Erratum to: Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ)

The environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), which involves a neurogenic-mediated mechanism. Plasma extravasation in trachea, main bronchus and lung was measured as the local ¹²⁵I-bovine albumin accumulation. RT-PCR quantification of TRPV1 and tachykinin (NK₁ and NK₂) receptor gene expression were investigated in main bronchus. Intratracheal injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in trachea and bronchus. 1,2-NQ markedly increased the DEP-induced responses in the rat airways in an additive rather than synergistic manner. This effect that was significantly reduced by L-732,138, an NK₁ receptor antagonist, and in a lesser extent by SR48968, an NK₂ antagonist. Neonatal capsaicin treatment also markedly reduced DEP and 1,2-NQ-induced oedema. Exposure to pollutants increased the TRPV1, NK₁ and NK₂ receptors gene expression in bronchus, an effect was partially suppressed by capsaicin treatment. In conclusion, our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ and takes place by neurogenic mechanisms involving up-regulation of TRPV1 and tachykinin receptors.     

Tachykinin Receptor Antagonists and Cough

Several potent and selective antagonists for tachykinin receptors are now available and appear as powerful tools to investigate the physiological and pathological roles of tachykinins and to identify the type of receptor involved in their effect. Indeed, a lot of studies have shown that tachykinin NK₂receptor antagonists (SR 48968, MEN 10627) are able to inhibit cough induced by citric acid, capsaicin or allergen challenge in the unanesthetized guinea-pig or mechanical stimulation of the trachea in the cat. The effects of tachykinin NK₁receptor antagonists are still debated, whereas an inhibitory effect of SR 142801, a tachykinin NK₃receptor antagonist, has been reported against citric acid-induced cough in the guinea-pig. Experiments with tachykinin receptor antagonists which do not cross the blood brain barrier suggest that the site of action of tachykinin receptor antagonists is most probably peripheral, but a central action, at least in an area not protected by the blood brain barrier, cannot be excluded. Finally, tachykinin NK₂receptor stimulation seems to be involved in sensitisation of cough reflex.     

Facilitation and inhibition by capsaicin of cholinergic neurotransmission in the guinea-pig small intestine

The effects of capsaicin on [³H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [³H]choline. Capsaicin concentration-dependently increased both basal [³H]acetylcholine release (pEC₅₀ 7.0) and muscle tone (pEC₅₀ 6.1). The facilitatory effects of capsaicin were antagonized by 1 μM capsazepine (pK _B 7.0 and 7.6), and by the combined blockade of NK₁ and NK₃ tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 μM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK₁ and NK₃ receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-N^G-nitroarginine (100 μM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [³H]acetylcholine release (pEC₅₀ 6.4) and twitch contractions (pEC₅₀ 5.9). The inhibitory effects were not affected by either capsazepine, NK₁ and NK₃ receptor antagonists, the cannabinoid CB₁ antagonist SR141716A or by L-N^G-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 μM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK₁ and NK₃ receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca²⁺ influx into cholinergic neurons.     

The Contractile Effect of Anandamide in the Guinea‐Pig Small Intestine is Mediated by Prostanoids but not TRPV1 Receptors or Capsaicin‐Sensitive Nerves

Although exogenous and endogenous cannabinoid receptor agonists have well‐documented inhibitory effects on gastrointestinal motility, a TRPV1 receptor‐mediated excitatory action of anandamide (arachidonoyl ethanolamide, AEA) in the guinea‐pig ileum strip has also been described. We used in vitro capsaicin desensitization for assessing the possible participation of sensory neurons in the contractile effect of anandamide on the guinea‐pig whole ileum, as well as autonomic drugs and a cyclooxygenase inhibitor for characterizing this response. Isolated organ experiments were used with isotonic recording. Contractions induced by anandamide (1 or 10 μM) were strongly inhibited by tetrodotoxin, indomethacin or atropine plus a tachykinin NK₁ receptor antagonist, but weakly to moderately reduced by atropine alone and partly diminished by the fatty acid amide hydrolase inhibitor URB 597. Neither capsaicin pre‐treatment nor the TRPV1 receptor antagonist BCTC, the ganglionic blocking drug hexamethonium or cannabinoid (CB₁ or CB₂) receptor antagonists, influenced the effect of anandamide. It is concluded that the capsaicin‐insensitive, neuronal excitatory effect of anandamide in the intestine is most probably mediated by cyclooxygenase products. Such a mechanism may also play a role at other sites in the mammalian body.     

