Modulation of PGF2{alpha}- and hypoxia-induced contraction of rat intrapulmonary artery by p38 MAPK inhibition: a nitric oxide-dependent mechanism

Greg A. Knock,1 Anushika S. De Silva,1 Vladimir A. Snetkov,1 Richard Siow,2 Gavin D. Thomas,1 Mitsuya Shiraishi,3 Michael P. Walsh,3 Jeremy P. T. Ward,1 and Philip I. Aaronson1

1Department of Asthma, Allergy and Respiratory Science and 2Cardiovascular Division, School of Medicine, King’s College London, London SE1 9RT, United Kingdom; and King’s College London, 3Smooth Muscle Research Group and Department of Biochemistry & Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada

Submitted 1 March 2005 ; accepted in final form 26 July 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The mechanisms through which p38 mitogen-activated protein kinase (p38 MAPK) is involved in smooth muscle contraction remain largely unresolved. We examined the role of p38 MAPK in prostaglandin F2{alpha} (PGF2{alpha})-induced vasoconstriction and in hypoxic pulmonary vasoconstriction (HPV) of rat small intrapulmonary arteries (IPA). The p38 MAPK inhibitors SB-203580 and SB-202190 strongly inhibited PGF2{alpha}-induced vasoconstriction, with IC50s of 1.6 and 1.2 µM, whereas the inactive analog SB-202474 was ~30-fold less potent. Both transient and sustained phases of HPV were suppressed by SB-203580, but not by SB-202474 (both 2 µM). Western blot analysis revealed that PGF2{alpha} (20 µM) increased phosphorylation of p38 MAPK and of heat shock protein 27 (HSP27), and this was abolished by SB-203580 but not by SB-202474 (both 2 µM). Endothelial denudation or blockade of endothelial nitric oxide (NO) synthase with N{omega}-nitro-L-arginine methyl ester (L-NAME) significantly suppressed the relaxation of PGF2{alpha}-constricted IPA by SB-203580, but not by SB-202474. Similarly, the inhibition of HPV by SB-203580 was prevented by prior treatment with L-NAME. SB-203580 (2 µM), but not SB-202474, enhanced relaxation-induced by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in endothelium-denuded IPA constricted with PGF2{alpha}. In {alpha}-toxin-permeabilized IPA, SB-203580-induced relaxation occurred in the presence but not the absence of the NO donor sodium nitroprusside (SNP); SB-202474 was without effect even in the presence of SNP. In intact IPA, neither PGF2{alpha}- nor SNAP-mediated changes in cytosolic free Ca2+ were affected by SB-203580. We conclude that p38 MAPK contributes to PGF2{alpha}- and hypoxia-induced constriction of rat IPA primarily by antagonizing the underlying Ca2+-desensitizing actions of NO.

p38 mitogen-activated protein kinase; pulmonary artery; prostaglandin F2{alpha}; intracellular calcium; calcium sensitization; heat shock protein 27


P38 MITOGEN-ACTIVATED PROTEIN KINASE (p38 MAPK) is a serine/threonine kinase that can be activated by cellular stress (17) and certain G protein-coupled receptors (4, 27, 38). p38 MAPK phosphorylation has been shown to be increased by contractile stimuli such as endothelin (ET)-1, angiotensin II, norepinephrine, and the thromboxane A2 analog U-46619, as well as pressure, in several smooth muscle preparations (4, 2628) including canine pulmonary artery (PA) (38). In each of these studies, SB-203580 [4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4'-pyridyl)-1H-imidazole], a selective blocker of the {alpha}- and {beta}-isoforms of p38 MAPK (9), markedly inhibited or abolished both agonist-induced p38 MAPK phosphorylation and tension development.

p38 MAPK has been proposed to regulate contraction via interaction with actin-based systems, rather than through elevation of cytosolic free Ca2+ ([Ca2+]i) or enhanced myosin phosphorylation at a given level of [Ca2+]i (myosin phosphatase-dependent Ca2+ sensitization) (33, 36). Acting through MAPK-activated protein kinase-2 (MAPKAPK2), p38 MAPK activation results in phosphorylation of heat shock protein 27 (HSP27) (2, 3, 1214, 22, 38), which is thought to contribute to the sustained constriction of smooth muscle by promoting actin polymerization. Recent evidence suggests that HSP27 may also regulate cross-bridge cycling and actin membrane attachment via interaction with proteins such as tropomyosin (2, 12). Interestingly, the action of protein kinase G (PKG) and protein kinase A, the key effector kinases of the endothelium-derived vasodilators NO and prostacyclin, also involves an interaction with thin filament regulatory proteins via phosphorylation of heat shock protein 20 (HSP20) (11, 31).

Using a relatively high single concentration of the inhibitor 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4'-pyridyl)1H-imidazole (SB-202190), structurally similar to SB-203580, Karamsetty et al. (18) presented evidence that p38 MAPK plays a major role in the sustained phase of the hypoxic constrictor response in main PA. It is well known that for hypoxic pulmonary vasoconstriction (HPV) to occur in isolated PA the presence of a subthreshold concentration of a contractile agonist is required. For this purpose, Karamsetty et al. (18) used phenylephrine, which causes a strong sustained contraction in main PA. They also found that SB-202190 suppressed this phenylephrine-induced contraction. Furthermore, although they suggested that hypoxia itself directly led to activation of p38 MAPK, the influence of other factors such as activity of endothelium-derived vasodilators, as well as potential nonselective actions of the inhibitor, were not examined.

