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J Appl Physiol 87: 993-998, 1999;
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Vol. 87, Issue 3, 993-998, September 1999

Role of protein kinase G in nitric oxide- and cGMP-induced relaxation of newborn ovine pulmonary veins

Yuansheng Gao, Srinivas Dhanakoti, Jean-Francois Tolsa, and J. Usha Raj

Department of Pediatrics, University of California, Los Angeles, School of Medicine, Harbor-UCLA Medical Center, Torrance, California 90509


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a variety of systemic blood vessels, protein kinase G (PKG) plays a critical role in mediating relaxation induced by agents that elevate cGMP, such as nitric oxide. The role of PKG in nitric oxide- and cGMP-induced relaxation is less certain in the pulmonary circulation. In the present study, we examined the effects of inhibitors of PKG on the responses of isolated fourth-generation pulmonary veins of newborn lambs (10 ± 1 days of age) to nitric oxide and cGMP. In vessels preconstricted with endothelin-1, nitric oxide and 8-bromo-cGMP (a cell-membrane-permeable cGMP analog) induced concentration-dependent relaxation. The relaxation was significantly attenuated by beta -phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Rp-8-Br-PET-cGMPS; a PKG inhibitor) and N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide [H-8; an inhibitor of PKG and protein kinase A (PKA)] but was not affected by KT-5720 (a PKA inhibitor). Biochemical study showed that PKG activity in newborn ovine pulmonary veins was inhibited by 8-Br-PET-cGMPS and H-8 but not by KT-5720. PKA activity was not affected by 8-Br-PET-cGMPS but was inhibited by H-8 and KT-5720. These results suggest that PKG is involved in relaxation of pulmonary veins of newborn lambs induced by nitric oxide and cGMP.

guanosine 3',5'-cylic monophosphate; beta -phenyl-1, N2-etheno-8-bromoguanosine 3',5'-monophosphorothioate; N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide; KT-5720; vasodilation; neonatal pulmonary circulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIUM-DERIVED NITRIC OXIDE, exogenous nitric oxide, and other nitrovasodilators cause smooth muscle relaxation by elevating intracellular cGMP content after activating soluble guanylate cyclase (23, 29). In various systemic blood vessels, studies show that relaxation induced by nitric oxide and cGMP is primarily mediated by cGMP-dependent protein kinase G (PKG) (12, 25, 27, 43). In porcine coronary arteries, there is a close correlation between the potencies of cGMP analogs in causing relaxation and the potencies of the analogs in activating PKG in vitro (38). In cultured aortic cells, 8-bromo-cGMP (8-BrcGMP), a cell-membrane-permeable cGMP analog (28), decreases intracellular Ca2+. This effect is less prominent in passaged cells, where the expression of PKG is reduced. When PKG is restored to the kinase-deficient cells, responsiveness to 8-BrcGMP is also restored (10). In PKG-deficient mice, relaxation of isolated aortic rings caused by endothelium-derived nitric oxide and 8-BrcGMP is abolished. Moreover, the mean arterial blood pressure in these PKG-deficient mice is higher than that in control mice. The hypotensive effect induced by exogenous nitric oxide is also abolished in PKG-deficient mice (33).

In the pulmonary circulation, the role of PKG in nitric oxide- and cGMP-induced smooth muscle relaxation is not well studied, and the study results are controversial (2, 8, 11, 32). In fetal lambs, the oxygen-induced pulmonary vasodilation is markedly attenuated by inhibitors of soluble guanylate cyclase and PKG (8). In pulmonary arterial smooth muscle cells of rats and humans, nitric oxide, cGMP, and cGMP analogs activate Ca2+-sensitive potassium channels. This effect is attenuated by inhibitors of PKG (2, 32). Because activation of the above-mentioned channels causes membrane hyperpolarization and thus vasodilation, these results suggest that PKG is involved in nitric oxide- and cGMP-induced pulmonary vasodilation. In another study, however, it has been found that hypoxia-induced vasoconstriction of the rat lung was augmented by an inhibitor of soluble guanylate cyclase but was not affected by inhibitors of PKG. It is postulated that PKG is not essential to nitric oxide-cGMP modulation of rat pulmonary vascular tone (11).

