The Mechanism Of Bitter Taste Receptor Mediated Relaxation Of Rat Aorta

Background:Bitter taste receptors?TAS2Rs?are a family of the G protein-coupled receptor?GPCR?.Early researches of TAS2Rs mainly focused on the gustatory system.The discovery of TAS2Rs in the digestive system,respiratory system,reproductive system and the cardiovascular system has been reported.Recent studies have reported that TAS2Rs express in vascular smooth muscle cells.TAS2Rs agonists,such as denatonium,chloroquine and quinine can relax guinea pig aorta pre-contracted by L-phenylephrine.However,there is no report on the expression of TAS2Rs in rat aorta,and the mechanism of TAS2Rs regulating the function of rat aorta are not clear yet.Aim:The aim of this study is to determine the expression levels of TAS2Rs on rat aorta and isolated vascular smooth muscle cells.Three TAS2Rs agonists,denatonium,chloroquine and quinine were selected to study their regulation of rat aorta function,and the underlying mechanism of TAS2Rs regulating the tension of rat aorta.Method:1.Rat aorta and isolated vascular smooth muscle cells were processed to detect the TAS2Rs mRNA expression by real-time fluorescence quantitative PCR.2.The functional effect of TAS2Rs activation on rat aortic contractility and the underlying mechanisms were investigated using isometric tension recordings.Three TAS2Rs agonists,denatonium,chloroquine and quinine were used and the rat aorta ring were pre-contracted by 60 mM KCl,10?M L-phenylephrine,or electrical filed stimulation?10 V,25 Hz?,respectively.3.The mechanisms underlying the TAS2Rs regulation of rat aortic contractility were studied by using TAS2Rs agonist in combination with inhibitors for,PLC?U-73221?,K⁺channel?tetraethylammonium,TEA?,L-type Ca²⁺channel?nifedipine?,or sarcoplasmic reticulum Ca²⁺-ATPase?SERCA??thapsigargin?.Result:1.Six TAS2Rs mRNA were detected in rat aorta tissue and freshly isolated vascular smooth muscle cells.The relative expression levels of TAS2R143,TAS2R135,TAS2R126,TAS2R120,TAS2R121 and TAS2R108 were 0.30±0.16%,0.19±0.08%,0.16±0.08%,0.07±0.04%,0.04±0.05%,and 0.07±0.03%in aorta tissue,and were 2.01±0.71%,1.22±0.90%,0.20±0.08%,0.19±0.08%,0.10±0.03%and 0.03±0.02%,in freshly isolated vascular smooth muscle cells respectively.The expression level of the reference gene,GAPDH,was 100%.2.Denatonium?50-1000?M?and chloroquine?10-300?M?can relax aorta ring pre-contracted by 60 mM KCl in a concentration-dependent manner.The maximal relaxation was 94.9±5.5%with 1000?M denatonium?P<0.05?,and was 87.2±3.9%with 300?M chloroquine?P<0.05?.3.Denatonium at low concentration of 20?M transiently reduced the tension of aorta ring pre-contracted by L-phenylephrine?10?M?;it significantly reduced the tension of aorta at high concentrations of 800 to 1500?M with the maximal relaxation of 96.0±4.9%?P<0.05?.Chloroquine?20-300 uM?and quinine?100-200?M?can relax the pre-contracted rat aorta in a concentration-dependent manner;the maximal relaxation was 100.5±3.7%with 300?M chloroquine?P<0.05?,and was 98.1±2.6%with 200?M quinine?P<0.05?,respectively.4.Denatonium?300?M?,chloroquine?200?M?,and quinine?200?M?inhibited the electrical field stimulation?10 V,25 Hz?induced aortic contractions by 81.1±7.0%,88.1±1.8%,and 85.9±3.0%,respectively?P<0.05?.5.The relaxation effect of chloroquine and quinine on aorta ring pre-contracted by L-phenylephrine?10?M?was endothelium-independent.Denatonium?20-500?M?can relax endothelium-denuded aorta pre-contracted by L-phenylephrine?10?M?in a concentration-dependent manner,while it transiently reduces and finaly increases the tention of the endothelium-intact aorta at same concentrations.6.The K⁺channel blocker,TEA attenuated the relaxation effect of chloroquine on aorta ring pre-contracted by L-phenylephrine.The reduction of the aortic tension caused by chloroquine?200?M?was 96.4±3.9%in the absence of TEA,and was 71.9±10.7%in the presence of TEA,respectively,?P<0.05?.TEA abolished the transient relaxation of aorta ring caused by denatonium.The SERCA inhibitor,thapsigargin attenuated the relaxation effect of chloroquine?300?M?from 100.5±3.7%in the absence of the inhibitor to 50.2±7.4%?P<0.05?.The L-type Ca²⁺channel inhibitor,nifedipine?1?M?,eliminated the contractive effect of denatonium and the relaxation effect of quinine on the aorta ring.In the presence of nifedipine,denatonium in the concentration range of 20 to 500?M relaxed the aorta ring pre-contracted by L-phenylephrine?10?M??P<0.05?.Conclusion:TAS2Rs express in rat aorta and isolated vascular smooth muscle cells and they might be involved in the vasodilation of rat aorta induced by TAS2Rs agonists.Three TAS2Rs agonists regulate the contractility of rat aorta ring with different mechanisms.Chloroquine might decrease the tension of rat aorta by activating potassium channels and promote intracellular calcium reabsorption to the sarcoplasmic reticulum.Quinine might decrease the tension of aorta by inhibiting L-type calcium channel.Denatonium might transiently decrease the tension of aorta by activating potassium channels.And denatonium at low concentrations causes vasoconstriction by activating L-type calcium channels.