Pharmacological characterisation of the plant sesquiterpenes polygodial and drimanial as vanilloid receptor agonists

This study was designed to assess the participation of transient receptor potential vanilloid 1 (TRPV1) in the biological effects induced by the plant-derived sesquiterpenes polygodial and drimanial. In rat isolated urinary bladder, polygodial and drimanial produced a tachykinin-mediated contraction that was inhibited by combination of NK(1) and NK(2) tachykinin receptor antagonists, SR 140333 and SR 48968. Furthermore, two different TRPV1 antagonists, capsazepine and ruthenium red prevented the contraction induced by both compounds. In addition, capsaicin, polygodial and drimanial displaced in a concentration-dependent manner the specific binding sites of [(3)H]-resiniferatoxin to rat spinal cord membranes, with a IC(50) values of 0.48, 4.2 and 3.2 microM, respectively. Likewise, capsaicin, polygodial and drimanial promoted an increase of [(45)Ca(2+)] uptake in rat spinal cord synaptosomes. In cultured rat trigeminal neurons, polygodial, drimanial and capsaicin were also able to significantly increase the intracellular Ca(2+) levels, effect that was significantly prevented by capsazepine. Together, the present results strongly suggest that the pharmacological actions of plant-derived sesquiterpenes polygodial and drimanial, seem to be partially mediated by activation of TRPV1. Additional investigations are needed to completely define the pharmacodynamic properties of these sesquiterpenes.     

The endogenous cannabinoid agonist, anandamide stimulates sensory nerves in guinea-pig airways

The endogenous cannabinoid agonist, anandamide produced a modest contractile response in guinea-pig isolated bronchus compared with the vanilloid receptor agonist capsaicin. The contractile response to both anandamide and capsaicin was inhibited by the vanilloid receptor antagonist, capsazepine. Furthermore, the NK(2)-selective antagonist, SR48968 but not the NK(1)-selective antagonist, SR140333 inhibited contractile responses to anandamide. The contractile response to anandamide was abolished in tissues desensitized by capsaicin. However, anandamide failed to cross-desensitize the contractile response to capsaicin. The contractile response to anandamide was not significantly altered in the presence of the CB(1) receptor antagonist, SR141716A, nor the amidase inhibitor, phenylmethylsulphonyl fluoride (PMSF) but was significantly increased in the presence of the neutral endopeptidase inhibitor, thiorphan. The cannabinoid agonist, CP55,940 failed to significantly attenuate the excitatory non-adrenergic non-cholinergic (eNANC) response in guinea-pig airways. In contrast, the ORL(1) receptor agonist, nociceptin, significantly inhibited this response. The results demonstrate that anandamide induces a modest contractile response in guinea-pig isolated bronchus that is dependent upon the activation of vanilloid receptors on airway sensory nerves. However, cannabinoid receptors do not appear to play a role in this regard, nor in regulating the release of neuropeptides from airway sensory nerves under physiological conditions.     

The differential contractile responses to capsaicin and anandamide in muscle strips isolated from the rat urinary bladder

The contractile responses to capsaicin and anandamide, exogenous and endogenous agonists for transient receptor potential vanilloid receptor 1 (TRPV1), respectively, were investigated in muscle strips isolated from the rat urinary bladder. Capsaicin and anandamide produced concentration-dependent contractions of the muscle strips. The contractile response induced by capsaicin disappeared within approximately 20 min. In contrast, anandamide produced contractile responses lasting at least for 30 min. Capsaicin produced additive contractile responses in anandamide-treated muscle strips. The contractile response to anandamide was attenuated, but not abolished in strips desensitized by capsaicin. The response to capsaicin was abolished in the presence of a TRPV1 antagonist, N-(4-tertiarybutylphenyl)-4-(3-chlorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), but not altered in the presence of either tetrodotoxin, atropine or indomethacin. In the presence of SR140333, a tachykinin NK₁ receptor antagonist or SR48968, an NK₂ receptor antagonist, the response to capsaicin was attenuated. The response to anandamide was partially attenuated in the presence of ONO8130, a prostanoid EP₁ receptor antagonist, URB597, a fatty-acid amide hydrolase inhibitor, BCTC, SR140333 or SR48968, and almost completely abolished by indomethacin. Neither tetrodotoxin, atropine, a cannabinoid CB₁ receptor antagonist, AM251, nor a cannabinoid CB₂ receptor antagonist, AM630, had any effect on the response to anandamide. These results indicate that capsaicin produces muscle contractions by stimulating the TRPV1 receptor, followed by release of neuropeptides that can activate tachykinin NK₁ and/or NK₂ receptors in the bladder and that the contractile response to anandamide is mediated at least in part by activation of prostanoid EP₁ receptors due to production of prostaglandins in addition to TRPV1 receptor activation.     

