Effect of oxygen on cyclic GMP-dependent protein kinase-mediated relaxation in ovine fetal pulmonary arteries and veins

Yuansheng Gao, Srinivas Dhanakoti, Earleen M. Trevino, Fred C. Sander, Ada M. Portugal, and J. Usha Raj

Department of Pediatrics, Harbor-UCLA Research and Education Institute Incorporated, University of California, Los Angeles School of Medicine, Torrance, California 90509

Submitted 2 December 2002 ; accepted in final form 8 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cyclic GMP-dependent protein kinase (PKG) plays an important role in regulating pulmonary vasomotor tone in the perinatal period. In this study, we tested the hypothesis that a change in oxygen tension affects PKG-mediated pulmonary vasodilation. Isolated intrapulmonary arteries and veins of near-term fetal lambs were first incubated for 4 h under hypoxic and normoxic conditions (PO2 of 30 and 140 mmHg, respectively) and then contracted with endothelin-1. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cell membrane-permeable analog of cGMP, induced a greater relaxation in vessels incubated in normoxia than in hypoxia. {beta}-Phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-PET-cGMPS), a selective inhibitor of PKG, attenuated relaxation induced by 8-BrcGMP (10-4 and 3 x 10-4 M). In the presence of Rp-8-Br-PET-cGMPS, the differential responses to 8-BrcGMP between hypoxia and normoxia treatment were abolished in veins but not in arteries. cGMP-stimulated PKG activity was present in arteries but not in veins after 4 h of hypoxia. Both vessel types showed significant increase in cGMP-stimulated PKG activity after 4 h of normoxia. PKG protein (Western blot analysis) and PKG mRNA levels (quantitative RT-PCR) were greater in veins but not in arteries after 4-h exposure to normoxia vs. hypoxia. These results demonstrate that oxygen augments cGMP-mediated vasodilation of fetal pulmonary arteries and veins. Furthermore, the effect of oxygen on response of the veins to cGMP is due to an increase in the activity, protein level, and mRNA of PKG.

protein kinase G; vasodilation; vein; perinatal lung


AT BIRTH, THERE IS an immediate decrease in pulmonary vascular resistance and an increase in pulmonary blood flow with the onset of breathing. This is followed by a further decrease in pulmonary vascular resistance in the subsequent hours to days. Many factors contribute to this immediate fall in pulmonary vascular resistance. Activation of the nitric oxide-cGMP pathway is one important mechanism by which the pulmonary circulation is regulated in the immediate newborn period (2, 6, 7, 12, 28, 29, 36, 39, 42). In the lungs of fetal lambs, an increase in oxygen tension causes an acute increase in the production of endothelium-derived nitric oxide (EDNO) (36, 42). Prolonged oxygenation is associated with increased endothelial nitric oxide synthase (eNOS) mRNA and protein expression in pulmonary endothelium (2, 29).

Activation of cGMP-dependent protein kinase (PKG) is important in mediating the dilator effects of agents such as EDNO, nitrosovasodilators, and natriuretic peptides, which elevate intracellular cGMP levels in vascular smooth muscle (13, 20, 24, 25, 30). In the pulmonary circulation of the fetus and newborn, PKG plays a key role in nitric oxide-cGMP-induced smooth muscle relaxation (7, 8, 14). However, it is not known whether oxygen modulates PKG activity and PKG-mediated relaxation. The present study was designed to determine the effects of oxygen on PKG-mediated relaxation. Our results show that cGMP-mediated relaxation is upregulated by oxygen in pulmonary arteries and veins of fetal lambs and that an increase in PKG activity and expression may contribute to the oxygen effect in the veins.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Pulmonary vessel preparations. Fifteen fetal lambs (131-145 days of gestation, term being 150 days of gestation; either sex) from 11 pregnant ewes from Nebeker Ranch (Lancaster, CA) were used. The fetus was delivered by cesarean section and killed by a lethal dose of pentobarbital (100 mg/kg) via the umbilical vein. Before the cesarean section, the pregnant ewe was anesthetized with ketamine hydrochloride (30 mg/kg im) and atropine sulfate (40 µg/kg im). After the fetuses were delivered, the ewe was killed with an overdose of pentobarbital (100 mg/kg iv). The animal handling and study protocols were reviewed and approved by the Harbor-UCLA Animal Care and Use Committee.

