Igor Dimitri Gama Duarte

Introduction: Our group has described the nitrergic pathway as a key analgesic mechanism of drugs and as part of the endogenous control of peripheral inflammatory pain. Studies have shown that epoxyeicosatrienoic acids (EETs) can activate different isoforms of nitric oxide synthase, leading to nitric oxide release and subsequent vasodilation. Aim: Therefore, the goal of this study was to determine whether the nitrergic pathway mediates the peripheral antinociceptive effects of EETs. Methods: The mechanical paw pressure test was used as an algesimetric method. Hyperalgesia was induced by intraplantar injection of prostaglandin E2 to assess the effects of exogenous EETs administration and its impact on inhibiting the nitrergic pathway. The EETs (5,6-, 8,9-, 11,12-, and 14,15-EET) and drugs targeting the nitrergic pathway were administered intraplantarly to male Swiss mice (n = 4). Statistical analysis was performed using ANOVA followed by Bonferroni post-test. Results: The peripheral antinociceptive effects of 5,6-, 8,9-, 11,12-, and 14,15-EET were antagonized by L-NOArg (12, 18, and 24 µg/paw), a non-selective nitric oxide synthase (NOS) inhibitor. The selective inhibitors of inducible (iNOS) (LNIL; 24 μg/paw) and endothelial (eNOS) (LNIO; 24 μg/paw) isoforms partially reduced the antinociceptive effects of EETs. In contrast, selective inhibition of neuronal NOS (nNOS) with LNPA (24 µg/paw) did not affect the peripheral antinociceptive effects of EETs. The soluble guanylate cyclase (sGC) inhibitor ODQ (50, 100, and 200 μg/paw) decreased the peripheral antinociceptive effects of EETs. Conversely, Zaprinast (50 μg/paw), a selective phosphodiesterase (PDE) inhibitor, enhanced the peripheral antinociceptive effects of low doses of EETs. The results suggest that EETs may be linked with iNOS and eNOS activation, but not nNOS, and this process leads to the production of nitric oxide, which stimulates sGC, raises cGMP, and ultimately produces the antinociceptive effect observed with these substances.

1. Dos Santos RG, Hallak JEC. Ayahuasca: pharmacology, safety, and therapeutic effects. CNS Spectr. 2024 Nov 20;30(1):e2. 2. Bouso JC, González D, Fondevila S, Cutchet M, Fernández X, Ribeiro Barbosa PC, et al. Personality, psychopathology, life attitudes and neuropsychological performance among ritual users of Ayahuasca: a longitudinal study. PLoS One. 2012;7(8):e42421. 3. Sarris J, Perkins D, Cribb L, Schubert V, Opaleye E, Bouso JC, et al. Ayahuasca use and reported effects on depression and anxiety symptoms: An international cross-sectional study of 11,912 consumers. Journal of Affective Disorders Reports. 2021 Apr 1;4:100098. 4. Dos Santos RG, Osório FL, Crippa JA, Hallak JE. Antidepressive and anxiolytic effects of ayahuasca: a systematic literature review of animal and human studies. Braz J Psychiatry. 2016 Mar;38(1):65–72. 5. Barker SA, McIlhenny EH, Strassman R. A critical review of reports of endogenous psychedelic N, N-dimethyltryptamines in humans: 1955-2010. Drug Test Anal. 2012 Jul-Aug;4(7-8):617–35. 6. Jiménez JH, Bouso JC. Significance of mammalian N, N-dimethyltryptamine (DMT): A 60-year-old debate. J Psychopharmacol. 2022 Aug;36(8):905–19. 7. Schimmelpfennig J, Jankowiak-Siuda K. Unique biological and physiological properties of endogenous N, N-dimethyltryptamine from the perspective of functioning of the nervous system. Neuropsychiatria i Neuropsychologia/Neuropsychiatry and Neuropsychology. 