Abstract

Epoxyaurapten is a component of coumarin, which was isolated from Aurantii Fructus Immaturus. The objective of this study was to determine the inhibition of epoxyaurapten on smooth muscle in vitro. The experiment of smooth muscle contraction directly monitored the contractions of the isolated proximal duodenum by frequency and amplitude at different epoxyaurapten concentrations and under different incubation times. The results showed that epoxyaurapten inhibited contraction in intestinal muscles in a dose- and time-dependent manner. The effects of epoxyaurapten on myosin were measured in the presence of Ca²⁺-calmodulin using the activities of 20 kDa myosin light chain (MLC₂₀) phosphorylation and myosin Mg²⁺-ATPase. The results demonstrated that MLC₂₀phosphorylation by myosin light chain kinase (MLCK) decreased with increasing epoxyaurapten concentration. The myosin Mg²⁺-ATPase activity also gradually deceased with increasing epoxyaurapten concentration. With prolonged incubation time, MLC₂₀phosphorylation by MLCK and myosin Mg²⁺-ATPase activities furthered declined. The effect of epoxyaurapten on MLCK expression was measured by western blot, and the results showed that epoxyaurapten inhibited the expression of MLCK in a dose- and time-dependent manner. The findings demonstrated that epoxyaurapten could effectively attenuate the contractions of intestinal smooth muscle and therefore could be developed as a potential therapy in the future.

Key words: Epoxyaurapten, myosin light chain kinase (MLCK), 20 kDa regulating myosin light chain (MLC₂₀)_, myosin Mg²⁺-ATPase, Aurantii Fructus Immaturus.

Abbreviation

SMCs, smooth muscle cells; PAGE, polyacrylamide gel electrophoresis;MLC₂₀, 20 kDa regulating myosin light chain; LC₂₀, unphosphorylated MLC₂₀; p-LC₂₀,mono-phosphorylated MLC₂₀; LC₁₇, 17 kDa myosin essential light chains.