Vol. 90, Issue 6, 2101-2108, June 2001
L-NAME enhances responses to atrial
natriuretic peptide in the pulmonary vascular bed of the cat
Albert L.
Hyman1,2,
Bracken J.
De Witt2,
Bulent
Gumusel3,4,
Quingzhong
Hao3,4,
Philip J.
Kadowitz2, and
Howard L.
Lippton3,4
1 Cardiopulmonary Research Laboratory, Department of
Surgery, and 2 Department of Pharmacology, Tulane University
School of Medicine, 3 H. L. Laboratories, and
4 Department of Pharmacology, Louisiana State University School
of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
This study investigated
the hypothesis that atrial natriuretic peptide (ANP) responses are
mediated by particulate guanylate cyclase in the pulmonary vascular bed
of the cat. When tone in the pulmonary vascular bed was raised
to a high steady level with the thromboxane mimic U-46619, injections
of ANP caused dose-related decreases in lobar arterial pressure. After
administration of HS-142-1, an ANP-A- and ANP-B-receptor antagonist,
vasodilator responses to ANP were reduced. The nitric oxide (NO)
synthase inhibitor
N
-nitro-L-arginine methyl ester
(L-NAME) enhanced ANP vasodilator responses, suggesting
that inhibition of NO modulates ANP responses. L-NAME
administration with constant 8-bromo-cGMP infusion attenuated the
increased vasodilator response to ANP, suggesting that supersensitivity to ANP occurs upstream to activation of a cGMP-dependent protein kinase. In pulmonary arterial rings, ANP produced concentration-related vasorelaxant responses with and without endothelium. Methylene blue,
L-NAME, or
N-monomethyl-L-arginine did not
alter ANP vasorelaxant responses. These data show that ANP
supersensitivity observed in the intact pulmonary vascular bed is not
seen in isolated pulmonary arterial segments, suggesting that it may
only occur in resistance vessel elements. These results suggest that
ANP responses occur through activation of ANP-A and/or -B receptors in
an endothelium-independent manner and are modulated by NO in resistance
vessel elements in the pulmonary vascular bed of the cat.
lung; guanosine 3',5'-cyclic monophosphate; methylene blue; atrial
natriuretic peptide receptors; N-monomethyl-L-arginine
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INTRODUCTION |
ATRIAL NATRIURETIC
PEPTIDE (ANP) is a naturally occurring peptide discovered by de
Bold et al. in 1981 (4). ANP is released under
physiological conditions from atrial myocytes in response to increases
in central venous pressure. The effects of ANP have been extensively
studied in a variety of species (11, 25). These studies
indicate that ANP is a natriuretic, diuretic, and vasodilatory peptide
involved in the regulation of volume and electrolyte balance and in the
regulation of systemic and pulmonary arterial pressure. ANP levels are
elevated in a number of pathological conditions, including
hypertension, cardiomyopathy, and congestive heart failure. ANP is
reported to cause arterial relaxation with increases in cGMP
accumulation by endothelium-independent mechanisms (9, 10,
24). ANP binds to specific receptors in target tissues,
including systemic vasculature, kidney, and lung (11, 25).
Receptor heterogeneity of the ANP effector system has been demonstrated
in vivo, as well as in vitro. At least three functionally distinct
subtypes of ANP receptors have been identified. The ANP-A and -B
receptors contain a guanylyl cyclase domain, and the other, ANP-C, has
only a short cytoplasmic tail and is thought to act as a clearance or
storage receptor by removing ANP from the circulation to regulate
plasma ANP levels (3). ANP binding to the ANP-A and/or -B
receptor is directly associated with increases in cGMP production by
activation of particulate guanylate cyclase. Analysis of the role of
the peptide in physiological and pathophysiological processes has been
impeded by the absence of a selective ANP receptor antagonist. HS-142-1
was isolated from the culture broth of Aureobasidium pullulans
var. melanigenum and found to be a potent, long-acting ANP-A- and ANP-B-receptor antagonist, and, although HS-142-1 has been
shown to inhibit ANP-induced increases in urine flow, sodium excretion,
and systemic hypotension in the rat, little if anything is known about
the ANP-receptor-mediating vasodilator responses in the pulmonary
vascular bed of the cat (16, 17, 19, 21). The present
study was, therefore, undertaken to investigate the hypothesis that ANP
produces vasodilation that is mediated by particulate guanylate cyclase
in the pulmonary vascular bed of the cat.
