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.