Cardiorespiratory responses to hypercarbia in tambaqui Colossoma macropomum: chemoreceptor orientation and specificity
1 Department of Physiological Sciences, Federal University of São
Carlos, Via Washington Luiz km 235, São Carlos, SP, 13565-905,
Brazil
2 Department of Biology, University of Ottawa, 150 Louis Pasteur, Ottawa,
ON, K1N 6N5 Canada
3 Department of Zoology, University of British Columbia, Vancouver, BC, V6T
1Z4 Canada
4 Department of Life Sciences, University of Toronto at Scarborough,
Toronto, ON, M1C 1A4 Canada
* Author for correspondence (e-mail: Katie.Gilmour{at}science.uottawa.ca)
Accepted 3 January 2005
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Summary |
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Key words: tambaqui, Colossoma macropomum, hypercarbia, blood pressure, ventilation, blood flow, acetazolamide, CO2, pH
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Introduction |
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These cardiorespiratory responses to hypercarbia appear to be triggered by
peripheral chemoreceptors that are located primarily, although probably not
exclusively in at least some species (Reid
et al., 2000; Milsom et al.,
2002
), on the gills. Bilateral denervation of the gills was
sufficient to abolish most or all of the hypercarbia-induced changes in
ventilation and/or cardiovascular variables in spiny dogfish Squalus
acanthias (McKendry et al.,
2001
), channel catfish Ictalurus punctatus
(Burleson and Smatresk, 2000
),
traira Hoplias malabaricus (Reid
et al., 2000
) and tambaqui Colossoma macropomum
(Sundin et al., 2000
;
Milsom et al., 2002
;
Florindo et al., 2004
).
Similarly, bilateral extirpation of the first gill arch prevented or greatly
attenuated ventilatory and cardiovascular responses to hypercarbia in rainbow
trout Oncorhynchus mykiss, pointing to the first gill arch as the
chief location of chemoreceptors involved in initiating cardiorespiratory
responses to hypercarbia in this species
(Perry and Reid, 2002
).
The branchial chemoreceptors in rainbow trout, Atlantic salmon Salmo
salar and dogfish appear to respond primarily to changes in water
CO2 tension specifically; neither alteration of water pH nor
manipulation of blood PCO2 were effective in triggering
cardiorespiratory responses (Perry and
McKendry, 2001; Perry and
Reid, 2002
). Although denervation studies have identified the
gills as the principal location of the chemoreceptors that initiate
cardiorespiratory responses to hypercarbia in tambaqui
(Sundin et al., 2000
;
Milsom et al., 2002
;
Florindo et al., 2004
), the
orientation of these receptors (whether they preferentially detect water or
blood) as well as their sensitivity to CO2 vs
H+, remain uncertain. The tambaqui is a hypoxia-tolerant
(P50=2.4 mmHg; Brauner
et al., 2001
) and hypercarbia-tolerant neotropical fish species
that is found throughout the Amazon basin, often in floodplain lakes that are
subject to large variations in O2, CO2 and pH. Owing to
the frequent occurrence of hypercarbic conditions in its natural environment
(water total dissolved CO2 may range from 0.82 to 1.79 mmol
l-1 depending on season and depth;
Reid et al., 2000
), and an
anatomy that renders this species amenable to surgical sectioning of nerves
innervating selected chemosensory areas, the tambaqui has been the focus of a
concerted research effort to identify the locations and roles of
chemoreceptors involved in respiratory reflexes to both hypoxia and
hypercarbia (Sundin et al.,
2000
; Milsom et al.,
2002
; Reid et al.,
2003
; Florindo et al.,
2004
). Previous studies have focused primarily on reflex changes
in ventilation and heart rate during hypoxic or hypercarbic exposures without
monitoring blood gas or acid-base status. Blood gas and acid-base data were
reported by Wood et al.
(1998
), but in the context of
acid-base regulation in response to an acid challenge, and so without
cardiorespiratory data. Thus, the objective of the present study was to
characterize more fully blood gas and acid-base status, as well as the
cardiovascular responses to hypercarbia, while testing the hypothesis that
cardiorespiratory responses to hypercarbia in tambaqui are triggered by
externally oriented branchial chemoreceptors that react specifically to
changes in water CO2 tension.
