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Why is there no carbonic anhydrase activity available to fish plasma? Lessard, Joanne 1994

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Why is there no carbonic anhydrase activity available to fish plasma?ByJoanne LessardB.Sc., Université Laval, Québec, Canada, 1991A thesis submitted in partial fulfilment of the requirements for the degree ofMaster of ScienceinThe Faculty of Graduate Studies(Department of Zoology)We accept this thesis as conforming to the required standardThe University of British ColumbiaDecember 1993© Joanne Lessard, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of i / .7The University of British Colum iaVancouver, CanadaDate ,DE-6 (2/88)AbstractCarbonic anhydrase (CA) is absent in the plasma of vertebrates. In vitro, CA in fishplasma will short-circuit the effect of catecholamines on the increase in red blood cell(RBC) pH and volume, both of which increase the hemoglobin affinity for 02. CA wasinfused into trout for a period of 6h during which the animal was submitted to deephypoxia (P02= 30-35 torr) and during recovery after exhaustive exercise. During hypoxia,02 content, lactate, catecholamines, hematocrit, hemoglobin and pH1 were similar to thesaline infused control group. On the other hand, cell volume was significantly higher andPHe, total CO2 and organic phosphates were significantly lower than the control group.The concentration of CA was not high enough to completely short-circuit the increase inpH1 and RBC volume caused by catecholamines. The lower pH in the CA infused animalscould enhance the activity of the Na/H pump which would keep the NTP low. CA inplasma, during hypoxia, did not cause the expected reduction in blood oxygen content butdid have a marked effect on plasma total CO2. During the recovery period of exhaustiveexercise, lactate, catecholamines, hematocrit, hemoglobin, MCHC, Po2,Hb02and pH1weresimilar to the saline infused animals. Total CO2 and PHe were significantly higher in theCA infused fish than in the saline infused ones. CA infusion, in this case, probably causedacid retention in the muscle. Acid efflux from the muscle would decrease pH or if the acidwas excreted at the gills, bicarbonate would be titrated and the stores would be lower thanin control animals. CA activity available to plasma would mean greater fluctuation ofplasma pH, at least in hypoxic conditions, and red blood cell pH in general. pH is aIIbalance between acid loading at the muscle and acid excretion at the gills or the kidneys,we cannot distinguish between a decrease and an increase of one of the two which resultedin a decrease of plasma pH. Fish have a large Haldane effect and HCO3 flux through thered cell ensures that the protons are excreted as CO2 and cannot bind again to hemoglobin.The absence of CA in the plasma ensures that HC03 flux through the red cell ismaintained.IIIAbstractList of TablesList of Figures.AcknowledgementsChapter 1General IntroductionChapter 2Carbonic Anhydrase Infusion inIntroductionMaterials and MethodsResultsDiscussionChapter 3Carbonic Anhydrase Infusion inIntroductionMaterials and MethodsResultsDiscussionTable of Content• . II..vIVII.Ix10111214241Rainbow Trout during HypoxiaRainbow Trout Following Exhaustive ExerciseChapter 4General Discussion282930314043IVReferences.48VList of TablesTable 1. Plasma catecholamines concentration for saline and carbonic anhydraseinfused rainbow trout during deep hypoxia 17Table 2. Plasma catecholamines concentration for saline and carbonic anhydraseinfused rainbow trout during recovery after exhaustive exercise 34VIList of FiguresFigure 1. Jacobs-Stewart cycle in red blood cell 9Figure 2. Lactate levels in hypoxic rainbow trout infused with carbonic anhydrase orsaline 18Figure 3. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on plasma pHand intracellular pH 19Figure 4. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on plasmaCO2 and CO2 partial pressure 20Figure 5. Effects of carbonic anhydrase infusion in hypoxic rainbow trout onhematocrit, hemoglobin concentration and mean cell hemoglobin concentration 21Figure 6. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on oxygencontent 22Figure 7. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on totalorganic phosphates and nucleotide triphosphates 23Figure 8. Lactate levels in rainbow trout infused with carbonic anhydrase or salinefollowing exhaustive exercise 35Figure 9. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on hematocrit, hemoglobin concentration and mean cellhemoglobin concentration 36Figure 10. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on the amount of oxygen bound to hemoglobin and oxygenpartial pressure of arterial blood 37VIIFigure 11. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on plasma pH and intracellular pH 38Figure 12. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on plasma CO2 and CO2 partial pressure 39Figure 13. Jacobs-Stewart cycle in the gills 47VIIIAcknowledgementsThe completion of this thesis would have been impossible without the help ofmany poeple:- the members of the Randall lab for their help in the lab as well as withdiscussions;- my supervisor, Dave Randall, for his patience and his acceptance of me in hislab following only telephone conversations;- to all the other labs from which I borrowed equipment and assistance;- to Richard Kinkead for his help on the writting of my thesis and to have kickedmy b... when I was conplaining too much;- to Rhonda Garland for her friendship and for all the healthy breaks;- and to Scott, the last but not the least, for his support and tolerance over mybitching and whining.IxChapter 1General Introduction1Carbonic anhydrase: general informationCarbon dioxide is the major end product of aerobic metabolism. When CO2 istransferred to the blood, most of it is hydrated to HCO3 according to the followingequation:CO2 + HO -‘ H2C03 HC03 + H.Because the ionization of H2C03 is very rapid, H2C03 is often omitted in this reaction.From this reaction, it is evident that when CO2 is added to a solution, pH decreases.Conversely, the addition of protons will affect CO2 concentrations. It is not surprising,therefore, that the dynamics of this reaction are an important component of acid-basebalance.The enzyme carbonic anhydrase (CA) catalyzes the reversible CO2hydration/dehydration reaction. In the absence of CA the CO2 equilibrium reaction is veryslow, about 30 seconds at 100 C (Nikimnaa, 1992). This enzyme has one of the highestknown turnover numbers (k= 1041O6 sec’) and is inhibited by a wide range of inhibitorswith acetazolamide as the most commonly used (Dodgson, 1992).There are 7 isoenzymes known in mammals (Dodgson, 1992). Two cytosolicisoenzymes are present in the red blood cells (CA I and CA II). CA II is also present inmany other tissues like kidneys and lungs. By definition, the CA present in skeletal muscleis CA III and the one membrane-bound is CA IV. CA V is the mitochondrial isoenzyme,CA VI is secreted in the saliva and CA VII is found in the salivary gland. In fish, carbonicanhydrase activity has been found in a number of different tissues including: red blood cell(Sanyal et al., 1982; Henry et al., 1988), muscle (Sanyal et al., 1982), swimbladder (Adamson2and Waxman, 1976), and gill epithelium (Dimberg et a!., 1988; Henry et a!.; 1988; Rahimet al., 1988).Carbonic anhydrase: its rolesIn general, CO2 diffuses faster through solutions if CA is present because CA keepsthe concentration gradient high. Studies done by Gros and associates have indicated thatCA-catalyzed facilitated diffusion is possibly of great importance in the functioning of theskeletal muscle (Geers and Gros, 1991). Without CA present, the buffering capacity of theC02-HCO3system cannot be fully utilized because the uncatalyze reaction is too slow(Thomas, 1983). It appears that there is CA wherever significant amounts of HC03 ormove across cell membrane (Maren, 1987). CA is also involved in intermediary metabolismmaintaining the supply of HC03 and H.In fish, CA is involved in many of the physiological roles of the gills as well asseveral functions in other tissues. In general, CA has three important functions in fish gills:1. Acid-base balance; CA supplies the proton for acid excretion at the gills. Lin andRandall (1991) showed that in the presence of acetazolamide, a CA inhibitor, acidexcretion was reduced. 