Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-5218
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ABSTRACT |
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Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3'5'-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.
isolated rat lung; nitric oxide; soluble guanylate cyclase; phosphodiesterase; pulmonary hypertension
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INTRODUCTION |
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CHRONIC EXPOSURE TO HYPOXIA results in structural as well as functional alterations in the pulmonary vasculature, leading to the development of pulmonary hypertension and right ventricular hypertrophy. The principal mediators of chronic hypoxia (CH)-induced pulmonary hypertension are polycythemia, pulmonary arterial remodeling, and arterial constriction. Among the corresponding functional alterations is responsiveness to vasoactive factors. Studies from our laboratory (23, 24, 26) and others (12, 20, 22, 28, 29) have shown that CH augments pulmonary vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent vasodilators. EDNO is synthesized in the vasculature by endothelial nitric oxide synthase (eNOS), and several studies have demonstrated that CH is associated with increased pulmonary eNOS levels, gene expression, and activity (12, 15, 20, 23, 24, 32, 39). Consistent with elevated eNOS levels, nitric oxide (NO) synthesis appears to be greater in lungs isolated from CH rats compared with normoxic controls (12, 20, 30, 36). Despite enhanced reactivity to EDNO-dependent vasodilators, some studies have demonstrated impaired responsiveness to exogenous NO following CH (7, 19, 24, 27, 38). However, it is unclear whether this attenuated reactivity results from acute effects of increased endogenous NO production or rather from decreased sensitivity of vascular smooth muscle (VSM) to NO. The present study investigates downstream effectors in the NO-dependent signal transduction pathway to determine the mechanism of decreased NO sensitivity in the pulmonary vasculature following CH.
Although studies suggest NO can act through cGMP-independent mechanisms
(4), the most prominent target in VSM for NO is soluble
guanylyl cyclase (sGC), where binding to the heme moiety activates the
enzyme, increasing intracellular cGMP. Levels of cGMP are further
regulated by the rate of degradation by cGMP-specific phosphodiesterase
type 5 (PDE5), which plays a significant role in modulating pulmonary
VSM tone (5, 6, 8, 13, 21, 40). Once formed, cGMP can
mediate VSM relaxation through several mechanisms involving a decrease
in intracellular calcium and/or decreased sensitivity of the
contractile apparatus to calcium (17). Therefore, we
hypothesized that attenuated NO-dependent pulmonary vasodilation
following CH is mediated by downregulation of VSM sGC expression and/or
activity, increased cGMP degradation by PDE5, or decreased sensitivity
of VSM to cGMP. To study this, we examined vasodilatory responses to
both NO donors and dissolved NO solutions in lungs isolated from
normoxic and CH rats. The NOS inhibitor
N-nitro-L-arginine
(L-NNA) was used in some experiments to eliminate any acute
influence of endogenously produced NO. Additional lungs from each group
were isolated to determine effects of sGC inhibition and PDE5
inhibition on NO-dependent vasodilation. Expression and activity of
pulmonary sGC were assessed by Western blot analysis and a cGMP
radioimmunoassay (RIA), respectively. Furthermore, we assessed effects
of CH on pulmonary VSM sensitivity to the membrane-permeable cGMP
analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP).
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METHODS |
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All protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine (Albuquerque, NM).
Experimental Groups
Female (200-250 g) Sprague-Dawley rats (Harlan Industries) were divided into two groups for each experiment. Animals designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ~380 mmHg for 4 wk. The chamber was opened three times per week to provide animals with fresh food, water, and clean bedding. On the day of experimentation, rats were removed from the hypobaric chamber and immediately placed in a Plexiglas chamber continuously flushed with a 12% O2-88% N2 gas mixture to reproduce inspired PO2 (~70 mmHg) within the hypobaric chamber. Age-matched control animals were housed at ambient barometric pressure (~630 mmHg). All animals were maintained on a 12:12-h light-dark cycle.CH-Induced Right Ventricular Hypertrophy and Polycythemia
Blood samples were obtained by direct cardiac puncture at the time of lung isolation for measurement of hematocrit. Right ventricular hypertrophy was assessed as an index of CH-induced pulmonary hypertension, as previously described (23, 24). Briefly, after isolation of the heart, the atria and major vessels were removed from the ventricles. The right ventricle (RV) was dissected from the left ventricle and septum, and each was weighed. The degree of right ventricular hypertrophy is expressed as the ratio of RV to total ventricle weight (T).Isolated Lung Preparation
