scholarly journals Upregulation of RGS4 and downregulation of CPI-17 mediate inhibition of colonic muscle contraction by interleukin-1β

2007 ◽  
Vol 293 (6) ◽  
pp. C1991-C2000 ◽  
Author(s):  
Wenhui Hu ◽  
Sunila Mahavadi ◽  
Fang Li ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Rho Kinase ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Inhibitory Effect ◽  
Protein Signaling ◽  
Sustained Contraction ◽  
Bowel Diseases ◽  
Inflammatory Bowel ◽  
Kinase Expression

The pro-inflammatory cytokine IL-1β contributes to the reduced contractile responses of gut smooth muscle observed in both animal colitis models and human inflammatory bowel diseases. However, the mechanisms are not well understood. The effects of IL-1β on the signaling targets mediating acetylcholine (ACh)-induced initial and sustained contraction were examined using rabbit colonic circular muscle strips and cultured muscle cells. The contraction was assessed through cell length decrease, myosin light chain (MLC20) phosphorylation, and activation of PLC-β and Rho kinase. Expression levels of the signaling targets were determined by Western blot analysis and real-time RT-PCR. Short interfering RNAs (siRNAs) for regulator of G protein signaling 4 (RGS4) were used to silence endogenous RGS4 in muscle strips or cultured muscle cells. IL-1β treatment of muscle strips inhibited both initial and sustained contraction and MLC20 phosphorylation in isolated muscle cells. IL-1β treatment increased RGS4 expression but had no effect on muscarinic receptor binding or Gαq expression. In contrast, IL-1β decreased the expression and phosphorylation of CPI-17 but had no effect on RhoA expression or ACh-induced Rho kinase activity. Upregulation of RGS4 and downregulation of CPI-17 by IL-1β in muscle strips were corroborated in cultured muscle cells. Knockdown of RGS4 by siRNA in both muscle strips and cultured muscle cells blocked the inhibitory effect of IL-1β on initial contraction and PLC-β activation, whereas overexpression of RGS4 inhibited PLC-β activation. These data suggest that IL-1β upregulates RGS4 expression, resulting in the inhibition of initial contraction and downregulation of CPI-17 expression during sustained contraction in colonic smooth muscle.

Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

2005 ◽  
Vol 288 (1) ◽  
pp. G23-G31 ◽  
Author(s):  
Jiean Huang ◽  
Huiping Zhou ◽  
Sunila Mahavadi ◽  
Wimolpak Sriwai ◽  
Vijay Lyall ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Dominant Negative ◽  
Phosphatase Inhibitors ◽  
Sustained Contraction ◽  
Pi Hydrolysis

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC50 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 Gq and G13, stimulated Gαq-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP3)-dependent Ca2+ release, and increased cytosolic free Ca2+. PI hydrolysis was blocked by expression of Gαq minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC50 = 1.0 ± 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca2+-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 MLC20 phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of Gαq or Gα13 minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC20 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 Gαq-mediated cascade involving Ca2+/calmodulin activation of MLC kinase and transient MLC20 phosphorylation and contraction as well as a sustained Gαq- and Gα13-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC20 phosphorylation and contraction.

Characterization of S1P1 and S1P2 receptor function in smooth muscle by receptor silencing and receptor protection

2006 ◽  
Vol 291 (4) ◽  
pp. G605-G610 ◽  
Author(s):  
Wenhui Hu ◽  
Sunila Mahavadi ◽  
Jiean Huang ◽  
Fang Li ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Signaling Pathways ◽  
Kinase Activity ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Small Interfering Rnas ◽  
Sustained Contraction ◽  
Sphingosine 1 Phosphate ◽  
Cultured Smooth Muscle Cells

