Perfusion of the isolated trout heart coronary circulation with red blood cells: effects of oxygen supply and nitrite on coronary flow and myocardial oxygen consumption
1 Institute of Biology, University of Southern Denmark, DK-5230 Odense M,
Denmark
2 Department of Biological Sciences, University of Naples Federico II,
Napoli, Italy
* Author for correspondence (e-mail: fbj{at}biology.sdu.dk)
Accepted 28 July 2005
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Summary |
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Key words: erythrocyte, haemoglobin, microcirculation, nitrite, nitric oxide, vasodilation
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Introduction |
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The purposes of the present study were to develop a method for RBC
perfusion of the trout heart coronary circulation and to evaluate the impact
of RBC perfusion on coronary flow and myocardial O2 consumption (as
compared with saline perfusion). Furthermore, we tested the hypothesis that
nitrite is converted to NO in the RBC-perfused trout heart coronary tree and
that this has an influence on coronary blood flow. NO of endothelial origin
has previously been implicated in vasodilatory mechanisms in trout coronaries
(Mustafa et al., 1997;
Mustafa and Agnisola,
1998
).
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Materials and methods |
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The fish were euthanized with a rapid stunning blow to the head, and a blood sample was taken from the caudal vessels in the tail with a heparinized syringe. Fish to be dissected were subsequently injected with 0.5 ml 100 IU ml-1 heparin into the caudal vessel.
Preparation of red blood cell suspensions for heart perfusion
Freshly drawn trout blood was centrifuged (4600 g, 5 min)
and the plasma was discharged. The RBCs were subsequently washed three times
in approximately seven times their volume of physiological saline. The saline
had the following composition (g l-1): 7.89 NaCl, 0.23 KCl, 0.23
MgSO4.7H2O, 0.016 NaH2PO4.
H2O, 0.28 Na2HPO4.2H2O, 0.37
CaCl2.2H2O, 1 glucose, 10 polyvinylpyrrolidone. This
saline was constructed to match the osmolality of plasma and has an increased
[NaCl] compared with earlier versions
(Farrell et al., 1986). The
measured osmolality of samples taken randomly throughout the experiments was
293.6±0.5 mOsmol kg-1 for plasma (N=11) and
292.3±0.5 mOsmol kg-1 (N=8) for the saline (means
± S.E.M.). Following the final wash, the RBCs were suspended
to the required haematocrit (Hct), and a minor amount of heparin was added
(1.3 IU ml-1 final concentration). The RBC suspension was placed in
two rotating glass tonometers, each containing 910 ml RBC suspension,
and equilibrated for 45 min at 15°C to a humidified gas mixture of
O2 (3 or 4%) and CO2 (0.5%), with N2 as the
balance gas. Gas mixtures were delivered from a Wösthoff (Bochum,
Germany) Digamix 5KM432X gas-mixing pump.
Preparation of hearts and the isolated heart perfusion set-up
The coronary circulation of a non-working, electrically paced isolated
heart was perfused under constant pressure. The heart was isolated and
cannulated as previously described
(Mustafa and Agnisola, 1994)
with some modifications. The heart was dissected out, and the coronary artery
was cannulated with a 5 mm-long, 0.30 mm outer diameter nylon tube. The atrium
was cannulated with a 2.5 mm-diameter, 14 mm-long cannula, which, outside the
atrium, continued into a 4.5 mm-diameter, 6 mm-long cannula. The ventral aorta
was cannulated with an occluded cannula. The coronaries were preperfused with
saline at a constant pressure (1 kPa) to wash out blood, and the heart was
mounted into a jacketed chamber maintained at 15°C
(Fig. 1). Next, the perfusion
with saline from a saline head pressure reservoir was started. The saline was
equilibrated to the same gas mixture as the RBCs
(Fig. 1). The mounted heart was
paced at a rate of 30 beats min-1 with two platinum electrodes
connected with a Grass S6 Stimulator (stimulus 10 V, 20 ms). Under these
conditions, the coronary perfusate drained into the atrium and then flowed out
from the atrium via the atrial cannula. An ISO-NOP electrode (World
Precision Instruments, Sarasota, FL, USA) was inserted through the atrial
cannula to continuously detect the NO levels in the effluent from the atrium.
