| Hydrogen sulfide(H2S) is an endothelium-derived hyperpolarizing factor that enhances relaxation of the peripheral vasculature. H2 S has been recognized as the third new gaseous signal molecule following nitric oxide(NO) and carbon monoxide(CO), and plays an important role in various systems. Recent studies have also reported its physiological functions in the central nervous system. In mammalian tissues, H2 S is produced through degradation of L-cysteine mainly by two mainenzymes: cystathionine-β-synthase(CBS) and cystathionine-γ-lyase(CSE). CBS is primarily found in the brain, whereas CSE is mainly expressed in the peripheral tissues. CBS has been found in regions of the brain, such as the nucleus tractus solitarii, paraventricular nucleus of the hypothalamus and rostral ventrolateral medulla(RVLM), which are responsible for the regulation of sympathetic nerve activity and thus arterial blood pressure.However, conflicting results have also been reported regarding the role of H2 S in regulating sympathetic nerve activity and blood pressure. Previous experiments have demonstrated that bilateral microinjection of sodium hydrosulfide(Na HS), an H2 S donor, into the RVLM decreases arterial blood pressure, and the CBS inhibitor hydroxylamine(HA)produces a pressor response. In contrary, another study has shown that the microinjection of Na HS into the RVLM has no effect on mean arterial pressure(MAP). Furthermore, conflicting results have been obtained with respect to the effects of H2 S in the PVN. Although the effect of H2 S or CBS inhibitors in the RVLM on cardiovascular function has been extensively investigated, most studies are conducted in acute experimental conditions and with the use of anesthetized animals. The long-term effects of increased H2 S production in the central nervous system on the regulation of sympathetic nerve activity and blood pressure in conscious rats remain unclear. Part I Overexpression of CBS by adenoviral vectors encoding c DNA of CBS leads to a long-term effect of increased H2 S productionObjective:H2S has been recognized as the third new gaseous signal molecule following NO and CO, and plays an important role in various systems. Sodium hydrosulfide(Na HS), as an H2 S donor, has been used in many previous studies. However, Na HS is an unstable and short-lived donor and does not mimic the slow and continuous process of H2 S production in vivo. Furthermore, Na HS in a water based solvent may be quickly oxidized by oxygen. In present study, CBS adenovirus expression system was established to investigate the effect of CBS on RVLM.Methods:An adenoviral vectors carrying CBS gene(Ad CBS, Gen Bank: M88344.1 Supplied from Gene Sil Biotechnology Company Ltd) or enhanced green fluorescent protein(Ad EGFP; as a control vector) were delivered into the RVLM by microinjection bilaterally.Results: To confirm effective overexpression of CBS in the RVLM with these viruses, a Western blot analysis of micropunches obtained from the RVLM was performed at day 0, 3, 7, 11, or 14 after the gene transfer. The expression of the CBS protein peaked at day 7 after the CBS overexpression and then declined over time in the RVLM of rats transfected with Ad CBS. In addition, the H2 S production was measured in the RVLM. The time course of the H2 S production was compatible with that of the expression of the CBS protein in the RVLM.Conclusion: the present study demonstrates a technique of transferring the CBS gene into the RVLM of rats for the first time in vivo. Part II Gene transfer of cystathionine β-synthase into RVLM increases hydrogen sulfide-mediated suppression of sympathetic outflow via KATP channel in normotensive ratsObjective:H2S is an endothelium-derived hyperpolarizing factor that enhances relaxation of the peripheral vasculature. Recent studies have also reported its physiological functions in the central nervous system. H2 S has been shown to have a sympathoinhibitory effect in the RVLM. The present study examined the function of CBS/H2 S system in the RVLM which plays a crucial role in the control of blood pressure and sympathetic nerve activity.Methods:In this study, the following two groups of rats were used. SD rats were randomly subjected to the RVLM microinjection of Ad EGFP or Ad CBS. Acute experiments were carried out at day 7 after gene transfer.Firstly, SBP and HR were measured before gene transfer and every other day after gene transfer in two groups of conscious rats(n=6 for each group). At the same time, CBS protein expression and H2 S production in the RVLM were measured in two groups(n=6 for every time) to determine the time period of gene transfer. The maximal effects were observed at day 7 after the CBS gene transfer. Therefore, the following experiments were carried out at the end of the 7 day after the gene transfer.Secondly, experiments were performed to examine if the endogenous H2S-mediated inhibitory effects in the RVLM on sympathetic outflow are enhanced in rats with gene transfer of CBS in the RVLM. After a stable 30 min recording of MAP, HR, and RSNA was obtained, artificial cerebrospinal fluid, as vehicle solution, were microinjected into