| Objective:1.To investigate the effect of niclosamide ethanolamine(NEN)on the resting tone of isolated rat coronary artery(RCA);to observe the relaxation effect and inhibitory effect of NEN on RCA contraction induced by KCl and U46619.2.To study the effect of NEN on mobilization by observing vasoconstrictor-induced constractions in the tissue solution deprived of Ca2+ followed by Ca2+ restoration;to explore the possible involvement of potassium channel and MAPK signaling pathway in the vasorelaxation of NEN on isolated RCA.3.To observe the effect of NEN on coronary flow of isolated rat hearts by Langendorff isolated heart perfusion system.To explore the effect of NEN on the blood pressure and heart rate in hypertensive and normotensive rats by carotid artery intubation.Methods:1.The male SD rats(220-250 g)were anesthetized.After exsanguination,the heart was immediately removed into chilled physiological salt solution and RCA was isolated.The RCA ring with a length of approximately 2 mm was cut and mounted on the microvascular tension recorder(DMT).The changes of RCA tension at resting state with different concentrations of NEN(0.5,1,1.5,2,2.5,and 3 ?mol/L),and the effect of RCA contraction induced by KCl(60 mmol/L),U46619(0.3 ?mol/L)after 20 min incubation of the different concentration of NEN were observed using the Chart recording system.2.KCl(60 mmol/L)and U46619(0.3 ?mol/L)were respectively add to the chamber to constrict the isolated RCA rings.When it comes to a plateau,NEN is added cumulatively to make the final concentrations of 0.5,1,1.5,2,2.5,3 ?mol/L,record the changes of RCA-ring tension pre-constricted by NEN.The control group followed by adding the appropriate volume ratio of solvent(DMSO)to observe the effect of the solvent on RCA rings.The maximum amplitude of RCA contraction caused by vasoconstrictors was taken as 100%.The relaxation percentage,maximal relaxation rate and the drug concentration(RCso)required for relaxation of 50%of NEN at different concentrations were calculated.3.The normal PSS was changed into Ca2+-free PSS solution,and then changed into Ca2+-free KCl(60 mmol/L)or U46619(0.3 ?mol/L)after 20 min incubation of different concentrations of NEN(1,1.5,2 ?mol/L).Restore calcium to 2.5 mmol/L when it comes to a plateaus.We used the method of remove and recovery calcium to record the RCA constrictions,and to compare the effects of NEN on RCA constrictions made by extracellular Ca2+ internal flow and internal Ca2+ release.4.When the contraction of RCA was stimulated to a plateau,the relaxation of NEN was recorded in the absence of inhibitor.The pre-incubation of inhibitor includs Kv blockers 4-AP(1 mmol/L),Kir,blockers BaCl2(1 mmol/L),Kca blockers TEA(1 mmol/L),KAPT blockers Gli(0.01 mmol/L),MAPK inhibitor PD98059(3 ?mol/L)or SB239063(3?mol/L).When RCA contracted and reached the plateau,NEN(2 ?mol/L)was added to observe the effects of the inhibitors on RCAs.The percentage of relaxation was calculated,taking the maximum constriction as 100%.5.The effect of NEN(1,1.5,2,2.5,3 ?mol/L)on coronary artery flow in isolated rat hearts by Langendorff isolated heart perfusion system.6.The effect of the femoral vein injection of NEN(1,1.5,2,2.5,3 ?mol/L)on blood pressure and heart rate of rats in the spontaneously hypertensive rat(SHR)group and the normal blood pressure control rat(WKY)group by using carotid artery intubation.Results:1.NEN(0.5-3 ?mol/L)had no significant effect on resting RCA vascular tone.The maximum amplitude of RCA contraction induced by KCl(60 mmol/L)was 100%.After adding NEN(0.5-3 ?mol/L),the percentages of RCA contraction were(0.12 ± 0.03)%,(0.23 ± 0.08)%,(0.31 ± 0.11)%,(0.18 ± 0.15)%,(0.22 ± 0.13)%,(0.26 ± 0.23)%.Compared with the solvent control group,the difference was not statistically significant(P>0.05).After preincubated with NEN(0.5-3 ?mol/L),the maximal contractile amplitude of RCA contracted with KC1(60 mmol/L)and U46619(0.3 ?mol/L)decreased(90.69 ± 0.98)%,(86.85 ± 1.26)%(P<0.05).2.The maximum contractile amplitude of contractile RCA were(5.04 ± 0.46)mN and(5.15 ± 0.79)mN respectively by KCl(60 mmol/L)and U46619(0.3 ?mol/L).NEN(0.5-3 ?mol/L)could relaxe KC1(60 mmol/L)and U46619(0.3 ?mol/L)pre-contracted RCA in a concentration-dependent manner and the maximum relaxation rates were(98.61±0.78)%and(95.51±1.02)%,and the RC50 were(1.85 ± 0.28)?mol/L,and(1.94± 0.41)?mol/L.3.The contractile amplitude of RCA induced by KC1(60 mmol/L)in Ca2+-free PSS was(0.11 ± 0.13)mN,and the contractile amplitude of re-calcium treatment was(3.92 ±1.13)mN.Preincubation with NEN(1,1.5,2 ?mol/L)significantly inhibited the RCA contraction induced by extracellular Ca2+ internal flow KCl(60 mmol/L),with amplitude of(3.73 ± 1.02)mN and(2.35 ± 0.61)mN,(1.60 ± 0.25)mN.The contractile amplitude of RCA induced by U46619(0.3 ?mol/L)was(0.55 ± 0.14)mN in calcium-free PSS and contracted RCA after re-calcium treatment,and the contraction amplitude was(3.44 ±1.13)mN.Preincubation with NEN(1,1.5,2 ?mol/L)significantly inhibited RCA contraction induced by Ca2+ influx after U46619(0.3 ?mol/L)resynchronization with amplitude of(3.28 ± 0.95)mN and(2.34 ± 0.16)mN,(1.71 ± 0.23)mN,respectively.The difference was statistically significant(P<0.05).NEN had no significant effect on the contraction caused by intracellular calcium release(P>0.05).4.After adding four kinds of potassium channel blockers 4-AP(1 mmol/L),BaCl2(1 mmol/L),TEA(1 mmol/L)and Gli(0.01 mmol/L),the maximal relaxation percentage of U46619(0.3 ?mol/L)pre-contracted coronary artery rings were(87.88 ± 5.63)%,(91.36 ± 2.68)%,(88.89 ± 3.70)%and(93.35 ± 4.20)%,respectively.Compared with the control group,all four potassium channel blockers had no significant effect on the relaxation of NEN(2 ?mol/L)(P>0.05).In the presence of MAPK inhibitor PD98059(3?mol/L)and SB239063(3 ?mol/L),the relaxation amplitude of NEN(2 ?mol/L)precontracted with KCl(60 mmol/L)decreased by(27.93 ± 0.71)%and(30.47 ± 0.82)%,respectively.The relaxation amplitude of pre-contracted RCA of U46619(0.3 ?mol/L)decreased by(25.92 ± 0.58)%and(27.09 ± 0.65)%(P<0.05).5.The CF of isolated rat hearts were(7.8 ± 1.1)ml/min,and the maximum CF increased by 12.7%,22.1%,30.7%,37.8%,46.2%respectively,after adding NEN(0.5,1,1.5,2,2.5,3 ?mol/L).6.The SP,DP,MAP and HR of the SHR group were(158.5 ± 7.6)mmHg,(124.1 ±9.3)mmHg,(147.0 ± 8.2)mmHg and(419.3 ± 17.9)beat/min,and the maximum SP,DP,MAP and HR of SHR group decreased by 22.6%,30.1%,24.7%and 14.5%respectively after intravenous injection of NEN(P<0.05).The SP,DP,MAP and HR of the WKY group were(158.5 ± 7.6)mmHg,(124.1 ± 9.3)mmHg,(147.0 ± 8.2)mmHg and(324.1 ±20.7)beat/min,and the maximum SP,DP,MAP and HR of WKY group decreased by 8.6%,10.4%,9.2%and 7.5%respectively after intravenous injection of NEN(P<0.05).Conclusion:1.NEN had no effect on resting tone of isolated RCA rings,and inhibited isolated RCA contractions induced by KCl and U46619.2.NEN relaxed RCA precontracted with KCl or U46619 in a concentration dependent manner.The relaxant effects of NEN on isolated RCA was mediated by the activation of MAPK signaling pathway and the inhibition of extracellular Ca2+ influx,but did not depend on potassium channel.3.NEN increase coronary flow of isolated rat hearts and reduced SP,DP,MAP and HR in hypertensive rats and normotensive rats. |
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