2. Ionic regulation; CA provides the counter-ions necessary for theexchange of Na and C1 across membranes. Several studies demonstrated a reduction intransepithelial Na or Cl- uptake after inhibition of branchial CA with acetazolamide(Maetz and Gracia-Romeu, 1964; Kersteller et aL, 1970; Kersteller and Kirsehner, 1972;Payan et aL, 1975; Henry et aL, 1988). 3. CO2 excretion; as in most vertebrates, there isa flux of HC03 into the red blood cell where it is rapidly dehydrated by CA to form CO23which diffuses across the respiratory epithelium (Perry, 1986).The red blood cell offishIt is generally accepted that the major pathway for CO2 excretion is HCO3 fluxthrough the red cell. In the blood, CO2 is carried mostly as HC03 but if the blood onlyrelied on the hydration of CO2 that occurs in the plasma, the amount of CO2 carried by theblood would be small because the reaction is uncatalyzed in the plasma. CO2 is hydratedmainly inside the red cell as it diffuses through the blood and HCO3 is transferred in theplasma in exchange for Cl through the anion exchanger. When the blood arrives at thegills, HC03 enters the red cell in exchange for C1 via a band III protein: the anionexchanger. Once inside, HC03 combines with a proton at the catalyzed rate to form CO2and H20, as describe in the reaction cited above. CO2 then diffuses out of the cell, throughthe gill epithelium and into the respiratory water. As CO2 is excreted, the concentrationof HC03 decreases inside the red cell and the influx of HCO3 into the red cell ismaintained. The protons come from the intracellular buffers and hemoglobin oxygenation.When oxygen binds to hemoglobin, protons are released and used in CO2 excretion, thisphenomena is called the Haldane effect. Fish have a large Haldane effect andconsequently large Bohr and Root effect. The Bohr effect is a reduction of hemoglobinoxygen affinity when protons bind to hemoglobin. The Root effect is a decrease in oxygencarrying capacity of hemoglobin when it binds protons.Catecholamines released into the blood in stress conditions, for example duringextreme hypoxia and exhaustive exercise (see Randall and Perry, 1992), activate a Na1jFI4exchanger across the RBC membrane, raising erythrocytic pH (Nikinmaa et al, 1990; Motaiset a!., 1990). Fish erythrocytes, like those of mammals, are not very permeable to protons(Forster and Steen, 1969). Acid can be transferred between the plasma and the erythrocyteby the cycling of CO2 and HC03 (Fig. 1). This is known as the Jacob-Stewart cycle. ThusNa/W exchange removes protons from the RBC and these will re-enter via the Jacobs-Stewart cycle, the rate limiting step being the uncatalyzed HCO3 dehydration reaction inthe plasma. As mentioned above, the rise of pH caused by catecholamines will increasehemoglobin-oxygen affinity and as consequence blood will carry more oxygen for the samewater Po2. pH is not the only factor that influences hemoglobin-oxygen affinity, otherligands such as ATP and more specifically GTP also decrease hemoglobin-oxygen affinitywhen they bind to hemoglobin.The problemAlthough the major pathway for CO2 excretion is the HC03 flux through the redcell, some crustaceans use extracellular CA for this purpose (Henry, 1988). In mammals,inhibition of extracellular CA produces a pH disequilibrium but this CA does not seem tobe involved in CO2 excretion (Heming et al., 1986). Since CA does not occur in the plasmaof all vertebrates studied to date, this extracellular CA means that some HC03 containedin the plasma has access to CA without going through the red cell. In salmonids, CA is notavailable to the plasma at the gills. Rahim et a!. (1988) showed using immunohistology thatthe basal membrane of the gill epithelium is devoid of CA activity. Henry et a!. (1988)concluded, from injecting CA inhibitor into intact animals, that there is very little, if any,5membrane-associated CA in the gills.Why is there this difference between mammals and fish? Why is there no carbonicanhydrase available to fish plasma? Motais et a!. (1989) and Nikinmaa et a!. (1990) haveshown that the addition of CA to the plasma, in vitro, will short circuit the action ofcatecholamines on RBC pH. It has been suggested, therefore, that the absence of CAactivity in plasma allows catecholamine regulation of erythrocytic pH. Thus, fish plasmais totally devoid of any CA activity and has erythrocytic pH regulation whereas mammalianplasma has access to CA activity but there is no RBC pH regulation by catecholamines.Motais et a!. (1989), however, showed that catecholamines could still raise trout RBCpH in vitro even in the presence of 0.5 mg/mI CA. These CA levels are an order ofmagnitude higher than those nonnally available to mammalian plasma (Effros et aL, 1980;Bidani et aL, 1983). Low levels of CA activity available to plasma, therefore, may notjeopardize catecholamine induced RBC pH regulation and the absence of CA from fishplasma may be correlated with factors other than RBC pH regulation per Se.Fish bodies may consist of more than two thirds muscle. These muscles arepredominantly glycolytic and large amounts of acid are released into the blood followingburst swimming. In the presence of carbonic anhydrase activity available to the plasma thisacid would be rapidly transferred into the RBC and, in addition, the acid would titrateplasma HC03 and be excreted as CO2. Both these processes would occur more slowly inthe absence of carbonic anhydrase activity available to plasma.The gills are not only a site for gas exchange and extracellular CA might interferein the other functions of the gills. When blood reaches the lungs of birds and mammals,6plasma HCO3 is transferred into the red cell (in exchange for Cl) where it is rapidlydehydrated to CO2. CO2 diffuses across the epithelium following the concentrationgradient. In mammals and birds, the CO2 gradient can be adjusted by changes inventilation. Thus hyperventilation will increase the CO2 concentration gradient, and CO2excretion will increase; the reverse is also true. Because CO2 is a weak acid in solution,removal or addition of this molecule will change the pH. Therefore, mammals and birdscan alter their acid-base balance via CO2 excretion. Because oxygen