SR 48968, A Novel Non-peptide Tachykinin NK-2-Receptor Antagonist,Selectively Inhibits the Non-Cholinergically Mediated Neurogenic Contraction of Guinea-pig Isolated Bronchial Muscle

Abstract— We have examined the actions of SR 48968 ((S)-N-methyl-N-[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl]benzamide), a novel non-peptide tachykinin NK-2-receptor antagonist, on the response evoked by electrical field stimulation or by acetylcholine and neurokinin A on guinea-pig isolated airway smooth muscle. Electrical field stimulation (1–32 Hz, 0·3 ms, 30 V for 20 s) evoked a biphasic response in a frequency-dependent manner, consisting of a cholinergically-mediated fast contraction followed by a non-adrenergically-mediated relaxation in tracheal muscle and by a noncholinergically-mediated slow contraction in bronchial muscle. SR 48968 (0·01–1 μm ) caused a concentration-dependent inhibition of non-cholinergically mediated contraction of bronchial muscle, without significant influence on cholinergically and non-adrenergically-mediated responses. Submaximal contractions of tracheal and bronchial muscles evoked by exogenous neurokinin A (10–300 Nm ) were markedly inhibited by SR 48968 (0·1–1 μm ), but those by exogenous acetylcholine (1–3 μm ) were slightly inhibited by the antagonist. The results indicate that in guinea-pig isolated bronchial muscle, SR 48968 selectively inhibited non-cholinergically mediated neurogenic contraction via antagonism of NK-2 receptors.     

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.     

Increased blocking activity of combined tachykinin NK1- and NK2-receptor antagonists on tachykinergic bronchomotor responses in the guinea-pig

1. The present study compared the effect of the administration of tachykinin NK1- and NK2-receptor antagonists alone and in combination on exogenous and endogenous tachykinin-induced contractions using three different guinea-pig airway preparations: isolated bronchus, isolated perfused lung and in vivo. 2. In the isolated bronchi, the tachykinin NK1-receptor antagonist CP 99994 (0.01-1 microM) produced concentration-dependent inhibition of contractions induced the tachykinin NK1-receptor agonists substance P (SP) and [Met-OMe11] SP ([Met-OMe11]SP), whereas the tachykinin NK2-receptor antagonist SR 48968 (0.1 microM) had no effect. SR 48968 (0.001-0.01 microM) concentration-dependently inhibited contractions induced by the tachykinin NK2-receptor agonists neurokinin A (NKA) and [beta-Ala8]-neurokinin A (4-10) ([betaAla8]-NKA) whereas CP 99994 (0.1 microM) did not inhibit the contractions. The contractile activity of capsaicin, an agent that releases endogenous tachykinins from sensory C-fibres, was inhibited in a concentration dependent manner by SR 48968 (0.001-0.03 microM) but not by CP 99994 (0.1 microM). Combination of CP 99994 and SR 48968 caused increased inhibitory effects on the concentration-response curves to SP, [Met-OMe1l]SP, NKA, [beta-Ala8]-NKA and capsaicin. 3. In isolated perfused lungs, SR 48968 concentration (0.01-10 microM) dependently inhibited NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction whereas CP 99994 (30 microM) had no effect on SP-, NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction. Combination of inactive concentrations of CP 99994 and SR 48968 produced an increased inhibitory effect on all previous stimuli-induced bronchoconstriction. 4. In in vivo guinea-pig studies, intravenous and oral pretreatment with SR 48968 (0.01-1 mg kg(-1) i.v. and 0.1-3 mg kg(-1) p.o., respectively), but not with CP 99994 (1 mg kg(-1) i.v. and 0.3-30 mg kg(-1) p.o., respectively), produced a dose-dependent inhibition of the bronchoconstrictor responses induced by NKA, [beta-Ala8]-NKA and capsaicin. CP 99994 intravenously (0.3 mg kg(-1)) and orally (3-10 mg kg(-1)) inhibited SP-induced bronchoconstriction only. Intravenous and oral low dose combinations of CP 99994 and SR 48968 produced an increased inhibition of SP-, NKA-, [beta-Ala8]-NKA- and capsaicin-induced bronchoconstriction, respectively. The present data indicate that combined tachykinin NK1- and NK2-receptor antagonist treatment compared with single antagonist treatment, using CP 99994 and SR 48968, produced an augmented blockade of tachykinin NK1-, NK2- and capsaicin-mediated contractions in guinea pig airways. These findings support the hypothesis that a dual NK1- and NK2-receptor antagonist may provide an advantage over single activity tachykinin NK1- or NK2-receptor antagonists in pulmonary obstructive diseases.     