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Materials and methods |
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Surgery was carried out on fish anaesthetized by immersion in an aerated
solution of benzocaine (ethyl-p-aminobenzoate; 100 mg l-1)
and then transferred to an operating table where the gills were irrigated
continuously with a more dilute anaesthetic solution (50 mg l-1).
For monitoring of blood gas and acid-base status using an extracorporeal
circulation (Thomas, 1994),
the caudal artery and caudal vein were cannulated
(Axelsson and Fritsche, 1994
);
in addition, the caudal artery cannula was used for measurements of blood
pressure (PDA) while the caudal vein cannula also served
for saline or drug administration. Following exposure of the haemal arch by
means of a lateral incision at the level of the caudal peduncle, flexible
polyethylene tubing (Clay-Adams PE50, Becton-Dickenson and Co., Sparks, MD,
USA) was inserted into the vessels in the anterior direction. Cannulae were
filled with heparinized (100 i.u. ml-1 ammonium heparin) modified
(4.5 mmol l-1 NaHCO3) Cortland saline
(Wolf, 1963
) and flushed
daily. To measure cardiac output, a 3S ultrasonic flow probe (Transonic
Systems, Ithaca, NY, USA) was placed around the ventral aorta, which was
accessed via an incision through the overlying epithelium within the
opercular chamber. The operculum was reflected forward, and a small (
1.5
cm) incision was made parallel to the ventral aorta in the epithelium near the
isthmus. Blunt dissection exposed the ventral aorta, and the flow probe was
then placed around the vessel using lubricating jelly (K-Y Personal Lubricant;
Johnson and Johnson, Montréal, Canada) as an acoustic couplant. With
this approach, disruption of the pericardium was avoided. Both incisions were
closed, and the cannulae and flow probe lead were secured to the skin, with
silk sutures. Ventilation was assessed by suturing brass plates (1
cm2) to the external surface of each operculum to measure
breath-by-breath displacement of the opercula with an impedance converter.
Finally, two holes were drilled through the snout between the nostrils using a
Dremel tool, and a flared cannula (PE 160) was fed through each hole and
secured in place with a cuff. These cannulae were used to deliver
CO2-equilibrated or acidified water into the flow of inspired
ventilatory water. After surgery, fish were revived and transferred to
individual holding boxes of opaque acrylic provided with flowing, aerated
water for at least 24 h of recovery before experimentation.
Experimental protocol
Experiments commenced with a 5 min `pre' period of recording baseline
ventilation and cardiovascular parameters. Tambaqui were then subjected to a
series of injections of CO2-equilibrated or acidified water (into
the inspired water stream), and CO2-equilibrated saline (into the
caudal vein), with 4 or 6 min intervals between injections. Water injections
(50 ml kg-1) were delivered over a 20 s period into a snout
cannula, and included aerated but otherwise untreated water (control) and
water pre-equilibrated with 1%, 3%, 5% or 10% CO2 in air.
Measurement of pH for water equilibrated to each of these CO2
levels revealed values of 6.3, 5.6, 5.3 and 4.9 pH units, respectively. To
differentiate between CO2-induced cardiorespiratory effects and
those triggered by the concomitant elevation of H+, each injection
of CO2-equilibrated water was followed by an injection of water
titrated with HCl to the corresponding pH value; vigorous aeration before and
after addition of HCl ensured removal of CO2. Saline (control), or
saline equilibrated with 5% or 10% CO2 in air, was delivered as a
bolus (2 ml kg-1 over 20 s) into the caudal vein. Because
CO2-enriched saline injections were without effect (see Results),
there was no further attempt to dissect the relative roles of CO2
vs H+.
Following the injection series, the extracorporeal blood circulation (see below) was initiated. Once the extracorporeal blood shunt was established and the measured variables had stabilized, baseline conditions for blood gases and acid-base status as well as ventilation and cardiovascular parameters under normoxic normocarbic conditions were recorded over a 5 min `pre' period. Blood pressure was monitored at regular (2-5 min) intervals by briefly (for 10-15 s) switching the caudal artery cannula from the extracorporeal blood loop to a pressure transducer using a T-junction and three-way valve. The water supplying the fish box was then rendered progressively hypercarbic by gassing a water equilibration column with 1%, 3%, and then 5% CO2 in air (Cameron flowmeter model GF-3/MP, Port Aransas, TX, USA) for 15 min at each level. Using this protocol, the final water PCO2 (PwCO2) values achieved at each step were 7.2±0.5, 15.5±0.9 and 26.3±2.5 mmHg, respectively (mean ± S.E.M., N=11). At the end of the final hypercarbic exposure, PwCO2 within the experimental chamber was rapidly returned to normocarbic conditions ('washed out') by increasing the flow of air-equilibrated water to the fish box.
