The Central Mechanism Of Chronic Intermittent Hypobaric Hypoxia On Renal Vascular Hypertension

Hypertension is one of the most commonly cardiovascular diseases and is a high risk factor of stroke and coronary artery disease. The cause of hypertension remains incompletely understood but emerging evidence demonstrates that it is closely associated with dysfunction of arterial baroreflex which plays a pivotal role in acute regulation of blood pressure. The nucleus tractus solitaries(NTS), located in the dorsal medulla, is the first relay center of baroreceptor afferent information and is served as initial integration of cardiopulmonary inputs. It is well documented that nitric oxide(NO) is capable of regulating baroreflex outputs by recruiting different mechanisms at lower brainstem. Chronic intermittent hypobaric hypoxia(CIHH) has been evidenced to exert a variety of biological roles including cardiovascular protective effects. For example, CIHH can moderately regulate arterial blood pressure, an effect through relaxation of artery and reduction of peripheral resistance due to an improvement of endothelial function and increase of NO production. Although our previous study demonstrated that CIHH can facilitate arterial baroreflex in anesthetized rats, it remains to be understood whether CIHH has access to rescuing dysfunction of baroreflex.The present study examined whether CIHH can regulate baroreflex outputs via Rho A/ROCK-NO signaling pathway in renal vascular hypertension(RVH) rats and revealed the mechanism responsible for the contribution of CIHH to anti-hypertensive effects using electrophysiological and biomolecular approaches. Using a RVH rat model, we sought to test the effect of CIHH on baroreflex outputs, to test if NO signaling in the NTS mediated facilitation of barereflex by CIHH and to explore the underlying mechanism, and to examine if Rho A/ROCK-NO signaling pathway at the NTS level participated in the effect of CIHH on baroreflex outputs. Part Ⅰ Effect of CIHH on blood pressure and renal sympathetic nerve activity in RVH ratsObjective: CIHH could facilitate the baroreflex in rats, suggesting that an anti-hypertensive effect of CIHH was due likely to facilitation of baroreflex. The aim of present study was to investigate the effect of CIHH on blood pressure(BP) and renal sympathetic efferent nerve activity(RSNA) in RVH rats.Methods: Adult male Sprague-Dawley rats(170-190g) used in this study were randomly divided into four groups: Sham-operated(SHAM) group, RVH group, CIHH treatment(CIHH) group, RVH and CIHH treatment(RVH+CIHH) group. Two-kidney-1-clip method was used to induce RVH. CIHH group was subjected to 28 days hypobaric hypoxia simulating 5000 m altitude, 6 hours daily in hypobaric chamber and in normal oxygen environment in the rest of time. BP of RVH+CIHH rats was raised and stayed at high level 3 week after two-kidney-1-clip method. These rats were then challenged by 28 days hypobaric hypoxia simulating 5000 m altitude, 6 hours daily in hypobaric chamber. RSNA, BP and HR was recorded using a chart software(Powe Lab,ADI instrument,Australia) and amplified(X1K) and filtered between 10 and 3 KHz with a Warner DP301 amplifier. Baroreflex loading and barereflex unloading were elicited by intravenous infusion of phenylephrine HCl(25μg) and infusion of sodium nitroprusside(10μg), respectively. Baroreflex curves were plotted by RSNA vs BP. Analysis was made to compare differences between each experimental group.Results: 1 The systolic BP of RVH and RVH + CIHH rats were significantly higher than that of CIHH and SHAM rats(P<0.01). The systolic BP of RVH + CIHH rats was significantly reduced after CIHH, compared with RVH rats(P<0.01).2 Compared with Sham rats, the baroreflex gain(Gmax) curves of the RVH rats shifted downwards but it shifted upwards in CIHH rats. The baroreflex gain(Gmax) curves were shifted up and right in the RVH+CIHH group compared with RVH rats. These results suggested that sympathetic outflows were potentiated in RVH rats but attenuated in CIHH rats.3 The calculated gain curve of HR was shifted down and right in RVH rats compared with SHAM rats(P<0.01), and the curve was unchanged in the CIHH group. There were no differences between RVH and RVH+CIHH rats, but HR was decreased significantly and the gain curve shifted downward. Results suggested that the baroreflex sympathetic inhibition decreased in RVH rats but increased in CIHH rats.Conclusion: 1 CIHH can decrease blood pressure in renovascular hypertensive rats.2 Baroreflex sympathetic inhibition was dramatically attenuated in RVH rats, suggesting dysfunction of baroreflex function; CIHH enhanced the baroreflex sympathetic inhibition, facilitated baroreceptor reflex, and rescued damaged baroreflex function in hypertensive rats. Part Ⅱ The involvement of NO signaling in the NTS in CIHH-stimulated facilitation of the arterial baroreflexObjective: Based on the above data CIHH enhanced the baroreflex sympathetic inhibition and facilitated baroreceptor reflex, to a great extent rescued the damage of baroreflex function in hypertensive rats, and exerted an anti-hypertensive role. The purpose of the part was to explore the effect and cellular mechanism of NO on CIHH facilitated the arterial baroreflex in NTS by methods of microinjection, whole-cell patch clamp and molecular biology techniques in the model of RVH rats.Methods: Microinjection of L-arg, L-NAME, DEA/NO and p CPT-c GMP into the NTS of rats. Recordings were made to test the alterations of BP, RSNA and HR. The evoked excitatory postsynaptic currents(e EPSCs) and spontaneous