Role of endothelin in the pathophysiology of migraine: A new view on an old player.

There is cumulating evidence that endothelin-1 (ET-1) may play a role in migraine, however controversial findings still impede a conclusion to be drawn. Herein we tested the hypothesis that endothelin ETB receptors are major contributors to migraine-like responses. ET-1, IRL-1620 (selective ETB receptor agonist) or CGRP were injected into the trigeminal ganglion (TG) of female Wistar rats, and the development of periorbital mechanical allodynia was assessed hourly with von Frey hairs. Twenty-four hours later, rats were exposed to an aversive light for 1 h, after which the reactivation of periorbital mechanical allodynia (indicating photic sensitivity) was assessed up to 4 h. Moreover, the effect of systemic Bosentan (ETA/ETB receptors antagonist) or the selective antagonists of ETA (BQ-123) and ETB (BQ-788) receptors injected into the TG were evaluated against CGRP-induced responses. ET-1 and IRL-1620 injection into the TG induced periorbital mechanical allodynia and photic sensitivity. Bosentan attenuated periorbital mechanical allodynia but failed to affect photic sensitivity induced by CGRP. Selective blockade of ETB receptors in the TG fully prevented the development of periorbital mechanical allodynia and photic sensitivity induced by CGRP, but ETA receptor blockade caused only a slight reduction of periorbital mechanical allodynia without affecting photic sensitivity. ETB receptor-operated mechanisms in the TG may contribute to migraine-like responses in female rats.

Blockade of peripheral endothelin receptors abolishes heat hyperalgesia and spontaneous nociceptive behavior in a rat model of facial cancer.

OBJECTIVE: To improve understanding of the pathophysiology of cancer-induced facial nociception, by evaluating the contribution of peripheral endothelin receptors in tumor-induced facial heat hyperalgesia, increased spontaneous grooming, as well as ongoing nociception in a rat model of facial cancer. DESIGN: The study was conducted using 396 rats. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rats' right vibrissal pad. Facial heat hyperalgesia and spontaneous grooming were assessed on day 6, while the conditioned place preference (CPP) test was performed on days 3-6 after tumor cells inoculation. Rats received local injections of the non-peptidic dual ETA/ETB endothelin receptors antagonist, bosentan (10 and 30 µg/50 µL), single or combined injections of peptidic ETA and ETB endothelin receptors antagonists (BQ-123 and BQ-788, at 20 ug/50 µL, each), or of lidocaine (1 mg/50 µl) and morphine (30 µg/50 µL). RESULTS: Bosentan, lidocaine and morphine local treatment all attenuated tumor-induced heat hyperalgesia (p < 0.05) and spontaneous facial grooming (p < 0.05). However, BQ-123 and BQ-788 did not modify tumor-induced heat hyperalgesia or the spontaneous facial grooming (p > 0.05). Whether this difference in effectiveness is due to receptor affinity or to pharmacokinetic factors still needs to be explored. Local injection of bosentan, lidocaine or morphine failed to control ongoing nociception, as evidenced by the absence of CPP in tumor-bearing rats (p > 0.05). CONCLUSION: Endothelins, acting through peripheral ETA and ETB receptors, may play a significant role on the development of heat hyperalgesia and increased spontaneous grooming associated to facial cancer in rats.

Facial hyperalgesia due to direct action of endothelin-1 in the trigeminal ganglion of mice.

