Studies On The Sensitivity Of BK_Ca To NO In Mesenteric Artery Smooth Muscle During Hypertension

Objective:Hypertension is the largest independent risk factor for many cardiovascular diseases such as coronary heart disease, stroke and so on. which exerts great effect on human health. Effectively decreasing blood pressure can significantly reduce the death rate of patients. During the development of hypertension, some factors that regulate blood pressure have been changed, including the activity of sympathetic and the expression of vasoactive substances (such as nitric oxide). Hypertension is characterized by that tension is increased in small arteries and peripHeral vascular resistance is consistently arisen. The mechanism for the development of hypertension has not yet been fully elucidated. Mesenteric artery is one of the main visceral vessels in human beings and accounts for the largest proportion among arteries. It is one of the main vessels to develop peripHeral resistance and is sensitive to vasoactive substances. It plays an important role in regulating blood pressure. Large conductance Ca2+-activated potassium channels (BKCa channels) are distributed broadly on vascular smooth muscle cell membrane and are the main negative feedback regulator in vascular smooth muscle. They play a very important role in the regulation of vascular tone. NO is an endogenous vasodilator and also one of the important cellular messengers to regulate the function of cardiovascular system by activating BKCa Current studies to elucidate the relationship between NO and hypertension have been focused on the change of NO expression while few papers report whether the sensitivity of BKca to NO is changed or not. Discussion of these issues thoroughly will help to reveal the mechanism of hypertension. In the present study, single-channel patch clamp, Real-time PCR and western blot techniques were used to detect the sensitivity change of BKca to NO in mesenteric artery vascular smooth muscle cells (VSMCs) during hypertension. We determined the change of mRNA and protein level of several regulatory factors in NO-BKCa pathway which are associated with the sensitivity change of BKCa to NO. Our study provided experimental data to further investigate if there is a functional change of ion channel during hypertension and so to elucidate the mechanism of hypertension. Methods:(1) Patch clamp:35 right mesenteric artery samples from patients who had rectal cancer excision were divided into two groups:the HT group (n=20) and the NT group (n=15). The isolated mesenteric artery was vertically cut into small segments (2 mm×2 mm) and incubated in enzymatic dissociation solution. Single smooth muscle cells were obtained by two-step enzyme digestion. Cells were picked up that attached to the dish bottom well with good refraction, stereoscopic appearance and smooth surface. All experiments were performed using single smooth muscle cells incubated in symmetric high potassium solution ([K+]o:[K+]i=140:140 mM). The single channel currents were recorded using patch clamp single channel recording technique. The currents were amplified and filtered (1 KHz) by patch clamp amplifiter (CEZ-2200), then imported into the computer. pClamp 6.0 software was used to record the digital data and Clampfit 10.1 software was used to analyze data. The effects of NO on BKca channels were observed in cell-attached and inside-out configuration. The sensitivity of BKCa to NO was compared between hypertension and normal blood pressure group. (2) Real-time PCR experiments:①32 right mesenteric artery samples from patients who had rectal cancer excision were divided into two groups:the HT group (n=12) and the NT group (n=20).②Extracted total RNA from the mesenteric artery tissues. The standards and standard curves were prepared by RT-PCR.③Compared the mRNA level of the sGCα,sGCβand cGKI gene between the two groups by SYBR Green I Real-time PCR. The house keeping gene GAPDH was used as the control. (3) Western blot:①32 right mesenteric artery samples from patients who had rectal cancer excision were divided into two groups:the HT group (n=16) and the NT group (n=16).②First, total protein was extracted from mesenteric tissue sample and protein concentration was determined. The protein samples were seperated through 12% polyacrylamide gel. The electropHoresis condition for stacking gel and separating gel were 60 V, 40min and 120 V,40min, respectively. Then the proteins in the gel were transferred to membrane and the transfer condition was 390 mA,50 min. Next, the membrane was blocked in 5% dried skim milk overnight at 4℃. Next day, the membrane was incubated with sGCα, sGCβor cGKI primary antibody for 5h at room temperature. Then the membrane was washed and incubated with HRP-goat-anti-rabbit-IgG secondary antibody (1:5000 dilution) for 1 h at room temperature. The images of sGCα, sGCβand cGKI specific bands were semi-quantitatively analyzed using Quantity One software. The protein amount of sGCα, sGCβand cGKI were normalized to GAPDH. Results:(1) The characteristics of BKCa channels in mesenteric artery smooth muscle cells were as following:①In symmetrical high K+ solution (140 mM), we recorded single channel conductance of BKCa on mesenteric artery smooth muscle cells in inside-out patch was 200±6.97 pS (n=9), The channels are voltage-dependent and calcium-sensitive.