Rats from each group were anesthetized with pentobarbital sodium (52 mg ip). After the trachea was cannulated with a 17-gauge needle stub, the lungs were ventilated with a Harvard positive-pressure rodent ventilator (model 683) at a frequency of 55 breaths/min and a tidal volume of 2.5 ml with a warmed and humidified gas mixture (6% CO2 in room air). Inspiratory pressure was set at 9 cmH2O, and positive end-expiratory pressure was set at 3 cmH2O. After a median sternotomy, heparin (100 units) was injected directly into the RV, and the pulmonary artery was cannulated with a 13-gauge needle stub. The preparation was immediately perfused with a physiological saline solution (in mM: 129.8 NaCl, 5.4 KC1, 0.83 MgSO4, 19 NaHCO3, 1.8 CaC12, and 5.5 glucose; all from Sigma) containing 4% (wt/vol) albumin (Sigma), meclofenamate (30 µM; Sigma), and L-NNA (300 µM; Sigma) at 0.8 ml/min with a Masterflex microprocessor pump drive (model 7524-10). Meclofenamate and L-NNA were added to acutely minimize the potential complicating influences of endogenous prostaglandins and NO on vascular reactivity. This dose of meclofenamate is approximately threefold higher than that previously shown to provide effective inhibition of prostaglandin synthesis in this preparation (9). We have previously demonstrated that the dose of L-NNA employed is effective in inhibiting EDNO-dependent pulmonary vasodilation in the isolated perfused rat lung (24, 26). Additional experiments were performed in the absence of L-NNA where noted.The LV was cannulated with a plastic tube (4-mm outer
diameter), and the heart and lungs were removed en bloc and
suspended in a humidified chamber maintained at 38°C. The perfusion
rate was gradually increased to 30 ml · min1 · kg
1 body weight
and maintained at this rate for the duration of the experiment. Twenty
milliliters of perfusate were washed through the lungs and discarded
before recirculation was initiated with 40 ml. Experiments were
performed with lungs in zone 3 conditions, achieved by
elevating the perfusate reservoir until pulmonary venous pressure
(Pv) was 3-4 mmHg. Pulmonary arterial pressure (Pa) and Pv were measured with Spectramed P23XL
pressure transducers and recorded on a Gould RS 3400 chart recorder.
Data were stored and processed with a computer-based data
acquisition/analysis system (AT-CODAS, Dataq Instruments).
After a 30-min stabilization period, the thromboxane analog
9,11-dideoxy-11,9
-epoxymethanoprostaglandin F2
(U-46619, Cayman Chemical) was added to the perfusate reservoir
until a stable arterial pressor response of ~10 mmHg was achieved.
U-46619 provides consistent and stable pressor responses in this
preparation (23, 24, 26), allowing assessment of
subsequent vasodilatory responses as outlined in the following
protocols. A double occlusion technique was employed for all protocols
to allow calculation of segmental vascular resistances as described
previously (23-26).
Isolated Lung Experiments
Vasodilatory responses to NO donors. To examine the effect of CH on NO-dependent pulmonary vasodilation, we assessed responses to two mechanistically independent NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and spermine NONOate. After attainment of a stable vasoconstrictor response to U-46619, a cumulative dose-response relationship to SNAP (1 and 10 µM; Sigma) was assessed in lungs from control and CH rats in the presence or absence of L-NNA. A stable vasodilatory response to the first dose of SNAP was allowed to develop before administration of the second dose. A similar dose-response relationship was generated for spermine NONOate (0.1 and 1 µM; Cayman Chemical) in separate sets of lungs from each group. The concentrations of each NO donor were determined to provide dose-dependent vasodilation in preliminary experiments. Parallel experiments were performed with N-acetylpenicillamine (1 and 10 µM; Sigma) and spermine (0.1 and 1 µM; Sigma) to test for possible nonspecific actions of SNAP and spermine NONOate, respectively.
Vasodilatory responses to pinacidil. To determine whether decreased responsiveness to NO following CH represents an attenuated reactivity to vasodilators in general, we examined responses to the ATP-sensitive K+ channel (KATP) activator pinacidil. A cumulative dose-response relationship to pinacidil (10 and 100 µM; Sigma) was assessed in U-46619-constriced lungs from both groups. A stable vasodilatory response to the first dose of pinacidil was allowed to develop before administration of the second dose.
Effects of sGC inhibition on NO-mediated vasodilation.
The contribution of endogenous cGMP in mediating vasodilatory responses
to NO donors in each group of rats was assessed using a heme
site-specific sGC inhibitor,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Sigma). ODQ (50 µM) or its vehicle (DMSO, 75 µl) was added to
the recirculating reservoir (40 ml) immediately after lung isolation
and was present throughout the experiment. This dose of ODQ has been
previously employed by other investigators to inhibit sGC in this
preparation (3). Responses to SNAP (1 and 10 µM) and
spermine NONOate (1 µM) were then determined as described above. To
demonstrate specificity of ODQ, we assessed vasodilatory responses to
the -adrenergic receptor agonist isoproterenol (10 nM) in the
presence or absence of ODQ (50 µM) in separate sets of lungs from
control animals.
Effect of PDE5 inhibition on NO-dependent vasodilation. cGMP is rapidly hydrolyzed to GMP by the PDE5 family of PDEs in pulmonary VSM (6, 18). To assess the possible role of increased PDE5 activity in the attenuated NO-dependent vasodilation following CH, we examined responses to SNAP (0.1, 0.5, and 1 µM) in lungs from each group after treatment with the selective PDE5 inhibitor, dipyridamole (10 µM), or its vehicle [ethanol (EtOH); 100 µl]. This dose of dipyridamole was determined to augment SNAP-induced pulmonary vasodilation in preliminary experiments.
