The GPR55 agonist lysophosphatidylinositol relaxes rat mesenteric resistance artery and induces Ca(2+) release in rat mesenteric artery endothelial cells

Abstract

Background and purpose: Lysophosphatidylinositol (LPI), a lipid signalling molecule, activates GPR55 and elevates intracellular Ca(2+). Here, we examine the actions of LPI in the rat resistance mesenteric artery and Ca(2+) responses in endothelial cells isolated from the artery.

Experimental approach: Vascular responses were studied using wire myographs. Single-cell fluorescence imaging was performed using a MetaFluor system. Hypotensive effects of LPI were assessed using a Biopac system.

Key results: In isolated arteries, LPI-induced vasorelaxation was concentration- and endothelium-dependent and inhibited by CID 16020046, a GPR55 antagonist. The CB1 receptor antagonist AM 251 had no effect, whereas rimonabant and O-1918 significantly potentiated LPI responses. Vasorelaxation was reduced by charybdotoxin and iberiotoxin, alone or combined. LPI decreased systemic arterial pressure. GPR55 is expressed in rat mesenteric artery. LPI caused biphasic elevations of endothelial cell intracellular Ca(2+). Pretreatment with thapsigargin or 2-aminoethoxydiphenyl borate abolished both phases. The PLC inhibitor U73122 attenuated the initial phase and enhanced the second phase, whereas the Rho-associated kinase inhibitor Y-27632 abolished the late phase but not the early phase.

Conclusions and implications: LPI is an endothelium-dependent vasodilator in the rat small mesenteric artery and a hypotensive agent. The vascular response involves activation of Ca(2+)-sensitive K(+) channels and is not mediated by CB1 receptors, but unexpectedly enhanced by antagonists of the 'endothelial anandamide' receptor. In endothelial cells, LPI utilizes PLC-IP3 and perhaps ROCK-RhoA pathways to elevate intracellular Ca(2+). Overall, these findings support an endothelial site of action for LPI and suggest a possible role for GPR55 in vasculature.

Figure 1

LPI-induced vasorelaxation of the rat small mesenteric artery precontracted with 10 μM methoxamine (Meth) in the presence and absence of the endothelium. (A) Relaxation was determined in the presence (n = 10) and absence (n = 6) of endothelium. Values are shown as means ± SEM. (B, C) Original traces demonstrating LPI-induced relaxation of rat small mesenteric arteries precontracted with methoxamine. (A) shows a vessel with an intact endothelium and (B) represents an endothelium-denuded vessel. Vertical lines denote addition of drugs at the concentrations indicated.

Figure 2

Concentration-dependent relaxation to LPI of methoxamine-induced tone in rat mesenteric resistance arteries. Responses were determined in the presence of functional endothelium. (A) Vessels were relaxed by LPI alone (n = 4), or in the presence of 10 μM indomethacin (n = 6). (B) Vessels were relaxed by LPI in the absence (n = 10) or presence (n = 4) of ODQ. Data are presented as means + SEM.

Figure 3

Role of K⁺ channels in LPI-induced vasorelaxation. (A) Relaxation by LPI of either methoxamine- or KCl-induced tone in rat isolated small mesenteric arteries with an intact endothelium. Tone was induced by either 10 μM methoxamine (n = 7) or 60 mM KCl (n = 6). (B) Relaxation was produced by LPI either alone (n = 7) or in the presence of either apamin (n = 4), charybdotoxin (n = 4), apamin plus charybdotoxin (n = 4), iberiotoxin (n = 4) or TRAM 34 (n = 5). (C) Relaxation was elicited by LPI alone (n = 6), or in the presence of 4-aminopyridine (n = 6) or glibenclamide (n = 4). **P < 0.01, significantly different from control; one-way anova with Bonferroni's post hoc test. Data are presented as means ± SEM.

Figure 4

Relaxation by LPI of methoxamine-induced tone in rat small mesenteric arteries. All responses were determined in the presence of functional endothelium. (A) Vasorelaxation was elicited by LPI either alone (n = 10), or in the presence of AM 251 (n = 5). (B) Vasorelaxation was elicited by LPI alone (n = 10), or in the presence of either O-1918 (n = 4) or rimonabant (n = 8). (C) Vessels were relaxed by LPI alone (n = 4), or in the presence of 2.5 μM of the GPR55 antagonist CID 16020046 (n = 4). Data are presented as means + SEM. In (A) and (B), *P < 0.05, significantly different from control; two-way anova with Bonferroni's post hoc test. In (C), ***P < 0.001, significantly different from control; two-tailed Student's t-test.

Figure 5

Involvement of the PLC-IP₃ and RhoA-ROCK pathways in LPI-induced vasorelaxation of rat mesenteric resistance arteries. Vessels were precontracted with U46619 (3 μM) alone or in combination with methoxamine (10 μM). Relaxation was elicited by LPI alone (n = 6), or in the presence of U73122 (n = 8), 2-APB (n = 7) or Y-27632 (n = 5). *P < 0.05, significantly different from control; one-way anova with Dunnett's multiple comparison test. Data are shown as means + SEM.

Figure 6

Ca²⁺ signals evoked by 10 μM LPI in endothelial cells isolated from the rat mesenteric arterial bed. Responses were obtained in Ca²⁺-free HBS. (A) A representative recording from a single cell showing the characteristic biphasic Ca²⁺ mobilization. (B) Images of the endothelial cells at 40× while being recorded for Ca²⁺ signalling using the MetaFluor system. Blue indicates basal calcium levels, whereas green and red demonstrate the increase in cytosolic Ca²⁺. Zero seconds represents the point where LPI was added.

Figure 7

Ca²⁺ signals evoked by LPI in endothelial cells isolated from the rat mesenteric arterial bed. Responses were recorded in Ca²⁺-free HBS. (A) The initial rapid phase (control, n = 27) which was abolished by thapsigargin (n = 15), 2-APB (n = 14) and U73122 (n = 39). The response was also significantly reduced by Y-27632 (n = 43). The late slow phase (control, n = 42) as shown in (B) was attenuated by thapsigargin (n = 31), 2-APB (n = 14) and Y-27632 (n = 49), whereas U73122 (n = 48) did not reduce the peak response. (C) The time to peak of the late phase was significantly shortened by U73122 (control, n = 28; U73122, n = 39). Drugs were pre-incubated for 10–15 min before the addition of LPI and were present throughout the period of the experiment. n indicates the number of cells obtained from two to three independent isolations. Data are presented as means + SEM. In A and B, ***P < 0.001, significantly different from control; one-way anova with Bonferroni's post hoc test. In C, ***P < 0.001, significantly different from control; unpaired two-tailed t-test.

Figure 8

Expression of mRNA transcripts for GPR55, CB₁ and CB₂ receptors in the rat mesenteric artery. (A) Representative real-time PCR measurement of the mRNA levels in the rat mesenteric artery for a housekeeping gene (GAPDH), and for GPR55, the CB₁ and the CB₂ receptors. Each curve is an average of duplicate readings from the same isolation. The dashed lines represent samples where reverse transcriptase was omitted (negative control). (B) Real-time PCR products of specific gene fragments from two separate samples of mRNA isolated from mesenteric artery (M) and testis (T). The expected sizes of amplicons were 341 bp for rat GPR55, 357 bp for the rat CB₁ receptor, 275 bp for the rat CB₂ receptor and 307 bp for rat GAPDH. Also shown are negative control samples where RNA was not reverse-transcribed to cDNA. (C) Quantified mRNA expression levels in both rat mesenteric artery and testis. Values are presented as mean expression, relative to the housekeeping gene, + SEM.