1,2-Naphthoquinone activates vanilloid receptor 1 through increased protein tyrosine phosphorylation, leading to contraction of guinea pig trachea

1,2-Naphthoquinone (1,2-NQ) has recently been identified as an environmental quinone in diesel exhaust particles (DEP) and atmospheric PM2.5. We have found that this quinone is capable of causing a concentration-dependent contraction of tracheal smooth muscle in guinea pigs with EC50 value of 18.7 microM. The contraction required extracellular calcium and was suppressed by L-type calcium channel blockers nifedipine and diltiazem. It was found that 1,2-NQ activated phospholipase A2 (PLA2)/lipoxygenase (LO)/vanilloid receptor (VR1) signaling. Additionally, 1,2-NQ was capable of transactivating protein tyrosine kinases (PTKs) such as epidermal growth factor receptor (EGFR) in guinea pig trachea, suggesting that phosphorylation of PTKs contributes to 1,2-NQ-induced tracheal contraction. Consistent with this notion, this action was blocked by the PTKs inhibitor genistein and the EGFR antagonist PD153035, indicating that contraction was, at least in part, attributable to PTKs phosphorylation that activates VR1, resulting in increased intracellular calcium content in the smooth muscle cells.     

Capsaicin‐ and Mustard Oil‐Induced Extracellular Signal‐Regulated Protein Kinase Phosphorylation in Sensory Neurons in vivo: Effects of Neurokinins 1 and 2 Receptor Antagonists and of a Nitric Oxide Synthase Inhibitor

Abstract: Stimulation of primary sensory neurons with capsaicin or mustard oil leads to phosphorylation of extracellular signal‐regulated protein kinase 1/2 (p‐ERK1/2) via activation of transient receptor potential V1 (TRPV1) or TRPA1, respectively. p‐ERK1/2 was determined by Western immunoblotting in the dorsal root ganglia and in the sciatic nerve of rats following either systemic or perineural capsaicin treatment, or mustard oil application to the hind paw skin. To investigate the possible involvement of neurokinin 1 (NK₁) and NK₂ receptors as well as of nitric oxide, the selective antagonists, SR140333 for NK₁ and SR48968 for NK₂, and the nitric oxide synthase inhibitor, N^G‐nitro‐L‐arginine methyl ester (L‐NAME), were employed. The increase of p‐ERK1/2 after systemic capsaicin treatment was markedly attenuated by SR140333, while only the increase in the dorsal root ganglia was impaired by SR48968; in contrast, inhibition of nitric oxide synthase had no effect. Perineural capsaicin induced an increase in p‐ERK1/2 in the ipsilateral sciatic nerve and in the dorsal root ganglia. This effect was not influenced by SR140333 or L‐NAME. We found for the first time that mustard oil application to the hind paw skin caused an increase in p‐ERK1/2 in the sciatic nerve and in the dorsal root ganglia and only the phosphorylation in the latter was attenuated by SR140333 while L‐NAME showed no effect. From the present results, it may be assumed that capsaicin‐ or mustard oil‐induced p‐ERK1/2 in sensory neurons is not solely directly linked to TRPV1 or TRPA1 channels, but under certain conditions NK₁‐ and NK₂‐mediated mechanisms are involved.     

