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

Abstract

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.

Keywords: Calcium mobilization; Cholecystokinin octapeptide; Interstitial cells of Cajal; Protein kinase C.

Figure 1

Identification of cultured interstitial cells of Cajal. A-C: Prolonging the culture to 4-7 d, the cultured interstitial cells of Cajal (ICC), which are identified by c-Kit immunofluorescence, had distinctive shapes such as spindle, triangular or stellar-like with two to five long processes. ICC were fixed with acetone and identified immunologically using a monoclonal c-Kit antibody and Alexa Fluor 488-conjugated secondary fluorescent antibody. Nuclei were stained with Hoechst 33258 dye (B, blue); C: A merged image of A and B; D: A light microscopic image of ICC.

Figure 2

The regulation of sulfated cholecystokinin-8 on [Ca²⁺]i in cultured interstitial cells of Cajal from the murine gastric antrum. A1: Fluorescent intensity image of Fluo-3/AM loaded cultured interstitial cells of Cajal (ICC) under normal conditions; A2-6: Fluorescent intensity gradually increased in the presence of cholecystokinin-8 (CCK-8S) (100 nmol/L); B-E: Effects of different concentrations of sulfated CCK-8S on mean [Ca²⁺]i. In each case, cells from at least five different cell cultures; F: Effects of CCK-8S were estimated as percentage of ΔF/F0, where F0 was derived from the averaged intensity of the first 10-30 frames minus the background in the cell-free region and ΔF is fluorescent intensity of the response minus F0. ^aP < 0.05, ^bP < 0.01 vs control.

Figure 3

Sulfated cholecystokinin-8 activates interstitial cells of Cajal through the cholecystokinin1 receptor. A: Compared with the control; B: Lorglumide significantly inhibited cholecystokinin-8 (CCK-8S)-induced increase in [Ca²⁺]i of interstitial cells of Cajal; C: Quantification of [Ca²⁺]i changes shown in A and B. Each experiment was repeated at least three times. ^bP < 0.01 vs control.

Figure 4

Sulfated cholecystokinin-8 induced calcium mobilization in cultured interstitial cells of Cajal. A, B: Pretreatment with 5 μmol/L thapsigargin (A) or 1 μmol/L xestospongin C (B) completely abolished sulfated cholecystokinin-8 (CCK-8S)-induced [Ca²⁺]i increases; C: Addition of nifedipine resulted in a smaller peak of [Ca²⁺]i in comparison with normal conditions; D: The CCK-8S-elicited [Ca²⁺]i increase in the calcium-free medium was lower than that in the calcium-containing buffer; E: Quantification of [Ca²⁺]i changes following addition of nifedipine or removal of extracellular Ca²⁺. The fluorescence was normalized as F1/F0 (F1: Maximal fluorescence after drug addition; F0: Basal fluorescence before drug addition). ^bP < 0.01 vs control.

Figure 5

Sulfated cholecystokinin-8 stimulation of interstitial cells of Cajal resulted in the protein kinase C-dependent phosphorylation of type III inositol 1,4,5-triphosphate receptor. A: Western blots of proteins were immunoprecipitated with type III inositol 1,4,5-triphosphate receptor (InsP₃R3)-specific antibody. The immunoprecipitated proteins were probed with antibody specific for phosphorylated Ser/Thr protein kinase C (PKC) substrate sequences. The sulfated cholecystokinin-8 (CCK-8S)-induced phosphorylation of InsP₃R3 was apparently inhibited by pretreatment with chelerythine. 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 phorbol-12-myristate-13-acetate (PMA) also resulted in an enhanced phosphorylation of InsP₃R3; B: The nitrocellulose membrane in A was stripped and reprobed with InsP₃R3 (1:1000) to determine the levels of InsP₃R3 immunoprecipitated. Each cell sample contained nearly equal amounts of InsP₃R3.

Figure 6

Effects of phorbol-12-myristate-13-acetate on sulfated cholecystokinin-8-evoked Ca²⁺ signaling in interstitial cells of Cajal. Fluo-3-loaded interstitial cells of Cajal were pretreated with various concentrations of Phorbol-12-myristate-13-acetate (PMA) (A: 10 nmol/L; B: 100 nmol/L; C: 1 μmol/L; D: 10 μmol/L) for 4 min before addition of sulfated cholecystokinin-8 (CCK-8S) (100 nmol/L). All could significantly reduce the CCK-8S-evoked [Ca²⁺]i response; E: Quantification of [Ca²⁺]i changes shown in A-D. Each experiment was repeated at least three times. ^bP < 0.01 vs control.

Figure 7

Effects of chelerythrine on Sulfated cholecystokinin-8-evoked Ca²⁺ signaling in interstitial cells of Cajal. Fluo-3-loaded interstitial cells of Cajal were pretreated with various concentrations of chelerythrine (A: 10 nmol/L; B: 100 nmol/L; C: 1 μmol/L; and D: 10 μmol/L) for 2 min before administration of cholecystokinin-8 (CCK-8S) (100 nmol/L). All could significantly increase the CCK-8S-evoked [Ca²⁺]i response; E: Quantification of [Ca²⁺]i changes shown in A-D. Each experiment was repeated at least three times. ^bP < 0.01 vs control.