OBJECTIVE: This study assessed the ability of endothelin-1 (ET-1) to evoke heat hyperalgesia when injected directly into the trigeminal ganglia (TG) of mice and determined the receptors implicated in this effect. The effects of TG ETA and ETB receptor blockade on alleviation of heat hyperalgesia in a model of trigeminal neuropathic pain induced by infraorbital nerve constriction (CION) were also examined. METHODS: Naive mice received an intraganglionar (i.g.) injection of ET-1 (0.3-3 pmol) or the selective ETB R agonist sarafotoxin S6c (3-30 pmol), and response latencies to ipsilateral heat stimulation were assessed before the treatment and at 1-h intervals up to 5 h after the treatment. Heat hyperalgesia induced by i.g. ET-1 or CION was assessed after i.g. injections of ETA R and ETB R antagonists (BQ-123 and BQ-788, respectively, each at 0.5 nmol). KEY FINDINGS: Intraganglionar ET-1 or sarafotoxin S6c injection induced heat hyperalgesia lasting 4 and 2 h, respectively. Heat hyperalgesia induced by ET-1 was attenuated by i.g. BQ-123 or BQ-788. On day 5 after CION, i.g. BQ-788 injection produced a more robust antihyperalgesic effect compared with BQ-123. CONCLUSIONS: ET-1 injection into the TG promotes ETA R/ETB R-mediated facial heat hyperalgesia, and both receptors are clearly implicated in CION-induced hyperalgesia in the murine TG system.

Blockade of endothelin receptors reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial carcinoma induced pain.

Pain reported by patients with head and neck cancer is characterized as persistent pain with mechanical allodynia. Pain management is inadequate for many patients, highlighting the need for improved therapies. We examined the hypothesis that the mixed endothelin ETA and ETB receptor antagonist, bosentan, reduces tumor-induced ongoing pain and evoked hypersensitivity in a rat model of facial cancer pain. Facial cancer was induced by inoculating a suspension of Walker-256 cells into the rat's right vibrissal pad. Tumor-bearing rats developed heat and tactile hypersensitivity along with increased spontaneous grooming behavior. Systemic morphine (2.5mg/kg, s.c.) blocked tumor-induced thermal and tactile hypersensitivity, with a lower dose (0.625mg/kg, s.c.) effective only against thermal hypersensitivity. Systemic bosentan blocked tumor-induced thermal hypersensitivity only at a high (300mg/kg, p.o.) dose, but failed to modify tactile hypersensitivity. Co-administration of the low doses of bosentan and morphine resulted in improved reduction of the tumor-induced heat and tactile hypersensitivity compared to either dose alone. Bosentan (100mg/kg, p.o.) reduced spontaneous grooming and induced conditioned place preference (CPP) selectively in tumor-bearing rats, suggesting that bosentan reduces tumor-induced ongoing pain at a lower dose than required to block tumor-induced hypersensitivity. This study provides evidence that endothelins may mediate tumor-induced ongoing pain and thermal hypersensitivity. In addition, bosentan enhanced morphine's effects on blocking tumor-induced heat and tactile hypersensitivity indicating that endothelin antagonists may be beneficial therapeutic targets that can be used to manage cancer-induced facial pain with opioid-sparing effects.

Mechanisms involved in facial heat hyperalgesia induced by endothelin-1 in female rats.

OBJECTIVE: Pronociceptive responses to endothelins in the trigeminal system seem to be mediated by ETA and ETB receptors, which have been shown to be expressed in neurons of the trigeminal ganglion of humans and rats. The present study aimed to evaluate the ability of endothelin-1 (ET-1) to induce facial heat hyperalgesia in female rats, the contribution of ETA and ETB receptors to this response, as well as the mechanisms underlying heat hyperalgesia induced by ET-1. DESIGN: ET-1 (100pmol/50µL) was injected into the upper lip and heat hyperalgesia was evaluated for up to 6h. Facial heat hyperalgesia induced by ET-1 was assessed in rats pre-treated locally with BQ-123 or BQ-788 (selective ETA and ETB receptor antagonists, respectively, 30nmol/50µL); BCTC (TRPV1 receptor antagonist; 300µg/50µL); anti-NGF (3µg/50µL); K252a (TrkA inhibitor, 1µg/50µL); or in rats that received intraganglionar resiniferatoxin injection (RTX, 200ng/10µL) to promote C-fibers ablation. RESULTS: ET-1 induced facial heat hyperalgesia that persisted up to 6h and was prevented by BQ-123, BQ-788 or by intraganglionar RTX injection. Likewise, local pre-treatment with BCTC abolished ET-1 induced facial heat hyperalgesia up to 3h. Local pre-treatment with anti-NGF or K252a was effective to prevent ET-1 induced heat hyperalgesia. CONCLUSIONS: In conclusion, ET-1 is able to induce heat hyperagelsia in trigeminal primary afferents of female rats, which is mediated by ETA and ETB receptors. Activation of TRPV1 receptors and NGF-signaling pathways may contribute to heat hyperalgesia induced by ET-1.

