Adenosinergic and cholinergic control mechanisms during hypoxia in the epaulette shark (Hemiscyllium ocellatum), with emphasis on branchial circulation
1 Physiology Programme, Department of Molecular Biosciences, University of
Oslo, PO Box 1041, NO-0316 Oslo Norway
2 Department of Zoophysiology, Göteborg University, SE-405 30
Göteborg, Sweden
3 Hypoxia and Ischemia Research Unit, School of Physiotherapy and Exercise
Science, Griffith University, PMB 50 Gold coast Mail Centre, Queensland, 9726
Australia
* Author for correspondence (e-mail: k.o.stenslokken{at}bio.uio.no)
Accepted 17 September 2004
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Summary |
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Key words: elasmobranch, blood pressure, cardiovascular, gill, shunt, bradycardia
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Introduction |
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The anatomy of the elasmobranch gill is somewhat different from that of
teleosts (Kempton, 1969;
Wright, 1973
;
Olson and Kent, 1980
;
Devries and Dejager, 1984
).
First, the two rows of gill filaments on each gill arch are separated by a
septum. Second, the afferent filament artery is not a vessel but a cavernous
body [also called medial afferent sinus
(Olson and Kent, 1980
) or
corpus cavernosum (Wright,
1973
)] at the free tip of the filament. However, as in teleosts,
the branchial circulation of elasmobranchs comprises an arterioarterial
(AA) and arteriovenous (AV) circulation
(Cooke, 1980
;
Olson and Kent, 1980
;
Devries and Dejager, 1984
).
Blood enters the lamellae, the respiratory unit of the gill, through afferent
lamellar arterioles from a cavernous body in series with the afferent filament
artery (AFA) in the gill filament. After being oxygenated, the blood leaves
the lamellae through efferent lamellar arterioles flowing into an efferent
filament artery (EFA) and, in the AA circulation, the blood enters the dorsal
aorta through the efferent branchial artery. Blood can enter the AV
circulation through anastomoses from either the afferent or the efferent side,
the precise arrangement may vary from species to species
(Randall, 1985
;
Olson, 2002b
).
It was initially suggested that the AV system may serve as a lamellar
bypass, hence creating a non-respiratory shunt
(Steen and Kruysse, 1964).
However, it has become evident that the AV system is a low-pressure system
that does not enable blood to re-enter the EFA. The AV circulation enters the
branchial veins and is, therefore, returned back to the heart. An increase in
the AV circulation has been observed in response to hypoxia in cod, and this
may serve to increase the blood supply to the heart and the ion-regulatory
cells of the filament epithelium (Sundin
et al., 1995
).
Most teleost fishes studied respond to hypoxia with an increase in
branchial vascular resistance (Rgill)
(Holeton and Randall, 1967;
Petterson and Johansen, 1982
;
Sundin and Nilsson, 1997
),
bradycardia and an increase in systemic pressure
(Fritsche and Nilsson, 1989
;
Fritsche and Nilsson, 1990
;
Bushnell and Brill, 1992
;
Sundin, 1995
). The increase in
Rgill is possibly related to a cholinergically mediated
constriction of a sphincter situated at the EFA near the base of the filament.
The site of action of acetylcholine (ACh) in the branchial vasculature was
first revealed by Smith
(1977
), and later confirmed by
cardiovascular (Farrell, 1981
;
Farrell and Smith, 1981
) and
immunohistochemical (Bailly and Dunel-Erb,
1986
) measurements. The increase in Rgill
caused by a constriction of the sphincter is thought to force more blood into
the AV circulation through post-lamellar arterioles
(Sundin, 1995
). The
bradycardia can be inhibited by the muscarinic receptor blocker atropine,
revealing that a vagal cholinergic innervation mediates this response. Hypoxic
bradycardia by vagal innervation has, for example, been well characterized in
dogfish (Taylor and Butler,
1982
; Barrett and Taylor,
1985
; Taylor,
1994
).
There are few studies on the branchial effects of hypoxia in elasmobranchs.
Unlike teleosts, which have an atropine-sensitive cholinergic innervation, the
increase in gill resistance in nerve-stimulated sharks appears to be entirely
due to contraction of striated muscles in the gill arch, because it is
abolished by pancuronium, a nicotinic receptor blocker
(Metcalfe and Butler, 1984;
Chopin and Bennett, 1995
). A
branchial cholinergic innervation involving muscarinic receptors has, as far
as we know, not hitherto been demonstrated in an elasmobranch.
Adenosine is thought to serve an important protective function in hypoxic
vertebrates, from mammals to teleost fish
(Mubagwa et al., 1996;
Lutz et al., 2003
). Adenosine
is formed from ATP in energy-deficient cells or released during purinergic
neurotransmission. In the heart, its negative inotropic and chronotropic
effects will reduce oxygen demand, and by inducing coronary vasodilatation,
adenosine will increase oxygen supply to the cardiac muscle
(Rongen et al., 1997
).
Moreover, adenosine stimulates glucose uptake in myocardial cells
(Wyatt et al., 1989
),
increasing the substrate for glycolysis.
Very little is known about the effect of adenosine on branchial circulation
in fish. There is evidence for the presence of A1 and A2
adenosine receptors in the ventral aorta of the spiny dogfish
(Evans, 1992). In the blacktip
reef shark (Carcharhinus melanopterus), adenosine constricts the
efferent branchial artery (Bennett,
1996
). In rainbow trout, injection of adenosine, and the
A1 receptor agonist CPA caused a 60% reduction of both the AFA and
the EFA, and increased Rgill with 150%
(Sundin and Nilsson, 1996
). In
the same study, cardiac output (
)
increased while fH fell after adenosine injection,
indicating an increase in stroke volume (SV).
