Vasodilator Activity Of Dipfluzine Metabolites In Isolated Rat Basilar Artery And Its Underlying Mechanisms

Since the beginning of hunman studies of drug treatment of diseases, drug metabolism is considerded one of the most important factors which influencing drug therapeutic effects. Investigating the metabolites of drugs is considered one indispensable part of drug metabolism research.Drugs proceed a serials of interactions with biosystem in the body, and then the structure of drugs changes. In this process, some drugs lost pharmacological activity, but a number of drugs produce pharmacologically active or toxic metabolites. These metabolites may be closely related to the therapeutic effects of drugs, and in some cases, are more active than its parent drug and play an important role of drug therapy. While some metabolites may be a longer half-life or performance of non-linear dynamics to eliminate, at this time, the possibility of accumulation and the resulting accumulation of toxic drug reaction must be taken to account. Sometimes metabolites have a activity outside of the therapeutic activity, toxic side effects, and even have potentially carcinogenic, teratogenic and mutagenic effects, which affecting the safety of drug treatment.Therefore, it is a great significance of studying the activity of drug metabolites for better design of new drug, clinical use of drugs to ensure the safety and efficacy.Dipfluzine (dipfluzine, Dip), a new derivative of cinnarizine (Cin), is first designed and synthesized by a College of Pharmacy, Hebei Medical University, according to the chemical structures of sectionⅣcalcium channel blocker cinnarizine and central dopamine receptor antagonist droperidol. It has a highly selective dilated effects on cerebral artery, and it can inhibit platelet aggregation and thrombosis, effectively prevent the brain edema induced by cerebral ischemia, reduce infacted size, improve the disorder of somatosensory evoked potentials and cerebral blood flow reduction suffered from cerebral ischemia, decrease the levels of excitatory amino acids raised by cerebral ischemia. These effects of dipfluzine were significantly stronger than cinnarizine, revealing a potential value of therapeutic drugs for cerebral vascular disease.Our laboratory has studied metabolic pathways and metabolites of dipfluzine in rats, indicating that it is metabolized principally through piperazine ring 1- and 4- N-dealkylation. Some metabolites further combine with glucuronic acid and / or sulfuric acid. According to the UV absorption peak of dipfluzine and its metabolites in rat urine, dipfluzine is calculated to be mainly metabolized in rats by the metabolites forms of 1-(4-fluorophenyl) -4-piperazinylbutanone, 4-hydroxybenzophenone, 4-fluoro-γ-hydroxybenzene -butanoic acid, diphenylmethanol, benzophenone and so on. At present, there is no research about vasodilator activity of dipfluzine metabolites.Part 1 vasodilator activity of dipfluzine metabolitesObjective: Our study is to identify and find the metabolites with vasodilator activity by comparing effects of the dipfluzine and its metabolites on high-K+ / 5-HT pre-contracted vessels.Methods: Basilar artery was acutely separated from the male rats which about 250 g. Its non-branching segment 0.5 mm with 2 mm was choosed, which was weared to two fine glass electrode of the bath of Pressure Myogragh System Model 120CP, and fastened with two fine nylon sutures. Lumen pressure was gived 70 mmHg, and 95% O2 +5% CO2 gas mixture was piped into the bath with temperature 37℃in the experiment process. After the artery was equilibrated for 1 hour, the effects of different concentrations of dipfluzine or its metabolites on the high-K+/ 5-HT pre-contracted vessels in vivo were observed by superfusion administration.Results:1 The stability of blood vessel testExperimental results indicated that the diameter changes of rat basilar arteries without external stimulation were 100.47%±1.12%, 99.67%±1.21%, 100%±1.86%, 100.83%±3.05%, and 