Kinetics of the acclimational responses of tench to ... - Springer Link

Journal of Comparative Systemic, Biochemica[, and EnvironPhysiology B m onta, Physiology

J Comp Physiol B (1985) 156:192203

9 Springer-Verlag 1985

Kinetics of the acclimational responses of tench to combined hypoxia and hypercapnia I. Respiratory responses Frank B. Jensen and Roy E. Weber Institute of Biology,Odense University, DK-5230 Odense M, Denmark Accepted July 7, 1985

Summary. Exposing tench to environmental hypoxia-hypercapnia reduces routine O2 consumption, sharply decreases arterial 02 tension and the Po2 difference between the water and the blood, and results in marked swelling of the erythrocytes. These changes are rapidly reversed upon return to normoxia. Hypoxic-hypercapnic conditions lower the blood N T P / H b ratio to a new steady state level within 24 h, by reducing G T P / H b but not ATP/ Hb. A similar selective reduction of eryhtrocytic GTP content forms the initial response of blood incubated in vitro to anoxic conditions. The swelling as well as the reduced G T P / H b ratio in the erythrocytes appear to improve 02 loading in the gills during environmental hypoxiahypercapnia.

sociated with a degree of hypercapnia. This particularly applies to stagnant waters, or waters partly covered by floating aquatic plants, as those often inhabited by the tench. Whereas normoxic hypercapnia has frequently been used to perturb acidbase balance and to induce regulatory acid-base responses (reviewed by Heisler 1984), the e c o p h y siologically relevant condition of combined hypoxia and hypercapnia has only received minor attention (Jensen and Weber 1982; Thomas 1983). Tench acclimated to hypoxia-hypercapnia show pronounced capacity for adaptive responses in the respiratory properties and acid-base status of the blood (Jensen and Weber 1982). In order to understand these adaptational processes, and the underlying mechanisms, we have studied the time courses of the compensatory changes induced by hypoxia-hypercapnia, in the metabolic rate and blood respiratory properties (present paper) and in the extra- and intracellular acid-base status of the blood (Jensen and Weber 1985 b).

Introduction The frequent occurrence in aquatic environments of hypoxia, which may drastically hamper metabolic performance, has resulted in many studies dealing with the acute effects of hypoxia in fish, and adaptational responses observed in the various aspects of its respiratory physiology (e.g. Holeton and Randall 1967; Wood and Johansen 1972; Weber and Lykkeboe 1978; Lomholt and Johansen 1979; Booth 1979; Soivio and Nikinmaa 1981). Due to the inherent reciprocality of environmental O2 and CO2 changes, originating in microbial, as well as animal, respiratory processes, however, environmental hypoxia will almost invariably be asSymbols and abbreviations: a arterial; GTP guanosine triphosphate; Hct hematocrit; I inspired; NTP nucleoside triphosphate; w water

Materials and methods In vivo e x p e r i m e n t s Animals and experimental set-up. These experiments were carried out in the summerof 1984 on tench (Tinca tinca), 541 • 53 g

(x• N=6) in weight, which were obtained from ponds near Gr~sten, Southern Jutland in Denmark. The fish were kept at least 14 days before experimentation in holding tanks with normoxicwater (Po2> 130 mmHg) at 16 ~ and were subjected to a 12 h light - 12 h dark rhythm. Followinganaesthetizationin MS 222 the fish were placed on an operating table and the dorsal aorta cannulated according to Soivio et al. (1975). The fish were then placed in an experimental chamber with recirculating normoxic water, and allowed to recover for 48 h. The recirculating system consisted of an equilibration aquarium that was thermostated at 16 ~ and connected, via gastight tubing, to the experimental chamber, which was submerged in another aquarium, that also was kept at 16 ~

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F.B. Jensen and R.E. Weber: Respiratory compensations in hypoxia-hypercapnia

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(Fig. 1). This second aquarium was covered in such a way, that visual disturbance of the fish was avoided, but that dim light was allowed to enter the fish in a 12 h-12 h day-night cycle, as in the holding tank. The total water volume of the recirculating system was 23 I. Water was pumped through the experimental chamber at a flow rate of 32 1- h - 1. During the normoxic period the equilibration aquarium was thoroughly flushed with fresh pre-equilibrated water. Water composition was: Ca ++ ~3.5 mM, Na + ~ / . 2 mM, CI- - 1 raM, total CO2 6 m M (for Po2, Pco~ and pH values see Fig. i and below). Normoxic-normocapnic (N) conditions were obtained by bubbling air through the equilibration aquaria, and hypoxichypercapnic (H-H) conditions by bubbling of 1% CO2, 20% air and 79% N2. Gas mixtures were delivered from a WSsthoff gas mixing pump.

