Evidence for the role of p38 MAP kinase in hypoxia-induced pulmonary vasoconstriction

M. R. Karamsetty1, J. R. Klinger1,2, and N. S. Hill1

1 Division of Pulmonary and Critical Care Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence 02903; and 2 Veterans Administration Medical Center, Providence, Rhode Island 02908


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitogen-activated protein (MAP) kinases regulate smooth muscle cell contraction. Hypoxia contracts pulmonary arteries by mechanisms that are incompletely understood. We hypothesized that hypoxic contraction of pulmonary arteries involves activation of the MAP kinases. To test this hypothesis, we studied the effects of SB-202190, a p38 MAP kinase inhibitor, PD-98059 and UO-126, two structurally different MEKK inhibitors, and anisomycin, a stimulator of p38 MAP kinase on acute hypoxia-induced contraction in rat conduit pulmonary artery rings precontracted with phenylephrine or KCl. Hypoxia induced a transient contraction, followed by a relaxation, and then a slowly developing sustained contraction. Hypoxia also significantly increased phosphorylation of p38 MAP kinase. SB-202190 did not affect the transient phase but abrogated the sustained phase of hypoxic contraction, whereas anisomycin enhanced both phases of contraction. SB-202190 also attenuated and anisomycin enhanced the phenylephrine-induced contraction. In contrast, PD-98059 and UO-126 had minimal effects on either hypoxic or phenylephrine-induced contraction. None of the treatments modified KCl-induced contraction. We conclude that p38, but not the ERK1/ERK2 MAP kinase pathway, mediates the sustained phase of hypoxic contraction in isolated rat pulmonary arteries.

phospho-p38; extracellular signal-regulated kinase 1/2; mitogen-activated protein kinase kinase; rat pulmonary artery


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MITOGEN-ACTIVATED PROTEIN KINASES (MAP kinases) are a family of serine/threonine kinases that are expressed in all eukaryotic cells including vascular smooth muscle and endothelial cells. The well-characterized MAP kinase family members include extracellular signal-regulated kinases (ERK1 and ERK2), c-Jun-NH2-terminal kinases/stress-activated protein kinase, and p38 kinase. They regulate cell proliferation and differentiation in response to a variety of growth factors and stress stimuli such as hypoxia. In addition, MAP kinases, particularly the p38 and ERK1/ERK2 kinases, regulate contraction of both vascular and nonvascular smooth muscle cells. For instance, p38 MAP kinase mediates angiotensin II-induced contraction of rat aorta (21, 29, 30) and endothelin-1-induced contraction in canine pulmonary artery (37, 38), whereas ERK1/ERK2 mediates PGF2alpha -induced contraction in iris sphincter smooth muscle (39).

The constrictor response of pulmonary arteries to acute hypoxia is a homeostatic mechanism that matches perfusion to ventilation within the lungs. The cellular mechanisms responsible for hypoxic pulmonary vasoconstriction are not fully understood but appear to include altered release of endothelium-derived relaxing and contracting factors, such as nitric oxide and endothelin-1, and inhibition of K+ channels on smooth muscle cells (32, 34). These events lead to membrane depolarization, Ca2+ entry that increases intracellular Ca2+, and smooth muscle contraction (32, 34).