The saline level in the chamber was kept constant by an overflow. Perfusate
outflow from the atrium was helped by the regular atrial beating induced by
electrical pacing. Because of the occluded aortic cannula, only the compact
layer of the ventricle wall was perfused in this preparation, leaving out any
possible contribution to NO release from the spongy myocardium.
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For RBC perfusions, the equilibrated RBC suspension was transferred from the tonometer to an RBC head pressure reservoir (Fig. 1) in a gas-tight Hamilton syringe. The atmosphere of the reservoir received gas from the gas-mixing pump to guarantee that the RBCs continued their gas equilibration. A three-way tap allowed the switch between saline and RBC perfusion (Fig. 1) and to draw the erythrocyte suspension from the reservoir directly to the coronary input upon initiation of RBC perfusion. To avoid RBC sedimentation in the reservoir, the RBC suspension was intermittently mixed by gently sucking it into the gas-tight syringe and re-injecting it into the reservoir.
Experimental protocols
In initial experiments, we tested perfusion of the trout heart coronary
circulation with RBC suspensions having Hct values of 15%. In some of
these experiments, the coronary resistance increased sharply after a while,
and the hearts developed a few visual dark spots, which was ascribed to
problems with precipitation or sedimentation of RBCs inside the vasculature.
These problems were not encountered at lower Hct, which accordingly was chosen
for the experimental protocols.
Protocol 1
Animal mass, 294±29 g; ventricle mass, 0.28±0.03 g (means
± S.E.M., N=6). In this protocol, the coronary
circulation was first perfused with saline at a specific input oxygen tension
(PO2). The preparation was allowed to stabilize
until the coronary resistance did not change between two successive
measurements made 5 min apart (which usually occurred within 1520 min).
Samples were then taken from the input and the atrium for perfusate
measurements (see below). The perfusion was subsequently shifted to RBC
suspension (typically having an Hct of 6%), and after a few minutes of
stabilization, input and atrium samples were taken, and pressure, flow and
resistance were determined. Finally, nitrite was added to the RBC suspension
in the RBC reservoir to an extracellular concentration of 400 µmol
l-1. The concentration was chosen to be lower than the millimolar
values in nitrite-exposed fish but higher than the low micromolar values in
fish from clean water (Stormer et al.,
1996
) to promote nitrite effects. Nitrite was added from a 140
mmol l-1 NaNO2 stock solution, and the amount added was
calculated taking into account the volume of RBC suspension and Hct. The
suspension was gently mixed (as above) and drawn into the input. After a few
minutes of perfusion with the nitrite-containing RBC suspension, further
samples were taken and measurements made.
Protocol 2
Animal mass, 315±51 g; ventricle mass, 0.31±0.04 g
(N=5). This protocol was the same as protocol 1, apart from the
presence of the nitric oxide synthase (NOS) inhibitor L-NA
(N-nitro-L-arginine; Sigma-Aldrich,
Steinheim, Germany) at a concentration of 10-4 mol l-1
in both the saline and the RBC suspension.
Protocol 3
Animal mass, 301±7 g; ventricle mass, 0.29±0.03 g
(N=3). This protocol involved saline perfusion in the absence and
presence of nitrite, to test the effect of nitrite on the preparation in the
absence of RBCs.
There were no significant differences in ventricle mass or animal mass between the three protocols. Samples from the input were typically taken and measured 23 min before samples from the atrium were taken.
Nitrite uptake and metHb formation in red blood cells
To study the uptake of nitrite into RBCs, washed RBCs were suspended in the
physiological saline and equilibrated in the tonometers (cf. above). In one
experiment (Hct=9.1%), RBCs were equilibrated to 3% O2/0.5%
CO2/96.5% N2 for 45 min, giving an intermediate
O2 saturation (close to 50%). Following withdrawal of a control
sample, nitrite was added from a 140 mmol l-1 NaNO2
stock solution to reach a concentration of 400 µmol l-1 (time
zero). Further samples were taken at predetermined times for measurements of
Hct, Hb, metHb and extracellular [NO2-]. After 107 min,
the gas supply was shifted to 0.5% CO2/99.5% N2 to fully
deoxygenate the cells, and at 150 min the gas supply was shifted to 30%
O2/0.5% CO2/69.5% N2 to oxygenate the cells.