Transplantation of Human Skin-Derived Mesenchymal Stromal Cells Improves Locomotor Recovery After Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a devastating neurologic disorder with significant impacts on quality of life, life expectancy, and economic burden. Although there are no fully restorative treatments yet available, several animal and small-scale clinical studies have highlighted the therapeutic potential of cellular interventions for SCI. Mesenchymal stem cells (MSCs)-which are conventionally isolated from the bone marrow-recently emerged as promising candidates for treating SCI and have been shown to provide trophic support, ameliorate inflammatory responses, and reduce cell death following the mechanical trauma. Here we evaluated the human skin as an alternative source of adult MSCs suitable for autologous cell transplantation strategies for SCI. We showed that human skin-derived MSCs (hSD-MSCs) express a range of neural markers under standard culture conditions and are able to survive and respond to neurogenic stimulation in vitro. In addition, using histological analysis and behavioral assessment, we demonstrated as a proof-of-principle that hSD-MSC transplantation reduces the severity of tissue loss and facilitates locomotor recovery in a rat model of SCI. Altogether, the study provides further characterization of skin-derived MSC cultures and indicates that the human skin may represent an attractive source for cell-based therapies for SCI and other neurological disorders. Further investigation is needed to elucidate the mechanisms by which hSD-MSCs elicit tissue repair and/or locomotor recovery.

Lipoxin A4 inhibits microglial activation and reduces neuroinflammation and neuropathic pain after spinal cord hemisection.

BACKGROUND: Spinal cord injury (SCI) is a severe neurological disorder with many disabling consequences, including persistent neuropathic pain, which develops in about 40 % of SCI patients and is induced and sustained by excessive and uncontrolled spinal neuroinflammation. Here, we have evaluated the effects of lipoxin A4 (LXA4), a member of a unique class of endogenous lipid mediators with both anti-inflammatory and analgesic properties, on spinal neuroinflammation and chronic pain in an experimental model of SCI. METHODS: Spinal hemisection at T10 was carried out in adult male CD1 mice and Wistar rats. To test if LXA4 can reduce neuroinflammation and neuropathic pain, each animal received two intrathecal injections of LXA4 (300 pmol) or vehicle at 4 and 24 h after SCI. Sensitivity to mechanical stimulation of the hind paws was evaluated using von Frey monofilaments, and neuroinflammation was tested by measuring the mRNA and/or protein expression levels of glial markers and cytokines in the spinal cord samples after SCI. Also, microglia cultures prepared from murine cortical tissue were used to assess the direct effects of LXA4 on microglial activation and release of pro-inflammatory TNF-α. RESULTS: LXA4 treatment caused significant reductions in the intensity of mechanical pain hypersensitivity and spinal expression levels of microglial markers and pro-inflammatory cytokines induced by SCI, when compared to rodents receiving control vehicle injections. Notably, the increased expressions of the microglial marker IBA-1 and of the pro-inflammatory cytokine TNF-α were the most affected by the LXA4 treatment. Furthermore, cortical microglial cultures expressed ALX/FPR2 receptors for LXA4 and displayed potentially anti-inflammatory responses upon challenge with LXA4. CONCLUSIONS: Collectively, our results suggest that LXA4 can effectively modulate microglial activation and TNF-α release through ALX/FPR2 receptors, ultimately reducing neuropathic pain in rodents after spinal cord hemisection. The dual anti-inflammatory and analgesic properties of LXA4, allied to its endogenous nature and safety profile, may render this lipid mediator as new therapeutic approach for treating various neuroinflammatory disorders and chronic pain with only limited side effects.

