Cannabinoid receptor-specific mechanisms to alleviate pain in sickle cell anemia via inhibition of mast cell activation and neurogenic inflammation

Key Points Inhibition of mast cells with cromolyn or imatinib results in reduced systemic inflammation and neurogenic inflammation in sickle mice. Pharmacological inhibition or genetic depletion of mast cells in sickle mice ameliorates chronic and hypoxia/reoxygenation-induced pain.

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Mast Cell Activation Contributes to Neurogenic Inflammation and Pain in Sickle Cell Disease

Blood ◽

10.1182/blood.v120.21.374.374 ◽

2012 ◽

Vol 120 (21) ◽

pp. 374-374

Author(s):

Lucile Vincent ◽

Julia Nguyen ◽

Derek Vang ◽

Oludare B Taiwo ◽

Kathryn Luk ◽

...

Keyword(s):

Mast Cell ◽

Cell Activation ◽

Neurogenic Inflammation ◽

Control Mouse ◽

Mast Cell Degranulation ◽

Mast Cell Activation ◽

Plasma Extravasation ◽

Cell Disease ◽

Gm Csf ◽

Skin Biopsies

Abstract Abstract 374 Sickle cell disease (SCD) is associated with inflammation, endothelial dysfunction and pain. We observed increased immunoreactivity (ir) of pro-inflammatory and vasoactive neuropeptides, substance P (SP) and calcitonin-gene related peptide (CGRP) accompanied by decreased mu opioid receptor (MOR)-ir in the skin of sickle as compared to control mice (Kohli et al., Blood 2010). SP activates mast cells (MC), which are tissue resident leukocytes, leading to the release of inflammatory cytokines, tryptase and neuropeptides. SP also stimulates vascular permeability resulting in plasma extravasation and neurogenic inflammation. We hypothesized that pain in SCD is associated with a persistent feed-forward cycle of mast cell degranulation and neurogenic inflammation characterized by increased release of SP and CGRP from activated nociceptors in the skin leading to neuroinflammation, plasma extravasation and pain. We examined this hypothesis using sickle (HbSS-BERK) and control (HbAA-BERK) mice expressing sickle and normal human hemoglobin, respectively; and MOR-knockout (MOR-KO) mice with their wild type 129S6 controls. We developed an ex-vivo system to analyze the release of inflammatory cytokines, mast cell degranulation markers (tryptase and beta-hexosaminidase) and neuropeptides in skin biopsies. Neurogenic inflammation was studied in vivo using the Miles' assay. Evans blue was injected into the tail vein and its extravasation in skin evoked by stimulation with SP and capsaicin was quantified. Skin biopsies from sickle mice exhibited constitutively enhanced release of several cytokines (IL6, MCP-1, TNFalpha, MIP-1alpha, GM-CSF, RANTES, etc), tryptase and the neuropeptides SP, and CGRP as compared to control mouse skin (p<0.05 for each). Increased RANTES and GM-CSF are suggestive of mast cell recruitment. Mast cell tryptase-ir was increased 2-fold while MOR-ir (but not delta- or kappa-OR-ir), was reduced by ∼50% in the skin of sickle as compared to control mice, suggestive of enhanced MC degranulation in sickle. In MC cultures prepared from sickle skin increased c-kit/CD117-, FCeR- and tryptase-ir were observed as compared to control mouse MCs. The plasma of sickle exhibited a ∼60–80% increase in MC degranulation markers, tryptase and beta-hexosaminidase, acute phase protein, serum amyloid protein, and neuropeptides, SP and CGRP, as compared to control mice (p<0.01 for each). These correlative molecular changes in the plasma and skin were accompanied by increased SP- and capsaicin-induced Evans blue dye leakage in the skin of sickle mice suggestive of neurogenic inflammation as compared to control (p<0.001 for each). MOR-KO mice also exhibited increased SP- and CGRP-ir in the skin and neurogenic inflammation, indicative of a contribution by MOR to the neuroinflamamtory process. In sickle mice treated with the mast cell stabilizer cromolyn sodium (CS), or the c-kit inhibitor, Imatinib, for 5 days, the inflammatory cytokine and neuropeptide release from the skin and the neurogenic inflammation were ameliorated as compared to vehicle (p<0.01). Additionally, morphine at a dose of 10 mg/Kg was ineffective in treating tonic cutaneous and thermal hyperalgesia, but effectively reduced hyperalgesia in CS and Imatinib treated sickle mice. Thus, MC degranulation contributes to neurogenic inflammation and pain in sickle mice. Imatinib treatment by itself reduced tonic hyperalgesia and significantly decreased GM-CSF release from the skin (p<0.05) correlative to the reduced white blood cell (WBC) count in sickle mice vs vehicle. In addition to inhibiting MC activity, Imatinib may be inhibiting protein tyrosine kinases involved in cytokine processing, vascular function and nociception. Together, our observations demonstrate that MCs contribute to a vicious cycle of pain and neurogenic inflammation mediated by increased neuropeptides in SCD. It is likely that mast cell inhibitors such as Imatinib may have a therapeutic effect on pain, inflammation and vascular dysfunction in SCD by reducing mast cell activation and neurogenic inflammation. Since, Imatinib decreased GM-CSF levels and WBC, it may even increase HbF levels, which are negatively regulated by GM-CSF in SCD. We therefore speculate that therapies targeting mast cells may potentiate therapeutic outcomes of analgesics, anti-inflammatory agents and Hydroxyurea® in SCD. Disclosures: No relevant conflicts of interest to declare.

