PKC dependent pathways appear to be of particular importance in the tonic contraction of smooth muscle. The calmodulin dependent pathway has been well characterized in gastrointestinal (GI) muscle while the PKC-dependent pathway has not. We propose to study the intracellular PKC-dependent, calmodulin independent mechanisms responsible for regulation of circular smooth muscle contraction in the rectosigmoid colon. Preliminary data indicate that in response to contractile agonists that seem to activate the PKC-dependent pathway, like carbachol, endothelin and bombesin, different molecular species of diacylglycerol (DAG) are produced and may derive from phosphatidylcholine, phosphatidylinositol and phosphatidylserine. In addition, these same agonists cause a dose- dependent increase in production of ceramide which results from hydrolysis of the membrane sphingolipid sphingomyelin. The involvement of the sphingomyelin pathway in GI smooth muscle contraction has not been examined previously. In addition, the PKC-mediated contraction induced by the same agonists ((carbachol, endothelin and bombesin), by ceramide or through direct activation of PKC by TPA causes a dose-dependent increase in mitogen activated protein (MAP) kinase activity. MAP kinase has been suggested as a mediator of PKC induced smooth muscle contraction. The observed increase in MAP kinase activity may be derived from DAG induced activation of PKC, from ceramide induced activation of PKC, or from their interactions. Furthermore, PKC-mediated contraction results in phosphorylation of proteins which are different from those phosphorylated during calmodulin dependent contraction. One of these proteins is a heat shock-like 27kd protein (HSP 27). Preliminary studies suggest that bombesin and ceramide induce phosphorylation of HSP27, cellular translocation and redistribution of MAP kinase associated with HSP27. The relaxant neuropeptide VIP causes a reduction in bombesin induced contraction, a reduction in MAP kinase activation and a reduction in HSP phosphorylation, suggesting that phosphorylation of HSP and PKC mediated contraction may be related. In addition there is a strong association and colocalization between HSP 27 and actin. We therefore propose to examine DAG and ceramide, kinases, and phosphorylated substrates which are activated in response to peptide induced, PKC-mediated contraction. These pathways which are responsible for a calmodulin independent contraction have not been clearly defined in gastrointestinal smooth muscle.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK042876-09
Application #
6177109
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Hamilton, Frank A
Project Start
1991-06-15
Project End
2002-08-31
Budget Start
2000-09-01
Budget End
2002-08-31
Support Year
9
Fiscal Year
2000
Total Cost
$244,226
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pediatrics
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Zakhem, Elie; Raghavan, Shreya; Bitar, Khalil N (2014) Neo-innervation of a bioengineered intestinal smooth muscle construct around chitosan scaffold. Biomaterials 35:1882-9
Raghavan, Shreya; Bitar, Khalil N (2014) The influence of extracellular matrix composition on the differentiation of neuronal subtypes in tissue engineered innervated intestinal smooth muscle sheets. Biomaterials 35:7429-40
Raghavan, Shreya; Gilmont, Robert R; Bitar, Khalil N (2013) Neuroglial differentiation of adult enteric neuronal progenitor cells as a function of extracellular matrix composition. Biomaterials 34:6649-58
Bitar, Khalil N; Zakhem, Elie (2013) Tissue engineering and regenerative medicine as applied to the gastrointestinal tract. Curr Opin Biotechnol 24:909-15
Zakhem, Elie; Raghavan, Shreya; Gilmont, Robert R et al. (2012) Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering. Biomaterials 33:4810-7
Somara, Sita; Bashllari, Daniela; Gilmont, Robert R et al. (2011) Real-time dynamic movement of caveolin-1 during smooth muscle contraction of human colon and aged rat colon transfected with caveolin-1 cDNA. Am J Physiol Gastrointest Liver Physiol 300:G1022-32
Bitar, K; Greenwood-Van Meerveld, B; Saad, R et al. (2011) Aging and gastrointestinal neuromuscular function: insights from within and outside the gut. Neurogastroenterol Motil 23:490-501
Hashish, Mohamed; Raghavan, Shreya; Somara, Sita et al. (2010) Surgical implantation of a bioengineered internal anal sphincter. J Pediatr Surg 45:52-8
Somara, Sita; Gilmont, Robert R; Varadarajan, Saranyaraajan et al. (2010) Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle. Am J Physiol Gastrointest Liver Physiol 299:G1164-76
Gilmont, Robert R; Somara, Sita; Bitar, Khalil N (2008) VIP induces PKA-mediated rapid and sustained phosphorylation of HSP20. Biochem Biophys Res Commun 375:552-6

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