Inhibition of spinal c-Jun-NH2-terminal kinase (JNK) improves locomotor activity of spinal cord injured rats.

Mitogen-activated protein kinases (MAPKs) have been implicated in central nervous system injuries, yet the roles within neurodegeneration following spinal cord injury (SCI) still remain partially elucidated. We aimed to investigate the changes in expression of the three MAPKs following SCI and the role of spinal c-jun-NH2-terminal kinase (JNK) in motor impairment following the lesion. SCI induced at the T9 level resulted in enhanced expression of phosphorylated MAPKs shortly after trauma. SCI increased spinal cord myeloperoxidase levels, indicating a local neutrophil infiltration, and elevated the number of spinal apoptotic cells. Intrathecal administration of a specific inhibitor of JNK phosphorylation, SP600125, given at 1 and 4h after SCI, reduced the p-JNK expression, the number of spinal apoptotic cells and many of the histological signs of spinal injury. Notably, restoration of locomotor performance was clearly ameliorated by SP600125 treatment. Altogether, the results demonstrate that SCI induces activation of spinal MAPKs and that JNK plays a major role in mediating the deleterious consequences of spinal injury, not only at the spinal level, but also those regarding locomotor function. Therefore, inhibition of JNK activation in the spinal cord shortly after trauma might constitute a feasible therapeutic strategy for the functional recovery from SCI.

Neuroprotective effects of lipoxin A4 in central nervous system pathologies.

Many diseases of the central nervous system are characterized and sometimes worsened by an intense inflammatory response in the affected tissue. It is now accepted that resolution of inflammation is an active process mediated by a group of mediators that can act in synchrony to switch the phenotype of cells, from a proinflammatory one to another that favors the return to homeostasis. This new genus of proresolving mediators includes resolvins, protectins, maresins, and lipoxins, the first to be discovered. In this short review we provide an overview of current knowledge into the cellular and molecular interactions of lipoxins in diseases of the central nervous system in which they appear to facilitate the resolution of inflammation, thus exerting a neuroprotective action.

Neuroprotective effect of the proanthocyanidin-rich fraction in experimental model of spinal cord injury.

OBJECTIVES: In this study, we evaluated the effect of the proanthocyanidins-rich fraction (PRF) obtained from Croton celtidifolius bark in an experimental animal model of spinal cord injury and cell death induced by glutamate. METHODS: Experiments were conducted using adult male Wistar rats (10 weeks old and weighing 270-300g). Experimental groups were randomly allocated into the following groups: spinal cord injury (SCI) + vehicle group: rats were subjected to SCI plus intraperitoneal administration of vehicle (saline 10 ml/kg); SCI + PRF: rats were subjected to SCI plus intraperitoneal administration of PRF (10 mg/kg) at 1 and 6 h after injury and sham operated. KEY FINDINGS: The treatment with the proanthocyanidin-rich fraction significantly improved not only motor recovery and grip force but also H2 O2 or glutamate-induced cell death and reactive oxygen species generation induced by glutamate in dorsal root ganglion cells. In this study we demonstrate that the neuroprotective effect triggered by the proanthocyanidins-rich fraction appears to be mediated in part by the inhibition of N-methyl-D-aspartate-type glutamate receptors. CONCLUSIONS: Taken together, our results demonstrate that PRF treatment ameliorates spinal cord injury and glutamatergic excitotoxicity and could have a potential therapeutic use.

Endothelin-1 induces itch and pain in the mouse cheek model.

