Signal Transduction Pathways Mediating CCK-8S-Induced Gastric Antral Smooth Muscle Contraction

Abstract

Aim: To investigate the functional and molecular mechanisms by which sulfated cholecystokinin octapeptide (CCK-8S) regulates calcium mobilization in gastric antral smooth muscle cells (SMCs) of rats. Methods: Isotonic contraction of antral strips was recorded using a polyphysiograph. Immunoprecipitation was used to determine the regulatory effect of protein kinase C (PKC) on regulating the phosphorylation of the type III inositol 1,4,5-triphosphate receptor (InsP₃R3) in gastric SMCs. Alterations in the intracellular calcium ([Ca²⁺]_i) concentration were assayed using fura-2/AM-loaded microspectrofluorometry, and the L-type calcium current (I_Ca-L) was recorded by patch-clamp techniques. Results: CCK-8S (5 × 10^–8 mol/l) significantly increased the mean contractile amplitude of circular muscle by 61.85 ± 12.67% and the frequency of longitudinal muscle by 57.91 ± 15.70% in gastric antral strips, which were suppressed by dexloxiglumide or thapsigargin (TG) and BAPTA-AM (BA). Treatment withchelerythrine (5 × 10^–8 mmol/l) significantly inhibited the CCK-8S-increased phosphorylation of InsP₃R3 in SMCs. The amplitudes of the CCK-8S-triggered [Ca²⁺]_i concentration oscillations were reduced in a dose-dependent manner when the SMCs were pretreated with increasing concentrations of PMA (from 10^–8 to 10^–5 mol/l). On removal of extracellular calcium or blocking I_Ca-L by nifedipine, a smaller but significant rise in the [Ca²⁺]_i concentration was still elicited by CCK-8S. When [Ca²⁺]_i was depleted by the administration of 10^–5 mol/l TG and 10^–5 mol/l BA or blocked by the calcium-dependent chloride current (I_Cl-Ca) by giving 5 × 10^–6 mol/l niflumic acid, the CCK-8S-intensified I_Ca-L (from –56.42 ± 6.57 to –88.54 ± 5.71 pA) was apparently inhibited by 90.34 ± 4.71% and 82.59 ± 4.24%. Conclusions: These results demonstrate that the CCK-8S-evoked [Ca²⁺]_i concentration increase in gastric antral SMCs depends on the release of [Ca²⁺]_i stores which are negatively regulated by PKC-mediated phosphorylation of InsP₃R3. Released calcium in turn activates I_Ca-L through the activation of I_Cl-Ca, ultimately resulting in the contraction of the gastric smooth muscle.