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
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ABSTRACT |
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protein kinase G; vasodilation; vein; perinatal lung
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.
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MATERIALS AND METHODS |
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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
-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 -[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
-[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 (, 75 kDa;
, 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 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
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 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
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
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.
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RESULTS |
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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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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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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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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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PKG I mRNAs. RQ RT-PCR yielded two distinctive
bands corresponding to the predicted sizes for mRNA fragments of PKG I
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
mRNA
than those incubated in hypoxia. There was no significant difference in PKG
I
mRNA content of arteries between hypoxia and normoxia treatments
(Fig. 6).
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DISCUSSION |
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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 and PKG I
).
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
(10,
23,
35). In our study, the protein
level of PKG type I and mRNA level of PKG I
(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 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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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