Download Role of Nitric Oxide in the Systemic and Pulmonary Circulation of

JOURNAL OF EXPERIMENTAL ZOOLOGY 286:683–689 (2000)
Role of Nitric Oxide in the Systemic and
Pulmonary Circulation of Anesthetized Turtles
(Trachemys scripta)
DANE A. CROSSLEY II,1,2 TOBIAS WANG,1,3 AND JORDI ALTIMIRAS1,4*
1
Center for Respiratory Adaptation, University of Aarhus, DK-8000 Århus C,
Denmark
2
Department of Biological Sciences, University of North Texas, Denton, Texas
76203-5220
3
School of Biological Sciences, University of Birmingham, Birmingham B15
2TT, United Kingdom
4
Department of Zoophysiology, University of Göteborg, SE 405 30 Göteborg,
Sweden
ABSTRACT
In reptiles the influence of local vascular factors on blood flow regulation is vaguely
understood. The aim of this study was to investigate the role of nitric oxide (NO) on vascular
function in anesthetized Trachemys scripta. The experimental protocol consisted of serial injections of sodium nitroprusside (SNP; 25 µg · kg–1), L-arginine (185 mg · kg–1) and L-NAME
(50 mg · kg–1). SNP induced a systemic vasodilation (0.05 to 0.02 kPa · min · kg · mL–1, P =
0.015), with no change in pulmonary vascular resistance (0.07 versus 0.08 kPa · min · kg ·
mL–1, P > 0.05). L-Arg had no effect on resistances but increased cardiac output by 17%. LNAME increased systemic resistance (33% increase; P = 0.01) while pulmonary resistance
was unchanged. These effects are consistent with in vivo and in vitro studies on the systemic
vasculature of different reptilian species, suggesting that NO has an important role in maintaining systemic vascular tone. The pulmonary vasculature did not respond to NO due to either a
lack of an endogenous NO tone or a relaxed state of the pulmonary vasculature. The importance of
NO-based mechanisms versus other neuro-humoral modulators in the reptilian circulation remains
uncertain. However, as established in prior studies, cholinergic control of the proximal pulmonary
artery is the main regulator of pulmonary resistance while systemic resistance depends on a more
complex suite of neural, humoral and local effectors that include NO. J. Exp. Zool. 286:683–689,
2000. © 2000 Wiley-Liss, Inc.
Vascular tone in reptiles is controlled by cholinergic and adrenergic neural efferents (Kirby
and Burnstock, ’69a; Comeau and Hicks, ’94) and
it is well established that pulmonary resistance
in turtles (Rpul) is actively controlled through a
vagal innervation of smooth muscle in the proximal pulmonary artery (e.g., Burggren, ’77; Milsom
et al., ’77). In anesthetized specimens, efferent
electrical stimulation of the left vagus increases
Rpul (Milsom et al., ’77: Comeau and Hicks, ’94),
and atropine injection decreases Rpul in conscious
animals (Hicks and Wang, ’98). Oppositely, adrenergic innervation of the pulmonary artery reduces Rpul (Burggren, ’77; Hicks, ’94). Neural
control of systemic resistance (Rsys) is also under
a dual autonomic control. In anesthetized turtles,
vagal efferent stimulation reduces Rsys, while vagal afferent stimulation increases Rsys. The effects
can be blocked by atropine and bretylium, respec© 2000 WILEY-LISS, INC.
tively, indicating the presence of a cholinergic vasodilator and an adrenergic vasoconstrictor tone
(Comeau and Hicks, ’94). These results are supported by the presence of adrenergic and cholinergic fibers in the systemic circulation of reptiles
(Kirby and Burnstock, ’69b, Berger and Burnstock, ’79).
In addition to central nervous control, hormones
and local factors affect blood flow distribution as
well as cardiac shunt patterns in reptiles. As an
example, increased levels of circulating catecholamines decrease Rpul, causing an increased pulmo·
nary blood flow (Q pul) (Donald et al., ’90). Recently,
Grant sponsor: The Danish Research Council; Grant sponsor: National Science Foundation; Grant number: IBN-9616138.