2023;18(1):1–10. 8. Nardai S, László M, Szabó A, Alpár A, Hanics J, Zahola P, et al. N,N-dimethyltryptamine reduces infarct size and improves functional recovery following transient focal brain ischemia in rats. Exp Neurol. 2020 May;327:113245. 9. Morales-Garcia JA, Calleja-Conde J, Lopez-Moreno JA, Alonso-Gil S, Sanz-SanCristobal M, Riba J, et al. N,N-dimethyltryptamine compound found in the hallucinogenic tea ayahuasca, regulates adult neurogenesis in vitro and in vivo. Transl Psychiatry. 2020 Sep 28;10(1):331. 10. Barker SA. N, N-dimethyltryptamine (DMT) in rodent brain: Concentrations, distribution, and recent pharmacological data. Prog Neuropsychopharmacol Biol Psychiatry. 2025 Mar 20;137:111259. 11. Cameron LP, Benson CJ, Dunlap LE, Olson DE. Effects of N, N-Dimethyltryptamine on Rat Behaviors Relevant to Anxiety and Depression. ACS Chem Neurosci. 2018 Jul 18;9(7):1582–90. 12. Kooijman NI, Willegers T, Reuser A, Mulleners WM, Kramers C, Vissers KCP, et al. Are psychedelics the answer to chronic pain: A review of current literature. Pain Pract. 2023 Apr;23(4):447–58. 13. Machado PD, Fonseca FCS, Azevedo MC, Filho SAV, Cavalcanti AD, Santos ODH, et al. Antinociceptive effects of Psychotria viridis Ruiz & Pav. on carrageenan-induced hyperalgesia in rats. Curr Top Pharmacol. 27: 1–8. 14. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain. 1983 Jun;16(2):109–10. 15. RANDALL LO, SELITTO JJ. A method for measurement of analgesic activity on inflamed tissue. Arch Int Pharmacodyn Ther. 1957 Sep 1;111(4):409–19. 16. Soares-Santos RR, Machado DP, Romero TL, Duarte IDG. Nitric oxide and potassium channels but not opioid and cannabinoid receptors mediate tramadol-induced peripheral antinociception in rat model of paw pressure withdrawal. Can J Physiol Pharmacol. 2024 Mar 1;102(3):218–27. 17. Wall PD, Melzack R, McMahon SB, Koltzenburg M, Tracey I, Turk DC. Wall and Melzack's textbook of pain. (No Title). 2006. 18. Hata AN, Breyer RM. Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol Ther. 2004 Aug;103(2):147–66. 19. Cozzi NV, Gopalakrishnan A, Anderson LL, Feih JT, Shulgin AT, Daley PF, et al. Dimethyltryptamine and other hallucinogenic tryptamines exhibit substrate behavior at the serotonin uptake transporter and the vesicle monoamine transporter. J Neural Transm (Vienna). 2009 Dec;116(12):1591–9. 20. Halberstadt AL. Recent advances in the neuropsychopharmacology of serotonergic hallucinogens. Behav Brain Res. 2015 Jan 15;277:99–120. 21. Nagai F, Nonaka R, Satoh Hisashi Kamimura K. The effects of non-medically used psychoactive drugs on monoamine neurotransmission in rat brain. Eur J Pharmacol. 2007 Mar 22;559(2-3):132–7. 22. Halman A, Kong G, Sarris J, Perkins D. Drug-drug interactions involving classic psychedelics: A systematic review. J Psychopharmacol. 2024 Jan;38(1):3–18. 23. Diniz DA, Petrocchi JA, Navarro LC, Souza TC, Castor MG, Perez AC, et al. Serotonin induces peripheral mechanical antihyperalgesic effects in mice. Eur J Pharmacol. 2015 Nov 15;767:94–7. 24. Carbonaro TM, Gatch MB. Neuropharmacology of N,N-dimethyltryptamine. Brain Res Bull. 2016 Sep;126(Pt 1):74–88. 25. Fontanilla D, Johannessen M, Hajipour AR, Cozzi NV, Jackson MB, Ruoho AE. The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator. Science. 2009 Feb 13;323(5916):934–7. 