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 Ca(2+) ([Ca(2+)]i) signalling was studied in smooth muscle cells using Ca(2+) 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 [Ca(2+)]i followed by a sustained elevation in [Ca(2+)]i. The emodin -induced increase in [Ca(2+)]i was unaffected by nifedipine, a voltage-gated Ca(2+)-channel antagonist, and the sustained phase of the rising of [Ca(2+)]i was attenuated by extracellular Ca(2+) removal with EGTA solution. Inhibiting Ca(2+) release from ryanodine-sensitive intracellular stores by ryanodine reduced the peak increase in [Ca(2+)]i. Using heparin, an antagonist of IP(3)R, almost abolished the peak increase in [Ca(2+)]i.

Conclusion: Emodin has a direct excitatory effect on circular smooth muscle cells in rat colon mediated via Ca(2+)/CaM dependent pathways. Furthermore, emodin-induced peak [Ca(2+)]i increase may be attributable to the Ca(2+) release from IP(3) sensitive stores, which further promote Ca(2+) release from ryanodine-sensitive stores through CICR mechanism. Additionally, Ca(2+) influx from extracellular medium contributes to the sustained increase in [Ca(2+)]i.

Figure 1

Contractile effect of emodin on isolated smooth muscle cell. Values were calculated as means ± SE from 3 experiments.

Figure 2

Effect of ML-7 on emodin-induced contraction of smooth muscle cells. Values were means ± SE of 3 experiments. ^aP < 0.05 by Student’s t-test.

Figure 3

Timecourse changes of [Ca²⁺]i induced by emodin in a circular colonic smooth muscle cells (analysed by BioRad Laserpix software).

Figure 4

Average peak and sustained changes in [Ca²⁺]i in response to emodin (50 μmol/L). ^aP < 0.05, ^bP < 0.001.

Figure 5

Average peak (top) and sustained (bottom) changes in [Ca²⁺]i in response to emodin under control conditions, in the presence of nifedipine, and EGTA solution. n = 15. ^aP < 0.05 vs emodin group.

Figure 6

Effect of ryanodine and heparin on the change in [Ca²⁺]i induced by emodin. n = 15 ^aP < 0.05 vs emodin group ^bP < 0.01 vs emodin group.