Inhibition of p38 MAPK-dependent bronchial contraction after ozone by corticosteroids

Abstract

We determined the role of p38 mitogen-activated protein kinase (MAPK) in the increased airway smooth muscle (ASM) contractile responses following ozone and modulation by corticosteroids. Mice were exposed to air or ozone (3 ppm for 3 h) and isometric contractile responses of bronchial rings to acetylcholine (ACh) were measured using a myograph in the presence of p38 MAPK inhibitor, SB239063 (10⁻⁶ M) or dexamethasone (10⁻⁶ M). Because MAPK phosphatase (MKP)-1 is a negative regulator of p38 MAPK, we also studied these effects in MKP-1(-/-) mice. Bronchial rings from ozone-exposed wild-type and MKP-1(-/-) mice showed increased contractile responses, with a leftward shift of the dose-response curve in MKP-1(-/-) mice. SB239063 inhibited bronchial contraction equally in air- and ozone-exposed C57/BL6 and MKP-1(-/-) mice. Dexamethasone inhibited ACh-induced bronchial contraction in both air- and ozone-exposed C57/BL6 mice, but not in air- or ozone-exposed MKP-1(-/-) mice. ACh-stimulated p38 MAPK and heat shock protein (HSP)27 phosphorylation, as measured by Western blotting, and this effect was suppressed by SB239063 in C57/BL6 and MKP-1(-/-) mice, but not by dexamethasone in either air- or ozone-exposed MKP-1(-/-) mice. p38 MAPK plays a role in maximal ACh-induced isometric contractile responses and increased contractility induced by ozone. Dexamethasone inhibits ACh-induced ASM contraction through phosphorylation of p38 MAPK and HSP27.

FIGURE 1

Acetylcholine (ACh)-induced isometric bronchial contractile tension in air- (●) and ozone-exposed (○) a) C57/BL6 mice (12 in air- and nine ozone-exposed) and b) mitogen-activated protein kinase phosphatase (MKP)-1^-/- mice (11 in air- and nine ozone-exposed). Data presented as mean±SEM. *: p<0.05 compared with air-exposed group; **: p<0.01 compared with air-exposed group.

FIGURE 2

Effect of SB239063 (10⁻⁶ M) on acetylcholine (ACh)-induced bronchial contractile responses in air-exposed a) C57/BL6 mice (n=12) and b) mitogen-activated protein kinase phosphatase (MKP)-1^-/- mice (n=11), and in ozone-exposed c) C57/BL6 mice (n=9) and d) MKP-1^-/- mice (n=9). Data presented as mean±SEM. □: air; ▲: air plus SB230963; ●: ozone; : ozone plus SB230963. *: p<0.05 compared with untreated mice; **: p<0.01 compared with untreated mice.

FIGURE 3

Inhibition of 5-hydroxytryptamine (5-HT)-induced bronchial contractile tension by a) p38 mitogen-activated protein kinase (MAPK) inhibitor SB239063 (10⁻⁶ M) in air-exposed mice (n=6). Inhibition of acetylcholine (ACh)-induced bronchial contraction by b) p38 MAPK inhibitor SD282 (10⁻⁶ M; n=3), c) extracellular signal-regulated kinase inhibitor PD98059 (10⁻⁶ M; n=6) and d) c-Jun N-terminal kinase inhibitor SP600125 (10⁻⁶ M; n=6) in air-exposed C57/BL6 mice. e) Effect of MAPK-activated protein kinase 2 inhibitor PF3644022 on ACh-induced bronchial contractile tension in air-exposed C57/BL6 mice. Data are presented as mean±SEM. □: air; ▲: air plus SB239063; : air plus SD282; ▼: air plus PD98059; : air plus SP600125; ■: control; :10⁻⁶ M PF3644022; ●:10⁻⁵ M PF3644022; ○: 10⁻⁶ M PF3644022. *: p<0.05 compared with untreated mice; **: p<0.01 compared with untreated mice.

FIGURE 4

Effect of dexamethasone (10⁻⁶ M) on inhibition of acetylcholine (ACh)-induced bronchial contractile tension both in a) air-exposed C57/BL6 (n=11) and b) MKP-1^-/- mice (n=11), and in ozone-exposed c) C57/BL6 mice (n=9) and d) MKP-1^-/- mice (n=9). Data are presented as mean±SEM. □: air; ▲: air plus dexamethasone; ●: ozone; : ozone plus dexamethasone. *: p<0.05 compared with untreated mice; **: p<0.01 compared with untreated mice.

FIGURE 5

Western blot analysis of ratio of a, b) phosphorylated p38 mitogen-activated protein kinase (MAPK) to nonphosphoryated p38 MAPK and c, d) phosphorylated heat shock protein (HSP)27 to nonphosphoryated HSP27 in bronchial preparations that were unstimulated (Con), acetylcholine (ACh)-stimulated, or ACh-stimulated and pretreated with SB239063 (10⁻⁶ M) or dexamethasone (Dex; 10⁻⁶ M) from air-exposed a, c) C57/BL6 and b, d) MAPK phosphatase (MKP)-1^-/- mice. Each panel shows representative Western blots of phosphorylated p38 MAPK and nonphosphoryated p38 MAPK, or phosphorylated HSP27 to nonphosphoryated HSP27, with individual results of n=6–7 in each group. *: p<0.05 compared with ACh alone; **: p<0.01 compared with ACh alone; ^#: not significant.

FIGURE 6

Western blot analysis of ratio of a, b) phosphorylated p38 mitogen-activated protein kinase (MAPK) to nonphosphoryated p38 MAPK and c, d) phosphorylated heat shock protein (HSP)27 to nonphosphoryated HSP27 in bronchial preparations that were unstimulated (Con), acetylcholine (ACh)-stimulated, or ACh-stimulated and pretreated with SB239063 (10⁻⁶ M) or dexamethasone (Dex; 10⁻⁶ M) from ozone-exposed a, c) C57/BL6 and b, d) MAPK phosphatase (MKP)-1^-/- mice. Each panel shows representative Western blots of phosphorylated p38 MAPK and nonphosphoryated p38 MAPK, or phosphorylated HSP27 to nonphosphoryated HSP27, with individual results of n=6–7 in each group. *: p<0.05 compared with ACh alone; **: p<0.01 compared with ACh alone; ^#: not significant.

FIGURE 7

Representative Western blot analysis of mitogen-activated protein kinase phosphatase (MKP)-1 and α-tubulin under control (Con) conditions, after addition of acetylcholine (ACh; 10⁻³ M), and after ACh (10⁻³ M) and dexamethasone (Dex; 10⁻⁶ M) to bronchial tissues from a) air- and b) ozone-exposed mice. *: p<0.05.