Collaborative action between noradrenergic and serotoninergic systems in peripheral antinociception in mice.

Noradrenaline (NA) and serotonin (5-HT) induce nociception and antinociception. This antagonistic effect can be explained by the dose and type of activated receptors. We investigated the existence of synergism between the noradrenergic and serotonergic systems during peripheral antinociception. The paw pressure test was performed in mice that had increased sensitivity by intraplantar injection of prostaglandin E2 (PGE2). Noradrenaline (80 ng) administered intraplantarly induced an antinociceptive effect, that was reversed by the administration of selective antagonists of serotoninergic receptors 5-HT1B isamoltan, 5-HT1D BRL15572, 5-HT2A ketanserin, 5-HT3 ondansetron, but not by selective receptor antagonist 5-HT7 SB-269970. The administration of escitalopram, a serotonin reuptake inhibitor, potentiated the antinociceptive effect at a submaximal dose of NA. These results, indicate the existence of synergism between the noradrenergic and serotonergic systems in peripheral antinociception in mice.

Participation of the cannabinoid system and the NO/cGMP/KATP pathway in serotonin-induced peripheral antinociception.

It has already been shown that serotonin can release endocannabinoids at the spinal cord level, culminating in inhibition of the dorsal horn. At the peripheral level, cannabinoid receptors modulate primary afferent neurons by inhibiting calcium conductance and increasing potassium conductance. Studies have shown that after the activation of opioid receptors and cannabinoids, there is also the activation of the NO/cGMP/KATP pathway, inducing cellular hyperpolarization. In this study, we evaluated the participation of the cannabinoid system with subsequent activation of the NO/cGMP/KATP pathway in the peripheral antinociceptive effect of serotonin. The paw pressure test of mice was used in animals that had their sensitivity to pain increased due to an intraplantar injection of PGE2 (2 µg). Serotonin (250 ng/paw), administered locally in the right hind paw, induced antinociceptive effect. CB1 and CB2 cannabinoid receptors antagonists, AM251 (20, 40 and 80 µg) and AM630 (25, 50 and 100 µg), respectively, reversed the serotonin-induced antinociceptive effect. MAFP (0.5 µg), an inhibitor of the FAAH enzyme that degrades anandamide, and JZL184 (3.75 µg), an inhibitor of the enzyme MAGL that degrades 2-AG, as well as the VDM11 (2.5 µg) inhibitor of anandamide reuptake, potentiated the antinociceptive effect induced by a low dose (62. 5 ng) of serotonin. In the evaluation of the participation of the NO/cGMP/KATP pathway, the antinociceptive effect of serotonin was reversed by the administration of the non-selective inhibitor of NOS isoforms L-NOarg (12.5, 25 and 50 µg) and by the selective inhibitor for the neuronal isoform LNPA (24 µg), as well as by the soluble guanylate cyclase inhibitor ODQ (25, 50 and 100 µg). Among potassium channel blockers, only Glibenclamide (20, 40 and 80 µg), an ATP-sensitive potassium channel blocker, reversed the effect of serotonin. In addition, intraplantar administration of serotonin (250 ng) was shown to induce a significant increase in nitrite levels in the homogenate of the plantar surface of the paw of mice. Taken together, these data suggest that the antinociceptive effect of serotonin occurs by activation of the cannabinoid system with subsequent activation of the NO/cGMP/KATP pathway.

Anandamide reduces the migration of lymphocytes to the intestine by CB2 activation and reduces TNF-α in the target organs, protecting mice from graft-versus-host disease.

Graft-versus-host disease (GVHD) is a serious inflammatory illness that often occurs as a secondary complication of bone marrow transplantation. Current therapies have limited effectiveness and fail to achieve a balance between inflammation and the graft-versus-tumor effect. In this study, we investigate the effects of the endocannabinoid anandamide on the complex pathology of GVHD. We assess the effects of an irreversible inhibitor of fatty acid amine hydrolase or exogenous anandamide and find that they increase survival and reduce clinical signs in GVHD mice. In the intestine of GVHD mice, treatment with exogenous anandamide also leads to a reduction in the number of CD3+, CD3+CD4+, and CD3+CD8+ cells, which reduces the activation of CD3+CD4+ and CD3+CD8+ cells, as assessed by enhanced CD28 expression, a T cell co-stimulatory molecule. Exogenous AEA was also able to reduce TNF-α and increase IL-10 in the intestine of GVHD mice. In the liver, exogenous AEA reduces injury, TNF-α levels, and the number of CD3+CD8+ cells. Interestingly, anandamide reduces Mac-1α, which lowers the adhesion of transplanted cells in mesenteric veins. These effects are mimicked by JWH133-a CB2 selective agonist-and abolished by treatment with a CB2 antagonist. Furthermore, the effects caused by anandamide treatment on survival were related to the CB2 receptor, as the CB2 antagonist abolished it. This study shows the critical role of the CB2 receptor in the modulation of the inflammatory response of GVHD by treatment with anandamide, the most prominent endocannabinoid.

