Involvements of calcium channel and potassium channel in Danshen and Gegen decoction induced vasodilation in porcine coronary LAD artery.

Danshen (Salviae Miltiorrhizae Radix) and Gegen (Puerariae Lobatae Radix) have been widely used in treating cardiovascular diseases for thousands of years in China. The present study was carried out to evaluate the effects of a Danshen and Gegen decoction (DG) on the vascular reactivity of a porcine isolated coronary artery and the underlying mechanisms involved. Porcine coronary rings were precontracted with 15 nM U46619. The involvement of endothelium-dependent mechanisms was explored by removing the endothelium; the involvement of potassium channels was investigated by the pretreatment of the artery rings with various blockers, and the involvement of the calcium channels was investigated by incubating the artery rings with Ca²⁺-free buffer and priming them with high [K⁺] prior to adding CaCl₂ to elicit contraction. The involvement of Ca²⁺ sensitization was explored by evaluating the Rho-activity expression. The results revealed that DG elicited a concentration-dependent relaxation on a U46619-precontracted coronary artery ring. These relaxation responses were not altered by the pretreatment of inhibitors of endothelium-related dilator synthases, cGMP and cAMP pathway inhibitors, potassium channel (BK(Ca), SK(Ca), K(V) and K(ATP)) blockers and endothelium removal. The K(IR) channel blocker BaCl₂ only slightly attenuated the DG-induced relaxation. However, the Ca²⁺-induced artery contraction was inhibited by DG. Additionally, the expression of the phosphorylated myosin light chain was inhibited by DG whereas the activity of RhoA was not affected. Therefore, DG could be a useful cardioprotective agent for vasodilation in patients who have hypertension.

Hu F ，Koon CM ，Chan JY ，Lau KM ，Kwan YW ，Fung KP ... -  《-》

The anti-hypertensive effect of Danshen (Salvia miltiorrhiza) and Gegen (Pueraria lobata) formula in rats and its underlying mechanisms of vasorelaxation.

Radix Salviae miltiorrhizae (Danshen) and Radix Puerariae lobatae (Gegen) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating cardiovascular disease. In the present study, an aqueous extract comprising Danshen and Gegen in the ratio of 7:3 (DG) was investigated for its anti-hypertension in vivo and vasodilative activities ex vivo. The anti-hypertensive effect of DG extract was investigated in spontaneously hypertensive rat (SHR) by measuring systolic blood pressure (SBP). Oral administration of DG extract was started at age of 6 weeks and 14 weeks for the preventive and therapeutic studies, respectively. Blood pressure was measured by tail-cuff method biweekly for 12 weeks. The ex vivo vasodilative activities of DG extract, its dependency on endothelium and the involvement of nitric oxide, prostacyclin and potassium channels were investigated using isolated rat aorta ring in organ bath. For in vivo study, systolic blood pressure was significantly reduced in DG extract-treated groups (90.2 and 300 mg/kg) as compared with the SHR control in both preventive and therapeutic studies. However, DG extract was unable to suppress or delay the onset of hypertension in the preventive study. For ex vivo study, the results showed that DG extract induced a concentration-dependent relaxation in aorta and persisted response was observed with the removal of endothelium. Besides, pretreatment with a non-selective potassium channel inhibitor tetraethylammonium (TEA) also significantly inhibited DG extract-induced vasodilation. Further investigations on specific potassium channel blockers revealed that ATP-sensitive potassium (K(ATP)) channel inhibitor glibenclamide, inward rectifier potassium (Kir) inhibitor barium chloride and voltage-dependent potassium (K(v)) channel inhibitor 4-aminopyridine, but not BK(Ca) channel inhibitor iberiotoxin, exerted significant inhibition on DG extract-induced vasodilation. The results of in vivo SHR animal model suggested that DG aqueous extract possessed blood pressure lowering effect on both pre- and post-hypertensive rats, which could be explained by its endothelium-independent vasodilation via the opening of K(ATP), Kir and K(v) channels.

Ng CF ，Koon CM ，Cheung DW ，Lam MY ，Leung PC ，Lau CB ，Fung KP ... -  《-》

Cerebral vasodilator properties of Danshen and Gegen: a study of their combined efficacy and mechanisms of actions.

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED50, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a KATP channel inhibitor glibenclamide, a non-selective K(+) channel inhibitor tetraethylammonium (TEA), and by a combination of K(+) channel inhibitors (glibenclamide+TEA+iberiotoxin+4-aminopyridine+barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of KATP currents and inhibited Ca(2+) influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl2-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of KATP channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.

Deng Y ，Ng ES ，Kwan YW ，Lau CB ，Cheung DW ，Koon JC ，Zhang Z ，Zuo Z ，Leung PC ，Fung KP ，Lam FF ... -  《-》

Mechanisms of the cerebral vasodilator actions of isoflavonoids of Gegen on rat isolated basilar artery.

