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

Abstract

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) (GTPγS)‐induced contraction in α‐toxin‐ or β‐escin‐permeabilized rabbit trachea. Although pre‐treatment with Y‐27632 dose‐dependently inhibited GTPγS (10 μM)‐induced Ca2+ sensitization of α‐toxin‐permeabilized trachea, a Y‐27632‐insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPγS response in the presence of Y‐27632 (100 μM). GF 109203X (5 μM) abolished 1 μM 4β‐phorbol 12, 13‐dibutyrate (PDBu)‐induced, but only partially inhibited the GTPγS‐induced Ca2+ sensitization. A combination of Y‐27632 (100 μM) and GF 109203X (5 μM) totally abolished the GTPγS response. GTPγS caused only a small contraction in the absence of Ca2+. Wortmannin (30 μM), 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 μM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 μM), but not by Y‐27632 (100 μM), in the presence of GTPγS and Ca2+. GTPγS, but not PDBu, contracted the β‐escin‐permeabilized trachea in the absence of Ca2+, but the presence of Ca2+‐independent MLCK. 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. British Journal of Pharmacology (1999) 128, 925–933; doi:

8-Bromo-cAMP decreases the Ca2+sensitivity of airway smooth muscle contraction through a mechanism distinct from inhibition of Rho-kinase

“…2, the contractile rate (T 1/2 ) was comparable between 8-BrcAMP-treated strips and control strips. The T 1/2 of the strips treated with a phosphatase inhibitor (such as microcystin-LR or calyculin A) is dependent on the MLCK- associated mechanisms in the presence of Ca 2ϩ (13,16). These results suggest that cAMP/PKA signaling preferentially affects MLCP-associated mechanisms of Ca 2ϩ sensitization in rabbit tracheal smooth muscle.…”

“…However, precise time-course studies suggest that initiation of contraction occurred within milliseconds and that the early phase of maintenance of force occurred within several seconds (25). Furthermore, we have demonstrated that MLCK inhibition with wortmannin but not Rho-kinase inhibition with Y-27632 slowed force developments under the same experimental conditions (13). The final amplitudes were not different.…”

“…Theoretically, GTP␥S may activate both trimetric and monometric G proteins, resulting in direct activation of Rho/Rho-kinase signaling and indirect activation of trimetric G protein-mediated PKC signaling. However, practically, PKC plays a minor role in GTP␥S-induced Ca 2ϩ sensitization in rabbit tracheal smooth muscle (13). Hepler et al (8) showed that purified G q had only a slight binding affinity to GTP␥S.…”

“…To clarify whether 8-BrcAMP-induced Ca 2ϩ desensitization is due to inhibition of MLCK-associated mechanisms, we abolished the MLCP activity of tracheal smooth muscle with calyculin A (300 nM) in rigor solutions and then observed ATP (4.5 mM, Mg 2ϩ salt)-triggered contraction. We knew that the rate of force would rise but the final amplitude would not, reflecting MLCK-associated mechanisms (13,17,19). After obtaining the maximum contraction at pCa 5.0, we relaxed the tracheal smooth muscle in G10 solution.…”

“…We have demonstrated that receptor-dependent, G protein-mediated Ca 2ϩ sensitization occurs in canine, rabbit, and human airway smooth muscles (28) and that a small G protein, RhoA, and its target protein, Rho-kinase, play a key role in G protein-mediated Ca 2ϩ sensitization of smooth muscle contraction (26), especially in the sustained phase. Rho/Rho-kinase signaling increases phosphorylation of MLC 20 through the inhibition of myosin light chain phosphatase (MLCP)-associated mechanisms, but it does not directly phosphorylate the MLC 20 of airway smooth muscle in situ (13).…”