). Because the rate of the uncatalyzed dehydration of HCO (rate constant >20 s; see Ref. 10) is low relative to the gill transit time (1-3 s; see Ref. 7), effective CO₂ excretion depends largely on the dehydration of HCO within the red blood cell (RBC), where it is catalyzed by carbonic anhydrase (CA) (11, 17, 28, 42). HCO ions gain access to the RBCs via the electroneutral Cl/HCO exchanger (anion exchanger 1 isoform, band 3 protein) on the RBC membrane (6, 22, 26, 37). The entry of HCO into the RBC via this process is believed to be the rate-limiting step in CO₂ excretion (9, 28). In addition to the CA present in vertebrate RBCs, mammals possess a pulmonary membrane-associated CA (CA IV) with an extracellular orientation that is absent from the gills of teleost fish [see the review by Henry and Swenson (18)]. Consequently, in teleosts, the slow rate of conversion of HCO to CO₂ (due to chemical equilibrium limitations) in plasma may result in apparent diffusion limitations.

In theory, in a diffusion-limited system, gas transfer efficiency (a measure of the ability of arterial blood gas tensions to reach equilibrium with the inspired medium) is affected by changes in blood transit (residence) time within the respiratory organ. Thus, for a gas where the time to diffusion equilibrium across the respiratory barrier exceeds the residence time, a decrease in transit time would be expected to lower the efficiency of transfer, whereas an increase in transit time would be expected to increase gas transfer efficiency. In contrast, gas transfer efficiency in a perfusion-limited system is maintained despite changes in blood flow until the residence time of blood at the gas exchange surface approaches the diffusion equilibrium time of the respiratory gas.

Although several studies have investigated the sensitivity of O₂ uptake to manipulation of cardiac output (b) in assessing the perfusion and diffusion limitations on O₂ transfer in fish (8, 27), few have measured the effects of changes in b on CO₂ excretion. Utilizing a spontaneously ventilating blood-perfused trout preparation, Perry (28) reported an increase in the arterial partial pressure of CO₂ (Pa_CO2) with elevation of b. Recently, Brauner and colleagues (5) observed that when b was increased during sustained exercise in rainbow trout, there was an associated elevation of Pa_CO2. These data suggest that transit-time limitations exist for CO₂ excretion in rainbow trout.

With this background, the objective of the present study was to test the hypothesis that CO₂ transfer efficiency in rainbow trout would be sensitive to changes in blood transit time in the gill because CO₂ excretion behaves as a diffusion-limited system, and that this sensitivity would reflect chemical equilibrium constraints. b was manipulated by vascular volume loading or by blood withdrawal; changes in b were assumed to inversely modify the blood residence time in the gills. Gas transfer efficiency was estimated by continuous online monitoring of arterial blood gases (because efficiency is defined as the difference between arterial and venous gas tensions divided by the difference between venous and inspired gas tensions). Intravenous administration of bovine CA, to reduce the need for RBC HCO dehydration (9), was used to assess the role of chemical equilibrium constraints in any apparent CO₂ transfer diffusion limitations.

	METHODS

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Experimental animals. Rainbow trout (Oncorhynchus mykiss) of either sex were obtained from Linwood Acres Trout Farm (Campbellcroft, Ontario). Fish were maintained on a 12:12-h light-dark photoperiod in large, circular fiberglass aquaria supplied with flowing, aerated, and dechlorinated tap water (from the city of Ottawa) at 13°C. Fish were allowed at least 2 wk to acclimate to the holding conditions before any experiments were performed and were fed to satiation on alternate days with a commercial trout pellet diet until 24 h before experimentation. Two groups of fish were used. In one group [n = 60, body wt 555 ± 16 g (mean ± 1 SE)], blood respiratory variables were measured in vivo by means of an extracorporeal blood shunt (see Experimental protocol). A second group of fish (n = 14, body wt 611 ± 36 g) acted as blood donors for blood-removal experiments. All experimental protocols were previously approved by the University of Ottawa Animal Care Committee in accordance with guidelines provided by the Canadian Council on Animal Care.

