Systemic administration of the G-protein-coupled receptor 18 (GPR18) agonist abnormal cannabidiol (Abn CBD) lowers blood pressure (BP). Whether GPR18 is expressed in the central nervous system (CNS) and plays a role in BP control is not known despite the abundance of the GPR18 ligand N-arachidonoyl glycine (NAGly) in the CNS. Therefore, we first determined whether GPR18 is expressed in the presympathetic tyrosine hydroxylase (TH) immunoreactive (ir) neurons of the brainstem cardiovascular regulatory nuclei. Second, we investigated the impact of GPR18 activation and blockade on BP and heart rate (HR) and neurochemical modulators of sympathetic activity and BP. Immunofluorescence findings revealed GPR18 expression in TH-ir neurons in the rostral ventrolateral medulla (RVLM). Intra-RVLM GPR18 activation (Abn CBD) and blockade (O-1918, 1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-,cyclohexen-1-yl] benzene) elicited dose-dependent reductions and elevations in BP, respectively, along with respective increases and decreases in HR in conscious male Sprague-Dawley rats. RVLM GPR18 activation increased neuronal adiponectin (ADN) and NO and reduced reactive oxygen species (ROS) levels, and GPR18 blockade reduced neuronal ADN and increased oxidative stress (i.e., ROS) in the RVLM. Finally, we hypothesized that the negligible hypotensive effect caused by the endogenous GPR18 ligand NAGly could be due to concurrent activation of CB 1 R in the RVLM. Our findings support this hypothesis because NAGly-evoked hypotension was doubled after RVLM CB 1 R blockade (SR141716, rimonabant). These findings are the first to demonstrate GPR18 expression in the RVLM and to suggest a sympathoinhibitory role for this receptor. The findings yield new insight into the role of a novel cannabinoid receptor (GPR18) in central BP control.

Direct activation of the endocannabinoid receptor G protein-coupled receptor 18 (GPR18) in the rostral ventrolateral medulla (RVLM) of conscious rats by abnormal cannabidiol (Abn CBD; trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) elevates local nitric oxide (NO) and adiponectin (ADN) levels and reduces oxidative stress and blood pressure (BP). However, the molecular mechanisms for GPR18-mediated neurochemical responses, including the nitric oxide synthase isoform that generates NO, and their potential causal link to the BP reduction are not known. We hypothesized that GPR18-mediated enhancement of Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and neuronal nitric oxide synthase (nNOS) phosphorylation, triggered by a reduction in cAMP, accounts for the NO/ADN-dependent reductions in RVLM oxidative stress and BP. Intra-RVLM GPR18 activation (Abn CBD; 0.4 mg) enhanced RVLM Akt, ERK1/2, and nNOS phosphorylation as well as ADN levels during the hypotensive response. Prior GPR18 blockade with O-1918 (1,3-dimethoxy-5-methyl-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]benzene) produced the opposite effects and abrogated Abn CBD-evoked neurochemical and BP responses. Pharmacological inhibition of RVLM phosphoinositide 3-kinase (PI3K)/Akt (wortmannin), ERK1/2 (PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one]), or nNOS (N v -propyl-Larginine), or activation of adenylyl cyclase (forskolin) virtually abolished intra-RVLM Abn CBD-evoked hypotension and the increases in Akt, ERK1/2, and nNOS phosphorylation and in ADN levels in the RVLM. Our pharmacological and neurochemical findings support a pivotal role for PI3K, Akt, ERK1/2, nNOS, and adenylyl cyclase, via modulation of NO, ADN, and cAMP levels, in GPR18 regulation of the RVLM redox state and BP in conscious rats.

Background and objectivesThe abnormal cannabidiol (Abn CBD) receptor GPR18 mediates peripheral vasodilatation. N‐arachidonoyl glycine (NAGly) is the endogenous ligand of GPR18. Despite NAGly's abundance in the CNS and GPR18 mediated peripheral vasodilation, there are no studies on the role of central GPR18 in blood pressure control. Therefore, we investigated if GPR18 is: (i) present in the brainstem cardiovascular regulatory nuclei, particularly in presympathetic tyrosine hydroxylase (TH) expressing neurons; (ii) co‐expressed with Cannabinoid 1 receptors (CB1R) in those neurons and, (iii) the impact of GPR18 activation and blockade on blood pressure (BP) and heart rate.Key resultsOur immunofluorescence studies demonstrated GPR18 expression in the rostral vento‐lateral medulla (RVLM). GPR18 is co‐localized with TH expressing neurons as well as with CB1R in RVLM neurons. Intra‐RVLM microinjection in conscious male Sprague Dawley rats demonstrated dose‐dependent reduction in BP following GPR18 activation with Abn CBD. By contrast, intra‐RVLM blockade of GPR18 with the antagonist O‐1918 caused increase in BP and abrogated the Abn CBD‐evoked reduction in BP.ConclusionsThe present studies are the first to demonstrate the expression of GPR18 in the RVLM and to suggest a role for this receptor in modulating central cardiovascular functions.

Background and objectivesVascular GPR18 activation causes vasodilation, and our preliminary findings are the first to show: (i) GPR18 expression in tyrosine hydroxylase (TH) immunoreactive neurons in the rostral ventrolateral medulla (RVLM); (ii) RVLM GPR18 activation (Abn CBD) and blockade (O‐1918) dose‐dependently reduced and increased BP, respectively, in conscious male Sprague Dawley rats. Here we tested the hypothesis that NO generation, via ERK1/2 activation, coupled with reduced reactive oxygen species (ROS) generation in RVLM underlies GPR18‐mediated hypotension. We complemented the integrative studies with signaling studies in differentiated PC12 cells (catecholamine expressing cells with neuronal phenotype) to elucidate the molecular mechanisms implicated in GPR18 signaling.Key results: Ex vivo studies, on RVLM neuronal tissues, collected during the hypotensive response elicited by intra‐RVLM GPR18 activation in conscious rats revealed increases in ERK phosphorylation and NO level and reduced ROS in RVLM while GPR18 blockade elicited opposite BP and molecular responses. In PC12 cells, GPR18 is associated with lipid rafts, and its activation replicated the molecular responses obtained in the RVLM.ConclusionGPR18 activation in the RVLM lowers BP by reducing oxidative stress and increasing NO, via ERK1/2 phosphorylation. Similar molecular findings in PC12 cells support the use of this model system in ongoing GPR18 signaling studies. Specifically, pharmacologic/genetic manipulation studies will elucidate the role of the lipid rafts and their major protein, caveolin, in GPR18 signaling.