Potential role for ET-2 acting through ETA receptors in experimental colitis in mice.

OBJECTIVE AND DESIGN: This study attempted to clarify the roles of endothelins and mechanisms associated with ETA/ETB receptors in mouse models of colitis. MATERIALS AND METHODS: Colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 1.5 mg/animal) or dextran sulfate sodium (DSS, 3%). After colitis establishment, mice received Atrasentan (ETA receptor antagonist, 10 mg/kg), A-192621 (ETB receptor antagonist, 20 mg/kg) or Dexamethasone (1 mg/kg) and several inflammatory parameters were assessed, as well as mRNA levels for ET-1, ET-2 and ET receptors. RESULTS: Atrasentan treatment ameliorates TNBS- and DSS-induced colitis. In the TNBS model was observed reduction in macroscopic and microscopic score, colon weight, neutrophil influx, IL-1ß, MIP-2 and keratinocyte chemoattractant (KC) levels, inhibition of adhesion molecules expression and restoration of IL-10 levels. However, A192621 treatment did not modify any parameter. ET-1 and ET-2 mRNA was decreased 24 h, but ET-2 mRNA was markedly increased at 48 h after TNBS. ET-2 was able to potentiate LPS-induced KC production in vitro. ETA and ETB receptors mRNA were increased at 24, 48 and 72 h after colitis induction. CONCLUSIONS: Atrasentan treatment was effective in reducing the severity of colitis in DSS- and TNBS-treated mice, suggesting that ETA receptors might be a potential target for inflammatory bowel diseases.

Neuropathic pain induced by spinal cord injury: Role of endothelin ETA and ETB receptors.

Spinal cord injury (SCI) is a devastating neurologic disorder that often inflicts neuropathic pain, which further impacts negatively on the patient's quality of life. Endothelin peptides, which exert their effects via endothelin A (ETAR) and endothelin B (ETBR) receptors, can contribute to sensory changes associated with inflammatory and neuropathic pain, but their role in nociception following SCI is unknown. At different time points after subjecting male Wistar rats to surgery for compression-induced T10 level SCI, the spinal cord levels of ETAR and ETBR were assessed by Western blot and immunohistochemistry, and the corresponding mRNAs by real-time PCR, alongside recordings of behavioural responses to mechanical stimulation of the hind paws with von Frey hairs. SCI was associated with development of hind paw mechanical allodynia from day 14 onwards, and up-regulation of ETAR and ETBR mRNA in the spinal cord and dorsal root ganglia, and of ETAR protein in the spinal cord. SCI increased ETAR protein expression in spinal grey matter. Treatment on day 21 after surgery with the ETAR selective antagonist BQ-123 (40 and 90 pmol, intrathecally) or the dual ETAR/ETBR antagonist bosentan (30 and 100mg/kg, orally) transiently reduced SCI-induced mechanical allodynia, but the ETBR antagonist BQ-788 was ineffective. Altogether, these data show that SCI upregulates ETAR expression in the spinal cord, which appears to contribute to the hind paw mechanical allodynia associated with this condition. Therapies directed towards blockade of spinal ETAR may hold potential to limit SCI-induced neuropathic pain.

Contribution and interaction of kinin receptors and dynorphin A in a model of trigeminal neuropathic pain in mice.

