Effects Of Exendin-4 And Apelin-13 On Colonic Motility In Rats And Their Underlying Mechanisms

Background:Several mechanisms are involved in the control of colonic motility,and elements that contribute to the regulation of colonic motor activity include interstitial cells of Cajal,enteric intrinsic nervous system and smooth muscles.Enteric nervous systems are organized in 2 main plexuses:the submucosal and the myenteric.Acetylcholine(Ach),tachykinins and so on exert excitatory stimuli,nitric oxide(NO),adenosine triphosphate(ATP)and vasoactive intestinal peptide(VIP)exert inhibitory stimuli,and these neurotransmitters regulate contraction and relaxation of colonic smooth muscle.The activities of ion channels closely correlate with the movement of smooth muscle.L-type calcium channels and large conductance Ca2+-activated K+(BKCa)channels are widely expressed in colonic smooth muscles,and serve as the main channels involved in the process of excitation-contraction coupling in gastrointestinal(GI)smooth muscle.Glucagon-like peptide-1(GLP-1)is an incretin that are produced in intestinal L cells,has many biological activities.Exendin-4(Ex-4),a full glucagon-like peptide-1 receptor(GLP-1R)agonist,shares 53%homology with GLP-1.Studies have reported that both exogenous and endogenous GLP-1 play important roles in GI tract,including regulating visceral sensitivity,regulating GI motor function and so on.Both GLP-1 and GLP-1R agonists on GI motor function have been reported,but most studies are focus on gastric and small intestinal motility,including increasing gastric volumes,delaying gastric emptying and inhibiting small bowel motility,however,the action of GLP-1 on colonic motility remains controversial,because both stimulatory and inhibitory activities have been reported,and the mechanism is not yet fully understood.Apelin is a novel peptide,its receptor is a putative receptor protein related to the angiotensin-II receptor 1(AT1)(APJ).Apelin has several subtypes,such as apelin-12,apelin-13,apelin-17,apelin-36,Apelin-13 is a subtype that has highest affinity for its receptor,APJ,and has many biological activities,including regulating blood pressure,exerting neuroprotective effect,anti-inflammatory,alleviating pain and so on.Apelin-13 play critical roles in GI tract,including protecting gastric mucosal,delaying gastric emptying,inhibiting colonic transit.In terms of GI motility,previous studies mainly focused on the effect of apelin-13 in vivo as a whole,however,only a handful of studies are involved in the action of apelin-13 on colonic motility,and the mechanism is unclear.The studies are designed to investigate the effects of Ex-4 and apelin-13 on colonic motility in vitro and related mechanisms.Part Ⅰ Effects of Ex-4 on colonic smooth muscle strips in ratsObjective:To investigate the effects of Ex-4 on spontaneous contractions of colonic smooth muscle strips and its underlying mechanism.Methods:The colonic tissue was obtained from healthy male Wistar rats.The colonic smooth muscle including whole layers was cut into strips(2×10 mm,width x length)vertically or along the mesenteric edge to obtain circular smooth muscle(CM)strips or longitudinal smooth muscle(LM)strips.The effects of Ex-4 spontaneous contractions of colonic smooth muscle strips were recorded by multichannel physiological signal acquisition system(RM6240B)and a four-chamber bath experimental system.The smooth muscle strips were incubated with GLP-1R antagonist,exendin-4(9-39)and nerve blocker,tetrodotoxin(TTX),Nco-Nitro-L-arginine(L-NNA),apamin,the alteration of the effects of Ex-4 was