*Correspondence to: Jordi Altimiras, Department of Zoophysiology,
University of Göteborg, Box 463, SE 405 30 Göteborg, Sweden.
E-mail: [email protected]
Received 9 June 1999; Accepted 22 October 1999
684
D.A. CROSSLEY II ET AL.
it has been shown that severe hypoxia leads to
·
an increased Rpul, a reduction in Q pul and an increased net right-to-left cardiac shunt (hypoxic
pulmonary vasoconstriction, Crossley et al., ’98).
Nevertheless, at present, little is known regarding the possible effects of other humoral and local factors on the reptilian circulation.
Among all known local factors that affect vascular tone in mammals, nitric oxide (NO) is of particular importance due to its generic activity and
ubiquity in many vascular beds (Moncada and
Higgs, ’93). NO is endogenously produced in vascular endothelial cells from L-arginine (L-Arg) via
nitric oxide synthase (NOS). The release of NO
relaxes subjacent smooth muscle cells and results
in vasodilation. In reptiles, exogenous NO administration (via the NO donor sodium nitroprusside)
greatly reduces Rsys (e.g., Miller and Vanhoutte,
’86; Altimiras et al., ’98). Further, an endothelium
dependent vascular relaxation following acetylcholine (Ach) injection has also been demonstrated
in garter snakes (Knight and Burnstock, ’93), and
the use of specific inhibitors of NO synthesis (LNA) raises systemic blood pressure in the turtle
Trachemys scripta (Söderström et al., ’97). Nevertheless, NO effects on central vascular blood flows
and the pulmonary circulation in particular have
not been studied in reptiles. Therefore, to further
characterize the local control of blood flows in reptiles, the present study focused on the role of NO
in the systemic and pulmonary vasculature in
anesthetized turtles.
MATERIALS AND METHODS
Experimental animals
Five freshwater turtles, Trachemys scripta Gray
(902 g; range, 472–1,600 g) were obtained from
Lemberger Inc. (Oshkosh, WI) and air freighted
to Aarhus University where they were maintained
in a 1 × 1 m fiberglass tank containing freshwater heated at 28°C. The animals had free access
to dry platforms and heating lamps allowing for
behavioral thermoregulation and were maintained on a 12 hr:12 hr light and dark cycle. Animals were fed fish several times a week, but food
was withheld for at least three days before experimentation.
Anesthesia, surgery and data recording
The surgical procedure was identical to that described previously (Crossley et al., ’98). Briefly,
turtles were anesthetized with sodium pentobarbital (Mebumal; 50 mg · kg–1 intraperitoneal) and
tracheotomized for artificial ventilation. During
the surgical procedure (60–90 min) each turtle was
ventilated every 5 min with room air using a 50
mL syringe. Using a bone saw, a 5 × 5 cm piece of
the plastron was removed to expose the central
blood vessels; eventual bleeding was stopped via
electrocauterization. The left carotid artery was
occlusively cannulated (PE-90; 1.27 mm O.D.; 0.86
mm I.D.) and the tip of the catheter forwarded
into the right aortic arch, while the common pulmonary was non-occlusively cannulated using the
Seldinger technique (White et al., ’89). Blood flows
were measured in the left aortic arch (LAo) and the
left pulmonary artery (LPA) using 2S transit-time
ultrasonic blood flow probes (Transonic System, Inc.,
Ithaca, NY). The catheters were connected to
Statham pressure transducers (calibrated against
static water columns) and a Beckman R611A recorder. Blood flow probes were connected to a Transonic dual channel blood flow meter (T206) for
measurements of instantaneous blood flow rates.
All signals were digitally stored at 50 Hz with an
AcqKnowledge MP 100 (version 3.2.3) data acquisition system.
