Reasons for performing study: Carbonic anhydrase (CA) catalyses the hydration/dehydration reaction of CO2 and increases the rate of Cl- and HCO3- exchange between the erythrocytes and plasma. Therefore, chronic inhibition of CA has a potential to attenuate CO2 output and induce greater metabolic and respiratory acidosis in exercising horses.
Objectives: To determine the effects of Carbonic anhydrase inhibition on CO2 output and ionic exchange between erythrocytes and plasma and their influence on acid-base balance in the pulmonary circulation (across the lung) in exercising horses with and without CA inhibition.
Methods: Six horses were exercised to exhaustion on a treadmill without (Con) and with CA inhibition (AczTr). CA inhibition was achieved with administration of acetazolamide (10 mg/kg bwt t.i.d. for 3 days and 30 mg/kg bwt before exercise). Arterial, mixed venous blood and CO2 output were sampled at rest and during exercise. An integrated physicochemical systems approach was used to describe acid base changes.
Results: AczTr decreased the duration of exercise by 45% (P<0.0001). During the transition from rest to exercise CO2 output was lower in AczTr (P<0.0001). Arterial PCO2 (P<0.0001; mean ± s.e. 71 ± 2 mmHg AczTr, 46 ± 2 mmHg Con) was higher, whereas hydrogen ion (P = 0.01; 12.8 ± 0.6 nEq/l AczTr, 15.5 ± 0.6 nEq/l Con) and bicarbonate (P = 0.007; 5.5 ± 0.7 mEq/l AczTr, 10.1 ± 1.3 mEq/l Con) differences across the lung were lower in AczTr compared to Con. No difference was observed in weak electrolytes across the lung. Strong ion difference across the lung was lower in AczTr (P = 0.0003; 4.9 ± 0.8 mEq AczTr, 7.5 ± 1.2 mEq Con), which was affected by strong ion changes across the lung with exception of lactate.
Conclusions: CO2 and chloride changes in erythrocytes across the lung seem to be the major contributors to acid-base and ions balance in pulmonary circulation in exercising horses.
Marked increase in CO2 production during exercise is evident from the large increase in pulmonary ventilation (VE) and pulmonary CO2 output (VCO2) (Kowalchuk et al. 1988a; Bayly et al. 1989). Elimination of CO2 by the lungs during exercise constrains the rise in hydrogen ion concentration ([H+]) in contracting muscles (Jones 1980; Kowalchuk et al. 1988b; Geers and Gros 2000). Classical studies by Hamburger (1891, 1918) suggest that when CO2 is being eliminated down its partial pressure gradient from the mixed venous blood to the alveolus the erythrocyte volume would decrease in association with efflux of chloride (Cl-) from erythrocytes to plasma. Movement of Cl- from erythrocytes (chloride shift = Hamburger shift) occurs by the Cl-/bicarbonate (HCO3-) exchanger (AE1, Band 3) (Bretcher 1971) and is responsible for Cl-, partial pressure of CO2 (PCO2), water and [H+] exchanges between erythrocytes and plasma (Hamburger 1918; Jacobs and Stewart 1942; Chow et al. 1976).
Effects of CO2 retention and acidosis on acid base and electrolyte balance across the lung in pulmonary circulation in man, horses or other animals at rest and during exertion have not yet been described. Using the integrated physicochemical systems approach, it is possible within each fluid compartment to describe the influence of 3 independent variables, strong ion difference (SID), PCO2 and total concentrations of weak acids and bases (Atot) on [H+] and [HCO3-], which are considered dependent variables (Stewart 1983). We hypothesised that Hamburger shift and the Jacobs-Stewart cycle play a critical role in acid base homeostasis across the lung. In the present study we used the integratedphysicochemical systems approach to describe acid base changes across the lung in pulmonary circulation in exercising horses without (control) and with CA inhibition (with related metabolic and respiratory acidosis [Swenson 1998, 2000]). Pulmonary circulation volume and acid-base/electrolyte dynamics across the lung should contribute to information with regards to exercise and nonexercise related adaptations in pulmonary diseases.
