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Adenosine receptor brockade and hypoxia tolerance in rainbow trout and Pacific hagfish Bernier, Nicholas J. 1994

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ADENOSINE RECEPTOR BLOCKADE AND HYPOXIA TOLERANCE IN RAINBOW TROUT AND PACIFIC HAGFISH by NICHOLAS J. BERNIER B.Sc, McGill University, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT OF THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1994 © Nicholas J. Bernier, 1994 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of j ^ O O L "P G>> The University of British Columbia Vancouver, Canada Date w* ^ DE-6 (2/88) ii ABSTRACT The physiological properties of adenosine may be essential in the control of energy metabolism for the survival of animals exposed to oxygen shortages. Accordingly, in this thesis the hypothesis that adenosine modulates the response of rainbow trout and Pacific hagfish to acute hypoxic exposure was tested. Three different experimental series were conducted to investigate the possible roles of adenosine in hypoxia tolerance. Rainbow trout were exposed to either a Pw02 of 30 torr for 6 h (SERIES I), or to 25 torr for 1 h (SERIES II). Pacific hagfish were exposed either to a Pw0 of 10 or 30 torr for 1 h (SERIES III). In all three series, the role of adenosine was assessed by comparing the response of animals treated with non-specific adenosine receptor blockers to control shams under hypoxic and normoxic conditions. Three different areas were investigated for adenosine's actions in these experiments: 1) the recruitment of anaerobic metabolism; 2) the stress response; and 3) the role of erythrocytes for oxygen capacitance. Relative to hypoxic sham fish, increases in plasma [lactate] with hypoxic exposure were greater in the animals injected with the adenosine receptor (AR) blocker theophylline in all three series. This response to AR blockade was also associated with a more rapid and pronounced metabolic acidosis in SERIES I & II. In hagfish, plasma [lactate] increased following exposure to a Pw0 of 10 torr but not 30 torr, and plasma acidosis was only observed in the animals exposed to a Pw0 of 10 torr and treated with theophylline. In SERIES II, only the tissues from the hypoxic theophylline treated trout had significant increases in tissue [lactate] when compared to the normoxic groups. Decreases in creatine charge were observed in the heart and red muscle, but not white muscle, of theophylline iii treated fish. The glycogen content of the heart also decreased following theophylline treatment. The tissue metabolites of hypoxic trout treated with enprofylline, an AR blocker with very weak affinity, were similar to the hypoxia sham fish, and the increase in plasma lactate was intermediate to the hypoxic theophylline and sham groups. Both AR blockers had no measurable effects on normoxic controls. These findings indicate that AR blockade results in a more rapid and pronounced recruitment of anaerobic metabolism following acute hypoxic exposure in rainbow trout and Pacific hagfish. In SERIES I, plasma [Cortisol] increased after 10 min of hypoxic exposure and remained elevated in the theophylline group. An increase in plasma [Cortisol] was observed after 30 min of hypoxia but was transient in the hypoxic sham trout. In SERIES II, after 10 min of hypoxic exposure, the plasma [adrenaline] in the theophylline and enprofylline treatments were respectively 16 and 4 fold higher than in the hypoxic sham treatment. This difference, although not as pronounced, was maintained after 60 min of acute hypoxia between the theophylline treatment and the two other hypoxic groups. In hagfish, whereas plasma [adrenaline] did not change following exposure to a Pw0 of 10 torr in the hypoxic sham group, the [adrenaline] increased 3.8 fold within 10 min in the theophylline group and returned to control levels by 60 min. AR blockade with methylxanthines had no effect on the concentrations of plasma Cortisol and catecholamines in the normoxic animals. These results indicate that adenosine receptor blockade modulates the primary stress response of hypoxic rainbow trout and Pacific hagfish. In rainbow trout, serial blood sampling in SERIES I resulted in a greater [Hb] decrease in the hypoxic group treated with theophylline than in the hypoxic sham group. iv In SERIES II, an increased in [Hb] was observed in the hypoxic sham group but not in the hypoxic groups treated with the AR blockers. In the hagfish experiment, AR blockade had no effect on the relative decrease in [Hb] in all the treatments. These results indicate that in hypoxic rainbow trout, AR blockade may prevent splenic release of rbc by abolishing the stimulatory effects of catecholamines on this tissue. Unlike rainbow trout, Pacific hagfish may not increase their [Hb] under acute hypoxic conditions. Results from these experiments show marked differences between rainbow trout and Pacific hagfish in their response to hypoxia, and in the strategy that each utilizes to resist such conditions. However, results also indicate that adenosine has an important protective role in both species, and that the actions of adenosine in fish, as in other vertebrates, may have a common tendency to reduce energy expenditure, while improving oxygen delivery. Specifically, adenosine may reduce the extent to which anaerobic metabolism is recruited upon acute hypoxic exposure, modulate the circulating levels of the primary stress hormones catecholamines and Cortisol, and play a role in maintaining the oxygen carrying capacity of the trout by modulating the splenic contribution of rbc. TABLE OF CONTENTS Abstract ii Table of Contents v List of Tables vi List of Figures vii List of Abbreviations ix Acknowledgements x General Introduction 1 Chapter One: Effects on metabolism 8 Introduction 9 Methods 10 Results 21 Discussion 33 Chapter Two: Effects on the stress response 47 Introduction 48 Methods 50 Results 51 Discussion 57 Chapter Three: Erythrocyte responses to adenosine receptor blockade 64 Introduction 65 Methods 66 Results 66 Discussion 79 General Discussion 84 Literature Cited 89 Appendix 1 104 vi LIST OF TABLES Table 1. Concentration of metabolites in heart, red muscle and white muscle after one hour of either normoxia or hypoxia (Pw02=25 torr) in rainbow trout at 7°C. Animals were infused with either saline or one of two adenosine receptor blockers: enprofylline or theophylline. 36 Table 2. Blood parameters of the Pacific hagfish and the rainbow trout. 67 Table 3. Haematocrit, mean cellular haemoglobin content, red blood cell pH, and the difference between whole blood pH and pHi of rainbow trout, in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were infused with either saline, or theophylline. 75 Table 4. Haematocrit, mean cellular haemoglobin content, red blood cell pH, and the difference between whole blood pH and pHi of rainbow trout, in relation to exposure duration to normoxia and hypoxia (Pw02=25 torr). Animals were infused with either saline, enprofylline, or theophylline. 77 Table 5. Blood pH, rbc pH, rbc transmembrane pH difference, haematocrit, haemoglobin, and mean cellular haemoglobin content of Pacific hagfish, in relation to exposure duration to normoxia and hypoxia (Pw02 = 10 or 30 torr). Animals were infused with either saline, or theophylline. 105 vii LIST OF FIGURES Figure 1. A schematic diagram of the generalized cellular pathways of adenosine formation, metabolism, and mode of action. 3 Figure 2. Pattern of change in water P 0 2 in a 7.5 1 fish box following transfer from a normoxic water source to a hypoxic water source with a P 0 2 of 25 torr. 14 Figure 3. Plasma pH of rainbow trout in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were injected with either saline or theophylline. 22 Figure 4. Plasma pH of rainbow trout in relation to exposure duration to normoxia and hypoxia (Pw02=25 torr). Animals were injected with either saline, enprofyUine, or theophylline. 24 Figure 5A. Plasma pH of Pacific hagfish in relation to exposure duration to hypoxia (PwO2=10 torr). Animals were injected with either saline, or theophylline. 27 Figure 5B. Plasma lactate of Pacific hagfish in relation to exposure duration to hypoxia (Pw02= 10 torr). Animals were injected with either saline, or theophylline. 27 Figure 6. Plasma lactate of rainbow trout in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were injected with either saline or theophylline. 29 Figure 7. Plasma lactate of rainbow trout in relation to exposure duration to normoxia and hypoxia (Pw02=25 torr). Animals were injected with either saline, enprofyUine, or theophylline. 31 Figure 8. Tissue lactate in white muscle, red muscle, and heart of rainbow trout exposed to 60 minutes of normoxia or hypoxia (Pw02=25 torr). Animals were injected with either saline, enprofyUine, or theophylline. 34 viii Figure 9. Plasma theophylline of rainbow trout injected with 4 mg/kg theophylline in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). 38 Figure 10. Plasma Cortisol of rainbow trout in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were injected with either saline or theophylline. 52 Figure 11 A. Plasma adrenaline of rainbow trout in relation to exposure duration to hypoxia (Pw02=25 torr). Animals were injected with either saline, enprofylline, or theophylline. 55 Figure 11B. Plasma noradrenaline of rainbow trout in relation to exposure duration to hypoxia (PwOz=25 torr). Animals were injected with either saline, enprofylline, or theophylline. 55 Figure 12A. Plasma adrenaline of Pacific hagfish in relation to exposure duration to hypoxia (PwO2=10 torr). Animals were injected with either saline, or theophylline. 58 Figure 12B. Plasma noradrenaline of Pacific hagfish in relation to exposure duration to hypoxia (PwO2=10 torr). Animals were injected with either saline, or theophylline. 58 Figure 13. Haemoglobin concentration of rainbow trout in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were injected with either saline or theophylline. 70 Figure 14. Haemoglobin concentration of rainbow trout in relation to exposure duration to normoxia and hypoxia (Pw02=25 torr). Animals were injected with either saline, enprofylline, or theophylline. 72 IX LIST OF ABBREVIATIONS Ax / A2: adenosine receptor subtype 1 & 2 ACTH: adrenocorticotrophic hormone ADO: adenosine AMP: adenosine monophosphate ATP: adenosine triphosphate AR: adenosine receptor CNS: central nervous system Cr / PCr: creatine / creatine phosphate Hb: haemoglobin Hct: haematocrit IMP: inosine monophosphate MCHC: mean cellular haemoglobin content NAD+ / NADH: nicotinamide adenine dinucleotide / reduced form the partial pressure of oxygen at which half the haemoglobin is saturated arterial blood oxygen partial pressure water oxygen partial pressure PCA: perchloric acid PDE: phosphodiesterase enzyme pHe: blood pH pHi: red blood cell pH rbc: red blood cell P50: PaQ2: PwO, X ACKNOWLEDGEMENTS I would like to thank first and foremost my supervisor Dr. Dave Randall, for giving me endless opportunities to learn, to explore, and to appreciate the field of research in comparative zoology. I gratefully acknowledge Peter Arthur, Juan Fuentes, Joelle Harris, Daniel Heath, Richard Kinkead, Steve Land, Joanne Lessard, Marc Mossey, and Tim West. Their collaboration, assistance, and teaching made the experiments and the analysis possible technically and humanly. For their time and patience, I thank them. I would also like to acknowledge the Zoology Department for a Teaching Assistantship, and the BC Science Council for a GREAT Award. 1 General Introduction. Most fish share a number of behavioral and metabolic features which allow them to acclimate to moderate hypoxia because they live in an environment where the availability of oxygen is quite variable. In response to low oxygen, fish reduce their locomotor activity (Nilsson, G. E. et al. 1993b) and seek out a lower thermal regime (Crawshaw et al. 1989; Schurmann et al. 1991; Steffensen, 1993). Rapid detection of declining oxygen tensions by oxygen-sensitive chemoreceptors located in the gills (Burleson et al. 1992), leads to quick adjustments in cardiovascular and ventilatory activity (Smith and Jones, 1978; Randall, 1982, 1990; Fritsche and Nilsson, 1993). These regulatory mechanisms improve oxygen extraction (Jensen et al. 1993), and reduce the need to recruit anaerobic metabolism as a compensatory measure to meet energy requirements (Van Den Thillart, 1982). The response of fish to acute hypoxia, while involving adjustments in behavioral and cardio-ventilatory activity, is also characterized by the release of catecholamines and corticosteroids (Barton and Iwama, 1991). This stress response further enhances blood oxygen transport (Randall and Perry, 1992), and mobilizes energy reserves to maintain energy turnover (Wright et al. 1989; Vijayan et al. 1991). While a few species can chronically tolerate acute hypoxia by using modified anaerobic metabolic pathways which do not result in metabolic acidosis (Nilsson, G. E., 1990; Van Den Thillard and Van Waarde, 1991), this is not the case for most fish species. Therefore, surviving acute hypoxia in most fish, becomes a balance between the need to meet the energy demands of vital functions, and the need to minimize the use of limited energy reserves to fuel anaerobic pathways and meet the energy shortfall. 