Early postnatal,but not late,exposure to chemical ambient pollutant 1,2-naphthoquinone increases susceptibility to pulmonary allergic inflammation at adulthood

High diesel exhaust particle levels are associated with increased health effects; however, knowledge on the impact of its chemical contaminant 1,2-naphthoquinone (1,2-NQ) is limited. We investigated whether postnatal and adult exposures to 1,2-NQ influence allergic reaction and the roles of innate and adaptive immunity. Male neonate (6 days) and adult (56 days) C57Bl/6 mice were exposed to 1,2-NQ (100 nM; 15 min) for 3 days, and on day 59, they were sensitized and later challenged with ovalbumin (OVA). Airway hyper-responsiveness (AHR) and production of cytokines, immunoglobulin E (IgE) and leukotriene B₄ (LTB₄) were measured in the airways. Postnatal exposure to 1,2-NQ activated dendritic cells in splenocytes by increasing expressing cell surface molecules (e.g., CD11c). Co-exposure to OVA effectively polarized T helper (Th) type 2 (Th2) by secreting Th2-mediated cytokines. Re-stimulation with unspecific stimuli (PMA and ionomycin) generated a mixed Th1 (CD4⁺/IFN-γ⁺) and Th17 (CD4⁺/IL-17⁺) phenotype in comparison with the vehicle-matched group. Postnatal exposure to 1,2-NQ did not induce eosinophilia in the airways at adulthood, although it evoked neutrophilia and exacerbated OVA-induced eosinophilia, Th2 cytokines, IgE and LTB₄ production without affecting AHR and mast cell degranulation. At adulthood, 1,2-NQ exposure evoked neutrophilia and increased Th1/Th2 cytokine levels, but failed to affect OVA-induced eosinophilia. In conclusion, postnatal exposure to 1,2-NQ increases the susceptibility to antigen-induced asthma. The mechanism appears to be dependent on increased expression of co-stimulatory molecules, which leads to cell presentation amplification, Th2 polarization and enhanced LTB₄, humoral response and Th1/Th2 cytokines. These findings may be useful for future investigations on treatments focused on pulmonary illnesses observed in children living in heavy polluted areas.     

MEN 11,420, a peptide tachykinin NK2 receptor antagonist, reduces motor responses induced by the intravesical administration of capsaicin in vivo

This study investigates the role of tachykinin NK₁ and NK₂ receptors in motor responses induced by the intravesical instillation of capsaicin in urethane-anaesthetized rats. SR 140,333 (1 μmol/kg, i.v.), a non-peptide NK₁ receptor antagonist, abolished urinary bladder contractions induced by the selective NK₁ receptor agonist [Sar⁹]SP-sulfone (0.1-100 nmol/kg, i.v.) without affecting those induced by the NK₂ receptor agonist [?Ala⁸]NKA(4-10). MEN 11,420 (100 nmol/kg, i.v.), a cyclic peptide NK₂ receptor antagonist, abolished bladder contractions induced by [?Ala⁸]NKA(4-10) (0.3-300 nmol/kg, i.v.) without modifying those induced by [Sar⁹]SP-sulfone. Intravesical instillation of capsaicin (6 nmol/0.6 ml/rat) produced a motor response consisting in a primary contraction followed by a series of high amplitude phasic contractions. The intravesical instillation of saline (0.6 ml/rat) produced a primary contraction of lower amplitude with respect to that induced by capsaicin and the total area under the curve was also lower in saline-instilled rats, however the number and the amplitude of phasic contractions was similar to that induced by capsaicin. MEN 11,420 (100 nmol/kg, i.v.) did not modify motor responses induced by the intravesical administration of saline. In contrast, in capsaicin-instilled rats, MEN 11,420 (100 nmol/kg, i.v.) reduced the primary contraction, the area under the curve and also the number of phasic contractions. SR 140,333 (1 μmol/kg, i.v.) reduced the primary contraction but not other parameters. The combination of SR 140,333 (1 μmol/kg, i.v.) and MEN 11,420 (100 nmol/kg, i.v.) produced an additive inhibitory effect on the primary contraction but not a further inhibition on other parameters with respect to that observed with MEN 11,420 alone. In hexamethonium (110 μmol/kg, i.v.)-pretreated animals the intravesical instillation of capsaicin produced a tonic contraction having greater amplitude and area than that induced by saline. MEN 11,420, but not SR 140,333, significantly reduced the bladder response to capsaicin in hexamethonium-pretreated rats. Again, the combined administration of MEN 11,420 and SR 140,333 did not produce further inhibitory effect in comparison to MEN 11,420 alone. It is concluded that the motor responses induced by the intravesical instillation of capsaicin are mediated by the activation of peripheral tachykinin NK₂ receptors. Received: 11 February 1997 / Accepted: 17 April 1997     