EPSCs(s EPSC) of barereflex sensitive NTS neurons were recorded via electrical stimulation within ipsilateral TS by a whole-cell patch clamp mode. Addition of L-arg and L-NAME was to test effect of endogenous NO on the electrophysiological activities of NTS neurons. Application of NO donor(DEA/NO) was to determine whether the effect of L-arg was due to the decrease of NO synthesis, likewise, application of p CPT-c GMP for testing whether the effect of L-arg is related to the downstream pathway. The expression of n NOS and e NOS in NTS was measured by Western blots.Results: 1 BP, HR and RSNA of each group were decreased after microinjection of L-arg and L-NAME. The change in BP, HR and RSNA was much smaller in RVH rats(P<0.05) but bigger in CIHH rats than other groups(P<0.05). There was no obvious change in degree of decrease between RVH+CIHH and SHAM rats(P>0.05). Following microinjection of DEA/NO, BP and RSNA of each group were increased with being a small increase in RVH rats(P<0.01) and a larger increase in RVH+CIHH rats than RVH rats(P<0.01). Similarly, microinjection of p CPT-c GMP(c GMP analogues) inhibited BP, HR and RSNA of each group with being a large drop in BP, HR and RSNA in RVH rats(P<0.01-0.05) and obvious difference in RVH+CIHH, CIHH and SHAM rats(P>0.05). These results indicated that CIHH can enhance baroreflex sympathetic inhibition by either endogenous or exogenous NO release. We could not rule out other putative molecular mechanisms underlying NO actions.2 Compared with SHAM rats, the average amplitude of e EPSCs of baroreflex sensitive NTS neurons was decreased in RVH rats(P<0.01). Compared with RVH rats, the amplitude of e EPSCs was increased significantly in CIHH rats and RVH+CIHH rats(P<0.01-0.05). Compared with SHAM rats, the frequency of s EPSCs recorded in RVH rats increased significantly(P<0.05); the s EPSC frequency of baroreflex sensitive NTS neurons in CIHH and RVH+CIHH rats was higher significantly than that of RVH rats(P < 0.05). Our results demonstrate that NO could potentiate excitatory transmission in NTS baroreflex sensitive NTS neurons after CIHH treatment.3 Compared with SHAM rats, the expression of n NOS and e NOS was increased in NTS of CIHH rats(P < 0.01) but was significantly decreased in NTS of RVH rats(P < 0.01). The expression of n NOS and e NOS increased significantly in RVH+CIHH rats than SHAM rats(P < 0.05).Conclusion: 1 The inhibition of sympathetic outflows evoked by endogenous and exogenous NO in the NTS were significantly reduced in RVH rats but significantly enhanced in CIHH rats.2 NO could potentiate the e EPSC and s EPSC baroreflex sensitive NTS neurons, this effect was weakened in RVH rats, but was significantly enhanced in CIHH rats.3 The protein expression of n NOS and e NOS was down-regulation in NTS of RVH rats, and was up-regulation in CIHH rats. Part Ⅲ The effect of Rho kinase on NO mediated NTS pressure sensitivity modulationObjective: Based on the above data, anti-hypertensive effect of CIHH was due to enhancement of the excitability of baroreflex-sensitive NTS neurons and depression of sympathetic outflows through NO signaling pathway. The following study was further to determine the role of CIHH in activation of Rho A/ROCK-NO signaling in the NTS.Methods: BP, RSNA and HR were measured following microinjection of L-arg alone, L-arg+Y-27632 and PTIO+Y-27632, respectively. TS-evoked EPSCs and spontaneous EPSC were recorded using a whole cell patch clamp. Analysis was then made to determine the differential influence of these results obtained in four group rats. The quantitative expression of Rho A in the NTS was measured using Western Blot.Results: 1 Microinjection of Y-27632 into NTS in RVH rats potentiated inhibitory responses of BP, RSNA and HR elicited by L-arg(P<0.01), whereas such inhibitory responses evoked by L-arg were attenuated in the presence of Y-27632 in CIHH rats( P < 0.01). The NO scavenger carboxy-PTIO abolished effect of Y-27632 on L-arg-elicited inhibitory actions( P < 0.01). Thereby, suggestion was that activation of Rho A/ROCK signaling pathway at the level of NTS is closely associated with attenuation of inhibited sympathetic outflows in RVH rats, while blockade of activation of Rho A/ROCK enhanced inhibited sympathetic outflows in CIHH ras, both of effects that were NO-dependent.2 In RVH rats, the ROCK inhibitor Y-27632 potentiated L-arg-evoked increased amplitude of e EPSC of baroreflex-sensitive NTS neurons but attenuated L-arg elicited increased frequency of s EPSC(P<0.01); In contrast, these effects of Y-27632 in RVH group were reversed in CIHH group(P<0.01). It was therefore suggested that ROCK mediated NO-stimulated potentiation of e EPSC of NTS neurons by CIHH.3 Compared with rats in SHAM group, the expression of Rho A was increased in NTS neurons of CIHH group rats but reduced in CIHH group rats(P<0.05);the expression of Rho A in RVH+CIHH rats was less than that in RVH rats.Conclusion: 1 Microinjection of the ROCK inhibitor Y-27632 into NTS resulted in potentiation of L-arg elicited inhibited sympathetic outflows in RVH rats but attenuation of such effects in CIHH rats. NO scavenger carboxy-PTIO abolished effect of Y-27632 on L-arg-induced inhibition.2 In the presence of Y-27632, L-arg-evoked increased e EPSC of baroreflex-sensitive NTS neurons was potentiated in RVH rats but weakened in CIHH rats. Likewise, in the presence of this blocker, L-arg-elicited potentiation of s EPSC was attenuated in RVH rats but enhanced in CIHH rats.3 The expression of Rho A of NTS neurons was up-regulated in RVH rats but down-regulated in CIHH rats.