Infraorbital nerve constriction (CION) causes hypersensitivity to facial mechanical, heat and cold stimulation in rats and mice and is a reliable model to study trigeminal neuropathic pain. In this model there is evidence that mechanisms operated by kinin B1 and B2 receptors contribute to heat hyperalgesia in both rats and mice. Herein we further explored this issue and assessed the role of kinin receptors in mechanical hyperalgesia after CION. Swiss and C57Bl/6 mice that underwent CION or sham surgery or dynorphin A (1-17) administration were repeatedly submitted to application of either heat stimuli to the snout or mechanical stimuli to the forehead. Treatment of the animals on the fifth day after CION surgery with DALBK (B1 receptor antagonist) or HOE-140 (B2 receptor antagonist), both at 0.01-1µmol/kg (i.p.), effectively reduced CION-induced mechanical hyperalgesia. Knockout mice for kinin B1, B2 or B1/B2 receptors did not develop heat or mechanical hyperalgesia in response to CION. Subarachnoid dynorphin A (1-17) delivery (15nmol/5µL) also resulted in orofacial heat hyperalgesia, which was attenuated by post-treatment with DALBK (1 and 3µmol/kg, i.p.), but was not affected by HOE-140. Additionally, treatment with an anti-dynorphin A antiserum (200µg/5µL, s.a.) reduced CION-induced heat hyperalgesia for up to 2h. These results suggest that both kinin B1 and B2 receptors are relevant in orofacial sensory nociceptive changes induced by CION. Furthermore, they also indicate that dynorphin A could stimulate kinin receptors and this effect seems to contribute to the maintenance of trigeminal neuropathic pain.

Endogenous cannabinoids induce fever through the activation of CB1 receptors.

BACKGROUND AND PURPOSE: The effects of centrally administered cannabinoids on body core temperature (Tc) and the contribution of endogenous cannabinoids to thermoregulation and fever induced by lipopolysaccharide (LPS) (Sigma Chem. Co., St. Louis, MO, USA) were investigated. EXPERIMENTAL APPROACH: Drug-induced changes in Tc of male Wistar rats were recorded over 6 h using a thermistor probe (Yellow Springs Instruments 402, Dayton, OH, USA) inserted into the rectum. KEY RESULTS: Injection of anandamide [(arachidonoylethanolamide (AEA); Tocris, Ellisville, MO, USA], 0.01-1 microg i.c.v. or 0.1-100 ng intra-hypothalamic (i.h.), induced graded increases in Tc (peaks 1.5 and 1.6 degrees C at 4 h after 1 microg i.c.v. or 10 ng i.h.). The effect of AEA (1 microg, i.c.v.) was preceded by decreases in tail skin temperature and heat loss index (values at 1.5 h: vehicle 0.62, AEA 0.48). Bell-shaped curves were obtained for the increase in Tc induced by the fatty acid amide hydrolase inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (Cayman Chemical Co., Ann Arbor, MI, USA) (0.001-1 ng i.c.v.; peak 1.9 degrees C at 5 h after 0.1 ng) and arachidonyl-2-chloroethylamide (ACEA; Tocris) (selective CB(1) agonist; 0.001-1 microg i.c.v.; peak 1.4 degrees C 5 h after 0.01 microg), but (R,S)-(+)-(2-Iodo-5-nitrobenzoyl)-[1-(1-methyl-piperidin-2-ylmethyl)-1H-indole-3-yl] methanone (Tocris) (selective CB(2) agonist) had no effect on Tc. AEA-induced fever was unaffected by i.c.v. pretreatment with 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indole-3-yl](4-methoxyphenyl) methanone (Tocris) (selective CB(2) antagonist), but reduced by i.c.v. pretreatment with N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; Tocris) (selective CB(1) antagonist). AM251 also reduced the fever induced by ACEA or LPS. CONCLUSIONS AND IMPLICATIONS: The endogenous cannabinoid AEA induces an integrated febrile response through activation of CB(1) receptors. Endocannabinoids participate in the development of the febrile response to LPS constituting a target for antipyretic therapy.

Endothelin-1 (1-31): from chymase-dependent synthesis to cardiovascular pathologies.

The mast cell-derived serine protease chymase is importantly involved not only in degradation, but in synthesis of bioactive peptides as well. Several studies suggest that chymase is the predominant enzyme in the production of angiotensin II (Ang II) from angiotensin-I in interstitial tissues. Interestingly, chymase has also been suggested to mature endothelin-1 (ET-1) from its precursor, big-ET-1 in vitro. The lack of availability of specific chymase inhibitors, beyond the chymotrypsin-like inhibitor chymostatin, currently hampers the investigation of the chymase/ET-1/Ang II paradigm in physiology and cardiovascular diseases. Nonetheless, the recent advent of highly selective chymase inhibitors is shedding new light on the role of this enzymatic pathway in the several inflammatory prone vascular diseases as summarized in the present review. Considering increasing evidence towards significant interactions between Ang II and ET-1 in cardiovascular diseases, the present review will address the role of chymase in the production of those two peptides. Whether chymase-dependent production of ET-1 plays an important role in cardiovascular pathologies will also be discussed.