tension is high in air,small changes in ventilation will not affect oxygen uptake. Oxygen concentration is lowerin water than in air and water-breathers regulate ventilation for oxygen uptake (Randalland Cameron, 1973). In addition, the solubility of CO2 in water is about 30 times greaterthan oxygen. Regulation of ventilation to adjust CO2 may compromise oxygen uptake inwater-breathers (Randall and Cameron, 1973). However, because CO2 excretion isrelatively easy compared with oxygen uptake, CO2 levels in water-breathers are low. Forall those reasons, acid-base balance adjustment by changes in CO2 excretion would be a badstrategy for water-breathers. In the latter, pH regulation is achieved by changes in plasmaHCO3 concentration modulated by Cl/HCO3 exchange across the gills (Randall andCameron, 1973; Janssen and Randall, 1975) and by protons excretion directly via a protonATPase (Lin and Randall, 1991). Gills are also the major site of osmoregulation comparedto lungs which have no function in this matter.In order to try to answer the question as to why carbonic anhydrase is not availableto fish plasma, two series of experiment were undertaken. The first study looked at thepossibility that CA available to the plasma in the gills would impair oxygen uptake because7of the accelerated Jacobs-Stewart which would impair the red cell pH regulation andtherefore oxygen transport. The second study looked at the possibility of maintainingHC03 stores under acidotic conditions which would be impossible if CA was available tothe plasma in the gills.802HCO;+If‘‘CatecholaminesCo2‘0Figure1.Jacobs-StewartcycleinredbloodcellChapter 2Carbonic Anhydrase Infusion in Rainbow Trout during Hypoxia10IntroductionThe object of experiments reported here was to infuse carbonic anhydrase into troutto obtain activity levels similar to that available to mammalian plasma in order to determinethe effect of plasma carbonic anhydrase activity on hemoglobin-oxygen affinity and plasmaacid base regulation during exposure to hypoxia. Hypoxia was used as a mechanism forpromoting the release of catecholamines and protons into the blood. In addition, hypoxiahave been shown to enhance the effect of catecholamines on red cells (Ferguson et al.,1989). The plasma acidosis was the result of anaerobic production of lactic acid in themuscle and the catecholamine induced movement of protons out of the RBC.Fish subjected to deep hypoxia increase ventilation and release catecholamines. Iftheir oxygen supply does not meet their energy demand, they switch to anaerobicmetabolism. It is well known now that if the hypoxia is prolonged, the NTP (ATP + GTP)content of the red cells decreases (see Thomas and Motais, 1990). Although everybodyagrees that NTP content is reduced in hypoxic fish, the time course of this decrease variedin different studies [1 or 2 weeks in eel (Wood and Johansen, 1973), 36 hours in carp(Likkeboe and Weber, 1978) and 1 hour in rainbow trout (Tetens and Likkeboe, 1985)].As mentioned in the general introduction, ATh and more specifically GTP bind tohemoglobin and decrease its affinity for oxygen. It has been concluded that reducing NTPcontent in the red cells maintains oxygen uptake during hypoxia (Wood and Johansen,1973).11Materials and MethodsFreshwater rainbow trout [Oncorhynchus mykiss (Walbaum)], weighing 300-600 g,were obtained from a local hatchery, and held outdoors at the University of BritishColumbia, in dechlorinated Vancouver tap water (8-12°C) for at least 2 weeks beforeexperimentation. The animals were fed once a week with commercial trout pellets, butfeeding was suspended 3 days prior to surgery. Under MS-222 anesthesia (1:10 000 inNaHCO3-buffered freshwater), fish were fitted with dorsal aortic catheters according toSoivio et a!. (1975). Following surgery, fish were allowed to recover for at least 48h indarkened Plexiglass box with recirculating water (8-10°C).Experimental protocolFor measurements at rest, one blood sample (1.2 ml) was taken from the dorsalaorta and immediately analyzed for plasma pH (pHj, blood and plasma total C02,hematocrit, hemoglobin and oxygen content. The remainder of the blood was centrifuged,plasma was separated from RBC’s and both were frozen in liquid nitrogen for furtheranalysis of lactate, catecholamines, adenylates and guanylates and red blood cell pH (pH1).These fish were not used for the hypoxia experiments because we did not want any stressprior to the infusion and the hypoxia exposure. At the same time that hypoxia was induced(30-35 Torr), one group of fish was infused with saline and another with a solution ofcarbonic anhydrase to achieve 0.3 g/1 in the animal for the duration of hypoxia exposure(6h). Blood samples were taken as described above at 10, 30, 120 and 360 minutes.12Analytical proceduresPlasma pH was determined with a microcapillary electrode (Radiometer G279/G2)coupled to a PHM84 pH meter. Total CO2 measurements were done using a gaschromatography method (Boutilier et a!., 1985) on samples obtained anaerobically. TotalCO2 inside the erythrocytes was obtained from the subtraction of total plasma CO2 fromtotal blood C02, taking into account the hematocrit. Pco2 was calculated from theHenderson-Hasselbach equation with solubility coefficient and pK calculated according toBoutilier et al., (1984). Hematocrit was measured using microhematocrit tubes centrifugedat 12 000 rpm for 5 minutes. Hemoglobin (Hb) was measured using Drabkin’s reagentfrom Sigma kit (kit #525-A). Mean cellular hemoglobin concentration (MCHC) wascalculated as HbIHct. The oxygen content of the blood was determined using the methodof Tucker (1967). Whole-blood lactate levels were analyzed using the L-lactatedehydrogenase/NAD method (Sigma kit #826-B). High pressure liquid chromatography(HPLC) was used to measure ATP, ADP, AMP, simultaneously with GTP, GDP, GMP.The procedure was carried out using an LKB 2152 HPLC controller and 2150 titaniumpump coupled to 2220 recording integrator. The separation was performed on anAquapore AX-300 7 m weak anion exchanger (Brownlee laboratories) eluting at 2 ml miii1 at 55°C (Schulte et a!., 1992). Analyses of plasma catecholamines levels were performedby HPLC with electrochemical detection, using a Brownlee Spheri-5 reverse-phase column(Technical Marketing, Richmond, BC), a Bioanalytical Systems LC-4A amperometricdetector (Mandel Scientific, Rockwood, Ont.) and a Spectra-Physics SP8700 solvent deliverysystem (Terochem Laboratories Ltd., Edmonton, Aib.), as described by Primmett et a!.13(1986). The freeze thaw method of Zeidler and Kim (1977) was used to measure pH1.Statistical methodsStatistical significance of data for 6 hour hypoxia was determined using a two-wayANOVA followed by a Dunnet test when comparing the hypoxia values with the restingvalues and an unpaired t-test when comparing CA infused values with control; both witha statistical significance level of 5%. Data are presented as mean ± standard error on themean (SEM).ResultsIn resting animals, catecholamines and lactate were similar to resting levelsmeasured by others (Nilsson, 1983; Tetens et al., 1988; Hart et al., 1989; Perry et al., 1989;Thomas and Perry, 1991; McDonald and Milligan, 1992). In both treatments,catecholamines increased significantly after 10 minutes of deep hypoxia reaching a peak at2h and