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MATERIALS AND METHODS |
In vivo.
Forty-two adult cats of either sex, weighing 2.4-4.5 kg (mean 3.4 kg), were sedated with ketamine hydrochloride (10-15 mg/kg im) and
were anesthetized with pentobarbital sodium (30 mg/kg iv). The animals
were strapped in the supine position to a fluoroscopic table, and
supplemental doses of anesthetic were administered as needed to
maintain a uniform level of anesthesia. The trachea was intubated with
a cuffed pediatric endotracheal tube, and the animals spontaneously
breathed room air enriched with 100% O2. Systemic arterial
(aortic) pressure was measured from a femoral artery, and intravenous
injections were made into a catheter positioned in the inferior vena
cava from a femoral vein.
For perfusion of the left lower lobe, a triple-lumen 28-cm-long 6-F
balloon perfusion catheter (Arrow International, Reading, PA) was
passed under fluoroscopic guidance from an external jugular vein into
the artery to the left lower lung lobe. After the animals were given
heparin (1,000 U/kg iv), the lobar artery was vascularly isolated by
distention of the balloon cuff on the perfusion catheter. The lung lobe
was perfused with a perfusion pump (model 1210; Harvard Apparatus,
Millis, MA) by way of the catheter lumen beyond the cuff, with blood
withdrawn from a femoral artery. The perfusion rate was adjusted so
that lobar arterial perfusion pressure approximated mean pressure in
the main pulmonary artery and was not changed thereafter. The flow rate
ranged from 29 to 51 ml/min. Left atrial pressure was measured with a
6-F double-lumen catheter (Arrow International) passed transseptally
into the left atrium. The catheter tip was positioned so that the left
atrial pressure port on the distal lumen was 1-2 cm into the lobar
vein and the second catheter port was near the venoatrial junction.
When necessary, blood could be withdrawn from the left atrium to keep
left atrial pressure constant. All vascular pressures were measured
with Statham P23 or Spectromed DTX Plus transducers (Spectromed,
Oxnard, CA) zeroed at right atrial level. Mean vascular pressures
obtained by electronic averaging were recorded on a Grass recorder
(model 7, Grass Instruments, Quincy, MA). Agonists were injected in
small volumes (30-100 µl) directly into the perfusion circuit
distal to the pump in a random sequence. Sufficient time was permitted between injections (usually 5-20 min) to allow lobar arterial perfusion pressure to return to baseline value.
In the present study, five sets of experiments were carried out. The
first set of experiments was undertaken to investigate the hypothesis
that ANP produces vasodilation that is mediated by the ANP-A and/or -B
receptor. The effects of HS-142-1, an ANP-A- and ANP-B-receptor
antagonist, on responses to ANP, acetylcholine, isoproterenol,
cromakalim, sodium nitroprusside, and prostacyclin were investigated in
the pulmonary vascular bed of the cat. When pulmonary lobar vascular
resistance was elevated by infusion of the stable prostaglandin analog
U-46619, the agonists were injected directly into the perfusion
circuit. The second set of experiments was undertaken to
investigate the hypothesis that nitric oxide synthesis may modulate
responses to ANP. In these experiments, vasodilator responses to ANP
were compared when lobar vascular tone was elevated with U-46619 alone
(15-200 ng/min) and when tone was increased by the administration
of N-nitro-L-arginine methyl
ester (L-NAME; 100 mg/kg iv), followed by an intralobar
infusion of U-46619 (10-100 ng/min). The dose of
L-NAME was determined in pilot experiments and has been
used in many previous studies (5, 6, 13, 14). In these
experiments, control responses to vasodilator agents were obtained when
lobar arterial pressure was raised to an average of 35 ± 2 mmHg
with U-46619 infused directly into the perfused lobar artery. The
U-46619 infusion was then terminated, and lobar arterial pressure was permitted to return to near-control value. L-NAME (100 mg/kg iv) was then administered as a slow intravenous bolus.
