Contractile effects and intracellular Ca2+ signalling induced by emodin in circular smooth muscle cells of rat colon

Abstract

AIM: To investigate whether emodin has any effects on circular smooth muscle cells of rat colon and to examine the mechanism underlying its effect.

METHODS: Smooth muscle cells were isolated from the circular muscle layer of Wistar rat colon and the cell length was measured by computerized image micrometry. Intracellular Ca²⁺ ([Ca²⁺]i) signalling was studied in smooth muscle cells using Ca²⁺ indicator Fluo-3 AM on a laser-scanning confocal microscope.

RESULTS: Emodin dose-dependently induced smooth muscle cells contraction. The contractile responses induced by emodin were inhibited by preincubation of the cells with ML-7 (an inhibitor of MLCK). Emodin caused a large, transient increase in [Ca²⁺]i followed by a sustained elevation in [Ca²⁺]i. The emodin –induced increase in [Ca²⁺]i was unaffected by nifedipine, a voltage-gated Ca²⁺-channel antagonist, and the sustained phase of the rising of [Ca²⁺]i was attenuated by extracellular Ca²⁺ removal with EGTA solution. Inhibiting Ca²⁺ release from ryanodine-sensitive intracellular stores by ryanodine reduced the peak increase in [Ca²⁺]i. Using heparin, an antagonist of IP₃R, almost abolished the peak increase in [Ca²⁺]i.

CONCLUSION: Emodin has a direct excitatory effect on circular smooth muscle cells in rat colon mediated via Ca²⁺/ CaM dependent pathways. Furthermore, emodin-induced peak [Ca²⁺]i increase may be attributable to the Ca²⁺ release from IP₃ sensitive stores, which further promote Ca²⁺ release from ryanodine-sensitive stores through CICR mechanism. Additionally, Ca²⁺ influx from extracellular medium contributes to the sustained increase in [Ca²⁺]i.

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