Camp. Biochem. Physiol. Vol. 82C, No. I, pp. 231-234, 1985 0306-4492/85 $3.00 + 0.00 Printed in Great Britain m\$:, 1985 Pergamon Press Ltd

PLASMA CATECHOLAMINES AND ERYTHROCYTE SWELLING FOLLOWING CAPTURE STRESS

IN A MARINE TELEOST FISH

N. LING and R. M. G. WELLS

Department of Zoology, University of Auckland, Auckland, New Zealand. Telephone: 737-999

(Received 8 March 1985)

Abstract-l. Plasma concentrations of adrenaline and noradrenaline were measured at rest from cannulated fish and following net capture.

2. Adrenaline and noradrenaline concentrations in capture-stressed fish averaged 36,740 pmol I-’ and 38,860 pmol I-’ respectively, whereas resting values were 1200 pmol I-’ for both amines.

3. Erythrocyte swelling and raised blood lactate were evident in stressed fish. 4. In z~itro effects of 5 mmol 1-l adrenaline on erythrocyte suspensions suggested that the catecholamine

had a direct effect on erythrocyte volume. 5. The significance of these results is discussed in relation to the oxygen transport properties of the

INTRODUCTION

Plasma catecholamine concentrations in fish appear

to be elevated following stress (Nakano and Tom-

linson, 1967; Butler et al., 1978; Epple et al., 1982; Le Bras, 1982). The levels of adrenaline increased S-20-fold and noradrenaline increased 2-5-fold in teleost plasma Following handling stress (Mazeaud and Mazeaud, 1981). Estimates of maximal cat- echolamine levels in stressed fish vary from 30 nmol I-’ in eels (Le Bras, 1982) to 1.8 mmol 1-l in dogfish (Butler et al., 1978). Dopamine, which has also been detected in fish plasma, appears unaffected by stress (Mazeaud and Mazeaud, 1981).

Values from resting fish are scarce because of the difficulties with the sensitivity and complexity of the catecholamine assay, and problems in sampling from unstressed and inactive fish. Butler et al. (1978) found the lowest levels in dogfish to be 4.6nmol I-’ for adrenaline and 5.3 nmol I-’ for noradrenaline. Using restrained rainbow trout, Ristori et al. (1979) deter- mined minimal resting concentrations of 2.95 and 2.30 nmoll-’ for plasma adrenaline and nor- adrenaline, respectively.

Cardiovascular responses to increased cat- echolamine concentrations are well established (see Nilsson, 1983). Secondary metabolic effects of adrenaline have also been described (Mazeaud and Mazeaud, 1981). It has recently been shown, how- ever, that adrenaline exerts a specific influence on trout erythrocytes. High adrenaline concentrations modify the intraerythrocytic environment and cause the celts to swell (Nikinmaa, 1982). These obser- vations are expected to have important consequences for blood oxygen transport.

In order to assess some of these consequences, we have measured plasma catecholamines and hema- tological indices in a marine teleost at rest and following capture. The hematological effects of ad- renaline were then examined in vitro to ascertain the possible degree of control of the blood oxygen trans-

port system which is manifested at the cellular (eryth- rocyte) level.

MATERIALS AND METHODS

Specimens of the marine teleost Girella trie~~pi~tu were captured in 100 mm mesh gill nets in a shallow mixed weed habitat in the harbour entrance close to the Leigh Marine Laboratory (36”18’S, 174”46’E) and placed in 1000 I. tanks under continuous seawater flow at 18°C. All fish were mature unsexed adults in the weight range 0.7-1.2 kg.

Fish were lightly anesthetized in buffered MS222 and non-occlusively cannulated with PE-50 tubing (Intramedic, Clay Adams) via the caudal vein. Cannulae were back-filled with heparinized saline and tested for patency at regular intervals.

Blood lactate and ATP were assayed using Sigma en- zymatic test chemicals and metabolic standards (bulletins 826-UV, 1981 and 36-UV, 1974; St Louis, Missouri). He- matocrit and hemoglobin concentrations were determined (Blaxhall, 1972) with the added step of centrifuging the met-cyanide hemoglobin derivative to remove nuclear de- bris. Mean corpuscular hemoglobin concentration (MCHC) was calculated by Hb concentration x IOO/Hct. Spec- trophotomet~c measurements were made using a Pye Uni- cam SP 1750 recording spectrophotometer.

