Roles Of ET-1on Chronic Intermittent Hypoxia-induced Hypertension In Rats

Obstructive sleep apnea syndrome (OSAS) is characterized by repeatedupper airway obstruction during sleep and affects between5%and20%of thepopulation. Many studies suggest that chronic intermittent hypoxia (CIH) as aconsequence of OSA is an independent risk factor in hypertension. Severalstudies have found an association between increased circulating endothelin-1(ET-1) and OSA. In Rats exposed to CIH, there were increased circulatingET-1and sensitivity of vasoconstriction to ET-1, whereas ET-1receptorantagonists (ETRA) reduced blood pressure. Therefore, elevated levels ofET-1combined with increased vasoconstrictor responses to this peptide maycontribute to the hemodynamic changes.It is well established that patients experiencing CIH as a consequence ofOSA exhibit increased muscle sympathetic nervous activity (SNA) andelevated plasma catecholamines. Carotid bodies are sensory organs fordetecting changes in arterial blood O₂and mediating the reflex increase insympathetic activity and blood pressure associated with CIH. Sectioning ofthe carotid sinus nerve bilaterally prevents the increase in arterial pressureassociated with CIH exposure. Although it has been proposed that CIH, byway of activating the chemoreceptor reflex, leads to sympathetic excitation,the underlying mechanisms are incompletely understood. Some studiesindicate that ET-1can efficiently augment chemosensitivity after CIH.Many studies suggest that CIH is an independent risk factor in hypertension,but the mechanism is unclear and under investigation. Based on the aboveinformation, we investigated:â‘ Effects of ET-1receptor antagonists onhaemodynamics and cardiac muscle histomorphology in CIH rats.â‘¡Effectsof ET-1on CSN discharge and possible molecular mechanisms in CIH rats.â‘¢Augmented endothelin vasoconstriction of isolated aortas in CIH rats: altered expression of ET-1receptors and vasocular endothelial function.Part1Effects of ET-1receptor antagonists on haemodynamics andcardiac muscle histomorphology in chronic intermittent hypoxia ratsObjective: To investigate the roles of ET-1receptor antagonists on decreaseof blood pressure and protection of myocardial injury in CIH rats.Methods: Male Sprague-Dawley rats (190-210g) were purchased from theLaboratory Animal Center (Hebei Medical University, China). The rats weredivided into six groups as follows: Normoxia (n=24), CIH (n=24),CIH+BQ123(n=8), CIH+BQ788(n=8), CIH+BQ123+BQ788(n=8),CIH+DMSO (n=8). CIH exposure: the hypoxic exposure was designed tomimic the cyclic hypoxia experienced by patients with OSA. Animals wereexposed to hypoxia during their normal rest period (light). Animals wereplaced in commercial hypoxic chambers. These were flushed with100%nitrogen to FIO2nadir of9%for1min and the FIO2was allowed to return to21%gradually over the remainder of each cycle. The exposure cycle wasrepeated every2min for8hours/day,7days/week for3weeks. Normoxic ratsunderwent identical handling and exposure but chambers were flushed withroom air rather than N2. Six group rats of eight in each group were injectedfrom vena caudalis. All drugs were given as follows: Normoxia (saline5ml/kg), CIH (saline5ml/kg), BQ123(50μg/kg), BQ788(50μg/kg),CIH+BQ123+BQ788(BQ12350μg/kg, BQ78850μg/kg), CIH+DMSO(0.5μl/kg). Drugs were administered every2days.The indices as follow:1Estimation of heart weight index: body weight (BW); heart weight index(HWI);1eft ventricular weight index (LVWI).2Measurement of plasma ET-1and serum No level3Measurement of systolic blood pressure (SBP) from arteria caudalis4Hemodynamics analysis: carotid artery systolic arterial pressure (SAP),carotid artery