The Effect Of Estrogen On BK_Ca Currents In Artery Smooth Muscle Cells Of Post-menopause Hypertension Women

Objective: Essential hypertension is one of common diseases which isvery harmful to health with main manifestations including increasingarterial tension and failure of resisting higher vascular tone and makingabnormal response to vasoactive substances．Mesenteric arteries are mainvisceral vessels of human beings，and they are the largest proportion ofvessels in body and the main vessels of forming peripheral resistance，which are sensitive to vasoactive substances，they play an important rolein regulating blood pressure． Large-conductance calcium-activatedpotassium channels (BK_Ca)，main negative feedback regulator in vascularsmooth muscle，is playing a key role in relaxation and contraction ofvessel. Estrogen （E） is an important endocrine hormone in women，andhas a wide range of biological effects. Epidemiologic studies indicate thatgender differences exist in essential hypertension. Premenopausal womenhave a much reduced incidence of hypertension compared withage-matched men. However, post-menopausal women develop increasedincidence from hypertension. Laboratory researches suggest that estrogenhas beneficial cardiovascular effects through their ability to modulate their function; however, these mechanisms remain incompletelyunderstood. To apply acute enzyme method to isolate women mesenteryartery smooth muscle cells and record large-conductance Ca²⁺-activatedpotassium channel currents and spontaneous transient outward currentsusing perforate whole cell patch technique, and to examine the effects ofE on BK_Caand STOCs of women mesenteric artery vascular smoothmuscle cells （VSMCs） of PNH, NH and EH groups, and to explore therelation among E, BK_Caand women essential hypertension, and toidentify that the mechanisms of effect of E on pre-menopause andpost-menopause women essential hypertension. Methods: The freemesenteric arteries were cut into small segments （2mm×2mm×1mm）under somatotype microscope and then transferred to enzymaticdissociation solution for37℃incubation．Single smooth muscle cell wasobtained by two-step enzyme digestion at37℃．We chose slick andrefracted and stereo smooth muscle cells for our experiment. The currentswere recorded by perforate whole cell patch technique. The currents wereamplified by patch clamp amplifier and deal by AD/DA translator, andimported into the computer. The Clampfit10.1, MiniAnalysis6.0andOriginPro8.0soft wares were used to analysis and deal data. Thedifference were observed and compared between pre-menopause womennon-hypertension group （PNH）, post-menopause womennon-hypertension group （NH） and post-menopause women essential hypertension group （EH） in BK_Cacurrents and STOCs after addingdrugs．Results：（1）The properties of BK_Cacurrents on mesenteric artery smoothmuscle cells by applying an acute enzyme method to isolate these cellswere as follows:â‘ T heBK_Camacroscopic currents hadvoltage-dependence and outward rectifying; The amplitude and frequencyof STOCs had voltage-dependence; STOCs were superimposedstochastically on BK_Camacroscopic currents （n=15）;â‘¡The BK_Camacroscopic currents and STOCs were inhibited by200nM Iberiotoxin（IbTX）, a specific BK_Cablocker.（2）The effects of estrogen on BK_CaandSTOCs of PNH group：100μM E could significantly activate on BK_CaofPNH group from0mV to60mV. At0mV and+60mV, the currentdensities of BK_Caof PNH group could respectively increase from1.98±0.39pA/pF、16.43±4.90pA/pF to4.45±1.25pA/pF（P<0.05, n=7）、32.36±6.86pA/pF（P<0.01, n=7）. At-20mV, The amplitude andfrequency of STOCs of PNH group could respectively increase from8.98±2.56pA、3.05±0.84Hz to16.88±3.90pA（P<0.05, n=7）、5.26±1.46Hz（P<0.01, n=7）after adding100μM E.（3） The effects of estrogen onBK_Caand STOCs of NH group：100μM E could significantly activate onBK_Caof NH group from0mV to60mV. At0mV and+60mV, the currentdensities of BK_Caof PNH group could respectively increase from1.99±0.41pA/pF、15.89±4.87pA/pF to3.26±1.32pA/pF（P<0.05, n=23）、 27.88±6.75pA/pF（P<0.01, n=23）. At-20mV, The amplitude andfrequency of STOCs of PNH group could respectively increase from8.56±2.43pA、2.98±0.77Hz to14.75±3.78pA（P<0.05, n=23）、4.70±1.57Hz（P<0.01, n=23）after adding100μM E.