The role of Transient Receptor Potential (TRP) channels in EDH-mediated vasorelaxation in Porcine Isolated Coronary Artery.

PS Wong, RE Roberts, MD Randall. University of Nottingham, Nottingham, UK

Recent studies have indicated that Transient Receptor Potential (TRP) channels could be a new target for pharmacological intervention for cardiovascular diseases (Earley & Brayden, Clin Sci (Lond) 119(1): 19-36, 2010). We have previously reported sex differences in endothelial function specifically in the EDH-mediated vasorelaxation in porcine isolated coronary arteries (PCAs) (Wong et. al., http://www.pa2online.org/abstract/abstract.jsp?abid=30846, 2012). Therefore, in this study, the effects of TRP channels inhibitors on PCAs from male and female pigs were compared.

Distal PCAs were mounted in a wire myograph and pre-contracted with U46619 (7nM-0.2µM), a thromboxane A2 mimetic. Concentration-response curves to bradykinin (0.01nM-1µM), an endothelium-dependent relaxant were constructed in the presence of various inhibitors. L-NAME (300µM) and indomethacin (10µM) were used to inhibit the synthesis of NO and prostanoids respectively. 2-Aminoethyl diphenylborinate (2-APB) (100µM) was used to inhibit TRPC and TRPM channels, whereas 1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF96365) (10µM) was used to inhibit only TRPC channels. 1-[4-[(2,3,3-Trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4carboxylic acid (Pyr3) (3µM) was used as a selective TRPC3 inhibitor. Rmax (maximum relaxation) and pEC50 values were analysed using 2-tailed, paired Student’s t-test, but for data where Rmax was not achieved, two-way ANOVA, followed by Bonferroni’s post hoc test was used.

The presence of 2-APB significantly inhibited bradykinin-induced vasorelaxation in PCAs from both male and female pigs. The Rmax was reduced from 103.5±4.4% (n=6) to Rmax=65.5±5.9% (n=6) in males and from Rmax=98.8±3.6% (n=6) to Rmax=75.4±5.6% (n=6) in females (both P<0.05, 2-tailed, paired Student’s t-test). In the presence of L-NAME and indomethacin, 2-APB essentially abolished the bradykinin-induced vasorelaxation in PCAs from both male and female pigs. SKF96365 in the absence of L-NAME had no effect on PCAs from female pigs but significantly inhibited the Rmax in males from Rmax=103.6± 4.3% (n=5) to Rmax=89.7±3.4% (n=5). In the presence of L-NAME and indomethacin, SKF96365 significantly inhibited Rmax from 95.0±3.5% (n=5) to Rmax of 69.7±3.0% (n=5) in males and from Rmax of 84.0±6.4% (n=10) to Rmax of 62.3±5.8% (n=10) in females. Pyr3 in the absence of L-NAME had no effect on the bradykinin-induced vasorelaxation in PCAs from female pigs but significantly shifted the EC50 4.2-fold to the right from pEC50=8.29±0.10 (n=10) to pEC50=7.67±0.06 (n=10) in male pigs. Similarly, in the presence of L-NAME, Pyr3 had no effect on the bradykinin-mediated response in PCAs from female pigs but significantly inhibited the bradykinin-induced vasorelaxation at 30nM bradykinin in male pigs (p<0.05, two-way ANOVA, followed by Bonferroni’s post hoc test).

In conclusion, the EDH-mediated vasorelaxation in PCAs from male and female pigs may involve TRPC and TRPM channels and that TRPC3 channels play a role in PCAs from male pigs but not in female pigs. Further work with more selective TRP channel inhibitors is required to determine the channel subtypes involved.