Vol. 280, Issue 4, R1197-R1205, April 2001
Control of vascular tone in notothenioid fishes is determined
by phylogeny, not environmental temperature
S.
Egginton,
M. E.
Forster, and
W.
Davison
Department of Zoology, University of Canterbury, Private Bag
4800, Christchurch, New Zealand
 |
ABSTRACT |
We examined potential
vasomotor control mechanisms in an Antarctic fish (Trematomus
bernacchii; usual core temperature approximately 
1°C),
comparing sensitivity to agonists by means of the cumulative dose
response and potency with reference to depolarization by 50 mM KCl. In
efferent branchial arteries, norepinephrine (NE) produced ~20% of
the maximal KCl tension and ~40% in the presence of
10
3M sotalol, suggesting a modest contribution of 
-
and 
-adrenergic tonus [half-maximal response (pEC50) = 6.29 ± 0.37 M]. Carbachol (CBC) and serotonin (5-HT) had
different sensitivities (pEC50 = 4.50 ± 0.40 and
6.82 ± 0.08 M, respectively) but similar potencies (21.6 ± 11.1 and 31.1 ± 5.3% of KCl). A related species from warmer waters around New Zealand, Paranotothenia angustata, had
similar vascular reactivity for NE (pEC50 = 5.48 ± 0.31 M), CBC (pEC50 = 4.94 ± 0.22 M), and
methysergide-sensitive vasoconstriction with 5-HT
(pEC50 = 6.22 ± 0.40 M). Agonist potencies were
9, 65, and 45% that of KCl, respectively. Bovichtus
variegatus, a member of the phylogenetic sister group to the
notothenioids, also gave broadly similar responses. In contrast,
Dissostichus mawsoni, a pelagic Antarctic notothenioid,
showed a dominance of vasodilatation over vasoconstriction, with
sensitive isoprenaline (pEC50 = 6.66 ± 0.05 M)
but weak serotonergic (5.2 ± 1.5% KCl) responses. The unusual
dominance of serotonergic control appears to be primarily a consequence
of evolutionary lineage rather than low environmental temperature, but
the pattern may be modified according to functional demand.
Antarctica; catecholamines; myography; serotonin
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INTRODUCTION |
FISHES THAT LIVE IN THE
SEAS around Antarctica comprise a fauna that is quite recent,
appearing about 30 million years ago, that arose as a result of a
uniquely isolated marine lake event leading to rapid radiation of the
perciform stem into available niches (10, 17). Most of the
biomass in the region consists of members of the suborder
Notothenioidei, which are almost exclusively the only fish caught in
coastal waters (16). Consequently, we probably know more
about the ecophysiology of Antarctic fishes than any other group except
the salmonids (12, 30). Despite this, many questions
remain unanswered. Icebound waters of the Southern Ocean are a uniquely
stable thermal environment, within which cold adaptation of fishes
could be expected to have occurred. This may have obviated the need to
retain the functional plasticity demanded by more variable ecosystems
during the process of polar cooling. In addition, physical isolation
caused by the opening of the Drake Passage provided the potential for
development of unique features resulting from endemic speciation.
Indeed, these notothenioid fishes show some unusual traits that
apparently equip them for life in subzero temperatures, and much effort
has gone into determining the metabolic capacity and elucidating the
regulatory mechanisms in this extreme thermal environment
(16). Importantly, they offer a unique opportunity among
marine families to explore the evolutionary physiology behind the
cardiovascular system, allowing comparisons among species with a close
phylogenetic relationship, but living at very different temperatures.
Do these traits actually represent cold adaptation (functionally
relevant responses to the environment that have become fixed in the
genome), or do they merely represent ancestral characteristics (are a
specialization of the notothenioids as a group)?
In fishes, most stressors (whether manmade or natural) cause increased
cardiac output due to the chronotropic and inotropic effects of
circulating catecholamines on myocytes, in addition to any direct
neural influences. The major effects on cardiac afterload reflect the
balance between -adrenoceptor-mediated dilatation in the branchial
circulation and -adrenoceptor-mediated vasoconstriction in the
systemic circulation. Under conditions of increased energy demand, this
tends to result in an increased dorsal aortic (DA) blood pressure (BP)
and increased perfusion of the gill secondary lamellae, allowing
improved oxygen uptake (37). In contrast, the available
data suggest that Antarctic notothenioids release little or no
catecholamines in response to net capture, surgery, handling, or forced
exercise (21). This strikingly weak primary stress
response may be a result of an inefficient chromaffin tissue (low
synthetic capacity) and/or to a downregulation of adrenergic control
(receptor density or efficacy). Injection of epinephrine (Epi) and
norepinephrine (NE) into Antarctic fish has shown that the potential
for adrenoceptor response is present, but that significant circulating
levels are not routinely produced as a result of low anabolic enzyme
activity (45).