We have previously shown that the sustained phase of HPV is endothelium dependent and largely dependent on activity of Rho-activated kinase (ROCK) (32), but there is some evidence to suggest a degree of interaction between the ROCK and p38 MAPK pathways (29). In light of the observations of Karamsetty et al. (18) in main PA, we examined whether p38 MAPK was also involved in modulating the contraction to hypoxia in rat small intrapulmonary arteries (IPA), which are believed to be of the greatest functional importance to pulmonary vascular resistance (37). Because IPA are relatively insensitive to phenylephrine, we used PGF2{alpha} to generate pretone and, therefore, also studied whether p38 MAPK played any role in contractions induced by this agonist.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissue preparation and measurement of isometric tension. Male Wistar rats (~250 g) were killed by cervical dislocation; the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The lungs were excised and placed in physiological salt solution (PSS) containing (in mM): 118 NaCl, 24 NaHCO3, 1 MgSO4, 0.435 NaH2PO4, 5.56 glucose, 1.8 CaCl2, and 4 KCl. Small IPA (internal diameter ~200–500 µm) were dissected free of surrounding adventitia and mounted in a wire myograph (Danish Myo Technology, Aarhus, Denmark), bathed in PSS at 37°C, and gassed with 95% air-5% CO2 (pH 7.35) as previously described (23, 37). Preparation viability was assessed from the response to 80 mM K+ PSS (KPSS, isotonic replacement of NaCl by KCl). Before experimentation, arteries were equilibrated with three 2-min exposures to KPSS. Where stated, we achieved endothelial denudation by rubbing the artery lumen with a human hair. Absence of endothelial function was confirmed by loss of relaxation to acetylcholine. Unless otherwise stated, IPA were precontracted with a submaximal concentration of PGF2{alpha} (20 µM, ~EC75). Time control experiments showed these constrictions were stable for >70 min; stability was not affected by any treatment used (data not shown). Once tension had reached a plateau (~10 min), we assessed the effects of MAPK inhibitors by cumulative-concentration response studies, allowing the response to stabilize between successive applications of agent.

Estimation of [Ca2+]i. IPA were loaded with the Ca2+-sensitive fluorophore fura PE3, via incubation of the vessels with the acetoxymethyl ester fura PE3-AM (4 µM) for 2 h at room temperature. Similar loading protocols have previously shown that subsequent measurements are of smooth muscle [Ca2+]i, with negligible contribution to the fluorescence signal from the endothelium (8, 20). IPA were continually gassed with 95% air-5% CO2 during this procedure. After loading, the arteries were washed with PSS, the temperature increased to 37°C, and the myograph was transferred to an inverted fluorescence microscope (Nikon Diaphot, Nikon UK). We assessed changes in [Ca2+]i by calculating the ratio of the light emitted through a >500-nm emission filter when the vessel was illuminated at 340 and 380 nm, respectively (F340/380) (Cairn spectrophotometer; Cairn Research, Newnham, Kent, UK).

Hypoxic protocol. As we have previously shown, a small degree of agonist-induced tone is required to facilitate the hypoxic response in rat IPA and mesenteric artery (23). The vessels were therefore exposed to sufficient PGF2{alpha} (3–6 µM) to induce a constriction of ~15% of that to KPSS for 20 min before and during the hypoxic challenge. We induced hypoxia by gassing with 1% O2-95% N2-5% CO2 for 45 min, after which time the vessels were reoxygenated and washed with PSS. In some experiments, oxygen tension in the myograph chamber was continuously monitored via a dissolved oxygen meter (Diamond General oxygen electrode, Ann Arbor, MI; Strathkelvin oxygen meter, Glasgow, UK). During the hypoxic challenge the chamber PO2 was typically 18–20 mmHg, compared with the control PO2 of 135–145 mmHg.

{alpha}-Toxin permeabilization of IPA. Isometric tension was recorded in {alpha}-toxin-permeabilized arteries, as described previously (8, 20). Briefly, IPA were mounted on a myograph as above but bathed in PIPES-buffered solution (pH 7.1), gassed with 100% air rather than 5% CO2, and incubated at 26°C rather than 37°C. To prevent involvement of intracellular Ca2+ stores, 10 µM cyclopiazonic acid was present throughout. We regulated pCa by adjusting the ratio of K2EGTA to CaEGTA. Permeabilization was performed with 60 µg/ml {alpha}-toxin in pCa 6.5. After reequilibration with Ca2+-free relaxing solution, a submaximal constriction was elicited by raising pCa to 7.0–6.9. Once this contraction reached a plateau, PGF2{alpha}, GTP (1 µM), and/or sodium nitroprusside (SNP) were added. Higher concentrations of agonists are commonly required in permeabilized compared with intact preparations, and in this case we used 100 µM PGF2{alpha}.

