Endocannabinoids, through opioids and prostaglandins, contribute to fever induced by key pyrogenic mediators.

This study aims to explore the contribution of endocannabinoids on the cascade of mediators involved in LPS-induced fever and to verify the participation of prostaglandins and endogenous opioids in fever induced by anandamide (AEA). Body temperature (Tc) of male Wistar rats was recorded over 6h, using a thermistor probe. Cerebrospinal fluid concentration of PGE2 and ß-endorphin were measured by ELISA after the administration of AEA. Intracerebroventricular administration of the CB1 receptor antagonist AM251 (5µg, i.c.v.), reduced the fever induced by IL-1ß (3ng, i.c.v.), TNF-α (250ng, i.c.v.), IL-6 (300ng, i.c.v.), corticotrophin release factor (CRH; 2.5µg, i.c.v.) and endothelin (ET)-1 (1pmol, i.c.v.), but not the fever induced by PGE2 (250ng, i.c.v.) or PGF2α (250ng, i.c.v.). Systemic administration of indomethacin (2mgkg(-1), i.p.) or celecoxib (5mgkg(-1), p.o.) reduced the fever induced by AEA (1µg, i.c.v.), while naloxone (1mgkg(-1), s.c.) abolished it. The increases of PGE2 and ß-endorphin concentration in the CSF induced by AEA were abolished by the pretreatment of rats with AM251. These results suggest that endocannabinoids are intrinsically involved in the pyretic activity of cytokines (IL-1ß, TNF-α, IL-6), CRH and ET-1 but not the PGE2 or PGF2α induced fevers. However, anandamide via CB1 receptor activation induces fever that is dependent on the synthesis of prostaglandin and opioids.

Contribution of peripheral endothelin ETA and ETB receptors in neuropathic pain induced by spinal nerve ligation in rats.

Endothelins (ETs) contribute to the sensory changes seen in animals models of inflammatory, cancer and diabetic neuropathic pain, but little is known about their nociceptive role following peripheral nerve injury. The current study evaluated mechanisms by which ETs can drive changes in nociceptive responses to thermal stimulation of the hind paw of rats induced by unilateral lumbar L5/L6 spinal nerve ligation (SNL) injury. SNL sensitizes rats to acetone-evoked cooling of and radiant heat application (Hargreaves test) to the ipsilateral hind paw (throughout 3-40 and 9-40 days after surgery, respectively). At 12 days after SNL, intraplantar (i.pl.) injection of endothelin-1 (ET-1, 10 pmol) induces greater overt nociception that was reduced only by treatment with the selective ET(A) peptidic antagonist (BQ-123, 10 nmol, i.pl), but unchanged by the selective ET(B) peptidic antagonist (BQ-788). Cold allodynia evoked by cooling the ipsilateral hind paw with acetone was reduced by i.pl. injection of both antagonists BQ-123 or BQ-788 (3 or 10 nmol). In contrast, heat hyperalgesia evaluated by Hargreaves method was reduced only by BQ-123. SNL enhanced the [Ca(+2)](i) increases induced by ET-1 (100 nM) in neurons from L5/L6 (injured) and L4 (intact) cultured dorsal root ganglion, but did not change the responses of non-neuronal cells. Furthermore, Western blot analysis revealed that SNL increased ET(A) and ET(B) receptor protein expression in spinal nerves. Thus, SNL induces marked hind paw hypersensitivity to thermal stimulation in part via up-regulation of peripheral sensory nerve pronociceptive ET(A) and ET(B) receptor-operated mechanisms.

Mechanisms operated by endothelin ETA and ETB receptors in the trigeminal ganglion contribute to orofacial thermal hyperalgesia induced by infraorbital nerve constriction in rats.