Additional experiments examined effects of PDE5 inhibition on the amplitude and duration of vasodilatory responses to bolus administration of NO-containing solutions in lungs from each group of rats. After treatment with dipyridamole (10 µM) or vehicle (EtOH, 100 µl), lungs were administered successive arterial boluses of NO solution (100 µl of 103-, 104-, and 105-fold dilutions from a saturated stock solution). Vasodilatory responses to NO solutions were transient, and therefore full recovery of each response was allowed before administration of subsequent doses. Parallel experiments were conducted using the more selective PDE5 inhibitor, T-1032 (1 µM; Sigma) (14), or its vehicle (methanol, 100 µl). Saturated NO solution was prepared by first deoxygenating double distilled water (ddH2O) by bubbling with 100% N2 for 20 min. The deoxygenated ddH2O was then bubbled with 100% NO gas (Matheson Gas Products) for 20 min at 0°C. The saturated NO solution was diluted to working concentrations with deoxygenated ddH2O in airtight Vacutainers.Vasodilatory responses to 8-Br-cGMP. Because attenuated NO-dependent pulmonary vasodilation following CH could potentially result from altered VSM reactivity to cGMP, additional experiments examined responses to the membrane-permeable cGMP analog 8-Br-cGMP (1 and 10 µM; Sigma) in U-46619-constricted lungs from each group in the presence or absence of L-NNA. 8-Br-cGMP produced slowly developing and progressive vasodilatory responses in lungs from control rats; therefore, assessments were made 20 min after administration. In contrast, responses to 1 µM 8-Br-cGMP in lungs from CH rats were minimal and stable during this period. Consequently, responses to each dose were assessed in separate lungs from each group of rats. Although 8-Br-cGMP is reportedly a PDE-resistant molecule (41), we further assessed vasodilatory responses to 1 µM 8-Br-cGMP in the presence of dipyridamole or its vehicle in each group of animals to confirm PDE resistance in this preparation.
cGMP RIA
Whole lung cGMP levels were assessed to examine whether attenuated responsiveness to NO following CH is associated with decreased cGMP synthesis. Lungs from normoxic and CH rats were isolated and prepared for experimentation as described above and treated with dipyridamole (10 µM). Lungs were quickly frozen in liquid N2 at maximal dilation to a 104-fold dilution of NO stock solution. A separate set of normoxic control lungs was treated with ODQ (50 µM) before administration of NO. Lungs were coarsely ground with a mortar and pestle. Acid extract homogenization buffer (0.1 N HCl, 450 µM IBMX) was added to each tissue sample and allowed to incubate for 1 h at room temperature. Tissue samples were homogenized with a Dounce homogenizer and centrifuged at 6,000 g for 10 min. Supernatant was collected and lyophilized overnight. Whole lung cGMP content was assessed using a standard RIA kit (Amersham) and expressed as femtomoles per milligram of lung tissue.sGC Western Blots
The enzyme sGC is a heterodimer consisting ofCalculations and Statistics
Pulmonary vascular resistance in isolated, perfused lungs was calculated as the difference between Pa and Pv divided by flow (30 ml · min ![]() |
RESULTS |
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CH-Induced RV Hypertrophy and Polycythemia
RV:T ratios were greater in CH rats (n = 88, 0.312 ± 0.003) compared with normoxic control rats (n = 94, 0.209 ± 0.002), thus demonstrating RV hypertrophy indicative of pulmonary hypertension. Furthermore, CH rats exhibited polycythemia as indicated by a significantly greater hematocrit in CH rats (n = 87, 61 ± 0%) compared with normoxic rats (n = 98, 43 ± 0%).Isolated Lung Experiments
Baseline vascular resistances and responses to U-46619.
Consistent with previous reports (23, 24, 26),
baseline vascular resistances were significantly greater in lungs
from CH (n = 62, 0.112 ± 0.003 mmHg · ml1 · min · kg)
rats compared with normoxic controls (n = 68, 0.071 ± 0.002 mmHg · ml
1 · min · kg). Because
the pulmonary circulation exhibits no detectable tone in this
preparation (25), these data provide functional evidence
for CH-induced vascular remodeling. U-46619 produced similar increases
in resistance between CH (0.331 ± 0.009 mmHg · ml
1 · min · kg) and
normoxic (0.356 ± 0.012 mmHg · ml
1 · min · kg) groups.
Furthermore, we observed no differences in the concentration of U-46619
required to elicit comparable vasoconstriction between CH (124 ± 7 nM) and normoxic (110 ± 27 nM) groups.
Vasodilatory responses to NO donors.
Vasodilatory responses to both 1 and 10 µM SNAP were
attenuated in lungs from CH rats (Fig.