AIMS: To date, suggestions that endothelin-1 (ET-1) causes nociception and pruritus are based on results in preclinical models in which responses to pruritic and nociceptive stimuli cannot be distinguished. This study reexamines these sensory effects of ET-1 in the new mouse cheek model, in which pruritogens and algogens evoke distinct behavioral responses. MAIN METHODS: Mice received intradermal (i.d.) injections of test substances into the left cheek and bouts of hind limb scratches or forepaw wipes, directed to the injection site, were considered indicative of pruritus and nociception, respectively. KEY FINDINGS: Histamine and capsaicin selectively evoked scratching and wipes, respectively, whereas ET-1 (3-60 pmol) promoted dose-dependent bouts of both behaviors. While scratching and wipe responses to ET-1 (30 pmol) were potentiated by BQ-788 (an ET(B) receptor antagonist) and reduced by co-injection of BQ-788 plus BQ-123 (an ET(A) receptor antagonist), BQ-123 alone inhibited scratching responses only. CTOP (µ-opioid receptor selective antagonist) only augmented scratching responses to ET-1, whereas DAMGO (µ-opioid receptor selective agonist) reduced both behaviors. Loratadine (histamine H(1) receptor antagonist) marginally reduced scratching, but markedly suppressed wipes. SIGNIFICANCE: These results demonstrate that ET-1 evokes pruritic and nociceptive behaviors in the mouse cheek model. Both responses to ET-1 appear to be mediated via ET(A) receptors and subjected to limitation by simultaneous ET(B) receptor activation. Local endogenous opioids acting on µ-opioid receptors selectively modulate the pruritic response to ET-1, whereas histamine, possibly derived from mast cells and acting on H(1) receptors, contributes importantly to the nociceptive effect of ET-1 in this model.

Endothelin and bradykinin: 'brothers-in-arms' in Chagas vasculopathies?

Reports of Chagas disease are increasing in non-endemic populations across the globe. Apart from vector eradication and prevention efforts by public health organizations, current pharmacological interventions are sparse and show important side effects. In this issue of the BJP, Andrade et al. elegantly demonstrate a new pharmacological paradigm whereby Trypanosoma cruzi host cell invasion requires significant cross-talk between receptors for kinins and endothelins. It is shown, for example, that acting via both ET(A) and ET(B) receptors, endothelin-1 (ET-1) cooperates with the (TLR2/CXCR2/B(2) kinin receptor) complex to activate inflammatory processes in response to invading trypomastigotes. This study by Andrade et al. prompts, however, several important questions, summarized in this Commentary, such as the putative role of chymase-dependent production of ET-1, the contentious protective role of ACE inhibitors in Chagasic patients, the unexplored role of de novo formed B(1) receptors for kinins triggered by cytokines and the putative role of compartmentalized calcium pools in host cell invasion by trypomastigotes.

Antinociception of ß-D-glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition.

ß-D-Glucan, a polysaccharide isolated from an edible mushroom Pleurotus pulmonarius (Fr.) Quel., presented antinociceptive activity in mice. This study evaluated the involvement of transient receptor potential (TRP) channels and protein kinase C (PKC) on antinociceptive effect of a (1→3),(1→6)-linked ß-D-glucan (GL) in mice. Intraperitoneal administration of GL potently inhibited nociceptive responses induced by intraplantar injections of capsaicin, cinnamaldehyde, menthol, acidified saline and phorbol myristate acetate (PMA). Moreover, Western blot analysis revealed that GL treatment also prevented PMA-induced PKCɛ activation. Collectively, present results demonstrate that GL could constitute an attractive molecule of interest for the development of new analgesic drugs.

Endothelins implicated in referred mechanical hyperalgesia associated with colitis induced by TNBS in mice.