101.14%±2.99%, respectively, at continuous perfusion 5 min, 10 min, 15 min, 30 min, and 60 min after the first high K+-PSS solution perfusion was washed out. There were no significant differences (P>0.05) comparing with 100%±0.00% of the initial vessel diameter prior to the first high K+-PSS solution perfusion. The contraction amplitudes of the vessel diameter induced by the high K+-PSS solution were recorded twice before and after above continuous perfusion of 1 h and were 63.84%±1.94% and 64.38%±3.64% respectively. There were significant contractions (P<0.05) through comparing with 100%±0.00% of the initial vessel diameter, but no significant difference between them (P>0.05). It suggested that the reactivity of the basilar artery can be stabilized for at least 60 min in our experimental conditions.2 Determination of the vasodilator activity of dipfluzine2.1 The inhibition of dipfluzine on vasoconstriction induced by high potassiumThe inhibition percentages of dipfluzine (10-10~10-3 mol/L) on the contraction of the basilar artery induced by 50mol/L KCl were: 0.0180±0.0069, 0.0364±0.0187, 0.0545±0.0329, 0.0651±0.0323, 0.2011±0.0287, 0.4802±0.0491, 0.5315±0.0513, 0.5363±0.0597, when the contraction amplitude of the vessels induced by 50 mol/L KCl was regarded as 100% in the perfusion without dipfluzine, and its IC50=1.8277E-6±2.2228E-7. Experimental results indicated that dipfluzine could concentration -dependently inhibit the contraction of rat basilar artery induced by high-K+ pre-stimulation, suggesting that a vasodilatory activity of dipfluzine was induced by blocking voltage-dependent calcium ion channels.2.2 The inhibition of dipfluzine on vasoconstriction induced by 5-HTThe inhibition percentages of dipfluzine (10-10~10-3 mol/L) on the contraction of the basilar artery induced by 5×10-7 mol/L 5-HT were: 0.0203±0.0092, 0.0404±0.0268, 0.1410±0.0538, 0.2184±0.0521, 0.5069±0.0499, 0.7903±0.0518, 0.9455±0.0583, 0.9412±0.0614, when the contraction amplitude of the vessels induced by 5×10-7 mol/L 5-HT was regarded as 100% in the perfusion without dipfluzine, and its IC50=9.5916E-7±2.3346E-7. Experimental results indicated that dipfluzine could concentration -dependently inhibit the contraction of rat basilar artery induced by of 5-HT pre-stimulation, suggesting that a vasodilatory activity of dipfluzine was also induced by blocking receptor-operated calcium ion channels.3 Determination of the vasodilator activity of dipfluzine metabolites3.1 The inhibition of 1-(4-fluorophenyl)-4-piperazinylbutanone on vaso -constriction induced by high potassiumThe inhibition percentages of 1-(4-fluorophenyl)-4-piperazinylbutanone (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 50mol/L KCl were: 0.0432±0.0114, 0.0748±0.0160, 0.2756±0.0270, 0.4504±0.0115, 0.5285±0.0136, when the contraction amplitude of the vessels induced by 50 mol/L KCl was 100% in the perfusion without 1-(4-fluorophenyl)-4- piperazinylbutanone, and its IC50=1.1454E-6±3.093E-7. Experimental results indicated that 1-(4-fluorophenyl)-4-piperazinylbutanone could concentration -dependently inhibit the contraction of rat basilar artery induced by high-K+ pre-stimulation, suggesting that a vasodilatory activity of 1-(4-fluorophenyl)-4 -piperazinylbutanone was induced by blocking voltage-dependent calcium ion channels.3.2 The inhibition of 1-(4-fluorophenyl)-4-piperazinylbutanone on vaso -constriction induced by 5-HTThe inhibition percentages of 1-(4-fluorophenyl)-4-piperazinylbutanone (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 5×10-7 mol/L 5-HT were: 0.0301±0.0106, 0.1661±0.0232, 0.7529±0.0302, 0.9667±0.0125, 0.9816±0.0099, when the contraction amplitude of the vessels induced by 5×10-7 mol/L 5-HT regarded as 100% in the perfusion without 1-(4-fluorophenyl)-4-piperazinylbutanone, and its IC50=3.9663E-6±1.1595E-9. Experimental results indicated that 1-(4-fluorophenyl)-4-piperazinylbutanone could concentration-dependently inhibit the contraction of rat basilar artery induced by 5-HT pre-stimulation, suggesting that a vasodilatory activity of 1-(4-fluorophenyl)-4-piperazinylbutanone was also induced by blocking receptor-operated calcium ion channels. 