Celina M.C. Lotufo

Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/KATP signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kγ/AKT protein kinase B (AKT) and culminated in increased activation of KATP channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Melatonin and N-acetylserotonin inhibit leukocyte rolling and adhesion to rat microcirculation

The hormone melatonin produced by the pineal gland during the daily dark phase regulates a variety of biological processes in mammals. The aim of this study was to determine the effect of melatonin and its precursor N-acetylserotonin on the microcirculation during acute inflammation. Arteriolar diameter, blood flow rate, leukocyte rolling and adhesion were measured in the rat microcirculation in situ by intravital microscopy. Melatonin alone or together with noradrenaline did not affect the arteriolar diameter or blood flow rate. Melatonin inhibited both leukocyte rolling and leukotriene B(4) induced adhesion while its precursor N-acetylserotonin inhibits only leukocyte adhesion. The rank order of potency of agonists and antagonist receptor selective ligands suggested that the activation of MT(2) and MT(3) melatonin binding sites receptors modulate leukocyte rolling and adhesion, respectively. The effect of melatonin and N-acetylserotonin herein described were observed with concentrations in the range of the nocturnal surge, providing the first evidence for a possible physiological role of these hormones in acute inflammation.

Fractalkine mediates inflammatory pain through activation of satellite glial cells

The activation of the satellite glial cells (SGCs) surrounding the dorsal root ganglion (DRG) neurons appears to play a role in pathological pain. We tested the hypothesis that fractalkine, which is constitutively expressed by primary nociceptive neurons, is the link between peripheral inflammation and the activation of SGCs and is thus responsible for the genesis of the inflammatory pain. The injection of carrageenin into the rat hind paw induced a decrease in the mechanical nociceptive threshold (hypernociception), which was associated with an increase in mRNA and GFAP protein expression in the DRG. Both events were inhibited by anti-fractalkine antibody administered directly into the DRG (L5) [intraganglionar (i.gl.)]. The administration of fractalkine into the DRG (L5) produced mechanical hypernociception in a dose-, time-, and CX3C receptor-1 (CX3CR1)–dependent manner. Fractalkine’s hypernociceptive effect appears to be indirect, as it was reduced by local treatment with anti–TNF-α antibody, IL-1–receptor antagonist, or indomethacin. Accordingly, the in vitro incubation of isolated and cultured SGC with fractalkine induced the production/release of TNF-α, IL-1β, and prostaglandin E2. Finally, treatment with i.gl. fluorocitrate blocked fractalkine (i.gl.)- and carrageenin (paw)-induced hypernociception. Overall, these results suggest that, during peripheral inflammation, fractalkine is released in the DRG and contributes to the genesis of inflammatory hypernociception. Fractalkine’s effect appears to be dependent on the activation of the SGCs, leading to the production of TNFα, IL-1β, and prostanoids, which are likely responsible for the maintenance of inflammatory pain. Thus, these results indicate that the inhibition of fractalkine/CX3CR1 signaling in SGCs may serve as a target to control inflammatory pain.

Stimulation of peripheral kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3Kγ/AKT/nNOS/NO signaling pathway.

BackgroundIn addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral μ-opioid receptor (MOR) activation are able to direct block PGE2-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE2-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated.ResultsLocal (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE2-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3Kγ/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3Kγ null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3Kγ (≅ 43%).ConclusionsThe present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3Kγ/AKT signaling. This study extends a previously study of our group suggesting that PI3Kγ/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.

Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions

We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells.

Inflammatory sensitization of nociceptors depends on activation of NMDA receptors in DRG satellite cells

Significance The present study provides evidence for a role of glutamate as a neuromodulator of the afferent nociceptive information. Our results show that the nociceptive impulse generated by an inflammatory event in the peripheral tissue is regulated in the dorsal root ganglia (DRG) by a system that involves satellite glial cells and glutamatergic NMDA receptors. To our knowledge, this work is one of the first demonstrations of the involvement of glutamate in a modulatory process in the DRG, a site where there are no synapses, in addition to its classical role as a neurotransmitter. The present study evaluated the role of N-methyl-d-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E2 (PGE2), which was significantly attenuated by injection of the NMDAR antagonist d(-)-2-amino-5-phosphonopentanoic acid (d-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE2-induced hyperalgesia in thepaw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE2-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE2-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.

