lysophosphatidylinositol and anandamide

Stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. Interestingly, endocannabinoids such as anandamide (which interacts with CB1 , CB2 , TRPV1 and GPR55 receptors) can regulate the activity of perivascular sensory nerves in dural blood vessels by modulating CGRP release. Yet, as no publication has reported whether this mechanism is operative in the healthy systemic vasculature, this study has specifically analysed the receptors mediating the potential inhibitory effects of the cannabinoid (CB) receptor agonists anandamide (non-selective), JWH-015 (CB2 ) and lysophosphatidylinositol (GPR55) on the rat vasodepressor sensory CGRPergic outflow (an index of systemic vasodilatation). Healthy pithed rats were pre-treated with consecutive i.v. continuous infusions of hexamethonium, methoxamine and the above agonists. Electrical spinal (T9 -T12 ) stimulation of the vasodepressor sensory CGRPergic outflow or i.v. injections of α-CGRP produced frequency-dependent or dose-dependent vasodepressor responses. The infusions of anandamide in a dose-dependent manner inhibited the vasodepressor responses by electrical stimulation (remaining unaffected by JWH-015 or lysophosphatidylinositol), but not those by α-CGRP. After i.v. administration of antagonists, the inhibition by 3.1 μg/kg min anandamide was: (i) potently blocked by 31-100 μg/kg NIDA41020 (CB1 ), (ii) unaffected by 180 μg/kg AM630 (CB2 ), 31 μg/kg cannabidiol (GPR55) or 31-100 μg/kg capsazepine (TRPV1) and (iii) slightly blocked by 310 μg/kg AM630. The above doses of antagonists were enough to block their respective receptors. These results suggest that anandamide-induced inhibition of the vasodepressor sensory CGRPergic outflow is mainly mediated by pre-junctional activation of CB1 receptors, with no pharmacological evidence for the role of CB2 , TRPV1 or GPR55 receptors.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cannabinoid Receptor Agonists; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Indoles; Lysophospholipids; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Vasodilation

The endocannabinoid system exhibits multiple functions in cardiovascular regulation mainly by cannabinoid (CB1 and CB2) receptors, vanilloid TRPV1 receptors and, probably, by the orphan G protein-coupled receptor 55 (GPR55). Hence, the role of these receptors was investigated in Wistar pithed rats on anandamide- and lysophosphatidylinositol (LPI)-induced inhibition of the vasopressor responses induced by preganglionic (T7-T9) stimulation of the vasopressor sympathetic outflow or i.v. bolus injections of noradrenaline. The corresponding frequency- and dose-dependent vasopressor responses were analyzed before and during i.v. continuous infusions of anandamide (CB1, CB2, TRPV1 and GPR55), JWH-015 (CB2) and LPI (GPR55) in animals receiving (i.v.) the antagonists NIDA41020 (CB1), AM630 (CB2), capsazepine (TRPV1) and/or cannabidiol (GPR55). Anandamide (0.1-3.1 μg/kg min) inhibited the vasopressor responses by electrical stimulation, but not those by noradrenaline; while LPI (5.6-10 μg/kg min) inhibited both responses. In contrast, JWH-015 (5.6-10 μg/kg min) failed to induce sympatho-inhibition. Anandamide-induced sympatho-inhibition was: (i) dose-dependently blocked by 31 and 100 μg/kg NIDA41020; (ii) slightly blocked by 310 μg/kg AM630 or 31 μg/kg cannabidiol; and (iii) unaffected by 310 μg/kg capsazepine. Moreover, LPI-induced inhibition of both vasopressor responses was blocked and abolished by 10 and 31 μg/kg cannabidiol, respectively, and weakly blocked by 100 μg/kg NIDA41020. Thus, the sympatho-inhibition by anandamide is primarily mediated by cannabinoid CB1 and, minimally, by cannabidiol-sensitive receptors. In contrast, LPI-induced inhibition of both responses seems to be mainly mediated by postjunctional cannabidiol-sensitive (presumably endothelial GPR55) receptors.

Topics: Animals; Arachidonic Acids; Blood Pressure; Desipramine; Electric Stimulation; Endocannabinoids; Heart Rate; Indoles; Lysophospholipids; Male; Norepinephrine; Polyunsaturated Alkamides; Rats; Rats, Wistar; Sympathetic Nervous System; Vasoconstrictor Agents

Both L-α-lysophosphatidylinositol (LPI) and 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) have been reported to activate the putative cannabinoid receptor, GPR55. Recent microsecond time-scale molecular dynamics (MD) simulations and isothiocyanate covalent labeling studies have suggested that a transmembrane helix 6/7 (TMH6/7) lipid pathway for ligand entry may be necessary for interaction with cannabinoid receptors. Because LPI and 2-AGPI are lipid-derived ligands, conformations that each assumes in the lipid bilayer are therefore likely important for their interaction with GPR55. We report here the results of 70 ns NAMD molecular dynamics (MD) simulations of LPI and of 2-AGPI in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These simulations are compared with a 70 ns simulation of the cannabinoid CB1 receptor endogenous ligand, N-arachidonoylethanolamine (anandamide, AEA) in a POPC bilayer. These simulations revealed that (1) LPI and 2-AGPI sit much higher in the bilayer than AEA, with inositol headgroups that can at times be solvated completely by water; (2) the behavior of the acyl chains of AEA and 2-AGPI are similar in their flexibilities in the bilayer, while the acyl chain of LPI has reduced flexibility; and (3) both 2-AGPI and LPI can adopt a tilted headgroup orientation by hydrogen bonding to the phospholipid phosphate/glycerol groups or via intramolecular hydrogen bonding. This tilted head group conformation (which represents over 40% of the conformer population of LPI (42.2 ± 3.3%) and 2-AGPI (43.7 ± 1.4%)) may provide a low enough profile in the lipid bilayer for LPI and 2-AGPI to enter GPR55 via the putative TMH6/7 entry port.

Topics: Arachidonic Acids; Cannabinoid Receptor Agonists; Endocannabinoids; Hydrogen Bonding; Ligands; Lipid Bilayers; Lysophospholipids; Molecular Conformation; Molecular Dynamics Simulation; Phosphatidylcholines; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Thermodynamics