3.3 The inhibition of 4-hydroxybenzophenone on vasoconstriction induced by high potassiumThe inhibition percentages of 4-hydroxybenzophenone (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 50 mol/L KCl were: 0.0402±0.0125, 0.0553±0.0128, 0.0765±0.0141, 0.0978±0.0118, 0.1042±0.0152, when the contraction amplitude of the vessels induced by 50 mol/L KCl was regarded as 100% in the perfusion without 4-hydroxybenzophenone. Experimental results indicated 4-hydroxybenzophenone did not inhibit the contraction of rat basilar artery induced by high-K+ pre-constriction.3.4 The inhibition of 4-hydroxybenzophenone on vasoconstriction induced by 5-HTThe inhibition percentages of 4-hydroxybenzophenone (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 5×10-7 mol/L 5-HT were: 0.0624±0.0176, 0.0981±0.0130, 0.1292±0.0106, 0.1347±0.0108, 0.1459±0.0086, when the contraction amplitude of the vessels induced by 5×10-7 mol/L 5-HT was regarded as 100% in the perfusion without 4-hydroxybenzophenone. Experimental results indicated 4-hydroxybenzo -phenone did not inhibit the contraction of rat basilar artery induced by 5-HT pre-constriction, and it further demonstrated that 4-hydroxybenzophenone has no vasodilatory activity .3.5 The inhibition of 4-fluoro-γ-hydroxybenzenebutanoic acid on vaso -constriction induced by high potassiumThe inhibition percentages of 4-fluoro-γ-hydroxybenzenebutanoic acid (10-8~10-4 mol/L) on contraction of induced by 50 mol/L KCl were: 0.0266±0.0075, 0.0374±0.0106, 0.0481±0.0115, 0.0595±0.0112, 0.0669±0.0131, when the contraction amplitude of the vessels induced by 50 mol/L KCl was regarded as 100% in the perfusion without 4-fluoro-γ-hydroxy -benzenebutanoic acid. Experimental results indicated that 4-fluoro-γ- hydroxybenzenebutanoic acid did not inhibit the contraction of rat basilar artery induced by high-K+ pre-constriction.3.6 The inhibition of 4-fluoro-γ-hydroxybenzenebutanoic acid on vaso -constriction induced by 5-HTThe inhibition percentages of 4-fluoro-γ-hydroxybenzenebutanoic acid (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 5×10-7 mol/L 5-HT were: 0.0516±0.0113, 0.0582±0.0147, 0.0665±0.0113, 0.0665±0.0107, 0.0699±0.0106, when the contraction amplitude of the vessels induced by 5×10-7 mol/L 5-HT was regarded as 100% in the perfusion without 4-fluoro-γ-hydroxybenzenebutanoic acid. Experimental results indicated that 4-fluoro-γ-hydroxybenzenebutanoic acid did not inhibit the contraction of rat basilar artery induced by 5-HT pre-constriction, and it further demonstrated that 4-fluoro-γ-hydroxybenzenebutanoic acid has no vaso -dilatory activity.3.7 The inhibition of diphenylmethanol on vasoconstriction induced by high potassiumThe inhibition percentages of diphenylmethanol (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 50 mol/L KCl were: 0.0193±0.0075, 0.0270±0.0117, 0.0340±0.0081, 0.0342±0.0158, 0.0455±0.0109, when the contraction amplitude of the vessels induced by 50mol/L KCl was regarded as 100% in the perfusion without diphenyl -methanol. Experimental results indicated that diphenylmethanol did not inhibit the contraction of rat basilar artery induced by high-K+ pre -constriction. 3.8 The inhibition of diphenylmethanol on vasoconstriction induced by 5-HT The inhibition percentages of diphenylmethanol (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 5×10-7 mol/L 5-HT were: 0.0526±0.0105, 0.0760±0.0105, 0.0775±0.0121, 0.0951±0.0117, 0.1060±0.0139, when the contraction amplitude of the vessels induced by 5×10-7 mol/L 5-HT was regarded as 100% in the perfusion without diphenylmethanol. Experimental results indicated that diphenylmethanol did not inhibit the contraction of rat basilar artery induced by 5-HT pre-constriction, and it further demonstrated that diphenylmethanol has no vasodilatory activity.3.9 The inhibition of benzophenone on vasoconstriction induced by