L-NAME increased lobar arterial and systemic arterial
pressure by 13 ± 1 and 44 ± 4 mmHg, respectively. After a
20- to 30-min period, the infusion of U-46619 was resumed at a rate
(10-100 ng/min) sufficient to raise lobar arterial pressure to
35 ± 1 mmHg, and responses to vasodilator agents were again
determined. Therefore, in this and all following experiments, responses
were obtained at similar levels of lobar arterial pressure during the
control and treatment periods. The third set of experiments was
undertaken to investigate the hypothesis that the nitric oxide synthase
modulation of ANP responses could be inhibited by administration of
cGMP. In the third set of experiments, vasodilator responses to ANP
were compared when lobar arterial pressure was raised with U-46619
alone and when tone was increased by intralobar infusions of U-46619
and 8-bromo-cGMP (8-BrcGMP) was infused into the lobar artery. In these
experiments, control responses to vasodilator agents were obtained when
lobar arterial pressure was raised to an average of 35 ± 1 mmHg
with U-46619. The lobar arterial pressure was permitted to return to
near-control value, and a constant intralobar infusion of 8-BrcGMP was
then started. The infusion rate of 8-BrcGMP, determined in pilot
experiments, decreased lobar arterial pressure by ~5 mmHg. Twenty to
thirty minutes after 8-BrcGMP, the infusion of U-46619 was increased
sufficiently to raise lobar arterial pressure to 35 ± 2 mmHg, and
responses to vasodilator agents were again determined. The fourth set
of experiments was similar to the previous set using a cAMP analog,
8-bromo-cAMP (8-BrcAMP), to investigate the hypothesis that the
responses seen with 8-BrcGMP were selective in nature. In the fifth set
of experiments, vasodilator responses to ANP, nitroglycerin, and sodium
nitroprusside were compared when lobar arterial pressure was raised
with U-46619 alone and when tone was increased by the administration of
L-NAME followed by a constant intralobar infusion of
U-46619 and 8-BrcGMP. In these experiments, lobar arterial pressure
averaged 35 ± 1 mmHg in the control period and 34 ± 2 mmHg
during the treatment period.
In vitro.
For in vitro studies, adult cats of either sex were sedated with
ketamine hydrochloride (10-15 mg/kg im) and were anesthetized with
pentobarbital sodium (30 mg/kg iv). The cat lungs were quickly removed
and immersed in cold (40°C) Krebs-Henseleit (KH) solution (composition in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, and 10 dextrose). The pulmonary artery was isolated,
and excess fat and connective tissue were removed. Vessels were cut
into 3- to 5-mm rings and mounted in organ baths containing 10-ml KH
solution. Two stainless steel hooks were inserted into the lumen of the
pulmonary artery: one was fixed, whereas the other was connected to a
transducer. The tissue bath solution was maintained at 37°C and
bubbled with a 95% O2-5% CO2 mixture. The
pulmonary arterial rings were equilibrated for 90 min with three
changes of KH solution, and an optimal tension of 2-3 g was
applied. Contractions were measured isometrically with
force-displacement transducers (FT03; Grass Instruments) and were
recorded on a Grass model 7 polygraph. The contractile ability of each
ring was then assessed by exposing the ring to 60 mM KCl, and then the
ring was washed and allowed to relax to baseline tension. Only when two
reproducible contractions could be elicited was the individual ring
used in further studies. The integrity of the endothelium was
determined by obtaining a maximal vasorelaxant response to acetylcholine. Three sets of experiments were carried out during the in
vitro portion of the study. The first set of experiments was undertaken
to investigate the hypothesis that responses to ANP are endothelium
independent in pulmonary arteries of the feline. The second set of
experiments investigated the hypothesis that ANP produces vasodilation
via a soluble guanylate cyclase-independent mechanism; methylene blue
(an inhibitor of soluble guanylate cyclase activation) was utilized.