Peripheral antinociceptive effect of pertussis toxin: activation of the arginine/NO/cGMP/PKG/ ATP‐sensitive K+ channel pathway

The aim of the present study was to determine the effect of pertussis toxin (PTX) on inflammatory hypernociception measured by the rat paw pressure test and to elucidate the mechanism involved in this effect. In this test, prostaglandin E2 (PGE2) administered subcutaneously induces hypernociception via a mechanism associated with neuronal cAMP increase. Local intraplantar pre‐treatment (30 min before), and post‐treatment (5 min after) with PTX (600 ng/paw1, in 100 µL) reduced hypernociception induced by prostaglandin E2 (100 ng/paw, in 100 µL, intraplantar). Furthermore, local intraplantar pre‐treatment (30 min before) with PTX (600 ng/paw, in 100 µL) reduced hypernociception induced by DbcAMP, a stable analogue of cAMP (100 µg/paw, in 100 µL, intraplantar), which indicates that PTX may have an effect other than just Gi/G0 inhibition. PTX‐induced analgesia was blocked by selective inhibitors of nitric oxide synthase (L‐NMMA), guanylyl cyclase (ODQ), protein kinase G (KT5823) and ATP‐sensitive K+ channel (Kir6) blockers (glybenclamide and tolbutamide). In addition, PTX was shown to induce nitric oxide (NO) production in cultured neurons of the dorsal root ganglia. In conclusion, this study shows a peripheral antinociceptive effect of pertussis toxin, resulting from the activation of the arginine/NO/cGMP/PKG/ATP‐sensitive K+ channel pathway.

Adrenal deficiency alters mechanisms of neutrophil mobilization

Deficiency of adrenal hormones promotes exacerbated neutrophil influx into inflammatory sites. We investigated the effect of adrenal deficiency on neutrophil mobilization comparing adrenalectomized (ADX) male Wistar rats to sham-operated (SO) or non-manipulated (N) animals, as controls. Seven days after surgeries, the number of neutrophils in peripheral blood was increased in ADX rats, by accelerating neutrophil maturation steps in the bone marrow. The investigation of adhesive properties on neutrophil membranes indicated reduced and increased expressions of L-selectin on cells present in the bone marrow and circulating blood, respectively. Similar levels of L-selectin mRNA in both cells from ADX or non-manipulated rats suggest that these effects do not depend on gene expression. Even though no differences in the expression of beta(2) integrin by neutrophils were detected, modulation on subsequent PMN activation may occur by adrenal hormones, since circulating neutrophils from ADX exhibit lower in vitro adherence to the endothelium. We conclude that adrenal hormones control the adhesive interactions of neutrophils with the bone marrow microenvironment and with the vascular endothelium chiefly by modulation of L-selectin on PMN membrane in a mechanism independent of L-selectin gene expression.

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 μL) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1β (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1β. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1β in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1+ cells of the DRG, significantly increased after carrageenan or IL-1β administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKCε expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 µg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1β in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.

261 THE PI3Kg/AKT SIGNALING PATHWAY MEDIATES PERIPHERAL ANTINOCICEPTIVE ACTION OF DIPYRONE

Methods: Male SD rats were subjected to implantations of PE-10 catheter into the lumbar subarachnoid space for drug injection. Five days after surgery, vehicles (normal saline or DMSO), or the selected drugs (MC, SB or AG) in 10mL vehicle were injected 10 min prior to BIC (10 mg). We assessed the degree of TA (graded 0, no response; 1, mild response; 2, moderate response; 3, strong response) at every 5 min for 30 min. Results were analyzed using one-way ANOVA followed by a Tukey test for multiple comparisons (P< 0.05). Results: Rats showed no TA with IT normal saline, DMSO, MC, SB or AG alone. IT BIC-induced strong TA reached its peak and kept plateau until 20 min. Pretreatment IT MC, SB and AG significantly attenuated BIC-induced TA in dose dependent manner. Conclusions: IT BIC induces functional loss of GABA(A) receptors, which inhibit the presynaptic glutamate release and hyperpolarize the postsynaptic neuron, result in neuronal synaptic hyperexcitation and TA. This BIC-induced TA could be facilitated by the neuro-microglia interaction that was probably regulated by p38MAPK downstream signaling in the spinal microglia.