In the epaulette shark, the only known anoxia-tolerant elasmobranch, we wanted to investigate circulatory and branchial responses to hypoxia. Given the important role of adenosine in hypoxia tolerance, we hypothesised an involvement of adenosinergic mechanisms in the cardiovascular system of this species. A second objective of this study was to characterize cholinergically mediated mechanisms during severe hypoxia.
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Materials and methods |
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The sharks were anaesthetised in a 50 l tank by adding benzocaine, dissolved in 96% ethanol (50 g l1) to the water, to a final concentration of 60 mg l1. After being weighed each fish was placed on a surgical table and was ventilated with aerated seawater floating through a tube inserted into the mouth. This water (25 l containing 30 mg benzocaine l1) was recirculated in a system held at room temperature (25°C).
An incision was made on the ventral side, posterior to the first gill slit, perpendicular to the midline. To record PVA, the first branchial artery was dissected free and a PE50 polyethylene cannula (Intramedic; Becton Dickinson, Sparks, MD, USA) with a small bubble 10 mm from the tip, was occlusively inserted and secured with a thread around the vessel behind the bubble. The incision was closed by sutures and the cannula was secured to the skin. To record dorsal aortic blood pressure (PDA), the shark was placed on its dorsal side and a PE50 cannula was inserted inside a large needle, which was used as a guide to reach the dorsal aorta by a blind penetration made 30 mm behind the anal opening. The cannula was secured to the skin by sutures. Both cannulas (containing heparin-containing elasmobranch saline: 280 mmol l1 NaCl and 350 mmol l1 urea) were connected to Gould Statham P23 Db pressure transducers (Gould-Statham, Oxnard, CA, USA). Pressure calibrations were performed each day against a static water column.
During pilot experiments, a Doppler flow probe (20 MHz, 45; Iowa Doppler
Products, Iowa City, IA, USA) around the aorta was placed in three individuals
to measure . However, in the epaulette
shark, the aorta sends off the first branchial arteries directly after the
conus arteriosus, so it was impossible to position the Doppler probe without
damaging the pericardium. Each time the pericardium was opened, both
PVA and PDA fell dramatically during
the first 10 min and continued to fall for about 2 h until the shark died.
Because of limited access to sharks and placing the Doppler after the first
branch would give an incorrect estimate of
, no further attempts were made to
measure
.
To observe microcirculatory changes in the gill, a digital video camcorder
(DCR-PC7E; Sony, Japan) was connected to an epi-illumination microscope (Leitz
Ortholux; Wetzlar, Germany) fitted with a Leitz Ultropak water immersion
objective (22x or 11x) as described by Nilsson et al.
(1995) and Sundin
(1995
).
After surgery, the shark was placed in a plastic chamber and continuously ventilated with seawater (60 l containing 30 mg benzocaine l1) and left to rest for 1 h before any experiments were conducted. The water level was adjusted with a standpipe to a level that covered the shark. A plexiglas column (1500 mm high, 80 mm diameter) was included in the water circulation. This column was bubbled with N2 to make the water severelty hypoxic ([O2]<0.3 mg l1) within 1 min, and adjusted with air to prevent anoxic conditions.
Experimental protocol
First the sharks (N=14) were exposed to two 20 minepisodes of
severe hypoxia to allow microscopic observation of afferent and efferent sides
of the filament. After the two initial hypoxic periods, sharks where divided
into two groups, one injected with adenosine and the other injected with
ACh.
Adenosine
Adenosine was injected into the afferent branchial artery. Cardiovascular
measurements and microscopic observations were done continuously on either the
afferent or the efferent side of the gill filament (N=7). After 30
min, the adenosine injection was repeated and the gill filament was observed
on the opposite side. Before the experiments, we tested for suitable doses and
1 µmol adenosine kg1 was chosen. Subsequently, the
non-specific adenosine receptor agonist aminophylline (10 mg
kg1) was injected, followed by two more hypoxic periods and
measurements (as above). The experiments ended with adenosine injections to
verify an efficient adenosine receptor blockade.
ACh
For ACh (Sigma; St Louis, MO, USA) a similar protocol as for adenosine was
used. ACh was injected twice into the ventral aorta (N=7) and
cardiovascular measurements and microscopic observations were done on the
afferent and the efferent side. Continuous measurements after each treatment
were made until the parameters were had returned to pre-injection or
pre-hypoxic levels. Before the experiments, we tested for suitable doses and 1
µmol ACh kg1 was chosen. Next the animals were treated
with atropine (2 mg kg1; Sigma). After 30 min, the ACh
injections were repeated to ensure that all muscarinic receptors were blocked.
The injections were subsequently followed by two additional hypoxic periods.
Finally, 5 mg kg1 of the nicotinic receptor antagonist
tubocurarine (Sigma) was injected and the two hypoxic periods and ACh
injections were repeated.
