Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries

Abstract

The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): RHC 802675 2.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control 2.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(2+)-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.

Fig. 1.

A: extracellular Ca^{2+ $\left({\text{Ca}}_{\text{e}}^{2+}\right)$}-induced relaxation of isolated, endothelium-replete (■) and endothelium-denuded (□) phenylephrine (PE)-contracted mesenteric arteries. B: acetylcholine (ACh)-induced relaxation of endothelium-replete (●) and endothelium-denuded (○) arteries precontracted with 5 μM PE. Values are means ± SE of 5 experiments. *,**Significantly different from endothelium replete.

Fig. 2.

Effect of the diacylglycerol (DAG) lipase (DAGL) inhibitor RHC 80267 on Ca²⁺-induced relaxation of isolated, PE-contracted mesenteric arteries. Arteries were mounted on a wire myograph in physiological salt solution (PSS) containing 1 mM Ca²⁺ and equilibrated for 30 min at 37°C with constant aeration. Relaxations were induced by increasing concentrations of ${\text{Ca}}_{\text{e}}^{2+}$ and were measured in the presence (▲) and absence (●) of 5 μM RHC 80267 and after washout (○) of the inhibitor. Values are means ± SE of 5 experiments. *,**Significantly different from control.

Fig. 3.

Effects of miconazole and quinacrine on Ca²⁺-induced relaxation of isolated, PE-contracted mesenteric arteries. Arteries were mounted on a wire myograph in PSS containing 1 mM Ca²⁺ and equilibrated for 30 min at 37°C with constant aeration. Relaxations to increasing concentrations of ${\text{Ca}}_{\text{e}}^{2+}$ were measured in the absence (●) or presence of 5 μM miconazole (■) or 5 μM quinacrine (○). Values are means ± SE of 5 experiments. *,**Significantly different from quinacrine and miconazole.

Fig. 4.

Effect of the specific phospholipase A2 (PLA2) inhibitor arachidonyltrifluoromethyl ketone (AACOCF₃) on Ca²⁺-induced relaxation of PE-contracted mesenteric artery. A: force tracings showing typical relaxation of vessel in the absence (Control) or presence of 1 μM AACOCF3. B: histogram showing inhibition of Ca²⁺-induced relaxation of mesenteric arteries by AACOCF3. Data are means ± SE of 4 separate experiments. *Significantly different from PE tension; #significantly different from control (P < 0.05).

Fig. 5.

A: force tracings showing the effect of graded concentrations of SR-141716A on Ca²⁺-induced relaxation of a PE-contracted mesenteric artery. B: histogram showing the concentration-dependent inhibition of Ca²⁺-induced relaxation of mesenteric arteries by SR-141716A, a CB1 receptor antagonist. Data are means ± SE of 4 separate experiments. *Significantly different from PE tension; #significantly different from controls (P < 0.05).

Fig. 6.

A: force tracings showing relaxation of an isolated, PE-contracted mesenteric artery segment to graded concentrations of Ca²⁺ and 2-arachidonoylglycerol (2-AG). B: effect of 100 nM iberiotoxin (IBTX) on 2-AG-induced relaxation of isolated, PE-precontracted mesenteric arteries. IBTX shifted the 2-AG concentration-effect curve to the right. C: i: force tracings showing the effect of 1 μM miconazole on 2-AG-induced relaxation. ii: Histogram showing the inhibition of relaxation by miconazole. Values are means ± SE of 4 separate experiments. *Significantly different from control (P < 0.05).

Fig. 7.

A: glycerated epoxyeicosatrienoic acid (GEET)-induced relaxation of isolated, PE-contracted mesenteric arteries mounted in a wire myograph in PSS containing 1 mM Ca²⁺ and equilibrated for 30 min at 37°C with constant aeration and responses to GEET isomers determined. B: effect of the Ca²⁺-activated K⁺ (KCa) channel inhibitor IBTX on 11,12-GEET-induced relaxation of isolated, PE-contracted mesenteric arteries incubated with 100 nM IBTX for 10 min before cumulative additions of 11,12-GEET. Values are means ± SE of 5 experiments. *Significantly different from control (P < 0.05).

Fig. 8.

Effects of the CB1 cannabinoid receptor antagonist SR-141716A and IBTX on 14,15-GEET-induced relaxation of isolated, PE-contracted mesenteric arteries. Arteries were mounted in a wire myograph in PSS containing 1 mM Ca²⁺ and equilibrated for 30 min at 37°C with constant aeration. Responses to 14,15-GEET (●) or 14,15-GEET in the presence of 1 μM SR-141716A (○) or 1 μM IBTX (▲) were determined. Values are means ± SE of 6 experiments. *Significantly different from control (P < 0.05).

Fig. 9.

Proposed model for perivascular nerve Ca²⁺-sensing receptor (CaR)-mediated vasodilator release. Agonist activation of the CaR at the nerve terminal leads to receptor coupling to Gαq and activation of phospholipase C (PLC) and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 then binds to its receptor (IP3R) in the endoplasmic reticulum (ER) membrane to release Ca²⁺ into the cytoplasm. Released Ca²⁺ then activates 1) Ca²⁺-sensitive N-acetyl transferase (N-AT) to generate anandamide (AEA) from phosphatidylcholine (PC) and phosphatidylethanolmine (PE) through N-arachidonoylphosphatidylethanolamine (NAPE) and 2) Ca²⁺-sensitive PLA2 to generate arachidonic acid (AA), a substrate for cytochrome P-450 (CYP), from AEA. DAG, on the other hand is metabolized by DAGL to give 2-AG, which in turn is metabolized by PLA2 to give AA and/or by CYP to give GEET. The AA produced is metabolized by CYP to give EET. Both EET and GEET then cross the plasma membrane to the adjacent smooth muscle layer to activate KCa channels, resulting in hyperpolarization of the muscle cells and vascular relaxation. AEA can also bind to the CB1 receptor (CB1R) or an AEA receptor in the plasma membrane to activate the cannabinoid pathway and cause relaxation.