Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to β-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.
- guanosine 3′,5′-cyclic monophosphate
- β-phenyl-1,N2-etheno-8-bromo-guanosine 3′,5′-cyclic monophosphorothioate Rp isomer
- vascular smooth muscle
- perinatal lungs
cyclic gmp-dependent protein kinase (PKG) is a key enzyme involved in vasodilatation induced by endogenous and exogenous nitric oxide (4, 6, 7, 8, 13, 17, 18). Nitric oxide activates soluble guanylyl cyclase, resulting in increased intracellular cGMP content. cGMP has many actions, one of which is to stimulate PKG activity, which ultimately results in a reduction in intracellular Ca2+ concentration and vasodilatation (17). Two types of PKG enzymes have been identified (type I and type II) in mammalian cells (6, 17). In blood vessels, type I PKG is the predominant form (6, 13, 17, 26). PKG type I has two isoforms (PKG-Iα and PKG-Iβ). So far, all studies seem to indicate that PKG type-Iα enzyme is the main isoform involved in nitric oxide-mediated vasodilatation (5, 15, 33).
In human mammary arteries, continuous nitroglycerin infusion induces nitrate tolerance, cross-tolerance to endothelium-derived nitric oxide, and a decrease in vasodilator-stimulated phosphoprotein serine-239 phosphorylation (an index of PKG activity; see Ref. 30). In rat and bovine aortic smooth muscle cells, continuous exposure to nitrovasodilators (S-nitroso-N-acetylpenicillamine and sodium nitroprusside) reduces PKG type I protein and mRNA levels (34). Also, in rat and bovine aortic smooth muscle cells, continuous exposure to nitric oxide or cyclic nucleotides suppresses PKG-Iα mRNA expression by decreasing the activity of the transcriptional factor, Sp1. Nitric oxide appears to directly affect the activity of this transcription factor while cyclic nucleotides act via cAMP-dependent protein kinase (PKA; see Ref. 33). In transgenic mice overexpressing nitric oxide synthase, relaxation of the aorta to ACh, sodium nitroprusside, atrial natriuretic peptide, and 8-bromo-cGMP is reduced. Furthermore, PKG protein levels and PKG enzyme activity are decreased in the systemic blood vessels of these transgenic mice (36). These results suggest that, in systemic vessels, decreased PKG activity may in part explain development of tolerance to nitric oxide (19, 23, 30, 33, 34, 36). The underlying mechanism by which PKG activity is downregulated is not well understood.
In patients with pulmonary hypertension, prolonged treatment with inhaled nitric oxide results in a decrease in endothelium-derived nitric oxide (EDNO)-mediated vasodilatation (29). In this study, we wished to determine whether PKG activity is downregulated in the pulmonary vasculature after continuous nitric oxide exposure and to determine the underlying mechanisms by which this occurs. Our data suggest that, in the pulmonary vasculature, continuous exposure to nitric oxide results in suppression of PKG activity and PKG protein levels by a cGMP- and PKG-dependent mechanism.
MATERIALS AND METHODS
Pulmonary vessel preparations. Seventeen newborn lambs (7–12 days old, either sex) from Nebeker Ranch (Lancaster, CA) were used. They were anesthetized with ketamine hydrochloride (30 mg/kg im) and killed with an overdose of pentobarbital sodium. The animal handling and study protocols were reviewed and approved by the Harbor-University of California Los Angeles Animal Care and Use Review Committee.
The lungs were immediately removed, and fourth-generation pulmonary veins (outside diameter: 1.5–2.0 mm) were dissected free of parenchyma and cut into rings (length: 3 mm) in ice-cold modified Krebs-Ringer bicarbonate buffer [composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 glucose].