Hypoxia-induced increases in intracellular Ca2+ are known to activate MAP kinases (3). In addition, hypoxia stimulates other upstream activators of MAP kinases such as protein kinase C (14, 35) and tyrosine kinases (31). Moreover, hypoxia increases sensitivity of the contractile apparatus to Ca2+ (23), a process that is known to involve MAP kinases (37). Therefore, we hypothesized that hypoxic contraction of pulmonary arteries involves activation of MAP kinase pathways. To test this hypothesis, we examined the effects of relatively selective inhibitors of p38 MAP kinase and the ERK pathway on hypoxia-induced pulmonary vasoconstriction in rat pulmonary artery rings. We also investigated the effects of a p38 MAP kinase stimulator on hypoxia-induced pulmonary artery contractions and effects of hypoxia on phosphorylation of p38. Our results demonstrate that hypoxia activates p38 MAP kinase and that this in turn plays a significant role in mediating the sustained phase of hypoxic contraction.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and materials. Adult male Sprague-Dawley rats (6-8 wk old, 250-275 g) were obtained from Harlan Sprague-Dawley Laboratories (Madison, WI). The following chemical reagents were used: L-phenylephrine, carbachol, (2R,3S,4S)-2-[(4-Methoxyphenyl)methyl]-3,4-pyrrolidinediol 3-acetate (anisomycin) (all from Sigma Chemicals, St. Louis, MO), potassium chloride (KCl; Fisher Scientific, Fairlawn, NJ), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD-98059), 1,4-diamino-2,3-dicyano-1,4bis(2-aminophenylthio)butadiene (UO-126), and 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]phenol (SB-202190) (all from Tocris Cookson, Ballwin, MO). All drug concentrations are expressed as the final molar concentration in the organ bath. PD-98059, UO-126, SB-202190, and anisomycin were dissolved in DMSO. All other drugs were dissolved in deionized water.

Isolated artery preparation. We anesthetized the rats with pentothal (100 mg/kg ip) and exsanguinated them by cutting the abdominal aorta. Heart and lungs were removed en bloc and placed in oxygenated Earle's balanced salt solution (EBSS) containing (in mM) 116.3 NaCl, 5.4 KCl, 0.83 MgSO4, 19.0 NaHCO3, 1.04 NaH2PO4, 1.8 CaCl2 · 2H2O, 5.5 D-glucose, and 0.03 phenol red as a pH indicator. The main intralobar pulmonary artery from the left lung and the middle lobe of the right lung (inner diameter ~1.5-2.0 mm) were isolated, cut into rings (2-3 mm long), and mounted in 10-ml organ chambers filled with EBSS and bubbled with 95% O2 and 5% CO2. Force was measured in grams using isometric force transducers (Grass FT03) and recorded on a Grass polygraph (Model 790).

Before starting the experimental protocols, we stretched the artery rings to a predetermined optimal passive load of 1 g and allowed them to stabilize for 60 min at this resting force. Then we checked the viability of the smooth muscle and endothelium by obtaining a contractile response to phenylephrine (10-6 M) and a subsequent relaxation response to carbachol (10-6 M).

Response to acute hypoxia. Pulmonary arterial rings with intact endothelium were precontracted with a submaximal concentration of phenylephrine (10-8-10-7 M). When the contractile response to phenylephrine reached a plateau, we rendered the rings hypoxic by bubbling the bath with an anoxic gas mixture (95% N2, 5% CO2) for 50 min. In a previous study, we determined that this procedure reduces PO2 in the bath to 10 torr (25). After hypoxia, we reintroduced oxygen by bubbling organ chambers with a 95% O2-5% CO2 gas mixture. In some experiments, pulmonary artery rings were precontracted with KCl (15-20 mM) instead of phenylephrine. The contractile responses to hypoxia were measured as the difference between the baseline to peak responses during hypoxic exposure for both the rapidly developing transient contraction and the slowly developing sustained contraction. The baseline was the steady-state contraction induced by phenylephrine or KCl before the exposure to hypoxia.