A regional difference of the effect of tachykinin on cholinergic response evoked by electrical field stimulation in the rabbit airway

1. Experiments were designed to determine whether regional differences exist in the effects of phosphoramidon (a metalloprotease inhibitor) and [d-Arg, ¹d-Pro,²d-Trp,^7,9Leu,¹¹]-substance P (a tachykinin antagonist: rpwwL-SP) on contractile responses to electrical field stimulation (EFS) in rabbit airways.2. EFS contractions were potentiated by phosphoramidon and were attenuated by rpwwL-substance P at low frequencies (less than 10 Hz).3. Potentiating effect of phosphoramidon was more pronounced in distal bronchus than trachea and was proportional to total proteinase activity.4. The rank order of inhibitory effect of rpwwL-SP was: trachea > proximal bronchus > distal bronchus, and inverse relationship was observed between the drug's inhibitory effect of drug and total proteinase activity in three different regions.5. Good correlation was observed between total proteinase activity and pD₂ value of neurokinin A in each airway region.6. In conclusion, tachykinin modulates acetylcholine release in the contractile response to EFS at low frequencies (less than 10Hz), and regional differences in the effects of the inhibitor and the antagonist on EFS-evoked contractions in the rabbit airway were suggested to be due to heterogenous distribution of the metalloprotease which metabolized tachykinins.     

Mechanisms involved in the contractile responses induced by the hydroalcoholic extract of Phyllanthus urinaria on the guinea pig isolated trachea: Evidence for participation of tachykinins and influx of extracellular Ca2+ sensitive to ruthenium red

• 1.1. The hydroalcoholic extract (HE) of stems, leaves and roots from P. urinaria (Euphorbiaceae) (1–3000 μ/ml), caused graded contraction in guinea pig trachea (GPT), being more effective in preparations without epithelium.
• 2.2. Response to HE was slightly affected by tetrodotoxin (0.3 μM) and nicardipine (1 μM), but was unaffected by w-conotoxin, atropine, mepyramine or staurosporine (all 1 μM). Indomethacin (3 μM) greatly inhibited HE contraction, but MK 571 (leukotriene D₄ and E₄ antagonist) caused partial inhibition; L-655,240 (thromboxane A₂ antagonist) and WEB 2086 (PAF antagonist) (all 1 μM) were ineffective.
• 3.3. Response to HE was markedly inhibited in a Ca²⁺-free solution and was partially affected in GPT desensitized to capsaicin (10 μM).
• 4.4. Capsazepine (capsaicin antagonist, 3 μM) antagonized the contraction from capsaicin, leaving the response to HE unaffected. In contrast, ruthenium red (an ionic channel antagonist coupled to vanilloid receptors of capsaicin) (0.1–3 μM) caused graded and equipotent noncompetitive inhibition of HE- and capsaicin-induced contractions, but had no effect on carbachol- and prostaglandin E₂-mediated responses.
• 5.5. FK 888 and SR 48968 (NK₁ and NK₂ receptor antagonists, respectively) (both 1 μM) antagonized, through a competitive mechanism, the contraction from SP and [β-ala⁸]NKA(4–10) respectively, but antagonized, through a noncompetitive mechanism, HE-mediated contraction.
• 6.6. We concluded that contraction to HE in GPT is modulated by the epithelium, depends on the release of a cyclo-oxygenase metabolite, and relies largely upon an extracellular Ca²⁺ influx that is highly sensitive to ruthenium red, but is insensitive to L and N-type of voltage-sensitive Ca²⁺ channel antagonists. In addition, NK₁ and NK₂ tachykinins, but not vanilloid receptors, play an important role in mediating its response.

