No hemoglobin but NO: the icefish (Chionodraco hamatus) heart as a paradigm
1 Department of Pharmaco-Biology, University of Calabria, 87030, Arcavacata
di Rende, CS, Italy
2 Department of Cellular Biology, University of Calabria, 87030, Arcavacata
di Rende, CS, Italy
3 Department of Cellular and Molecular Biology, University of Perugia,
06126, Perugia, Italy
4 Zoological Station `A. Dohrn', Villa Comunale, 80121, Napoli,
Italy
* Author for correspondence (e-mail: tota{at}unical.it)
Accepted 13 July 2004
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Summary |
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Key words: nitric oxide, heart, Antarctic teleost, icefish, Chionodraco hamatus, myocardial performance, nitric oxide synthase (NOS), immunocytochemistry
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Introduction |
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While the pathway of NO formation is one of the oldest evolutionary
signaling systems in animals (Feelish and Martin, 1995), in fish there is
surprisingly scant information regarding the cardiocirculatory roles of NO. We
have documented direct local cardiac and vascular actions of NO in various
teleosts (Anguilla anguilla; heart, Imbrogno et al.,
2001,
2003
; branchial vasculature,
Pellegrino et al., 2002
;
Salmo salar; heart, Gattuso et
al., 2002
; icefish Chionodraco hamatus; branchial
vasculature, Pellegrino et al.,
2003
). The Antarctic teleost, the hemoglobinless Chionodraco
hamatus, is an intriguing animal with which to explore aspects of unity
and diversity regarding the physiological roles of NO. This species is member
of the family Channichthyidae or icefish. Icefish belong to the cold-adapted
diversification of the perciform suborder Nototheniodei, the dominant fish
group, in terms of both abundance and number of species, of the Antarctic
ichthyofauna. They are characterized by extreme stenothermia
(Hofmann et al., 2000
) and
evolutionary loss of haemoglobin (Hb;
Ruud, 1954
) as well as, in
some species, cardiac myoglobin (Moylan
and Sidell, 2000
). The absence of Hb appears not to be crucial for
the animal's fitness in the highly oxygenated and thermally stable Antarctic
habitat (i.e. the `blind cave fish phenomenon';
Somero et al., 1998
). Multiple
coupled compensations at different levels of organization allow for efficient
oxygen delivery to icefish tissues. A notable example of compensation at the
subcellular level is the dramatic proliferation of mitochondria in the
myotomal (Egginton and Sidell,
1989
) and cardiac (Johnston
and Harrison, 1987
; Tota et
al., 1991a
; O'Brien and
Sidell, 2000
) muscle. At the systemic level, the
cardio-circulatory compensations appear to be crucial
(Hemmingsen et al., 1972
;
Tota et al., 1991b
), and
include a low-resistance vascular tree, large blood volume (24 times
greater than in red-blooded teleosts) coupled with remarkable large
heart-to-body mass ratio (Johnston et al.,
1983
; Tota et al.,
1991a
), and a cardiac output (CO) greater than that of most fishes
(Tota et al., 1991b
). This
large CO is attained with impressively large stroke volume
(VS), displaced at high flow rates, low heart rate
(fH) and low ventral aortic pressure
(PVA) (Hemmingsen et
al., 1972
; Tota et al.,
1991b
). Accordingly, the icefish heart works as a typical volume
pump (Tota and Gattuso, 1996
).
Conceivably, in view of its modulation of myocardial inotropism, relaxation
and energetics in the mammalian heart
(Shah and McCarthy, 2000
),
intracardiac NO could have a paracrine/autocrine role for optimising such
specialized volume pump function in the icefish. Furthermore, at the whole
organismal level, it is of interest to explore NO functions in the icefish,
since in this animal one of the major mechanisms for disarming NO bioactivity
(the NOHb reaction) is lacking.
In this study, we used an isolated and perfused working heart preparation of the icefish C. hamatus to demonstrate the responses to NO-donors and inhibitors, together with the release of NO (in terms of nitrite) detected by electrochemical assay in the cardiac effluent. Moreover, we have documented the presence of cardiac NOS by morphological methods (i.e. NADPH-diaphorase activity and immunolocalization). On the whole, these results demonstrate that NO, produced within the heart, directly modulates myocardial performance through a cGMP-mediated mechanism, thus emphasizing the potential for nitrergic autoregulation of cardiac function in icefish.
