Characterization of protein kinase pathways responsible for Ca²⁺sensitization in rat ileal longitudinal smooth muscle

Abstract

2ϩsensitization in rat ileal longitudinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 293: G699-G710, 2007. First published July 26, 2007; doi:10.1152/ajpgi.00214.2007.-We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca 2ϩ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 M microcystin induced LC20 diphosphorylation and contraction of ␤-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 M, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca 2ϩ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle. ileum; protein kinase C; myosin phosphatase; mitogen-activated protein kinase; protein kinase C-potentiated phosphatase inhibitor protein of 17 kDa; myosin-targeting subunit of myosin light chain phosphatase SMOOTH MUSCLE CONTRACTION is a dynamic and highly regulated process. The contractile state of smooth muscle is mainly regulated by phosphorylation of the 20-kDa myosin regulatory light chain (LC 20 ] i )} is the primary determinant of smooth muscle contraction. Force can be further increased through signaling pathways that modulate MLCK and/or MLCP activities. The Ca 2ϩ sensitivity of contraction can be affected by any change in the ratio of MLCK:MLCP activity. A decrease in MLCP activity will shift the balance in favor of MLCK, resulting in a greater degree of LC 20 phosphorylation and contraction. The phenomenon of ...

Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics

“…MLC 20 phosphorylation is regulated by the relative activity of MLC kinase (MLCK) and MLC phosphatase (MLCP); and the activity of MLCK and MLCP is modulated by the small molecular weight (MW) proteins, calmodulin (CaM; MW ϭ 17) and CPI-17 (MW ϭ 17), respectively (37). Loss of CaM (27) and CPI-17 (26) has been implicated in the depressed contractile response of skinned or stringently permeabilized smooth muscle.…”

Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

“…ROK, ILK, and PKC have all been suggested to play a physiologically important role in the regulation of vascular smooth muscle contractility (4,12). Conventional PKC isozymes (PKC-␣, -␤, and -␥), ERK1/2, and p38MAPK have been shown to play a central role in regulating MLCP activity during Ca 2ϩ -independent contraction in rat ileal smooth muscle (15,18). AV25 did not affect Ca 2ϩ -independent contraction of vascular smooth muscle, and our experiments with PKC and ROK inhibitors revealed that these protein kinases did not contribute to the AV25-induced Fig.…”

“…5A). We have recently linked PKC and MAPK pathways to Ca 2ϩ -independent, microcystin-induced contraction in rat ileal smooth muscle (18). AV25 potentiation was enhanced by pretreatment with broadspecificity PKC inhibitor, GF109203x (Fig.…”

Epithelial relaxation mediated by the myosin phosphatase regulator Mypt1 is required for brain ventricle lumen expansion and hindbrain morphogenesis

“…Like non-muscle myosin, smooth muscle is also regulated by Mypt1 and myosin phosphatase (Pfitzer, 2001), and processes akin to epithelial relaxation may exist in vascular smooth muscle (Hirano, 2007), smooth muscle of the urinary bladder (Ding et al, 2009;Poley et al, 2008), gastrointestinal smooth muscle (Huang et al, 2005;Ihara et al, 2007) and in secretion of bile by the gall bladder (CamelloAlmaraz et al, 2009).…”