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Cardioventilatory effects of acclimatization to aquatic hypoxia in channel catfish
Respiratory Physiology & Neurobiology 131 (2002) 223– 232 www.elsevier.com/locate/resphysiol
Cardioventilatory effects of acclimatization to aquatic hypoxia in channel catfish Mark L. Burleson *, Anna L. Carlton, Philip E. Silva Department of Biology, Uni6ersity of Texas at Arlington, Box 19498, Arlington, TX 76019, USA Accepted 14 March 2002
Abstract The mechanisms responsible for altering cardioventilatory control in vertebrates in response to chronic hypoxia are not well understood but appear to be mediated through the oxygen-sensitive chemoreceptor pathway. Little is known about the effects of chronic hypoxia on cardioventilatory control in vertebrates other than mammals. The purpose of this study was to determine how cardioventilatory control and the pattern of response is altered in channel catfish (Ictalurus punctatus) by 1 week of moderate hypoxia. Fish were acclimatized for 7 days in either normoxia (PO2 150 Torr) or hypoxia (PO2 75 Torr). After acclimatization, cardioventilatory, blood-gas and acid/base variables were measured during normoxia (PO2 14891 Torr) then at two levels of acute (5 min) hypoxia, (PO2 72.69 1 and 50.4 90.4 Torr). Ventilation was significantly greater in hypoxic acclimatized fish as was the ventilatory sensitivity to hypoxia (D ventilation/DPO2). The increase in ventilation and hypoxic sensitivity was due to increases in opercular pressure amplitude, gill ventilation frequency did not change. Heart rate was greater in hypoxic acclimatized fish but decreased in both acclimatization groups in response to acute hypoxia. Heart rate sensitivity to hypoxia (D heart rate/DPO2) was not affected by hypoxic acclimatization. The ventilatory effects of hypoxic acclimatization can be explained by increased sensitivity to oxygen but the effects on heart rate cannot. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Acclimatization, hypoxia; Control of breathing, cardioventilatory response to hypoxia; Fish, channel catfish (Ictalurus punctatus); Hypoxia, cardioventilatory response; Pressure, blood
1. Introduction Chronic hypoxia elicits numerous physiological changes that help vertebrates maintain metabolism during prolonged periods of reduced O2 availability. One poorly understood effect of * Corresponding author. Tel.: +1-817-272-2426; fax: + 1817-272-2855 E-mail address: [email protected] (M.L. Burleson).
chronic hypoxia is augmentation of hypoxic reflexes. For example, it has been shown in mammals that chronic hypoxia significantly alters the pattern and intensity of the ventilatory response to hypoxia. These changes depend on the time-scale of the hypoxic exposure (see Powell et al., 1998 for review). Chronic hypoxia (hours to days, depending on the species studied) results in a timedependent increase in ventilation, relative to the initial increase, termed ventilatory acclimatization
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to hypoxia (VAH) (Powell et al., 1998). An important component of VAH is increased ventilatory sensitivity to hypoxia (Powell et al., 2000b). Most evidence indicates that VAH is an adaptive response to hypoxia mediated by changes in the peripheral oxygen-sensitive pathway (Bisgard, 2000) and increased central nervous system gain (Powell et al., 2000a). Despite recent advances in this area, the mechanisms responsible for VAH and other cardioventilatory effects of chronic hypoxia are not well understood. Little is known about the cardioventilatory effects of chronic hypoxia in vertebrates other than mammals and the evolutionary origin of VAH is unknown (Powell et al., 1998). Therefore, important insights into the physiological mechanisms stimulated by chronic hypoxia may be learned from experiments on water-breathing vertebrates. Hypoxia is a relatively rare environmental challenge for terrestrial vertebrates except for those comparatively few species and individuals that live at or ascend to high altitudes. Aquatic hypoxia, on the other hand, is a frequent and often chronic condition faced by fishes and other organisms that respire water (Val et al., 1999; Wannamaker and Rice, 2000). Chronic aquatic hypoxia occurs naturally and has been demonstrated to impact fish populations in environments such as swamps (Chapman et al., 1999) and springs (McKinsey and Chapman, 1998). Another relevant environmental factor is that the solubility difference of O2 and CO2 in water dictates that cardioventilatory control in water-breathers is dominated by O2 rather than CO2 as in air-breathers. Thus, the physiological effects of chronic hypoxia should be more critical to the survival of water-breathing vertebrates than air-breathers. In fact, acclimatization to hypoxia has been shown to increase the time that zebra fish survive lethal hypoxia levels (Rees et al., 2001). The purpose of this study was to examine how acclimatization to moderate hypoxia (75 Torr for 7 days) affects blood-gas and cardiovascular and ventilatory responses of channel catfish (Ictalurus punctatus, Rafinesque) to two levels of acute, hypoxia ( 75 and 50 Torr). Channel catfish offer a robust model in which to
study acclimatization to hypoxia because they have a comparatively weak ventilatory response to CO2, lack central chemoreceptors (Burleson and Smatresk, 2000) and routinely experience acute and chronic hypoxia in their natural environment.
2. Material and methods
2.1. Experimental animals Channel catfish (4769 23 g, n=17) were purchased from a local commercial supplier and transported to the University of Texas at Arlington. They were housed in large (1300 L) fiberglass holding tanks filled with filtered, dechlorinated tap water in a temperature controlled (20 °C) room on a 12/12-h light–dark cycle and fed commercial fish food. Fish were held for at least 1 week before experimentation.
2.2. Surgery Catfish were anesthetized in MS-222 (ethyl-maminobenzoate, 0.01% solution) dissolved in dechlorinated tap water. After transfer to a surgery table, the fish were artificially ventilated with oxygenated anesthetic solution. Dorsal aorta (DA) and opercular cannulae were implanted as described previously (Burleson and Smatresk, 1990). Recovery was accomplished by artificially ventilating the fish with dechlorinated tap water without anesthetic. Fish were allowed to recover overnight before beginning acclimatization.
2.3. Acclimatization Following surgery and recovery, the fish were kept without feeding for 6 days in glass aquaria filled with 40 L of dechlorinated tap water and maintained under the same light and temperature conditions as the holding tanks. Sections of PVC pipe, 40×15 cm2, were put into the aquaria to provide cover for the fish. The partial pressure of oxygen in the water (PwO2) for hypoxic acclimatized fish (n=8) was maintained at 50% air satu-
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ration ( 75 Torr) by bubbling a 50/50 mix of nitrogen and air (controlled by flow meters) through the aeration/filtration system in the aquaria. Plastic covers on the aquaria restricted gas exchange between the water and atmosphere. Normoxic acclimatized fish (n =9) were kept in normally aerated aquaria with an oxygen tension near air saturation (150 Torr). Oxygen saturation levels in the acclimatization aquaria were measured daily with an oxygen meter (YSI). On the sixth day after surgery, fish were transferred to a experimental chamber which was a darkened Plexiglass chamber equipped with a continuous flow of dechlorinated tap water (2 L/m) and allowed to adjust overnight. Oxygen tensions were controlled with a stripping column and a gas-mixing flowmeter (Cameron Instruments) which allowed rapid changes in PwO2. During this time, the water in the chamber was kept at the same partial pressure as in the acclimatization aquaria; PwO2 was monitored using an oxygen electrode and meter (Cameron Instruments).