Organ chamber study. Rings of pulmonary veins were incubated for 20 h in serum-free DMEM (GIBCO; supplemented with 2 mM glutamate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B; 37 ± 0.5°C, 20% O2-5% CO2, pH = 7.4) in the presence of solvent or DETA NONOate [DETA NO; 2,2′-(hydroxynitrosohydrazono)bis(ethanamine), 10-5 M]. In some experiments, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one [ODQ, 3 × 10-5 M; an inhibitor of soluble guanylyl cyclase (11)], Rp-8-Br-PET-cGMPS [β-phenyl-1,N2-etheno-8-bromoguanosine-3′,5′-cyclic monophosphorothioate, Rp-isomer, 3 × 10-5 M; a selective PKG inhibitor (2)], or KT-5720 [5 × 10-5 M; selective inhibitor of PKA (14)] was coincluded with DETA NO or solvent. To eliminate the possible involvement of endogenous prostanoids and endothelium-derived nitric oxide, indomethacin (10-5 M) and nitro-l-arginine (10-4 M) were included in the medium and present throughout the experiments (10).
After incubation, vessel rings were washed three times and suspended in organ chambers filled with 10 ml of the modified Krebs-Ringer bicarbonate solution maintained at 37 ± 0.5°C and aerated with 95% O2-5% CO2 (pH = 7.4). Two stirrups passed through the lumen suspended each ring. One stirrup was anchored to the bottom of the organ chamber; the other one was connected to a strain gauge (model FT03C; Grass Instrument, Braintree, MA) for the measurement of isometric force (9, 10).
At the beginning of the experiment, each vessel ring was stretched to its optimal resting tension. This was achieved by stepwise stretching until the active contraction of the vessel ring to 100 mM KCl reached a plateau. The optimal resting tension of pulmonary veins of newborn lambs was ∼0.3 g/mm2 cross-sectional area of smooth muscle. The cross-sectional area of smooth muscle of each vessel ring was determined as previously described (8, 9). After the vessels were brought to their optimal resting tension, 1 h of equilibration was allowed.
Effects of DETA NO (10-8 to 10-4 M) and β-phenyl-1,N2-etheno-8-bromo-cGMP [8-Br-PET-cGMP; 10-8 to 3 × 10-5 M; a cell membrane permeable analog of cGMP (28)] were determined in vessels constricted with endothelin-1 (3 × 10-9 M). The concentration-response curves to DETA NO and 8-Br-PET-cGMP were constructed in a cumulative fashion. In a preliminary study, some vessels were preconstricted with U-46619 (3 × 10-8 M) to a similar tension to that induced with endothelin-1. Relaxation of these vessels to DETA NO and 8-Br-PET-cGMP was not significantly different from those preconstricted with endothelin-1 (data not shown; n = 5, P > 0.05).
All experiments were carried out in a parallel manner. For each vessel ring, only one vasodilator was tested under control conditions or in the presence of one inhibitor. In some vessels, ODQ, Rp-8-Br-PET-cGMPS, or KT-5720 was included during 20 h incubation with DETA NO or solvent. After incubation and three washes, the inhibitor was added again before constriction with endothelin-1 and remained in contact with the tissue throughout the experiment.
PKG activity assay. Isolated pulmonary veins of newborn lambs were incubated in serum-free DMEM as described earlier in Organ chamber study. They were then homogenized in a buffer containing 50 mM Tris·HCl (pH 7.4), 10 mM EDTA, 2 mM dithiothreitol, 1 mM isobutylmethylxanthine, 100 μM nitro-l-arginine, and 10 μM indomethacin. The homogenate was sonicated and centrifuged at 13,000 g for 10 min at 4°C. Supernatants were assayed for PKG activity by measuring the incorporation of 32P from [γ-32P]ATP into a specific PKG substrate, BPDEtide (Biomol Research Laboratories, Plymouth Meeting, PA). Aliquots (20 μl) of supernatant were added to a mixture (total volume, 50 μl) containing 50 mM Tris·HCl (pH 7.4), 20 mM MgCl2, 0.1 mM isobutylmethylxanthine, 10 μM indomethacin, 100 μM nitro-l-arginine, 150 μM BPDEtide, 1 μM PKI (a synthetic PKA inhibitor; Peninsula Laboratories, Belmont, CA), and 0.2 mM [γ-32P]ATP (specific activity 3,000 Ci/mmol). The mixture was incubated at 30°C for 10 min in the presence or absence of 3 μM exogenous cGMP. The reaction was terminated by spotting 40-μl aliquots of mixture on phosphocellulose papers (2 × 2 cm; P81 Whatman) and placing them in ice-cold 75 mM phosphoric acid. The filter papers were washed, dried, and counted in a liquid scintillation counter. Assays were performed in triplicate with appropriate controls. After subtracting control counts, counts obtained indicate PKG activity, which is expressed as picomoles of 32P incorporated into PKG substrate per minute per milligram protein. Protein content in supernatant was measured by Bradford's procedure, using BSA as a standard. Preliminary experiments confirmed the linearity of PKG activity at the protein concentration used within the incubation time (4, 7).