the RVLM. Subsequently, HA(10, 25 and 50 m M, 100nl) or AOA(1 m M, 100nl) were microinjected into the RVLM, and RSNA, MAP, and HR were recorded. One dose was tested per rat.Lastly, to testify the possibility that the increased suppression of sympathetic outflow may be caused by KATP channels, we investigated subunits of KATP channels protein in RVLM by Western blot analysis. In two groups of rats, the samples were quickly collected for the measurement of the SUR1, SUR2, Kir6.1 and Kir6.2 protein expression in the RVLM at day 7 after gene transfer(n=6 for each group).Result:CBS protein production was increased by 83% in rats treated with Ad CBS compared to rats treated with Ad EGFP at day 7 after the gene transfer. In addition, the level of H2 S was significantly higher in rats transfected with Ad CBS than in rats transfected with Ad EGFP(114±7.5 versus 73.1±5.6, pg/mg; P<0.05) at day 7 after the gene transfer.There were no significant differences in SBP and HR between the two groups in the conscious state. Next, we examined the effect of microinjection of HA on the RSNA, MAP and HR at day 7 after the gene transfer in both groups of animals. The magnitudes of the increases in the RSNA, MAP and HR evoked by HA were significantly increased in the Ad CBS treated group compared to the Ad EGFP treated group(RSNA: 21.3±3.7% versus 14.2±3.2%; MAP: 17.0±2.1 versus 9.7±1.3 mm Hg; HR: 24.6±4.2 versus 14.7±3.1, bmp; P<0.05). No change was noted in the responses of RSNA, MAP and HR to microinjection of HA into the RVLM between noninjected normal rats and rats injected with Ad EGFP(n=6; data not shown). In addition, the responses to microinjection of AOA, another CBS inhibitor, were consistent with those of HA.To determine if the endogenous H2S-mediated inhibitory effect is dependent on KATP channel function, the RSNA, MAP and HR responses to the RVLM injection of HA were determined, after the pretreatment with pinacidil(KATP channel activator) in rats. After tested the initial effect of HA on RSNA, MAP, and HR, and these variables returned to baseline level, pinacidil was microinjected into the RVLM followed by HA. Fifteen minutes after pinacidil injection, another dose of HA was injected into the RVLM. Pretreatment with pinacidil completely abolished the increases in response to the administration of HA in two groups.To determine if KATP channels are involved in the mechanism of the potentiation of the increases in RSNA, MAP, and HR in response to the administration of HA in Ad CBS injected rats, glibenclamide was injected into the RVLM. Microinjection of glibenclamide into the RVLM at day 7 after the gene transfer elicited a small but significant increases(change in RSNA: 11.4±2.1 %; change in MAP: 14.1±2.7 mm Hg; change in HR: 22.7±2.7 bpm, P<0.05) in rats transfected with Ad CBS. In contrast, microinjection of glibenclamide did not alter RSNA, MAP and HR(change in RSNA: 4.2±2.1 %; change in MAP: 4.5±1.2 mm Hg; change in HR: 6.1±1.3 bpm, P<0.05) in rats transfected with Ad EGFP.Western blot analysis showed that Kir6.1 and SUR2 proteins expression was increased, whereas SUR1 and Kir6.2 showed no change in the RVLM in Ad CBS injected rats compared to Ad EGFP injected rats at day 7 after the gene transfer.Conclusion: the present study demonstrates a technique of transferring the CBS gene into the RVLM of rats for the first time in vivo. Furthermore, it shows that the endogenous H2S-mediated effect in the RVLM is more effective in suppressing sympathetic nerve activity in rats injected with Ad CBS compared to rats injected with Ad EGFP, and the effect may be mediated via KATP channels. Taken together, these findings provide new insights into the role of the new gaseous transmitter H2 S as a potential target for cardiovascular gene therapy. These findings may be useful for which certain pathological conditions that exhibit heightened sympathetic activity. Part III Cystathionine β-synthase Gene Transfer into Rostral Ventrolateral Medulla Exacerbates Hypertension via Nitric Oxide in Spontaneously Hypertensive RatsObjective:RVLM plays a crucial role in the central regulation of cardiovascular functions. CBS is a major hydrogen sulfide-generating enzyme that has been identified mainly in the brain. The present study was designed to examine CBS expression and determine its roles and mechanisms of regulating sympathetic outflow and blood pressure in the RVLM in spontaneously hypertensive rats(SHR).Methods:In this study, the following four groups of rats were used. WKY rats were randomly subjected to the RVLM microinjection of Ad EGFP(Ad EGFP treated WKY) or Ad CBS(Ad CBS treated WKY). Similarly, SHR were randomly subjected to the RVLM microinjection of Ad EGFP(Ad EGFP treated SHR) or Ad CBS(Ad CBS treated SHR). Acute experiments were carried out at day 7 after gene transfer.Firstly, SBP and HR were measured before and after gene transfer in four groups of conscious rats(n=6 for each group). At the same time, CBS protein expression and H2 S production in the RVLM were measured in four groups(n=6 for every time) to determine the time period of gene transfer. The H2 S level was determined with enzyme-linked immunosorbent assay(ELISA). The maximal effects were observed at day 7 after the CBS gene transfer. Therefore, the following experiments were carried out at the end of the 7 day after the gene transfer.Secondly, urine sample was collected from metabolic cage for measured urinary norepinephrine concentrations before the gene transfer and at day 7 after the gene transfer; and then calculated the urinary norepinephrine excretion for 24 hours.