2.4. Protocol On the seventh day of acclimatization the cannulae were led out a small hole in the experimental chamber and connected to pressure transducers. DA blood and opercular pressures were recorded from the DA and opercular cannulae, respectively. Outputs from the transducer amplifiers and O2 electrode were recorded with a computer data acquisition system (Windaq). Fish that were acclimatized to hypoxia were switched to normoxia (1489 1 Torr) and monitored until ventilation stabilized ( 30 min). Normoxic acclimatized fish were simply kept in normoxia. The fish were then exposed to two levels of acute (5 min) hypoxia and the responses recorded as hypoxic response 1 (72.69 1 Torr) and hypoxic response 2 (50.49 0.4 Torr). Blood and opercular pressures were recorded for the last 3-min during each level of oxygenation. A blood sample (0.4 ml) was removed from the DA cannula immediately after each recording period for analysis of blood-gas and acid/base variables and hematocrit. At the conclusion of the experiment the fish was euthanized by an overdose of the anesthetic.
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2.5. Blood-gas and pH measurements Total CO2 content (CaCO2) of the sample was determined using a Capni-Con (Cameron Instruments), while total O2 content (CaO2) of the blood was measured with an Oxycon (Cameron Instruments). Arterial oxygen tension (PaO2) and pH were measured with a Cameron Blood Gas Meter with associated electrodes. Hematocrit was calculated as percent packed red blood cell volume after centrifugation.
2.6. Cardio6ascular and 6entilatory measurements Gill ventilation frequency (fG, beats/min), opercular pressure amplitude (Pop, cmH2O), heart rate (fH, beats/min) and DA blood pressure were obtained from recordings of blood and opercular pressure. Mean arterial pressure (MAP) was calculated as the sum of systolic pressure and diastolic pressure divided by two. All cardioventilatory variables were averaged over the 3-min. period preceding the blood sample. The ventilatory sensitivity to hypoxia was quantified by the following procedure. First a ventilatory index (Vi) was calculated by multiplying fG and opercular pressure (Vi= fG× Pop). Since Pop is proportional to the volume of each breath, Vi is analogous to minute ventilation. Ventilatory sensitivity to hypoxia (Fig. 3) was calculated by dividing the change in Vi between normoxia and the two levels of hypoxia by the change in PwO2 between normoxia and the two levels of hypoxia (DVi/DPwO2). fH sensitivity to hypoxia (Fig. 5) was similarly calculated (DfH/ DPwO2).
2.7. Statistical analyses The effects of acclimatization and acute hypoxic exposure were analyzed with a 2-way ANOVA using a commercial software program (Statistica). Specific effects (i.e. the effects of hypoxia within one acclimatization group or between groups at specific hypoxia levels) were analyzed using the least significant difference test for planned post hoc comparisons or the Scheffe´ test for unplanned comparisons (PB0.05).
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3. Results Acclimatization of chronically cannulated fish in aquaria to either normoxia or hypoxia had no noticeable effect on the outward appearance, health or behavior of the fish. Hypoxic fish were breathing faster than normoxic fish but did not display any other behavioral responses to hypoxia such as aquatic surface respiration which is breathing the oxygenated surface film of water during aquatic hypoxia (Kramer and McClure, 1982).
3.1. Ventilatory and cardio6ascular 6ariables in normoxia: post-acclimatization compared to control During normoxia there was no statistically significant difference in either fG or Pop between acclimatization groups (Figs. 1 and 2). MAP varied little (19.891.7 – 22.7 9 2.1 cmH2O overall) and there was not a statistically significant difference in MAP between acclimatization groups at any PwO2 level. The only cardioventilatory variable that was significantly different between acclimatization groups during normoxia was fH which was significantly greater in hypoxic acclimatized fish (Fig. 4).
Fig. 1. fG responses of hypoxia acclimatized and normoxia acclimatized controls to two levels of acute aquatic hypoxia. fG increased significantly in both groups in response to hypoxia but there was no significant difference between the groups. Error bars are 1 SEM. (*) indicates significant differences between acclimatization groups.
Fig. 2. Pop responses of hypoxia acclimatized and normoxia acclimatized controls to two levels of acute aquatic hypoxia. Pop increased significantly in both groups in response to hypoxia. Pop was not significantly different between groups during normoxia but is significantly different at the two levels of acute hypoxia. (*) indicates significant differences between acclimatization groups.
3.2. Ventilatory and cardio6ascular 6ariables: responses to acute hypoxia Acclimatization to aquatic hypoxia significantly affected the ventilatory response to acute hypoxia (Figs. 1 and 2). Hypoxic acclimatized fish had increased ventilatory sensitivity to hypoxia. Although there were no differences in ventilatory variables during normoxia, differences arose when fish were acutely exposed to hypoxia. Hypoxia stimulated ventilation in both groups of fish (Figs. 1 and 2). The increase was due primarily to increased Pop (Fig. 2). fG increased significantly in both groups during acute hypoxia (Fig. 1) but the increase was not as large as seen for Pop. Acclimatization, however, did not affect fG and was not significantly different between acclimatization groups at any level of hypoxia (Fig. 1). Pop was significantly greater in hypoxic acclimatized fish during both levels of acute hypoxia. The percent change in Pop in response to acute hypoxia was greater in the hypoxic acclimatized fish. Normoxic fish increased Pop 66% in hypoxic response 1 and 154% in hypoxic response 2. Hypoxic fish increased Pop 91 and 165% respectively. Ventilatory sensitivity to hypoxia (Fig. 3) shows that fish acclimatized to hypoxia were significantly more sensitive to aquatic hypoxia than normoxic acclimatized fish.
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Fig. 3. Ventilatory sensitivity to hypoxia in hypoxia acclimatized and normoxia acclimatized fish. Asterisks indicate significant differences in ventilatory sensitivity between acclimatization groups. Ventilatory sensitivity to hypoxia was significantly greater in hypoxic acclimatized fish. (*) indicates significant differences between acclimatization groups.
MAP stayed remarkably stable during acute hypoxic exposures. There were no significant changes in either acclimatization group in response to acute hypoxia. There was a statistically significant effect of hypoxic acclimatization on fH (Fig. 4). fH was significantly higher in hypoxia acclimatized fish at all aquatic PO2 levels. There was a significant bradycardia in response to acute hypoxia in both acclimatization groups and although there was a
Fig. 4. fH responses of hypoxia acclimatized and normoxia acclimatized controls to two levels of acute aquatic hypoxia. fH was significantly elevated in hypoxic acclimatized fish. Acute aquatic hypoxia caused a significant bradycardia in both groups. (*) indicates significant differences between acclimatization groups.
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Fig. 5. fH sensitivity to hypoxia in hypoxia acclimatized and normoxia acclimatized fish. The fH sensitivity of the two acclimatization groups was not significantly different.
large difference in fH between the acclimatization groups, the percent change in fH in response to the two levels of acute hypoxia were similar with about a 20% drop in hypoxic response 1 and a 40% drop in hypoxic response 2. This similarity is confirmed when these data are expressed as sensitivity to hypoxia (Fig. 5) and show that acclimatization to hypoxia did not have a significant effect on the fH sensitivity to hypoxia.