The third set of experiments was undertaken to investigate the
hypothesis that ANP responses are independent of
L-arginine. To investigate the role of nitric oxide in
mediating responses to ANP, L-NAME and
N-monomethyl-L-arginine
(L-NMMA; substrate analog) were used. All agents were added
directly to the organ bath in a cumulative concentration manner. The
concentration of all drugs was reported as the final molar
concentration in the organ chamber.
Preparation of drugs and statistics.
Acetylcholine chloride, bradykinin, ANP, glyceryl trinitrate,
isoproterenol, sodium nitroprusside, 8-BrcGMP, 8-BrcAMP, dibutyryl-cAMP (DBcAMP), and prostacyclin (Sigma Chemical, St. Louis, MO) were dissolved in 0.9% saline. HS-142-1 was dissolved and diluted with saline. U-46619
(11
,9-epoxymethano-9
,11-dideoxyprostaglandin F2
; Upjohn, Kalamazoo, MI) was dissolved in 100%
ethanol at a concentration of 10 mg/ml, and further dilutions were made in normal saline. Methylene blue, L-NAME hydrochloride, and
L-NMMA (Sigma Chemical) were dissolved in normal saline
immediately before use. Cromakalim (SmithKline Beecham, Sussex, UK) was
dissolved in 20% ethanol in saline at a concentration of 1 mg/ml, and
further dilutions were made in 0.9% saline.
Blood gases and pH were measured with a Corning model 178 analyzer and
were in the normal range. All hemodynamic data are expressed in
absolute units and are presented as means ± SE. Responses represent peak changes, unless otherwise noted. These data were analyzed by using a one-way analysis of variance and Scheffé's F test or a paired t-test. P < 0.05 was the criterion for statistical significance.
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RESULTS |
Under baseline conditions, when tone in the pulmonary vascular bed
was at resting levels (12-15 mmHg), injections of ANP into the
perfused lobar artery in doses of 0.1 and 1.0 µg had no significant effect on lobar arterial pressure (data not shown). However, when lobar
arterial pressure was raised to a high steady value (35 ± 1 mmHg)
with an infusion of U-46619, intralobar injections of ANP in doses of
0.1-1 µg caused significant dose-related decreases in lobar
arterial pressure (Fig. 1). Left atrial
pressure was unchanged at all doses of the peptide studied (data not
shown). The effects of the ANP-receptor antagonist HS-142-1 on
pulmonary vasodilator responses to ANP were investigated, and these
data are also summarized in Fig. 1. The decreases in lobar arterial pressure in response to ANP (0.1-1 µg) under elevated tone
conditions were reduced significantly after administration of HS-142-1
in a dose of 10 mg/kg iv (Fig. 1A). The inhibitory effects
of the ANP-receptor antagonist on vasodilator responses to ANP were
overcome when larger doses of the peptide were administered (Fig.
1A). The duration of the actions of HS-142-1 was assessed by
comparing responses to ANP 2 h after administration of the
antagonist. There was little, if any, tendency for vasodilator
responses to ANP to return toward control 2 h after administration
of ANP-receptor blocking agent in a dose of 10 mg/kg iv (Fig.
1B). Pulmonary vasodilator responses to acetylcholine,
isoproterenol, cromakalim, sodium nitroprusside, and prostacyclin in
these experiments were not altered after administration of HS-142-1,
indicating that the ANP-receptor blockade was selective and that
responsiveness of the vascular bed did not change over the 2-h period
during which responses were studied (data not shown). Administration of
HS-142-1 in a dose of 10 mg/kg iv had no significant effect on mean
systemic arterial, lobar arterial, or left atrial pressure (data not
shown).
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Fig. 1.