②Under the outside out patches (Vm=+30 mV),200 nM of IbTX almost completely blocked the channel activity. (2) We compared the sensitivity change of BKCa channels to NO between the HT and NT group:①Under the cell-attached patches (Vm=+40 mV), in the NT group, exogenous NO (SNP) enhanced the open probability of BKCa channel (NPo) from 0.007±0.002 to 0.015±0.004 (n=9, P<0.05), the mean open time (To) of BKCa channels was increased from 5.339±1.389 (ms) to 10.241±2.290 (ms) (n=9, P<0.05), and the mean close time of BKca channels was reduced from 1289.492±299.291 (ms) to 901.174±176.264 (ms) (n=9, P<0.05). In the HT group, only the mean open time of the BKCa channels was increased from 6.010±1.557 (ms) to 9.356±1.482 (ms) (n=9, P<0.05) while there was no statistical significant difference for the open probability and the mean close time. There were no changes for the amplitude (Amp) of BKca channels before and after treatment.②In inside-out patch (Vm=+40 mV), in the NT group, after applying exogenous NO (SNP) to the cells, the open probability of BKCa channel (MPo) was increased from 0.004±0.001 to 0.014±0.003 (n=9, P<0.05), the mean open time (To) of BKca channels was increased from 4.837±0.978 (ms) to 6.731±1.555 (ms) (n=9, P<0.05), and the mean close time of BKCa channels was reduced from 1852.972±587.168 (ms) to 762.020±213.296 (ms) (n=9, P<0.05). In the HT group, only the mean open time of the BKca channels was increased from 8.110±1.992 (ms) to 10.271±2.345 (ms) (n=9, P<0.05) while there was no statistical significant difference for the open probability and the mean close time.There were no changes for the amplitude (Amp) of BKca channels before and after treatment.③Compared with the NT group, in the HT group, after:applying exogenous NO (SNP) to the cells, the increasing rate of the open probability of BKCa channels and the decreasing rate of the mean closing time were lower. (3) The mRNA change of the sGCα, sGCβand cGKI gene in the HT group.①The quality of the total RNA:The ratio of A260/A280 was between 1.80-2.10 and the ratio of A260/A230 was between 1.90-2.20 measured by UV spectropHotometer. The RNA concentration was higher than 100ng/ul. Seperated by 1% gel electropHoresis, the 28S band and the 18S band were obvious, the gray scale of the 28S band was more than that of the 18S band. The 5S band was weak and had no tail.②The standard curve and the melting curve:the standard curves had good linear correlation and the correlation coefficient R2is greater than 0.99. The standard curve of the sGCa gene was y=-0.30x+7.28 (R2=0.999), the standard curve of the sGCP gene was y=-0.30x+7.44 (R2=0.998), the standard curve of the cGKI gene was y=-0.29x+7.60 (R2=1.000), and the standard curve of the GAPDH gene was y=-0.30x+7.40 (R2=0.999). The shape of the melting curves exhibited unimodality. The Tm values of the products coincided with the predictive values.③The respective ratio of sGCa/GAPDH between the HT and NT group was 0.1035±0.0322 (n=9) and 0.1843±0.0210 (n=15), P<0.05; the respective ratio of sGCβ/VGAPDH between the HT and NT group was 0.0393±0.0064 (n=5) and 0.0712±0.0103 (n=8), P<0.05; the respective ratio of cGKI/GAPDH between the HT and NT group was 0.0271±0.0108 (n=8) and 0.0706±0.0164 (n=7), P<0.05; (4) The change of protein expression of sGCa, sGCPβand cGKI in the HT group:The relative protein expression level of sGCa in the HT and NT group was 0.5510±0.0220 (n=15) and 0.6483±0.0468 (n=9), respetively. The relative protein expression level of sGCβin the HT and NT group was 0.4583±0.0209 (n=16) and 0.7690±0.0392 (n=15), respetively. The relative protein expression level of cGKI in the HT and NT group was 0.5418±0.1028 (n=13) and 0.6692±0.0249 (n=11), respetively. The relative protein expression level of sGCa, sGCβand cGKI in the HT group was all significantly lower than that in the NT group, P<0.05. Conclusions:(1) Both in cell-attached and inside-out configuration, BKca in human mesenteric arterial smooth muscle cells are activated by NO. (2) Both in cell-attached and inside-out configuration, the sensitivity of BKCa channels to NO is significantly lowered in the HT group compared to the NT group. (3) During hypertention, the mRNA of the sGCa, sGCp and cGKI in the NO-BKCa channels was down regulated. (4) During hypertension, the protein expression of sGCa, sGCβand cGKI in the NO-BKca channels was downregulated. The downregulation of mRNA and protein of sGCa, sGCβand cGKI in the BKCa channels caused the reaction reduction of the BKca channels to NO during hypertension.