The internal anal sphincter (IAS) plays a vital role in continence and defecation. Disorders of the IAS underlie many clinical abnormalities, such as fecal incontinence and constipation. Disorders of the IAS occur more frequently in the rapidly expanding population of the elderly, and thus give increased urgency to understanding of the basic mechanism regulating the IAS function. The objectives of this research proposal are: 1) to investigate the role of local renin-angiotensin system (RAS) and prostanoids (PGF2 alpha and thromboxanes) in the basal tone of the IAS, and 2) to identify intracellular molecular mechanisms for the genesis of basal tone of the IAS smooth muscle. Correlations between intracellular biochemistry and contraction-relaxation of the IAS will be monitored by changes in: 1) force of contraction (in vitro studies), 2) morphometric analyses of smooth muscle cells (SMC), 3) quantitation of G-proteins, 4) activities of Rho kinase (ROK), PKC, myosin light chain kinase (MLCK) and MLC-phosphatase, and 5) the state of phosphorylation of myosin light chain (p-MLC20). In addition, we will determine immunocytochemical localization, direct release, and enzymatic activities involved in the biosynthesis of Ang II and prostanoids. Some studies will involve monitoring of the intraluminal pressures in the IAS. The proposed studies will also use the novel RhoA siRNA and RhoA mutants (in vitro smooth muscle strips as well as in the freshly isolated SMC) to finally determine the role of RhoA/ROK in the signal transduction and maintenance of the I AS smooth muscle tone. These novel molecular approaches will also be exploited to determine the role of RAS and prostanoids in the IAS tone. Rats will be the primary animal model for the majority of the studies. Murine models with targeted disruption of specific genes for AT1-R (AT1-R-A), COX-1 and COX-2 (COX-1-/- and COX-2-/-), FP-R (FP-R-/-), TP-R (TP-R-/-), and H-Ras (H-Ras-/-) will be used to determine the role of Ang II, cyclooxygenases and prostanoids, and H-Ras respectively, in the IAS tone. The main focus of the grant is to determine the role of RhoA/ROK signal transduction machinery for the basal tone in the IAS, and its contraction following the neurohumoral agonists. Doing so will provide important information on the extracellular, and the intracellular molecular mechanisms governing the basal tone in the IAS. This information will have significant implications in the pathophysiology and therapeutic potentials of the IAS dysfunction. [unreadable] [unreadable] [unreadable]