remained elevated (Table 1). In both groups, lactate was significantly elevatedrelative to the resting fish at 10 minutes and kept increasing for the next six hours (Fig. 2).Hypoxia had little effect on plasma pH in saline infused fish (Fig. 3). In the groupof fish infused with carbonic anhydrase, however, plasma pH initially increased and thendecreased for the next six hours of hypoxic exposure. Erythrocytic pH1 did not varysignificantly during hypoxia in either treatment (Fig. 3).14Total CO2 content of the plasma decreased significantly more in the CA treatedanimals than in saline treated ones (Fig. 4a). No significant changes were observed in RBCtotal CO2 during exposure to hypoxia with or without the infusion of carbonic anhydrase(data not shown). The calculated equilibrium Pco2 decreased significantly in the CAinfused group compared with both normoxic and hypoxic saline infused values (fig. 4b).Pco2 showed a small decrease in the saline infused group during hypoxia compared withnormoxic values which was not significant.Hematocrit initially increased peaking at 30 minutes of hypoxia (Fig. 5a).Hemoglobin was constant during the first six hours of exposure to hypoxia (Fig. 5b). Theratio of hemoglobin and hematocrit (MCHC), which is an indication of RBC volume,differed between the two groups (Fig. 5c). Initially, hypoxia produced an increase in RBCvolume (as indicated by the decrease in MCHC) which was significantly greater than thenormoxic values in both treatments. In the saline treatment, RBC volume remainedconstant for the next six hours. In the CA infused group RBC volume continued toincrease and became significantly different from the saline group at 6h. In both treatments,02 content dropped dramatically within the first 10 minutes. It then remained constantwith no significant difference between the two for the next six hours of hypoxia (fig. 6).Adenylates and guanylates are expressed as a ratio with hemoglobin to avoid theeffect of changes in RBC volume. CA infused fish showed a significant decrease in thetotal pool of organic phosphates after 30 minutes when compared to resting values (Fig.7a). In the saline infused group, there is also a decrease which is not significantly differentfrom the resting values. NTP (ATP + GTP) followed the same pattern except that the15concentration at 6h in the CA infused group is significantly different from the salineinfused group that is NTP level do not return to resting values in CA infusion.16Table1.Plasmacatecholamineconcentrationsfor salineandcarbonicanhydrase(bold)infusedfishduringnormoxiaandatspecifictimeafterhypoxiaexposure.Significanceof differencefromnormoxiccontrol:.(n=7)normoxiahypoxiahypoxiahypoxia120hypoxia10minutes30minutesminutes360minutesadrenaline(ng/ml)4.15(1.35)Saline16.2±53*13.2±2.7*21.6±6.7*22.4±93*CA10.2±2.2*14.1±43*23.1±9•5*19.3±6.9*noradrenaline3.99(1.40)(ng/ml)22.7±7.817.2±3.8*25.2±6.2*17.6±7.9Saline11.1±2.1*18.1±6.9*31.1±15.9*23.7±10.7CA0 60 120 180 240 300 360TIME (mm)Figure 2. Lactate levels in hypoxic rainbow trout infused with carbonic anhydrase or saline.* indicates significant difference between hypoxia and normoxia.188.1 *:* • Ccrbonic onhydrosez-______________C-)_____73I I I I I0 60 120 180 240 300 360Time (mm)Figure 3. Effects of carbonic aithydrase infusion in hypoxic rainbow trout on plasma pH andintracellular pH. * indicates significant difference between hypoxia and normoxia.indicates significant difference between saline and carbonic anhydrase infused animals.199• Corboniç onhydroseo Soline**3.53.0—S‘- 2.503 2.01.5S*5I Io 60 120 180 240 300 360Time (mm)Figure 4. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on plasma CO2and CO2 partial pressure. * indicates significant difference between hypoxia and normoxia.** indicates significant difference between saline and carbonic anhydrase infused animals.2036• Corbonc onhydrose34 - 0 Soline3200CEa,8TTTCCfltI0Ea,2826-D24k%jC)C-)22a20 I0 60 120 180 240 300 350Time (mm)Figure 5. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on hematocrit,hemoglobin concentration and mean cell hemoglobin concentration. * indicates significantdifference between hypoxia and normoxia. ** indicates significant difference between salineand carbonic anhydrase infused animals.211.0-. 0.8CC,>‘x 0.600.40 60 120 180 240 300 360Time (mm)Figure 6. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on oxygencontent. * indicates significant difference between hypoxia and normoxia.2240__• Corbonic onhydroseo Soline.rc.,.ttC.,00.< 3000I I I I I I25201•-,--..0z15—SI I I I I I0 60 120 180 240 300 360Time (mm)Figure 7. Effects of carbonic anhydrase infusion in hypoxic rainbow trout on total organicphosphates and nucleotide triphosphates. * indicates significant difference between hypoxiaand normoxia. ** indicates significant difference between saline and carbonic anhydraseinfused animals.23DiscussionCarbonic anhydrase was infused continuously during the six hours of hypoxia andthe calculated concentration of CA in the plasma, assuming no loss, was nearly an orderof magnitude higher than that available to plasma in the mammalian lung (Effros et a!.,1980). Lessard et al. (1993) showed that the concentration of CA was still 60% of theinitial values after 60 minutes of a bolus injection indicating a slow rate of removal of CAactivity from the plasma. Thus the plasma CA activity, assuming some loss, was probablysimilar to that available to plasma flowing through the mammalian lung. This activity ofCA was insufficient to affect the regulatory effects of catecholamines on RBC pH as therewas no difference in either RBC pH or blood oxygen content between the saline and CAinfused groups during the first six hours of hypoxic exposure (Fig. 3 and 6). The fact thatthere was a catecholamine effect is indicated by the marked increase in RBC volume inboth groups (Fig. Sc). These results are in contrast to the in vitro experiments of Nikinmaaet a!., (1990) who added 3g/l (rather than 0.3g11) CA to the blood and short-circuited theeffects of catecholamines on RBC pH. Motais et a!. (1989), also in vitro, used a lowerconcentration (0.5gfl) than Nikinmaa, but similar to ours, and observed an increase of pH1in response to the addition of catecholamines. So it seems that to short-circuit the rise inpH1 the concentration of CA needed is higher than that used in this study and, therefore,that available to mammalian plasma in the lungs. However, PHe has a very differentbehaviour at the onset of hypoxia between saline and CA infused animals which make itdifficult to interpret the regulatory effects of catecholamines on pH1. Nevertheless, oxygen24content was similar in both groups indicating a comparable hemoglobin-oxygen affinity.Lastly, the enzyme carbonic anhydrase has one of the highest turnover numbers and forthat reason the concentration of CA might not be the limiting factor. The difference in theresults between the study of Nikinmaa et a!. (1990) and Motais et a!. (1989) and this studycould be due to different experimental conditions, especially plasma buffering, which wouldaffect catalyzed HCO3 dehydration rates.The increase in CA activity in fish plasma in these experiments had a marked effecton plasma total CO2 content. PaCO2 in the saline infused fish is determined by the rateof C1JHCO3 exchange across the RBC membrane