Cardiovascular responses to hypoxia and anaemia in the toad Bufo marinus
1 Department of Zoophysiology, Institute of Biological Sciences, University
of Aarhus, Denmark
2 Department of Biological Sciences, California State University, Hayward,
CA 94542, USA
* Author for correspondence (e-mail: johnnie.andersen{at}biology.au.dk)
Accepted 28 November 2002
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Summary |
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Key words: amphibian, Bufo, cardiovascular, respiratory, anaemia, hypoxia, cardiac shunt
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Introduction |
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The specific oxygen stimulus that triggers the cardiorespiratory response
to hypoxia remains controversial. At issue is the degree to which arterial
oxygen tension [physically dissolved oxygen in arterial blood
(PaO2)], haemoglobin oxygen saturation [the
percentage of haemoglobin molecules with bound oxygen (HbO2sat)]
and/or oxygen concentration {the sum of dissolved and haemoglobin bound
oxygen, ([O2])} represent the primary signal for stimulating these
homeostatic adjustments. For example, several studies on reptiles have
suggested that HbO2sat is the primary stimulus for the ventilatory
response to hypoxia because ventilation is well-correlated with arterial
HbO2sat (Glass et al.,
1983; Dupré et al.,
1989
). Other studies on amphibians have noted cardiovascular
changes in response to anaemia, by either anaemia or carbon monoxide exposure,
without associated changes in ventilation
(Wang et al., 1994
;
Branco and Glass, 1995
). This
suggests that different oxygen signals partial pressure and
concentration may exist for cardiovascular and ventilatory adjustments
to hypoxia and anaemia.
In the present study, we investigate the cardiovascular response to reductions in both arterial [O2] and PaO2 by measuring blood flows and arterial blood gases during exposure to hypoxia (progressively reducing inspired oxygen from 0.21 to 0.05) before and after inducing anaemia that reduced haematocrit by approximately 50%.
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Materials and methods |
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Surgical procedures
Toads were anaesthetised by immersion into a 1.0 g l-1
benzocaine solution (ethyl p-amino benzoate, Sigma E 1501), and
surgery started when the corneal reflex disappeared. The lungs were deflated
by opening the glottis using a small piece of plastic tubing, and the animal
was placed on a surgical table and covered with wet paper towels. Toads were
artificially ventilated with air every 15 min through a small piece of soft
rubber tubing inserted through the glottis. A left lateral incision
(approximately 2-3 cm) was made in the body wall ventral to the parotoid
gland. From this incision, the left systemic and pulmocutaneous arteries were
exposed by blunt dissection of connective tissue between the abdominal and
forelimb muscles (see Hedrick et al.,
1999). Transonic 2S blood flow probes (Transonic Systems Inc.,
Ithaca, NY, USA) were placed around the left systemic and left pulmocutaneous
arteries, and the space between probe and artery was filled with an acoustic
coupling gel (Berner Lab, Helsinki, Finland). The leads from the flow probes
were threaded back through the incision and tied to the skin at several
positions on the dorsal surface of the toad to ensure that the probes remained
in position. The right femoral artery was occlusively cannulated through an
incision in the hind leg to sample arterial blood and to measure systemic
arterial blood pressure. The catheter was secured to the dorsal surface of the
animal by silk sutures.
Surgery normally lasted less than 60 min and all toads regained normal
righting reflexes within 45 min of being placed under running tapwater. All
toads were treated with enrofloxacin (Baytril Bayer AG, Leverkusen, Germany; 2
mg kg-1, intramuscular) to prevent infections. When the toads had
regained normal reflexes, each individual animal was transferred to an
experimental chamber (40 cmx30 cmx20 cm) containing wet paper
towels and a dry area. These experimental chambers were maintained within a
climatic chamber at a constant temperature of 25°C, which is close to the
preferred body temperature (24°C) of Bufo marinus in the
laboratory (Johnson, 1972).
Toads were visually and audibly shielded from disturbances during measurements
and withdrawal of arterial blood.