2 A large literature, centred on the hypoxia-sensitive terrestrial mammals, has attributed a regulatory role for energy consumption in hypoxic conditions to adenosine (Mullane and Williams, 1990; Ribeiro, 1991). Altogether, the many actions of adenosine have a common tendency to redress an imbalance between energy demand and availability (Newby et al. 1990). The common life-preserving functions of adenosine in the bacterium Myxococcus xanthus (Newby, 1984), the turtle Pseudemys scripta (Nilsson, G. E. and Lutz, 1992), and the rat (Stefanovich, 1988), indicates that this metabolite may also have a role in the hypoxia tolerance of fish. The most important source of adenosine formation results from the breakdown of cytosolic ATP in response to an imbalance between ATP generation and utilization during hypoxia (fig. 1; Stone et al. 1990). Since the [ATP] in resting cells is approximately 50 times higher than that of AMP, a small decrease in ATP can give rise to a significant accumulation of AMP (Meghji, 1991). In oxidative tissues, such as the brain, heart, and red muscles, further degradation of AMP can lead to an increase in adenosine formation. In glycolytic tissues, such as white muscle, however, IMP is usually the only product of ATP breakdown. The transmethylation pathway, which involves the formation of SAM (S-adenosylmethionine) and SAH (S-adenosylhomocysteine), can contribute up to 90% of cellular adenosine during normoxia, but less than 20% under hypoxic conditions (Stone et al. 1990). Whereas cytoplasmic production of adenosine has been demonstrated in a number of mammalian aerobic tissues (Stone et al. 1990), and in the brain of some lower vertebrates (Nilsson, G. E. and Lutz, 1992), adenosine production has not been measured in fish. 3 Figure 1. A schematic diagram of the generalized cellular pathways of adenosine formation, metabolism, and mode of action. Abbreviations used: Al & A2, adenosine receptor subtypes; ADO, adenosine; HYPO, hypoxanthine; INO, inosine; N, nucleoside transporter; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine. 4 SAH -• glycolytic ^ ' enzymes ( ( f Ion Channels 2 messengers & protein kinases ADO-^ \NO-+ HYPO 5 Once formed adenosine can be further deaminated to inosine and hypoxanthine. However, when a favourable transcellular gradient is established, adenosine is released to the extracellular space via a nucleoside transporter (fig. 1; Geiger and Fyda, 1990). In humans adenosine has a plasma half-life of less than 10 sec (Mullane and Williams, 1990). As a result, once in the extracellular space, adenosine has very localized effects, binding to receptors that are in close proximity to the site of release. Adenosine can bind to one of several subtypes of adenosine receptors, the main ones being the Ax and A2 subtypes (Collis and Hourani, 1993). The metabolite-receptor complex can then activate various secondary messengers and/or protein kinases (Morgan, 1991). These are involved in the regulation of the intracellular [Ca2+], the phosphorylation of key glycolytic enzymes, and the permeability of the cell membrane by direct and indirect effects on ion channels (Stiles, 1991; Rudolphi et al. 1992). Investigations into the possible cardiovascular effects of adenosine in fish, have reported complex positive and negative responses that are dose and species specific (see Nilsson, S. and Holmgren, 1993b for review). In fact, in vitro differences in the action of adenosine between mammalian and fish vessels, led to the hypothesis that the coronary artery of fish may lack specific receptors for adenosine, and instead have a more general non-specific purinergic receptor system (Small et al. 1990). In mammals, purinergic receptors can be distinguished into two types, Vx for adenosine, and P2 for ATP (Burnstock, 1978). A useful discrimination between the two purinergic receptor subtypes is the ability of methylxanthines, such as theophylline, to block adenosine effects (Px receptors), but not ATP effects (P2 receptors; Burnstock, 1978; 1983). 6 Based on agonist affinity and their second messenger system, adenosine receptors (Pj) have been classified as Al and A2 subtypes, with further subdivisions (Collis and Hourani, 1993). In fish, such classification of purinergic receptors has not been carried out. However, the presence of substantial amounts of Al adenosine receptors in brain membranes of hagfish (Eptatretus deani), rainbow trout (Oncorhynchus mykiss), and a number of other fish species, with binding affinities and specificities similar to those of mammals, argues for the presence of specific adenosine receptors and a physiological relevance of adenosine as a neuromodulator in fish (Siebenaller and Murray, 1986, 1990; Murray and Siebenaller, 1987; Lucchi et al. 1992). A role for adenosine in the control of metabolism in fish has been studied in the anoxia-tolerant crucian carp (Carassius carassius, Nilsson, G. E., 1991). Intraperitoneal treatment of anoxic crucian carp with the adenosine receptor antagonist aminophylline, resulted in a threefold increase in the ethanol production rate (Nilsson, G. E., 1991), the main metabolic end-product in anoxic Carassius (Shoubridge and Hochachka, 1980). My thesis tested the hypothesis that adenosine has a modulatory role in the hypoxia tolerance of rainbow trout and Pacific hagfish (Eptatretus stouti). Three different areas were investigated for adenosine's actions: 1) the recruitment of anaerobic metabolism; 2) the stress response; and 3) the role of erythrocytes for oxygen capacitance. In all the experiments, the role of adenosine was assessed by comparing the response of animals treated with non-specific adenosine receptor blockers with sham control fish, under hypoxic and normoxic conditions. 7 Comparisons were made between Pacific hagfish and rainbow trout because of the known differences in their ability to tolerate hypoxia. Hagfish, with one of the lowest standard metabolic rate of vertebrates (Munz and Morris, 1965), tissues with a large anaerobic potential (Sidell et al. 1984), and resting cardiac energy demands which can be met almost entirely by anaerobic metabolism (Forster et al. 1991), can tolerate hypoxic and even anoxic habitats. While rainbow trout, which must adopt a number of cardiovascular and ventilatory strategies to try to maintain oxygen uptake (Randall, 1982) and meet the oxygen requirements of their tissues (Dunn and Hochachka, 1986), are relatively hypoxia intolerant. In view of these differences, and given the possible protective role of adenosine in hypoxia outline above, differences may also be expected in the possible physiological actions of adenosine in the hypoxia tolerance of rainbow trout and Pacific hagfish. Although hagfish and rainbow trout differ in many respects, they both produce lactate as an end-product of anaerobic metabolism, and seem to lack the ability to enter a state of metabolic arrest under severe hypoxic conditions (Boutilier et al. 1988; Davison et al. 1990; Forster, 1990). Hence, to extend survival during acute hypoxia, they must both contend with the problems of metabolic acidosis, and the maintenance of vital functions on a fixed energy budget. 8 Chapter 1 Effects on metabolism 9 Introduction Environmental hypoxia in freshwater and marine habitats has a variety of origins and is of fairly common occurrence (Heath, A. G., 1987). Most fish species can tolerate moderate hypoxia, but only a few survive extended periods of severe hypoxia or anoxia (Van Den Thillard and Van Waarde, 1985). Upon exposure to reduced oxygen availability, numerous physiological processes are recruited with a common goal to maximize oxygen transport capacity (Randall, 1982; Fritsche and Nilsson, 1993; Rantin, 1993). Extensive use of anaerobic metabolism to cope with periods of severe hypoxia is a last resort option given the inefficiency, reduced capacity, and metabolic waste problems associated with these pathways (Hochachka, 1991). Hence, without the ability to depress metabolism, most fish species exposed to severe hypoxia must maximize the use of their precious oxygen stores, while minimizing the use of anaerobic metabolism to compensate the energy deficit. This tight energy budgeting can be achieved by selectively favouring vital over non-vital metabolic functions, and by closely adjusting the rate of energy consumption to the metabolic supply of each tissue. In mammals, adenosine plays a key role under conditions of severe hypoxia, by modulating various physiological processes which have a common tendency to redress an imbalance between energy demand and availability (Berne et al. 1982; Newby, 1984; Newby et al. 1990). There is also evidence that adenosine may be important in the control of anaerobic metabolism of the anoxic crucian carp (Nilsson, G. E., 1991). However, fish from the genus Carassius such as crucian carp, are an exception amongst fish, having the 10 ability to produce and excrete ethanol as the main metabolic end product during anoxia (Johnston and Bernard, 1983), and reducing energy consumption by up to 70% under anoxic conditions (Van Waversveld et al. 1989). While adenosine may also be involved in the modulation of the cardiovascular system of several fish species (Nilsson, G. E. et al. 1993a; Nilsson, S. and Holmgren, 1993b), its possible metabolic role has not been investigated in fish that do not produce ethanol under severe hypoxia. The objective of this study was to assess the possible role of adenosine in metabolic regulation during severe hypoxia in fish. To this end, rainbow trout and Pacific hagfish were given the adenosine receptor blocker theophylline under hypoxic and normoxic conditions, and their response monitored in comparison to control shams. In one experimental series an additional adenosine receptor blocker, enprofylline, was used to separate endogenous actions of adenosine from possible adenosine-independent actions of these blockers. I chose to investigate hagfish and rainbow trout in view of their marked difference in hypoxia tolerance (Thomas and Hughes, 1982; Boutilier et al. 1988; Axelsson et al. 1990; Forster et al. 1992), but common anaerobic metabolic strategy which produces lactate as the end-product (Dunn and Hochachka, 1986; Davison et al. 1990). Methods Three different experimental series involving the exposure of fish to hypoxic conditions were conducted. Rainbow trout {Oncorhynchus mykiss) were exposed to either a PwOz of 30 torr for 6 h (SERIES I), or to 25 torr for 1 h (SERIES II). Pacific hagfish (Eptatretus stouti) were either exposed to a PwOz of 10 or 30 torr for 1 h (SERIES III). 11 Experimental protocols, sampling regimes, and analytical procedures described below were similar for all three experiments. Specifics regarding the conditions of a given experimental series are given only when they differ from the common approach. Experimental Animals Rainbow trout of either sex were obtained from West Creek Trout Farm (Aldergrove, B.C.), and kept in 2,000 1 outdoor fibreglass tanks supplied with flow-through dechlorinated tap water. The fish were acclimated to these conditions for a minimum of three weeks prior to the experiments. They were fed with a commercial trout food and kept on a maintenance ration. SERIES I experiments were carried out in June at a mean water temperature of 10°C, while SERIES II experiments were carried out in February at a mean water temperature of 7°C. The trout for these experiments had mean body weights of 461 ± 24 g (SERIES I) and 1,144 ± 32 g (SERIES II). Pacific hagfish (mean body weight 183 ± 7 g) were collected from Trevor Channel on the west coast of Vancouver Island with baited traps, at a depth of approximately 30 m. They were kept at Bamfield Marine Station in an 800 1 fibreglass tank with flow-through salt water (30%o). The fish were acclimated to this water for at least two weeks prior to the experiments. Both acclimation and experiments were carried out at a mean water temperature of 10°C. The fish were starved over the duration of the study. Surgical Procedure Trout were anaesthetized in a buffered (NaHC03) MS-222 solution at a concentration of 1:10,000, and transferred to an operating table where they were forced ventilated with a buffered, cooled, and aerated MS-222 solution of 1:16,000. The dorsal 12 aorta was chronically cannulated with polyethylene (PE) 50 tubing (Clay Adams) using the technique of Soivio et al. (1972), and allowed to recover in a flow-through black perspex box. The cannulae were filled and flushed with heparinized (50 IU/ml sodium heparin) Cortland saline (Wolf, 1963). The trout were undisturbed for 48 (SERIES I) and 72 h (SERIES II) prior to the hypoxic exposure experiments. The hagfish were anaesthetized in a sea water solution of MS-222 (1:400) for approximately 10 min, and transferred to an operating table for cannulation. The operating table used was filled with water and chilled prior to surgery. The surgery involved making a 2.5 cm incision along the midline of the ventral surface between gill apertures 7 and 10, dissecting out the ventral aorta from the surrounding tissue, stopping the circulation momentarily, and securing a T-shape PE cannula into the ventral aorta via a small incision made into the wall of this vessel. The cannula consisted of a 5 mm piece of PE 240 tubing which allowed blood to flow through the ventral aorta, and a long piece of PE 50 tubing from which blood samples could be withdrawn. Once the cannula was in place, a small hole was made through the skin on one side of the ventral incision in order to guide the cannula to the outside. The cannula was then secured in place and the incision closed with skin sutures. The surgery took between 10 and 15 min to complete, at which time the hagfish were placed in a flow-through black perspex box to recover for 48 h before the start of the experiments. Sampling was only carried out if the hagfish were inactive and coiled once this recovery period had elapsed, a behaviour which was characteristic of resting hagfish kept in captivity. 