     

Analysis of the Mechanisms Underlying the Contractile Response Induced by the Hydroalcoholic Extract of Phyllanthus urinaria in the Guinea-pig Urinary Bladder In-vitro

The hydroalcoholic extract of Phyllanthus urinaria (Euphorbiaceae), substance P and substance P methyl ester all caused graded contractions in the guinea-pig urinary bladder. Responses to hydroalcoholic extract and substance P were markedly inhibited in calcium-free Krebs solution, this effect being reversed by reintroduction of calcium in the medium. The contraction in response to hydroalcoholic extract was unaffected by atropine, propranolol, prazosin, yohimbine, tetrodotoxin, w-conotoxin, nicardipine, HOE 140, guanethidine, staurosporine, phorbol ester or indomethacin, excluding the involvement of nervous mediated responses, or action via cholinergic, adrenergic, kinins, cyclo-oxygenase metabolites, protein kinase C or activation of L or N-type calcium channels. The selective NK₁ tachykinin antagonist (FK 888), but not NK₂ (SR 48968) antagonized substance P-induced contraction, but both drugs failed to effect Phyllanthus urinaria-induced contraction. Prolonged desensitization of guinea pig urinary bladder with capsaicin (10 μM) or preincubation of guinea-pig urinary bladder with capsazepine did not affect contraction caused by hydroalcoholic extract. Ruthenium red almost completely abolished capsaicin-induced contraction, but had no effect on hydroalcoholic extract-mediated contraction. Substance P and the hydroalcoholic extract caused marked potentiation of the twitch response in the preparations field stimulated. The facilitatory effect of substance P, but not that of hydroalcoholic extract, was prevented by the NK₁ (FK 888), but not by NK₂ (SR 48968) antagonist. We concluded that contraction induced by hydroalcoholic extract of Phyllanthus urinaria in the guinea pig urinary bladder involves direct action on smooth muscle and relies on the mobilization of extracellular calcium influx unrelated to activation of L- and N-type calcium channels or activation of protein kinase C mechanisms. In addition contraction caused by the hydroalcoholic extract of Phyllanthus urinaria in guinea-pig urinary bladder does not involve the activation of tachykinin or vanilloid receptors.     

Endothelin-1 affects capsaicin-evoked release of neuropeptides from rat vas deferens

