The Effects Of Estrogen On BK_Ca Currents In Artery Smooth Muscle Cells During Hypertension

Objective:Hypertension is a common disease which is harmful to health, with main manifestations including increased arterial tension, failure of resisting higher vascular tone and making normal response to vasoactive substances. Mesenteric artery, main visceral vessels of human beings, the largest proportion of vessels in body, is the main vessel of forming peripheral resistance, which is sensitive to vasoactive substances, so has significant effects in regulating blood pressure. Large-conductance calcium-activated potassium channels (BKca), main negative feedback regulator in vascular smooth muscle, is playing a key role in relaxation. Estrogen (E2) is an important endocrine hormone in women, and has a wide range of biological effects. Epidemiologic studies indicate that gender differences exist in hypertension. Premenopausal Women have a much reduced incidence of hypertension compared with age-matched men. However, menopausal women develop increased incidence from hypertension. Laboratory researches suggest that estrogen has beneficial cardiovascular effects through their ability to modulate function; however, these mechanisms remain incompletely understood. In the present study, single channel patch-clamp techniques were used to examine the effects of E2 and the inhibitor of ER (ICI 182780) on BKCa in mesenteric artery vascular smooth muscle cells (VSMCs) during hypertension, and to explore the relation among E2, ER, BKCa and hypertension. Methods:The free mesenteric artery was cut into small segments (2 mm×2 mm) and then transferred to enzymatic dissociation solution for incubation. Single smooth muscle cells were obtained by two-step enzyme digestion at 37℃. We chose the smooth muscle cells stereo and slick for experiment. The currents were recorded by single channel patch-clamp techniques. Single (CEZ-2200), then imported into the computer. The pClamp 6.0 software was used to record data. The difference between hypertension group (HT) and normotension (NT) in BKCa currents adding drugs before and after was observed and compared. Results:1. In symmetrical high K+ solution (140 mM), We recorded single channel currents of BKCa channels on human mesenteric artery smooth muscle cells in inside-out patch. Single channel conductance of NT group was 176.3±21.1 pS (n=7) and that of HT group was 182.9±29.2 pS (n=6). There was no distinct difference (P>0.05).2. The voltage-dependence of BKCa.:In cell-attached patch and inside-out patch, the Open Probability (Po), Amplitude (Am), Mean Open Time (To) increased and Mean Close Time (Tc) of NT (n=7) and HT (n=6) decreased obviously with the increasing of holding potential (+10 mV,+20 mV,+30 mV,+40 mV,+50 mV,+60 mV) (PNT<0.01, PHT<0.01).3. The Ca2+-dependence of BKCa:In inside-out patch, the Po, To increased and Tc decreased with the increasing of calcium concentration (10-8M,10-7M,5×10-7M,10-6M, 10-5M). The different calcium concentration had no influence on the Am. There was distinct difference in Po,To between NT (n=7) and HT (n=6) (P<0.05).4. In outside-out patch clamp, the BKCa currents were suppressed by 200 nM Iberiotoxin (IbTX), a specific BKCa blocker.5. The effects of E2 and ICI 182780 on BKCa:①In cell-attached patch, E2 (100μM) activated BKCa channels apparently both in NT and HT. The Po of NT (n=7) and HT (n=6) increased from 0.0224±0.0004 to 0.089±0.009 and from 0.030±0.007 to 0.062±0.004, respectively. The distinct difference exist in NT and HT themselves and between them.②In cell-attached patch clamp, there was a inhibitory effect on BKCa after adding ICI 182780 subsequently. The Po of NT and HT decreased to 0.031±0.001 and 0.045±0.011, respectively. The difference in NT was obvious, but was not in HT and between them. (PNT<0.01, PHT>0.05, PNT/HT>0.05).③After the step②, there was no remarked effects on BKCa when adding E2 (100 uM) again. Conclusions:1. The BKCa currents of human mesenteric artery smooth muscle cells were voltage-dependent and Ca2+-dependent, however, the Ca2+ sensitivity of BKCa in HT was lower than that in NT.2. The BKCa was activated by E2 greatly, but inhibited partly by ICI 182780 subsequently. ER was involved mainly in the mechanisms of E2-induced relaxation in mesenteric artery.3. The response to E2 and subsequent ICI 182780 in HT was lower than that in NT.④E2 activated BK through ER partly.