Mechanisms of cholecystokinin-induced calcium mobilization in gastric antral interstitial cells of Cajal.

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 Ca(2+) concentration ([Ca(2+)]i). A confocal laser scanning microscope was used to monitor [Ca(2+)]i changes. The selective CCK(1) receptor antagonist lorglumide, the intracellular Ca(2+)-ATPase inhibitor thapsigargin, the type III inositol 1,4,5-triphosphate (InsP(3)) receptor blocker xestospongin C and the L-type voltage-operated Ca(2+) channel inhibitor nifedipine were used to examine the mechanisms of [Ca(2+)]i elevation caused by CCK-8S. Immunoprecipitation and Western blotting were used to determine the regulatory effect of PKC on phosphorylation of type III InsP(3) receptor (InsP(3)R3) 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 [Ca(2+)]i in ICC increased and 100 nmol/L CCK-8S significantly increased the mean [Ca(2+)]i by 59.30% ± 4.85% (P < 0.01). Pretreatment of ICC with 5 μmol/L lorglumide inhibited 100 nmol/L CCK-8S-induced [Ca(2+)]i increment from 59.30% ± 4.85% to 14.97% ± 9.05% (P < 0.01), suggesting a CCK(1)R-mediated event. Emptying of intracellular calcium stores by thapsigargin (5 μmol/L) prevented CCK-8S (100 nmol/L) from inducing a [Ca(2+)]i increase. Moreover, pretreatment with xestospongin C (1 μmol/L) could also abolish the CCK-8S-induced effect, indicating that Ca(2+) release from InsP(3)R-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 [Ca(2+)]i could be still elicited by CCK-8S. These data suggest that the [Ca(2+)]i release is not stimulated or activated by the influx of extracellular Ca(2+) in ICC, but the influx of extracellular Ca(2+) can facilitate the [Ca(2+)]i increase evoked by CCK-8S. CCK-8S increased the phosphorylation of InsP(3)R3, which could be prevented by chelerythrine. Pretreatment with lorglumide (5 μmol/L) could significantly reduce the CCK-8S intensified phosphorylation of InsP(3)R3. In the positive control group, treatment of cells with PMA also resulted in an enhanced phosphorylation of InsP(3)R3. 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. 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 [Ca(2+)]i in ICC via the CCK(1) receptor. This effect depends on the release of InsP(3)R-operated Ca(2+) stores, which is negatively regulated by PKC-mediated phosphorylation of InsP(3)R3.