Microparticles induce pulmonary artery contraction through activation of acid sphingomyelinase and inhibition of Kv currents

Javier Moral-Sanz1,2, Bianca Barreira2, Laura Moreno1,2, Enrique Moreno2, Raffaella Soleti3, Daniel Morales-Cano1,2, Ramaroson Andriantsitohaina3, Angel Cogolludo1,2, Carmen Martinez3, Francisco Perez-Vizcaino1,2. 1Universidad Complutense, Pharmacology, Fac. Medicine, 28040 Madrid, Spain, 2Ciber Enferemedades Respiratorias, (Ciberes), Spain, 3INSERM UMR U1063, IBS-IRIS, Université d\'Angers F-49100, France

Microparticles (MPs) are membrane vesicles from damaged or activated cells which are associated with atherosclerosis, cancer and inflammatory and autoimmune diseases. Long term exposure to MPs induces vascular hyporeactivity in aortae via the Fas/FasL pathway (Agouni et al., 2011) and endothelial dysfunction in pulmonary arteries (PA, Tual-Chalot et al., 2010). We hypothesized that MPs have acute effects on pulmonary vascular contractility and Kv channel function.

Male Wistar rats (250-300 g) were anesthetized with ketamine/xilacine and killed by decapitation. Resistance PA (300-500 μm internal diameter) were dissected and PA myocytes were isolated by enzymatic digestion. Vascular reactivity was assessed using isometric wire myographs. The responses induced by MPs from apoptotic T lymphocytic cells (10 μg protein/ml) or the Fas L (10 μg/ml) were measured after 30 minutes and expressed as a percentage of an initial stimulation with KCl (80 mM). Some experiments were performed in the presence of nifedipine (1 μM), tiron (1 μM), an anticeramide antibody (200ng/mL) or inhibitors of the acid (D-609, 100 μM) or neutral sphingomyelinases (GW4869, 10μM). Potassium currents and membrane potential were recorded using the patch clamp technique in freshly isolated PASMC. Reactive oxygen species (ROS) were measured in human PASMC in culture incubated with dichlorofluorescein. Statistical analysis was carried out by ANOVA followed by Dunnett’s test and P<0.05 was considered statistically significant.

MPs reduced Kv current amplitude (37 ± 6% inhibition at +30 mV; n = 7; P<0.01) and produced membrane depolarization in PASMC (from -32 ± 2 to -20 ± 4 mV; n = 6; P<0.05). Moreover, MPs-induced a slow developing contraction in PA (8 ± 2% of KCl contraction after 30 min) that was blunted by the L-type Ca2+ channel blocker nifedipine (0.8 ± 2% KCl; n = 5; P<0.01). MPs-induced contraction was attenuated by the anticeramide antibody (2.4 ± 2.1%; n=7) and by D-609 (1.5 ± 1.7 %; n=6), but not by GW4869 (7.8 ± 1.7%); as compared to parallel controls (12 ± 2% KCl). Similar to MPs, FasL-induced contraction (14.5 ± 3.9%; n=7) was attenuated by the anticeramide antibody (1.6 ± 1.6%; n=7) and by D-609 (3.8 ± 2.2%; n=6), but not by GW4869 (7.2 ± 1.9%; n=4). MPs also increased ROS in human PASMC in culture and the contractile effect in PA was abolished by the ROS scavenger tiron (3 ± 1% vs 15 ± 3% in parallel control). MPs expressed FasL and PA expressed Fas as measured by Western blot.

In conclusion, MPs from apoptotic T lymphocytes produce pulmonary vasoconstriction involving the activation of Fas, acid sphingomyelinase, increase in ROS and inhibition of Kv channels.

Agouni et al., 2011. PLoS One. 2011;6(11):e27809. Epub 2011 Nov 15

Tual-Chalot et al., 2010. Am J Respir Crit Care Med. 2010 Jul 15;182(2):261-8.

Supported by SAF2010-22066, SAF2011- 28150.