Western blotting. Isolated IPA were incubated with 20 µM PGF2{alpha} for 5 min (controls) or preincubated for 10 min with 2 µM SB-203580 or SB-202474 and then treated with PGF2{alpha} for a further 5 min. We then snap froze them by immersing them in liquid nitrogen and homogenizing them in SDS sample buffer containing protease and phosphatase inhibitors (Sigma) using a Wheaton microtissue grinder. Samples were centrifuged at 6,000 rpm for 1 min and loaded onto 4–12% NUPAGE Bis-Tris gels, electrophoretically separated (200 V for 1 h), and transferred to nitrocellulose membranes in 25 mM Tris, 192 mM glycine, and 20% methanol using a mini Trans-blot unit (100 V for 1.5 h at 4°C). The membranes were washed in Tris-buffered saline (TBS; 20 mM Tris·HCl, pH 7.5, and 500 mM NaCl) and blocked with 5% skim milk in TBS for 1 h at room temperature. The membranes were then probed with phospho-p38 MAPK or phospho-HSP27 antibodies (Cell Signaling) at 1:1,000 dilution in 1% milk in TBS containing 0.1% Tween (TBS-Tween) overnight at 4°C. The membranes were washed 6 x 10 min in TBS-Tween and probed with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000 dilution) for 1 h at room temperature and then exposed to West Femto chemiluminescent substrate (Pierce Biotechnology). Band intensity was quantified using ImageJ software. To analyze total protein, the membranes were stripped for 1 h at room temperature and reprobed with pan-p38 MAPK and pan-HSP27 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). The bands were visualized using West Pico chemiluminescent substrate (Pierce Biotechnology) as described above.

cGMP assay. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical veins and grown to confluence in M-199 containing 20% fetal calf serum. Once confluent, cells were equilibrated for 10 min in HEPES-buffered saline, pH 7.4, containing 100 µM L-arginine [to provide substrate for endothelial nitric oxide synthase (eNOS)] and 500 µM 3-isobutyl-1-methylxanthine (to inhibit all phosphodiesterase activity), were preincubated with 2 µM SB-203580 or left free of inhibitor (15 min), and then were treated with 20 µM PGF2{alpha} for a further 5 min at 37°C. ATP (100 µM) was used as a positive control. Cells were then immediately lysed with ice-cold 0.5 M HCl, and the lysate was collected for assessment of cGMP content. This provided a measurement of both NO production and guanylyl cyclase activity (34), using an ACE competitive enzyme immunoassay kit (Cayman Chemical) (30).

Data analysis. Changes in [Ca2+]i are represented in terms of the change in the fura PE3 fluorescence ratio at 340/380 nm. Mean changes are expressed as a percentage of the maximum ratio change seen during the final 80 mM KPSS challenge during the run-up procedure (R340/380). Although not linearly related to [Ca2+]i, this provides a reliable qualitative index of changes in [Ca2+]i. Values for IC50 and maximum response were derived by nonlinear curve fitting of concentration-response data (SigmaPlot; SPSS, Chicago, IL). Results are expressed as means ± SE, and means were compared by ANOVA for repeated measures or paired or unpaired Student’s t-test as appropriate (SigmaStat, Jandel). A difference was deemed significant if P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Relaxation of PGF2{alpha}-contracted IPA by MAPK inhibitors. To study the role of p38 MAPK in PGF2{alpha}- and hypoxia-induced contractions, we first established the range of concentrations over which SB-203580 is likely to be acting specifically via inhibition of p38 MAPK activity by constructing relaxation concentration-response curves to SB-203580 and SB-202190, as well as to 4-ethyl-2-(p-methoxyphenyl)-5-(4'-pyridyl)-1H-imidazole (SB-202474, a structural analog of SB-203580 that is inactive against p38 MAPK) (24). We also examined the effect of the MAPKK (MEK) blocker PD-98059. PGF2{alpha} (20 µM, ~EC70)-induced constriction of intact IPA was inhibited by SB-203580 with an IC50 of 1.6 ± 0.3 µM and maximum effect of 80 ± 5% (n = 12), whereas SB-202474 was ~30-fold less potent (Fig. 1A). SB-203580 had only a minor inhibitory effect on KPSS-induced constriction (Fig. 1A), suggesting that relaxation of PGF2{alpha}-induced constriction was unlikely to be due to inhibition of voltage-gated Ca2+ channels. SB-202190 also relaxed PGF2{alpha}-contracted IPA, with an IC50 of 1.2 ± 0.2 µM (n = 8, Fig. 1B), whereas the MEK inhibitor PD-98059 had only a minor effect at relatively high concentrations (Fig. 1B). These results implicate a role for p38 MAPK activation, but not for p42/44 MAPK, in PGF2{alpha}-induced constriction of IPA.



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Fig. 1. Relaxant effects of MAPK inhibitors on PGF2{alpha}-induced constriction of intact intrapulmonary arteries (IPA). A: concentration-dependent relaxation of PGF2{alpha} (20 µM)-contracted intact IPA by SB-203580 ({bullet}, n = 12) and SB-202474 ({circ}, n = 10), and of 80 mM K+ PSS (KPSS)-contracted intact IPA by SB-203580 ({blacksquare}, n = 5). B: concentration-dependent relaxation of PGF2{alpha} (20 µM)-contracted intact IPA by SB-202190 ({blacktriangledown}, n = 8) and PD-98059 ({square}, n = 4). Symbols are means ± SE.

