胆囊收缩素对结肠平滑肌及其细胞膜通道电流和膜电位影响的研究
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摘要
背景：胆囊收缩素(cholecystokinin, CCK)是一种双重分布在中枢神经系统和胃肠道的脑肠肽,并以神经递质的形式参与了多种生理及病理过程。硫酸化胆囊收缩素八肽(sulfated cholecystokinin octapeptide, CCK-8S)是具有CCK完全生物学功能的最小活性片段。已知CCK主要通过两种受体发挥其不同的生理及药理作用,即CCK1受体(cholecystokinin-1 receptor)和CCK2受体(cholecystokinin-2 receptor)。前期关于CCK的研究多偏重于其在胆囊、胰腺及心肺功能活动中的调节作用。近年来,随着功能性胃肠病发病率日益增高,有关CCK调节胃肠动力的作用引起人们的高度重视。多项研究显示功能性胃肠病患者血清中CCK存在异常,但有关CCK对胃肠平滑肌的具体作用机制报道较少。
第一部分
目的：探讨CCK-8S对豚鼠近端结肠平滑肌肌条自发性收缩频率和强度的影响及可能的作用机制。
方法：取6 cm左右豚鼠近端结肠,在持续充以95%02和5%CO2的Ca-freePSS中去除黏膜后剪成8 mm×10 mm平滑肌肌条,并将纵行肌与环行肌分离。将肌条浸没于37℃含钙台式液中,一端固定于垂直灌流槽,另一端与张力换能器相连,并持续充以95%O2和5%C02的混合气体。待肌条出现自发节律性收缩并稳定后采用RM6240多道生理信号采集处理系统记录给药前后收缩频率和强度。
结果：CCK-8S (10-7 mol/L)作用后,豚鼠结肠平滑肌纵行与环形肌条的收缩幅值分别为对照组的156.53%±11.92%(n=15,P=0.038)和165.93%±12.98%(n=15,P=0.019)；结肠平滑肌纵行肌条的收缩频率为对照组的131.69%±13.58%(n=15,P=0.023),而环形肌条的收缩频率无明显变化,为对照组的102.19%±8.96%(n=15,P=0.087)。当预先将灌流液中分别加入CCK1受体拮抗剂devazepide、L-型钙通道阻断剂nifedipine、大电导钙依赖钾通道阻断剂iberiotoxin、钙泵抑制剂thapsigargin (TG)和钙离子螯合剂BAPTA-AM (BA)后,CCK-8S对结肠平滑肌肌条的增强效应被抑制(n=15,均P<0.05)；而预先加入CCK2受体拮抗剂CI 988后,CCK-8S的增强效应仍然存在(n=15,P>0.05)。
结论：CCK-8S通过CCK1受体能促进豚鼠近端结肠纵,环行平滑肌自发性收缩强度及纵行平滑肌收缩频率；但对环行平滑肌收缩频率无明显影响。其机制与促进平滑肌细胞内钙的释放、胞膜上大电导钙依赖钾通道及L-型钙通道的激活有关。
第二部分
目的：探讨CCK-8S对单个豚鼠结肠平滑肌细胞静息电位(：resting potential, RP)、动作电位(action potential, AP)、大电导钙依赖钾通道电流(large conductance potassium currents,IBKca)和L-型钙通道电流(L-type calcium currents, ICa-L)的影响及可能的作用机制。
方法：取6 cm左右近端结肠,在持续充以95%02和5%CO2的Ca-free PSS中去除黏膜后剪成2 mm×6 mm大小,置于无钙消化液中,37℃振荡孵育20-30min,漂洗,吹打成细胞悬液。将细胞悬液接种于装有倒置显微镜的灌流槽中,贴壁后,用含钙台式液以2-3 ml／min速度持续灌流,并选取贴壁良好,折光性好,胞膜光滑清晰的杆状细胞进行高阻封接。用EPC-10膜片钳放大器记录结肠平滑肌细胞AP和RP给药前后的变化；在穿孔全细胞膜片钳模式下记录单个细胞IBKca和ICa-L给药前后的变化。
结果：1.CCK-8S (10-7 mol/L)作用后,结肠平滑肌细胞RP、AP峰值及AP快速复极时间分别为对照组的48.6±5.3%、138.6%±3.2%和63.1±8.7%(n=8,P=0.032、0.015和0.026),该效应可被预先加入的devazepide和／或nifedipine阻断(均n=8,P<0.05),而预先向灌流液中加入CI 988后,CCK-8S对RP、AP峰值及AP快速复极时间的作用仍存在。2.从-60 mV去极化至+100 mV,CCK-8S可剂量依赖性升高IBKca(P<0.05,at 10-8,10-7和10-6mol／L；ECso=3.5×10-8mol／L)。CCK-8S(10-7mol／L)升高IBKca为对照组的118.7%±2.1%(从916±183 pA至1088±226 pA；n=8,P=0.029),该效应可被预先向灌流液中加入的devazepide抑制(908±109pA,n=8,P=0.012 vsCCK-8S组,P=0.083vs对照组),但CI 988无明显抑制作用(1052±196 pA,n=8,P=0.098 vsCCK-8S组)。