Experimental protocol
Experiments began 24-48 h after surgery; arterial blood gases and
acidbase parameters of Bufo marinus stabilise within 24 h of
surgery (Andersen and Wang,
2002). The blood flow probe leads and catheter were connected to
blood flow and pressure measuring equipment located outside the experimental
chamber housing the toad, and haemodynamic variables were allowed to stabilise
for 30-90 min. Thereafter, initial cardiovascular measurements were made, a
control (normoxic) blood sample was withdrawn, and the toads were exposed to
progressive hypoxia [fraction of oxygen in the inspired air
(FIO2)=0.15, 0.10, 0.07 and 0.05]. The hypoxic
gas mixtures were prepared by mixing pure N2 and air with a Dameca
gas mixer, and the PO2 of the mixed gases
entering and leaving the chamber was monitored by a gas analyser (model 602;
Criticare Systems, Inc., Waukesha, WI, USA). The incoming gas mixtures passed
through a water column placed inside the climatic chamber to ensure the
appropriate temperature and high humidity in the experimental chamber. Each
gas level was maintained for 30 min, and a blood sample was removed for
analysis at the end of each level. Following the exposure to hypoxia, toads
were bled to reduce [O2] by approximately 50%. Plasma was reinfused
with toad Ringer (composition given by
Prosser, 1970
) in order to
maintain blood volume. After approximately 24 h, the hypoxic protocol above
was repeated with the anaemic animals.
A second group of control animals (N=6) were exposed to hypoxia on successive days without anaemia exposure. These animals were equipped with flow probes to measure haemodynamic variables as described above, but were not equipped with an arterial catheter, so measurements of blood gases were not performed.
Measurements of blood flows and blood pressures and calculations of
cardiac shunt patterns
The blood flow probes were connected to a dual-channel flow meter (model
T201; Transonic Systems Inc., Ithaca, NY, USA). The femoral catheter was
connected to a disposable pressure transducer (model PX600; Baxter Edward,
Irvine, CA, USA), and the signal was amplified using an in-house-built
preamplifier. The transducer was calibrated daily against a static water
column. Signals from the blood pressure transducer and the blood flow meter
were collected digitally using an AcqKnowledge MP 100 data-acquisition system
(version 3.2.3; BioPac Systems, Inc., Santa Barbara, CA, USA) at 50 Hz.
The left and right side of the truncus arteriosus and the pulmocutaneous
arteries in Bufo marinus are of similar diameter, and blood flows are
similar when probes are placed in ipsilateral or bilateral positions
(West and Burggren, 1984).
Therefore, we assumed that flows were bilaterally equal. Total blood flow in
the pulmocutaneous (
pc) and
systemic (
sys) arteries was
obtained by doubling measured values in the left pulmocutaneous artery and
left systemic artery, and total blood flow
(
tot) was calculated as
pc+
sys.
Heart rate (fH) was calculated directly from the blood flow trace,
and stroke volume (Vs) was calculated as
tot/fH. The net
shunt flow (net
shunt) was
calculated as
pc
sys,
and the cardiac shunt pattern was also expressed as
pc/
sys.
Blood gas analysis
Arterial blood was analysed for oxygen tension
(PaO2), pH, haematocrit, blood haemoglobin
concentration ([Hb4]), oxygen content ([O2]) and total
carbon dioxide content of plasma ([CO2]).
PO2 and pH were measured with Radiometer
(Copenhagen, Denmark) electrodes maintained in a BMS 3 electrode set-up at
25°C, and the output from the electrodes was displayed on a Radiometer PHM
73. Haematocrit was determined in duplicate as the fractional red cell volume
after centrifugation (12 000 r.p.m. for 3 min), and [Hb4] was
measured in triplicate after conversion to cyanmethaemoglobin and applying a
millimolar extinction coefficient of 10.99 at 540 nm
(Zijlstra et al., 1983).
Arterial [O2] was measured as described by Tucker
(1967
), with a correction
described by Bridges et al.
(1979
), and plasma
[CO2] was measured according to Cameron
(1971
). The Tucker and Cameron
chambers were maintained at 40°C. Haemoglobin-bound oxygen
(HbO2) was calculated as arterial
[O2](
O2xPaO2),
where
O2 is the blood oxygen solubility
(Christoforides and Hedley-Whyte,
1969
). Haemoglobin saturation (HbO2sat) was calculated
as HbO2/[Hb], under the assumption that all haemoglobin was
functional.