Experimental protocol
Turtles were maintained ventral side up and
artificially ventilated using a Harvard ventilator
(HI 665) at a tidal volume of 20 mL and frequency
of 24 min–1. Under these conditions, tracheal pressure was approximately 0.8 kPa. The Harvard
ventilator was connected to a Wosthoff gas mixing pump (Bochum, Germany) that delivered a gas
mixture 3 kPa CO2 (balance air) to mimic the arterial blood PCO2 normally observed in vivo (e.g.,
Glass et al., ’83). Following surgery, the animals
were left for approximately 40 min to ensure
stable blood pressures and flows. The experiments
were conducted at 22–23°C and all turtles were
killed by vascular injections of KCl after completion of the experiments. All experimental procedures were carried out in accordance to NIH
guidelines.
All drugs were injected as single boluses through
the catheter in the left carotid artery; preliminary
experiments showed that injections through the pulmonary artery yielded identical results. The doses
of SNP and L-NAME were chosen according to other
studies in reptiles (Söderström et al., ’97; Altimiras
et al., ’98) and preliminary trials. The effects
shown in Figure 1 were consistently observed in
all animals and are in agreement with other studies and with the current knowledge on the pharmacology of NO in vertebrates, indicating that
NITRIC OXIDE IN TURTLE CARDIOVASCULAR CONTROL
685
Fig. 1. Original traces of animal four displaying the basic
changes in flows and pressures in the systemic and pulmonary circulation after injection of the different drugs (arrows
mark the time of injection). SNP, sodium nitroprusside 25 µg
· kg–1; L-Arg, L-arginine 185 mg · kg–1; and L-NAME, Nωnitro-L-arginine methyl ester 50 mg · kg–1.
such doses were appropriate for a qualitative characterization of the role of NO in vascular regulation. L-Arg was injected as a close-to-saturation
solution (L-Arg; 185 mg · kg–1) in order to avoid
long infusion times. This dose is within the range
employed in several mammalian studies (Loeb and
Longnecker, ’92; Fineman et al., ’94; Ogilvie and
Zborowska-Sluis, ’95).
The following experimental protocol was used.
First, NO levels were exogenously increased via
injection of the NO donor sodium nitroprusside
(SNP; 25 µg · kg–1), to estimate the capacity of
NO to relax the vascular beds. This was followed by injection of L-arginine (L-Arg; 185 mg
· kg–1), the metabolic substrate of the reaction
for NO production. Subsequently, NO synthesis was inhibited by injection of the NOS inhibitor L-NAME (50 mg · kg–1) in order to assess
the existence of a NO endogenous tone. Blood
flows and pressures returned to baseline values before 10 min when SNP and L-Arg were
injected. The effects of L-NAME were permanent and reached stable values after 5 min. In
an additional set of experiments (N = 4 animals), the order of injection of L-Arg and LNAME was reversed in order to measure the
effects of L-Arg under blockade of the nitric oxide
synthase.
As in previous studies we estimated systemic
·
·
·
blood flow (Q sys) as 2.85 × Q Lao, while Q pul was es·
timated as 2 × Q LPA (Wang and Hicks, ’96). Pulmonary and systemic resistances were calculated
as the mean blood pressure relative to blood flow
·
·
(Rpul = Ppul/Q pul and Rsys = Psys/Q sys) under the assumption that both atrial pressures are zero. Beatto-beat heart rate (fH) was calculated on basis of
·
the instantaneous Q LAo profile; total cardiac out·
put (Q) as the sum of systemic and lung blood
·
·
flow (Q sys + Q pul), and total stroke volume (Vstot)
as total cardiac output divided by fH.
A paired ANOVA design was employed to assess significant effects of treatment versus preinjection values. A fiducial limit of significance of
P = 0.05 was applied and all data are presented
as mean ± 1 SE.
Calculations and statistics
All recordings were analyzed using AcqKnowledge
data analysis software (version 3.2.3). For each
treatment mean values for blood flows in the left
·
aortic arch and the left pulmonary artery (QLAo and
·
QLPA, respectively) as well as systemic and pulmonary blood pressures (Psys and Ppul, respectively)
were analyzed over for a 3–5 min period.