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Materials and methods |
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European eels Anguilla anguilla L. (N=7) of both sexes, weighing 229±31 g, were provided by a local hatchery and kept at room temperature (1820°C) without feeding for 56 days.
Isolated and perfused working heart preparation
The animals were anaesthetized in benzocaine (0.2 g l1)
for 15 min. Each animal was opened ventrally behind the pectoral fins. The
ventral aorta was cannulated and the heart removed without the pericardium and
placed in an ice-chilled dish filled with saline for the atrium cannulation
procedure. A polyethylene cannula was secured in the atrium at the junction
with the sinus venosus. The cannulated heart was transferred to a perfusion
chamber filled with saline and connected with a perfusion apparatus (as
described by Tota et al.,
1991b). Perfusion was immediately started; the heart received
saline from an input reservoir pumped against an afterload pressure given by
the height of an output reservoir. Isolation time was 5 min. In these
experiments the chamber was designed to allow the development of subambient
pressures during ventricle contraction, thus permitting suction filling of the
atrium, which is an important mechanism operating in teleosts
(Farrell et al., 1988
). The
chamber was completely filled with perfusate and covered with an unsecured
Plexiglas lid (Fig. 1A). A thin
layer of neoprene, placed between the large upper lid of the main part of the
chamber and the lid, allowed slow capillary movements of the medium into and
out of the chamber in response to the volume changes of the heart during its
cycle. It is important to use this type of chamber for isolated heart
preparations in those teleosts where it is difficult to isolate the heart with
an intact pericardium in a reasonably short time. The parietal pericardium in
the icefish is firmly connected to the perimisium of the muscles surrounding
the heart (Tota et al.,
1991a
).
|
All the experiments were performed in a cold thermostatted cabinet (LKB
2021 Maxicoldlab, Malbo, Sweden), which allowed the heart and perfusion system
to be maintained at near zero temperatures. The fish were weighed before each
experiment and the blotted wet masses of whole hearts (atrium, ventricle and
bulbus) and ventricles were determined at the end. For the detection of NO in
the cardiac effluent (see below), we used for comparison a similar in
vitro isolated and perfused working heart preparation from a temperate
teleost counterpart, i.e. the European eel Anguilla anguilla (for
details, see Imbrogno et al.,
2001).
Saline
The icefish perfusate was a modified version of Cortland saline
(Wolf, 1963), with an
increased NaCl content to bring the ionic concentration up to the level found
in channichthyid blood (Holeton,
1970
). Its composition (in mmol l1) was: NaCl
252.4, KCl 5.0, MgSO4 7H2O 2.0, dextrose 5.56,
CaCl2 2.3, NaH2PO4 H2O 0.2,
Na2HPO4 2H2O 2.3. The saline was aerated and
the pH adjusted to 7.84 at 1°C, by using about 0.5 g of
NaHCO3.
The composition of the eel perfusate (in mmol l1) was:
NaCl 115.17, KCl 2.03, KH2PO4 0.37, MgSO4
2.92, (NH4) 2SO4 50, CaCl2 1.27, glucose
5.55, Na2HPO4 1.90; pH was adjusted to 7.77.9 at
room temperature (1820°C), by using 1 g of NaHCO3
(Imbrogno et al., 2001). The
saline was equilibrated with a mixture of 99.5% O2:0.5%
CO2.
Measurements and calculations
Hearts were stabilized under basal conditions for 1520 min, before
drug treatment. Pressure was measured through T-tubes placed immediately
before the input cannula and after the output cannula, and connected to two
MP-20D pressure transducers (Micron Instruments, Simi Valley, CA, USA) in
conjunction with a Unirecord 7050 (Ugo Basile, Comerio, Italy). Pressure
measurements (input and output; expressed in kPa) were corrected for cannula
resistance. Heart rate was calculated from pressure recordings. Cardiac output
was collected over 1 min and weighed; values were corrected for fluid density
and expressed as volume measurements. The afterload (mean aortic pressure) was
calculated as 2/3 diastolic pressure + 1/3 maximum pressure. Stroke volume
(VS = cardiac output/heart rate; ml kg1)
was used as a measure of ventricular performance; changes in stroke volume
were considered inotropic effects. Cardiac output and stroke volume were
normalized per kg wet body mass. Ventricular stroke work
(WS = afterload preload x stroke
volume/ventricle mass; mJ g1) served as an index of systolic
functionality.
