Bitter Taste Receptor Activation Relaxes Myometrium And Prevents Preterm Labor

Preterm labor(PTL)is the leading cause of neonatal mortality and morbidity,with few treatment options.Worldwide nearly 15 million PTBs and 1 million neonatal deaths resulting from PTBs occur annually.Moreover,PTB is a major risk factor for many lifelong disabilities and diseases in the progeny,such as asthma,diabetes and developmental disorders.The human and financial impact of PTB on the family and our health care system are staggering.Therefore,unless high risk factors to fetus and/or mother are clearly identified,alleviating myometrial contractions with tocolytics have been one of the basic mechanisms used to delay or even prevent PTB.Although being used in clinics,tocolytics,for example nifedipine,β-adrenergic receptor agonists,indomethacin and magnesium sulfate,have many side effects or safety concerns to either mother,fetus or both.Moreover,they are often not effective in extending pregnancy for more than 48 hours,and haven’t been able to reduce the incidence of PTBs.Hence,finding new molecular targets that mediate myometrial relaxation,and developing new classes of effective and safe tocolytics are necessary for better PTB management.Animal abortion is a complex problem in animal production with few therapeutic options.For livestock,the economic loss will be reduced if we are able to save the fetus through a cheap way..While for endangered animals,especially those with high value,every effort are made to rescue the preterm birth.Thus,gaining insight into the mechanism of animal abortion is necessary in animal production.Bitter taste receptors(Tas2R,or T2R family)are a family of G-protein coupled membrane receptors.It has long been known that specialized epithelial cells in the taste buds of the tongue detect bitter tastants via Tas2Rs and initiate the sensation of bitterness.However,emerging evidence has gradually brought attention to cells in extra-oral tissues where bitter tastants can generate different biological responses tailored to their location.Recently,Tas2Rs have become an attractive target for developing smooth muscle relaxants.It remains little explored whether bitter tastants can exert such potent relaxation in other types of smooth muscle.Given the central role of myometrial contraction in PTB,and lack of any major advancement in tocolytics for PTB in the last three decades,,we were prompted to determine whether Tas2R activation was able to relax myometrium and whether these receptors served as targets for tocolytics for preterm labor.Here,we report that myometrial cells express bitter taste receptors(Tas2Rs)and their canonical signaling components.Bitter tastants relax myometrium pre-contracted by uterotonics more completely than current commonly used tocolytics(i.e.,nifedipine,indomethacin and MgSO4)and at a lower dose than MgSO4 and indomethacin.Using isolated single mouse myometrial cells,we found that chloroquine reverses the rise in[Ca2+]i and cell shortening induced by uterotonics,and this reversal effect was inhibited by pertussis toxin and by genetic deletion of α-gustducin.In human myometrial cells,knockdown of Tas2R14 but not Tas2R10 inhibited chloroquine’s reversal effect on an oxytocin-induced rise in[Ca2+]i.Finally,chloroquine prevented mouse PTL induced by bacterial endotoxin lipopolysaccharide or progesterone receptor antagonist RU486 more often than current commonly used tocolytics.Collectively,our results reveal that activation of the canonical Tas2R signaling system in myometrial cells exerts profound relaxation and targeting Tas2Rs promises to be an attractive approach to developing more effective tocolytics than those currently used for PTL management.Our results are presented in detail as follows:1.Myometrial cells express TAS2Rs and their signaling elements.We first examined whether Tas2Rs are presented in myometrial cells from pregnant mouse and human.Of 35 mouse Tas2Rs,we detected mRNA transcripts for Tas2R126,135,137 and 143 in myometrium from pregnant mice at day 18.Out of a total of 25 human Tas2Rs we detected the transcripts for Tas2R4,5,10,13,and 14 in freshly collected human myometrial tissuesand for Tas2R3,4,5,7,8,10,13,14,31,39,42,43,45,and 50 in cultured human myometrial cells hTERT-HM.We also detected mRNAs for the elements in the Tas2R signaling pathway in myometrial cells from mouse and human.These results indicate that myometrial cells from both mouse and human express the canonical Tas2R signaling cascade,raising a possibility that bitter tastants act on this pathway to generate their responses in these cells.2.Bitter tastants relax myometrium more completely than currently used tocolyticsTo assess the effects of bitter tastants on myometrial contractility,we used freshly isolated mouse uterine strips and measured their force generation under isometric conditions.Oxytocin(OT),a hormone critical for parturition,was used a contractile agent.Bitter tastants chloroquine,denatonium and 1,10-phenanthroline dose-dependently suppressed the OT-induced contraction