Experimental protocol. An extracorporeal blood shunt (41) was used to continuously monitor arterial or venous O₂ tension (Pa_O2 and Pv_O2, respectively) and CO₂ tension (Pa_CO2 and Pv_CO2, respectively). Blood withdrawn from the caudal artery or vein using a peristaltic pump was passed through an external circuit containing electrodes that measured partial pressures of O₂ and CO₂ (PO₂ and PCO₂, respectively) before being returned to the fish via the other cannula. The flow rate through the external loop, which contained ~1 ml of blood (<4% of a fish's blood volume), was 0.6 ml/min. Immediately before the intravascular cannulae were attached, the extracorporeal shunt was rinsed for 10-15 min with heparinized (540 IU/ml) saline to prevent blood from clotting in the tubing [a combination of PVC peristaltic pump tubing (internal diameter = 0.6 mm) and PE 50 tubing] and electrode chambers. After the extracorporeal blood shunt was initiated, ~20 min were required to obtain stable readings for blood gas variables as well as blood flow and ventilation parameters. Upon stabilization, experiments commenced with a 10-min period of baseline recording followed by one of four procedures.

1

1

Analytical procedures. In experiments utilizing the extracorporeal blood shunt, arterial or venous blood PCO₂ and PO₂ were monitored using CO₂ and O₂ electrodes (Cameron Instruments) housed in thermostatted cuvettes and connected to a blood gas analyzer (Cameron Instruments). The O₂ electrode was calibrated by pumping a zero solution (2 g/l sodium sulfite) or air-saturated water continuously through the circuit until stable readings were recorded. The CO₂ electrode was calibrated in a similar manner using mixtures of 0.5 and 1.0% CO₂ in air that were provided by a gas-mixing flowmeter (model GF-3/MP, Cameron Instruments). Electrodes were calibrated before each experiment. The frequency and amplitude of opercular displacements were assessed as indices of ventilation by use of a custom-built impedance converter that detected and quantified the changes in impedance between the brass plates attached to the opercula (32). b was determined by connecting the ultrasonic flow probe to a small animal-blood flow meter (model T106, Transonic Systems). All analog signals (blood gases, impedance values, and b) were converted to digital data and stored by interfacing with a data acquisition system (Biopac Systems) using Acknowledge data acquisition software (with sampling rate set at 30 Hz) and a Pentium PC. Hematocrit was determined in duplicate by centrifuging microcapillary tubes at 5,000 g for 10 min.

Statistical analyses. All data are presented as means ± 1 SE. For experiments using the extracorporeal blood shunt, means for blood gases, ventilatory data, and b were compiled for 2-min periods from 10 min before until 60 min after intravascular volume changes. Owing to the variation within the population and the small magnitude of the observed effects, data for individual fish for blood gases, ventilatory variables, and b were normalized by subtracting from each data point the value at time 0 (the point of blood volume modification). The statistical significance of differences in initial absolute values among treatments was assessed by one-way ANOVA or unpaired Student's t-tests, as appropriate. Two-way repeated-measures ANOVA followed by the Bonferroni post hoc multiple comparisons test, as appropriate, was used to assess the statistical significance of differences in blood gases, ventilatory variables, and blood flow with time and treatment. The fiducial level of significance was considered to be 5%, and a commercial software package (Sigmastat version 2.03) was used to perform all statistical analyses. The sigmoidal curve in Fig. 6A was fitted to the data using a curve-fitting option in a commercial software package (SigmaPlot 2000, SPSS).

	RESULTS

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Absolute values for cardiorespiratory variables before the modification of b are presented in Table 1. Apart from the expected differences between venous and arterial blood values, the only significant difference among the groups was a higher initial Pa_O2 in fish that were later subjected to blood withdrawal than in those that were subsequently volume loaded. The effectiveness of volume manipulations in eliciting changes in b is illustrated in Figs. 1A and 2A. Addition to the circulation of 10 ml/kg saline containing 3% BSA increased b by on average 50% over an untreated control group, whereas plasma removal was associated with a 52% decrease in b. These changes in b in turn were accompanied by significant changes in Pa_CO2. The injection of saline resulted in a significant elevation of Pa_CO2 (maximum increase = 0.28 ± 0.03 mmHg; Fig. 1B); Pa_O2 was not affected (Fig. 1C). Although not statistically significant, saline injection tended to decrease the hematocrit (from 22.3 ± 1.7 to 18.8 ± 1.3%; n = 10). The removal of plasma resulted in a significant decrease in Pa_CO2 (maximum change = 0.40 ± 0.08 mmHg; Fig. 2B). In this case, a small but significant fall in Pa_O2 occurred (Fig. 2C). Although not statistically significant, plasma removal tended to increase the hematocrit (from 26.5 ± 2.1 to 31.5 ± 4.4%). All measured variables remained constant throughout the experimental period in the untreated control fish.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effects of vascular volume loading (10 ml/kg; , n = 11) on changes in cardiac output (b; A), arterial partial pressure of CO2 (PaCO2; B), and arterial partial pressure of O2 (PaO2; C) in rainbow trout (Oncorhynchus mykiss). Control fish were not treated (, n = 6). Dashed vertical line at time 0 indicates the point of saline injection. Data are means ± 1 SE; horizontal line, P < 0.05, significant differences from the final preinjection values (time 0); P < 0.05, significant differences from the corresponding value in the control group.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Effects of blood plasma removal (8-12 ml; , n = 7) on changes in b (A), PaCO2 (B), and PaO2 (C) in rainbow trout. Control fish (same data as in Fig. 1) were untreated (, n = 6). Dashed vertical line at time 0 indicates the beginning of blood removal. Data are means ± 1 SE; horizontal line, P < 0.05, significant differences from final preremoval values (time 0); P < 0.05, significant differences from the corresponding value in the control group.

