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Aim To research the effects of total flavones of rhododendra(TFR)on transient receptor potential vanilloid receptor 4(TRPV4)in cerebral basilar arteries(CBA)of rats subjected to ischemia/reperfusion(IR)injury.Methods The model of total brain IR was established by four-artery occlusion(4-VO)method in rats.Arterial pressure perfusion and cell membrane potential recording methods were used for surveying the dilatation and hyperpolarization of TFR and ruthenium red(RR,an inhibitor of TRPV4)in the KCl-preconstricted CBA ex vivo in rats subjected to IR.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot were utilized to investigate the TRPV4 mRNA and protein expressions of TFR and RR in cerebrovascular endothelial cells of CBA in vivo in rats subjected to IR.Results 11～2 700 mg·L-1 TFR significantly induced concentration-dependent hyperpolarization and dilatation in the KCl-preconstricted CBA in rats subjected to IR.TFR still produced degenerative hyperpolarization and dilatation by removal of endothelium in CBA,which was remarkably attenuated as compared with endothelium-intact group(P<0.01).After removal of NO and PGI2 vasodilatation,TFR obviously elicited the hyperpolarization and dilatation that were further decreased by RR(an inhibitor of TRPV4)in IR CBAs.TFR pretreatment apparently increased the level of TRPV4 mRNA and protein expressions in IR CBAs.These effects were restrained by RR,an inhibitor of TRPV4.Conclusions TFR could mediate endothelium-dependent and endothelium-independent effects.The endothelium-derived dilatation may be related to the increase of endothelium activity and endothelium-derived hyperpolarizing factor(EDHF)generation and release that have been promoted by TFR,and secondarily activating TRPV4,which results in Ca2+ inflow and subsequent hyperpolarization of vascular smooth muscle cell membrane and vasorelaxation.
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OBJECTIVE: To investigate the dilatation and mechanism of hyperoside (Hyp) in middle cerebral arteries (MCA) of rats subjected to cerebral ischemia reperfusion (CIR). METHODS: Rat isolated MCA segments were used for surveying vasomotoricity in a pressurized chamber. Transmembrane potential was recorded by using glass microelectrodes to evaluate MCA vascular smooth muscle cell hyperpolarization. (1×10-6-1×10-4) mol·L-1 Hyp was used to investigate the effects on vasodilatation and hyperpolariza-tion in MCA of rats subjected to CIR. And the effects of nitric oxide synthase inhibitor (N-nitro-L-arginine-methyl-ester, L-NAME, 3×10-5 mol·L-1) or L-NAME plus prostaglandin I2 synthetase inhibitor (indomethacin, Indo, 1×10-5 mol·L-1) on vasorelaxation and hyperpolarization induced by Hyp were observed, respectively. Auto ELISA Detector and nitrate reductase methods were utilized to detect the H2 S and NO content in the cerebrum of rats. RESULTS: Hyp remarkably induced dose-dependent vasodilatation and hyperpolarization in 1×10-7 mol·L-1 U46619-preconstricted MCA of CIR rats. Hyp-mediated effects were notably attenuated after removal of endothelium in CIR MCA as compared with endothelium-intact group (P-5 mol·L-1) plus Indo (1×10-5 mol·L-1), the vasodilatation and hyperpolarization evoked by Hyp were significantly attenuated in sham operation group and CIR MCAs. Compared with the residual effects in sham vessels, those of CIR MCAs were remarkably potentiated (P-3 mol·L-1), an inhibitor of Ca2+-activated potassium channel, or PPG (1×10-4 mol·L-1), an inhibitor of the endogenous H2S synthese-CSE could markedly restrain Hyp-induced non-NO and non-PGP relaxation and hyperpolarization in sham and CIR vessels. As compared with CIR group, pretreatment with Hyp increased the H2S contents while decreased the NO contents. CONCLUSION: Hyp has the potential to evoke endothelium-dependent and endothelium-inde pendent effects in CIR MCAs. In these responses to Hyp, NO-mediated response is downregulated while endothelium-derived hyperpolarizing factor (EDHF) is upregulated, ie, endogenous H2S, is upregulated. Hyp can also protect the brain against cerebral ischemia injury by promoting H2S contents and decreasing NO contents of brain tissues.
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Objective To study the effect of Perfadex and Wisconsin Unviersity solution on the function of pulmonary artery endothelium.Methods Small lobe pulmonary arteries were dissected from nine porcine lungs.The artery from each lung was cut into six rings in 2mm.Two of them were randomly incubated in Krebs,Perfadex or Wisconsin Unviersity solution (UW solution) at 4℃ for 4 hours.Endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation (percentage of-7.5 logM U46619 precontraction) induced by bradykinin or calcium ionophone A23187 in the present of indomethacin,L-NNA and oxyhemoglobin were measured at 37 ℃ in the organ chambers.Results Vasoconstriction induced by U46619 is no significant difference among three groups (P＞ 0.05).Compared with Krebs group,the relaxation induced by bradykinin or A23187 was significantly decreased in Perfadex group (P＜0.01),while there is no significant difference in UW group (P＞0.05).Conclusion Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in endothelium-mediated relaxation of porcine pulmonary artery.EDHF-mediated relaxation is impaired when the lung preserved with Wisconsin Unviersity solution,wheras its function is not affected by Perfadex solution.