Peripheral kinin B(1) and B(2) receptor-operated mechanisms are implicated in neuropathic nociception induced by spinal nerve ligation in rats.

The kinin system can contribute distinctly to the sensory changes associated with different models of nerve injury-induced neuropathic pain. This study examines the roles of kinin B(1) and B(2) receptor-operated mechanisms in alterations in nociceptive responses of rats submitted to unilateral L5/L6 spinal nerve ligation (SNL) injury. Behavioural responses to ipsilateral hind paw stimulation with acetone (evaporation-evoked cooling), radiant heat (Hargreaves method) or von Frey hairs revealed that SNL rats developed long-lasting cold allodynia (from Days 3 to 40 post-surgery, peak on Day 6), heat hyperalgesia (stable peak from Days 9 to 36) and tactile allodynia (stable peak from Days 3 to 51). SNL rats manifested nocifensive responses to intraplantar injections on Day 12 of the selective B(1) receptor agonist des-Arg(9)-bradykinin (DABK) and augmented responses to the selective B(2) receptor agonist bradykinin (BK; each at 0.01-1nmol/paw). Systemic treatment of SNL rats with des-Arg(9)-Leu(8)-BK or HOE 140 (peptidic B(1) and B(2) receptor antagonists, respectively; 0.1-1mumol/kg, i.p.) selectively blocked responses triggered by DABK and BK (1nmol/paw) and alleviated partially and transiently established cold allodynia, heat hyperalgesia and (to a lesser extent) tactile allodynia. Western blot analysis revealed enhanced expression of kinin B(1) and B(2) receptor protein in ipsilateral L4-L6 spinal nerve and hind paw skin samples collected on Day 12 after SNL surgery. These results indicate that peripheral pronociceptive kinin B(1) and B(2) receptor-operated mechanisms contribute significantly to the maintenance of hind paw cold and mechanical allodynia and heat hyperalgesia induced by L5/L6 SNL in rats.

Antidepressant-like effects of the nociceptin/orphanin FQ receptor antagonist UFP-101: new evidence from rats and mice.

Receptor antagonist and knockout studies have demonstrated that blockade of signalling via nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) has antidepressant-like effects in mice submitted to the forced swimming test (FST). The aim of the present study was to explore further the antidepressant-like properties of the NOP antagonist UFP-101 in different species (mouse and rat) and using different assays [FST and tail suspension test (TST)], and to investigate the mechanism(s) involved in its actions.UFP-101 (10 nmol i.c.v.) reduced immobility time of Swiss mice in the TST (mean+/-SEM) from 179+/-11 to 111+/-10 s. N/OFQ (1 nmol i.c.v.) was without effect per se, but fully prevented the effect of UFP-101. The spontaneous immobility time of NOP(-/-) CD1-C57BL/6J-129 mice in the TST was much lower than that of wild-type (NOP(+/+)) littermates (75+/-11 vs. 144+/-17 s) or of Swiss mice. UFP-101 (10 nmol i.c.v.) decreased immobility time (-65%) and increased climbing time (71%) in rats submitted to the FST. In rat brain slices, N/OFQ (100 nM) triggered robust K(+)-dependent hyperpolarizing currents in locus coeruleus and dorsal raphe neurons. UFP-101 (3 microM) fully prevented N/OFQ-induced currents, but was inactive per se. Fluoxetine, desipramine (both 30 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.) reduced immobility time of mice in the FST. The serotonin synthesis inhibitor p-chlorophenylalanine methylester (PCPA, 4 x 100 mg/kg per day i.p.) prevented the antidepressant-like effects of fluoxetine and UFP-101 (but not desipramine), whereas N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine (DSP-4, neurotoxic for noradrenergic neurons; 50 mg/kg i.p., 7 days beforehand), suppressed only the effect of desipramine. Neither pretreatment affected spontaneous immobility time per se.Thus, UFP-101 exhibits pronounced antidepressant-like effects in different species and animal models, possibly by preventing the inhibitory effects of endogenous N/OFQ on brain monoaminergic (in particular serotonergic) neurotransmission. Participation of the N/OFQ-NOP receptor system in mood modulation sets new potential targets for antidepressant drug development.