Finally, the carbonic anhydrase inhibitor acetazolamide was used to investigate the cardiorespiratory effects of external vs internal hypercarbia. After the 5 min `pre' period of recording baseline conditions, acetazolamide (30 mg kg-1) was administered via the caudal vein cannula and blood acid-base and cardiorespiratory parameters were monitored for 30 min. Fish were then exposed to hypercarbia by gassing the water equilibration column with 3% CO2 in air (final PwCO2=13.6±0.5, N=7), and at the end of the 15 min hypercarbic exposure, CO2 was again rapidly washed out of the system by increasing the flow of air-equilibrated water to the fish box. Acetazolamide was prepared by dissolving the drug in saline with added NaOH and then slowly titrating the pH down to a level as close as possible to physiological (final pH of the acetazolamide solution was approximately 8.5).
Analytical techniques
An extracorporeal blood circulation
(Thomas, 1994) was used to
continuously monitor blood gas and acid-base variables. Blood was withdrawn at
a rate of 0.5 ml min-1 from the caudal artery cannula using a
peristaltic pump, and passed through an external circuit (of
1 ml volume)
containing PO2, PCO2 and pH electrodes
before being returned to the fish via the caudal vein cannula. To
prevent clotting, the circuit was rinsed with heparinised (540 i.u.
ml-1) saline for 10-15 min prior to initiating blood flow. Arterial
blood pH (pHa), PCO2 (PaCO2) and
PO2 (PaO2) were measured using Metrohm
(model 6.0204.100, Brinckman Instruments, Canada, Ltd., Mississuaga, ON,
Canada; pH) and Cameron Instruments (CO2, O2) electrodes
housed in thermostatted cuvettes and connected to a blood gas analyser (BGM
200; Cameron Instruments). A second peristaltic pump was used to withdraw
water at a rate of 3.5 ml min-1 from the mouth of the fish
via one of the two buccal cannulae. This water was passed through
thermostatted cuvettes containing pH (Metrohm model 6.0204.100) and
PCO2 (Cameron Instruments) electrodes connected to a blood
gas analyser (Cameron Instruments) for the measurement of water pH (pHw) and
PwCO2. Before each experiment, the pH electrodes were
calibrated by pumping precision buffer solutions through the circuits until
stable readings were recorded. A similar procedure was used to calibrate the
O2 and CO2 electrodes with a zero solution (2 g
l-1 sodium sulphite; O2 electrode only) and/or water
equilibrated with appropriate gas mixtures (supplied by a GF-3/MP gas mixing
flowmeter; Cameron Instruments).
Blood pressure was measured by connecting the caudal artery cannula to a
pressure transducer (Model 1050BP, UFI, Morro Bay, CA, USA) linked to an
amplifier (Biopac DA 100, Santa Barbara, CA, USA). The pressure transducer was
calibrated daily against a static column of water. Blood flow was determined
by attaching the factory-calibrated ultrasonic flow probe to a blood flowmeter
(model T106; Transonic Systems, Ithaca, NY, USA). The frequency and amplitude
of opercular displacements were monitored as indices of ventilation using an
impedance converter (model 911, Biocom Inc; Culver City, CA, USA) that
detected and quantified the changes in impedance between the brass plates
attached to the opercula (Peyraud and
Ferret-Bouin, 1960).