Western analysis of PKG protein. An affinity-purified polyclonal antibody detecting a 75-kDa protein, corresponding to the apparent molecular mass of PKG type I on SDS-PAGE immunoblots in human, mouse, and Xenopus origins (Stressgen, Victoria, Canada), was used.
Tissue lysates were prepared from isolated pulmonary veins incubated for 20 h in serum-free DMEM as described earlier in the presence of solvent, DETA NO (10-5 M), 8-Br-PET-cGMP (10-5 M), 8-Br-cGMP (10-4 M), or 8-Br-cAMP (10-4 M). In some experiments, ODQ (3 × 10-5 M) or Rp-8-Br-PET-cGMPS (3 × 10-5 M) was present in the incubation medium.
The lysates each containing 20 μg protein were subjected to SDS-PAGE, electrotransferred to nitrocellulose. Nonspecific binding of antibody was blocked by washing with Tris-buffered saline (TBS) buffer containing 10% milk for 1 h. The blot was then subjected to two brief washes with TBS plus 0.5% Tween 20 and incubated in TBS plus 0.1% Tween 20 and a 1:5,000 dilution of PKG antibody for 1 h. After two more washes in TBS plus 0.1% Tween 20, the blot was incubated for 40 min in secondary antibody, washed, and developed using the chemiluminescent detection method (Amersham ECL). The amount of PKG protein present in blots was quantified by densitometry using an Eagle Eye II Still Video System (Stratagene, La Jolla, CA) and normalized to scanning signals of actin (Oncogene, La Jolla, CA) (8).
Relative quantitative RT-PCR for PKG-Iα mRNAs. Total RNA was extracted from pulmonary vessels using Trizol reagent (Life Technologies, Grand Island, NY) according to the manufacturer's protocol. The vessels were preincubated for 20 h in serum-free DMEM as described earlier in the presence of solvent, DETA NO (10-5 M), or 8-Br-PET-cGMP (10-5 M). In some experiments, ODQ (3 × 10-5 M), Rp-8-Br-PET-cGMPS (3 × 10-5 M), or KT-5720 (5 × 10-5 M) was present in the incubation medium.
Total RNA (100 ng) was reverse transcribed in a 20-μl volume using 250 ng random primers and 20 units of SuperScript II RT (Invitrogen, Carlsbad, CA). PCR was carried out using primers for PKG-Iα (sense, 5′-CTGGAGGAAGACTTTGCCAAGATTC-3′ and anti-sense, 5′-TCGGATTTGGTGAACTTCCGGAATG-3′, which match GenBank Accession X160886 at bp 16–40 and 269–245, respectively).
Relative quantitative (RQ) RT-PCR was carried out using the Competitive Quantitative RT-PCR Kit components and protocol (Ambion, Austin, TX). PCR reactions (50 μl) were performed on a RoboCycler thermal cycler (Stratagene) using 20 units ThermanAce DNA polymerase (Invitrogen), primers for PKG-Iα, and 0.75 μl Classic 18S primer with 1.75 μl 18S Competimer primer (Ambion). PCR was carried out for 32 cycles (30 s at 94°C,45sat55°C, and 60 s at 72°C), which was determined to be in the linear range of amplification for both PKG-Iα and the competed 18S products. PCR products were visualized using 1.5% agarose gel electrophoresis and quantified by image analysis using an Eagle Eye II Still Video System (Stratagene). The quantities of mRNA for PKG-Iα were expressed as relative units to 18S.