Sphingosine-1-phosphate (S1P) induces an initial Ca2+-dependent contraction followed by a sustained Ca2+-independent, RhoA-mediated contraction in rabbit gastric smooth muscle cells. The cells coexpress S1P1 and S1P2 receptors, but the signaling pathways initiated by each receptor type and the involvement of one or both receptors in contraction are not known. Lentiviral vectors encoding small interfering RNAs were transiently transfected into cultured smooth muscle cells to silence S1P1 or S1P2 receptors. Phospholipase C (PLC)-β activity and Rho kinase activity were used as markers of pathways mediating initial and sustained contraction, respectively. Silencing of S1P1 receptors abolished S1P-stimulated activation of Gαi3 and partially inhibited activation of Gαi1, whereas silencing of S1P2 receptors abolished activation of Gαq, Gα13, and Gαi2 and partially inhibited activation of Gαi1. Silencing of S1P2 but not S1P1 receptors suppressed S1P-stimulated PLC-β and Rho kinase activities, implying that both signaling pathways were mediated by S1P2 receptors. The results obtained by receptor silencing were corroborated by receptor inactivation. The selective S1P1 receptor agonist SEW2871 did not stimulate PLC-β or Rho kinase activity or induce initial and sustained contraction; when this agonist was used to protect S1P1 receptors so as to enable chemical inactivation of S1P2 receptors, S1P did not elicit contraction, confirming that initial and sustained contraction was mediated by S1P2 receptors. Thus S1P1 and S1P2 receptors are coupled to distinct complements of G proteins. Only S1P2 receptors activate PLC-β and Rho kinase and mediate initial and sustained contraction.

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1and S1P2

2004 ◽  
Vol 286 (5) ◽  
pp. C1130-C1138 ◽  
Author(s):  
Huiping Zhou ◽  
Karnam S. Murthy
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Rt Pcr ◽  
Sustained Contraction ◽  
Gastric Smooth Muscle ◽  
Rho Kinase Inhibitor ◽  
Sphingosine 1 Phosphate

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC20) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P1and S1P2receptors. S1P activated Gq, G13, and all Giisoforms and stimulated PLC-β1, PLC-β3, and Rho kinase activities. PLC-β activity was partially inhibited by pertussis toxin (PTX), Gβ or Gαqantibody, PLC-β1 or PLC-β3 antibody, and by expression of Gαqor Gαiminigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of Gα13or Gαqminigene and abolished by expression of both. S1P stimulated Ca2+release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC501 nM). Initial contraction and MLC20phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and Gαqor Gβ antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC20phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P2and S1P1involving concurrent activation of PLC-β1 and PLC-β3 via Gαqand Gβγi, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca2+release and MLCK-mediated MLC20phosphorylation, and 2) sustained contraction exclusively mediated by S1P2involving activation of RhoA via Gαqand Gα13, resulting in Rho kinase- and PKC-dependent MLC20phosphorylation.

DIFFERENTIAL EXPRESSION OF NEUROTROPHINS IN (DSS)-INDUCED COLITIS IN SMOOTH MUSCLE OF RAT COLON

2016 ◽  
Vol 78 (5-5) ◽  
Author(s):  
Mohammad Al-Qudah ◽  
Doaa Abu Shammala ◽  
Ahmed Al-Dwairi ◽  
Othman Al-Shboul
Keyword(s):  
Smooth Muscle ◽  
Expression Pattern ◽  
Muscle Tissue ◽  
Circular Muscle ◽  
Sodium Sulphate ◽  
Rat Colon ◽  
Smooth Muscle Tissue ◽  
Bowel Diseases ◽  
Inflammatory Bowel

There is an increasing recognition of the role of neurotrophins in the mature gastrointestinal tract both at the physiological and pathological levels. However, their expression and role in smooth muscle in the GIT system is under investigated. The aim of this study is to elucidate the expression of the four neurotrophins in smooth muscle tissue of the rat colon and to test the effect of dextran sodium sulphate (DSS) - induced colitis on the expression pattern of these factors. Using specific ELISA kits for each neurotrophin revealed that the four neurotrophins are differentially expressed in the longitudinal and circular muscle layers of the rat colon and that DSS-induced colitis alters this expression pattern. These results indicate that smooth muscle tissue contributes to the pool of neurotrophins in the GIT and might play a role in the pathogenesis of colitis. Understanding the interactions of neurotrophins produced from smooth muscle and colitis could provide new avenues to tackle the dysfunction associated with inflammatory bowel diseases such as colitis.