3.3. Blood-gas 6ariables Blood-gas values measured in this study (Table 1) were similar to previously reported values for this species at similar PwO2 levels (Burleson and Smatresk, 1990; McKim et al., 1994; Burleson and Smatresk, 2000). PaO2 dropped predictably and significantly in both groups of fish in response to acute aquatic hypoxia. However, hypoxic acclimatization did not significantly affect PaO2 and these values are almost identical in each group (Table 1). Although CaO2 during normoxia was significantly higher in normoxic acclimatized fish, hypoxic acclimatized fish were better able to maintain CaO2 in response to acute hypoxia. CaO2 decreased significantly in normoxic acclimatized fish in response to acute hypoxia but did not in hypoxic acclimatized fish. CaO2 of normoxic acclimatized fish decreased 29% in hypoxic response 1 and 59% in hypoxic response 2. In contrast, CaO2 was better maintained in hypoxic acclimatized fish, dropping only 9 and 35%, respectively.
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Hematocrit increased significantly during hypoxia in both acclimatization groups but there was no difference between acclimatization groups (Table 1). Neither acute hypoxia nor hypoxic acclimatization had any effect on blood pH despite large differences in CaCO2 (Table 1). CaCO2 was significantly lower in hypoxic acclimatized fish and significantly decreased in both acclimatization groups in response to acute hypoxia. In normoxic acclimatized catfish CaCO2 decreased 14% in hypoxic response 1 and 27% in hypoxic response 2. Decreases in hypoxic acclimatized fish were 17 and 19%, respectively.
4. Discussion The results of this study show that acclimatization to moderate aquatic hypoxia (PwO2 75 Torr) for 1 week significantly alters the ventilatory response to acute hypoxia in channel catfish. fH is higher in fish acclimatized to hypoxia but the fH
sensitivity to hypoxia is no different from normoxic fish. While the ventilatory effects of hypoxic acclimatization may be due to increased O2 sensitivity the data indicate that the cardiac effects are probably not. The few studies that have previously examined the effects of chronic hypoxia in fishes have used severe hypoxia, usually below the species-specific critical PwO2 (the PwO2 below which O2 consumption begins to decline) and much longer acclimatization time periods. Severe hypoxia can elicit stress responses, for example, elevated blood catecholamine levels, that may have effects secondary to reflexes mediated by O2-sensitive chemoreceptors. The level of hypoxia used in this study, 75 Torr, is above critical PwO2, 65–70 Torr, for channel catfish (Burggren and Cameron, 1980) and the fact that MAP, hematocrit and CaO2 did not differ between the acclimatization groups suggests that this level of hypoxia was not severe enough to stimulate the release of catecholamines to the extent that they might affect cardioventilatory variables.
Table 1 Blood-gas values in response to acute hypoxia PwO2 (Torr)
Acclimatization group Normoxic
Hypoxic
148.39 0.9 (normoxia)
PaO2 (Torr) CaO2 (mmol/L) pH CaCO2 (mmol/L) Hct
64.5 95.1 0.56 90.09a 7.17 90.14 5.46 9 0.24 14.4 9 1.5
64.3 96.4 0.43 9 0.04 7.14 9 0.18 3.72 9 0.42a 15.1 91.4
72.6 91.0 (hypoxic response 1)
PaO2 CaO2 pH CaCO2
37.0 9 5.8b 0.40 9 0.07 7.42 9 0.13 4.67 90.27
35.1 9 2.2 0.39 90.04 7.05 9 0.08 3.09
Hct
15.7 9 1.9
90.30a,b 17.7 91.4
PaO2 CaO2 pH CaCO2 Hct
23.7 93.7 0.23 9 0.05 7.49 9 0.23 3.98 9 0.22 17.4 9 2.0
22.4 92.8 0.28 90.07 7.23 9 0.15 3.0 90.24a,b 18.1 91.0
50.49 0.4 (hypoxic response 2)
Values for dorsal aortic blood in normoxic fish and fish acclimatized to hypoxia. a Significant difference between acclimatization groups. b Significant difference from normoxic values.
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4.1. Ventilation Previous studies on fishes suggested that acclimatization to hypoxia could affect ventilation. Lomholt and Johansen (1979) examined the effects of severe chronic hypoxia (30 Torr, 4 weeks) in carp (Cyprinus carpio) and report that the pattern of ventilation was altered by hypoxic acclimatization. Normoxic acclimated carp showed periodic ventilation but hypoxic acclimated fish ventilated continuously even during aquatic normoxia. However, despite continuous ventilation, the hypoxic carp had the same ventilation volume as normoxic fish during normoxia and reduced ventilation in hypoxia. These data argue against acclimatization to hypoxia increasing the ventilatory sensitivity to hypoxia in fish, however, other studies have shown that hypoxic acclimatization can stimulate ventilation. In flounder (Platichthys flesus) ventilatory frequency and amplitude were significantly greater in hypoxic-acclimated (30 Torr, 3 weeks) fish during both aquatic normoxia and hypoxia (Kerstens et al., 1979). Johnston et al. (1983) report increased ventilation in an airbreathing catfish, Clarias mossambicus, acclimatized to hypoxia (18 Torr, 27 days). These studies indicate that acclimatization to hypoxia increases ventilation. The effects of hypoxic acclimatization on airbreathing fish are variable. One study indicates that the sensitivity to oxygen may increase after hypoxic acclimatization. Synbranchus marmoratus, an air-breathing eel switches to aerial respiration at higher PwO2 levels after acclimatization to hypoxic water (20–25 Torr, 6 weeks) (Eduardo et al., 1979). Another study, however, on this species found that there was no change in air-breathing nor gill ventilation rate after hypoxic acclimatization (B 20 Torr, 10 weeks) (Graham and Baird, 1984). Acclimatization to hypoxic water (15– 40 Torr, 14–21 days) increases the volume of each air breath in Ancistrus chagresi (Graham, 1983) but does not change air-breathing thresholds for this species or Hypostomus plecostomus (Graham and Baird, 1982). It appears that acclimatization to aquatic hypoxia has little effect on ventilatory variables in air-breathing fish, however, it could be argued that these air-breathing fish were never truly hypoxic because they had access to air.
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The pattern of the ventilatory response to hypoxia suggests that effects of hypoxic acclimatization on ventilation in channel catfish is mediated by an increased sensitivity to oxygen as observed in mammals. Both peripheral (Bisgard, 2000) and central (Powell et al., 2000a) mechanisms have been proposed to contribute to VAH in mammals. Where this altered sensitivity occurs in channel catfish cannot be determined from these data. Channel catfish acclimatized to hypoxia show increased ventilatory sensitivity to hypoxia and the large increase in Pop and more modest increases in ventilatory rate is the typical pattern of response to hypoxia exhibited by most fishes (Kerstens et al., 1979; Johnston et al., 1983). Like the ventilatory response to acute hypoxia, hypoxic acclimatization significantly stimulated opercular pressure. Some mammals show similar pattern in their response to hypoxic acclimation. Increased ventilation in humans and rats acclimatized to hypoxia is due to primarily large changes in tidal volume and more modest increases in frequency (Forster et al., 1974; Aaron and Powell, 1993).
