A major role for the rho-associated coiled coil forming protein kinase in G-protein-mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea - PubMed (original) (raw)

A major role for the rho-associated coiled coil forming protein kinase in G-protein-mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea

K Iizuka et al. Br J Pharmacol. 1999 Oct.

Abstract

1 G protein-mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho-associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y-27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-induced contraction in alpha-toxin- or beta-escin-permeabilized rabbit trachea. 2 Although pre-treatment with Y-27632 dose-dependently inhibited GTPgammaS (10 microM)-induced Ca2+ sensitization of alpha-toxin-permeabilized trachea, a Y-27632-insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPgammaS response in the presence of Y-27632 (100 microM). 3 GF 109203X (5 microM) abolished 1 microM 4beta-phorbol 12, 13-dibutyrate (PDBu)-induced, but only partially inhibited the GTPgammaS-induced Ca2+ sensitization. A combination of Y-27632 (100 microM) and GF 109203X (5 microM) totally abolished the GTPgammaS response. 4 GTPgammaS caused only a small contraction in the absence of Ca2+. Wortmannin (30 microM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+-induced contraction. ATP-triggered contraction of the strip which had been treated with calyculin A (1 microM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 microM), but not by Y-27632 (100 microM), in the presence of GTPgammaS and Ca2+. 5 GTPgammaS, but not PDBu, contracted the beta-escin-permeabilized trachea in the absence of Ca2+, but the presence of Ca2+-independent MLCK. 6 We conclude that ROCK plays a primary role in G-protein-mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.

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Figures

Figure 1

Putative mechanism of phosphorylation of mysoin light chain in smooth muscle. In ATP free (rigor) solutions Y-27632, wortmannin, and calyculin A inhibit ROCK, MLCK, and SMPP-1M, respectively, although ROCK, MLCK, and other kinase(s) cannot phosphorylate the substrates even when Ca2+ and GTPγS are present because ATP is absent. Once SMPP-1M has been inhibited by calyculin A, Rho/ROCK-mediated signalling cannot affect SMPP-1M activity. ATP application triggers phosphorylation of MLC20, resulting in contraction. The rate of force rise, but not the final amplitude, reflects ROCK, MLCK and other kinase(s) activities towards MLC20. CaM, calmodulin; MLC20, 20 kDa myosin light chain; MLCK, myosin light chain kinase; ROCK, Rho-associated coiled coil forming protein kinase; SMPP-1M, smooth muscle protein phosphatase 1 associated with myosin.

Figure 2

Effect of subsequent addition of Y-27632 on GTPγS-induced Ca2+ sensitization. When the GTPγS-induced Ca2+ sensitization reached a peak, a high concentration of Y-27632 was applied to the strip. The traces are representative of five experiments.

Figure 3

Time course of GTPγS-induced Ca2+ sensitization in the presence or absence of Y-27632. After stable pCa 5.0 response was obtained, the α-toxin-permeabilized tracheal strips were pre-incubated for 30 min without or with Y-27632 at indicated concentrations. When the submaximal contraction at pCa 6.5 became stable, GTPγS (10 μ

) was added to the strips, and the serial changes in tension were observed for 30 min. Y-27632 was present during GTPγS-induced Ca2+ sensitization. Developed force was normalized to the initial pCa 5.0 response. *P<0.05 vs control. (_n_=4–7).

Figure 4

Complete inhibition of PDBu- but not GTPγS-induced Ca2+ sensitization by GF 109203X. After stable pCa 5.0 response was obtained, the α-toxin-permeabilized tracheal strips were preincubated for 30 min with either GF 109203X (5 μ

, b) or 0.5% dimethyl sulphoxide (DMSO, a). When the submaximal contraction at pCa 6.5 became stable, PDBu (1 μ

) was added to the strips, followed by application of GTPγS (10 μ

) as indicated. The traces are representative of four experiments.

Figure 5

Lack of effect of GF 109203X on Ca2+-induced contraction of Triton X-100-permeabilized trachea. The trachea was contracted by a solution of pCa 5.0 containing calmodulin (CaM, 5 μ

), and relaxed in a relaxing solution (G1). When the pCa 6.2-induced contraction became stable, GF 109203X followed by wortmannin were added to the strip. The trace was representative of four experiments. All solutions except for pCa 5.0 contained 1 μ

CaM.

Figure 6

GTPγS-induced Ca2+ sensitization in the presence of Y-27632, GF 109203X, or both. After stable pCa 5.0 response was obtained, the α-toxin-permeabilized tracheal strips were preincubated for 30 min without or with either GF 109203X, Y-27632, or both. When the submaximal contraction at pCa 6.5 became stable, GTPγS (10 μ

) was added to the strips, and the changes in tension were observed for 30 min. The reagents were present during GTPγS-induced Ca2+ sensitization, and control experiments were carried out in the presence of vehicle 0.5% dimethyl sulphoxide (DMSO), water, or both. Developed force was normalized to the initial pCa 5.0 response. *P<0.05 vs control. (_n_=4–11).

Figure 7

Complete inhibition of high Ca2+ alone and low Ca2+ plus GTPγS-induced contractions by wortmannin. After obtaining the pCa 5.0 response, the strips were treated with wortmannin (30 μ

) (b) or the vehicle (0.3% dimethyl sulphoxide, DMSO) (a) for 30 min in a relaxing solution (G1), followed by exposure of high Ca2+ (pCa 5.0) and its washing. Then, GTPγS (10 μ

) was added to the strips in G1, and quick transferring the strips to the pCa 6.5 solution containing GTPγS. The traces are representative of six experiments.

Figure 8

The effects of wortmannin and Y-27632 on ATP-triggered contraction of calyculin A-treated trachea. After the pCa 5.0 response was obtained, the α-toxin-permeabilized tracheal strips were incubated in an ATP free Ca2+ free solution (G10) for 10 min to remove ATP from the strip, and then incubated in a pCa 6.5 rigor solution containing calyculin A (1 μ

) for 60 min. GTPγS (10 μ

) was present as indicated. Contraction was initiated by addition of ATP (a). Time matched experiments were carried out in the presence of wortmannin (30 μ

), Y-27632 (100 μ

), or both 60 min before and during contraction as indicated*. Data are summarized in (b) (_n_=4–5).

Figure 9

Requirement of MLCK activity but not Ca2+ itself for GTPγS-induced contraction in β-escin-permeabilized trachea. After stable pCa 5.0 plus 5 μ

calmodulin (CaM) response was obtained, the β-escin-permeabilized strips were contracted either by Ca2+-independent MLCK (IMLCK, 2 μ

) in G10 (buffered by 10 m

EGTA) (b) or by pCa 6.0 (a). PDBu (1 μ

), GTPγS (10 μ

), and CaM (5 μ

) were added sequentially to each strip as indicated. Unless noted otherwise all solutions contained CaM (0.1 μ

). Data are summarized in (c). Submax. contraction means either pCa 6.0- or IMLCK-induced force prior to addition of Ca2+ sensitizing agents. The responses to pCa 6.0, IMLCK, PDBu, GTPγS, and CaM were normalized to the initial maximum contraction of each strip. **P<0.01, (Mann-Whitney _U_-test, _n_=4). In separate set of experiments, Y-27632 was added to the peak of contraction induced by GTPγS in the presence of IMLCK, followed by application of calyculin A. The traces are representative of four experiments (d).