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Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model

Annals of Dermatology ◽

10.5021/ad.2016.28.1.22 ◽

2016 ◽

Vol 28 (1) ◽

pp. 22 ◽

Cited By ~ 15

Author(s):

Gaewon Nam ◽

Se Kyoo Jeong ◽

Bu Man Park ◽

Sin Hee Lee ◽

Hyun Jong Kim ◽

...

Keyword(s):

Atopic Dermatitis ◽

Mast Cell ◽

Cell Activation ◽

Cannabinoid Receptor ◽

Mast Cell Activation ◽

Cannabinoid Receptor 1

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Mast Cell Activation Syndrome as a Significant Comorbidity in Sickle Cell Disease

The American Journal of the Medical Sciences ◽

10.1097/maj.0000000000000325 ◽

2014 ◽

Vol 348 (6) ◽

pp. 460-464 ◽

Cited By ~ 10

Author(s):

Lawrence B. Afrin

Keyword(s):

Mast Cell ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Cell Activation ◽

Mast Cell Activation ◽

Cell Disease ◽

Mast Cell Activation Syndrome ◽

Activation Syndrome

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Natural α,β-unsaturated lactones inhibit neuropeptide-induced mast cell activation in an in vitro model of neurogenic inflammation

Inflammation Research ◽

10.1007/s00011-020-01380-8 ◽

2020 ◽

Vol 69 (10) ◽

pp. 1039-1051

Author(s):

Roberto Carlos Coll ◽

Patricia María Vargas ◽

María Laura Mariani ◽

Alicia Beatriz Penissi

Keyword(s):

Mast Cell ◽

Cell Activation ◽

Neurogenic Inflammation ◽

In Vitro Model ◽

Mast Cell Activation ◽

Vitro Model ◽

Unsaturated Lactones

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Characteristics and Potential Biomarkers for Chronic Pain in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood.v126.23.986.986 ◽

2015 ◽

Vol 126 (23) ◽

pp. 986-986 ◽

Cited By ~ 11

Author(s):

Nina Kuei ◽

Niren Patel ◽

Hongyan Xu ◽

Leigh Wells ◽

Latanya Bowman ◽

...