Involvement of the cannabinoid system in chronic inflammatory intestinal diseases: opportunities for new therapies.

The components of the endogenous cannabinoid system are widely expressed in the gastrointestinal tract contributing to local homeostasis. In general, cannabinoids exert inhibitory actions in the gastrointestinal tract, inducing anti-inflammatory, antiemetic, antisecretory, and antiproliferative effects. Therefore, cannabinoids are interesting pharmacological compounds for the treatment of several acute intestinal disorders, such as dysmotility, emesis, and abdominal pain. Likewise, the role of cannabinoids in the treatment of chronic intestinal diseases, such as irritable bowel syndrome and inflammatory bowel disease, is also under investigation. Patients with chronic intestinal inflammatory diseases present impaired quality of life, and mental health issues are commonly associated with long-term chronic diseases. The complex pathophysiology of these diseases contributes to difficulties in diagnosis and, therefore, in the choice of a satisfactory treatment. Thus, this article reviews the involvement of the cannabinoid system in chronic inflammatory diseases that affect the gastrointestinal tract and highlights possible therapeutic approaches related to the use of cannabinoids.

Cannabidiol Enhances Intestinal Cannabinoid Receptor Type 2 Receptor Expression and Activation Increasing Regulatory T Cells and Reduces Murine Acute Graft-versus-Host Disease without Interfering with the Graft-versus-Leukemia Response.

Cannabidiol (CBD) is a highly lipidic phytocannabinoid with remarkable anti-inflammatory effects. The aim of this study was to evaluate CBD's effects and mechanisms of action in the treatment of mice subjected to acute graft-versus-host disease (aGVHD). aGVHD was induced by the transplantation of bone marrow cells and splenocytes from C57BL-6j to Balb-c mice. The recipient mice were treated daily with CBD, and the treatment reduced mouse mortality by decreasing inflammation and injury and promoting immune regulation in the jejunum, ileum, and liver. Analysis of the jejunum and ileum showed that CBD treatment reduced the levels of C-C motif chemokine ligand (CCL) 2, CCL3, CCL5, tumor necrosis factor α, and interferon γ (IFNγ). CCL3 and IFNγ levels were also decreased in the liver. Mechanistically, CBD also increased the number of cannabinoid receptor type 2 (CB2) receptors on CD4+ and forkhead box P3+ cells in the intestine, which may explain the reduction in proinflammatory cytokines and chemokines. Antagonists of the CB2 receptor reduced the survival rates of CBD-treated mice, suggesting the participation of this receptor in the effects of CBD. Furthermore, treatment with CBD did not interfere with the graft-versus-leukemia response. CBD treatment appears to protect aGVHD mice by anti-inflammatory and immunomodulatory effects partially mediated by CB2 receptor interaction. Altogether, our study suggests that CBD represents an interesting approach in the treatment of aGVHD, with potential therapeutic applications in patients undergoing bone marrow transplantation. SIGNIFICANCE STATEMENT: This study provides for the first time a mechanism by which cannabidiol, a phytocannabinoid with no psychoactive effect, induces immunomodulation in the graft-versus-host disease. Enhancing intestinal cannabinoid receptor type 2 (CB2) receptor expression on CD4+ and forkhead box P3+ cells and increasing the number of these regulatory cells, cannabidiol decreases proinflammatory cytokines and increases graft-versus-host disease mice survival. This effect is dependent of CB2 receptor activation. Besides, cannabidiol did not interfere with graft-versus-leukemia response, a central response to avoid primary disease relapse.

Evidence for the involvement of opioid and cannabinoid systems in the peripheral antinociception mediated by resveratrol.

