Gastrointestinal (GI) motility is sexually dimorphic. In women, GI motility varies with hormonal changes during the menstrual cycle and pregnancy. However, there is a gap in the knowledge and understanding the role of estrogen and the mechanism by which it regulates sex- and cycle-dependent changes in GI motility and dysmotility. Unlike classical nuclear receptors ERα, ERβ, activation of membrane-bound G protein-coupled estrogen receptor (GPER) by estrogen initiates rapid cellular events. We have obtained preliminary evidence to show that GPER is expressed in smooth muscle and is coupled to AC/cAMP/PKA pathway to mediate muscle relaxation in the stomach and the colon. Smooth muscle from GPER KO mice exhibited loss of relaxation in response to GPER agonist providing evidence for a role of GPER in smooth muscle relaxation. GI transit was delayed in female mice of high-estrogen proestrus and estrus phases compared to low-estrogen diestrus phase, and male mice. These sex- and cycle-dependent changes in GI transit are associated with changes in the expression and function of GPER. Based on these preliminary data, studies are proposed to test the hypothesis that sex- and cycle-dependent changes in GI transit are due to changes in expression and function of GPER coupled to activation of AC/cAMP/PKA pathway and smooth muscle relaxation (Specific Aim 1). GI motility disorders are also sexually dimorphic. There is greater prevalence of diabetes-associated gastroparesis and constipation in women compared to men. The contribution of impaired smooth muscle function and role of GPER in smooth muscle has not been fully explored in sexual dimorphism in GI dysmotility in diabetes. Preliminary studies showed that expression of GPER was decreased in smooth muscle from diabetic mice and the decrease is mediated via oxidative stress-induced changes in epigenetic regulation via a decrease in trimethylation of H3 at lysine residue 4 (H3K4me3) and acetylation of H3 at lysine residue 27 (H3K27ac) at the promoter region of GPER gene. RNA-seq data combined with heatmap analysis showed an increase in the expression of selective histone deacetylase (HDACs) in smooth muscle from diabetic mice. An increase in oxidative stress was also obtained in smooth muscle from GPER KO mice suggesting the increase in oxidative stress in diabetes could be due to a decrease in GPER expression. Based on the preliminary data, studies ae proposed to test the hypothesis that an increase in oxidative stress in diabetes downregulates GPER expression via decrease in H3K4me3 and H3K27ac of GPER promoter, and loss of GPER-mediated protection against oxidative stress leads to decrease in cAMP/cGMP signaling, muscle relaxation and GI transit (Specific Aim 2). Knowledge gained from our study is intended to provide insights into the cellular mechanisms involved in sex-and cycle-dependent changes in GI motility. The unknown underlying etiology of female sex-specific prominence of diabetes-associated GI ...