Six race fit Standardbred horses (3 female, 3 geldings), with a mean age of 5.6 years (range 5–6), mean weight of 450 kg (range 412–482 kg) and mean peak O2 uptake of 160 ml/kg bwt/min (range 141–179 ml/kg bwt/min) were used. Horses were recruited from a private racing stable with owner consent. The study protocols were approved by the Animal Care committee according to the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, Ontario).
Five days prior to the experiment each horse was familiarised to the treadmill. During the first 3 days horses were given repeated walking exercise on the treadmill for 20 min daily (15 min walk, 5 min slow pace) at 10% treadmill inclination, followed by 2 days of exercise with the respiratory mask fitted over their nose, which was used later in the experiment for gas exchange measurements. Before every treadmill exercise horses were weighed, fitted with a safety harness, hobbles and heart rate meter (Equistat Model HR-8A)1.
On Day 6, peak O2 uptake (VO2peak) was determined for each horse, which comprised of 3 treadmill exercise periods: warm-up, incremental exercise and recovery. During the warm-up period the horses were walked on horizontal treadmill with no inclination for 5 min at 2–3 m/s and then trotted for 5 min at 4–5 m/s. At the end of the 10 min warming-up period the treadmill was inclined to 10% and the speed increased to 8 m/s. The incremental exercise consisted of a stepwise increase of velocity of 1 m/s every 60 s. An open flow-through system was used for collection of pulmonary gases throughout the entire exercise protocol. Peak O2 uptake was determined as the point at which no further increase in VO2 occurred, despite an increase in speed, or a level of exercise where the horse could no longer maintain pace with the treadmill speed.
Experiments were carried out 72 h following the determination of VO2peak in a randomised manner. A washout period of at least 8 days was given. During this time horses rested and were hand walked for 15 min daily.
Control experiment (Con)
The skin at the site of catheter insertion for blood collection was clipped, desensitised with lidocaine 2.5% and prilocaine 2.5% cream (EMLA)2 and aseptically prepared. A Pulmonary Swan-Ganz catheter3 and 50 cm long central venous polyethylene blood catheter (#240)4 were placed aseptically via the left and right jugular vein into the pulmonary artery for core body temperature measurements and mixed central venous blood sampling, respectively. Correct catheter placement was assured by observing characteristic pressure waveforms on an oscilloscope (Criticare 1100)5. A 20 gauge catheter (Insyte-W)6 was inserted into the facial or transverse facial artery. A 30 cm long extension tubing with a 3 way stopcock was connected to i.v. and intra-arterial catheters. Catheters and extension tubing were all sutured securely to the skin. Thirty minutes before the experiment horses were given 8 ml of tap water/kg bwt via the nasogastric tube. This is a similar volume to that given to horses when treated with Acz. Horses then rested on the treadmill until their heart rate reached their respective resting value.
The high intensity exercise experiment followed, which consisted of a warm-up phase as outlined in the VO2peak testing in the pre-experimental protocol. The subsequent treadmill speed was then set to the speed velocity producing 80% of the VO2peak determined previously during the VO2peak testing. Horses were exercised until fatigued. During recovery, horses were walked on the treadmill at 2–3 m/s until all blood samples were collected or until horses cooled down.
Acetazolamide treatment (AczTr)
Horses were given Acz (Apo-acetazolamide)7 orally at a dose of 10 mg/kg bwt t.i.d. for 3 days before the experiment. Acetozolamide tablets were crushed and mixed with a mixture of molasses and corn oil, and administered orally with a Toomey syringe8. Horses then underwent the same procedure as described in the control experimental protocol. Thirty minutes before the experiment, horses were given 30 mg/kg bwt of Acz mixed in 8 ml of tap water/kg bwt via a nasogastric tube.