Data treatment and statistics
Cardiovascular variables were sampled using Labview (version 5.0, National
Instruments, Austin, TX, USA). Sampling frequency used was 30 Hz and mean
values were subsequently calculated at 30 s (hypoxia) and 15 s (adenosine and
ACh) intervals. Data are presented as means ± S.E.M. In
Fig. 1, only data for every 1
min 30 s are shown to allow separation of the S.E.M. From the
pulsed blood pressure signals, fH was derived using a
Labview-based calculation program. Evaluations of statistically significant
differences were done with Grap Pad Instat (3.01; Graph Pad Software, San
Diego, CA, USA) and a repeated measure analysis of variance (ANOVA) was
performed to detect statistically different changes during the hypoxic and
drug injection periods. A Dunnet post-test was used to evaluate which time
interval was significantly different from pre-injection values. In the
figures, significant differences (P<0.05) over a time interval are
indicated with a line. To detect significant differences between non-treated
(control) and treated (aminophylline, atropine and tubocurarine) sharks, the
control values at each time point of a cardiovascular variable in each animal
were subtracted from the sum of the treatment values, and a repeated measure
ANOVA was performed to detect significant differences and indicated in the
figures with an asterisk. A Student's t-test was used on the last
pre-hypoxic value to detect differences between groups before and after
treatment with aminophylline. In the duplicate exposures (hypoxia, ACh and
adenosine) only the first set of pressure and fH data were
used for statistical analysis.
|
Measurements of microvascular changes in the branchial vasculature were done on a TV screen using a slide calliper for EFA diameter measurements and a stopwatch to detect difference in the velocity of erythrocytes between different exposures. The pre-exposure (hypoxia and drug injected) values were set to 100% and differences were compared using a non-parametric ANOVA (Freidmann's test) followed by Dunn post-test.
A Fisher's exact test was used to evaluate differences in blood flow in the longitudinal vessels in the filament between treated and non-treated individuals. Fig. 4 shows number of sharks with blood flow as a proportion of the total number of sharks at each time point. Ventilation frequency was measured with a stopwatch, and a repeated measure ANOVA was performed to detect significant differences. All post-exposure values highlighted in the results are minimum or maximum values.
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Results |
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The anaesthetised shark displayed ventilatory movements, although they were shallower than in awake sharks. The ventilation frequency initially increased and then decreased during hypoxia (Fig. 5A).
|
Treatment with aminophylline (10 mg kg1) had a large effect both on PVA and on PDA resting values, which were significantly reduced from 2.82±0.12 kPa to 1.95±0.17 kPa, and from 2.06±0.09 kPa to 1.3±0.09 kPa respectively (P<0.05; Fig. 1B,C). There was no significant reduction in fH after aminophylline treatment, but there was a significant difference indicated by the ANOVA only, between aminophylline-treated and non-treated sharks during hypoxia (although the post-test failed to detect differences at specific time points). The initial reduction in fH appeared to be slower after aminophylline treatment, but fH decreased more towards the end of the hypoxic period after aminophylline treatment. Even if blood pressures were lower in aminophylline-treated animals, they showed the same response pattern as in control animals during hypoxia, with significant reductions in PVA, PDA and fH (Fig. 1). There was no significant difference between aminophylline-treated and non-treated sharks in PVA or PDA responses to hypoxia.
After aminophylline injections, only two out of seven hypoxic sharks showed a commenced blood flow in the longitudinal vessels (Fig. 4A). Aminophylline abolished the spontaneous ventilatory movements in the shark.
In the second group of sharks, injection of atropine (2 mg kg1) had neither effects on normoxic fH and blood pressures, nor on the hypoxia induced bradycardia and reduced blood pressures (Fig. 1). Similarly, atropine did not inhibit the commenced blood flow into the longitudinal vessels and the biphasic change (an initial increase followed by a decrease) in ventilatory frequency observed during hypoxia (data not shown). In fact, the only significant effect of atropine on the hypoxic responses was a reduction in the pressure difference (PVAPDA) over the gills (Fig. 1D; P<0.05).
When adenosine was injected, fH fell from 58.60±1.10 beats min1 to 48.87±4.49 beats min1 (P<0.05; Fig. 6A). Simultaneously, PVA fell from 2.73±0.13 kPa to 2.12±0.11 kPa (P<0.05; Fig. 6B) and PDA from 2.01±0.08 kPa to 1.39±0.11 kPa (P<0.05; Fig. 6C). Thus, adenosine injections mimicked the hypoxic cardiovascular response patterns. There was no change in the pressure fall over the gills (PVAPDA; Fig. 6D). Adenosine also initiated blood flow in the longitudinal blood vessels (Fig. 4B). Most of the responses to injected adenosine were abolished after aminophylline treatment, but adenosine still caused a significant reduction in blood pressures and fH. The post-test revealed a significant difference between treated and non-treated sharks in PVA between 60 s and 720 s, and in PDA between 30 s and 300 s (P<0.05).
|
Adenosine also induced a significant and biphasic response in ventilation frequency with an initial increase, followed by a decrease (Fig. 5B), which was opposite to the pattern elicited by hypoxia (Fig. 5A). In aminophylline treated sharks, adenosine injection did not initiate breathing movements again.
After injecting 1 µmol kg1 ACh into normoxic sharks,
fH fell from 58.18±0.82 beats
min1 to 29.93±6.91 beats min1
within 30 s (Fig. 6E;
P<0.05). There was also an increase in PVA
from 2.84±0.13 kPa to 4.13±0.41 kPa
(Fig. 6F) and a decrease in
PDA from 2.25±0.18 kPa to 1.72±0.17 kPa
(Fig. 6G; P<0.05)
resulting in a large pressure fall
(PVAPDA) over the gills
(Fig. 6H). Visual observations
of the branchial vasculature in the microscope detected no changes in the
diameter of the EFA after ACh injections. However, the speed of the
erythrocytes in the EFA was significantly reduced by 46.7±4.2%
(P<0.05) during the first minute after the injection
(Fig. 3). Blood flow in the
longitudinal vessels was commenced by ACh injection in three out of six
individuals (data not shown). Another interesting observation was that in the
animals where ACh transiently paused the heart beat, the thickness of the
filament appeared to decrease, and simultaneously the filament bent downwards.
This supports the idea that the cavernous body functions as a supportive
hydrostatic skeleton in the free tip of the shark gill filament
(Devries and Dejager, 1984).