Endothelins, acting through specific endothelin ET(A) and/or ET(B) receptors, participate in nociceptive processing in models of cancer, inflammatory and neuropathic pain. The present study investigated which cell types express endothelin receptors in the trigeminal ganglion, and the contribution of mechanisms mediated by endothelin ET(A) and ET(B) receptors to orofacial heat hyperalgesia induced by unilateral constriction of the infraorbital nerve (CION). Both receptor types were identified by immunohistochemistry in the trigeminal ganglion, ET(A) receptors on small-sized non-myelinated and myelinated A-fibers and ET(B) receptors on both satellite glial cells and small-sized non-myelinated neuronal cells. CION promoted ipsilateral orofacial heat hyperalgesia which lasted from Day 2 until Day 10 after surgery. Ongoing CION-induced heat hyperalgesia (on Day 4) was reduced transiently, but significantly, by systemic or local treatment with antagonists of endothelin ET(A) receptors (atrasentan, 10 mg/kg, i.v.; or BQ-123, 10 nmol/lip), endothelin ET(B) receptors (A-192621, 20 mg/kg, i.v.; or BQ-788, 10 nmol/ lip), or of both ET(A)/ET(B) receptors (bosentan, 10 mg/kg, i.v.; or BQ-123 plus BQ-788, each at 10 nmol/lip). On the other hand, CION-induced heat hyperalgesia was transiently abolished over the first 90 min following i.p. injection of morphine hydrochloride (2.5 mg/kg), but fully resistant to reversal by indomethacin (4 mg/kg, i.p.) or celecoxib (10 mg/kg, i.p.). Thus, heat hyperalgesia induced by CION is maintained, in part, by peripheral signaling mechanisms operated by ET(A) and ET(B) receptors. Endothelin receptors might represent promising therapeutic targets for the control of trigeminal neuropathic pain.

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-alpha, and CXCL-1.

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET-1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan-induced arthritis. Intra-articular (i.a.) administration of selective ET(A) or ET(B) receptor antagonists (BQ-123 and BQ-788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 microg/cavity) markedly reduced knee-joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ET(A) or ET(B) receptor blockade suppressed zymosan-induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ET(A)/ET(B) with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ-123 or BQ-788 (i.a.; 15 pmol/cavity) also decreased zymosan-induced TNF-alpha production within 6 h, keratinocyte-derived chemokine/CXCL1 production within 24 h, and leukotriene B(4) at both time-points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan-induced inflammation, i.a. injection of ET-1 (1-30 pmol/cavity) or sarafotoxin S6c (0.1-30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee-joint synovial tissue expressed ET(A) and ET(B) receptors. These findings suggest that endogenous ETs contribute to knee-joint inflammation, acting through ET(A) and ET(B) receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

Central endothelin ET(B) receptors mediate IL-1-dependent fever induced by preformed pyrogenic factor and corticotropin-releasing factor in the rat.

Blockade of central endothelin ET(B) receptors inhibits fever induced by LPS in conscious rats. The contribution of ET(B) receptor-mediated mechanisms to fever triggered by intracerebroventricular IL-6, PGE2, PGF(2alpha), corticotropin-releasing factor (CRF), and preformed pyrogenic factor derived from LPS-stimulated macrophages (PFPF) was examined. The influence of natural IL-1 receptor antagonist or soluble TNF receptor I on endothelin (ET)-1-induced fever was also assessed. The selective ET(B) receptor antagonist BQ-788 (3 pmol icv) abolished fever induced by intracerebroventricular ET-1 (1 pmol) or PFPF (200 ng) and reduced that caused by ICV CRF (1 nmol) but not by IL-6 (14.6 pmol), PGE2 (1.4 nmol), or PGF(2alpha) (2 nmol). CRF-induced fever was also attenuated by bosentan (dual ET(A)/ET(B) receptor antagonist; 10 mg/kg iv) but unaffected by BQ-123 (selective ET(A) receptor antagonist; 3 pmol icv). alpha-Helical CRF(9-41) (dual CRF1/CRF2 receptor antagonist; 6.5 nmol icv) attenuated fever induced by CRF but not by ET-1. Human IL-1 receptor antagonist (9.1 pmol) markedly reduced fever to IL-1beta (180 fmol) or ET-1 and attenuated that caused by PFPF or CRF. Murine soluble TNF receptor I (23.8 pmol) reduced fever to TNF-alpha (14.7 pmol) but not to ET-1. The results of the present study suggest that PFPF and CRF recruit the brain ET system to cause ET(B) receptor-mediated IL-1-dependent fever.

Endothelin-1 (1-31) is an intermediate in the production of endothelin-1 after big endothelin-1 administration in vivo.

