The Mechanism Of Endothelium-derived Hyperpolairzing Factor/Hydrogen Sulphide For The Cerebral Vasodilation To Total Flavones Of Rhododendra Flower In Cerebral Ischemia-reperfusion Rat

Endothelium-derived hyperpolarizing factor （EDHF） is the third kind of relaxingfactor and autacoids, which derived from vascular endothelium after nitric oxide （NO）and prostaglandin I2（PGI2, prostacyclin）, described as the non-NO and non-PGI2factor.EDHF is likely to be an important modulator in the regulation of organ blood flow andvascular resistance during normal physiological states and play an even greater rolefollowing pathological conditions such as organ ischemia, hypoxia and acidosis.Currently, the appearance of EDHF has been proved in variant blood vessels of differentspecies, but until at present the chemical nature of EDHF, particularly in cerebralarteries, is still unclear. Recent study in our laboratory has found that in cerebral basilararteries （CBAs） and middle cerebral arteries （MCAs） from normal rats, H₂S waspossibly induced in EDHF responses.H₂S is recognized as the third kind of novel gaseous transmitter after nitric oxide（NO） and carbon monoxide, participated in regulating a vast number of physiologicaland pathological processes in vitro. It has been shown that vascular endothelium caninduce production of endogenous H₂S, which leads to the hyperpolarization andvasorelaxation responses.Total flavones of rhododendra flower （TFR） is the active part extracted from theflower of rhododendra. Our studies have shown that TFR is provided withantispasmodic, analgesic and anti-inflammatory role. TFR can produce vascularrelaxation in CBA and MCA from normal rats. And in this study, we try to study thechanges of EDHF-and NO-mediated response in the isolated CBA and MCA of ratsubjected to cerebral ischemia-reperfusion （CIR）. And EDHF/H₂S-mediated cerebrovascular relaxation generated by TFR in MCA of CIR rat and its underlyingmechanisms.Purpose:1. To examine the changes of EDHF-and NO-mediated relaxation in the isolated CBAand MCA of rat subjected to CIR.2. To evaluate the dilatation of the non-NO and non-PGI2to TFR in MCA of ratsubjected to CIR and its underlying mechanisms.3. To investigate the effect on calcium-activated potassium channels in vascular smoothmuscle cell （VSMC） from MCA of rat subjected to CIR.4. To research the effect of TFR on cerebral H₂S content in rat subjected to CIR.Methods:1. The model of cerebral ischemia reperfusion injury was produced by four-vessleocclusion （4-VO） in rat.2. Isolated artery segments from CBA and MCA of rat subjected to CIR were suspendedin baths, which contained PSS solution, and the diameter was measured. The dilatationeffects of a cumulative application of ACh, TFR, L-Cys and NaHS were observed,respectively. And the effects of TEA （an inhibitor of Kca） and PPG （an inhibitor ofcystathionine-γ-lyase） on the non-NO and non-PGI2relaxation of TFR, L-Cys andNaHS were also investigated.3. The transmembrane resting potentials of VSMCs from CBA and MCA of ratsubjected to CIR were recorded by using microelectrode technology, which was utilizedto observe the hyperpolarization effects of a cumulative application of ACh, TFR,L-Cys and NaHS. The influence of TEA and PPG on the the non-NO and non-PGI2hyperpolarization induced by TFR, L-Cys and NaHS were evaluated, respectively.4. The outward potassium current in VSMC from MCA of rat subjected to CIR wasexaminated by making use of whole-cell patch clamp recording. The effects of TFR and TEA were observed.5. The magnetic activated cell sorting technique was utilized to purify and obtain theendothelial cells from MCA of rat subjected to CIR. The separated and purifiedendothelial cells were used to examine the expression of cystathionine-γ-lyase （CSE）mRNA by RT-PCR method, which is a synthase of the endogenous production of H₂S inthe endothelial cells. ELIASA method was used to detect the H₂S content in thecerebrum.Results:1. In the presence of3×10-5mol/L L-NAME, ACh （10-7¹0-4.5mol/L） producedconcentration-dependent relaxation effects of VSMC in30mmol/L KCl-preconstrictedCBA of sham and model rat. The maximum diastolic ratio of CBA of rat subjected tocerebral ischemia-reperfusion （model） was61.8±5.3%, as compared with that of thesham vessels, there was noticeably different. Co-application of L-NAME （3×10-5mol/L）and Indo （10-5mol/L）, ACh also induced significant relaxation in both sham and modelrats. And