As with most vertebrates, the fish heart usually operates under an
inhibitory vagal tone, the degree of which varies according to
physiological demand and environmental conditions. For example, the
typical response to environmental hypoxia in fish is a reflex bradycardia and increased ventilation that are largely under vagal control, while vagal tone is increased with increasing temperature of
acclimation (42). In contrast, the very low resting heart rates of Antarctic fishes in the oxygen-saturated waters at 0°C are
due to exceptionally high levels of vagal tone (3). These species are unusual in that they do not display a hypoxic bradycardia, presumably because of the existing dominance of the cholinergic inhibition, and in fact often show a slight tachycardia. The spleen is
almost entirely under cholinergic control (32). Gill
perfusion also appears to be under cholinergic control
(4), although both - and -adrenergic responses can
be seen, and we have recently detailed an extensive serotonergic
control of branchial blood flow (22, 26). It is unclear
what, if any, selective advantage this confers. It is possible that the
balance of vascular control mechanisms may reflect lifestyle, with the
benthic Trematomus bernacchii having one order of magnitude
greater excitatory adrenergic tone (~30%) than the cryopelagic
Pagothenia borchgrevinki (3).
The cardiovascular responses that support increased aerobic activity in
Antarctic fish are therefore unusual among teleosts. They can, however,
cope with the increased demand of forced exercise. For example,
P. borchgrevinki increases blood oxygen carrying capacity
(13), although less active species show more modest responses (14, 19), and there is effective redistribution of cardiac output during functional hyperemia (20). These
data suggest that 1) alternate control pathways to the
normal adrenergic system may be operating in these animals and
2) the ecotype may influence the form of cardiovascular physiology.
We wished to determine whether the apparent downregulation of
adrenergic and upregulation of serotonergic vasoconstrictor activity
represented true cold adaptation. In particular we hypothesized that
this form of cardiovascular control represents a basic phylogenetic trait that is modified by the ecology of individual species. To test
these possibilities, we examined vessels from the benthic Antarctic
notothenioid T. bernacchii and compared the results with
those obtained from 1) Paranotothenia angustata,
a closely related species, that presumably spread north during an
episode of global cooling and remained there when the ice retreated to Antarctica and now inhabit waters some 12-14°C warmer
(15); 2) Bovichtus variegatus,
another species found within New Zealand waters, whose family, the
Bovichtidae, is the genetic outlier within the Notothenioidei (6,
39); and 3) Dissostichus mawsoni, an
active notothenioid piscivore that occupies the midwater niche in
McMurdo Sound, Antarctica.
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MATERIALS AND METHODS |
Animals.
Fish were caught on baited hooks through holes cut in the annual fast
ice in McMurdo Sound, Antarctica (77°51'South, 166°45'East). The
pelagic D. mawsoni (Norman) (body mass 27.8 ± 1.4 kg,
n = 4) were caught between 300 and 500 m, the
cryopelagic P. borchgrevinki (Boulenger) (109.3 ± 6.9 g, n = 4) were caught immediately beneath the
sea ice swimming over deep water, and the benthic T. bernacchii (Boulenger) (161.2 ± 13.2 g,
n = 8) off the bottom in shallow water near Scott Base.
All fish were held in laboratory aquariums with running seawater pumped
directly from the Sound at 1.0 ± 0.2°C and a natural
photoperiod of 24-h daylight. They were allowed at least
48 h to recover from the effects of capture and transportation and
were killed by a sharp blow to the head (P. borchgrevinki, T. bernacchii) or anesthetized in MS-222 (D. mawsoni)
before dissection.
P. angustata, or Maori Chief (1,580 ± 105 g,
n = 12), and Bovichtus variegatus, or
Thornfish (92.7 ± 21.7 g, n = 3), were caught by baited trap off the South Island, New Zealand. They were
transported back to the University of Canterbury aquarium and held
without feeding in recirculating, filtered seawater at 12 ± 0.5°C and a 12:12-h light-dark photoperiod for 1-2 wk before experimentation. All fish were killed by MS-222 overdose.
Physiology.