A data acquisition system (Biopac Systems) with Acknowledge data
acquisition software (sampling rate set at 40 Hz) and a personal computer were
used to convert all analogue signals (blood gases and pH, water
PCO2 and pH, blood pressure, blood flow and impedance
recordings) to digital data. With this system, continuous data recordings were
obtained for PaCO2, PaO2, pHa,
PwCO2, pHw, mass-specific blood flow
(b), heart rate
(fH; automatic rate calculation from the pulsatile
b (trace), mean
PDA (arithmetic mean), systemic vascular resistance
(Rs;
PDA/
b and
ventilation amplitude (VAMP; the difference between
maximum and minimum impedances). Note that PDA and
Rs were recorded only intermittently in experiments where
the extracorporeal blood shunt was utilised. In addition, cardiac stroke
volume (VS) was calculated by dividing mass-specific blood
flow by heart rate, and ventilation frequency (fV) was
determined from the impedance trace.
Statistical analyses
Data are reported as means ± 1
S.E.M. For experiments involving water or
saline injections, mean ventilatory and cardiovascular data were compiled for
10 s intervals over the 20 s before and 100 s after the injection, except
during the injection itself. For experiments employing the extracorporeal
blood circulation, mean blood gas, water gas, acid-base, ventilatory and
cardiovascular data were compiled over 2 min periods at selected intervals,
apart from PDA and Rs, for which data
were compiled only during the 10-15 s periods of recording. Data were analysed
for statistical significance by one-way repeated measures analysis of variance
(RM-ANOVA) followed by post hoc multiple comparisons using the
Holm-Sidak method, as appropriate. Where assumptions of normality or equal
variance were violated, equivalent non-parametric analyses were employed. The
commercial package SigmaStat v3.0 (SPSS Inc.) was used to carry out
statistical analyses, and the fiducial limit of significance in all cases was
5%.
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Results |
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Rapid removal of CO2 from the water resulted in the equally rapid return of ventilation (Fig. 1B,C), heart rate (Fig. 2E) and stroke volume (Fig. 2F) towards pre-exposure levels. Blood flow increased significantly during the rapid washout, by 16-23% (Fig. 2D), owing to the persistent elevation of cardiac stroke volume. The greater blood flow was accompanied by a significant lowering of systemic resistance (Fig. 2C). Although blood gas and acid-base variables also recovered during the lowering of water CO2 (Fig. 1D-F), these changes were slower to occur, such that even 20 min after initiation of the rapid washout, PaCO2 remained significantly (Wilcoxon signed rank test, P=0.008) elevated over the pre-exposure value by 6.62±2.20 mmHg (N=8), while pHa was depressed by 0.18±0.04 units (N=8). Notably, neither ventilation amplitude (Wilcoxon signed rank test, P=0.469) nor frequency (paired Student's t-test, P=0.094) was significantly different from the baseline value at this time, suggesting that ventilation, at least, was more sensitive to changes in PwCO2 than to those in PaCO2.
Acetazolamide treatment
This observation was confirmed by administration of the carbonic anhydrase
inhibitor acetazolamide, a treatment that generated a significant respiratory
acidosis (Fig. 3D,F) in the
absence of any change in PwCO2
(Fig. 3A). A single bolus
injection of acetazolamide caused PaCO2 to increase
approximately threefold over the subsequent 30 min, at the same time lowering
pHa by 0.26±0.02 units (N=8), yet was without significant
effect on ventilation (Fig.
3B,C) or most cardiovascular variables
(Fig. 4); systemic resistance
(Fig. 4C) and cardiac stroke
volume (Fig. 4F) did differ
statistically from the pre-injection value at single points in each case. By
contrast, exposure of acetazolamide-treated fish to 15 min of hypercarbia
(first broken line), in which PwCO2 reached
13.6±0.5 mmHg, produced changes in ventilation and cardiovascular
variables that were virtually identical to those observed in untreated fish
exposed to a similar level of hypercarbia, even though the respiratory
acidosis was more profound in treated fish; VAMP and
fV rose by 110% and 24%, respectively, and heart rate fell
by 20% (Fig. 4E), but blood
flow was maintained (Fig. 4D)
owing to the concomitant 19% increase in stroke volume. A small and transient
increase in blood pressure also occurred
(Fig. 4B); this response was
not observed in untreated fish.