To ensure that the increase in Pa_CO2 in volume-loaded fish was not a consequence of changes in tissue CO₂ production, the effects on arterial blood gases of increasing b were compared with those on venous blood gases. Although volume loading induced similar increases in b in both groups of fish, venous PCO₂ was unaffected (Fig. 3).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of vascular volume loading (10 ml/kg) on changes in b (A) and PCO2 (B) described as PaCO2 (, n = 11) or PvCO2 (, n = 6) in rainbow trout. Data for fish in which arterial blood was monitored () are replotted from Fig. 1. Dashed vertical line at time 0 indicates the point of saline injection. Data are means ± 1 SE; horizontal line, P < 0.05 and *P < 0.05, significant differences from the final preinjection values (time 0); P < 0.05, significant differences from the corresponding value in the control group (in which arterial blood was monitored).

Absolute values for b, blood gases, and ventilation parameters were similar before the elevation of blood volume for control and CA-treated fish (Table 2). Despite b being elevated to the same extent as in the control group (Fig. 4A), pretreatment of fish with CA prevented the increase in Pa_CO2 that is normally associated with volume loading (Fig. 4B). A significant but transient fall in Pa_O2 immediately after volume loading was observed for both groups and was significantly larger in control fish than in CA-treated fish (Fig. 4C).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of vascular volume loading (10 ml/kg) on changes in b (A), PaCO2 (B), and PaO2 (C) in rainbow trout pretreated with bovine carbonic anhydrase (10 mg/kg) (, n = 9) or saline vehicle alone (control; , n = 9). Dashed vertical line at time 0 indicates the beginning of volume loading. Data are means ± 1 SE; *P < 0.05, significant differences from the final preloading values (time 0); P < 0.05, significant differences from the corresponding value in the control group.

Volume loading was accompanied by significant increases in ventilation (Fig. 5). In control fish, both frequency and amplitude were increased, whereas only ventilation amplitude was increased in the CA-treated fish. Further, the elevation of ventilation amplitude was significantly greater in the control fish than in the CA-treated fish (Fig. 5B).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of vascular volume loading (10 ml/kg) on changes in ventilation frequency (Vf; A) and ventilation amplitude (Vamp; B) in rainbow trout pretreated with bovine carbonic anhydrase (10 mg/kg) (, n = 9) or saline vehicle alone (control; , n = 9). Dashed vertical line at time 0 indicates the beginning of volume loading. Data are means ± 1 SE; *P < 0.05, significant differences from the final preloading values (time 0); P < 0.05, significant differences from the corresponding value in the control group.

Figure 6 depicts the relationship between changes in b and arterial blood gas tensions for all fish subjected to volume loading or blood withdrawal except those pretreated with CA. Over the entire range of blood flows obtained, a significant sigmoidal relationship between Pa_CO2 and b (r² = 0.75; P < 0.05) was observed (Fig. 6A). Furthermore, Pa_CO2 demonstrated a significant linear relationship with b (r² = 0.72; P < 0.05) within the narrower range of b changes of 3 to 11 ml · min¹ · kg¹ (as illustrated in Fig. 6A, inset). Figure 6B presents the relationship between changes in b and the corresponding changes in Pa_O2; no significant correlation was observed (r² = 0.07; P > 0.05). Among fish treated with CA, no significant correlation between Pa_CO2 and b was detected (r² = 0.24; P > 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Relationship between b and the changes in PaCO2 (n = 36; A) and PaO2 (n = 33; B) in rainbow trout. Changes in b were elicited by vascular volume loading or plasma removal. Note the significant sigmoidal correlation (r2 = 0.75; P < 0.05) between b and PaCO2. Inset in A represents the linear portion of the curve (r2 = 0.72; n = 20) shaded in gray. b and PaO2 were not significantly correlated (r2 = 0.07).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of the present study constitute the first direct in vivo evidence that Pa_CO2 is sensitive to changes in b over the physiological range. Because the sensitivity of Pa_CO2 (and hence CO₂ transfer efficiency) to changes in b is a hallmark of a diffusion-limited system (43), the current results confirm the indirect findings of previous studies, i.e., that CO₂ excretion in teleost fish behaves as a diffusion-limited process. The observation that the transit-time limitations on Pa_CO2 were relieved by providing plasma CO₂ reactions with access to CA activity speaks to the key role played by chemical equilibrium limitations and reveals that diffusion per se does not constrain CO₂ excretion. These data were collected for rainbow trout, a teleost fish; it was chosen for the present study because, apparently uniquely among vertebrates, teleost fish lack plasma-accessible CA at the gas-exchange surface and are therefore ideal model organisms in which to conduct experiments involving the manipulation of CA availability (18).

