SIGNALLING PATHWAYS INVOLVED IN THE HYDROGEN PEROXIDE-INDUCED VASOCONSTRICTION OF RAT INTRAMYOCARDIAL CORONARY ARTERIES

E Santiago2, C Contreras1, A García-Sacristán1, L Rivera1, B Climent1, D Prieto1. 1Universidad Complutense de Madrid, Departamento de Fisiologia, Facultad de Farmacia, 28040-Madrid, Spain, 2Hospital Universitario Puerta de Hierro-Majadohonda, Servicio de Farmacia Hospitalaria, Spain

Reactive oxygen species (ROS) are important second messenger molecules involved in multiple signaling pathways within the vascular wall under both physiological and pathophysiological conditions. Dysregulated ROS production and/or metabolism –oxidative stress- promote enhanced vasoconstriction, growth and inflammation associated to cardiovascular diseases such as hypertension, diabetes and atherosclerosis (Paravicini & Touyz, Diabetes Care 2: S170, 2008). In human and porcine coronary microvessels, H2O2 has been demonstrated to be an endothelium-derived hyperpolarizing factor (EDHF) released by flow (Liu et al., Circ Res 93:573, 2003). Since opposing vasoactive effects have been reported for H2O2 depending on the species, vascular bed and experimental conditions, the aim of the present study was to assess the mechanisms underlying H2O2 vasoactive effects in rat intramyocardial arteries in vitro.

Second- third-order branches of the left descending coronary artery from male Wistar rats were mounted on microvascular myographs and the effects endothelium removal and inhibitors of the cyclooxygenase and various kinase pathways were assessed on the H2O2-induced vasoactive effects. Simultaneous measurements of [Ca2+]i and tension were also performed to elucidate the Ca2+ signaling mechanisms of H2O2.

On coronary arteries precontracted with serotonin, H2O2 (1-300 µM) elicited further contractions (maximal response, Emax, of 19 ± 7%, n=8), whereas in mesenteric arteries this agent caused concentration-dependent relaxations (Emax 90 ± 2%, n=7). H2O2 also induced contractions on basal tension of coronary arteries whose magnitude was correlated to the vessel diameter. This contractile response was significantly enhanced by raising the extracellular K+ concentration to 30 mM (76 ± 9%, n=20, vs 18 ± 7%, n=8, of the response elicited by a high K+ solution (KPSS), on K30-precontracted and on basal tension arteries, respectively, p<0.0001) and blunted by catalase pretreatment. H2O2–elicited contractions were significantly reduced by endothelial cell removal (42 ± 12%, n=5, p< 0.01 vs control), by indomethacin (47 ± 11, n=8, p<0.001 vs control) and by the thromboxane receptor antagonist ICI192 (46 ± 7%, n=7; p<0.001 vs control). Furthermore, inhibition of the mitogen activated protein (MAP) kinase and of Rho kinase significantly reduced H2O2-evoked contractions to 41 ± 6% (n=8, p< 0.001 vs control) and to 22 ± 3% (n=5, p< 0.001 vs control), respectively. H2O2 (100 µM) elicited simultaneous sustained increases in [Ca2+]i and tension averaging 84 ± 14% and 72 ± 12% (n=4), respectively, of the responses elicited by KPSS.

The present data demonstrate that H2O2 is an endothelium-dependent vasoconstrictor in rat intramyocardial coronary arteries which activates various intracellular signaling pathways including mobilization of [Ca2+]i, release of a cyclooxygenase-derived prostanoid, probably TXA2, and stimulation of the MAP and Rho kinase pathways.

This work was supported by grant SAF 2009-10448 from MICINN