Published online Dec 28, 2012. doi: 10.3748/wjg.v18.i48.7184
Revised: November 2, 2012
Accepted: November 6, 2012
Published online: December 28, 2012
Processing time: 179 Days and 0.1 Hours
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 mechanisms 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 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 [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.