A Study On The Correlation Between Effects Of Angⅱon BK_Ca In Mesenteric Artery Smooth Muscle Cells And Protein Expression Of AT2R During Hypertension

Objective:Hypertension (HT) is a common and frequent cardiovascular disease, and leads increasing threaten to human health. The continuous increasing tension of resistance vessel is the fundamental pathological features of HT, and the ion channels on vascular smooth muscle cells (VSMCs) are involved in the regulation of vascular tension. Activation of large conductance calcium-activated potassium channels (BKCa) is one of the important mechanisms involved in vascular smooth muscle relaxation, which exerts a negative feedback effect. As an effective endogenous vasoconstrictor substances, AngⅡplays a cruial role in contracting vessel by acting on angiotensinⅡtype 1 receptor (AT1R); However, recent studies have found that AngⅡcan relaxes mesenteric artery, which is contradictory with the above. AngⅡrelaxes mesenteric artery via stimulation of the angiotensinⅡtype 2 receptor (AT2R) with subsequent opening of BKCa, leading to membrane repolarization and vasodilation. The novel concept concerning AngⅡserves as an endothelium-independent vasodilatory effect will expand our understanding of the pathogenesis and development of hypertension. It is necessary to investigate the change of AT2R under the hypertension condition and BKCa change response to AngⅡUsing Patch clamp single channel recording technique and Western blot analysis, we will know the relationships among AngⅡ, AT2R and hypertension, so more experimental data were provided to elucidate the blood vessel protection function of AT2R. Methods:(1) Patch clamp single channel recording technique:21 right Mesenteric Artery samples from patients who accept rectal cancer excision were divided into two groups:the HT group (8 not taking antihypertensive medicine) and the NT group (n=13). The isolated mesenteric artery was cut into small segments (2 mm×2 mm) and then placed in the solution containing enzymes. Single vascular smooth muscle cells were obtained by two-step enzyme digestion at 37℃.Chose stereo and slick vascular smooth muscle cells for experiment. The current was recorded on single myocyte in symmetric high potassium solution by patch clamp single channel recording technique. Single channel current was amplified and filtered by patch clamp amplifier (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. Through a 10-minute pretreatment with 10μM valsartan,the different effects between HT and NT of AngⅡon BKCa channels were observed and compared in cell-attached and inside-out patch, and then to confirm whether AngⅡvia AT2R mediated stimulation of BKCa.(2) Western blot: 27 right Mesenteric Artery samples from patients who accept rectal cancer excision were divided into two groups:the HT group (n=14, including 6 not taking antihypertensive medicine and 8 taking antihypertensive medicine) and the NT group(n=13).The human mesenteric arteries mixed with extraction buffer to get AT2R total protein. BCA Protein Assay Kit was applied to measure the proteins concentration. Equal amounts of protein (30μg for AT2R) from various samples were subjected to SDS-Polyacrylamide gel electrophoresis and electroblotting. The membrane was incubated with primary monoclonal antibodies for the AT2R (1:800 dilution).After the incubation with the primary antibodies, the membrane was washed and incubated in goat anti-rabbit IgG-HPR (secondary antibody) (1:5000 dilution). The images were analyzed by Quantity One software. The amount of AT2R protein was normalized to GAPDH to correct for loading. Results:(1) The properties of single channel currents of BKCa channels in human mesenteric artery smooth muscle cells were as follows:①Single channel currents of BKCa were recorded in inside-out patch. The value of single channel conductance was 220.10±10.90pS (n=11).②The channels had distinct voltage dependent and calcium dependent characteristics.③In outside-out patch (Vm=+30 mV),the activation of BKCa could be blocked by 200 nM IbTX completely.(2)In inside-out patch (Vm=+40 mV), AngⅡhad no significant effect on BKCa in the NT group.(3) In cell-attached patch (Vm =+40 mV), the effects of AngⅡon BKCa in the NT group and the HT group (not taking antihypertensive medicine):①In the NT group, AngⅡstimulates BKCa activity significantly:Ang II enhanced BKCa open probability (NPO), from 0.072±0.022 to 0.224±0.054 (n=13, P<0.005). AngⅡcould increase the mean open time (To) of BKCa markedly, from 7.850±2.001 ms to 30.701±8.316 ms (n=13,P<0.005).But there were no significant changes in amplitude (Amp) of current and the mean close time (Tc) of BKCa.②In the HT group (not taking antihypertensive medicine), AngⅡhad no significant effect on BKCa.③In the NT group, Blockade of AT2R2, PD123,319 prevented the stimulatory effect of AngⅡ:NPO of BKCa decreased from 0.391±0.112 to 0.120±0.087(n=5, P<0.05), To of BKCa from 8.947±2.107 ms to 1.738±0.238 ms (n=5, P<0.05); Tc of BKCa from 12.952±3.426 ms increase to 48.339±10.481 ms (n=5, P<0.05) ms; There were no significant changes in amplitude of current of BKCa. (4) The protein amount of the AT2R in mesenteric artery smooth muscle cells among the NT group and the HT group(not taking antihypertensive medicine) and the HT group (taking antihypertensive medicine) was 1.2046±0.0564 (n=13),0.9893±0.0543 (n=6) and 1.3197±0.0491 (n=8). The expression level of AT2R protein of the NT group was greater than the HT group (not taking antihypertensive medicine) (P<0.05), The expression level of AT2R protein of the HT group(taking antihypertensive medicine) was greater than the HT group (not taking antihypertensive medicine) (P<0.05),but it had no significant difference between the NT group and the HT group(taking antihypertensive medicine) (P>0.05).The expression level of AT2R protein of the HT group (taking antihypertensive medicine)was greater than the HT group (not taking antihypertensive medicine) (P<0.05), Conclusions:(1) AngⅡhad no significant influence on BKCa activity of human mesenteric artery VSMCs in inside-out patch.(2) In cell-attached patch, AngⅡcan activate BKCa channel which mediated by AT2R. Furthermore, the effect occurs only in normotension controls rather than untreated hypertension patients. (3) AT2R is downregulated by hypertension and antihypertensive treatments restore AT2R protein expression. We speculate that the different protein of AT2R results in the response difference of BKCa channel which mediated by AT2R.