Capsaicin-sensitive neurones release a number of neuropeptides, such as substance P, neurokinin A, somatostatin and calcitonin gene-related peptide (CGRP), which exert a number of effects on smooth muscle tissues. Endothelin-1 was thought to potentiate the capsaicin-evoked release of neuropeptides from sensory neurones of the rat. We have investigated the neuromodulatory effects of endothelin-1 on capsaicin-induced release of neurotransmitters from rat vas deferens. Capsaicin and human α calcitonin gene-related peptide (human αCGRP) reduced the rat vas deferens twitch responses induced by electrical field stimulation. Human β calcitonin gene-related peptide-(8–37) [human βCGRP-(8–37)] (1 μM), a selective αCGRP receptor antagonist, antagonized the inhibitory effects of both drugs. Endothelin-1 concentration dependently evoked an increase in basal tone of the musculature and potentiated the amplitude of the electrically stimulated responses, blocking inhibitory effects of capsaicin but not of human αCGRP. Moreover, endothelin-1 did not markedly change the inhibitory effects of papaverine (0.1–100 μM) or isoprenaline (1 nM–100 μM) on responses to electrical field stimulation. FR 139317 [(N,N-hexamethylene) carbamoyl-Leu-d-Trp(N-Me)-d-2-Pya], a selective endothelin ET_A receptor antagonist, administered 30 min before endothelin-1 restored the capsaicin effects whereas BQ 788 [Dmpc-γ-MeLeu-d-Trp-(1-methoxycarbonyl)-d-Nle], a selective endothelin ET_B receptor antagonist, was completely ineffective. The endothelin-1-induced block of the capsaicin effect was resistant to tetrodotoxin (1 μM) and 30-min pre-treatment with MEN 10.627 (cyclo[(Met-Asp-Trp-Phe-Dap-Leu) cyclo (2β–5β)]), a selective tachykinin NK₂ receptor antagonist, did not abolish the endothelin-1 effect on the inhibitory response to capsaicin. These results suggest that endothelin-1 selectively inhibits the capsaicin-induced release of neurotransmitters from rat vas deferens and these effects are mediated via endothelin ET_A receptors but not by tachykinin release.     

Pharmacological characterization of senktide-induced tail whips

The tachykinin NK₃ receptor shows promise as a novel target for antipsychotics, but knowledge of downstream activity following tachykinin NK₃ receptor activation is lacking. To determine the practical utility of senktide-induced tail whips in mice as a tool for determining and characterizing downstream activity following tachykinin NK₃ receptor activation, mice were injected with 0.05 nmol of senktide i.c.v. and the number of tail whip bouts was counted for 20 min. Strain differences were observed, with NMRI mice showing a stronger tail whip response than C57Bl/6J mice. Tachykinin NK₃ receptor specificity was confirmed by the absence of the senktide-induced tail whip response in tachykinin NK₃ receptor knockout mice. Effects of tachykinin receptor pharmacological agents were tested by pretreatment with tachykinin NK₃ receptor antagonists (SB222200, talnetant and osanetant), which attenuated senktide-induced tail whips, and the tachykinin NK₁ receptor antagonist MK869, which had no effect on senktide-induced tail whips. Pharmacological interactions with other neurotransmitter systems were determined by pretreatment with dopamine D₁, D₂, and D₃ receptor antagonists, atypical antipsychotics, serotonin 5HT_1a receptor antagonists, serotonin 5HT_2a/c receptor antagonists, benzodiazepine and putative anxiolytics, antidepressants, and an anticholinergic. Senktide-induced tail whips were attenuated by dopamine D₂ receptor antagonists, atypical antipsychotics, serotonin 5HT_2a/c antagonists, and benzodiazepine anxiolytics, but unaffected by drugs from other classes. Thus, the senktide-induced tail whip response is easily quantifiable, specific to the tachykinin NK₃ receptor, and provides valuable information on the downstream pharmacology of tachykinin NK₃ receptor activation.     

Involvement of NK1 and NK2 Receptors in Pulmonary Responses Elicited by Non-adrenergic,Non-cholinergic Vagal Stimulation in Guinea-pigs