Fourth and fifth generation pulmonary arteries and veins [as defined by Weibel and Taylor (43), designating the left and right main branch of pulmonary arteries and veins as the first generation] were dissected from the lungs and cut into rings (length, 5 mm; diameter, 1.3-2.0 mm for arteries and 0.8-1.5 mm for veins) in ice-cold modified Krebs-Ringer bicarbonate buffer [(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. Vessel rings were suspended in organ chambers filled with 10 ml of buffer maintained at 37 ± 0.5°C. Buffer solution in the organ chamber was aerated constantly with O2, N2, and CO2. The proportions of O2 and N2 varied so that the PO2 in the solution could be set at 140 mmHg (normoxia) or 30 mmHg (hypoxia). The proportion of CO2 was 5%, and the PCO2 was kept constant to 36 mmHg. The pH was kept at 7.4. The PO2, PCO2, and pH were measured with a pH/blood gas analyzer (Stat Profile Plus 3; Nova Biomedical, Waltham, MA).

To measure the isometric tension, we passed two stirrups through the vessel lumen. One stirrup was anchored to the bottom of the chamber; the other one was connected to a strain gauge (model FT03C; Grass Instrument, Quincy, MA). The resting tension of vessel rings was manually stretched to ~0.5 g/mm2 cross-sectional area of smooth muscle (CSASM) and ~0.3 g/mm2 CSASM for the arteries and veins, respectively, which are their optimal resting tension. The CSASM of each vessel ring was determined as previously described (41).

To eliminate the confounding effects of endogenous prostanoids and EDNO, indomethacin (10-5 M) and nitro-L-arginine (10-4 M) were added to the vessel bath (17, 18). In our preliminary study, using pulmonary arteries and veins denuded of endothelium, we found no significant difference in 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP)-induced relaxation between control and nitro-L-arginine group (data not shown; n = 4, P > 0.05).

After the vessels were exposed to normoxia (140 mmHg) or hypoxia (30 mmHg) for 1, 2, 4, 8, and 20 h, the effect of 8-BrcGMP (3 x 10-5-3 x 10-4 M), a cell membrane-permeable analog of cGMP (26), was determined after the vessel tension was raised with endothelin-1 (3 x 10-9 M for veins and 6 x 10-9-10-8 M for arteries) to comparable levels. In our preliminary study, we determined the response of pulmonary arteries of fetal lambs to 8-BrcGMP after the vessels were contracted with endothelin-1 at different concentrations under normoxia conditions. We found that the relaxation was not significantly different among those contracted with endothelin-1 at concentrations from 3 x 10-9 to 10-8 M (data not shown, n = 4-5, P > 0.05).

The concentration-response curves to 8-BrcGMP were constructed in a cumulative fashion. Experiments were performed under control conditions and in the presence of {beta}-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp isomer [Rp-8-Br-PET-cGMPS, 3 x 10-5 M, an inhibitor of PKG (4)]. In our preliminary study, we found that PKG inhibitor (Rp-8-Br-PET-cGMPS) at 3 x 10-5 M completely abolishes the near-maximal stimulation in PKG activity of fetal pulmonary arteries and veins caused by cGMP (5 x 10-6 M). Moreover, the PKG inhibitor at higher concentrations (10-4 M) did not cause further attenuation of relaxation of the arteries and veins to 8-BrcGMP, suggesting that the PKG inhibitor at the concentrations used in the present study can fully inhibit PKG activity of both vessel types. The PKG inhibitor was added before vessels were contracted with endothelin-1 and was present throughout the experiment.

To determine the role of PKG in endogenous cGMP-mediated relaxation, the effect of 2,2'-(hydroxynitrosohydrazono) bis(ethanamine) [DETA NONOate, a stable NO donor (27)] was examined under control, in the presence of Rp-8-Br-PET-cGMPS (3 x 10-5 M) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one [ODQ, 3 x 10-5 M, an inhibitor of soluble guanylyl cyclase (19)] under normoxia conditions. All experiments were carried out in a parallel manner.