Arterial carbon dioxide tension (PaCO2) was
calculated from pH and plasma [CO2] using the rearranged
HendersonHasselbalch equation, and the plasma solubility of
CO2 (CO2) was provided by Boutilier et
al. (1979
). Assuming that the
carbonate concentration is negligible, plasma [HCO3-]
was calculated as [CO2]
(
CO2xPCO2).
Data analysis, statistical analysis and presentation
For each hypoxic level, a continuous recording of 3-8 min was analysed for
mean blood flows (sys and
pc), mean blood pressure
and heart rate (obtained from the systemic blood flow trace). All recordings
were analysed using AcqKnowledge data-analysis software (version 3.5.7).
A two-way analysis of variance with repeated measures (RM-ANOVA) was used to identify significant effects of hypoxia and anaemia on measured variables. Because anaemia and exposure to an FIO2 of 0.10 in toads with normal haematocrit resulted in similar reductions in arterial [O2] (Fig. 1), we performed an additional one-way RM-ANOVA to evaluate the specific effects of anaemia and hypoxia on the measured variables. In all analyses, differences among means were analysed post hoc using a StudentNewmanKeuls (SNK) multiple comparison test. All statistical analyses were performed using SigmaStat statistical software (version 2.03; SPSS Science, Chicago, IL, USA), and the level of significance was chosen at the P<0.05 level. All data are presented as means ± 1 S.E.M.
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Results |
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In the toads with normal haematocrit, hypoxia elicited an increase in arterial pH (pHa; Fig. 2A) that was associated with a reduction in PaCO2 (Fig. 2C) and followed the non-bicarbonate buffer line (Fig. 2D) until the most severe hypoxic exposure, where a metabolic acidosis contributed to a reduction in pHa. Arterial acidbase parameters were not affected by anaemia during normoxia (Fig. 2; Table 1), but the anaemic toads exhibited a larger reduction in pHa than did toads with normal haematocrit during hypoxia. The more severe acidosis was due to a metabolic acidosis and a smaller reduction in PaCO2 during hypoxia (Fig. 2).
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Effects of hypoxia and anaemia on distribution of blood flows and
cardiac parameters
In toads with normal haematocrit,
pc was lower than
sys during normoxia, which
is indicative a net RL cardiac shunt. Hypoxia caused an almost
threefold increase in
pc
(Fig. 3A,B).
sys, however, did not
change (Fig. 3C,D), and
exposure to hypoxia was, therefore, associated with a pronounced increase in
pc/
sys
(Fig. 3E,F) and a reversal to a
net LR cardiac shunt (Fig.
3G,H). The increase in
tot was primarily caused by
an increased fH, but an increased VS also contributed
(Fig. 4). Blood pressure
increased slightly at the most severe hypoxic exposures, but these changes
were not statistically significant.
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Anaemia was associated with a doubling of
pc in normoxic toads, but
sys was not affected
(Fig. 3). Anaemia, therefore,
caused an increase in
pc/
sys
and a reduction in the net RL cardiac shunt. The increased
tot caused by anaemia was
attributed to a combination of an increased fH and an increased
VS (Fig.
4). When exposed to hypoxia, the anaemic toads exhibited further
increases in
pc, so
pc remained elevated in
comparison with toads with normal haematocrit at any given level of hypoxia.
As in toads with normal haematocrit,
sys did not increase during
hypoxia, and hypoxia was associated with a progressive increase in the net
LR shunt and a large increase in
pc/
sys
(Fig. 3). Blood pressure did
not change after anaemia and remained stable during hypoxia in the anaemic
toads. The control group, which was exposed to two periods of hypoxia with no
manipulation of haematocrit, exhibited a decrease in
sys (P=0.04)
between the first and second day (Fig.
5), while all other parameters were unaffected by the repeated
hypoxic exposure.
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As shown in Fig. 1A, toads with normal haematocrit at FIO2=0.10 had an arterial [O2] that is similar to the arterial oxygen concentration of anaemic animals in normoxia (1.47±0.21 mmol l-1 and 1.41±0.22 mmol l-1, respectively; see Table 1 for an additional comparison of haematology and blood gases between these groups). It is illustrative, therefore, to compare the cardiovascular status of anaemic animals with the status of toads with normal haematocrit during normoxia and when exposed to an FIO2 of 0.10. These comparisons are shown by the bar graphs inserted on the right panel of Figs 3 and 4.
