A Role For The Sodium Pump In Hydrogen Peroxide-Induced Relaxation In The Porcine Isolated Coronary Artery

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

Hydrogen peroxide (H2O2) has been proposed to act as an endothelium-derived hyperpolarising factor (EDHF) in human, rat, mouse, porcine and canine arteries (Shimokawa, 2010 Pflugers Arch 459(6):915-922, Wheal et. al., 2012 Eur J Pharmacol 674(2-3):384-390). In disease states, EDHF may act as a ‘back up’ system to compensate the loss of the nitric oxide pathway (Luksha et al., 2009 Atherosclerosis 202(2):330-344). Here, the mechanism of action of exogenous H2O2 in porcine coronary arteries (PCAs) isolated from female pigs and the role of H2O2 as an EDHF in the cardiovascular system were investigated. Distal PCAs were mounted in a wire myograph, pre-contracted with U46619 (2nM-50µM), a thromboxane A2 mimetic or KCl (60mM) to examine the role of H2O2 as an EDHF. Concentration-response curves to H2O2 (10nM-1mM), bradykinin (0.01nM-1µM), sodium nitroprusside (SNP) (10nM-10µM) or verapamil (1nM-10µM) were constructed. L-NAME (300µM) and indomethacin (10µM) were used to inhibit the synthesis of NO and prostanoids respectively. PEG-catalase (300Uml-1) was used to breakdown H2O2 and carbenoxolone (100µM), a gap junction inhibitor was used to study the role of gap junctions. Tetraethylammonium (TEA) (10mM) was used as a non-selective potassium (K+) channel inhibitor and glibenclamide (1µM) was used as an ATP-sensitive K+ channel inhibitor. Apamin (500nM), TRAM-34 (10µM) and iberiotoxin (100nM), small, intermediate and large-calcium activated K+ channels inhibitors respectively were used to examine the role of K+ channels in the H2O2–induced relaxation. Ouabain (500nM) was used to inhibit the sodium-potassium pump activity and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10µM) was used to selectively block guanylyl cyclase activity. Data were analysed using 2-tailed, paired Student’s t-test or one-way ANOVA, followed by Bonferroni’s post hoc test. H2O2 caused concentration-dependent relaxations with a maximum relaxation (Rmax) of 99.8±11.5% (mean±SEM), pEC50=4.17±0.15, n=4) and removal of endothelium (Rmax=97.8±3.5%,pEC50=3.96± 0.03,n=5) produced a comparable curve to the control supporting previous report that H2O2–induced relaxation is endothelium-independent. Addition of L-NAME and indomethacin (Rmax=87.5±2.7%, pEC50=7.6±0.05,n=5), in the absence or presence of carbenoxolone (Rmax=94.4±8.0%,pEC50=4.08± 0.10,n=4), ODQ (Rmax=127±25%,pEC50=3.7±0.2,n=4), glibenclamide (Relaxation at 1mM H2O2 (R)=97.7±2.1%,pEC50=4.1±0.04,n=6), barium (R=103.9± 3.8%,n=5), iberiotoxin (Rmax =119±16%, pEC50=3.8±0.2,n=4), TRAM-34 (Rmax=99.7±6.8%,pEC50 =3.8±0.07,n=6) or apamin (Rmax=105±6.9%, pEC50=3.9±0.06,n=6) had no effect on the H2O2–induced relaxation. TEA caused a 2.5-fold shift in the H2O2-induced relaxation (P<0.05) while high extracellular K+ (R=40.8±8.5%,n=5) or ouabain (R=47.5±8.6%,n=6) significantly inhibited the H2O2-induced relaxation (P<0.05). PEG-catalase significantly inhibited the relaxation at higher concentration of H2O2 (0.1–1.0mM) (P<0.05). Ouabain (Relaxation at 1µM of bradykinin R=8.88±3.31%,n=7) significantly inhibited the bradykinin-induced relaxation (P<0.05) in the presence (R=70.1±10.8%,n=7) and absence (R=100.7±0.5%, n=7) of L-NAME and indomethacin. Ouabain (n=6) caused a significant rightward shift (20-fold) in the concentration-response curve to SNP. Ouabain had no effect in the verapamil-induced relaxation (n=6). In conclusion, the relaxation induced by H2O2 is primarily mediated via ouabain-sensitive sodium-potassium pumps in the porcine coronary artery.