PKG activity assay. Isolated pulmonary arteries and veins of fetal lambs were incubated for 4 h under normoxic or hypoxic conditions (PO2 at 140 and 30 mmHg, respectively) as described earlier in the section Organ chamber study. They were then homogenized in a buffer containing 50 mM Tris·HCl (pH 7.4 at 22°C), 10 mM EDTA, 2 mM dithiothreitol (DTT), 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. We assayed supernatants for PKG activity by measuring the incorporation of 32P from {gamma}-[32P]ATP into a specific PKG substrate, BPDEtide (Biomol Research Laboratories, Plymouth Meeting, PA). Aliquots (10 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 {gamma}-[32P]ATP (specific activity 3,000 Ci/mmol). The mixture was incubated at 30°C for 10 min in the presence or absence of 5 µM exogenous cGMP. We terminated the reaction by spotting 0.04-ml aliquots of mixture onto phosphocellulose papers (2 x 2 cm, P81 Whatman) and placing them in ice-cold 75 mM phosphoric acid. The filter papers were washed, dried, and counted with a liquid scintillation counter. Assays were performed in triplicate with appropriate controls. After control counts are subtracted, counts obtained in the presence or absence of cGMP represent PKG activity, which is expressed as picomoles of 32P incorporated into PKG substrate per minute per milligram of protein (8, 14). Protein content in supernatant was measured by Bradford's procedure, using bovine serum albumin as a standard (3). Preliminary experiments confirmed the linearity of PKG activity with increase in protein concentration and incubation time.

Western analysis of PKG protein. An affinity-purified polyclonal antibody, which recognizes both isoforms of PKG I ({alpha}, 75 kDa; {beta}, 78 kDa), on SDS-PAGE immunoblots, in human, mouse, and Xenopus origins (StressGene, Victoria, Canada) was used. Tissue lysates were prepared from pulmonary arteries and veins incubated for 4 h under normoxic or hypoxic conditions (PO2 at 140 and 30 mmHg, respectively) as described earlier. The lysates, each containing 20 µg of protein, were subjected to SDS-PAGE and 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, 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 by 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). The blot was subsequently stripped and reprobed with antiactin antibody, to which PKG values were normalized (22).

Relative quantitative reverse transcription-polymerase chain reaction for PKG I{alpha} 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 4 h under normoxic or hypoxic conditions (PO2 at 140 and 30 mmHg, respectively) as described earlier. We synthesized cDNA from total RNA by first adding 2 µl RNA into 11.5 µl of buffer containing 250 ng of random hexamers and 10 units of RNase inhibitor (Life Technologies), incubating the mixture at 70°C for 10 min, and then quickly chilling it on ice. Then, 4.0 µl5x First Strand Buffer (Life Technologies), 2.0 µl DTT (0.1 M), 1.0 µl dNTP (10 mM), 0.5 µl RNase inhibitor (20 U/µl), and 1 µl Moloney monkey leukemia virus reverse transcriptase (200 U/µl, Life Technologies) were added. The reaction mixture was incubated at 42°C for 1 h and at 90°C for 10 min. The cDNA products were amplified by PCR with sense and antisense primers for PKG I{alpha} 5'-ctggaggaagactttgccaagattc -3' (16-40: accession no. X16086 [GenBank] ) and 5'-tcggatttggtgaacttccggaatg-3' (269-245: accession no. X16086 [GenBank] ), respectively.