Although small significant increases in PVA and
PDA occurred after atropine treatment
(Fig. 6F,G), atropine blocked
the ACh-induced increase in pressure difference over the gills
(PVAPDA;
Fig. 6H). Atropine inhibited
the commenced blood flow in the longitudinal vessels and the reduced speed of
erythrocytes in the EFA (Fig.
2B).
Injection of tubocurarine in atropine treated animals had no significant effect on any of the cardiovascular values measured in either normoxic or hypoxic conditions (Fig. 1). By contrast, the spontaneous ventilatory movements disappeared after injection of tubocurarine verifying a successful inhibition of nicotinic receptors.
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Discussion |
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The longitudinal vessels are probably the same vessels as those described
in spiny dogfish as vessels draining blood from interlamellar vessels
(Olson and Kent, 1980;
Devries and Dejager, 1984
).
The interlamellar vessels form a sheet of vessels that run parallel to and
between the inner margins of the lamellae. The blood supply for the
intralamellar vessels varies from species to species. Most fishes appear to
have post-lamellar anastomoses (Olson,
2002a
,b
).
However, pre-lamellar anastomoses exists in eel
(Hughes et al., 1982
) and in
spiny dogfish, they are also present
(Olson and Kent, 1980
), but
not in the smallspotted catshark (Dunel
and Laurent, 1980
; Randall,
1985
). The longitudinal vessels in the spiny dogfish are connected
with the intralamellar vessels system and display a marked constriction as
they pass between afferent lamellar arterioles
(Devries and Dejager, 1984
).
The control mechanisms of these anastomoses have been unknown, but the present
results suggest that they are under adenosinergic control. The intralamellar
sinus is in series with the diaphragmal sinus, which drains into the anterior
cardinal vein and jugular vein, thereby returning blood directly to the heart
(Devries and Dejager, 1984
).
The portion of
that normally enters
the AV circulation varies between species, but has been estimated to 8% in
Atlantic cod (Gadus morhua;
Sundin and Nilsson, 1992
), 7%
in trout (Oncorhynchus mykiss;
Ishimatsu et al., 1988
), and
up to 30% in eel (Hughes et al.,
1982
). A full understanding of the AV system is lacking. However,
this circulation is probably important for the many functions of the gill
tissue, including ion-balance, acidbase regulation and metabolism of
circulating hormones (Laurent,
1984
; Olson, 1991
,
1996
,
2002a
;
Ishimatsu et al., 1992
;
Goss et al., 1998
;
Randall and Brauner, 1998
).
Our observation of a hypoxia-induced flow in the longitudinal vessels suggests
an increased drainage into the AV circulation. Thus, this may be a mechanism
for increasing the supply of blood to non-respiratory functions of the gills
during energy deficiency. The fact that this blood is delivered directly back
to the heart, may also function to protect the heart during hypoxia.
Hypoxic bradycardia
Hypoxia induced a profound bradycardia, and a decrease in
PVA and PDA, in the epaulette shark.
Unfortunately, we were not able to measure
directly because of difficulties in
attaching a Doppler flow probe without damaging the pericardium, but a
reduction in blood flow velocity in the EFA was observed during hypoxia
indicating a fall in
. A reduced
during hypoxia has, in fact, been
observed in two dogfish species, Scyliorhinus stellaris
(Piiper et al., 1970
) and
spiny dogfish (Davie and Farrell,
1991
). Thus, it is possible that PVA and
PDA fell during hypoxia because of a reduced
(Fig. 2A). There was no
significant change in the blood pressure fall over the gills. Both an increase
and decrease in Rgill during hypoxia has been observed in
elasmobranchs (Satchell, 1962
;
Piiper et al., 1970
;
Butler and Taylor, 1971
;
Butler and Taylor, 1975
;
Kent and Peirce, 1978
). The
increase in Rgill displayed by hypoxic teleosts may result
in lamellar recruitment and also serve to increase the overflow to the AV
circulation (Sundin,
1995
).
Interestingly, the hypoxic bradycardia in the epaulette shark could not be blocked by the cholinergic muscarinic-receptor antagonist atropine (Fig. 1A). In this respect, the epaulette shark differs from other elasmobranchs and teleosts exposed to hypoxia, which invariably show an atropine-sensitive reduction in fH. Because ACh injections into the ventral aorta of the epaulette shark reduced fH by 50%, and as this response was abolished by atropine treatment, it proves that this species indeed has muscarinic receptors on the heart, but these are apparently not stimulated by nervous release of ACh during hypoxia.
Other neurotransmitters, in addition to ACh, have been found to elicit a
negative chronotropic effect during electrical stimulation of the vagus nerve
in the toad, Bufo marinus
(Courtice and Delaney, 1994).
Cardiac nerve endings in this toad contain ACh, somatostatin and galanin,
which all are co-released upon nerve stimulation. However, there seems to be
no chronotropic effect of other substances than ACh in the shark
Heterodontus portusjacksoni
(Preston and Courtice, 1995
),
making it less likely that neuropeptides are mediating bradycardia in the
epaulette shark. The slow onset of the hypoxia-induced bradycardia in the
epaulette shark also argues for a non-nervous origin of the response. In the
epaulette shark, the bradycardia was not fully developed until after 15 min,
while in spiny dogfish (Scyliorhinus canicula), atropine-blockable
bradycardia sets in within 1 min of hypoxia
(Taylor et al., 1977
).
In teleosts, acidosis reduces by
suppressing both fH and SV
(Farrell and Jones, 1992
;
Driedzic and Gesser, 1994
).
