MEK modulates force-fluctuation-induced relengthening of canine tracheal smooth muscle

Abstract

Tidal breathing, and especially deep breathing, is known to antagonise bronchoconstriction caused by airway smooth muscle (ASM) contraction; however, this bronchoprotective effect of breathing is impaired in asthma. Force fluctuations applied to contracted ASM in vitro cause it to relengthen, force-fluctuation-induced relengthening (FFIR). Given that breathing generates similar force fluctuations in ASM, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. Thus it is of considerable interest to understand what modulates FFIR, and how ASM might be manipulated to exploit this phenomenon. It was demonstrated previously that p38 mitogen-activated protein kinase (MAPK) signalling regulates FFIR in ASM strips. Here, it was hypothesised that the MAPK kinase (MEK) signalling pathway also modulates FFIR. In order to test this hypothesis, changes in FFIR were measured in ASM treated with the MEK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Increasing concentrations of U0126 caused greater FFIR. U0126 reduced extracellular signal-regulated kinase 1/2 phosphorylation without affecting isotonic shortening or 20-kDa myosin light chain and p38 MAPK phosphorylation. However, increasing concentrations of U0126 progressively blunted phosphorylation of high-molecular-weight caldesmon (h-caldesmon), a downstream target of MEK. Thus changes in FFIR exhibited significant negative correlation with h-caldesmon phosphorylation. The present data demonstrate that FFIR is regulated through MEK signalling, and suggest that the role of MEK is mediated, in part, through caldesmon.

FIGURE 1

Experimental protocol. Tracheal smooth muscle strips were exposed to U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) or vehicle (control) for 45 min, after which the oscillation protocol was then repeated. The difference (Δ) in force-fluctuation-induced relengthening (FFIR) after versus before treatment was determined. Load oscillation was performed at 32±16% of the maximum force of contraction (F_max). Near the end of the second contraction sequence, tissues were flash-frozen in liquid nitrogen For the vehicle-treated (control) muscle strip (a and c), there was no appreciable ΔFFIR. MEK inhibition with U0126 (b and d) significantly increased ΔFFIR relative to vehicle treated control strips. Representative traces are shown. L_ref: reference length.

FIGURE 2

Effect of varying concentrations of the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophe-nylthio]butadiene). The difference (Δ) in force-fluctuation-induced relengthening (FFIR) was compared to that of acetylcholine-contracted canine tracheal smooth muscle strips that received vehicle alone (n=14). Data are presented as mean ± SEM. MEK inhibition modulates FFIR in a concentration-dependent manner (p <0.001 (n=6 per concentration); ANOVA). *: p<0.05 versus no drug treatment; ^# : p<0.05 versus 0, 3 and 15 μM U0126.

FIGURE 3

Effect of mitogen-activated protein kinase kinase inhibition on isometric responsiveness and isotonic shortening in acetylcholine (ACh)-contracted canine tracheal smooth muscle (TSM) strips. a) U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) at concentrations of 15 (□; n=3) and 30 μM (○; n=5) had no significant effect on isometric responsiveness compared to control strips (●; n=4) (p=0.486; ANOVA). b) 30 μM U0126 (n=6) treatment resulted in a substantial increase in change in force-fluctuation-induced relengthening compared to control (n=14), but did not affect isotonic shortening of the TSM strips after treatment (p=0.951; unpaired t-test). Data are presented as mean ± SEM. L_ref: reference length.

FIGURE 4

Effect of mitogen-activated protein kinase kinase inhibition on caldesmon phosphorylation in canine tracheal smooth muscle (TSM) strips. a) Relative caldesmon phosphorylation levels with 3 (n=6), 15 (n=5) and 30 μM (n=6) U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene) in comparison to those in TSM strips that received vehicle alone (n=10); and b) representative corresponding western blot of control- and U0126-treated tissues. Caldesmon demonstrated reduced phosphorylation following treatment with 15 and 30 μM U0126 compared to both vehicle-treated and 3-μM strips (p=0.002; ANOVA). c) The change in force-fluctuation-induced relengthening (ΔFFIR; ●) varied inversely with relative caldesmon phosphorylation (□) in strips contracted with acetylcholine in the presence of U0126 (data derived from figures 2 and 4a). Data are presented as mean ± SEM. h-caldesmon: high-molecular-weight caldesmon; L_ref: reference length. *: p<0.05 versus no drug treatment; ^#: versus 0 and 3 μM U0126; ^¶: versus 0, 3 and 15 μM U0126.

FIGURE 5

Schematic model of the signalling pathways involved in smooth muscle contraction. Acetylcholine (ACh) activates the G-protein-coupled M₂ and M₃ muscarinic receptors, resulting in the activation of several signalling cascades. M₂ activates Raf/mitogen-activated protein kinase (MAPK) kinase (MEK) signalling, which, in turn, activates extracellular signal-regulated kinase (ERK) 1/2 and their downstream regulators of contraction, including high-molecular-weight caldesmon (h-caldesmon). Through activation of myosin light chain kinase (MLCK) and ERK1/2, intracellular Ca²⁺ and integrin-linked kinase (ILK) promote phosphorylation (P) of 20-kDa myosin light chain (MLC20). Only a few of the likely effector molecules involved in this pathway are depicted. DAG: diacylglycerol; PKC: protein kinase C; IP₃: inositol 1,4,5-trisphosphate; CaM: calmodulin; PI3K: phosphatidylinositol-3′-kinase; Hsp27: 27-kDa heat shock protein.