Catecholamine assay

Plasma concentrations of adrenaline and noradrenaline were determined by radioenzymatic assay according to the method of Peuler and Johnson (1977). Catecholamines were labeled by transfer of the tritiated methyl group from S-adenosyl methionine using the enzyme catechol O-methyl transferase. The O-methyl derivatives were extracted in toluene and alcohol, transfered to acetic acid solution, separated by thin layer chromatography and measured by standard liquid scintillation counting techniques. The sensi- tivity of the method was approximately 150 pmol 1-l.

Since heparin was found to interfere with the assay, it was substituted by ethyleneglycoltetraacetic acid (EGTA). 100mg EGTA and 30mg reduced glutathione were added

to I ml glass-distilled water and the pH adjusted to 7.2. This solution was used in the proportion of 20yi/ml-i whole blood.

231

232 N. LING and R. M. G. WELLS

Table I. Plasma catecholamines and hematology of Girella tricuspidafa

Hct Hb MCHC ATP/Hb Lactate A NA Individual (%) k/l) k/l) (M/M) @M) (PM) (PM)

Post-capture

A B

23.0 67.4 293 I .64 30.7 57,8l I 60,904 22.0 62.7 285 1.76 4.9 11,528 23,995

C D

Cannulated

E

36.4 90.1 247 1.51 32.4 15,912 30,201 18.4 52.5 285 2.43 1.5 61,709 40,349

19.5 63.9 328 2.05 0.3 <I50 <I50 F 21.8 67.1 308 2.15 0.6 <I50 195

For resting levels, 1.5 ml blood samples were drawn anaerobically from chronically implanted cannulae some 48 hr after capture and anesthesia. Post-stress levels were determined on samples drawn anaerobically by acute caudal venepuncture 3 hr after capture. These fish had not been cannulated or anesthetized. Samples were drawn into syr- inges containing 30 pl EGTA solution, immediately centri- fuged at 1300 g for 5 min and the plasma stored at - 78°C until assayed within 2 weeks.

In vitro erythrocytic response lo adrenaline

Approximately I ml heparinized blood samples were taken from resting fish, immediately centrifuged (13OOg for 5 min), and the huffy coat removed by aspiration. The erythrocytes were then washed twice in teleost Ringer’s (after Cala, 1977) and incubated for 1 hr at 18°C to stabilize osmotically. Temperature was maintained constant at 18°C throughout this part of the study, since it was found that abrupt temperature change or refrigerated centrifugation induced an unacceptable level of hemolysis. Hematocrits were adjusted to 15-20x.

Following incubation, the cells were divided into 100 ~1 aliquots. To each was added either 10 ~1 Ringer’s (control) or lop1 Ringer’s containing adrenaline to a final concen- tration of 5.0mmoll~‘. The pH of the suspensions was measured in a Radiometer system before and after incu- bation.

Hematocrit and MCHC were determined following incu- bation and 5min after addition of Ringer’s solutions. Values determined prior to addition of Ringer’s were ad- justed for dilution. All measurements were obtained in quadruplicate.

RESULTS

Plasma catecholamines and hematology

Results of catecholamine determinations are sum- marized in Table 1. Levels of adrenaline (A) and noradrenaline (NA) in resting fish were, in general, below the threshold of sensitivity for the assay. Levels in capture-stressed fish, however, were substantially higher, averaging 36,740 pmol 1-l for adrenaline and 38,860 pmol 1-l for noradrenaline. Also given are other hematological parameters of the individual fish studied showing the expected increase in plasma lactate and erythrocyte swelling (decreased MCHC) in stressed fish. These differences are obviously statis- tically significant by any non-parametric ranking test.

In vitro effects of adrenaline

Figure 1 shows the effects of 5 mmol 1-l adrenaline on Hct and MCHC. A significant increase in Hct (P < 0.001) was observed and a concomitant de- crease in MCHC (P < 0.001) indicated erythrocyte swelling since the in vitro concentration of hemo- globin was constant. All samples treated with adrena- line showed considerable hematocrit increases (mean

Hct increase = 6.84 & 1.05%) and erythrocyte swelling (mean MCHC Increase = 6.35 + 1.30%). The pH was 7.6 and remained constant throughout incubation.