To complement previous data regarding cardiovascular control in vivo,
the relative degree of adrenergic and cholinergic tone was established
by monitoring heart rate (HR) and BP in P. angustata after
infusion of the muscarinic and -adrenergic receptor antagonists, atropine and sotalol, respectively. Afferent branchial arteries were
cannulated by the method described by Axelsson et al. (3). Pressures and HR were monitored using Bell and Howell type 4-327 pressure transducers and a Devices MX4 recorder and preamplifiers. Drugs were administered via the indwelling arterial cannulas. In three
of the five fish used, an initial single bolus injection of Epi was
given to check the pressor response, and a period of 30 min was allowed
before atropine injection for the adrenergic effect to dissipate.
Myography.
The ventral (VA) and DA aortae were exposed by removing the skin from
the floor and roof of the mouth, respectively. Vessels were exposed by
blunt dissection and ligatures applied to aid retrieval and prevent
recoil when sectioned. Afferent (ABA) and efferent (EBA) branchial
arteries were cut free some distance away from their origin with the
aortae to minimize variations in vessel composition. In some specimens,
the hypobranchial artery (Hypo) was located superficially with respect
to the ventricle and cut before its deep insertion into the branchial
vasculature. The subclavian artery arose immediately caudal to the
fifth EBA. Thus the vascular architecture was similar in all
notothenioid species so far examined (23). All vessels
were stored in physiological saline on ice (Antarctic species) or at
4°C (New Zealand species) and used over a 2-day period; no
differences in vessel response were noted between days 1 and
2, although a progressive decline in maximal tension was
observed in vessels from D. mawsoni over a period of 3 days
(data not shown).
We gently cleaned short lengths of vessel (~2 mm) of connective
tissue, being careful to avoid stretching the preparation, and mounted
them in a dual Mulvaney myograph chamber (type 410A, JP Trading) using
two strands of stainless steel wire (40-µm thick). Branchial vessels
thus prepared weighed ~0.4-0.6 mg (B. variegatus), 1.8-1.9 mg (P. angustata), 0.6-0.7 mg (T. bernacchii), 1.0 mg (P. borchgrevinki), and
4.2-4.8 mg (D. mawsoni). The saline composition was as
follows: 258 mM NaCl, 7.47 mM KCl, 4.05 mM CaCl2, 0.79 mM
MgCl2, 7.31 mM Na HEPES, 2.75 mM HEPES, 1 g/l glucose, pH
8.2 at 0°C (32) and was gassed with air before and
during the experiments. All experiments were performed in laboratories
with relatively constant ambient temperatures. The myographs had been
modified to contain a more extensive heat exchange circuit than
required for use with mammalian vessels, such that cooled water
circulating around the myograph compartments held the saline bathing
the vessels at either 1 ± 0.4°C (Antarctica) or 12 ± 0.2°C (New Zealand). Blood vessels were exposed to 2-4 mN
of tension, depending on type, for 1-2 h before exposure to drugs
until a stable baseline was established. During this incubation
period, the vessels were rinsed with saline twice, which appeared to
aid stability of the preparation. Limited time in the Antarctic and
scarcity of the fish species around New Zealand meant that we were
unable to run pairs of vessels with and without endothelium, and
consequently all data were gathered from intact vessels alone.
Pilot studies suggested, however, that endothelium-dependent dilatation
(in response to low doses of acetylcholine) was weak. The adrenergic response therefore represented the balance between -constrictory and
-dilatory influences, the extent of which was quantified by blockade
with the -antagonist sotalol. Cumulative dose-response curves were
constructed to determine vessel sensitivity to various agonists, and
the maximal tension generated compared with that following
depolarization with 50 mM KCl to assess the relative potency (% TKCl). Vessel sensitivity was expressed as the negative logarithm of agonist concentration that elicited a pEC50,
calculated using Prism (GraphPad Software).
Reagents.
Acetylcholine, L-epinephrine bitartrate, carbachol
(carbamylcholine chloride; CBC), L-isoprenaline bitartrate,
L-norepinephrine bitartrate, and serotonin (5-HT) were
obtained from Sigma (St. Louis, MO) and sotalol hydrochloride from
Bristol Myers Squibb (Copenhagen, Denmark). Inorganic chemicals were of
reagent grade or better.
Statistical analysis.
Data are presented as means ± SE (number of animals).
Changes in tension were analyzed by repeated-measures ANOVA,
other data by factorial ANOVA and protected least significance
difference post hoc test used to assess the level of significance.
| |
RESULTS |
Cardiovascular tonus in notothenioid fishes.
BP and HR were measured in five fish with an average mass of 1.956 ± 0.186 kg. After muscarinic and adrenergic blockade, the intrinsic HR
in P. angustata was 47.3 ± 1.8 beats/min at 12°C; the cholinergic and adrenergic tonuses of the heart were calculated to
be 35.1 ± 9.1 and 15.5 ± 2.5%, respectively. Resting VA BP averaged 4.02 ± 0.25 kPa. Before injection of the antagonists, a
single bolus injection of Epi (104 M at 100 µl/kg)
raised resting HR by 28.7 ± 10.2% and provoked a mean rise in BP
of 51.2 ± 5.9%.