Pulmonary gas collection
Pulmonary gas exchange was measured at rest, during exercise and during the recovery period. Before exercise the respiratory mask was fitted on the horse's nose. An open flow through system was used for collection of expired gases throughout the entire exercise protocol (Wagner et al. 1989). The expired gas was drawn into the O2 analyser (Model S-3A/1)9, which measured the concentration of inspired O2 and expired CO2. Both inspired O2 and expired CO2 were recorded in 10 s periods throughout the experiment. For analysis, the average of 3 measurements in a 30 s period was used, which coincided with blood sampling intervals.
Blood sampling and blood analysis
Horses rested on the treadmill until their heart rate achieved their respective resting value. Resting arterial and mixed venous blood was then collected simultaneously under anaerobic conditions twice in a 5 min interval. Further sampling was performed in 60 s intervals during the exercise period until fatigue. During the recovery period sampling was performed starting after the treadmill was stopped (0 min) and then at 1, 2, 3, 5, 10 and 15 min into the recovery period. Prior to each sampling 10 ml of blood was withdrawn from catheters and discarded. Blood samples were collected into lithium-heparinised syringes (S-Monovette)10, stored on ice, and analysed in duplicates with the Stat Profile M Analyser11 immediately after the treadmill protocol ended. Stat Profile M Analyser uses ion selective electrodes for analysis of Na+, K+, Cl-, as well as for pH and PCO2; amperometry for PO2 and La-; conductivity for packed cell volume (PCV) and conductivity/reflectance for haemoglobin (Hb). The pH, PCO2, HCO3- and PO2 sample values were corrected for the horses’ core body temperature (Model COM-2)12 measured in the pulmonary artery. Total plasma protein was measured using a clinical refractometer. The highest coefficient of variance measured was 2.4%.
Plasma [H+] was calculated from the measured pH as the antilog.
Strong ion difference was calculated as the sum of strong cations minus the sum of the strong anions:
Plasma [Atot] was calculated using a conversion factor of 0.21 mmol/l of plasma (Stämpfli et al. 1999).
Changes in the plasma volume across the lung (ΔPVV-A) were calculated from changes in plasma protein [PP] across the lung (Dill and Costill 1974):
where [PPv] is the plasma protein concentration in venous blood and [PPa] the plasma protein in arterial blood.
Changes in erythrocyte volume (ΔEVV-A) across the lung were calculated during and after exercise from changes in [Hb] and PCV in venous ([Hbv], PCVv) and arterial blood ([Hba], PCVa) (Strauss et al. 1951; Harrison 1985):
All venoarterial differences for plasma ions and proteins were corrected for ΔPVv-a using the equation (McKenna et al. 1997):
A similar correction was made for erythrocyte ion concentration using ΔEVV-A (McKenna et al. 1997).
Design: split-plot with horses as blocks and repeated measures over time. Ventilation, gas exchange, and plasma acid-base variables were analysed using a 2-way repeated-measures ANOVA. The main effects of treatment and time as well as treatment × time interaction were included in the model with appropriate covariance structure to account for making repeated measures on the same animal. A significant F ratio was further analysed using Tukey post hoc analysis. A statistical significance level of P<0.05 was used, and data are expressed as mean ± s.e.
Treatment effect on exercise
Exercise duration at 80% VO2peak was significantly shorter in AczTr (2.6 ± 0.2 min) compared to Con (4.7 ± 0.2 min, P<0.0001).
Resting VCO2 and VO2 did not differ between treatments. VCO2 and VO2 increased during exercise (P = 0.001). During exercise, VCO2 and VO2 were lower in AczTr compared to Con (P<0.05). VO2 did not return to resting value until the last min of recovery in Con and AczTr. VCO2 returned to resting value by the 15th min of recovery in Con and AczTr (Table 1).
