of rainbow trout (Oncorhynchus mykiss) to acute external hypercapnia. ... specifically with acidosis in animals with an intact respiratory drive (i.e. not reduced by ... Each fish was allowed to recover from anesthesia and surgery for 48h before.
J. exp. Biol. 175, 115–126 (1993) Printed in Great Britain © The Company of Biologists Limited 1993
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PROPRANOLOL IMPAIRS THE HYPERVENTILATORY RESPONSE TO ACUTE HYPERCAPNIA IN RAINBOW TROUT R. KINKEAD1, S. AOTA1, S. F. PERRY2 and D. J. RANDALL1 1Department of Zoology, University of British Columbia, 6270 University Boulevard,
Vancouver, British Columbia, Canada V6T 1Z4 and 2Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, K1N 6N5 Canada Accepted 12 October 1992
Summary This study investigated the possible role of catecholamines in the ventilatory response of rainbow trout (Oncorhynchus mykiss) to acute external hypercapnia. The ventilatory response to hypercapnia [partial pressure of CO2 in water (PwCO∑=0.76kPa)] of fish pretreated with the selective b-adrenoceptor antagonist, D,L-propranolol, was compared with that of D-propranolol (an isomer with minimal b-antagonistic activity) and saline pretreated fish (sham). A sustained 3.6-fold increase in gill ventilation volume (V˙W) was observed in the sham and D-propranolol-treated groups during the 30min interval of hypercapnia. Fish pre-treated with D,L-propranolol displayed a blunted hyperventilatory response to hypercapnia (1.9-fold increase at 30min). These results indicate that the bcomponent of an adrenergic response is involved in the usual hyperventilatory response to external hypercapnia. It is suggested that the impaired hyperventilatory response of the D,L-propranolol-treated group reflects an inhibition of central adrenergic mechanism(s) involved in the hyperventilatory reflex to respiratory acidosis.
Introduction The role of catecholamines in the regulation of breathing in fish has been a topic of extensive debate and investigation in recent years (Peyraud-Waitzenegger, 1979; Peyraud-Waitzenegger et al. 1980; Aota et al. 1990; Kinkead and Perry, 1990, 1991; Playle et al. 1990; Kinkead et al. 1991; Aota and Randall, 1992; for reviews, see Randall and Taylor, 1991; Perry et al. 1992). The discrepant results of these studies, however, have hindered the development of a general model of the physiological significance of these hormones in the control of piscine ventilation. For instance, Aota et al. (1990) showed that b-adrenoceptor blockade with D,L-propranolol impaired the hyperventilatory response to severe hypoxia (3.2–8.0kPa) and intravascular infusion of acid, whereas other investigators using similar experimental protocols observed no effects on the ventilatory response to hypoxia in rainbow trout (9.6kPa; Kinkead and Perry, 1990) or Atlantic cod, Gadus morhua (5.7kPa; Kinkead et al. 1991). Aside from the differences in Key words: control of breathing, hypercapnia, propranolol, catecholamines, Oncorhynchus mykiss, rainbow trout.
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the levels of hypoxia utilized in the different studies, with corresponding dissimilar effects on blood respiratory status and circulating catecholamine levels, the experimental protocol used by Aota et al. (1990) also induced acidemia. Since depression of wholeblood pH was not observed by Kinkead and Perry (1990) and Kinkead et al. (1991), this may explain the discrepancies between those studies. Thus, it is possible that catecholamines play a role in the regulation of ventilatory responses associated specifically with acidosis in animals with an intact respiratory drive (i.e. not reduced by external hyperoxic conditions). In the present study, the role of catecholamines in the ventilatory response to acidemia was assessed by pre-treating fish with the selective b-adrenoceptor antagonist D,Lpropranolol prior to exposure to external hypercapnia. This experimental condition was chosen for its ability to stimulate ventilation (Dejours, 1973; Janssen and Randall, 1975; Smith and Jones, 1982; Perry and Wood, 1989) and at the same time mobilize catecholamine release into the circulation (Perry et al. 1989) and induce respiratory acidosis. Materials and methods Experimental animals Rainbow trout [Oncorhynchus mykiss (Walbaum)] of either sex, weighing between 150 and 416g [mean mass=334±14g (standard error of the mean; S.E.M.)] were obtained from a local supplier. Fish were maintained in large fiberglass tanks supplied with flowing, dechlorinated and aerated tapwater. The temperature of the holding and experimental water for the control (normocapnic) group was 15˚C (June); for all other groups, holding and experimental water temperature varied between 11 and 9˚C (November–December). Fish were fed daily with floating commercial trout pellets. Food was withheld 48h before the experiments commenced. Animal preparation In all experimental groups, trout were anesthetized in a 1:10000 (w/v) solution of ethyl-m-aminobenzoate (MS 222; Syndel Laboratories; adjusted to pH7.5 with NaHCO3) and then placed onto an operating table that allowed continuous irrigation of the gills with anesthetic solution. An indwelling cannula was implanted into the dorsal aorta using flexible polyethylene tubing (Clay-Adams PE 50) to permit