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Discussion |
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We did not measure blood flows in all arteries leaving the heart, but the
left and right truncus arteriosus have similar diameters, and blood flows are
similar on the two sides (West and
Burggren, 1984). Therefore, we calculated
pc and
sys by doubling the values
measured on the left pulmocutaneous artery and the left aortic arch. The probe
on the pulmocutaneous artery was placed before the small cutaneous artery
branches off the larger pulmonary artery, so we cannot distinguish flows
between these two circuits. Cutaneous flow ranges between 10% and 20% of
pc in Bufo, and
absolute changes in flows are small compared with pulmonary flow
(West and Burggren, 1984
). On
the systemic side, we underestimate
sys because the probe was
positioned after the point where the carotid arteries emerge from the systemic
arch. The flows in the carotid arteries are less than 6%
(West and Smits, 1994
) but
will cause some underestimation of
pc/
sys
relative to the study by Gamperl et al.
(1999
).
The heart rates reported for Bufo marinus differ enormously among
studies depending on the degree of instrumentation. In our study, fH
of undisturbed normoxic toads was elevated compared with previous studies on
toads instrumented with ECG leads or a femoral cannula (e.g.
Dumsday, 1990;
Wang et al., 1994
).
Nevertheless, our values for fH,
tot and net cardiac shunt
patterns are consistent with values reported previously on toads instrumented
with flow probes (Gamperl et al.,
1999
; Hedrick et al.,
1999
).
Ventilatory response to reduced oxygen levels
Bufo marinus exhibits a vigorous ventilatory response to hypoxia
(e.g. Boutilier et al., 1979;
Kruhøffer et al., 1987
;
Wang et al., 1994
), which is
believed to stem from stimulation of oxygen-sensitive receptors on the major
arteries (see West and Van Vliet,
1992
; cf. Jones and Chu,
1988
). The hypoxic ventilatory response of toads is mediated by
reduction in PaO2, as toads breathing normoxic
air do not increase ventilation when blood [O2] is reduced by
anaemia or inhalation of CO (Wang et al.,
1994
; Branco and Glass,
1995
). Furthermore, afferent nerve activity from the carotid nerve
of Bufo marinus is not affected by blood [O2]
(Van Vliet and West, 1992
).
Our study is consistent with PaO2 being the
major determinant for hypoxic ventilatory responses because anaemia did not
alter PaCO2 during normoxia, suggesting that
lung ventilation did not change with reduced haematocrit
(Fig. 2C; see also
Katz, 1980
).
Cardiovascular responses to hypoxia and anaemia
The toads with normal haematocrit were characterised by a large net
RL cardiac shunt during normoxia, which is consistent with previous
studies on resting and undisturbed toads
(Gamperl et al., 1999;
Hedrick et al., 1999
). When
exposed to hypoxia, the toads with normal haematocrit responded with an
increased fH and a large increase in
pc, while
sys did not change. This
response resulted in progressive reduction in the RL cardiac shunt and
a reversal to a net LR shunt during severe hypoxia. A large reduction
in the net RL shunt has previously been documented in Bufo
marinus (Gamperl et al.,
1999
) and serves to increase systemic oxygen transport because
arterial [O2] is maximised by elimination of RL cardiac
shunts (e.g. Wang and Hicks,
1996
). Cholinergic blockade by infusion of atropine results in
similar cardiovascular changes to those observed during hypoxia
(Gamperl et al., 1999
), and
circulating catecholamines increase only during exposure to severe hypoxia
(Andersen et al., 2001
). Thus,
most of the cardiovascular response to hypoxia is probably caused by release
of vagal tone on the heart and pulmonary artery
(de Saint-Aubain and Wingstrand,
1979
; West and Burggren,
1984
).
Our study shows that toads respond to reduced haematocrit by increasing
fH and pc and by
reducing the net RL cardiac shunt (Figs
3B,F,H,
4D). Thus, even though anaemia
did not affect PaO2, the cardiovascular
response was qualitatively similar to the response elicited by hypoxia. When
exposed to hypoxia, the anaemic toads exhibited an additional increase in
pc and also developed a
large net LR cardiac shunt. The cardiovascular response to anaemia
cannot be ascribed to habituation or exposure to hypoxia on the previous day,
because all haemodynamic variables in normoxia and hypoxia were similar
(although
sys was slightly
elevated) during the second experimental day of the control animals, where
haematocrit was not manipulated (Fig.
