G. Vianen

Substrate mobilization and hormonal changes in rainbow trout (Oncorhynchus mykiss, L) and common carp (Cyprinus carpio, L) during deep hypoxia and subsequent recovery

Common carp (at 20°C) and rainbow trout (at 15°C) were fitted with an indwelling cannula in the dorsal aorta. The fish were exposed to a controlled decline of waterpO2 followed by 90 min deep hypoxia at 0.3 kPa (carp) or 4.8 kPa (trout). Thereafter, normoxic recovery was monitored in both species for 48 h. At regular intervals blood samples were analysed for glucose, lactate, free fatty acids, adrenaline, noradrenaline and cortisol. The oxygen restriction was maximal in both species and resulted in a significant increase of plasma lactate levels. In carp, adrenaline, noradrenaline and cortisol levels increased to 2, 50, and 753 ng·ml-1 respectively during anoxia, whereas in trout these hormones increased to 12, 8 and 735 ng·ml-1 respectively during hypoxia. In hypoxic trout, the plasma levels of glucose (3 mol·l-1) were increased modestly whereas levels of free fatty acids (0.25 mmol·l-1) were decreased to 0.15 mmol·l-1. In carp, however, a marked and prolonged hyperglycaemia (from 5 to 10 mmol·l-1) and a significant continuous depression of plasma levels of free fatty acids (from 0.4 to 0.2 mmol·l-1) were observed indicating a difference in metabolic organization. It is suggested that hyperglycaemia is likely to be the result of hepatic glycogenolysis, stimulated by circulating catecholamines and a stimulation of gluconeogenesis by cortisol during recovery. The mechanism for the decline of plasma levels of free fatty acids is most probably a reduction of lipolytic activity, which appears to be an adaptation to hypoxia.

Adrenergic regulation of lipid mobilization in fishes; a possible role in hypoxia survival

Lipids are for fish the major energy source. The nutritional conditions of most species vary throughout the year considerably. Thus storage and mobilization of lipids have to be tightly controlled, yet little is known about its control. Though several hormones are known to have a lipolytic effect, short term regulation of lipolysis is known for catecholamines only. Catecholamines are usually released under stress conditions, including hypoxia. In mammals these hormones have a strong lipolytic action, causing high plasma fatty acids levels during hypoxia. In contrast to mammals, several fish species show a decrease of plasma FFA-levels during hypoxia and anoxia. However, some studies gave contrasting results when catecholamines were administrated to different fish species. The reason for this may be due to opposing effects of catecholamines on lipolysis in different tissues. From catecholamine administration experiments in cannulated carp there is evidence that norepinephrine inhibits lipolysis via β1- and β3- adrenoceptors while β2-adrenoceptors are involved in stimulation of lipolysis. Thus the opposite responses of different β-adrenoceptors may explain the conflicting in-vivo results obtained with fish. In vitro studies with adipocytes from different fish species confirm that activation of β-adrenoceptors suppresses lipolysis, while the opposite occurs in hepatocytes. Inhibiting β1- and β3- adrenoceptors in adipocytes were shown to be involved. Under hypoxia β-oxidation is inhibited, resulting in accumulation of fatty acids together with intermediates of the β-oxidation. This process may cause severe cellular damage in mammalian tissues, which ‘apparently’ does not occur in fishes. Fishes encounter (environmental) hypoxia on a regular basis, while for mammals hypoxia/anoxia is a pathological phenomenon. Hence the suppression of lipolysis in fishes under hypoxia (by β1- and β3- adrenoceptors) may be considered as a survival mechanism lost in higher vertebrates.

Plasma lactate and stress hormones in common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) during stepwise decreasing oxygen levels

By measuring the lactate response it is possible to determine whether a teleost is able to adapt to a certain oxygen level. It is hypothesized that recovery will occur at oxygen levels above the critical oxygen level (PO2)(crit) reflected by a transient lactate increase. In contrast, continuous lactate accumulation occurs at oxygen levels below the (PO2)(crit), which will be lethal in case of prolonged exposure. Since catecholamines as well as cortisol increase the availability of glucose, it is expected that these stress hormones are involved in the activation of the anaerobic metabolism. Common carp and rainbow trout were cannulated and exposed to stepwise decreasing oxygen levels. At each oxygen level blood samples were taken at several time-points and analyzed for plasma lactate, adrenaline, noradrenaline and cortisol. The results show that both individual and inter-specific differences in lactate response occur during exposure to hypoxia. These differences can be associated with observed differences in behaviour. Whereas carp stayed quiet during the hypoxia treatment, trout displayed individually different behaviour. In contrast to the passive responders, the active responding trout did not survive as a result of continuous lactate accumulation. Interestingly, both in carp and trout a strong correlation exists between the lactate and catecholamine levels. This may indicate that these stress hormones are of importance for the metabolic changes occurring during anaerobic activation.

