| [Objective] Essential hypertension accounted for more than 95% of total incidence of hypertension and its complications,such as stroke,heart failure,hypertensive kidney damage and fundus lesions and so on,have been recognized as a threat to human health.The cause of essential hypertension is not clear,thus it is a main research field of cardiovascular field for long.A large number of studies have shown that an increase in sympathetic nervous activity is one of the pathological mechanisms responsible for essential hypertension.The control center of sympathetic nervous system locates in the rostral ventrolateral medulla(RVLM)of brain stem,which is the key area of maintaining resting blood pressure and sympathetic nerve system activity.Previous evidence have shown that enhanced oxidative stress in the RVLM is an important mechanisms involved in the overactivitity of sympathetic nervous system in hypertension.However,the exact mechanisms of enhanced oxidative stress in the RVLM are unclear.Studies have demonstrated that nitric oxide synthase(NOS)uncoupling is an important contributor to oxidative stress.NOS uncoupling is produced by an NOS dimer with normal biological functions depolymerizing to two NOS monomer.Uncoupled NOS fails to catalytic synthesis of NO,but it transfers electrons from reduced form of nicotinamide-adenine dinucleotide phosphate(NADPH)oxidase to O2 and form superoxide anion(?O2-),which results to oxidative stress.The major goal of this study was to determine the role of NOS uncoupling in the RVLM in mediating oxidative stress and cardiovascular dysfunction in hypertension and further investigate how to improving NOS uncoupling.Angiotensin-1-7(Ang1-7)exerts a variety of cardiovascular protective effects,which is opposite to Angiotensin-?(Ang?),but it is not clear whether Ang1-7 can reduce oxidative stress by improving NOS uncoupling in the RVLM.In our previous study,we found that microinjection of Ang? into the RVLM improved the n NOS uncoupling via promoting the expression of type 1 guanosine triphosphate(GTP)cyclization hydrolase(GTPCH1),the rate-limiting enzyme for the synthesis of tetrahydrobiopterin(BH4).BH4 is a necessary cofactor for the formation of NOS dimer.It is not clear whether Ang1-7 in the RVLM presents a protective cardiovascular function through improving the n NOS uncoupling.Studies have shown that Ang1-7 can raise the content of the second messengercyclic adenosine monophosphate(c AMP),it is reported that an increase in the content of c AMP can promote the increasing in content of BH4 and the m RNA transcription of GTPCH1.Therefore,this study further tested whether ACE2/Ang1-7/Mas axis improves the NOS uncoupling in the RVLM via the c AMP signaling pathway.In this study,we carried out the experiments following several aspects.First,we compared the ratio of NOS dimer/monomer expression,GTPCH1 expression,and BH4 content in the RVLM between spontaneously hypertensive rats(SHR)and Wistar-Kyoto(WKY)rats.Second,we observed the effect of overexpression of GTPCH1 in the RVLM on the NOS uncoupling-derived oxidative stress and sympathetic nerve activity/blood pressure in hypertensive rats.Third,we examined if overexpression of ACE2 in the RVLM improves NOS uncoupling and oxidative stress.Finally,we determined if ACE2/Ang1-7/Mas axis improves NOS uncoupling via increasing the content of c AMP in CATH.a neuron.[Methods] In this study,male SHR and WKY rats,and the culture CATH.a neuron were performed.Blood pressure(BP),heart rate(HR),and renal sympathetic nerve activity(RSNA)of SHR and WKY rats were recorded under the conscious and/or anaesthetized state.The GTPCH1-adenoviral vector and ACE2-lentiviral vector were constructed and cloned by Allbright and Invabio Biotech,respectively.High performance liquid phase(HPLC)was performed to detect the contents of BH4 in the RVLM and 24-h urinary norepinephrine(NE);Low-temperature western blot was performed to determine the expression of NOS-dimer/monomer,and general western blot was performed to determine the expression of GTPCH1 and NOS.The level of ?O2-was measured by dihydroethidium(DHE)fluorescent probe.The levels of Ang?,Ang1-7 and c AMP in the RVLM or CATH.a were detected by enzyme-linked immunosorbent assay(ELISA).