In lamb lungs, under basal conditions, pulmonary veins contribute ~36%, whereas arteries contribute ~32%, to total pulmonary vascular resistance (36). When stimulated with thromboxane, platelet-activating factor, and endothelin, and during hypoxia, pulmonary veins constrict equally or more than arteries (34, 35, 41, 42). In fetal, newborn, juvenile, and adult sheep, nitro-L-arginine causes contractions of isolated pulmonary veins but not of arteries (3, 17, 18, 40). In fetal and newborn lambs, endothelium-derived nitric oxide and sodium nitroprusside cause greater relaxation of pulmonary veins than of arteries (17, 18, 40). Relaxation of ovine pulmonary veins to nitric oxide is preceded by the elevation of the intracellular cGMP concentration and is largely eliminated by the inhibition of guanylate cyclase (17, 18). These studies suggest that the effect of nitric oxide in ovine pulmonary veins is primarily mediated by cGMP (17, 18).

In the present study, we examined the effect of PKG inhibitors on the relaxation that was induced by nitric oxide and cGMP in isolated pulmonary veins of newborn lambs and on the PKG activity of these vessels. Our results suggest a significant role for PKG in nitric oxide- and cGMP-mediated relaxation of the pulmonary veins of newborn lambs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fifteen newborn lambs (7-13 days old, either sex) from Nebeker Ranch (Lancaster, CA) were used. They were anesthetized with ketamine hydrochloride (30 mg/kg im) and then killed with an overdose of pentobarbital. The lungs were immediately removed, and fourth-generation pulmonary veins (outside diameter: 1.5-2.5 mm) were dissected free of parenchyma and cut into rings (length: 3 mm).

Organ chamber study. Vessel rings were suspended in organ chambers filled with 10 ml of modified Krebs-Ringer bicarbonate solution [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] maintained at 37 ± 0.5°C and aerated with 95% O2-5% CO2 (pH = 7.4). Each ring was suspended by two stirrups passed through the lumen. One stirrup was anchored to the bottom of the organ chamber; the other one was connected to a strain gauge (model FT03C, Grass Instruments, Quincy, MA) for the measurement of isometric force (17).

At the beginning of the experiment, each vessel ring was stretched to its optimal resting tension. This was achieved by stepwise stretching, in 0.1-g increments, 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 0.28 ± 0.06 g (n = 15).

After the vessels were brought to their optimal resting tension, 1 h of equilibration was allowed. Then, indomethacin [10-5 M; an inhibitor of cyclooxygenase (7)] and nitro-L-arginine [10-4 M; an inhibitor of nitric oxide synthase (31)] were administrated to eliminate the possible involvement of endogenous prostanoids and endothelium-derived nitric oxide (16, 31). In our preliminary study, we found no difference in relaxation induced by nitric oxide or 8-BrcGMP between newborn ovine pulmonary veins without endothelium and those with endothelium treated with nitro-L-arginine. With regard to the inhibition of cyclooxygenase, we determined the effect of indomethacin on the release of 6-keto-PGF1alpha (the stable breakdown product of PGI2) by radioimmunoassay as described previously. {The antisera to 6-keto-PGF1alpha and [3H]6-keto-PGF1alpha were purchased from New England Nuclear, Boston, MA (16)}. Under basal conditions, the release of 6-keto-PGF1alpha within 30 min from newborn ovine pulmonary veins with endothelium was significantly greater than that from veins without endothelium (266.4 ± 85.4 vs. 29.0 ± 7.8 pg/mg tissue; n = 7 animals/group, P < 0.05). In the presence of indomethacin (10-5 M), the basal release of 6-keto-PGF1alpha was 9.6 ± 2.7 and 11.4 ± 2.8 pg/mg tissue for veins with and without endothelium (n = 7), respectively. There is no significant difference between these values (P > 0.05).