Owing to its high capacitance in water and tissue, the permeation coefficient of CO₂ is ~30-fold greater than that for O₂. Previous studies, however, have suggested that CO₂ excretion in fish behaves as a diffusion-limited system whereas O₂ uptake is perfusion limited (5, 8, 25, 27, 33, 39). In a diffusion-limited system, gas transfer efficiency is inversely related to b, because transit time through the respiratory structure is largely set by b. However, capillary recruitment and/or distension, both of which would tend to decrease blood flow velocity, likely lessen the impact of increased b on transit time (19, 34). In contrast, gas transfer efficiency in a perfusion-limited system remains constant during changes in transit time within the physiological range because of the rapid rate of equilibration of gases between the respiratory medium and the blood. A perfusion-limited system could, however, become diffusion limited if the residence time of the blood in the gas exchange surface was decreased so as to approach the diffusion equilibration time of the respiratory gas. Because the partial pressures of gases in the blood leaving the exchange surface provide an index of transfer efficiency (provided that as in the present study, inspired and venous gas tensions remain constant), changes in arterial blood gas tensions with changes in b (hence transit time) can readily demonstrate diffusion limitations. This approach was used in the present study. The teleost gill lacks plasma-accessible CA (35) and therefore blood exiting the gill is in a state of acid-base disequilibrium [reviewed by Gilmour (12)]. Consequently, there are downstream increases in PCO₂ in the arterial blood owing to the continuing uncatalyzed dehydration of plasma HCO (15). Fortunately, the magnitude of these changes is negligible (i.e., PCO₂ = 0.04 mmHg), and thus the measurement of Pa_CO2 can be used as an accurate estimate of postbranchial PCO₂.

In the present study, O₂ uptake fit the criterion of a perfusion-limited system in that Pa_O2 was largely constant despite significant changes in b and the presumed concomitant changes in the transit time of blood through the gills. In some instances, Pa_O2 declined immediately after the volume loading. Frequently, saline injections into the venous circulation induced a sudden and profound apparent reflex decrease in b lasting 10-40 s (P. R. Desforges, unpublished observations; see Fig. 5). It is possible that the immediate reduction in Pa_O2 in volume-loaded fish may have been related to this injection artifact. Importantly, however, the reductions in Pa_O2 (unlike the increases in Pa_CO2) were transient and not synchronized with the long-lasting increases in b. Moreover, correlation analysis revealed that there was no significant relationship between Pa_O2 and b (see Fig. 6B). Thus, despite the unexplained transient decline in Pa_O2 in volume-loaded fish, we are confident that the overall results of the current study support the notion of perfusion-limited O₂ transfer.

Whereas O₂ uptake is perfusion limited, the significant relationship between Pa_CO2 and b indicated that CO₂ excretion was effectively diffusion limited. Several additional lines of evidence from recent in vivo studies support this contention. Julio and colleagues (23) observed a significant increase in Pa_CO2 (Pa_O2 was unchanged) in trout experiencing a 30% reduction in gill surface area as a result of selective gill-arch ligation. Similarly, hormonally induced (using cortisol and growth hormone) thickening of the blood-to-water diffusion barrier also caused a specific impairment of CO₂ transfer (3). Unlike the present investigation, however, these previous studies did not focus specifically on the assessment of diffusion and perfusion limitations; thus their interpretation was confounded by uncontrolled changes to ventilation and the absence of blood flow measurements.