Maximal vessel tonus.
The mass of 2-mm branchial vessel rings used in myography scaled with
body mass, presumably reflecting wall thickness, showed a sevenfold
range among species. However, the maximal specific tension generated
during KCl contraction was greater in the species with the lower body
mass, indicating that a greater proportion of the vessel was comprised
of noncontractile material as wall thickness increased (Table
1). For example, in P. angustata, Hypo, DA, and VA generated 5.37 ± 0.92, 1.57 ± 0.32, and 0.77 ± 0.09 mN/mg, respectively. Branchial arteries
from the two temperate species exhibited greater KCl contraction than
from the Antarctic species.
Vascular reactivity in Antarctic notothenioids.
Branchial vessels of T. bernacchii had different vascular
reactivities that revealed a weak adrenergic vasoconstriction with similar - and -mediated potencies, e.g., the EBA showed 22.6 ± 9.3% of the KCl response with NE and 38.1 ± 9.3% relative
potency of KCl (TKCl) in the presence of 103M
of the -blocker sotalol. Vascular reactivity was qualitatively similar in the ABA, being 8.8 ± 1.7 vs. 19.0 ± 4.1% TKCl, respectively (Table
2). This indicates, respectively, a 55 and 42% -adrenergic component of the intact vessel response to NE.
Carbachol, the stable analog of acetylcholine, was similarly potent
(21.6 ± 11.1% TKCl) but less sensitive
(pEC50 = 4.5 ± 0.4 M) than NE (Tables 2 and
3). The dominant agonist in ABA was 5-HT
but showed a similar responsiveness to that of NE in EBA (Table 3),
whereas the potency was similar for both EBA and ABA of ~30%
TKCl (Table 2).
To compare these data with those from a previous study on P. borchgrevinki (26), we also examined the relative
potency of the agonist response normalized to KCl contraction. This
revealed a greater difference between potential cholinergic and
serotonergic tonus in EBA of 15.5 ± 7.1 and 70.1 ± 15.2%
TKCl, respectively. The vasoconstrictor response to 5-HT
was biphasic, giving a weak dilatation at low (109 to
108 M) concentrations, with a peak constrictor response
around 105 M, followed by tachyphylaxis at higher doses
(Fig. 1).
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Fig. 1.
Cumulative dose-response curves of afferent (ABA) and
efferent branchial arteries (EBA) from the temperate and Antarctic
notothenioids, Paranotothenia angustata (A
and C) and Trematomus bernacchii (B
and D), for the dominant agonists norepinephrine (NE;
A and B) and serotonin (5-HT; C and
D).
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Vascular reactivity in cold temperate notothenioids.
Qualitatively, both P. angustata and B. variegatus were similar to T. bernacchii in agonist
sensitivity but showed a greater difference in vessel potency among
agonists. P. angustata in particular showed surprisingly
little differentiation among vessel types for agonist potencies, which
were in the order cholinergic > serotonergic > adrenergic
(Table 2), and sensitivities were in the order serotonergic > adrenergic > cholinergic (Table 3). The variability among VA, DA,
and Hypo potency for 5-HT (60.21 ± 11.97, 23.21 ± 2.52, 62.88 ± 8.28% TKCl) and carbachol (33.62 ± 8.63, 66.44 ± 4.78, 83.25 ± 4.05% TKCl), as
well as sensitivity for 5-HT (6.31 ± 0.04, 6.44 ± 0.24, 5.87 ± 0.12 M) and carbachol (4.61 ± 0.12, 4.18 ± 0.17, 4.91 ± 0.12 M, respectively) are probably of little
functional relevance. Noradrenergic sensitivity (pEC50 = 5.5 ± 0.3 M) and potency (8.5 ± 1.4% TKCl)
of EBA were both less than those of T. bernacchii.
Application of sotalol also revealed a similar influence of - and
-adrenergic influences on branchial vascular reactivity (Table
4), increasing vessel tonus to 38.1 ± 9.3% of the maximal KCl response. The cholinergic response was
again insensitive (pEC50 = 4.9 ± 0.2 M), but in
this species was very potent (65.8 ± 9.3% TKCl). The
vasoconstrictor response to 5-HT was more clearly biphasic, giving a
weak dilatation (2-10% of baseline tonus) at low concentrations,
with a peak constrictor response ~104 M (Fig.