Blockade of nociceptin/orphanin FQ-NOP receptor signalling produces antidepressant-like effects: pharmacological and genetic evidences from the mouse forced swimming test.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, regulates several central functions such as pain transmission, learning and memory, fear and anxiety and feeding and locomotor activity. It has been recently reported that NOP receptor antagonists induce antidepressant-like effects in the mouse forced swimming test (FST), i.e. reduce immobility time. This assay was used in the present study for further investigating the involvement of the NOP receptor in depression states. In male Swiss mice, intracerebroventricular injection (i.c.v) of the novel NOP receptor antagonist, UFP-101 (1-10 nmol) dose-dependently reduced the immobility time (control 192 +/- 14 s, UFP-101 91 +/- 15 s). The effect of 3 or 10 nmol UFP-101 was fully or partially reversed, respectively, by the coadministration of 1 nmol N/OFQ, which was inactive per se. NOP receptor knockout mice showed a reduced immobility time compared with their wild-type littermates (wild-type 215 +/- 10 s, knockout 143 +/- 12 s). Moreover, i.c.v. injected UFP-101 (10 nmol) significantly reduced immobility time in wild-type mice but not in NOP receptor knockout animals. In conclusion, these results, obtained using a combined pharmacological and genetic approach, indicate that blockade of the N/OFQ-NOP receptor signalling in the brain produces antidepressant-like effects in the mouse FST. These findings support the NOP receptor as a candidate target for the development of innovative antidepressant drugs.

Diabetes-induced changes in responsiveness of rat bladder and vas deferens to peptides in vitro: susceptibility to reversal by insulin.

Changes in responsiveness of the vas deferens and urinary bladder to bradykinin (BK) receptor agonists (Tyr8-BK and des-Arg9-BK), substance P (SP), and endothelin-1 (ET-1) were assessed 8 weeks after streptozotocin (STZ)-induced diabetes. Preparations from control or STZ-treated (60 mg/kg i.p.) male rats were tested for contractile and neurogenic twitch potentiating (TP, in VD only) effects of all four agonists (1 nM to 0.3 or 3 microM). In diabetic VD, contractile effects of Tyr8-BK, des-Arg9-BK, and SP were enhanced, but ET-1 effects were unchanged. In contrast, TP by des-Arg9-BK was unaffected, that by Tyr8-BK was decreased, and those by SP and ET-1 were increased. In diabetic UB, only contractions to des-Arg9-BK and SP were enhanced. Following insulin replacement (human, 1-3 U/day s.c.), starting 1 week after STZ, TP induced by Tyr8-BK and des-Arg9-BK in VD were further inhibited, but all other changes in both preparations were reversed at least partially. Insulin treatment of nondiabetic rats, however, also affected VD (but not UB) responsiveness, such that contractions to Tyr8-BK and TP by ET-1 were increased, but TP by Tyr8-BK was decreased. Thus, STZ-induced type I diabetes causes important alterations in responsiveness of non-vascular smooth muscle tissues of the rat to BK, SP, and ET-1. Long term insulin replacement, at doses normalising glycaemia, effectively reversed most changes in VD or UB responsiveness, but it is unclear if this is truly due to blocking of STZ-induced changes, since the treatment also affected responsiveness of nondiabetic tissues.

A comparative study of the antipyretic effects of indomethacin and dipyrone in rats.