Previous studies from our laboratory using exogenously administered neurokinin (NK) agonists have shown that both NK₁- and NK₂-receptor subtypes are involved in plasma extravasation in the guinea-pig airways. In the present study, we have extended these observations using antidromic vagal stimulation to stimulate sensory c-fibres as a means of eliciting the release of endogenous tachykinins in propranolol- and atropine-treated guinea-pigs. Antidromic vagal stimulation (5 ms, 30 s) induced frequency-dependent (1–10 Hz) bronchoconstriction that was completely abolished by co-administration of the NK₁-selective antagonist CP-99,994 ((2s-methoxy-benzyl)-(2-phenyl-piperidin-3s-yl)-amine), and the NK₂-selective antagonist SR-48,968 ((S)-N-methyl-N-[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide), each at a dose sufficient to block NK₁ and NK₂ receptors, respectively (each at 0.3 mg kg^?1, i.v.). In contrast, SR-48,968 when given alone only partially blocked the vagal stimulation-induced bronchospasm, whereas CP-99,994 had no effect. Significant increases (2–3-fold) in plasma extravasation of [¹²⁵I]fibrinogen in the trachea, main bronchi, distal airways and oesophagus following vagal stimulation (5 Hz, 5 min, 10 V, 5 ms) were observed. Pretreatment with the neutral endopeptidase inhibitor, thiorphan (1 mg kg^?1, i.v.), and the angiotensin-converting enzyme inhibitor, enalapril (1 mg kg^?1, i.v.), potentiated both vagal stimulation-induced bronchoconstriction and plasma leakage in all tissues examined. This potentiation was due to reduced metabolism of endogenously released tachykinins since enhanced plasma overflow of immuno-reactive substance P was observed following vagal stimulation in thiorphan- and enalapril-treated guinea-pigs. CP-99,994 substantially blocked plasma leakage in all parts of the airways and in the oesophagus. In comparison, SR-48,968 had no significant effect in the trachea and the oesophagus but partially inhibited plasma leakage in the main bronchi and distal airways. Co-administration of both CP-99,994 and SR-48,968 abolished the residual plasma leakage in these two regions. These results support the hypothesis that both NK₁ and NK₂ receptors are involved in tachykinin-induced pulmonary responses in the airways.     

Hydrogen sulfide causes vanilloid receptor 1-mediated neurogenic inflammation in the airways

Hydrogen sulfide (H(2)S) is described as a mediator of diverse biological effects, and is known to produce irritation and injury in the lung following inhalation. Recently, H(2)S has been found to cause contraction in the rat urinary bladder via a neurogenic mechanism. Here, we studied whether sodium hydrogen sulfide (NaHS), used as donor of H(2)S, produces responses mediated by sensory nerve activation in the guinea-pig airways. NaHS evoked an increase in neuropeptide release in the airways that was significantly attenuated by capsaicin desensitization and by the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine. In addition, NaHS caused an atropine-resistant contraction of isolated airways, which was completely prevented by capsaicin desensitization. Furthermore, NaHS-induced contraction was reduced by TRPV1 antagonism (ruthenium red, capsazepine and SB366791), and was abolished by pretreatment with the combination of tachykinin NK(1) (SR140333) and NK(2) (SR48968) receptor antagonists. In anesthetized guinea-pigs, intratracheal instillation of NaHS increased the total lung resistance and airway plasma protein extravasation. These two effects were reduced by TRPV1 antagonism (capsazepine) and tachykinin receptors (SR140333 and SR48968) blockade. Our results provide the first pharmacological evidence that H(2)S provokes tachykinin-mediated neurogenic inflammatory responses in guinea-pig airways, and that this effect is mediated by stimulation of TRPV1 receptors on sensory nerves endings. This novel mechanism may contribute to the irritative action of H(2)S in the respiratory system.     

Inflammatory oedema induced by Lachesis muta muta (Surucucu) venom and LmTX-I in the rat paw and dorsal skin

The ability of crude venom and a basic phospholipase A₂ (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H₁ antagonist mepyramine (6 mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), cyclooxygenase inhibitor indomethacin (5 mg/kg), nitric oxide synthesis inhibitor l-NAME (100 nmol/site), tachykinin NK₁ antagonist SR140333 (1 nmol/site) and bradykinin B₂ receptor antagonist Icatibant (0.6 mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5 mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while l-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF.     