2). The vessel constriction was blocked
by the 5-HT1/5-HT2 receptor antagonist
methysergide, although we were unable to block the dilator response
that seems to be the dominant systemic effect of 5-HT infusion
(40). Interestingly, there was also a greater potency for
5-HT in P. angustata than that seen in T. bernacchii (55.0 ± 4.9% TKCl). In two
specimens, the influence of other potential agonists was explored.
Isoprenaline gave an insensitive and weak dilatation (<2% KCl
response), whereas 107 M endothelin-1 (ET-1) was very
potent and produced tensions of >200% TKCl for both ABA
and EBA.
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Fig. 2.
Representative 5-HT dose-response curves for ABA of
P. angustata, illustrating the variability of the
vasodilatation at low concentrations.
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B. variegatus was similar to P. angustata in that
both CBC and 5-HT were more potent agonists than NE (Tables 2 and 4). However, in EBA, the -adrenergic component was higher (80.4 ± 1.4% of the KCl response, n = 5), and in ABA, 5-HT
sensitivity was greater (pEC50 = 7.0 ± 0.1 M)
than in the corresponding vessels of P. angustata. The
reciprocal potency of the adrenergic and serotonergic systems among
branchial vessels was most evident in this species (Fig.
3).
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Fig. 3.
Reciprocity in adrenergic (A) and serotonergic
(B) vasoconstriction among branchial vessels of
Bovichtus variegatus. *P < 0.05, **P < 0.01 between vessel type.
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Influence of ecotype on vascular reactivity.
In contrast to the four benthic notothenioids studied (above), EBA of
the pelagic D. mawsoni had a weak constrictor response to
5-HT (5.5 ± 0.6% TKCl) and CBC (9.2 ± 8.2%
TKCl). These vessels showed a decline in tension with NE
(28.2 ± 9.1% TKCl) and isoprenaline (80.3 ± 39.7% TKCl), with a pEC50 for isoprenaline
of 6.22 ± 0.40 M, suggesting a -dilator dominated branchial
vascular reactivity (Table 4 and Fig.
4).
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Fig. 4.
The response of branchial vessels for D. mawsoni to major agonists, showing a relatively greater potency of
cholinergic to serotonergic vasoconstriction and a dominance of
adrenergic vasodilatation in EBAs.
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DISCUSSION |
At any given hematocrit, the blood in fishes living at subzero
temperatures will have a higher viscosity than in temperate zone
species; this higher viscosity increases the afterload on the cardiac
pump and might limit tissue perfusion. It is reasonable to
suppose that, under these conditions, fishes living in the extremely
cold waters around Antarctica developed a low vascular resistance to
offset increased blood viscosity and hence reduce the power output
required of the heart. Notothenioids appear to have a lower peripheral
resistance compared with other fishes (3, 23). Indeed,
this may be necessary, given the failure of the cardiac pump at
relatively low afterload (43). Although blood viscosity
has been reported to be unusually low (44), selection
pressure is unlikely to have acted on this parameter to maximize
cardiovascular performance (18). Possibly of greater importance is the larger vessels found within the systemic circulation (23) and the control of vascular reactivity that underlies
peripheral resistance. Previous data suggest that there has been a
downregulation of adrenergic control during the radiation of the
notothenioids, both in terms of catecholamine release and weak in vivo
responses to exogenous catecholamines, relying more on neural than
humoral control of the cardiovascular system (21).
However, redistribution of cardiac output during the functional
hyperemia accompanying exercise (20) demonstrates that
control is effective, and this has been shown to be mainly due to a
cholinergic tonus (4). In contrast to other teleosts
subjected to forced swimming, these species actually show an increased
branchial resistance (4). This leads to the paradoxical
conclusion that a dominance of vasoconstrictory responses in the
control of both branchial and systemic resistances is associated with
the subzero temperatures at which the animals are found
(26).
Central cardiovascular control.
