title: Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors.
abstract: The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC(50) 0.7 +/- 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated G(q) and G(13), stimulated G alpha(q)-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release, and increased cytosolic free Ca(2+). PI hydrolysis was blocked by expression of G alpha(q) minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC(50) = 1.0 +/- 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca(2+)-dependent contraction and myosin light-chain (MLC)(20) phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC(20) phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of G alpha(q) or G alpha(13) minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC(20) phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a G alpha(q)-mediated cascade involving Ca(2+)/calmodulin activation of MLC kinase and transient MLC(20) phosphorylation and contraction as well as a sustained G alpha(q)- and G alpha(13)-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC(20) phosphorylation and contraction.
authors.affiliation: Department of Physiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Jiean
authors.initials: J
authors.lastname: Huang
authors.forename: Huiping
authors.initials: H
authors.lastname: Zhou
authors.forename: Sunila
authors.initials: S
authors.lastname: Mahavadi
authors.forename: Wimolpak
authors.initials: W
authors.lastname: Sriwai
authors.forename: Vijay
authors.initials: V
authors.lastname: Lyall
authors.forename: Karnam S
authors.initials: KS
authors.lastname: Murthy
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D005765:Gastrointestinal Agents / Q000494:pharmacology*; D005769:Gastrointestinal Motility / Q000502:physiology*; D006868:Hydrolysis; D007421:Intestine, Small / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D009037:Motilin / Q000494:pharmacology*; D009119:Muscle Contraction / Q000502:physiology*; D009130:Muscle, Smooth / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000494:pharmacology; D011817:Rabbits; D011964:Receptors, Gastrointestinal Hormone / Q000502:physiology*; D018013:Receptors, Neuropeptide / Q000502:physiology*; D015398:Signal Transduction; D013270:Stomach / Q000502:physiology; D054460:rho-Associated Kinases
chemical_list: D005765:Gastrointestinal Agents; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D011964:Receptors, Gastrointestinal Hormone; D018013:Receptors, Neuropeptide; C050802:motilin receptor; D009037:Motilin; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C