当向电极内液加入IP3受体阻断剂肝素(10-5mol／L)或向细胞外液加入蛋白激酶C拮抗剂staurosporine(10-6mol／L)后,CCK-8S(10-7mol／L)对IBKca均没有影响(879±117 pA,n=8,P=0.074vs对照组,P=0.016 vsCCK-8S组；887±120 pA,n=8,P=0.069 vs对照组,P=0.041 vs CCK-8S组)。3.从-60 mV去极化至+10 mV,CCK-8S可剂量依赖性升高ICa-L(P<0.05,at 10-8,10-7和10-6mol／L；EC50=3.2×10-8mol／L)。CCK-8S(10-7mol／L)升高ICa-L为对照组的140%±5.3%(从60±8 pA至84±11 pA,n=8,P=0.012),该效应可分别被预先向灌流液中加入的devazepide(61±9 pA；at+10 mV；n=8,P=0.023 vs CCK-8S组,P=0.079 vs对照组),TG和BA抑制(7±5 pA；at+10 mV,n=8,P=0.006 vs CCK-8S组),但CI 88无明显抑制作用(84±11pA；n=8,P=0.079 vs CCK-8S组)。当向电极内液加入IP3受体阻断剂肝素(10-5mol／L)或向细胞外液加入蛋白激酶C拮抗剂staurosporine(10-6mol／L)后,CCK-8S(10-7mol／L)对ICa-L均没有影响(63±12 pA,65±10 pA；at+10 mV；n=8；P=0.183,0.215vs正常组,P=0.042,0.032·vs CCK-8S组)。结论：CCK-8S通过CCK1受体促进近端结肠平滑肌细胞内钙库释放钙及增强肌细胞膜ICa-L,促进细胞外钙内流,从而使平滑肌细胞RP下降,AP幅值增加；通过增强细胞膜IBKca使平滑肌细胞AP快速复极时间缩短,从而增加了单位时间内AP次数,促进结肠平滑肌细胞收缩。其作用机制与激活IP3介导的PKC途径有关。
第三部分
目的：检测胆囊收缩素受体基因在豚鼠近端结肠平滑肌的表达。方法：取新鲜的豚鼠近端结肠平滑肌组织,在装有生理盐水的烧杯中去除肠内容物,然后在持续充以95%02和5%CO2的Ca-free PSS中去除粘膜层及粘膜下层,取50-100 mg新鲜组织在冰浴中匀浆,离心后,吸取上清,应用逆转录多聚酶链反应(reverse transcription polymerase chain reaction, RT-PCR)检测CCK1受体及CCK2受体mRNA在豚鼠近端结肠平滑肌组织中的表达。结果：豚鼠近端结肠平滑肌组织中有CCK1受体mRNA表达(0.83±0.15,n=15,P=0.263vsβ-actin),扩增产物片段大小为630bp,但未见CCK2受体mRNA的表达。
结论：豚鼠近端结肠平滑肌中分布的CCK受体主要为CCK1受体,这是CCK促进结肠平滑肌收缩的分子基础。
Background
Cholecystokinin(CCK), a gut-brain peptide, plays an important role in regulating a variety of physiological functions and involving in many pathophysiological processes. It has been reported that sulfated cholecystokinin octapeptide(CCK-8S) is the predominant and major molecular form of CCK peptides for biological activity and it actions on the gastrointestinal tract through two receptor subtypes, designated as CCK1 receptor and CCK2 receptor. Previous studies investigating the targets for CCK have focused mainly on the gallbladder, pancreas, heart and lung. However, recent researches have shown that CCK and its related peptides are implicated in the pathophysiology of functional digestive diseases. Several early reports also have indicated that either an altered release of CCK or abnormal responses to this peptide could be responsible for the symptoms of functional digestive diseases. However, the direct electrophysiological effects of CCK on the contractile activity of gut remains unclear.