high potassiumThe inhibition percentages of benzophenone (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 50 mol/L KCl were: 0.0426±0.0095, 0.0583±0.0120, 0.0627±0.0156, 0.0633±0.0121, 0.0773±0.0120, when the contraction amplitude of the vessels induced by 50 mol/L KCl was regarded as 100% in the perfusion without benzophenone. Experimental results indicated that benzophenone did not inhibit the contraction of rat basilar artery induced by high-K+ pre-constriction.3.10 The inhibition of benzophenone on vasoconstriction induced by 5-HTThe inhibition percentages of benzophenone (10-8~10-4 mol/L) on the contraction of the basilar artery induced by 5×10-7 mol/L 5-HT were: 0.0574±0.0130, 0.0749±0.0098, 0.0782±0.0130, 0.0947±0.0112, 0.0965±0.0135, when the contraction amplitude of the vessels induced by 5×10-7 mol/L 5-HT was regarded as 100% in the perfusion without benzophenone. Experimental results indicated that benzophenone did not inhibit the contraction of rat basilar artery induced by 5-HT pre-constriction, and it further demonstrated that benzophenone has no vasodilatory activity.Conclusion: Among many metabolites of dipfluzine, only 1-(4fluoro -phenyl)-4-piperazinylbutanone, not the others, like its parent compound, possesses concentration-dependent inhibitory effects on vasoconstriction of rat basilar artery induced by high potassium/5-HT. These results indicated that its mechanisms of dilating blood vessels may be related to blocking voltage-dependent and receptor-operated calcium channels.Part 2 The mechanisms of vasodilatation of dipfluzine metaboliteObjective: Our study is to investigate the mechanisms of vasodilatation of dipfluzine and its active metabolite in high-K+/5-HT pre-contracted basilar arteries with or without endothelium.Methods: The inhibition of dipfluzine and its active metabolite on the contractions of the basilar arteries with or without endothelium induced by high-K+/5-HT. The effects of endothelium derived relaxing factors inhibitors on the inhibitory effects of dipfluzine and its active metabolite were determined in the vessels with intact endothelium. The effects of potassium ion channels inhibitors on the inhibitory effects of dipfluzine and its active metabolite were detected in the vessels of denuded-endothelium.Results:1 The relationship between the vasodilatation of dipfluzine and calcium channels1.1 The inhibition of dipfluzine on the contraction of denuded-endothelium basilar artery induced by 5-HTThe inhibition percentages of dipfluzine (10-8~10-4 mol/L) on the contraction of denuded-endothelium basilar artery induced by 5-HT were: 0.1529±0.0260, 0.1844±0.0112, 0.3939±0.0151, 0.6263±0.0158, 0.7329±0.0261, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100% in the perfusion without dipfluzine. Experimental results indicated that the inhibition of dipfluzine on vasoconstriction induced by 5-HT in the basilar artery with intact endothelium was significantly greater (P<0.05) than that in the denuded-endothelium basilar artery (the first part of the experiment has been done). It suggested that the mechanisms of dipfluzine -induced vasodilatation might be not only due to blocking receptor-dependent calcium ion channel, but also related to endothelium derived relaxing factors.1.2 The inhibition of dipfluzine on the contraction of denuded-endothelium basilar artery induced by high potassiumThe inhibition percentages of dipfluzine (10-8~10-4 mol/L) on the contraction of denuded-endothelium basilar artery induced by KCl were: 0.0361±0.0116, 0.0488±0.0133, 0.0928±0.0170, 0.2573±0.0251, 0.3534±0.0339, when the contraction amplitude of the vessels induced by KCl was regarded as 100% in the perfusion without dipfluzine. Experimental results indicated that the inhibition of dipfluzine on vasoconstriction induced by KCl in the basilar artery with intact endothelium was significantly greater (P<0.05) than that in the denuded-endothelium basilar artery (the first part of the experiment has been done). It suggested that the