1A) compared with the
normoxic control group. In addition, vasodilatory responses were
reduced at both doses of spermine NONOate (0.1 and 1 µM) after
CH (Fig. 1B). N-acetylpenicillamine
(n = 2, 1 and 10 µM) and spermine (n = 2, 0.1 and 1 µM) exhibited no apparent vasoactive properties in
lungs from normoxic rats (data not shown). In the absence of L-NNA, vasodilatory responses to 1 µM SNAP were
similarly attenuated in lungs from CH rats (Table
1). Because vasodilatory
responses were similar for arterial and venous segments of the
pulmonary vasculature as assessed by the double occlusion method,
segmental responses are not shown for these and all subsequent
protocols.
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Vasodilatory responses to pinacidil.
In contrast to NO-dependent responses, CH lungs demonstrated augmented
vasodilatory responses to the KATP channel activator, pinacidil, compared with lungs from normoxic rats (Fig.
2).
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Effects of sGC inhibition on NO-mediated vasodilation.
The sGC inhibitor ODQ effectively blocked vasodilatory responses to
SNAP (1 and 10 µM, Fig. 3A)
and spermine NONOate (1 µM, Fig. 3B) in lungs from both CH
and normoxic rats. There was no effect of ODQ on vasodilatory responses
to the cAMP-dependent dilator isoproterenol (DMSO vehicle: 83.97 ± 1.82%, n = 4; ODQ: 78.52 ± 3.77%,
n = 4). Together, these data demonstrate the
specificity of ODQ for sGC and suggest NO-dependent vasodilation is
mediated through cGMP in this preparation.
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Effects of PDE5 inhibition on NO-dependent vasodilation.
The selective PDE5 inhibitor dipyridamole significantly augmented
vasodilatory responses to SNAP (0.5 and 1 µM) in lungs from CH rats
(Fig. 4). In contrast, dipyridamole
augmented reactivity to SNAP in lungs from control animals only at the
1-µM concentration. Although decreased responsiveness to 1 µM SNAP
persisted in lungs from CH vs. normoxic rats after treatment with
dipyridamole, reactivity to 0.5 µM SNAP was not different between
groups after PDE5 inhibition. No significant differences in responses
to 0.1 µM SNAP were observed between groups (Fig. 4).
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Vasodilatory responses to 8-Br-cGMP.
Similar to NO-dependent reactivity, lungs from CH rats exhibited
significantly smaller vasodilatory responses to 1 µM 8-Br-cGMP compared with those of normoxic rats in the presence (Fig.
7A) or absence of
L-NNA (Table 1). However, no differences in reactivity were
observed between groups at the 10 µM dose of 8-Br-cGMP (Fig. 7A and Table 1). Dipyridamole had no effect on vasodilatory
responses to 8-Br-cGMP in lungs from either group (Fig. 7B).
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cGMP RIA
There were no differences in cGMP levels in response to a 104-fold dilution of NO stock solution between lungs from CH and normoxic rats in the presence of dipyridamole. However, lungs from normoxic rats treated with ODQ demonstrated significantly lower levels of cGMP compared with normoxic lungs pretreated with dipyridamole (Fig. 8A).
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sGC Western Blot
Immunoreactive sGC- ![]() |
DISCUSSION |
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The present study examined potential mechanisms by which CH attenuates NO-mediated pulmonary vasodilation. The major findings from this study are: 1) CH attenuates pulmonary vasodilation to exogenous NO; 2) vasodilatory responses to the KATP channel agonist pinacidil are augmented after CH; 3) pulmonary vasodilatory responses to NO are cGMP dependent; 4) although PDE5 inhibition produced a greater potentiation of NO-mediated vasodilation in lungs from CH vs. normoxic rats, CH-induced attenuation of NO-dependent responsiveness persisted after PDE5 inhibition; 5) decreased pulmonary vasoreactivity to NO after CH does not appear to be associated with reduced sGC expression or activity; and 6) CH similarly inhibited 8-Br-cGMP-mediated pulmonary vasodilation. These data suggest the attenuated pulmonary vasodilatory response to NO after CH is mediated by both increased PDE5 activity and decreased VSM sensitivity to cGMP.
Previous studies from our laboratory (23, 24, 26) and others (12, 20, 22, 28, 29) suggest that CH augments EDNO-dependent pulmonary vasodilation, a response associated with upregulation of pulmonary eNOS mRNA and protein levels (12, 15, 20, 23, 24, 32, 39) as well as increased synthesis of NO (12, 20, 36). Despite this enhanced reactivity to EDNO-mediated agonists, we have recently reported that CH attenuates vasodilatory responsiveness to exogenous NO in lungs isolated from female rats (24). However, considering this impaired responsiveness to NO after CH could potentially be explained by acute effects of increased endogenous NO synthesis and thus greater basal activity of NO-associated signal transduction pathways in VSM, the present study examined responses to both NO donors and authentic NO after nitric oxide synthase (NOS) inhibition with L-NNA in lungs isolated from normoxic and CH female rats. We found that CH-induced attenuation of reactivity to both SNAP and spermine NONOate (Fig. 1) as well as authentic NO (Figs. 5 and 6) persisted after NOS inhibition, suggesting this change in reactivity to NO after CH represents decreased VSM sensitivity to NO. However, the mechanisms responsible for this effect of CH on NO-dependent pulmonary vasoreactivity are not presently understood.