Experimental protocols
Basal conditions
Isolated perfused icefish hearts were allowed to equilibrate to conditions
simulating an in vivo resting state for up to 1520 min.
Baseline hemodynamic parameters are listed in
Table 1. In all experiments the
control conditions were a mean output pressure of about 1.4 kPa, with a
cardiac output set to 50 ml min1 kg1 wet
body mass by appropriately adjusting the filling pressure. The heart generated
its own rhythm. Cardiac parameters were simultaneously measured during
experiments. Hearts that did not stabilize within 20 min of perfusion were
discarded. The experiments were done at 3°C and were completed within 2 h;
when perfused at constant temperature the isolated heart performance was
stable for at least 3 h (Tota et al.,
1991a).
|
In the case of the eel heart preparation, the experiments were performed at 18°C.
Test condition
After the 15 min control period, the treated hearts were perfused for 20
min with Ringer's solution enriched with either L-arginine or
3-morpholinosydnonimine (SIN-1) or SIN-1 plus superoxide dismutase (SOD) or
L-N5-N-iminoethyl-L-ornithine
(L-NIO) or 8-bromo-guanosine 3'5'-cyclic monophosphate
(8Br-cGMP) or 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ). To construct
a concentrationresponse curve (L-arg, SIN-1), the
preparation was exposed for 20 min to physiological saline containing a given
drug in increasing concentrations. Each preparation was tested for at most
three concentrations of the drug. In the case of pre-treatment with superoxide
dismutase, the preparations were exposed to this agent for 20 min before and
during SIN-1 treatment. SIN-1 was used in a darkened perfusion apparatus to
limit drug degradation.
Data analysis and statistics
The results are expressed as means ± S.E.M. Because each
heart represented its own control, the statistical significance of differences
was assessed on parameter changes using the paired Student's t-test
(P<0.05). Percentage changes were evaluated as means ±
s.e.m. of percent changes obtained from individual experiments.
Drugs and chemicals
All the solutions were prepared in double-distilled water (except ODQ,
prepared in ethanol); dilutions were made in Ringer's solution just before
use. L-arginine, SIN-1, SOD, L-NIO, ODQ and 8Br-cGMP were purchased
from Sigma Chemical Company (St Louis, MO, USA).
Determination of nitrite by electrochemical assay
The electrochemical apparatus used in this work has been previously
described (Palmerini et al.,
1998,
2002
). The assay measures NO
and its derivatives (nitrosothiols and nitrite) in the nmolar range. Briefly,
it consisted of a reaction vessel (5 ml) equipped with an injector, maintained
at a constant temperature and supplied with a flow of N2 at 5 ml
min1. NO formed in the vessel was carried by the
N2 to an amperometric sensor
(Fig. 1A), where NO was first
transformed into NO2 using a small trap filled with an acidic
solution of permanganate (50 mmol l1 KMnO4, 0.5
mol l1 HClO4). The presence of NO in the reaction
vessel was recorded vs time t as a peak of electric current
I. The apparatus was calibrated by injecting known amounts of
standard nitrite solution into the reaction vessel, which also contained 0.1
mol l1 CuCl2 and 0.1 mol l1
cysteine in a final volume of 2 ml. The lowest detectable amount of NO was
510 pmol. To measure nitrites, 100 µl aliquot samples of the
specimen, i.e. either cardiac perfusate or plasma or hemolysate from icefish
or eel, were injected into the reaction vessel of the electrochemical
apparatus, which also contained 0.1 mol l1 CuCl2
and 0.1 mol l1 cysteine. Specimens of plasma and hemolysate
were obtained from heparinized blood collected from the caudal vein. Plasma
was obtained following centrifugation at 800 g; the pellets
were suspended in hypotonic solution and then centrifuged at 10 000
g to remove cell debris. Cardiac perfusates were obtained
during perfusion experiments: after stabilization, cardiac perfusates of eight
icefish and five eel hearts were collected (20 ml) and immediately stored at
80°C before use. Data are presented as mean ±
S.E.M. (N=5 determinations).