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Objective: To study the effect of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses of relaxation and hyperpolarization of vascular smooth muscle cell (VSMC) of rat cerebral basilar artery (CBA) subjected to cerebral ischemia/reperfusion (I/R) to total flavones in rhododendra (TFR). Methods: The model of global cerebral I/R in rats was made by 4-vessel occlusion (4-VO). The vasodilation and resting membrane potential (RMP) of VSMC of rat CBA were detected in vitro. Results: In the presence of 3×10-5 mol/L Nω-nitro-L-arginine-methyl-ester (L-NAME, an inhibitor of nitric oxide synthase) and 1×10-5 mol/L Indomethacin (Indo, an inhibitor of PGI2 synthesis), the global cerebral I/R markedly enhanced 1×10-7-1×10-5 mol/L acetylcholine (Ach)-elicited relaxation and hyperpolarization of RMP of VSMC in rat CBA. In the presence of L-NAME and Indo, 11-2 700 mg/L TFR induced significant and dose-dependent hyperpolarization of RMP of VSMC and relaxation of rat CBA subjected to global cerebral I/R. The hyperpolarization and relaxation were obviously inhibited by tetraethylammonium (an inhibitor IKCa at 1 mmol/L) and 1×10-4 mol/L dl-propargylglycine (PPG), an inhibitor of endogenous hydrogen sulfide (H2S) synthase. Conclusion: Global cerebral I/R could enhance the non-NO-non-PGI2-mediated responses of hyperpolarization and vasorelaxation in rat CAB. In rat CAB subjected to global cerebral I/R, TFR could significantly induce this non-NO-non-PGI2 hyperpolarization and relaxation, the so-called EDHF response that might be mediated by endogenous H2S.
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BACKGROUND: Besides prostacyclin and nitric oxide, the endothelium-derived hyperpolarizing factor (EDHF), which is another distinct endothelium-dependent vasodilator, is involved in relaxing the vascular smooth muscle cells. The myoendothelial gap junction (MEGJ) and female sex hormone play important roles in the EDHF-mediated responses. Therefore, this study was designed to determine the influence of gender on the gap junctional distribution and endothelium-dependent vasodilation in the rat mesenteric arteries. METHODS: Male and female Sprague-Dawley rat were euthanized and the tertiary branch of the mesenteric artery was harvested. Immunohistochemistry and confocal microscopic examination of the arterial wall were performed after treating them with specific antibodies to delineate the distribution of connexin 43, a gap junctional protein. Segments of the mesenteric artery, 5 mm in length, were connected to two tungsten wires under isometric tension. The arterial segments were suspended in a modified Krebs solution (37 degrees C) aerated with 95% O2 and 5% CO2 in a vertical water-jacketed temperature-controlled tissue bath. The standard dose-response curve for acetylcholine (10(-9)-10(-5) M) was drawn in the presence of the NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M) plus indomethacin (10(-5) M) and/or gap junctional inhibitor, carbenoxolone (10(-4) M). RESULTS: In the female rat mesenteric artery, the gap junctional plagues were more prevalent particularly along the endothelial layer. The inhibition of the relaxation response to acetylcholine was depressed in the presence of L-NAME plus indomethacin and augmented in the presence of carbenoxolone when compared with the male rat mesenteric arteries (P < 0.05). CONCLUSIONS: Gender differences in the rat mensenteric arteries have an effect on the expression of connexin 43 and the release of EDHF through MEGJ may play a key role in controlling the female arterial tone.
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Animals , Female , Humans , Male , Rats , Acetylcholine , Antibodies , Arteries , Baths , Carbenoxolone , Connexin 43 , Dilatation , Epoprostenol , Gap Junctions , Immunohistochemistry , Indomethacin , Mesenteric Arteries , Muscle, Smooth, Vascular , NG-Nitroarginine Methyl Ester , Nitric Oxide , Nitric Oxide Synthase , Rats, Sprague-Dawley , Relaxation , Tungsten , VasodilationABSTRACT
Cardioplegic (and organ preservation) solutions were initially designed to protect the myocardium (cardiac myocytes) during cardiac surgery (and heart transplantation). Due to the differences between the cardiac myocytes and vascular (endothelial and smooth muscle) cells in structure and function, the solutions may have adverse effect on coronary vascular cells. However, such effect is often complicated by many other factors such as ischemiareperfusion injury, temperature, and perfusion pressure or duration. In evaluation of the effect of a solution on the coronary endothelial function, a number of points should be taken into consideration. First, the overall effect on endothelium should be identified. Second, the effect of the solution on the individual endothelium-derived relaxing factors (nitric oxide, prostacylin, and endothelium-derived hyperpolarizing factor) must be distinguished. Third, the effect of each major component of the solution should be investigated. Fourth, the effect of a variety of new additives in the solution may be studied. In the last decades, we have focused our research on the endothelial function during open heart surgery and have for the first time found that high potassium concentration impairs the EDHF-mediated function. This review attempts to discuss the above issues based on available literature in order to provide information for further development of cardioplegic or organ preservation solutions.