DISCUSSION

Plasma catecholamines

Resting levels of the catecholamines adrenaline and noradrenaline in G. tricuspidata were appreciably lower than those reported in other teleosts. Mean values for adrenaline and noradrenaline in cannu-

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30 -

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L.J

PRE POST

INCUBATION INCUBATION

Fig. 1. Effect of 5 mM adrenaline on hematocrit and MCHC of erythrocyte suspensions at 18°C. Individual responses are

shown in addition to the mean values k SEM.

Catecholamines in fish plasma 233

lated eels were 131Ok380 and 3370&410pmol1-‘, respectively (Le Bras, 1982). Comparable levels for trout restrained in small dark boxes were 2950 and 2300 pmol I-‘, respectively (Ristori ef al., 1979). Our resting values approximate to those found in the plasma of cyclostomes (Mazeaud and Mazeaud, 1981) which have an exceptionally low metabolic rate. One possible reason for the higher resting levels observed in other teleost species is that the fish were cannulated via the dorsal aorta. Occlusion of part of this vessel may sufficiently elevate dorsal aortic pres- sure to cause catecholamine release from the head kidney.

Catecholamine levels in stressed G. tricuspidata compared favorably with levels in other species (Maz- eaud et a/., 1977; Butler et al., 1978; Le Bras, 1982), although the degree of stress sustained by capture and exercise may not be comparable.

Blood lactate concentrations appear to be a good indicator of post-capture stress (Dando, 1969). Post- capture lactate levels in G. tricuspidata, however, did not correlate with catecholamine concentrations (Table 1). Specimen C showed the highest level of blood lactate and died within 5 hr of capture, despite the fact that catecholamine concentrations were not excessively high, indicating that such levels are not necessarily good indicators of the maximum degree of stress or whether a fish is likely to survive a particular stress. They are, however, a reasonable indication of whether or not a fish is physiologically stressed.

It is interesting to note that in G. tricuspidata levels of adrenaline and noradrenaline are similar both during stress and resting conditions. Large differences in the concentrations of the two catecholamines have been found in other species following stress (Maz- eaud and Mazeaud, 1981), with one or the other the predominant amine. This does not take into account, however, the possibility that rates of turnover for either amine may differ significantly, as has been shown to occur in rainbow trout. Mazeaud (1979) found that the half-life of adrenaline was 66 min compared with 163 min for noradrenaline. Ungell and Nilsson (1979) found that the concentration of tritiated adrenaline injected into cod decreased ex- tremely rapidly within the first 10 min, and thereafter decreased at a slower rate. If these assumptions hold true for G. tricuspidata, adrenaline levels during the period of capture stress might be expected to be an order of magnitude higher than those measured here, and significantly higher than noradrenaline. Further, adrenaline has been shown to exert a more potent effect than noradrenaline for the humoral control of most adrenergic responses studied (Holmgren, 1977; Ask et a/., 1980; Forster, 1981). Levels determined in the present study, however, might represent maxi- mum attainable plasma concentrations due to de- pletion of catecholamine stores in the head kidney which is presumably the major site for release into the blood stream.

(Nikinmaa, 1982). Although the concentration of adrenaline used in our experiment was higher than that found in vivo, it was chosen to elicit a maximal response and diminish the effects of amine turnover in the closed incubation system.

Erythrocyte swelling will be expected to influence intracellular hemoglobin-ATP binding and thereby increase the hemoglobin-oxygen affinity (Weber, 1982). Such effects have been previously demon- strated for the erythrocytes of hypoxically stressed trout (Soivio and Nikinmaa, 1981) but not demon- strated directly for capture-stressed fish. An increased hemoglobin-oxygen affinity would however, tend to offset the Bohr effect (sensitivity of hemoglobin- oxygen binding to pH) mediated through lactate dumping in the blood stream. Elevation of plasma catecholamines during stress may therefore induce changes in erythrocyte volume and hemoglobin- oxygen affinity, in addition to the well established vasoactive and ionoregulatory responses attributed to these amines.

Acknowledgements-The authors wish to thank the Auck- land University Research Committee for funding through Project Grant No. 141-Z-168. We are indebted to Linley Hunter, Department of Psychiatry, for assistance with cate- cholamine determinations.

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