Serotonin-induced production of superoxide anion in rat intrapulmonary arteries: A new physiological role for mitochondria

Abstract

Introduction We have investigated signalling pathways implicated in the 5-HT-induced contraction dependent on O2 • production by focusing on: – sources of reactive oxygen species, especially, NADPH oxidases (Nox) and mitochondria; – caveoli. Methods Experiments were conducted on IPA and pulmonary arterial smooth muscle cells (PA-SMC) of male Wistar rats. O2 • levels were measured by electron paramagnetic resonance (EPR) in rat IPA. Cytosolic and mitochondrial Ca2+ levels were assessed in cultured pulmonary arterial smooth muscle cells (PA-SMC) with Fluo-4 (2 μM) and Rhod-2 (1 μM) respectively. Isometric contractions were recorded on IPA rings with an organ bath system. Immunofluorescent labellings of 5-HT2A receptors, caveolin 1 (Cav 1) and Cx 43 were performed on transversal sections of rat IPA as well as en face view of internal elastic lamina of opened IPA and visualised by confocal microscopy. Results With electron paramagnetic resonance on rat IPA, we showed that 5-HT-induced O2 • production was inhibited by ketanserin, an inhibitor of the 5-HT2A receptor, absence of extracellular calcium, two blockers of voltage-independent calcium permeable channels (RHC80267 and LOE-908), a blocker of the mitochondrial complex I (rotenone), a Nox inhibitor (apocynin) and a blocking peptide of PKCɛ. We demonstrated that, 5-HT increased mitochondrial [Ca2+], depolarized mitochondria and decreased mitochondrial respiratory rate. Fluorescent immunolabeling on transversal sections and whole IPA highlighted the localization of 5-HT2A receptors and caveolin-1, specific protein of caveoli, in smooth muscle, endothelium and fenestrae of the internal elastic lamina. Moreover, disruption of caveoli with methyl-β-cyclodextrin, significantly decreased the contraction to 5-HT. Conclusion We have demonstrated that 5-HT-induced specific production of O2 • is dependent on Nox and mitochondria under physiological conditions. As O2 • is often overproduced in cardiovascular diseases, such process may be important in the development of pulmonary hypertension.