It is possible that the unusually high degree of oxygen solubility in
polar waters may have altered the nature of centrally mediated
temperature compensation of HR, i.e., the degree of cardiorespiratory interaction. In contrast to other species, resting HR is determined by
a very high inhibitory cholinergic tone of 55% in P. borchgrevinki and a massive 80% in T. bernacchii
(3), with the lower value associated with higher HR and BP
in the more active species. At 47 beats/min, the intrinsic HR in the
benthic temperate species, P. angustata, was more than
double that of the benthic Antarctic species, T. bernacchii,
which had a corresponding value of 22 beats/min at 0°C
(3). The expected relative tachycardia in line with the
12°C environmental temperature difference resulted in an effective
interspecific Q10 of ~1.8, which is comparable to the
value obtained for P. borchgrevinki of 1.96 by Axelsson et
al. (3) over a narrower range of acclimation temperature from 1 to +5°C. Resting VA BPs in P. angustata were
higher than those in P. borchgrevinki and T. bernacchii, but this difference could reflect the smaller mass of
the Antarctic species. The cholinergic tonus on the heart of P. angustata at 35% is considerably less than that of P. borchgrevinki and T. bernacchii, which supports the
notion that there is an unusually high cholinergic tone of 50-80%
on the heart of Antarctic fishes (12). However, we need to
be aware that a second study (4) of the two Antarctic
species gave resting HRs that were almost double that of the earlier
study, which suggests that holding conditions could influence the
degree of cholinergic tonus. The adrenergic tonus in P. angustata was intermediate between that of the two Antarctic
species (3). Although the adrenergic tonus on the heart
was not high, the response to a bolus injection of Epi indicated that
circulating catecholamines can exert a marked effect on the
cardiovascular system of P. angustata. However, the marked
reduction in synthetic capacity for catecholamines among notothenioids
suggests that this is likely to be a control mechanism invoked only in
extremis (45). There was little variability in BP within
species following pharmacological blockade, demonstrating an effective
accommodation of altered HR by adjustments in cardiac contractility
and/or vascular resistance, and providing evidence for a barostatic
compensation typical of most vertebrates.
Peripheral cardiovascular control.
Acute cold exposure decreases sensitivity to vasoactive compounds,
which in extreme cases may as a consequence lead to shock due to poor
control over peripheral resistance. In mammals we showed that
adaptation can occur on chronic cold exposure, of particular interest
being the recovery of an effective adrenergic constrictor response
(9). It is therefore reasonable to assume that there would
be no impairment of vascular reactivity in animals that live in
permanently cold environments and that the mechanisms involved may
offer some insight into the limitations on tissue perfusion during
sustained hypothermia. The established role of BP regulation by the
sympathico-adrenal system of vertebrates is evident in fishes, where
circulating catecholamines released from chromaffin tissue and
autonomic nerves affect both HR and vascular resistance
(5). Gill blood flow is regulated by vasoconstriction due
to both vagal cholinergic and sympathetic adrenergic fibers, acting via
muscarinic and -adrenoreceptors, respectively. Vasodilatation, or
decreased vascular resistance, is mediated via -adrenoreceptor stimulation. Other substances that are known to affect active control
in fishes are the purines, neuropeptides and serotonin (5-HT)
(33). We should note that both afferent and
efferent branchial arteries are relatively large conductance vessels
and should not be considered resistance vessels. Xu and Olsson
(46) reported greater sensitivities of isolated vessels to
catecholamines than was found in perfused splanchnic vascular beds of
rainbow trout, reflecting this difference. Our experiments were
designed to be directly comparable with published work on the branchial vessels of teleost fish, where smaller vessels have received little attention.
The presence of both adrenergic and cholinergic control of the
branchial vasculature has been demonstrated in P. borchgrevinki (4). The dominance of - over
-adrenoreceptor responses in these highly stenothermal species may
reflect an extension of the seasonal plasticity in eurythermal species
from temperate latitudes, which show a transition in relative
importance of - to -adrenoreceptor subtypes from summer to winter
(38). Both agonist systems, however, were less potent than
monoamine-induced vasoconstriction by 5-HT (26).
Serotonergic storage sites in gills appear to be ubiquitous among
fishes and, in P. borchgrevinki, may play a significant
nonadrenergic, noncholinergic role in branchial vascular control
(40). We wished to establish the extent to which these
unusual features of Antarctic notothenioids represent adaptation to
life in very cold waters.
The similar - and -adrenergic potencies in the branchial arteries
of T. bernacchii do not follow the pattern of dominant winter constrictor vs. summer dilator influences, nor do they indicate
an enhanced vascular -adrenergic response at low temperatures (7, 25). However, -adrenergic vasoconstrictor activity
predominates over -adrenergic vasodilatation in efferent branchial
vessels of the cryopelagic Antarctic species P. borchgrevinki (26) and in the EBAs of the temperate
species B. variegatus, suggesting that low environmental
temperature per se cannot have exerted the primary selection pressure
for differential adrenoreceptor expression. A complementary sensitivity
of the afferent and efferent branchial vessels to vasoactive agents may
be expected to be functionally important, with a postbranchial
vasoconstriction increasing lamellar BP and prebranchial vasodilatation
resulting in lamellar recruitment, potentially delivering more highly
oxygenated blood to active tissue (24, 34). The differing
responses among sympatric species may therefore be a result of ecotypic
modification of a basic notothenioid phenotype.