|
|
The recovery of ventilation and cardiovascular variables in response to the rapid lowering of PwCO2 (second broken line) was particularly striking in acetazolamide-treated fish because it occurred on the backdrop of a sustained, severe, respiratory acidosis (Figs 3 and 4). The significant decreases in ventilation variables (Fig. 3B,C) and stroke volume (Fig. 4F) as well as the rise in heart rate (Fig. 4E) tracked changes in PwCO2 rather than PaCO2 (Fig. 4A). The lack of correspondence between VAMP and PaCO2 is illustrated by a representative data recording for an individual fish (Fig. 5). As in untreated fish, blood flow increased significantly with the onset of rapid water CO2 washout (Fig. 4D), but the extent of the blood flow increase (7-13%) was smaller in acetazolamide-treated tambaqui and was not accompanied by a significant decrease in systemic resistance (Fig. 4C).
|
Injections of CO2-enriched saline
As a final test of the potential for changes in blood CO2
tension to elicit cardiorespiratory responses, tambaqui were injected with
saline equilibrated with 5% or 10% CO2 in air. Assuming complete
mixing of the saline bolus (2 ml kg-1 delivered over 20 s) with
venous blood, estimating venous PCO2 to be approximately 4
mmHg (based on the measured PaCO2 of 3mmHg), and
using the cardiac output measured under resting conditions (21 ml
min-1 kg-1), these internal injections of 5% or 10%
CO2-equilibrated saline would be expected to yield transient
PCO2 values of 11.5 or 20mmHg, respectively, in the blood
at the gill. Increases in blood pressure (7-11%), blood flow (20-24%) and
systemic resistance (6-11%) occurred in response to these injections but were
attributable to volume loading, since similar increases (9%, 31% and 4%,
respectively) were also observed upon injection of air-equilibrated saline
(Table 1). No specific effect
of internally injected CO2 was detected for any measured variable
(Table 1).
|
Injections of CO2-enriched or acidified water
The effects of injecting CO2-enriched water into the flow of
ventilatory water were compared with reactions to the injection of acidified
water to distinguish between the roles of CO2 and H+ in
eliciting cardiorespiratory responses to hypercarbia. A marked,
PCO2-dependent bradycardia accompanied the injection of
CO2-enriched water into the buccal cavity of tambaqui
(Fig. 6A-D). At the peak of the
response (20-30 s after beginning the injection), fH
was decreased by 20-49% for injections of water equilibrated with 1-10%
CO2. Because cardiac stroke volume was maintained or increased only
slightly (data not shown), the bradycardia resulted in significant 14-45%
reductions in cardiac output with injection of all levels of
CO2-enriched water (Table
2). The fish also exhibited significant
PCO2-dependent increases in systemic resistance
(Fig. 6E-H), which were
reflected in significant increases in blood pressure
(Table 2). The different
responses of systemic resistance to bolus injections of
CO2-enriched water (Fig.
6E-H) vs exposure to hypercarbic water (Figs
2C,
4C) were striking. By contrast
with the effect of CO2, injection of acidified water was generally
without significant effect, apart from a small (11%) and transient depression
of heart rate that occurred only in response to injection of the most acidic
(pH 4.9) water (Fig. 6C), and a
correspondingly slight 5% depression of cardiac output
(Table 2). Ventilatory
responses to CO2-enriched water injections were more sporadic;
VAMP increased significantly only with injection of 10%
CO2, while significant frequency responses were limited to
injections of 5% and 10% CO2
(Table 2). Responses to the
parallel injections of acidified water were either insignificant or of
substantially lower magnitude (Table
2). Injection of air-equilibrated water was without significant
effect in all cases (data not shown).
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Discussion |
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The cardiorespiratory responses elicited by hypercarbia may be initiated by
branchial chemoreceptors that detect changes in water CO2/pH,
and/or by receptors that monitor blood CO2/pH levels, because
exposure to elevated ambient CO2 causes a rise in blood
PCO2 and a concomitant fall in blood pH
(Fig. 1). Thus, three
experimental approaches were employed to discern between external and internal
orientation of the branchial CO2/pH receptors. First, tambaqui were
treated with acetazolamide to inhibit red blood cell carbonic anhydrase
activity. Assuming that CO2 excretion in tambaqui follows the
pathway mapped out for teleost fish in general (e.g.