26. Keiser MJ, Setola V, Irwin JJ, Laggner C, Abbas AI, Hufeisen SJ, et al. Predicting new molecular targets for known drugs. Nature. 2009 Nov 12;462(7270):175–81. 27. Riba J, McIlhenny EH, Valle M, Bouso JC, Barker SA. Metabolism and disposition of N,N-dimethyltryptamine and harmala alkaloids after oral administration of ayahuasca. Drug Test Anal. 2012 Jul-Aug;4(7-8):610–6. 28. Ruffing DM, Domino EF. Effects of selected opioid agonists and antagonists on DMT- and LSD-25-induced disruption of food-rewarded bar pressing behavior in the rat. Psychopharmacology (Berl). 1981;75(3):226–30. 29. Romero TR, Pacheco Dda F, Duarte ID. Xylazine induced central antinociception mediated by endogenous opioids and μ-opioid receptor, but not δ-or κ-opioid receptors. Brain Res. 2013 Apr 19;1506:58–63. 30. Petrocchi JA, de Almeida DL, Paiva-Lima P, Queiroz-Junior C, Caliari MV, Duarte IDG, et al. Peripheral antinociception...

Background: The overall pain experience results from the balance between the nociceptive pathway and the body’s endogenous modulation of nociception. The interaction of these systems reduces nociception. Therefore, this study aimed to evaluate how the opioid and dopaminergic systems collaborate to inhibit pain at the peripheral level. Methods: Swiss mice (30–40 g) had their pain sensitivity increased through paw administration of the prostaglandin E2 (2 μg). They then received opioid and dopaminergic receptor antagonists and agonists, along with an inhibitor of endogenous opioid peptide degradation and a dopamine (DA) reuptake inhibitor. The nociceptive threshold was measured using the paw withdrawal test. Groups were compared using one-way analysis of variance (ANOVA), with p < 0.05 considered significant. Results: The nonselective opioid receptor antagonist naloxone (50 μg/paw) and the selective κ nor-BNI (200 μg/paw; nor-Binaltorphimine) and δ naltrindole (60 μg/paw) receptor antagonists reversed the antinociception caused by peripheral administration of DA (80 ng/paw), but not the μ-opioid receptor antagonist CTOP (20 μg/paw; D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). The antinociception induced by a submaximal dose of DA (5 ng/paw) was enhanced by bestatin (400 μg/paw), an inhibitor of endogenous opioid peptide degradation. Conversely, peripheral antinociception from submaximal doses of the μ-, δ-, and κ-opioid agonists DAMGO (0.25 μg/paw; [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin), SNC 80 (5 μg/paw; (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), and bremazocine (200 ng/paw) was increased by the DA reuptake inhibitor GBR 12783 (16 μg/paw; 1-(2-Diphenylmethoxyethyl)-4-(3-phenyl-2-propenyl)-piperazine). Furthermore, the antinociception from these agonists’ maximum doses was reversed by dopaminergic D2 (remoxipride, 4 μg/paw) and D3 (U99194, 16 μg/paw; 2,3-Dihydro-5,6-dimethoxy-N, N-dipropyl-1H-inden-2-amine) receptor antagonists, but not by the D4 (L-745, 870, 16 μg/paw; 3-(4-[4-Chlorophenyl]piperazin-1-yl)-methyl-1H-pyrrolo[2,3-b]pyridine trihydrochloride) receptor antagonist. Conclusions: Overall, the data suggest that opioid-mediated antinociception depends on the activation of the dopaminergic system. This demonstrates that pain modulation can be enhanced through the interaction of these systems. Controlling pain at a peripheral level by activating endogenous pathways could be a promising approach to pain management.

