Effects and its possible mechanism of Radix Saposhnikoviae on rat colonic smooth muscle in vitro

Abstract

Objective To determine the effect of different concentrations of Radix Saposhnikoviae (RS) on the contraction of smooth muscle strips and the Ca 2+ mobilization of cultured smooth muscle cells of rat colon and its possible mechanism of action. Methods Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell- experiments, intracellular Ca 2+ was assessed using fluorescent intensity (FI) of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results In the in vitro experiment, RS (0.02, 0.2, 2, 20 g/L) inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations ( P < 0.01) for both Peak (the maximal contraction amplitude) and Area (the area under curves). Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant ( P < 0.01), as was the inhibition of the Area values in all RS groups ( P < 0.05). Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect ( P < 0.01). In experiments using primary smooth muscle cell cultures in Ca 2+ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values ( P < 0.05), and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant ( P < 0.01). However, in Ca 2+-free buffer, FS had no significant effect on cell fluorescence. Conclusion RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α-adrenoceptors, but not β-adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca 2+ from extracellular sources, but may had no effect on the release of Ca 2+ from sarcoplasmic reticulum and endoplasmic reticulum.