Enhancement Of Voltage-gated K⁺Currents And Depression Of L-type Voltage-gated Ca²⁺ Currents Are Involved In Quercetin-induced Vasorelaxation In Rat Coronary Artery

BackgroundQuercetin is as a typical fruit-derived flavonoid, which is widely distributed infruits, vegetables, botanic flowers and leaves, such as apple, sea buckthorn, broccoli,onions, tea, etc. Quercetin has been reported to possess many biological activities,including antioxidation, antiplatelet aggregation, antiinflammatory,immunomodulation, anticancer as well as reduction of cardiac ischemia-reperfusioninjury. Quercetin could induce vasodilation in rat aorta, rat mesenteric arteries, ratportal vein and porcine coronary arteries, whether quercetin could exert relaxationeffect on coronary artery (CA) of rat has not been studied.Calcium influx mainly through L-type voltage gated calcium channels (LVGC),which is prerequisite for the physiological function of vasomotor regulation ofvascular smooth muscle cells (VSMCs). At the same time, potassium channels couldmaintain normal cell membrane potential and regulate of vascular tone. There aremainly four kinds of potassium channels on vascular smooth muscle: voltage-gatedK+channels (Kv), Ca2+-activated K+channels (KCa), inward rectifier K+channels (KIR)and ATP-activated K+channels (KATP). However, Kv channels are the highestexpression, Kv channels play an important role in regulating cell resting potential andsmall artery diameter. The present study was designed to observe the effects ofquercetin on coronary dilation of rat and coronary flow and hemodynamic parametersof isolated rat heart, and whether LVGC and KVare involved in quercetin-inducedvasorelaxation in rat coronary arteries (RCAs). Part1The myogenic response of quercetin in rat coronary arteryObjectives1. To observe the myogenic response of quercetin in isolated RCAs, and to elucidatethe effect of quercetin on the inhibitory vasoconstriction and relaxantion in RCAs.2. In the myogenic experiment, we used the potassium channels blockers, nitric oxidesynthase inhibitor and endothelium denudation to study the relaxant effect ofquercetin on RCAs, and whether potassium channels, nitric oxide (NO) andendothelium are involved in myogenic response of quercetin.3. In the calcium-free solution, the effect of quercetin on extracellular calcium inducecontraction was observed.Methods1. After sacrifice, the heart was removed and transferred immediately into chilled(4°C) HEPES solution bubbled with100%O2. RCAs were isolated, cut into2mm-long rings and mounted on a wire myograph (Multi Myograph System-610M,DMT, Denmark) using two tungsten wires (25μm in diameter) in a tissue chambercontaining of5.0mL HEPES solution bubbled with100%O2,37°C. The rings werenormalized, stretched to a state equal to80mmHg, equilibrated for at least1h andwashed with HEPES solution (37°C) every15min. The tension changes of rings wererecorded by Chart7.03on the computer. After equilibration1h, the rings werecontracted with60mM KCl, then were relaxed with Ach, three times repeatedly.When the contraction or relaxation difference did not exceed10%, we thought that therings were good at the vascular reactivity. We discarded the arterial rings in case thatthe contraction was not repeatable or not sustained, or the contraction was less than2mN.2. High K+HEPES solution or U46619was cumulatively added to the chamber toconstruct a concentration-contraction curve for KCl (20,28,39,55,77,108mM) orU46619(0.03,0.1,0.3,1,3μM). When successive curves were repeatable, the arterialrings were preincubation with quercetin (3,10,30μM) for15min beforereconstruction of the curves for KCl or U46619in the presence of quercetin(quercetin was always in the chamber in this period). The maximal contraction induced by108mM KCl or3μM U46619in the absence of quercetin was taken as100%and the percentage of contraction by each concentration of stimulator in theabsence or presence of quercetin was calculated.3. When the contraction induced by60mM KCl or1μM U46619was sustained,quercetin (1,3,10,30,100μM) or vehicle were added cumulatively to theendothelium-intact or-denudation RCAs. The maximal contraction induced by60mM KCl or1μM U46619in the absence of quercetin was taken