Abstract
Serotonin (5-HT) is a potent vasoconstrictor agonist and contributes to several vascular diseases including systemic or pulmonary hypertension and atherosclerosis. Although superoxide anion () is commonly associated to cellular damages due to overproduction, we previously demonstrated that, in physiological conditions, also participates to the 5-HT contraction in intrapulmonary arteries (IPA). Here, we focused on the signaling pathways leading to production in response to 5-HT in rat IPA. Using electron paramagnetic resonance on rat IPA, we showed that 5-HT (100 μM)-induced production was inhibited by ketanserin (1 μM—an inhibitor of the 5-HT2 receptor), absence of extracellular calcium, two blockers of voltage-independent calcium permeable channels (RHC80267 50 μM and LOE-908 10 μM) and a blocker of the mitochondrial complex I (rotenone—100 nM). Depletion of calcium from the sarcoplasmic reticulum or nicardipine (1 μM—an inhibitor of the L-type voltage-dependent calcium channel) had no effect on the 5-HT-induced production. levels were also increased by α-methyl-5-HT (10 μM—a 5-HT2 receptors agonist) whereas GR127935 (1 μM—an antagonist of the 5-HT1B/D receptor) and citalopram (1 μM—a 5-HT transporter inhibitor) had no effect on the 5-HT-induced production. Peroxynitrites were increased in response to 5-HT (100 μM). In isolated pulmonary arterial smooth muscle cells loaded with rhod-2 or mitosox probes, we respectively showed that 5-HT increased both mitochondrial calcium and levels, which were both abrogated in absence of extracellular calcium. Mitochondrial levels were also abolished in the presence of rotenone (100 nM). In pulmonary arterial smooth muscle cells loaded with TMRM, we showed that 5-HT transiently depolarized the mitochondrial membrane whereas in the absence of extracellular calcium the mitochondrial membrane depolarisation was delayed and sustained in response to 5-HT. 5-HT decreased the mitochondrial respiratory rate measured with a Clark oxygen electrode. Altogether, in physiological conditions, 5-HT acts on 5-HT2 receptors and induces an production dependent on extracellular calcium and mitochondria.
Highlights
Several agonists acting on seven transmembrane domain receptors (G-protein coupled receptors) are involved in vascular tone
In rat intrapulmonary arteries (IPA), that 5-HT increases superoxide anion (O2) levels in smooth muscle and such O2 participates to pulmonary vasoconstriction (Billaud et al, 2009)
Given the importance of calcium in the contraction to 5-HT in rat IPA, we have studied the role of intracellular calcium by depleting the calcium from the sarcoplasmic reticulum with a calcium free Krebs– HEPES–bicarbonate (KHB) solution containing a calcium chelator (0.4 mM EGTA), an activator of ryanodine receptors and an inhibitor of the sarcoplasmic reticulum Ca2+ATPase to prevent calcium re-uptake in the sarcoplasmic reticulum
Summary
Several agonists acting on seven transmembrane domain receptors (G-protein coupled receptors) are involved in vascular tone. The mitogenic effect of 5-HT in the pulmonary artery (PA) is dependent on 5-HT uptake through the 5-HT transporter (Maclean and Dempsie, 2010) and the activation of 5HT receptors whereas the contractile effect of 5-HT is mainly due to its action on 5-HT2A and 5-HT1B/D receptors (Rodat-Despoix et al, 2008). Since the mitochondrial electron transport chain (METC) is known to be sensitive to calcium to produce O2 (Denton et al, 1978; Archer et al, 1993; Yuan et al, 1993; Leach et al, 2001; Dromparis and Michelakis, 2013; Freund-Michel et al, 2014; Yumnam et al, 2016) we have investigated, in the present study, the signaling pathways involved in 5-HT-induced O2 increase in rat IPA by focusing on the mitochondria as an important source of ROS and the role of intra and extracellular calcium
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