Korean J Physiol Pharmacol.  2017 Nov;21(6):609-616. 10.4196/kjpp.2017.21.6.609.

Ardipusilloside-I stimulates gastrointestinal motility and phosphorylation of smooth muscle myosin by myosin light chain kinase

• Xu Z
• Liang H
• Zhang M
• Tao X
• Dou D
• Hu L
• Kang T

• KMID: 2395255
• DOI: http://doi.org/10.4196/kjpp.2017.21.6.609

Abstract

Ardipusilloside-I is a natural triterpenoid saponin, which was isolated from Ardisia pusilla A. DC. The aim of the study was to evaluate the stimulation of ardipusilloside-I on gastrointestinal motility in vitro and in vivo. The experiment of smooth muscle contraction directly monitored the contractions of the isolated jejunal segment (IJS) in different contractile states, and the effects of ardipusilloside-I on myosin were measured in the presence of Ca²âº-calmodulin using the activities of 20 kDa myosin light chain (MLCâ‚‚â‚€) phosphorylation and myosin Mg²âº-ATPase. The effects of ardipusilloside-I on gastro emptying and intestinal transit in constipation-predominant rats were observed, and the MLCK expression in jejuna of constipated rats was determined by western blot. The results showed that, ardipusilloside-I increased the contractility of IJS in a dose-dependent manner and reversed the low contractile state (LCS) of IJS induced by low Ca²âº, adrenaline, and atropine respectively. There were synergistic effects on contractivity of IJS between ardipusilloside-I and ACh, high Ca²âº, and histamine, respectively. Ardipusilloside-I could stimulate the phosphorylation of MLCâ‚‚â‚€ and Mg²âº-ATPase activities of Ca²âº- dependent phosphorylated myosin. Ardipusilloside-I also stimulated the gastric emptying and intestinal transit in normal and constipated rats in vivo, respectively, and increased the MLCK expression in the jejuna of constipation-predominant rats. Briefly, the findings demonstrated that ardipusilloside-I could effectively excite gastrointestinal motility in vitro and in vivo.

Keyword

Ardipusilloside-I; Gastrointestinal motility; Myosin light chain kinase; Myosin Mg²⁺-ATPase

MeSH Terms

Animals
Ardisia
Atropine
Blotting, Western
Epinephrine
Gastric Emptying
Gastrointestinal Motility*
Histamine
In Vitro Techniques
Muscle, Smooth*
Myosin Light Chains*
Myosin-Light-Chain Kinase*
Myosins*
Phosphorylation*
Rats
Saponins
Atropine
Epinephrine
Histamine
Myosin Light Chains
Myosin-Light-Chain Kinase
Myosins
Saponins

Figure

• 

  Fig. 1 Effects of ardipusilloside-I on gastrointestinal motility (X±s, n=10). (A) The chemical structure of ardipusilloside-I. (B, C) The effects of ardipusilloside-I on gastric emptying and intestinal transit in normal rats and in constipated rats respectively. **p<0.01 compared with NC group. ##p<0.01 compared with MC group.

• 

  Fig. 2 Effects of ardipusilloside-I on the contractility of isolated jejunal segment (IJS) in normal contractile state (X±s, n=6). (A) Representative traces and (B) statistical analysis of concentration- response relation of ardipusilloside-I on the contractility of IJS. The contractile amplitude of IJS in normal contractile state was set as 100% (NC). **p<0.01 compared with the NC.

• 

  Fig. 3 Effects of ardipusilloside-I on the contractility of isolated jejunal segment (IJS) in different contractile states (X±s, n=6). (A1) Representative traces and (A2) statistical analysis of ardipusilloside-I on the contractility of IJS in 3 low contractile states pre-induced with low Ca2+ (1.25 mM), adrenaline (1 µM), and atropine (1 µM), respectively. (B1) Representative traces and (B2) statistical analysis of ardipusilloside-I on the contractility of IJS in 3 high contractile states pre-induced with ACh (acetylcholine, 1 µM), high Ca2+ (5 mM), and histamine (5 µM) respectively. **p<0.01 compared with the NC, ##p<0.01 compared with the contractile amplitudes in low contractile states (LCS) and in high contractile states (HCS) before ardipusilloside-I treatment, respectively.

• 

  Fig. 4 Effects of ardipusilloside-I on phosphorylation and Mg2+-ATPase activity of myosin in vitro and content of MLCK in vivo (X±s, n=6). The assays were performed in the assay as described in Materials and Methods. (A1) was glycerol electrophoresis results of ardipusilloside-I on the extent of myosin phosphorylation in a Ca2+-dependent way. LC20, unphosphorylated MLC20; p-LC20, mono-phosphorylated MLC20; LC17, 17 kDa myosin essential light chain. Lane 0 was unphosphorylation control, lane 1 was phosphorylation of myosin without ardipusilloside-I administered, lane 2~5 represented phosphorylation of myosin after 12.5, 25, 50, 100 µM ardipusilloside-I administered. (A2) was the extent of myosin phosphorylation, which was analyzed by using Scion Image Software. Mono-phosphorylation was chosen as the control and calculated as 100%, and other data were the relative values compared to the control. **p<0.01 compared with phosphorylation without ardipusilloside-I. (B1) and (B2) were the Mg2+- ATPase activities of Ca2+-dependent phosphorylated myosin in the absence and in the presence of actin, respectively. The Mg2+-ATPase activity of unphosphorylated myosin was calculated as 100% in the absence of ardipusilloside-I and actin. Other data obtained were the relative values compared to it. *p<0.05, **p<0.01 compared with the corresponding controls without ardipusilloside-I. (C1) and (C2) Representative bands and statistical analysis (columns) of MLCK protein contents in the absence and presence of ardipusilloside-I (25, 50, 100 mg/kg). Data obtained from normal control (NC) was set to 100%. **p< 0.01 compared with the NC, ##p< 0.01 compared with the constipated rats of MC group.

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