as100%and thepercentage of contraction by each concentration of stimulator in the absence orpresence of quercetin or vehicle was calculated.4. When the contraction induced by60mM KCl or1μM U46619reached a sustainedplateau,4-aminopyridine (4-AP, a KVblocker), iberiotoxin (Iber, a KCablocker),BaCl2(a KIRblocker) or glibenclamide (Glib, a KATPblocker) was added respectivelyto the chamber. After10min, based on the arterial tone was stable in the presence ofan inhibitor, quercetin (1,3,10,30,100μM) was cumulatively added to the abovechamber. Relaxation was expressed as a percentage of the precontraction induced by60mM KCl or1μM U46619, respectively.5. When the contraction induced by60mM KCl was repeatable, the ring was rinsedand incubated with Ca2+-free HEPES solution (containing1mM EGTA) for20min.When the tone of the ring restored to the baseline, the solution in above chamber waschanged to Ca2+-free HEPES solution. After10min, the ring was stimulated with60mM KCl or1M U46619, after10min,2.5mM CaCl2was added to the chamber.Quercetin was added to the chamber (the bath concentration of quercetin was keptconstant through the following experiment), after15min, the ring was stimulated with60mM KCl or1μΜ U46619in the Ca2+-free HEPES solution, after10min,2.5mMCaCl2was added to the chamber. The contraction induced by60mM KCl,1μΜU46619and2.5mM CaCl2were recorded in the absence or presence of quercetin.Results1. KCl (20,28,39,55,77,108mM) and U46619(0.03,0.1,0.3,1,3μM) inducedcontraction concentration-dependently. The maximal contractions were4.910.38 mN and4.830.41mN, respectively. The values of EC50were31.87mM for KCland0.19M for U46619, respectively. Pretreatment with quercetin (3,10,30M)shifted the concentration curves downwards to the right. The values of IC50were48.80M for KCl and88.30M for U46619, respectively.At3μM quercetin, there were no significant difference between quercetin groupand control group on KCl or U46619-contraction (P>0.05). At10μM, quercetindepressed the maximal contraction of KCl or U46619by35.4%and21.3%,respectively. There were significant difference between quercetin group and controlgroup on KCl or U46619-contraction (P <0.05). At30μM, quercetin depressed themaximal contraction of KCl or U46619by79.9%and54.7%, respectively. Therewere significant difference between quercetin group and control group on KCl orU46619-contraction (P <0.05).2. Quercetin (1,3,10,30,100μM) elicited concentration-dependent relaxation inendothelium-intact or-denudation RCAs, and endothelium has no effect on relaxationof quercetin in RCAs.Vehicle did not significantly affect the tone of endothelium-intact RCAs; themaximal relaxation of quercetin in RCAs precontracted by either60mM KCl or1M U46619were (96.34±7.35)%and (82.47±7.24)%, respectively. The values ofRC50were22.87M for KCl and41.42M for U46619, respectively.Compared endothelium-denudation RCAs with endothelium-intact RCAs, themaximal relaxation of quercetin did not change significantly (P>0.05).3. Preincubation with Iber, Glib, BaCl2or L-NAME, there have no effect on therelaxation of quercetin in RCAs, but preincubation with4-AP (1mM) attenuated therelaxation on KCl-precontraction by42.88%(P <0.05). Preincubation with4-AP (1mM) attenuated the relaxation on U46619-precontraction by28.82%(P <0.05).4. In Ca2+-free HEPES solution,60mM KCl failed to produce significant contraction(phasic) and restoration of2.5mM CaCl2evoked a full contraction (tonic). Quercetin(3,10,30μM) inhibited2.5mM CaCl2-induced contraction in aconcentration-dependent manner and the calculated IC50was9.55M. Quercetin didnot significantly affect the phasic for U46619, but reduced tonic component of the contraction and the calculated IC50was22.5M.Part2Effects of quercetin on [Ca2+]inand LVGC currents and Kvcurrents of rat coronary artery smooth muscle cellsObjectives1. To study the effect of quercetin on [Ca2+]influorescence intensity of RCA VSMCs.2. In order to clarify the mechanism of quercetin relaxation in RCAs, we recordedLVGC currents and Kv currents of RCA VSMCs.Methods1. Rat coronary artery smooth muscle cells isolation:Coronary artery smooth muscle cells were obtained using two steps enzymatic:Firstly, RCAs were placed in enzymatic solution I (HEPES solution containing0.1mM CaCl2,1mg/mL albumin,0.5mg/mL papain and1mg/mL dithioerythritol)bubbled with100%O2and incubated for30min at37oC. The RCAs were transferredto enzymatic solution II (HEPES solution containing1mg/mL albumin,0.5mg/mLcollagenase F and0.5mg/mL collagenase H) bubbled with100%O2and incubatedfor15min at37oC.2.