Role Of 17-β Estradiol In The Nitric Oxide Production Of Interstitial Cells Of Cajal

Background:Slow transit constipation (STC) is traditionally considered and classified as a colonic motor disorder with a character of measurably delayed movement of materials through the colon. Previous large studies have uniformly demonstrated that the prevalence of slow-transit constipation (STC) in females is higher and the severity is greater than in males. Most of the patients that require surgical treatment for intractable constipation also are women. Factors such as pelvic surgery, childbirth, central neuronal disease and spinal injury etc may be explanations for some of female patients but not for all; especially those whose symptoms started in childhood or adolescence. They are termed"idiopathic"STC and considered as an almost exclusively female disease.Female sex hormones have been suggested as important causative factors because pregnancy has a major effect on motility throughout the gastrointestinal tract and constipation is a common complication of pregnancy. Among the female hormones, estrogen and progesterone are candidates because constipation has connection with estrogen and progesterone during pregnancy and colonic transit time is longer during the luteal phase with highest level of estrogen and progesterone than during the follicular phase of the menstrual cycle. Further study reveals over expression of progesterone receptors cause down-regulation of contractile G proteins and up-regulation of inhibitory G proteins lead to an impaired colonic muscle contraction and may contribute to STC in females. The mechanism responsible for estrogen and the colonic motility disorder, however, remains unexplained.Interstitial cells of Cajal (ICC) have been suggested as pacemaker cells in the gastrointestinal tract. They generate and propagate electrical slow waves. And slow waves are coupled within ICC networks, and are transmitted to the smoothmuscle cells through gap junctions. ICC also show a highly branched morphology and form networks associated with the enteric nervous system in the gastrointestinal musculature. Recent studies have shown functional evidence about the role of ICC as mediators of neurotransmission, Synthesized by nitric oxide synthase (NOS) from the amino acid L-arginine, Nitric oxide (NO) is a major nonadrenergic noncholinergic inhibitory neurotransmitter in the gastrointestinal tract. ICC play an important role in NO-mediated inhibitory signal effects. On the other hand, ICC of several species have been shown to contain NOS and produce NO. ICC may possess a means to amplify NO signaling in GI muscles.The various effects of estrogen are mediated by estrogen receptors(ERs). There are tw o kinds of estrogen receptors, ERαand ERβ, both of which are members of superfamily of st eroid hormone receptors. ERαand ERβhave considerable homology and, like all other steroid hormone receptors, are transcriptional factors. The classicalm model of steroid ac tionin involves rapid diffusion of the hormone into the target cell and combination with a hi gh affinity cytosolic/nuclear receptor. The hormone-receptor complex then binds to s pecific DNAs equences, the hormone response elements, resulting in altered transcription of specific mRNA and subsequent protein synthesis. Most effects of steroids are mediated by this genomic pathway, which is called "genomic effects".However, there is increasing evidence that estrogens can rapidly modulate cell functions through nongenomic actions. These alternative mechanisms involve short-term activation of signals generated from cytoplasmic and/or cell membrane receptors, inducing downstream regulatory cascades such as the mitogen-activated protein kinases (MAP kinases), the phosphatidylinositol 3-kinase (PI3-kinase) and various tyrosine kinase cascades through nongenomic mechanisms. Some of these nongenomic effects seem to be ascribed to ERs located in the plasma membrane since they can be reproduced by estrogen forms that do not pass across the plasma membrane such as the estrogen-bovine serum albumin conjugates. Estrogens exert cardioprotective effects linked at least in part to increased NO production by endothelial nitric oxide synthase (NOS III). As estrogens activate rapidly several kinase cascades, we postulated that nongenomic estrogen action may modulate NOS activity by increasing of NOS activity in ICC. In the present study we investigated the effect of estrogen on ICC and now report evidence from molecular