4.2. Cardio6ascular Hypoxic bradycardia in channel catfish, and most other fish studied, is mediated by externally oriented oxygen-sensitive chemoreceptors in the gills (Burleson and Smatresk, 1990; Sundin et al., 2000). These branchial receptors are homologous to mammalian carotid body oxygen-sensitive chemoreceptors (Burleson and Milsom, 1993). Since VAH in mammals is believed to be due to increased oxygen chemoreceptor sensitivity, it was predicted that fish acclimatized to aquatic hypoxia would have lower fH than normoxic controls or larger decreases in fH in response to acute hypoxia. Although both groups showed bradycardia in response to acute hypoxic exposure, hypoxic acclimatized fish had significantly higher fHs than normoxic controls at all levels of aquatic PO2. The reason for this difference is not clear. The percent decrease in fH in response to the two levels of acute hypoxia was the same in acclimatized fish and controls and the fH sensitivity to hypoxia in hypoxic acclimatized fish was the same as con-
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trols. These data suggest that acclimatization to hypoxia might not have changed the sensitivity of the oxygen-sensitive chemoreceptors that mediate hypoxic bradycardia. The mechanisms responsible for the difference in the ventilatory and cardiac effects of hypoxic acclimatization are unknown but several hypotheses are presented below. Chronic hypoxia has been shown to elevate circulating catecholamines in both humans (Antezana et al., 1994) and fish (Montpetit and Perry, 1998) and could explain the elevated fHs in hypoxia acclimatized fish. Although circulating catecholamines were not measured in this study, the moderate level of hypoxia used to acclimatize fish was specifically chosen in advance to avoid causing the release of catecholamines that occurs in response to severe hypoxia. While the chronotropic effects of increased circulating catecholamines could have elevated fH in the hypoxia acclimatized catfish, evidence of this should have been reflected in other physiological variables. For example, if the release of catecholamines into the blood was stimulated by hypoxic acclimatization in this study then there should have been significant differences in MAP, hematocrit and CaO2 between the acclimatization groups. Thus, it appears that the elevated fH observed in hypoxic acclimatized catfish in this study was probably not due to an increase in circulating catecholamines. Another potential explanation for these results is that chronic hypoxia may have affected autonomic nervous control of the heart. fH in fishes, like most other vertebrates, is under both sympathetic and parasympathetic control (Nilsson, 1983). Therefore, the effect of hypoxic acclimatization on fH in channel catfish could be due to either an increase in adrenergic tone or a decrease in cholinergic tone. Changes in cardiac autonomic control after acclimatization to hypoxia have been documented in mammals. Although resting fH in mammals is elevated by increased sympathetic activity in response to hypoxic acclimatization, the effects of exogenous catecholamines are obtunded (see Wagner, 2000 for review). Part of this response may be due to down-regulation of myocardial b-adrenergic receptors which has been shown to occur in rats acclimatized to hypoxia (Kacimi et al., 1992). There is evidence, however,
that a similar process may not occur in fish. Forty-eight h of aquatic hypoxia (60 Torr) does not change the number or affinity of b-adrenergic receptors on rainbow trout erythrocytes (Reid and Perry, 1995). Gamperl et al. (1998) show that after 6 h of hypoxia there is no change in myocardial b-adrenergic receptor density in rainbow trout. While these data suggest that down-regulation of adrenergic receptors may not occur in fish, it may be that acclimatization to hypoxia takes longer in fish, especially species such as trout at temperatures between 5 and 15 °C. The other possibility is that cholinergic control of the heart could have been modified. Hypoxic acclimatization down-regulates muscarinic receptors in rat hearts (Kacimi et al., 1993). The effects of chronic hypoxia on catecholamine release in rainbow trout show that chromaffin cells become more sensitive to cholinergic stimulation (Montpetit and Perry, 1998). Although the effects of the autonomic changes observed in these previous studies do not always predict what was observed in channel catfish, they illustrate the plasticity of autonomic mechanisms in response to chronic hypoxia and point the way for further experimentation.
4.3. Blood-gases In addition to cardioventilatory reflexes, chronic hypoxia can also stimulate a number of hematological changes that increase the bloodoxygen transport capabilities therefore have the potential to affect cardioventilatory reflex responses to hypoxia. Previous studies have shown that hypoxic acclimatization of fish for more than 1 week to severe hypoxia, usually below the species critical PO2, increases hemoglobin-oxygen affinity by reduced inorganic phosphate levels, b-adrenergic erythrocyte Na+/H+ exchange and respiratory alkalosis (see Nikinmaa, 1992 for review) and increases hematocrit. In the present study, acclimatization of channel catfish to moderate hypoxia, above the critical PO2, had only modest effects on blood-gas variables. Although CaO2 was significantly higher in normoxic acclimatized fish during normoxia, this difference disappeared when aquatic PO2 was reduced. Thus, it
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appears that the level of hypoxia used in this study did not stimulate the hematological modifications that are seen in fishes in response to severe hypoxia. Hypoxic acclimatized fish were better able to maintain CaO2 in response to acute hypoxia and had lower CaCO2, a respiratory alkalosis, probably as a result of augmented ventilation.
4.4. Summary In summary, the results of this study show that 7 days of acclimatization to moderate aquatic hypoxia causes an increase in ventilation and fH in channel catfish. The ventilatory effects of acclimatization to hypoxia indicate increased O2 sensitivity but the cardiac effects do not. The mechanisms underlying these responses are currently unknown but may be due to changes in the oxygen-sensitive chemoreceptors, central integration of their activity and/or changes in autonomic control of the heart.
Acknowledgements The authors thank Elham Ighani-Husseinabadi for technical assistance. This research was supported by the Ronald McNair Scholars Program, (P. Silva) the Louis Stokes Alliance for Minority Participation (E. Ighani-Husseinabadi) and the Department of Biology at the University of Texas at Arlington.
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McKim, J.M., Nichols, J.W., Lien, G.J., Bertelsen, S.L., 1994. Respiratory-cardiovascular physiology and chloroethane gill flux in the channel catfish, Ictalurus punctatus. J. Fish Biol. 44, 527 – 547. McKinsey, D.M., Chapman, L.J., 1998. Dissolved oxygen and fish distribution in a Florida spring. Env. Biol. Fish 53, 211 – 223. Montpetit, C.J., Perry, S.F., 1998. The effects of chronic hypoxia on the acute adrenergic stress response in the rainbow trout (Oncorhynchus mykiss). Physiol. Zool. 71, 377– 386. Nikinmaa, M., 1992. Membrane transport and control of hemoglobin-oxygen affinity in nucleated erythrocytes. Physiol. Rev. 72, 301 –321. Nilsson, S., 1983. Autonomic Nerve Function in the Vertebrates. Springer, New York. Powell, F.L., Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory response. Respir. Physiol. 112, 123 – 134. Powell, F.L., Huey, K.A., Dwinell, M.R., 2000a. Central nervous system mechanisms of ventilatory acclimatization to hypoxia. Respir. Physiol. 121, 223 –236.