Although no measurement of blood pH has been done in the epaulette shark, it
is likely that the sevenfold increase in lactate observed at low oxygen
tensions in this shark (Routley et al.,
2002
) acidifies the blood. This may be one of the reasons for the
observed reduction in fH in the epaulette shark. In sea
raven (Hemitripterus americanus) and the ocean pout (Macrozoarces
americanus), a reduction in blood pH from 7.9 to 7.4 reduced
by 12% and 18%, respectively, by
suppressing both SV and fH
(Farrell et al., 1983
).
Interestingly, an -adrenoceptor-mediated negative chronotropy has
been observed in the spiny dogfish (Capra
and Satchell, 1977
) and we can not rule out this mechanism
underlying the hypoxia-induced bradycardia. However, 30 min of hypoxia did not
elevate the plasma catecholamines in the spiny dogfish
(Perry and Gilmour, 1996
).
Effects of ACh
Upon ACh injections, the blood flow came almost to a complete stop in the
EFA between heartbeats. Still no constriction of the EFA was observed in the
outer portion of the filament, which strongly suggests a constriction further
down the EFA, possibly at the EFA sphincter. By contrast, marked
vasoconstrictions could be observed in the distal portions of AFA and EFA in
rainbow trout (Sundin and Nilsson,
1997). Thus, it appears that muscarinic receptors are not spread
throughout the epaulette shark gill vasculature, but may be concentrated more
basally or in the EFA sphincter. ACh injections ultimately lead to a large
increase in PVA, whereas PDA remained
unchanged. Taken together, this indicates a substantial increase in
Rgill. Efferent branchial artery (EBA) rings from blacktip
reef shark and the lemon shark (Negaprion queenslandicus) have been
found to constrict in response to ACh in an atropine-sensitive manner
(Bennett, 1993
,
1996
). It is possible that also
the observed increase in Rgill in the epaulette shark
could be related to constriction of the EBA.
ACh injections induced blood flow in the longitudinal vessels in the
filament tip in three out of six individuals. Although an adenosinergic
mechanism is probably involved (see below), it is possible that also the large
increase in Rgill (induced by ACh) `forces' more blood
into the AV system. As hypothesized by Randall
(1982), expansion of the
elastic high-pressure vessels during increased branchial pressure probably
squeezes the extensively valved low-pressure system, augmenting flow in the AV
vessels. Thus, an adenosine-induced dilation of the AV system in the epaulette
shark (see below) may work in concert with a cholinergic increase in branchial
pressure to increase oxygenated blood supply to the gill tissues during
hypoxia. In addition, when a larger portion of
enters the AV circulation, the heart
muscle will receive more oxygenated venous return in addition to the blood
supplied by the coronary system.
Effects of adenosine
Adenosine injections mimicked the hypoxic responses in the epaulette shark
by reducing fH, PVA and
PDA (Fig.
6AC). Interestingly, adenosine also induced blood flow in
the longitudinal vessels. Most of the effect was blocked by the unspecific
adenosine receptor antagonist aminophylline. In mammals, adenosine has a
cardio-protective role during hypoxia
(Mubagwa et al., 1996), and it
is possible that it also has such a function in other vertebrates. In rainbow
trout, adenosine has negative chronotropic and inotropic effects
(Aho and Vornanen, 2002
). There
is evidence for the presence of A1 and A2 receptors in
the ventral aorta of spiny dogfish (Evans,
1992
), and adenosine has negative inotropic and chronotropic
effects in smallspotted catshark (Meghji
and Burnstock, 1984
).
Adenosine constricts the branchial circulation in teleosts
(Colin and Leray, 1981;
Sundin and Nilsson, 1996
;
Sundin et al., 1999
). Using
epi-illuminating microscopy, Sundin and Nilsson
(1996
) observed a 60%
reduction of the EFA diameter in rainbow trout, probably mediated by the
A1 receptor, because the response was mimicked by the specific
A1 agonist CPA. However, utilizing the same technique we could not
observe any constriction in the EFA in the epaulette shark after adenosine
injections.
Interestingly, adenosine initiated a flow of blood in the longitudinal
vessels. This observation, together with the finding that aminophylline could
block the same blood flow during hypoxia, makes adenosine a likely candidate
for mediating this hypoxic response. A likely scenario is that adenosine, as a
result of hypoxic energy deficiency, functions to reduce
fH and possibly to increase blood flow directly from the
gill back to the heart. Unlike other fishes, the reduction in
fH is not mediated by muscarinic receptors and must,
therefore, be mediated by some other mechanism acting on the heart.
Interestingly, the ability of adenosine to reduce the activity of adenylyl
cyclase through A1 receptors and to activate an outward potassium
current IKAdo appear to be sufficient to explain
the negative chronotropic and inotropic effects of adenosine in mammals
(Shryock and Belardinelli,
1997). However, aminophylline did not inhibit the hypoxic
bradycardia, indicating that adenosine is not involved in this response in the
epaulette shark. The epaulette shark may have a large adenosinergic tone on
blood vessels because aminophylline injections significantly reduced
PVA and PDA, indicating a general
vasodilatation. However, as we do not have a measurement of
, we cannot rule out a reduction in
SV, because fH was not affected in
aminophylline-treated normoxic animals. However, removal of a possible
positive adenosinergic tone on SV by injecting aminophylline is not
likely, because we observed a negative chronotropic effect of injecting
adenosine in the epaulette shark.