Table 1. Respiratory changes, haemoglobin, packed cell volume, plasma protein
All values are means ± s.e. (n = 6). VO2, oxygen consumption. VCO2, pulmonary carbon dioxide Output. PCO2, blood CO2 partial pressure in arterial (PaCO2) and venous blood (PvCO2). PO2, Blood O2 partial pressure in arterial (PaO2) and venous blood (PvO2). Hb, haemoglobin in arterial (Hba) and venous blood (Hbv). PCV, packed cell volume in arterial (PCVa) and venous blood (PCVv). PP, plasma protein in arterial (PPa) and venous blood (PPv). FTG, fatigue. Con, Control. AczTr, Acetazolamide treatment. #, resting, exercise and/or recovery values different between Con and AczTr. *, fatigue and recovery values different from rest. @, variables also expressed in figures.
Resting arterial CO2 partial pressure (PaCO2) in AczTr was lower than in Con (P<0.001). During exercise, PaCO2 rose sharply in AczTr. This was not observed in Con, where PaCO2 remained similar to resting value throughout exercise. PaCO2 during exercise was higher in AczTr compared to Con (P = 0.04). During recovery, PaCO2 decreased below the resting value in Con (P<0.05) and returned to resting value by the end of recovery. In AczTr PaCO2 returned slowly towards resting value (P<0.05), but, in contrast to Con, never decreased below the resting value (Table 1, Fig 1).
Resting venous CO2 partial pressure (PvCO2) in Con and AczTr were not different. During exercise, PvCO2 increased in Con and AczTr (P<0.0001) and was higher in AczTr compared to Con (P = 0.04). During recovery, in Con PvCO2 returned immediately to resting value, whereas in AczTr did not recover until 10 min. Throughout recovery PvCO2 was higher in AczTr compared to Con (P = 0.004) (Table 1, Fig 2).
Resting arterial O2 partial pressure (PaO2) was higher in AczTr than in Con (P<0.001). During exercise, PaO2 decreased in Con and AczTr (P<0.05). PaO2 was higher in AczTr compared to Con during exercise and recovery (P<0.01) (Table 1).
Compared to Con, resting venous O2 partial pressure (PvO2) was higher in AczTr (P<0.001). During exercise, PvO2 decreased (P<0.05) to similar values in Con and AczTr. During recovery PvO2 rose above resting values in Con and AczTr (P<0.05). PvO2 returned to resting value by the first minute of recovery in Con and by the fifth minute in AczTr. During recovery PvO2 was higher in AczTr than in Con (P<0.01) (Table 1).
Haemoglobin, PCV and plasma protein
At rest no differences were observed between Con and AczTr in Hb, PCV and plasma protein (PP) in arterial and venous blood.
Arterial and venous Hb (Hba and Hbv) increased at the onset of exercise in Con and AczTr (P<0.05). During recovery, Hba and Hbv returned to resting value after 5 min of recovery. During exercise and recovery Hba and Hbv were lower in AczTr compared to Con (P≤0.001) (Table 1).
Packed cell volume increased after the initiation of exercise in arterial (PCVa) and venous blood (PCVv) in both Con and AczTr (P<0.05). PCVa and PCVv increased at the onset of exercise in Con and AczTr (P<0.05). During recovery, PCVa and PCVv did not return to resting values. PCVa and PCVv during exercise and recovery were higher in Con than in AczTr (P<0.03) (Table 1).
A rise in [PP] was observed during exercise in Con and AczTr, in arterial and venous blood (PPa and PPv) (P<0.05). During recovery PPa and PPv returned to resting value after the cessation of exercise. No difference between Con and AczTr PPa and PPv were observed during exercise and recovery (Table 1).
Erythrocyte, plasma volume changes across the lung
Erythrocyte and plasma volume changes across the lung were calculated from the arterial and venous concentration changes in PCV, Hb and PP as outlined in the methods.
At rest ΔEVV-A did not change at rest in Con and AczTr. During exercise ΔEVV-A decreased in Con, but not in AczTr (P<0.05). ΔEVV-A during exercise was lower in AczTr (P = 0.03) compared to Con. During recovery ΔEVV-A decreased to resting value after the cessation of exercise (Table 2).