 
Inhibition of PGF2{alpha}-induced p38 MAPK and HSP27 phosphorylation by SB-203580. As shown in Fig. 2A, treatment of IPA with PGF2{alpha} (20 µM) caused a significant increase in phosphorylation of both p38 MAPK (Fig. 2B) and its downstream effector HSP27 (Fig. 2C). The data presented in Fig. 1A suggest that the selective action of SB-203580 against p38 MAPK reaches a maximum at <2 µM, if it can be assumed that its nonselective effects are similar to those of the inactive analog SB-202474. Consistent with this assumption, the PGF2{alpha}-induced increase in p38 MAPK phosphorylation was abolished by 2 µM SB-203580 but unaffected by SB-202474 (Fig. 2B). Similar results were obtained for HSP27 phosphorylation (Fig. 2C). SB-203580 did not significantly reduce the levels of phospho-p38 MAPK or phospho-HSP27 in unstimulated IPA. In light of these results, in most subsequent experiments where only a single concentration of inhibitor was used, we chose 2 µM.



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Fig. 2. Western blot analysis of phosphorylation of p38 MAPK and heat shock protein (HSP) 27 in intact IPA. A: representative blots of intact IPA [unstimulated, PGF2{alpha} (20 µM), SB-203580 (2 µM), or PGF2{alpha} + SB-203580] with anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-HSP27, or anti-HSP27. Total p38 MAPK and HSP27 levels were unaffected by the various treatments. B and C: cumulative data showing the quantification of phosphorylation of p38 MAPK and HSP27. *P < 0.01 vs. control, n = 16 (B); *P < 0.05 vs. control, n = 21 (C); {dagger}P < 0.05 vs. PGF2{alpha} treatment, n = 5 (B); {dagger}P < 0.05 vs. PGF2{alpha} treatment, n = 8 (C). SB-202474 had no significant effect on PGF2{alpha}-induced phosphorylation of p38 MAPK (n = 7) (B) or HSP27 (n = 8) (C).

 
Effect of p38 MAPK inhibition on HPV. As we have previously reported for rat small IPA (23, 32), application of hypoxia to IPA precontracted with PGF2{alpha} to a "pretone" level equivalent to 10–15% of an 80 mM KPSS contraction caused a transient vasoconstriction superimposed on a sustained vasoconstriction (Fig. 3A). In the presence of 2 µM SB-203580, wherein the concentration of PGF2{alpha} was raised to compensate for blockade of pretone by the p38 MAPK inhibitor, both phases of HPV were depressed (% block: first phase, 47.0 ± 11.7, P < 0.01; second phase, 41.2 ± 5.8, P < 0.01, n = 8) (Fig. 3A). Furthermore, the specificity of this action to p38 MAPK blockade was confirmed by the lack of effect of the inactive analog SB-202474 (2 µM) on either phase of HPV (Fig. 3B).



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Fig. 3. Effects of p38 MAPK inhibition on hypoxic pulmonary vasoconstriction (HPV). A: control, {bullet}; SB-203580 (2 µM), {circ}; SB-203580 caused significant inhibition of both the transient (P < 0.01, n = 8) and sustained phase of HPV (P < 0.01, ANOVA). B: in contrast, the inactive analog SB-202474 (2 µM) had no significant effect on either phase of HPV (control, {blacksquare}; SB-202474, {square}, n = 6). Symbols are means ± SE.

 
Although the results of the above experiments are consistent with those of Karamsetty et al. (18) in that they showed attenuation of HPV by SB-203580, they also revealed that PGF2{alpha}-induced contraction was also inhibited, and to a similar extent as HPV. To understand the mechanisms by which p38 MAPK inhibition suppresses contraction, we first explored in more depth the effects of SB-203580 on the PGF2{alpha}-induced contraction.

Effects of p38 MAPK inhibition on PGF2{alpha}-induced rises in [Ca2+]i. To determine whether SB-203580 was causing relaxation of PGF2{alpha}-induced vasoconstriction by suppressing the elevation of [Ca2+]i, we studied the effect of SB-203580 on [Ca2+]i measured simultaneously with tension in fura PE3-loaded IPA. SB-203580 fluoresces under UV illumination; to circumvent this, SB-203580 was applied before addition of PGF2{alpha} so that its effect on baseline fluorescence could stabilize first. Under these conditions, despite significantly inhibiting tension, SB-203580, even at 4 µM, did not alter the [Ca2+]i response to PGF2{alpha} (n = 5, Fig. 4). Neither tension nor [Ca2+]i responses to KPSS were altered by SB-203580 (n = 2, data not shown).



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Fig. 4. Effect of SB-203580 (4 µM) on [Ca2+]i and tension in response to PGF2{alpha} (20 µM) in fura-PE3-loaded IPA. Left: representative experiment showing changes in [Ca2+]i [expressed in terms of fluorescence ratio at 340/380 nm (R340/380), top trace] and isometric tension (bottom trace). The baseline was adjusted for SB-203580 fluorescence at the arrow. Mean data for 5 experiments is shown on the right; SB-203580 did not affect the PGF2{alpha}-induced change in R340/380 but nevertheless significantly inhibited PGF2{alpha}-induced tension in the same arteries (*P < 0.05).

