Mechanism Of EDHF In The Relaxation To Acetylcholine In Rat Basilar Arteries And The Effects Of Total Flavones Of Rhododendra

9 Endothelium-dependent hyperpolarizing factor (EDHF) is the third relaxing factor derived from vascular endothelium besides NO and PGI2. At present, EDHF responses are reported in a wide variety of arteries from different species including humans. One purpose of the present article was to evaluate whether or not there is the role of EDHF in the basilar artery in rats. From the proposed EDHF to now, the chemical nature of EDHF was also a puzzle. Especially in cerebral arteries, there are still unkown. Total flavones of Rhododendra (TFR) was extracted from the flower of Cuckoo. The essential components of TFR are quercetin, hyperin and other flavonoids. It has been preliminary assessed to be of protection against myocardial and cerebral ischemia. In the present study the partial mechanism of the effects of TFR against myocardial and cerebral were studied in this paper.Purposes:1. To evaluate whether there was EDHF in the rat basilar arteries.2. To evaluate whether H2S was the chemical nature of EDHF.3. To evaluate the relaxation effects of TFR on rat basilar arteries, and to assess whether the efffects of EDHF were involved in the mechanism.4. To evaluate the effects of TFR on the expression of iNOS in myocardial ischemia reperfusion rats.Methods: 1. The basilar arteries in vitro were used to observe the relaxation effects to drugs or blockers. In this model it was observed that the relaxation effects of ACh, EDHF, NaHS, L-cys, and TFR. And the influence of PPG to the relaxation of EDHF and TFR was also observed. The luminal perfusate of each artery was collected and the content of H2S was evaluated.2. Transmembrane potentials were recorded to evaluate the hyperpolarization effects of drugs and the blockers. In this model the hyperpolarization effects of ACh, EDHF and NaHS were observed. And the influence of PPG to the effects of EDHF was also observed.3. Endothelial cells of rat basilar arteries were primary cultured, and used to detect the expression of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). And the effect of ACh and TFR on the expression of CSE was also evaluated.4. On the model of myocardial ischemia and reperfusion in rat, all animals were subjected to 30 minutes occlusion of the left anterior descending coronary artery (LAD). The protective effects of TFR preconditioning were observed in this model. In this model the IS/AAR, and the contents of cTnI and NO in rat serum were evaluated. The expression of iNOS in myocardium were evaluated by the western blot method.Results:1. In the rat basilar arteries, preconstricted by 30mmol/L KCl or 10-7mol/L U46619 in vitro, ACh (10-7-10-4.5mol/L) had the concentration-dependent relaxations, and the effects disappeared by removal of the vascular endothelial cells.2. 3×10-5mol/L L-NAME and 10-5mol/L Indo could partly inhibit the relaxation of ACh to the rat basilar arteries, but there were still partial relaxation effect (P<0.01 vs Vehicle). After preperfusion of L-NAME and Indo there also had the effects of ACh to hyperpolarize the smooth muscle cells. These results suggested that the effect of EDHF existed in rat basilar arteries.3. 10-5-10-2.5mol/L NaHS had concentration-dependent relaxations on KCl- or U46619- preconstricted rat basilar arteries. And NaHS could induce concentration-dependent hyperpolarization to the smooth muscles of rat basilar arteries.4. 10-4mol/L PPG, the inhibitor of CSE, could markedly inhibit the effects of relaxation and hyperpolarization to EDHF.5. 10-5-10-2.5mol/L L-cys could induce concentration-dependent relaxations on KCl- or U46619- preconstricted endothelium-intact rat basilar arteries. And the relaxation was significantly attenuated by removal of endothelium.6. The luminal perfusate was collected to measure the contents of H2S after the end of perfusion of drugs or vehicle. It was found that there had the produce of H2S in the normal condition, and after perfusion of ACh the contents of H2S were increased, which was also found after perfusion in combination of L-NAME and Indo. PPG could inhibit the increase of H2S contents.7. The method of RT-PCR was used to evaluate the expression of CSE and CBS in the primary cultured endothelial cells of rat basilar arteries. It was found that in the primary cultured endothelial cells there was CSE not CBS expressing. And the expression of CSE in the primary cultured endothelial cells could be increased by ACh.8. TFR could induce concentration-dependent relaxations on U46619- preconstricted endothelium-intact rat basilar arteries. The relaxation of TFR on rat basilar arteries was significantly attenuated by removal of endothelium or pre-perfusion of L-NAME and Indo. Which suggestted that the effect of EDHF was included in the relaxation of TFR. 9. After perfusion of TFR the contents of H2S were increased, which was also found after perfusion in combination of L-NAME and Indo. PPG could inhibit the increase of H2S contents. It was found that TFR could increase the expression of CSE in the primary cultured endothelial cells. These results further suggested that the effect of EDHF was included in the relaxation of TFR.10. In the model of myocardial ischemia and reperfusion in rat, all animals were subjected to 30 minutes occlusion of the left anterior descending coronary artery(LAD) and 60 minutes of reperfusion. TFR preconditioning could markedly inhibit the increase of cTnI in rat serum, and the reduction of serum NO contents. TFR (20, 40mg/kg) preconditioning could markedly decrease IS/AAR, and TFR (40 mg/kg) preconditioning could significantly increase the expression of inducible nitricoxide synthase (iNOS) in rat myocardium.Conclusions:1. In rat basilar arteries, acetylcholine could induce the no-NO, no-PGI2, endothelium-dependent relaxation, which suggested EDHF may be existing in rat basilar arteries.2. It was found that it was CSE not CBS expressing in the primary cultured endothelial cell. And the inhibitor of CSE could markedly inhibit the effects of hyperpolarization and relaxation of EDHF. It was also found that NaHS had concentration-dependent hyperpolarization and relaxation effect in rat basilar arteries. Theses results suggested that H2S may be the chemical nature of EDHF.3. TFR has the effects of relaxation in rat basilar arteries and the effect of EDHF was included in the relaxation of TFR, which may be one of the mechanisms of TFR against cerebral ischemia injury. TFR preconditioning could induce the expression of iNOS in myocardium and increase the content of NO in rat serum, which may be the mechanism of TFR against myocardial ischemia injury.