Drugs. The following drugs were used (unless otherwise specified, all were obtained from Sigma, St. Louis, MO): 8-(2-Aminophenylthio)-cGMP (8-APT-cGMP; Biolog Life Science Institute, La Jolla, CA), 8-bromo-cAMP (8-Br-cAMP), 8-bromo-cGMP (8-Br-cGMP), 8-Br-PET-cGMP (Biolog Life Science Institute), DETA NO, endothelin-1 (American Peptide, Sunnyvale, CA), indomethacin, isobutyl methylxanthine, KT-5720, nitro-l-arginine, ODQ, and Rp-8-Br-PET-cGMPS (Biolog Life Science Institute).
Indomethacin (10-5 M) was prepared in equal molar Na2CO3. This concentration of Na2CO3 did not significantly affect the pH of the solution in the organ chamber. ODQ and KT-5720 were dissolved in DMSO (final concentration <0.2%). Preliminary experiments showed that DMSO at the concentration used had no effect on contraction to endothelin-1 and relaxation induced by the nitric oxide donor and cGMP analogs. The other drugs were prepared using distilled water.
Data analyses. Data are shown as means ± SE. When mean values of two groups were compared, Student's t-test for unpaired observations was used. When the mean values of the same group before and after stimulation were compared, Student's t-test for paired observations was used. Comparison of mean values of more than two groups was performed with the one-way ANOVA test with the Student-Newman-Keuls test for post hoc testing of multiple comparisons. All these analyses were performed using a commercially available statistics package (SigmaStat; Jandel Scientific, San Rafael, CA). Statistical significance was accepted when the P value (2 tailed) was <0.05. In all experiments, n represents the number of animals.
Organ chamber studies. After 20 h incubation with DETA NONOate [DETA NO, 10-5 M; a stable nitric oxide donor, (22)] and three washes, rings of pulmonary veins of newborn lambs were constricted with endothelin-1 (3 × 10-9 M) before testing their responses to nitric oxide and the cGMP analog. The increase in tension of various groups evoked by endothelin-1 was not significantly different from each other (1.65 ± 0.27 to 1.76 ± 0.17 g/mm2 cross-sectional area of smooth muscle; P > 0.05, n = 6 for each group).
DETA NO and 8-Br-PET-cGMP induced a significantly smaller relaxation in veins after 20 h of incubation with DETA NO (10-5 M) than with solvent (Figs. 1 and 2). The attenuation of 8-Br-PET-cGMP-induced relaxation after incubation with DETA NO was prevented by including ODQ [3 × 10-5 M, an inhibitor of soluble guanylyl cyclase (11)] but not by the addition of KT-5720 [5 × 10-5 M, a PKA inhibitior (14)] in the incubation medium (Fig. 2). Incubation with ODQ or KT-5720 alone for 20 h had no significant effect on relaxation of the vessels to 8-Br-PET-cGMP (data not shown, n = 5).
In some experiments, pulmonary veins were incubated for 20 h with Rp-8-PET-cGMPS [3 × 10-5 M; a PKG inhibitor (2)] or DETA NO (10-5 M) plus Rp-8-PET-cGMPS (3 × 10-5 M). The inclusion of PKG inhibitor not only attenuated the subsequent relaxation of the veins to 8-Br-PET-cGMP but also eliminated the differential response of the vessels that were incubated with or without DETA NO (Fig. 3).
PKG activity. Pulmonary veins incubated for 20 h with DETA NO (10-5 M) showed a significantly diminished cGMP-stimulated PKG activity. The effect of DETA NO incubation was blocked by ODQ (3 × 10-5 M) but not by KT-5720 (5 × 10-5 M) in incubation medium. There were no significant differences in PKG activity between these groups when the assay was performed in the absence of exogenous cGMP (Fig. 4).
PKG protein. Incubation with DETA NO (10-5 M) or 8-Br-PET-cGMP (10-5 M) for 20 h significantly suppressed the PKG protein level in pulmonary veins. The inclusion of ODQ (3 × 10-5 M) in the incubation medium prevented the reduction of PKG protein level caused by DETA NO (10-5 M) but not by 8-Br-PET-cGMP (10-5 M), whereas Rp-8-Br-PET-cGMPS (3 × 10-5 M) prevented the reductions caused by both DETA NO and 8-Br-PET-cGMP (Fig. 5).
