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. 2005 Oct 15;568(Pt 2):539-51.
doi: 10.1113/jphysiol.2005.094292. Epub 2005 Aug 4.

Anandamide elicits an acute release of nitric oxide through endothelial TRPV1 receptor activation in the rat arterial mesenteric bed

Inés M Poblete  1 , María Luz Orliac, René Briones, Edda Adler-Graschinsky, J Pablo Huidobro-Toro
Affiliations

Anandamide elicits an acute release of nitric oxide through endothelial TRPV1 receptor activation in the rat arterial mesenteric bed

Inés M Poblete et al. J Physiol. .

Abstract

In the isolated rat mesenteric bed, the 1 min perfusion with 100 nm anandamide, a concentration that did not evoke vasorelaxation, elicited an acute release of 165.1 +/- 9.2 pmol nitric oxide (NO) that was paralleled by a 2-fold increase in cGMP tissue levels. The rise in NO released was mimicked by either (R)-(+)-methanandamide or the vanilloid receptor agonists resiniferatoxin and (E)-capsaicin but not by its inactive cis-isomer (Z)-capsaicin. The NO release elicited by either anandamide or capsaicin was reduced by the TRPV1 receptor antagonists 5'-iodoresiniferatoxin, SB 366791 and capsazepine as well as by the cannabinoid CB(1) receptor antagonists SR 141716A or AM251. The outflow of NO elicited by anandamide and capsaicin was also reduced by endothelium removal or NO synthase inhibition, suggesting the specific participation of endothelial TRPV1 receptors, rather than the novel endothelial TRPV4 receptors. Consistently, RT-PCR showed the expression of the mRNA coding for the rat TRPV1 receptor in the endothelial cell layer, in addition to its expression in sensory nerves. The participation of sensory nerves on the release of NO was precluded on the basis that neonatal denervation of the myenteric plexus sensory nerves did not modify the pattern of NO release induced by anandamide and capsaicin. We propose that low concentrations of anandamide, devoid of vasorelaxing effects, elicit an acute release of NO mediated predominantly by the activation of endothelial TRPV1 receptors whose physiological significance remains elusive.

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Figures

Figure 1
Figure 1. Anandamide, 100 nm, elicits a surge of NO and increases cGMP tissue levels but does not vasodilate the arterial mesenteric bed of the rat
A, representative tracing illustrates the lack of vasodilatation induced by a 4-min perfusion with 100 nm anandamide (AEA) in mesenteries precontracted with 20 μm phenylephrine. B, prototype experiment shows the time course of NO release induced by a 1-min exposure to 100 nm AEA (filled rectangle at min 4). Stimulated NO release was quantified as the integrated NO recovered above the corresponding baseline values, shown in grey. C, tissue levels of cGMP were measured in mesenteric bed homogenates either in the absence (open bars) or immediately after the 1-min perfusion with 100 nm AEA (filled bars). Both the basal and the 100 nm AEA-stimulated increase in cGMP tissue levels were also measured after a 20-min pretreatment with 3 μm ODQ, the inhibitor of soluble guanylyl cyclase. Results in C are the mean ±s.e.m. of 4–6 experiments per group. *P < 0.05 when compared with the corresponding basal value, **P < 0.01 when compared with the corresponding values in the absence of ODQ.
Figure 2
Figure 2. Concentration–response curves for anandamide and its structurally related analogues, resiniferatoxin and the capsaicin isomers
A, release of NO above baseline levels induced by a 1-min perfusion with increasing concentrations of either anandamide (AEA, n = 30) or its synthetic analogue (R)-(+)-methanandamide (MTA, n = 16) or the endogenous anandamide analogue N-arachidonylglycine (N-arach.glycine, n = 15). B, the release of NO elicited by a 1-min perfusion with increasing concentrations of vanilloid receptor agonists: resiniferatoxin (RTX, n = 20), (E)-capsaicin (n = 23) or its inactive cis isomer (Z)-capsaicin (n = 13) was compared to the AEA concentration–response curve. Symbols represent mean values, bars the s.e.m.
Figure 3
Figure 3. Effects of TRPV1 and cannabinoid CB1 receptor antagonists on the release of NO elicited by resiniferatoxin, anandamide or capsaicin
A, 5′-iodoresiniferatoxin-induced (5′-I-RTX) and AM 251 blocked concentration-dependently the NO release elicited by a 1-min perfusion with 1 nm resiniferatoxin (RTX, n = 18). B, comparative study of the potency of SB 366791, capsazepine or SR 141716A to block the NO production elicited by a 1-min pulse application of 100 nm anandamide (AEA). C, capsazepine, SR 141716A and AM 251 reduced concentration-dependently the NO production elicited by a 1-min perfusion with 100 nm capsaicin. Left side columns denote the NO produced in separate control experiments by a 1-min perfusion with 1 nm resiniferatoxin (RTX), or 100 nm of either anandamide (AEA) or capsaicin alone (panels A, B and C, respectively). The antagonists were perfused 30 min before and during the 1-min incubation with the agonists. Results are the mean ±s.e.m. of at least 4 separate experiments per concentration of each antagonist assayed in the presence of either anandamide or capsaicin.
Figure 4
Figure 4. RT-PCR analysis of the mRNA coding for the vanilloid TRPV1 receptor expressed in rat mesenteries
Representative ethidium bromide stained agarose gel of reversed transcribed and PCR-amplified fragments of the expected size for the vanilloid TRPV1 receptor mRNA. RT-PCR analysis was performed by using TRPV1-specific primers to examine expression in mRNA extracted from mesenteries isolated from adult controls and adult chronically denervated rats (neonatal capsaicin treatment) either with intact (E+), or without endothelium (E−). Total rat tongue mRNA was used as a positive control tissue. While GAPDH was used as an internal standard to control gel loading, CD31 served as the endothelium marker. Left lane indicates the molecular weight markers. The mRNA used for the TRPV1, CD31 and GAPDH identification proceeded from a same rat mesentery. Identical results were attained in three protocols using separate intact or endothelium denuded rat mesenteries.
Figure 5
Figure 5. Chronic sensory nerve denervation reduced myenteric CGRP immunoreactivity but not the anandamide- or capsaicin-induced NO release
A, representative whole-mount sections of myenteric CGRP-immunostained nerve terminals from adult control (left image) and denervated adult rats (right panel). Neonatal capsaicin treatment was performed on days 1 and 2 after birth as described under methods. B, control values of NO release elicited by 1-min exposure to either anandamide (AEA, left panel) or capsaicin (CAP, right panel) are depicted as hatched bars. Open bars represent the outflow of NO after chronic denervation. The vanilloid TRPV1 receptor antagonist 100 nm capsazepine (CPZ), or 0.3 μm ruthenium red (RR), the TRPV1 channel blocker, was added 20 min before and during the 1-min incubation with the agonists. When indicated (E−) the endothelium was removed by perfusing 0.1% saponin for 55 s. Results are the mean ±s.e.m. of at least 4 separate experiments per group. P < 0.01 when compared to the NO release induced by the agonists alone in denervated mesenteries.
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