The Mechanism Of G Protein-coupled Bitter Taste Receptor Agonist Regulation Of Rat Aortic Smooth Muscle Contractility

Background:Bitter taste receptors（Tas2rs）belong to the G protein-coupled receptor（GPCR）.Early studies have shown that activating Tas2rs initiate the dissociation of heterotrimeric G protein Gαgustducin/Gβ1γ13,and the taste-specific G protein（Gαgustducin）activates phosphodiesterases（PDEs）to accelerate the hydrolysis of cyclic adenosine monophosphate（cAMP）,it leads to the decrease of cytosolic level of cAMP in taste cells;Gβ1γ13 dimer can activate phospholipase Cβ2（PLCβ2）to generate a second messenger inositol 1,4,5-triphosphate（IP₃）.IP₃ activates IP₃R on the endoplasmic reticulum（ER）,releasing Ca²⁺from the ER into the cytoplasm,thereby increasing the concentration of free Ca²⁺in the cytoplasm in taste cells.Recent studies have identified the expression of Tas2rs in a variety of non-lingual tissues including vascular smooth muscle（VSM）,pulmonary smooth muscle and airway smooth muscle.However,there are no reports on the physiological function and mechanism of Tas2rs on vascular smooth muscle,especially in rat aortic vascular smooth muscle.Aim:The current study aims to determine the expression of Tas2rs and gustducin in rat aortic smooth muscle tissue and to investigate the effect of Tas2rs agonist denatonium on the tone of isolated denuded aorta rings.Method:1.Denuded rat aorta rings were processed to detect the Tas2rs mRNA expression by real-time fluorescence quantitative PCR.2.Denuded rat aorta rings were used to detect gustducin protein expression by immunofluorescence.3.The functional effect of Tas2rs activation on rat aortic contractility and the underlying mechanisms were investigated using isometric tension recordings.A canonical tas2rs agonist,denatonium was used.4.The mechanisms underlying the Tas2rs regulation of rat aortic contractility were studied by using Tas2rs antagonist adenosine 5′-monophosphate（5′-AMP）,cAMP-hydrolyzing PDE inhibitors（IBMX）,PDE3 inhibitor（cilostamide）,PDE4 inhibitor（rolipram）,cAMP-synthesizing enzyme activator forskolin,by the Gβγinhibitor（gallein）,PLCβ2 inhibitor（U-73122）,sarco-endoplasmic reticulum Ca²⁺ATPases inhibitor（cyclopiazonic acid,CPA）and a L-type calcium channels（LTCCs）blocker（nifedipine）respectively.Result:1.The mRNAs of Tas2rs and their coupled G proteins expressed in rat aorta tissue:Our RT-qPCR results revealed the mRNA expression of 6 Tas2rs（Tas2r40,108,126,135,137,143）and Gαgustducin,Gβ1,Gβ3,and Gγ13 in the denuded rat aorta tissue.These data demonstrated that Tas2r signaling cascade may exist in the rat aorta,particularly in VSMCs,and suggest that Tas2rs may have a role in the regulation of aorta function.2.Gαgustducin expressed in VSMCs of rat aorta rings:we perform a double immunostaining on rat aorta ring sections.Our result showed that the immunostaining to gustducin（green）was present in VSMCs labeled withα-SMA（red）located in the tunica media of rat aorta rings.3.Denatonium increased the isometric tension of denuded rat aorta rings:In the denuded aorta ring,the addition of single concentration of denatonium increased the tension of aorta.We used denatonium in the range of 10μM to 100μM,denatonium-induced maximal tonic contractions were similar at 20μM,50μM and 100μM except that at 10μM.However,the time needed for denatonium to increase the tension to the maximum became shorter at higher concentrations.These suggest that Tas2rs agonist denatonium can enhance the tone of aortic smooth muscle.4.The potentiation effect of denatonium on the isometric tension of VSM was Tas2r-dependent:denatonium（50μM,100μM）induced tension increase can be blocked by a Tas2r antagonist 5’-AMP（5 mM）,which target the cell-surface receptor.5.PDE Blockade eliminated denatonium-induced tension increase in denuded aorta rings:Denatonium（100μM）increased the aortic tension by 70.3%±2.7%（Normalized to 60mM KCl induced tension increase）and the subsequent addition of a broad spectrum PDE inhibitor,IBMX（100μM）,eliminated denatonium-induced tension;the washout of IBMX recovered the tension to 42.3%±2.8%.The selective inhibition of PDE3 by cilostamide（2μM）or PDE4 by rolipram（20μM）alone attenuated the denatonium-induced tension from 78.2%±1.7%to 20.2%±2.6%,and from 76.9%±1.8%to 54.7%±4.7%,respectively.Pretreatment with a cocktail containing cilostamide（2μM）and rolipram（20μM）slightly relaxed the denuded aorta ring with the initial tension of 15mN and diminished the ability of denatonium to evoke a tension increase.These demonstrate that the activation of cAMP-hydrolyzing PDEs was necessary for denatonium to increase the tension of aorta.PDE3 contributed more than PDE4 in denatonium-induced tonic contractions（20.2%±2.6%vs 54.7%±4.7%,P<0.0001）.To eliminate the possibility that cGMP might be involved in the function of denatonium,we pretreated the denuded aorta with a cGMP-specific PDE5 inhibitor,sildenafil,the blocked of PDE5 did not affect the denatonium-induced tension increase.6.The adenylyl cyclase activator,forskolin,prevented denatonium-induced tension increase in aorta:Pretreatment with forskolin（100 nM,300 nM,10μM）attenuated denatonium（100μM）-induced contraction in the aorta.The post treatment with forskolin（300 nM,1μM,10μM）significantly relaxed the denatonium-induced contraction.The subsequent addition of forskolin（10μM）following denatonium eliminated the tension increase from 68.5%±4.5%to-3.5%±0.8%and the washout of forskolin recovered the tension to 24.5%±1.9%.These results indicate that the denatonium-evoked tension increase may related with the change of intracellular cAMP level.7.In order to determine whether denatonium was able to affect the total intracellular cAMP level we measured cAMP in the denuded aorta treated with denatonium and forskolin as in the organ bath experiments.Denatonium did not decrease the basal cAMP levels in the aorta without forskolin treatment.Forskolin at 100 nM and 300 nM did not raise the total cAMP levels.The increase of total cAMP level was detected only in the tissues treated with 10μM forskolin.8.The inhibition of Gβγand PLCβ2 with gallein and U-73122,respectively,did not have any negative impact on the tonic function of denatonium.9.The depletion of intracellular sarco-endoplasmic reticulum Ca²⁺store via the inhibition of sarco-endoplasmic reticulum Ca²⁺-ATPases（SERCA）with cyclopiazonic acid（CPA）did not hinder the denatonium-evoked tension increase.However,the denatonium-induced tonic contraction was completely abolished by the L-type Ca²⁺channel（LTCC）blocker,nifedipine.These data suggested that the denatonium-induced tonic contraction might dependent on the influx of Ca²⁺rather than the intracellular Ca²⁺release.Conclusion:Our results revealed the expression of Tas2rs and the taste specific G-protein gustducin in rat aortic smooth muscle,and for the first time,demonstrated that the denatonium-induced tonic contraction was dependent on the activation of Tas2r and PDEs,rather than the activation of Gβγand PLCβ2.