periodic blood sampling and drug injection (see below). The fish was then fitted with a latex mask sutured around the mouth and attached to the divider of an opaque van Dam box (Cameron and Davis, 1970) supplied with flowing, aerated water. This installation separates the box into two compartments. When the water level is uniform in the box, the movement of water from the cephalic (anterior) to the caudal (posterior) compartment causes an overflow which can be monitored, allowing a direct measurement of ventilation volume (V˙W). Each fish was allowed to recover from anesthesia and surgery for 48h before experiments commenced. During the first 24h, recuperation was facilitated by a slight pressure head in the cephalic compartment (approximately 1cm). The pressure difference between the cephalic and caudal compartments was eliminated during the final 24h of
Propranolol impairs hyperventilation during hypercapnia
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recovery. The dorsal aortic cannula was flushed at least once daily with 0.2ml of heparinized (10unitsml21 ammonium heparin; Sigma) Cortland saline. Experimental protocol Experimental conditions Series I: ventilatory adjustments to external hypercapnia. The condition of external normocapnia (blood sampling, experimental manipulating control) or hypercapnia was achieved rapidly (approximately 5min) by gassing the cephalic chamber of the van Dam box, as well as a counter-current gas exchange column (which supplied water to the cephalic chamber of the box), either with air (PwCO∑ approximately 0.04kPa, N=7) or a pre-analyzed commercial 1% CO 2 in air gas mixture to achieve a target PwCO∑ of 0.8kPa (hypercapnic+saline series: PwCO∑=0.88±0.03kPa, N=6). PwCO∑ of the cephalic compartment was continuously monitored throughout the experiment using a radiometer PCO∑ electrode housed in a thermostatted cuvette. Fish exposed to hypercapnic water, in this series, acted as a sham group for the fish treated with b-adrenoceptor antagonists (series III). Therefore, these animals were injected via the dorsal aortic cannula with 0.2ml of saline (pH7.8) 2h before the initiation of hypercapnia. Series II: assessment of the non-specific effects of propranolol on the ventilatory adjustments to hypercapnia. The racemic mixture of propranolol (D,L) is commonly used to abolish the b component of an adrenergic response. Unfortunately, this drug also has local anesthetic properties that can be mistaken for its blocking effects on badrenoceptors. The local anesthetic properties of propranolol are not stereospecific (Barrett and Cullum, 1968). This contrasts with b-antagonistic activity since Dpropranolol has less than one-hundredth of the potency of the L- or D,L - isomer in blocking the heart rate responses to isoprenaline infusion in rats (Barrett and Cullum, 1968). Hence, to ensure that the results observed in series III were due solely to an inhibited b-adrenergic response, fish were injected with D-propranolol (Sigma Chemical Company) 2h prior to being exposed to acute external hypercapnia (D-propranolol series: PwCO∑=0.88±0.03kPa, N=6). The drug was dissolved in Cortland saline immediately before use (final pH of the drug solution=7.8) and injected (approximately 0.2ml) into the dorsal aortic cannula of fish at a dose of 2mgkg21 bodymass. The cannula was flushed with an additional 0.2ml of saline after each injection to ensure complete delivery of Dpropranolol to the circulation. Series III: Effect of b-adrenoceptor blockade on the ventilatory adjustments to hypercapnia. Fish were injected with the b-adrenoceptor antagonist D,L-propranolol (Sigma Chemical Company) 2h prior to being exposed to acute external hypercapnia (D,L-propranolol series: PwCO∑=0.77±0.04kPa, N=6) to ensure adequate antagonistic activity (Nilsson, 1983). The preparation and injection protocol were similar to the one described for series II. Blood sampling Three blood samples of 750 ml each were taken from all fish. An initial sample was withdrawn from the dorsal aortic cannula before the onset of the experiment (0min), a
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second one after 30min of exposure to the experimental condition (30min) and a final sample was taken 30min after cessation of the experiment (recovery). After each blood sample, an equivalent volume of heparinized (10unitsml21) saline was injected into the fish to restore blood volume. Arterial blood was analyzed immediately after sampling to determine the partial pressure of oxygen (PaO∑), arterial blood pH (pHa) and oxygen content (CaO∑). Remaining blood was centrifuged and the plasma (250 ml) was immediately frozen in liquid N2 before being stored at 270˚C for subsequent determination of catecholamine levels. Ventilatory measurements Gill ventilation volume of each fish was monitored in the following sequence: two preexperimental measurements (Pre 1 and Pre 2) were taken 10min apart. Another measurement was taken 10min later (0min), just prior to withdrawal of the first blood sample. The animal then was exposed to the particular experimental condition (normocapnia or hypercapnia) for 30min, at which time a second blood sample was withdrawn. During that period, V˙W was monitored every 10min (see below). The fish was allowed to recover under normocapnic conditions (PwO∑