PartⅠ
Objective To investigate the effects of sulfated cholecystokinin octapeptide (CCK-8S) on the spontaneous contractile activity of guinea-pig proximal colonic smooth muscle strips.
Methods The proximal colon (6 cm) was removed, cleaned and opened along the mesenteric border and then placed in Ca2+-free PSS bubbled with carbogen (95% O2/5% CO2). The smooth muscle strips (8×10 mm) were obtained after the mucosa and submucosa were excised and the longitudinal muscle(LM) and circular muscle(CM) strips were segregated. Each fresh smooth muscle strip was mounted in an organ bath and connected to an isometric force transducer. The organ baths contained 10 ml Tyrode's buffer and were constantly warmed by a circulating water jacketed at 37℃and bubbled with carbogen (95% O2/5% CO2). One end of the strip was fixed to a hook on the bottom of the chamber, while the other end was connected by a thread to an external isometric force transducer at the top. Each muscle strip was placed under a resting preload of 1.0g and allowed to equilibrate for 60 min with solution change every 20 min. The relative effects on the contractile amplitude and frequency of LM and CM strips were observed before and after the addition of drugs.
Results The mean contractile amplitude of CM and LM strips stimulated by CCK-8S (10-7 mol/L) were 156.53%±11.92% and 165.93%±12.98% respectively of that of the control group(n=15, P=0.038,0.019);the mean frequency of LM stimulated by CCK-8S (10-7 mol/L) was 131.69%±13.58% of that of the control group(P=0.023), but CCK-8S had little influence on the frequency of CM(n=15, P=0.087). CCK-8S-intensified effects on proximal colonic strips were significantly attenuated when these strips were pretreated with CCK1 receptor antagonist devazepide (10-7 mol/L), L-type calcium channel inhibitor nifedipine (10-5 mol/L) or Ca2+-ATPase inhibitor TG (10-5 mol/L) and intracellular calcium chelator BA (10-5 mol/L) (n=15, P＜0.05 for each group). Pretreating CM and LM strips with iberiotoxin (10-6 mol/L), a selective BKCa channel blocker, did not inhibit the CCK-8S-induced increase in the contractile amplitude of CM and LM strips (n=15, P=0.096,0.078 vs CCK-8S group), but decreased their frequencies (n=15, P= 0.036,0.041 vs CCK-8S group), whereas superfusion with the CCK2 receptor antagonist CI 988 (10-7 mol/L) did not block the CCK-8S-intensified effect on CM and LM strips (n=15, P＞0.05 for each group).
Conclusion CCK-8S promotes the contraction of the longitudinal muscle and circular muscle of guinea-pig proximal colon by CCK1 receptor mostly owing to increasing the release of intracellular Ca2+, large conductance potassium currents and L-type calcium currents, but CCK-8S has little effect on the frequency of the circular muscle.
PartⅡ
Objective To examine the effect and its mechanism of CCK-8S on resting potential(RP), action potential(AP), large conductance potassium currents(IBKCa) and L-type calcium currents(ICa-L).
Methods The proximal colon (about 6 cm long) was removed, cleaned and opened along the mesenteric border and then placed in Ca2+-free PSS bubbled with carbogen (95% O2/5% CO2). The strips of proximal colon were pinned to the base of the sylgard surface of a Petri dish and the mucosa together with submucosa was carefully dissected away. The tissue was cut into small strips (about 2 mm wide and 6 mm long) and placed in Ca2+-free PSS solution and incubated for 25 min at 37℃. After completion of digestion, the segments were washed and then triturated gently to create a cell suspension. Several drops of cell suspensions were placed in a recording chamber that was mounted with an inverted microscope. Cells were superfused with CaPSS (3 ml/min) after adhering to the coverslip. RP and AP were recorded by EPC-10 amplifier. Whole-cell currents were recorded by using a nystatin-perforated whole-cell patch-clamp configuration with an EPC-10 amplifier. Drugs were added subsequently to the cell suspension to observe relative effects on AP, RP, IBKCa and ICa-L when their values were stable.
