| BackgroundCholecystokinin(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.PartⅠObjective 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. PartⅡObjective 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.PartⅢObjective To detect the expression of cholecystokinin receptors mRNA in guinea-pig colonic smooth muscle cellsMethods 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. |
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