Powell, F.L., Dwinell, M.R., Aaron, E.A., 2000b. Measuring ventilatory acclimatization to hypoxia: comparative aspects. Respir. Physiol. 122, 271 – 284. Rees, B.B., Sudradjat, F.A., Love, J.W., 2001. Acclimatization to hypoxia increases survival time of zebrafish, Danio rerio, during lethal hypoxia. J. Exp. Zool. 289, 266 – 272. Reid, S.G., Perry, S.F., 1995. The effects of hypoxia, in vivo, on red blood cell beta-adrenoceptors in the rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 14, 519 – 524. Sundin, L., Reid, S.G., Rantin, F.T., Milsom, W.K., 2000. Branchial receptors and cardiorespiratory reflexes in a neotropical fish, the tambaqui (Colossoma macropomum). J. Exp. Biol. 203, 1225 – 1239. Val, A.L., Silva, M.N.P., Almeida-Val, V.M.F., 1999. Hypoxia adaptation in fish of the Amazon: a never-ending task. S. Afr. J. Zool. 33, 107 – 114. Wagner, P.D., 2000. Reduced maximal cardiac output at altitude — mechanisms and significance. Respir. Physiol. 120, 1 – 11. Wannamaker, C.M., Rice, J.A., 2000. Effects of hypoxia on movements and behavior of selected estuarine organisms from the southeastern United States. J. Exp. Mar. Biol. Ecol. 249, 145 – 163.
Cardioventilatory effects of acclimatization to aquatic hypoxia in channel catfish Mark L. Burleson *, Anna L. Carlton, Philip E. Silva Department of Biology, Uni6ersity of Texas at Arlington, Box 19498, Arlington, TX 76019, USA Accepted 14 March 2002
Abstract The mechanisms responsible for altering cardioventilatory control in vertebrates in response to chronic hypoxia are not well understood but appear to be mediated through the oxygen-sensitive chemoreceptor pathway. Little is known about the effects of chronic hypoxia on cardioventilatory control in vertebrates other than mammals. The purpose of this study was to determine how cardioventilatory control and the pattern of response is altered in channel catfish (Ictalurus punctatus) by 1 week of moderate hypoxia. Fish were acclimatized for 7 days in either normoxia (PO2 150 Torr) or hypoxia (PO2 75 Torr). After acclimatization, cardioventilatory, blood-gas and acid/base variables were measured during normoxia (PO2 14891 Torr) then at two levels of acute (5 min) hypoxia, (PO2 72.69 1 and 50.4 90.4 Torr). Ventilation was significantly greater in hypoxic acclimatized fish as was the ventilatory sensitivity to hypoxia (D ventilation/DPO2). The increase in ventilation and hypoxic sensitivity was due to increases in opercular pressure amplitude, gill ventilation frequency did not change. Heart rate was greater in hypoxic acclimatized fish but decreased in both acclimatization groups in response to acute hypoxia. Heart rate sensitivity to hypoxia (D heart rate/DPO2) was not affected by hypoxic acclimatization. The ventilatory effects of hypoxic acclimatization can be explained by increased sensitivity to oxygen but the effects on heart rate cannot. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Acclimatization, hypoxia; Control of breathing, cardioventilatory response to hypoxia; Fish, channel catfish (Ictalurus punctatus); Hypoxia, cardioventilatory response; Pressure, blood
1. Introduction Chronic hypoxia elicits numerous physiological changes that help vertebrates maintain metabolism during prolonged periods of reduced O2 availability. One poorly understood effect of * Corresponding author. Tel.: +1-817-272-2426; fax: + 1817-272-2855 E-mail address: [email protected] (M.L. Burleson).
chronic hypoxia is augmentation of hypoxic reflexes. For example, it has been shown in mammals that chronic hypoxia significantly alters the pattern and intensity of the ventilatory response to hypoxia. These changes depend on the time-scale of the hypoxic exposure (see Powell et al., 1998 for review). Chronic hypoxia (hours to days, depending on the species studied) results in a timedependent increase in ventilation, relative to the initial increase, termed ventilatory acclimatization
1569-9048/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 9 - 9 0 4 8 ( 0 2 ) 0 0 0 1 9 - 8
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to hypoxia (VAH) (Powell et al., 1998). An important component of VAH is increased ventilatory sensitivity to hypoxia (Powell et al., 2000b). Most evidence indicates that VAH is an adaptive response to hypoxia mediated by changes in the peripheral oxygen-sensitive pathway (Bisgard, 2000) and increased central nervous system gain (Powell et al., 2000a). Despite recent advances in this area, the mechanisms responsible for VAH and other cardioventilatory effects of chronic hypoxia are not well understood. Little is known about the cardioventilatory effects of chronic hypoxia in vertebrates other than mammals and the evolutionary origin of VAH is unknown (Powell et al., 1998). Therefore, important insights into the physiological mechanisms stimulated by chronic hypoxia may be learned from experiments on water-breathing vertebrates. Hypoxia is a relatively rare environmental challenge for terrestrial vertebrates except for those comparatively few species and individuals that live at or ascend to high altitudes. Aquatic hypoxia, on the other hand, is a frequent and often chronic condition faced by fishes and other organisms that respire water (Val et al., 1999; Wannamaker and Rice, 2000). Chronic aquatic hypoxia occurs naturally and has been demonstrated to impact fish populations in environments such as swamps (Chapman et al., 1999) and springs (McKinsey and Chapman, 1998). Another relevant environmental factor is that the solubility difference of O2 and CO2 in water dictates that cardioventilatory control in water-breathers is dominated by O2 rather than CO2 as in air-breathers. Thus, the physiological effects of chronic hypoxia should be more critical to the survival of water-breathing vertebrates than air-breathers. In fact, acclimatization to hypoxia has been shown to increase the time that zebra fish survive lethal hypoxia levels (Rees et al., 2001). The purpose of this study was to examine how acclimatization to moderate hypoxia (75 Torr for 7 days) affects blood-gas and cardiovascular and ventilatory responses of channel catfish (Ictalurus punctatus, Rafinesque) to two levels of acute, hypoxia ( 75 and 50 Torr). Channel catfish offer a robust model in which to
study acclimatization to hypoxia because they have a comparatively weak ventilatory response to CO2, lack central chemoreceptors (Burleson and Smatresk, 2000) and routinely experience acute and chronic hypoxia in their natural environment.
2. Material and methods
2.1. Experimental animals Channel catfish (4769 23 g, n=17) were purchased from a local commercial supplier and transported to the University of Texas at Arlington. They were housed in large (1300 L) fiberglass holding tanks filled with filtered, dechlorinated tap water in a temperature controlled (20 °C) room on a 12/12-h light–dark cycle and fed commercial fish food. Fish were held for at least 1 week before experimentation.
2.2. Surgery Catfish were anesthetized in MS-222 (ethyl-maminobenzoate, 0.01% solution) dissolved in dechlorinated tap water. After transfer to a surgery table, the fish were artificially ventilated with oxygenated anesthetic solution. Dorsal aorta (DA) and opercular cannulae were implanted as described previously (Burleson and Smatresk, 1990). Recovery was accomplished by artificially ventilating the fish with dechlorinated tap water without anesthetic. Fish were allowed to recover overnight before beginning acclimatization.
