Lysophosphatidylinositol (LPI) mediates vasorelaxation of the rat mesenteric resistance artery and induces calcium release in rat mesenteric artery endothelial cells

Yousuf M Al Suleimani, C Robin Hiley. Department of Pharmacology, University of Cambridge, CB2 1PD Cambridge, United Kingdom.

Lysophosphatidylinositol (LPI) is a novel cell membrane-derived lipid signalling molecule that can activate the orphan G protein-coupled receptor, GPR55, and initiate cellular responses via distinct intracellular pathways (Brown & Hiley, 2009). In human endothelial cells, LPI has been reported to induce elevation of intracellular Ca2+ via activation of GPR55 (Oka et al., 2007). Here we examine the actions of LPI in the rat resistance mesenteric artery as well as the Ca2+ responses in endothelial cells isolated from it.

Vascular responses were assessed in third order branches of rat (Wistar males 250-350g) superior mesenteric artery, preconstricted with methoxamine, using a wire myograph (see White & Hiley, 1997). For endothelial cell isolation, rats were killed with a pentobarbital overdose and a cannula placed in the superior mesenteric artery at its aortic origin. The mesentery was removed and placed in PBS at 37°C and its vasculature perfused with PBS at 1ml min-1 for 15 min at 37°C. This was followed by perfusion with collagenase type A (0.2%; 1ml min-1; 37°C) for 40 min. Perfusate was collected every 10 min and centrifuged at 1200g for 10 min. The pellet was resuspended in EBM-2 medium supplemented with growth factors. Isolated cells were plated on coverslips coated with 0.5% gelatin and incubated for 3-5days, then they were used for Ca2+ imaging. The cells were incubated for 1 h with fura-2AM (2 μM in HBS; 20°C), then washed and incubated for a further 30 min in HBS. Single-cell fluorescence imaging was performed using a MetaFluor system (Rosker et al., 2009).

LPI produced concentration- and endothelium-dependent vasorelaxation (with endothelium: n = 10, relaxation at 10 μM [R10μM], 68.1±4.5%; without: n = 6, R10μM , 13.2±4.2%; P<0.01). Indomethacin (10 μM) had no significant effect on the relaxation (with indomethacin: R10μM , 69.3±6.3%, n = 6; without: R10μM , 66.8±8.9%, n = 4). Precontraction with 60 mM KCl abolished the response and it was markedly reduced by charybdotoxin (50 nM), iberiotoxin (50 nM) or a combination of apamin+charybdotoxin (both 50 nM) but not by apamin alone (R10μM, charybdotoxin: 24.1±7.7%, n = 4; charybdotoxin+apamin: 27.1±8.1%, n = 4; iberiotoxin: 43.4±8.6%, n = 4; P<0.01). The CB1 receptor antagonist AM251 (10 μM) did not affect relaxation to LPI, whereas two antagonists at a putative endothelial “anandamide receptor”, rimonabant (3 μM) and O-1918 (10 μM; Offertáler et al., 2003), significantly potentiated LPI responses (rimonabant: R10μM, 89.7±4.5%, n = 7; O-1918: R10μM, 92.9±1.7%, n = 4; P<0.01). In isolated endothelial cells, LPI (10 μM) caused biphasic elevations in intracellular Ca2+, with an initial rapid (20s after agonist stimulation) phase lasting for 100s and a later phase (observed at 250s after the first). The responses were independent of extracellular Ca2+. Pretreatment with thapsigargin (1 μM) or 2-APB (100 μM) abolished both phases. The PLC inhibitor U73122 (10 μM) attenuated the initial phase and enhanced the later one, whereas the ROCK inhibitor Y27632 (50 μM) failed to inhibit the fast phase but completely abolished the slow phase.

In conclusion, LPI is an endothelium-dependent vasodilator in the rat small mesenteric artery. The response involves activation of Ca2+-sensitive K+ channels (KCa) (mainly IKca and BKca), and is not mediated by CB1 receptors, but is enhanced by blockade of endothelial “anandamide” receptors. In endothelial cells from the mesenteric artery, LPI utilizes both PLC-IP3 and ROCK-RhoA pathways to elevate intracellular Ca2+ which may account for its observed vasodilator effect. Overall, these findings support the existence of a novel endothelial site where LPI mediates its action.

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