Vasorelaxant Effect And Mechanism Of Luteolin On Rat Intrarenal Artery

Part one The vasotonic effect of luteolin on isolated rat intrarenal arteriesObjective:1.To observe the effect of luteolin（Lut）on the resting tension of isolated rat intrarenal arteries（IRAs）;to observe inhibitiory effect of Lut on rat IRAs contraction induced by thromboxane analogue（U46619）,potassium chloride（KCl）,phenylephrine（PE）,vasopressin（VP）and calcium ion(Ca²⁺)and vasorelaxation effect of Lut on U46619,KCl,PE and VP-induced contraction of rat IRAs.2.To explore the relationship between the vasorelaxation of Lut on IRAs and intracellular and extracellular calcium by decalcification-calcium method.3.Observing the effect of without Cl^-solution on Lut-induced vasorelaxation on rat IRAs.4.To study the effect of Lut on rat IRAs by applying Ca²⁺-activated Cl^-channels（CaCCs）blocker CaCC_inh-A01.Methods:1.Adult male SD rat（220-250 g）was anesthetized by intraperitoneal injection of sodium pentobarbital（40 mg/kg）.Kidney immediately removed and IRAs was isolated.The vascular rings were mounted on wire myograph using two tungsten wires（40μm）.Before the formal experiment,IRAs were equilibrated in the bath for 1 h and changed every 15 min.The vessels were contracted repeatedly with 60 mM KCl.If the difference was less than 10%,and the maximum contraction amplitude was greater than 2 mN,and the activity of the vascular ring was considered to be good,and the experiment was started after 30 min of equilibrated.2.Using the PowerLab recording system,the changes of IRAs tension at resting state with different concentrations of Lut（1-100μM）were observed.The concentration contraction curve was established by using U46619（0.01-1μM）,KCl（20-108 mM）,VP（0.1-10μM）,PE（0.1-10μM）and Ca²⁺（0.03-10 mM）.After 15-30 min incubation of Lut（10,30,100μM）,the concentration contraction curve was re-established.The maximum contraction caused by vasoconstrictors was taken as 100%.The maximum inhibitory rate and the drug concentration required to inhibit the contraction of 50%(IC₅₀)were calculated.3.0.3μM U46619,60 mM KCl,1μM PE and 1μM VP were add to the chamber.When it comes to a platform,Lut was added to the chamber cumulatively to make the final concentrations of 1,3,10,30,100μM.Establishing the concentration-diastolic curve of Lut,observed the effect of different concentrations of Lut on the tension of pre-contracted IRAs.The control group followed by adding an equal volume of DMSO to observe the influence of solvent on the experiment.The maximum amplitude of the IRAs contraction was taken as 100%,and maximal relaxation rate and the drug concentration(RC₅₀)required for relaxation of 50%were calculated.4.Ca²⁺-free PSS with EGTA was added into chamber for 15 min,so that the extracellular fluid reached a calcium-free state,and then added Ca²⁺-free PSS without EGTA at the same time and then added 0.3μM U46619 or 60 mM KCl into Ca²⁺-free PSS without EGTA,and finally restored calcium to 2.5 mM when it comes to a plateaus.After incubation with different concentrations of Lut（10,30μM）.The method of remove and recovery calcium was used to study the effect of Lut on vasoconstriction caused by extracellular calcium influx and intracellular calcium release.5.0.3μM U46619 or 60 mM KCl was added into chamber and when IRAs contracted and reached plateau,Lut was added.Pre-incubation with the normal PSS without Cl^-for 30min,in which NaCl,KCl,CaCl₂,MgCl₂ were respectively replaced with sodium gluconate,potassium gluconate,calcium gluconate and magnesium sulfate;The IRAs were stimulated with U46619 or KCl and Lut was added again when reached the platform to observe the effects of without Cl^-on IRAs.The maximum contraction of U46619 or KCl was taken as100%,and the relaxation rate with or without Cl^-was calculated.6.0.3μM U46619 or 60 mM KCl was added into chamber and when IRAs contracted and reached plateau,the relaxation of Lut was recorded.The IRAs were rinseied and stabilized for 30 min,then established vasoconstriction again.After reaching the platform,10μM CaCC_inh-A01 was pre-incubated for 10 