8-Br-PET-cGMP activates PKG-Iα and -Iβ subtypes with nearly similar potency, whereas 8-APT-cGMP is a selective PKG type Iα activator (32). A 20-h incubation with 8-Br-PET-cGMP caused a greater reduction of PKG protein level in pulmonary veins than that by 8-APT-cGMP (Fig. 6).
A reduced PKG protein level in pulmonary veins was also observed after 20 h of incubation with 8-Br-cGMP (10-4 M) but not with 8-Br-cAMP (10-4 M), cell membrane analogs of cGMP and cAMP, respectively (20). The effect of 8-Br-cGMP was prevented by the inclusion of Rp-8-Br-PET-cGMPS (3 × 10-5 M) in the incubation medium (Fig. 7).
PKG-Iα mRNAs. RQ RT-PCR yielded two distinctive bands, corresponding to the predicted sizes for mRNA fragments of PKG-Iα and the 18S Internal Standards (255 and 488 bp, respectively) according to the DNA ladders. Pulmonary veins incubated for 20 h with DETA NO (10-5 M) or 8-Br-PET-cGMP (10-5 M) showed a decreased content of PKG-Iα mRNA than those incubated with solvent (Fig. 8). The effect of incubation with DETA NO was blocked by coincubation with ODQ (3 × 10-5 M) or Rp-8-Br-PET-cGMPS (3 × 10-5 M), whereas the effect of incubation with 8-Br-PET-cGMP was blocked by Rp-8-Br-PET-cGMPS (3 × 10-5 M; Fig. 8). The effect of 20 h incubation with 8-Br-PET-cGMP (10-5 M) was not significantly affected by the inclusion of KT-5720 (PKA inhibitor, 5 × 10-5 M) in the incubation medium (relative density of the PKG-Iα mRNA fragments normalized to 18S: 0.79 ± 0.07 vs. 0.82 ± 0.06; n = 4, P > 0.05).
We have previously reported that, in perinatal ovine lungs, nitric oxide-induced vasodilatation is predominantly mediated by cGMP (7–10). The present study demonstrated that continuous exposure of pulmonary veins of newborn lambs to nitric oxide for 20 h resulted in development of tolerance to nitric oxide and also to 8-Br-PET-cGMP, a cell membrane permeable analog of cGMP (32). Furthermore, the cross-tolerance to cGMP caused by nitric oxide was prevented by ODQ [an inhibitor of soluble guanylyl cyclase (11)], indicating that cGMP is important in the development of tolerance to nitric oxide. This study was performed in pulmonary veins of neonatal lambs because they exhibit greater relaxation responses to nitric oxide compared with pulmonary arteries (8, 10).
There are three major targets for cGMP action in eukaryotic cells: PKG, cGMP-gated ion channels, and cGMP-binding cyclic nucleotide phosphodiesteraes (6, 17). In ovine perinatal lungs, we have shown that PKG plays a major role in nitric oxide and cGMP-induced vasodilation (4, 7, 8). In the present study, prolonged exposure to nitric oxide markedly reduced PKG activity in pulmonary veins. This effect was blocked by ODQ, an inhibitor of guanylyl cyclase, suggesting that it is the elevated cGMP level present during continuous exposure to nitric oxide that suppresses PKG activity and thus results in decreased relaxation responses of the vessels to nitric oxide and cGMP. Our results also show that tolerance to cGMP after continuous exposure of the vessels to nitric oxide was prevented by Rp-8-Br-PET-cGMPS, an inhibitor of PKG (2). This suggests that, during incubation with nitric oxide, a prolonged stimulation of PKG by elevated cGMP levels reduces the activity of the enzyme itself. Such a negative feedback mechanism appears to be responsible for the development of tolerance in pulmonary veins to nitric oxide and cGMP.