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.     

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.     

Antisecretory and relaxatory effects of tachykinin antagonists in the guinea-pig intestinal tract.

Existing models used to study the mechanism of action and antagonism of tachykinergic effects on intestinal contraction and secretion suffer from technical problems and have not been fully characterized using specific tachykinin antagonists. Contraction of ileal segments by substance P, colonic circular muscle by beta-alanine-neurokinin A, and longitudinal muscle by senktide were used as models for neurokinin-induced contraction in the guinea-pig. Guinea-pig colonic epithelial tissue was stimulated by substance P and senktide to assess NK1- and NK3-mediated secretion. Using these models the potency of therapeutically useful compounds was determined. NK1 and NK2 activation directly contracted smooth muscle, while NK1-mediated secretion was nerve-mediated. NK3 stimulation of contraction and secretion was neurally mediated, involving cholinergic nerves and 5-HT release. NK1-mediated contraction and secretion were antagonized by SR140333 (pD'2 = 9.29 and pKb = 8.53); NK2-mediated contraction was antagonised by SR48968 (pD'2 = 8.35) and NK3-mediated contraction and secretion were antagonized by SB223412 (pKb = 8.97 and 8.79). The mixed antagonist MDL103392 blocked NK1- and NK2-mediated contraction with pKb values of 7.92 and 6.71 respectively and NK1-mediated secretion with a pKb value of 6.57. This data characterizes existing tachykinin antagonists, and should orientate the development of improved compounds as therapies for intestinal disease.     

Prevention by the tachykinin NK2 receptor antagonist, SR 48968, of antigen-induced airway hyperresponsiveness in sensitized guinea-pigs.

The involvement of tachykinins in antigen-induced airway hyperresponsiveness (AHR) was characterized pharmacologically in guinea-pigs sensitized to ovalbumin with antagonists of tachykinin NK1 and NK2 receptors, namely SR 140333 and SR 48968, respectively. AHR was illustrated by increased sensitivity to bronchoconstriction provoked by aerosolized acetylcholine in anaesthetized, ventilated animals, administrated 48 h after ovalbumin aerosol challenge. SR 48968 (1 mg kg-1, i.p.), when given once 30 min before the antigen challenge, prevented AHR, whereas SR 140333 did not. These findings suggest that the tachykinin NK2 receptor antagonist, SR 48968, may be useful for investigating mechanisms of tachykinins in the development of airway hyperresponsiveness.     

Respiratory action of capsaicin microinjected into the nucleus of the solitary tract: involvement of vanilloid and tachykinin receptors.