Keyword(s):

Chronic Pain ◽

Mast Cell ◽

Sickle Cell Disease ◽

Substance P ◽

Cell Activation ◽

Neurogenic Inflammation ◽

Mast Cell Activation ◽

Cell Disease ◽

Opioid Use ◽

Pressure Pain

Abstract Vaso-occlusive episodes (VOE) or pain crises are a hallmark of sickle cell disease (SCD), with increasing recognition that a significant portion of SCD patients develop chronic pain. In the landmark PiSCES study (Smith et al), patients reported pain on 55% days, with ~30% reporting pain on >90% days. Thus, the episodic, nociceptive pain (VOE) in younger patients, evolves into a chronic pain syndrome, with neuropathic and centralized components in some adults. Kutlar et al (Blood, 2014), reported on the association of different pain related phenotypes (pain diaries, frequency of hospitalizations/ED visits, pressure pain threshold) with polymorphisms in candidate genes in 167 SCD patients, providing evidence that multiple signaling pathways and mechanisms are likely involved. In this study, 12 SCD subjects with "chronic pain", defined by reported pain >50% of days in pain diaries collected over 6 months, were enrolled (SCD-CP). 17 SCD patients who did not have chronic pain (SCD-NCP), and 9 non-SCD African-Americans (C) were enrolled as controls. Informed consent was obtained. Age, gender, Hb F levels, HU usage, and pressure pain algometer readings were recorded from SCD subjects. 8 ml of blood (EDTA) was collected from subjects at "steady state" and from normal controls. Plasma was separated and kept at -80 C until the assay. Plasma tryptase and Substance P levels were assayed by ELISA using kits from Biomatik, Inc. (catalog # EKU07922) and Enzo Life Sciences (Catalog #ADI-900-018), respectively. SCD-CP patients were significantly older than SCD-NCP: mean age 41 vs 32.2 (p=0.033). The pressure pain algometer readings did not differ significantly between SCD-CP and SCD-NCP at three sites (trapezius, ulna, masseter, p= 0.67-0.74). There were 12/17 patients on HU (70.6%) among SCD-NCP, and 6/12 (50%) among SCD-CP (p=0.435). Similarly, Hb F levels were not significantly different (14.7% in SCD-CP, vs 11.7% in SCD-NCP, p=0.446). Opioid use (average morphine equivalent as mg/day) was significantly higher in the chronic pain group (11.45 mg/day, vs 2.92 mg/day, p=0.015). Plasma tryptase and substance-P levels are shown in the table: Table 1. Tryptase (pg/ml) Substance-P (pg/ml) SCD-CP 1388.6 ±519.8 7221.1±7742.7 SCD-NCP 1023.64±221.04 5983.1±3473.0 Control 768.9±416.16 3939.7±1350.1 The difference in substance-P levels did not reach significance across groups by ANOVA (p=0.337) or in pairwise comparison between groups. However, tryptase levels were significantly different across groups by ANOVA (p=0.00615). Pairwise comparisons between two groups showed that tryptase levels were significantly different between SCD-CP and controls (p=0.0053). These results highlight characteristics of SCD patients with chronic pain: they are older, have a higher use of opioids, and have significantly higher tryptase levels. These observations support previous findings that age is a significant factor in transition to chronic pain in SCD. Higher dose of opioid use in SCD-CP could result from dose escalation to control pain; conversely, it could be argued that higher opioid use itself could be a factor in development of chronic pain through opioid-induced hyperalgesia. To our knowledge, this is the first study of plasma tryptase levels in SCD, in relation to different pain phenotypes. Tryptase is released into plasma with degranulation of mast cells and leads to inflammation, anaphylaxis, urticaria, and neuropathic pain. It binds PAR2 (protease activated receptor 2), releasing inflammatory mediators and substance P, inducing neurogenic inflammation. Elevated tryptase levels are found in systemic mastocytosis, and the newly recognized Mast Cell Activation Syndrome (MCAS). Vincent et al (Blood, 2013) showed that mast cell activation played an important role in neurogenic inflammation and chronic pain in a mouse model of SCD. They also demonstrated that inhibition of mast cell activation, via c-kit knockout or with imatinib or cromolyn sodium improved neurogenic inflammation and chronic pain. Two recent case reports (Murphy et al, Stankovic et al) document significant improvement in pain in SCD patients who developed CML, during treatment with imatinib. These observations, and the findings of our pilot study, not only suggest a novel mechanism and biomarker for chronic pain in SCD, but also a potential therapeutic target by inhibition of mast cell activation via c-kit pathway, or stabilization with cromolyn. Disclosures No relevant conflicts of interest to declare.

