| Background:Bitter taste receptors（Tas2rs）are a member of the G protein coupled receptor（GPCR）A family.Tas2rs were originally thought to mediate aversive reactions in humans and mammals to protect them from toxic substances.With the researches advancing,Tas2rs are found in other systems outside the oral cavity of humans and rodents,such as the gastrointestinal tract,airway,uterus,blood vessels,sinoatrial node and heart.This might indicate that Tas2rs have other functions besides taste perception.Tas2rs can activate phosphodiesterases（PDEs）,which leads to a decrease in the rhythm of sinoatrial node and an increase in aortic tone in rats.Detrusor underactivity（DUA）is a symptom in which the contraction strength of detrusor smooth muscle is reduced or the contraction duration is reduced,resulting in the inability to effectively empty the bladder in time.At present,the pathogenesis of DUA is still unclear completely,and there is no clear diagnostic criteria and treatment guide.Therefore,it is of great significance to study drug interventions to enhance the contractility of detrusor smooth muscle.Cyclic adenosine monophosphate（c AMP）can regulate the excitability and contractility of DSM by activating protein kinase A（PKA）to phosphorylate ion channels and other proteins.The c AMP-hydrolyzing phosphodiesterases（PDEs）can inhibit the activities of c AMP and PKA in cells,and increase the excitability and contractility of DSM.Recently,it has been reported in the literature that there is Tas2rs m RNA expression in human and mouse detrusor smooth muscle.However,the expression of Tas2rs at the protein level is still unclear,and there is no report on whether there is Tas2rs in rat detrusor smooth muscle.Whether Tas2rs can cause the activation of downstream PDE and increase the excitability and contractility of DSM needs to be studied urgently.Objective:To clarify the m RNA and protein expression of Tas2rs and its coupled G protein subunits in rat detrusor smooth muscle;to study the effect and mechanism of Tas2rs in regulating rat detrusor smooth muscle contractility.Methods:We used the RT-PCR technology to study the m RNA expression of Tas2rs and G protein subunits in rat detrusor smooth muscle tissue and cells.Western blot technology was employed to detect the protein expression of Tas2rs and G protein in rat DSMs with antibodys available on the market.We used immunoprecipitation-mass spectrometry technology and peptide blocking experiment to further confirm the existence of Tas2r protein in DSMs.Immunofluorescence experiment was performed to detect the expression and distribution of Tas2r protein and G protein in DSM cells.The isometric DSM tension recording was performed to study the effects of the Tas2r agonists diphenidol,quinine,and chloroquine on the DSM contraction.Tas2r antagonist 5’AMP,non-selective PDEs inhibitor 3-isobutyl-1-methylxanthine（IBMX）,combination of PDE3 inhibitor cilostamide and PDE4 inhibitor rolipram,AC agonist forskolin,muscarinic receptor antagonist atropine,Gβγinhibitor gallein and PLC inhibitor U73122were used to study the mechanism of diphenidol’s effect on DSM contractility.Results:1.The PCR results showed 6 subtypes of Tas2r m RNA（Tas2r40,108,126,135,137 and 143）and 5 subtypes of Tas2r m RNA（Tas2r108,126,135,137 and 143）expressed in rat DSMs and DSM cells,respectively.There were four Tas2rs-coupled G protein subunits Gnat3（Gαgustducin）,Gnb1（Gβ1）,Gnb3（Gβ3）and Gng13（Gγ13）expressed in DSMs and DSM cells.Western blot results showed that Tas2r108,Tas2r137and Gβ3 protein were expressed in DSMs.The combination of immunoprecipitation mass spectrometry and peptide blocking experiment further proved the specificity of Tas2r137antibody.The results of immunofluorescence experiment showed that Tas2r137 and Gαgustducin protein expressed in DSM cells,and Tas2r137 and Gαgustducin protein have a certain co-localization relationship.2.The bitter compound diphenidol concentration-dependently enhanced the spontaneous and 20 m M KCl-induced phasic contraction of rat DSM strips,as assessed by increased contraction force,contraction amplitude,contraction duration（P<0.05）,It could also reduce contraction frequency（P<0.05）,but have no effect on the tone（P>0.05）.Similar effects on the DSM strips could also be found when treated with quinine and chloroquine.The spontaneous contraction or KCl-induced contraction of DSM strips could be attenuated after the removal of diphenidol（P<0.05）.3.Pre-incubation with 5’AMP could dose-dependently reduce the spontaneous contraction amplitude,force and duration increased by diphenidol（30μM）（P<0.01）.After diphenidol（30μM）increased spontaneous phasic contraction of DSM stripes,5’AMP dose-dependently reduced the contraction amplitude,force and the contraction duration（P<0.01）.Diphenidol（10μM）increased the contraction caused by KCl,and5’AMP（1 m M）could completely eliminate the phasic contraction.4.Atropine（1μM）had no significant effect on both the spontaneous contraction and the KCl-induced contraction（P>0.05）,indicating that effects of diphenidol on the contration of DSMs were independent of its role as an M receptor antagonist.5.The non-selective PDEs inhibitor IBMX,or the combination of PDE3 inhibitor ciloxamide and PDE4 inhibitor rolipram could inhibit the enhancement in spontaneous contraction and KCl-induced contraction of DSMs by diphenidol（P<0.05）.This indicated that effect of diphenidol on contraction of detrusor smooth muscle is dependent on the activation of PDEs.6.Forskolin（10μM）,an adenylate cyclase agonist,completely diminished the effect of diphenidol on spontaneous contraction and KCl-induced contraction of DSMs,indicating that the effect of diphenidol on detrusor smooth muscle contraction is related to c AMP.7.The Gβγinhibitor Gallein and PLC inhibitor U73122 had no significant effect on diphenidol’s increased phasic contraction（P>0.05）,indicating that the effect of diphenidol on contraction of DSMs has nothing to do with the Gβγ-PLC-IP3signaling pathway.Conclusion:In this study,we first indentified the expression of Tas2rs and their conjugated G protein both in the m RNA and protein level in the smooth muscle of rat bladder.Tas2r agonists diphenidol,quinine and chloroquine increased phasic contraction of rat DSMs.Diphenidol activated Tas2rs,and the Tas2r-coupled Gαgustducin led to the activation of PDEs,especially PDE3 and PDE4 subtypes.This decreased c AMP levels in DSM cells,and enventually increased the phasic contractile function of rat DSMs.This study elucidated the mechanism by which Tas2rs regulated the contraction of rat DSMs,and provided a new idea for the treatment of detrusor underactivity. |
