The P2Y1 receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin

Abstract

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y₁ receptor (P2Y₁R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y₁R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y₁R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y₁R protein expression. In addition, assays with the Ca²⁺ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca²⁺ modulation. P2Y₁R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y₁ receptor signaling.

Keywords: Endothelial cells; Inflammation; Melatonin; P2Y1 receptor; Purinergic signaling.

Fig. 1

Endothelial P2Y₁R activation stimulates leukocyte adhesion to mesenteric endothelial cells. Rat endothelial cells were left untreated (“basal” group) or were treated with the P2Y₁R agonist 2MeSATP (60 μM; black bar) for 4 h, followed by the addition of mononuclear cells. Alternatively, endothelial cells were pre-incubated with the P2Y₁R antagonist MRS2179 (0.3 μM) for 30 min before treatment with 2MeSATP (gray bar). Leukocyte adhesion to endothelial cells was estimated by direct counting by light microscopy. Data are expressed as mean ± SEM. N = 3 independent experiments performed in triplicates. ***P < 0.001 vs. 2MeSATP, by one-way ANOVA followed by Newman-Keuls test

Fig. 2

Melatonin inhibits P2Y₁R-mediated leukocyte adhesion to mesenteric endothelial cells. Rat endothelial cells were left untreated (basal group) or were treated with the P2Y₁R agonist 2MeSATP (60 μM, for 4 h), followed by the addition of mononuclear cells (black bar). Alternatively, endothelial cells were pre-incubated with melatonin (30 nM) for 30 min prior to treatment with 2MeSATP (in the presence of melatonin; gray bar). Mononuclear cell adhesion to endothelial cells was estimated by direct counting by light microscopy. Data are expressed as mean ± SEM. N = 7–9 replicates performed, with 2–3 independent experiments. **P < 0.01 vs. 2MeSATP, by one-way ANOVA followed by Newman-Keuls test

Fig. 3

Melatonin MT receptors mediate the inhibitory effect of melatonin on P2Y₁R-dependent leukocyte adhesion to mesenteric endothelial cells. Rat endothelial cells were left untreated (basal group) or were treated with the P2Y₁R agonist 2MeSATP (60 μM, for 4 h), followed by the addition of mononuclear cells (black bar). Alternatively, endothelial cells were pre-incubated with the MT receptor antagonist luzindole (10 μM (gray bar) or 30 μM (hatched gray bar)) for 30 min prior to incubation with melatonin (30 nM, in the presence of luzindole) for a further 30 min, and before treatment with 2MeSATP (4 h). Data are expressed as mean ± SEM. N = 7 replicates, from 3 independent experiments. ***P < 0.001 vs. 2MeSATP or melatonin plus 2MeSATP; *P < 0.05 vs. melatonin plus 2MeSATP, by one-way ANOVA followed by Newman-Keuls test

Fig. 4

Melatonin does not alter endothelial P2Y₁ receptor (P2Y₁R) expression. Western blotting analysis of P2Y₁R expression in endothelial cells. a P2Y₁R and β-actin protein expression in rat mesenteric endothelial cells (20 μg of protein/lane, in 10% SDS-PAGE gels). b Densitometry analysis of P2Y₁R bands (relative to β-actin) in blots (n = 3 independent experiments using three different cultures) from endothelial cells left untreated (basal (B1, B2, B3)) or treated with 30 nM melatonin (M1, M2, M3) for 4 h, showing that melatonin treatment did not alter P2Y₁R expression. Data are expressed as mean ± SEM (P = 0.78, by Student’s t-test)

Fig. 5

The P2Y₁ receptor-mediated TNF-α production by endothelial cells is inhibited by melatonin. Mesenteric endothelial cells were treated with 60 μM 2MeSATP (4 h; black bar) in the absence or presence of the P2Y₁R antagonist MRS2179 (0.3 μM; gray bar) or melatonin (30 nM; hatched gray bar), both added 30 min before the agonist. Alternatively, cells were left untreated (basal; white bar). Data are expressed as mean ± SEM. *P < 0.005 vs. 2MeSATP (by one-way ANOVA followed by Newman-Keuls test; n = 3–5 different cultures)