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Neurogenic inflammation in mice deficient in heparin-synthesizing enzyme

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00917.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. H884-H888 ◽

Cited By ~ 5

Author(s):

Tine V. Karlsen ◽

Vegard V. Iversen ◽

Erik Forsberg ◽

Lena Kjellén ◽

Rolf K. Reed ◽

...

Keyword(s):

Mast Cell ◽

Cell Activation ◽

Neurogenic Inflammation ◽

Inflammatory Responses ◽

Fluid Pressure ◽

Mouse Skin ◽

Mast Cell Activation ◽

Protein Extravasation ◽

Plasma Protein Extravasation ◽

Rats And Mice

Mast cell activation, or neurogenic inflammation, is known to induce lowering of interstitial fluid pressure (Pif) and plasma protein extravasation (PPE) in several tissues from both rats and mice. To examine a possible role of connective tissue mast cells (CTMCs) in these inflammatory responses, we used mice with dysfunctional CTMCs due to lack of the N-deacetylase/ N-sulfotransferase-2 enzyme (NDST-2–/–). Pif and PPE were measured after challenge with compound 48/80 (C48/80), and Pif alone was measured after treatment either with capsaicin, substance P (SP), or calcitonin gene-related peptide (CGRP). Measurements of Pif in anesthetized (fentanyl/fluanison and midazolam, 1:1) mice were performed in paw skin with glass capillaries connected to a servo-controlled counterpressure system. PPE was measured with microdialysis by using hollow plasmapheresis fibers (cutoff at 3,000 kDa) placed subcutaneously on the back. Intravenous administration of C48/80 lowered Pif significantly ( P < 0.05) in NDST-2–/– mice (–1.67 ± 0.42 mmHg) compared with vehicle (–0.57 ± 0.17 mmHg) but the lowering was significantly ( P < 0.05) less compared with that of the NDST-2+/+ mice (–2.31 ± 0.47 mmHg). PPE was increased 300% after treatment with C48/80 in NDST-2+/+ mice, whereas there was no increase in PPE in NDST-2–/– mice. Capsaicin, SP, and CGRP lowered Pif significantly ( P < 0.05) compared with vehicle and to the same extent in both NDST-2+/+ and NDST-2–/– mice. We can conclude that although NDST-2–/– mice demonstrate an altered response in Pif after mast cell activation, there was no similar alteration after neurogenic inflammation. Therefore, we suggest that neurogenic inflammation in mouse skin is not exclusively dependent on intact CTMCs.

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Acupuncture Led Attenuation of Inflammatory and Nociceptive Peripheral and Central Microenvironment in Sickle Mice

Blood ◽

10.1182/blood.v126.23.2169.2169 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2169-2169

Author(s):

Ying Wang ◽

Jianxun Lei ◽

Yann Y Lamarre ◽

Ritu Jha ◽

Fei Peng ◽

...

Keyword(s):