RESULTS
Examples of pulmonary and systemic flows and
pressures for one individual during the different
pharmacological treatments are shown in Figure
1, while the mean values are depicted in Figures
2–4. SNP induced a clear vasodilation in the systemic vasculature and the effects were not blocked
with L-NAME (Fig. 2). Rsys dropped from 0.046 ±
0.003 to 0.024 ± 0.003 kPa · min · kg · mL–1, as·
sociated to a drop in Psys while Q syswas maintained. Rpul was not altered by SNP injections
(0.071 ± 0.015 vs. 0.080 ± 0.007 kPa · min · kg ·
mL–1). No changes in heart rate or stroke volume
were observed (Table 1).
After injection of L-Arg, a transient systemic hypotension was observed in four out of five animals.
The hypotensive effect lasted less than 1 min and
a slight systemic hypertension developed after 5 min
(2.56 ± 0.32 kPa versus 2.9 ± 0.37 kPa, P = 0.035)
686
D.A. CROSSLEY II ET AL.
Fig. 2. Effects of 25 µg · kg–1 SNP injections on pressures,
flows, and resistances in the pulmonary and systemic circu-
lation. Data plotted as mean ± 1 SE. *Significant difference
from pre-injection values (P < 0.05).
with no changes in the resistance of the systemic
·
·
or pulmonary vasculature (Fig. 3). Qpul and Qsys increased significantly (P = 0.019 and P = 0.03, respectively) as did total stroke volume and total
cardiac output (17% increase from 81 ± 12.9 mL
· min–1 · kg–1 to 95 ± 13.7 mL · min–1 · kg–1; Table 1).
L-NAME increased systemic resistance (33% increase; P < 0.05; Fig. 4), resulting in an increased
·
Psys and decreased Q sys Despite the slight significant pulmonary hypertension (2.09 ± 0.31 to 2.56
± 0.46 kPa), Rpul was unchanged (0.055 ± 0.009 to
0.069 ± 0.017 kPa · min · kg · mL–1; P = 0.17). No
changes in heart rate or stroke volume were observed (Table 1).
Fig. 3. Effects of 185 mg · kg–1 L-Arg injections on pressures, flows, and resistances in the pulmonary and systemic
circulation. Data plotted as mean ± 1 SE. *Significant difference from pre-injection values (P < 0.05).
Fig. 4. Effects of 50 mg · kg–1 L-NAME injections on pressures, flows, and resistances in the pulmonary and systemic
circulation. Data plotted as mean ± 1 SE. *Significant difference from pre-injection values (P < 0.05).
DISCUSSION
Critique of methods
The present study was performed on anesthetized animals to preclude the large cardiovascu-
NITRIC OXIDE IN TURTLE CARDIOVASCULAR CONTROL
687
TABLE 1. Cardiac output, heart rate and stroke volumes after the different treatments shown in Figures 2–41
·
Treatment
fH
Q
Vstot
SNP
SNP (after L-NAME)
L-Arg
L-NAME
C
I
C
I
C
I
C
I
30.9 ± 1.94
31.2 ± 1.76
31.4 ± 2.69
32.3 ± 2.62
31.3 ± 1.82
31.3 ± 2.06
31.3 ± 2.04
31.5 ± 2.15
ns
ns
ns
ns
84 ± 13.2
75 ± 14.7
102 ± 18.8
99 ± 17.7
81 ± 12.9
95 ± 13.7
96 ± 14.1
95 ± 15.8
ns
ns
*
ns
2.7 ± 0.33
2.3 ± 0.39
3.2 ± 0.32
3.0 ± 0.32
2.5 ± 0.32
3.0 ± 0.30
3.0 ± 0.33
2.9 ± 0.30
ns
ns
*
ns
N
(4)
(4)
(4)
(4)
(5)
(5)
(5)
(5)
·
Values are mean ± SE; N, number of animals; fH, heart rate (beats · min–1); Q,, cardiac output (mL · min–1 · kg–1); Vstot, stroke volume (mL ·
–1
kg ); SNP, sodium nitroprusside; L-NAME, N-nitro-L-arginine methyl ester; L-Arg, L-arginine; ns, no significant differences between control
(C) and injection (I).
