Chlorogenic Acid Relaxes Rat Ileal Smooth Muscles by Mechanisms Independent of Rho Kinase Mediated Effects on Myosin Light Chain Phosphorylation

Abstract

Chlorogenic acid (CGA) is a polyphenolic compound found in fruits and vegetables and generally known to have antioxidant, anti-inflammatory and antithrombotic effects. Studies have shown that CGA induces nitric oxide-sGC-PKG pathway to cause relaxation of the vascular smooth muscles. But the effects of CGA on ileal smooth muscles are still unknown. In our previous experiments, we have shown that CGA increases basal tone with no effect on relaxation of rat ileal smooth muscles when they are depolarized by 140 mM potassium kreb's buffer. The present study is designed to evaluate if CGA acts through the rho-kinase mediated pathway in relaxing rat ileal smooth muscles when challenged with acetylcholine. Effects of CGA (10 μM) on basal tension and maximum contractile tension induced by acetylcholine (0.5μM) in isolated rat ileal rings (3mm) were determined using tissue bath apparatus. CGA effects on myosin light chain phosphorylation at maximum contractile tension in response to acetylcholine (0.5 μM) were also measured by freezing the ileal rings and extracting the protein and running Western blots. The role of rho-kinase in regulating smooth muscle relaxation is tested by incubating ileal rings with rho-kinase inhibitor Y-27632 (1 μM) and measuring the basal tension, maximum contractile tension and myosin light chain phosphorylation as described above. The results show that basal tension increases from 100% (control group) to 130.8 ± 19.0% and 121.7 ± 30.6 % when treated with CGA alone and the combination of CGA and Y-27632 respectively. The peak contractile tension in response acetylcholine changes from 100% (control group) to 50.4 ± 12.13 % and 55 ± 4.05 % when treated with CGA alone and the combination of CGA and Y-27632 respectively. The results from Western blots show that myosin light chain phosphorylation didn't change significantly between CGA (41.8 ± 5.5%) and the combination of CGA and Y-27632 (37.7 ± 3.7%) treatments. These results suggest that CGA alone and in combination with rho-kinase inhibitor is involved in increasing the basal tone of ileal smooth muscles when compared with the control group. In contrast, CGA significantly decreased the acetylcholine induced peak contractile tension both in presence and absence of rho-kinase inhibitor with out any corresponding changes in myosin light chain phosphorylation. Therefore, we conclude that CGA relaxes ileal smooth muscles by mechanisms independent of rho-kinase mediated effects on myosin light chain phosphorylation. Further studies on effects of CGA on activity of soluble guanylate cyclase and phosphodiesterase activity may provide some insights on mechanism of action of CGA on smooth muscles.