In another set of experiments (N=4), RBC suspensions (Hct 20%)
were equilibrated in parallel to 99.5% air/0.5% CO2 (oxygenated
RBCs) or 99.5% N2/0.5% CO2 (deoxygenated RBCs).
Following withdrawal of control samples, nitrite was added to an extracellular
concentration of 3 mmol l-1 (calculated by taking into account the
measured Hct), and further samples were then taken at predetermined times.
Uptake of nitrite into the RBCs was assessed from the time-dependent decrease
in extracellular [NO2-].
Measurements
The coronary pressure was continuously monitored through a saline-filled
sidearm with a Uniflow Pressure Transducer (Baxter Uniflow, Bentley
Laboratories Europe BV, Uden, Holland) connected to a computer for direct data
acquisition. Pressure was expressed in kPa and was referenced to the saline
level in the chamber and corrected for cannula resistance.
The coronary flow was determined by measuring the time it took 0.05 ml of
perfusate to pass through the coronary artery, using a measuring system
similar to that reported by Agnisola et al.
(1994), modified for
computer-driven automatic control and data acquisition
(Fig. 1).
NO was measured with an ISO-NOP 2 mm sensor connected to an ISO-NO Mark II meter (World Precision Instruments), and the signal was sampled on a PC using the Duo.18TM data recording system (World Precision Instruments) at a sampling rate of 3 samples s-1. The electrode was calibrated by decomposition of the NO donor SNAP (S-nitroso-N-acetylpenicillamine; Sigma) as described in the instruction manual. The reading from the electrode (pA) was converted to concentration (nmol l-1) via the standard curve and multiplied by coronary flow to calculate rate of NO production.
Oxygen tension (PO2) and pH in saline and
RBC suspension samples from the input and the atrium were measured at 15°C
by Radiometer (Copenhagen, Denmark) electrodes in a BMS3 electrode set-up,
with the signals displayed on Radiometer PHM 73 and PHM 84 meters and REC 80
recorders. Osmolality was measured with a Gonotec Osmomat 030 (Berlin,
Germany). Nitrite was measured spectrophotometrically by a method based on the
Gries reaction (Jensen, 1992).
The fraction of metHb was evaluated by the three wavelength method of Benesch
et al. (1973). Total Hb concentration was measured by the cyanmethaemoglobin
method, using an extinction coefficient of 11 mmol l-1
cm-1 at 540 nm. Hct was determined by centrifugation (2 min at 13
700 g) in glass capillaries. Oxygen content
(CO2) in saline samples was calculated from the
measured PO2 and an O2 solubility
coefficient of 0.0408 µl O2 ml-1 torr-1.
CO2 of RBC suspension samples was measured by
the Tucker (1967
) method.
Myocardial O2 consumption
(
O2) was
calculated by multiplying the difference in CO2
between input and atrium with coronary flow and dividing by ventricle mass.
Myocardial O2 extraction (EO2) was
determined as
(CO2,inputCO2,atrium)/CO2,input.
The O2 saturation (SO2) of
functional Hb was calculated according to:
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Statistics
Results are expressed as means ± S.E.M., unless otherwise
stated. Means were compared using one-way analysis of variance (ANOVA) with
the Tukey post hoc test. One sample t-test was used to
evaluate whether a mean was significantly different from zero. Results on
nitrite uptake and metHb formation in oxygenated and deoxygenated RBCs in
vitro were evaluated by two-way ANOVA for repeated measures. Differences
were considered significant at P<0.05. Two-variable regression
analysis and multiple regression analysis were used to evaluate the
relationship between myocardial oxygen consumption and coronary flow, oxygen
supply and oxygen extraction.
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Results |
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![]() | (2) |
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![]() | (3) |
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NO production
A representative NO trace from the experiments is shown in
Fig. 4. The signal included
significant noise as well as artefacts from the sampling procedure and
measurements of flow, but the underlying NO signal could be effectively
isolated by performing a 400-point adjacent-averaging smoothing of the raw
data. After stabilisation of the signal during saline perfusion, a switch to
RBC perfusion was performed. This typically caused an increased signal that
subsequently tended to stabilise. Nitrite addition caused a further increase.