A: influence of HS-142-1 on decreases in lobar
arterial pressure in response to atrial natriuretic peptide. The
peptide was injected into the perfused lobar artery in doses of
0.1-1 µg under elevated tone conditions (control). B:
duration of the inhibitory actions of HS-142-1 on responses to atrial
natriuretic peptide in the pulmonary vascular bed under elevated tone
conditions (control). Responses were compared before and after 2-h
administration of the atrial natriuretic peptide antagonist in a dose
of 10 mg/kg iv. Tone was increased with U-46619. Values are means ± SE; n, no. of animals. * Significantly different from
control, P < 0.05.
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The effect of the nitric oxide synthase inhibitor L-NAME on
pulmonary vasodilator responses to ANP was investigated, and responses to ANP were compared when tone in the pulmonary vascular bed was increased with U-46619 alone (control) and with U-46619 and
L-NAME. When lobar arterial pressure had attained a peak
value after administration of L-NAME, the U-46619 infusion
was again started, and the infusion rate was adjusted so that an
average pressure similar to that obtained during the control period was
attained. The administration of L-NAME in a dose of 100 mg/kg iv resulted in a significant increase in the pulmonary
vasodilator response to ANP (Fig. 2), nitroglycerin (Fig. 3), and sodium
nitroprusside (Fig. 3), whereas no change was seen in response to the
endothelium-independent vasodilator agonists cromakalim (data not
shown), isoproterenol (data not shown), prostacyclin (data not shown),
8-BrcGMP (Fig. 3), or DBcAMP (Fig. 3). After administration of
L-NAME, a significant decrease in vasodilator responses to
the endothelium-dependent agonists bradykinin and acetylcholine was
observed (data not shown). After administration of L-NAME,
the increase in vasodilator response to ANP, nitroglycerin, and sodium
nitroprusside was reproducible for up to 30 min and did not show
evidence of tachyphylaxis (data not shown).
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Fig. 2.
Influence of
N-nitro-L-arginine methyl ester
(L-NAME) on decreases in lobar arterial pressure in
response to atrial natriuretic peptide. Responses were compared before
(control) and after administration of L-NAME in a dose of
100 mg/kg iv. Values are means ± SE; n, no. of
animals. * Significantly different from control, P < 0.05.
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Fig. 3.
Influence of L-NAME and L-NAME
with constant infusion of 8-bromo-cGMP (8-BrcGMP) on decreases in lobar
arterial pressure in response to nitroglycerin (GTN; A) and
sodium nitroprusside (SNP; B). Responses were compared at
similar levels of baseline tone before (control) and after
administration of L-NAME and L-NAME with
constant infusion of 8-BrcGMP. C: influence of
L-NAME on decreases in lobar arterial pressure in response
to 8-BrcGMP and dibutyryl-cAMP (DBcAMP). Responses were compared at
similar levels of baseline tone before (control) and after
administration of L-NAME. Values are means ± SE;
n, no. of animals. * Significantly different from
control, P < 0.05.
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To investigate the role of the endothelium and the subsequent
activation of soluble guanylate cyclase in mediating vasodilator responses to ANP, responses were obtained in isolated feline pulmonary arterial rings with intact endothelium and without endothelium. In the
first set of experiments, ANP produced concentration-dependent relaxation of pulmonary arterial rings with and without endothelium precontracted with 15 µM U-46619, suggesting that ANP-induced vasorelaxation is not dependent on the presence of the endothelium (Fig. 4). In a separate set of
experiments, the role of soluble guanylate cyclase activation was
investigated. In pulmonary arterial rings with intact endothelium
precontracted with U-46619, addition of ANP
(10
9-107 M) produced
concentration-dependent relaxation (Fig.
5). After administration of methylene
blue, responses to ANP were not significantly altered. In the next set
of experiments, the influence of two nitric oxide synthase inhibitors
on responses to ANP were investigated. After administration of
L-NMMA or L-NAME (300 µM), a dose that significantly inhibited endothelial-dependent responses to
acetylcholine (data not shown), no significant change in the arterial
vasorelaxant response to ANP was observed compared with control (Fig.
6).
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Fig. 4.