Spinal Cord ◽

Mast Cell ◽

Pain Relief ◽

Substance P ◽

Inflammatory Response ◽

Cell Activation ◽

Culture Medium ◽

Neurogenic Inflammation ◽

Mast Cell Activation ◽

Skin Biopsies

Abstract Background: Inflammation, neurogenic inflammation and pain remain challenging to treat in sickle cell disease (SCD). Alternative therapies including acupuncture have been used for centuries to reduce pain and ameliorate underlying pathobiology of many disorders. We examined the mechanisms underlying acupuncture therapy in sickle mice. To prevent the influence of anesthetics and constraint on the pathobiology we developed electroacupuncture (EA) treatment for awake/conscious freely moving mice to simulate treatment conditions in patients, and then examined the peripheral and central mechanisms of neuroinflammation and nociception. Methods: HbSS-BERK sickle and HbAA-BERK control mice were treated with four EA treatments (every 3rd day, frequency: 4 or 10 Hz, pulse width: 100 microsecond, duration: 30 min) at acupoint GB30. Untreated and sham-EA treated (acupuncture without electrical stimulation) were used as controls. Hyperalgesia was evaluated daily by determining mechanical threshold, deep tissue hyperalgesia and thermal hyperalgesia using von Frey filaments, grip force, and cold plate, respectively. Blood and tissues were collected for analysis after four sessions of treatment. Skin biopsies were incubated overnight and culture medium was analyzed for mast cell activation marker tryptase, and neuromodulatory marker substance P. Results: Varied analgesic response to EA treatment was observed in sickle mice. About 86% treated mice equally showed positive (>50% pain relief) or moderate (20-30% pain relief) response and 14% were non-responsive (<20% pain relief) to EA. In positive responders, EA significantly reduced white blood cells (p<.001 Vs moderate- and non-responders), serum amyloid protein (p<.01 Vs untreated), IL-1beta (p<.05 Vs untreated, p<.01 Vs non-responders), and substance P (p<.05 Vs untreated and p<.001 Vs non-responders and p<.05 Vs moderate-responders). Concurrently, spinal cord analysis of EA treated positive-responders showed reduced substance P (p<.05 Vs untreated and non-responders), IL-1 beta (p<.01 Vs untreated), TNF alpha (p<.05 and p<.01 Vs moderate- and non-responders, respectively). Consistent with this central and peripheral anti-inflammatory response, culture medium from skin biopsies of positive responders demonstrated reduced substance P (p<.01 Vs moderate- and non-responders) and tryptase (p<.01 Vs untreated, moderate- and non-responders), and significantly less toluidine blue stained degranulating mast cells in the skin (p<.05 Vs untreated and non-responders) suggestive of attenuation of mast cell and peripheral nervous system activation. Functionally, capsaicin and substance P-induced neurogenic inflammation were significantly attenuated in positive-responders vs non-responders (p<.05) or untreated (p<.05). Peripheral and central attenuation of inflammatory and neurogenic response to EA was accompanied by inhibition of nociceptive signaling in the spinal cord. Spinal phosphorylation of p38 MAPK decreased in EA treated mice (p<.05 Vs sham-EA and untreated control; and positive-responders Vs non-responders). Conclusions: EA treatment on conscious free-moving mice simulates clinical conditions in patients and excludes the potential influence due to restraint or anesthetics. EA leads to peripheral and central neuromodulation and anti-inflammatory response by attenuating mast cell activation, substance P, and cytokine release in the periphery and by abrogating spinal nociceptive signaling of p38MAPK and inflammation. Together, these molecular and cellular effects lead to EA-induced attenuation of neurogenic inflammation and hyperalgesia in sickle mice. Importantly, these data explain the cause of variable effectiveness of EA in SCD. Disclosures No relevant conflicts of interest to declare.

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Mast Cells Contribute to Brain Microvascular Permeability in Sickle Cell Disease

Blood ◽

10.1182/blood-2018-99-117246 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 269-269 ◽

Cited By ~ 2

Author(s):