Consistent with our present findings are reports of impaired NO-dependent vasorelaxation in isolated conduit pulmonary arteries from CH male rats compared with normoxic controls (7, 19, 27, 38). However, these results are at odds with previously published observations from our laboratory and others demonstrating no significant effect of CH on vasodilatory responsiveness to NO-donors (26) or inhaled NO (25, 8) in isolated lungs from male rats. Furthermore, Isaacson et al. (12) reported that CH enhanced vasodilatory responses to arterial boluses of saturated NO solution in isolated lungs. The reasons for these discrepancies are not presently clear but could be consequences of different preparations employed, a gender difference, and/or the presence of NOS inhibition in the current study. However, this effect of CH appears to be independent of ovarian hormones, as evidenced by similar CH-induced inhibition of NO-dependent vasodilation in lungs from ovarian-intact and ovariectomized rats (24). Furthermore, unpublished observations from our laboratory have demonstrated a similar inhibition of reactivity to both 1 and 10 µM SNAP after NOS inhibition in lungs from CH vs. normoxic male rats (n = 6/group), further suggesting this response to CH is not gender specific. In contrast to these effects of CH on NO-mediated responses, we observed augmented vasodilation to the KATP channel agonist pinacidil after hypoxic exposure (Fig. 2), as previously reported in isolated pulmonary arteries from male rats (27, 38). The mechanism of this enhanced responsiveness to pinacidil after CH is not clear but may be secondary to CH-induced pulmonary VSM membrane depolarization (33, 34, 35) and, therefore, greater inhibition of calcium influx through voltage-dependent calcium channels after pinacidil-induced membrane hyperpolarization. Nonetheless, these data support the view that attenuated NO-dependent responsiveness after CH is not associated with a generalized decrease in reactivity to vasodilators but may be specific to the NO pathway.
Although NO appears to mediate VSM relaxation, at least in part,
through cGMP-independent mechanisms in some vascular preparations (4), the primary target of NO in the pulmonary circulation is sGC (4, 31). Activation of sGC leads to cGMP synthesis, which results in VSM relaxation through various mechanisms involving a
decrease in intracellular calcium and desensitization of the contractile apparatus to calcium (17). Consistent with a
primary role of sGC activity in mediating NO-dependent pulmonary
vasodilation, we found that the heme site-specific sGC inhibitor ODQ
abolished vasodilatory responses to SNAP and spermine NONOate in lungs
from both normoxic and CH rats (Fig. 3). However, ODQ did not alter vasodilatory responses to the -adrenergic agonist isoproterenol, suggesting ODQ is selectively blocking sGC and not cAMP-dependent vasodilatory pathways.
cGMP hydrolysis is regulated primarily by cGMP-specific PDEs (PDE5) in pulmonary VSM (5, 6, 18). In addition, selective PDE5 inhibitors are potent vasodilators in the hypertensive pulmonary circulation, suggesting an important role for PDE5 in regulation of pulmonary vascular tone (5, 6, 8, 13, 21, 40). Interestingly, PDE5 expression (2) and activity (2, 18, 21) appear to be increased after pulmonary hypertension. Considering this, we hypothesized that increased PDE5 activity is mediating the impaired vasodilation to NO donors in lungs from CH rats. Although the selective PDE5 inhibitors dipyridamole and T-1032 largely augmented vasodilatory responses to SNAP and arterial boluses of NO solutions in lungs from CH rats, these inhibitors appeared to have minimal effects on NO-dependent responses in lungs from normoxic rats. Although it is possible that other PDE isoforms contribute to cGMP hydrolysis and diminished vasodilation to NO after CH, these results nevertheless suggest increased PDE5 activity is, at least in part, responsible for this effect of CH. However, the maintained attenuation of reactivity to NO in CH lungs after PDE5 inhibition (Figs. 4-6) suggests that other mechanisms may be involved, including decreased sGC expression/activity or reduced VSM sensitivity to cGMP.
Considering that impaired reactivity to NO after CH could result from
decreased pulmonary sGC expression, we further examined relative
expression levels of sGC- in whole lung homogenates from each group
of rats. However, Western blot analyses revealed a tendency for
upregulation of sGC-
after CH, although this did not reach
statistical significance (Fig. 8, B and C,
P = 0.051). These findings are consistent with recent
studies demonstrating elevated sGC expression and activity in pulmonary
hypertensive lambs (2) as well as increases in lung sGC
protein, mRNA levels, and increased sGC activity in whole lung tissue
from male rats after 21 days of hypoxic exposure (16).