Influence of endothelial cell removal on the pulmonary
vasorelaxant response to atrial natriuretic peptide on feline pulmonary
arterial (PA) rings precontracted with U-46619. Values are means ± SE; n, no. of rings from separate animals.
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Fig. 5.
Influence of methylene blue on the pulmonary vasorelaxant
response to atrial natriuretic peptide on feline PA rings precontracted
with U-46619. Control, responses without methylene blue. Values are
means ± SE; n, no. of rings from separate animals.
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Fig. 6.
Influence of L-NAME and
N-monomethyl-L-arginine
(L-NMMA) on the pulmonary vasorelaxant response to atrial
natriuretic peptide on feline PA rings precontracted with U-46619.
Control, response without L-NAME or L-NMMA.
Values are means ± SE; n, no. of rings from separate
animals.
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In addition to investigating the effects of inhibition of nitric oxide
synthase, the role of the endothelium, and the activation of soluble
guanylate cyclase, the effects of cGMP-dependent protein kinase
activation and of L-NAME on responses to ANP were studied in the pulmonary vascular bed of the cat. In experiments with 8-BrcGMP
before L-NAME, vasodilator responses to ANP were compared when tone was increased with U-46619 and, to a similar level, by an
infusion of both U-46619 and 8-BrcGMP. During treatment with 8-BrcGMP,
decreases in lobar arterial pressure in response to ANP (0.1-1
µg) were not significantly changed compared with responses obtained
during the U-46619 infusion in the control period (Fig.
7A). In experiments with
8-BrcGMP in the presence of L-NAME, vasodilator responses
to ANP were compared when tone was increased with U-46619 and to a
similar level after administration of L-NAME (100 mg/kg iv)
with constant infusion of U-46619 and 8-BrcGMP. Treatment with
L-NAME and 8-BrcGMP did not change the decreases in lobar
arterial pressure in response to ANP (0.1-1 µg) compared with
responses to ANP during the U-46619 infusion control period (Fig.
7B). However, responses to nitroglycerin and sodium
nitroprusside were significantly increased compared with responses
obtained in the U-46619 infusion control period (Fig. 3). In similar
experiments, the effects of constant infusion of 8-BrcAMP in the
presence of L-NAME were also investigated, and vasodilator
responses to ANP were compared when tone was increased with U-46619,
with L-NAME, and with L-NAME and constant
infusion of both U-46619 and 8-BrcAMP. During treatment with 8-BrcAMP
in the presence of L-NAME, decreases in lobar arterial
pressure in response to ANP (0.1-1 µg) were not significantly
different compared with the L-NAME treatment period but
were significantly decreased compared with responses obtained in the
U-46619 infusion control period (data not shown).
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Fig. 7.
A: influence of 8-BrcGMP on decreases in lobar
arterial pressure in response to atrial natriuretic peptide. Responses
were compared at similar levels of baseline tone before (control) and
after infusion of 8-BrcGMP. B: influence of
L-NAME and constant infusion of 8-BrcGMP on decreases in
lobar arterial pressure in response to atrial natriuretic peptide.
Responses were compared at similar levels of baseline tone before
(control) and after administration of L-NAME with constant
infusion of 8-BrcGMP. Values are means ± SE; n, no. of
animals.
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DISCUSSION |
Results of the present investigation show that ANP produces
dose-related decreases in lobar arterial pressure when tone in the
pulmonary vascular bed is elevated with U-46619 and that responses to
the peptide are inhibited by HS-142-1, an ANP-A- and ANP-B-receptor antagonist. Inasmuch as blood flow was maintained constant and left
atrial pressure was unchanged, the decreases in lobar arterial pressure
reflect decreases in lobar vascular resistance. The inhibitory effects
of HS-142-1 on vasodilator responses to ANP were overcome when larger
doses of the peptide were injected after administration of the ANP-A-
and ANP-B-receptor antagonist. The shift to the right of the ANP
dose-response curve was parallel after administration of HS-142-1,
suggesting that the blockade was competitive in nature. The duration of
action of the ANP-A- and ANP-B-receptor antagonist was long, and there
was little tendency for responses to the peptide to return toward
control value over the 2-h period in which responses were investigated.