OBJECTIVE: Compare the antipyretic effects of dipyrone and indomethacin. MATERIALS AND METHODS: Fever was induced in rats by i. v. LPS or i. c. v. interleukins (IL), prostaglandins (PG), arachidonic acid (AA), pre-formed pyrogenic factor (PFPF), tumour necrosis factor-alpha (TNF-alpha) or corticotrophin releasing hormone (CRH). Dipyrone and indomethacin were administered i.p., arginine vasopressin V1-receptor antagonist, d(CH2)5 Tyr(Me)AVP, into the ventral septal area. Cyclooxygenase (COX-1/-2) blocking activity was assessed in transfected COS-7 cells. CRH release from isolated hypothalami was determined by ELISA. RESULTS: Indomethacin or dipyrone reduced LPS, IL-1beta, IL-6 or TNF-alpha induced fever and CRH release from rat hypothalamus. Only dipyrone inhibited IL-8, PFPF or PGF2alpha fever. Only indomethacin inhibited fever induced by AA or IL-1beta, plus AA. Neither antipyretic affected fever caused by PGE2 or CRH. d(CH2)5Tyr(Me)AVP only blocked antipyresis induced by indomethacin. Dipyrone at a very high concentration (10 mM) inhibited only COX-1, while indomethacin (0.1 microM) blocked COX-1 and COX-2 in COS-7 cells. CONCLUSION: The antipyretic effect of dipyrone differs from that of indomethacin in that it does not depend on AVP release or inhibition of PG synthesis.

Changes in paw oedema triggered via bradykinin B(1) and B(2) receptors in streptozotocin-diabetic rats.

The present study investigated hind paw oedema mediated by bradykinin B(1) and B(2) receptors in streptozotocin-diabetic rats. Paw oedema induced by intraplantar (i.pl.) injection of bradykinin or the selective bradykinin B(2) receptor agonist, Tyrosine(8)-bradykinin ([Tyr(8)]bradykinin) (both 3 nmol/paw), was significantly reduced at 4 weeks after streptozotocin treatment (34 +/- 8% and 40 +/- 7%). At 6 weeks after streptozotocin, when paw oedema caused by substance P or prostaglandin E(2) (both 10 nmol/paw) was unchanged, inhibition of bradykinin B(2) receptor-mediated oedema was maximal (66 +/- 6% and 72 +/ -2%, for bradykinin and [Tyr(8)]bradykinin, respectively). The selective bradykinin B(1) receptor agonist, [des-Arg(9)]bradykinin (100 nmol/paw), induced only slight paw oedema in non-diabetic controls. Responses to [des-Arg(9)]bradykinin were markedly enhanced 8 weeks after streptozotocin (from 0.09 +/- 0.01 to 0.38 +/- 0.05 ml), less so at 10 weeks (0.22 +/- 0.03 ml), and returning to basal values at 12 weeks (0.11 +/- 0.03 ml). Treatment with insulin protamine zinc (1-3 U/day/7 weeks, s.c.) did not reverse the inhibition of responses to [Tyr(8)]bradykinin or the potentiation of responses to [des-Arg(9)]bradykinin seen at 8 weeks. Thus, streptozotocin-induced diabetes induces long-lasting alterations in oedematogenic responsiveness to kinins in the rat, characterized by marked reduction of oedema involving activation of bradykinin B(2) receptors, associated with enhancement of bradykinin B(1) receptor-mediated oedema.

Receptors mediating endothelin-1-induced contractions of rabbit gallbladder.