Usually, the increase in the signal occurred after a delay
(Fig. 4), which reflected the
time needed for the perfusate to travel the coronary circulation and reach the
measurement position at the atrial level. These changes to a new quasi-steady
plateau indicated that a new balance between NO production and degradation was
established and probably reflected an increased NO production. The NO signal
change also depends on the prevailing coronary flow, which can change with
treatment. The NO current at a given plateau was therefore converted to NO
concentration via the standard curve, and the change in NO production
rate related to a given experimental change was obtained as the difference
between the multiplication product of concentration and flow after and before
the change.
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Perfusion of the coronary circulation with saline alone was associated with
an NO production that was significantly different from zero
(Fig. 5A). The NOS inhibitor
L-NA significantly obliterated this NO production
(Fig. 5A), suggesting that it
resulted from endothelial NOS activity. Switching to RBC perfusion appeared to
increase the NO production but, due to a large variability between
preparations, the NO production rate was not significantly different
from zero (Fig. 5B).
Interestingly, there was a significant linear decrease in
NO production
with ventricle mass that seemed to explain the variability between
preparations (Fig. 6). L-NA
tended to inhibit the
NO production
(Fig. 5B). When nitrite was
added during RBC perfusion, the
NO production rate increased to values
that were significantly different from zero, but in this case there was no
difference between absence and presence of L-NA
(Fig. 5C). There was no sign of
haemolysis in centrifuged samples of the RBC perfusates, which is important
because extracellular Hb would have scavenged the produced NO
(Gladwin et al., 2004
).
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Nitrite was added to a nominal extracellular concentration of approximately 400 µmol l-1 during RBC perfusion. Values measured at the input were lower (Fig. 7), suggesting a rapid initial influx of NO2- to the RBCs, which was confirmed by in vitro experiments (see below). As the RBC passed through the coronary circulation, a further slight decrease in extracellular [NO2-] occurred, and this was paralleled by a significant increase in metHb, as measured in the atrium output (Fig. 7). Hct values during these experiments were 6.3±0.6 for input samples and 5.6±0.7 for atrium samples (N=12 in each case), which were not statistically different.
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In vitro RBC nitrite uptake
When nitrite was added to an RBC suspension in the tonometer under
conditions similar to those prevailing during RBC perfusion of the coronaries
(i.e. with respect to [NO2-],
SO2, Hct and pH), extracellular
[NO2-] showed a major and rapid decline below the added
value (400 µmol l-1) and then continued to decrease at a slower
rate (Fig. 9A). This reflected
a rapid initial entry of nitrite into the RBCs followed by a slower continued
entry. The entry of nitrite caused a moderate increase in RBC metHb content
(Fig. 9C). A change in Hb
O2 saturation from about 50% to full deoxygenation, followed by a
subsequent full oxygenation, had no major influence on
[NO2-] and metHb
(Fig. 9A,C). To further study a
possible oxygenation dependency of nitrite uptake, nitrite was added to
oxygenated and deoxygenated RBC suspensions at a higher extracellular
concentration (3 mmol l-1) and haematocrit (Hct 20%). This was
followed by a rapid initial decrease in extracellular
[NO2-], with a subsequent slower continued decrease, and
there was no significant difference between oxygenated and deoxygenated RBC
suspensions (Fig. 9B). The
entry of nitrite into the RBCs caused a significant increase in metHb with
time, and metHb values tended to be higher in oxygenated than deoxygenated
RBCs, but this difference was not significant
(Fig. 9D).
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Discussion |
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Basically, O2
is the product of the ingoing O2 concentration, the flow and the
O2 extraction coefficient
(
O2=CO2,inputx
xEO2).
With saline as the perfusion medium, both
CO2,input and
EO2 are low
(Fig. 2A,C), and changes in
flow have little impact on
O2
(Fig. 2B). Perfusion with RBCs
elevates the O2 capacitance of the medium, which elevates
CO2,input
(Fig. 2A), increases
EO2 (Fig.
