Development of Methodology and Data Acquisition

Infantile hypertrophic pyloric stenosis (IHPS): A study of its pathophysiology utilizing the newborn 1 hph-1 mouse model of the disease 2 3 Running Title: Rho-kinase and infantile pyloric stenosis 27 28 29 30 31 Abstract 33 Infantile hypertrophic pyloric stenosis (IHPS) is a common disease of unknown etiology. The 34 tetrahydrobiopterin (BH4)-deficient hph-1 newborn mouse has a similar phenotype to the human 35 condition. hph-1 and wild-type control animals' pyloric tissue agonist-induced contractile properties, 36 reactive oxygen species (ROS) generation, cGMP, neuronal nitric oxide synthase (nNOS) content, as well 37 as, ROCK-2 expression and activity were evaluated. Primary pyloric smooth muscle cells from wild-type 38 newborn animals were utilized to evaluate the effect of tetrahydrobiopterin (BH4) deficiency. One-39 week old hph-1 mice exhibited a 4-fold increase (P<0.01) in the pyloric sphincter muscle contraction 40 magnitude, but similar relaxation values, when compared with wild-type animals. The pyloric tissue 41 nNOS expression and cGMP content were decreased, while the rate of nNOS uncoupling increased 42 (p<0.01) in one-week old hph-1 mice, when compared with wild-type animals. These changes were 43 associated with increased pyloric tissue ROS generation and elevated ROCK-2 expression/activity 44 (p<0.05). At 1-3 days of age and during adulthood, the hph-1 mice's gastric emptying rate was not 45 altered and there were no genotype differences in pyloric tissue ROS generation, nNOS expression or 46 ROCK-2 activity. BH4 inhibition in pyloric smooth muscle cells, resulted in increased ROS generation 47 (p<0.01) and ROCK-2 activity (p<0.05). Oxidative stress upregulated ROCK-2 activity in pyloric, but no 48 changes were observed in newborn fundal tissue in vitro. We conclude that ROS-induced upregulation 49 of ROCK-2 expression accounts for the increased pyloric sphincter tone and nNOS downregulation in the 50 newborn hph-1 mice. The role of ROCK-2 activation in the pathogenesis of IHPS warrants further study. 51 52