Aditya M Mittal ◽

Huy Tran ◽

Varun Sagi ◽

Aithanh Nguyen ◽

Kathryn Luk ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Er Stress ◽

Sickle Cell ◽

Cell Activation ◽

Cell Activity ◽

Mast Cell Activation ◽

Bbb Permeability ◽

The Brain

Abstract Mast cells are in close proximity to the vasculature and cause endothelial activation, plasma extravasation, and vascular dysfunction (Gupta & Harvima, Immunol Rev 2018). Vascular dysfunction in sickle cell disease (SCD) is accompanied by increased expression of P-selectin. Treatment with Crizanlizumab, an antibody against P-selectin, led to significantly less sickle cell-related pain crises (Ataga et al., NEJM 2017) highlighting the role of endothelial P-selectin in vasoocclusive crises (VOC). Earlier studies demonstrated that mast cell activation with morphine or ischemia/reperfusion stimulates endothelial E- and/or P-selectin expression. However, it is unknown how mast cells stimulate endothelial selectin expression in SCD. Endothelial dysfunction contributes significantly to the pathobiology of SCD including VOC and may play a critical role in increased blood-brain barrier (BBB) permeability, which may contribute to stroke, another major comorbidity of SCD. One of the known triggers of endothelial dysfunction, inflammation and oxidative stress is endoplasmic reticulum (ER) stress. We hypothesize that in a sickle microenvironment, mediators derived from activated mast cells stimulate endothelial P-selectin expression via ER stress leading to increased BBB permeability. We examined the ability of mast cells to stimulate P-selectin expression and BBB permeability via ER stress in a sickle microenvironment. We isolated MCs from HbAA-BERK and HbSS-BERK, control and sickle mice, respectively; incubated them in vitro and collected mast cell conditioned media (MCCM) from HbAA MCs and HbSS MCs. Normal mouse brain microvascular endothelial cells (mBMECs) were treated with unconditioned MCCM, HbAA MCCM, or HbSS MCCM to examine the effect of mast cell activation on endothelium. We observed increased mast cell activity in HbSS mice evinced by significantlyhigher plasma and skin histamine levels, compared to HbAA mice (p< 0.02 for both). Mast cells from HbSS mouse skin showed significantly increased expression of histamine compared to HbAA skin mast cells (p< 0.04). mBMECs incubated with HbAA and HBSS MCCM exhibited about 3- and 6-fold fold increases in P-selectin expression, compared to unconditioned culture medium, respectively (p< 0.0001 for both). Therefore, mast cells in culture release substances that stimulate P-selectin expression which is further increased by mast cells from sickle (HbSS) microenvironment. Preincubation of mBMEC with 5 microM salubrinal, an inhibitor of dephosphorylation of elongation initiation factor-a, which reduces ER stress, significantly inhibited HbSS MCCM-induced P-selectin expression on mBMEC (p< 0.0001) to the level induced by HbAA-MCCM. In contrast, salubrinal did not inhibit HbAA-MCCM-induced P-selectin expression on mBMEC, suggesting that in a sickle microenvironment mast cells contribute to P-selectin expression via ER stress. We next examined mast cell activity on endothelial permeability in vitro on mBMEC monolayers and in vivo in the brain of HbSS mice. mBMECs incubated with HbSS MCCM showed a significant increase in Evans blue leakage compared to unconditioned or HbAA MCCM (p<0.0001 for both), which was inhibited by preincubation of mBMEC with 5 microM salubrinal prior to incubation with HbSS MCCM (p< 0.0001). In vivo female HbSS mice showed a significantly increased leakage in the brain of FITC-dextran injected through tail vein compared to HbAA mice (p< 0.01). HbSS mice treated with 1 mg/kg salubrinal demonstrated inhibition of FITC-dextran leakage in the brain compared to vehicle (p< 0.05). Thus, ER stress contributes to increased BBB permeability in HbSS mice. We observed activated degranulating mast cells in the brain parenchyma of HbSS mice. In HbAA mice, quiescent mast cells were confined to the meninges of the brain but not seen in the parenchyma. Together, these data suggest that mast cell activation contributes to BBB permeability in a sickle microenvironment via ER stress-mediated P-selectin expression. In turn, this mast cell-initiated activity in the brain may underlie the pathobiology of stroke in SCD. Inhibitors of mast cells and P-selectin have been tested clinically leading to reduced VOC in SCD without known adverse events. Therefore, mast cell activation-induced P-selectin via ER stress may serve as a treatable target for reducing the risk of stroke in SCD. Disclosures Gupta: Novartis: Honoraria; Tau tona: Consultancy.