and ventilation (Perry, 1986). In the CAinfused group PaCO2 will be determined largely by ventilation because plasma HC03dehydration is catalyzed and does not have to pass through the anion exchanger to beexcreted. The end result is that the equilibrium PaCO2 level is higher in the saline infusedfish compared with the CA infused fish (Fig. 4). Hypoxic exposure results in an increasein ventilation and therefore a small reduction in PaCO2 (Thomas et a!., 1988), whichcontributes to the initial reduction in total CO2 observed in fish exposed to hypoxia (Fig.4). The subsequent decrease in total C02, especially in CA infused fish, is probably relatedto acid titration of plasma HC03. The increase in plasma lactate concentration duringhypoxia was the same in both saline and CA infused groups (Fig. 2), indicating thatapproximately the same amount of metabolic acid was produced by both groups. Bloodtotal CO2 decreased more rapidly in the CA, compared with the saline infused group (Fig.4), presumably because plasma HCO3 is titrated to a greater extent by the acid releasedby anaerobic metabolism in the carbonic anhydrase infused animals. CA infusion caused25a rapid removal of protons via HC03 dehydration. This resulted in a marked alkalosis notseen in the saline infused group. Much of the changes in HC03 concentration was in thefirst 30 minutes of hypoxia and infusion, but the subsequent rate of change in HCO3concentration was much the same in both groups for the next six hours indicating that CAwas no longer the determining factor. During this time blood pH in saline infused fishremained constant, that is proton entry into the blood was the same as proton excretion.In the CA infused animals blood pH fell after the initial alkalosis, i.e. in the blood, protonentry exceeded proton excretion at the gills. It is possible, therefore, that CA infusion mayhave resulted in increased acid removal from muscle.Red cell volume increased upon catecholamine stimulation in both groups (Fig. Sc).This can be accounted for by the entry of ions, mainly Na and Cl, with water followingosmotically (Fievet et a!., 1988; Baroin et a!., 1984). CA infusion, however, caused a furtherincrease in RBC volume after 30 minutes exposure to hypoxia. This could be due toincreased activity of the Na71I exchanger, which has been shown to be enhanced by a fallin blood pH (Nikinmaa et a!., 1990) like that seen following CA infusion in hypoxic fish(Fig. 3). The levels of organic phosphates remained depressed in the CA infused group (fig.7) probably because of increased ATP utilization by the Na/H exchanger. RBC swellingresulted in the initial increase in hematocrit observed when fish were exposed to hypoxia,any release of red blood cells from the spleen was offset by RBC removal during sampling.Why then is there no carbonic anhydrase activity available to blood plasma in fish?It is not simply to permit catecholamine induced pH regulation of the red blood cellbecause this occurs in the presence of low levels of CA. In general, the absence of CA26activity reduces the titration of HCO3 when protons are liberated from muscle into theblood. The red blood cells are protected from this acidosis because the acid is transferredto the RBC more slowly than it would be if CA was present. In addition, the transfer ofacid across the gill epithelium is not short-circuited by a rapid HCO3 hydration/dehydrationreaction in the plasma causing a rapid cycling of CO2 between water and plasma. Theuncatalyzed C02/HCO3 reaction in the plasma, therefore, facilitates blood pH regulationdue to acid transfer across the gill epithelium.Fish tolerate a plasma acidosis but protect the RBC from pH changes and maintainoxygen transfer by having no CA activity available to the plasma. This also prevents largechanges in plasma HC03 and allows the animal to adjust plasma HCO3 levels in order toregulate pH. This is important because fish do not regulate pH by changes in PaCO2viaventilation. Mammals adjust PaCO2 via ventilation and have carbonic anhydrase activityavailable to the plasma as it flows through the lungs.27Chapter 3Carbonic Anhydrase Infusion in Rainbow Trout Following Exhaustive Exercise28IntroductionIn the study presented in chapter 2, it was thought that CA would short-circuit thepH regulation of the red blood cells by catecholamines and as a result would impair oxygenuptake. CA in plasma did not cause the expected reduction in blood oxygen content duringhypoxia but did have a marked effect on plasma total CO2 and PHe The CO2 decrease wasexplained by the titration of HC03 by protons released during anaerobic metabolism andthe adrenergic extrusion from the red cells and the subsequent excretion of molecular CO2at the gills. It was concluded that the decrease in PHe following CA infusion was due toan imbalance in acid excretion at the gills and acid load at the tissues. So CA infusionchanges the movement of acid in the animal.In chapter 2, it was reported that total CO2 content of the plasma decreased byabout 60% of the resting value during hypoxia. Thus, if CA is available to the plasma, thestore of HC03 is rapidly depleted in acidotic conditions. In the light of this finding, thepresent experiment was conducted to see if, in fact, the HCO3 reserve would be depletedwith a different pattern of acidosis. In hypoxia, the acid is released slowly during anaerobicmetabolism. If it is true that the greater decrease in total CO2 following CA infusionduring hypoxia was due to the HC03 titration, then, the HC03 decrease would be moredrastic if the acid should be released in a shorter time as in recovery after exhaustiveexercise. Furthermore, exhaustive exercise promotes the release of catecholamines and anincrease in ventilation as in deep hypoxia.To further investigate the fact that CA is not available to the plasma in fish gills, we29infused CA during the recovery from exhaustive exercise and monitored the acid-basebalance and oxygen transport in the blood of trout.Materials and MethodsFreshwater rainbow trout [Oncorhynchus mykiss (Walbaum)], weighing 700-850 g,were obtained from a local hatchery, and held outdoors at the University of BritishColumbia, in dechlorinated Vancouver tap water (6-7°C) for at least 2 weeks beforeexperimentation. The animals were fed once a week with commercial trout pellets, butfeeding was suspended 3 days prior to surgery. Under MS-222 anesthesia (1:10 000 inNaHCO3-buffered freshwater), fish were fitted with dorsal aortic catheters according toSovio et al. (1975). Following surgery, fish were allowed to recover for at least 48h in aBrett type respirometer with recirculating water (6-7°C).Experimental protocolThe animal was swim to exhaustion by gradually increasing the speed of the waterin a swim tunnel. At exhaustion, the animal was transferred to a dark Plexiglass box anda blood sample (800 l) was immediately taken from the dorsal aorta. Plasma pH (pHj,plasma total C02, hematocrit, hemoglobin, oxygen content and oxygen partial pressure(Po2)were immediately analyzed. The remainder of the blood was centrifuged, plasma wasseparated from RBC’s and both were frozen in liquid nitrogen for further analysis oflactate, catecholamines and red blood cell pH (pH1). Just before exhaustion, one group of30fish was injected with lml/kg of saline and another with