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BACKGROUND: In addition to nitric oxide (NO) and prostacyclin (PGI2), there is another endothelium-derived mechanism of smooth muscle relaxation, which is associated with an endothelium-derived hyperpolarizing factor (EDHF). To assess the role of gap junctions in endothelium dependent hyperpolarization, we investigated the relationship between distribution of myoendothelial gap junction (MEGJ) and relative importance of the EDHF pathway in the regulation of vascular tone. METHODS: Immunohistochemistry and confocal microscopic examination of the mesenteric arterial wall of male Sprague-Dawley rat following treatment with specific antibodies were performed to delineate the distribution of connexin 43, a gap junctional protein. The standard dose-response curve for acetylcholine (10-9-10-5 M) of the mesenteric artery was regarded as the release of EDHF in the presence of the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME, 10-4 M) and indomethacine (10-5 M). The effects of the gap junction inhibitors such as 18alpha-glycyrrhetinic acid (18alpha-GA; (2 x 10-4 M)) and carbenoxolone (3 x 10-4 M) was assessed regarding relaxtion to acetylcholine, contraction to phenylephrine (5 x 10-6 M) in the proximal and distal mesenteric arteries. RESULTS: In the distal artery, gap junctional plaques were more prevalent, and the relaxation response to acetylcholine was augmented and the contraction response to phenylephrine was depressed compared with the proximal artery. In both the proximal and distal mesenteric arteries, acetylcholine-induced relaxations attributable to EDHF were near completely blocked by 18alpha-GA and carbenoxolone to the same degree. Regardless of the presence of L-NAME plus indomethacin, 18alpha-GA significantly augmented the contraction response to phenylephrine. CONCLUSIONS: The vasomotor regulatory response by EDHF in the rat mesenteric arteries may be explained by extensive heterocellular coupling through MEGJs. Moreover, the release of EDHF through MEGJ may have a essential role in the regulation of resistor arterial tone.
Subject(s)
Animals , Humans , Male , Rats , Acetylcholine , Antibodies , Arteries , Carbenoxolone , Connexin 43 , Endothelium , Epoprostenol , Gap Junctions , Immunohistochemistry , Indomethacin , Mesenteric Arteries , Muscle, Smooth , NG-Nitroarginine Methyl Ester , Nitric Oxide , Nitric Oxide Synthase , Phenylephrine , Rats, Sprague-Dawley , RelaxationABSTRACT
AIM:To study the effects of Hydrogen Peroxide(H2O2)and 11,12-epoxyeicosatrienoic acid(11,12-EET)on EDHF-mediated relaxation in the rat basilar arteries.METHODS: The relaxant effects of acetylcholine(ACh),H2O2,11,12-EET,and catalase(CAT) on rat arteria basilaris in vitro were detected by vasomotoricity experiment in vitro.RESULTS: In the rat basilar arteries,preconstricted by 30 mmol/L KCl in vitro,ACh(10-7-10-4.5 mol/L) had the concentration-dependent relaxation effect.3?10-5 mol/L N?-nitro-L-arginine-methyl-ester(L-NAME) and 10-5 mol/L indomethacin(Indo) could partly inhibit the relaxation effect of ACh to the rat basilar arteries,but non-No/non-PGI2-mediated relaxation was still significant(P
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Until now, although the molecule of EDHF has not been cloned, some characteristics of EDHF has been clarified by scientists. It is different from NO and prostacyclin, and it is considered to play an important role in the regulation of vascular tone by stimulating the channels of K + and Ca 2+ .
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AIM: To investigate whether endogenous endothelium-derived hyperpolarizing factors(EHDFs) produced by CYP epoxygenases BM3?F87V,2C11OR or CYP2J2 transfection was able to protect endothelial cells against apoptosis induced by tumor necrosis factor alpha.METHODS: Three or four passages of cultured bovine aortic endothelial cells(BAECs) were transfected with epoxygenases or the empty vector(pCB6).Cell viability was detected by MTT assay.Apoptosis of transfected endothelial cells was evaluated by DNA ladder assay,flow cytometry and morphological observations under fluorescence microscopy.RESULTS: Overexpression of CYP epoxygenases BM3?F87V,2C11OR,CYP2J2 increased cell viability respectively observed by MTT assay.The percentage of cells undergoing apoptosis was decreased in 2C11OR-,BM3F87V-or 2J2-transfected cells compared to the vector as evaluated by DNA fragment assay,flow cytometry analysis and morphological observations under fluorescence microscopy.CONCLUSION: Overexpression of CYP epoxygenases BM3?F87V,2C11OR or 2J2 increases cell viability and protects endothelial cells against TNF-?-induced apoptosis.These findings suggest new targets to investigate the endothelium-associated disorders and provide novel therapeutic strategies to treat them by modulating cytochrome P450 epoxygenases.