Table 2. Volume changes across the lung
All values are means ± s.e. (n = 6). ΔEVV-A, erythrocyte volume changes across the lung. ΔPVV-A, plasma volume changes across the lung. FTG, fatigue. Con, Control. AczTr, Acetazolamide treatment. #, resting, exercise and/or recovery values different between Con and AczTr. *, fatigue and recovery values different from rest. Positive value indicates a net release of fluid from the compartment across the lung. Negative value indicates a net uptake of fluid from the compartment across the lung.
At rest plasma volume across the lung (ΔPVV-A) was not different between Con and AczTr. Treatment had no effect on ΔPVV-A during exercise and recovery (Table 2).
Erythrocyte and plasma ion changes across the lung (Table 3)
Table 3. Changes across the lung
All values are means ± s.e. (n = 6). e[La-]V-A (mEq/L), erythrocyte lactate changes across the lung. e[Cl-]V-A (mEq/L), erythrocyte chloride changes across the lung. e[Na+]V-A (mEq/L), erythrocyte sodium changes across the lung. e[K+]V-A (mEq/L), erythrocyte potassium changes across the lung. p[La-]V-A (mEq/L), plasma lactate changes across the lung. p[Cl-]V-A (mEq/L), plasma chloride changes across the lung. p[Na+]V-A (mEq/L), plasma sodium changes across the lung. p[K+]V-A (mEq/L), plasma potassium changes across the lung. [H+]V-A (nEq/L), plasma hydrogen changes across the lung. [HCl3-]V-A (mEq/L), plasma bicarbonate changes across the lung. e[SID]V-A (mEq/L), erythrocyte strong ion difference changes across the lung. p[SID]V-A (mEq/L), plasma strong ion difference changes across the lung. p[Atot]V-A (mmol/L), plasma concentration of week electrolytes changes across the lung. #, resting, exercise and/or recovery values different between Con and AczTr. *, fatigue and recovery values different from rest. Positive value indicates a net release of the ion from the compartment across the lung. Negative value indicates a net uptake of ion from the compartment across the lung. @, variables changes across the lung also expressed in figures.
Lactate: Erythrocyte (e[La-]V-A) and plasma (p[La-]V-A) [La-] changes across the lung were not significantly different between Con and AczTr.
Chloride: At rest erythrocyte [Cl-] changes across the lung (e[Cl-]V-A) were not different between Con and AczTr. During exercise e[Cl-]V-A was lower in Con compared to AczTr (P<0.001). Similar was not observed during recovery.
At rest plasma [Cl-] changes across the lung (p[Cl-]V-A) were not different between Con and AczTr. Treatment had no effect on p[Cl-]V-A during exercise and recovery.
Sodium: At rest erythrocyte [Na+] changes across the lung (e[Na+]V-A) were lower in AczTr compared to Con (P = 0.05). During exercise e[Na+]V-A decreased in Con (P<0.05) but not in AczTr. During recovery in Con e[Na+]V-A rose to resting value immediately after the cessation of exercise. During recovery e[Na+]V-A was not different between Con and AczTr.
At rest plasma [Na+] changes across the lung (p[Na+]V-A) were not significantly different between Con and AczTr. During exercise p[Na+]V-A was higher in Con compared to AczTr (P = 0.006). During recovery p[Na+]V-A was not different between Con and AczTr.
Potassium: At rest erythrocyte [K+] changes across the lung (e[K+]V-A) were lower in AczTr compared to Con (P = 0.001). During exercise e[K+]V-A increased in Con (P<0.05), but did not change in AczTr. During recovery in Con e[K+]V-A returned to resting value immediately after the cessation of exercise. During recovery e[K+]V-A was not different between Con and AczTr.
Plasma [K+] changes across the lung (p[K+]V-A) at rest, during exercise and recovery were not significantly different between Con and AczTr.
Hydrogen: At rest [H+] changes across the lung ([H+]V-A) were not different between Con and AczTr. During exercise [H+]V-A increased in Con and AczTr (P<0.05). Exercise [H+]V-A was significantly lower in AczTr than in Con (P = 0.01). In contrast to exercise values, during recovery [H+]V-A was higher in AczTr compared to Con (P = 0.03). [H+]V-A returned to resting values after the first and fifth minutes of recovery in Con and AczTr, respectively (Fig 2).
