| Backgrounds and ObjectivesHypertension is a common disease. It is reported by World Health Statistics2012(World Health Statistics2012by World Health Organization) that1/3adults sufferedfrom hypertension around the globe, half of the total number of deaths from strokeand heart disease was caused by hypertension. Study on the pathogenesis of essentialhypertension has been the focus of medical research for long. A lot of research datashowed, hypertension can be caused by abnormal renin-angiotensin system (RAS).Angiotensin1-7[Ang-(1-7)] is a new member of the RAS.Ang-(1-7) is generated from angiotensin1(Ang I) and angiotensin2(Ang II) underthe catalysis of angiotensin converting enzyme (ACE) and angiotensin convertingenzyme2(ACE2). Ang-(1-7) is a polypeptide of seven amino acids, which cancombined with the specific membrane receptors, the Mas receptors (MasR), inducingthe effects of lowering blood pressure, slowing down the heart rate, decreasing renalsympathetic nerve activity (RSNA), decreasing myocardial fibrosis and remodeling.These effects antagonize with Ang II, thus maintaining the homeostasis ofcardiovascular system. Hypertension can be caused by the unbalance betweenAng-(1-7) and Ang II. Enhancing the effect of Ang-(1-7) to rebalance Ang-(1-7) andAng II is an effective strategy for treatment of hypertension.The cardiovascular regulation center in central nervous system has an important rolein the regulation of blood pressure. Cardiovascular center is mainly located in thebrainstem, some of the important regions are the nucleus of the solitary tract (NTS), the rostral ventrolateral medulla (RVLM) and the caudal ventrolateral medulla(CVLM). The NTS is the first region in central control of cardiovascular activity.NTS neurons project nerve fibers to the other centers to regulate the sympathetic andvagal nerve activity. NTS plays a key role in the regulation of cardiovascular functionby maintaining blood pressure, sympathetic tone and mediating baroreflex sensitivity.NTS was closely related with the onset and development of hypertension. Centralnervous system has a complete RAS for its own, and the local production of Ang-(1-7)in the central nervous system plays an important role in cardiovascular regulation.Nitric oxide (NO) is an important messenger both systemically and in the CNS. Studyof peripheral circulation system has proved that the effects of Ang-(1-7) can bemediated by nitric oxide. NO plays an important role in cardiovascular regulation bymodulating the activity of specific neurons in NTS. The mechanism remains unclear.Is the effect of Ang-(1-7) in NTS mediated by NO? What is the specific signalingpathway? Based on the above questions, the aim of the present study was to determinethe role of phosphatidylinositol3-kinase (PI3K) signaling pathways in mediating theeffect of Ang-(1-7) on NO generation in the NTS.Methods300-350g male Sprague-Dawley (SD) rats were used in this study. The effects ofAng-(1-7) on blood pressure, heart rate, renal sympathetic nerve activity(RSNA) andarterial baroreflex(ABR) were observed. We observed whether the effects of Ang-(1-7)were blocked by pretreatment of the nitric oxide synthase (NOS) inhibitor L-NAME.NO production and NOS activity were detected after treatment of Ang-(1-7) in NTS. Western blot analysis was used for detecting the effects of Ang-(1-7) on Akt, nNOSand eNOS phosphorylation in NTS. MasR blocker (A779), PI3K inhibitor (LY294002)and Akt inhibitors (TCN) were used to observe the pathway of Ang-(1-7) action.Local infection of lenti-virus in NTS was used to interfer the expression of PI3K.Fifteen days after the infection, effects of Ang-(1-7) on Akt, nNOS and eNOSphosphorylation were determined by Western blot. Immunofluorescence was used todetermine the co-localization of MasR and nNOS is NTS.Results1. Ang-(1-7) enhances