the maximal dilations in model vessels were significantly potent than that insham vessels （P <0.01）. ACh-induced non-NO and non-PGI2vasorelaxation was almostcompletely abolished by PPG （10-4mol/L） in an L-NAME-Indo-treated CBA.2. ACh still exerted dose-dependent dilatation in isolated MCAs pretreated withL-NAME. The maximum diastolic ratio in the model vessels was obviously higher thanin the sham vessels （P<0.01）. After the administration of L-NAME （3×10-5mol/L） andIndo （10-5mol/L）, the maximal vasorelaxation of MCAs from the two groups wereapparently attenuated. In contrast to the maximum diastolic ratio of the sham vessels49.6±4.8%, that of the model reduced less （P <0.01）. PPG could eliminate theACh-elicited non-NO and non-PGI2effect.3. ACh generated concentration-dependent hyperpolarizations of the transmembraneresting potentials of VSMC from CBA in the presence of L-NAME plus Indo. However,the maximal change in membrane potential in model rat was-10.1±1.3mV and markedly different with that of the sham （P<0.05）. And the non-NO and non-PGI2hyperpolarizing effect was blocked by PPG.4. When L-NAME （3×10-5mol/L） and Indo （10-5mol/L） were added to perfusate, adose-dependent hyperpolarization was still elicited by ACh （10-7¹0-4.5mol/L） in MCApreconstricted with30mmol/L KCl from model rat. The maximal changes in membranepotentials of the sham and model were-10.7±0.8mV and-12.5±1.2mV, respectively,there was apparently discriminative （P<0.05）. The hyperpolarizing response to ACh waseliminated by PPG.5. After blockade of NO and PGI2production, L-Cys （10-5¹0-2.5mol/L）, the substrateof endogenous H₂S synthesis, induced dose-dependent relaxation and hyperpolarizationin MCA of rat subjected to CIR, and these were abrogated by PPG （10-4mol/L）, aninhibitor of the endogenous H₂S synthese-CSE. Similarly, NaHS （10-5～10-2.5mol/L）, adonor of exogenous H₂S, could significantly elicit concentration-dependentvasorelaxation and hyperpolarization of MCA of rat subjected to CIR, which wereobviously abolished by1×10-3mol/L TEA.6. TFR （11²700mg/L） evoked dose-dependent vasodilation and hyperpolarizationeffects in both sham MCA and model MCA of rat subjected to CIR, which wereprecontracted by30mmol/L KCl. L-NAME （3×10-5mol/L） and Indo （10-5mol/L） couldpartly restrain the relaxation and hyperpolarization by TFR in MCA of rat subjected toCIR, but there were still apparently relaxant effect （P <0.01, vs Vehicle）.7. In the presence of L-NAME （3×10-5mol/L） plus Indo （10-5mol/L）, TFR-inducedvasorelaxation and hyperpolarization were notably inhibited by10-4mol/L PPG, abloker of endogenous H₂S synthese-CSE. TEA （1×10-3mol/L）, an inhibitor ofCa²⁺-activated potassium channel, could markedly restrain TFR-elicited non-NO andnon-PGI2relaxation and hyperpolarization.8. An outward current was induced in VSMC from MCA of rat subjected to CIR. At300²700mg/L, TFR could remarkably enhance the outward current, and the enhancement could be prevented by1×10-3mol/L TEA, an inhibitor of Ca²⁺-activatedpotassium channel.9. The expression of CSEmRNA was observed by RT-PCR semi-quantitative techniquesin the purified and isolated endothelial cells from MCA of rat subjected to CIR.Furthermore, CIR increased the level of CSEmRNA expression （P <0.01, vs Control）.10. TFR （50,100mg/kg） markedly enhanced the CSEmRNA expression, which wasupregulated in the endothelial cells from MCA of rat subjected to CIR （P <0.01, vs NS）.11. The contents of H₂S were disclosed in the cerebrum of rat subjected to CIR byELIASA method. CIR increased the contents of H₂S, and TFR significantly promotedthe increase in the range of25to100mg/kg.Conclusions:1. In rat subjected to CIR, ACh induced EDHF-mediated vasorelaxation andhyperpolarization of VSMCs in a concentration-dependent manner.2. The effect of EDHF may be upregulated in CBA and MCA of rat subjected to CIR,which simultaneously that of NO possibly downregulated.3. In MCA of rat subjected to CIR, TFR apparently generated the non-NO andnon-PGI2-mediated effects of vasorelaxation and hyperpolarization of VSMCs, whichmay be relevant to Ca²⁺-activated potassium channel; this suggests that the non-NO andnon-PGI2respones induced by TFR is probably related to EDHF, which might beproduced by endogenous H₂S.4. TFR evidently increased the level of CSEmRNA expression in MCA endothelial cellsand H₂S content in the cerebrum of rat subjected to CIR.5. TFR could markedly enhance Ca²⁺-actived potassium current.