2C) and effects a large influence of myocardial flow on
O2 delivery (Fig.
2B). Thus, RBC perfusion of the coronaries comes closer to the
in vivo situation where CO2 and
EO2 are high and where the major method to
increase
O2 is
via increased myocardial flow. A parallel can be drawn to the human
heart, where coronary blood flow and myocardial
O2 are closely
matched (Tune et al., 2002
).
In rainbow trout, coronary blood flow increases in vivo both during
hypoxia and during exercise, which improves myocardial O2 supply
and cardiac performance (Gamperl et al.,
1995
).
Through the measurements of corresponding values of
SO2 and PO2
before and after the coronary circulation, we constructed the overall in
situ O2 equilibrium curve
(Fig. 3). The
P50 was slightly higher than reported in normoxic rainbow
trout (Tetens and Lykkeboe,
1981). This is explained by the Bohr effect (increase in
P50 with pH decrease), because we used an equilibration
PCO2 (3.74 mmHg) that was slightly higher than
the normal arterial PCO2 value (23
mmHg), and our input pH (7.772) was therefore lower than the arterial pH above
7.9 in rainbow trout at 15°C (Wang et
al., 1998
). The input and atrium
SO2PO2
values show that there was a major decrease in
SO2 as result of the RBC transit through the
microvasculature. Thus, the condition of low
SO2, which is required for deoxyHb-mediated
reduction of nitrite to NO to take place, was present.
NO production
There was a significant NO production when the coronary vessels were
perfused with hypoxic saline, and this NO production was fully inhibited by
L-NA (Fig. 5A). This shows that
the trout coronaries produce NO under hypoxic conditions and that inhibition
of NO synthase obliterates this production. The parallel recordings of
coronary flow furthermore attest a vasodilatory role to this NO production
(cf. below). The NO probably resulted from endothelial NOS activity in the
endothelium lining the vessels. A contribution from inducible NOS cannot be
excluded, although this isoform usually mediates responses that are slower and
wider than those observed here (Mershon et
al., 2002).
In the presence of RBCs, NO produced in the endothelium and in the RBCs
runs the risk of being scavenged by Hb
(Gladwin et al., 2004). On the
other hand, if an NO signal is produced in the blood to be picked up by
vascular smooth muscle, then it should also be possible to harvest that signal
at the NO electrode membrane. Indeed, we registered an increased signal after
switch to RBC perfusion and after NO2- addition
(Fig. 4). The amperometric NO
sensor method was also found to be reliable for recordings of NO production in
human RBC suspensions (Carvalho et al.,
2004
). The
NO production from RBC perfusion per se
showed large variation (Fig.
5B), which appeared accounted for by differences in ventricle mass
(Fig. 6). One possible
explanation is that RBC perfusion causes a higher shear stress in smaller
hearts due to their smaller vessel dimensions, which might effect a
compensatory increase in endothelial NO production. Sheer stress is known to
increase NO release from the endothelium in the mammalian coronary circulation
(Stepp et al., 1999
), and
sheer stress-mediated NO-release has been implicated in the vasodilatory
response of the trout coronaries to adenosine
(Mustafa and Agnisola,
1998
).
Addition of nitrite during RBC perfusion increased the NO production
rate, and, importantly, this NO production was unaffected by L-NA
(Fig. 5C). Thus, in contrast to
the NO production during saline perfusion, the nitrite-induced NO production
was not due to endothelial NOS activity, suggesting that it occurred
via the RBCs or cardiac myocytes. The potential role of RBCs in blood
flow regulation via deoxyHb-mediated reduction of nitrite to NO
(Cosby et al., 2003
) implies a
sequence of events. First, nitrite should enter the RBCs. Secondly, because
nitrite reacts with oxyHb to form nitrate and metHb and with deoxyHb to form
NO and metHb, a significant desaturation of Hb is required, and some metHb
should be formed. Thirdly, to exert an effect on the vasculature, the NO
formed should escape the RBCs. All these prerequisites appeared to be
fulfilled: there was a rapid entry of nitrite into the RBCs
(Fig. 9), a gradient in
[NO2-] and rise in metHb between input and atrium
(Fig. 7), a significant
decrease in HbO2 saturation in the coronary circulation
(Fig. 3), and an NO signal was
registered (Fig. 5C). Thus, the
data support the idea that NO is produced from nitrite in the RBCs. It cannot
be excluded, however, that the heart itself may generate NO from nitrite by
means of xanthine oxidoreductase activity, as reported in human and rat hearts
(Webb et al., 2004
).