 
Effects of p38 MAPK inhibition on PGF2{alpha}-induced Ca2+ sensitization. PGF2{alpha} induces contraction partly via enhancing the sensitivity of the smooth muscle to [Ca2+]i (Ca2+ sensitization) (4, 21, 27, 28, 35, 38). To evaluate whether SB-203580 caused relaxation of PGF2{alpha}-induced vasoconstriction of IPA by suppressing Ca2+ sensitization, we examined whether SB-203580 relaxed the contraction elicited by PGF2{alpha} (100 µM) in {alpha}-toxin-permeabilized IPA clamped at pCa ~6.9. SB-203580, even at 10 µM, was not able to cause significant relaxation of IPA under these conditions (n = 8, not significant; but see below and Fig. 9).



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Fig. 9. Effects of p38 MAPK inhibition on PGF2{alpha}-induced contraction in {alpha}-toxin-permeabilized IPA. AC: example traces of contractions to 100 µM PGF2{alpha} in pCa 6.9 and 1 µM GTP. Alone, 10 µM SB-203580 had little effect (A), but in the presence of 100 µM sodium nitroprusside (SNP) it caused a significant relaxation (B). 10 µM SB-202474 had little effect even in the presence of SNP (C). D: summarized data for 6–8 IPA; neither the response to SB-203580 alone nor SB-202474 in the presence of SNP was significantly different from zero, whereas that to SB-203580 in the presence of SNP was significantly different from both SB-203580 alone and SB-202474 in the presence of SNP (*P < 0.01, 1-way ANOVA).

 
Because preliminary experiments showed that endothelium-dependent relaxation is inoperative following {alpha}-toxin-permeabilization in this preparation, the above experiments do not rule out the possibility that inhibition of p38 MAPK might suppress contraction in intact arteries by enhancing the release or effect of endothelial vasodilators that could affect Ca2+ sensitization. To explore this possibility, we examined whether SB-203580-induced relaxation was affected by removal of the endothelium.

Effects of endothelium removal and inhibition of NOS. As shown in Fig. 5A, endothelial denudation markedly suppressed relaxation of PGF2{alpha}-evoked constrictions by SB-203580, with a large rightward shift in the SB-203580 concentration-response relationship (IC50: 14 ± 3 µM, n = 10, P < 0.001 compared with intact IPA). However, it had no effect on relaxation to the inactive analog SB-202474, and in the absence of the endothelium, the concentration-response relationships for SB-203580 and SB-202474 were the same (Fig. 5A).



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Fig. 5. Effect of endothelial denudation and endothelial nitric oxide synthase (eNOS)/cyclooxygenase inhibition on SB-203580-induced relaxation of IPA contracted with 20 µM PGF2{alpha}. A: SB-203580 on endothelium-intact ({bullet}, n = 12) and endothelium-denuded IPA ({circ}, n = 10); and SB-202474 on endothelium-intact ({blacktriangleup}, n = 10) and endothelium-denuded IPA ({triangleup}, n = 6). B: effects of preincubation with 1 mM L-NAME ({blacksquare}, n = 13) or 1 µM indomethacin ({square}, n = 11). The data for SB-203580 alone on intact IPA are reproduced from Fig. 1A for comparison. Symbols = means ± SE.

 
These results suggest that the relaxation to low concentrations of SB-203580, which are more likely to cause selective block of p38 MAPK activation, was endothelium dependent. In support of this conclusion, the relaxation to SB-203580 over a similar concentration range was also suppressed by the eNOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME, 1 mM), albeit to a smaller extent (IC50: 8.7 ± 1.7 µM, n = 13, P < 0.001 compared with control; Fig. 5B). Inhibition of cyclooxygenase with indomethacin (1 µM) also caused a small but significant increase in IC50 of the SB-203580-induced relaxation (IC50: 4.7 ± 1.0 µM, n = 11, P < 0.05 compared with control; Fig. 5B); indomethacin alone did not alter the response to PGF2{alpha}. To confirm the involvement of NO and the cGMP/PKG pathway in SB-203580-induced relaxation, we also examined the response to SB-203580 in the presence of the PKG inhibitor KT-5823 (1 µM). KT-5823 reduced 2 µM SB-203580-induced relaxation to a similar extent as L-NAME [control (SB-203580 alone): 44.8 ± 3.6% relaxation, n = 12; +L-NAME, 25.4 ± 2.5%, P < 0.01 vs. control, n = 13; +KT-5823, 25.9 ± 3.8%, P < 0.001 vs. control, n = 8].

Because these results suggested that the vasorelaxant effect of p38 MAPK inhibition was largely due to enhancement of the release or effect of endothelium-derived NO, we reexamined the suppression of HPV by SB-203580 following inhibition of eNOS with 1 mM L-NAME. L-NAME alone had no significant effect on HPV, as we have previously reported in this preparation [Ref. 23, and see discussion in Aaronson et al. (1)]. However, in the presence of L-NAME, SB-203580 was without effect on either phase of HPV (Fig. 6), strongly suggesting that its suppressive action on HPV, as on PGF2{alpha}-induced contraction, is mediated via NO.



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Fig. 6. Effect of SB-203580 (2 µM) on HPV following inhibition of eNOS. A: example trace of HPV in the presence of 1 mM N{omega}-nitro-L-arginine methyl ester (L-NAME); B: HPV in the same IPA, but following addition of 2 µM SB-203580. C: summarized data from 8 such experiments (L-NAME, {bullet}; L-NAME + SB-203580, {circ}). Symbols = means ± SE.