Perry, 1986;
Tufts and Perry, 1998
),
carbonic anhydrase will catalyze the interconversion of CO2 and
HCO3- within the red blood cell, a step that is critical
to the transfer of CO2 from tissues to blood, and from blood to
ventilatory water. In acetazolamide-treated fish, this step would be slowed to
the uncatalyzed rate, thereby causing CO2 retention
(Henry and Heming, 1998
). As
expected, treatment of tambaqui with acetazolamide evoked the classic response
(e.g. Hoffert and Fromm, 1973
)
of a profound respiratory acidosis, in which PaCO2
approximately tripled in 30 min while pHa fell by 0.26 units
(Fig. 3D,F). Yet despite this
marked internal hypercapnia, the typical cardiorespiratory responses to
CO2/pH of hyperventilation and bradycardia (Figs
1B,C,
2E) were not observed until the
acetazolamide-treated tambaqui were exposed to external hypercarbia (Figs
2B,C,
3E). These findings argue
strongly in favour of branchial CO2/pH chemoreceptors with a solely
external orientation. Observations from the two additional experimental
approaches were in agreement with this conclusion, allaying concerns about any
non-specific side effects of the drug treatment. Chemoreceptor impairment, in
particular, was considered a possibility because carbonic anhydrase plays a
role in CO2 chemoreception in both invertebrates and vertebrates
(e.g. Iturriaga et al., 1991
;
Swenson and Hughes, 1993
;
Erlichman et al., 1994
;
Coates et al., 1998
; see also
review by Iturriaga, 1993
),
although the very similar responses of acetazolamide-treated and untreated
tambaqui to PwCO2 values of 14-15 mmHg rendered this
prospect unlikely.
Externally oriented branchial CO2/pH chemoreceptors would account for the close correspondence between cardiorespiratory adjustments and changes in water PCO2 during the rapid washout of CO2 from the water following hypercarbic exposures, as well as the independence of these responses from arterial PCO2. The CO2 electrode response time was likely faster for water than for blood measurements, owing to the different viscosities of these liquids. However, any concern that the apparent tracking of cardiorespiratory responses to water rather than blood PCO2 simply reflected different response times was alleviated by the particularly marked differences in the time courses of PwCO2 and cardiorespiratory variable changes during washout in acetazolamide-treated tambaqui (Fig. 5).
The observation that injection of CO2-laden water into the
buccal cavity triggered cardiorespiratory reactions
(Fig. 6,
Table 2) that were not detected
in response to the injection of a bolus of CO2-enriched saline into
the caudal vein (Table 1) was
also consistent with an external orientation for the branchial
CO2/pH chemoreceptors. In the latter experiment, it was assumed
that externally oriented receptors would be preferentially stimulated by
injecting CO2-enriched water into the mouth, whereas
CO2-enriched saline injections would preferentially stimulate
internally oriented receptors. Although exclusive stimulation of internally or
externally oriented receptors is likely to be impossible with this approach
owing to the potential for CO2 diffusion across the gill
epithelium, the extent of activation of blood-oriented receptors by external
(water) relative to internal (saline) injections was probably trivial.
Similarly, the absence of response to internal injection suggested that
water-oriented receptors were activated to a trivial extent by
CO2-enriched saline. Alternative explanations for the lack of
response to CO2-enriched saline injection are that the
PCO2 increase achieved by the injection of
CO2-equilibrated saline was insufficient (in length or magnitude)
to trigger internally oriented CO2 receptors, or that the
CO2 was converted to HCO3- and/or excreted
during transit through the circulation and gills. These possibilities cannot
be ruled out, but the most parsimonious explanation of the data in the context
of the results for the two other experimental approaches is that the
cardiorespiratory responses to hypercarbia in tambaqui are linked to the
activation of branchial CO2/pH chemoreceptors that are oriented
only towards the external (water) milieu. In dogfish
(Perry and McKendry, 2001),
Atlantic salmon (Perry and McKendry,
2001
) and rainbow trout
(McKendry and Perry, 2001
;
Perry and Reid, 2002
),
externally oriented branchial CO2/pH chemoreceptors were also
deduced from data generated using experimental approaches similar to those of
the present study. The existence of internally oriented receptors that detect
changes in blood PCO2 and/or pH was suggested by earlier
studies in which indirect correlative relationships between ventilation and
blood PCO2 or acid-base status were constructed (e.g.
Heisler et al., 1988
;
Graham et al., 1990
;
Wood and Munger, 1994
; see
also review by Gilmour, 2001
).