Furthermore, this change in sGC expression appeared to be localized
primarily to small pulmonary arteries as assessed by
immunohistochemistry and in situ hybridization (16). In
the current study, we also did not detect differences in NO-stimulated
cGMP levels between lungs from CH and normoxic rats after PDE5
inhibition (Fig. 8A), suggesting that the maintained
attenuation of NO-mediated dilation after PDE5 inhibition in CH lungs
is a function of decreased VSM reactivity to cGMP, as opposed to
reduced cGMP synthesis.
Finally, we tested the hypothesis that a decrease in pulmonary VSM sensitivity to cGMP mediates CH-induced attenuation of NO-dependent reactivity. Consistent with this hypothesis, we observed decreased vasodilation to the relatively stable and PDE-resistant cGMP analog 8-Br-cGMP in lungs from CH rats compared with lungs from normoxic rats (Fig. 7A and Table 1). Dipyridamole had no effect on vasodilatory responses to 1 µM 8-Br-cGMP in lungs from normoxic and CH rats, suggesting that 8-Br-cGMP is PDE5 resistant in our preparation (Fig. 7B). These data are in agreement with other studies, which have shown an inhibitory effect of CH on responsiveness to 8-Br-cGMP in rat main pulmonary arteries and isolated lungs from neonatal pigs (1, 27). These findings suggest that the reduction in vasodilatory responsiveness to NO after CH involves a decrease in VSM sensitivity to cGMP.
CH could desensitize pulmonary VSM to cGMP through several mechanisms, including changes in the activity of downstream targets of cGMP. The most well recognized of these is protein kinase G, which mediates VSM relaxation through activation of multiple Ca2+-lowering mechanisms as well as Ca2+ desensitization of VSM (17). Other potential targets include cGMP-inhibitable cAMP PDEs (PDE3) (10, 37) and protein kinase A (PKA) (11, 31), although the relative contribution of the cAMP/PKA pathway to NO-dependent pulmonary responses is not well understood. Alternatively, impaired NO-mediated responsiveness after CH could be due to altered Ca2+ sequestration, influx or efflux mechanisms, or a change in sensitivity of the contractile machinery to Ca2+ (17). However, any such changes are likely specific to the NO/cGMP pathway, considering that CH augmented pulmonary vasodilation to the cGMP-independent agonist pinacidil (Fig. 2).
In summary, we have investigated the possibility that alterations in cGMP formation/degradation or reactivity to cGMP influence the attenuated NO-dependent pulmonary vasodilatory response observed after CH. Our findings suggest that whereas NO-dependent dilation is cGMP mediated, altered reactivity to NO after CH is not likely a function of decreased pulmonary sGC expression or activity. Rather, this effect of CH appears to result from increased cGMP degradation by PDE5 and decreased pulmonary VSM sensitivity to cGMP. These findings could have important clinical implications with respect to pulmonary vasoreactivity in patients with hypoxic pulmonary hypertension secondary to chronic lung disease. Further research is necessary to determine whether diminished pulmonary vasodilation to NO after CH is mediated by changes in activity/expression of intracellular cGMP targets.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the expert technical assistance of Anna Holmes and Minerva Murphy.
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FOOTNOTES |
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This work was supported by a Scientist Development Grant from the American Heart Association (T. C. Resta) and by National Institutes of Health Grants RR-164808 (T. C. Resta) and GM-08139 (N. L. Jernigan).
T. C. Resta is a Parker B. Francis Fellow in Pulmonary Research.
Address for reprint requests and other correspondence: N. L. Jernigan, Dept. of Cell Biology and Physiology, Univ. of New Mexico Health Sciences Center, 915 Camino de Salud NE, Albuquerque, NM 87131-5218 (E-mail: njernigan{at}salud.unm.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.
First published January 18, 2002;10.1152/ajplung.00273.2001
Received 19 July 2001; accepted in final form 16 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berkenbosch, JW,
Baribeau J,
and
Perreault T.
Decreased synthesis and vasodilation to nitric oxide in piglets with hypoxia-induced pulmonary hypertension.
Am J Physiol Lung Cell Mol Physiol
278:
L276-L283,
2000
2.
Black, SM,
Sanchez LS,
Mata-Greenwood E,
Bekker JM,
Steinhorn RH,
and
Fineman JR.
sGC and PDE5 are elevated in lambs with increased pulmonary blood flow and pulmonary hypertension.
Am J Physiol Lung Cell Mol Physiol
281:
L1051-L1057,
2001
3.
Carter, EP,
Sato K,
Morio Y,
and
McMurtry IF.
Inhibition of KCa channels restores blunted hypoxic pulmonary vasoconstriction in rats with cirrhosis.
Am J Physiol Lung Cell Mol Physiol
279:
L903-L910,
2000
4.
Carvajal, JA,
Germain AM,
Huidobro-Toro JP,
and
Weiner CP.
Molecular mechanism of cGMP-mediated smooth muscle relaxation.