The entry of nitrite into the RBCs deserves some attention. In carp, tench
and whitefish, nitrite preferentially permeates the RBC membrane at low
O2 saturation (Jensen,
1992,
2003
), which appears to supply
nitrite for subsequent deoxyHb-mediated reduction to NO in an appropriate way.
In rainbow trout, on the other hand, there was no significant oxygenation
dependency of nitrite transport (Fig.
9). This contrasts sharply with the other teleost fishes examined
but compares with the situation in pig erythrocytes
(Jensen, 2005
). It appears
that in some species (carp, tench, whitefish) nitrite transport is governed by
a major oxygenation-dependent change in membrane permeability, whereas in
others (pig, trout) nitrite quickly equilibrates across the membrane, after
which nitrite entry is governed by intracellular nitrite removal via
its reactions with Hb (cf. Jensen,
2005
).
Effects on coronary flow
Coronary flow was significantly higher in the absence of L-NA than in the
presence of L-NA during both saline perfusion and RBC perfusion
(Fig. 8C,F). This shows that
the NO released from the coronary endothelium produced vasodilation in the
absence of L-NA, whereas inhibition of NOS-catalyzed NO production by L-NA
induced vasoconstriction. The data therefore corroborate that endothelial NO
plays a role in regulating flow in the saline perfused trout coronary
circulation (Mustafa et al.,
1997) and extends the conclusion to RBC perfusions, where
CO2 is higher. The mean values for coronary
flow (Fig. 8C) are some
34% of the in vivo cardiac output in resting normoxic trout at
15°C (
38 ml min-1 kg-1;
Wood and Shelton, 1980
) and
are accordingly higher than in vivo coronary flow in normoxic
salmonids (
1% of cardiac output;
Axelsson and Farrell, 1993
;
Gamperl et al., 1994
). This is
not surprising given the hypoxic conditions and the lower viscosity and oxygen
capacitance in saline and low-Hct erythrocyte media than in whole blood
(Farrell, 1987
). In particular,
as shown in the present study, hypoxia will induce NO release and
vasodilation.
Addition of nitrite during RBC perfusion did not change coronary flow, and
this was the case both in the absence and the presence of L-NA
(Fig. 8C,F). Thus, the NO
produced from nitrite was unable to induce further vasodilation in the absence
of L-NA or to overcome the relative vasoconstriction in L-NA-treated
preparations. The formation of NO from nitrite in RBCs with subsequent
vasodilation may require that PO2 is at or
below P50, i.e. that
SO250%
(Gladwin et al., 2004
). In our
experiments, SO2 in the inflowing RBC
suspension was around or above 50% (Fig.
3). One possible explanation for the absent vasodilation may
therefore be that most NO was produced from nitrite following deoxygenation in
the capillaries, whereby the NO was found in capillaries, post-capillary
vessels and the atrium, where it had little likelihood of acting on coronary
arterioles. If the RBCs function as a sensor of O2 conditions
(via degree of deoxygenation) and mediator of increased blood flow
(via NO release) in the normal arterialvenous circulation,
then an essential question is how the signal is propagated to the major
resistance vessels. In the microcirculation, RBCs may experience a decline in
PO2 before reaching the capillaries, in which
case NO released from RBCs could exert an effect on arterioles. It is also
possible that a signal generated in the capillaries can be conducted to
upstream arterioles, as in skeletal muscle microvasculature
(Murrant and Sarelius, 2000
).
The present data do not support this happening in the trout coronary
circulation, but this could be a characteristic of the particular
microvascular bed. It may be rewarding to test other microcirculations in
fish, such as skeletal muscle vasculature, where nitrite-induced vasodilation
is known to occur in mammals (Cosby et al.,
2003
), or cerebral microcirculation, where nitric oxide-dependent,
acetylcholine vasodilation has been demonstrated in rainbow trout and crucian
carp (Söderström et al.,
1995
; Hylland and Nilsson,
1995
).