Relative quantitative reverse transcription-polymerase chain reaction (RQ RT-PCR) was performed on a Stratagene thermal cycler (RoboCycler Gradient 96; La Jolla, CA) according to a protocol by Ambion (Austin, TX). In brief, a 50-µl reaction mixture contains 5 µl 10x ThermalAce Buffer (Invitrogen, Carlsbad, CA), 1.0 µl 50x dNTPs (10 mM each, Invitrogen), 20 units ThermalAce DNA Polymerase (Invitrogen), 2.0 µl cDNA, 25 pM each for PKG I{alpha} sense and antisense oligomers, 0.75 µl classic 18S primers, and 1.75 µl 18S competimers (Ambion). The mixture was initially subjected to heating for 2 min at 94°C, then 32 cycles of 30 s at 94°C, 45 s at 55°C, and 60 s at 72°C. Preliminary studies showed that 32 cycles is in the linear range of the PCR product amplification and that the ratio for 18S primers to 18S competimers produces similar yields for the 18S internal standard and PKG I{alpha} mRNA. PCR products were separated on 1.5% agarose gels (containing 0.08% ethidium bromide) and quantified by densitometry with the Eagle Eye II still video system (Stratagene). The quantities of mRNA for PKG I{alpha} 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-BrcGMP, DETA NONOate, endothelin-1 (American Peptide, Sunnyvale, CA), indomethacin, isobutylmethylxanthine, nitro-L-arginine, ODQ, and Rp-8-Br-PET-cGMPS (Biolog Life Science Institute, La Jolla, CA).

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 was dissolved in DMSO (final concentrations <0.06%). Preliminary experiments showed that DMSO at the concentration used has no effect on contraction to endothelin-1 and relaxation induced by DETA NONOate and 8-BrcGMP in pulmonary arteries and veins of fetal lambs. The other drugs were prepared with 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 one-way ANOVA test with Student-Newman-Keuls test for post hoc testing of multiple comparisons. All these analyses were performed with a commercially available statistics package (SigmaStat; Jandel Scientific, San Rafael, CA). Statistical significance was accepted when the P value (two tailed) was <0.05. In all experiments, n represents the number of animals.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Organ chamber studies. Experiments were carried out in the presence of indomethacin (10-5 M) plus nitro-L-arginine (10-4 M) to exclude the confounding effects of endogenous nitric oxide and prostaglandins (17, 18). These inhibitors caused an increase in resting tension by 0.36 ± 0.08 g/mm2 CSASM (n = 11) in pulmonary veins of fetal lambs that were incubated in normoxia but not in those incubated in hypoxia. They had no effect on the resting tension of pulmonary arteries. In the presence of these inhibitors, Rp-8-Br-PET-cGMPS [3 x 10-5 M, a selective inhibitor of PKG (4)] and ODQ [3 x 10-5 M, a selective inhibitor of soluble guanylyl cyclase (19)] had no significant effect on the resting tension of the vessels (data not shown; n = 5-6 for each group, P > 0.05).

Under normoxic conditions (140 mmHg), DETA NONOate induced concentration-dependent relaxation of pulmonary arteries and veins after the vessel tension was raised to a similar level with endothelin-1 (Table 1). Relaxation to DETA NONOate was greater in veins than in arteries (Fig. 1; P < 0.05), a phenomenon reported earlier by us and others (17, 18, 38). Relaxation induced by the nitric oxide donor of arteries and veins was significantly inhibited by Rp-8-Br-PET-cGMPS (3 x 10-5 M) and was nearly abolished by ODQ (3 x 10-5 M) (Fig. 1).


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Table 1. Tension in pulmonary arteries and veins increased by endothelin-1 before the effects of DETA NONOate were determined

 


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Fig. 1. Relaxations of pulmonary arteries (PA) and veins (PV) of fetal lambs to 2,2'-(hydroxynitrosohydrazono)bis(ethanamine) (DETA NONOate) under normoxia (PO2 140 mmHg). Vessels were preconstricted to a similar tension with endothelin-1 (see Table 1). Data are shown as means ± SE; n = 5 for each group. *Significant difference between control and treated with {beta}-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-PET-cGMPS, 3 x 10-5 M); {dagger}significant difference between control and treated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 3 x 10-5 M) (P < 0.05). PKG-I, PKG inhibitor.

 

Relaxation of pulmonary vessels induced by 8-BrcGMP was determined after different lengths of exposure to hypoxia (30 mmHg) or normoxia (140 mmHg). Endothelin-1 was used to raise the vessel tension to a similar level before the administration of 8-BrcGMP (Table 2).