NOS localization
NADPH-diaphorase
The protocol for the NADPH-diaphorase histochemical method for localizing
NOS activity was modified by Hope et al.
(1991). The hearts of three
C. hamatus were removed immediately after sacrifice, flushed with
ice-cold saline and fixed in 4% paraformaldehyde solution for 57 h. The
tissues were cryoprotected by infiltration with 30% sucrose solution for
34 days. Then they were embedded in an optimal cutting temperature
(OCT) compound, rapidly frozen in liquid nitrogen and cut at the level of the
ventricle using a cryostat (Microm HM505E; Walldorf, Germany). The transverse
ventricular sections (5 µm thick) were postfixed in phosphate buffer (PB)
enriched with 4% paraformaldehyde for 30 min. The sections were then incubated
for 1 h at 37°C in Tris-HCl (0.1 mol l1, pH 7.5)
containing Triton X-100 (0.3%), Nitro-blue Tetrazolium (NBT, 0.6 mmol
l1), NADPH (1 mmol l1) and sodium azide (1
mmol l1). Parallel sections were incubated in the same
buffer without NADPH as a control. After incubation, the ventricular tissue
was observed under an optical photomicroscope (ZEISS Axioscope; Thornwood, NY,
USA).
Immunofluorescence
Immediately after sacrifice, the hearts of three C. hamatus were
removed, washed in ice-cold saline solution and blocked in diastole by
perfusion with a high potassium hyperosmotic saline. The tissues were embedded
in OCT, fixed in liquid nitrogen and stored at 80°C until use.
Frozen sections (7 µm thick) were cut at the cryostat (Microm HM505E),
postfixed with acetone for 10 min and stored at 20°C. Before
immunostaining, the slides were washed with Tris-HCl buffered saline (TBS),
and then incubated with 1:100 anti-eNOS or anti-iNOS antibodies (mouse
monoclonal, FITC-conjugated; BD Transduction Laboratories, Lexington, KY, USA)
for 24 h at 4°C. To stop the reaction the sections were washed with TBS
then the slides mounted with mounting medium (Vector Laboratories, Burlingame,
CA, USA) and observed under a confocal laser microscope (TCS-SP2, Leika
Microsystem, Wetziar, Germany). Negative controls were obtained using parallel
ventricular sections treated in the same manner, excluding antibodies (data
not shown).
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Results |
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NOcGMP mechanism
Chronotropic effect
As shown in Table 2, in
perfused icefish heart preparations, NO (L-arg, SIN-1, SIN-1+SOD)
and cGMP (8Br-cGMP) donors, the NOS inhibitor L-NIO, and the sGC
inhibitor ODQ did not affect the heart rate. These data suggest that in the
icefish NO is not involved in the regulation of heart rate.
|
Inotropic effect
The doseresponse curve of L-arginine, the authentic
NO-synthase substrate, at concentrations from 107 to
105 mol l1, shows a significant positive
inotropism at 106 mol l1 and
105 mol l1
(Fig. 2). When
L-lysine was used as control
(Amrani et al., 1992), there
was no effect (data not shown). Treatment with the NOS inhibitor
L-NIO (105 mol l1) induced a
significant negative inotropism (Fig.
4). Of the two NO donors, SNP and SIN-1, SNP
(105 mol l1) induced a positive inotropic
effect, similar to L-arginine (data not shown), while the
doseresponse curve of SIN-1
(107105 mol l1)
(Fig. 3A) showed significant
negative inotropism at 105 mol l1. It is
known that SIN-1 simultaneously generates NO and superoxide anions, which
react instantaneously to form peroxynitrite
(Beckman and Koppenol, 1996
).