The increase in Pa_CO2 that was observed in the present study during volume loading could not be explained by changes in prebranchial PCO₂ because venous PCO₂ was unaltered in the volume-loaded fish. Similarly, although volume loading was associated with an increase in ventilation, such a response would be expected to lower Pa_CO2 (21). Interestingly, the fish that were pretreated with CA exhibited a smaller increase in ventilation after volume loading. Thus ventilatory adjustments clearly did not contribute to the changes in Pa_CO2 that accompanied the variations in b, nor could they explain the effect of CA on these changes. Finally, dilution of the blood after volume loading may have reduced the circulating concentration of RBC Cl/HCO exchangers and thus impeded overall CO₂ excretion. Given the small change in hematocrit (from 22.3 to 18.8%) that accompanied volume loading, this possibility seems unlikely. Indeed, such a variation in hematocrit is both common within trout populations and without any apparent impact on gas transfer. Much larger reductions in hematocrit (to ~5%) are required to impair CO₂ excretion (46).

The present study has demonstrated that the overall process of chemical conversion of HCO to CO₂ is responsible for the apparent diffusion limitations on CO₂ excretion and its sensitivity to transit-time changes. Although HCO is dehydrated rapidly at the catalyzed rate by RBC CA, the RBC Cl/HCO exchanger (20), by providing the pathway for HCO entry into the RBC, sets the rate at which catalyzed HCO dehydration occurs (6, 22, 26, 29, 30). As the slowest step in the cascade of events that comprise CO₂ excretion, Cl/HCO exchange is also believed to be the rate-limiting step in this process (28). Experimental support for this hypothesis was recently provided by Desforges and colleagues (9), who demonstrated that addition of CA to the plasma of trout in vivo caused a significant and rapid lowering of Pa_CO2. Thus it is not an intrinsically low activity of RBC CA per se that is the basis for CO₂ chemical equilibrium limitations in teleosts. Rather, CO₂ excretion is constrained by the relatively slow delivery of substrate (HCO).

The dehydration of plasma HCO requires equimolar quantities of H⁺ that are largely derived from non-HCO buffers. Despite the low buffering capacity () of trout plasma [approx. 3 mmol · l¹ · pH unit¹; (46)], the addition of extracellular CA fully relieved the transit-time limitations imposed by the elevated b. This finding suggests that inadequate buffering did not limit the catalyzed dehydration of HCO in the plasma, at least at the rates required to eliminate the transit-time limitations. Similarly, Desforges and colleagues (9) demonstrated that the capacity of injected exogenous CA to lower Pa_CO2 in trout was unrelated to  within the range 3.9 to 12.1 mmol · l¹ · pH unit¹. Thus unlike in mammals, where low plasma  is believed to restrict the contribution of pulmonary endothelial CA to <10% of total CO₂ excretion (1, 39), the lower rates of CO₂ excretion in fish may allow for a greater contribution of extracellular HCO dehydration when CA is added to the plasma. Indeed, in dogfish, a species that is known to posses plasma-accessible gill-membrane-bound CA (13, 16), the extracellular dehydration of HCO contributes significantly to overall CO₂ excretion (14).

During exercise in trout, b can rise by as much as fourfold, and this increase is associated with an estimated reduction in gill transit time from 3 to 1 s (36). Based on the results of the present study, such changes in b during exercise would be expected to impose transit-time limitations on CO₂ transfer and hence cause an elevation of Pa_CO2 (independently of the potential impact of elevated CO₂ production). Indeed, in most fish species that have been examined, exercise results in a marked elevation of Pa_CO2 without any concomitant reduction in Pa_O2 (see Ref. 45). The possible cause(s) of the postexercise respiratory acidosis have been debated previously (45), and only recently has diffusion limitation been suggested to be a causative factor (5). Given the results of the present study, the simplest explanation is that CO₂ transfer, by behaving as a diffusion-limited system, is constrained by the reduced transit time such that Pa_CO2 rises. Pa_O2 is unaffected by the transit-time reductions (as in the present study) because O₂ transfer behaves as a perfusion-limited system. It is less clear to what extent transit-time reductions would affect Pa_CO2 in exercising mammals. It has been argued that during intense exercise, pulmonary transit time may fall below the time required to complete RBC Cl/HCO exchange (1) and thus may impede CO₂ excretion. Whether the activity of pulmonary endothelial CA could supplement CO₂ excretion under such conditions to enable Pa_CO2 to be maintained is uncertain. Indeed, the contribution made to CO₂ excretion by pulmonary endothelial CA IV remains unclear, with estimates ranging from <10% to >40% (2, 24, 40), although the consensus appears to be that its contribution is limited to <10% in vivo owing to limited H⁺ availability in the plasma (18).

In conclusion, the present study utilized experimental manipulation of the blood transit time through the gill in vivo to demonstrate apparent diffusion limitations on CO₂ excretion but not O₂ uptake. Because CA eliminated the apparent diffusion limitation on CO₂ excretion, the results indicate that rather than a true diffusion limitation, CO₂ excretion is constrained by a chemical equilibrium limitation.