*Significant different from pre-injection values (P < 0.05).
1
lar changes associated with spontaneous breathing. Although anesthesia may blunt NO dependent
processes, pentobarbital does not appear to affect
the NO-dependent tone in mammals and studies of
endothelium-related vasoactive substances are routinely conducted under pentobarbital anaesthesia
(e.g., Loeb and Longnecker, ’92; Sprague et al., ’92).
Further, previous studies with pentobarbital anesthesia have shown a clear hypertensive effect on
the systemic vasculature of Trachemys scripta after L-NA infusion (Söderström et al., ’97), similar
to the present study, which suggests that NO-tone
is conserved during anesthesia.
Our experimental protocol consisted of serial
drug injection. It is possible, therefore, that there
may have been cumulative effects from previous
drugs. While the serial injections may account for
the fact that the control values for pulmonary and
systemic blood flows increased significantly during the experiment, this possibility is unlikely because SNP is quickly degraded and L-Arg rapidly
metabolized. In any event, a comparison between
pre- and post-injection values suffice to qualitatively describe NO-related vascular changes, and
it seems unlikely that our experimental protocol
will yield erroneous results.
Role of NO in the systemic circulation
In the present study, injection of the NO-donor
SNP decreased Rsys, while NOS inhibition increased
Rsys (Figs. 2 and 4). These effects are consistent with
previous reports on SNP and nitroglycerin injections
in various reptiles (Millard and Moalli, ’80; Stephens
et al., ’83) and with the observation that L-NA lead
to an increased systemic blood pressure in turtles
(Söderström et al., ’97). Furthermore, in vitro studies on the vasoactivity of isolated systemic vessels
of reptiles to acetylcholine and diverse NO donors
indicate vasodilator effects of NO. In the garter
snake Thamnophis sirtalis parietalis there is a dosedependent relaxation of the pre-constricted aorta
with acetylcholine and sodium nitroprusside (Knight
and Burnstock, ’93). In the caiman, ACh-dependent
vasodilation requires an intact endothelium (Miller
and Vanhoutte, ’86), suggesting a direct role of NO.
Thus, with respect to the systemic vasculature, reptiles appear to exhibit a similar NO-tone as that
demonstrated in mammals (Loeb and Longnecker,
’92; Scrogin et al., ’98).
Infusion of L-Arg, the substrate for endogenous
NO synthesis, did not cause vasodilation in the
present study. This may be because NO synthesis
in vivo is limited by other factors such as the
abundance of the co-substrate NADPH or cofactors such as FAD, FMN, heme, calmodulin, and
tetrahydrobiopterin as cofactors (Umans and Levi,
’95). A limited supply of any of these substances
could limit the reaction irrespective of the amount
of L-Arg administered. L-Arg did have a positive
inotropic effect on the heart. This effect, however,
seemed unrelated to NO production given the lack
of cardioactive effects by SNP, in analogy with effects previously reported in man (Hishikawa et
al., ’92). This is further supported from data obtained in other individuals of T.scripta (N = 4)
under identical experimental conditions. As shown
in Table 2, L-Arg induced similar cardiovascular
changes after NOS blockade, namely an increased
cardiac output via an increased heart rate and
stroke volume. Thus, the positive inotropic effects
of L-Arg are unrelated to NO production because
they occur in the presence or absence of NOS
blockade.
Role of NO in the pulmonary circulation
The present study shows that inhibition of NOS
with an analogue of L-Arg (L-NAME) does not affect the vascular tone in the pulmonary vascula-
688
D.A. CROSSLEY II ET AL.