In blood vessels, type I PKG is the predominant form (6, 13, 17, 26), which has two isoforms (PKG-Iα and PKG-Iβ). Studies to date suggest that the type-Iα enzyme is the major isoform involved in PKG-mediated vasodilation (5, 15, 33). Our results from Western analysis and RT-PCR show that prolonged exposure to nitric oxide and cGMP analogs caused a decrease in levels of PKG type I protein and type Iα mRNA. This effect was abrogated by inhibition of soluble guanylyl cyclase, which is consistent with our vessel tension studies and PKG activity assays. The reduction in PKG protein and mRNA levels after continuous exposure to nitric oxide and cGMP analogs was blocked by Rp-8-Br-PET-cGMPS, an inhibitor of PKG (2). These findings provide further evidence that the development of tolerance to nitric oxide may result from PKG-mediated downregulation of the enzyme itself.
In smooth muscle cells, both PKG-Iα and -Iβ isoforms are highly expressed (6, 13). In our study, incubation for 20 h with 8-APT-cGMP, a highly selective activator of PKG-Iα, significantly suppressed PKG protein level in the veins. This indicates that PKG-Iα alone is sufficient for PKG-mediated downregulation of the enzyme itself. On the other hand, 8-Br-PET-cGMP, an activator of both PKG-Iα and -Iβ, caused a greater decrease in the PKG protein level than did 8-APT-cGMP. It is possible that PKG-Iβ may also be involved in the downregulation of PKG enzyme. It should be noted that many factors may contribute to the differential effect between these cGMP analogs, such as their differences in permeability and in their resistance to degradation by phosphodiesterases (31).
In rat and bovine aortic smooth muscle cells, continuous exposure to nitric oxide or cyclic nucleotides suppresses PKG-Iα expression by decreasing activity of the transcription factor Sp1. Nitric oxide acts directly on this transcription factor, whereas the cyclic nucleotides act via PKA. In that study, it was noted that cAMP was more potent than cGMP in suppressing PKG-Iα expression (33). In our study, the decreased relaxation to the cGMP analog that we observed and the decrease in PKG protein level after continuous exposure to nitric oxide were not affected by KT-5720 at a concentration that we have previously shown to be effective in inhibiting PKA activity in pulmonary veins of newborn lambs (7). Furthermore, 20 h incubation of the veins with 8-Br-cGMP but not 8-Br-cAMP, at the same concentration (10-4 M), suppressed PKG protein level. In addition, KT-5720 had no effect on the decrease in PKG-Iα mRNA levels. Taken together, these results suggest that PKA does not play an important role in the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.
Nitric oxide inhalation therapy is used in the treatment of a number of pulmonary vasculature disorders, such as persistent pulmonary hypertension of the newborn and respiratory distress syndrome associated with pulmonary hypertension (21, 27, 28). However, clinical reports indicate that discontinuation of inhaled nitric oxide after prolonged periods of administration may result in rebound pulmonary hypertension (12, 16, 25, 37). The underlying mechanisms are likely to be multifactorial and may include an increase in plasma endothelin-1, an increase in superoxide production, and a decrease in endogenous nitric oxide synthase activity (1, 3, 24, 25, 35). In the pulmonary circulation, prolonged treatment with inhaled nitric oxide inhibits EDNO-mediated vasodilation (29). Our present study suggests that a decrease in PKG activity may be involved in this phenomenon.
In summary, our data show that prolonged exposure of the pulmonary vasculature to nitric oxide and cGMP induces tolerance to their effects in the pulmonary vessels. This tolerance is the result of a downregulation of PKG resulting from prolonged stimulation of PKG activity and gene expression by elevated cGMP levels. It seems that cAMP and PKA do not play a significant role in this phenomenon.
We thank Mary Lee Ryba and Nik Phou for excellent secretarial assistance.
This study was supported in part by National Institutes of Health (NIH) Grants HL-059435 and HL-075187, a special fund for promotion of education from the Ministry of Education, China, and National Natural Science Foundation of China Grant no. 30370523. A. D. Portugal was supported in part by NIH Grant 5 R25 GM-56902, and E. M. Trevino was supported by NIH Grant R25 GM-62252.
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