Experimental protocol. Following 48 t~ of normoxic-normocapnic exposure (Po2= 143 mmHg, Pco~ =0.7 mmHg) the gas supply to the equilibration aquarium was switched to the hypoxic-hypercapnie mixture. This was defined as time zero in all experiments. Po2 now started to decline and Pco2 to increase, reaching stable levels of Po~ =28 mmHg and Pco, = 7.5 mmHg after about 4 h. Water pH concomitantly declined from pH 8.4 to pH 7.5 (Fig. 1). The hypoxic-bypercapnic exposure was maintained for 48 h, whereupon normoxic-normocapnic conditions were reestablished for an additional 24 h. Blood samples were taken through the cannula at --2 h (normoxic sample), ].75 h (transition), 4, 6, 9, 24, 47 h (hypoxia-hypercapnia), 49.75 and 72 h (normoxia again). Volume

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of blood samples were 0.5 or 0.7 ml, depending on the number of variables measured.

Measurements. Po2 and pH of water entering the experimental chamber were monitored in all experiments, using a Radiometer (Copenhagen) BMS 3 electrode assembly, coupled to PHM 71 and 72 and thermostatted at 16 ~ during the whole experiment, giving high and long-term stability of the electrodes. Water Pco2 was calculated from measurements of total CO/ content (Cameron 1971) and pH, using tabulated relationships of components in the carbon dioxide system at variable pH, temperature and ionic staength (Rebsdorf 1972). Oxygen consumption was determined around --3, 7.75, 28, 46, 49, 51 and 72 h from measurements of water flow rate and Po2 in inflowing and outflowing water, using the mean value of 4-5 determinations over a period of 40 min at each measuring time. Arterial Po2 and pH was determined with the above described BMS 3 electrode assembly. Hematocrit values (Hct) were determined by centrifugation (3 rain at 12,000 rev.miu -1) in glass capillaries. Blood hemoglobin concentration was measured spectrophotometrically as cyanmethemoglobin. Total nucleoside triphosphate (NTP) concentrations were determined enzymatically (Sigma Bulletin No. 366-UV) and apportioned between ATP and GTP via thin-layer chromatography (Johansen et al. 1976). Erythrocytic concentrations were calculated from the corresponding Hct values. The data are presented as means _+SE. Significance of dif-

F.B. Jensen and R.E. Weber: Respiratory compensations in hypoxia-hypercapnia ferences, relative to normoxic control values, were evaluated using the Student's t-test.

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In vitro experiments For determination of the dynamics of NTP, ATP and GTP changes in vitro, blood samples were pooled from 3 specimens of tench from the same stock of fish as used in the in vivo experiments. The blood was divided into two 3 ml portions and incubated in Eschweiler (Kid, FRG) tonometers at 16 ~ under either normoxic (99.6% air, 0.4% CO2) and anoxic (99.6% N2, 0.4% COz) conditions, or normoxic and hypoxic (1.6% air, 98% N > 0.4% COz) conditions. Gas mixtures were supplied from cascaded W6sthoff gas mixing pumps. Blood samples were removed at specified times during the incubation and analysed for Hb, Hct, NTP, ATP and GTP as described above. Oxygen equilibrium curves at varying ATP, GTP and pH conditions were obtained using a modified diffusion chamber method on purified hemoglobin solutions (Jensen and Weber 1985a). The described cofactor conditions were obtained by mixing appropriate amounts of two Hb solutions buffered at slightly different pH, but of equal organic phosphate composition, until pH measured showed the desired pH. Results

