Abstract
To explore the effects and possible mechanism of 17β-estradiol on the expression of small conductance Ca(2+) activated K(+) channel 3 (SK3) in rat colonic smooth muscle cells (SMC). The SMC isolated from male SD rats by enzymolysis were cultured. And double immunofluorescence staining was used to detect the co-expression of SK3 and α-actin. Colonic SMC were cultured with different concentrations of 17β-estradiol for 24 h or with 50 nmol/L 17β-estradiol at different time points respectively. The expressions of SK3 in colonic SMC were measured by real-time quantitative reverse transcription (qRT)-PCR and Western blotting. The effects of estrogen receptor (ER) inhibitor ICI 182780, albumin bovine serum-17β-estradiol (BSA-E2), ERα selective agonist propyl pyrazole triol (PPT) and ERβ selective agonist diarylpropionitrile (DPN) on SK3 expression were observed. Double immunofluorescence staining showed that SK3 and α-actin co-expressed in cultured colonic SMC. The expression of SK3 of 17β-estradiol at different concentration (10, 50 nmol/L) significantly higher than the control group (protein: 0.217 ± 0.030 and 0.321 ± 0.077 vs 0.103 ± 0.063, mRNA: 1.872 ± 0.606 and 2.967 ± 0.659 vs 0.813 ± 0.202, all P < 0.05). And 50 nmol/L was the most effective in vitro concentration. The peak expression of SK3 appeared at 12 and 24 hour (2.91 and 3.30-fold in protein vs 3.46 and 3.37-fold in mRNA respectively, all P < 0.05). The protein levels of SK3 in ICI 182780 plus 17β-estradiol group was less than 17β-estradiol group (0.111 ± 0.050 vs 0.351 ± 0.084, P < 0.05). But it was not influenced by BSA-E2. The expressions of SK3 in PPT and E2 groups were both higher than control group (0.270 ± 0.071, 0.309 ± 0.052 vs 0.087 ± 0.018, both P < 0.05) . However DPN had no effect on SK3 protein levels. SK3 is localized in rat colonic SMC. And 17β-estradiol increases its expression in an ERα-dependent manner.
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