The hyperpolarization of neuronal membranes through the activation of potassium and chloride channels is a significant mechanism involved in the antinociceptive effects of various drugs. Thus, in this study, we aimed to determine whether potassium or chloride channels mediate the peripheral antinociceptive effects of epoxyeicosatrienoic acids (EETs). The mechanical paw pressure test was utilized as an algesimetric method. Hyperalgesia was induced by intraplantar injection of prostaglandin E2. The EETs (5,6-, 8,9-, 11,12-, and 14,15-EET) and the channel blockers were administered intraplantar in male Swiss mice (n=4). Statistical analysis was performed using ANOVA followed by Bonferroni post-test. Glibenclamide (Glib; 80 μg/paw), a blocker of ATP-sensitive K+ channels; tetraethylammonium (TEA; 30 μg/paw), a blocker of voltage-gated K+ channels; dequalinium (DQ; 50 μg/paw), a blocker of small conductance Ca2+-activated K+ channels; and paxilline (Pax; 20 μg/paw), a blocker of high-conductance Ca2+-activated K+ channels, did not alter the peripheral antinociception induced by 5,6-, 8,9-, 11,12-, and 14,15-EET. All blockers, when administered alone, did not change the nociceptive threshold of the animals. Niflumic acid (Nifl. Ac.; 32 μg/paw), a selective calcium-activated chloride channels blocker, did not reverse the peripheral antinociceptive effect of 5,6-, 8,9-, 11,12-, and 14,15-EET. In conclusion, these results suggest that the peripheral antinociceptive effects of 5,6-, 8,9-,11,12-, and 14,15-EET do not appear to be related to the opening of potassium or chloride channels.

Orofacial pain represents a significant portion of the complaints from patients seeking treatment at pain management centers worldwide. Although the treatment for orofacial pain is primarily pharmacological, there has been an increase in reports showing significant clinical results from non-pharmacological therapies, including electroacupuncture (EA). Recently, EA has been recognized as an effective and affordable non-pharmacological strategy with minimal side effects. However, the mechanisms underlying its pain-relieving (antinociceptive) effects remain poorly understood. The present study aimed to evaluate the roles of α2 adrenoreceptors and GABA in the antinociception induced by electroacupuncture. Male Wistar rats underwent EA stimulation at the acupoint St36 for 20 minutes at a frequency of 100 Hz and an intensity of 0.5 mA. To assess the thermal nociceptive response, a stimulus was applied to the vibrissae (whiskers) of the rats, and the time taken for facial withdrawal was measured before the EA stimulation and at 15-minute intervals afterward until the effects diminished. EA at acupoint St36 for 20 minutes effectively reversed thermal nociception, with this effect lasting for 150 minutes. The antinociceptive effects of EA were blocked by the pre-injection of yohimbine, an antagonist of α2 adrenoreceptors, at doses of 2 and 4 mg/kg. However, it was primarily observed at the onset, indicating that α2 adrenoreceptors play a role in the initial antinociceptive effect of EA. To investigate the involvement of GABA in this effect, intraperitoneal injections of a GABAB antagonist (sacoflen) and a GABA reuptake inhibitor (guvacine) were administered 10 minutes before EA. Neither of these drugs affected the antinociceptive effect produced by EA. Thus, this study suggests that GABA does not participate in the antinociception induced by electroacupuncture.