In vivo experiments. U p o n exposure to combined hypoxia and hypercapnia, routine oxygen consumption decreased significantly ( P < 0.05) from a normoxic value of 45 ml 02" h - ~. k g - ~ to a value of about 30 ml O 2 . h - l . k g -1 at 7.75 h, which remained constant for the remainder of the hypoxichypercapnic period (Fig. 2). When normoxia was reestablished, a sharp transient increase in lz%, representing a respiratory overshoot, was observed. At 3 h after the normoxic return, however, the l/o~ was only slightly above the initial value (Fig. 2). The arterial oxygen tension decreased gradually from 5 2 m m H g in normoxia to a value of 7 m m H g at 4 h (P P > 0 . 0 5 ) (Fig. 3). After the return of normoxia, a rapid decrease in Hct to values lower than the initial normoxic value occurred. Blood hemoglobin concentration also increased to a peak value at 4 h, then decreased for the rest of the hypoxic-hypercapnic period to values that were

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Fig. 2. Oxygen uptake of tench during exposure to normoxia (N), hypoxia-hypercapnia (H-H) and subsequent return to normoxia (N)

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I i I I I 40 50 60 70 B0 Time, h Fig. 3. Time-dependent changes of oxygen tension, hematocrit (Hct) blood Hb concentration and erythrocytic Hb concentration in arterial blood of tench during the experimental cycle of normoxia ~hypoxia-hypercapnia ~ normoxia I

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Fig. 4. Oxygen gradient (APo~(I, a)) between inspired water and arterial blood of tench in normoxia (N), hypoxia-hypercapnia (H-H) and reestablished normoxia (NR)

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not significantly different from the normoxic value. Hb subsequently decreased to values below the initial value upon return to normoxia (Fig. 3). Hypoxia-hypercapnia gradually decreased cellular Hb concentration from 4.9 to 3.8 retool.1 cells- t (p < 0.01) in about 6 h. This level was maintained constant during the whole hypoxic-hypercapnic period, then rose rapidly to values slightly above 5 mmol'l cells-t upon return to normoxia (Fig. 3). The erythrocytic concentration of total nucleoside triphosphates measured in 3 specimens decreased from about 8 to about 5 mmol. 1 cells-1. The decrease in GTP concentration was more pronounced than in ATP concentration. When expressed as organic phosphate/Hb ratio, it is seen, that the decrease in N T P / H b is due to a decrease in GTP/Hb only, whereas ATP/Hb is kept constant (Fig. 5). The slight decrease in the cellular ATP concentration thus must be solely due to swelling of the erythrocytes, which keeps the ATP/ Hb ratio constant, whereas the decrease in GTP/ Hb reflects an actual decrease in the amount of GTP. The decreases in N T P and N T P / H b ap-

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peared to be completed within 24 h of combined hypoxia and hypercapnia (Fig. 5). In vitro experiments. In tench blood incubated in vitro under anoxia (Po2 = 0 mmHg, Pco2 = 3 mmHg) cellular N T P started to decline after approximately 2 h, and continued to decrease until an apparently stable level of 0.65 mmol'l cells -1 (7.5% of initial value) was obtained at 10-11 h (Fig. 6 B). The half time of this decrease was about 4 h. The initial decrease in the erythrocytic N T P concentration was due to a marked reduction in GTP concentration after about 2 h, whereas the ATP concentration was almost constant up to 4 h. Only after 4 h, when GTP concentration had reached very low levels, did the ATP concentration start to fall markedly (Fig. 6 B). By the end of the anoxic incubation GTP was barely detectable, whereby the low final N T P concentration was practically only ATP. The subsequent shift to normoxic conditions for 15 h only partially restored the N T P content from 7.5% to 22% of the intitial value (not illustrated). In the normoxic (control) incubation both total N T P and ATP and

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Fig. 7. Oxygen equilibrium curves of tench hemoglobin at various pH, GTP/Hb and ATP/Hb conditions. The conditions for curves I and III approximate the observed erythrocytic characteristics in normoxic and hypoxic-hypercapnic (from 24 h to 47 h) tench, respectively. Temperature, 16 ~ Tetrameric Hb concentration, 0.2 mM. See text for further explanation