Plasma bicarbonate: At rest [HCO3-] changes across the lung ([HCO3-]V-A) were not different between Con and AczTr. During exercise [HCO3-]V-A remained similar to resting value in Con and AczTr. During exercise treatment had a significant effect on [HCO3-]V-A (P = 0.009). During recovery [HCO3-]V-A was not different between Con and AczTr (Fig 2).
Independent acid-base variables changes across the lung
Strong ion difference: At rest, erythrocyte [SID] changes across the lung (e[SID]V-A) were not significantly different between Con and AczTr. During exercise and recovery treatment had a significant effect on e[SID]V-A (P<0.05) (Table 3, Fig 3).
At rest plasma [SID] changes across the lung (p[SID]V-A) were not significantly different between Con and AczTr. During exercise in Con p[SID]V-A increased (P<0.05) and was higher than in AczTr (P = 0.001). During recovery in Con p[SID]V-A returned to resting value immediately upon cessation of exercise. Recovery p[SID]V-A was not different between Con and AczTr (Table 3, Fig 3).
Partial pressure of CO2, an independent acid-base variable, in arterial and venous blood is documented under respiratory changes (Table 1, Fig 1).
Total concentrations of weak acids and bases (Atot): At rest, during exercise and recovery [Atot] difference across the lung ([Atot]V-A) was not different between Con and AczTr (Table 3, Fig 3).
In the present study, exercise at 80% VO2peak was performed after 3 days administration of Acz to describe acid-base and ion balances in pulmonary circulation in horses. Similar to previous reports, an adequate CA inhibition was achieved as compensatory mechanisms could not accommodate for slow mobilisation of CO2 from HCO3- across the lung (Rose et al. 1990; Hodgson et al. 1991; Kowalchuk et al. 1992, 1994). On the basis of our experimental results, the following conclusions can be made: 1) chronic CA inhibition affects exercise time to fatigue; 2) chronic CA inhibition greatly influences the acid base homeostasis across the lung; 3) acid base disturbance across the lung is caused by retention of CO2 and inhibition of Cl- flux across the erythrocyte membrane; 4) volume regulation of erythrocytes and acid base changes across the lung complement each other; and 5) Atot and La- have no influence on acid base status across the lung.
Lungs are the major regulators of acid base homeostasis during exercise (Jones and Heigenhauser 1996). CO2 elimination requires transformation of intravascular HCO3- to molecular CO2 via the Jacobs-Stewart cycle. Bicarbonate is transported into the red blood cell in exchange for Cl- by band 3-mediated anion exchange (Bretcher 1971). Chloride moves across the erythrocyte membrane to maintain a homeostatic relationship between [H+] inside and outside the erythrocyte. Hydrogen ion combines with intraerythrocytic HCO3- to generate molecular CO2, which diffuses into plasma and is expired with ventilation. The rate limiting step in the reaction is the hydration/dehydration of CO2/HCO3-, which is catalysed by CA (Jacobs and Stewart 1942; Maren 1967; Chow et al. 1976; Klocke 1988; Jennings 1989). Carbonic anhydrase catalyses the reversible reaction involving the hydration/dehydration of CO2 (Maren 1967) as shown by:
The acceleration of CO2 hydration/dehydration by erythrocytic CA is essential. The rate of hydration of respiratory CO2 in blood and its dehydration and excretion are too fast to be accomplished by the uncatalysed reactions. Carbonic anhydrase reduces the reaction half-time from 5–8 s to 5–10 ms (Maren 1967; Geers and Gros 2000).