mechanisms of dipfluzine-induced vasodilatation might be not only due to blocking voltage -dependent calcium ion channel, but also related to endothelium derived relaxing factors.2 The relationship between the vasodilatation of dipfluzine and endothelium derived relaxing factors2.1 The effects of L-NAME on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4 mol/L dipfluzine on 5-HT pre-contracted vessels was 0.9632±0.0301 before incubated with L-NAME and 0.7362±0.0200 after incubated with L-NAME, respectively, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that L-NAME significantly reduced (P<0.01) the vasodilatation of dipfluzine. It suggested that the vasodilatation of dipfluzine might be related to the release of endothelium derived relaxing factor NO.2.2 The effects of MB on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4mol/L dipfluzine on 5-HT pre-contracted vessels was 0.9693±0.0232 before incubated with MB, an inhibitor of guanylate cyclase, and 0.7309±0.0127 after incubated with MB, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that MB significantly reduced (P<0.01) the vasodilatation of dipfluzine, which further demonstrated that the vasodilatation of dipfluzine might be related to the release of endothelium derived relaxing factor NO.2.3 The effects of INDO on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4mol/L dipfluzine on 5-HT pre-contracted vessels was 0.9575±0.0293 before incubated with INDO (indomethacin, an inhibitor of cycloxygenase), and 0.9389±0.0167 after incubated with INDO, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. INDO could inhibit the synthesis of prostacyclin. The results indicated that the vasodilatation of dipfluzine might not be related to the release of endothelium derived relaxing factor prostacyclin. 2.4 The effects of SKF-525A on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4mol/L dipfluzine on 5-HT pre-contracted vessels was 0.9496±0.0290 before incubated with SKF-525A, and 0.8983±0.0202 after incubated with SKF-525A, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. SKF-525A is the inhibitors of cytochrome P450. The results indicated that SKF-525A significantly reduced (P<0.01) the vasodilatation of dipfluzine. It suggested that the vasodilatation of dipfluzine might be related to the release of endothelium derived relaxing factor EDHF.3 The relationship between the vasodilatation of dipfluzine and potassium ion channels3.1 The effects of calcium-activated potassium ion channel inhibitor TEA on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4mol/L dipfluzine on 5-HT pre-contracted vessels was 0.7344±0.0277 before incubated with TEA, and 0.7111±0.0303 after incubated with TEA, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that the vasodilatation of dipfluzine might not be related to the open of calcium-activated potassium ion channel.3.2 The effects of voltage-sensitive potassium channel inhibitor 4-AP on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4mol/L dipfluzine on 5-HT pre-contracted vessels was 0.7447±0.0336 before incubated with 4-AP, and 0.7369±0.0164 after incubated with 4-AP, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that the vasodilatation of dipfluzine might not be related to the open of voltage-sensitive potassium ion channel.3.3 The effects of inward rectifier potassium ion channel inhibitor BaCl2 on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4mol/L dipfluzine on 5-HT pre-contracted vessels was 0.7344±0.0304 before incubated with BaCl2, and 0.7334±0.0218 after incubated with BaCl2, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that the vasodilatation of dipfluzine might not be related to the open of inward rectifier potassium ion channel.3.4 The effects of ATP-sensitive potassium ion channel inhibitor glibenclamide on dipfluzine-induced vasodilatationThe inhibitory rate of 10-4mol/L dipfluzine on 5-HT pre-contracted vessels was 0.7423±0.0383 before incubated with glibenclamide, and 0.5354±0.0192 after incubated with glibenclamide, when the contraction amplitude of the vessels induced by 5-HT was 100%. The results indicated that glibenclamide significantly reduced (P<0.01) the vasodilatation of dipfluzine, which suggested that the vasodilatation of dipfluzine might be related to the the open of ATP-sensitive potassium ion channel. 