GTP concentrations were practically constant during the whole incubation period (Fig. 6A). In sharp contrast to the anoxic incubation, hypoxic incubation (P% = 2.5 mmHg, Pc% = 3 mmHg) for up to 24 h, produced no changes in either total NTP, ATP or GTP concentrations (not shown). The in vivo changes in organic phosphates (Fig. 5) and intracellular pH (Jensen and Weber 1985b) observed under hypoxia-hypercapnia profoundly affects the 02 equilibrium curve, as judged from curves obtained on Hb solutions at ATP/Hb, GTP/Hb and pH conditions close to those observed in vivo. As the direct effect of CO2 in the presence of organic phosphates is negligible, these curves may be expected to reflect blood 02 affinities (Jensen and Weber 1982). Under normoxic conditions (curve I, Fig. 7) the P5o value was 6.1 mmHg, which compares well with the mean value of 6.2 m m H g obtained on whole blood from normoxic fish (Jensen and Weber 1982). When pH and ATP/Hb are kept constant, but GTP/Hb is decreased to the approximate hypoxic-hypercapnic value, the Pso decreased to 5.2 m m H g (curve II, Fig. 7). IfpH, together with GTP/Hb, was changed to a value close to the hypoxic-hypercapnic value, then the increase in oxygen affinity was even greater (P~o = 3.8 m m H g - curve III, Fig. 7). The oxygen equilibrium curve of the Hb at an intermediate pH in absence of both ATP and GTP is shown for comparison (curve IV, Fig. 7). Discussion

Exposure to combined hypoxia and hypercapnia decreases routine 02 consumption (Fig. 2). This parallels observations in many other fish subjected to acute, severe hypoxia (e.g. Lomholt and Johan-

201

sen 1979; Kerstens et al. 1979), whereas pure hypercapnia had no effect on 12o2, except for transient initial increases in dogfish and trout (Randall et al. 1976; Thomas et al. 1983). One reason for the decrease here observed in I7o2 is a generally lower level of spontaneous activity in the hypoxiahypercapnia exposed fish. This decrease in 1/o2 occurs, however, in the face of an apparent increase in the cost of ventilation. In normoxia, ventilatory movements are scarcely visible, whereas pronounced increases in both ventilatory frequency and especially stroke volume are clearly evident under hypoxic-hypercapnic conditions. A marked increase in water convection requirement per unit of O2 uptake l>w/l?%, can thus be predicted under these conditions. Arterial Po2 values are considerably lower than the theoretically values, approaching PI% (i.e. counter current gas exchange with complete equilibration between blood and inspired water). Thus in normoxia PIo2-Pao2 ( = A P o 2 ( I , a)) is about 90 mmHg (Fig. 4), which is similar to that in carp (Itazawa and Takeda 1978), but higher than that in trout (Holeton and Randall 1967). Reasons for a high AP% (I, a) in tench might be blood shunting through basal channels of the secondary lamellae in the gills (P/irt et al. 1984) and inequalities in the ventilation-perfusion ratio. In combined hypoxia-hypercapnia, however, AP%(I, a) decreases to 20mmHg, and the apparent conductance Go2(I, a), for the 02 flux from inspired water to arterial blood, increases. Several factors may contribute to these changes, e.g. increased functional surface area of the gills resulting from lamellar recruitment (Booth 1979), decreased diffusion distance (Soivio and Tuurala 1981), changes in ventilation-perfusion flow ratios and shunting characteristics (Tuurala et al. 1984), as well as the changes in blood 02 binding characteristics. The initial peak in both blood Hb concentration and Hct at 4 h of hypoxia-hypercapnia (Fig. 3) reflects a transitory increase in the number of erythrocytes in the dorsal aorta. This resembles the response seen in rainbow trout after adrenaline injection, and may be explained by an adrenaline mediated increase in red cell number in the dorsal aorta, via plasma skimming in the gills (Nikinmaa 1982a, b). A contribution from other processes such as the liberation of erythrocytes from the spleen or water shifts between extra- and intracellular compartments can, however, not be ruled out. But the response is short-lived, and in the remainder of the hypoxic-hypercapnic period blood Hb concentration is kept approximately at the normoxic level (Fig. 3), as is also seen in long-term