Carbonic anhydrase activity after treatment with Acz was not measured in this study. The dose was formulated based on previous study by Rose et al. (1990) and based on pharmacokinetics of Acz in horses (Alberts et al. 2000). According to previous reports, the dose used in this study should inhibit >90% of activities of CA isozymes (Maren 1967; Swenson and Maren 1978). The horses generally tolerated treatment with Acz well. Chronic treatment produced mild depression approximately 10–12 h post treatment, as well as increased urination, and decreased faecal output. During this period horses also showed moderate reduction in appetite. No changes in vital parameters were observed.
On Day 2 of treatment with Acz, subjective and laboratory estimation of the horse's hydration status did not reveal abnormalities at any time of treatment. This is in agreement with observations made by Maren (1956) who reported that renal bicarbonaturia induced by Acz is markedly reduced by acidosis: the greater the acidosis, the less the diuretic effect of CA inhibition. This is also in agreement with observation by Yin et al. (1995) who reported that when the kidneys were exposed to a high [Cl-], glomerular filtration rate is reduced and Na+ excretion decreased.
During exercise an inadequate hydration/dehydration reaction slowed the equilibration between CO2 species in pulmonary capillaries, which further increased PaCO2. The slowing of the CA reaction means that equilibration is not complete during the transit through the pulmonary capillary. The reflection of these activities is also inhibition of HCO3- transport. Unhydrated CO2 and small pSIDV-A in this study provided means to regulate the decreased rate of HCO3- transport. In fact the decrease in the rate of HCO3- dehydration after treatment with Acz increased the e[HCO3-] and consequently reduced the p[HCO3-] (Sterling et al. 2001).
Erythrocyte volume decrease across the lung remained unchanged in AczTr during exercise. There are several active and passive mechanisms that regulate erythrocyte volume (Fievet et al. 1990; Gibson et al. 1993, 1995, 2000; Honess et al. 1996; Speake et al. 1997; Juel et al. 1999), which is evident as the erythrocyte regulatory volume decrease in peripheral tissues, and by the erythrocyte regulatory volume increase across the lung (Vengust et al. 2006a,b). Based on our results it seems that incomplete or absent volume decrease across the lung in AczTr is mainly due to depressed eCl- and slowed CO2 dehydration/hydration reaction (Jacobs-Stewart cycle).
Plasma volume was relatively stable across the lung in Con and AczTr indicating that water is moving directly into pulmonary interstitium, which is in agreement with a report from Hansen (1961). In addition to water, treatment with Acz attenuated efflux of Cl- from erythrocytes during exercise. Cl- changes across the lung via Cl-/HCO3- exchanger (AE1) governed the acid base and volume changes in concert with depressed hydration/dehydration of PCO2. AE1 is regulated minimally by [H+] within physiological ranges (Humphreys et al. 1994); therefore, e[H+] is unlikely to impair the anion exchanger significantly. Carbonic anhydrase is metabolically closely interlinked with the activity of anion exchanger (AE1), with which it forms a capnometabolon (Kifor et al. 1993). An indirect influence of CA inhibition and CHO3--CO2 disequilibria on AE1 is likely and it is evident by e[Cl-]V-A.
Physiologically, the erythrocyte regulatory volume decrease is also governed by the erythrocyte K+-Cl- cotransport mechanisms (Gibson et al. 1993, 1995; Honess et al. 1996), which contribute substantially to Cl- efflux from erythrocytes and are influenced by PO2 and e[H+] through the Haldane effect. It is possible that this element of erythrocyte volume regulation was stopped or slowed by Acz from displacing solutes from erythrocytes into plasma. This is evident in the present study during the steady state stage of exercise and fatigue in e[Cl-] and e[K+]. It is also possible that the absolute increase in e[H+] in AczTr stimulated erythrocyte membrane Na+/H+ pump (Gibson et al. 1993, 1995; Honess et al. 1996) before and during initial stages of exercise to function towards erythrocyte volume increase across the lung via increasing the e[Na+], which is evident at rest. According to Na+ and K+ activity across the lung during exercise it seems that mechanisms/activities were expressed that counteracted erythrocyte volume regulation malfunction.