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Table 2. Tension in pulmonary arteries and veins increased by endothelin-1 before the effects of 8-BrcGMP were determined

 



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Fig. 2. Relaxations of PA and PV of fetal lambs to 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 10-4 M) following incubation for varying time periods under hypoxia and normoxia conditions (PO2 30 and 140 mmHg, respectively). Vessels were preconstricted to a similar tension with endothelin-1 (Table 2). Data are shown as means ± SE; n = 6 for each group. *Significant difference between vessels incubated under hypoxia and normoxia; {dagger}significantly different from those incubated for 2 h (P < 0.05).

 


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Fig. 3. Relaxations of PA and PV of fetal lambs to 8-BrcGMP after 4 h incubation under hypoxia and normoxia conditions (PO2 30 and 140 mmHg, respectively). Vessels were preconstricted to a similar tension with endothelin-1 (Table 2). Data are shown as means ± SE; n = 6 for each group. *Significant difference between vessels incubated under hypoxia and normoxia under control conditions; {dagger}significant difference between vessels incubated under hypoxia and normoxia in the presence of Rp-8-Br-PET-cGMPS [PKG inhibitor (PKGI); 3 x 10-5 M] (P < 0.05).

 
There was no significant difference in relaxation of pulmonary arteries and veins to 8-BrcGMP (10-4 M) among vessels exposed to different lengths (1, 2, 4, 8, 20 h) of normoxia. However, the relaxation was attenuated after 2 h of hypoxia exposure, reaching maximal attenuation within 4 h (Fig. 2). In pulmonary arteries and veins incubated under hypoxia (O2, 30 mmHg) for 4 h and then followed by normoxia (O2, 140 mmHg) for 4 h, the relaxation caused by 8-BrcGMP (10-4 M) was not significantly different from that incubated under normoxia (O2, 140 mmHg) for 8 h (data not shown; n = 4, P > 0.05).

Pulmonary arteries and veins, after 4 h of exposure to normoxia, showed a concentration-dependent relaxation to 8-BrcGMP. The relaxation was significantly attenuated by Rp-8-Br-PET-cGMPS (3 x 10-5 M; n = 6, P < 0.05). After exposure to 4 h of hypoxia, relaxation of arteries and veins to 8-BrcGMP at 10-4 and 3 x 10-4 M but not lower concentration (3 x 10-5 M) was attenuated by Rp-8-Br-PET-cGMPS (3 x 10-5 M; n = 6, P < 0.05) (Fig. 3). The relaxation of arteries incubated for 4 h of normoxia in response to 8-BrcGMP was significantly greater than that of arteries incubated under 4-h hypoxia, both under control conditions and in the presence of Rp-8-Br-PET-cGMPS (3 x 10-5 M). In veins, the differential response to 8-BrcGMP between hypoxia and normoxia treatment occurred under control conditions but not in the presence of Rp-8-Br-PET-cGMPS (3 x 10-5 M) (Fig. 3).

PKG activity. In the absence of exogenous cGMP, PKG activity in pulmonary arteries after 4-h hypoxia was not significantly different from that of vessels incubated in normoxia. There was a moderate but statistically significant increase in PKG activity in veins incubated in normoxia compared with that in hypoxia (Fig. 4).



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Fig. 4. PKG activity in pulmonary vessels after 4 h incubation under hypoxia and normoxia conditions (PO2 30 and 140 mmHg, respectively). (-) cGMP, without cGMP; (+) cGMP, in the presence of cGMP at 5 x 10-6 M. Data are shown as means ± SE; n = 4 for each group. *Significantly different from those without cGMP; {dagger}significantly different from those incubated under hypoxia (P < 0.05).

 

Addition of exogenous cGMP (5 x 10-6 M) increased PKG activity markedly from baseline values in arteries but had no effect in veins after 4-h hypoxia. cGMP-stimulated PKG activity of all vessel types was significantly greater after 4-h normoxia than hypoxia (arteries: 94.9 ± 2.4 vs. 65.6 ± 4.3 pmol·min-1·mg protein-1; veins: 59.1 ± 6.7 vs. 14.5 ± 2.4 pmol·min-1·mg protein-1; n = 4 for each group, P < 0.05) (Fig. 4).