1. The respiratory response to microinjection of capsaicin into the commissural nucleus of the solitary tract (cNTS) of urethane-anaesthetized rats was investigated in the absence and presence of the competitive vanilloid (capsaicin) antagonist, capsazepine, and selective tachykinin NK1, NK2 and NK3 antagonists (RP 67580, SR 48968 and SR 142801, respectively). 2. Microinjection of capsaicin reduced respiratory frequency but not tidal volume (VT), leading to an overall reduction in minute ventilation (VE). The effect was dose-dependent between 0.5 and 2 nmol capsaicin. Doses greater than 2 nmol produced apnoea. Tachyphylaxis was observed following repeated injection of capsaicin (1 nmol, 30 min apart). 3. Capsazepine (1 nmol) had no effect on frequency or VT when injected alone but completely blocked the respiratory response to capsaicin (1 nmol). 4. RP 67580 (1 but not 5 nmol) alone depressed frequency and VT slightly. Moreover, RP 67580 appeared to potentiate the bradypnoeic effect of capsaicin. In contrast, SR 48968 and SR 142801 (1 and 5 nmol) alone had no significant effect on respiration. However, both agents significantly attenuated the reduction in frequency produced by capsaicin. 5. In conclusion, microinjection of capsaicin into the cNTS decreases overall ventilation, primarily by reducing frequency. The action of capsaicin appears from the data to be mediated by vanilloid receptors since it is blocked by the competitive vanilloid antagonist capsazepine and is subject to tachyphylaxis. However, since NK2 (SR 48968) and NK3 (SR 142801) receptor antagonists block the actions of capsaicin, we propose that capsaicin acts also by releasing tachykinins from central afferent terminals in the cNTS.     

Tachykinin NK(2) receptors facilitate acetylcholine release from guinea-pig isolated trachea

The release of newly synthesised [3H]acetylcholine was evoked by electrical field stimulation (5 Hz, 600 pulses) of epithelium-deprived guinea-pig trachea strips after sensory neuropeptides depletion with 3 microM capsaicin. The selective tachykinin NK(2) receptor agonist [betaAla(8)]neurokinin A-(4-10) increased in a concentration-dependent manner the electrically-induced release of [3H]acetylcholine. The facilitatory effect was antagonised by the selective non-peptide tachykinin NK(2) receptor antagonist, SR 48968 (apparent pK(B) 8.9). The tachykinin NK(1) and NK(3) receptor agonists substance P methyl ester and senktide (both 10 and 100 nM), respectively, did not affect the evoked release of [3H]acetylcholine. It is concluded that the cholinergic nerves of guinea-pig trachea are endowed with prejunctional facilitatory tachykinin receptors of the NK(2) subtype.     

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.     

Reduced inflammation in genetically hypertensive rat airways is associated with reduced tachykinin NK(1) receptor numbers

The airways of the genetically hypertensive rat (GH) are hyperinnervated by substance P-containing sensory nerves and exhibit reduced inflammatory responsiveness to substance P and to capsaicin. The present study measured tracheal inflammation to resiniferatoxin (1.0 microgram/kg i.v.), a capsaicin analogue, which lacks the hypotensive action of capsaicin itself, alone or after the neuronal nitric oxide synthase inhibitor 1-(2-trifluoromethylphenyl)imidazole (TRIM) (50 mg/kg i.p.). The inflammatory response to resiniferatoxin alone was 50% lower in untreated GH than in control rats, a similar strain difference to that seen previously with capsaicin. Pre-treatment with TRIM had no effect on inflammation in either strain. Binding kinetics of the tachykinin NK(1) receptor antagonist [3H](S)-1-(2-[3-(3, 4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4- phenyl-l-azoniabicyclo[2,2,2,]octane chloride ([3H]SR140333)(0.125-16.0 nM) showed 50% reduction of B(max) in GH versus control tracheae (74+/-13 cf.165+/-26 fmol/mg protein). Our results indicate that the reduced neurogenic inflammatory responsiveness in GH rats can be attributed entirely to reduced tachykinin NK(1) receptor numbers.     

Contractile effect of (+)-glaucine in the isolated guinea-pig ileum.

The intestinal effects of (+)-glaucine [(S)-1,2,9,10-tetramethoxyaporphine] were studied using the guinea-pig ileum. (+)-Glaucine (10-300 microM) induced ileal contractions. The contraction was not affected by tetrodotoxin, atropine, hexamethonium, propranolol, naloxone, methysergide, N(G)-nitro-L-arginine methyl ester, SR141716A (a cannabinoid CB1 receptor antagonist) or SR140333 (a tackykinin NK1 receptor antagonist) plus SR48968 (a tackykinin NK2 antagonist). (+)-Glaucine-induced contraction was reduced by indomethacin, nordihydroguaiaretic acid or bisindolylmaleimide I and abolished by verapamil and nifedipine. These results suggest that (+)-glaucine-induced contraction involves activation of voltage-dependent Ca2+ channels and protein kinase C and could be mediated by the release of arachidonic acid metabolites.     