We have examined the responsiveness of strips of rabbit gallbladder (RGB) to endothelin (ET) receptor agonists, and its susceptibility to blockade by selective antagonists. Endothelin-1 (ET-1; 0.1-100.0 nM) caused graded tonic contractions with a CK50 (concentration causing response equivalent to 50% of KCl 80 mM) of 3.4 nM and EH (response to highest concentration) of 186 +/- 22, being 40-fold less potent than cholecystokinin-8 (CCK-8), 103-fold more potent than carbachol, but equipotent to ET-3, sarafotoxin S6c (S6c) and IRL 1620. The selective ETA receptor antagonists BQ-123 (3 microM) and A-127722-5 (0.3 microM) did not block responses to ET-1, but BQ-123 depressed responses to 30-100 nM ET-3 by about 35%. The ETB receptor antagonist BQ-788 (1 microM) shifted the curve to S6c by only fivefold. In rabbit aorta and at these same concentrations, BQ-123 and A-127722-5 markedly shifted (> or = 100-fold) the curve for ET-1-induced contraction, whereas BQ-788 shifted that for S6c 40-fold. Higher concentrations of all three antagonists contracted the RGB. Thus, although RGB responses to ETs and selective ETB receptor agonists seem to be largely mediated via ET, receptors, they are remarkably insensitive to blockade by both selective ETA and ETB receptor antagonists, as previously reported in the guinea pig gallbladder. Finally, through yet unknown mechanisms, high concentrations of ET receptor antagonists induce marked RGB contractions. It remains to be seen whether this finding is predictive of adverse biliary tract side-effects of such drugs in the clinic.

Influences of indomethacin on contractions induced by endothelins in guinea pig isolated gallbladder.

Endothelins (ETs) potently contract guinea pig gallbladder (GPGB) via endothelin-A and -B (ETA and ETB) receptors. This study assesses the possible participation of eicosanoids in the mediation of responses of the GPGB (Krebs' solution, 37 degrees C, 0.5 g load) triggered through each receptor type. Indomethacin (INDO; 5.6 microM) shifted the curve to endothelin-1 (ET-1) (0.1-100 nM) to the right, enhancing the CK50 (concentration causing 50% of response to KCl 80 mM) from 0.9 to 6.8 nM and reducing its EH (response to 100 nM) from 170 +/- 13 to 123 +/- 9 (% of response to 80mM KCl). INDO strongly depressed responses to sarafotoxin S6c (S6c; control CK50 0.9 nM), reducing its EH from 108 +/- 5 to 21 +/- 4. Neither BQ-123 nor BQ-788 (1 microM) changed responses to ET-1, but each markedly reduced responsiveness to ET-3 (control: CK50 of 9.7 nM and EH of 153 +/- 14; BQ-123: approximately = 100 nM and 44 +/- 12; BQ-788 approximately = 100 nM and 65 +/- 18). In the presence of BQ-123, INDO further depressed responses to ET-3 (EH 26 +/- 6), whereas in the presence of BQ-788, such responses were strongly enhanced (EH 126 +/- 9). These findings strongly suggest that contractions of GPGB caused via ETB receptors are mediated to a large extent by contractile eicosanoids, whereas those caused (at least by ET-3) via ETA receptors are limited by relaxant eicosanoids. The cellular sources and nature of the eicosanoids released by ETs remain to be established.

Mixed endothelin ET(A) and ET(B) antagonist bosentan inhibits oleic acid-induced lung plasma extravasation in mouse.

The possible participation of endogenous endothelins (ETs) in enhancement of plasma extravasation induced by oleic acid was assessed in mice. Oleic acid (0.5-2%/kg, i.v.) increased accumulation of Evans blue in lungs in dose-dependent fashion, with a clearcut peak at 1 h (lung Evans blue content: control 0.17 +/- 0.01; oleic acid 1%/kg 0.63 +/- 0.04 microg per 10 mg wet tissue). Pretreatment with the mixed endothelin-ET(A) and ET(B) (ET(A)/ET(B)) receptor antagonist bosentan (30 mg/kg, i.v., 30 min before oleic acid) markedly reduced lung Evans blue content (to 0.24 +/- 0.04), as did pretreatments with prazosin (1 mg/kg), meloxicam (5 mg/kg) and dexamethasone (1 mg/kg/day, for 3 days). Thus, ETs play a pivotal role in the increase in lung microvascular permeability caused by oleic acid in the mouse. The ET receptors involved in the pulmonary vascular changes associated with this experimental model of adult respiratory distress syndrome (ARDS), as well as the relationship between ETs and the sympathetic system, eicosanoids and cytokines remain to be clarified.

Participation of endogenous endothelins in delayed eosinophil and neutrophil recruitment in mouse pleurisy.