This study evaluated the contribution of endothelins to changes in sensitivity to mechanical stimulation of the lower abdomen and hind paw associated with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. The frequency of withdrawal responses to 10 consecutive applications of von Frey probes to the lower abdomen (0.07 g) or hind paw (0.4 g) was assessed in male BALB/c mice before and after intracolonic TNBS injection (0.5 mg in 100 microL of 35% ethanol). TNBS (0.5 mg) induced referred mechanical hyperalgesia in the abdomen (response frequencies at 24 h: saline 11.0% +/- 3.1%, TNBS 48.0% +/- 6.9%) and hind paw (frequencies at 24 h: saline 12.5% +/- 4.7%, TNBS 47.1% +/- 7.1%) lasting up to 72 and 48 h, respectively. Mice receiving 1.0 or 1.5 mg TNBS assumed hunch-backed postures and became immobile during abdominal mechanical stimulation, suggestive of excessive ongoing pain. Atrasentan (ETA receptor antagonist; 10 and 30 mg/kg, i.v.) given 24 h after TNBS abolished hind paw and abdominal mechanical hyperalgesia for 2-3 h. A-192621 (ETB receptor antagonist; 20 mg/kg, i.v.) attenuated abdominal mechanical hyperalgesia at the 3 h time point only. Thus, endothelins contribute importantly to abdominal and hind paw referred mechanical hyperalgesia during TNBS-induced colitis mainly through ETA receptor-signaled mechanisms.

Endothelins as pronociceptive mediators of the rat trigeminal system: role of ETA and ETB receptors.

The trigeminal nerve is comprised of three main divisions, ophthalmic, maxillary and mandibular, each providing somatosensory innervation to distinct regions of the head, face and oral cavity. Recently, a role for endothelins in nociceptive signaling in the trigeminal system has been proposed. The present study aimed to gain better insight into the participation of the endothelin system in trigeminal nociceptive transmission. Herein ET-1 and ET-3 mRNA was detected in the rats' trigeminal ganglion (TG). Fluorescent labeling of TG neurons revealed that ET(A) and ET(B) receptors are distributed along the entire TG, but ET(A) receptor expression slightly predominated within the three divisions. TRPV1 receptors were also detected throughout the entire TG, and a significant proportion of TRPV1-positive neurons (approximately 30%) co-expressed either ET(A) or ET(B) receptors. Our behavioral data showed that ET-1 (3 to 30 pmol/site) induced overt nociceptive responses after injection into the upper lip or temporomandibular joint (TMJ) and hyperalgesic actions when applied to the eye, while ET-3 and the selective ET(B) receptor agonist IRL-1620 (each at 3 to 30 pmol/site) showed only the first two effects. Injection of BQ-123, but not BQ-788 (ET(A) and ET(B) receptor antagonists, respectively, 10 nmol/site each, 30 min beforehand), into the ipsilateral upper lip abolished ET-1 induced facial grooming, but both antagonists markedly reduced the nociceptive responses induced by ET-1 injected into the TMJ. Taken together, these findings suggest that endothelins, acting through ET(A) and/or ET(B) receptors, may play an important role in mediating pain resulting from activation of most trigeminal nerve branches.

Kinin B(1) and B(2) receptors contribute to orofacial heat hyperalgesia induced by infraorbital nerve constriction injury in mice and rats.

Mechanisms coupled to kinin B(1) and B(2) receptors have been implicated in sensory changes associated to various models of neuropathy. The current study aimed to investigate if kinins also participate in orofacial thermal hyperalgesia induced by constriction of the infraorbital nerve (CION), a model of trigeminal neuropathic pain which displays persistent hypersensitivity to orofacial sensory stimulation, in rats and mice. Male Swiss mice (30-35g) or Wistar rats (200-250g; n=6-10 per group in both cases) underwent CION or sham surgery and were submitted repeatedly to application of heat ( approximately 50 degrees C) to the ipsilateral or contralateral snout, delivered by a heat source placed 1cm from the vibrissal pad. Decreases in latency to display head withdrawal or vigorous snout flicking were considered indicative of heat hyperalgesia. CION caused long-lasting heat hyperalgesia which started on Day 2 after surgery in both species and lasted up to Day 17 in mice and Day 10 in rats. Administration of DALBK or HOE-140 (peptidic B(1) and B(2) receptor antagonists, respectively; each at 3nmol in 10microl) onto the exposed infraorbital nerve of mice at the moment of surgery delayed the development of the thermal hyperalgesia. Systemic treatment on Day 5 (mice) or Day 4 (rats) with Des-Arg(9), Leu(8)-Bradykinin (DALBK, B(1) receptor antagonist, 0.1-1micromol/kg, i.p.) or HOE-140 (B(2) receptor antagonist, 0.001-1micromol/kg, i.p.) transiently reduced heat hyperalgesia in both species. Due to the peptidic nature of DALBK and HOE-140, it is likely that their effects reported herein resulted from blockade of peripheral kinin receptors. Thus, mechanisms operated by kinin B(1) and B(2) receptors, contribute to orofacial heat hyperalgesia induced by CION in both mice and rats. Perhaps kinin B(1) and B(2) receptor antagonists might constitute effective preventive and curative treatments for orofacial thermal hyperalgesia induced by nerve injury.

