Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway

Abstract

Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gβγ i3 with activation of phospholipase C-β3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Gα q/11 with activation of phospholipase C-β1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44 + − 5 %). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr 38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28 + − 3 %). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC 20 ) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKCdependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC 20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLC 20 phosphorylation and contraction.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

“…The pathway in circular smooth muscle, as previously shown, involves sequential activation of G q and/or G 13 , p115 RhoGEF, and RhoA (20,37,38,41,42). RhoA initiates two pathways involving activation of Rho kinase and PKC, both of which participate in inhibition of MLCP: Rho kinase via phosphorylation of MYPT1 at Thr 696 , and PKC, via phosphorylation of CPI-17, an endogenous inhibitor of MLCP, at Thr 38 (41,42). In addition, Rho kinase activates ZIP kinase (4,11).…”

“…Studies in intestinal circular smooth muscle have shown that muscarinic m2 receptors are coupled to pertussis toxin (PTx)-sensitive G i3 , whereas m3 receptors are coupled to G q and G 13 (4,41). The specific proteins coupled to m2 and m3 receptors in longitudinal smooth muscle have not been identified.…”

“…DMEM-10 medium was replaced every 3 days for 2-3 wk until confluence was attained. All experiments were done on cells in the first passage (41,56).…”

“…The initial transient phase is followed by a sustained contraction mediated by inhibition of MLCP and activation of a Ca 2ϩ -independent MLCK (e.g., zipper-interacting kinase, ZIP kinase) (4, 56). The signaling pathway involves sequential activation of G␣ 13 , p115RhoGEF, RhoA/Rho kinase, and RhoA/PKC leading to inhibition of MLCP upon phosphorylation of its regulatory subunit, MYPT1 by Rho kinase, and phosphorylation of CPI-17, an endogenous inhibitor of MLCP, by PKC (4,41,42). ZIP kinase is activated downstream of Rho kinase and induces sustained MLC 20 phosphorylation and muscle contraction enhanced by a low ambient MLCP activity (23,36).…”

“…The initial phase in circular smooth muscle is mediated by sequential activation of G q and phospholipase C (PLC-␤1), resulting in inositol 1,4,5-trisphosphate (IP 3 )-induced Ca 2ϩ release via IP 3 receptor IP 3 R-1/Ca 2ϩ channels, Ca 2ϩ /calmodulin-dependent activation of MLCK, and phosphorylation of MLC 20 (20,37,38,41). The signal for the initial phase is rapidly terminated by "regulator of G protein signaling" 4 (RGS4), which accelerates inactivation of G␣ q , and by Ca 2ϩ reuptake into intracellular Ca 2ϩ stores via the sarcoendoplasmic reticulum Ca 2ϩ -ATPase pump, SERCA2 (7,15,16,20,40).…”