J Cell Physiol
184:
409-420,
2000[ISI][Medline].
5.
Clarke, WR,
Uezono S,
Chambers A,
and
Doepfner P.
The type III phosphodiesterase inhibitor milrinone and type V PDE inhibitor dipyridamole individually and synergistically reduce elevated pulmonary vascular resistance.
Pulm Pharmacol
7:
81-89,
1994[ISI][Medline].
6.
Cohen, AH,
Hanson K,
Morris K,
Fouty B,
McMurtry IF,
Clarke W,
and
Rodman DM.
Inhibition of cyclic 3'-5'-guanosine monophosphate-specific phosphodiesterase selectively vasodilates the pulmonary circulation in chronically hypoxic rats.
J Clin Invest
97:
172-179,
1996
7.
Crawley, DE,
Zhao L,
Giembycz MA,
Liu S,
Barnes PJ,
Winter RJ,
and
Evans TW.
Chronic hypoxia impairs soluble guanylyl cyclase-mediated pulmonary arterial relaxation in the rat.
Am J Physiol Lung Cell Mol Physiol
263:
L325-L332,
1992
8.
Eddahibi, S,
Raffestin B,
Le Monnier de Gouville AC,
and
Adnot S.
Effect of DMPPO, a phosphodiesterase type 5 inhibitor, on hypoxic pulmonary hypertension in rats.
Br J Pharmacol
125:
681-688,
1998[Abstract].
9.
Feddersen, CO,
Chang S,
Czartalomna J,
and
Voelkel NF.
Arachidonic acid causes cyclooxygenase-dependent and -independent pulmonary vasodilation.
J Appl Physiol
68:
1799-1808,
1990
10.
Haynes, J, Jr,
Kithas PA,
Taylor AE,
and
Strada SJ.
Selective inhibition of cGMP-inhibitable cAMP phosphodiesterase decreases pulmonary vasoreactivity.
Am J Physiol Heart Circ Physiol
261:
H487-H492,
1991
11.
Haynes, J, Jr,
Robinson J,
Saunders L,
Taylor AE,
and
Strada SJ.
Role of cAMP-dependent protein kinase in cAMP-mediated vasodilation.
Am J Physiol Heart Circ Physiol
262:
H511-H516,
1992
12.
Isaacson, TC,
Hampl V,
Weir EK,
Nelson DP,
and
Archer SL.
Increased endothelium-derived NO in hypertensive pulmonary circulation of chronically hypoxic rats.
J Appl Physiol
76:
933-940,
1994
13.
Jeffery, TK,
and
Wanstall JC.
Phosphodiesterase III and V inhibitors on pulmonary artery from pulmonary hypertensive rats: differences between early and established pulmonary hypertension.
J Cardiovasc Pharmacol
32:
213-219,
1998[ISI][Medline].
14.
Kotera, J,
Fujishige K,
Michibata H,
Yuasa K,
Kubo A,
Nakamura Y,
and
Omori K.
Characterization and effects of methyl-2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5).
Biochem Pharmacol
60:
1333-1341,
2000[ISI][Medline].
15.
Le Cras, TD,
Xue C,
Rengasamy A,
and
Johns RA.
Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung.
Am J Physiol Lung Cell Mol Physiol
270:
L164-L170,
1996
16.
Li, D,
Zhou N,
and
Johns RA.
Soluble guanylate cyclase gene expression and localization in rat lung after exposure to hypoxia.
Am J Physiol Lung Cell Mol Physiol
277:
L841-L847,
1999
17.
Lucas, KA,
Pitari GM,
Kazerounian S,
Ruiz-Stewart I,
Park J,
Schulz S,
Chepenik KP,
and
Waldman SA.
Guanylyl cyclases and signaling by cyclic GMP.
Pharmacol Rev
52:
375-414,
2000
18.
Maclean, MR,
Johnston ED,
McCulloch KM,
Pooley L,
Houslay MD,
and
Sweeney G.
Phosphodiesterase isoforms in the pulmonary arterial circulation of the rat: changes in pulmonary hypertension.
J Pharmacol Exp Ther
283:
619-624,
1997
19.
Maruyama, J,
and
Maruyama K.
Impaired nitric oxide-dependent responses and their recovery in hypertensive pulmonary arteries of rats.
Am J Physiol Heart Circ Physiol
266:
H2476-H2488,
1994
20.
Muramatsu, M,
Tyler RC,
Rodman DM,
and
McMurtry IF.
Thapsigargin stimulates increased NO activity in hypoxic hypertensive rat lungs and pulmonary arteries.
J Appl Physiol
80:
1336-1344,
1996
21.
Oka, M.
Phosphodiesterase 5 inhibition restores impaired ACh relaxation in hypertensive conduit pulmonary arteries.
Am J Physiol Lung Cell Mol Physiol
280:
L432-L435,
2001
22.
Oka, M,
Hasunuma K,
Webb SA,
Stelzner TJ,
Rodman DM,
and
McMurtry IF.