and functional studies indicating multiple components of a novel estrogen signal transduction cascade in ICC. Our hypothesis is that estrogen stimulates production of NO via activation of NOS in ICC by a mechanism involving the MAPK signaling system.Objective: The aims of this study were:(1) To demonstrate that plasma membrane estrogen binding sites are present on ICC and that cell impermeant estrogens rapidly trigger multiple key signaling cascades; To explore the correlation between key signaling cascades and membrane estrogen binding sites;(2) To study the acute effects of 17β-estradiol (E2) treatment on ICC; To investigate hormone-activated rapid responses in ICC such as NO production, NOS activity;(3) To explore the correlation between key signaling cascades and those hormone -activated rapid responses; To demonstrate estrogen play an important role in aetiolgy of female STC.Methods:Enzymatic digestion and ficoll density centrifugation were used to dissociate ICC from murine small intestine. ACK2, the antibody of c-kit, was used to identify the cultured ICC of murine small intestine. Both light microscope and fluorescence microscope were used to observe ICC of murine small intestine during the primary culture. E2-BSA covalently linked to FITC, was used to identify E2 binding cite on the membrane of ICC. Rapid effects of estrogens on Nitric Oxide (NO) production in ICCs using E2 and its membrane impermeant albumine conjugated form (E2-BSA) were tested by NO fluorescence studies and NOS activity assay.Results:(1) Immunofluorescence showed the staining pattern of nonfixed, nonpermeabilized ICC incubated with E2-BSA-FITC. This punctate membrane staining pattern is remarkably similar to that previously described in human breast cancer cell (MCF-7) and. human vascular endothelial cells (HUVEC). (2) Identically prepared ICC, fixed and permeabilized, didn't demonstrate typical nuclear E2 binding. We could only detect labeling of E2 binding sites on cell surface but not in cell nuclei.(3) No fluorescence is found among cells incubated with BSA-FITC and cells preincubated with ER antagonist ICI 182,780. The results suggested that FITC labeled bingding cite is E2 specific.(4) The plasma membrane from ICC were isolated from the postnuclear supernatant fraction and then were assayed for to verify its purity. The Activity of Na+-K+-ATPase in membrane fraction and cytosolic fraction were 10.23±1.54μmolpi/mg/h and 0.72±0.31μmolpi/mg/h respectively.The results indicated that the purified membrane fraction consisted of plama membrane.(5) The radioligand binding assay was analyzed by Scatchard software, The Bmax of mER was 45.75 fmol/mg protein and the KD was 0.7173 nmol/L.(6) Western blot assay showed that both E2 and E2-BSA rapidly induced phosphorylation of ERK1/2 within 5 min, E2-induced phosphorylation of ERK1/2 reach its peak at 15min and rapidly declined thereafter. The effect of E2 on ERK1/2 activation was abolished by a selective estrogen receptor antagonist, ICI182,780.(7) E2 rapidly induced phosphorylation of ERK1/2 within 5 min, E2-induced phosphorylation of ERK1/2 reach its peak at 15min and rapidly declined thereafter. The effect of E2 on ERK1/2 activation was abolished by a selective estrogen receptor antagonist, ICI182780.(7) Western blot assay not only confirmed the expression of neuronal nitric oxide synthase (nNOS) in ICC, but also showed E2 had no effect on the expression nNOS protein in short time.(8) NOS assay showed that both E2 and E2-BSA rapidly stimulated NOS activity, The NOS acitivity reach its peak at 15min and rapidly declined thereafter. The effect of E2 and E2BSA on nNOS activation were abolished by ICI182,780, a selective estrogen receptor antagonist; and UO126, an inhibitor of ERK1/2.(9) In accordance with the results of NOS assay, immunofluorescence showed both E2 and E2-BSA rapidly increased the NO production of ICC. These effects were abolished by ICI182,780 and UO126.Conclusions:(1) A form of the estrogen receptor is present within the cell membrane of ICC and capable of binding to extracellular E2. No nuclear estrogen receptor can be detected in ICC. The Bmax of mER was 45.75 fmol/mg protein and the KD was 0.7173 nmol/L.(2) Membarne estrogen receptor mediates the requisite downstream signal transduction pathways including ERK1/2, which enable stimulation of nNOS.(3) Estrogen increase nitric oxide production by rapidly activating nNOS, which is mediated by membrane estrogen receptor and independent on nNOS protein synthesis.