Role of ET(A) and ET(B) endothelin receptors on endothelin-1-induced potentiation of nociceptive and thermal hyperalgesic responses evoked by capsaicin in rats.

Increasing evidence indicates that endothelin-1 (ET-1) activates nociceptive neurons and sensitizes them to different noxious stimuli, but involvement of TRPV1-dependent mechanisms in mediation of such effects is not yet fully understood. Here we report that intraplantar (i.pl.) injection of ET-1 (10 pmol) into the hind paw of rats induced overt nociceptive behavior over the first hour, followed by a slowly developing thermal hyperalgesia, lasting from 3 to 8h after injection. Both effects were also induced by similar injections of capsaicin (10-1000 pmol), but these responses were shorter lasting than those caused by ET-1. Local pre-treatment with the TRPV1 antagonist capsazepine (30 nmol, i.pl.) reduced only the thermal hyperalgesia induced by ET-1, but fully suppressed both responses to capsaicin (1000 pmol). Injection of a sub-threshold dose of ET-1 (0.1 pmol, i.pl.) prior to capsaicin (1 pmol, i.pl.) markedly sensitized the hind paw to the overt nociceptive and thermal hyperalgesic effects of the later. The potentiation of capsaicin-induced nociception by ET-1 was abolished by prior i.pl. injection of BQ-123 (ET(A) receptor antagonist, 10 nmol), but unaffected by BQ-788 (ET(B) receptors antagonist, 10 nmol), whereas the enhancement of capsaicin-induced hyperalgesia by ET-1 was attenuated by both antagonists. Therefore, differently to what has been reported in mice, in rats TRPV1 receptors contribute selectively to thermal hyperalgesia, but not overt nociception, induced by ET-1. Importantly, although ET-1 augments capsaicin-induced overt nociception and thermal hyperalgesia, potentiation of the former relies solely on ET(A) receptor-mediated signaling mechanisms, whereas both receptors contribute to the latter.

Anti-inflammatory effects of a triterpenoid isolated from Wilbrandia ebracteata Cogn.

Wilbrandia ebracteata (WE), a Brazilian medicinal plant used in folk medicine for the treatment of rheumatic diseases, displays anti-inflammatory properties and constitutes a rich source of cucurbitacins and cucurbitacin-related compounds. The current study investigated the potential anti-inflammatory properties of Dihydrocucurbitacin B (DHCB), a cucurbitacin-derived compound isolated from roots of WE, in some in vivo and in vitro experimental models. Intraperitoneal treatment of mice with DHCB reduced both carrageenan-induced paw edema (0.3, 1 and 3 mg/kg caused inhibitions of 26, 44 and 56 % at 2 h after stimulation, respectively) and pleurisy (10 mg/kg inhibited leukocyte numbers and LTB(4) levels in the pleural fluid by 51 and 75% at 6 h after cavity challenge, respectively). In vitro, DHCB (up to 10 microg/mL) failed to modify LTB(4) production by human neutrophils or PGE(2) production by COS-7 cells transfected with COX-1, but PGE(2) production by COX-2 transfected COS-7 cells was markedly inhibited (by 72%). The levels of COX-1 or COX-2 proteins in IL-1alpha-stimulated NIH3T3 cells were unaffected by DHCB. The results corroborate the potential anti-inflammatory properties ascribed to W. ebracteata Cogn. in folk medicine and suggest that they might be attributed, at least in part, to the capacity of one of this plants main constituents, DHCB, to inhibit COX-2 activity (but not its expression) during inflammation.