During the experiments, the epaulette shark displayed shallow rhythmic
ventilatory movements that showed an initial increase, followed by a
ventilatory depression, in response to hypoxia. A similar response has been
reported previously in un-anaesthetized epaulette sharks, although at a higher
frequency (Routley et al.,
2002). The respiratory rate we recorded was most likely lower
because of the anaesthesia, but because the same response pattern was
observed, the mechanisms are probably similar. Interestingly, a reversed
biphasic response pattern, an initial decrease followed by an increase, was
observed when adenosine where injected
(Fig. 5). The ventilatory
movement ceased when aminophylline was injected either suggesting that
adenosine is important for the respiratory drive, or that unspecific effects
of aminophylline interferes with respiratory functions. An increase in
ventilation frequency as a response to hypoxia is widespread among fishes but,
to our knowledge, this is the first indication of an involvement of adenosine
in a respiratory reflex in fish. In rat, injection of adenosine into the
circulation inhibits respiration through vagal pulmonary C fibres
(Kwong et al., 1998
). As
adenosine levels are likely to increase during hypoxia, a role of adenosine in
ventilatory control in fish during hypoxia needs further attention.
Conclusion
Taken together, our results indicate a role for adenosine in the hypoxic
survival of the epaulette shark. Adenosinergic control of blood flow into the
longitudinal vessels is likely to be involved in increasing in the portion of
that flows through the AV circulation
during hypoxia. This could be a mechanism aimed at increasing blood supply to
heart and gill tissue during hypoxic challenges. Another major finding is that
despite the presence of muscarinic receptors on the heart, the epaulette shark
responds to hypoxia with a large and highly unusual atropine insensitive
bradycardia. Because the elasmobranchs belong to an evolutionary `old' line in
vertebrate evolution, and as this shark can survive several hours of severe
hypoxia at high temperatures, it appears to be a good model for revealing
evolutionary conserved mechanisms in hypoxic survival.
Abbreviations
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Aho, E. and Vornanen, M. (2002). Effects of adenosine on the contractility of normoxic rainbow trout heart. J. Comp. Physiol. B 172,217 -225.[Medline]
Bailly, Y. and Dunel-Erb, S. (1986). The sphincter of the efferent filament artery in teleost gills: I. Structure and parasympathetic innervation. J. Morphol. 187,219 -237.
Barrett, D. J. and Taylor, E. W. (1985). Spontaneous efferent activity in branches of the vagus nerve controlling heart rate and ventilation in the dogfish. J. Exp. Biol. 117,433 -448.[Abstract]
Bennett, M. B. (1993). Vascular reactivity of the efferent branchial arteries of the lemon shark, Negaprion queenslandicus. Comp. Biochem. Physiol. C 105,535 -541.[CrossRef]
Bennett, M. B. (1996). Efferent branchial artery reactivity in the blacktip reef shark, Carcharhinus melanopterus (Carcharhinidae: Elasmobranchii). Comp. Biochem. Physiol. C 114,165 -170.
Bushnell, P. G. and Brill, R. W. (1992). Oxygen transport and cardiovascular responses in skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) exposed to acute hypoxia. J. Comp. Physiol. B 162,131 -143.[Medline]
Butler, P. J. and Taylor, E. W. (1971). Response of dogfish (Scyliorhinus canicula L) to slowly induced and rapidly induced hypoxia. Comp. Biochem. Physiol. A 39,307 -323.[CrossRef]
Butler, P. J. and Taylor, E. W. (1975). Effect of progressive hypoxia on respiration in dogfish (Scyliorhinus canicula) at different seasonal temperatures. J. Exp. Biol. 63,117 -130.[Abstract]
Capra, M. F. and Satchell, G. H. (1977). The differential haemodynamic response of the elasmobranch, Squalus acanthias, to the naturally occurring catecholamines, adrenaline and noradrenaline. Comp. Biochem. Physiol. C 58, 41-47.[CrossRef][Medline]
Chopin, L. K. and Bennett, M. B. (1995). The regulation of branchial blood-flow in the blacktip reef shark, Carcharhinus melanopterus (Carcharhinidae: Elasmobranchii). Comp. Biochem. Physiol. A 112, 35-41.[CrossRef]
Colin, D. A. and Leray, C. (1981). Vasoactivities of adenosine analogues in trout gill (Salmo gairdneri R.). Biochem. Pharmacol. 30,2971 -2977.[CrossRef][Medline]
Cooke, I. R. C. (1980). Functional aspects of the morphology and vascular anatomy of the gills of the endeavour dogfish, Centrophorus scapratus (McCulloch) (elasmobranchii:squalidae). Zoomorphologie 94,167 -183.
Courtice, G. P. and Delaney, D. J. (1994). Effect of frequency and impulse pattern on the noncholinergic cardiac response to vagal stimulation in the toad, Bufo marinus. J. Auton. Nerv. Syst. 48,267 -272.[CrossRef][Medline]
Davie, P. S. and Farrell, A. P. (1991). Cardiac performance of an isolated heart preparation from the dogfish (Squalus acanthias) the effects of hypoxia and coronary artery perfusion. Can. J. Zool. 69,1822 -1828.
Devries, R. and Dejager, S. (1984). The gill in the spiny dogfish, Squalus acanthias respiratory and nonrespiratory function. Am. J. Anat. 169, 1-29.[Medline]
Driedzic, W. R. and Gesser, H. (1994). Energy
metabolism and contractility in ectothermic vertebrate hearts Hypoxia,
acidosis, and low temperature. Physiol. Rev.
74,221
-258.
Dunel, S. and Laurent, P. (1980). Functional organisation of the gill vasculature in different classes of fish. In Epithelial Transport in The Lower Vertebrates: Transports épithéliaux chez les vertébrés inférieurs: proceedings of the memorial symposium to Jean Maetz held at the Station Zoologique of Villefranche-sur-Mer, 26-27 June 1978 (ed. B. Lahlou), pp. 37-58. Cambridge, UK and New York: Cambridge University Press.