OBJECTIVE: Investigate the role of endothelins in leukocyte recruitment in allergic and non allergic inflammation. METHODS: Pleurisy was induced in mice by intrathoracic injection of ovalbumin (OVA; in sensitized animals), E. coli LPS, carrageenan, Mycobacterium bovis (BCG) or zymosan. Animals were treated with BQ-123 or BQ-788 (1.5-150 pmol/cavity), or intravenously with bosentan (30 mg/kg). RESULTS: None of the ET receptor antagonists modified early neutrophil recruitment (at 4 h) induced by OVA, LPS, carrageenan, BCG or zymosan or plasma leakage caused by carrageenan or zymosan. Mononuclear and eosinophil accumulation triggered by OVA were reduced by BQ-123 (150 pmol/cavity) or bosentan (68 and 43% inhibition of eosinophilia), but unaffected by BQ-788. BQ-123 and bosentan also inhibited LPS increases in neutrophil (by 67 and 40%) and eosinophil (by 63 and 74%) at 24 h. CONCLUSIONS: Endothelins, acting via ETA receptors, play a role in late eosinophil and neutrophil accumulation (24 h), but not in the acute (4 h) neutrophilic response.

Endothelin-1-induced ET(A) receptor-mediated nociception, hyperalgesia and oedema in the mouse hind-paw: modulation by simultaneous ET(B) receptor activation.

Endothelin-1 causes ET(A) receptor-mediated enhancement of capsaicin-induced nociception in mice. We have assessed if this hyperalgesic effect of endothelin-1 is also accompanied by other pro-inflammatory effects, namely nociception and oedema, and characterized the endothelin ET receptors involved. Intraplantar (i. pl.) hind-paw injection of endothelin-1 (0.3 - 30 pmol) induced graded nociceptive responses (accumulated licking time: vehicle, 20. 5+/-3.3 s; endothelin-1 at 30 pmol, 78.1+/-9.8 s), largely confined to the first 15 min. Endothelin-1 (1 - 10 pmol) potentiated ipsilateral capsaicin-induced (0.1 microgram, i.pl.; at 30 min) nociception (vehicle, 40.2+/-2.6 s; endothelin-1 at 10 pmol, 98.4+/-5.8 s, but 30 pmol was inactive), and caused oedema (increase in paw weight 5 min after capsaicin: vehicle, 46.3+/-2.3 mg; endothelin-1 at 30 pmol, 100.3+/-6.1 mg). Selective ET(B) receptor agonists sarafotoxin S6c (up to 30 pmol) and IRL 1620 (up to 100 pmol) were inactive, whereas endothelin-3 (up to 30 pmol) induced only modest oedema. ET(A) receptor antagonists BQ-123 (1 nmol, i.pl. ) or A-127722-5 (6 micromol kg(-1), i.v.) prevented all effects of endothelin-1 (10 pmol), but the ET(B) receptor antagonist BQ-788 (1 or 10 nmol, i.pl.) was ineffective. BQ-788 (10 nmol, i.pl.) unveiled hyperalgesic effects of 30 pmol endothelin-1 and endothelin-3. Sarafotoxin S6c (30 pmol, i.pl.) did not modify endothelin-1-induced (10 pmol) nociception or oedema, but abolished hyperalgesia. Thus, endothelin-1 triggers ET(A) receptor-mediated nociception, hyperalgesia and oedema in the mouse hind-paw. Simultaneous activation of ET(B) receptors by endothelin-1 or selective agonists can limit the hyperalgesic, but not the nociceptive or oedematogenic, effects of the peptide.

ET(B) receptor blockade potentiates the pressor response to big endothelin-1 but not big endothelin-2 in the anesthetized rabbit.

