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AIM:To investigate the effect of sulfated cholecystokinin-8(CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal(ICC) and its possible mechanisms.METHODS:ICC were isolated from the gastric antrum of mice and cultured.Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC.The responsiveness of ICC to CCK-8S was measured using Fluo-3/AM based digital microfluorimetric measurement of intracellular Ca2+ concentration([Ca2+]i).A confocal laser scanning microscope was used to monitor [Ca2+]i changes.The selective CCK1 receptor antagonist lorglumide,the intracellular Ca2+-ATPase inhibitor thapsigargin,the type Ⅲ inositol 1,4,5-triphosphate(InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2+ channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2+]i elevation caused by CCK-8S.Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type Ⅲ InsP3 receptor(InsP3R3) in ICC.Protein kinase C(PKC) activator phorbol 12-myristate 13-acetate(PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca2+]i increment of ICC.RESULTS:ICC were successfully isolated from the gastric antrum of mice and cultured.Cultured ICC were identified by immunofluorescence staining.When given 80 nmol/L or more than 80 nmol/L CCK-8S,the [Ca2+]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca2+]i by 59.30% ± 4.85%(P < 0.01).Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca2+]i increment from 59.30% ± 4.85% to 14.97% ± 9.05%(P < 0.01),suggesting a CCK1R-mediated event.Emptying of intracellular calcium stores by thapsigargin(5 μmol/L) prevented CCK-8S(100 nmol/L) from inducing a [Ca2+]i increase.Moreover,pretreatment with xestospongin C(1 μmol/L) could also abolish the CCK-8S-induced effect,indicating that Ca2+ release from InsP3R-operated stores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC.On the other hand,by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine,a smaller but significant rise in the [Ca2+]i could be still elicited by CCK-8S.These data suggest that the [Ca2+]i release is not stimulated or activated by the influx of extracellular Ca2+ in ICC,but the influx of extracellular Ca2+ can facilitate the [Ca2+]i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3,which could be prevented by chelerythrine.Pretreatment with lorglumide(5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group,treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA(10 nmol/L-10 μmol/L) apparently inhibited the effect of CCK-8S and the effect of100 nmol/L PMA was most obvious.Likewise,the effect of CCK-8S was augmented by the pretreatment with chelerythrine(10 nmol/L-10 μmol/L) and 100 nmol/L chelerythrine exhibited the maximum effect.CONCLUSION:CCK-8S increases [Ca2+]i in ICC via the CCK1 receptor.This effect depends on the release of InsP3R-operated Ca2+ stores,which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.
AIM: To investigate the effect of sulfated cholecystokinin-8 (CCK-8S) on calcium mobilization in cultured murine gastric antral interstitial cells of Cajal (ICC) and its possible mechanisms. METHODS: ICC were isolated from the gastric antrum of mice and cultured. Immunofluorescence staining with a monoclonal antibody for c-Kit was used to identify ICC. The responsiveness of ICC to CCK-8S was measured using Fluo-3 / AM based digital microfluorimetric measurement of intracellular Ca2 concentration ([Ca2 +] i) scanning microscope was used to monitor [Ca2 +] i changes. The CCK1 receptor antagonist lorglumide, the intracellular Ca2 + -ATPase inhibitor thapsigargin, the type III inositol 1, 4,5-triphosphate (InsP3) receptor blocker xestospongin C and the L-type voltage-operated Ca2 + channel inhibitor nifedipine were used to examine the mecha-nisms of [Ca2 +] i elevation caused by CCK-8S. Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphatidylcholine horylation of type III InsP3 receptor (InsP3R3) in ICC. Protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and inhibitor chelerythrine were used to assess the role of PKC in the CCK-8S-evoked [Ca 2+] i increment of ICC.RESULTS: ICC were successfully isolated from the gastric antrum of mice and cultured. Cultured ICC were identified by immunofluorescence staining. When given 80 nmol / L or more than 80 nmol / L CCK-8S, the [Ca2 +] i (P <0.01) .Pretreatment of ICC with 5 μmol / L lorglumide inhibited 100 nmol / L CCK-8S-induced ICC increased and 100 nmol / L CCK-8S significantly increased the mean [Ca 2+] i by 59.30% ± 4.85% CCK-8S (100 nmol / L) was increased from 59.30% ± 4.85% to 14.97% ± 9.05% (P <0.01) / L) from inducing a [Ca2 +] i increase. Moreover, pretreatment with xestospongin C (1 μmol / L) could also abolish the CCK-8S-induced effect, indicating that Ca2 + release from InsP3 R-operatedstores appeared to be a major mechanism responsible for CCK-8S-induced calcium mobilization in ICC. On the other hand, by removing extracellular calcium or blocking the L-type voltage-operated calcium channel with nifedipine, a smaller but significant rise in the [ Ca2 +] i could be still elicited by CCK-8S.These data suggest that the [Ca2 +] i release is not stimulated or activated by the influx of extracellular Ca2 + in ICC, but the influx of extracellular Ca2 + can facilitate the [Ca2 +] i increase evoked by CCK-8S.CCK-8S increased the phosphorylation of InsP3R3, which could be prevented by chelerythrine. Treatment with lorglumide (5 μmol / L) could significantly reduce the CCK-8S intensified phosphorylation of InsP3R3.In the positive control group, treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP3R3.Pretreatment with various concentrations of PMA (10 nmol / L-10 μmol / L) apparently inhibited the effect of CCK-8S and the effect of 100 nmol / L PMA was most obvious. Likewis e, the effect of CCK-8S was augmented by the pretreatment with chelerythrine (10 nmol / L-10 μmol / L) and 100 nmol / L chelerythrine showed the maximum effect. CONCLUSION: CCK-8S increases [Ca 2+] i in ICC via the CCK1 receptor. This effect depends on the release of InsP3R-operated Ca2 + stores, which is negatively regulated by PKC-mediated phosphorylation of InsP3R3.