EDRF suppresses an unidentified vasoconstrictor mechanism in hypertensive rat lungs.
Am J Physiol Lung Cell Mol Physiol
264:
L587-L597,
1993
23.
Resta, TC,
Chicoine LG,
Omdahl JL,
and
Walker BR.
Maintained upregulation of pulmonary eNOS gene and protein expression during recovery from chronic hypoxia.
Am J Physiol Heart Circ Physiol
276:
H699-H708,
1999
24.
Resta, TC,
Kanagy NL,
and
Walker BR.
Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression.
Am J Physiol Lung Cell Mol Physiol
280:
L88-L97,
2001
25.
Resta, TC,
Sanders TC,
Eichinger MR,
Crowley MR,
and
Walker BR.
Segmental vasodilatory effectiveness of inhaled NO in lungs from chronically hypoxic rats.
Respir Physiol
114:
161-173,
1998[ISI][Medline].
26.
Resta, TC,
and
Walker BR.
Chronic hypoxia selectively augments endothelium-dependent pulmonary arterial vasodilation.
Am J Physiol Heart Circ Physiol
270:
H888-H896,
1996
27.
Rodman, DM,
Yamaguchi T,
Hasunuma K,
O'Brien RF,
and
McMurtry IF.
Effects of hypoxia on endothelium-dependent relaxation of rat pulmonary artery.
Am J Physiol Lung Cell Mol Physiol
258:
L207-L214,
1990
28.
Roos, CM,
Frank DU,
Xue C,
Johns RA,
and
Rich GF.
Chronic inhaled nitric oxide: effects on pulmonary vascular endothelial function and pathology in rats.
J Appl Physiol
80:
252-260,
1996
29.
Russell, PC,
Emery CJ,
Cai YN,
Barer GR,
and
Howard P.
Enhanced reactivity to bradykinin, angiotensin I and the effect of captopril in the pulmonary vasculature of chronically hypoxic rats.
Eur Respir J
3:
779-785,
1990[Abstract].
30.
Sato, K,
Morio Y,
Morris KG,
Rodman DM,
and
McMurtry IF.
Mechanism of hypoxic pulmonary vasoconstriction involves ETA receptor-mediated inhibition of KATP channel.
Am J Physiol Lung Cell Mol Physiol
278:
L434-L442,
2000
31.
Sausbier, M,
Schubert R,
Voigt V,
Hirneiss C,
Pfeifer A,
Korth M,
Kleppisch T,
Ruth P,
and
Hofmann F.
Mechanisms of NO/cGMP-dependent vasorelaxation.
Circ Res
87:
825-830,
2000
32.
Shaul, PW,
North AJ,
Brannon TS,
Ujiie K,
Wells LB,
Nisen PA,
Lowenstein CJ,
Snyder SH,
and
Star RA.
Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.
Am J Respir Cell Mol Biol
13:
167-174,
1995[Abstract].
33.
Shimoda, LA,
Sylvester JT,
and
Sham JS.
Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes.
Am J Physiol Lung Cell Mol Physiol
277:
L431-L439,
1999
34.
Smirnov, SV,
Robertson TP,
Ward JP,
and
Aaronson PI.
Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells.
Am J Physiol Heart Circ Physiol
266:
H365-H370,
1994
35.
Suzuki, H,
and
Twarog BM.
Membrane properties of smooth muscle cells in pulmonary arteries of the rat.
Am J Physiol Heart Circ Physiol
242:
H900-H906,
1982[ISI].
36.
Tyler, RC,
Muramatsu M,
Abman SH,
Stelzner TJ,
Rodman DM,
Bloch KD,
and
McMurtry IF.
Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension.
Am J Physiol Lung Cell Mol Physiol
276:
L297-L303,
1999
37.
Wagner, RS,
Smith CJ,
Taylor AM,
and
Rhoades RA.
Phosphodiesterase inhibition improves agonist-induced relaxation of hypertensive pulmonary arteries.
J Pharmacol Exp Ther
282:
1650-1657,
1997
38.
Wanstall, JC,
and
O'Donnell SR.
Responses to vasodilator drugs on pulmonary artery preparations from pulmonary hypertensive rats.
Br J Pharmacol
105:
152-158,
1992[Abstract].
39.
Xue, C,
and
Johns RA.
Upregulation of nitric oxide synthase correlates temporally with onset of pulmonary vascular remodeling in the hypoxic rat.
Hypertension
28:
743-753,
1996
40.
Ziegler, JW,
Ivy DD,
Fox JJ,
Kinsella JP,
Clarke WR,
and
Abman SH.
Dipyridamole, a cGMP phosphodiesterase inhibitor, causes pulmonary vasodilation in the ovine fetus.
Am J Physiol Heart Circ Physiol
269:
H473-H479,
1995
41.
Zimmerman, AL,
Yamanaka G,
Eckstein F,
Baylor DA,
and
Stryer L.
Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments.
Proc Natl Acad Sci USA
82:
8813-8817,
1985[Abstract].