Endothelin ET(B) receptor antagonist reduces mechanical allodynia in rats with trigeminal neuropathic pain.

Trigeminal neuropathic pain, which is associated with marked orofacial mechanical allodynia, is frequently refractory to currently available drugs. Because endothelins (ETs) can contribute to nociceptive changes in animal models of inflammatory, cancer, and diabetic neuropathic pain, the present study evaluated the influence of ET(A) and ET(B) receptor antagonists on orofacial mechanical allodynia in a rat model of trigeminal neuropathic pain. Unilateral constriction (C) of the infraorbital nerve (ION) caused pronounced and sustained bilateral mechanical allodynia, evaluated by application of von Frey hairs to the vibrissal pad. Mechanical allodynia on postoperative days 12-15 after nerve injury was abolished for up to 90 mins by subcutaneous administration of 2.5 mg/kg morphine, but was fully refractory to intravenous (iv) administration of 10 mg/kg of the dual ET(A) plus ET(B) or selective ET(A) receptor antagonists, bosentan and atrasentan, respectively. In sharp contrast, iv administration of 20 mg/kg of the selective ET(B) receptor antagonist, A-192621, caused a net 61 +/- 15% reduction of mechanical threshold, lasting 2 hrs. Co-injection of atrasentan plus A-192621 did not modify ION injury-induced mechanical allodynia. Injection of 10 pmol ET-1 into the upper lip of naive rats caused ipsilateral mechanical allodynia lasting up to 5 hrs. Thus, ET(B) receptor-mediated mechanisms contribute to orofacial mechanical allodynia induced by CION injury, but, some-how, functional ET(A) receptors are required for expression of the antiallodynic effect of ET(B) receptor blockade.

Mechanical hyperalgesia induced by endothelin-1 in rats is mediated via phospholipase C, protein kinase C, and MAP kinases.

In addition to causing overt nociception, intraplantar (ipl) endothelin (ET)-1 injection into the rat hind paw induces hyperalgesia to mechanical stimuli, mediated via local ET(B) receptors coupled to protein kinase (PK) C, but not PKA. The present study further examines the intracellular signaling mechanisms underlying this effect of ET-1. ET-1 (30 pmol) or phospate-buffered saline (PBS) was injected ipl in rats and the threshold of responsiveness to mechanical stimulation was assessed repeatedly each hour up to 8 hrs and 24 hrs, using the dynamic plantar aesthesiometer test, which detects the minimal pressure required to evoke paw withdrawal. Different groups were treated, 15 mins before ET-1 administration, with ipsilateral injection of selective inhibitors of either phospholipase (PL) A2 (1 nmol PACOCF3), PLC (30 pmol U73122), PKC (1 nmol GF109203X), p38 mitogen-activated protein kinase (MAPK; 30 nmol SB203580), extracellular signal-regulated kinase (ERK1/2; 30 nmol PD98059), c-Jun N-terminal kinase (JNK; 30 nmol SP600125), or vehicle, to assess their influence on the hyperalgesic response. The mechanical hyperalgesia caused by ET-1 started 2 hrs after injection, peaked at 5 hrs (PBS, 29 +/- 0.5 g; ET-1, 17 +/- 1.3 g) and lasted up to 8 hrs. The inhibitors of PLC, PKC, p38 MAPK, ERK1/2, and JNK caused long-lasting reductions of the mechanical hyperalgesia (inhibitions at 4 hrs of 100%, 90%, 97%, 90%, and 100%, respectively), but the PLA2 inhibitor reduced hyperalgesia only at 4 hrs (by 58%). Thus, mechanical hyperalgesia triggered by ET-1 in the rat hind paw depends importantly on signaling pathways involving PLC, PKC, p38 MAPK, ERK1/2, and JNK, whereas the contribution of PLA2 is relatively minor.