Evans, D. H. (1992). Evidence for the presence of A1 and A2 adenosine receptors in the ventral aorta of the dogfish shark, Squalus acanthias. J. Comp. Physiol. B 162,179 -183.[Medline]
Farrell, A. P. (1981). Cardiovascular changes in the lingcod (Ophiodon elongatus) following adrenergic and cholinergic drug infusions. J. Exp. Biol. 91,293 -305.
Farrell, A. P. and Jones, D. R. (1992). The heart. In Fish Physiology, vol.12 , part A (ed. W. S. Hoar, D. J. Randall and A. P. Farrell), pp. 1-88. New York: Academic Press.
Farrell, A. P. and Smith, D. G. (1981). Microvascular pressures in gill filaments of lingcod (Ophiodon elongatus). J. Exp. Zool. 216,341 -344.
Farrell, A. P., Macleod, K. R., Driedzic, W. R. and Wood, S. (1983). Cardiac performance in the in situ perfused fish heart during extracellular acidosis interactive effects of adrenaline. J. Exp. Biol. 107,415 -429.[Abstract]
Fritsche, R. and Nilsson, S. (1989). Cardiovascular responses to hypoxia in the Atlantic cod, Gadus morhua.Exp. Biol. 48,153 -160.[Medline]
Fritsche, R. and Nilsson, S. (1990). Autonomic nervous control of blood pressure and heart rate during hypoxia in the cod, Gadus morhua. J. Comp. Physiol. B 160,287 -292.
Goss, G. G., Perry, S. F., Fryer, J. N. and Laurent, P. (1998). Gill morphology and acid-base regulation in freshwater fishes. Comp. Biochem. Physiol. A 119,107 -115.
Holeton, G. F. and Randall, D. J. (1967). Changes in blood pressure in rainbow trout during hypoxia. J. Exp. Biol. 46,297 -305.[Medline]
Hughes, G. M., Peyraud, C., Peyraud-Waitzenegger, M. and Soulier, P. (1982). Physiological evidence for the occurrence of pathways shunting blood away from the secondary lamellae of eel gills. J. Exp. Biol. 98,277 -288.[Abstract]
Ishimatsu, A., Iwama, G. K. and Heisler, N. (1988). In vivo analysis of partitioning of cardiac output between systemic and central venous sinus circuits in rainbow trout a new approach using chronic cannulation of the branchial Vein. J. Exp. Biol. 137,75 -88.[Abstract]
Ishimatsu, A., Iwama, G. K., Bentley, T. B. and Heisler, N. (1992). Contribution of the secondary circulatory system to acid-base regulation during hypercapnia in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 170, 43-56.
Kempton, R. T. (1969). Morphological features of functional significance in the gills of the spiny dogfish, Squalus acanthias. Biol. Bulletin 136,226 -240.
Kent, B. and Peirce, E. C., 2nd (1978). Cardiovascular responses to changes in blood gases in dogfish shark, Squalus acanthias. Comp. Biochem. Physiol. C 60, 37-44.[CrossRef][Medline]
Kwong, K., Hong, J. L., Morton, R. F. and Lee, L. Y.
(1998). Role of pulmonary C fibers in adenosine-induced
respiratory inhibition in anesthetized rats. J. Appl.
Physiol. 84,417
-424.
Laurent, P. (1984). Gill internal morphology. In Fish Physiology, vol. 10 (ed. W. S. Hoar and D. J. Randall), pp. 73-172. Orlando: Academic Press.
Lutz, P. L., Nilsson, G. E. and Prentice, H. M. (2003). The brain without oxygen: causes of failure physiological and molecular mechanisms for survival. Dordrecht, Boston, London: Kluwer Academic Publishers.
Meghji, P. and Burnstock, G. (1984). The effect of adenyl compounds on the heart of the dogfish, Scyliorhinus canicula.Comp. Biochem. Physiol. C 77,295 -300.[CrossRef][Medline]
Metcalfe, J. D. and Butler, P. J. (1984). On the nervous regulation of gill blood flow in the dogfish (Scyliorhinus canicula). J. Exp. Biol. 113,253 -267.
Mubagwa, K., Mullane, K. and Flameng, W. (1996). Role of adenosine in the heart and circulation. Cardiovasc. Res. 32,797 -813.[CrossRef][Medline]
Nilsson, G. E., Løfman, C. and Block, M.
(1995). Extensive erythrocyte deformation in fish gills observed
by in vivo microscopy: apparent adaptations for enhancing oxygen
uptake. J. Exp. Biol.
198,1151
-1156.
Nilsson, G. E. and Østlund-Nilsson, S. (2004). Hypoxia in paradise: widespread hypoxia tolerance in coral reef fishes. Proc. R. Soc. Lond. B Biol. Lett. Sup. 271,S30 -S33.
Olson, K. R. (1991). Vasculature of the fish gill Anatomical correlates of physiological functions. J. Electron Microsc. Tech. 19,389 -405.[Medline]
Olson, K. R. (1996). Secondary circulation in fish: anatomical organization and physiological significance. J. Exp. Zool. 275,172 -185.[CrossRef]
Olson, K. R. (2002a). Gill circulation: regulation of perfusion distribution and metabolism of regulatory molecules. J. Exp. Zool. 293,320 -335.[CrossRef][Medline]
Olson, K. R. (2002b). Vascular anatomy of the fish gill. J. Exp. Zool. 293,214 -231.[CrossRef][Medline]
Olson, K. R. and Kent, B. (1980). The microvasculature of the elasmobranch gill. Cell Tissue Res. 209,49 -63.[Medline]
Perry, S. F. and Gilmour, K. M. (1996).