[Ca2+]inmeasurement:Isolated RCA VSMCs were loaded with Fluo-4-AM and perfused with thedifferent solutions (HEPES solution,40mM KCl,40mM KCl+quercetin (10,30,100μM), HEPES solution and40mM KCl) in order of time precedence. Thefluorescence intensity of RCA VSMCs were recorded under various conditions.Fluorescence ratios (F/F0) were calculated to reflect the [Ca2+]in.3. Electrophysiological measurements:Using whole-cell voltage clamp, we recorded and investigated the effects ofquercetin on LVGC currents and Kv currents of RCA VSMCs.Results1. Quercetin reduced KCl-induced elevation of the intracellular Ca2+concentration ina concentration-dependent manner.40mM KCl increased fluorescence intensity ratio (F/F0, fluorescence after vs before40mM KCl) by97.4%. Application of quercetin(10,30,100M) decreased the intracellular Ca2+fluorescence intensity increment by30.1%,60.8%and86.5%, respectively.2. Bay K8644(1M) increased LVGC currents and increased the peak currents by31.9%; nifedipine (1M) decreased LVGC currents and decreased the peak currentsby75.6%. These results proved that the currents recorded were LVGC currents.Quercetin (3,10,30μM) shifted the I-V curve of LVGC currents of RCA VSMCsupwards to the right in a concentration-dependent manner. At test potential of+10mV,quercetin (3,10,30M) decreased LVGC currents by28.7%,57.1%and76.8%,respectively.3. At test potential of+60mV, the stable peak of Kv currents was345.26±23.57pAand current density was32.52±5.24pA/pF (n=12). Quercetin (10,30,100μM)shifted the I-V curve of KVcurrents upward in a concentration-dependent manner. Attest potential of+60mV, quercetin (10,30,100M) increased the KVcurrents ofRCA VSMCs by20.1%,38.1%and73.3%, respectively.Part3Effects of quercetin on coronary flow and hemodynamicparameters of isolated rat heartObjectiveTo study the effects of quercetin on coronary flow (CF) and hemodynamicparameters (LVDP，LVEDP，+dp/dtmax，-dp/dtmax) of isolated rat heart by Langendorffapparatus.MethodsThe isolated rat heart was perfused firstly with Tyrode’s solution usingLangendorff apparatus, subsequently, it was perfused with Tyrode’s solutioncontaining different concentrations of quercetin (0.3,1,3,10,30μM). At the sametime, coronary flow and hemodynamics parameters were recorded.ResultsThis experiment showed that quercetin increased the vaule of CF, LVDP, +dp/dtmaxand-dp/dtmax, but it had no effect on LVEDP.1. When the isolated rat hearts were perfused with Tyrode’s solution, the vaule of CFwas7.8±1.2mL/min, then they were perfused with Tyrode’s solution containingdifferent concentration of quercetin (0.3,1,3,10,30μM), quercetin increased the CFin a concentration-dependent manner, the maximal value of CF was13.0±1.3mL/min and the EC50was3.04μM.2. Quercetin increased LVDP and+dp/dtmaxof isolated rat hearts in aconcentration-dependent manner, the basal value were64±3mmHg (LVDP) and1368±124mmHg/s (+dp/dtmax), respectively. With increasing concentrations ofquercetin (0.3,1,3,10,30μM), the value of LVDP and+dp/dtmaxwere graduallyincreased, the maximal value were78±2mmHg and1692±196mmHg/s,respectively.3. Quercetin increased-dp/dtmaxof isolated rat hearts in a concentration-dependentmanner, the basal value was1143±126mmHg/s. With increasing concentrations ofquercetin (0.3,1,3,10,30μM), the value of-dp/dtmaxwas gradually increased, themaximal value was1375±151mmHg/s. The basal value of LVEDP was10±1mmHg, quercetin had no effect on LVEDP.Conclusions1. Quercetin elicited relaxation in RCA in a concentration-dependent manner. Therelaxation effect of quercetin on RCA was non-endothelial dependent, and wasassociated with increased Kv currents and depressed LVGC currents of RCA VSMCs.2. Quercetin increased coronary flow of isolated rat heart, this result was consistentwith the relaxation effect of quercetin on RCA in myogenic experiment. Quercetinhad a positive inotropic effect on isolated rat heart.