（4） The effects ofestrogen on BK_Caand STOCs of EH group：100μM E could notsignificantly activate on BK_Caat-50mV to+60mV. There was activeeffect only+60mV. At0mV and+60mV, the current densities of BK_CaofEH group could respectively increase from1.35±0.44pA/pF、4.48±1.47pA/pF to1.48±0.55pA/pF（P>0.05, n=17）、7.17±2.47pA/pF（P<0.05,n=17）. At-20mV, The amplitude and frequency of STOCs of EH groupcould respectively increase from5.67±1.73pA、1.28±0.43Hz to6.58±1.34pA（P>0.05, n=17）、1.90±0.99Hz（P<0.05, n=17） after adding100μM E.（5） Comparisons of effects of estrogen on BK_Camacroscopiccurrents and STOCs between PNH, NH and EH groups.â‘ At+60mV,the current densities of BK_Caof PNH group increased0.97±0.40timesafter adding100μM E. At-20mV, the amplitude and frequency of STOCsof PNH group respectively increased0.88±0.43and0.72±0.39times afteradding100μM E.â‘¡At+60mV, the current densities of BK_Caof NHgroup increased0.75±0.47times after adding100μM E. At-20mV, theamplitude and frequency of STOCs of NH group respectively increased0.72±0.32and0.58±0.34times after adding100μM E.â‘¢At+60mV, thecurrent densities of BK_Caof EH group increased0.60±0.33times after adding100μM E. At-20mV, the amplitude and frequency of STOCs ofEH group respectively increased0.16±0.11and0.48±0.30times afteradding100μM E.（6） Effects of ICI182780on BK_Caand STOCs ofwomen mesenteric artery smooth muscle cells. At+60mV, the currentdensities of BK_Caof women mesenteric artery smooth muscle cells couldincrease from15.89±4.87pA/pF to27.88±6.75pA/pF（P<0.01, n=23）.There was inhibitory effect on BK_Caafter adding ICI182780subsequently. The current densities of BK_Caof women mesenteric arterysmooth muscle cells could decrease to20.15±6.21pA/pF （P<0.05, n=23）.At-20mV, The amplitude and frequency of STOCs of women mesentericartery smooth muscle cells could increase from8.56±2.43pA、2.98±0.77Hz to14.75±3.78pA（P<0.05, n=23）、4.70±1.57Hz（P<0.01,n=23）after adding100μM E. There was inhibitory effect on STOCs afteradding ICI182780subsequently. The amplitude and frequency of STOCsof women mesenteric artery smooth muscle cells could respectivelydecrease to10.87±3.45pA （P<0.05, n=23） and3.52±0.98Hz （P<0.05,n=23）.（7） The effects of L-NAME on BK_Caand STOCs of womenmesenteric artery smooth muscle cells. BK_Cacould obviously be activatedafter adding100μM E on women mesenteric artery smooth muscle cells.At+60mV, the current densities of BK_Caof women mesenteric arterysmooth muscle cells could increase from17.52±4.25pA/pF to25.83±5.27pA/pF（P<0.05, n=4）.There was inhibitory effect on BK_Caafter adding 600μM L-NAME subsequently. At+60mV, the current densities of BK_Cadecreased to14.25±3.57pA/pF （P<0.05, n=4）; At-20mV, the amplitudeand frequency of STOCs respectively could increase from7.24±3.27pAand2.04±0.74Hz to9.85±2.62pA （P<0.05, n=4） and2.25±0.65Hz（P<0.05, n=4）after adding100μM E. The amplitude and frequency ofSTOCs respectively decreased to6.24±1.86pA （P<0.05, n=4） and1.65±0.54Hz （P<0.05, n=4）. Conclusions:â‘´T heBK_Camacroscopiccurrents of mesenteric artery SMC had voltage-dependence and outwardrectifying; STOCs were induced by BK_Ca, the amplitude and frequency ofSTOCs had voltage-dependence and randomness.⑵Estrogen couldactivate BK_Camacroscopic currents and the amplitude and frequency ofSTOCs on PNH group, NH group, EH group. Compared with PNH group,the effect of estrogen on BK_Caand the amplitude and frequency ofSTOCs in NH was lower. That suggest that E play an important role inpre-menopause women’ heart protection. Compared with NH group, theeffect of estrogen on BK_Caand the amplitude and frequency of STOCs inEH was lower. These data suggest that the responsiveness of effect ofestrogen was lower on blood vessel after hypertension and menopausewas a factor of happening of hypertension.（3）E could make womenmesenteric artery relax, and the effect could partly be inhibited by ICI182780, so BK_Caand ER were involved mainly in the mechanisms ofE-induced relaxation in mesenteric artery．（4） Nitric oxide synthesis L-NAME could partly inhibit the relaxation effect of E on womenmesenteric artery，so NO took part in the mechanism of the relaxationeffect of E on mesenteric artery.