The precursor of endothelin-1, big endothelin-1, can be hydrolyzed by chymase to generate endothelin-1 (1-31) in vitro. In the present study, we explored the processes involved in the production of endothelin-1 (1-31) as well as its pharmacodynamic characteristics in the rabbit in vivo. Endothelin-1 (1-31) (1 nmol/kg, injected into the left cardiac ventricle) induced a monophasic increase of mean arterial blood pressure similarly to big endothelin-1 (1-38), whereas endothelin-1 induces a biphasic response. Phosphoramidon, a dual neutral endopeptidase and endothelin-converting enzyme inhibitor, blocked both pressor responses to endothelin-1 (1-31) and big endothelin-1 but not those afforded by endothelin-1. Thiorphan, a neutral endopeptidase inhibitor, markedly inhibited the response to endothelin-1 (1-31) but only weakly reduced that of big endothelin-1. In contrast, CGS 35066, an endothelin-converting enzyme inhibitor, was significantly more efficient against the pressor response to big endothelin-1 than to endothelin-1 (1-31). Furthermore, injection of big endothelin-1 concomitantly with phosphoramidon induced an increase in endothelin-1 (1-31) plasma levels. Finally, intracardiac-administered endothelin-1 (1-31) induced an increase of endothelin-1 plasma levels, which are markedly reduced by phosphoramidon and thiorphan but not by CGS 35066. Our results thus demonstrate that endothelin-1 (1-31) is an alternate intermediate in the production of endothelin-1 after big endothelin-1 administration in the rabbit in vivo.

Endothelins induce ETB receptor-mediated mechanical hypernociception in rat hindpaw: roles of cAMP and protein kinase C.

The present study assesses the capacity of endothelins to induce mechanical hypernociception, and characterises the receptors involved and the contribution of cAMP and protein kinases A (PKA) and C (PKC) to this effect. Intraplantar administration of endothelin-1, endothelin-2 or endothelin-3 (3-30 pmol) induced dose- and time-dependent mechanical hypernociception, which was inhibited by BQ-788 (N-cys-2,6-dimethylpiperidinocarbonyl-l-gamma-methylleucyl-d-1-methoxycarboyl-d-norleucine; endothelin ET(B) receptor antagonist), but not BQ-123 (cyclo[d-Trp-d-Asp-Pro-d-Val-Leu]; endothelin ET(A) receptor antagonist; each at 30 pmol). The selective endothelin ET(B) receptor agonist BQ-3020 (N-Ac-Ala(11,15)-endothelin-1 (6-21)) fully mimicked the hypernociceptive effects of the natural endothelins. Treatments with indomethacin, atenolol or dexamethasone did not inhibit endothelin-1-evoked mechanical hypernociception. However, endothelin-1-induced mechanical hypernociception was potentiated by the cAMP phosphodiesterase inhibitor rolipram (4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone) and inhibited by the PKC inhibitors staurosporine and calphostin C, but was unaffected by the PKA inhibitor H89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide). Thus, endothelins, acting through endothelin ET(B) receptors, induce mechanical hypernociception in the rat hindpaw via cAMP formation and activation of the PKC-dependent phosphorylation cascade.

Effects of endothelin ETA receptor antagonism on granulocyte and lymphocyte accumulation in LPS-induced inflammation.

Endothelin peptides play active roles in different aspects of inflammation. This study investigates the contribution of endogenous endothelins to lipopolysaccharide (LPS) pulmonary inflammation by assessing the influence of ET(A) receptor antagonism on leukocyte accumulation, granulocyte adhesion molecule expression, and chemokine/cytokine modulation. Local pretreatment with BQ-123 or A-127722 (150 pmol), two selective and chemically unrelated endothelin ET(A) receptor antagonists, inhibits neutrophil and eosinophil accumulation in LPS-induced pleurisy at 24 h but not neutrophil migration at 4 h. The effect of endothelin antagonism on neutrophil accumulation at 24 h was concomitant with inhibition of eosinophil and CD4 and CD8 T lymphocyte influx. It is surprising that the ET(A) receptor blockade did not inhibit the accumulation of gammadelta T lymphocytes, cells that are important for granulocyte recruitment in this model. Blockade of ET(A) receptors did not influence the expression of adhesion molecules (CD11b, CD49d) on granulocytes but abrogated the increase in tumor necrosis factor alpha levels 4 h after LPS stimulation and also markedly inhibited increases in levels of interleukin-6 and keratinocyte-derived chemokine/CXC chemokine ligand 1 but not eotaxin/chemokine ligand 11. Thus, acting via ET(A) receptors, endogenous endothelins play an important role in early cytokine/chemokine production and on granulocyte and lymphocyte mobilization in LPS-induced pleurisy.

