The objective of this proposal is to characterize the distinctive signaling pathways that initiate, sustain, and terminate contraction of longitudinal colonic smooth muscle and identify the changes in expression and activity of specific targets that determine hypercontractility of this muscle during inflammation. Contraction in longitudinal muscle is initiated by cPLA2-dependent Ca2+ influx leading to Ca2+- and cyclic ADP ribose-induced Ca2+ release via ryanodine (RYR2) receptors. Preliminary studies showed that initial contraction was terminated by inactivation of MLCK via a novel pathway involving sequential activation of CaMK kinase and AMPK. Preliminary studies also showed that sustained contraction was mediated via sequential activation of G12, LARG, RhoA/Rho kinase leading to inhibition of MLC phosphatase. The pathways for relaxation via PKA and PKG in longitudinal muscle were also distinct and involved inhibitory phosphorylation of cPLA2 and ADP ribosyl cyclase during initial contraction and stimulatory phosphorylation of the MLCP activator, telokin, during sustained contraction. Preliminary studies in IL-1?-treated colonic longitudinal muscle strips in vitro and muscle strips from TNBS-colitis in vivo showed similar changes in the expression and/or activity of specific signaling targets that resulted in hypercontractility during inflammation. These targets included: (i) s-nitrosylation and activation of RYR2 via iNOS, leading to increase in Ca2+ release, and NF-?B/PKA-mediated inactivation of AMPK, leading to increase in MLCK activity, MLC20 phosphorylation, and initial contraction;and (ii) up-regulation of LARG via an iNOS/JNK/AP-1 pathway, and down-regulation of telokin via an NF-?B pathway, leading to further inhibition of MLCP and increase in sustained MLC20 phosphorylation and contraction. Preliminary studies also showed that relaxation mediated by PKA and PKG in longitudinal muscle is attenuated during inflammation. The targets included: (i) s-nitrosylation of sGC and ACIII leading to inhibition of cGMP and cAMP synthesis, and (ii) up-regulation of PDE1A1 via NF-?B, and ERK1/2- mediated activation of PDE4D5, leading to augmentation of cGMP and cAMP degradation.
The specific aims are to characterize novel signaling pathways that initiate, sustain, and terminate contraction in colonic longitudinal muscle, identify the specific targets whose expression and/or activity is altered during inflammation, and determine their regulation via iNOS/JNK/AP-1, NF-:B, and ERK1/2. These targets include RYR2, AMPK, LARG, and telokin (Aims I and II);and sGC, ACIII, PDE1A1, and PDE4D5 (Aim III). The innovative aspects of this proposal lie in the discovery of mechanisms of hypercontractility of longitudinal muscle during inflammation.

Public Health Relevance

The objective of this proposal is to characterize the distinctive signaling pathways that initiate, sustain, and terminate contraction and relaxation of longitudinal intestinal smooth muscle and identify the changes in expression and activity of specific targets that determine hypercontractiliy of this muscle during inflammation. The project involves a comprehensive analysis of the mechanisms by which inflammatory cytokines augment contraction and inhibit relaxation at cellular level, both in vitro and in an established model of colonic inflammation.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01DK015564-43
Application #
8636425
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Serrano, Jose
Project Start
Project End
Budget Start
Budget End
Support Year
43
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Nalli, Ancy D; Kumar, Divya P; Mahavadi, Sunila et al. (2014) Hypercontractility of intestinal longitudinal smooth muscle induced by cytokines is mediated by the nuclear factor-?B/AMP-activated kinase/myosin light chain kinase pathway. J Pharmacol Exp Ther 350:89-98
Al-Shboul, Othman; Nalli, Ancy D; Kumar, Divya P et al. (2014) Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation. Am J Physiol Cell Physiol 306:C1129-41
Nalli, Ancy D; Kumar, Divya P; Al-Shboul, Othman et al. (2014) Regulation of G??i-dependent PLC-?3 activity in smooth muscle: inhibitory phosphorylation of PLC-?3 by PKA and PKG and stimulatory phosphorylation of G?i-GTPase-activating protein RGS2 by PKG. Cell Biochem Biophys 70:867-80
Mahavadi, Sunila; Sriwai, Wimolpak; Huang, Jiean et al. (2014) Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/?-arrestin activation of ERK1/2 and Src kinase. Am J Physiol Gastrointest Liver Physiol 306:G535-45
Hurst, Norm R; Kendig, Derek M; Murthy, Karnam S et al. (2014) The short chain fatty acids, butyrate and propionate, have differential effects on the motility of the guinea pig colon. Neurogastroenterol Motil 26:1586-96
Al-Qudah, M; Anderson, C D; Mahavadi, S et al. (2014) Brain-derived neurotrophic factor enhances cholinergic contraction of longitudinal muscle of rabbit intestine via activation of phospholipase C. Am J Physiol Gastrointest Liver Physiol 306:G328-37
Huang, Jiean; Nalli, Ancy D; Mahavadi, Sunila et al. (2014) Inhibition of G?i activity by G?? is mediated by PI 3-kinase-?- and cSrc-dependent tyrosine phosphorylation of G?i and recruitment of RGS12. Am J Physiol Gastrointest Liver Physiol 306:G802-10
Bhattacharya, Sayak; Mahavadi, Sunila; Al-Shboul, Othman et al. (2013) Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1. Am J Physiol Cell Physiol 305:C334-47
Ahmed, Rashad; Mahavadi, Sunila; Al-Shboul, Othman et al. (2013) Characterization of signaling pathways coupled to melatonin receptors in gastrointestinal smooth muscle. Regul Pept 184:96-103
Alkahtani, Reem; Mahavadi, Sunila; Al-Shboul, Othman et al. (2013) Changes in the expression of smooth muscle contractile proteins in TNBS- and DSS-induced colitis in mice. Inflammation 36:1304-15

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