Effects of nitric oxide (1.6 × 10-9 to 5 × 10-7 M) and 8-BrcGMP [10-8 to 3 × 10-4 M; a cell-membrane-permeable analog of cGMP (28)] were determined in vessels preconstricted with endothelin-1 (3 × 10-9 to 10-8 M) to a similar tension. The concentration-response curves to nitric oxide or 8-BrcGMP were constructed in a cumulative fashion. Each concentration was added only when the response to the prior one became stable. Experiments were performed under control conditions and in the presence of 1H-[1,24]oxadiazolo4,3-a]quinoxalin-1-one [ODQ; 3 × 10-5 M; an inhibitor of soluble guanylate cyclase (19)], beta -phenyl-1, N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate [Rp-8-Br-PET-cGMPS; 3 × 10-5 and 10-4 M; an inhibitor of PKG (6)], N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide [H-8; 10-4 M; an inhibitor of PKG and protein kinase A (PKA) (22)], and KT-5720 [5 × 10-5 M; an inhibitor of PKA (24)]. These inhibitors were administrated before vessels were contracted with endothelin-1 and remained in contact with the tissue throughout the experiment. 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.

PKG activity assay. Isolated pulmonary veins of newborn lambs were homogenized in a buffer containing 50 mM Tris · HCl (pH 7.4 at 22°C), 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 [alpha -32P]ATP into the specific PKG substrate BPDEtide (Biomol Research Laboratories, Plymouth Meeting, PA) (20). 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, 5 µM PKI (a synthetic PKA inhibitor; Peninsula Laboratories, Belmont, CA), and 0.2 mM [alpha -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. Reaction was terminated by spotting 0.04-ml aliquots onto 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 with a liquid scintillation counter (37). Assays were performed in triplicate with appropriate controls. After control counts were subtracted, counts were obtained in the presence or absence of cGMP to indicate PKG activity, which is expressed as picomoles 32P incorporated into PKG substrate per minute per milligram protein. Protein content in supernatant was measured by Bradford's procedure, using bovine serum albumin as a standard (4). Preliminary experiments confirmed the linearity of PKG activity at the protein concentration used within the incubation time.

PKA activity assay. The PKA assay was carried out in a fashion similar to that for PKG, except that the PKG substrate BPDEtide was replaced with a specific PKA substrate (Kemptide, 130 µM, Peninsula Laboratories), cGMP was replaced with cAMP (2 µM), and PKI was omitted. The linearity of PKA activity at the protein concentration used within the incubation time was determined in preliminary experiments.

Preparation of nitric oxide. A gas bulb sealed with a silicone rubber injection septum was filled with nitric oxide from a cylinder (Union Carbide, Chicago, IL). An appropriate volume (0.25 and 2.5 ml) was removed with a syringe and injected into another gas bulb filled with 250 ml of distilled water, which had been gassed with helium for over 3 h, giving stock solutions of nitric oxide of 4.2 × 10-5 and 10-4 M (17).

Drugs. The following drugs were used (unless otherwise specified, all were obtained from Sigma Chemical, St. Louis, MO): 8-BrcGMP, endothelin-1 (American Peptide, Sunnyvale, CA), H-8 (Biomol Research Laboratories), indomethacin, isobutylmethylxanthine, KT-5720 (a gift from Dr. Douglas Palmer of Kamiya Biochemical, Seattle, WA), nitro-L-arginine, ODQ, and Rp-8-Br-PET-cGMPS (Rp isomer; Biolog Life Science Institute, La Jolla, CA).

H-8 and KT-5720 were dissolved in DMSO (final concentrations: <0.2%). Preliminary experiments showed that DMSO at the concentration used had no effect on contraction to endothelin-1 and relaxation induced by nitric oxide and 8-BrcGMP in pulmonary vessels of newborn lambs. Indomethacin (10-5 M) was prepared in equimolar Na2CO3. This concentration of Na2CO3 did not significantly affect the pH of the solution in the organ chamber. The other drugs were prepared by 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 one-way ANOVA with the Student-Newman-Keuls test for post hoc testing of multiple comparison. All these analyses were performed by 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Organ chamber studies. Experiments were carried out in the presence of indomethacin plus nitro-L-arginine to exclude the involvement of endogenous nitric oxide and PGs (7, 16, 17, 31). Indomethacin (10-5 M) plus nitro-L-arginine (10-4 M) caused an increase in resting tension by 0.81 ± 0.10 g (n = 15) in pulmonary veins of newborn lambs. In the presence of indomethacin and nitro-L-arginine, ODQ [3 × 10-5 M; an inhibitor of soluble guanylate cyclase (19)] and Rp-8-Br-PET-cGMPS [3 × 10-5 and 10-4 M; a highly selective inhibitor of PKG (6)] had no significant effect on the resting tension. H-8 [10-4 M; an inhibitor of PKG and PKA (22)] and KT-5720 [5 × 10-5 M; a selective inhibitor of PKA (24)] reduced the resting tension by 0.32 ± 0.05 and 0.29 ± 0.02 g, respectively (n = 6 animals/group).