Although responses to ANP were reduced significantly up to 2 h
after administration of HS-142-1, vasodilator responses to
acetylcholine were not changed, suggesting that the responsiveness of
the vascular bed was not impaired by treatment with the ANP-A- and
ANP-B-receptor antagonist. The specificity of the blocking effects of
HS-142-1 was further investigated by studying the effects of the
antagonist on responses to agonists that dilate the pulmonary vascular
bed by diverse mechanisms, and the results of these experiments show
that responses to isoproterenol, cromakalim, sodium nitroprusside, and
prostacyclin were not altered. The long duration and specificity of the
inhibitory actions of HS-142-1 in the pulmonary vascular bed extend the
results of previous studies of systemic arterial pressure in the
anesthetized rat, of experiments in bovine adrenocortical cells, and of
experiments in neuronal cell lines (17, 21, 22). The long
duration of action of the ANP-A- and ANP-B-receptor antagonist on
responses to ANP is probably due to the polysaccharide nature, which
makes the antagonist resistant to degradation by neutral endopeptidase
and other peptidases present in the lung (21). The results
of experiments with HS-142-1 suggest that ANP dilates the pulmonary
vascular bed by stimulating ANP-A and/or -B receptors on undefined
resistance vessel elements; that the inhibitory effects of HS-142-1 are
selective, competitive, and long in duration; and that the antagonist
had little agonist activity.
Results of the present study show that L-NAME increases
systemic and lobar arterial pressures in the intact-chest cat. These data are consistent with results of studies in the pulmonary
circulation of the cat, lamb, fetal lamb, and rabbit in which nitric
oxide synthase inhibitors increased pulmonary vascular resistance
(1, 2, 5-8, 13, 20). These studies are consistent
with the hypothesis that tonic release of nitric oxide may serve to
regulate baseline vascular tone in the pulmonary circulation. In
addition to increasing lobar vascular resistance in the cat,
L-NAME significantly increased vasodilator responses to
ANP, nitroglycerin, and sodium nitroprusside. The enhanced vasodilator
response to agents that release nitric oxide is not observed in all
models, and the mechanism is uncertain, but it has been hypothesized
that the removal of the basal nitric oxide-mediated vasodilator tone in
the cardiovascular system leads to a "specific supersensitivity" of
soluble guanylate cyclase (13, 15). The results of the
present study with nitroglycerin and sodium nitroprusside are
consistent with previous studies and with the hypothesis that
inhibition of nitric oxide synthesis with agents such as
L-NAME will enhance responses to nitrovasodilators. The
observation that vasodilator responses to 8-BrcGMP, DBcAMP, cromakalim,
isoproterenol, or prostacyclin are not reduced suggests that
L-NAME did not alter endothelium-independent responses
mediated by agents with increased cGMP or cAMP levels, activate cGMP-
or cAMP-dependent protein kinases, stimulate -adrenergic or
prostacyclin receptors, or open ATP-sensitive potassium channels.
Moreover, results demonstrating that vasodilator responses to
bradykinin and acetylcholine are reduced by L-NAME suggest
that they are mediated in part by the release of nitric oxide and the
activation of soluble guanylate cyclase in the pulmonary vascular bed
(4, 6, 13).
The results showing that responses to ANP are enhanced after
L-NAME administration extend previous observations in
certain studies showing enhancement of vasodilator responses to agents that release nitric oxide (13, 15). ANP produces
smooth-muscle relaxation through increases in cGMP by activation of
ANP-A and/or -B receptors and the subsequent stimulation of particulate
guanylate cyclase (11, 25). The observation that responses
to ANP are enhanced by nitric oxide synthase inhibitors may suggest
that the "supersensitivity" seen with the stimulation of soluble
guanylate cyclase after administration of an arginine analog may also
be induced by the ANP-stimulated particulate guanylate cyclase in the
presence of L-NAME. Furthermore, the observation that
responses to 8-BrcGMP (a lipophilic cGMP analog) and DBcAMP (a
lipophilic cAMP analog) were not significantly different after
administration of L-NAME suggests that the supersensitivity
to ANP is selective for the particulate guanylate cyclase pathway
and occurs at the level of the ANP-A or -B receptor, membrane-bound
guanylate cyclase.
