The internal anal sphincter (IAS) maintains 70% of anal resting tone, which is critical for maintaining fecal continence. Relaxation of contraction is an essential feature of defecation, and this is regulated by input from inhibitory nerves. Reduced and increased anal tone contribute to fecal incontinence and defecatory disorders respectively. Our understanding of the mechanisms which maintain normal IAS contraction is limited. Recent studies have emphasized the role of the Rho kinase pathway, but there are only 4 reports which have investigated the role of membrane potential (Em) in the regulation of this clinically important region of the GI tract. Our preliminary data suggest that L-type Ca2+ channels (Cav1.2), regulated by Em, are fundamental to the delivery of Ca2+ to the contractile apparatus and for determining the pattern of contractile activity in the IAS. Preliminary experiments also suggest that at least a portion of the regulation of contraction by Rho kinase may involve changes in Em. Our immunohistochemical studies of the IAS have revealed that interstitial cells of Cajal (ICC) are densely distributed throughout the muscularis of the IAS. However, the contribution of these cells to Em and electrical rhythmicity in the IAS is unknown. Thus, our overall hypothesis for these studies is that contraction in the IAS and the changes elicited by inhibitory motor nerves is primarily regulated by Em due to its effects upon Cav1.2 activity and that ICC play an important role in this process by modulating Em in adjacent SMCs. To examine this hypothesis Em and contraction will be measured in isolated segments of the monkey and mouse IAS. ICC and smooth muscle cells (SMC) will also be dispersed from these tissues to evaluate ion channel activity and sorted using fluorescence activated cell sorting (FACS) to evaluate gene expression. The Rho kinase pathway will be assessed using biochemical techniques and the distribution of nitrergic nerves and their relationship to ICC and SMC will be determined using immmunohistochemical and immunocytochemical techniques. Novel approaches involving newly developed transgenic mice models will also be used to study and eliminate ICC. Dysfunction of the IAS leads to substantial social isolation, loss of employment, and institutionalization as well as extracting billions of dollars in health care costs in the United States. In spite of this large health care problem the IAS is the least studied region of the GI tract. The proposed study will provide new insights into the mechanisms which control muscle contraction and hence anal pressure. Such information is critical for developing new strategies to help prevent as well as diagnose and treat IAS dysfunction.