The myogenic tone in the internal anal sphincter (IAS) plays a crucial role in rectoanal continence. Indeed, IAS dysfunction has been associated with a number of rectoanal motility disorders, such as incontinence, Hirschsprung's disease, hemorrhoids, and recurrent anal fissures. The IAS disorders especially affect the rapidly expanding population of the elderly. Therefore, there is an increased urgency in understanding the molecular mechanisms regulating the IAS smooth muscle (SM) function. In the first aim of the proposal we will determine the relative contribution of RhoA/Rho kinase (RhoA/ROCK) compared to protein kinase C (PKC) in the IAS tone of intact human IAS vs. rectal smooth muscle (RSM), in their basal states. This will be accomplished by determination of correlations between changes in the basal tone, ROCK and PKC enzymatic activities, phosphorylated vs. nonphosphorylated states, mRNA levels, and cellular distribution of RhoA/ROCK, PKC, CPI-17, MYPT1, and MLC20. These parameters will be examined via force measurements, WB analyses, qPCR, and confocal microscopy. In the second aim, we plan to determine the nature of interaction between RhoA/ROCK and PKC pathways following their activation, in the human IAS. For this, we will compare the effects of ROCK and PKC activators, before and after treatment with their inhibitors, on the functional and signal transduction for these pathways. In addition, after the identification of ROCK and PKC isozymes most relevant to the human IAS tone, we will determine the effects of isozyme-specific siRNA-induced gene silencing;and site-directed MYPT1 mutagenesis, on the IAS tone while tracking the above signal transduction pathways. In the third am, we will investigate the role of RhoA/ROCK in the pathogenesis and targeted therapy of the hypertensive IAS, using appropriate animal models. Here, we will determine the upregulation of RhoA/ROCK pathway-related machinery in the SHR and EDNRB-/- rats with the IAS dysfunction (vs. normal animals). Following this, we will perform restiutive studies after the topical anal application of ROCK II siRNA. This will be done in conjunction with the intraluminal manometry and the IAS SM function. The major strength of the proposal is its focus on the molecular mechanisms that regulate the basal tone in the intact human IAS, and on development of innovative approaches to targeted therapy of the IAS dysfunction via the use of appropriate animal models. The information obtained will directly impact our present knowledge of the pathophysiology, and in the evolution of effective therapy for a number of debilitating anorectal motility disorders.

Public Health Relevance

Rectoanal incontinence and other anorectal dysfunctions involving the autonomic smooth muscle of the internal anal sphincter (IAS) affect a significant part of the US population. Currently, because of the lack of knowledge in the molecular mechanisms regulating the human IAS, there is no satisfactory treatment for these debilitating conditions. The proposed research is vital in the advancement of the present understanding of the pathophysiology, and in the evolution of effective therapy of such disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK035385-29
Application #
8313851
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Hamilton, Frank A
Project Start
1985-07-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
29
Fiscal Year
2012
Total Cost
$348,750
Indirect Cost
$123,750
Name
Thomas Jefferson University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Krishna, Chadalavada Vijay; Singh, Jagmohan; Kumar, Sumit et al. (2014) Heme oxygenase-1 upregulation modulates tone and fibroelastic properties of internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 307:G595-601
Singh, Jagmohan; Kumar, Sumit; Krishna, Chadalavada Vijay et al. (2014) Aging-associated oxidative stress leads to decrease in IAS tone via RhoA/ROCK downregulation. Am J Physiol Gastrointest Liver Physiol 306:G983-91
Rattan, Satish (2013) Smooth muscle-specific myosin phosphatase target subunit 1 (MYPT1): an important piece of the puzzle. Gastroenterology 145:1494-5
Gibbons, Ahmara V; Lin, Jieru E; Kim, Gilbert W et al. (2013) Intestinal GUCY2C prevents TGF-* secretion coordinating desmoplasia and hyperproliferation in colorectal cancer. Cancer Res 73:6654-66
Romer, Anthony I; Singh, Jagmohan; Rattan, Satish et al. (2013) Smooth muscle fascicular reorientation is required for esophageal morphogenesis and dependent on Cdo. J Cell Biol 201:309-23
Singh, Jagmohan; Rattan, Satish (2013) Role of PKC and RhoA/ROCK pathways in the spontaneous phasic activity in the rectal smooth muscle. Am J Physiol Gastrointest Liver Physiol 304:G723-31
Rattan, Satish; Singh, Jagmohan (2012) RhoA/ROCK pathway is the major molecular determinant of basal tone in intact human internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 302:G664-75
Singh, Jagmohan; Maxwell 4th, Pinckney J; Rattan, Satish (2011) Immunocytochemical evidence for PDBu-induced activation of RhoA/ROCK in human internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 301:G317-25
de Godoy, Marcio A F; Rattan, Satish (2011) Role of rho kinase in the functional and dysfunctional tonic smooth muscles. Trends Pharmacol Sci 32:384-93
Rattan, S; Singh, J (2011) Basal internal anal sphincter tone, inhibitory neurotransmission, and other factors contributing to the maintenance of high pressures in the anal canal. Neurogastroenterol Motil 23:3-7

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