Thirty minutes after the administration of different inhibitors or solvent, vessels were constricted with endothelin-1 (3 × 10-9 to 10-8 M) to a similar tension level (Table 1).

                              
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  		Table 1.   Tension levels in pulmonary veins raised by endothelin-1 before determination of vasodilating effects of nitric oxide and 8-BrcGMP

In vessels preconstricted with endothelin-1, nitric oxide and 8-BrcGMP induced concentration-dependent relaxations of pulmonary veins. The relaxations were inhibited by Rp-8-Br-PET-cGMPS (3 × 10-5 M) and H-8 (10-4 M) but not by KT-5720 (5 × 10-5 M). ODQ (3 × 10-5 M) inhibited relaxation induced by nitric oxide but not that by 8-BrcGMP (Figs. 1 and 2). The effect of Rp-8-Br-PET-cGMPS at different concentrations on 8-BrcGMP-induced response was also examined. Relaxation induced by the cGMP analog at 3 × 10-4 M under control conditions, in the presence of Rp-8-Br-PET-cGMPS at 3 × 10-5 M, and at 10-4 M was 92.6 ± 1.9, 62.6 ± 7.2, and 65.1 ± 3.7%, respectively (n = 5 animals/group). There is a significant difference between the control values and those from veins treated with the PKG inhibitor (P < 0.05); however, the values from vessels treated with Rp-8-Br-PET-cGMPS at 3 × 10-5 M were not significantly different from those treated at 10-4 M (P > 0.05).


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  		Fig. 1.   Relaxation induced by nitric oxide in pulmonary veins of newborn lambs. Vessels were preconstricted to a similar tension with endothelin-1 (3 × 10-9 to 10-8 M). ODQ, 1H-[1,24]oxadiazolo[4,3-a]quinoxalin-1-one (3 × 10-5 M); Rp-8-Br-PET-cGMPS, beta -phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (3 × 10-5 M); H-8, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (10-4 M); KT-5720, 5 × 10-5 M. Values are means ± SE; n = 6 animals/group. * Significant difference between control and vessels treated with Rp-8-Br-PET-cGMPS or H-8. dagger  Significant difference between vessels treated with ODQ and those treated with Rp-8-Br-PET-cGMPS or H-8, P < 0.05.



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  		Fig. 2.   Relaxation induced by 8-BrcGMP in pulmonary veins of newborn lambs. Vessels were preconstricted to a similar tension with endothelin-1 (3 × 10-9 to 10-8 M). ODQ, 3 × 10-5 M; Rp-8-Br-PET-cGMPS, 3 × 10-5 M; H-8, 10-4 M; KT-5720, 5 × 10-5 M. Values are means ± SE; n = 6 animals/group. * Significant difference between control and vessels treated with Rp-8-Br-PET-cGMPS or H-8, P < 0.05.

PKG and PKA activity. The basal activity of PKG in pulmonary veins of newborn lambs was 10.5 ± 3.9 pmol · min-1 · mg protein-1 (n = 4). It was not significantly affected by Rp-8-Br-PET-cGMPS (3 × 10-5 M), H-8 (10-4 M), or KT-5720 (5 × 10-5 M).

The basal activity of PKA was 212 ± 38 pmol · min-1 · mg protein-1 (n = 5), which is greater than that of PKG (P < 0.05). The basal activity of PKA in pulmonary veins was not affected significantly by Rp-8-Br-PET-cGMPS (3 × 10-5 M) and KT-5720 (5 × 10-5 M) but was reduced by H-8 (10-4 M) (see Fig. 4).

cGMP at 5 × 10-6 M caused a greater than fourfold increase in PKG activity. The increase was inhibited by Rp-8-Br-PET-cGMPS (3 × 10-5 M) and H-8 (10-4 M) but not by KT-5720 (5 × 10-5 M) (Fig. 3). cAMP at 2 × 10-6 M caused a greater than twofold increase in PKA activity. The increase in PKA activity was not affected significantly by Rp-8-Br-PET-cGMPS (3 × 10-5 M) but was inhibited by H-8 (10-4 M) and KT-5720 (5 × 10-5 M) (Fig. 4).