8-BrcGMP is a lipophilic cGMP analog that directly activates
cGMP-dependent protein kinase (12). ANP responses were not significantly different after administration of L-NAME with
constant infusion of 8-BrcGMP, demonstrating that 8-BrcGMP infusion was able to inhibit the increased vasodilation that was seen after the
L-NAME treatment period, whereas constant infusion of
8-BrcGMP alone did not significantly alter responses to ANP. These data further support the hypothesis that the supersensitivity to ANP occurs
at a level upstream to cGMP-dependent protein kinase activation. During
the L-NAME treatment period, responses to nitroglycerin and
sodium nitroprusside were significantly enhanced compared with
responses obtained in the control period; however, during constant
infusion of 8-BrcGMP with L-NAME, responses to
nitroglycerin and sodium nitroprusside were not significantly changed
compared with responses in the L-NAME treatment period.
These data show that 8-BrcGMP does not have the ability to inhibit the
increased vasodilator response seen in response to agents that are
nitric oxide donors. The reason for the difference between effects on ANP and on nitroglycerin or sodium nitroprusside is unknown but may be
related to the action of soluble guanylate cyclase in the endothelial
cell and the action of particulate guanylate cyclase in the vascular
smooth muscle cell.
The results of this study show that ANP relaxed precontracted rings of
feline pulmonary artery with and without endothelium and are consistent
with previous studies of the bovine pulmonary circulation and of the
systemic circulation of a variety of species (10, 11, 18,
25). Previous reports have shown that ANP causes
endothelium-independent relaxation of arteries and cGMP, but not cAMP,
accumulation (10). The observation that methylene blue, an
inhibitor of soluble guanylate cyclase or superoxide radical inducer
(6, 13), failed to alter arterial relaxant responses to
ANP is consistent with reports that stimulation of ANP receptors
activates the particulate rather than the soluble form of guanylate
cyclase and that this activation is methylene blue insensitive.
Previous studies have reported that responses to ANP and ANP receptors
exhibit heterogeneity. L-NAME and the arginine analog L-NMMA did not significantly alter responses to ANP in
precontracted feline pulmonary rings. The reason for the difference in
results between the in vitro and in vivo responses of ANP is unknown
but may be due to differences in the inhibitory function domain within the ANP receptor (23). These results may suggest a
fundamental difference in response to ANP in the conduit-type pulmonary
arterial segment used in vitro and the resistance vessel segments that regulate tone and responses in vivo in the feline pulmonary vascular bed.
In summary, the present results show that, under elevated tone
conditions, ANP has potent pulmonary vasodilator activity and that
responses to the peptide are blocked in a selective manner by the
ANP-receptor antagonist HS-142-1, indicating that responses are
mediated by ANP-A and/or -B receptors. The present data show that
vasodilator responses to ANP are increased by L-NAME,
indicating that inhibition of basal release of nitric oxide modulates
responses to ANP. The observation that ANP-induced vasodilator
responses were not enhanced after administration of L-NAME
and constant infusion of 8-BrcGMP suggests that supersensitivity to ANP
occurs upstream to the activation of cGMP-dependent protein kinase.
Furthermore, although the reason for the difference is unknown, the
present data show that the supersensitivity to ANP is not observed in isolated pulmonary arterial segments, suggesting that this
supersensitivity may be observed only in resistance vessel elements in
the intact pulmonary vascular bed of the cat.
| |
ACKNOWLEDGEMENTS |
This study was supported in part by National Heart, Lung, and Blood
Institute Grant HL-62000 and a grant from the American Heart Association.
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
P. J. Kadowitz, Dept. of Pharmacology, SL83, Tulane Univ. School
of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail:
pkadowi{at}tulane.edu).
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 5 November 1999; accepted in final form 8 December 2000.
| |
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