Results 1.The amplitude of RP and AP and fast repolarization time (repolarizing to 90% of the peak value of AP, T90) induced by CCK-8S (10-7 mol/L) were 48.6%±5.3%,138.6%±3.2% and 63.1%±8.7% respectively of that of the control group (n=8,P=0.032,0.015 and 0.026), which were significantly inhibited when these cells were pretreated with devazepide (10-7 mol/L) and/or nifedipine (10-5 mol/L)(n=8, P＜0.05 for each group), whereas CCK-8S had little influence.2. IBKCa was evoked by using a depolarizing step pulse from a holding potential of -60 mV to+100 mV. CCK-8S increased IBKCa in a concentration-dependent manner (P＜ 0.05,at 10-8,10-7 and 10-6 mol/L;EC50=3.5×10-8mol/L).The peak amplitude of IBKCa was enhanced to 118.7%±2.1%(from 916±183 pAto 1088±226 pA,n=8, P=0.029)of that of the control group,which was blocked by pretreatment with 10-7 mol/L devazepide(908±109 pA,n=8,P=0.012 vs CCK-8S group,P=0.083 vs control group).However,the CCK2 receptor antagonist CI 988 exerted little effect (1052±196 pA,n=8,P=0.098 vs CCK-8S group).When heparin(10-6 mol/L),an inhibitor of IP3 receptors,was added to the pipette solution,CCK-8S(10-7 mol/L)did not enhance IBKCa(879±117 pA;n=8,P=0.074 vs control group,P=0.016 vs CCK-8S group).Pretreating cells with the PKC inhibitor staurosporine(10-6 mol/L) also attenuated the CCK-8S-induced effect on IBKCa(887±120 pA;n=8,P=0.069 vs control group,P=0.041 vs CCK-8S group).3.ICa-L was evoked by using a depolarizing step pulse from a holding potential of -40 mV to +30 mV.CCK-8S enhanced ICa-L in a concentration.dependent manner(P＜0.05,at 10-8,10-7 and 10-6 mol/L;EC50=3.2×10-8 mol/L).The peak amplitude of ICa-L was increased to 140%±5.3％(from 60±8 pA to 84±11 pA;n=8,P=0.012)of that of the control group which was blocked by pretreatment with 10-7 mol/L devazepide(61±9 pA;at+10 mV;n=8,P=0.023 vs CCK-8S group,P=0.079 vs control group)or 10-5 mol/L TG and BA(7±5 pA;at+10 mV,n=8,P=0.006 vs CCK-8S group),but CI 988 had little effect(84±11 pA;n=8,P=0.079 vs CCK-8S group).When 10-6 mol/L heparin was added to the pipette solution,CCK-8S(10-7 mol/L)had no effect on ICa-L (63±12 pA;at+10 mV,n=8,P=0.183 vs control group,P=0.042 vs CCK-8S group).When cells were pretreated with 10-6 mol/L staurosporine,CCK-8S(10-7 mol/L)did not increase ICa-L(65±10 pA;n=8,P=0.215 vs control group;P= 0.032 vs CCK-8S group).
Conclusion CCK-8S promotes Ca2+ release from sarcoplasmic reticulum and increases Ca2+ influx through L-type calcium channels,via the IP3-PKC signal transduction pathway by activation of CCK1 receptor.In addition,the decrease in AP duration is caused by the acceleration of fast repolarization of AP through increased IBKCa, and the increase in AP amplitude results from the enhancement of ICa-L, which both ultimately contribute to the contraction induced by CCK-8S.
PartⅢ
Objective To detect the expression of cholecystokinin receptors mRNA in guinea-pig colonic smooth muscle cells
Methods The fresh proximal colon was taken from the guinea pigs and intestinal contents were removed in the beaker with physiological saline and then the colon was placed in Ca2+-free PSS bubbled with carbogen (95% O2/5% CO2). The colonic smooth muscle tissue weighing 50-100 mg was obtained after the mucosa and submucosa were excised and then the supernatant was taken out after homogenate in ice bath and following centrifugalization. Then the expression of CCK1 receptor and CCK2 receptor mRNA was detected by reverse transcription polymerase chain reaction.