The dominant agonist in T. bernacchii is 5-HT, being 1.4- and 3.7-fold more potent than NE in EBA and ABA, respectively. As with
the adrenergic response, so the serotonergic vasoconstrictor activity
is equally potent on both ABA and EBA. This is contrary to the usual
teleost pattern of controlling branchial oxygen uptake by regulating
outflow (via EBA resistance) rather than inflow (ABA tonus), as was
found in P. borchgrevinki (26), suggesting that
the low metabolic rate of Antarctic fishes (11) may have reduced the functional requirement for peripheral vascular control. The
relative serotonergic dominance may represent a parallel with the
cold-induced increase in 5-HT receptor efficacy in mammalian vessels
(8, 28), although the magnitude of the response is surprising. Although 5-HT is an apparently ubiquitous vasoactive agent
among vertebrates, the Antarctic teleosts show a sensitivity that is
1,000-fold greater than that found in trout (41), which represents an impressive upregulation of the serotonergic system in
these animals (40).
Given the importance of cholinergic control in regulating
cardiovascular performance (3, 4) and gill perfusion
(26, 31), it was not surprising that acetylcholine and its
stable analog (CBC) were similarly potent as 5-HT, although the
response was less sensitive. That the relative potency of 5-HT was 50% lower in P. borchgrevinki than T. bernacchii
again suggests that ecotype may play a modifying role. In addition, in
vivo estimates of branchial resistance demonstrated both -adrenergic
and cholinergic vasomotor tonus, -adrenergic-mediated vasodilator
mechanisms, and possible involvement of the renin-angiotensin system
(4). However, such data reflect a balance between the
effect of agonists/antagonists on both cardiac myocytes and vascular
smooth muscle. Examining the potential vascular component by means of
ex vivo myography, Forster et al. (26) demonstrated a low
cholinergic sensitivity and the absence of an angiotensin II effect on
branchial vascular tonus. This suggests that an apparent downregulation
of adrenergic and upregulation of serotonergic vasoconstrictor activity
may be a common feature of the notothenioids. The important question is
whether or not this is an integral part of the cold adaptation that is
assumed to have occurred during endemic speciation. We addressed this
question by comparing data from related species to determine whether
the pattern of cardiovascular control may be an ancestral
characteristic (i.e., due to phylogeny) or whether it may correspond to
the particular lifestyle of these species (i.e., a consequence of ecology).
Phylogenetic patterns.
Although the stem nototheniids in Antarctica underwent speciation
isolated from the rest of the world's fish stocks once South America
separated from the Antarctic Peninsula and the circumpolar current
became established, we may be observing an ancestral characteristic that is unrelated to cardiovascular function in the cold.
To test this possibility we examined P. angustata, a related
species, possessing nonexpressed antifreeze genes (A. L. DeVries,
personal communication), presumed to have spread north during an
episode of global cooling and remained around the South Island of New Zealand when the ice retreated to Antarctica. We also investigated B. variegatus as an example of the phylogenetic sister group
to the notothenioids, the Bovichtidae, which lack antifreeze genes, and
hence we can assume that this family has not experienced subzero temperatures in its evolutionary past (17). The similar
inter- and intraspecific Q10s for cardiac function is
indicative of a common form of cardiovascular control. In addition,
despite having core temperatures some 12-14°C warmer than
T. bernacchii, the vasoconstrictor response to various
agonists were qualitatively similar. In particular, there was
strikingly little differentiation among vessel types examined. This may
be interpreted as a reduced reliance on vasoconstrictor control in a
species at the northern limit of its distribution, demanding a low
peripheral resistance to accommodate the high cardiac output associated
with an elevated metabolic rate. The biphasic response to 5-HT was a
repeatable phenomenon, even though the initial vasodilatation was
modest. This may represent an efficient mechanism by which a vascular bed could change from a low- to high-resistance channel, by increasing production of a tonically released agonist rather than inducing the
production of a different metabolic pathway. 5-HT-induced vasoconstriction could be blocked by the general
5-HT1/5-HT2 receptor antagonist
methysergide, which in P. borchgrevinki was shown to be
a 5-HT2-dependent response (40). This
monoamine-induced branchial vasoconstriction appears to be widespread
among teleosts, being also described in cod, trout, and eel
(40), and, in Antarctic fishes at least, is likely to be a
significant component of the proposed excitatory nonadrenergic,
noncholinergic branchial vascular control (36).