202

F.B. Jensen and R.E. Weber: Respiratorycompensationsin hypoxia-hypercapnia

acclimation to hypoxia-hypercapnia (Jensen and Weber 1982). Thus a persistent quantitative strategy of improving blood O2 transport is not used. After the return to normoxia the Hb level is slightly lower than the initial normoxic value, which, however, may be expected as a result of the repetitive blood sampling. The increase in Hct in hypoxia-hypercapnia is associated with a reduced intracellular Hb concentration (Fig. 3). This strongly indicates swelling of the erythrocytes, which is a common phenomenon in fish in hypoxia and other "stressing" conditions (Soivio and Nikinmaa 1981). The swelling is maximal after 6-9 h, and thereafter intracellular Hb concentration remains constant for the remainder of the hypoxic-hypercapnic exposure. Red cell swelling persists also in tench acclimated to hypoxia-hypercapnia for 0.5-3 months (Jensen and Weber 1982). Upon return to normoxia, however, the cells rapidly shrink (cellular Hb concentration increases - Fig. 3), illustrating a capacity for rapid, reversible volume changes in fish erythrocytes. Swelling of trout erythrocytes can be induced by fl-adrenergic stimulation both in vivo and in vitro (Nikinmaa 1982b). As the concentrations of adrenaline and noradrenaline increase in hypoxia (e.g. Butler et al. 1978), the present swelling response is most probably initiated by the binding of fi-agonists to the red cell membrane. This is associated with an elevation of intracellular pH, and a decrease in the gradient of protons across the erythrocytic membrane (Nikinmaa 1982b), as observed in the red cell of hypoxic-hypercapnic tench (Jensen and Weber 1985b). fl-adrenergic swelling thus will increase oxygen affinity via the Bohr shift, and perhaps by the reduction in the cellular concentrations of Hb and NTP per se (Nikinmaa 1983; Weber etal. 1976; Lykkeboe and Weber 1978; Soivio and Nikinmaa 1981). Further enhancement of blood O2 loading is obtained by the later NTP/Hb decrease. In hypoxia-hypercapnia the NTP/Hb ratio is decreased to a new steady-state level within about 24 h (Fig. 5). This is a rapid time course in comparison with that reported in vivo for other fish exposed to hypoxia only, where it typically takes one week (Greaney and Powers 1977; Weber and Lykkeboe 1978; Soivio et al. 1980). The rapidity of the response in tench may contribute to its success to colonize in natural environments of variable but frequently very low 02 tensions. The decrease in NTP/Hb is due to a selective reduction in GTP/Hb leaving ATP/Hb unaltered (Fig. 5). This also characterizes the fully acclimated state (Jensen and Weber 1982). Selective decrease of GTP is advantageous, as the effect of GTP on the O2 affinity of

fish Hb is greater than that of ATP (Weber et al. i975; Jensen and Weber 1982). This mechanism keeps ATP available for energy requiring processes in the cell. In this connection it is striking, that other fish, such as carp (Weber and Lykkeboe 1978), eel (Weber et al. 1975) and lungfish (Johansen et al. 1976) which experience hypoxic conditions in their natural life cycles, also have large parts of their erythrocytic NTP represented by GTP; whereas a fish like the rainbow trout, living in well aerated waters, has very low GTP levels (Tetens and Lykkeboe 1981). High normoxic GTP levels available for selective reduction thus appears to be part of a successful adaptation to periodic hypoxic environments. Although the beneficial effect of a selective reduction of GTP/Hb is easily recognized, the regulation of the processes involved is still an open question. When tench blood is subjected to anoxic conditions, erythrocytic NTP appears to be depleted as a result of impeded oxidative phosphorylation (Fig. 6 B). This tallies with findings in erythrocytes from killifish and trout (Greaney and Powers 1977; Tetens and Lykkeboe 1981). Significant in the present results, however, is the extended depletion of GTP that occurs before a significant ATP depletion is seen (Fig. 6B). These in vitro changes support the changes seen in vivo. The causal factor for the in vivo GTP decrease might, however, not be the low Po2 per se, as an incubation of blood at an oxygen tension of 2.5 m m H g (approaching venous Po2 in hypoxic-hypercapnic fish) did not affect either erythrocytic ATP or GTP concentrations. The in vivo response might thus be initiated by a hormone, as suggested by Tetens and Lykkeboe (1981). The dynamics of the changes seen in the anoxic incubation suggests, however, that the preservation of high ATP concentration and selective reduction of GTP, is an inherent characteristic at the cellular level. The in vivo alterations in the cellular physicochemical microenvironment will strongly improve arterial O2 loading of the hemoglobin. Both the direct allosteric effect of the GTP/Hb decrease, as well as the Bohr shift due to the decreased proton concentration (Jensen and Weber 1985b) significantly raises O2 affinity (Fig. 7). From the present H b - O 2 equilibrium curves, and the curves obtained on whole blood (Jensen and Weber 1982), some estimates of the blood Oz transport can be made. In acute hypoxia-hypercapnia extracellular pH decreases, whereas erythrocytic pH is rapidly restored to the normoxic level due to the swelling response (Jensen and Weber 1985b). Thus the normoxic H b - - O 2 equilibrium curve can be used to estimate oxygen saturation. This curve predicts an