2.3. Acclimatization Following surgery and recovery, the fish were kept without feeding for 6 days in glass aquaria filled with 40 L of dechlorinated tap water and maintained under the same light and temperature conditions as the holding tanks. Sections of PVC pipe, 40×15 cm2, were put into the aquaria to provide cover for the fish. The partial pressure of oxygen in the water (PwO2) for hypoxic acclimatized fish (n=8) was maintained at 50% air satu-
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ration ( 75 Torr) by bubbling a 50/50 mix of nitrogen and air (controlled by flow meters) through the aeration/filtration system in the aquaria. Plastic covers on the aquaria restricted gas exchange between the water and atmosphere. Normoxic acclimatized fish (n =9) were kept in normally aerated aquaria with an oxygen tension near air saturation (150 Torr). Oxygen saturation levels in the acclimatization aquaria were measured daily with an oxygen meter (YSI). On the sixth day after surgery, fish were transferred to a experimental chamber which was a darkened Plexiglass chamber equipped with a continuous flow of dechlorinated tap water (2 L/m) and allowed to adjust overnight. Oxygen tensions were controlled with a stripping column and a gas-mixing flowmeter (Cameron Instruments) which allowed rapid changes in PwO2. During this time, the water in the chamber was kept at the same partial pressure as in the acclimatization aquaria; PwO2 was monitored using an oxygen electrode and meter (Cameron Instruments).
2.4. Protocol On the seventh day of acclimatization the cannulae were led out a small hole in the experimental chamber and connected to pressure transducers. DA blood and opercular pressures were recorded from the DA and opercular cannulae, respectively. Outputs from the transducer amplifiers and O2 electrode were recorded with a computer data acquisition system (Windaq). Fish that were acclimatized to hypoxia were switched to normoxia (1489 1 Torr) and monitored until ventilation stabilized ( 30 min). Normoxic acclimatized fish were simply kept in normoxia. The fish were then exposed to two levels of acute (5 min) hypoxia and the responses recorded as hypoxic response 1 (72.69 1 Torr) and hypoxic response 2 (50.49 0.4 Torr). Blood and opercular pressures were recorded for the last 3-min during each level of oxygenation. A blood sample (0.4 ml) was removed from the DA cannula immediately after each recording period for analysis of blood-gas and acid/base variables and hematocrit. At the conclusion of the experiment the fish was euthanized by an overdose of the anesthetic.
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2.5. Blood-gas and pH measurements Total CO2 content (CaCO2) of the sample was determined using a Capni-Con (Cameron Instruments), while total O2 content (CaO2) of the blood was measured with an Oxycon (Cameron Instruments). Arterial oxygen tension (PaO2) and pH were measured with a Cameron Blood Gas Meter with associated electrodes. Hematocrit was calculated as percent packed red blood cell volume after centrifugation.
2.6. Cardio6ascular and 6entilatory measurements Gill ventilation frequency (fG, beats/min), opercular pressure amplitude (Pop, cmH2O), heart rate (fH, beats/min) and DA blood pressure were obtained from recordings of blood and opercular pressure. Mean arterial pressure (MAP) was calculated as the sum of systolic pressure and diastolic pressure divided by two. All cardioventilatory variables were averaged over the 3-min. period preceding the blood sample. The ventilatory sensitivity to hypoxia was quantified by the following procedure. First a ventilatory index (Vi) was calculated by multiplying fG and opercular pressure (Vi= fG× Pop). Since Pop is proportional to the volume of each breath, Vi is analogous to minute ventilation. Ventilatory sensitivity to hypoxia (Fig. 3) was calculated by dividing the change in Vi between normoxia and the two levels of hypoxia by the change in PwO2 between normoxia and the two levels of hypoxia (DVi/DPwO2). fH sensitivity to hypoxia (Fig. 5) was similarly calculated (DfH/ DPwO2).
2.7. Statistical analyses The effects of acclimatization and acute hypoxic exposure were analyzed with a 2-way ANOVA using a commercial software program (Statistica). Specific effects (i.e. the effects of hypoxia within one acclimatization group or between groups at specific hypoxia levels) were analyzed using the least significant difference test for planned post hoc comparisons or the Scheffe´ test for unplanned comparisons (PB0.05).
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3. Results Acclimatization of chronically cannulated fish in aquaria to either normoxia or hypoxia had no noticeable effect on the outward appearance, health or behavior of the fish. Hypoxic fish were breathing faster than normoxic fish but did not display any other behavioral responses to hypoxia such as aquatic surface respiration which is breathing the oxygenated surface film of water during aquatic hypoxia (Kramer and McClure, 1982).
3.1. Ventilatory and cardio6ascular 6ariables in normoxia: post-acclimatization compared to control During normoxia there was no statistically significant difference in either fG or Pop between acclimatization groups (Figs. 1 and 2). MAP varied little (19.891.7 – 22.7 9 2.1 cmH2O overall) and there was not a statistically significant difference in MAP between acclimatization groups at any PwO2 level. The only cardioventilatory variable that was significantly different between acclimatization groups during normoxia was fH which was significantly greater in hypoxic acclimatized fish (Fig. 4).
Fig. 1. fG responses of hypoxia acclimatized and normoxia acclimatized controls to two levels of acute aquatic hypoxia. fG increased significantly in both groups in response to hypoxia but there was no significant difference between the groups. Error bars are 1 SEM. (*) indicates significant differences between acclimatization groups.
Fig. 2. Pop responses of hypoxia acclimatized and normoxia acclimatized controls to two levels of acute aquatic hypoxia. Pop increased significantly in both groups in response to hypoxia. Pop was not significantly different between groups during normoxia but is significantly different at the two levels of acute hypoxia. (*) indicates significant differences between acclimatization groups.
3.2. Ventilatory and cardio6ascular 6ariables: responses to acute hypoxia Acclimatization to aquatic hypoxia significantly affected the ventilatory response to acute hypoxia (Figs. 1 and 2). Hypoxic acclimatized fish had increased ventilatory sensitivity to hypoxia. Although there were no differences in ventilatory variables during normoxia, differences arose when fish were acutely exposed to hypoxia. Hypoxia stimulated ventilation in both groups of fish (Figs. 1 and 2). The increase was due primarily to increased Pop (Fig. 2). fG increased significantly in both groups during acute hypoxia (Fig. 1) but the increase was not as large as seen for Pop. Acclimatization, however, did not affect fG and was not significantly different between acclimatization groups at any level of hypoxia (Fig. 1). Pop was significantly greater in hypoxic acclimatized fish during both levels of acute hypoxia. The percent change in Pop in response to acute hypoxia was greater in the hypoxic acclimatized fish. Normoxic fish increased Pop 66% in hypoxic response 1 and 154% in hypoxic response 2. Hypoxic fish increased Pop 91 and 165% respectively. Ventilatory sensitivity to hypoxia (Fig. 3) shows that fish acclimatized to hypoxia were significantly more sensitive to aquatic hypoxia than normoxic acclimatized fish.
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Fig. 3. Ventilatory sensitivity to hypoxia in hypoxia acclimatized and normoxia acclimatized fish. Asterisks indicate significant differences in ventilatory sensitivity between acclimatization groups. Ventilatory sensitivity to hypoxia was significantly greater in hypoxic acclimatized fish. (*) indicates significant differences between acclimatization groups.
