| BackgroundConstipation, one of the most common gastrointestinal tract disorders, is affecting people’s physical and mental health and quality of life. Recently, more and more studies reported that the symptoms and incidence of constipation are sex-dependent, suggesting that colonic motility is associated with female hormone. Muscle strip experiments in vitro have confirmed that estrogen rapidly inhibits colonic smooth muscle contraction, and animal studies have determined that estrogen induces colonic dilatation and prevents gastric emptying. Furthermore, ion channel is one of the key factors affecting gastrointestinal motility. Throughout the years, much attention has been paid to the functional small conductance Ca2+ activated K+(SK) channels. SK channels, which are Ca2+-sensitive, non-voltage-dependent, selective K+ channels, are involved in the inhibition of colonic contractile activity. Estrogen not only directly affects the function of SK channels, but also mediates classical actions on SK expression in GnRH neurons, myometrial cells and so on. However, it is still not well understood whether SK channels involve in the estrogen (17β-estradiol)-induced colonic relaxation or not.Objective1. To determine effects of SK channels on rat colonic relaxation caused by estrogen (17β-estradiol).2. To examine the effect and the possible mechanism of estrogen (17β-estradiol) on the expression of small conductance Ca2+ activated K+ channels 3 (SK3) in rat colonic smooth muscle cells (SMCs).Methods1. Tissue preparation:Colon tissues were isolated from distal colon region of male Sprague-Dawley (SD) rats and the circular colon smooth muscle layer was carefully separated from the mucosal layer. The muscle was then dissected into strips measuring approximately 0.3×0.8 cm.(1) Effects of E2 and apamin on colonic carbachol (CCh)-induced contractions:As soon as a transient plus produced by CCh, drugs such as vehicle DMSO, 17β-estradiol (E2) or apamin (SK channels blocker), respectively, were added directly to the bath solution, and the time course of muscle activity was recorded.(2) Effects of apamin on the response to estradiol:As soon as a transient plus produced by CCh, circular muscle strips were allowed to preincubate for an additional 10 min with apamin before exposure to E2.2. Isolation and culture of colonic SMCs:The colon was taken from male SD rats and rinsed repeatedly with Hepes-Ringer solution. The serosa and mucosa were removed and the remaining muscularis was cut into pieces. Then the colonic SMCs was achieved by enzymolysis and cultured with DMEM.(1) Expression of SK channels in colonic SMCs: ① Double-immunohistochemical staining of rat colonic SMCs for a-Actin, SK2 and SK3.② Total RNA was analyzed by qRT-PCR for SK2 and SK3 transcripts.(2) Effect of E2 on the expression of SK3 in colonic SMCs:Colonic SMCs were cultured with different concentrations of E2 for 24 h or with 50 nmol/L E2 for different periods of time. The expression of SK3 in colonic SMCs were measured by qRT-PCR and Western blot.(3) Role of nuclear estrogen receptors (nERs) in E2-induced SK3 expression:Colonic SMCs were treated with E2, ICI 182780 (estrogen receptor inhibitor) or Albumin Bovine Serum conjugated 17β-estradiol (BSA-E2), and then the expression of SK3 was measured by Western blot.(4) Role of ERα in E2-induced SK3 expression:Colonic SMCs were treated with E2, PPT (ERa selective agonist) or DPN (ERβ selective agonist), and then the expression of SK3 was measured by Western blot.Results1. E2 inhibits colonic contractility while the tissues responded to apamin, an SK channels inhibitor, with a transient increase of tension, after CCh-induced peak contractions.2. Preincubated with apamin partially prevent the E2-induced relaxation.3. Double immunofluorescence staining showed that two subtyps of SK channels, SK2 and SK3 are co-expressed with a-Actin, respectively, in colonic SMCs. But the mRNA quantitative ratio of SK transcriptional expression was SK3> SK2.4. E2 treatment significantly increased the expression of SK3. The peak expression of SK3 was showed at the 12th and 24th hour after stimulating with 50 nmol/L E2, which was considered as the most effective concentration in vitro.5. The expression of SK3 was down regulated by ICI 182780, an estrogen receptor inhibitor, but was not influenced by BSA-E2.6. ERa selective agonist PPT treatment significantly promoted SK3 expression, exerting an effect similar to that of E2, while the addition of ER|3 selective agonist DPN had no effect on SK3 expression.Conclusions1. E2 treatment inhibits rat colonic circular muscle contraction, which was partially blocked following apamin pretreatment. In other words, the activated apamin-sensitive SK channels was one of machanisms involved in the relaxing effect of E2.2. Two subtypes of apamin-sensitive SK channels SK2 and SK3 are located in rat colonic SMCs, with different mRNA levels(SK3> SK2).3. E2 increased the expression of SK3 in a ERa-deoendant manner. |
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