The Function And Expression Of ANO1 In The Blood Vessels Of Spontaneously Hypertensive Rats

Essential hypertension(EH) is not only a kind of disease, but also a risk factor which leads to atherosclerosis, stroke and other cardiovascular and cerebrovascular diseases. Therefore, how to effectively control the occurrence and development of EH is essential for the prevention of cardio- and cerebrovascular diseases. However, the clinical prevention of hypertension has been limited because the pathogenesis of EH has not been clear, until ANO1, a member of anoctamins family, was proved to be a classical calcium activated chloride channel. ANO1 has opened a new chapter in research on the role of classical calcium activated chloride channels in cardiovascular system. The latest data show that ANO1 has been involved in the regulation of systematic arterial blood pressure. However, whether ANO1 is involved in the pathogenesis of EH has not been reported at present. Therefore, using spontaneously hypertensive rats(SHRs) as an animal model of EH, we explored the role and function of ANO1 in the occurrence and development of EH:(1) to identify the functional expression of ANO1 in different beds of the blood vessels in SHRs;(2) to observe the effects of ANO1 on the blood pressure of SHRs and the possible mechanisms.Research contents and experimental methods1.Using the method of western blotting, we observed the expression of ANO1 in the different vascular tissues in SHRs. ANO1-dependent calcium activated chloride currents were recorded in acutely isolated vascular smooth muscle cells(VSMCs) by the whole cell patch clamp configuration.2. The glycosylation of ANO1 protein was confirmed with glycosidase F and western blotting.3. The expression and function of ANO1 was observed with the primary culture of VSMCs.4. By vascular ring perfusion, examined the effect of ANO1 on vasomotoricity of rat mesenteric artery rings.5. We further studied the effect of the functional expression of ANO1 on systolic blood pressure(SBP) and diastolic blood pressure(DBP) in SHRs by siRNA interference technology.6. We observed the role of ANO1 in the proliferation of VSMCs induced by angiotensinⅡ(AngⅡ); with the primary culture of VSMCs and the siRNA interference, the intracellular calcium signal mediated by ANO1 expression was further tested.Results1. Compared with Wistar-Kyoto(WKY)rats as control, the protein expression of ANO1 were significantly increased in carotid artery, thoracic aorta, mesenteric artery(including its branches) and femoral artery(including its branches) in SHRs(P<0.01).We observed that the protein size of ANO1 in blood vessels was about 150 kDa on western blottings. The protein expression of ANO1 showed similar results in primary culture of VSMCs isolated from aorta and mesenteric arteries of SHRs and WKY rats.2. Compared with the primary culture of VSMCs from aorta and mesenteric artery of WKY rats, we obtained more potent calcium activated chloride current in VSMCs from SHRs. The result suggests that ANO1 was functional calcium activated chloride channel in blood vessels of SHRs. Calcium activated chloride currents were weakened in VSMCs from SHRs mesenteric artery interfered by siRNA-ANO1.The result shows that calcium activated chloride current recorded in VSMCs was mediated by ANO1.3. The protein extracted from aorta of SHRs and WKY rats was treated with glycosidase F; the molecular weight of ANO1 dropped from 150kDa± to 128kDa±, due to the treatment. The result indicates that ANO1 is glycosylated in rat blood vessels.4. The mesenteric artery rings of SHRs and WKY rats were incubated with different concentrations of T16Ainh-A01(ANO1 specific inhibitor), from 1, 2, 5, 10, 20 to 30μmol/L, and then were contracted by Phenylephrine(PE, 1μmol/L). T16Ainh-A01 at 10μmol/L significantly inhibited WKY rats mesenteric artery rings contraction induced by PE, while T16Ainh-A01 at 20μmol/L showed similar inhibitory effect on the contraction of SHRs mesenteric artery rings. The result indicates that overexpression of ANO1 leaded to higher concentration of T16Ainh-A01 required to relax the rings contracted by PE. T16Ainh-A01 at 10μmol/L could attenuate intracellular calcium fluorescence signal intensity induced by PE in primary culture of VSMCs isolated from the mesenteric artery of WKY rats, suggesting that the inhibition of ANO1 might reduce the intracellular calcium signaling which contributes to the vasodilation.5. T16Ainh-A01 was injected via tail veins and the changes of SBP and DBP were observed every 60 minutes in SHRs and WKY rats at age of 12 weeks. We found that T16Ainh-A01 at 10μmol/L significantly lowered SBP and DBP of WKY rats, while T16Ainh-A01 at 20μmol/L showed similar effect in SHRs. Injection of siRNA-ANO1, through tail vein which attenuated endogenous expression of ANO1 in SHRs, could significantly lowered SBP and DBP of SHRs(P<0.01). The results showed that the knockdown of endogenous ANO1 could lower the SBP and DBP and, delay the development of hypertension, which is involved in the pathological process of SHRs.6. In primary culture of WKY rat mesenteric arterial SMCs, AngⅡ promoted the expression of ANO1 in a concentration and time dependent manner. Telmisartan, an Ang II type 1 receptor(AT1R) specific inhibitor, significantly inhibited the expression of ANO1 induced by AngⅡ, while PD123319, the specific inhibitor of AT2 R, had no inhibitory effect. Therefore, AngⅡ promoted expression of ANO1 was mediated by AT1 R. PI3K/Akt is downstream signaling molecules of AT1 R. Compared with WKY rats mesenteric artery, the level of phosphorylated Akt was increased in SHRs; AngⅡalso contributed to Akt phosphorylation in a time-dependent manner. LY294002, a PI3 K specific inhibitor, significantly inhibited the expression of ANO1 induced by AngⅡ in primary culture of WKY rat mesenteric arterial SMCs. The results showed that AngⅡ promoted the expression of ANO1 in VSMCs via the AT1R-PI3K-Akt signaling pathway.7. Compared with WKY rats, proliferation cell nuclear antigen(PCNA)was increased in SHRs mesenteric artery. Using MTT method, the cell vitality was detected. AngⅡ improved the vitality of VSMCs derived from WKY rat mesenteric artery; T16Ainh-A01 significantly inhibited the process in a concentration dependent manner. We further detected the expression of PCNA by Western blotting, and found T16Ainh-A01 inhibited the expression of PCNA in VSMCs from SHRs mesenteric artery. At the same time, T16Ainh-A01 also reduced the expression of PCNA in WKY rat VSMCs induced by AngⅡ. Intracellular calcium fluorescent intensity induced by AngⅡwas attenuated when the expression of ANO1 was reduced by siRNA-ANO1.In conclusion, the high expression of ANO1 in the blood vessels of SHRs contributed to the occurrence and development of hypertension. The role ANO1 played in hypertension through two aspects: on the one hand, ANO1 enhanced vasoconstriction through increasing the concentration of intracellular calcium; on the other hand, ANO1 promoted the proliferation of VSMCs and speeded vascular remodeling in SHRs. This study has provided a new target for the effective prevention of EH.