diastolic arterial pressure (DAP), carotid artery mean arterialpressure (MAP), heart rate (HR), left ventricular systolic pressure (LVSP), leftventricular end diastolic pressure (LVEDP), maximum ascending and descending rate of ventricular pressure (±dp/dtmax).5Hematoxylin-eosin (HE) staining of myocardial tissue.Results:1Effects of ETRA on BW, HWI and LVWI in CIH ratsBW was decreased in CIH group as compared to Normoxia group (P <0.05), HWI and LVWI were no significantly difference among groups.2Effect of ETRA on ET-1level in plasma and NO level in serumET-1level in plasma was increased and NO level was decreased in serum inCIH rats as compared to Normoxia group (P <0.01), and NO level in serumwas decreasd in CIH+BQ788as compared to CIH group (P <0.01).3Effects of ETRA on SAP of arteria caudalis in CIH ratsCompared with Normoxia group, SAP of arteria caudalis was increased inCIH group on day15, day18, and day21of CIH exposure (P <0.05or P <0.01). BQ123and BQ123and BQ788can decrease the CIH-induced SAPaugment (P <0.05or P <0.01).4Effects of ETRA on SAP, DAP and MAP of arteria caudalis in CIH ratsCompared with Normoxia group, SAP, DAP and MAP of arteria caudaliswere increased in CIH group (P <0.01). SAP, DAP and MAP of arteriacaudalis were decreased in CIH+BQ123and CIH+BQ123+BQ788ascompared to CIH group (P <0.01)。5Effects of ETRA on hemodynamic parameters in CIH ratsLVSP and±dp/dtmax were significantly increased in CIH group comparedto Normoxia group, and treatments of BQ123or BQ123+BQ788inducedmarkedly decrease (P <0.01).6Effects of ET-1and ETRA on LVSP in CIH ratsAdministration of ET-1from femoral vein increased LVSP significantlymore in CIH group than that in Normoxia group (P <0.05or P <0.01).Treatments with BQ123or BQ123+BQ788can decrease the LVSP responseto ET-1significantly more in CIH rats than that in normoxic rats (P <0.05orP <0.01).Administration with BQ788increased LVSP in Normoxia group (P <0.05), but had no effect in CIH group.7Effects of ETRA on histomorphology of left ventricle in CIH ratsMyocardial cell atrophy, spotty necrosis and endothelial cell necrosis,exfoliation were found in CIH and CIH+DMSO groups. However, comparedwith CIH group, myocardial tissue damages in CIH+BQ123andCIH+BQ123+BQ788groups were significantly attenuated. CIH inducedmyocardial injury was more serious associated with lamellar necrosis andfibrosis in CIH+BQ788group.Conclusions: Both the selective ET_Areceptor antagonist BQ123and thecombination of ET_Aand the ET_Breceptor antagonists BQ123+BQ788candecrease blood pressure and improve myocardial injury effectively.Part2Effects of ET-1on carotid sinus nerve discharge and possiblemolecular mechanisms in chronic intermittent hypoxia ratsObjective: To investigate the role of ET-1on CSN discharge in CIH ratsand clarify the possible molecular mechanismsMethods: Male Sprague-Dawley rats (190-210g) were purchased from theLaboratory Animal Center (Hebei Medical University, China). The rats weredivided into two groups: Normoxia and CIH group. The raising and hypoxicexposure were same as Part1.The indices as follow:1Physiological studiesUnder a surgical operating microscope, the left CSN was isolated carefully.The effects of ET-1on CSN discharge were examined with perfused isolatedcarotid bodies.2Western blottingCarotid bodies from noamoxic rats and CIH rats at different time pointsafter exposure to CIH (15min,1h,3h,5h,8h,2w2h and3w) were harvested,and the expression of ET_A, ET_B, p38MAPK, phospho-p38MAPK,phospho-CaMKII and phospho PKC protein were determined by WesternBlotting.Results: 1Effects of CIH exposure on CSN dischargeAverage data showed that the CSN discharge was significantly enhanced inCIH group compared with