TABLE 2. Main cardiovascular parameters induced by
L-Arg in 4 anaesthetized turtles pre-treated with L-NA under
the same experimental conditions of this study1
Control
·
Q pul
Ppul
Rpul
·
Q sys
Psys
Rsys
fH
Vstot
27.3 ± 9.4
2.9 ± 0.4
1.277 ± 0.213
22.2 ± 1.9
3.9 ± 0.7
1.716 ± 0.259
37.5 ± 1.2
1.32 ± 0.30
L-Arg
P
28.1 ± 9.2
2.6 ± 0.4
1.089 ± 0.175
28.7 ± 4.6
3.4 ± 0.6
1.180 ± 0.089
39.0 ± 1.2
1.44 ± 0.32
ns
ns
ns
ns
ns
ns
*
*
·
·
Values are mean ± SE. Q pul, Q sys, pulmonary and systemic blood
–1
–1
flow (mL · min · kg ); Ppul, Psys, pulmonary and systemic pressure
(kPa); Rpul, Rsys, pulmonary and systemic resistance (kPa · min · kg ·
mL–1); fH, heart rate (beats · min–1); Vstot, stroke volume (mL · kg–1).
ns, no significant differences pre- and post- L-Arg injection.
*Significant difference (P < 0.05).
1
ture of anaesthetized turtles (i.e., Rpul is not affected). Furthermore, exogenously administrated
NO (via SNP injections) has no effect on the lung
despite its marked vasodilator effects on the systemic vasculature.
No or little NO tone of the pulmonary circulation has also been reported in mammals (Persson
et al., ’90; McMahon et al., ’91; Perrella et al., ’91;
Bhattacharya and Bhattacharya, ’92; Nishiwaki
et al., ’92; Barer et al., ’93; McCormack and Paterson, ’93; Russ and Walker, ’93). Nevertheless, the
role of NO in the mammalian lung remains debated and it appears that the pulmonary endothelium is able to release NO, and that the
expression of a NO tone depends on whether the
vasculature is preconstricted. Thus, if the pulmonary vasculature is pre-constricted pharmacologically or by hypoxic gas mixtures, administration
of NO via inhalation or injection of NO donors
leads to a vasodilation. This is the case in intact
perfused lungs, anesthetized preparations and
during pathologic states of pulmonary hypertension (Archer et al., ’90; Pepke-Zaba et al., ’91;
Nishiwaki et al., ’92) but apparently not during
normal resting conditions (Van Camp et al., ’94).
It is possible, therefore, that the lack of NO related tone can be explained, at least partially, by
the dilated state that appear to characterize the
pulmonary vasculature of anaesthetized turtles
(Crossley et al., ’98). Nevertheless, the hemodynamic variables in the present study are close to
in vivo values (Shelton and Burggren, ’76; Wang
and Hicks, ’96), and it seems that a very low NO
related tone is a characteristic of the chelonian
pulmonary vasculature. A lack of an acetylcholine-dependent endogenous NO tone has also been
reported in excised lung tissue of the South
American lungfish Lepidosiren paradoxa and in
the gas bladder in trout (Staples et al., ’95). In
trout, sodium nitroprusside lowers systemic resistance and dorsal aortic pressure but gill resistance remains unchanged (Olson et al., ’97).
Functional measurements of constitutive NOS activity in the big-head carp support this conclusion. NOS activity was important in many organs
such as the brain, the gastrointestinal tract, the
swim bladder, and the heart but absent in the gills
(Wong et al., ’98). However, in the gas bladder of
the air-breathing teleost Hoplerythrinus unitaeniatus there is a marked NO mediated tone and
the perfusion of some gas exchange organs may,
therefore, be controlled through NO effects (Staples et al., ’95).
In conclusion, while the importance of NO-based
mechanisms versus other neuro-humoral modulators in the vascular resistance of reptiles cannot be discerned without future research, it seems
that pulmonary resistance is primarily under cholinergic and adrenergic control whereas systemic
resistance is determined by a suite of neural, humoral, and local effectors including NO.
ACKNOWLEDGMENT
D.C. was partially supported by NSF grant IBN9616138 to Dr. W.W. Burggren.
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