Consequences of catecholamine release on ventilation and blood oxygen
transport during hypoxia and hypercapnia in an elasmobranch (Squalus
acanthias) and a teleost (Oncorhynchus mykiss). J.
Exp. Biol. 199,2105
-2118.
Petterson, K. and Johansen, K. (1982). Hypoxic vasoconstriction and the effects of adrenaline on gas exchange efficiency in fish gills. J. Exp. Biol. 97,263 -272.
Piiper, J., Baumgarten, D. and Meyer, M. (1970). Effects of hypoxia upon respiration and circulation in the dogfish Scyliorhinus stellaris. Comp. Biochem. Physiol. 36,513 -520.[CrossRef][Medline]
Preston, E. and Courtice, G. P. (1995). Physiological correlates of vagal nerve innervation in lower vertebrates. Am. J. Physiol. 268,R1249 -R1256.[Medline]
Randall, D. J. (1982). The control of respiration and circulation in fish during exercise and hypoxia. J. Exp. Biol. 100,275 -288.
Randall, D. J. (1985). Shunts in fish gills. In Cardiovascular Shunts: Phylogenetic, Ontogenetic and Clinical Aspects: Proceedings of The Alfred Benzon Symposium 21 Held at the Premises of The Royal Danish Academy of Sciences and Letters, Copenhagen 17-21 June 1984 (ed. K. Johansen and W. Burggren), pp.71 -82. Copenhagen: Munksgaard.
Randall, D. J. and Brauner, C. (1998). Interactions between ion and gas transfer in freshwater teleost fish. Comp. Biochem. Physiol. A 119, 3-8.
Renshaw, G. M. C., Kerrisk, C. B. and Nilsson, G. E. (2002). The role of adenosine in the anoxic survival of the epaulette shark, Hemiscyllium ocellatum. Comp. Biochem. Physiol. B 131,133 -141.[CrossRef][Medline]
Rongen, G. A., Floras, J. S., Lenders, J. W. M., Thien, T. and Smits, P. (1997). Cardiovascular pharmacology of purines. Clin. Sci. 92,13 -24.[Medline]
Routley, M. H., Nilsson, G. E. and Renshaw, G. M. C. (2002). Exposure to hypoxia primes the respiratory and metabolic responses of the epaulette shark to progressive hypoxia. Comp. Biochem. Physiol. A 131,313 -321.
Satchell, G. H. (1962). Intrinsic vasomotion in the dogfish gill. J. Exp. Biol. 39,503 -512.[Medline]
Shryock, J. C. and Belardinelli, L. (1997). Adenosine and adenosine receptors in the cardiovascular system: Biochemistry, physiology, and pharmacology. Am. J. Cardiol. 79, 2-10.[Medline]
Smith, D. G. (1977). Sites of cholinergic vasoconstriction in trout gills. Am. J. Physiol. 233,R222 -R229.[Medline]
Steen, J. B. and Kruysse, A. (1964). The respiratory function of teleostean gills. Comp. Biochem. Physiol. 12,127 -142.[CrossRef][Medline]
Sundin, L. I. (1995). Responses of the branchial circulation to hypoxia in the Atlantic cod, Gadus morhua.Am. J. Physiol. 268,R771 -R778.[Medline]
Sundin, L. and Nilsson, S. (1992). Arteriovenous branchial bloodflow in the Atlantic cod, Gadus morhua. J. Exp. Biol. 165,73 -84.
Sundin, L. and Nilsson, G. E. (1996). Branchial and systemic roles of adenosine receptors in rainbow trout: an in vivo microscopy study. Am. J. Physiol. 271,R661 -R669.[Medline]
Sundin, L. and Nilsson, G. E. (1997). Neurochemical mechanisms behind gill microcirculatory responses to hypoxia in trout: in vivo microscopy study. Am. J. Physiol. 272,R576 -R585.[Medline]
Sundin, L., Nilsson, G. E., Block, M. and Lofman, C. O. (1995). Control of gill filament blood flow by serotonin in the rainbow trout, Oncorhynchus mykiss. Am. J. Physiol. 268,R1224 -1229.[Medline]
Sundin, L., Axelsson, M., Davison, W. and Forster, M. E.
(1999). Cardiovascular responses to adenosine in the antarctic
fish Pagothenia borchgrevinki. J. Exp. Biol.
202,2259
-2267.
Taylor, E. W. (1994). The evolution of efferent vagal control of the heart in vertebrates. Cardioscience 5,173 -182.[Medline]
Taylor, E. W. and Butler, P. J. (1982). Nervous
control of heart rate activity in the cardiac vagus of the dogfish.
J. Appl. Physiol. 53,1330
-1335.
Taylor, E. W., Short, S. and Butler, P. J. (1977). Role of cardiac vagus in response of dogfish Scyliorhinus canicula to hypoxia. J. Exp. Biol. 70,57 -75.
Wise, G., Mulvey, J. M. and Renshaw, G. M. C. (1998). Hypoxia tolerance in the epaulette shark (Hemiscyllium ocellatum). J. Exp. Zool. 281, 1-5.[CrossRef]
Wright, D. E. (1973). The structure of the gills of the elasmobranch, Scyliorhinus canicula (1). Z. Zellforsch. Mikroscop. Anat. 144,489 -509.[CrossRef]
Wyatt, D. A., Edmunds, M. C., Rubio, R., Berne, R. M., Lasley, R. D. and Mentzer, R. M. (1989). Adenosine stimulates glycolytic flux in isolated perfused rat hearts by A1-adenosine receptors. Am. J. Physiol. 257,H1952 -H1957.[Medline]