Vasorelaxtion And Mechanism Of Trans-ferulic Acid On Rat Isolated Coronary Artery

Objective:(1) To observe the effects of different concentrations of trans-Ferulic acid(1~3mmol/L) in the resting tone of isolated rat coronary artery rings(RCAs).(2) To observe the relaxant and inhibitory effect of trans-Ferulic acid on the contraction induced by KCl, TXA 2 analogues U46619 and ET-1 in isolated RCAs.(3) To study the possible involvement of endothelium, potassium channels(4-AP、Gli、Ba Cl2 and TEA), NOS(L-NAME) and COX-2 prostoids synthesis inhibitor(Indo) in trans-Ferulic-acid induced vasorelaxation in isolated RCAs.(4) To study effect of trans-Ferulic acid on the rat coronary arterys contration induced either by intracellular calcium release or by extracellular calcium influx.Methods:(1) Procedures of isolated RCA rings: When the young male SD rats(200 ~ 250 g)were anesthesia and decapitated, the heart were take out and transferred immediately into chilled(4℃) Physiological saline solution(PSS) solution, fixed with some pins, bubbled with 100% O2, and keep p H7.40. The RCAs were blunt dissection by a dissecting microscope, cut into 2 mm-long rings, the inner diameter of 200~300 microns and mounted on a wire myograph(Multi Myograph System-610 M, DMT). The resting tone changes of different concentrations trans-Ferulic acid(1, 1.5, 2, 2.5, 3 mmol/L)induced in isolated RCAs were recorded by Power Lab and DMT systems. To facilitate the calculation,the maximal contraction induced by KCl(60 mmol/L)was taken as 100%.(2) KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L) were cumulatively added to the chamber to contraction the isolated RCAs. When the contraction curve returned to baseline, trans-Ferulic acid(1, 1.5, 2 mmol/L)were added to the chamber,incubation of 15~20 min,the maximal contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L)in the absence of trans-Ferulic acid was taken as 100%respectively and calculate the percentage of contraction by KCl(60 mmol/L), U46619(1μmol/L) or ET-1(0.01 μmol/L) with trans-Ferulic acid.(3) When the contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L) was sustained, trans-Ferulic acid(1, 1.5, 2, 2.5, 3 mmol/L) were added cumulatively to the endothelium-intact or denudation RCAs. The maximal contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L) in the absence of trans-Ferulic acid was taken as 100% and the percentage of contraction by each concentration of stimulator in the presence of trans-Ferulic acid was calculated.(4) When the contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L) was sustained, add four kinds of potassium channel blockers(TEA, Gli,Ba Cl2, 4-AP), NOS inhibitors(L-NAME) and COX-2 prostoids synthesis inhibitors(Indo).After 15 minites incubation, the contraction was sustained again, then add trans-Ferulic acid(1, 1.5, 2, 2.5, 3 mmol/L) accumulatively. The maximal contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L) in the absence of trans-Ferulic acid was taken as 100% respectively, calculate the percentage of diastolic after joining tool medicine.(5) When the contraction induced by KCl(60 mmol/L) or U46619(1 μmol/L)was sustained, we used the method of remove and recovery calcium to record the contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) and Ca Cl2(2.5 mmol/L) in absence or presence of trans-Ferulic acid.Results:(1) In the resting tone, trans-Ferulic acid(1, 1.5, 2, 1.5, 3 mmol/L) had no obvious effect on isolated RCA rings. The maximal contraction induced by KCl(60 mmol/L) was taken as 100%. With the increase of trans-Ferulic acid concentration, contraction percentage of isolated RCA rings in turn is(0.11 ± 0.02)%、(0.14 ± 0.09)%、(0.23 ±0.12)%、(0.37 ± 0.15)%、(0.58 ± 0.24)%. Compared with solvent control group, there was no significant difference(P > 0.05).(2) KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L) induced contraction of isolated RCA rings and the maximum contraction amplitude are respectively(2.69 ±0.16) m N,(4.48 ± 0.42) m N,(6.74 ± 0.35) m N. When the incubation concentration of trans-Ferulic acid was 1.5mmol/L, the contraction induced by KCl(60 mmol/L),U46619(1 μmol/L) or ET-1(0.01 μmol/L) had no significant difference compared with controlgroup(P>0.05). When the incubation concentration was 2mmol/L, the maximum contraction amplitude induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01μmol/L) was reduced by 50.12%, 43.38% and 36.25%, respectively. It has a statistically significant difference compared with solvent control group(P<0.05). When the incubation concentration was 2.5 mmol/L, the maximum contraction amplitude induced by KCl(60mmol/L),U46619(1 μmol/L) or ET-1(0.01 μmol/L) was reduced by 94.19%、95.97%�'�97.23%. Compared with solvent control group, has a significant difference(P < 0.05).(3) Trans-Ferulic acid(1, 1.5, 2, 1.5, 3 mmol/L) elicited concentration-dependent relaxation in endothelium-intact or –denudation isolated RCA rings, and endothelium has no relaxation of trans-Ferulic acid in isolated RCA rings. Trans-Ferulic acid could diastolic the contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L)on isolated RCA rings, The maximum relaxation rate is respectively(98.39 ± 2.92)%,(95.03 ± 2.86)%,(91.58 ± 2.10)%.(4) Pre-incubation with TEA, Gli, Ba Cl2, L-NAME or Indo, there have no effect on the relaxation of trans-Ferulic acid on isolated RCA rings, but with 4-AP attenuated the relaxation on KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L) by 16.36%,20.46%, 17.55%. Compared with the control group, they had statistically significant difference(P < 0.05).(5) Pre-incubation trans-Ferulic acid(1, 1.5, 2 mmol/L) can significantly inhibit calcium dependent contract induced by KCl(60 mmol/L) when the absence of calcium PSS liquid restore the calcium content, the maximum shrinkage rate is respectively(58.35± 1.61)%,(32.90 ± 1.63)%,(2.95 ± 0.59)%. Trans-Ferulic acid can inhibit U46619(1μmol/L) in the absence of calcium in PSS liquid caused by transient vasoconstriction(phasic contraction) and the contraction after calcium(tonic contraction), the maximum shrinkage rate is respectively(69.35 ± 1.52)%,(40.10 ± 1.02)%,(6.74 ± 0.36)%;(40.89 ±0.91)%,(13.74 ± 0.43)%,(5.43 ± 0.17)%.Conclusion:(1) Trans-Ferulic acid has no effect on resting tone of RCA, but inhibits the contraction induced by KCl(60 mmol/L), U46619(1 μmol/L) or ET-1(0.01 μmol/L).(2) Trans-Ferulic acid relaxes pre-contracted RCA in a concentration-dependent manner. Netiher endothium, NO synthesis nor COX-2 prostoids synthesis is involved in vasorelaxation induced by trans-Ferulic acid in RCAs.(3) The present results suggested that activation of Kv but neither KCa, KATP nor KIR and inhibition of both intracellular calcium release and extracellular calcium influx may be involved intran- Ferulic acid induced coronary vasorelaxtion.