Internal Anal Sphincter Tone in Humans

39 The extracellular signal which triggers activation of rho-associated kinase (RhoA/ROCK), the major 40 molecular determinant of basal internal anal sphincter (IAS) smooth muscle tone in humans, is not 41 known. Using human IAS tissues, the presence of the biosynthetic machineries for Ang II, TXA2 and 42 PGF2α were identified. These end products of the renin-angiotensin system (Ang II) and arachidonic acid 43 (TXA2 and PGF2α) pathways and their effects in human IAS vs. rectal smooth muscle (RSM) were 44 studied. A multipronged approach utilizing immunocytochemistry (ICC), Western blot (WB) analyses, 45 and force measurements was implemented. Additionally, a systematic analysis of the effects of respective 46 inhibitors and agonists along different steps of biosynthesis of these end products were evaluated either 47 individually or in combination. To further describe the molecular mechanism for the IAS tone via these 48 pathways, we monitored RhoA/ROCK activation, and its signal transduction cascade. Data showed 49 characteristically higher expression of biosynthetic machineries of RAS and AA pathways in the IAS as 50 compared with the RSM. Additionally, specific inhibition of AA pathway caused ~80% decrease in the 51 IAS tone, while that of RAS lead to ~20% decrease. Signal transduction studies revealed that the end 52 products of both arachidonic acid (AA) and renin-angiotensin system (RAS) pathways cause increase in 53 the IAS tone via activation of RhoA/ROCK. Both AA and RAS (via the release of their end products 54 TXA2, PGF2α, and Ang II respectively), provide extracellular signals which activate RhoA/ROCK for the 55 maintenance of the basal tone in human IAS. 56 57 58