The anandamide transport inhibitor AM404 activates vanilloid receptors
Introduction
The anandamide transport inhibitor N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide (AM404) has been developed to prevent inactivation of anandamide by a cellular re-uptake mechanism and thereby prolong the biological effects of anandamide (Beltramo et al., 1997). This mechanism of inactivation of anandamide has been suggested to be of importance, e.g., in blood pressure regulation, since AM404 potentiated hypotensive responses to anandamide in guinea-pigs (Calignano et al., 1997). Initially, we attempted to investigate the influence of the anandamide transporter on the vasodilator action of anandamide in rat isolated hepatic artery. Anandamide relaxes this blood vessel via activation of vanilloid receptors present on perivascular sensory nerves and the subsequent release of vasodilator peptides such as calcitonin-gene related peptide (CGRP) (Zygmunt et al., 1999). However, we found that pre-treatment of rat hepatic arteries with AM404 did not potentiate the vasodilation induced by anandamide, but rather inhibited this response. The unsaturated fatty acyl chain combined with a vanillyl-like moiety makes AM404 structurally similar to both anandamide and capsaicin (Fig. 1). We therefore explored the possibility that this compound acts as a vanilloid receptor agonist causing receptor desensitization and/or depletion of sensory neruopeptides. This could be the reason why prior treatment of hepatic arteries with AM404 inhibited rather than potentiated the vasodilator effect of anandamide.
Section snippets
Recording of tension
Experiments were performed on hepatic arteries from female Wistar–Hannover rats (200–250 g) as described (Zygmunt et al., 1998). Briefly, the arteries were cut into ring segments and mounted in tissue baths, containing aerated physiological salt solution (5% CO2 and 95% O2, 37°C, pH 7.4). Relaxant responses were studied in preparations contracted with phenylephrine. When stable contractions were obtained, AM404 was added cumulatively to determine concentration–response relationships. The
Effect of capsaicin and CGRP-(8-37) on AM404-induced vasodilation
AM404 induced concentration-dependent relaxations in hepatic arteries of the rat (Fig. 2A,B). The pEC50 and Emax values were 7.4±0.1% and 97±2%, respectively (n=10). Pre-treatment of preparations with capsaicin (10 μM) abolished AM404-induced relaxations (Fig. 2A). Likewise, the CGRP receptor antagonist CGRP-(8-37) at 3 μM abolished relaxations induced by AM404 (Fig. 2A).
Effect of capsazepine and SR141716A on AM404-induced vasodilation
The vanilloid receptor antagonist capsazepine (3 μM) caused a significant right-ward shift of the concentration–response
Discussion
The present study shows that AM404 is a vasodilator and activator of VR1 expressed in Xenopus oocytes. Pre-treatment of rat hepatic arteries with capsaicin abolished AM404-induced relaxations indicating that sensory nerves are involved in the vasodilator action of AM404. The fact that the CGRP receptor antagonist CGRP-(8-37) abolished vasodilation induced by AM404 suggests that CGRP is the mediator of such relaxations. Indeed, anandamide, which releases CGRP from sensory nerves in the rat
Acknowledgements
This work was supported by grants from the Swedish Medical Research Council (no. 13423) and the Medical Faculty of Lund (ALF). H.C. and D.J. were supported by the National Institutes of Health and the McKnight Foundation for Neuroscience. P.M.Z. was supported by the Swedish Medical Research Council (no. 13024). SR141716A was a generous gift from Sanofi Winthrop.
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