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  		Fig. 3.   Effects of Rp-8-Br-PET-cGMPS (3 × 10-5 M), H-8 (10-4 M), and KT-5720 (5 × 10-5 M) on protein kinase G activity of pulmonary veins. Values are means ± SE; n = 4 animals/group. (-)cGMP, Without cGMP; (+)cGMP, in presence of cGMP (5 × 10-6 M). * Significantly different from basal, P < 0.05.



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  		Fig. 4.   Effects of Rp-8-Br-PET-cGMPS (3 × 10-5 M), H-8 (10-4 M), and KT-5720 (5 × 10-5 M) on protein kinase A activity of pulmonary veins. Values are means ± SE; n = 5 animals/group. (-)cAMP, without cAMP; (+)cAMP, in presence of cAMP (2 × 10-6 M). * Significantly different from basal, P < 0.05. dagger  Significantly different from basal control values, P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the perinatal pulmonary circulation, vasodilation induced by nitric oxide is mainly due to an increase in the intracellular cGMP content resulting from activation of soluble guanylate cyclase (14, 17, 18, 39). In the present study, relaxation induced by nitric oxide was largely eliminated by ODQ, an inhibitor of soluble guanylate cyclase (19). These results are consistent with our previous studies and those of other investigators (14, 17, 18, 39). Relaxation induced by 8-BrcGMP, a cell-membrane-permeable analog of cGMP (28), was not significantly affected by ODQ. Studies show that ODQ acts selectively on soluble guanylate cyclase (19). Thus it would not be expected to have a direct effect on the action of cGMP.

In various systemic blood vessels, relaxation induced by nitric oxide and cGMP is believed to be mediated by PKG (12, 25, 43). In our present study, Rp-8-Br-PET-cGMPS, a potent and selective inhibitor of PKG (6), significantly attenuated relaxation of pulmonary veins to nitric oxide and 8-BrcGMP. Also, the cGMP-stimulated activity of PKG in pulmonary veins was largely eliminated. Similar results were also obtained with H-8, a potent but less selective inhibitor of PKG (22). These results suggest that, in newborn ovine pulmonary veins, PKG plays an important role in mediating nitric oxide- and cGMP-induced relaxation. It is notable that, although the cGMP-stimulated PKG activity was largely inhibited by PKG inhibitors, the basal PKG activity was not altered by these inhibitors. The basal PKG activity was measured in the presence of nitro-L-arginine (an inhibitor of nitric oxide synthase). Therefore, it is likely that very little endogenous cGMP existed in the preparation and that basal PKG activity was very low. This might contribute to the lack of detectable effect of these PKG inhibitors.

Besides acting as a PKG inhibitor, H-8 is also a potent PKA inhibitor (22). Therefore, it may affect the relaxation induced by nitric oxide and cGMP via its inhibition of PKA activity (9). In the present study KT-5720, a selective inhibitor of PKA (24), had no significant effect on PKG activity but markedly inhibited the cAMP-stimulated PKA activity of pulmonary veins. Furthermore, KT-5720 showed no effect on relaxation induced by nitric oxide and 8-BrcGMP. Hence, a role for PKA seems unlikely. In our study, both KT-5720 and H-8 reduced the resting tension. The underlying mechanism is not clear. The reduction of the resting tension by these inhibitors could not result from their inhibition of PKA, because the inhibition of the enzyme should cause vasoconstriction rather than vasodilation.

