Mechanism of Relaxation to Antimycin A, Mitochondrial Complex III inhibitor, in the Porcine Coronary Artery

HM Almukhtar, PA Smith, RE Roberts. University Of Nottingham, Nottingham, UK

Traditionally mitochondria are known as the energy generating centre of the cells. However, there is growing consensus that mitochondria actively participate in intracellular signalling, such as production of reactive oxygen species (ROS) and regulation of the intracellular Ca2+ concentration (1). Moreover, ROS could play an important supportive role in a variety of vascular cell signalling processes, including activation of nitric oxide synthase (NOS), modulation of intracellular Ca2+, and AMP kinase activation (2). We have previously demonstrated that antimycin A, a complex III inhibitor, causes acute relaxation of the precontracted porcine coronary artery (3). However, the mechanism of this relaxation remains to be elucidated.

Proximal coronary artery segments were isolated from porcine hearts obtained from a local abattoir and set up in Krebs-Henseleit buffer (NaCl 118, KCl 4.8, CaCl2.H2O 1.3, NaHCO3 25.0, KH2PO4 1.2, MgSO4.7H2O, glucose 11.1 (in mM) in isolated tissue baths for isometric tension recording. Endothelium-intact segments were precontracted with the thromboxane mimetic U-46619 or KCl, in the presence or absence of L-NAME (a nitric oxide synthase inhibitor, 300µM), TEA (a non-selective K+ channel inhibitor, 10mM), 4-AP (a voltage-dependent K+ channel blocker, 3mM), or dorsomorphin (an AMPK inhibitor, 10µM). The role of ROS in the relaxation response was investigated by pre-incubation with the hydrogen peroxide scavenger (ebselen, 3µM), the superoxide dismutase inhibitor (DETCA, 10mM), or the mitochondrial complex I inhibitor (rotenone, 10µM). To assess the effect of antimycin on Ca2+ influx, concentration–response curves to CaCl2 were constructed in calcium-free, high potassium Krebs’ solution in the absence or presence of antimycin A (10µM).

L-NAME, TEA and 4-AP had no effect on the antimycin A-induced relaxation, indicating that NO and K+ channels are not involved. Dorsomorphin significantly attenuated the relaxation response (31±4% relaxation after 120 mins) compared to control (63.6±9.3% relaxation; mean ± SEM from 10 different animals; P <0.001 (2- way ANOVA followed by Bonferroni post-hoc test)). Both ebselen (30.0±7.3% relaxation, n=6) and DETCA (19.9±16.6% relaxation, n=7) produced significant inhibition of the relaxation response (p<0.005, 2-way ANOVA). Similarly, rotenone abolished the antimycin A-induced dilation (15.2±5.6% relaxation, n=6) (p<0.001, 2-way ANOVA). Pre-incubation with antimycin A also inhibited the calcium-induced contractile responses (antimycin Rmax = 20.5±8.3 compared to control Rmax = 41.61±13.44, n=10; P <0.001, 2- way ANOVA). Collectively, this study demonstrates that antimycin A acutely dilates coronary resistance vessels via ROS generation at mitochondrial complex III and inhibition of extracellular Ca2+ influx. ROS may activate AMP kinase, which in turn regulates Ca2+- influx, although future studies are required to determine if this is the case.

(1) Michelakis et al. (2002). Circ Res 90: 1307-15

(2) Cardaci et al. (2012). J Cell Sci 125: 2115-25

(3) Almukhtar HM et al. (2012) pA2 online