The precursor of endothelin-1, big endothelin-1, is considered to be a more reliable marker of systemic production of vasoactive peptide. However, it is largely unclear whether ET(B) receptor-dependent clearance and endothelium-derived relaxing factors affect the precursor in a similar manner to mature ET-1. These ET(B)-dependent modulations of big ET-1 and big ET-2 pressor properties were therefore studied in the anesthetized rabbit. When injected into the left cardiac ventricle, ET-1 and ET-2 (0.01 to 1 nmol/kg) each induced biphasic responses (a depressor followed by a pressor response), whereas big ET-1 and big ET-2 (0.1 to 3 nmol/kg) caused only protracted pressor responses. The highest dose of big ET-1 caused significantly greater responses than ET-1, ET-2, or big ET-2. A selective ET(A) receptor antagonist, BQ-123 (1 mg/kg), markedly reduced pressor responses to all 4 peptides, whereas blockade of ET(B) receptors with BQ-788 (0.25 mg/kg) sharply potentiated the responses to ET-1, ET-2, and big ET-1, but not to big ET-2. Indomethacin (10 mg/kg) sharply potentiated the pressor response to ET-1 (1 nmol/kg), but not big ET-1, at all time points. In control animals, ET-1, but not big ET-1, also triggered an indomethacin-sensitive increase in circulating prostacyclin. Finally, systemically administered big ET-1, but not big ET-2, induced a phosphoramidon-sensitive increase in plasma IR-ET. Our results suggest a significant limiting role of ET(B) receptors on pressor responses to big ET-1. In contrast, the same receptor entities do not modulate the hemodynamic properties of the ET-2 precursor, given that, unlike big ET-1, it is poorly converted in the pulmonary or systemic circulation in anesthetized rabbits.

Role of endothelins on lymphocyte accumulation in allergic pleurisy.

Endothelins participate in different aspects of inflammatory reactions, including edema formation and eosinophil accumulation in allergic reaction. In this study, we demonstrated a role for endogenous endothelins in eosinophil and T lymphocyte recruitment and cytokine secretion in a murine model of allergic inflammation. Intrathoracic stimulation with endothelin-1 triggered a neutrophil accumulation at 4 h, concomitant with an increase of CD4+ and CD8+ T lymphocyte populations. Antigen challenge in sensitized animals leads to an increase in eosinophil and mononuclear cell numbers at 24 h. Treatment with ETA receptor antagonist (BQ123) inhibited antigen-induced eosinophil and mononuclear cell migration, whereas the selective ETB receptor antagonist BQ-788 was ineffective. The latter effect of BQ-123 was due to inhibition of CD4+ and CD8+ T lymphocytes. Treatment with BQ-123 also inhibited interleukin-5 levels in the exudate and plasma as well as intracellular staining of interleukin-4, interleukin-5, and interferon-gamma in CD4+ lymphocytes. These findings suggest that endogenous endothelins contribute to allergic inflammation by modulating lymphocyte recruitment and cytokine production.

Effects of endothelin ET(B) receptor agonists and antagonists on the biphasic response in the ileum.

In the guinea-pig ileum, both sarafotoxin S6c and IRL1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21) induced a concentration-dependent biphasic effect (relaxation and contraction), but distinct tachyphylaxis of the tissue. Cross-tachyphylaxis and additivity experiments evidenced distinct receptors for these agonists. BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]), an endothelin ET(A) receptor antagonist, did not affect the response induced by either agonist. PD145065 [Ac-(D-Bhg-Leu-Asp-Ile-Ile-Trp) (D-Bhg = 5H-dibenzyl[a,d]cycloheptene-10,11-dihydroglycine)], an endothelin ET(A)/ET(B) receptor antagonist, inhibited the contractions induced by IRL1620 and sarafotoxin S6c in competitive and noncompetitive manner, respectively. RES-701-1 [cyclic(Gly1-Asp9)(Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-P he-Phe-Asn-Tyr-Tyr-Trp)], an endothelin ET(B1) receptor antagonist, inhibited both components of the response induced by IRL1620, whereas it inhibited mainly the relaxation induced by low sarafotoxin S6c doses. Apamin and suramin had different effects towards the agonists. Our results suggest that two endothelin ET(B) receptors with distinct signal transduction mechanism mediate the biphasic response: (1) the endothelin ET(B1) receptor: sensitive to RES-701-1 and PD145065 and (2) the endothelin ET(B2) receptor: less sensitive to RES-701-1 and PD145065.