Results CCK1 receptor mRNA was positively expressed in guinea-pig colonic smooth muscle cells (0.83±0.15, n=15, P=0.263 vsβ-actin), whereas the expression of CCK2 receptor mRNA was not detected.
Conclusion CCK1 receptor is the main type of cholecystokinin receptors in smooth muscle cells of guinea-pig proximal colon, which is the molecular identity of CCK-induced contraction in colon.
第一部分
目的：探讨CCK-8S对豚鼠近端结肠平滑肌肌条自发性收缩频率和强度的影响及可能的作用机制。
方法：取6 cm左右豚鼠近端结肠,在持续充以95%02和5%CO2的Ca-freePSS中去除黏膜后剪成8 mm×10 mm平滑肌肌条,并将纵行肌与环行肌分离。将肌条浸没于37℃含钙台式液中,一端固定于垂直灌流槽,另一端与张力换能器相连,并持续充以95%O2和5%C02的混合气体。待肌条出现自发节律性收缩并稳定后采用RM6240多道生理信号采集处理系统记录给药前后收缩频率和强度。
结果：CCK-8S (10-7 mol/L)作用后,豚鼠结肠平滑肌纵行与环形肌条的收缩幅值分别为对照组的156.53%±11.92%(n=15,P=0.038)和165.93%±12.98%(n=15,P=0.019)；结肠平滑肌纵行肌条的收缩频率为对照组的131.69%±13.58%(n=15,P=0.023),而环形肌条的收缩频率无明显变化,为对照组的102.19%±8.96%(n=15,P=0.087)。当预先将灌流液中分别加入CCK1受体拮抗剂devazepide、L-型钙通道阻断剂nifedipine、大电导钙依赖钾通道阻断剂iberiotoxin、钙泵抑制剂thapsigargin (TG)和钙离子螯合剂BAPTA-AM (BA)后,CCK-8S对结肠平滑肌肌条的增强效应被抑制(n=15,均P<0.05)；而预先加入CCK2受体拮抗剂CI 988后,CCK-8S的增强效应仍然存在(n=15,P>0.05)。
结论：CCK-8S通过CCK1受体能促进豚鼠近端结肠纵,环行平滑肌自发性收缩强度及纵行平滑肌收缩频率；但对环行平滑肌收缩频率无明显影响。其机制与促进平滑肌细胞内钙的释放、胞膜上大电导钙依赖钾通道及L-型钙通道的激活有关。
第二部分
目的：探讨CCK-8S对单个豚鼠结肠平滑肌细胞静息电位(：resting potential, RP)、动作电位(action potential, AP)、大电导钙依赖钾通道电流(large conductance potassium currents,IBKca)和L-型钙通道电流(L-type calcium currents, ICa-L)的影响及可能的作用机制。
方法：取6 cm左右近端结肠,在持续充以95%02和5%CO2的Ca-free PSS中去除黏膜后剪成2 mm×6 mm大小,置于无钙消化液中,37℃振荡孵育20-30min,漂洗,吹打成细胞悬液。将细胞悬液接种于装有倒置显微镜的灌流槽中,贴壁后,用含钙台式液以2-3 ml／min速度持续灌流,并选取贴壁良好,折光性好,胞膜光滑清晰的杆状细胞进行高阻封接。用EPC-10膜片钳放大器记录结肠平滑肌细胞AP和RP给药前后的变化；在穿孔全细胞膜片钳模式下记录单个细胞IBKca和ICa-L给药前后的变化。
结果：1.CCK-8S (10-7 mol/L)作用后,结肠平滑肌细胞RP、AP峰值及AP快速复极时间分别为对照组的48.6±5.3%、138.6%±3.2%和63.1±8.7%(n=8,P=0.032、0.015和0.026),该效应可被预先加入的devazepide和／或nifedipine阻断(均n=8,P<0.05),而预先向灌流液中加入CI 988后,CCK-8S对RP、AP峰值及AP快速复极时间的作用仍存在。2.从-60 mV去极化至+100 mV,CCK-8S可剂量依赖性升高IBKca(P<0.05,at 10-8,10-7和10-6mol／L；ECso=3.5×10-8mol／L)。CCK-8S(10-7mol／L)升高IBKca为对照组的118.7%±2.1%(从916±183 pA至1088±226 pA；n=8,P=0.029),该效应可被预先向灌流液中加入的devazepide抑制(908±109pA,n=8,P=0.012 vsCCK-8S组,P=0.083vs对照组),但CI 988无明显抑制作用(1052±196 pA,n=8,P=0.098 vsCCK-8S组)。