BLOOD GAS PARAMETERS AND THE RESPONSES OF ERYTHROCYTES IN CARP EXPOSED TO DEEP HYPOXIA AND SUBSEQUENT RECOVERY

The quantitative importance of the adrenergic response of carp erythrocytes during severe oxygen restriction is not clear at present. Quantitative differences between in vivo and in vitro studies suggest that the response of carp erythrocytes may be dependent on the actual hypoxic condition. To our knowledge, a clear picture of the blood gas status, erythrocytic responses and catecholamines measured simultaneously in carp exposed to deep severe hypoxia or anoxia has not yet been reported. Therefore, we studied the physiological response of carp exposed to deep hypoxia at 0.3 kPa and subsequent recovery. Carp were fitted with an indwelling cannula in the dorsal aorta for repeated blood sampling and the blood was analysed for hematocrit, hemoglobin, mean cellular hemoglobin content, intra- and extracellular pH,pO2,PCO2, total CO2 and catecholamines. Large fluctuations in arterialpO2 levels were observed in normoxic control carp, probably caused by the alternating breathing pattern of carp. Even at waterpO2 levels of 0.3 kPa, arterialpO2 levels were maintained at about 0.2–0.3 kPa. Catecholamine levels were increased during deep hypoxia with noradrenaline as the predominant catecholamine. Hematological variables showed that the number of circulating erythrocytes was increased during hypoxia. The intracellular pH of carp red cells was maintained at pre-exposure values despite a considerable decrease of pHe. In this in vivo study, a marked decrease of the proton gradient across the red cell membrane (pHe-pHi), as high as 0.35 pH units, was observed, which is quantitatively similar to that usually observed in salmonids during hypoxia. It is suggested that the regulation of the carp erythrocytic pHi is probably caused to a major extent by deoxygenation of hemoglobin (Haldane effect) while adrenergic activation of the red cells is likely to contribute significantly to the observed reduction of the proton gradient. These mechanisms result in the persistence of a capacity for aerobic metabolism in carp of about 10–20% of the energy metabolism despite environmentalpO2 values of 2–3 mm Hg.

Beta-adrenergic control of plasma glucose and free fatty acid levels in the air-breathing African catfish Clarias gariepinus, Burchell 1822

SUMMARY In several water-breathing fish species, β-adrenergic receptor stimulation by noradrenaline leads to a decrease in plasma free fatty acid (FFA) levels, as opposed to an increase in air-breathing mammals. We hypothesised that this change in adrenergic control is related to the mode of breathing. Therefore, cannulated air-breathing African catfish were infused for 90 min with noradrenaline or with the nonselective β-agonist, isoprenaline. To identify the receptor type involved, a bolus of either a selective β1-antagonist (atenolol) or a selectiveβ 2-antagonist (ICI 118,551) was injected 15 min prior to the isoprenaline infusion. Both noradrenaline and isoprenaline led to an expected rise in glucose concentration. Isoprenaline combined with both theβ 1- and β2-antagonist led to higher glucose concentrations than isoprenaline alone. This could indicate the presence of a stimulatory β-adrenoceptor different from β1 andβ 2-adrenoceptors; these two receptors thus seemed to mediate a reduction in plasma glucose concentration. Both noradrenaline and isoprenaline led to a significant decrease in FFA concentration. Whereas theβ 1-antagonist had no effect, the β2-antagonist reduced the decrease in FFA concentration, indicating the involvement ofβ 2-adrenoceptors. It is concluded that the air-breathing African catfish reflects water-breathing fish in the adrenergic control of plasma FFA and glucose levels.

Diel fluctuations in blood metabolites in cannulated African catfish (Clarias gariepinus, Burchell 1822)

African catfish were cannulated in the dorsal aorta to study diurnal changes in blood metabolites. Cannulation of the branchial artery was tested but proved to be less successful. A clear diel fluctuation in the two major blood metabolites, free fatty acids (FFA) and glucose, was observed. Compared to the initial value at 8.30 a.m., the plasma FFA levels dropped by ca. 50% within 2 hours, after which the FFA concentration stayed relatively constant. Minimum values of 0.26±0.04 mM were reached at 12.30. The FFA concentration recovered to the initial value within the following 3 hours. The fluctuation in plasma glucose levels showed a comparable course but there was a phase-shift by 2 hours. The most astonishing finding of our study was the almost complete absence of glucose in the plasma of African catfish (0.05 ± 0.03 mM), a never reported phenomenon for any fish species. This study demonstrates the relatively low level of control of plasma glucose levels as compared to plasma FFA levels in African catfish.