F.B. Jensen and R.E Weber: Respiratory compensations in hypoxia-hypercapnia

arterial O2-saturation of approximately 50% at the arterial Po2 of 7 mmHg in hypoxia-hypercapnia. Without the swelling response the high Pc% and low plasma pH would have decreased pHi and thus blood O2 affinity, decreasing arterial 02 saturation well below 50%. In the course of 24 h, GTP/Hb decreases and pHi is further increased, resulting in an oxygen equilibrium curve for which arterial saturation is increased to about 65-70% at P % = 7 mmHg. Venous P% in normoxic tench is around 7 mmHg (Eddy 1974). Since the present hypoxichypercapnic load produced an arterial P% of 7 mmHg, the venous Po2 has to be decreased, probably to a value between 2 and 3 mmHg. This reduces the gradient driving the 02 flux from the tissue capillaries to the cells, which may dictate a lower 12o2(Fig. 2), and explain the slight increase observed in blood lactate concentration (Jensen and Weber 1985 b) in hypoxic-hypercapnic tench. References Booth JH (1979) The effects of oxygen supply, epinephrine, and acetylcholine on the distribution of blood flow in trout gills. J Exp Biol 83:31-39 Butler PJ, Taylor EW, Capra MF, Davison W (1978) The effect of hypoxia on the levels of circulating catecholamines in the dogfish Scyliorhinus canicula. J Comp Physiol 127:325-330 Cameron JN (1971) Rapid method for determination of total carbon dioxide in small blood samples. J Appl Physiol 31:632-634 Eddy FB (1974) Blood gases of the tench (Tinca tinca) in well aerated and oxygen-deficient waters. J Exp Biol 60:71-83 Greaney GS, Powers DA (1977) Cellular regulation of an allosteric modifier of fish haemoglobin. Nature 270 : 73-74 Heisler N (1984) Acid-base regulation in fishes. In: Hoar WS, Randall DJ (eds) Fish physiology, vol XA. Academic Press, New York, London, pp 315-401 Holeton GF, Randall DJ (1967) The effect of hypoxia upon the partial pressure of gases in the blood and water afferent and efferent to the gills of rainbow trout. J Exp Biol 46: 317-327 Itazawa Y, Takeda T (1978) Gas exchange in the carp gills in normoxic and hypoxic conditions. Respir Physiol 35:263-269 Jensen FB, Weber RE (1982) Respiratory properties of tench blood and hemoglobin. Adaptation to hypoxic-hypercapnic water. Molec Physiol 2:235 250 Jensen FB, Weber RE (1985) Proton and oxygen equilibria, their anion sensitivities and interrelationships in tench hemoglobin. Molec Physiol 7:41-50 Jensen FB, Weber RE (1985b) Kinetics of the acclimational responses of tench to combined hypoxia and hypercapnia. II. Extra- and intraceltular acid-base status in the blood. J Comp Physiol 205-2i i Johansen K, Lykkeboe G, Weber RE, Maloiy GMO (1976) Respiratory properties of blood in awake and estivating lungfish, Protopterus arnphibius. Respir Physiol 27:335 345 Kerstens A, Lomholt JP, Johansen K (1979) The ventilation, extraction and uptake of oxygen in undisturbed flounders, Platichthysflesus: Responses to hypoxia acclimation. J Exp Biol 83:169-179 Lomholt JP, Johansen K (1979) Hypoxia acclimation in carp