In isolated normotensive rat lungs, inhibition of soluble guanylate cyclase with ODQ augments hypoxic pulmonary vasoconstriction, whereas Rp-8-pCPT-cGMPS [Rp-isomer; an inhibitor of PKG (5)] had no effect. In hypertensive rat lungs, ODQ increased perfusion pressure but Rp-8-pCPT-cGMPS and H-8 had no effect. It was thus postulated that PKG was not essential for nitric oxide- and cGMP-mediated relaxation in rat pulmonary circulation (11). This is at variance with the conclusion reached from our present study. Some explanations can be put forward for the different conclusions of these two studies. For instance, in the study in rats, inhibition of PKG in the pulmonary vasculature was indirectly assessed by measurement of PKG-specific phosphorylation of inositol-1,4,5-trisphosphate receptor in the whole lungs. Because the pulmonary vasculature is only a small portion of the whole lungs, data obtained from the whole lung tissues may not correspond to effective inhibition of PKG in the pulmonary vasculature at the concentrations of the PKG inhibitors used. In mammalian cells, there are two major PKG isoforms, designated type I and type II (12, 25, 27). In the rat study, the PKG inhibitor that was used, Rp-8-pCPT-cGMPS, is a type II PKG inhibitor (13). It is 14- to 523-fold less potent in inhibiting type I PKG than is Rp-8-Br-PET-cGMPS (5, 6, 13). Because type I PKG is the predominant isoform in vascular smooth muscle (12), Rp-8-pCPT-cGMPS may not be an appropriate agent for inhibition of PKG activity in pulmonary vessels. We found that Rp-8-pCPT-cGMPS at 3 × 10-5 M had no effect on nitric oxide- and 8-BrcGMP-induced relaxation of pulmonary veins of newborn lambs (unpublished observations). Several other factors could also contribute to the difference in findings, such as differences in species (lambs vs. rats), ages (newborn vs. adult), and preparations (isolated pulmonary veins vs. isolated perfused lungs). Because our study was conducted in normal preparations whereas the rat study was in a hypoxic and/or hypertensive preparation, it is also possible that cGMP could be acting primarily via a PKG-independent mechanism during hypoxia and/or hypertension (11).

In the present study, PKG inhibitors largely eliminated PKG activity of the pulmonary vein preparations. However, these inhibitors at similar or higher concentrations only partially attenuated the relaxation induced by nitric oxide or 8-BrcGMP. These data suggest that some mechanisms that are independent of PKG may be involved. cGMP may directly act on cyclic nucleotide-gated cation channels (25). Presently, the involvement of these channels in the pulmonary circulation has not been studied. cGMP may also modulate vasoactivity by directly acting on phosphodiesterases. In pulmonary arteries, endothelium-derived nitric oxide potentiates cAMP-mediated vasodilation of pulmonary vessels by inhibiting the cGMP-inhibitable phosphodiesterase (15), thus reducing degradation of cAMP. The endogenous level of cAMP in ovine pulmonary vessels is mainly due to the action of PGs on adenylate cyclase and is largely reduced when cyclooxygenase is inhibited (16). In the present study, the vessels were pretreated with indomethacin. Hence, the relaxation induced by nitric oxide and 8-BrcGMP in the presence of the PKG inhibitor appears not to be attributable to a potentiated cAMP effect, i.e., due to inhibition of cGMP-inhibitable phosphodiesterases.

In the perinatal period, the nitric oxide-cGMP pathway plays a pivotal role in the regulation of pulmonary vascular tone (1, 14, 17, 21, 23, 26, 30, 44). The downstream mechanisms of this pathway are not well understood. The present study suggests that PKG, but not PKA, is involved in nitric oxide- and cGMP-induced relaxation of newborn ovine pulmonary veins. Our data also show that a substantial part of the relaxation was independent of PKG. The underlying mechanisms remain to be determined.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Douglas Palmer (Kamiya Biochemical, Seattle, WA) for providing KT-5720 and to Becky Saldana for secretarial assistance.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-38438 and HL-59435 and la Foudation Emma Mushamp, Switzerland.

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. §1734 solely to indicate this fact.

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: yuansheng_gao{at}prl.humc.edu).

Received 30 October 1998; accepted in final form 24 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abman, S. H., B. A. Chatfield, D. M. Rodman, S. L. Hall, and I. F. McMurtry. Maturational changes in endothelium-derived relaxing factor activity of ovine pulmonary arteries in vitro. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L280-L285, 1991[Abstract/Free Full Text].

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3.   Bansal, V., H. Toga, and J. U. Raj. Tone dependent nitric oxide production in ovine vessels in vitro. Respir. Physiol. 93: 249-260, 1993[Medline].

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