当向电极内液加入IP3受体阻断剂肝素(10-5mol／L)或向细胞外液加入蛋白激酶C拮抗剂staurosporine(10-6mol／L)后,CCK-8S(10-7mol／L)对IBKca均没有影响(879±117 pA,n=8,P=0.074vs对照组,P=0.016 vsCCK-8S组；887±120 pA,n=8,P=0.069 vs对照组,P=0.041 vs CCK-8S组)。3.从-60 mV去极化至+10 mV,CCK-8S可剂量依赖性升高ICa-L(P<0.05,at 10-8,10-7和10-6mol／L；EC50=3.2×10-8mol／L)。CCK-8S(10-7mol／L)升高ICa-L为对照组的140%±5.3%(从60±8 pA至84±11 pA,n=8,P=0.012),该效应可分别被预先向灌流液中加入的devazepide(61±9 pA；at+10 mV；n=8,P=0.023 vs CCK-8S组,P=0.079 vs对照组),TG和BA抑制(7±5 pA；at+10 mV,n=8,P=0.006 vs CCK-8S组),但CI 88无明显抑制作用(84±11pA；n=8,P=0.079 vs CCK-8S组)。当向电极内液加入IP3受体阻断剂肝素(10-5mol／L)或向细胞外液加入蛋白激酶C拮抗剂staurosporine(10-6mol／L)后,CCK-8S(10-7mol／L)对ICa-L均没有影响(63±12 pA,65±10 pA；at+10 mV；n=8；P=0.183,0.215vs正常组,P=0.042,0.032·vs CCK-8S组)。结论：CCK-8S通过CCK1受体促进近端结肠平滑肌细胞内钙库释放钙及增强肌细胞膜ICa-L,促进细胞外钙内流,从而使平滑肌细胞RP下降,AP幅值增加；通过增强细胞膜IBKca使平滑肌细胞AP快速复极时间缩短,从而增加了单位时间内AP次数,促进结肠平滑肌细胞收缩。其作用机制与激活IP3介导的PKC途径有关。
第三部分
目的：检测胆囊收缩素受体基因在豚鼠近端结肠平滑肌的表达。方法：取新鲜的豚鼠近端结肠平滑肌组织,在装有生理盐水的烧杯中去除肠内容物,然后在持续充以95%02和5%CO2的Ca-free PSS中去除粘膜层及粘膜下层,取50-100 mg新鲜组织在冰浴中匀浆,离心后,吸取上清,应用逆转录多聚酶链反应(reverse transcription polymerase chain reaction, RT-PCR)检测CCK1受体及CCK2受体mRNA在豚鼠近端结肠平滑肌组织中的表达。结果：豚鼠近端结肠平滑肌组织中有CCK1受体mRNA表达(0.83±0.15,n=15,P=0.263vsβ-actin),扩增产物片段大小为630bp,但未见CCK2受体mRNA的表达。
结论：豚鼠近端结肠平滑肌中分布的CCK受体主要为CCK1受体,这是CCK促进结肠平滑肌收缩的分子基础。
Background
Cholecystokinin(CCK), a gut-brain peptide, plays an important role in regulating a variety of physiological functions and involving in many pathophysiological processes. It has been reported that sulfated cholecystokinin octapeptide(CCK-8S) is the predominant and major molecular form of CCK peptides for biological activity and it actions on the gastrointestinal tract through two receptor subtypes, designated as CCK1 receptor and CCK2 receptor. Previous studies investigating the targets for CCK have focused mainly on the gallbladder, pancreas, heart and lung. However, recent researches have shown that CCK and its related peptides are implicated in the pathophysiology of functional digestive diseases. Several early reports also have indicated that either an altered release of CCK or abnormal responses to this peptide could be responsible for the symptoms of functional digestive diseases. However, the direct electrophysiological effects of CCK on the contractile activity of gut remains unclear.
