Actions of 3-methyl-N-oleoyldopamine, 4-methyl-N-oleoyldopamine and N-oleoylethanolamide on the rat TRPV1 receptor in vitro and in vivo
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.
References (28)
Activation of TRPV1 by the satiety factor oleoylethanolamide
Journal of Biological Chemistry
(2003)- et al.
Investigation of the role of TRPV1 receptors in acute and chronic nociceptive processes using gene-deficient mice
Pain
(2005) - et al.
N-oleoyldopamine, a novel endogenous capsaicin-like lipid that produces hyperalgesia
Journal of Biological Chemistry
(2003) - et al.
Anandamide: some like it hot
Trends in Pharmacological Sciences
(2001) - et al.
Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists
Journal of Biological Chemistry
(2005) - et al.
Concentration-dependent dual effect of anandamide on sensory neuropeptide release from isolated rat tracheae
Neuroscience Letters
(2003) Cannabinoid receptors and pain
Progress in Neurobiology
(2001)- et al.
Analgesic properties of oleoylethanolamide (OEA) in visceral and inflammatory pain
Pain
(2007) Anandamide and the question of its functional role for activation of capsaicin receptors
Trends in Pharmacological Sciences
(2000)- et al.
Direct evidence for activation and desensitization of the capsaicin receptor by N-oleoyldopamine on TRPV1-transfected cell line, in gene deleted mice and in the rat
Neuroscience Letters
(2004)
Relative roles of protein kinase A and protein kinase C in modulation of TRPV1 receptor responsiveness in rat sensory neurons in vitro and peripheral nociceptors in vivo
Neuroscience
Ethical guidelines for investigation of experimental pain in conscious animals
Pain
Anandamide regulates neuropeptide release from capsaicin-sensitive primary sensory neurons by activating both the cannabinoid 1 receptor and the vanilloid receptor 1 in vitro
European Journal of Neuroscience
Effect of resiniferatoxin on the noxious heat threshold temperature in the rat: a novel heat allodynia model sensitive to analgesics
British Journal of Pharmacology
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2016, Life SciencesCitation Excerpt :Heat threshold determination is indeed a more sensitive method when compared to the latency-based Hargreaves' plantar test for detecting the antihyperalgesic effects of TRPV1 antagonists [69] and similarly suitable for measuring potency of conventional analgesics [3,12,14,25]. With our novel methods it was possible to (i) reveal the role of protein kinases in TRPV1 responsiveness [73]; (ii) assess the nociceptive effects of TRPV1 agonist N-oleoyl-dopamine derivatives [4]; (iii) study the local sensory desensitizing actions of TRPV1 agonists in the rat [13]. Out of the thermosensitive TRP channels, the activation threshold of TRPV1 (43 °C) measured in transfected cells and in isolated primary sensory neurons corresponds best to the noxious heat threshold of humans [59].
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R. Almási and É. Szőke contributed equally to this work.