The Regulation Of G Protein Coupled Estrogen Receptor (GPER) Expression In Colonic Myenteric Plexus And Mechanisms Of GPER Participating In Colon Transit

ObjectivesClinical and epidemiological studies show that the colonic transit speed changes for women during menstrual cycle and pregnancy. Constipation was found more frequently in women than in man.These phenomenon indicate that estrogen may play an important role in the regulation of colonic movement. However, mechanisms are still unclear. Thus, firstly we aimed to localize the novle membranous estrogen receptor-GPER (G protein coupled estrogen receptor) in the colon of female C57BL /6 mice and explore the variation of GPER expression in myenteric neurons over estrous cycle, as well as the potential mechanism. Secondly, we tried to investigate whether GPER was involved in regulating colonic motor functions and explored the underlying physiological mechanisms. In the end, we studied the role of estrogen-GPER pathway in the development of slow transit constipation.Methods and ContentsFollowing the requirements of experiments female or male adult C57BL/6 mice were used.Part One:The expression and localization of GPER were examined by RT-PCR, western blot, immunohistochemistry and immunofluorescence. GPER expression over estrous cycle was tested by real-time PCR and Western blot. The time-dependent effect of exogenous estrogen on GPER expression was tested by RT-PCR and western blot following exposure to 17β-Estradiol (E2) at different time points in myenteric neurons. In order to determine the potential mechanisms that estrogen regulated GPER expression, several inhibitors were employed, i.e.G15 as the selective GPER bloker and ICI as the classical nuclear estrogen receptor. Following pre-treated with the inhibitor the GPER expression evoked by E2 was evaluated by RT-PCR and western blot.Part Two:The role of GPER in modulating colonic motor functions was assessed by the bead propulsion test in vivo. We detected the fluctuation of colon transit time (CTT) over estrus cycle or following acute E2 supplement in ovariectomized mice and the effect of GPER on colonic transmission in vivo. Organ bath experiments was used to record the movement of colon circular muscle in vitro and to detect the effect of selective activation of GPER on carbachol induced contraction of colon muscular strips in the presence and absence of tetrodotoxin or the neuronal nitric oxide synthase (nNOS) inhibitor N-propyl-L-arginine(NPLA). Immunofluorescence, nitrate reductase method and laser confocal microscope were employed to test the influence of estrogen and selective GPER agonists G1 on generation of nitric oxide (NO) in myenteric neurons. In order to identify the role and mechanism of E2 on nNOS expression in colon tissue we tested the nNOS protein expression following exogenous estrogen treatment and estrogen supplement in OVX mice with immunohistochemistry and western blot. Finally, we established the slow transit costipation model with intragastric cold water stress to observe the role of E2-GPER pathway in colon transit time in slow transit costipation.ResultsPart One:1. GPER was expressed in the submuscoal ganglion, myenteric ganglion and in the epithelial cells in intestinal crypts.2. GPER expression fluctuated depending on serum estrogen level over estrous cycle.3. Pre-treated with exogenous estrogen, GPER expression in myenteric neurons increased at 6 hours and peaked at 12 hours.4. Selectively blocking GPER didn’t affect the role of exogenous estrogen on GPER expression. On the contrary, selective estrogen nuclear receptor inhibitor blocked the increasing of GEPR mRNA and protein expression induced by exogenous estrogen.Part Two:1. The colonic transit time (CTT) in proestrus and estrus was significantly longer than that in diestrus. In vivo treatment with the selective GPER blocker G15 significantly shortened CTT in proestrus and estrus. In ovariectomized mice, acute estrogen supplementation increased CTT, which could be abolished by G15 co-administration.2. The GPER agonist G1 caused a concentration-dependent inhibition of carbachol-induced circular muscle strips contraction, which was abolished by tetrodotoxin and NPLA。3. Both E2 and selective GPER agonists stimulated NO production in isolated longitudinal muscle myenteric plexus and cultured myenteric neurons, which was dependent on nNOS.4. Immunofluorescence labeling showed co-localization of GPER with nNOS in the myenteric plexus.5. E2 replacement therapy increased nNOS expression in the colon of ovariectomy mice. Following exogenous E2 treatment, nNOS protein expression increased in myenteric nerve plexus. Selective GPER blocker G15 could blocke the effect of E2 on nNOS expression.6. Estrogen supplementation further increased CTT in a model of slow transit constipation, which could be abolished by G15 co-administration.ConclusionsGPER is expressed in colonic myenteric neurons and the expression of GPER appears to be positively regulated by the circulating estrogen level. Estrogen nuclear receptor may be involved in the GPER expression induced by E2.GPER may be involved in mediating the inhibitory effect of estrogen on the colonic motility. Activation of GPER exerts an inhibitory effect on colonic motility by promoting NO release from myenteric nitrergic nerves depending on nNOS. Activation of GPER in myenteric neurons may have an important role in the pathogenesis of slow transit constipation.