Despite all the development of modern medicine, around 100 compounds derived from natural products were undergoing clinical trials only at the end of 2013. Among these natural substances in clinical trials, we found the resveratrol (RES), a pharmacological multi-target drug. RES analgesic properties have been demonstrated, although the bases of these mechanisms have not been fully elucidated. The aim of this study was to evaluate the involvement of opioid and cannabinoid systems in RES-induced peripheral antinociception. Paw withdrawal method was used and hyperalgesia was induced by carrageenan (200 µg/paw). All drugs were given by intraplantar injection in male Swiss mice (n = 5). RES (100 µg/paw) administered in the right hind paw induced local antinociception that was antagonized by naloxone, non-selective opioid receptor antagonist, and clocinnamox, µOR selective antagonist. Naltrindole and nor-binaltorfimine, selective antagonists for δOR and kOR, respectively, did not reverse RES-induced peripheral antinociception. CB1R antagonist AM251, but not CB2R antagonist AM630, antagonized RES-induced peripheral antinociception. Peripheral antinociception of RES intermediate-dose (50 µg/paw) was increased by: (i) bestatin, inhibitor of endogenous opioid degradation involved-enzymes; (ii) MAFP, inhibitor of anandamide amidase; (iii) JZL184, inhibitor of 2-arachidonoylglycerol degradation involved-enzyme; (iv) VDM11, endocannabinoid reuptake inhibitor. Acute and peripheral administration of RES failed to affect the amount of µOR, CB1R and CB2R. Experimental data suggest that RES induces peripheral antinociception through µOR and CB1R activation by endogenous opioid and endocannabinoid releasing.

Noradrenaline induces peripheral antinociception by endogenous opioid release.

BACKGROUND: The aim of this study was to investigate this involvement in not inflammatory model of pain and which opioid receptor subtype mediates noradrenaline-induced peripheral antinociception. Noradrenaline is involved in the intrinsic control of pain-inducing pro-nociceptive effects in the primary afferent nociceptors. However, inflammation can induce various plastic changes in the central and peripheral noradrenergic system that, upon interaction with the immune system, may contribute, in part, to peripheral antinociception. METHODS: Hyperalgesia was induced by intraplantar injection of prostaglandin E2 (PGE2, 2µg) into the plantar surface of the right hind paw and the paw pressure test to evaluated the hyperalgesia was used. Noradrenaline (NA) was administered locally into right hind paw of Wistar rat (160-200g) alone and after either agents, α2-adrenoceptor antagonist yohimbine, α1-adrenoceptor antagonist prazosin, ß-adrenoceptor antagonist propranolol, µ-opioid antagonist clocinnamox, δ-opioid antagonist naltrindole and κ-opioid antagonist nor-binaltorfimina. In addition, the enkephalinase inhibitor bestatin was administered prior to NA low dose. RESULTS: Intraplantar injection of NA induced peripheral antinociception against hyperalgesia induced by PGE2. This effect was reversed, in dose dependent manner, by intraplantar injection of yohimbine, prazosin, propranolol, clocinnamox and naltrindole. However, injection of nor-binaltorfimina did not alter antinociception of NA after PGE2 hyperalgesia. Bestatin intensified the antinociceptive effects of low-dose of NA. CONCLUSION: Besides the α2-adrenoceptor, the present data provide evidence that, in absence of inflammation, NA activating α1 and ß-adrenoceptor induce endogenous opioid release to produce peripheral antinociceptive effect by µ and δ opioid receptors.

Serotonin induces peripheral antinociception via the opioidergic system.

PURPOSE: Studies conducted since 1969 have shown that the release of serotonin (5-HT) in the dorsal horn of the spinal cord contributes to opioid analgesia. In the present study, the participation of the opioidergic system in antinociceptive effect serotonin at the peripheral level was examined. METHODS: The paw pressure test was used with mice (Swiss, males from 35 g) which had increased pain sensitivity by intraplantar injection of PGE2 (2 µg). Serotonin (250 ng), administered locally to the right paw of animals, produces antinociception in this model. RESULTS: The selective antagonists for mu, delta and kappa opioid receptors, clocinnamox clocinnamox (40 µg), naltrindole (60 µg) and nor-binaltorfimina (200 µg), respectively, inhibited the antinociceptive effect induced by serotonin. Additionally, bestatin (400 µg), an inhibitor of enkephalinases that degrade peptides opioids, enhanced the antinociceptive effect induced by serotonin (low dose of 62.5 ng). CONCLUSIONS: These results suggest that serotonin possibly induce peripheral antinociception through the release of endogenous opioid peptides, possible from immune cells or keratinocytes.

Inhibition of 5-lipoxygenase alleviates graft-versus-host disease.