 
Effects of p38 MAPK inhibition on cGMP production in endothelial cells. To determine whether p38 MAPK inhibition might be altering NO release or guanylyl cyclase activity, we measured cGMP production as an indirect measure of NO production in HUVECs. As shown in Fig. 7, cell lysate cGMP concentration was significantly enhanced (approximately two- to threefold) by treatment with 20 µM PGF2{alpha}. However, preincubation with 2 µM SB-203580 before PGF2{alpha} application had no additional effect on cGMP concentration (Fig. 7).



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Fig. 7. Effect of PGF2{alpha} and SB-203580 on cGMP production in human umbilical vein endothelial cells (HUVECs). cGMP concentration in cell lysates obtained from HUVECs treated with 20 µM PGF2{alpha} in the absence or presence of 2 µM SB-203580; 100 µM ATP is shown for comparison as a positive control for stimulation of eNOS. SB-203580 had no significant effect on the increase in cGMP induced by PGF2{alpha}. *P < 0.01 compared with control (untreated); n = 8–10.

 
Effects of p38 MAPK inhibition on sensitivity of endothelium-denuded IPA to an NO donor. Because SB-203580 did not affect production of cGMP, at least in HUVECs, we next examined whether p38 MAPK inhibition might be potentiating the effects of NO on the vascular smooth muscle (VSM). To do this, we studied the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on PGF2{alpha}-induced contraction of endothelium-denuded IPA, in the absence and presence of SB-203580 or SB-202474. As shown in Fig. 8, 2 µM SB-203580 significantly enhanced the SNAP-induced relaxation, causing both a leftward shift in the IC50 [control (SNAP alone): 0.28 ± 0.11 µM, n = 8; +SB-203580, 0.12 ± 0.03 µM, n = 8, P < 0.05] and an enhancement of the apparent maximum relaxation (control: 56.0 ± 8.4%; +SB-203580, 82.8 ± 6.0%, P < 0.05). The same concentration of SB-202474 on the other hand did not significantly alter the response to SNAP (IC50, 0.58 ± 0.13 µM; maximum relaxation, 48.2 ± 10.6%, n = 8), indicating that these actions of SB-203580 were likely to be specific to p38 MAPK inhibition.



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Fig. 8. Effects of SB-203580 on S-nitroso-N-acetylpenicillamine (SNAP)-induced relaxation of endothelium-denuded IPA contracted with 20 µM PGF2{alpha}. Control, {circ} (n = 8); 2 µM SB-203580 ({bullet}, n = 8); 2 µM SB-202474, {blacksquare} (n = 8). Symbols = means ± SE.

 
We further investigated which of the actions of NO thought to be involved in relaxation of VSM were being influenced by p38 MAPK. We first examined the potential influence of p38 MAPK inhibition on SNAP-induced suppression of both contraction and [Ca2+]i responses to the stable thromboxane A2 analog U-46619 (100 nM) in endothelium-denuded IPA. U-46619 produces [Ca2+]i responses via the prostanoid TP receptor, which is also stimulated by PGF2{alpha}. We used U-46619 in preference to PGF2{alpha} because our recent experiments (Snetkov V, Knode G, Baxter L, Ward J, and Aaronson P, unpublished observations) have shown that the latter also causes a rise in [Ca2+] mediated via FP receptors, but which is not coupled to contraction. Thus by using U-46619 any effects of SB-203580 on [Ca2+]i would be more likely to influence contraction. Despite enhancing SNAP-induced relaxation [control (SNAP alone): 63.5 ± 5.7%, +SB-203580: 80.9 ± 3.8%, P < 0.05, n = 4], 2 µM SB-203580 did not potentiate the associated SNAP-mediated suppression of the rise in [Ca2+]i induced by U-46619 (control: 67.7 ± 9.2% R340/380, +SB-203580: 56.8 ± 8.2%, n = 4, not significant).

These results suggested that SB-203580 was not enhancing the response to NO by potentiating its ability to reduce [Ca2+]i. Alternatively, p38 MAPK inhibition might be enhancing NO-mediated Ca2+ desensitization. Because endothelial release of NO was not operative following {alpha}-toxin permeabilization, we studied this possible effect by applying SB-203580 (10 µM) to permeabilized IPA contracted with 100 µM PGF2{alpha} at pCa 6.9, in the presence of the NO donor SNP (100 µM) to generate an ambient level of NO sufficient to cause a partial relaxation (not shown). In contrast to its lack of effect when applied in the absence of SNP (Fig. 9, A and D), SB-203580 in the presence of SNP caused a significant relaxation (Fig. 9, B and D). Conversely, SB-202474 at this concentration had no effect (Fig. 9, C and D).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim of this study was to clarify the role of p38 MAPK in agonist- and hypoxia-induced contraction of rat small IPA. Our results suggest that the contribution of p38 MAPK activation to smooth muscle contraction elicited by either PGF2{alpha} or hypoxia is endothelium dependent and occurs primarily via suppression of the actions of endothelium-derived vasodilators, particularly NO. Inhibition of p38 MAPK would therefore relieve this suppression and result in vasorelaxation.