PartⅠ
Objective To investigate the effects of sulfated cholecystokinin octapeptide (CCK-8S) on the spontaneous contractile activity of guinea-pig proximal colonic smooth muscle strips.
Methods The proximal colon (6 cm) was removed, cleaned and opened along the mesenteric border and then placed in Ca2+-free PSS bubbled with carbogen (95% O2/5% CO2). The smooth muscle strips (8×10 mm) were obtained after the mucosa and submucosa were excised and the longitudinal muscle(LM) and circular muscle(CM) strips were segregated. Each fresh smooth muscle strip was mounted in an organ bath and connected to an isometric force transducer. The organ baths contained 10 ml Tyrode's buffer and were constantly warmed by a circulating water jacketed at 37℃and bubbled with carbogen (95% O2/5% CO2). One end of the strip was fixed to a hook on the bottom of the chamber, while the other end was connected by a thread to an external isometric force transducer at the top. Each muscle strip was placed under a resting preload of 1.0g and allowed to equilibrate for 60 min with solution change every 20 min. The relative effects on the contractile amplitude and frequency of LM and CM strips were observed before and after the addition of drugs.
Results The mean contractile amplitude of CM and LM strips stimulated by CCK-8S (10-7 mol/L) were 156.53%±11.92% and 165.93%±12.98% respectively of that of the control group(n=15, P=0.038,0.019);the mean frequency of LM stimulated by CCK-8S (10-7 mol/L) was 131.69%±13.58% of that of the control group(P=0.023), but CCK-8S had little influence on the frequency of CM(n=15, P=0.087). CCK-8S-intensified effects on proximal colonic strips were significantly attenuated when these strips were pretreated with CCK1 receptor antagonist devazepide (10-7 mol/L), L-type calcium channel inhibitor nifedipine (10-5 mol/L) or Ca2+-ATPase inhibitor TG (10-5 mol/L) and intracellular calcium chelator BA (10-5 mol/L) (n=15, P＜0.05 for each group). Pretreating CM and LM strips with iberiotoxin (10-6 mol/L), a selective BKCa channel blocker, did not inhibit the CCK-8S-induced increase in the contractile amplitude of CM and LM strips (n=15, P=0.096,0.078 vs CCK-8S group), but decreased their frequencies (n=15, P= 0.036,0.041 vs CCK-8S group), whereas superfusion with the CCK2 receptor antagonist CI 988 (10-7 mol/L) did not block the CCK-8S-intensified effect on CM and LM strips (n=15, P＞0.05 for each group).
Conclusion CCK-8S promotes the contraction of the longitudinal muscle and circular muscle of guinea-pig proximal colon by CCK1 receptor mostly owing to increasing the release of intracellular Ca2+, large conductance potassium currents and L-type calcium currents, but CCK-8S has little effect on the frequency of the circular muscle.
PartⅡ
Objective To examine the effect and its mechanism of CCK-8S on resting potential(RP), action potential(AP), large conductance potassium currents(IBKCa) and L-type calcium currents(ICa-L).
Methods The proximal colon (about 6 cm long) was removed, cleaned and opened along the mesenteric border and then placed in Ca2+-free PSS bubbled with carbogen (95% O2/5% CO2). The strips of proximal colon were pinned to the base of the sylgard surface of a Petri dish and the mucosa together with submucosa was carefully dissected away. The tissue was cut into small strips (about 2 mm wide and 6 mm long) and placed in Ca2+-free PSS solution and incubated for 25 min at 37℃. After completion of digestion, the segments were washed and then triturated gently to create a cell suspension. Several drops of cell suspensions were placed in a recording chamber that was mounted with an inverted microscope. Cells were superfused with CaPSS (3 ml/min) after adhering to the coverslip. RP and AP were recorded by EPC-10 amplifier. Whole-cell currents were recorded by using a nystatin-perforated whole-cell patch-clamp configuration with an EPC-10 amplifier. Drugs were added subsequently to the cell suspension to observe relative effects on AP, RP, IBKCa and ICa-L when their values were stable.