Interestingly, the vasodilatory response cannot be ascribed to
5-HT1, 5-HT2, 5-HT3, or
5-HT4 receptor subtypes (40), although this
seems to be the major systemic effect of 5-HT infusion. Data for
agonist potency broadly resemble those for sensitivity, but with an
even greater potency for 5-HT than found in T. bernacchii,
suggesting that the notothenioid pattern of cardiovascular control has
not been greatly modified by environmental temperature. Perhaps the
strongest evidence for the influence of phylogeny being dominant over
the influence of environmental temperature is the powerful 5-HT
response in the afferent branchial arteries of B. variegatus.
Notothenioids are labriform swimmers during routine locomotion and
switch to subcarangiform mode only during short periods of burst
activity (2, 27). The functional demand of the pectoral muscles may have prompted retention of the primitive hypobranchial system of delivering oxygenated blood, a feature that is particularly well developed in the icefishes (23). The data for
specific tension development and reactivity to CBC and 5-HT from
P. angustata suggest that functional control of the
hypobranchial system may be similar to that of branchial arteries,
especially the ABA with which it has its anatomical origin. The
significance of this finding is that the hypobranchial system
may preserve blood flow to the locomotory pectoral muscles under
conditions of increased oxygen demand, where constriction of the EBA
would lead to restricted flow down the subclavian artery.
Interestingly, phylogenetic influences on vascular phenotype may be
seen. In the EBA there is a negative correlation between vasoconstrictor pEC50s of CBC and 5-HT and between
%TKCl of 5-HT and NE, but a positive correlation between
%TKCl of 5-HT and NE in ABAs (Fig.
5). However, a study of broader range of
ecotypes and species is required before too much is read into such
apparent relationships.
View larger version (11K):
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|
Fig. 5.
The relationship between agonist potencies in branchial
arteries, showing a positive correlation between the capacity for
serotonergic and adrenergic vasoconstrictor in ABAs among species.
|
|
Ecological considerations.
The species so far examined are largely bottom-dwelling, sit-and-wait
predators (29), the presumed ancestral type
(16), which might explain the homogeneity in
cardiovascular control. Even the cryopelagic species P. borchgrevinki is often found resting on ledges within the platelet
ice and so may not be continuously swimming, or even very active
(1, 27). To examine what influence a more active lifestyle
may have on the nature of cardiovascular control, we were very
fortunate to be able to collect EBA samples from D. mawsoni,
a rarely caught predator that lives off fish caught in open water.
Although we have data from only one class of vessel, the potential key
role in controlling systemic oxygen delivery makes the EBA appropriate
for such comparisons. These vessels in D. mawsoni show a
weak constrictor response to 5-HT but were very sensitive to the
selective agonist isoprenaline, i.e., the branchial vessels would
appear to be dominated by -adrenergic vasodilatation rather than
vasoconstrictor influences, as befits its active lifestyle. In
contrast, P. borchgrevinki had a much more powerful
serotonergic constrictor response and weaker isoprenaline-induced vasodilatation (26).
In conclusion, the unusual dominance of serotonergic over adrenergic
control of vascular resistance is unlikely to have developed as an
integral part of the process of cold adaptation, as it appears to
reflect evolutionary lineage rather than low environmental temperature.
Importantly, however, this basic pattern may be modified according to
the functional demand placed on the cardiovascular system.
Perspectives
Although much is known about how the cardiovascular system is
controlled at moderate to high body temperatures, we know relatively little about how it is maintained in the cold. This report details interspecific differences in vascular control within a group of fish
whose distribution includes an area of constant subzero temperature. It
is typically very difficult to distinguish between evolutionary adaptation (strictly, traits that confer reproductive fitness), phenotypic plasticity (responsive expression of different phenotypes), and apparent fitness (retention of ancestral characteristics not subjected to natural selection) in the face of environmental changes. Concern about the possible shifts in global climate make this a topical
issue and one of increasing importance if we are to be able to assess
the impact such changes are likely to have on species distribution and survival.
| |
ACKNOWLEDGEMENTS |
This work was supported by the Natural Environment Research Council
(UK) and Antarctica New Zealand, and the award of an Erskine Fellowship
(to S. Egginton) by the University of Canterbury. Professor A. DeVries
kindly provided specimens of D. mawsoni for sampling.
| |
FOOTNOTES |
Present and permanent address for S. Egginton: Department of
Physiology, University of Birmingham, Birmingham B15 2TT, UK.
Address for reprint requests and other correspondence: S. Egginton, Dept. of Physiology, Univ. of Birmingham Medical School, Vincent Dr., Edgbaston, Birmingham B15 2TT, UK (E-mail:
s.egginton{at}bham.ac.uk).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 May 2000; accepted in final form 4 January 2001.
| |
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ABSTRACT
INTRODUCTION