Increasingly, however, the weight of evidence from experiments designed to
distinguish directly between internal and external stimuli suggests that
reflex cardiorespiratory responses to hypercarbia are mediated by externally
oriented chemoreceptors. In this regard, the situation for CO2/pH
sensing differs from that for O2, in that a population of
internally oriented O2 chemoreceptors exists; these receptors are
distributed over all gill arches and linked specifically to ventilatory
reflexes (Burleson et al.,
1992
; Burleson,
1995
).
The present study also included an experiment to discern between the
specific effects of CO2 vs H+ in the initiation
of cardiorespiratory responses to hypercarbia; namely, a comparison of
responses to the injection of CO2-enriched water into the buccal
cavity with those elicited by CO2-free water acidified to the pH of
the corresponding CO2-laden water injection. The results clearly
demonstrated that CO2 itself was the key factor controlling
cardiorespiratory function (Fig.
6, Table 2).
Although the injection of acidified water was accompanied by significant
changes in heart rate, blood flow, ventilation amplitude and ventilation
frequency, these effects were in general restricted to injection of the most
acidic water (pH 4.9) and were in all cases of much smaller magnitude (11%,
5%, 15% and 16%, respectively) than those produced by injection of the
corresponding CO2-laden water (49%, 45%, 44% and 34%,
respectively). The data imply that minor cardiorespiratory reactions may occur
with strongly acidic stimuli, but that the chemoreceptors respond
predominantly to CO2 rather than to protons. The small magnitude of
the effects, coupled with the need for intense acidic stimuli, probably
accounted for the absence of response to acid injection in a previous study on
tambaqui (Sundin et al.,
2000). Acid injections into the inspired water were also found to
be without significant effect in traira
(Reid et al., 2000
) and
dogfish (Perry and McKendry,
2001
), while the slight impact of acid injection on ventilation
amplitude in Atlantic salmon was attributed to CO2 formed when
HCO3- ions in seawater were titrated by the added
H+ (Perry and McKendry,
2001
). Earlier studies similarly reported that environmental
acidification in the absence of elevated PwCO2 had little
effect on ventilation in rainbow trout
(Janssen and Randall, 1975
;
Neville, 1979
;
Thomas and Le Ruz, 1982
) and
taken as a whole, these data suggest that the cardiorespiratory responses to
hypercarbia are mediated by externally oriented branchial chemoreceptors that
respond specifically to CO2. Nevertheless, protons produced by the
hydration of CO2 that diffuses into the cell probably play a role
in signal transduction within chemoreceptor cells in fish, as in mammals (e.g.
Iturriaga et al., 1991
,
1993
;
Gonzalez et al., 1994
).
In addition to contributing information on receptor orientation and
stimulus modality to the existing data on chemoreceptor localization in
tambaqui, the present study more fully characterizes the cardiovascular
reflexes of tambaqui to hypercarbia, as previous work focused on changes in
heart rate and ventilation (Sundin et al.,
2000; Milsom et al.,
2002
; Florindo et al.,
2004
). In the presence of ambient CO2 tensions elevated
to at least 7 mmHg (lower CO2 tensions were not tested), tambaqui
consistently exhibited bradycardia, greater cardiac stroke volume and
hyperventilation, marked by increases of both frequency and amplitude.
Arterial blood pressure rose in some cases, presumably in response to
increased systemic resistance, and despite simultaneous reductions in cardiac
output. This pattern of cardiorespiratory reflexes to hypercarbia emphasises
equally the relatively conserved nature of some responses and the highly
variable nature of others. For example, hyperventilation is a common response
to hypercarbia, both among the studies on tambaqui
(Sundin et al., 2000
;
Milsom et al., 2002
;
Florindo et al., 2004
) and
within fish in general (see review by
Gilmour, 2001
). In some
species, including tambaqui (this study;
Florindo et al., 2004
) and the
elasmobranchs examined to date (Randall et
al., 1976
; Graham et al.,
1990
; Perry and Gilmour,
1996
; McKendry et al.,
2001
; Perry and McKendry,
2001
), increases in ventilation amplitude are more important
contributors to the hyperventilatory response than are frequency adjustments,
but overall there is a high degree of interspecific variation in the relative
importance of frequency vs amplitude changes
(Gilmour, 2001
). Bradycardia
also is a common response to hypercarbia (see review by
Perry and Gilmour, 2002
), yet
while tambaqui responded to higher CO2 tensions (5%) in all studies
with bradycardia (Sundin et al.,
2000
; Milsom et al.,
2002
; Florindo et al.,
2004
), conflicting results were obtained using lower levels of
CO2. For example, Florindo et al.