lml/kg of carbonic anhydrasesolution of 10 000 units. After the fish was transferred in the Plexiglass box, saline or CAwas continuously infused for the duration of the experiment (6h). Blood samples weretaken as described above at 30, 60, 120 and 360 minutes of the recoveiy period.Analytical proceduresThe procedures used for the present experiment were the same as those describedin chapter 2.Statistical methodsStatistical significance of data was determined using a two-way ANOVA followedby a Dunnet test when comparing the hypoxia values with the resting values and anunpaired t-test; both with a statistical significance level of 5%. Data are presented as mean± standard error on the mean (SEM).ResultsCatecholamines were released before or at exhaustion as they were initially high andslowly decrease thereafter. CA infusion had no effect on catecholamines release (table 2).At zero time, immediately after exercise, lactate was above resting levels (see chapter 2),peaked at 2 hours and then decreased. There was no significant difference between salineand CA infused animals (fig. 8).31Hematocrit was initially very high and slowly decreased for the rest of theexperiment (Fig. 9c). Hemoglobin was also high at the beginning of the recovery periodand showed a steady decrease reflecting the hematocrit (Fig. 9b). A significant differencewas not observed in these parameters between treatments (fig. 9a-b). The ratio ofhemoglobin and hematocrit (MCHC), which is an indication of RBC volume, was initiallylow (indicating an increase in volume) and increased to return to resting values at 6 hours(Fig. 9a).The amount of oxygen bound to hemoglobin (02/}Ib) increased slightly right afterexhaustion and remained high for the rest of the recovery period (Fig. lOa). CA infusedfish had a similar pattern to the saline infused group. Oxygen partial pressure was higherin control fish than in CA infused fish and there was a significant difference at 60 minutes(Fig. lob). By 2 hours, Po2 in the CA infused group had reached the control values andthere was no significant difference thereafter (fig. lob).As expected, plasma pH dropped below resting levels at the onset of recovery (Fig.11). In the saline infused group the drop was greater than in the CA infused group andthe difference was significant at time zero. Then, PHe recovered at the same rate in thetwo groups but since PHe in the control group was lower at the beginning of the recoveryperiod, it was always lower the in the CA infused group. In the latter group, P11e overshootthe resting levels and was significantly higher than the saline infused group. On the otherhand, pH1 did not vary significantly during the recovery period in either treatment (fig. 11).32Total CO2 content of the plasma decreased significantly more in the saline treatedanimals than in CA treated ones (Fig. 12a). In the CA infused fish, total CO2 decreasedas well but was significantly different from the saline infused group at 1 and 2 hours. After2 hours total CO2 increased to reach the time zero values and there no significantdifference between the two groups (fig. 12a). The calculated equilibrium Pco2 wassignificantly different in the CA infused fish compared with the sham right at the beginningof the recovery period (Fig. 12b). In the saline infused fish, Pco2 was more than twicehigher than the resting values (see chapter 2, fig. 4). At thirty minutes, Pco2 levels weresimilar in the two groups, 2-3 torrs.33Table2.Plasmacatecholamineconcentrationsforsalineandcarbonicanhydrase(bold)infusedfishduringrecoveryafterexhaustiveexercise.Significanceof differencefromtimezeroafterexhaustiveexercise:•0minutes30minutes60minutes120minutes360minutesadrenaline(ng/ml)Saline64.4±39.910.8±4.523.9±12.98.9±2.927.1±17.2CA35.9±11.912.5±3.1*16.9±5.1*13.4±5.2*31.9±17.5noradrenaline(ng/mI)8.1±2.91.5±0.5*2.3±1.3*1.2±0.5*2.3±1.5*Saline6.6±1.41.9±0.5*2.3±0.7*1.9±0.7*2.8±1.4*CA201816120-4JC-)o 10860 60 120 180Time (mm)Figure 8. Lactate levels in rainbow trout infused with carbonic anhydrase or salinefollowing exhaustive exercise. * indicates significant difference between hypoxia andnormoxia.240 300 36035323028C.)z 26C-)24_11‘- 10C090E0Z 845401.C.)o 3530250 60 120 180 240 300 360Time (mm)Figure 9. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on hematocrit, hemoglobin concentration and mean cell hemoglobinconcentration. * indicates significant difference between hypoxia and normoxia.36SI I. I I1.6 *—.. 1.4-o=E 1• Corbonic onhydrose1.0 0 Soline0.8 I I I0 60 120 180 240 300 360Time (mm)Figure 10. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on the amount of oxygen bound to hemoglobin and oxygen partialpressure of arterial blood. * indicates significant difference between hypoxia and normoxia.** indicates significant difference between saline and carbonic anhydrase infused animals.378.20 8.0 -CEC’,Ca -C-)7.20 60 120 180 240 300 360Time (mm)Figure 11. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on plasma pH and intracellular pH. * indicates significant differencebetween hypoxia and normoxia. ** indicates significant difference between saline andcarbonic anhydrase infused animals.• Carbonic onhydraseo Saline***I I I I I I38zDz1II0 60 120 180 240 300 360lime (mm)Figure 12. Effects of carbonic anhydrase infusion in rainbow trout during recovery afterexhaustive exercise on plasma CO2 and CO2 partial pressure. * indicates significantdifference between hypoxia and normoxia. ** indicates significant difference betweensaline and carbonic anhydrase infused animals.39DiscussionRBC volume increased upon catecholamine stimulation in both experimental groups.The increase was slightly, though not significantly greater, in the CA infused animals.These findings are in accordance with the ones presented in chapter 2. As expected, RBCvolume returned to resting levels by 6 hours since catecholamine concentrations decreasedand the PHe increased close to resting values (see chapter 2). Hematocrit was very highat the beginning of the recovery period probably due to RBC recruitment from the spleenand RBC swelling. It then decreased to resting levels due to RBC shrinkage and sampling.During the entire recovery period CA had no effect on the amount of oxygen boundto hemoglobin. This was also observed in the other study presented in the precedingchapter where the oxygen content of the CA infused fish was similar to the control group.A lower Po2 was observed in the first hour of the recovery period in CA infused animals.This could result from a disequilibrium in the CO2 hydration/dehydration reaction whichis normally uncatalyzed in the plasma. As the blood flows through the gills, CO2 isexcreted and CO2 hydration is enhanced. As the blood flows away from the gills, molecularCO2 is formed and trapped in the blood. CO2 then enters the RBC and is rapidlydehydrated by CA present inside the cell. The protons produced from this dehydrationreaction bind to hemoglobin and dislodge the oxygen from hemoglobin, thus increasing Po2.In the presence of CA, CO2 formation is accelerated and the higher Po2 is not observedbecause the CO2 hydration/dehydration reaction is at equilibrium at the gills. In bothgroups, Pa02 and Hb02 tended to be low immediately following exercise. Primmett et at.40(1986) observed a decrease in gill ventilation frequency associated