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- how it affects 02 uptake, ventilation, and Oz extraction from water. Physiol Zool 52:38-49 Lykkeboe G, Weber RE (1978) Changes in the respiratory properties of the blood in the carp, Cyprinus carpio, induced by diurnal variation in ambient oxygen tension. J Comp Physiol 128:117-125 Nikinmaa M (1982a) The effects of adrenaline on the oxygen transport properties of Salrno gairdneri blood. Comp Biochem Physiol 71A: 353-356 Nikinmaa M (1982b) Effects of adrenaline on red cell volume and concentration gradient of protons across the red cell membrane in the rainbow trout, Salrno gairdneri. Molec Physiol 2:287-297 Nikinmaa M (1983) Adrenergic regulation of haemoglobin oxygen affinity in rainbow trout red ceils. J Comp Physiol 152:67 72 Pfirt P, Tuurala H, Nikinmaa M, Kiessling A (1984) Evidence for a non-respiratory intralamellar shunt in perfused rainbow trout gills. Comp Biochem Physiol 79A:29-34 Randall DJ, Heisler N, Drees F (1976) Ventilatory response to hypercapnia in the larger spotted dogfish Scyliorhinus stellar&. Am J Physiol 230:590-594 Rebsdorf A (]972) The carbon dioxide system in freshwater. A set of tables for easy computation of total carbon dioxide and other components of the carbon dioxide system. Freshwater Biol Lab, Hiller6d, Denmark Soivio A, Nyholm K, Westman K (1975) A technique for repeated sampling of the blood of individual resting fish. J Exp Biol 63:207-217 Soivio A, Nikinmaa M, Westman K (1980) The blood oxygen binding of hypoxic Salmo gairdneri. J Comp Physiol 136: 83-87 Soivio A, Nikinmaa M (1981) The swelling of erythrocytes in relation to the oxygen affinity of the blood of the rainbow trout, Salmo gairdneri Richardson. In: Pickering AD (ed) Stress and Fish. Academic Press, London, pp 103-119 Soivio A, Tuurala H (1981) Structural and circulatory responses to hypoxia in the secondary lamellae of Salmo gairdneri gills at two temperatures. J Comp Physiol 145: 37-43 Tetens V, Lykkeboe G (1981) Blood respiratory properties of rainbow trout, Salmo gairdneri: Responses to hypoxia acclimation and anoxic incubation of blood in vitro. J Comp Physiol 145 : 117-125 Thomas S (1983) Changes in blood acid-base balance in trout (Salmo gairdneri Richardson) following exposure to combined hypoxia and hypercapnia. J Comp Physiol 152: 53-57 Thomas S, Fievet B, Barthelemy L, Peyraud C (1983) Comparison of the effects of exogenous and endogenous hypercapnia on ventilation and oxygen uptake in the rainbow trout (Salrno gairdneri R.). J Comp Physiol 15]:185-190 Tuurala H, P/irt P, Nikinmaa M, Soivio A (1984) The basal channels of secondary lamellae in Salmo gairdneri gills A non-respiratory shunt. Comp Biochem Physiol 79A: 3539 Weber RE, Lykkeboe G, Johansen K (1975) Biochemical aspects of the adaptation of hemoglobin-oxygen affinity of eels to hypoxia. Life Sci 17:1345-1350 Weber RE, Wood SC, Lomholt JP (1976) Temperature acclimation and oxygen-binding properties of blood and multiple haemoglobins of rainbow trout. J Exp Biol 65:333-345 Weber RE, Lykkeboe G (1978) Respiratory adaptations in carp blood. Influences of hypoxia, red cell organic phosphates, divalent cations and CO2 on hemoglobin-oxygen affinity. J Comp Physiol 128:127-137 Wood SC, Johansen K (1972) Adaptation to hypoxia by increased HbO2 affinity and decreased red cell ATP concentration. Nature New Biol 237 :278-279