recored.Results:Ex-4 inhibited the spontaneous contractile activities of both CM strips and LM strips in a dose-dependent manner.The basal amplitude of CM strips before adding Ex-4 was 0.70±0.13 g,after adding 2.0 μM,4.0 μM,6.0 μM Ex-4,the mean amplitudes were reduced to 0.62±0.09 g,0.32±0.08 g and 0.09±0.01 g(P<0.05 vs.normal,n=5),the percent inhibition of mean amplitudes was 9.3%±3.5%,53.5%±4.7%,and 84.5%±3.7%.The frequency of spontaneous contractions was inhibited by 61.5%± 9.3%in the presence of the highest Ex-4 concentration(6.0 μM;P<0.05 vs.normal,n=5).The basal amplitude of LM strips before adding Ex-4 was 1.10±0.08 g,after adding 2.0 μM,4.0 μM,6.0 μM,8.0 μM Ex-4,the mean amplitudes were reduced to 1.00±0.09 g,0.85±0.07 g,0.45±0.11 g and 0.20±0.05 g(P<0.05 vs.normal,n=5),the percent inhibition of mean amplitudes was 7.8%±1.6%,21.7%±2.6%,59.2%±8.7%and 81.74%.The frequency of spontaneous contractions was inhibited by 45.0%±10.0%in the presence of the highest Ex-4 concentration(8.0 μM;P<0.05 vs.normal,n=5).The basal amplitude of muscle strips were not significantly altered after incubating with 1.0 μM exendin-4(9-39),a GLP-1R antagonist,for 15 minutes and the inhibitory effects of Ex-4 disappeared.The basal amplitude of muscle strips were slightly increased after incubating with 1μM TTX,500 μM L-NNA,1 μM apamin,but without significant differences and the inhibitory effects of Ex-4 were partly blocked.Conclusions:Ex-4 inhibited the spontaneous contractions of colonic smooth muscle in a dose-dependent manner via a nitrergic and a purinergic neural pathway through NO and ATP release.Part Ⅱ The molecular mechanism of Ex-4 on colonic smooth muscle cells of ratsObjective:To investigate the molecular mechanism of Ex-4 on colonic smooth muscle cells.Methods:The colonic tissue was obtained from healthy male Wistar rats.The smooth muscle cells were isolated by collagenase digestion after the mucosa and submucosa were removed.Using the whole-cell patch clamp technique,the effects of Ex-4 on the current of L-type calcium channels(ICa,L),the steady-state activation and the steady-state inactivation of L-type calcium channels and the current of BKCa channels(IBK,ca)were investigated under voltage-clamp mode.Results:ICa,L reached its peak value at 0 mV,and the currents mostly were inhibited by nifedipine(μM),a specific L-type calcium channel blocker(P<0.05 vs.normal,n=3).The average current density at 0 mV was-4.75 ± 0.37pA/pF,and successively decreased to-4.22 ± 0.44 pA/pF and-3.38±0.46 pA/pF after adding 0.2 μM and 2.0 μM Ex-4,significant differences were observed at dose of 2.0 μM(P<0.05 vs.normal,n=6).The inhibitory effect of Ex-4 on ICa,L reached a steady level after the agent was applied for 1-2 minutes and was partially reversed after Ex-4 was washed out.Although Ex-4 inhibited ICa,L,the shape of the I-V curves was not changed.The steady-state activation and steady-state inactivation of L-type calcium channels were not significantly altered by Ex-4 at a dose of 2.0 μM.IBK.ca reached its peak value at 60 mV,the currents were inhibited by beriotoxin(IbTX,300 nM),a specific BKCa channel blocker.The average current density at 60 mV was 18.4±0.83 pA/pF,decreased to 17.4 ± 0.79 pA/pF and 13.8 ±1.12 pA/pF after adding 0.2 μM and 2.0 μM Ex-4,significant differences were observed at dose of 2.0 μM(P<0.05 vs.normal,n=6).The inhibitory effect of Ex-4 on IBK,ca reached a steady level after the agent was applied for 2 minutes and was partially reversed after Ex-4 was washed out.Although Ex-4 inhibited IBK,ca,the shape of the I-V curves was not changed.Conclusions:Ex-4 