13 Experimental protocol, blood sampling and drug injections All experiments were carried out on fish kept in black perspex boxes supplied with an aerated normoxic water source, or hypoxic water of known oxygen partial pressure. Oxygen-stripping was produced by bubbling nitrogen gas through ceramic air stones (Point Four Systems) in a 100 1 reservoir. Water flow from the reservoir to the fish box was kept constant for the various treatments within each of the three series of experiments, and gas flow through the reservoir was adjusted to achieve the desired P02 . During transfer from normoxic to hypoxic conditions Pw02 decreased exponentially (figure 2), and 95% of the transformation from one state to the other was achieved within 10 min. The sampling regime used throughout each hypoxic trial was as follows: 1) removal of a blood sample to assess resting control values; 2) injection of either an adenosine receptor (AR) blocker or saline; 3) transfer from normoxic water to hypoxic water from the reservoir; and 4) removal of blood samples at set times following the transfer of water sources. The sampling regime used throughout each normoxic trial did not involve step #3 . Two different AR blockers were used in this study: theophylline (1,3-dimethylxanthine) and enprofylline (3-propylxanthine; Sigma, St-Louis, MI). At low doses (e.g. 1-5 mg/kg), the actions of methylated xanthines have been attributed to their ability to block AR (Bruns, 1988). However, methylxanthines can also inhibit cyclic nucleotide phosphodiesterase, the enzyme that breaks down cyclic AMP to 5'-AMP (Rail, 1990). Taking advantage of their different properties, theophylline and enprofylline have been used in concert to delineate endogenous actions of adenosine (Persson, 1988). Enprofylline is approximately six times more potent than theophylline as a competitive inhibitor of cyclic 14 Figure 2. Pattern of change in water P 0 2 in a 7.5 1 fish box following transfer from a normoxic water source to a hypoxic water source with a P0 2 of 25 torr. Transfer between water sources occurred at time 0, and the hypoxic water flow rate was 3.43 1/min. These boxes were used in SERIES II. 15 bO ffi 6 6 C\2 O OH *-. 0) -+-3 ctf ^ 160 140 120 100 80 60 40 20 0 0 10 20 30 40 50 60 Time (min) 16 nucleotide phosphodiesterase (Ukena et al. 1985, 1993). On the other hand, while both drugs do not discriminate between the two main AR subtypes (Ax & A2), enprofylline has a much lower affinity than theophylline for both receptor subtype (Ukena et al. 1985). In SERIES I four different experimental groups of 6 fish each were used to investigate the effects of AR blockade on hypoxia tolerance: 1) normoxic sham; 2) normoxic theophylline; 3) hypoxic sham; and 4) hypoxic theophylline. Throughout the course of a trial, blood samples of 700 /A were taken at time 0, 10, 30, 120, and 360 min. Each blood sample was replaced by an equivalent volume of Cortland saline. Theophylline was dissolved in saline to a concentration of 8 mg/ml, and given as a bolus injection at a dose of 4 mg/kg, followed by 0.2 ml of saline. SERIES II involved five different experimental groups of 8 fish: 1) normoxic sham; 2) normoxic enprofylline; 3) hypoxic sham; 4) hypoxic enprofylline; and 5) hypoxic theophylline. Throughout the course of a trial, blood samples of 900 /xl were taken at time 0, 10, 30, and 60 min. Each blood sample was replaced by an equivalent volume of saline. Enprofylline and theophylline were dissolved in saline to a concentration of 1.5 mg/ml, and infused at a rate of 0.5 ml/min. Both AR blocker were injected at a dose of 4 mg/kg, followed by 0.2 ml of saline. Immediately following the 60 min sampling time, fish were killed by a 2 ml injection of Somnitol (65 mg/ml sodium pentobarbitol). As soon as ventilation ceased, a cross-sectional slice was taken immediately posterior to the dorsal fin and freeze-clamped with aluminum tongs pre-cooled in liquid nitrogen. The fish was then beheaded, the heart removed from the pericardial chamber and freeze-clamped. Tissue samples were stored at -80°C until homogenized. The time between removal of the 17 terminally anaesthetized fish from the holding box and freeze clamping of the tissues was kept less than 30 sec. SERIES III involved six different experimental groups of 6 fish: 1) normoxic sham; 2) normoxic theophylline; 3) 30 torr Pw02 hypoxic sham; 4) 30 torr Pw02 hypoxic theophylline; 5) 10 torr Pw02 hypoxic sham; and 6) 10 torr PwOz hypoxic theophylline. Throughout the course of a trial, blood samples of 500 /A were taken at time 0, 10, 30, and 60 min. Each blood sample was replaced by an equivalent volume of hagfish saline (3.0 % NaCl and heparin 50 IU/ml). Theophylline was dissolved in hagfish saline to a concentration of 6 mg/ml, and given as a bolus injection at a dose of 4 mg/kg, followed by 0.2 ml of hagfish saline. In all three experimental series, blood samples were collected in 1.5 ml micro centrifuge tubes. From this initial sample, aliquots of whole blood were taken for measurement of arterial blood P 0 2 and arterial blood 0 2 content in SERIES II. The blood was then spun down at 11,000 rpm for 2 min, and plasma removed for measurement of blood pH (pHe), and later measurement of plasma [lactate]. The plasma aliquot for determination of lactate was deproteinized with ice-cold 0.6 N perchloric acid (PCA), spun down at 11,000 rpm for 1 min, and the supernatant frozen in liquid nitrogen and stored at -80°C for later analysis. In a separate experiment, the in vivo concentration of plasma theophylline following bolus injection of the drug was assessed in rainbow trout (avg. wt.: 441 ± 12g) either kept in normoxic water (n=6) or exposed to a Pw02 of 30 torr (n=6) for a period of 12 h at 12.5°C. Throughout the course of a trial, blood samples of 150 fx\ were taken at time 0, 5, 18 10, 20, 30, 60, 120, 240, 360, 540, 720 min. Each blood sample was replaced by an equivalent volume of saline. Theophylline was dissolved in Cortland saline to a concentration of 8 mg/ml, and given at a dose of 4 mg/kg, followed by 0.2 ml of saline. Finally, while plasma [theophylline] was not monitored in SERIES I-III, aliquots of plasma were taken in SERIES II to assess plasma [K+], an indicator of possible theophylline overdose. Analytical techniques Measurement of water oxygen tension, arterial blood P02 , and arterial blood 0 2 content were made using thermostatted Radiometer P0 2 electrodes (E5046; Radiometer, Copenhagen, Denmark) with Radiometer PHM71 acid-base analyzers. Arterial blood 0 2 content was measured using the method described by Tucker (1967), using 50 /xl blood samples collected in a gastight Hamilton syringe, and with the chamber thermostatted at 40°C. The P 0 2 electrodes were calibrated using nitrogen gas and air-saturated water. Whole blood pH was measured using a thermostatted Radiometer G297/G2 glass capillary electrode with a PHM71 acid-base analyzer. Calibration of the pH electrode was made using Radiometer Precision Buffer Solution Standards S1519 and S1500. Whole-blood [lactate] was determined using the NAD+-linked assay described by Bergmeyer (1985), and modified for use with microtitration plates and a Titertek Multiskan spectrophotometer (Flow Laboratories, Missisauga, Ontario). Plasma [theophylline] was determined by 125I-labeled theophylline radioimmunoassay (Clinical Assay No. 1592; Baxter Healthcare Corp., Cambridge, MA). Plasma [K+] was measured on a Perkin-Elmer Atomic Absorption Spectrophotometer, model 2380. 19 Tissue metabolite preparation and assay The frozen cross-sections of skeletal muscle were immersed in a shallow bath of liquid nitrogen and samples (approximately 0.8 g) of red and white muscle were dissected free from skin and bone. These frozen muscle samples and heart samples of similar size, were ground to a fine powder in a mortar cooled in liquid nitrogen. The powder was then transferred to pre-weighed chilled test tubes containing 4 ml of ice-cold 0.6 N PCA and re-weighed. The mixtures were homogenized with 3 X 20 sec passes of an Ultra-turrax tissue homogenizer. Homogenization of the tissues was done with the tubes in a slurry of salt water and ice. While stirring at low speed one aliquot of 250 /x\ was removed and frozen at -80°C for later glycogen analysis. The remaining homogenate was centrifuged at 12,000 g for 5 min (2°C). A 1 ml aliquot of the supernatant was removed and stored at -80°C for later determination of tissue ATP, PCr and Cr. Lastly, a further 500 /A aliquot of the supernatant was removed and neutralized with 3 M KHC0 3 / 0.5 M triethanolamine, spun again at 7,500 g for 5 min, and the resulting supernatant frozen at -80°C for later determination of lactate. Glycogen was measured in digested aliquots of tissue homogenate, and is presented as the difference between the concentration of glucosyl units/g tissue post digestion and prior to digestion. Glycogen homogenates were digested by incubating for 4 h at 40°C with amyloglucosidase (Boehringer Mannheim, 10 mg/ml) in acetate buffer (Bergmeyer, 1985). The enzymatic reaction was halted with 70% PCA and glucose was determined in the extract after neutralization with 3 M KHC03 . Tissue glucosyl units, lactate, and Cr, were determined using the standard NADH- or NAD+-coupled enzymatic procedures described 20 in Bergmeyer (1985), and modified for use with microtitration plates and a Titertek Multiskan spectrophotometer. Concentrations of PCr and ATP were determined using high performance liquid chromatography (HPLC) based on Harmsen et al. (1982) with modifications. The HPLC incorporates Waters 625 LC system controller coupled to Waters 996 photodiode array detector set at 210 nm. (Millipore Corp.) 50 fil aliquots of filtered tissue homogenates were passed through a 250 X 4.6 mm Partisil 10 SAX strong anion exchanger column (Whatman, Maidstone, Great Britain) at a flow rate of 1.5 ml/min. Elution was isocratic for the first 5 min, using 0.01 M H3P04 (pH 2.85; Solvent A), followed by a linear gradient from solvent A to 0.75 M KH2P04 (pH 4.40; solvent B) over 15 min. These conditions were then maintained for 5 min, followed by a linear gradient from solvent B to solvent A over 1 min. The column was re-equilibrated for 8 min with solvent A before the next run. Integration of the separated metabolites was done with the Millennium 2010 Chromatography Manager software program (version 1.1, Millipore Corp.). Concentrations were calculated based on linear standard curves constructed for both metabolites. As one possible measure of the energy state of the tissues, the creatine charge was calculated as [PCr]/([Cr]+[PCr]) according to Connett (1988). Statistical Analysis All data are presented as mean ± one standard error. The statistical significance of observed effects of treatment exposure within a group were tested by one-way repeated measures ANOVA. To compare pre-treatment means with means at subsequent sampling times Dunnett's test was used. Where appropriate, the statistical significance of observed 21 differences between the means from all treatments at a particular sampling time were tested by one-way ANOVA. Since the tissue and plasma lactate means were positively correlated with the variance, the nonparametric Kruskal-Wallis one-way ANOVA on ranks test had to be used to determine differences between the means from all treatments at a particular sampling time. To isolate which group(s) differed from the others, Student-Newman-Keuls test was used. The significance level for all statistical test was P < 0.05. Results The mean control Pa0 2 and 0 2 content values for all five experimental groups of rainbow trout in SERIES II were 123.4 ± 1.7 mmHg and 14.6 ± 0.4 vols % respectively. No changes were observed from these means in the normoxic sham and normoxic enprofylline groups throughout the 60 min trials. In the hypoxic sham, enprofylline, and theophylline groups, Pa0 2 and 0 2 content fell to 16.2 ± 1 . 1 mmHg and 5.7 ± 0.3 vol % from the control values after only 10 minutes of hypoxic exposure. Pa0 2 and 0 2 content remained at these low values in all three hypoxic groups for the remainder of the hypoxic challenge. Compared with normoxic and hypoxic sham animals, hypoxic rainbow trout given the AR blockers theophylline or enprofylline showed a rapid and pronounced blood acidosis (fig. 3 and 4). In SERIES I, whereas pHe had significantly decreased after 10 min of hypoxia and remained low for the duration of the trial in the hypoxic theophylline group, pHe did not decrease significantly until 360 min in the hypoxia sham group (fig. 3). In SERIES II, pHe also decreased significantly after 10 minutes of hypoxia in the theophylline 22 Figure 3. Plasma pH (pHe) of rainbow trout in relation to exposure duration to normoxia (open symbols) and hypoxia (closed symbols; PwO2=30 torr). Animals were injected with either saline (circles) or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment, b indicates a significant difference from both normoxic treatments at given sampling time. * indicates a significant difference from hypoxic sham treatment at given sampling time (p<0.05). Values are means ± 1 SEM. 23 8.2 r O Normoxic Sham • Normoxic Theophylline • Hypoxic Sham • Hypoxic Theophylline CD 8.1 8.0 7.9 7.8 7.7 7.6 7.5 _i i i • ' 0 30 -i i i i i 120 360 Time (min) 24 Figure 4. Plasma pH (pHe) of rainbow trout in relation to exposure duration to normoxia (open symbols) and hypoxia (closed symbols; Pw02=25 torr). Animals were injected with either saline (circles), enprofyUine (triangles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment, b indicates a significant difference from both normoxic treatments at given sampling time. * indicates a significant difference from hypoxic sham treatment at given sampling time (p<0.05). Values are means ± 1 SEM. O Normoxic Sham A Normoxic Enprofylline • Hypoxic Sham A Hypoxic Enprofylline • Hypoxic Theophyli ine Time (min) 26 and enprofylline groups (fig. 4). Recovery towards the normoxic values was only observed in the enprofylline group, and pHe was unchanged throughout the hypoxic exposure in the sham groups. Normoxic groups injected with theophylline (fig. 3) or enprofylline (fig. 4), showed no changes in pHe compare to normoxic sham animals. In SERIES III, a significant blood acidosis was only observed in the hypoxic theophylline group exposed to a Pw02 of 10 torr (fig. 5A). The mean pHe of the sham and theophylline injected hagfish exposed to a Pw02 of 30 torr were 7.95±0.01 and 7.97±0.01 respectively. These values remained the same throughout the trials, and were similar to the mean pHe of the normoxic sham (7.96±0.01) and normoxic theophylline (7.96±0.01) groups. Relative to hypoxic sham fish, increases in plasma [lactate] with hypoxic exposure were greater in the animals injected with the AR blockers (fig 5B, fig. 6, and fig. 7). In SERIES III no changes were observed in the mean lactate concentrations of the normoxic sham (1.43±0.11 mM), normoxic theophylline (1.33±0.08 mM), 30 torr Pw02 hypoxic sham (1.61±0.12 