Results 1.The amplitude of RP and AP and fast repolarization time (repolarizing to 90% of the peak value of AP, T90) induced by CCK-8S (10-7 mol/L) were 48.6%±5.3%,138.6%±3.2% and 63.1%±8.7% respectively of that of the control group (n=8,P=0.032,0.015 and 0.026), which were significantly inhibited when these cells were pretreated with devazepide (10-7 mol/L) and/or nifedipine (10-5 mol/L)(n=8, P＜0.05 for each group), whereas CCK-8S had little influence.2. IBKCa was evoked by using a depolarizing step pulse from a holding potential of -60 mV to+100 mV. CCK-8S increased IBKCa in a concentration-dependent manner (P＜ 0.05,at 10-8,10-7 and 10-6 mol/L;EC50=3.5×10-8mol/L).The peak amplitude of IBKCa was enhanced to 118.7%±2.1%(from 916±183 pAto 1088±226 pA,n=8, P=0.029)of that of the control group,which was blocked by pretreatment with 10-7 mol/L devazepide(908±109 pA,n=8,P=0.012 vs CCK-8S group,P=0.083 vs control group).However,the CCK2 receptor antagonist CI 988 exerted little effect (1052±196 pA,n=8,P=0.098 vs CCK-8S group).When heparin(10-6 mol/L),an inhibitor of IP3 receptors,was added to the pipette solution,CCK-8S(10-7 mol/L)did not enhance IBKCa(879±117 pA;n=8,P=0.074 vs control group,P=0.016 vs CCK-8S group).Pretreating cells with the PKC inhibitor staurosporine(10-6 mol/L) also attenuated the CCK-8S-induced effect on IBKCa(887±120 pA;n=8,P=0.069 vs control group,P=0.041 vs CCK-8S group).3.ICa-L was evoked by using a depolarizing step pulse from a holding potential of -40 mV to +30 mV.CCK-8S enhanced ICa-L in a concentration.dependent manner(P＜0.05,at 10-8,10-7 and 10-6 mol/L;EC50=3.2×10-8 mol/L).The peak amplitude of ICa-L was increased to 140%±5.3％(from 60±8 pA to 84±11 pA;n=8,P=0.012)of that of the control group which was blocked by pretreatment with 10-7 mol/L devazepide(61±9 pA;at+10 mV;n=8,P=0.023 vs CCK-8S group,P=0.079 vs control group)or 10-5 mol/L TG and BA(7±5 pA;at+10 mV,n=8,P=0.006 vs CCK-8S group),but CI 988 had little effect(84±11 pA;n=8,P=0.079 vs CCK-8S group).When 10-6 mol/L heparin was added to the pipette solution,CCK-8S(10-7 mol/L)had no effect on ICa-L (63±12 pA;at+10 mV,n=8,P=0.183 vs control group,P=0.042 vs CCK-8S group).When cells were pretreated with 10-6 mol/L staurosporine,CCK-8S(10-7 mol/L)did not increase ICa-L(65±10 pA;n=8,P=0.215 vs control group;P= 0.032 vs CCK-8S group).
Conclusion CCK-8S promotes Ca2+ release from sarcoplasmic reticulum and increases Ca2+ influx through L-type calcium channels,via the IP3-PKC signal transduction pathway by activation of CCK1 receptor.In addition,the decrease in AP duration is caused by the acceleration of fast repolarization of AP through increased IBKCa, and the increase in AP amplitude results from the enhancement of ICa-L, which both ultimately contribute to the contraction induced by CCK-8S.
PartⅢ
Objective To detect the expression of cholecystokinin receptors mRNA in guinea-pig colonic smooth muscle cells
Methods The fresh proximal colon was taken from the guinea pigs and intestinal contents were removed in the beaker with physiological saline and then the colon was placed in Ca2+-free PSS bubbled with carbogen (95% O2/5% CO2). The colonic smooth muscle tissue weighing 50-100 mg was obtained after the mucosa and submucosa were excised and then the supernatant was taken out after homogenate in ice bath and following centrifugalization. Then the expression of CCK1 receptor and CCK2 receptor mRNA was detected by reverse transcription polymerase chain reaction.
Results CCK1 receptor mRNA was positively expressed in guinea-pig colonic smooth muscle cells (0.83±0.15, n=15, P=0.263 vsβ-actin), whereas the expression of CCK2 receptor mRNA was not detected.
Conclusion CCK1 receptor is the main type of cholecystokinin receptors in smooth muscle cells of guinea-pig proximal colon, which is the molecular identity of CCK-induced contraction in colon.
引文
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