Thus, the RBC perfused trout coronary circulation did not respond to nitrite under normal and moderately hypoxic conditions, probably because deoxyHb-mediated NO generation occurred after the resistance vessels. During severe hypoxia, NO production from nitrite can be predicted to occur before the arterioles. In this situation, vasodilation may occur, because our results show that NO (of endothelial origin) is vasoactive in the trout coronary circulation.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agnisola, C. (in press). Role of nitric oxide in the control of coronary resistance in teleosts. Comp. Biochem. Physiol.
Agnisola, C., Venzi, R., Houlihan, D. and Tota, B. (1994). Coronary flow-pressure relationship in the working isolated fish heart: trout (Oncorhynchus mykiss) versus torpedo (Torpedo marmorata). Phil. Trans. R. Soc. Lond. B 343,189 -198.
Axelsson, M. and Farrell, A. P. (1993). Coronary blood flow in vivo in the coho salmon (Oncorhynchus kisutch). Am. J. Physiol. 264,R963 -R971.[Medline]
Carvalho, F. A., Martins-Silva, J. and Saldanha, C. (2004). Amperometric measurements of nitric oxide in erythrocytes. Biosens. Bioelectron. 20,505 -508.[CrossRef][Medline]
Christoforides, C. and Hedley-Whyte, J. (1969).
Effect of temperature and haemoglobin concentration on solubility of
O2 in blood. J. Appl. Physiol.
27,592
-596.
Cosby, K., Partovi, K. S., Crawford, J. H., Patel, R. P., Reiter, C. D., Martyr, S., Yang, B. K., Waclawiw, M. A., Zalos, G., Xu, X. et al. (2003). Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat. Med. 9,1498 -1505.[CrossRef][Medline]
Ellsworth, M. L. (2000). The red blood cell as an oxygen sensor: what is the evidence? Acta Physiol. Scand. 168,551 -559.[CrossRef][Medline]
Farrell, A. P. (1987). Coronary flow in a perfused rainbow trout heart. J. Exp. Biol. 129,107 -123.[Abstract]
Farrell, A. P., MacLeod, K. R. and Chancey, B. (1986). Intrinsic mechanical properties of the perfused rainbow trout heart and the effects of catecholamines and extracellular calcium under control and acidotic conditions. J. Exp. Biol. 125,319 -345.[Abstract]
Gamperl, A. K., Pinder, A. W., Grant, R. R. and Boutilier, R.
G. (1994). Influence of hypoxia and adrenaline administration
on coronary blood flow and cardiac performance in seawater rainbow trout
(Oncorhynchus mykiss). J. Exp. Biol.
193,209
-232.
Gamperl, A. K., Axelsson, M. and Farrell, A. P. (1995). Effects of swimming and environmental hypoxia on coronary blood flow in rainbow trout. Am. J. Physiol. 269,R1258 -R1266.[Medline]
Gladwin, M. T., Shelhamer, J. H., Schechter, A. N., Pease-Fye,
M. E., Waclawiw, M. A., Panza, J. A., Ognibene, F. P. and Cannon, R. O.,
III (2000). Role of circulating nitrite and
S-nitrosohemoglobin in the regulation of regional blood flow in humans.
Proc. Natl. Acad. Sci. USA
97,11482
-11487.
Gladwin, M. T., Crawford, J. H. and Patel, R. P. (2004). The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation. Free Radic. Biol. Med. 36,707 -717.[CrossRef][Medline]
Hylland, P. and Nilsson, G. E. (1995). Evidence that acetylcholine mediates increased cerebral blood flow velocity in crucian carp through a nitric oxide-dependent mechanism. J. Cer. Blood Flow Met. 15,519 -524.
Jensen, F. B. (1992). Influence of haemoglobin conformation, nitrite and eicosanoids on K+ transport across the carp red blood cell membrane. J. Exp. Biol. 171,349 -371.
Jensen, F. B. (2003). Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol. 135A,9 -24.