5). To evaluate the extent to which the cardiovascular response
correlates with PaO2 versus arterial
[O2], we compared the responses of toads with normal haematocrit
exposed to an FIO2 of 0.10 with
those induced by a similar reduction in [O2] caused by anaemia at
normoxic PaO2 (bar graphs in Figs
3 and
4). This analysis indicates
that reduced arterial [O2] can explain most of the cardiovascular
response that is observed when hypoxia reduces both arterial [O2]
and PaO2. However, the cardiovascular response
was more pronounced when similar reductions in arterial [O2] were
achieved by hypoxia. This may be explained by an additive effect of reduced
PaO2, which would be consistent with the more
pronounced cardiovascular response when the anaemic toads were exposed to
hypoxia. It is also possible that the increased ventilation during hypoxia, as
opposed to the lack of ventilatory response to anaemia
(Wang et al., 1994
), may be
associated with increased
pc through stimulation of
stretch receptors in the lungs and feed-forward mechanisms
(Wang et al., 1999
). Finally,
the higher PaCO2 of anaemic toads during
hypoxia may have augmented
pc, because hypercapnia is
associated with increased
pc
(West and Smits, 1994
;
Gamperl et al., 1999
).
The cardiovascular response to reduced arterial [O2] at normal
PaO2 may be explained by the presence of
oxygen-sensitive chemoreceptors that specifically affect the cardiovascular
system and are stimulated by reductions in blood [O2]. A receptor
that can sense oxygen bound to haemoglobin within the red cells is highly
unlikely, but PO2-sensitive chemoreceptors
located in an under-perfused tissue would be able to sense reduced arterial
[O2] as reductions in PO2. This is
believed to be the case for the aortic bodies in mammals (Lahiri et al.,
1980,
1981a
,b
)
that primarily affect the cardiovascular system
(Daly and Ungar, 1966
;
Daly, 1997
;
Jones and Daly, 1997
).
Alternatively, the PO2-sensitive chemoreceptor
could be located in the venous circulation or, as suggested previously, on the
pulmocutaneous artery, which is perfused predominantly by venous systemic
blood (Wang et al., 1997
,
in press
). In addition,
receptors have been located on the pulmocutaneous artery
(Ishii et al., 1985
), but
their reflex roles remain uncertain (West
and Van Vliet, 1992
; Wang et
al., in press
).
While the existence of a separate group of oxygen-sensitive chemoreceptors
affecting the cardiovascular system may explain the responses to anaemia,
other, not mutually exclusive, explanations are possible. Several compounds
have been suggested to regulate local blood flow in an [O2]- or
[Hb]-dependent manner. These compounds include the release of ATP
(Ellsworth et al., 1995) and
arachidonic acid metabolites (Harder et
al., 1996
) from red blood cells. Haemoglobin is an effective
scavenger of nitric oxide, and reduced [Hb] might result in more nitric oxide
available for local vasodilation (Stamler
et al., 1997
). Anaemia and associated reductions in tissue
PO2 may, therefore, have induced some
vasodilation in the systemic circulation that, in turn, would induce
barostatic responses where increased heart rate and cardiac output act to
maintain blood pressure. Furthermore, a reduction in haematocrit from 20% to
11%, as achieved with our anaemia protocol, would be expected to reduce
viscosity by approximately 40% (Hillman et
al., 1985
), which would contribute to an apparent reduction in
systemic vascular resistance. However, anaemia primarily affected
pc and fH, so a
classic barostatic response does not seem to explain the observations. This is
further substantiated by the fact that Bufo marinus does not exhibit
barostatic responses to reduced blood pressure
(West and Van Vliet,
1992
).
In conclusion, the cardiovascular and ventilatory responses to oxygen shortage of toads seem to differ with respect to the specific oxygen stimulus. Hypoxia elicited large changes in both ventilatory and cardiovascular parameters, whereas anaemia only had effects on cardiovascular parameters. Future investigations are needed to elucidate which receptor groups are responsible for this difference in oxygen modality.
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Acknowledgments |
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