[Results] 1 An increase in NOS uncoupling in SHR compared to WKY rats It was found that SHR showed a significant decrease in the levels of BH4(0.088±0.032 vs 0.279±0.056 ?g/g),the ratio of n NOS-dimer/monomer(0.06±0.16 vs 1.00±0.23),the ratio of p-n NOS/T-n NOS(0.63±0.08 vs 1.00±0.09),and GTPCH1(0.70±0.04 vs 1.00±0.05)compared with WKY rats(P < 0.05).2 Overexpression of GTPCH1 in the RVLM of SHR improved cardiovasculardysfunction In this part,experiment rats were divided into 4 groups: WKY-GFP,WKY-GTPCH1,SHR-GFP,and SHR-GTPCH1.2.1 Overexpression of GTPCH1 in the RVLM of SHR reduced BP and sympathetic never activity Baseline BP and HR in WKY and SHR for overexpression of GTPCH1 in the RVLM were 102±4 vs 146±5 mm Hg and 300±23 vs 290±25 bmp,respectively.Nine days after overexpression of GTPCH1 in the RVLM,BP of SHR-GTPCH1 was reduced by an average of 15 mm Hg compared with SHR-GFP(P<0.05).HR was not different statistically.NE content in 24-h urine was reduced significantly in the SHR-GTPCH1 group compared with SHR-GFP group(1.3±0.05 vs 1.0±0.04 ?g)(P<0.05).2.2 Overexpression of GTPCH1 in the RVLM of SHR reduced the n NOS uncoupling and oxidative stress After 9 days of overexpression of GTPCH1 in the RVLM,it was found that the ratio of n NOS dimer/monomer in the SHR-GTPCH1 group was increased significantly compared with the SHR-GFP group(1.69±0.12 vs 0.49±0.05)(P<0.05).The level of ?O2-of SHR-GTPCH1 group was significantly reduced compared with the SHR-GFP group(1.6±0.08 vs 2.3±0.09 A.U.)(P<0.05).3 Overexpression of ACE2 in the RVLM reduced NOS uncoupling in SHR This part experiment rats were divided into 4 groups: WKY-GFP,WKY-ACE2,SHR-GFP and SHR-ACE2.3.1 Overexpression of ACE2 in the RVLM of SHR reduced BP and sympathetic never activity After 6 weeks of overexpression of ACE2 in the RVLM,SHR-ACE2 showed an decrease in BP 151±8 vs 170±13 mm Hg,HR 365±27 vs 389±32 bpm,and RSNA(25±2 vs 40±3 % of Maximum RSNA)compared with the SHR-GFP group(P<0.05).3.2 Overexpression of ACE2 in the RVLM of SHR reduced n NOS uncoupling The ratio of Ang1-7/Ang ?(0.40±0.009 vs 0.22±0.02),the ratio of n NOS dimer/monomer(6.3±1.6 vs 3.3±0.4)and the expression level of GTPCH1(0.24±0.08 vs 0.08±0.01)in the SHR-ACE2 group were significantly higher than in the SHR-GFP group(P<0.05).3.3 Overexpression of ACE2 in the RVLM of SHR increased the level of c AMP SHR-ACE2 group showed an increase in the level of c AMP(16.7±1.7 vs 12.9±1.8pmol/mg)compared with the SHR-GFP group(P<0.05).4 Ang1-7 improved n NOS uncoupling via up-regulation of c AMP 4.1 Ang1-7 up-regulated the level of c AMP and expression of GTPCH1 in CATH.a neuron This part of the experiment was divided into 4 groups: Control,DMSO,Forskolin and Ang1-7 group.It was found that the level of c AMP(4.8±0.83 vs 2.19±0.98 pmol/mg)and the expression level of GTPCH1(2.38±0.05 vs 1.0±0.04)were significantly higher in the Ang1-7 group than Control group(P<0.05).4.2 Bupivacaine-HCl inhibited the effects of Ang1-7 in CATH.a neuron In this section,experiments were divided into 4 groups: Control,DMSO,Bupivacaine-HCl,and Bupivacaine-HCl+Ang1-7 group.Bupivacaine-HCl,a c AMP inhibitor,effectively reduced the level of c AMP(0.55±0.14 vs 2.5±0.48 pmol/mg)and the expression level of GTPCH1(0.46±0.02 vs 1.1±0.01)compared with Control group(P<0.05).The Bupivacaine-HCl+Ang1-7 group showed a significant decrease in the level of c AMP content(0.75±0.34 vs 2.5±0.48 pmol/mg)and GTPCH1 expression(0.55±0.14 vs 1.1±0.006)compared with the Control group(P<0.05).[Conclusion] The current data obtained from this study suggest that the NOS uncoupling-derived oxidative stress plays an important role in mediating high blood pressure and sympathetic overactivity in SHR.The activity of ACE2/Ang1-7/Mas axis is capable of improving the NOS uncoupling by up-regulation of the c AMP,and then reduces the oxidative stress and adjusts cardiovascular dysfunction in hypertension. |
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