We have shown that both SB-203580 and SB-202190 inhibited PGF2{alpha}-induced constriction with IC50s close to 1 µM, whereas SB-202474 (a structural analog of SB-203580 and SB-202190 that is ineffective as a blocker of p38 MAPK) only caused significant relaxation at concentrations >10 µM. In addition, SB-203580, but not SB-202474 (both at 2 µM), abolished PGF2{alpha}-induced phosphorylation of both p38 MAPK and of its downstream effector protein HSP27. SB-203580 is the most extensively used p38 MAPK inhibitor and has been shown to inhibit constrictions induced by several agonists in several vascular beds (4, 21, 27, 28, 35, 38). Importantly, in each of these studies, the agonist(s) causing SB-203580-inhibitable constriction also caused SB-203580-sensitive phosphorylation of p38 MAPK and/or HSP27. These observations are consistent with the hypothesis that agonist-induced p38 MAPK activation acts via HSP27 phosphorylation to cause constriction, as originally suggested for bombesin-stimulated constriction of rabbit rectosigmoid smooth muscle (3).

The selectivities of SB-203580 and SB-202190 have been extensively investigated, and in an in vitro assay system (7) both drugs caused near complete block of p38-{alpha} and -{beta} MAPK activity at 10 µM without significantly affecting the activities of 22 other kinases, including MAPKAPK-1/2, ROCK-II, PKC-{alpha}, ERK-2, MKK-1, and JNK-1{alpha}. In cell-based systems, the IC50 for SB-203580 against p38 MAPK is ~0.6 µM (e.g., Ref. 6), similar to that obtained in the present study (1.6 µM). SB-203580 activates phospholipase A2 (10), but this does not explain our results since SB-202190 has no effect on phospholipase A2 and yet still inhibited PGF2{alpha}-induced constriction with a potency similar to SB-203580. SB-203580 is also reported to inhibit cyclooxygenase (COX-1 and COX-2) in platelets with an IC50 close to 2 µM (5). However, this is unlikely to be a major factor here, because PD-98059 also inhibits COX with equal potency (5) but was a poor blocker of PGF2{alpha}-induced constriction. MAPK inhibitors also inhibit phosphodiesterase activity in rat pinealocytes (15). However, these effects only occur at concentrations >1 µM, and SB-202474 was markedly more effective than SB-203580. This effect, if it occurs in IPA, is therefore unlikely to explain our observed effects of SB-203580 at ≤2 µM, although it may explain the relaxation induced by SB-202474 at 3, 10, and 30 µM and the residual relaxation to higher SB-203580 concentrations after endothelial denudation.

Considering all the above and the fact that SB-203580 but not SB-202474 abolished PGF2{alpha}-induced p38 MAPK and HSP27 phosphorylation, we find it reasonable to suggest that at the concentrations used in this study, SB-203580 and SB-202190 are selective for p38 MAPK in this preparation.

To our knowledge this is the first study to address the role of the vascular endothelium in the effect of SB-203580 on constriction. Removal of the endothelium caused a >10-fold rightward shift in the concentration-response curve to SB-203580, such that it matched that of the inactive analog SB-202474. Preincubation of IPA with L-NAME also caused a significant rightward shift in the SB-203580 concentration-response curve, and a similar effect was observed with the PKG inhibitor KT-5823. Treatment with the COX inhibitor indomethacin had a similar, though much smaller, effect. This supports the concept that the bulk of SB-203580-induced relaxation was endothelium dependent.

In light of the observed dependence of SB-203580-mediated relaxation on the presence of an intact endothelium and on NO, we hypothesized that p38 MAPK inhibition might be either affecting the release of NO from the endothelium or altering the sensitivity of the underlying smooth muscle to NO. We evaluated the first of these possibilities by measuring the effects of SB-203580 and SB-202474 on cGMP production in primary cultured HUVECs. This particular method is useful because the endothelial cell acts as both producer of NO and sensor of NO production via an autocrine action on cGMP production. It was not surprising that stimulation with PGF2{alpha} alone resulted in increased production since the stimulation of NO production by contractile agonists is well documented (e.g., see Ref. 19) and indeed specifically with PGF2{alpha} in IPA (16). However, the combination of SB-203580 and PGF2{alpha} caused a rise in cGMP similar to that induced by PGF2{alpha} alone, suggesting that under the conditions used in our experiments SB-203580 was not affecting the release of NO or its ability to stimulate guanylate cyclase. On the other hand, SB-203580 did alter the apparent sensitivity of endothelium-denuded IPA to exogenous NO, as provided by the NO donor SNAP. Furthermore, this action is likely to be specific to p38 MAPK inhibition since SB-202474 was without effect. We infer from this that p38 MAPK is contributing to PGF2{alpha}-induced contraction by interfering with the underlying relaxing actions of NO.

We also attempted to determine at which point the p38 MAPK pathway was acting to inhibit NO/cGMP-mediated vasorelaxation. NO relaxes smooth muscle partly by increasing the pumping of Ca2+ out of cells and into the sarcoplasmic reticulum, as well as via the opening of K+ channels and hyperpolarization, all of which are reflected by a drop in [Ca2+]i (25). However, SB-203580 did not itself affect the PGF2{alpha}-mediated rise in [Ca2+]i, nor did it influence the SNAP-induced reversal of the U-46619-mediated rise in [Ca2+]i. This implied that it must be acting by decreasing the sensitivity of the contractile apparatus to Ca2+.

