The Study On Vasorelaxation Effect Of Cyclovirobuxine-D And Its Derivatives

Cyclovirobuxine D (CVB-D) is a kind of active alkaloid that isolated from plant Buxus microphylla. The chemical structure of CVB-D is belonged to the derivatives of pregnane and its molecular formula is C26H46N2O. The study of modern pharmacology indicated that CVB-D owned effects of anti-myocardial ischemia and anti-arrhythmia. Furthermore, it has the ability of strengthening the cardiac function and cutting down the pressure of aorta et al. CVB-D is mainly used for treating ischemic heart disease and arrhythmia, or treating for hyperlipoidemia and cardiac insufficiency. Moreover, it has the effects on cerebral arteriosclerosis and modulating blood pressure. Although CVB-D exhibited good pharmacodynamic action, its poor bioavailability, narrow safety margin and poor solubility limited the clinical application. In order to raise the bioavailability of CVB-D, enhangce its effect, raise the indication of therapy and improve the partition coefficient, researchers did structure modulation on its base structure. Vasodilation is important to the anti-myocardial ischemia. To screen the medicine value of derivatives of CVB-D, we compared the vasorelaxant effects among them and their prototype in isolated rat thoracic aorta rings. We also investigate the underlying vasorelaxation mechanism of the derivatives.Potassium chloride (KCl) or phenylephrine (PE) is commonly used to prepare the model of vasoconstriction. Potassium of high concentration can induce extracellular calcium influx to contract smooth muscle through opening voltage dependent calcium channel. PE can activate receptor operated channel to make extracellular calcium influx to constrict the vessel ring. It also produced more inositol triphosphate(IP3) to activate specific IP3 receptor channel of sarcoplasmic reticulum, then induce vasoconstriction by increasing Ca²⁺ releasing from reservoir of intracellular calcium.In order to observe the effects exerted on the maintenance of contractile tention during the contraction of vascular induced by KCl or PE, preconstricted model induced by KCl or PE was applied to evaluate the vasorelaxation effects of CVB-D and its derivatives at various concentrations (range from 1×10-5 to 6×10-4 mol·L-1). To KCl or PE -precontracted aorta rings, CVB-D and CVB-D3 showed dose-dependent relaxation effect at the given range of concentrations. Whereas CVB-D3 had stronger vesorelaxant effect on the rings preconstricted by KCl or PE than CVB-D.In order to observe the effects on the initiation of contractile tention during the contraction of vascular induced by KCl or PE, KCl or PE -induced contraction was also recorded after the aorta rings were preincubated with CVB-D or CBV-D3 at the concentration of 6×10-4 mol·L-1. Either CVB-D or CVB-D3 preincubation could inhibit KCl or PE -induced contraction, dose-response curve to the right, and the inhibitive effect of CVB-D3 was stronger than CVB-D. It suggest that both CVB-D and CVB-D3 could inhibit the maintenance and initiation of contractile tention during the vasoconstriction. At the same time,it is determined that CVB-D3 is a kind of derivatives of CVB-D that owned stronger effect on dilating the vessel.During the experiments of vasodilation or inhibition to the vasoconstriction, CVB-D3 showed stronger effects on the aorta rings with intact endothelium in comparison with the aorta rings removed endothelium. It showed that the vasorelaxation of CVB-D3 was partially contributed by endothelium. Vasorelaxation induced by endothelium rely on the generation of EDRF containing NO and PGI2 and dependent on the hyperpolarization induced by EDHF. To examine the role of production of NO, cGMP or prostaglandins on the vasorelaxant effects of CVB-D3 to the aorta rings preconstricted by PE, the rings were pretreated with non-selective NO synthase (NOS) inhibitor Nω-nitro -L -arginine -methyl -ester (L-NAME), soluble guanylate cyclase inhibitor 1H -[1], [2], [4] -oxadiazolo[4, 3 -a]quinoxalin -1 -one (ODQ) or cyclooxygenase inhibitor Indomethacin (Indo) respectively. After the pretreatment of L-NAME or ODQ with endothelium -intact rings, the dose-response curves of CVB-D3 to the PE -precontracted rings were inhibited significantly, but the maximum vasorelaxation effect did not affected. It suggests that the vasodilation is relavant to promoting releasing NO and subsequently to activating sGC to dilate aorta rings by producing more cGMP. While Indo did not suppress the vasorelaxation effect of CVB-D3. It suggests thet the vasodilation of CVB-D3 is not dependent on the generation of prostaglandin.Hyperpolarization of SMC is a main way in dilation response. Many endothelium-derived factors, such as NO could relax the vessel rings through activating various potassium channel and hyperpolarized VSMC. The involvement of Potassium ion channel opening activities in the relaxant effect of CVB-D3 was assessed by obtaining concentration-response curves to CVB-D3 in PE-precontracted rings preincubated with a Voltage dependent Potassium channel (Kv) blocker 4-aminopyridine (4-AP), an ATP-sensitive K+ channel (KATP) blocker glybenclamide or a non-selective K+ channel blocker tetraethylammonium(TEA). After 30 min incubation of 4-AP, Gly or TEA, the concentration-effect curves of CVB-D3 were not attenuated compaed with control group respectively, meanwhile, the maximal effect was not altered. It suggests that the vasorelaxation of CVB-D3 was not involved in the activation of potassium channel.The relaxation of VSM could be induced by EDRF or EDRF-activated relaxing factor that is not dependent on endothelium. While it could be caused by decreasing extracellular calcium influx and calcium release through blocking calcium channel. The contraction of VSM is closely linked to the intracellular free calcium concentration of the VSMC. The intracellular free calcium is mainly come from extracellular calcium influx and calcium release. In order to investigate the relationship between vasorelaxation of CVB-D3 and calcium influx, different concentrations of CVB-D3 in the medium without Ca²⁺ (containing 26μmol·L^-1 EDTA) and with high concentration K+, the effects on CaCl2 -induced contraction to the endothelium -denuded rings were recorded compared to the control group. To approach the relationship between vasorelaxation of CVB-D3 and calcium release, the effect on phasic and tonic contraction induced by PE was also described after preincubation with or without CVB-D3 (3×10^-4 mol·L^-1) in the medium without Ca²⁺ (containing 26μmol·L^-1 EDTA). CVB-D3 exhibited dose-dependent inhibitive effect on the contraction induced by cumulative addition of CaCl2 to the rings in the medium without Ca²⁺ (containing 26μmol·L^-1 EDTA) and with high concentration K+. At the concentration of 3×10^-4 mol·L^-1, CVB-D3 could suppress the phasic and tonic contraction initiated by PE apparently in the medium without Ca²⁺. It suggests that the inhibitive effect on VSMC extracellular calcium influx and calcium release are involved in the mechanism of vasorelaxation of CVB-D3.In summary, the vasoconstriction models of KCl and PE were applied to compare the vasorelaxation on the rat's aorta rings between CVB-D and it's derivatives. From the screened compounds,we investigated the possible mechanism. The results showed that CVB-D and CVB-D3 have similar vasorelaxant effect, but CVB-D3 exerted a higher maximum effect than CVB-D. It suggests that CVB-D3 is a potential compound on vasorelaxation. CVB-D3 induced endothelium -dependent and -independent relaxation in rat thoracic rings like CVB-D. The vessel relaxation effect of CVB-D3 is likely associated with the release of NO or via NO-cGMP pathway relaxing vessel rings by activating sGC of smooth muscle cells. It is probably be involved in inhibiting extracellular calcium influx and calcium release.