4 The relationship between the vasodilatation of 1-(4-fluorophenyl)-4 -piperazinylbutanone and calcium channels4.1 The inhibition of 1-(4-fluorophenyl)-4-piperazinylbutanone on the contraction of denuded-endothelium basilar artery induced by 5-HTThe inhibition percentages of 1-(4-fluorophenyl)-4-piperazinylbutanone (10-8~10-4 mol/L) on the contraction of denuded-endothelium basilar artery induced by 5-HT were: 0.0334±0.0122, 0.1411±0.0217, 0.6163±0.0218, 0.7630±0.0234, 0.8054±0.0191, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100% in the perfusion without 1-(4-fluorophenyl)-4-piperazinylbutanone. Experimental results indicated that the inhibition of 1-(4-fluorophenyl)-4-piperazinylbutanone on vaso -constriction induced by 5-HT in the basilar artery with intact endothelium was significantly greater (P<0.05) than that in denuded-endothelium basilar artery (the first part of the experiment has been done). It suggested that the mechanisms of 1-(4-fluorophenyl)-4-piperazinylbutanone vasodilatation might be not only due to blocking receptor-dependent calcium ion channel, but also related to endothelium derived relaxing factors.4.2 The inhibition of 1-(4-fluorophenyl)-4-piperazinylbutanone on the contraction of denuded-endothelium basilar artery induced by high potassiumThe inhibition percentages of 1-(4-fluorophenyl) -4-piperazinylbutanone (10-8~10-4 mol/L) on the contraction of denuded-endothelium basilar artery induced by KCl were: 0.0375±0.0168, 0.0561±0.0167, 0.1785±0.0210, 0.3492±0.0299, 0.4290±0.0326,when the contraction amplitude of the vessels induced by KCl was regarded as 100% in the perfusion without 1-(4-fluorophenyl)-4-piperazinyl-butanone. Experimental results indicated that the inhibition of 1-(4-fluorophenyl)-4-piperazinylbutanone on vaso -constriction induced by KCl in the basilar artery with intact endothelium was significantly greater (P<0.05) than that in denuded-endothelium basilar artery (the first part of the experiment has been done). It suggested that the mechanisms of 1-(4-fluorophenyl)-4-piperazinylbutanone vasodilatation might be not only due to blocking voltage-dependent calcium ion channel, but also related to endothelium derived relaxing factors.5 The relationship between the vasodilatation of 1-(4-fluorophenyl)-4 -piperazinylbutanone and endothelium derived relaxing factors5.1 The effects of L-NAME on 1-(4-fluorophenyl)-4-piperazinylbutanone -induced vasodilatationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4-piperazinyl -butanone on 5-HT pre-contracted vessels was 0.9633±0.0185 before incubated with L-NAME, and 0.7755±0.0519 after incubated with L-NAME, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that L-NAME significantly reduced (P<0.01) the vasodilatation of 1-(4-fluorophenyl)-4-piperazinylbutanone. It suggested that the vasodilatation of 1-(4-fluorophenyl)-4-piperazinylbutanone might be related to the release of endothelium derived relaxing factor NO.5.2 The effects of MB on 1-(4-fluorophenyl)-4-piperazinylbutanone- induced vasodilatationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4–piperazinyl -butanone on 5-HT pre-contracted vessels was 0.9775±0.0148 before incubated with MB, and 0.8598±0.0230 after incubated with MB, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that MB significantly reduced (P<0.01) the vasodilatation of 1-(4-fluorophenyl)-4-piperazinylbutanone, which further demonstrated that the vasodilatation of 1-(4-fluorophenyl)-4-piperazinyl butanone might be related to the release of endothelium derived relaxing factor NO.5.3 The effects of INDO on 1-(4-fluorophenyl)-4-piperazinylbutanone -induced vasodilatationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4-piperazinyl -butanone on 5-HT pre-contracted vessels was 0.9691±0.0231 before incubated with INDO, and 0.9701±0.0175 after incubated with INDO, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that the vasodilatation of 1-(4-fluorophenyl) -4-piperazinylbutanone might not be related to the release of endothelium derived relaxing factor prostacyclin.5.4 The effects of SKF-525A on 1-(4-fluorophenyl)-4-piperazinylbutanone -induced vasodilatationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4-piperazinyl -butanone on 5-HT pre-contracted vessels was 0.9697±0.0179 before incubated with SKF-525A, and 0.5374±0.0370 after incubated with SKF-525A, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that SKF-525A significantly reduced (P<0.01) the vasodilatation of 1-(4-fluorophenyl)-4-piperazinyl butanone. It suggested that the vasodilatation of 1-(4-fluorophenyl)-4 -piperazinylbutanone might be related to the release of endothelium derived relaxing factor EDHF.6 The relationship between the vasodilatation of 1-(4-fluorophenyl)-4 -piperazinylbutanone and potassium ion channels6.1 The effects of calcium-activated potassium ion channel inhibitor TEA on 1-(4-fluorophenyl)-4-piperazinylbutanone-induced vasodilatationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4-piperazinyl -butanone on 5-HT pre-contracted vessels was 0.8007±0.0202 before incubated with TEA, and 0.8032±0.0339 after incubated with TEA, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that the vasodilatation of 1-(4-fluorophenyl)-4 -piperazinylbutanone might not be related to the open of calcium-activated potassium ion channel.6.2 The effects of voltage-sensitive potassium channel inhibitor 4-AP on 1-(4-fluorophenyl)-4-piperazinylbutanone-induced vasodilatationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4-piperazinyl -butanone on 5-HT pre-contracted vessels was 0.7990±0.0171 before incubated with 4-AP, and 0.6930±0.0197after incubated with 4-AP, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that 4-AP significantly reduced (P<0.01) the vasodilatation of 1-(4-fluorophenyl)-4-piperazinylbutanone, which suggested that the vasodilatation of 1-(4-fluorophenyl)-4-piperazinylbutanone might be related to the open of voltage-sensitive potassium ion channel.6.3 The effects of inward rectifier potassium ion channel inhibitor BaCl2 on 1-(4-fluorophenyl)-4-piperazinylbutanone-induced vasodilatationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4-piperazinylbutan -one on 5-HT pre-contracted vessels was 0.7982±0.0366 before incubated with BaCl2, and 0.8001±0.0200 after incubated with BaCl2, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that the vasodilatation of 1-(4-fluorophenyl)-4-pipera -zinylbutanone might not be related to the open of inward rectifier potassium ion channel.6.4 The effects of ATP-sensitive potassium ion channel inhibitor glibenclamide on 1-(4-fluorophenyl)-4-piperazinylbutanone-induced vasodila -tationThe inhibitory rate of 10-4mol/L 1-(4-fluorophenyl)-4-piperazinyl -butanone on 5-HT pre-contracted vessels was 0.7952±0.0220 before incubated with glibenclamide, and 0.4082±0.0297 after incubated with glibenclamide, when the contraction amplitude of the vessels induced by 5-HT was regarded as 100%. The results indicated that glibenclamide significantly reduced (P<0.01) the vasodilatation of 1-(4-fluorophenyl)-4-piperazinyl -butanone, which suggested that the vasodilatation of 1-(4-fluorophenyl)-4 -piperazinylbutanone might be related to the the open of ATP-sensitive potassium ion channel.Conclusion: The mechanisms of vasodilatation of dipfluzine and its metabolite 1-(4-fluorophenyl)-4-piperazinylbutanone were not only related to receptor-dependent and voltage-dependent calcium ion channel of smooth muscle cell, but also related to the release of NO and EDHF from endothelial cells and opening of ATP-sensitive potassium ion channel, and the mechanism of vasodilatation of 1-(4-fluorophenyl)-4-piperazinylbutanone was related to voltage-sensitive potassium ion channel.