Therefore, to evaluate and clarify the SIN-1 negative inotropic effect, we
tested the effects of SIN-1 (105 mol l1)
in the presence of SOD (10 i.u. ml1), an antioxidant enzyme
that competes with NO binding to the superoxide anions. The negative inotropic
effect of SIN-1 was completely reversed in the presence of SOD
(Fig. 3B), while SOD per
se had no effect on cardiac contractility (data not shown). Consequently,
we can state that the SIN-1 derived NO induces a positive inotropic effect,
similar to SNP and L-arginine. To evaluate cGMP-related mechanisms,
the cardiac preparations were exposed to either a stable and lipid-soluble
analogue of cGMP, 8Br-cGMP (105 mol l1) or
to a specific inhibitor of guanylate cyclase, ODQ (105 mol
l1). Administration of 8Br-cGMP elicited a significant
positive inotropic effect while ODQ elicited negative inotropism
(Fig. 4).
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Determination of nitrite
Nitrite concentrations in plasma and blood cell hemolysates were higher
than in the cardiac effluents and were comparable in both icefish and eel
(Table 3). In all samples,
nitrosothiols of both low and high mass were absent or undetectable.
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NOS localization
NADPH-diaphorase
An intense dark blue staining resulting from NADPH-diaphorase activity was
observed in the ventricular endocardial endothelium (EE) cells
(Fig. 5A, arrowheads). A less
intense reaction was observed in the cardiac muscle fibers
(Fig. 5A, arrow). The
specificity of this staining was confirmed by its complete absence in the
control sections (data not shown).
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Immunofluorescence
Morphological examination of C. hamatus ventricular sections
incubated with anti-eNOS or with anti-iNOS antibodies revealed an intense iNOS
labelling present throughout the ventricle. By contrast, eNOS immunoreactivity
was not detectable in this cardiac region (data not shown). As shown in
Fig. 5B, iNOS labelling was
densely localized in the cytoplasm of the myocardiocytes while it was absent
in the EE cells. The specificity of the binding was confirmed by staining of
negative controls.
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Discussion |
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NOcGMP mechanism
Chronotropic effect
NO, tonically released in the heart, may be involved in the regulation of
intrinsic heart rate FH by different mechanisms, including
cholinergic antagonism of the adrenergic-mediated positive chronotropism and
direct NO-cGMP-mediated stimulation of the If pacemaker
current (Musialek and Casadei,
2000, and references therein). Both NO-induced positive and
negative chronotropic effects have been reported in several mammals including
man (Musialek and Casadei,
2000
). In the isolated working frog Rana esculenta heart,
SNP elicited a negative chronotropic effect, while SIN-1 had no influence
(Sys et al., 1997
). In fish,
McGeer and Eddy (1996
)
reported positive chronotropism in the trout Oncorhynchus mykiss
exposed to SNP, while in Atlantic salmon embryos the same NO donor elicited a
negative chronotropic effect (Eddy et al.,
1999
). In the isolated and spontaneously beating icefish heart, we
have shown that both endogenous (L-arginine) and exogenous (SIN-1)
NO have no effect on heart rate. The lack of significant chronotropic effects
following exposure to either the NOS inhibitor L-NIO or the sGC inhibitor ODQ,
or the analogue of cGMP, 8Br-cGMP, suggests that in C. hamatus the
intrinsic FH is not influenced by NO-cGMP signalling. In
the same icefish heart preparation a negative chronotropism was induced by
L-NMMA (NG-monomethyl-L-arginine;
Pellegrino et al., 2003
). This
discrepancy may be related to differences between the two NOS inhibitors,
L-NIO being, in comparison with L-NMMA, more selective,
potent and per se without effect on FH
(McCall et al., 1991
). While
the cholinergic control of FH in several red-blooded
Antarctic teleosts appears well developed compared to most other species
(Axelsson et al., 1992
), the
situation in icefish may be different. For example, in the icefish C.
aceratus, Hemmingsen et al.
(1972
) found that atropine
produced only a slight increase in FH, consistent with a
relatively low vagal tone. Whether this intrinsic low FH
is related to the extreme stenothermia of this fish and/or to the constraints
of a low-speed volume pump cardiac design
(Tota and Gattuso, 1996
)
remains to be established.