mM), and 30 torr PwOz hypoxic theophylline (1.57±0.10 mM) groups. In the sham and theophylline hagfish groups exposed to a Pw02 of 10 torr (fig. 5B), however, there was a gradual increase in plasma lactate resulting in a 5 and 8 fold increase respectively. In SERIES I, plasma lactate increased in both hypoxic groups, and the hypoxic theophylline group had a significantly higher concentration than the hypoxic sham group at the 30 min sampling time (fig. 6). In SERIES II, the increase in plasma lactate was most in the hypoxic theophylline group, intermediate in the hypoxic enprofylline group, and lowest in the hypoxia sham group (fig. 7). As with pHe, the injections of theophylline (fig. 6) or enprofylline (fig. 7) in normoxic groups, had no effect on resting control 27 Figure 5A. Plasma pH (pHe) of Pacific hagfish in relation to exposure duration to hypoxia (PwO2=10 torr). Animals were injected with either saline (circles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment (p<0.05). Values are means ± 1 SEM. Figure 5B. Plasma lactate of Pacific hagfish in relation to exposure duration to hypoxia (PwO2=10 torr). Animals were injected with either saline (circles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment. * indicates a significant difference from hypoxic sham treatment at given sampling time (p<0.05). Values are means ± 1 SEM. A. B. 12 10 8 6 4 2 0 • Hypoxic Sham • Hypoxic Theophylline 10 20 30 40 50 Time (min) 10 20 30 40 50 Time (min) 60 60 29 Figure 6. Plasma lactate of rainbow trout in relation to exposure duration to normoxia (open symbols) and hypoxia (closed symbols; PwO2=30 torr). Animals were injected with either saline (circles) or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment, b indicates a significant difference from both normoxic treatments at given sampling time. * indicates a significant difference from hypoxic sham treatment at given sampling time (p<0.05). Values are means ± 1 SEM. 30 24 r - v 21 B^ 18 15 12 9 6 3 0 0 30 O Normoxic Sham D Normoxic Theophylline • Hypoxic Sham • Hypoxic Theophylline 120 Time (min) ab a b =g J i i i i i i i 360 31 Figure 7. Plasma lactate of rainbow trout in relation to exposure duration to normoxia (open symbols) and hypoxia (closed symbols; Pw02=25 torr). Animals were injected with either saline (circles), enprofyUine (triangles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment, b indicates a significant difference from both normoxic treatments at given sampling time. * indicates a significant difference from hypoxic sham treatment at given sampling time (p<0.05). Values are means ± 1 SEM. 32 O Normoxic Sham A Normoxic Enprofylline • Hypoxic Sham A Hypoxic Enprofylline • Hypoxic Theophyll ine 10 20 30 40 50 Time (min) 33 concentrations of plasma lactate. In SERIES II, only the tissues from the hypoxic theophylline group had significant increases in tissue [lactate] when compared to the normoxic groups (fig. 8). Significant changes in tissue metabolites other than lactate, were also only associated with the hypoxic group infused with theophylline (table 1). Compared with normoxic groups, significant decreases in [glycogen], creatine charge, and [creatine phosphate] were observed in heart tissue of theophylline-injected rainbow trout. A significant decrease in creatine charge was also observed in the red muscle of the theophylline group. The in vivo plasma [theophylline] decreased exponentially in the first 120 min following bolus injection of the drug (fig. 9). The concentration decreased more gradually, and followed a linear pattern between 120 and 720 min. Relative to normoxic fish, the concentrations at times 10, 20, 30, and 60 min were significantly higher in the hypoxic trout. In SERIES II, the mean plasma [K+] at time 0 ranged from a low of 2.33 ±0.30 mM in the normoxic sham group to a high of 3.19±0.46 mM in the hypoxic enprofylline group. No significant differences in plasma [K+] were observed within or between groups throughout the 60 min trials. Discussion While the effects of the two methylxanthines used in this study, theophylline and enprofylline, are varied as in other systems (see Bruns, 1988), our data clearly indicate that the differences seen between the hypoxic treatments result from adenosine receptor blockade. Injected at a dose of 4 mg/kg, theophylline had a maximum in vivo concentration 34 Figure 8. Tissue lactate in white muscle, red muscle, and heart of rainbow trout exposed to 60 minutes of normoxia or hypoxia (PwOz=25 torr). Animals were injected with either saline, enprofyUine, or theophylline, a indicates a significant difference from normoxic sham treatment, b indicates a significant difference from both normoxic treatments (p<0.05). Values are means ± 1 SEM. 35 18 16 14 12 10 8 6 4 2 I-k i J 0Lli Y//A Normoxic S h a m Normoxic Enprofylline Hypoxic S h a m Hypoxic Enprofyl l ine Hypoxic Theophyl l ine I JL White Muscle Red Muscle Hear t 36 TABLE 1. Concentration of metabolites in heart, red muscle and white muscle after one hour of either normoxia or hypoxia (Pw02=25 torr) in rainbow trout at 7°C. Animals were either infused with either saline or with one of two adenosine receptor blockers: enprofylline (enpro.) or theophylline (theo.). Values are means ± 1 SEM in ;u,mol/g wet weight. Tissue Experimental Creatine Creatine Creatine ATP Glycogen Condition phosphate charge* Heart Normoxic Sham Normoxic En pro. Hypoxic Sham Hypoxic Enpro. Hypoxic Theo. 5.06 ±0.35 4.76 ±0.60 4.17±0.34 4.81±0.30 5.90±0.32 4.12±0.37 4.47±0.23 2.95±0.45 3.41 ±0.46 2.88 ±0.24* 0.45 ±0.03 0.52±0.03 0.40±0.04 0.40±0.05 0.33 ±0.03* 2.15 ±0.10 2.10±0.10 1.87±0.08 2.11±0.15 2.02±0.07 60.25 ±2.86 62.46±3.53 53.06 ±5.96 53.96 ±3.53 41.90 ±5.30"* Red muscle Normoxic Sham Normoxic Enpro. Hypoxic Sham Hypoxic Enpro. Hypoxic Theo. 5.12±0.50 5.80±0.49 7.18 ±1.24 5.94±0.63 6.91±0.54 5.85 ±0.50 6.85±0.64 5.70±0.46 4.48 ±0.28 5.10±0.86 0.53±0.01 0.54 ±0.02 0.47±0.03 0.44 ±0.04 0.41±0.04"6 2.16 ±0.09 2.29±0.10 2.24±0.15 2.00±0.07 2.09±0.17 ' Creatine charge = [PCr]/[PCr]+[Cr] a. Significantly different from normoxic (p<0.05) b. Significantly different from normoxic enpro. (p<0.05) 24.25±1.77 21.93±1.60 23.81 ±1.85 16.23±1.74 18.69±3.24 White muscle Normoxic Sham 20.32±1.73 13.53±0.77 0.41±0.03 4.66±0.20 33.08±2.44 Normoxic Enpro. 23.74±0.85 13.55±1.08 0.36±0.02 4.89±0.20 33.07±2.03 Hypoxic Sham 24.84±1.21 ll.14dbl.73 0.30±0.04 4.33±0.34 32.51±1.87 Hypoxic Enpro. 20.44±0.85 13.55±1.08 0.36±0.02 4.89±0.20 30.06±3.30 Hypoxic Theo. 23.38±0.92 12.18±1.25 0.34±0.03 4.76±0.25 28.16±1.19 - J 38 Figure 9. Plasma theophylline of rainbow trout injected with 4 mg/kg theophylline in relation to exposure duration to normoxia (circles) and hypoxia (triangles; PwO2=30 torr). Time 0 values are controls. * indicates a significant difference between the hypoxic and normoxic treatments (p<0.05). Values are means ± 1 SEM. 39 • Norznoxia V Hypoxia B CD o CD E-" ctf a w cti 0 120 240 360 480 600 720 TIME (min) 40 of 73 fiM (13.2 /xg/ml) after 5 min and decreased exponentially thereafter. Given that the IC50 (the concentration required for 50% inhibition) values of theophylline for the various phosphodiesterase isozymes are in the range of 155-630 /xM, but its affinity for Ax and A2 adenosine receptors is in the 10 //M range (Ukena et al. 1993), the in vivo concentration used in all our experimental series are not likely to result from phosphodiesterase inhibition. Hypokalaemia is the most consistent metabolic abnormality associated with theophylline toxicity (Kearney et al. 1985; Memon, 1993), but the theophylline and enprofyUine dosage used in our experiments had no effect on the plasma [K+]. Irrespective of the parameter measured, the normoxic treatments given the AR blockers were similar in every respect to the normoxic sham indicating no phosphodiesterase inhibition. Similarly, aminophylline (which forms theophylline) injected intraperitoneally at a dose of 75 mg/kg had no effect on the routine oxygen consumption of crucian carp (Nilsson, G. E., 1991). The effects of the methyxanthines were only observed under severe hypoxic conditions, when anaerobic metabolism was recruited to compensate an imbalance between energy supply and demand. These conditions favour the production of adenosine (Meghji, 1991), indicating that the methylxanthines were acting on adenosine receptors. Significant differences were also observed between the high affinity AR blocker theophylline and the low affinity receptor blocker enprofyUine. In every instance where AR blockade treatment caused a significant departure from the response in the hypoxic sham group, the response due to theophylline treatment was greater than to enprofyUine treatment, further supporting the conclusion that the effects observed were due to AR blockade. Responses to norepinephrine, isoprenaline, histamine, and prostaglandins are not 41 antagonized by theophylline (see Burnstock, 1978 for references). Furthermore, the apparent binding of theophylline to P2 purinergic receptors (receptors for ATP; Small et al. 1990; Meghji and Burnstock, 1984), can be explained by the breakdown of ATP to adenosine before acting on the Px purinergic receptors (receptors for adenosine; Meghji and Burnstock, 1984). Adenosine is generated from ATP by the existence of 5'-nucleotidase enzyme in close proximity to adenosine receptors (Bruns, 1980). Despite the various cardiovascular and respiratory adaptations of fish to maintain oxygen uptake under conditions of reduced oxygen availability (Randall, 1990; Fritsche and Nilsson, 1993; Rantin, 1993), the capacity of the oxygen transport system still becomes limiting during severe hypoxia. In SERIES II for example, the rainbow trout exposed to a Pw02 of 25 torr showed a 61% decrease in blood oxygen content after 10 min of hypoxia. Failure to meet the oxidative requirements of the tissues during severe hypoxia activates anaerobic metabolism, leading to an increase in production of the anaerobic end product lactate in all tissues (Heath, A. G. et al. 1980; Van Waarde et al. 1983; Dunn and Hochachka, 1986), and an increase in lactate turnover rate (Dunn and Hochachka, 1987). In all three experimental series presented in this study, the accumulation of plasma lactate following acute hypoxic exposure was significantly greater in the animals receiving AR blockers than in the sham hypoxic treatments. Measurement of heart, white and red muscle tissue [lactate] in SERIES II, also showed an increase in glycolytic activity following theophylline treatment under hypoxic conditions. Therefore, although lactate turnover was not measured, results suggest an increase in lactate production with theophylline treatment. The activation of anaerobic metabolism in the hypoxic groups, is associated with the 42 development of a metabolic acidosis. The acidosis results from an imbalance between H+ production from ATP hydrolysis and H+ consumption by the fermentation of glycogen (Hochachka and Mommsen, 1983). Although there was a significant increase in plasma [lactate] in the hypoxic sham group of SERIES I & II, and in the 10 torr Pw02 hypoxic sham group of SERIES III, a significant plasma acidosis only developed in SERIES I after 360 min of hypoxia. In the hypoxic sham treatments, the metabolic acidosis is compensated by a respiratory alkalosis resulting from an increase in gill ventilation (Thomas and Hughes, 1982; Tetens and Lykkeboe, 1985). In sharp contrast to this general pattern, the animals injected with the AR blockers were characterized by a rapid and pronounced plasma acidosis. Since there was no difference in Pa0 2 and 0 2 content between the 3 hypoxic treatments in SERIES II, the magnitude of the hyperventilatory response was also probably the same between these 3 treatments. Therefore, a respiratory alkalosis would also buffer changes in plasma pH in the AR-blocked animals. Furthermore, in all three experimental series no differences were observed between the various hypoxic groups in the magnitude of rbc alkalization following ^-adrenergic stimulation of the Na+/H+ exchanger (see chapter 3 for results). While this rapid movement of protons from the rbc to the plasma also significantly contributes to the metabolic acidosis (Fievet et al. 1987, 1988), it can not explain the abrupt and marked metabolic acidification observed in the AR-blocked animals. Instead, the rapid increase in anaerobic metabolism observed in the fish who received the AR blockers, results in a metabolic acidosis which exceeds the buffering capacity of the blood and the compensatory alkalosis offered by the hyperventilation (Thomas and Hughes, 1982). 