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XANTATINA INHIBE LA ACTIVACIÓN DE MASTOCITOS INDUCIDA POR NEUROPÉPTIDOS PRO-INFLAMATORIOS

Revista Argentina de Anatomía Clínica ◽

10.31051/1852.8023.v2.n1.13859 ◽

2016 ◽

Vol 2 (1) ◽

pp. 16-24

Author(s):

Patricia M. Vargas ◽

Elia Martino ◽

Teresa H. Fogal ◽

Carlos E. Tonn ◽

Alicia B. Penissi

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Substance P ◽

Cell Activation ◽

Neurogenic Inflammation ◽

Light And Electron Microscopy ◽

Serotonin Release ◽

Mast Cell Activation ◽

Morphological Studies ◽

Peritoneal Mast Cells

Los mastocitos son células del tejido conectivo que participan en la génesis y modulación de las respuestas inflamatorias. Previamente hemos demos-trado que xanthatina (xanthanólido sesquiterpeno aislado de Xanthium cavanillesii Schouw) inhibe la activación de mastocitos inducida por secretagogos experimentales. Sin embargo, se desconoce su efecto sobre la activación de mastocitos inducida por estímulos fisiopatológicos. Estos estímulos incluyen, entre otros, los neuropéptidos pro-inflamatorios sustancia P y neurotensina, responsables de una de las principales vías de inflamación neurogénica. El objetivo del presente trabajo fue estudiar el efecto de xanthatina sobre la activación de mastocitos inducida por sustancia P y neurotensina. Mastocitos peritoneales de rata se incubaron con: 1) PBS (basal); 2) sustancia P (100 µm); 3) neurotensina (50 µm); 4) xanthatina (8-320 µm)+sustancia P; 5) xanthatina (8-320 µm)+neurotensina. Se llevaron a cabo los siguientes estudios: análisis dosis-respuesta de la liberación de serotonina inducida por neuropéptidos proinflamatorios, vitalidad celular, morfología mastocitaria por microscopía óptica y electrónica, análisis de estabilidad de xanthatina por cromatografía en capa fina. Los ensayos de liberación de serotonina y los estudios morfológicos mostraron la efectividad de xanthatina para estabilizar mastocitos. El presente estudio provee la primer evidencia a favor de la hipótesis de que xanthatina inhibe la liberación de serotonina inducida por sustancia P y neurotensina a partir de mastocitos peritoneales. Este sesquiterpeno podría representar una nueva alternativa fármacológica en la regulación de la activación mastocitaria para el tratamiento de las inflamaciones neurogénicas. Mast cells are connective tissue cells involved in the genesis and modulation of inflammatory responses. We have previously shown that xanthatin (xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw) inhibits mast cell activation induced by experimental secretagogues. However, the effect of xanthatin on mast cell activation induced by pathophysiological stimuli remains unknown. These stimuli include, among others, the pro-inflammatory neuropeptide substance P and neurotensin, responsible for one of the main pathways of neurogenic inflammation. The present study was designed to examine the effects of xanthatin on mast cell activation induced by pro-inflammatory peptides, such as substance P and neurotensin. Rat peritoneal mast cells were incubated with: 1) PBS (basal); 2) substance P (100 µm); 3) neurotensin (50 µm); 4) xanthatin (8-320 µm)+substance P; 5) xanthatin (8-320 µm)+neurotensin. Concentration-response studies of mast cell serotonin release evoked by pro-inflammatory neuropeptides, evaluation of mast cell viability and morphology by light and electron microscopy, and drug stability analysis by thin layer chromatography were performed. Serotonin release studies, carried out together with morphological studies, showed the effectiveness of xanthatin to stabilize mast cells. The present study provides the first strong evidence in favour of the hypothesis that xanthatin inhibits substance P - and neurotensin-induced serotonin release from peritoneal mast cells. Our findings may provide an insight into the design of novel pharmacological agents which may be used to regulate the mast cell response in neurogenic inflammation.

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