| Objective:Hypertension is one of high risk diseases displaying increased arterial tension, and failure of resisting higher vascular tone and making normal response to vasoactive substances. As one of the main human visceral vessels, mesenteric artery holds the largest proportion of vessels in the body and forms peripheral resistance. In addition, mesenteric artery is sensitive to vasoactive substances, and therefore has a significant effect on the regulation of the blood pressure. Large-conductance calcium-activated potassium channels (BKCa), the main negative feedback regulator in vascular smooth muscle, plays a key role in the relaxation. Testosterone (TES) is a steroid and sex hormone released by body's endocrine system, and has a wide range of biological effects. The incidence of cardiovascular diseases is higher in men than in pre-menopausal women. TES had been thought to be a risk factor for cardiovascular. However, several studies have shown that TES-induced vasorelaxation may be involved in many cardiovascular protective effects. The mechanisms of protective effects remain unclear; but the activation of K+ channels in vascular smooth muscle cells (VSMCs) is possible to be involved in these effects. In this study, vascular rings and whole-cell patch clamp techniques are used to examine the effects of TES on BKCa in mesenteric artery VSMCs of hyper-tension patients, and therefore to explore the potential role of TES during hypertension. Methods:(1)Vascular rings:The mesenteric artery was quickly detached in solution I, the free artery was cut into vascular rings (0.3-0.4 cm). Vascular rings were suspended with two silver wires and fixed in a 37℃chamber with 95%O2 and 5%CO2. Vascular rings were linked to electro-physiolograph with tension transducer and biological amplifier. Observe the effects of TES on the tension of mesenteric artery vascular rings between hypertension group (HT) and normotension group (NT). KCl was used to test activity of vascular rings and cause contraction. (2)Patch clamp: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℃. Chosen the smooth muscle cells stereo and slick for experiment. The currents were recorded by a whole-cell, amphotericin-perforated configuration of patch-clamp techniques. The currents were amplified and filtered by patch clamp amplifier, and the digital datas were recorded by a Lenovo-compatible personal computer running Clampex 10.1 software and further analyzed by Clampfit 10.1 and MiniAnalysis6.0 software. The difference between HT and NT in BKCa macroscopic currents and spontaneous transient outward currents (STOCs) was observed and compared. Results:(1)The effects of TES on the tension of mesenteric artery vascular rings in HT and NT:①The increased tension by KCl (40 mM) was 2.26±0.91 in HT (n=10) and 2.00±0.49 in NT (n=10), indicated no significant difference between two groups (P>0.05);②The tension of mesenteric artery vascular rings was decreased by treating with TES, and IbTX (100 hM) could partly reverse it (n=5);③The decreased tension by TES in HT (57.98±19.67%, n=12) was lower than that in NT (74.67±24.24%, n=15) (P<0.05). (2)The features of BKCa currents in mesenteric artery smooth muscle cells:①The BKCa macroscopic currents were voltage-dependent and outwardly rectifying; The amplitude and frequency of STOCs were also voltage-dependent; STOCs were superimposed stochastically on the whole-cell BKCa macroscopic currents (n=10);②The BKCa macroscopic currents and STOCs were suppressed by 100 nM Iberiotoxin (IbTX), a specific BKCa blocker. (3) Comparisons of BKCa macroscopic currents and STOCs between HT and NT:①The current density of BKCa was 6.80±4.93 pA/pF (+60 mV) in HT (n=8) and 13.30±2.25 pA/pF (+60 mV) in NT (n=12). The statistical test shows that the current density in HT is lower than the one in NT (P<0.05);②The amplitude and frequency of STOCs (-20 mV) in HT (27.93±3.26 pA,1.52±0.28 Hz, n=5) were lower than that in NT (61.26±2.75 pA,2.27±0.47 Hz, n=5) (P<0.05). (4)The effects of TES on BKCa and STOCs:①The BKCa macroscopic currents were increased after added TES in an identical cell;②The current density of BKCa was 13.00±2.20 pA/pF (+60 mV), changed to 24.53±4.61 pA/pF (+60 mV) after added TES. The current density of BKCa was increased by TES (n=6) (P<0.05);③The amplitude and frequency of STOCs increased after added TES in an identical cell. Conclusions:(1)The BKCa macroscopic currents of mesenteric artery VSMCs were voltage-dependent and outwardly rectifying; STOCs can be induced by BKCa, and therefore the amplitude and frequency of STOCs were voltage-dependent and stochastic. (2)By compared with NT, the current density of BKCa as well as the amplitude and frequency of STOCs were significantly lower in HT. (3)Both the BKCa macroscopic currents of mesenteric artery VSMCs and the amplitude and frequency of STOCs were increased after treated with TES. (4)BKCa was involved in the mechanisms of TES-induced relaxation in mesenteric artery. (5)The response of TES in HT was lower than that in NT.
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