43 Glycolysis and PCr breakdown are the major pathways for anaerobic energy production in fish (Van Den Thillard and Van Waarde, 1985). While the first phase of muscle metabolism during hypoxia is supported by PCr hydrolysis in vertebrates (Hochachka and Mommsen, 1983), phosphagen stores are limited and anaerobic glycolysis is used as a backup system for ATP generation when PCr reserves are depleted (Hochachka, 1991). Significant depletion of glycogen and PCr stores in fish, are usually only observed after prolong exposure to severe hypoxic or anoxic conditions (Jorgensen and Mustafa, 1980a, b; Van Waarde et al. 1983, 1990; Dunn and Hochachka, 1986; Boutilier et al. 1988). Although the hypoxic treatment in SERIES II resulted in a significant increase in plasma [lactate] in all three hypoxic groups, the exposure to a Pw02 of 25 torr for 60 min was not severe enough to result in significant changes of any tissue metabolites in the hypoxic sham and hypoxic enprofylline groups. Significant decreases in energy reserves were only observed in the oxidative tissues, heart and red muscle, of animals treated with theophylline. No appreciable changes were observed in the glycogen and PCr stores of white muscle following hypoxic theophylline treatment. This apparent difference between the aerobic and anaerobic tissues in maintaining their energy reserves, may be related to differences in the possible interactions between adenosine and these tissues. In fish glycolytic muscles, a tight inverse stoichiometric relationship exits between ATP and IMP concentrations (Mommsen and Hochachka, 1988; Schulte et al. 1992), making the formation of adenosine in these tissues very unlikely. In mammals, the capacity for adenosine formation is much less in glycolytic tissues in comparison to oxidative tissues (Newby et al. 1990; Stone et al. 1990). Hence, since adenosine has very localized actions 44 (Stiles, 1991), it is possible that adenosine effects on fish white muscle are minimal but there are marked effects in heart and red muscle. Altogether, our results indicate that adenosine receptor blockade may reduce the hypoxia tolerance of rainbow trout and Pacific hagfish, by increasing the rate of anaerobic metabolism during acute hypoxic exposure. Similar results were obtained with crucian carp, where AR blockade caused the rate of ethanol excretion during anoxia to increase up to three times (Nilsson, G. E., 1991). As an explanation for his results, Nilsson (1991) suggested that AR blockade prevented the decrease in neuronal activity mediated by adenosine and its associated decrease in energy consumption. We have no reason to believe that a similar scenario is not also at play in our experiments, however unlike the experiments carried in anoxia with crucian carp, a limited amount of oxygen was available to the fish in this study. Besides its various inhibitory effects in the CNS, adenosine is also an important neuromodulator of the respiratory and cardiovascular systems in mammals (Ribeiro, 1991). Although the cardiovascular effects of adenosine in fish are complex and unresolved, the physiology of the heart, the coronary arteries, the branchial and brain vasculature, are modulated by adenosine under hypoxic conditions (Nilsson, G. E. et al. 1993a; Nilsson, S. and Holmgren, 1993b). Adenosine receptor blockade may prevent these presumably adaptive changes in cardiovascular control, and thereby alter the distribution of the very limited oxygen and fuel supply under severe hypoxia. Given the inefficiency and limited capacity of anaerobic metabolism to compensate reductions in aerobic metabolism, small impairments of the cardiovascular system may result in a large amplification of the anaerobic metabolic pathways. 45 Severe hypoxia in fish is also characterized by marked increases in the circulating concentrations of the catabolic stress hormone catecholamines (Boutilier et al. 1988; Perry et al. 1991, 1993) and Cortisol (see chapter 2). Circulating catecholamines and Cortisol can mobilize energy reserves in order to maintain or increase energy turnover. In hypoxic fish, catecholamines have been shown to have glycogenolytic and/or gluconeogenic effects on liver metabolism (Mommsen et al. 1988; Wright et al. 1989). Similarly, Cortisol has direct or permissive lipolytic, glycogenolytic, and/or gluconeogenic effects on the liver of fish (Vijayan et al. 1991; Vijayan and Leatherland, 1992), and enhances peripheral proteolysis (Woo and Cheung, 1980). In mammals, both hormones are involved in the control of glycogen metabolism in skeletal muscle, heart, and liver (Green et al. 1980; Coderre et al. 1991). Overall, the catabolic properties of catecholamines and Cortisol help fish to meet the increase in energy demand required to fuel the acute physiological responses required to maintain oxidative processes in hypoxia. As such, the magnitude of their actions may limit the ability of fish to conserve energy resources over prolonged periods of acute hypoxia. Results presented in chapter 2, show that adenosine receptor blockade increases the circulating concentrations of these hormones in rainbow trout and Pacific hagfish. Furthermore, in mammals adenosine can inhibit the secretion of catecholamines from the adrenal medulla (Chern et al. 1987, 1992), and function as a negative-feedback modulator of /3-adrenoceptor-mediated contractile and glycogenolytic responses in the myocardium (Dobson et al. 1987). Hence, the increase in anaerobic metabolism and decrease in tissue energy reserves observed with AR blockade in this study, may also be the result of an uncoordinated stress response with a much higher catabolic potential. 46 Results obtained in this study also reflect the marked differences in hypoxia tolerance between Pacific hagfish and rainbow trout. While anaerobic metabolism was recruited after only 10 min at a Pw02 of 30 torr in rainbow trout, there were no sign of any shortfall in aerobic metabolism in the Pacific hagfish exposed to a similar Pw02 for 60 min. So although the two species have very distinct abilities to tolerate hypoxia, our results show that adenosine receptor blockade reduces this tolerance once anaerobic metabolism is recruited. 47 Chapter 2 Effects on the stress response 48 Introduction An acute change in an environmental factor, such as 0 2 level, elicits a response in the hypothalamus which stimulates both the pituitary-interrenal axis and the chromaffin cells, respectively inducing the release of corticosteroids and catecholamines (Mazeaud et al. 1977). These hormones stimulate several physiological and metabolic processes, which all together form the stress response (Seyle, 1950). Although the response is adaptive, since it enables organisms to meet the immediate increased energy demands to resist the stress, it can also be maladaptive (Barton and Iwama, 1991). Chronic activation of this generalised endocrine response leads to exhaustion, and may impair biological processes from the cellular to the organismal level (Adams, 1990). Given the potency of the hormones involved and the multiplicity of their targets, control over the magnitude and the timing of the primary response to stress is key to its effectiveness. The adrenocorticotrophic hormone (ACTH) stimulates the release of Cortisol from the interrenal cells of the head kidney in fish. The release of ACTH from the pituitary gland is activated by the corticotropin-releasing factor (CRF), a peptide secreted by neurons originating in the hypothalamus (Donaldson, 1981). The release of ACTH is further controlled by negative feedback of Cortisol on the hypothalamic-pituitary axis (Fryer and Peter, 1977). In teleosts the predominant mechanism responsible for catecholamine mobilization is thought to be through neuronal stimulation of the chromaffin cells (Nilsson et al. 1976). However, another important stimulus for catecholamine release is a direct localized hypoxaemia of the chromaffin tissue (Perry et al. 1991; Perry and Reid, 1992). This 49 mechanism may be especially important in hagfish, where chromaffin cells lack extrinsic innervation (Perry et al. 1993). The release of adrenaline and noradrenaline from the chromaffin tissue, may be further controlled within the head kidney via both positive (Hathaway et al. 1989) and negative feedback of catecholamine overflow (Perry et al. 1991). The complexity of the regulation of catecholamine secretion is indicated by the multiplicity of receptor types involved in the modulation of this response in the mammalian adrenal medulla. These include: nicotinic and muscarinic cholinoceptors, opioid peptide, substance P, bradykinin, histamine Hx, dopamine Dx & D2, prostaglandin E2, angiotensin II, GABAA & GABAB, as well as adenosine receptors (see Oset-Gasque et al. 1993 for references). Since, adenosine is a potent modulator of neural activity in the CNS and the peripheral nervous system (Stone and Bartrup, 1991), a hypothetical role for this metabolite in the modulation of the primary stress response may be expected. Adenosine has been shown to have anti-adrenergic actions in various tissues (Dobson et al. 1987; Schimmel et al. 1987). In particular, in mammals, adenosine has been shown to modulate the secretion of catecholamines from the adrenal medulla (Chern et al. 1987, 1992), and the activity of the pituitary-adrenocortical axis (Anand-Srivastava et al. 1989; Feuilloley et al. 1992). This study investigates whether adenosine plays a modulatory role in the primary stress response of fish. To this end rainbow trout and Pacific hagfish were exposed to severe hypoxia, and changes in the circulating concentrations of Cortisol and catecholamines monitored following infusion or either adenosine receptor blockers or saline. 50 Methods The three same experimental series described in chapter 1, with the same set of fish, were used in this study. Rainbow trout were exposed to either a Pw02 of 30 torr for 6 h (SERIES I), or to 25 torr for 1 h (SERIES II). In SERIES III, Pacific hagfish were exposed to a PwOz of 10 or 30 torr for 1 h. Reference should be made to the methods section of chapter 1, for a detailed description of the experimental animals, surgical procedures, experimental protocol, sampling regime, and description of the drugs, since these were the same for both studies. In SERIES I, plasma aliquots were removed from each blood sample, frozen in liquid nitrogen, and stored at -80°C for later determination of plasma [Cortisol]. In SERIES II and SERIES III, plasma aliquots were also removed from each blood sample and frozen as above, for later determination of plasma adrenaline (A) and noradrenaline (NA) concentrations. Analytical techniques Plasma [Cortisol] were determined by 125I-labeled Cortisol radioimmunoassay (Clinical Assay No. 529; Baxter Healthcare Corp., Cambridge, MA). This procedure is based on the competitive binding principles of radioimmunoassay, and has been validated for use with salmonids (Heath, 1992). Plasma A and NA levels were determined on alumina-extracted plasma samples using high pressure liquid chromatography (HPLC) based on Woodward (1982). The HPLC incorporates a Waters 460 Electrochemical Detector using a glassy carbon electrode, a reverse-phase Waters Plasma Catecholamine Column, a Waters Model 510 HPLC Pump 51 with pulse dampeners and a Waters U6K Universal Liquid Chromatograph Injector (Waters Chromatography Division of Millipore Ltd.). Concentrations were calculated by an integrator (Waters 746 Data module) connected on-line to the electrochemical detector. Statistical Analysis All data are presented as mean ± one standard error. The statistical significance of observed effects of treatment exposure within a group were tested by one-way repeated measures ANOVA. To compare pre-treatment means with means at subsequent sampling times Dunnett's test was used. Where appropriate, the statistical significance of observed differences between the means from all treatments at a particular sampling time were tested by one-way ANOVA. Since the plasma adrenaline and noradrenaline mean values were positively correlated with the variance, the nonparametric Kruskal-Wallis one-way ANOVA on ranks test had to be used to determine differences between the means from all treatments at a particular sampling time. To isolate which group(s) differed from the others, Student-Newman-Keuls test was used. The significance level for all statistical test was P < 0.05. Results In SERIES I, plasma [Cortisol] increased significantly after 30 min of hypoxia in the sham group, but after only 10 min in the theophylline treated group (fig 10). The increase in plasma [Cortisol] after 30 min of hypoxia was significantly higher in the theophylline than in the sham group, and whereas the [Cortisol] returned to control levels in the hypoxic sham group, they remained elevated in the hypoxic theophylline treatment. Plasma [Cortisol] 52 Figure 10. Plasma Cortisol of rainbow trout in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were injected with either saline or theophylline. Time 0 values are controls, a indicates a significant difference from control in given treatment (p<0.05). Values are means ± 1 SEM. CO 1 B S o o O O CM CO o rt n H n D o o o lO to o o K) O in CM o o CN o m (jlIl/Sll) pST^JOQ 19UIS19IJ 54 remained low and unchanged throughout the 360 min trials in the normoxic groups injected with either saline or theophylline. In SERIES II, the one hour hypoxic exposure trials resulted in significant increases in plasma [adrenaline] in all three hypoxic treatments (fig. 11 A). Overall, the increase in [adrenaline] was most pronounced in the hypoxic theophylline group, intermediate in the hypoxic enprofylline group, and smallest in the hypoxia sham group. After 10 min of hypoxic exposure, the [adrenaline] in the theophylline and enprofylline treatments were respectively 16- and 4-fold higher than in the hypoxic sham treatment. The difference between the three groups was much less after 30 min of hypoxia, and only the theophylline treatment had a significantly higher [adrenaline] than the two other treatments after 60 min. No changes were observed in the resting [adrenaline] values throughout the 60 min sampling regime, either within or between the two normoxic treatments. The mean values were 1.73±0.35 and 2.77±0.34 nM in the normoxic sham and normoxic enprofylline groups respectively. Relative to their control values, plasma [noradrenaline] also increased in all three hypoxic treatments of SERIES II, but their were no significant differences between the 3 at any given sampling time (fig. 11B). No significant changes were observed in the two normoxic treatments, either within a group throughout the trial or between the two groups. The mean [noradrenaline] were 3.35±0.59 and 3.33±0.39 nM in the normoxic sham and normoxic enprofylline groups respectively. In the experiment involving hagfish (SERIES III), although plasma [adrenaline] remained at the control time 0 value in the 10 torr Pw02 hypoxic sham group throughout 55 Figure 11 A. Plasma adrenaline of rainbow trout in relation to exposure duration to hypoxia (Pw02=25 torr). Animals were injected with either saline (circles), enprofylline (triangles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment. * indicates a significant difference from hypoxic sham treatment at given sampling time (p<0.05). Values are means ± 1 SEM. Figure 11B. Plasma noradrenaline of rainbow trout in relation to exposure duration to hypoxia (Pw02=25 torr). Animals were injected with either saline (circles), enprofylline (triangles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment (p<0.05). Values are means ± 1 SEM. A. 56 a • 1—1 CO 