MAP stayed remarkably stable during acute hypoxic exposures. There were no significant changes in either acclimatization group in response to acute hypoxia. There was a statistically significant effect of hypoxic acclimatization on fH (Fig. 4). fH was significantly higher in hypoxia acclimatized fish at all aquatic PO2 levels. There was a significant bradycardia in response to acute hypoxia in both acclimatization groups and although there was a
Fig. 4. fH responses of hypoxia acclimatized and normoxia acclimatized controls to two levels of acute aquatic hypoxia. fH was significantly elevated in hypoxic acclimatized fish. Acute aquatic hypoxia caused a significant bradycardia in both groups. (*) indicates significant differences between acclimatization groups.
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Fig. 5. fH sensitivity to hypoxia in hypoxia acclimatized and normoxia acclimatized fish. The fH sensitivity of the two acclimatization groups was not significantly different.
large difference in fH between the acclimatization groups, the percent change in fH in response to the two levels of acute hypoxia were similar with about a 20% drop in hypoxic response 1 and a 40% drop in hypoxic response 2. This similarity is confirmed when these data are expressed as sensitivity to hypoxia (Fig. 5) and show that acclimatization to hypoxia did not have a significant effect on the fH sensitivity to hypoxia.
3.3. Blood-gas 6ariables Blood-gas values measured in this study (Table 1) were similar to previously reported values for this species at similar PwO2 levels (Burleson and Smatresk, 1990; McKim et al., 1994; Burleson and Smatresk, 2000). PaO2 dropped predictably and significantly in both groups of fish in response to acute aquatic hypoxia. However, hypoxic acclimatization did not significantly affect PaO2 and these values are almost identical in each group (Table 1). Although CaO2 during normoxia was significantly higher in normoxic acclimatized fish, hypoxic acclimatized fish were better able to maintain CaO2 in response to acute hypoxia. CaO2 decreased significantly in normoxic acclimatized fish in response to acute hypoxia but did not in hypoxic acclimatized fish. CaO2 of normoxic acclimatized fish decreased 29% in hypoxic response 1 and 59% in hypoxic response 2. In contrast, CaO2 was better maintained in hypoxic acclimatized fish, dropping only 9 and 35%, respectively.
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Hematocrit increased significantly during hypoxia in both acclimatization groups but there was no difference between acclimatization groups (Table 1). Neither acute hypoxia nor hypoxic acclimatization had any effect on blood pH despite large differences in CaCO2 (Table 1). CaCO2 was significantly lower in hypoxic acclimatized fish and significantly decreased in both acclimatization groups in response to acute hypoxia. In normoxic acclimatized catfish CaCO2 decreased 14% in hypoxic response 1 and 27% in hypoxic response 2. Decreases in hypoxic acclimatized fish were 17 and 19%, respectively.
4. Discussion The results of this study show that acclimatization to moderate aquatic hypoxia (PwO2 75 Torr) for 1 week significantly alters the ventilatory response to acute hypoxia in channel catfish. fH is higher in fish acclimatized to hypoxia but the fH
sensitivity to hypoxia is no different from normoxic fish. While the ventilatory effects of hypoxic acclimatization may be due to increased O2 sensitivity the data indicate that the cardiac effects are probably not. The few studies that have previously examined the effects of chronic hypoxia in fishes have used severe hypoxia, usually below the species-specific critical PwO2 (the PwO2 below which O2 consumption begins to decline) and much longer acclimatization time periods. Severe hypoxia can elicit stress responses, for example, elevated blood catecholamine levels, that may have effects secondary to reflexes mediated by O2-sensitive chemoreceptors. The level of hypoxia used in this study, 75 Torr, is above critical PwO2, 65–70 Torr, for channel catfish (Burggren and Cameron, 1980) and the fact that MAP, hematocrit and CaO2 did not differ between the acclimatization groups suggests that this level of hypoxia was not severe enough to stimulate the release of catecholamines to the extent that they might affect cardioventilatory variables.
Table 1 Blood-gas values in response to acute hypoxia PwO2 (Torr)
Acclimatization group Normoxic
Hypoxic
148.39 0.9 (normoxia)
PaO2 (Torr) CaO2 (mmol/L) pH CaCO2 (mmol/L) Hct
64.5 95.1 0.56 90.09a 7.17 90.14 5.46 9 0.24 14.4 9 1.5
64.3 96.4 0.43 9 0.04 7.14 9 0.18 3.72 9 0.42a 15.1 91.4
72.6 91.0 (hypoxic response 1)
PaO2 CaO2 pH CaCO2
37.0 9 5.8b 0.40 9 0.07 7.42 9 0.13 4.67 90.27
35.1 9 2.2 0.39 90.04 7.05 9 0.08 3.09
Hct
15.7 9 1.9
90.30a,b 17.7 91.4
PaO2 CaO2 pH CaCO2 Hct
23.7 93.7 0.23 9 0.05 7.49 9 0.23 3.98 9 0.22 17.4 9 2.0
22.4 92.8 0.28 90.07 7.23 9 0.15 3.0 90.24a,b 18.1 91.0
50.49 0.4 (hypoxic response 2)
Values for dorsal aortic blood in normoxic fish and fish acclimatized to hypoxia. a Significant difference between acclimatization groups. b Significant difference from normoxic values.
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4.1. Ventilation Previous studies on fishes suggested that acclimatization to hypoxia could affect ventilation. Lomholt and Johansen (1979) examined the effects of severe chronic hypoxia (30 Torr, 4 weeks) in carp (Cyprinus carpio) and report that the pattern of ventilation was altered by hypoxic acclimatization. Normoxic acclimated carp showed periodic ventilation but hypoxic acclimated fish ventilated continuously even during aquatic normoxia. However, despite continuous ventilation, the hypoxic carp had the same ventilation volume as normoxic fish during normoxia and reduced ventilation in hypoxia. These data argue against acclimatization to hypoxia increasing the ventilatory sensitivity to hypoxia in fish, however, other studies have shown that hypoxic acclimatization can stimulate ventilation. In flounder (Platichthys flesus) ventilatory frequency and amplitude were significantly greater in hypoxic-acclimated (30 Torr, 3 weeks) fish during both aquatic normoxia and hypoxia (Kerstens et al., 1979). Johnston et al. (1983) report increased ventilation in an airbreathing catfish, Clarias mossambicus, acclimatized to hypoxia (18 Torr, 27 days). These studies indicate that acclimatization to hypoxia increases ventilation. The effects of hypoxic acclimatization on airbreathing fish are variable. One study indicates that the sensitivity to oxygen may increase after hypoxic acclimatization. Synbranchus marmoratus, an air-breathing eel switches to aerial respiration at higher PwO2 levels after acclimatization to hypoxic water (20–25 Torr, 6 weeks) (Eduardo et al., 1979). Another study, however, on this species found that there was no change in air-breathing nor gill ventilation rate after hypoxic acclimatization (B 20 Torr, 10 weeks) (Graham and Baird, 1984). Acclimatization to hypoxic water (15– 40 Torr, 14–21 days) increases the volume of each air breath in Ancistrus chagresi (Graham, 1983) but does not change air-breathing thresholds for this species or Hypostomus plecostomus (Graham and Baird, 1982). It appears that acclimatization to aquatic hypoxia has little effect on ventilatory variables in air-breathing fish, however, it could be argued that these air-breathing fish were never truly hypoxic because they had access to air.