Normoxia group (P <0.01).2Effects of CIH exposure on CSN discharge to ET-1Dose response curves of CSN discharge indicated CIH group showed amore robust response at all doses (50,100,200,400pmol/L) compared withNormoxia group (P <0.05or P <0.01).The protein expression of carotid bodies ET_Aand ET_Breceptors in CIHgroup was dramatically increased, compared with that of the nomoxia group(P <0.01). Meanwhile, the selective ET_Areceptor antagonist BQ123essentially abolished the CSN discharge to ET-1. However, the selective ET_Breceptor antagonist BQ788had no effect on the CSN discharge to ET-1both inthe CIH and Normoxia group.3Effects of CIH exposure on p38MAPK, p-p38MAPK, p-PKC and CaMKâ…¡protein Expression in the carotid bodiesCompared with Normoxia group, the expression of p38MAPK in carotidbodies was increased at3w after exposure to CIH; The expression ofp-p38MAPK was increased at all time points; The expression of p-PKC wasincreased at1h,3h,8h,2w2h,3w and the expression of p-CaMKâ…¡ wasincreased at1h,8h,2w2h,3w (P <0.01or P <0.05).4Effects of signal pathway inhibitors on ET-1-induced augment of CSNdischarge in CIH ratsAdministration of the selective L-type calcium channel blocker nifedipine,selective PLC blocker U73122, selective PKC blocker chelerythine chloride,selective p38MAPK blocker SB203580and calcium free perfusion all candecrease the CSN discharge response to ET-1significantly more in CIH groupthan that in Normoxia group (P <0.01). But the selective CaMKâ…¡ blockerKN-62increased the CSN discharge to ET-1in CIH rats and Normoxia rats (P<0.01), the change percentage had no significantly difference between the twogroups (P>0.05).Conclusions: In summary, the present studies demonstrate that the increased of CSN discharge which induced by CIH may associate with theelevated expression of ET-1in carotid body, and the ET_Areceptor is the majorsubtype in the process; PLC/PKC/p38MAPK signaling pathways, L-typecalcium channels and CaMKII may mediate the ET-1-induced augmentationof CSN discharge in CIH rats.Part3Augmented endothelin vasoconstriction of isolated aortas inchronic intermittent hypoxic rats: altered expression of endothelin-1receptors and vasocular endothelial functionObjective: To investigate the effects of CIH on ET-1receptor expressionand vasocular endothelial function of isolated aortas in rats.Methods: Male Sprague-Dawley rats (190-210g) were purchased from theLaboratory Animal Center (Hebei Medical University, China). The rats weredivided into two groups: Normoxia group and CIH group. The raising andhypoxic exposure were same as Part1. After the exposure cycle wascompleted, animals were randomly assigned to either physiologicalinvestigation or molecular studies.The indices as follow:1Vessel tension studyThe tests include dose-response curves about ET-1(0.1to100nmol/L) onaorta, relaxation to acetylcholine (ACh10^-6mol/L) in aorta rings contractedwith phenylephrine (PE10^-6mol/L), and the effects of the selective ET_Areceptor antagonist (BQ-12310μmol/L), ET_Breceptor antagonist (BQ-78810μmol/L) and NOS inhibitor (L-NAME10μmol/L) on ET-1inducedvasoconstriction.2Hematoxylin-eosin (HE) staining of aortas.3RT-PCR: Expressions of ET-1, ET_Aand ET_Bin aortas were examined byRT-PCR.4Immunohistochemical analysis: The expression and localization of ET-1,ET_Aand ET_Breceptors were analyzed by immunohistochemistry.5Western Blotting: The expression of eNOS protein was determined byWestern Blotting. 