0) < cd 6 to cd E B Hypoxic Sham Hypoxic Enprofylline Hypoxic Theophylline 10 20 30 40 Time (min) 50 60 0) a •i—i CD I* cd o S3 85 E CO cd E 2 tf Time (min) 57 the trial, [adrenaline] increased 3.8 fold within 10 min in the hypoxic theophylline-treated fish and returned to control levels by 60 min (fig 12A). Relative to their control values, [adrenaline] did not change in all the other treatments, and the mean values were 1.99±0.19, 2.59±0.21, 2.47±0.22, and 2.33±0.19 nM in the normoxic sham, normoxic theophylline, 30 torr Pw02 hypoxic sham, and 30 torr Pw02 hypoxic theophylline groups respectively. Plasma [noradrenaline] increased in the two hagfish groups exposed to a Pw02 of 10 torr, but whereas the increase was significant after 10 min in the theophylline-treated fish, it took 60 min for the [noradrenaline] to increase above the control value in the sham group (fig 12B). No changes were observed in the plasma [noradrenaline] of all the other treatments, and the mean values were 3.53±0.44, 3.24±0.25, 4.16±0.61, and 3.42±0.33 nM in the normoxic sham, normoxic theophylline, 30 torr Pw02 hypoxic sham, and 30 torr Pw02 hypoxic theophylline groups, respectively. Discussion Adenosine receptor blockade with methylxanthines had no effect on the concentrations of Cortisol and catecholamines in the normoxic animals. These results are similar to those observed in several mammalian studies (Andersson et al. 1984; Whyte et al. 1987; Ishizaki et al. 1988; Schwertschlag et al. 1993), but also contrast with others (Vestal et al. 1983; Higbee et al. 1987). The control plasma [Cortisol] in SERIES I are characteristic of levels measured in fish which have recovered for 48 hr from cannulation and confinement (Gamperl et al. 58 Figure 12A. Plasma adrenaline of Pacific hagfish in relation to exposure duration to hypoxia (PwO2=10 torr). Animals were injected with either saline (circles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment. * indicates a significant difference from hypoxic sham treatment at given sampling time (p<0.05). Values are means ± 1 SEM. Figure 12B. Plasma noradrenaline of Pacific hagfish in relation to exposure duration to hypoxia (Pw02= 10 torr). Animals were injected with either saline (circles), or theophylline (squares). Time 0 values are controls, a indicates a significant difference from control in given treatment (p<0.05). Values are means ± 1 SEM. Hypoxic Sham Hypoxic Theophylline 9 8 7 6 5 4 3 2 1 0 -*" • « i 0 10 20 30 40 50 60 Time (min) 10 20 30 40 50 60 Time (min) 60 1994), but are considerably higher than the resting or unstressed levels measured in uncannulated fish (Barton and Iwama, 1991). The control catecholamine values for rainbow trout (SERIES II) are in accordance with reported values (Randall and Perry, 1992), and although catecholamine levels have not been previously reported for Pacific hagfish, the control values of SERIES III are similar to the concentrations reported in Atlantic hagfish (Perry et al. 1993). In response to severe hypoxic stress, rainbow trout and Pacific hagfish showed significant increases in the primary stress indicators Cortisol and catecholamines. This hormonal response to hypoxia has been well documented in fish (Ristori and Laurent, 1989; Barton and Iwama, 1991; Perry et al. 1993), and constitutes an important component of the complex biological response necessary to maintain homeostasis. Theophylline treatment, in all the hypoxic groups, resulted in greater circulating concentrations of these primary stress hormones. In fish, hypoxemia is the dominant factor initiating the release of catecholamines (Perry et al. 1991; Randall and Perry, 1992). Hence, differences in the oxygen-carrying capacity between the various hypoxic treatments could have accounted for the observed differences in circulating [catecholamine]. Oxygen content measurements in SERIES II does not support this hypothesis, since all three hypoxic groups were shown to have similar oxygen content values throughout the hypoxic exposure (see chapter 1). In mammals, high [theophylline] has been shown to stimulate the release of catecholamines, particularly adrenaline, from isolated perfused adrenal glands (Peach, 1972; Poisner, 1973). The ability of theophylline to inhibit phosphodiesterase (PDE), and thereby 61 elevate tissue cAMP concentrations, was originally considered as a possible mechanism. However, PDE inhibition was achieved with [theophylline] that have toxic in vivo effects in mammals (Aronson et al. 1992; Schwertschlag et al. 1993). At low levels, theophylline blocks all adenosine receptor subtypes. Since adenosine has the capacity to depress synaptic transmission (Stone and Bartrup, 1991), and suppress the release of many neurotransmitters in the peripheral nervous system (Fredholm and Dunwiddie, 1988), it is possible that adenosine receptor blockade may modulate regulation of the pituitary-interrenal axis and the sympathoadrenal system. Given that the chromaffin tissues are by far the most important source of circulating catecholamines in fish (Randall and Perry, 1992), modulation of the sympathetic innervation to this tissue may have some significance in the control of circulating catecholamines. Experiments on the Atlantic cod have shown that neural innervation of the chromaffin tissue may be a requirement for noradrenaline secretion, but not for adrenaline (Perry et al. 1991). Hagfish chromaffin cells receive no extrinsic innervation (Nilsson, S. and Holmgren, 1993a), so, while the modulation of synaptic transmission by adenosine may be a useful hypothesis for the control of noradrenaline release in rainbow trout, some other mechanism must be involved to explain adrenaline secretion in the rainbow trout theophylline treatment and in the hagfish experiment. Similarities in the response profile of catecholamines in the hypoxic rainbow trout and Pacific hagfish treated with theophylline, points to a similar mode of action by this adenosine receptor blocker in both species. The profile for adrenaline is characterized by an early peak, with a subsequent return to basal concentration. By contrast the increase 62 in noradrenaline is more gradual, less significant, and remains elevated throughout the treatment. In studies involving man, adrenaline is also elevated to a greater degree over basal levels than noradrenaline following theophylline treatment (Vestal et al. 1983; Higbee et al. 1987; Ishizaki et al. 1988), and [adrenaline] may also peak prior to termination of the treatment (Ishizaki et al. 1988). Depletion of adrenaline from the chromaffin cells as an explanation for the observed profile is unlikely, since the amount of catecholamine released into the circulation represents only a small percentage of the total level of stored catecholamines in fish (Reid and Perry, 1994). The differences between the pattern in the circulating concentrations of adrenaline and noradrenaline in fish treated with theophylline, may be further evidence for the presence of two populations of chromaffin cells containing predominantly one of the two catecholamines and responsive to different release stimuli (Perry et al. 1991; Reid and Perry, 1994). Adenosine receptor blockade may prevent an inhibitory feedback mechanism of catecholamine secretion. Adenosine, formed with the release of catecholamine from chromaffin cell vesicles, inhibits catecholamine secretion from bovine adrenal medulla cells by inhibiting calcium flux (Chern et al. 1987, 1992). Hence, in this system adenosine may act as a negative feedback regulator of catecholamine secretion. Investigations on the interactions between adenosine and the mammalian pituitary-adrenocortical axis have produced some conflicting results. Both stimulatory and inhibitory roles have been attributed to adenosine in the release of ACTH from the pituitary gland (Anand-Srivastava et al. 1989; Scaccianoce et al. 1989). In vitro, adenosine reduces both 63 basal and ACTH-stimulated corticosterone release from the rat adrenal gland (Scaccianoce et al. 1989), but does not modulate adrenal steroidogenesis in the frog (Feuilloley et al. 1992). Although these modulatory actions of adenosine have not yet been investigated in fish, results obtained in this study support a mechanism based on the involvement of adenosine receptors. In SERIES II, differences in the [adrenaline] in the three hypoxic groups reflect the degree to which adenosine receptors are antagonized by the treatments. Whereas the saline treatment had the lowest [adrenaline], the levels were intermediate with the weak adenosine receptor blocker enprofylline, and most significant in the higher affinity receptor blocker theophylline. The lack of an effect of the adenosine receptor blockers in all the normoxic treatments, is also consistent with the hypothesis that methylxanthines will impair the regulation of stress hormones under conditions that may result in the formation of adenosine. However, the possibility that uptake, release, and metabolism of catecholamines and Cortisol are inhibited by methylxanthines can not be excluded. In summary, my results have shown that adenosine receptor blockade modulates the primary stress response of rainbow trout and Pacific hagfish. Most significantly, plasma adrenaline levels during severe hypoxia rise faster and to a much greater extent in both species following adenosine receptor blockade. Given the importance of these hormones in regulating the overall stress response, an inadequate control over the circulating concentrations of catecholamines and Cortisol in response to an acute challenge may rapidly lead to a state of exhaustion. 64 Chapter 3 Erythrocyte responses to adenosine receptor blockade 65 Introduction In the absence of a capability to depress metabolism, increasing oxygen transport capacity is the most efficient way for a fish to compensate for an acute reduction in 0 2 availability. This can be accomplished by increasing ventilation, improving gill 0 2 diffusing capacity, and increasing blood 0 2 capacitance (Jensen et al. 1993). The latter can be increased by elevation of haemoglobin concentration ([Hb]) and Hb 0 2 affinity. Under conditions of acute hypoxia, the 0 2 carrying capacity can be increased via catecholamine-stimulated contraction of, and release of red blood cells (rbc) from, the spleen (Holmgren and Nilsson, 1975; Yamamoto et al. 1985; Perry and Kinkead, 1989). This response can increase [Hb] by up to 30-35% but is quite variable, being substantial in some species and almost insignificant in others (Weber and Jensen, 1988). Fish can also increase their 0 2 carrying capacity in severe hypoxia by regulation of their rbc pH (Tetens and Christensen, 1987; Nikinmaa, 1992). An increase in the concentration of circulating catecholamines with acute hypoxia, stimulates the rbc Na+/H+ exchanger via ^-adrenergic receptors, and results in a net outflow of H+ which raises rbc pH (Nikinmaa, 1982). Alkalinization of the rbc increases Hb O, affinity because the haemoglobin has a Bohr shift (Tufts and Randall, 1989). Coupled with the H+ outflow is an inflow of Na+, followed by CI", which together cause the cell to swell by osmosis. Adrenergic activation of the Na+/H+ exchanger also leads to a rapid decline in ATP and other phosphates (Ferguson and Boutilier, 1989), which may further enhance oxygen binding by reducing their negative allosteric effects (Nikinmaa, 1992). The focus of this study was to investigate the possible interactions between 66 adenosine and the adrenergic control of two mechanisms used by fish to increase blood 0 2 capacitance under severe hypoxia: 1) rbc pH regulation and 2) splenic release of rbc. Hence, the response to adenosine receptor blockers under normoxic and hypoxic conditions were monitored, and their responses compared with those obtained with sham groups given saline. Throughout the study, comparisons are also made between rainbow trout and Pacific hagfish, two species with marked differences in haematological characteristics (table 2). Relative to rainbow trout, hagfish have a large blood volume with a low Hct, [Hb], oxygen content and buffering capacity. Hagfish haemoglobin is monomeric, has a very high affinity, a small Bohr effect, and no Root effect. Other important differences between these two species are the extremely low metabolic rate of the Pacific hagfish (Munz and Morris, 1965; Farrell, 1991), the absence of a spleen (Satchell, 91), and no Band III protein on their rbc membrane (Ellory et al. 1987; Brill et al. 1992). Methods The three same experimental series described in chapter 1, and the same set offish, were used in this study. Rainbow trout were exposed to either a Pw02 of 30 torr for 6 h (SERIES I), or to 25 torr for 1 h (SERIES II). In SERIES III, Pacific hagfish were exposed to a PwOz of 10 or 30 torr for 1 h. Reference should be made to the methods section of chapter 1, for a detailed description of the experimental animals, surgical procedures, experimental protocol, 67 Table 2. Blood parameters of the Pacific hagfish and the rainbow trout. Blood Parameters Hct (%) Hb (gdl1) MCHC (gdl1) Blood Volume (%) Buffering Capacity (slykes) P50 (mmHg) Bohr factor Root effect Oxygen Content (vols %) Pacific Hagfish Eptatretus stouti 14.71 3.11 21.11 *182 *4.02 1.8 at 18°C4 -0.206 no2 *2.39 Rainbow Trout Onchorhynchus mykiss 30.31 8.71 28.71 5-62 9.73 19.0 at 15°C5 -0.547 yes8 14.61 References: 1. Value from this study; 2. Satchell (1991); 3. Mattsoff and Nikinmaa (1988); 4. Manwell (1958); 5. Milligan and Wood (1987); 6. Li et al. (1972); 7. Eddy (1971); 8. Boutilier et al. (1986); 9. Wells et al. (1986). * Value for Eptatretus cirrhatus. 