PKG protein. In vessels incubated under hypoxic conditions for 4 h, the PKG protein level in arteries was greater than that in veins. After 4-h incubation in normoxia, the PKG protein level of veins was significantly greater than that in hypoxia. For arteries, there was no significant difference in PKG protein levels between hypoxia and normoxia treatments (Fig. 5).



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Fig. 5. Top: Western blots of PKG and actin of the homogenate of PA and PV of fetal lambs under hypoxia and normoxia (PO2 30 and 140 mmHg, respectively). Bottom: densitometric scanning of PKG protein normalized to actin. Data shown: means ± SE; n = 6 for each group. *Significant different from arteries; {dagger}significant different from veins incubated under hypoxia (P < 0.05).

 

PKG I{alpha} mRNAs. RQ RT-PCR yielded two distinctive bands corresponding to the predicted sizes for mRNA fragments of PKG I{alpha} and the 18S internal standards (255 and 488 bp, respectively). Pulmonary veins incubated in normoxia for 4 h showed a greater content of PKG I{alpha} mRNA than those incubated in hypoxia. There was no significant difference in PKG I{alpha} mRNA content of arteries between hypoxia and normoxia treatments (Fig. 6).



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Fig. 6. Relative quantitative RT-PCR of mRNA fragments of PKG I{alpha} and 18S internal standards (253 and 489 bp, respectively) in fetal PA and PV preincubated in hypoxia and normoxia (PO2 30 and 140 mmHg, respectively; 4 h). Top: representative gel image. Bottom: densitometric scanning of mRNA fragments of PKG I{alpha} normalized to 18S. Data shown: means ± SE; n = 5 for each group. *Significant different from arteries; {dagger}significant different from veins incubated under hypoxia (P < 0.05).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In the immediate newborn period, oxygen modulates pulmonary vasoactivity by stimulation of endothelial nitric oxide production (6, 7, 28, 36, 39, 42). After prolonged exposure to higher oxygen concentrations, the expression of nitric oxide synthase (eNOS) protein and mRNA in endothelial cells of pulmonary arteries has been shown to be upregulated (2, 29). EDNO mediates vasodilation by causing an increase in intracellular cGMP levels (21). It is not known whether oxygen affects cGMP-mediated pulmonary vasodilation. In the present study, we found that relaxation of pulmonary arteries and veins of fetal lambs to 8-BrcGMP was attenuated after 2 h of exposure to hypoxia, reaching maximal attenuation within 4 h. Moreover, the diminished relaxation to the cGMP analog of pulmonary vessels observed under 4-h hypoxia was reversed after 4-h normoxia, suggesting that higher oxygen tension can upregulate, whereas lower oxygen tension can downregulate, cGMP-mediated response in pulmonary vasculatures.

In the present study, the relaxation of pulmonary vessels incubated under normoxic conditions in response to 8-BrcGMP was significantly inhibited by Rp-8-Br-PET-cGMPS, a selective inhibitor of PKG (4). These results are similar to those we previously obtained in pulmonary vessels of newborn lambs (8, 14). Our present study shows that relaxation of pulmonary vessels induced by DETA NONOate, a stable nitric oxide donor (27), was inhibited not only by ODQ (an inhibitor of soluble guanylyl cyclase) (19) but also by Rp-8-Br-PET-cGMPS, a PKG inhibitor (4). It suggests that, in fetal pulmonary vasculature, PKG plays an important role in endogenous cGMP-mediated response (13, 20, 21, 24).