Neuromuscular structures in opossum esophagus: role of interstitial cells of Cajal

1984 ◽  
Vol 246 (3) ◽  
pp. G305-G315 ◽  
Author(s):  
E. E. Daniel ◽  
V. Posey-Daniel
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Interstitial Cells Of Cajal ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Interstitial Cells ◽  
Complete Relaxation ◽  
Granular Vesicles ◽  
Cells Of Cajal

The structures of the lower esophageal sphincter (LES) and body circular muscle (BCM) from opossum were compared as to neural and muscular structures and the structural relations of interstitial cells of Cajal to nerves and muscle cells. Both LES and BCM were densely innervated by nerves with varicosities containing many small agranular vesicles and a few large granular vesicles. These nerves were more closely related structurally to the interstitial cells of Cajal than to smooth muscle cells. More gap junctions were observed between smooth muscle cells and between interstitial cells of Cajal and smooth muscle cells in BCM than in LES. Those between smooth muscle cells were larger in BCM. Complete relaxation of the LES strip by isoproterenol reduced these differences but did not eliminate them. The finding that interstitial cells of Cajal often had gap-junction contacts to smooth muscle and close associations with nerves is consistent with the hypothesis that interstitial cells are intercalated between the nerves and muscles and may mediate nerve responses. These findings also suggest that LES muscle cells may be less well coupled electrically than BCM muscle cells.

Role of HERG-like K+ currents in opossum esophageal circular smooth muscle

1999 ◽  
Vol 277 (6) ◽  
pp. C1284-C1290 ◽  
Author(s):  
Hamid I. Akbarali ◽  
Hemant Thatte ◽  
Xue Dao He ◽  
Wayne R. Giles ◽  
Raj K. Goyal
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Circular Smooth Muscle ◽  
Inward Currents

An inwardly rectifying K+ conductance closely resembling the human ether-a-go-go-related gene (HERG) current was identified in single smooth muscle cells of opossum esophageal circular muscle. When cells were voltage clamped at 0 mV, in isotonic K+ solution (140 mM), step hyperpolarizations to −120 mV in 10-mV increments resulted in large inward currents that activated rapidly and then declined slowly (inactivated) during the test pulse in a time- and voltage- dependent fashion. The HERG K+ channel blockers E-4031 (1 μM), cisapride (1 μM), and La3+ (100 μM) strongly inhibited these currents as did millimolar concentrations of Ba2+. Immunoflourescence staining with anti-HERG antibody in single cells resulted in punctate staining at the sarcolemma. At membrane potentials near the resting membrane potential (−50 to −70 mV), this K+ conductance did not inactivate completely. In conventional microelectrode recordings, both E-4031 and cisapride depolarized tissue strips by 10 mV and also induced phasic contractions. In combination, these results provide direct experimental evidence for expression of HERG-like K+ currents in gastrointestinal smooth muscle cells and suggest that HERG plays an important role in modulating the resting membrane potential.

scholarly journals Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway

2013 ◽  
Vol 304 (5) ◽  
pp. G527-G535 ◽  
Author(s):  
Senthilkumar Rajagopal ◽  
Divya P. Kumar ◽  
Sunila Mahavadi ◽  
Sayak Bhattacharya ◽  
Ruizhe Zhou ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Kinase Activity ◽  
Muscle Relaxation ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Receptor Activation ◽  
Gastric Smooth Muscle ◽  
Selective Ligand ◽  
Gastric Muscle ◽  
Kinase Pathway

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5−/− mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2′-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

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