(n=6 for each group).Thirdly, experiments were performed to examine whether or not the pressor response attributable to the decrease in NO level in the RVLM caused by overexpression of the CBS, L-NMMA was microinjected into the RVLM after gene transfer in four groups. After a stable 30 min recording of MAP, HR, and RSNA was obtained, artificial cerebrospinal fluid, as vehicle solution, were microinjected into the RVLM. Subsequently, L-NMMA(1 m M, 100nl) were microinjected into the RVLM, and RSNA, MAP, and HR were recorded. One dose was tested per rat.Lastly, we investigated NOS protein expression, NOx levels, and GABA production in the RVLM. In four groups of rats, the samples were quickly collected for the measurement in the RVLM at day 7 after gene transfer(n=6 for each group).Results:CBS expression peaked at day 7 after CBS gene transfer and declined over time, as indicated by green fluorescence. Localization of CBS gene expression in the RVLM was indicated by enhanced green fluorescence protein expression.Western blot analysis revealed that CBS protein expression was lower in the RVLM in SHR transfected with Ad EGFP compared to WKY transfected with Ad EGFP. In addition, the expression of the CBS protein was significantly increased in the RVLM in Ad CBS treated rats compared to Ad EGFP treated and nontreated rats at day 7 after gene transfer. However, the increase in the CBS protein caused by overexpression of CBS was not different between SHR and WKY. We also measured the production of H2 S in the RVLM at day 7 after the gene transfer. The level of H2 S was significantly higher in rats transfected with Ad CBS than in Ad EGFP treated rats. The basal level of H2 S was lower in the SHR treated with Ad EGFP than that in the Ad EGFP treated WKY. There was no difference in the H2 S concentration in the groups treated with Ad CBS. The SBP and HR were significantly increased between day 5 to day 10 in SHR, but not in WKY, after overexpression of CBS in the RVLM. Baseline SBPs and HRs did not differ between the Ad EGFP transfected rats and nontreated rats.The 24-hour urinary norepinephrine excretion was significantly higher in the SHR groups than those in the WKY groups before gene transfer. At day 7 after the gene transfer, the urinary norepinephrine excretion was much higher in the Ad CBS treated SHR than the Ad EGFP treated SHR, but this effect was not observed in the WKY groups. However, urinary norepinephrine excretion in both the SHR and WKY groups showed no change before and after gene transfer in the Ad EGFP treated rats.NNOS protein expression in the RVLM showed no significant difference between the SHR and WKY groups. However, n NOS protein expression in the RVLM was significantly lower in SHR transfected with Ad CBS than the SHR transfected with Ad EGFP, but this effect was not observed in the WKY groups. The expression of e NOS and i NOS protein did not differ among the groups. The NOx level was decreased in SHR, which was exacerbated after the CBS gene transfer, but this effect was not observed in the WKY groups. The release of GABA in the RVLM was decreased in Ad EGFP treated SHR compared to Ad EGFP treated WKY. Moreover, our present data showed a significant decrease in GABA release in SHR transfected with Ad CBS compared to SHR transfected with Ad EGFP. There was no difference in GABA concentration in the WKY groups. Bilateral microinjection of L-NMMA into the RVLM produced increases in the RSNA, ABP, and HR at day 7 after gene transfer in all four groups. The pressor response evoked by microinjection of L-NMMA into the RVLM was blunt in SHR transfected with Ad EGFP compared to WKY transfected with Ad EGFP. Furthermore, pressor responses were markedly smaller in Ad CBS treated SHR than those in Ad EGFP treated SHR, but no significant differences were observed in the WKY groups. Microinjection of the vehicle control(100 n L, artificial cerebrospinal fluid) into the RVLM showed no effects on the RSNA, ABP, and HR.Conclusion: The present study demonstrates that overexpression of CBS in the RVLM increases the SBP, HR and urinary norepinephrine excretion in the conscious state in SHR. These effects may be mediated by the NOS/NO system within the RVLM in SHR. These findings suggest that an interaction between the CBS/H2 S and NOS/NO systems is involved in excessive central sympathetic activation and that the decreased CBS expression in the RVLM may be an important compensatory mechanism to counter the down-regulation of the NOS/NO system in SHR. These results provide new insights into the central mechanisms of blood pressure regulation in hypertension within the RVLM of SHR. |
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