Pressor and pulmonary responses to ET-1(1-31) in guinea-pigs.

1. Endothelin-1(1-31) (ET-1(1-31); 0.25 to 4 nmol kg(-1); i.v.) induced, in the guinea-pig, graded increases in MAP and an indomethacin-sensitive enhancement of pulmonary insufflation pressure (PIP). At all doses, ET-1(1-31) induced a monophasic pressor response, except at 4 nmol kg(-1), which caused a rapid and transient response (first phase: over first 10 min after injection) followed by a more slowly-developing and sustained (second phase: between 10 and 45 min after injection) increase in MAP. ET-1(1-31) was 4 to 10 fold less potent than ET-1 on PIP responses. 2. Phosphoramidon (5 and 10 mg kg(-1)) reduced both pressor and PIP effects of ET-1(1-31). Thiorphan (0.25 and 2.5 mg kg(-1)) did not affect the pressor responses to ET-1(1-31) although its PIP effects were markedly reduced by the NEP inhibitor. A selective endothelin-converting enzyme (ECE) inhibitor, CGS 35066 (1 mg kg(-1)), significantly reduced the second phase pressor response and increase in PIP triggered by ET-1(1-31). 3. The second (but not the first) pressor phase of ET-1(1-31) (4 nmol kg(-1)) was markedly reduced by BQ-123 (selective ET(A) antagonist), whereas the increase of PIP was significantly reduced by BQ-788 (selective ET(B) antagonist). Co-administration of BQ-123 plus BQ-788 abolished ET-1(1-31)-induced increase in PIP, but blockade of the second pressor phase afforded by BQ-123 was now reversed. 4. In guinea-pig isolated perfused lungs, ET-1(1-31) (50 nM) induced the release of prostacyclin and thromboxane A(2), which was inhibited by BQ-788 (5 nM) or thiorphan (25 microM), but not BQ-123 (1 microM). 5. These results suggest that ET-1(1-31) enhances MAP. Its sustained, but not transient, pressor effects are mediated via ET(A) receptor activation. Furthermore, ET-1(1-31) increases airway resistance in vivo and triggers prostacyclin and thromboxane A(2) release from perfused lungs predominantly via ET(B) receptor activation. ET-1(1-31) failed to display any selectivity of action towards either ET(A) or ET(B) receptors in these models. 6. We suggest that, in order to raise MAP, ET-1(1-31) requires conversion to ET-1, predominantly by ECE and to a lesser extent neutral endopeptidase 24.11, whereas the reverse holds true regarding its pharmacological effects in airways.

Influence of indomethacin on effects of endothelin-1 on guinea pig isolated rings of common bile duct and sphincter of Oddi.

The effects of endothelin-1 on motility of guinea pig extra-hepatic biliary tract portions were studied. Endothelin-1 (< or =100 nM) failed to contract rings of hepatic, cystic, proximal or distal common bile ducts, or choledochal or papillary halves of sphincter of Oddi. At 100 nM, endothelin-1 or sarafotoxin S6c (selective endothelin ET(B) receptor agonist) inhibited contractions of choledochal (but not papillary) sphincter of Oddi to carbachol (1 microM) by 63+/-5 and 45+/-9%, respectively. In distal common bile duct, indomethacin (5.6 microM) unmasked potent contractile effects of endothelin-1 [EC(50) 7.8 (5.5-11.1) nM; E(MAX) 80+/-6% of response to 80 mM KCl] and enhanced the contractile potency of carbachol (585-fold at EC(50) level), but not cholecystokinin C-terminal octapeptide. Inhibition of cholinergic responsiveness of the choledochal sphincter of Oddi by endothelin-1 was reduced by BQ-123 (1 microM; endothelin ET(A) receptor antagonist; cyclo[DTrp-DAsp-Pro-DVal-Leu]) and abolished by either BQ-123 plus BQ-788 (1 microM; endothelin ET(B) receptor antagonist; N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarboyl-D-norleucine) or indomethacin. Thus, eicosanoids of the cyclo-oxygenase pathway (i.e. prostanoids) suppress endothelin-1-induced contractions of distal common bile duct and mediate endothelin ET(A) and ET(B) receptor-dependent inhibition of cholinergic responsiveness of the choledochal portion of the sphincter of Oddi.