Effects of MAP kinase inhibitors on hypoxia-induced contraction. To determine the role of MAP kinases in the response to hypoxia, we incubated parallel rings for 20 min with two structurally different inhibitors of MEKK, PD-98059 (10 µM) or UO-126 (10 µM), or with a selective inhibitor of p38 MAP kinase, SB-202190 (10 µM). The pulmonary artery rings treated with MAP kinase inhibitors were subsequently precontracted with phenylephrine or KCl and subjected to hypoxia in the continued presence of the drugs. The concentrations of MAP kinase inhibitors used in this study were previously shown to inhibit ~90% of enzyme activity (6). The functional response to hypoxia in drug-treated rings was compared with that of parallel vehicle (0.1% vol/vol DMSO)-treated rings. To further explore the contribution of p38 MAP kinase, we examined the effects of anisomycin (1 µM), a stimulator of p38 MAP kinase (1, 8), on the response to acute hypoxia. In some experiments at the end of the hypoxia protocol, pulmonary artery rings were flash-frozen in liquid nitrogen and stored at -80°C for later analysis.

Effects of MAP kinase inhibitors on phenylephrine- and KCl-induced contraction. To determine whether effects of MAP kinase inhibitors are specific to hypoxia or generalized to other contractile stimuli, we studied the effects of MAP kinase inhibitors on concentration-response curves to phenylephrine (10-10-10-5 M) and KCl (10-80 mM). We treated rings with vehicle or 10 µM PD-98059, UO-126, or SB-202190 added to the bath 20 min before obtaining concentration-response curves. The effects of anisomycin (1 µM) were also assessed on phenylephrine-induced contractions.

Effects of hypoxia on p38 MAP kinase activity. MAP kinases become phosphorylated upon activation. To determine whether hypoxia activates p38 MAP kinase, we performed Western analysis using a phospho-specific antibody that recognizes phosphorylated p38 (Cell Signaling, Beverly, MA), according to the manufacturer's instructions. To ensure that the changes in phosphorylated p38 immunolabeling reflected changes in protein phosphorylation rather than a greater total amount of p38 protein, we stripped these blots and reprobed with antibodies that recognize total p38 protein (Cell Signaling).

In brief, pulmonary arteries were homogenized in 5 volumes of radioimmunoprecipitation assay lysis buffer containing 1× protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN). The homogenate was centrifuged at 10,000 g for 30 min. The supernatant was collected and assayed for protein using the BCA kit (Pierce, Rockford, IL). Equal amounts of protein (15 µg) were loaded in each well of 4-20% Tris-glycine gels (BioWhittaker Molecular Applications, Rockland, ME), electrophoretically separated, and blotted onto a nitrocellulose membrane. The membrane was then washed, blocked with 5% milk, and probed with antibodies overnight at 4°C. The immunoreactive bands were visualized using a secondary antibody conjugated to horseradish peroxidase and a chemiluminescent substrate (Cell Signaling). The intensity of the bands was quantified with NIH Image analysis software and expressed as arbitrary densitometric units.

Data analysis. The contractile responses to hypoxia, phenylephrine, and KCl are expressed as force in grams. The potency was calculated as the negative logarithm of the concentration causing a 50% response. Results were analyzed by Student's t-test or analysis of variance followed by the Bonferroni test where appropriate. Differences were considered significant when P < 0.05. Values are means ± SE, and n refers to the number of animals used.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Contractile responses to acute hypoxia in phenylephrine- and KCl-precontracted pulmonary arteries. Under normoxic conditions, phenylephrine (10-8-10-7 M) induced a stable contractile force of 0.15 ± 0.05 g. Superimposed hypoxia elicited a rapidly developing transient contraction (0.11 ± 0.02 g, n = 11) followed by relaxation and then a slowly developing sustained contraction (0.05 ± 0.04 g, n = 11) over a period of 50 min. Reintroducing oxygen reversed the hypoxia-induced contraction (Fig. 1A). Hypoxia elicited a similar contractile response in KCl-contracted rings, with a magnitude of hypoxic contraction that was not significantly different from that observed in phenylephrine-contracted rings (forces developed during the transient and sustained contractions were 0.08 ± 0.02 and 0.09 ± 0.04 g, respectively, Fig. 1B).