Leukotriene B4 (LTB4), a proinflammatory mediator produced by the enzyme 5-lipoxygenase (5-LO), is associated with the development of many inflammatory diseases. In this study, we evaluated the participation of the 5-LO/LTB4 axis in graft-versus-host disease (GVHD) pathogenesis by transplanting 5-LO-deficient leukocytes and investigated the effect of pharmacologic 5-LO inhibition by zileuton and LTB4 inhibition by CP-105,696. Mice that received allogeneic transplant showed an increase in nuclear 5-LO expression in splenocytes, indicating enzyme activation after GVHD. Mice receiving 5-LO-deficient cell transplant or zileuton treatment had prolonged survival, reduced GVHD clinical scores, reduced intestinal and liver injury, and decreased levels of serum and hepatic LTB4 These results were associated with inhibition of leukocyte recruitment and decreased production of cytokines and chemokines. Treatment with CP-105,696 achieved similar effects. The chimerism or the beneficial graft-versus-leukemia response remained unaffected. Our data provide evidence that the 5-LO/LTB4 axis orchestrates GVHD development and suggest it could be a target for the development of novel therapeutic strategies for GVHD treatment.

κ-Opioid receptor participates of NSAIDs peripheral antinociception.

NSAIDs represent some of the most widely prescribed drugs for relief of short-term fever, pain and inflammation. The participation of the opioid system in the peripheral is poorly understood. The aim of this study was evaluate the role of opioid system in the peripheral antinociception by diclofenac and dipyrone. To test this hypothesis, opioid receptor antagonists were evaluated using the rat paw pressure test, in which pain sensitivity is increased by intraplantar injection of prostaglandin E2 (PGE2, 2µg). Diclofenac (20µg/paw) and Dipyrone (40µg/paw) administered locally into the right paw elicited an antinociceptive effect. It was used naloxone (50µg/paw), a non-selective opioid receptor antagonist, which antagonized peripheral antinociception induced by diclofenac and dipyrone. Selectively, it was evaluated the µ-, δ- and κ-opioid receptor antagonists, respectively, clocinnamox (40µg/paw), naltrindole (50µg/paw) and nor-binaltorphimine (20, 40 and 80µg/paw). Our data indicated that only the κ-opioid antagonist was capable to reverse the peripheral antinociception by NSAIDs. The present results provide evidence that the opioid system participated in the diclofenac and dipyrone-induced peripheral antinociception by indirect activation of κ-opioid receptor probable by release of endogenous opioids such as dynorphins.

Natural Diterpenes from Coffee, Cafestol, and Kahweol Induce Peripheral Antinoceception by Adrenergic System Interaction.

Cafestol and kahweol are diterpenes found only in the non-saponified lipid fraction of coffee. They are released during boiling and retained in the filtration process. Previous studies have shown peripheral antinociception induced by endogenous opioid peptides released by these diterpenes. Considering that the activation of the opioid system leads to a noradrenaline release, the aim of this study was to verify the participation of the noradrenergic system in the peripheral antinociception induced by cafestol and kahweol. Hyperalgesia was induced by an intraplantar injection of prostaglandin E2 (2 µg). Cafestol or kahweol (80 µg/paw) were administered locally into the right hindpaw alone, and after the agents α 2-adrenoceptor antagonist yohimbine (5, 10 and 20 µg/paw), α 2 A-adrenoceptor antagonist BRL 44 408 (40 µg/paw), α 2B-adrenoceptor antagonist imiloxan (40 µg/paw), α 2 C-adrenoceptor antagonist rauwolscine (10, 15 and 20 µg/paw), α 2D-adrenoceptor antagonist RX 821 002 (40 µg/paw), α 1-adrenoceptor antagonist prazosin (0.5, 1 and 2 µg/paw), or ß-adrenoceptor antagonist propranolol (150, 300 and 600 ng/paw), respectively. Noradrenaline reuptake inhibitor reboxetine (30 µg/paw) was administered prior to cafestol or kahweol low dose (40 µg/paw) and guanetidine 3 days prior to the experiment (30 mg/kg, once a day), depleting the noradrenaline storage. Intraplantar injection of cafestol or kahweol (80 µg/paw) induced a peripheral antinociception against hyperalgesia induced by PGE2. This effect was reversed by intraplantar injections of yohimbine, rauwolscine, prazosin and propranolol. Reboxetine injection intensified the antinociceptive effect of cafestol or kahweol low-dose, and guanethidine reversed almost 70 % of the cafestol or kahweol-induced peripheral antinociception. This study gives evidence that the noradrenergic system participates in cafestol and kahweol-induced peripheral antinociception with the release of endogenous noradrenaline.