Jensen, F. B. (2005). Nitrite transport into pig erythrocytes and its potential biological role. Acta Physiol. Scand. 184,243 -251.[CrossRef][Medline]
Kosaka, H. and Tyuma, I. (1987). Mechanism of autocatalytic oxidation of oxyhemoglobin by nitrite. Environ. Health Perspect. 73,147 -151.[Medline]
Mershon, J. L., Baker, R. S. and Clark, K. E. (2002). Estrogen increases iNOS expression in the ovine coronary artery. Am. J. Physiol. 283,H1169 -H1180.
Murrant, C. L. and Sarelius, I. H. (2000). Coupling of muscle metabolism and muscle blood flow in capillary units during contraction. Acta Physiol. Scand. 168,531 -541.[CrossRef][Medline]
Mustafa, T. and Agnisola, C. (1994). Vasoactivity of prostanoids in the trout (Oncorhynchus mykiss) coronary system: modification by noradrenaline. Fish Physiol. Biochem. 13,249 -261.
Mustafa, T. and Agnisola, C. (1998).
Vasoactivity of adenosine in the trout (Oncorhynchus mykiss) coronary
system: involvement of nitric oxide and interaction with noradrenaline.
J. Exp. Biol. 201,3075
-3083.
Mustafa, T., Agnisola, C. and Hansen, J. K. (1997). Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss) coronary system. J. Comp. Physiol. B 167,98 -104.
Nagababu, E., Ramasamy, S., Abernethy, D. R. and Rifkind, J.
M. (2003). Active nitric oxide produced in the red cell under
hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction.
J. Biol. Chem. 278,46349
-46356.
Okamoto, M., Tsuchiya, K., Kanematsu, Y., Izawa, Y., Yoshizumi, M., Kagawa, S. and Tamaki, T. (2005). Nitrite-derived nitric oxide formation following ischemia-reperfusion injury in kidney. Am. J. Physiol. 288,F182 -F187.
Pawloski, J. R., Hess, D. T. and Stamler, J. S. (2001). Export by red blood cells of nitric oxide bioactivity. Nature 409,622 -626.[CrossRef][Medline]
Söderström, V., Hylland, P. and Nilsson, G. E. (1995). Nitric oxide synthase inhibitor blocks acetylcholine induced increase in brain blood flow in rainbow trout. Neurosci. Lett 197,191 -194.[CrossRef][Medline]
Stepp, D. W., Nishikawa, Y. and Chilian, W. M.
(1999). Regulation of shear stress in the canine coronary
microcirculation. Circulation
100,1555
-1561.
Stormer, J., Jensen, F. B. and Rankin, J. C. (1996). Uptake of nitrite, nitrate, and bromide in rainbow trout, Oncorhynchus mykiss: effects on ionic balance. Can. J. Fish. Aquat. Sci. 53,1943 -1950.[CrossRef]
Tetens, V. and Lykkeboe, G. (1981). Blood respiratory properties of rainbow trout, Salmo gairdneri: Responses to hypoxia acclimation and anoxic incubation of blood in vitro. J. Comp. Physiol. 145,117 -125.[CrossRef]
Tucker, V. A. (1967). Method for oxygen content
and dissociation curves on microlitre blood samples. J. Appl.
Physiol. 23,410
-414.
Tune, J. D., Richmond, K. N., Gorman, M. W. and Feigl, E. O.
(2002). Control of coronary blood flow during exercise.
Exp. Biol. Med. 227,238
-250.
Wang, T., Knudsen, P. K., Brauner, C. J., Busk, M., Vijayan, M. M. and Jensen, F. B. (1998). Copper exposure impairs intra- and extracellular acid-base regulation during hypercapnia in the fresh water rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B 168,591 -599.[Medline]
Webb, A., Bond, R., McLean, P., Uppal, R., Benjamin, N. and
Ahluwalia, A. (2004). Reduction of nitrite to nitric oxide
during ischemia protects against myocardial ischemia-reperfusion damage.
Proc. Natl. Acad. Sci. USA
101,13683
-13688.
Wood, C. M. and Shelton, G. (1980). Cardiovascular dynamics and adrenergic responses of the rainbow trout in vivo. J. Exp. Biol. 87,247 -270.[Abstract]
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