(2004
) observed increases in
heart rate at 1-2.5% CO2, in contrast to the bradycardia observed
in the present study (Fig. 2E)
and the lack of heart rate response observed by Sundin et al.
(2000
) at similar
CO2 tensions. It is possible that this discrepancy reflects
differences in the length of hypercarbic exposure, as the first measurement
time utilized by Florindo et al.
(2004
) was 60 min, while the
hypercarbic period in the present study was limited to 15 min.
Although hyperventilation and bradycardia are common responses to
hypercarbia, they are by no means universal. White sturgeon Acipenser
transmontanus, for example, responded to hypercarbia with a tachycardia
(Crocker et al., 2000), while a
number of species, including channel catfish
(Burleson and Smatresk, 2000
)
and brown bullhead (see table 2 in
Gilmour, 2001
; table 1 in
Perry and Gilmour, 2002
) were
resistant to hypercarbia, either failing to change heart rate or ventilation,
or exhibiting very attenuated responses. To some extent, this variation may
reflect species differences in sensitivity to CO2. For example,
neither the European eel Anguilla anguilla nor the closely related
American eel Anguilla rostrata responded to CO2 tensions
of 5-6mmHg (McKenzie et al.,
2002
; see table 2 in Gilmour,
2001
; table 1 in Perry and
Gilmour, 2002
), but adjustments of both heart rate and ventilation
were exhibited by European eels exposed to PwCO2 values of
10-80 mmHg (McKenzie et al.,
2002
). Similarly, it was necessary to raise water
PCO2 to 14 mmHg before hyperventilatory responses appeared
in carp Cyprinus carpio (Soncini
and Glass, 2000
), and to
38 mmHg to observe significant
hypercarbic responses in traira (Reid et
al., 2000
). Like eel, carp and traira, the results of earlier
studies indicated that tambaqui are relatively insensitive to changes in water
CO2 tension (Sundin et al.,
2000
). The results of the present study and that of Florindo et
al. (2004
), however, indicated
that tambaqui are more sensitive to CO2 than originally thought.
Variation in the methods used to expose fish to hypercarbia may account for
this difference.
The remaining cardiovascular adjustments to hypercarbia, including blood
pressure, systemic resistance, cardiac output and stroke volume, exhibit a
high degree of interspecific variation among fish, encompassing essentially
all possible response patterns (see table 2 in
Perry and Gilmour, 2002). The
increases in arterial blood pressure and systemic resistance, coupled with
constant or slightly reduced cardiac output observed for tambaqui under some
conditions in the present study (Fig.
6, Table 2), were
reminiscent of the responses of the salmonid fish that have been examined to
date (Perry et al., 1999
;
McKendry and Perry, 2001
;
Perry and McKendry, 2001
). The
appearance of these responses, however, was dependent upon the method of
CO2 delivery (injection of a bolus of CO2-enriched water
into the mouth vs exposure to hypercarbic water), perhaps because of
preferential stimulation of different receptor populations. In addition,
Milsom et al. (2002
) reported
the existence in tambaqui of receptors sensitive to CO2 that had an
inhibitory influence on ventilation frequency. The chemoreceptor control of
cardiorespiratory function during hypercarbia in fish is clearly complex,
likely involving multiple receptor populations in a variety of locations
(although with a branchial concentration) that are linked to different
cardiorespiratory parameters with positive and/or negative influences.
Resolving this complexity is an ongoing challenge, particularly because the
patterns of chemoreceptor control as well as the cardiorespiratory responses
to hypercarbia vary among fish species for reasons that remain elusive.
Despite the variability, CO2 has emerged as an important modulator
of cardiorespiratory function in fish, with responses mediated, in all species
that have been examined to date, by receptors that are oriented towards the
external environment with sensitivity specifically to CO2.
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