NO generation and NOS activity via Mas/PI3K/Aktpathway in the NTSTo detect the effect of Ang-(1-7) on NO production and NOS activity in the NTS, ratswere received microinjections of vehicle (aCSF), Ang-(1-7), A779plus Ang-(1-7),LY294002plus Ang-(1-7) or TCN plus Ang-(1-7) in NTS. We found that NOproduction and NOS activity in the NTS were significantly increased by an average of34%and38%, respectively (P<0.05), in rats which were acutely injected withAng-(1-7)(25pmol) compared with the vehicle injection (aCSF,50nl). However, theAng-(1-7)-induced increase in NO generation and NOS activity were completelyprevented in the presence of the Ang-(1-7) Mas receptor antagonist A779(500pmol),LY294002(100pmol) or TCN (1mmol). In addition, we confirmed that injection ofalone A779, LY294002or TCN into the NTS had little effect on baseline level of NOproduction.2. Ang-(1-7) stimulates NOS phosphorylation via Mas/PI3K/Akt pathway in the NTSTo detect the signaling involved in the effect of Ang-(1-7) on NO release in the NTS,NOS phosphorylation was measured by western blot. We found that, in the NTS, thephosphorylation of nNOS at Ser1416and eNOS at Ser1177was significantlyincreased after acute injection of Ang-(1-7)(25pmol)(P<0.05), which was abolishedin the presence of A779(500pmol) or LY294002(100pmol). In addition, we alsofound that the phosphorylation of Akt, a downstream of PI3K, was significantlyincreased after injection of Ang-(1-7) into the NTS(P<0.05).3. Ang-(1-7) enhances the cardiovascular effects evoked by NOS inhibitor inthe NTSTo determine the effect of Ang-(1-7) on release of NO involved in cardiovascularresponse, several groups were used for monitoring changes in BP, HR, and RSNAevoked by the NOS inhibitor L-NAME following microinjection of Ang-(1-7) into theNTS. We first confirmed that25pmol of Ang-(1-7) injected into the NTSsignificantly decreased basal BP, HR, and RSNA(P<0.05), whereas it enhanced thebaroreflex sensitivity. Increases in BP, HR, and RSNA evoked by L-NAME (10nmol)injected into the NTS were enhanced5min after injection of Ang-(1-7)(25pmol)(P<0.05). Furthermore, co-injection of Ang-(1-7)(25pmol) and A779(500pmol) hadlittle effect on increases in BP, HR, and RSNA normally induced by L-NAMEinjected into the NTS. Moreover, it was found that the L-NAME-induced actions werecompletely abolished in the presence of LY294002.4. Blockade of NOS prevents the cardiovascular effects of Ang-(1-7) in the NTS Ten rats (two groups: no difference of baseline BP and HR) were used for determiningthe NO mechanism responsible for cardiovascular effects evoked by Ang-(1-7) in theNTS. Pretreatment with the NOS inhibitor L-NAME (10nmol)10min prior to NTSinjection with Ang-(1-7) prevented the reduction in BP, HR, and RSNA normallyevoked by Ang-(1-7).5. After lenti virus infected the NTS for15days, Ang-(1-7) was given in NTSfor subsequent molecular detections.PI3K Knock Down siRNA interference lenti virus (LenVi-PI3K KD) or scramblelenti virus (LenVi-GFP) were used for locally infecting the NTS. After lenti viralinfection in the NTS for15days, bilateral injection of Ang-(1-7)(150pmol) wasgiven in the NTS for detection of Western Blot or NO production. Western blot resultsshowed the Ang-(1-7) induced increase in eNOS, nNOS and Akt phosphorylation andNO production was inhibited by LenVi-PI3K KD(P<0.05). On the contrary,LenVi-GFP had no effect on Ang-(1-7).6. Immunofluorescence for detecting the co-localization of MasR and nNOSImmunofluorescence pictures indicated MasR and nNOS were expressed in the sameNTS neural cells, suggesting that nNOS may have close links to Ang-(1-7)/MasR.Conclusion: Our findings suggest that Ang-(1-7) is capable of stimulating therelease of nitric oxide in the NTS through the MasR-PI3K-Akt-NOS signalingpathway. |
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