Lactate concentration remained stable across the lung and was not influenced by CO2 species disequilibria or other strong ion activity. A relative stability of [La-] during exercise in all compartments across the lung shifts the primary role in acid base balance to other strong ions.
Plasma SIDV-A had a positive value in all treatments indicating a reduction in SID across the lung. This is in greater part driven by an influx of Cl- into erythrocytes to initiate the Jacobs-Stewart cycle (Jacobs and Stewart 1942; Jennings 1989), which substantially decreases e[SID]V-A. Carbonic anhydrase inhibition prevented efflux of Cl- from erythrocytes, hence the substantially decreased e[SID]V-A in AczTr. Other strong ions, with the exception of La-, showed the tendency to counteract changes caused by CO2 retention and Cl- flux attenuation, which was also evident in e[SID]V-A and p[SID]V-A. It seems obvious that compensatory mechanisms could not accommodate for the absence of CA activity (Kowalchuk et al. 1994).
Regulation of p[H+] may be quantitatively expressed through changes in PCO2, [SID] and Atot (Stewart 1983). At rest p[H+]V-A was similar in Con and AczTr indicating the increased elimination of CO2 with increased VE kinetics in AczTr (Swenson and Maren 1978; Ward et al. 1983; Kowalchuk et al. 1994). During exercise in Con, despite a concomitant pSID decrease across the lung, p[H+]V-A remained strongly positive (indicating a [H+] reduction in plasma across the lung). This was due to a substantial PCO2 reduction and Cl- efflux (and SID increase) across the lung. Inversely, in AczTr p[H+]V-A was affected by CO2 species retention and eCl- shift impairment.
Total concentrations of weak acids (Atot) did not change across the lung in Con or AczTr. Coates et al. (1984) reported that lymph protein to plasma protein ratio changes during exercise are due to an increase in perfused surface area in lungs indicating no net loss of protein from pulmonary circulation in intact lungs. This is in agreement with our findings.
As stated above, the slowing of the CA reaction means that equilibration among CO2 species and solutes is not complete during the transit through the pulmonary capillary. Carbonic anhydrase inhibition slows or attenuates Jacobs-Stewart cycle and anion exchange mechanism, increases the disequilibria, prevents prompt equilibration across the lung and causes slow equilibration processes in vitro. The equilibration, however, continues in the arterial blood or in vitro in a slower rate. Therefore, during the time that blood is stored in vitro and by the time measurements are made, full equilibration occurs between all forms of CO2 in blood: the measured CO2 species is then higher in vitro than in vivo (Kowalchuk et al. 1994; Cardenas et al. 1998).
Based on the findings of this study the cascade of acid-base equilibrium across the lung is initiated by Hamburger shift and Jacobs-Stewart cycle. The cascade was altered by the CA inhibition. Therefore, CO2 and Cl- changes in erythrocytes across the lung seem to be the major contributors to acid-base and ions balance in exercising horses, and probably in other mammalian species.
This study was supported by the Ontario Ministry of Agriculture and Food - Equine Program, Department of Clinical Studies at the University of Guelph, and the Slovenian Research Agency grant P4-0053.
Conflicts of interest
The authors have declared no conflicts of interest.
1 EQB Inc., Unionville, Pennsylvania, USA.
2 AstraZeneca Pharmaceuticals LP, Wilmington, Delaware, USA.
3 Baxter Healthcare Corp., Irvine, California, USA.
4 Becton Dickinson, Sparks, Maryland, USA.
5 Criticare Systems Inc., Waukesha, Wisconsin, USA.
6 Infusion Therapy Systems Inc., Sandy, Utah, USA.
7 Apotex Inc., Toronto, Ontario, Canada.
8 C.R. Bard Inc., Covington, Georgia, USA.
9 Ametek, Pittsburgh, Pennsylvania, USA.
10 Sarstedt AG & Co., Nümbrecht, Germany.
11 Nova Biomedical Corporation, Waltham, Massachusetts, USA.
12 Baxter Healthcare Corp., Deerfield, Illinois, USA.