The Effect Of Small-Conductance Ca²⁺-Activated K⁺ Channel 3 Expression On Relaxation Of Rat Colonic Smooth Muscle Regulated By Estrogen

BackgroundChronie constipation?CC?is one of the most common global chronic diseases.The morbidity of CC is 6%?80 million people?in China.And studies reported that the symptoms and incidence of CC are sex-dependent,indicating that female hormone may be one of the factors that cause CC.Muscle strip experments in vitro have confirmed that estrogen rapidly in hibits colonic dilatation and prevents gastric emptying.Physiological effects of estrogens are mediated by the classical nuclear estrogen receptor alpha and estrogen receptor beta,which mediate both genomic and rapid signaling events.In addition,estrogens induce rapid non-genomic responses through a membrane-associated G protein-coupled estrogen receptor?GPER?.Ion channel is one of the key factors affecting gastrointestinal motility.Small-Conductance Ca²⁺-Activated K⁺Channel?SK?,which are Ca²⁺-sensitive,non-voltage-dependent,selsctive K⁺channels,are involved in the inhibition of colonic contrcile activity.SK family include SKI,SK2 and SK3 which express abundantly in colonic smooth muscel cell?SMC?.Our previous study has indicted that 17?-estradiol increases SK3 expression in an ERa-dependant manner.Furthermore other experiments prove that KCNN3 transcription which is SK3 coding gene can be regulated by specificity protein transcription factors?Sp?1 and 3 in response to stimulation by estrogen in uterin SMC.Based on the above,our study would investigate the effect of SK3 expression on relaxation of rat colonic smooth muscle regulated by estrogen to provide a new strategy for female CC patients.Objectives1.To investigate the expression of SK3 and constrction of smooth muscel in rat colonic smooth muscel regulated by estrogen?17?-estrodiol,E2?.2.To investigate the effect of E2 and SK3 overexression on calcium mobilization in rat colonic SMC.3.To investigate the effect of Sp1 and Sp3 expression regulated by E2.Methods1.Animal groups and intervention:female Sprague-Dawley?SD?rats were randomly divided into 4 groups,and all groups were under ovariectomy before.A 3 0-mm silicone tube filled with different solution was subcutaneously implanted in different groups of rats.Group C was filled with the solvent;Group E was E2 in com oil?0.3 mg/mL?which can keep serum levels of E2 at physiological levels;Group EI was ICI 182780?estrogen receptor[ER]antagonist?plus E2 and group BSA-E2 was with BSA-E2.The contraction of muscle strips and levels of the SK3 protein expression in the colonic smooth muscle were detected 14 days after tube implanted.2.Isolation and culture of colonic SMCs:The colon was taken from male SD rats.Smooth muscle layer was removed from the serosa and mucosa and then was cut into pieces.SMCs were isolated by enzymatic digestion and cultured with DMEM.?1?Effect of E2 on calcium mobilization in rat colonic SMCs:Colonic SMCs were cultured with E2?50nmol/L?for 24h and Ca²⁺concentration in cells were detected after CCh stimulating by laser scanning confocal microscope.?2?Effect of SK3 overexression on calcium mobilization in rat coloruc SMCs:Ca²⁺concentration in cells were detected after CCh stimulating by laser scanning confocal microscope after SK3 overexpression in rat colonic SMCs.?3?the effect of Sp1 and Sp3 expression regulated by E2:Sp1 and Sp3 expression were detected after E2 stimulating SMCs by western blot and qPCR..Results?1?The expression level of SK3 in colonic muscle tissues:Compared with the other groups,the expression of SK3 in colonic muscle tissues in group E was significant increased?1.46±0.344 vs 0.67±0.143,0.51±0.066,0.62±0.225,P<0.05?;While there was no significant difference in group BSA-E2 compared with group C and El?1.46±0.344 vs 0.67±0.143,0.51±0.066,0.62±0.225,P<0.05?.?2?The constrction of rat smooth muscle:Compared with the other groups,the constraction of group E ignificantly decreased?1.88±0.244 vs 4.09±0.405,4.70±1.269,4.57±0.392,P<0.05?;While there was no significant difference in group BSA-E2 compared with group C and El?4.57±0.392 vs 4.09±0.405,4.70±1.269,P>0.05?.?3?The effect of E2 and SK3 overexression on calcium mobilization in rat colonic SMC:Compared with the control group,E2 can inhibit the increase Ca²⁺ in SMCs after CCh stimulating?1.085±0.68 vs 2.021±0.384,P=0.001?,and so as the SK3 overexpression?1.478±0.260 vs 0.690±0.140,1.070±0.149,P<0.05?.?4?The effect of E2 on Sp1 and Sp3 expression:Compared with control group the RNA expression of Sp1 increased significantly?1.44±0.012 vs 0.76±0.169,P<0.05?.The protein expression also increased but there was no significant difference?1.62±0.060 vs 1.42±0.171,1.40±0109,P>0.05?;While the RNA and protein expression both decreased significantly?1.34±0.246 vs 3.70±0.339,4.97±0.564,P<0.01?.Conclusions?1?E2 treatment increased SK3 expression and inhibit rat colon smooth muscle constrction through by the classical nuclear estrogen receptor.?2?The increase of SK3 expession regulated by E2 deceased calcium mobilization in rat colonic SMCs.?3?E2 treatment increased Sp1 expression and decreased Sp3 expression which would both increased SK3 expression.