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Fig. 1.   Hypoxia-induced constrictor responses in rat pulmonary arteries contracted with submaximal concentrations of phenylephrine (10-8-10-7 M, n = 7, A) and potassium chloride (KCl, 15-20 mM, n = 7, B). Arrows indicate the beginning and end of hypoxia exposures. With both, a transient contraction occurs during the first 5 min after inducing hypoxia, followed by relaxation and then sustained contraction. Values are means ± SE.

Effects of p38 MAP kinase inhibitor/activator on hypoxia-induced vasoconstriction. To determine the role of p38 MAP kinase in hypoxic pulmonary vasoconstriction, we treated pulmonary artery rings with the p38 MAP kinase inhibitor SB-202190 (10 µM) or the stimulator anisomycin (1 µM) for 20 min before the hypoxic response. SB-202190 attenuated the phenylephrine-induced contraction, so higher concentrations of phenylephrine (10-6 M) were used to match the precontractile tone extant before hypoxic exposure. SB-202190 had no effect on the initial transient hypoxic contraction but completely abolished the slowly developing sustained contraction after both phenylephrine (Fig. 2A) and KCl (Fig. 2B) precontraction. DMSO alone had no significant effect on the response to hypoxia (data not shown).


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Fig. 2.   Effects of the p38 MAP kinase inhibitor SB-02190 (10 µM, n = 8) on the transient and sustained phases of hypoxia-induced contraction in rat pulmonary arteries contracted with submaximal concentrations of phenylephrine (10-8-10-6 M, A) and KCl (15-20 mM, B). Insets: response to hypoxia in absence and presence of SB-202190. Values are means ± SE. *P < 0.05 and **P < 0.01 compared with controls.

To determine whether the inhibition of the sustained phase of hypoxic contraction was caused by irreversible injury to the contractile apparatus, we washed the tissue free of SB-202190 and re-exposed the vessel to hypoxia in the absence of drug. The sustained phase of the hypoxic contraction was restored to that seen in vehicle-treated controls (data not shown). In contrast to the attenuation of the hypoxic contractile response in response to the p38 MAP kinase inhibitor, treatment with anisomycin, the stimulator, significantly enhanced both the transient and sustained phases of hypoxic contraction in phenylephrine-contracted rings (Fig. 3).


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Fig. 3.   Effects of the p38 MAP kinase stimulator anisomycin (1 µM, n = 4) on the transient and sustained phases of hypoxia-induced contraction in rat pulmonary arteries contracted with submaximal concentration of phenylephrine (10-8 M). Values are means ± SE. **P < 0.01 and ***P < 0.001 compared with controls.

Effects of hypoxia on p38 MAP kinase activity. Because inhibitors attenuated and stimulators enhanced the sustained phase of hypoxic contraction, the effect of hypoxia on p38 MAP kinase activity in pulmonary arteries was determined. Phosphorylated p38 was detectable in phenylephrine-contracted pulmonary artery rings during normoxia. Hypoxia (50 min) induced a threefold increase in the phosphorylated form of p38 MAP kinase compared with normoxia (Fig. 4, A and C), but there was no significant change in total p38 MAP kinase protein (Fig. 4B).


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Fig. 4.   Effects of hypoxia on phosphorylation of p38 MAP kinase. Western analysis for phosphorylated (A) and total p38 MAP kinase (B) in phenylephrine-precontracted pulmonary arteries exposed to normoxia or hypoxia (50 min). C: hypoxia-induced phosphorylation was calculated by densitometric analyses and expressed relative to total p38 protein. Values are means ± SE. *P < 0.05 compared with normoxic control. - and + represent protein extracts from untreated and anisomycin-treated C6 cells, respectively. The lane at far left represents molecular weight markers.

Effects of ERK pathway inhibitors on hypoxia-induced vasoconstriction. To determine the role of the ERK pathway on hypoxic contractile responses, we treated pulmonary artery rings with the MEKK inhibitors PD-98059 (10 µM) or UO-126 (10 µM) for 20 min before hypoxic exposure. Neither inhibitor affected the transient or sustained phases of hypoxic contraction in pulmonary artery rings precontracted with phenylephrine (Figs. 5A and 6A) or KCl (Figs. 5B and 6B).