with exhaustion. Thefrequency of gill ventilation was not measured in the present experiment but a decrease inthis parameter could reduce the oxygen content of the blood.In the saline infused animals, CO2 tension at equilibrium immediately followingburst swimming was higher than in resting animals (fig. 4 and fig. 10). Pco2 was similarbetween the two groups for the rest of the recovery period. This high Pco2 without acorresponding decrease in Pa02 has been observed in many other studies (Turner et al.,1983; Milligan and Wood, 1986; McDonald et a!., 1989). The Pco2 presented here werecalculated reflecting equilibrium values and are probably not tensions that animals areexperiencing. Thomas and Perry (1991) showed that there is still an increase in Pco2 afterexhaustive exercise while recording continuously using an extracorporal loop. It has beenpostulated that adrenergic stimulation reversed the Pco2 gradient between red cells andplasma (CO2 entering the cells under those conditions) and that the red cells played a smallrole in CO2 excretion when catecholamines are present (Wood and Perry, 1991). Thiswould lead to an increase in Pco2 despite the hyperventilation. In addition, Primmett et a!.(1986) reported that ventilation was reduced immediately after exhaustive exercise. Sincecatecholamines were present after exhaustive exercise, ventilatory adjustments could explainthe Pco2 increase in the saline infused animals. In CA infused animals, Pco2 at equilibriumdecreased, probably because CO2 excretion does not have to go through the red cells whenCA is present in the plasma. Perry and Wood (1989) reported that the respiratory acidosiswas reduced after exhaustive exercise in the presence of CA, supporting the resultspresented here.41If the argument that acid loading titrate HC03 to form C02, which is excreted atthe gills, is true, then HC03 should have decreased more in the CA animals than in thecontrol animals. This is not the case as CO2 content in the plasma was higher in the CAinfused fish than in the controls. Plasma pH was also higher in CA infused animals thanin saline infused fish. We can presume that muscle acidification was the same in bothgroups of animals because plasma lactate concentrations were similar. If the acidificationof the muscles was the same, then either the acid was retained in the muscle or acid isexcreted at a higher rate across the gills. It is more likely that acid was retained in themuscles when CA was present in the plasma because there were no limitation for HCO3dehydration in the gills. In other words, CO2 and acid equivalents can be excreted fasterbecause of the presence of CA. This could not be the case because if acid was excretedat a higher rate to raise the PHe then total CO2 levels would be lower in the CA infusedanimals than in the saline ones. This difference in total CO2would result from the titrationof HC03 under acidotic conditions. It appears as though CA reduces the acid transfer inthe plasma from the muscle. The mechanisms of this retention are yet to be discovered.42Chapter 4General Discussion43Carbonic anhydrase did not have the expected effects in both studies. In hypoxia,CA did not short-circuit the regulatory effects of catecholamines on red blood cell pH thusmaintaining Hb-02 affinity. CA infusion, however, greatly reduced HC03 concentrationsin the plasma and created an acidosis which could not be explained by CO2 removal oraddition. The hypothesis of the second study was that in the face of rapid acid excretion(recovery after exhaustive exercise compared with deep hypoxia) HC03 stores would berapidly depleted. The opposite was observed; HC03 stores in the CA infused animals werealways higher than in the saline infused animals. Obviously, CA in the plasma changed therate at which acid is transferred within and/or excreted by the body.The patterns of acid formation were different in the two studies. During hypoxia,CO2 production probably remained the same or maybe might have even decreased whileacid production increased due to anaerobic metabolism. After exhaustive exercise, CO2production and acid release from the muscle, were greatly increased. CO2 loading in theblood from the muscle would be enhanced by CA in the plasma because the gradient fromthe muscle to the blood would be kept lower. That is to say that as soon as CO2 istransferred to the blood it is the hydrated to form HCO3,maintaining the Pco2 gradient.This in turn will lower the blood pH and might decrease acid excretion from the muscle.The inverse may also be true: H are released in the blood, shifting the reaction to CO2formation and decreasing the gradient for CO2 excretion in the blood. At the gills one canimagine the same interaction: one product of excretion limiting the excretion of the other.CA available to the plasma at the gill level might inhibit acid excretion or osmoregulationby altering the turnover of protons and/or CO2. Fish are often exposed to acid waters,44especially in freshwater. CA present in the plasma could potentially enhance acid uptakeby cycling CO2while maintaining the transepithelial acid gradient (fig 13). The same thingwould probably happen if the fish was submitted to an hypercapnic environment: a largertransfer of CO2 in the blood due to CA that maintains the gradient. In the lungs, thesesituations do not appear since lungs do not play any function in excretion of proton acrossthe membrane nor in ion regulation.The reasons why CA is not available to the plasma at the gills could not bedetermined by the two studies presented herein. In the presence of CA activity, there isprobably reduced HC03 flux through the red blood cell during the oxygenation anddeoxygenation process. HC03 dehydration consumes the protons produced by hemoglobinoxygenation. A reduced HC03 flux through the red cell will result in larger oscillations inerythrocyte pH during oxygenation of the hemoglobin and may impair blood oxygensaturation in fish with a marked Root shift. This does not happen in hypoxic conditionsbecause of the reduced hemoglobin-oxygen saturation. The hypoxia was severe and the pHmight not affect the oxygen binding at such low saturation of the hemoglobin. The a-chains of hemoglobin are acethylated in fish and therefore do not bind protons (Jensen,1989). The 13-chains do bind protons and they are also the two last chains to bind oxygenin the oxygenation process. Therefore, at the level of hypoxia at which the experiment wasdone might be too low to detect any difference in oxygenation between saline and CAinfused animals. During recovery from exhaustive exercise, impaired oxygenation mighthave been the case as the oxygen stores in the first minutes of recovery were lower,although not significantly, in the CA infused fish.45The question remains as to why is there no carbonic anhydrase available to fishplasma? The reason could be phylogenic; that is, membrane bound CA did not occur inthe evolution of fish. Since we know so little about the location and distribution of CA inlower vertebrates like fish we cannot rule out this possibility. CA infusion did notdrastically impair oxygenation under all experimental conditions. In addition, it did notdeplete HCO3 stores in all acidotic conditions. CA infusion, however, did change thepattern of acid loading at the muscle