Gegen (root of Pueraria lobata) is used in traditional Chinese medicine for treatment of cardiovascular diseases. In this study, the relaxant actions of three of its isoflavonoids; puerarin, daidzein, and daidzin, were investigated on rat-isolated cerebral basilar artery. Rat basilar artery rings were precontracted with 100 nM U46619. Involvement of endothelium-dependent mechanisms was investigated by mechanical removal of the endothelium and inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzymes. Adenylyl cyclase- and guanylyl cyclase-dependent pathways were investigated using their respective inhibitors 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ22536) and 1H-[1,2,4]oxadiazolo [4,3-[alpha]]-quinoxalin-1-one (ODQ). K(+) channels were investigated by pretreatment of the artery rings with various K(+) channel inhibitors, and Ca(2+) channels were investigated in artery rings incubated with Ca(2+)-free buffer and primed with 100 nM U46619 for 5 min prior to adding CaCl(2) to elicit contraction. Puerarin, daidzein, and daidzin produced concentration-dependent relaxation of the artery rings with concentration that produced 50% inhibition (IC(50)) of 304 ± 49 μM, 20 ± 7 μM, and 140 ± 21 μM, respectively. Removal of the endothelium produced no change on their vasorelaxant responses except the maximum response (I(max)) to puerarin was inhibited by 28%. The NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 100 μM) also produced 45% inhibition on the puerarin-induced vasorelaxant response, but not the COX inhibitor flurbiprofen (10 μM). SQ22536 (100 μM) and ODQ (100μM) did not affect the vasodilator responses to puerarin, daidzein and daidzin, but glibenclamide (1μM), tetraethylammonium (TEA, 100mM) or a combination of K(+) channel inhibitors (100nM iberiotoxin+1mM 4-aminopyridine+100 μM barium chloride+1 μM glibenclamide+100mM TEA) reduced their I(max). The contractile response to CaCl(2) was attenuated by 61% and 34% in the presence of daidzein and daidzin, respectively, whereas, puerarin did not significantly affect the contraction. The vasorelaxant action of daidzein and daidzin involved opening of K(+) channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. There is no evidence supporting involvement of endothelium-derived relaxing factors (EDRFs) in their actions. In contrast, puerarin produced vasodilatation via an endothelium-dependent mechanism involving nitric oxide production and an endothelium-independent pathway mediated by the opening of K(+) channels. The cerebral vasodilator activities of all these three isoflavonoids may be beneficial to patients with obstructive cerebrovascular diseases.

Deng Y ，Ng ES ，Yeung JH ，Kwan YW ，Lau CB ，Koon JC ，Zhou L ，Zuo Z ，Leung PC ，Fung KP ，Lam FF ... -  《-》

Vasorelaxant effects of Cerebralcare Granule® are mediated by NO/cGMP pathway, potassium channel opening and calcium channel blockade in isolated rat thoracic aorta.

Cerebralcare Granule (CG), one of the famous classical recipes in traditional Chinese medicine, is developed from the "Decoction of Four Drugs". It has been used for treatment of cerebrovascular related diseases, such as hypertension. It is well known that vasodilatation plays a very important role in hypertensive. Despite the popular medicinal use of CG, little data was available to its activity and mechanism involved in vasodilatation. Therefore, we aimed to investigate the vasorelaxant effects of CG on isolated rat thoracic aorta so as to assess some of the possible mechanisms. The present study was performed to examine the vasodilative activity of CG and its mechanisms in isolated rat thoracic aorta. CG was studied on isolated rat thoracic aorta in vitro, including endothelium-intact and endothelium-denuded aortic rings. In present study, specific inhibitors including NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), cyclooxygenase (COX) inhibitor indomethacin (INDO), non-selective K+ channel inhibitor tetraethylammonium chloride (TEA), Kir channel inhibitor BaCl2, KATP channel inhibitor Glibenclamide (Gli) and cholinergic receptor antagonist atropine were used, they were added 20 min before NE contraction and then added CG-induced vasodilation. Removal of endothelium or pretreatment of aortic rings (intact endothelium) with L-NAME (0.1 mM) or INDO (0.01 mM) significantly blocked the CG induced relaxation. Pretreatment with the non-selective K+ channel inhibitor TEA (1 mM), or the Kir channel inhibitor BaCl2 (0.1 mM), neither of them had no influence on the CG-induced response (p>0.05). However, pretreatment with the KATP channel inhibitor Gli (0.01 mM) produced significant inhibition on the CG-induced response (p<0.01). Besides, CG also inhibited the contraction triggered by NE in endothelium-denuded rings in Ca2+-free medium. CG (0.4, 0.8 and 3.2 mg/mL) produced rightward parallel displacement of CaCl2 curves and reduced the maximum contraction induced by 30 mM CaCl2 to 31.1±9.3%, 18.8±6.9% and 9.4±4.5%, respectively. The relaxation, induced by CG on endothelium-intact rat aortic rings pre-contracted with NE, was significantly attenuated in the presence of atropine (EC50=3.7 mg/mL, p<0.01). Our results suggest that CG induces relaxation in rat aortic rings through an endothelium-dependent pathway mediated by NO/cGMP pathway and an endothelium-independent pathway involving blockade of Ca2+ channels, inhibition of Ca2+ mobilization from intracellular stores, opening of KATP channel. In addition, the muscarinic receptor stimulation is also one of the vasorelaxant mechanisms.

Qu Z ，Zhang J ，Gao W ，Chen H ，Guo H ，Wang T ，Li H ，Liu C ... -  《-》