NK1 receptor-mediated endothelium-dependent relaxation and contraction with different sensitivity to post-receptor signaling in pulmonary arteries

In rabbit intrapulmonary arteries, substance P (SP) has been reported to induce endothelium-dependent relaxation (EDR) and endothelium-dependent contraction (EDC) via tachykinin NK₁ receptors, and endothelium-independent contraction (EIC) via tachykinin NK₂ receptors. The present study pharmacologically examined whether these opposite responses (EDR and EDC) are mediated by the same NK₁ receptor. Five tachykinin agonists, including septide, a reportedly atypical NK₁ agonist, caused concentration-dependent EDR in the presence of NK₂ antagonist (SR-48968) + TXA₂ synthetase inhibitor (ozagrel), which blocked EIC and EDC, in pre-contracted arteries, and concentration-dependent EDC in the presence of NK₂ antagonist (SR-48968) + nitric oxide synthase inhibitor (l-N^G-nitro-arginine methyl ester), which blocked EIC and EDR, in non-contracted arteries. The EC₅₀ values of these agonists for EDR were smaller than those for EDC, indicating that the affinities of NK₁ agonists to NK₁ receptors are different between EDR and EDC. However, the rank order of their potency for EDR and EDC was the same: SP = septide > SP methyl ester (SPME) > neurokinin A > neurokinin B. [Ala⁵, β-Ala⁸]-α-neurokinin fragment 4–10 (NK₂ agonist) and senktide (NK₃ agonist) caused no responses. Two structurally different NK₁ antagonists, CP-99994 and SR-140333, shifted the concentration–EDC and –EDR curves of SPME, a selective NK₁ agonist, and septide rightward and suppressed their maximal responses in a similar concentration-dependent manner, indicating that the affinities of NK₁ antagonists to NK₁ receptors are similar between EDR and EDC. U-73122, a phospholipase C inhibitor, and thapsigargin, 2,5-di-tert-butylhydroquinone, and ruthenium red, all intracellular Ca²⁺ release blockers, inhibited SP-induced EDR and EDC. Effective concentrations of ionomycin (Ca²⁺ ionophore) causing EDR were also lower than those causing EDC. Taken together, SP-induced EDR and EDC are mediated by activation of the same NK₁ receptor followed by an increase in intracellular Ca²⁺, and sensitivity to Ca²⁺ may be higher in the EDR than EDC pathway.     

A method to measure dual NK1/NK2-antagonist activity in dogs

The major pulmonary effects of tachykinins are produced by activation of both NK₁- and NK₂-receptors. A variety of animal models have been used to profile activity of the tachykinins, particularly rodents and guinea pigs, but little information exists regarding methods to evaluate NK₁- and NK₂-receptor antagonist activity in dogs. This study describes a simple method in dogs to measure NK₁- and NK₂-receptor agonist and antagonist activity of drugs in the same preparation. We measured pulmonary resistance (R_L), dynamic lung compliance (C_Dyn), minute volume (MV), and mean arterial blood pressure (MAP) before and after challenge with aerosolized NKA (1%) and i.v. SP (100 ng/kg) to quantify responses to the tachykinin challenge. Challenge with NKA produced an increase in R_L and a decrease in C_Dyn, and this bronchospasm was inhibited by the NK₂-antagonist SR 48968 (ID₅₀ R_L = 1.3 mg/kg and ID₅₀ C_Dyn = 1.3 mg/kg, p.o.). The NK₁-antagonist, CP 99994 was inactive against NKA-induced bronchospasm at doses up to 10 mg/kg, p.o. When the dogs were challenged with SP, there was a fall in MAP and an increase in MV and both responses were inhibited by CP 99994 (ID₅₀ MV = 2.3 mg/kg and ID₅₀ BP = 4.5 mg/kg, p.o.), but not by SR 48968 at doses up to 3 mg/kg, p.o. These results identify that NK₂-receptors mediate the bronchoconstrictor effect of NKA, and NK₁-receptors mediate the hypotension and respiratory stimulation due to SP in dogs. This method offers many advantages for evaluating the effects of tachykinin antagonists including the fact that it is relatively simple to perform and has the capacity to assess both NK₁ and NK₂ antagonist activity in the same preparation.