and, or, acid excretion at the gills. Fish plasma is notvery well buffered and a large Haldane effect is an important contribution to buffering pHeffects associated with CO2 transport. The evolution of a large Haldane effect could beassociated with large Bohr and Root effects. That is these effects may be structurallylinked together in the hemoglobin molecule. It is not clear whether selective forces haveoperated on the Haldane, Bohr or Root effects. Independent of the mechanism ofevolution, the result will lead to a large release of protons during oxygenation. If theanimal wants to maintain its oxygen carrying capacity, those protons have to be excretedor buffered. Fish hemoglobin has a low buffering capacity when compared to othervertebrates (Jensen, 1989) so the protons released upon oxygenation have to be removedin order to achieve full saturation of hemoglobin. HC03 flux through the red cell ensuresthat the protons are excreted as CO2 and cannot bind again to hemoglobin. The absenceof CA in the plasma ensures that HC03 flux through the red cell is maintained.The results of these two studies do not allow us to distinguish between enhancementor inhibition of acid excretion at the gills or at the muscles. Further experiments areneeded to determine where the acid is going during CA infusion.46GILL+Co2Figure13.Jacobs-StewartcycleinthegillsWATERCo2 HC03+BLOODCAEPITHELIUMHCO +CACo2 HC03+ATPase—H—1ReferencesBaroin, A., Gracia-Romeu, F., Lamarre, T. and Motais, R. (1984) Hormone-induced cotransport with specific pharmacological properties in erythrocytes of rainbowtrout, Salmo gairdneri. 3. Physiol. 350, 137-157.Bidani, A., Mathew, S.J. and Crandall, E.D. (1983) Pulmonary vascular carbonicanhydrase activity. J. Appl. Physiol. 55, 75-83.Boutilier, R.G., Heming, T.A. and Iwama, G.K. (1984) Physiochemical parameters foruse in fish respiratory physiology. In Fish Physiology, vol XA (ed W.S. Hoar andD.J. Randall), pp. 403-430. New York. Academic Press.Boutilier, R.G., Iwama, G.K., Heming, T.A. and Randall, D.J. (1985). The apparent pKof carbonic acid in rainbow trout blood plasma between 5 and 15°C. Resp.Physiol. 61, 237-254.Dimberg, K. (1988) Inhibition of carbonic anhydrase in vivo in freshwater-adaptedrainbow trout during long term hypercapnia in hard, bicarbonate rich freshwater.3. Comp. Physiol. 157: 405-412.Dodgson, S.J. (1991) The carbonic anhydrases: Overview of their importance in cellularphysiology and in molecular genetics. In: The Carbonic Anhydrases. (S.J.Dodgson, R.E. Tashian, G. Gros and N.D. Carter eds) Plenum Press, New York,p. 3-14.Effros, R.M., Shapiro, L. and Silverman, P. (1980). Carbonic anhydrase activity of rabbitlungs. 3. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 49, 589-600.48Ferguson, R.A., Tufts, B.L. and Boutilier, R.G. (1989) Energy metabolism in trout redcells: Consequences of adrenergic stimulation in vivo and in vitro. J. Exp. Biol.143, 133-147.Fievet, B., Claireaux, G., Thomas, S. and Motais, R. (1988) Adaptive responses of troutto acute hypoxia III. Ion movements and pH changes in the red blood cell. Resp.Physiol. 74, 99-114.Forster, R.E. and Steen, J.B. (1969). Rate limiting processes in the Bohr shift in humanred cells. J. Physiol. 196, 541-502.Geers, C. and Gros, G. (1991) Muscle carbonic anhydrase: Function in musclecontraction and in the homeostasis of muscle pH and Pco2. In: The CarbonicAnhydrases. (S.J. Dodgson, R.E. Tashian, G. Gros and N.D. Carter eds) PlenumPress, New York, p. 227-240.Hart, B.B., Stanford, G.G., Ziegler, M.G., Lake, C.R. and Chernow, B. (1989).Catecholamines: Study of interspecies variation. Crit. Care Med. 17, 1203-1222.Heming, T.A., Geers, C., Gros, G., Bidani, A. and Crandall, E.D. (1986) Effects ofdextran-bound inhibitors on carbonic anhydrase activity in isolated rat lungs. J.Appl. Physiol. 61(5), 1849-1856.Henry, R.P. (1988) Multiple functions of carbonic anhydrase in the crustacean gill. J.Exp. Zool. 248, 19-24.Henry, R.P., Smartresk, N.J. and Cameron, .J.N (1988) The distribution of branchialcarbonic anhydrase and the effects of erythrocyte carbonic anhydrase inhibition inthe channel catfish, Ictalurus punctatus. 3. exp. Biol. 134, 201-218.49Janssen, R.G. and Randall, D.J. (1975) The effects of changes in pH and PcoZ in bloodand water on breathing in rainbow trout, Salmo gairdneri. Resp. Physiol. 25, 235-245.Jensen, F.B. (1989) Hydrogen ion equilibria in fish hemoglobins. J. Exp. 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Evidence for NH4/Na andHCO31 1 exchanges. J. Gen. Physiol. 47, 1209-1227.McDonald, D.G. and Milligan, C.L. (1992). Chemical and physical properties of theblood. In “Fish Physiology” (W.S. Hoar, and D.J. Randall, eds.), Vol. 12, pp.50Academic Press, New York.McDonald, D.G., Tang, Y. and Boutilier, R.G. (1989) Acid and ion transfer across thegill of fish: Mechanisms and regulation. Can. J. Zool. 67, 3046-3054.Motais, R., Fievet, B. and Gracia-Romeu, F. (1989) Effect of Na/H antiport activationon pH for erythrocytes suspended in a HC03 containing saline. studia biophysica134, 121-126.Motais, R., Fievet, B., Gracia-Romeu, F. and Thomas, S. (1989) Na-H exchange andpH regulation in red blood cells: role of uncatalyzed H2C03 dehydration. Am. 3.Physiol. 256, C728-C735.Motais, R., Scheuring, F., Borgese, F. and Gracia-Romeu, F. (1990). Characteristics ofJ3-adrenergic-activated Natproton transport in red blood cells. In “Progress inCell Research” (J.M. Ritchie, P.J. Magistretti, and L. 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(1982) A comparison of CO2excretion in spontaneouly ventilating blood-perfused trout preparation and saline-perfused gill preparations: contribution of the branchial epithelium and red bloodcell. J. exp. Biol. 101, 47-60.Perry, S.F., Kinkead, R., Gallaugher,P. and Randall, D.J. (1989) Evidence thathypoxemia promotes catecholamine release during hypercapnic acidosis inrainbow trout (Salmo gairdneri). Resp. Physiol. 77, 351-364.Perry, S.F. and Wood, C.M. (1989) Control and coordination of gas transfer in fishes.Can. J. Zool. 67, 2961-2970.Primmett, R.N., Randall, D.J., Mazeaud, M. and Boutilier, R.G. (1986) The role ofcatecholamines in erythrocyte pH regulation and oxygen transport in rainbowtrout (Salmo gairdneri) during exercise. J. exp. Biol. 122, 139-148.Rahim, S.M., Delaunoy, J.-P. and Laurent, P. (1988) Identification andimmunocytochemical localization of two different carbonic anhydrase isoenzymesin teleostean fish erythrocytes and gill epithelia. 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(1967) Method for oxygen content and dissociation curves on microliterblood samples. J. Appi. Physiol. 23(3), 410-414.Turner, J.D., Wood, C.M. and Clark, D. (1983) Lactate and proton dynamics in therainbow trout (Salmo gairdneri). J. Exp. Biol. 104, 247-268.Wood, S.C. and Johansen, K. (1973) Organic Phosphates metabolism in nucleated redcells: Influence of hypoxia on eel Hb-02 affinity. Neth. J. Sea Res. 7, 328-338.Wood, C.M. and Perry, S.F. (1991) A new in vitro assay for carbon dioxide excretion bytrout red blood cells: Effects of catecholamines. J. Exp. Biol. 157, 349-366.Zeidler, R. and Kim, D.H. (1977). Preferencial hemolysis of postnatal calf red cellsinduced by internal alkalinization. J. gen. Physiol. 70, 385-401.54


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