of mouse myometrium.As a comparison,we also examined the effects of four currently used tocolytics(i.e.,MgSO4,indomethacin,nifedipine and albuterol).Compared with MgSO4 and indomethacin,bitter tastants relax myometrium both more completely and at a lower dose.Chloroquine at 1 mM can fully reverse both KCI-and PGF2a-induced contraction of the uterus from day 18 pregnant mice.We found that chloroquine and 1,10-phenanthroline almost fully reversed oxytocin-and KCl-induced contraction of human myometrium.This reversal effect is much greater than all four clinically used tocolytics in OT-induced contraction,and greater than MgS04,indomethacin and albuterol in KCl-induced contraction.These contraction bioassays indicated that bitter tastants could relax late pregnant uterus contracted by uterotonics better than currently used tocolytics targeting different receptors enzymes,and Ca2+channels in myometrial cells.3.The mechanism underlining bitter tastants relax pre-contracted myometriumWe examined the effect of ChQ on Ca2+ signaling in myometrial cells at rest.In freshly isolated myometrial cells from day 18 pregnant mice,ChQ alone modestly increased global[Ca2+]i;but to a level much less than that caused by oxytocin alone.We found pertussis-toxin,galleon,2-ABP and U73122 inhibited the ChQ-induced increases in[Ca2+]i.To assess whether the same signaling pathway is operative in human myometrial cells,we carried out the same series of experiments using cultured human myometrial cells hTERT-HM.These results indicate that bitter tastants activate the Tas2R signaling transduction pathway to modestly release Cato from internal stores.Then,we went on to study the hypothesis that bitter tastants reversed the uterotonic-induced rise in[Ca2+]i,resulting in myometrial relaxation.ChQ largely reversed oxytocin induced increase in[Ca2+]i and shorten in length.To examine whether ChQ reverses the OT-induced[Ca2+]i rise through the activation of Tas2Rs,we first used strategies to interfere with gustducin because all Tas2Rs are coupled with this G-protein.ChQ’s reversal effects on the OT-induced rise in[Ca2+]i and cell shortening were significantly reduced in gnat3-/-cells compared to the wild type cells.These results strongly suggest that gustducin plays an important role in ChQ-induced responses in myometrial cells.To further confirm that ChQ activates Tas2Rs to generate responses in myometrial cells,we examined the effects of Tas2R knockdowns using shRNA in human cultured myometrial cells.Using shRNA,we knocked-down Tas2R14 and Tas2R10,separately,in cultured human myometrial cells.Interestingly,the shRNA for Tas2R10 exerted no effect on the ChQ-induced rise in[Ca2+]i in resting cells,while the shRNA for Tas2R14 produced an inhibition of the ChQ-induced rise in[Ca2+]i.The shRNA for Tas2R10 exerted no effect on ChQ’s effect on the OT-induced rise in[Ca2+]i,but the shRNA for Tas2R14 significantly inhibited ChQ’s effect on the OT-induced rise in[Ca2+]i.These results indicate that Tas2R14 mediates the ChQ-induced effect on[Ca2+]i in human myometrial cells.4.Bitter tastant chloroquine protects mouse preterm births better than currently used tocolytics.The strong relaxation produced by bitter tastants implies that these compounds may be able to prevent or treat PTB.To assess this,we first established a mouse model of preterm labor with bacterial endotoxin lipopolysaccharide(LPS).When administering ChQ to the uteri 3 hrs after the LPS injection,more than 50%mice delivered their litters on day 19,i.e.,full term birth.As a comparison,MgSO4 and albuterol was applied to the uteri 3 hrs after LPS.Only albuterol was able to rescue the PTB by 50%.We then established another mouse model with progesterone receptor antagonist RU486.When administering ChQ to the uteri 3 hrs after the RU486 injection,of 10 mice,six gave birth to the pups on day 17(i.e,no protection from RU486),two delivered on day 19(i.e.,full term),and two on day 20(i.e.,delayed labor).These results suggest that the bitter tastant chloroquine can provide better protection in preventing LPS-or RU486-induced PTB in mice than currently used tocolytics.To test whether gustducin is involved in ChQ-induced protection from PTB,we injected LPS or RU486 followed 3 hrs later by ChQ into gnat3-/-mice.In the gnat3-/-mice administered LPS,the ChQ treatment resulted in 60%of the mice delivering on day 17,and another 40%on day 18,and all the litters were dead.In the gnat3-/-mice administered RU486,the ChQ treatment resulted in 50%of the mice delivering on day 17,and another 50%on day 18,and all the pups were dead when delivered.These results indicate that gustducin is a critical molecule mediating ChQ-induced protection from PTBs in mice.Collectively,our results reveal that activation of the canonical Tas2R signaling system in myometrial cells produces profound relaxation of myometrium pre-contracted by a broad spectrum of contractile agonists,and targeting Tas2Rs is an attractive approach to developing more effective tocolytics than those currently used for PTB management.