If, as has been suggested, the p38 MAPK/HSP27 axis is enhancing agonist-induced contraction via regulation of actin polymerization, cross-bridge cycling, or membrane attachment (2, 12), this should be largely independent of [Ca2+]i. Indeed, a role for p38 MAPK in mediating agonist-induced contraction via Ca2+ sensitization has already been suggested by Yamboliev et al. (38). They found that ET-1-induced contraction in {alpha}-toxin-permeabilized canine main PA was inhibited by ~30% by 10 µM SB-203580. They also provided more direct evidence for the involvement of HSP27 in VSM by demonstrating that an antibody to HSP27 inhibited constriction to ET-1 by ~20% in saponin-permeabilized canine PA. Massett et al. (26) have similarly observed that although pressure-induced myogenic tone was partially blocked by SB-203580, smooth muscle [Ca2+]i was unaffected. It has also been suggested that HSP27 may play an important role in sustained constriction of smooth muscle because it mediates association between RhoA and ROCK and thus maintains activation of ROCK-mediated Ca2+ sensitization (29). However, in the present study we were only able to elicit a relaxation to SB-203580 in permeabilized arteries, when 100 µM SNP was added to the buffer.

Taking into account the endothelium dependency of the relaxation to SB-203580 in intact IPA, we infer from this that the endothelium either was damaged during the {alpha}-toxin permeabilization or was unable to release NO because [Ca2+]i was clamped to pCa 6.9. Indeed, preliminary experiments showed that permeabilized IPA did not relax to acetylcholine and did not contract to L-NAME as do intact PGF2{alpha}-constricted IPA. In any case, the restoration in permeabilized IPA of SB-203580-mediated, but not SB-202474-mediated, relaxation in the presence of an NO donor confirmed our conclusion that p38 MAPK activity is contributing to PGF2{alpha}-induced contraction via an inhibition of an underlying NO-mediated relaxation and, in addition, that this occurs via a pathway acting upon the sensitivity of the smooth muscle to [Ca2+]i. Consistent with this concept, it has been suggested that cyclic nucleotide-mediated relaxation via activation of PKG involves phosphorylation of HSP20 (31) and that upon stimulation of the p38 MAPK pathway an increased phosphorylation of HSP27 is directly associated with a concomitant decrease in HSP20 phosphorylation (11). It is possible therefore that the degree of tone for a given contractile stimulus may be partially determined by an interaction between PGF2{alpha}-induced p38 MAPK/HSP27 activity and NO-mediated PKG/HSP20 activity at the level of the smooth muscle contractile machinery. It is unclear as to whether the divergence between our results and those of Yamboliev et al. (38) reflects species, preparation (main PA vs. IPA), or agonist-related differences, or retention of endothelial function in their permeabilized preparation.

Karamsetty et al. (18) suggested that activation of p38 MAPK may play a major role in the sustained phase of HPV, which we have previously suggested is dependent on the endothelium and ROCK-mediated Ca2+ sensitization (32, 37). Karamsetty et al. (18) showed that hypoxia increased p38 MAPK phosphorylation in rat main PA and that both this and the sustained phase of HPV were suppressed by 10 µM SB-202190. Our results with SB-203580 on HPV in rat small IPA were broadly similar, although in our study the effect on the transient phase was larger and that on the sustained phase smaller. The ineffectiveness of SB-202474 does indeed suggest that this effect is mediated via specific inhibition of p38 MAPK. However, blockade of eNOS with L-NAME abolished the effect of SB-203580, strongly suggesting that its suppressive action on HPV, like that on PGF2{alpha}-induced vasoconstriction, was largely mediated by a permissive enhancement of the actions of NO on the smooth muscle rather than any effect on the mechanisms of HPV per se.

Conclusions. We investigated the role of p38 MAPK in PGF2{alpha}-induced vasoconstriction and HPV of rat small IPA. SB-203580 and SB-202190 caused relaxation of PGF2{alpha}-contracted rat IPA at low concentrations, at which they are most likely to exert a selective action on p38 MAPK, via an endothelium- and largely NO-dependent mechanism. Our data imply that activation of p38 MAPK inhibits the actions of NO and possibly other endothelium-derived vasodilators on the smooth muscle, and this inhibition involves a pathway that regulates the sensitivity of the contractile apparatus to intracellular Ca2+. Our results do not support a direct or specific role for p38 MAPK in sustained HPV, though its putative suppressive action on NO-mediated relaxation would enhance HPV. We speculate instead that p38 MAPK-dependent suppression of NO mediated vasodilatation could provide a physiological brake for the action of NO during agonist-induced vasoconstriction and HPV, thereby allowing greater development of tension. Considering the importance of both endothelium-derived NO and ROCK-mediated Ca2+ sensitization for the regulation of pulmonary vascular resistance in health and disease, and their known interaction (36), this novel influence of p38 MAPK could potentially be of therapeutic importance.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Wellcome Trust Grant 062554 and partly by the CIHR.


    ACKNOWLEDGMENTS
 
The authors are grateful to Dr. David Wilson for constructive comments on the manuscript.

M. Shiraishi was the recipient of Fellowships from the Heart & Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR). M. P. Walsh is an AHFMR Medical Scientist, holder of a Canada Research Chair (Tier I) in Biochemistry, and Director of the Canadian Institutes of Health Research (CIHR) Group in Regulation of Vascular Contractility.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. P. T. Ward, Dept of Asthma Allergy & Respiratory Medicine, 2nd Fl. New Hunts House, King’s College London, London SE1 1UL, UK (e-mail: jeremy.ward{at}kcl.ac.uk)

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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 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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