6Measurement of NO level in aorta.Results:1. Effects of CIH on ET-1mediated contractionET-1(0.1–100nmol/L) produced a dose-dependent contraction in bothendothelium-intact and denuded normoxic and CIH rings. Compared withendothelium-intact aortas in normoxic rats, the absolute contraction inducedby ET-1in the CIH aortas was markedly increased (P <0.01), but it wasdecreased compared with endothelium-denuded normaxic aortas (P <0.01).2. Effects of CIH on endothelium–dependent vasodilationACh produced a relaxation to PE induced contraction, however, ACh onlyfaint relaxation from endothelium-denuded normoxic rats. The ACh-inducedrelaxation in the CIH aortas was significantly decreased compared withendothelium-intact normoxic rats (P <0.01), indicating that endothelialfunction was reduced. In contrast, the ACh-induced relaxation in the CIHaortas was greater than that in the endothelium-denuded normoxic rats (P <0.01).3. Effects of ET_Aand ET_Breceptor antagonists on ET-1-induced contraction inthe CIH aortaET_Areceptor antagonist BQ123almost completely inhibited ET-1-mediatedcontractions in aortas from endothelium-denuded, endothelium-intactnormoxic rats and CIH rats (P <0.01). ET_Breceptor antagonist BQ788alsosignificantly inhibited ET-1-mediated contractions in aortas fromendothelium-denuded, endothelium-intact normoxic rats and CIH rats (P <0.01). Compared with the endothelium-intact normoxic rat aortas, percentageof ET-1induced vasoconstriction to precontract with BQ788in aortas of CIHrats and endothelium-denuded normoxic rats were increased (P <0.01),whereas it was decreased from CIH rats versus endothelium-denudednormoxic rats (P <0.01).4. Effects of NOS inhibitor on ET-1-induced contraction in CIH aortaPretreatment with L-NAME significantly increased the contraction causedby ET-1in the endothelium-intact aortas of nomoxic rats (P <0.01). In contrast, the contraction evoked by ET-1was not affected by L-NAME inaortas of CIH rats and endothelium-denuded normoxic rats (P>0.05).Compared with the aortas of endothelium-intact normoxic rat, percentage ofET-1induced vasoconstrictions to precontract with L-NAME in aortas of CIHrats and endothelium-denuded normoxic rats was decreased (P <0.01).5. Aortic morphological changes after CIH exposureThe photomicrograph showed histopathological changes of endothelialmonolayer with cellular shrinkage of endochylema, denudation of someendothelial cells, as well as smooth muscle cell proliferation and edema.6. Effects of CIH on expression of ET-1, ET_A, and ET_BreceptorsImmunoreactivity for ET-1increased both in the media and intima of theaorta after exposure to CIH. In normal rats, ET_Areceptor was localized in themedia of the aorta with predominant distribution. Immunoreactivity for ET_Areceptor increased in the media of the aorta after exposure to CIH. Also, itshowed positive staining for ET_Breceptors in the intima and weak staining inthe media of aorta. Immunoreactivity for ET_Breceptor decreased in the intimaand media of the aorta after exposure to CIH.7. Effects of CIH on aortic mRNA expressions of ET-1, ET_Aand ET_BThe ET-1, ET_AmRNA levels in the aorta significantly increased in the CIHrats compared with the normoxic rats (P <0.01), whereas the ET_BmRNAlevels were significantly reduced (P <0.01).8. Effects of CIH on NO production and eNOS expressionThe aortic NO level in CIH rats was dramatically reduced (P <0.05),compared with that in the nomoxic rats, and the expression of aortic eNOS inCIH rats was dramatically reduced (P <0.01), compared with that of thenomoxic rats.Conclusions: These results indicate that increased ET_Areceptor in aortamediates a potent vasoconstrictor response and plays an important role in thepathogenesis of CIH. Meanwhile CIH induced endothelial damage anddecreased ET_Breceptor in endothelial cells might be associated with thedecrease in endothelium-dependent vasodilatation in aortas, which enhanced aortic vasoconstriction.