Inotropic effect
Both the endogenous NOS substrate L-arginine and the exogenous
NO-donor SIN-1 (in the presence of SOD) elicited a significant
concentration-dependent positive inotropism. In particular, experiments where
SOD competed with NO binding to superoxide anions
(Beckman and Koppenol, 1996),
unambiguously indicate that SIN-1-derived NO induces a positive inotropic
effect, as with L-arginine. A similar positive inotropism elicited
by the NO-donor SNP was reported in the same isolated working C.
hamatus heart preparation (Pellegrino
et al., 2003
). The significant negative decrease of
VS and WS following both NOS and sGC
inhibition by their specific inhibitors L-NIO and ODQ, respectively, along
with the increased VS and WS elicited
by the GC donor 8Br-cGMP, are all consistent with the presence of a
significant NO-cGMP-mediated positive inotropic influence on the basal (i.e.
unstimulated) mechanical performance of the icefish heart. The continuous
generation of relatively low basal levels (nanomolar concentrations) of NO
from the beating heart is considered critical for the functional integrity of
the myocardial pump in mammals (Moncada et
al., 1991
; Pinsky et al.,
1997
). Both positive and negative inotropic actions of NO (and
cGMP) at the cardiomyocyte level have been reported in various preparations,
but the reasons for these opposite responses to NO, and the underlying
mechanisms, remain to be identified
(Massion and Balligand, 2003
,
and references therein). It is well acknowledged, however, that in any given
situation, these direct nitrergic effects depend, among other factors, upon
the amount of NO generated, the type and sub-localization of NOS isoenzyme
involved, the target tissue (e.g. atrial or ventricular cardiocytes), the
microenvironment (e.g. antioxidant status and prevailing redox balance) and
the animal species examined (Shah and
MacCarthy, 2000
). Of note, in the icefish the
NOcGMP-mediated positive inotropism contrasts with the
NOcGMP-mediated negative inotropism shown in the isolated working heart
preparations of teleost (Anguilla anguilla,
Imbrogno et al., 2001
;
Salmo salar, Gattuso et al.,
2002
) and amphibian (Rana esculenta,
Sys et al., 1997
) hearts.
These hearts exhibit the same trabeculated myoarchitecture and intracardiac
blood supply as the icefish heart (Tota
and Gattuso, 1996
) and were studied under identical experimental
conditions. Therefore, the opposite inotropic responses between the above
mentioned species and icefish to NO and cGMP cannot be attributed to the
experimental hierarchic level of investigation, nor to macroscopic differences
in cardiac structure; but they may indicate species-specific differences at
more subtle ultrastructural (e.g. mitochondria, intracellular NOS isoforms
localization), biochemical (e.g. phosphodiesterase types and activities) or
molecular (e.g. ion channel) levels. For example, we could speculate that the
very expanded mitochondrial compartment in icefish cardiomyocytes
(Fig. 1D) could reasonably be a
target of NO. In fact, NO can act as a competitive inhibitor of cytochrome
oxidase, with consequent decrease of mitochondrial respiration, while
increased peroxynitrite formation in mitochondria may cause calcium release
via the pyridine nucleotide dependent pathway (see
Giulivi, 2003
, and references
therein).
The icefish heart as source of NO
Determination of free NO in cells and biological samples using conventional
methods, normally within the nanomolar range, has been hampered by the
chemical instability of the molecule in water solutions (half-life about
35 s), where traces of oxygen transform NO into nitrite, thus
reflecting the amount of NO released by the tissues
(Beckman and Koppenol, 1996;
Palmerini et al., 2002
, and
references therein). Due to its low concentrations, however, measurement of
nitrite requires high-sensitivity techniques. For this reason, we previously
developed an electrochemical assay that allows the determination in biological
fluids of nitrite and nitrosothiols in the nanomolar range
(Palmerini et al., 2002
). The
method exploits a specific solid-state amperometric sensor for the rapid
determination of NO in its gaseous phase based on the reduction of nitrite
and/or nitrosothiols without any additional purification steps (Palmerini et
al., 1998
,
2002
).