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Fig. 5.   Effects of the MEKK inhibitor PD-98059 (10 µM, n = 7-8) on the transient and sustained phases of hypoxia-induced contraction in rat pulmonary arteries contracted with submaximal concentrations of phenylephrine (10-8-10-7 M, A) and KCl (15-20 mM, B). Values are means ± SE.



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Fig. 6.   Effects of the MEKK inhibitor UO-126 (10 µM, n = 6-8) on transient and sustained phases of hypoxia-induced constrictor responses in rat pulmonary arteries contracted with submaximal concentrations of phenylephrine (10-8-10-7 M, A) and KCl (15-20 mM, B). Values are means ± SE.

Effects of MAP kinase inhibitors on phenylephrine- or KCl-induced contraction. As we have previously observed (25), phenylephrine induced a concentration-dependent contraction in pulmonary artery rings with a potency of 7.63 ± 0.14 (n = 7). The maximum contraction of 0.27 ± 0.04 g (n = 7) occurred at a concentration of 3 × 10-6 M phenylephrine. Pretreatment with SB-202190 caused a significant inhibition of the contractile response to phenylephrine as shown by a decrease in potency (6.75 ± 0.09, n = 6, P < 0.01) and maximum contraction (0.14 ± 0.04, n = 6, P < 0.05, Fig. 7A). Pretreatment with PD-98059 significantly inhibited contractions induced by phenylephrine (3 × 10-8-3 × 10-7 M) (Fig. 7B) and decreased its potency (6.83 ± 0.10, n = 7, P < 0.01). However, the maximum contraction was not affected by pretreatment with PD-98059 (Fig. 7B). Pretreatment with UO-126 had no effect on phenylephrine-induced contraction (Fig. 7C). On the other hand, pretreatment with anisomycin significantly enhanced contractile responses to phenylephrine (Fig. 8). In contrast to phenylephrine, contractile responses to KCl were not significantly affected by pretreatment with SB-202190 (Fig. 9A), PD-98059 (Fig. 9B), or UO-126 (Fig. 9C).


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Fig. 7.   Effects of the MAP kinase inhibitors SB-202190 (10 µM, n = 6, A), PD-98059 (10 µM, n = 7, B), and UO-126 (10 µM, n = 8, C) on concentration-response curves to phenylephrine in rat pulmonary arteries. Values are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle-treated controls.



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Fig. 8.   Effects of the p38 MAP kinase stimulator anisomycin (1 µM, n = 3) on concentration-response curves to phenylephrine in rat pulmonary arteries. Values are means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with vehicle-treated controls.



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Fig. 9.   Effects of the MAP kinase inhibitors SB-202190 (10 µM, n = 7, A), PD-98059 (10 µM, n = 7, B) and UO-126 (10 µM, n = 6, C) on concentration-response curves to KCl in rat pulmonary arteries. Values are means ± SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our findings demonstrate that hypoxia activates the p38 MAP kinase pathway and that this pathway mediates the sustained phase of hypoxic contraction in isolated rat pulmonary arteries. Our observation that hypoxia stimulates the phosphorylation of p38 MAP kinase in isolated pulmonary artery rings is in accordance with previous studies showing similar effects of hypoxia in PC12 cells (4), myocardium (19, 27), bovine pulmonary artery fibroblasts (26), and pulmonary arteries isolated from chronically hypoxic rats (13). Most importantly, our observation that SB-202190, a relatively specific p38 MAP kinase inhibitor, completely abrogates the sustained phase of hypoxic contraction provides direct evidence for a role of the p38 MAP kinase pathway in hypoxia-induced pulmonary vasoconstriction. Further supporting this conclusion is the evidence that anisomycin, a p38 MAP kinase stimulator, significantly enhances the contractile response to acute hypoxia. Collectively, these findings support the hypothesis that the contractile response to hypoxia involves activation of p38 MAP kinase.