The present study aimed to evaluate the possible peripheral H₂O₂-induced antinociception and determine the involvement of opioidergic, cannabinoidergic and nitrergic systems, besides potassium channels in its antinociceptive effect. Prostaglandin E₂ was used to induce hyperalgesia in male Swiss mice using the mechanical paw pressure test. H₂O₂ (0.1, 0.2, 0.3 µg/paw) promoted a dose-dependent antinociceptive effect that was not observed in contralateral paw. Female mice also showed antinociception in the model. The partial H₂O₂-induced antinociception was potentiated by the inhibitor of catalase enzyme, aminotriazole (40, 60, 80 µg/paw). The antinociception was not reversed by opioid and cannabinoid receptor antagonists naloxone, AM 251 and AM 630. The involvement of nitric oxide (NO) was observed by the reversal of H₂O₂-induced antinociception using the non-selective inhibitor of nitric oxide synthases L-NOarg and by inhibition of iNOS (L-NIL), eNOS (L-NIO) and nNOS (L-NPA). ODQ, a cGMP-forming enzyme selective inhibitor, also reversed the antinociception. The blockers of potassium channels voltage-gated (TEA), ATP-sensitive (glibenclamide), large (paxillin) and small (dequalinium) conductance calcium-activated were able to revert H₂O₂ antinociception. Our data suggest that H₂O₂ induced a peripheral antinociception in mice and the NO pathway and potassium channels (voltage-gated, ATP-sensitive, calcium-activated) are involved in this mechanism. However, the role of the opioid and cannabinoid systems was not evidenced.

Introduction: Tissue injury results in the release of inflammatory mediators, including a cascade of algogenic substances, which contribute to the development of hyperalgesia. During this process, endogenous analgesic substances are peripherally released to counterbalance hyperalgesia. The present study aimed to investigate whether inflammatory mediators TNF-α, IL-1β, CXCL1, norepinephrine (NE), and prostaglandin E2 (PGE2) may be involved in the deflagration of peripheral endogenous modulation of inflammatory pain by activation of the cholinergic system. Methods: Male Swiss mice were subjected to paw withdrawal test. All the substances were injected via the intraplantar route. Results: The main findings of this study were as follows: (1) carrageenan (Cg), TNF-α, CXCL-1, IL1-β, NE, and PGE2 induced hyperalgesia; (2) the acetylcholinesterase enzyme inhibitor, neostigmine, reversed the hyperalgesia observed after Cg, TNF-α, CXCL-1, and IL1-β injection; (3) the non-selective muscarinic receptor antagonist, atropine, and the selective muscarinic type 1 receptor (m1AChr) antagonist, telenzepine, potentiated the hyperalgesia induced by Cg and CXCL-1; (4) mecamylamine, a non-selective nicotinic receptor antagonist, potentiated the hyperalgesia induced by Cg, TNF-α, CXCL-1, and IL1-β; (5) Cg, CXCL-1, and PGE2 increased the expression of the m1AChr and nicotinic receptor subunit α4protein. Conclusion: These results suggest that the cholinergic system may modulate the inflammatory pain induced by Cg, PGE2, TNF-α, CXCL-1, and IL1-β.

Tramadol, an analgesic classified as an “atypical opioid”, exhibits both opioid and non-opioid mechanisms of action. This study aimed to explore these mechanisms, specifically the opioid-, cannabinoid-, nitric oxide-, and potassium channel-based mechanisms, which contribute to the peripheral antinociception effect of tramadol, in an experimental rat model. The nociceptive threshold was determined using paw pressure withdrawal. To examine the mechanisms of action, several substances were administered intraplantarly: naloxone, a non-selective opioid antagonist (50 µg/paw); AM251 (80 µg/paw) and AM630 (100 µg/paw) as the selective antagonists for types 1 and 2 cannabinoid receptors, respectively; nitric oxide synthase inhibitors L-NOArg, L-NIO, L-NPA, and L-NIL (24 µg/paw); and the enzyme inhibitors of guanylatocyclase and phosphodiesterase of cGMP, ODQ, and zaprinast. Additionally, potassium channel blockers glibenclamide, tetraethylammonium, dequalinium, and paxillin were used. The results showed that opioid and cannabinoid receptor antagonists did not reverse tramadol's effects. L-NOarg, L-NIO, and L-NPA partially reversed antinociception, while ODQ completely reversed, and zaprinast enhanced tramadol's antinociception effect. Notably, glibenclamide blocked tramadol's antinociception in a dose-dependent manner. These findings suggest that tramadol's peripheral antinociception effect is likely mediated by the nitrergic pathway and sensitive ATP potassium channels, rather than the opioid and cannabinoid pathways.