68 sampling regime, and description of the drugs, since these were the same for both studies. In all three experimental series, blood samples were collected in 1.5 ml micro centrifuge tubes. From this initial sample aliquots of whole blood were taken for measurement of haematocrit (Hct) and haemoglobin concentration ([Hb]), and the remaining blood was spun down at 7,500 g for 2 min. The remaining packed red blood cells (rbc) were frozen for later measurement of intracellular pH (pHi) of rbc. Analytical Procedures Hct was determined by centrifuging the blood in heparinized capillary tubes for 5 minutes at 11,500 rpm in a Damon IEC MB microhaematocrit centrifuge. Hb concentration was measured using a Sigma Total Haemoglobin (525-A) assay kit and the relative absorbency measured at 540 nm in a Shimadzu UV-160 visible recording spectrophotometer. Mean cellular [Hb] (MCHC) was calculated as ([Hb]/Hct)*100. Intracellular red cell pH was measured using a thermostatted Radiometer G297/G2 glass capillary electrode with a PHM71 acid-base analyzer, using the fast freeze-thaw technique (Zeidler and Kim, 1977). Calibration of the pH electrode was made using Radiometer Precision Buffer Solution Standards S1519 and S1500. Statistical Analysis All data are presented as means ± one standard error. The statistical significance of observed effects of treatment exposure within a group were tested by one-way repeated measures ANOVA. To compare pre-treatment means with means at subsequent sampling times Dunnett's test was used. Where appropriate, the statistical significance of observed differences between the means from all treatments at a particular sampling time were tested 69 by one-way ANOVA. To isolate which group(s) differed from the others, Student-Newman-Keuls test was used. The significance level for all statistical test was P < 0.05. Results In SERIES 1, removal of five 700 /A blood samples from rainbow trout (mean wt: 461 ± 24 g) resulted in a significant decrease in the [Hb] of all the experimental groups (fig. 13). Relative to time 0 values, the 360 min [Hb] values had decreased significantly by 30.2, 30.3, and 29.6% in the normoxic sham, normoxic theophylline, and hypoxic theophylline groups, respectively. In the hypoxic sham group the [Hb] was maintained through the first 4 sampling times, and decreased by 14.2% at 360 min. There was no difference between the mean body weight of all four groups. In SERIES II, removal of four 900 /xl blood samples from rainbow trout (mean wt: 1,144 ± 32 g) did not significantly decrease the [Hb] in any of the experimental groups (fig. 14). Whereas, a 15.2% increase in [Hb] was recorded after 60 min of hypoxic exposure in the sham group, no change in [Hb] was observed in the hypoxic enprofylline and hypoxic theophylline groups. Once again, there were no significant difference between the mean body weight of all five experimental groups used in this series. In the hagfish experiment (SERIES III; mean wt: 183 ± 7 g), removal of four 500 //,1 blood samples decreased the mean control [Hb] by 10.6% overall. This decrease in Hb was matched by a decrease of 11.5% in the Hct values. Of the six experimental groups, the decrease in [Hb] and Hct was only significant in the 30 torr PwO, hypoxic theophylline and 10 torr Pw02 hypoxic sham groups. No changes were observed in the MCHC values under 70 Figure 13. Haemoglobin (Hb) concentration of rainbow trout in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were injected with either saline or theophylline (theo.). Time 0 values are controls, a indicates a significant difference from control in given treatment (p<0.05). Values are means ± 1 SEM. 71 1 1 r 10 -T 9 -8 -7 -1 0 min ESI 10 min 30 min VTA 120 min 360 min Normoxic Normoxic Theo. Hypoxic Hypoxic Theo. 72 Figure 14. Haemoglobin (Hb) concentration of rainbow trout in relation to exposure duration to normoxia and hypoxia (Pw02=25 torr). Animals were injected with either saline, enprofylline (enpro.), or theophylline (theo.). Time 0 values are controls, a indicates a significant difference from control in given treatment (p<0.05). Values are means ± 1 SEM. 73 I I 0 min ££3 10 min B%£1 30 min Y77A 60 min 1 1 10 9 I-8 H Normozic Normoxic Hypoxic Hypoxic Hypoxic Enpro. Enpro. Theo. 74 normoxic or hypoxic conditions. The [Hb], Hct, and MCHC values for SERIES III are presented in Appendix 1. In SERIES I, there was a gradual decrease in the Hct values throughout the 360 min trials, and no change in the MCHC values of the two normoxic treatments (table 3). In the hypoxic sham treatment, the Hct values were significantly higher than the control value from time 30 min onwards, and the 360 min value was 14.9% higher than the control value. An initial increase in Hct values was also observed in the hypoxic theophylline treatment, however the 360 min value was 5.1% lower than the control value. The MCHC values in the hypoxic sham and theophylline groups decreased gradually throughout the 360 min trial. Significant decreases in Hct and no changes in MCHC values was observed in the two normoxic treatments of SERIES II (table 4). Compared to their respective control time 0 values, their was a significant increase in Hct in all 3 hypoxic treatments. In the hypoxic sham group, a decrease in MCHC was only seen at the 30 min sampling period. In contrast, in the two hypoxic groups treated with an AR blocker, the decrease in MCHC was progressive throughout the hypoxic exposure and the values significantly lower than in the hypoxic sham group at 360 min. In SERIES I and SERIES II, rbc pH (pHi) and the rbc transmembrane pH difference (pHe-pHi) remained unchanged throughout the normoxic treatments (table 3 & 4). Under hypoxic conditions, pHi increased significantly in all the treatments of both series, however whereas the rbc pH remained elevated for the 60 min hypoxic exposure in SERIES II, it returned to control values by 360 min in SERIES I. A significant decrease in rbc transmembrane pH difference was also observed in all the hypoxic treatments of both 75 Table 3. Haematocrit, mean cellular haemoglobin content (MCHC), red blood cell pH (pHi), and the difference between whole blood pH (pHe) and pHi of rainbow trout, in relation to exposure duration to normoxia and hypoxia (PwO2=30 torr). Animals were infused with either saline, or theophylline (theo.). Values are means ± 1 SEM. 76 Experimental Condition Normoxic Sham Normoxic Theo. Hypoxic Sham Hypoxic Theo. Time (min) 0 10 30 120 360 0 10 30 120 360 0 10 30 120 360 0 10 30 120 360 Haematocrit (%) 30.0±0.7 25.8±l.la 23.4±l.la 22.9±1.3a 20.6+1.1* 29.3±0.7 26.3±0.8 25.8±0.9 23.6±0.8a 19.8±1.0a 26.3±1.3 28.6+1.2 32.1±1.0a 34.1±2.1a 30.9±2.8a 27.6±1.7 30.3 ±2.0 33.9±2.4a 32.0±1.6" 26.2±0.5 MCHC (gdl1) 32.3±0.6 31.6±0.8 31.9±0.4 32.2±0.9 32.8±0.5 31.1±1.0 31.0+1.1 30.6±1.0 31.3±1.2 32.3 ±0.8 34.0±1.4 32.2±1.7 28.8±1.2a 27.2±1.5a 25.7±1.6a 32.9±1.8 28.5±1.2a 25.5±1.2a 24.6±l.la 24.3±1.3a pHi 7.46+0.04 7.42±0.03 7.38±0.02 7.40±0.02 7.38±0.02 7.42±0.02 7.42±0.02 7.37±0.02 7.41±0.03 7.42±0.03 7.44 ±0.02 7.69±0.02a 7.71±0.03ac 7.69±0.05a 7.52±0.06 7.39±0.01 7.68±0.02a 7.62±0.03a 7.60±0.02a 7.49±0.04 pHe-pHi 0.56±0.07 0.68±0.03 0.65 ±0.03 0.67±0.05 0.74 ±0.03 0.60 ±0.04 0.60±0.04 0.60±0.04 0.63 ±0.06 0.61±0.06 0.66±0.04 0.32±0.06a 0.31±0.07a 0.29±0.06a 0.29±0.06a 0.63±0.06 0.07±0.05ab 0.11±0.04ab 0.10±0.06ab 0.17±0.03a a. Significantly different from control time 0 value (p<0.05) b. Significantly different from hypoxic sham (p<0.05) c. Significantly different from hypoxic theo. (p<0.05) 77 Table 4. Haematocrit, mean cellular haemoglobin content (MCHC), red blood cell pH (pHi), and the difference between whole blood pH (pHe) and pHi of rainbow trout, in relation to exposure duration to normoxia and hypoxia (Pw02=25 torr). Animals were infused with either saline, enprofylline (enpro.), or theophylline (theo.). Values are means ± 1 SEM. 77 Table 4. Haematocrit, mean cellular haemoglobin content (MCHC), red blood cell pH (pHi), and the difference between whole blood pH (pHe) and pHi of rainbow trout, in relation to exposure duration to normoxia and hypoxia (Pw02=25 torr). Animals were infused with either saline, enprofylline (enpro.), or theophylline (theo.). Values are means ± 1 SEM. 78 Experimental Condition Normoxic Sham Normoxic Enpro. Hypoxic Sham Hypoxic Enpro. Hypoxic Theo. Time (min) 0 10 30 60 0 10 30 60 0 10 30 60 0 10 30 60 0 10 30 60 Haematocrit (%) 29.5±1.5 27.3±1.4a 26.9±1.2a 27.4±1.2a 30.6±1.0 29.7±1.3 28.9±1.2a 27.9±1.0a 30.3±1.3 31.8±1.0 35.5±0.9a 37.5±0.9a 30.3±1.3 31.4±1.8 36.7±1.4a 37.8±l.la 30.6±1.6 32.0±1.3 36.0±1.0a 38.1±1.6a MCHC 29.0±0.6 29.4±0.6 29.5±0.7 28.5 ±0.5 29.1 ±0.5 28.9±0.4 29.1±0.5 29.3±0.4 27.6±1.2 26.5±0.8 25.3±1.0a 26.3±0.8 29.4±0.7 26.4±0.5a 24.5±0.5a 23.4±0.4ab 28.6±0.3 26.4±0.5a 24.4±0.4a 23.2±0.6ab pHi 7.45 ±0.02 7.45±0.03 7.43 ±0.03 7.44±0.03 7.37±0.02 7.40 ±0.03 7.44 ±0.02 7.41+0.01 7.43 ±0.03 7.67±0.03a 7.65±0.03a 7.63±0.03a 7.46+0.02 7.70±0.03a 7.69±0.04a 7.67±0.04a 7.44 ±0.02 7.64±0.03" 7.61±0.04a 7.62±0.03a pHe-pHi 0.46 ±0.03 0.45 ±0.03 0.50 ±0.03 0.49±0.03 0.53±0.02 0.50 ±0.03 0.49±0.02 0.54±0.03 0.48 ±0.03 0.29±0.02a 0.31±0.04a 0.32±0.04a 0.47±0.04 0.03±0.07ab 0.09±0.08ab 0.21±0.07a 0.53 ±0.04 0.07±0.07ab 0.16±0.08a 0.17±0.07a a. Significantly different from control time 0 value (p<0.05) b. Significantly different from hypoxic sham (p<0.05) 79 series, but the values were consistently smaller in the groups treated with an adenosine receptor blockers. Unlike the results obtained with rainbow trout, no increase in rbc pH was observed in the hagfish that were exposed to either 30 torr or 10 torr Pw02 for 60 min. On the contrary, pHi decreased significantly in the 10 torr Pw02 hypoxic theophylline treatment. A small but significant decrease in rbc transmembrane pH difference was also only seen in the 10 torr Pw02 hypoxic theophylline group. The rbc pH and rbc transmembrane pH difference values are presented in Appendix I. Discussion AR blockade and erythrocytes Relative to the sham groups, AR blockers had no effect during normoxic conditions on any of the parameters measured in this chapter, indicating no action of adenosine in normoxia. The decrease in Hct observed with serial blood sampling in all three experimental series, also followed a similar pattern in the normoxic sham and AR blocker treated animals. With hypoxic exposure in SERIES I & II, the rbc transmembrane pH difference was significantly less in the groups treated with the adenosine receptor blockers than in the sham groups. This difference may reflect direct or indirect effects of AR blockade on the rbc. Although adenosine has been shown to play a role in the regulation of glycolysis in vertebrate erythrocytes (Fievet et al. 1987; Kaloyianni et al. 1993), and fish rbc possess a nucleoside transport mechanism which may make adenosine available for rbc energy 80 metabolism (Fincham et al. 1991), there is no evidence that these processes are mediated via adenosine receptors. Therefore, a direct mechanism to explain the effects of AR blockade on pH regulation does not appear likely. Indirect effects of AR blockade on fish erythrocytes may arise through changes in plasma pH and circulating [catecholamines] following hypoxic exposure. Theophylline and enprofylline treatment enhance metabolic acidosis which develops with hypoxic exposure (see chapter 1). Since at a given sampling time there was no difference in rbc pH between the different hypoxic groups, the greater decrease in pHe with AR blockade will result in a smaller rbc transmembrane pH difference in those treatments. Lower pHe values can also increase the alkalinization of rbc observed in hypoxia, because the sensitivity of the rbc Na+/H+ exchanger to adrenergic stimulation increases as a function of decreasing pHe (Borgese et al. 1987). Compared to the hypoxic sham trout, AR blockade also results in higher circulating concentrations of adrenaline (see chapter 2). It is possible that these higher [adrenaline] will also enhance the /3-adrenergic sensitive Na+/H+ exchanger activity (Nikinmaa, 1982) and further raise rbc pH. The possibility that AR blockade indirectly enhances the activity of the rbc Na+/H+ exchanger in rainbow trout, may also explain the greater decrease in MCHC values observed after treatment with the blockers than in the sham groups. The greater Na+ influx resulting from an enhanced rbc Na+/H+ exchanger activity would cause more osmotic swelling of the cells (Nikinmaa, 1992), and further decrease MCHC values. In the hagfish experimental series, although plasma noradrenaline increased significantly in both 10 torr Pw02 hypoxic exposure groups and adrenaline in the 81 theophylline treated group (see chapter 2), no significant regulation of rbc pH or changes in MCHC values were observed in this study. Although the [catecholamine] may not have been sufficiently high to activate a /3-adrenergic sensitive Na+/H+ exchanger, in vivo [adrenaline] much smaller than the ones recorded in this study results in adrenergic rbc pH regulation in rainbow trout (Perry and Kinkead, 1989). One possibility to explain this results, is the absence of a rbc Na+/H+ exchange transport pathway in hagfish rbc. This is supported by the inability of hagfish rbc to respond to intracellular acidification, or to osmotic swelling when exposed to a hypotonic medium (Nikinmaa et al. 1993). Hagfish erythrocytes are also unusual in virtually lacking the anion exchange pathway (Ellory et al. 1987); they do however possess other ion transport systems (Ellory and Wolowyk, 1991). AR blockade and [Hb] of the blood The increase in [Hb] observed in the hypoxic sham rainbow trout group of SERIES II, has been observed in several studies with similar acute hypoxic exposure (Tetens and Lykkeboe, 1985; Boutilier et al. 1988; Claireaux et al. 1988). With severe hypoxia, [Hb] may increase by haemoconcentration because of fluid shifting from the blood to lactate-loaded muscles (Milligan and Wood, 1986a, b). Increasing catecholamine concentrations with hypoxic exposure can also lead to an elevation in [Hb] via an adrenaline induced diuresis (Vermette and Perry, 1987). In vitro and in vivo studies have also shown that recruitment of red blood cells can result from contraction of the spleen by stimulation of ^-adrenoceptors (Nilsson, S. and Grove, 1974; Vermette and Perry, 1988; Perry and Kinkead, 1989). Contraction of the spleen is probably the dominant response causing an 82 increase in [Hb] following an increase in plasma catecholamines, since adrenergic elevation of blood Hb is absent in splenectomized trout (Perry and Kinkead, 1989). Relative to the hypoxic sham groups of