The p38 MAP kinase pathway has been shown to regulate actin assembly (10) and the initial rate and maximum force of contraction of isolated canine pulmonary artery smooth muscle (37). Consistent with the idea that p38 is involved in arterial smooth muscle contraction, our data show that inhibition of p38 MAP kinase with SB-202190 blunts phenylephrine-induced contraction of rat pulmonary arteries. This observation led us to speculate that stimulating p38 MAP kinase would enhance the contractile response to phenylephrine. Indeed, we found that the p38 MAP kinase stimulator anisomycin significantly increased the contractile response to phenylephrine. However, the role of p38 MAP kinase in pulmonary artery contraction appears to be agonist as well as species dependent. For instance, inhibiting p38 MAP kinase had no effect on norepinephrine (11) or 8-iso-PGE2-induced contraction (12) in canine pulmonary artery rings.

Inhibition of hypoxic contraction by SB-202190 is not attributable to attenuation of phenylephrine-induced contraction because SB-202190 also abolishes the sustained phase of hypoxic contraction in KCl-precontracted rings without attenuating the KCl-induced contraction. Furthermore, the effects of SB-202190 are reversible, because the sustained phase of hypoxic contraction was restored after rinsing of drug from the organ bath. Moreover, the stimulator of p38 MAP kinase anisomycin enhances the response to acute hypoxia. Together, these data support the idea that p38 MAP kinase is centrally involved in the signaling mechanisms of hypoxia- and phenylephrine-induced pulmonary vasoconstriction.

Analysis of the effects of SB-202190 and anisomycin on phenylephrine- and KCl-induced contractions provides insight into the mechanisms by which p38 MAP kinase mediates pulmonary vasoconstriction. Phenylephrine induces contraction of arterial smooth muscle not only by raising intracellular Ca2+ but also by activating certain signal transduction pathways, such as PKC and possibly Rho kinase (36), that enhance the sensitivity of the contractile apparatus to intracellular Ca2+. KCl induces contraction primarily by depolarizing the cell and subsequent Ca2+ influx through voltage-gated Ca2+ channels but does not involve sensitization of myofilaments to intracellular Ca2+. Because phenylephrine-induced but not KCl-induced contraction was attenuated by SB-202190 and augmented by anisomycin, p38 MAP kinase is likely to be involved in the sensitization of myofilaments to intracellular Ca2+. This explanation is also supported by a previous study on a permeabilized pulmonary artery preparation showing that p38 MAP kinase inhibition blocks the Ca2+-sensitizing effect of endothelin-1 (37). These observations also provide an explanation for why SB-202190 blocks the sustained, but not the transient, phase of hypoxic contraction. The transient contraction results primarily from depolarization of the smooth muscle cell membranes and Ca2+ influx through voltage-gated Ca2+ channels, whereas the sustained phase of hypoxic contraction has been associated with increased sensitization of myofilaments to intracellular Ca2+ (23).

Although the molecular mechanisms by which hypoxia activates the p38 MAP kinase pathway are not entirely known, the fact that the sustained phase of hypoxic contraction is endothelium dependent (16, 18, 25) in pulmonary arteries suggests that an endothelium-derived factor released in response to hypoxia might be involved in stimulating the p38 MAP kinase pathway. Reactive oxygen species have also been shown to activate the p38 MAP kinase pathway in rat aortic rings (21). Because hypoxia is known to generate reactive oxygen species by activating NADPH oxidase in isolated rat and rabbit lungs and in isolated rat pulmonary arteries (9, 15, 20, 40), it is conceivable that reactive oxygen species activate the p38 pathway. Hypoxia also activates tyrosine kinase (31) in isolated pulmonary arteries, offering additional pathways by which hypoxia might activate MAP kinases. Alternatively, hypoxia may also involve Rho kinase activation. Hypoxia activates Rho kinase in cultured pulmonary artery smooth muscle cells (33), and Rho kinase activation mediates the sustained phase of hypoxia-induced pulmonary vasoconstriction (24, 33). Further studies are required to show whether the p38 MAP kinase pathway is parallel to the Rho kinase pathway or whether they act in concert with each other. Another potential pathway may involve calcium-dependent isoforms of PKC that are upstream activators of p38 MAP kinase in mesangial cells (28). Although PKC mediates hypoxic pulmonary vasoconstriction in pulmonary arteries and isolated perfused rabbit lungs (35), it is less likely to be involved in isolated rat pulmonary arteries (23).