inhibited ICa,L,but not altered the shape of the I-V curves and not affect steady-state activation and steady-state inactivation,likewise,Ex-4 inhibited IBK,Ca,but not altered the shape of the I-V curves.Part Ⅲ Effects of apelin-13 on colonic motility in rats and its molecular mechanismObjective:To investigate the effects of apelin-13 on spontaneous contractions of colonic smooth muscle strips and its molecular mechanism on colonic smooth muscle cells.Methods:The colonic tissue was obtained from healthy male Wistar rats.The colonic smooth muscle including whole layers was cut into strips(2×10 mm,width ×length)vertically or along the mesenteric edge to obtain circular smooth muscle(CM)strips or longitudinal smooth muscle(LM)strips.The effects of apelin-13 spontaneous contractions of colonic smooth muscle strips were recorded by multichannel physiological signal acquisition system(RM6240B)and a fourchamber bath experimental system.The smooth muscle cells were isolated by collagenase digestion after the mucosa and submucosa were removed.Using the whole-cell patch clamp technique the effects of apelin-13 on L-type calcium channels and BKCa channels under voltage-clamp mode.Results:Apelin-13 inhibited the spontaneous contractions of both LM strips and CM strips in a dose-dependent manner.Prior to the addition of apelin-13,the basal amplitude of LM strips was 0.97±0.08 g,after the addition of apelin-13 at concentrations of 0.5 μM,1.0 μM,1.5 μM and 2.0 μM,the mean amplitude was decreased to 0.84±0.07 g,0.66±0.05 g,0.43±0.03 g and 0.23±0.02 g(P<0.05 vs.control,n=5),the percent inhibition of mean amplitudes was 13.2%±0.7%,31.9%±1.7%,55.2%±2.4%,76.3%±0.9%;Prior to the addition of apelin-13,the basal amplitude of CM strips was 0.85 ± 0.13 g,after the addition of apelin-13 atconcentrations of 0.5 μM,1.0 μM,1.5 μM and 2.0 μM,the mean amplitude was decreased to 0.72±0.10 g,0.57±0.09 g,0.34±0.03 g,0.19±0.02 g(P<0.05 vs.control,n=5),the percent inhibition of mean amplitudes was 14.8%±2.0%,32.4%±2.7%,56.12%±4.4%,76.6%±1.9%.The basal amplitude of muscle strips were not significantly altered after incubating with 2.0μM ML221,a APJ receptor antagonist,for 15 minutes and the inhibitory effect of apelin-13 was mostly disappeared.The basal amplitude of muscle strips were slightly increased after incubating with 1 μM TTX for 15 minutes,but without significant differences and the inhibitory effects of apelin-13 still exist.Apelin-13 inhibited ICa,L in a dose-dependent manner,but did not alter the steady-state activation and steady-state inactivation of L-type calcium channels.Prior to the addition of apelin-13,the current density at 0 mV was 3.94 ±0.22 pA/pF,after the addition of apelin-13 at concentrations of 0.5 μM,1.0 μM,1.5μM and 2.0 μM,the current density was reduced to-3.33±0.26 pA/pF,-2.71±0.23 pA/pF,-2.16±0.27 pA/pF and-1.67±0.24 pA/pF(P<0.05 vs.control,n=7).Likewise,Apelin-13 inhibited IBK,ca in a dose-dependent manner.Prior to the addition of apelin-13,the current density at 60 mV was 15.27±1.46 pA/pF,after the addition of apelin-13 at concentrations of 0.5 μM,1.0 μM,1.5 μM and 2.0 μM,the current density was reduced to 13.44±1.23 pA/pF,11.73±0.89 pA/pF,10.49±0.72 pA/pF and 8.91±0.41 pA/pF(P<0.05 vs.control,n=6).Conclusions:Apelin-13 inhibited the spontaneous contractions of colonic smooth muscle in a dose-dependent manner.The inhibitory effect was mainly depended on the colonic smooth muscle cells,and enteric nervous system had little effect.L-type calcium channels and BKCa channels are involved in the regulation of apelin-13 on colonic motility.