In pulmonary veins, the differential response in 8-BrcGMP-induced relaxation between vessels exposed to normoxia and those to hypoxia was abolished by Rp-8-Br-PET-cGMPS, suggesting that the cGMP-mediated response is primarily by acting on PKG. Such a notion is supported by the striking finding that cGMP-stimulated PKG activity occurred in normoxia but not in hypoxia-treated veins. PKG exists as two forms (type I and type II) in cells, and PKG type I has two isoforms (PKG I{alpha} and PKG I{beta}). Vascular smooth muscle cells contain mainly PKG type 1 (13, 20, 24). Recent studies indicate that cGMP-mediated vasodilation is mainly mediated by PKG I{alpha} (10, 23, 35). In our study, the protein level of PKG type I and mRNA level of PKG I{alpha} (measured by Western analysis and RT-PCR, respectively) were greater in veins exposed to normoxia than exposed to hypoxia. Together, these results suggest that cGMP-mediated relaxation of fetal ovine pulmonary veins is upregulated by oxygen. It should be pointed out that, compared with the greater-than-threefold difference in cGMP-stimulated PKG activity of the vein between normoxia and hypoxia treatments, the changes in PKG protein and mRNA levels associated with the oxygen tension were moderate. Thus in addition to acting on transcriptional and translational steps, oxygen may affect PKG activity by other mechanisms.

Unlike in the veins, the differential responses of arteries to the cGMP analog associated with oxygen tension were not abolished by Rp-8-Br-PET-cGMPS, the PKG inhibitor (4). These results are consistent with the findings that oxygen did not affect the protein level of PKG type I and mRNA level of PKG I{alpha} of the arteries. However, they are at variance with the data obtained in PKG activity assay showing that cGMP-stimulated PKG activity was greater by ~45% in arteries after 4-h exposure to normoxia compared with hypoxia. One explanation may be that oxygen exposure induced some phenomenon in arteries that counteracted the change in PKG activity. Alternatively, in the presence of the selective PKG inhibitor, which does not inhibit cAMP-dependent protein kinase (PKA), 8-BrcGMP could cross activate PKA and thus cause relaxation of arteries (8). The fact that hypoxia attenuated cGMP-induced relaxation of pulmonary arteries even in the presence of PKG inhibitor suggests a more important role for PKG-independent mechanisms of cGMP-mediated vasodilation in arteries. It is known that cGMP may cause vasodilation without activating PKG, for instance, by directly acting on cyclic nucleotide-gated cation channels (7, 24). It should be pointed out that the arteries we studied are conduit arteries. Heterogeneity exists along the pulmonary tree. The effect of oxygenation on cGMP-mediated relaxation may differ in resistance-level arteries. For instance, our previous study found that nitric oxide-mediated relaxation is more pronounced in smaller-sized than in large-size arteries (15).

In the pulmonary circulation during the perinatal period, veins exhibit greater vasoactivity than arteries in response to a variety of stimuli (1, 17, 18, 31, 32, 38, 41). We and other investigators have shown that relaxation induced by endogenous and exogenous nitric oxide is more pronounced in pulmonary veins than in arteries in fetal and newborn lambs as well as newborn pigs (1, 17, 18, 38). A greater response to vasoactive agents occurs not only in perinatal but also in adult lungs of various species. In lambs and adult rats and ferrets, pulmonary veins constrict as vigorously as or more than arteries during hypoxia (32, 33, 45). In rats, ferrets, lambs, and dogs, platelet-activating factor causes greater constrictions in pulmonary veins than in arteries (5, 16, 37, 40). In fetal and newborn lambs, newborn and adult pigs, and adult cows, relaxation induced by EDNO and nitrovasodilators is more pronounced in pulmonary veins than in arteries (1, 9, 11, 17, 18, 44).

Oxygenation plays an important role in regulating perinatal pulmonary vasoactivity (2, 6, 7, 12, 28, 29, 34, 36). The present study suggests that the oxygen-associated upregulation of PKG-activity plays a larger role in cGMP-mediated relaxation of pulmonary veins than that of arteries in fetal lambs. Because veins contribute substantially to total pulmonary vascular resistance (31-33), our results may indicate that oxygen-mediated modulation of PKG activity in veins may be important in the postnatal adaptation of the pulmonary circulation.


    DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
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This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-59435.


    ACKNOWLEDGMENTS
 
We thank Mary Lee Ryba and Nik Phou for excellent secretarial assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Gao, Harbor-UCLA Medical Center, Research and Education Institute, 1124 W. Carson St., RB-1, Torrance, CA 90502 (E-mail: ygao{at}gcrc.rei.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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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