By using a temperate fish (A. anguilla) as the counterpart to
C. hamatus, we found comparable concentrations of nitrite in plasma
and hemolysate of both icefish and eel. Nitrite was also present in the
cardiac effluents from both species (Table
3). This is the first demonstration that, like the mammalian heart
(Pinsky et al., 1997), the
contracting fish heart releases a substantial amount of NO under basal
conditions, challenging studies regarding alternative NO carriers in the
absence of Hb.
Morphological evidence of NOS
Using two different morphological methods (i.e. NADPH-diaphorase and
immuno-fluorescence) we have demonstrated NOS expression in the heart of
C. hamatus.
The NADPH-diaphorase histochemical method revealed an intense NOS activity in the EE cells lining the ventricular trabeculum and a less intense reaction in the ventricular myocardiocytes.
NOS immunolocalization in icefish was conducted using two mouse monoclonal
antibodies, anti-eNOS and anti-iNOS. In spite of the remarkable phylogenetic
distance between mouse and icefish, mouse anti-iNOS specifically bound the
icefish antigen, thus allow identification of the myocardial NOS as iNOS. The
intense staining observed in the ventricular EE cells with NADPH-diaphorase
was not confirmed by the application of anti eNOS or iNOS antibodies. Since EE
cells mainly express the eNOS isoform
(Brutsaert et al., 1998), we
can hypothesise that the lack of eNOS immunostaining is due to the low
specificity of the mouse antibody toward the icefish endocardial NOS isoform.
On the other hand, the absence of iNOS labelling in the EE cells may suggest
that the EE isoform is indeed eNOS. Due to the elevated binding specificity of
monoclonal antibody, the particular epitope of interest of the eNOS molecule
may have undertaken minor amino acid sequence modifications so as to be
unavailable for reaction (i.e. negative staining). Further studies should
explore the possibility that in the icefish the iNOS isoform could have been
conserved during evolution while the eNOS isoform could have encountered
structural modifications.
It is well known that iNOS is expressed under inflammatory and immune
stimuli (Vallance et al.,
2000). In the absence of any induced stimulation, our data suggest
a basal expression of this isoform in icefish ventricular myocytes. Recent
data have demonstrated cross-reactivity between antibodies directed against
different NOS isoforms, including that localized in the mitochondria (mtNOS;
Ghafourifar and Richter,
1997
). Since icefish ventricular myocardiocytes contain a notably
large mitochondrial compartment, it is possible that mtNOS contributes to the
observed high iNOS immunostaining. Taken together with the physiological
evidence for a tonic NOcGMP-mediated inotropism, these
immunofluorescence results suggest that a ventricular myocardial NOS isoform
(either iNOS and/or mtNOS) is involved in the basal release of NO in the
icefish heart effluent.
It has been suggested that mitochondrial NOS, as the source of NO acting as
competitive inhibitor of cytochrome c oxidase, can play an important
`local' role by helping average oxygen utilization between cells at different
distances from the capillaries (i.e. allowing the oxygen supply to diffuse
further into the tissue; Thomas et al.,
2001; Giulivi,
2003
). In icefish cardiomyocytes the increased mitochondria, often
arranged in clusters, have been viewed as oxygen conduits and are particularly
important for maintaining oxygen fluxes in species lacking Hb
(O'Brien and Sidell, 2000
).
Therefore, it is possible that in C. hamatus the presence of mtNOS in
the myocardiocytes could be an important molecular strategy for expanding the
mechanism by which these cells consume oxygen.
In conclusion, in the icefish, the nitrergic regulation of cardiac performance, the detection of nitrite as marker of NO released in the cardiac effluent, along with the morphological localization of cardiac NOS, all indicate an important role of NO in modulating heart function and, putatively, oxygen gradients in the absence of hemoglobin. These data are also of evolutionary interest since they suggest that the genetic retention of the NOS system in the cold-adapted Notothenioids has permitted retention of nitrergic effector functions located at the interface of organismal and molecular physiology, including molecular lines of defence against the hemoglobinless condition.
List of symbols and abbreviations
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Acknowledgments |
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References |
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