The Chronotropic Function Of Bitter Taste Receptor Agonists And The Underlying Mechanism In The Rat Heart

Background:Bitter taste receptors（Tas2rs）,the members of G-protein-coupled receptors,mediate the bitter taste and express in extra-oral tissues.Previous studies have shown that Tas2r mRNAs express in the whole heart and cultured cardiomyocytes of neonatal rats and that Tas2rs mediate the negative inotropic effect on the murine heart in a G-protein-dependent manner.However,the regulation and mechanism of Tas2rs on cardiac function have not been well characterized.In gustatory tissues,the agonist-binding activates the Tas2r-coupled heterotrimeric G-proteins,GαgustducinGβγ,leading to the separation of Gαgustducin from Gβγ.Gαgustducin can increase the degradation of cAMP by activating phosphodiesterase（PDEs）.The localized cAMP in the pacemaker cells is critical for generating the spontaneous action potentials in the sinoatrial（SA）node which travel through atria to ventricles and initiate the rhythmic beating of the heart.Gβγactivates phospholipase Cβ2（PLCβ2）and consequently manipulates intracellular Ca²⁺signaling.Our previous studies have shown that Tas2r agonists,quinine,chloroquine,and denatonium,decreased the heart rate of the Langendorff-perfused isolated rat hearts.However,the underlying mechanisms were unclear.Aim:This study aimed to determine the Tas2r mRNA expression in the SA node of rats,and to investigate the regulation of Tas2r agonists on the heart rate and SA node beating rate,and to reveal the underlying mechanisms.Methods:The current study clarified the regulation of Tas2r agonists on the heart rate of rats by electrocardiogram;determined the mRNA expression of Tas2rs in the SA node by real-time quantitative polymerase chain reaction（RT-qPCR）;investigated the effect of Tas2r agonists on the SA node beating rate and the underlying mechanisms by recording SA node beating in combination with pharmacological tools;studied the functional interplay between Tas2rs andβ-adrenergic receptors.Results:1.The electrocardiogram showed that the tail vein injection of quinine and chloroquine decreased the heart rate,and prolonged the RR,PR,QRS,QTc intervals and P duration（P<0.05）,suggesting that quinine and chloroquine decreased the SA node activity and slowed down the cardiac electrical conduction.2.The PCR experiments revealed the mRNA expression of 8 subtypes of Tas2rs（Tas2r38,108,120,121,126,135,137,143）and their coupled G-proteins（Gαgustducin,Gβ1,Gβ3,and Gγ13）in the SA node of rats.3.Quinine,chloroquine,and denatonium decreased the SA node beating rate in a concentration-dependent manner with pEC₅₀ values of 4.907±0.045,4.968±0.030,5.828±0.063（P<0.05）,respectively.However,Tas2r140 does not express in the SA node;its agonists,benzoin and benzamide,did not affect on the SA node beating rate（P>0.05）.These data indicated that Tas2r agonists exerted their negative chronotropic effect by attenuating the SA node activity.4.The treatment with IBMX,a broad-spectrum PDE inhibitor,decreased the pEC₅₀values of quinine and chloroquine for inhibiting the SA node beating from 4.907±0.045and 4.968±0.030 to 4.658±0.059 and 4.842±0.083,respectively（P<0.05）.PDEs,especially PDE3 and PDE4,are critical cAMP-hydrolyzing enzymes controlling cAMP levels in the SA node.Cilostamide and rolipram are selective inhibitors for PDE3 and PDE4,respectively.The treatment with a cocktail containing cilostamide and rolipram decreased the pEC₅₀ values of quinine and chloroquine for inhibiting the SA node beating from 4.907±0.045 and 4.968±0.030 to 4.658±0.059 and 4.532±0.069,respectively （P<0.05）.However,the treatment with IBMX or a cocktail containing cilostamide and rolipram changed the pEC₅₀ value of denatonium for inhibiting the SA node beating from5.828±0.063 to 6.106±0.111,and from 5.828±0.063 to 5.977±0.206,respectively（P>0.05）.The cAMP measurement results showed that quinine and chloroquine did not affect the total intracellular cAMP level（P>0.05）.These data demonstrated that PDEs,particularly,PDE3 and PDE4,played an important role in the negative chronotropic effects of quinine and chloroquine,but not of denatonium.5.The Tas2r108 antagonist abscisic acid and the Gβγinhibitor gallein decreased the pEC₅₀ values of quinine for inhibiting the SA node beating from 4.907±0.045 to 4.645±0.085（P<0.05）,and from 4.907±0.045 to 4.676±0.079（P<0.05）,respectively.However,gallein did not have any impact on the concentration-response curve of chloroquine（P>0.05）.These data indicated that the effect of quinine on the SA node beating rate was related to Tas2r108 and Gβγ.6.The L-type Ca²⁺channel（LTCC）opener FPL64176 decreased the pEC₅₀ value of chloroquine for inhibiting the SA node beating from 4.968±0.030 to 4.719±0.212（P<0.05）.FPL64176 rescued the SA node from the arrest caused by quinine（50μM）or chloroquine（20μM）（P<0.05）.These data suggested that quinine and chloroquine reduced the SA node beating rate in an LTCC-dependent manner.Furthermore,quinine decreased the frequency of Ca²⁺transients in the SA node（P<0.05）.7.In the presence of quinine or chloroquine,the E_max of isoprenaline for increasing the SA node beating decreased from 391.3±16.8 bpm to 303.7±17.2 bpm and 344.9±22.4bpm;the pEC₅₀ of isoprenaline decreased from 8.381±0.135 to 7.746±0.175 and 7.305±0.257,respectively（P<0.05）.These data showed that Tas2r agonists suppressed the response of the SA node to isoprenaline,and that Tas2r agonists made isoprenaline less effective and less potent to induce tachycardia.Conclusion:This study,for the first time,determined that Tas2rs expressed in the SA node of rats;Tas2r agonists,quinine and chloroquine,exerted their negative chronotropic effects by slowing down the pace of the SA node and the cardiac electrical conduction,which were PDE-and LTCC-dependent.Furthermore,the effect of quinine on the SA node was Tas2r108-and Gβγ-dependent.Quinine and chloroquine could counteract with theβ-adrenergic receptors and made isoprenaline less effective and less potent to induce tachycardia.