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The pattern of the ventilatory response to hypoxia suggests that effects of hypoxic acclimatization on ventilation in channel catfish is mediated by an increased sensitivity to oxygen as observed in mammals. Both peripheral (Bisgard, 2000) and central (Powell et al., 2000a) mechanisms have been proposed to contribute to VAH in mammals. Where this altered sensitivity occurs in channel catfish cannot be determined from these data. Channel catfish acclimatized to hypoxia show increased ventilatory sensitivity to hypoxia and the large increase in Pop and more modest increases in ventilatory rate is the typical pattern of response to hypoxia exhibited by most fishes (Kerstens et al., 1979; Johnston et al., 1983). Like the ventilatory response to acute hypoxia, hypoxic acclimatization significantly stimulated opercular pressure. Some mammals show similar pattern in their response to hypoxic acclimation. Increased ventilation in humans and rats acclimatized to hypoxia is due to primarily large changes in tidal volume and more modest increases in frequency (Forster et al., 1974; Aaron and Powell, 1993).
4.2. Cardio6ascular Hypoxic bradycardia in channel catfish, and most other fish studied, is mediated by externally oriented oxygen-sensitive chemoreceptors in the gills (Burleson and Smatresk, 1990; Sundin et al., 2000). These branchial receptors are homologous to mammalian carotid body oxygen-sensitive chemoreceptors (Burleson and Milsom, 1993). Since VAH in mammals is believed to be due to increased oxygen chemoreceptor sensitivity, it was predicted that fish acclimatized to aquatic hypoxia would have lower fH than normoxic controls or larger decreases in fH in response to acute hypoxia. Although both groups showed bradycardia in response to acute hypoxic exposure, hypoxic acclimatized fish had significantly higher fHs than normoxic controls at all levels of aquatic PO2. The reason for this difference is not clear. The percent decrease in fH in response to the two levels of acute hypoxia was the same in acclimatized fish and controls and the fH sensitivity to hypoxia in hypoxic acclimatized fish was the same as con-
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trols. These data suggest that acclimatization to hypoxia might not have changed the sensitivity of the oxygen-sensitive chemoreceptors that mediate hypoxic bradycardia. The mechanisms responsible for the difference in the ventilatory and cardiac effects of hypoxic acclimatization are unknown but several hypotheses are presented below. Chronic hypoxia has been shown to elevate circulating catecholamines in both humans (Antezana et al., 1994) and fish (Montpetit and Perry, 1998) and could explain the elevated fHs in hypoxia acclimatized fish. Although circulating catecholamines were not measured in this study, the moderate level of hypoxia used to acclimatize fish was specifically chosen in advance to avoid causing the release of catecholamines that occurs in response to severe hypoxia. While the chronotropic effects of increased circulating catecholamines could have elevated fH in the hypoxia acclimatized catfish, evidence of this should have been reflected in other physiological variables. For example, if the release of catecholamines into the blood was stimulated by hypoxic acclimatization in this study then there should have been significant differences in MAP, hematocrit and CaO2 between the acclimatization groups. Thus, it appears that the elevated fH observed in hypoxic acclimatized catfish in this study was probably not due to an increase in circulating catecholamines. Another potential explanation for these results is that chronic hypoxia may have affected autonomic nervous control of the heart. fH in fishes, like most other vertebrates, is under both sympathetic and parasympathetic control (Nilsson, 1983). Therefore, the effect of hypoxic acclimatization on fH in channel catfish could be due to either an increase in adrenergic tone or a decrease in cholinergic tone. Changes in cardiac autonomic control after acclimatization to hypoxia have been documented in mammals. Although resting fH in mammals is elevated by increased sympathetic activity in response to hypoxic acclimatization, the effects of exogenous catecholamines are obtunded (see Wagner, 2000 for review). Part of this response may be due to down-regulation of myocardial b-adrenergic receptors which has been shown to occur in rats acclimatized to hypoxia (Kacimi et al., 1992). There is evidence, however,
that a similar process may not occur in fish. Forty-eight h of aquatic hypoxia (60 Torr) does not change the number or affinity of b-adrenergic receptors on rainbow trout erythrocytes (Reid and Perry, 1995). Gamperl et al. (1998) show that after 6 h of hypoxia there is no change in myocardial b-adrenergic receptor density in rainbow trout. While these data suggest that down-regulation of adrenergic receptors may not occur in fish, it may be that acclimatization to hypoxia takes longer in fish, especially species such as trout at temperatures between 5 and 15 °C. The other possibility is that cholinergic control of the heart could have been modified. Hypoxic acclimatization down-regulates muscarinic receptors in rat hearts (Kacimi et al., 1993). The effects of chronic hypoxia on catecholamine release in rainbow trout show that chromaffin cells become more sensitive to cholinergic stimulation (Montpetit and Perry, 1998). Although the effects of the autonomic changes observed in these previous studies do not always predict what was observed in channel catfish, they illustrate the plasticity of autonomic mechanisms in response to chronic hypoxia and point the way for further experimentation.
4.3. Blood-gases In addition to cardioventilatory reflexes, chronic hypoxia can also stimulate a number of hematological changes that increase the bloodoxygen transport capabilities therefore have the potential to affect cardioventilatory reflex responses to hypoxia. Previous studies have shown that hypoxic acclimatization of fish for more than 1 week to severe hypoxia, usually below the species critical PO2, increases hemoglobin-oxygen affinity by reduced inorganic phosphate levels, b-adrenergic erythrocyte Na+/H+ exchange and respiratory alkalosis (see Nikinmaa, 1992 for review) and increases hematocrit. In the present study, acclimatization of channel catfish to moderate hypoxia, above the critical PO2, had only modest effects on blood-gas variables. Although CaO2 was significantly higher in normoxic acclimatized fish during normoxia, this difference disappeared when aquatic PO2 was reduced. Thus, it
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appears that the level of hypoxia used in this study did not stimulate the hematological modifications that are seen in fishes in response to severe hypoxia. Hypoxic acclimatized fish were better able to maintain CaO2 in response to acute hypoxia and had lower CaCO2, a respiratory alkalosis, probably as a result of augmented ventilation.
4.4. Summary In summary, the results of this study show that 7 days of acclimatization to moderate aquatic hypoxia causes an increase in ventilation and fH in channel catfish. The ventilatory effects of acclimatization to hypoxia indicate increased O2 sensitivity but the cardiac effects do not. The mechanisms underlying these responses are currently unknown but may be due to changes in the oxygen-sensitive chemoreceptors, central integration of their activity and/or changes in autonomic control of the heart.
Acknowledgements The authors thank Elham Ighani-Husseinabadi for technical assistance. This research was supported by the Ronald McNair Scholars Program, (P. Silva) the Louis Stokes Alliance for Minority Participation (E. Ighani-Husseinabadi) and the Department of Biology at the University of Texas at Arlington.
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