min and then Lut was added again to observe the effects of inhibitor on IRAs.The maximum contraction of U46619 or KCl was taken as 100%,and the relaxation rate of Lut in the presence or absence of inhibitors was calculated.Results:1.Lut（1-100μM）had no significant effect on resting isolated rat IRAs,but pre-incubation with Lut（10,30,100μM）could make U46619,KCl,PE,VP and Ca²⁺maximum amplitude decreased,and the maximum inhibition rates were（99.94?0.00）%,（97.88?0.01）%,（100.05?0.01）%,（99.95?0.04）%and（97.82?0.02）%（P<0.05）.IC₅₀were12.31,24.45,14.07,18.37 and 16.49μM.Compared with the control group,Lut（10,30,100μM）inhibited contraction induced by U46619,KCl,PE,VP and Ca²⁺on isolated rat IRAs.2.Lut（1-100μM）concentration-dependently relaxed IRAs ring precontracted with0.3μM U46619,60 mM KCl,1μM PE and 1μM VP.The maximum relaxation rates were（96.01?0.05）%,（90.97?0.05）%,（97.00?0.02）%,（101.58?0.04）%（P<0.05）and RC₅₀were5.63,11.66,4.67,2.92μM,respectively.3.In the calcium-free PSS,the contractile amplitude of the RIAs induced by 0.3 uM U46619 was weak,and the maximum contractile amplitude after re-calcium treatment was（2.33?0.62）mN.Pre-incubation with Lut（10,30μM）could inhibit the contraction of isolated rat RIAs induced by re-calcium,and the contractile amplitudes were（1.09?0.13）mN and（0.03?0.12）mN（P<0.05）;In the calcium-free PSS,the contractile amplitude of the RIAs induced by 60 mM KCl was low,and the contractile amplitude of re-calcium treatment was（4.08?1.03）mN.Pre-incubation with Lut（10,30μM）,the contractile amplitudes were（2.52?0.51）mN and（0.67?0.48）mN（P<0.05）.4.The maximal relaxation precentage of 0.3μM U46619 or 60 mM KCl pre-contracted IRAs was（65.75?0.16）%or（67.22?0.14）%.After pre-incubation with without Cl^-PSS 30 min,the relaxation percentage was（42.25?0.21）or（47.61?0.03）%（P<0.05）.5.The maximal relaxation precentage of 0.3μM U46619 or 60 mM KCl pre-contracted IRAs was（69.63?0.21）%or（56.12?0.19）%.After pre-incubation with 10μM CaCC_inh-A01,the relaxation precentage was（25.53?0.12）%or（43.13?0.15）%（P<0.05）.Conclusion:1.Lut has no effect on the resting tone of isolated rat IRAs,and inhibits contractions induced by U46619,KCl,PE,VP and Ca²⁺,and Lut relaxed IRAs pre-contracted by U46619,KCl,PE and VP in a concentration dependent manner.2.The relaxant effects of Lut may be related to calcium antagonism,mainly to inhibit extracellular calcium influx.3.Without Cl^-and CaCCs blocker can attenuate the relaxation of Lut on isolated IRAs,and its may be mediated by inhibition of CaCCs.Part two Effect of luteolin on CaCCs of acutely isolated rat intrarenal arteries smooth musclesObjective:To observe the effect of Lut on CaCCs current of rat IRASMCs.Methods:1.IRASMCs isolation:After the IRAs were separated,it was placed in cell separation solution immediately,in which it contained 1 mg/ml bovine serum albumin,1 mg/ml dithioerythritol,and 0.5 mg/ml papain.After incubation for 30 min with saturated O₂ at37°C),IRAs were then transferred to cell separation solution containing 1 mg/ml bovine serum albumin,0.5 mg/ml collagenase H,0.5 mg/ml collagenase F and incubated for 5-10min.We can add an equal volume of pre-warmed cell separation solution and incubate for2-5 min.Finally,after centrifugation at 800 rpm for 6 min,the supernatant was discarded,and isolated IRASMCs was ready for the whole cell patch clamp electrophysiology experiment within 8 h.2.Using whole-cell patch clamp technique to observe the effect of Lut on CaCCs current of rat IRASMCs.After the normal CaCCs current recording was stabilized,a CaCCs-specific blocker 10μM CaCC_inh-A01 was added to the cell bath.Normal CaCCs current was recorded again,CaCCs current was recorded after 30μM Lut was added into bath for 2 min.Results:At a test potential of+100mV,the stable peak current of IRASMCs was 516.13±92.81 pA and the current density was 39.21±8.39 pA/pF.10μM CaCCinh-A01 inhibited the peak current density,and the inhibition rate was（53.65±10.69）%（P<0.05）.30μM Lut significantly decreased the current density by（55.92±0.20）%（P<0.05）.Conclusion:30μM Lut can inhibit CaCCs current in IRASMCs.