SERIES I & II, adenosine receptor blockade negated the effects of acute hypoxia on [Hb]. While AR blockade resulted in significantly higher plasma [adrenaline] than sham treatment in SERIES II (chapter 2), and a dose-dependent relationship between plasma adrenaline and arterial blood Hb has been observed in rainbow trout (Perry and Kinkead, 1989), no change in [Hb] was observed following AR blockade. Changes in [Hb] resulting from water influx into tissues and hemoconcentration can not explain these results, since the animals treated with theophylline in SERIES II had significantly higher tissue [lactate] than the hypoxic sham group (see chapter 1), a situation favouring osmotic swelling of the tissues. Given the relative importance of the adrenoceptor-sensitive recruitment of rbc from the spleen to the final [Hb] discussed above, these results indicate that adenosine receptor blockade may prevent splenic release of rbcs during hypoxia. This hypothesis is supported by in vitro studies demonstrating mediation of rat splenic contraction by adenosine Ar receptor activation (Fozard and Milavec-Krizman, 1993). However, the effects of adenosine receptor agonists on the rat isolated spleen are not the result of direct or indirect interactions with the a^-adrenoceptors of that tissue (Fozard and Milavec-Krizman, 1993). Therefore, whereas contraction of the spleen is activated independently by adenosine and catecholamines in the rat, a somewhat different mechanism involving an interaction between adenosine and catecholamine receptors may be present in rainbow trout. In hagfish, the [Hb] decreased in all the experimental groups, irrespective of the 83 degree of hypoxia or [catecholamine]. Treatment with the adenosine receptor blocker theophylline was also without any effect on the changes in blood [Hb]. These results are consistent with the absence of a spleen in hagfish. In conclusion, adenosine receptor blockade does not appear to have any direct effects on the pH and volume regulation of rainbow trout erythrocytes. The indirect effects of adenosine receptor blockade observed under hypoxic conditions, i.e. greater metabolic acidosis and plasma [catecholamine], can explain the smaller rbc transmembrane pH difference and smaller MCHC values observed in the AR blockade groups. Hagfish erythrocytes do not appear to regulate rbc pH or volume under hypoxic conditions, they also appear unresponsive to adrenergic stimulation, and AR blockade had no direct effects on these cells. In rainbow trout, it appears that AR blockade may prevent splenic release of rbc under hypoxic conditions by abolishing the stimulatory effects of catecholamines on this tissue. These results indicate that adenosine may be an essential modulator of the adrenergic activation of the spleen observed under acute hypoxic conditions. In comparison to rainbow trout, the apparent absence of any physiological regulation of rbc pH, MCHC, or [Hb] in Pacific hagfish, reflect the very different strategies adopted by these two species in acute hypoxia tolerance. Rainbow trout try to maximize their blood oxygen capacitance during severe hypoxia in order to lessen the recruitment of anaerobic pathways, and sustain a fairly high metabolic rate. Hagfish maintain their relatively low oxygen capacity under acidotic conditions by combining a very high Hb 0 2 affinity with no Root effect, and accumulate high [lactate] in their tissues, supplementing their very low metabolic requirements by anaerobic pathways. 84 General Discussion Throughout this thesis, I have shown that adenosine receptor blockade had a number of diverse effects on the physiological response of rainbow trout and Pacific hagfish to hypoxia. These observations are consistent with the broad physiological actions attributed to adenosine in mammals, which have a common tendency to redress any imbalance between energy demand and availability (Newby et al. 1990). Comparison of the results obtained from fish treated with either the adenosine receptor blockers or saline, indicate that adenosine may increase hypoxia tolerance by reducing the recruitment of anaerobic metabolism. However, adenosine may stimulate glycogenolysis in the ischemic mammalian heart and brain (Magistretti et al. 1986; Janier et al. 1993), tissues which have been extensively investigated for the protective effects of adenosine. The protective role of adenosine in the ischemic brain results from a depression of neural firing, synaptic transmission, and release of excitatory neurotransmitters, combined with an increase in the supply of oxygen and glucose through vasodilatory effects (Stefanovich, 1988; Rudolphi et al. 1992). In the ischemic heart, adenosine increases glycolytic flux via its potentiating effects on myocardial glucose uptake (Law and Raymond, 1988), improves oxygen supply by increasing coronary vasodilation (Berne, 1980), has depressing effects on cardiac electrical and contractile activity (Belardinelli et al. 1987; West et al. 1987; Belardinelli and Shryock, 1992), and inhibits the myocardial effects of catecholamines (Dobson et al. 1987). The common link between these various studies is the ability of adenosine to reduce metabolic demand and preserve energy resources while maintaining energy supply. Thus adenosine receptor blockade may have prevented a direct 85 inhibitory effect of adenosine on anaerobic metabolism, but evidence from other studies portrays a more complex scenario. One way by which adenosine may have an effect on the recruitment of anaerobic metabolism, is by modulating the circulating levels of the hormones secreted in response to hypoxic stress. Adenosine receptor blockade during hypoxia resulted in an increase in the concentration of catecholamines and Cortisol. Given the powerful catabolic properties of these hormones, and their key role in the activation of glycogen metabolism, metabolic saving may be achieved by reducing their effects. In mammals, adenosine appears to have antiadrenergic actions in the heart (Mullane and Williams, 1990) and the brain (Stone and Bartrup, 1991), and also inhibits catecholamine secretion from chromaffin cells (Chern et al. 1987), however, its potential role in modulating the stress response in vivo has not received much attention. In fish, it does appear to play some role in this regard during hypoxia. Adenosine may also increase the aerobic capacity of rainbow trout during acute hypoxia. Although differences in 0 2 content between the various hypoxic groups were not observed, the smaller [Hb] in the trout treated with an AR blocker may reduce the capacity for aerobic metabolism. A small reduction in the energetic contribution from aerobic metabolism would require a substantial activation of anaerobic pathways to sustain energy turnover. While adenosine may stimulate rbc release from the spleen (Fozard and Milavec-Krizman, 1993), its potential role in vivo for the regulation of aerobic capacity remains to be investigated. The various effects observed following adenosine receptor blockade, also suggest a 86 broad distribution of adenosine receptors in fish. This is supported by the observation that adenosine receptors are found in most mammalian tissues (Ramkumar et al. 1988; Reddington and Lee, 1991; Collis and Hourani, 1993). Given the short half-life, and the very localized effects of adenosine, and given the results of this study it would appear that adenosine production and adenosine receptors may be found in at least the heart, red muscle, spleen, and possibly the chromaffin and interrenal cells of the head kidney of rainbow trout. Results from the experiment carried on hagfish, suggest the presence of adenosine receptors at least on the chromaffin cells. The response to hypoxia tolerance of the Pacific hagfish (Eptatretous stouti) was investigated in this thesis for the first time. Compared to most teleosts, the hypoxia tolerance of E. stouti was remarkable (Van Den Thillard and Van Waarde, 1985). Anaerobic metabolism was only seen at a Pw02 of 10 torr, and was not accompanied by a significant metabolic acidosis after 60 min of hypoxic exposure. These results are consistent with previous observations on Myxine glutinosa, and Eptatretus cirrhatus. While hagfish may have a limited ability to improve 0 2 extraction with declining 0 2 tensions, they can sustain a very large accumulation of tissue and plasma lactate during prolonged and acute hypoxia (Hansen and Sidell, 1983; Forster, 1990; Forster et al. 1992). Moreover, their capacity to regulate acid-base balance and to tolerate proton loads, may also be greater than other marine teleost (McDonald et al. 1991). The hypoxia tolerance of M. glutinosa and E. cirrhatus is best illustrated by the remarkable ability of their cardiovascular systems to maintain normal function during prolong hypoxia or even anoxia (Hansen and Sidell, 1983; Axelsson et al. 1990; Forster, 1991). Altogether, the hypoxia tolerance in hagfish results 87 from their very low metabolic rate (Munz and Morris, 1965; Forster, 1990), and their large potential for anaerobic metabolism. In contrast, the hypoxia tolerance of rainbow trout was significantly less than that observed for Pacific hagfish. The relatively large and rapid increase in plasma lactate at a Pw0 2 of 30 torr, and the gradual development of a metabolic acidosis after 360 min of exposure, indicate that rainbow trout are not particularly tolerant to environmental hypoxia in comparison to other teleosts (Van Den Thillard and Van Waarde, 1985). As reported in several studies, the energy status of the heart, red and white muscles are relatively unaffected by mild or acute hypoxia of short duration (Jorgensen and Mustafa, 1980b; Claireaux and Dutil, 1992), but drop substantially during longer exposure periods and anoxia (Van Waarde et al. 1983, 1990; Dunn and Hochachka, 1986). In contrast to the situation usually observed in hagfish (Forster et al. 1992), rainbow trout characteristically enhanced their blood oxygen transport capacity by elevating Hb 0 2 affinity through rbc pH regulation, and increasing their [Hb] (Boutilier et al. 1988; Fievet et al. 1988). These catecholamine-mediated responses are an essential part of the strategy used by trout to improve oxygen extraction (Boutilier et al. 1988; Thomas et al. 1992), and to minimize the recruitment of anaerobic metabolism. Finally, although there are marked differences between rainbow trout and Pacific hagfish in their tolerance to hypoxia, and in the strategy that both utilize to resist periods of oxygen shortage, results obtained throughout this thesis indicate that adenosine has an important protective role in both species. Given that hagfish may be an early offshoot from the primitive vertebrate stock, literally hundreds of millions of years ago (Hardisty, 1979; 88 Barback, 1991), it is rather surprising to see the degree of similarity between the two species in their response to adenosine receptor blockade. This long conservative history in the possible physiological actions of adenosine, lends support to the original idea of Newby (1984) of a primitive and ubiquitous life-preserving function for adenosine. In conclusion, the physiological role of adenosine during hypoxia in fish, may be fairly similar to the situation observed in other vertebrates. 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Fish Biol. 21, 429-432. WRIGHT, P. A., PERRY, S. F. and MOON, T. W. (1989). Regulation of hepatic gluconeogenesis and glycogenolysis by catecholamines in rainbow trout during environmental hypoxia. /. Exp. Biol. 147, 168-188. YAMAMOTO, K.-L, ITAZAWA, Y. and KOBAYASHI, H. (1985). Direct observation of fish spleen by an abdominal window method and its application to exercised and hypoxic yellowtail. Jap. J. Ichthyol. 31, 427-433. ZEIDLER, R. and KIM, H. D. (1977). Preferential hemolysis of postnatal calf red cells induced by internal alkalinization. /. Gen. Physiol. 70, 385-401. 104 APPENDIX 1 105 Table 5. Blood pH (pHe), red blood cell pH (pHi), rbc transmembrane pH difference (pHe-pHi), haematocrit (Hct), haemoglobin (Hb), and mean cellular haemoglobin content (MCHC) of Pacific hagfish, in relation to exposure duration to normoxia and hypoxia (Pw02 = 10 or 30 torr). Animals were infused with either saline, or theophylline (theo.). Values are means ± 1 SEM. Experimental Condition Normoxic Sham Normoxic Theo. Hypoxic Sham, PwO2=30 torr Hypoxic Theo., PwO2=30 torr Time (min) 0 10 30 60 0 10 30 60 0 10 30 60 0 10 30 60 pHe 7.96 ±0.02 7.97±0.02 7.96±0.03 7.94 ±0.03 7.95 ±0.04 7.98 ±0.03 7.95 ±0.02 7.94±0.02 7.93 ±0.02 7.95 ±0.01 7.97±0.01 7.94±0.01 7.94 ±0.02 7.98 ±0.02 7.98±0.01 7.98 ±0.01 pHi 7.13 ±0.02 7.14±0.02 7.15±0.01 7.11±0.01 7.15±0.02 7.15±0.01 7.16±0.01 7.15±0.01 7.18 ±0.02 7.18 ±0.02 7.19±0.02 7.18±0.02 7.13±0.01 7.14±0.02 7.14±0.02 7.14±0.02 pHe-pHi 0.82±0.03 0.83±0.02 0.81±0.03 0.83 ±0.03 0.80±0.02 0.83 ±0.03 0.80±0.02 0.80±0.02 0.75 ±0.02 0.77±0.02 0.78±0.03 0.77±0.03 0.81±0.02 0.84±0.02 0.84±0.02 0.83±0.02 Hct % 14.3±1.1 13.8 + 1.2 13.6±1.4 12.5 ±1.5 15.0±1.2 14.9±1.1 14.6 ±0.9 14.1±1.2 12.9 ±0.9 12.3±0.7 12.0±0.7 12.1±1.0 14.0±0.9 12.7±0.7 12.3±0.6 11.7±0.7 Hb gdl' 3.1±0.3 2.9 ±0.2 3.0±0.3 2.8±0.3 3.3±0.1 3.1±0.2 3.1±0.2 3.0±0.2 2.7±0.3 2.6 ±0.2 2.5 ±0.2 2.5 ±0.2 3.0±0.2 2.6±0.1 2.5±0.1 2.5±0.1 MCHC gdl"1 21.8±0.7 21.4±0.7 21.7±0.8 22.9±0.5 22.4 ±1.2 20.6±0.5 21.7±1.0 21.2±0.5 20.4±0.8 21.2±1.0 21.0±0.8 20.7±0.8 21.4±0.3 20.8 ±0.4 20.8±0.7 12.7±0.6 Experimental Time Condition (min) Hypoxic Sham, 0 PwO2=10torr 10 30 60 Hypoxic Theo., 0 PwO2=10torr 10 30 60 pHe pHi 7.93 ±0.02 7.16 ±0.02 7.93±0.02 7.16±0.02 7.92±0.03 7.16±0.02 7.85 ±0.03 7.11 ±0.02 7.93±0.02 7.13±0.00 7.91±0.02 7.13±0.01 7.82±0.04a 7.10±0.01 7.77±0.02a 7.07±0.01a pHe-pHi Hct % 0.77±0.02 16.3±1.3 0.77±0.01 16.1±1.1 0.76±0.02 14.6±1.0 0.74±0.02 12.3 ±0.7 0.80±0.02 15.8±1.5 0.78±0.02 16.9±1.5 0.72±0.03 16.9±1.3 0.70±0.03a 15.3±1.4 Hb MCHC gdl' gdl' 3.3±0.2 20.8 ±0.9 3.2±0.2 20.3±0.9 3.0±0.2 20.8±0.8 2.6±0.2 21.6±1.1 3.1±0.3 19.9±1.2 3.4±0.3 20.0±0.6 3.4±0.3 20.1±0.5 3.0±0.3 19.7±0.7 a. Significantly different from control time 0 value (p<0.05) 

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