Previous studies in nonvascular smooth muscle preparations indicate that the ERK1/ERK2 MAP kinase pathway controls PGF2alpha -induced contraction in iris sphincter smooth muscle (39) and histamine-induced contraction in tracheal smooth muscle (17). Moreover, hypoxia is known to activate ERK1/ERK2 pathways in bovine pulmonary artery fibroblasts (5, 26) and human microvascular endothelial cells (22). In our experiments with pulmonary artery rings, however, the MEKK inhibitors PD-98059 and UO-126 failed to block either the transient or sustained phases of hypoxic contraction.

Although PD-98059 and UO-126 are both MEKK inhibitors, PD-98059 attenuated the contractile response to lower concentrations of phenylephrine, whereas UO-126 had no effect. The mechanisms underlying these differential effects of the inhibitors are not known. PD-98059 inhibits cyclooxygenase and subsequent thromboxane production in platelets (2), offering a possible mechanism by which PD-98059 could inhibit phenylephrine-induced contraction. However, our preliminary studies showed that the cyclooxygenase inhibitor meclofenamate has no effect on phenylephrine-induced contraction (unpublished observations), making it unlikely that the cyclooxygenase inhibition explains the inhibitory effects of PD-98059 on phenylephrine-induced contraction. Moreover, the maximal contractile force generated by higher concentrations of phenylephrine was not modified by PD-98059. These observations are consistent with previous studies showing that PD-98059 does not modify phenylephrine-mediated contractions in canine pulmonary arteries (37) or histamine- or phorbol ester-mediated contraction in swine carotid arteries (7). Thus, although we cannot entirely exclude a role for other MAP kinase pathways in the mediation of hypoxia- and phenylephrine-induced contraction in isolated pulmonary arteries, our data indicate that the ERK pathway is less important than the p38 pathway.

In conclusion, our data suggest an important role for p38 MAP kinase in mediating hypoxia-induced contraction of the rat pulmonary artery. The MAP kinases have also been implicated in the pathogenesis of chronic hypoxia-induced pulmonary hypertension (13). Therefore, it is tempting to speculate that MAP kinase inhibitors might have a therapeutic role in the treatment of pulmonary hypertension. However, further studies are needed to elucidate the in vivo effects of p38 MAP kinase inhibitors on the development of chronic hypoxia-induced pulmonary hypertension.


    ACKNOWLEDGEMENTS

We thank Drs. R. M. Wadsworth and K. Harnett for critical reading of the manuscript. We also thank M. Benson and V. Hovanesian for technical support.


    FOOTNOTES

This study was supported by a beginner's grant-in-aid from the American Heart Association, New England Affiliate (M. R. Karamsetty), and National Heart, Lung, and Blood Institute Grant HL-40505 (N. S. Hill).

Address for reprint requests and other correspondence: M. R. Karamsetty, Pulmonary and Critical Care Div., 750 Washington St., New England Medical Center #257, Boston, MA 02111 (E-mail: mkaramsetty{at}lifespan.org).

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.

June 5, 2002;10.1152/ajplung.00475.2001

Received 13 December 2001; accepted in final form 30 May 2002.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 283(4):L859-L866