Elsevier

Life Sciences

Volume 82, Issues 11–12, 12 March 2008, Pages 644-651
Life Sciences

Actions of 3-methyl-N-oleoyldopamine, 4-methyl-N-oleoyldopamine and N-oleoylethanolamide on the rat TRPV1 receptor in vitro and in vivo

https://doi.org/10.1016/j.lfs.2007.12.022Get rights and content

Abstract

N-oleoyldopamine (OLDA) has been identified as an agonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor. A related fatty acid amide, N-oleoylethanolamide (OEA), was found to excite sensory neurons and produce visceral hyperalgesia via activation of the TRPV1 receptor, however, a recent study described this agent as an antinociceptive one. The aim of the present paper was to characterize two newly synthesized derivatives of N-oleoyldopamine, 3-methyl-N-oleoyldopamine (3-MOLDA) and 4-methyl-N-oleoyldopamine (4-MOLDA) as well as OEA with regard to their effects on the TRPV1 receptor. Radioactive 45Ca2+ uptake was measured in HT5-1 cells transfected with the rat TRPV1 receptor and intracellular Ca2+ concentration was monitored by fura-2 microfluorimetry in cultured trigeminal sensory neurons. Thermonociception was assessed by determining the behavioral noxious heat threshold in rats. 3-MOLDA induced 45Ca2+ uptake in a concentration-dependent manner, whereas 4-MOLDA and OEA were without effect. 4-MOLDA and OEA, however, concentration-dependently reduced the 45Ca2+ uptake-inducing effect of capsaicin. In trigeminal sensory neurons, 3-MOLDA caused an increase in intracellular Ca2+ concentration and this effect exhibited tachyphylaxis upon repeated application. Again, 4-MOLDA and OEA failed to alter intracellular Ca2+ levels. Upon intraplantar injection, 3-MOLDA caused an 8–10 °C drop of the noxious heat threshold in rats which was inhibited by the TRPV1 receptor antagonist iodo-resiniferatoxin. 4-MOLDA and OEA failed to alter the heat threshold but inhibited the threshold drop induced by the TRPV1 receptor agonist resiniferatoxin. These data show that 3-MOLDA behaves as an agonist, whereas 4-MOLDA and OEA appear to be antagonists, at the rat TRPV1 receptor.

Introduction

The transient receptor potential (TRP) family of ion channels includes the vanilloid (V) subfamily in which only one member, the TRPV1 receptor, is activated by capsaicin, the pungent agent of the red pepper (for a review see Dhaka et al., 2006). Since cloning of this receptor (Caterina et al., 1997) it has been shown that this ligand-gated non-selective cation channel expressed by nociceptive primary afferent neurons is activated by not only capsaicin but also other substances including resiniferatoxin (RTX), low pH (protons), lipoxygenase products and arachidonoylacylamides as well as by noxious heat (for a review see Pingle et al., 2007). Furthermore, since the TRPV1 receptor is indirectly activated also from other receptors like the bradykinin B2 receptor, it is often referred to as an integrator molecule of polymodal nociceptors.

The fatty acid amide arachidonylethanolamide, also known as anandamide, the endogenous ligand for the cannabinoid receptors (for a review see Pertwee, 2001), was also shown to activate the TRPV1 receptor (Zygmunt et al., 1999, Smart et al., 2000). There has been a long debate about its possible role as the natural endogenous agonist for the TRPV1 receptor (Szolcsányi, 2000, Di Marzo et al., 2001). Recently other related fatty acid amides, N-arachidonoyl dopamine and N-oleoyldopamine (OLDA, Fig. 1), were identified as TRPV1 receptor agonists (Huang et al., 2002, Chu et al., 2003, Szolcsányi et al., 2004) whereas N-arachidonoyl serotonin was described as an antagonist at this receptor (Maione et al., 2007). Parallel to this, a further fatty acid amide, N-oleoylethanolamide (OEA, Fig. 1), was also reported to activate sensory neurons and produce hyperalgesia via the TRPV1 receptor (Ahern, 2003, Wang et al., 2005, LoVerme et al., 2006). In contrast, OEA was found antinociceptive in two models of visceral and inflammatory pain both in the mouse and rat (Suardíaz et al., 2007).

The major aim of the present paper was to characterize two newly synthesized methylated derivatives of OLDA, 3-methyl-N-oleoyldopamine (3-MOLDA) and 4-methyl-N-oleoyldopamine (4-MOLDA) (Fig. 1), with regard to their effect on the rat TRPV1 receptor. In vitro and in vivo methods were employed: measurement of 45Ca2+ uptake in TRPV1 receptor-transfected non-neuronal cells, monitoring intracellular Ca2+ concentration in cultured rat trigeminal sensory neurons and assessment of thermonociception by determining the behavioral noxious heat threshold in unrestrained rats. Owing to the above-mentioned contradictory data obtained with OEA, its effects on the TRPV1 receptor were also investigated. Finally, the effect of anandamide with regard to TRPV1 was also examined but only in the in vivo behavioral model as it had previously been studied extensively in vitro.

Section snippets

Measurement of radioactive 45Ca2+ uptake in HT5-1 cells transfected with the rat TRPV1 receptor

The experiments were performed essentially as has been described previously (Szolcsányi et al., 2004). HT5-1 cells transfected with the rat TRPV1 receptor (Sándor et al., 2005) were plated in 15 μl cell culture medium onto Microwell Minitrays (Sigma Inc). The next day, the cells were washed 5 times with HEPES (10 mM, pH 7.4) buffered Hank's balanced salt solution containing 2 mM CaCl2. When testing compounds for TRPV1 receptor agonistic activity, the cells were incubated in 10 μl of the same

Effects of 3-MOLDA, 4-MOLDA and OEA in the 45Ca2+ uptake test and the inhibitory effects of 4-MOLDA and OEA on the capsaicin-induced 45Ca2+ uptake

In the first series of experiments 3-MOLDA, 4-MOLDA and OEA were tested for agonistic activity in HT5-1 cells expressing the rat TRPV1 receptor in the concentration range from 0.1 to 100 µM. The reference TRPV1 receptor agonist capsaicin was also tested and drug-induced 45Ca2+ uptake values are presented relative to the maximum response obtained with 330 nM capsaicin. The concentration–response curves obtained are shown in Fig. 2. 3-MOLDA even at the highest applied concentration (100 µM)

Discussion

The present study has shown that of the three fatty acid amides investigated both in vitro and in vivo, 3-MOLDA behaved as a TRPV1 receptor agonist while 4-MOLDA and OEA appeared to be antagonists of the TRPV1 receptor. According to behavioral data, anandamide failed to activate the TRPV1 receptor, rather it exerted an inhibitory effect on the action of the TRPV1 receptor agonist RTX via activation of CB1 cannabinoid receptors.

In the 45Ca2+ uptake assay in TRPV1 receptor-transfected

Conclusion

The present study investigated various fatty acid amides in relation to the rat TRPV1 receptor employing measurement of 45Ca2+ uptake in TRPV1 receptor-transfected HT5-1 cells and intracellular Ca2+ concentration in cultured rat trigeminal sensory neurons as well as assessment of thermonociception by determination of the behavioral noxious heat threshold in rats. While 3-MOLDA proved to be an agonist of the rat TRPV1 receptor, the chemically only slightly different 4-MOLDA exerted effects

Acknowledgements

The authors thank Ms Cecília Disztl and Mrs Katalin Gógl for expert technical assistance. This work was supported by Hungarian Grants ETT 348/2006 and ETT 285/2006.

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    R. Almási and É. Szőke contributed equally to this work.

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