In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist

(S)1- {;2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl };-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR140333) is a new non-peptide antagonist of tachykinin NK₁ receptors. SR140333 potently, selectively and competitively inhibited substance P binding to NK₁ receptors from various animal species, including humans. In vitro, it was a potent antagonist in functional assays for NK₁ receptors such as [Sar⁹,Met(O₂)¹¹]substance P-induced endothelium-dependent relaxation of rabbit pulmonary artery and contraction of guinea-pig ileum. Up to 1 μM, it had no effect in bioassays for NK₂ ([βAla⁸]neurokinin A-induced contraction of endothelium-deprived rabbit pulmonary artery) and NK₃ ([MePhe⁷]neurokinin B-induced contraction of rat portal vein) receptors. The antagonism exerted by SR140333 toward NK₁ receptors was apparently non-competitive, with pD'₂ values (antagonism potency evaluated by the negative logarithm of the molar concentration of antagonist that produces a 50% reduction of the maximal response to the agonist) between 9.65 and 10.16 in the different assays. SR140333 also blocked in vitro [Sar⁹,Met(O₂)¹¹]substance P-induced release of acetylcholine from rat striatum. In vivo, SR140333 exerted highly potent antagonism toward [Sar⁹,Met(O₂¹¹]substance P-induced hypotension in dogs (ED₅₀ = 3 μg/kg i.v., bronchoconstriction in guinea-pig (ED₅₀ = 42 μg/kg i.v.) and plasma extravasation in rats (ED₅₀ = 7 μg/kg i.v.). Finally, it also blocked the activation of rat thalamic neurons after nociceptive stimulation (ED = 0.2 μg/kg i.v.).     

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.     

Neurogenic plasma leakage in mouse airways

1. This study sought to determine whether neurogenic inflammation occurs in the airways by examining the effects of capsaicin or substance P on microvascular plasma leakage in the trachea and lungs of male pathogen-free C57BL/6 mice. 2. Single bolus intravenous injections of capsaicin (0.5 and 1 micromol kg(-1), i.v.) or substance P (1, 10 and 37 nmol kg(-10, i.v.) failed to induce significant leakage in the trachea, assessed as extravasation of Evans blue dye, but did induce leakage in the urinary bladder and skin. 3. Pretreatment with captopril (2.5 mg kg(-1), i.v.), a selective inhibitor of angiotensin converting enzyme (ACE), either alone or in combination with phosphoramidon (2.5 mg kg(-1), i.v.), a selective inhibitor of neutral endopeptidase (NEP), increased baseline leakage of Evans blue in the absence of any exogenous inflammatory mediator. The increase was reversed by the bradykinin B2 receptor antagonist Hoe 140 (0.1 mg kg(-1), i.v.). 4. After pretreatment with phosphoramidon and captopril, capsaicin increased the Evans blue leakage above the baseline in the trachea, but not in the lung. This increase was reversed by the tachykinin (NK1) receptor antagonist SR 140333 (0.7 mg kg(-1), i.v.), but not by the NK2 receptor antagonist SR 48968 (1 mg kg(-1), i.v.). 5. Experiments using Monastral blue pigment as a tracer localized the leakage to postcapillary venules in the trachea and intrapulmonary bronchi, although the labelled vessels were less numerous in mice than in comparably treated rats. Blood vessels of the pulmonary circulation were not labelled. 6. We conclude that neurogenic inflammation can occur in airways of pathogen-free mice, but only after the inhibition of enzymes that normally degrade inflammatory peptides. Neurogenic inflammation does not involve the pulmonary microvasculature.     

NK2 receptors mediate tachykinin-induced contractions of rat uterus during the oestrous cycle.

We examined tachykinin-induced contractions of uteri from rats during the oestrous cycle. The potencies of substance P, neurokinin A, neurokinin B and the tachykinin NK2 receptor-selective agonist, [Lys5, MeLeu9, Nle10] neurokinin A-(4-10), and of the non-peptide tachykinin NK1, NK2 and NK3 receptor antagonists (S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pip eridin-3-yl]ethyl]-4phenyl-1-azonia-bicyclo[2.2.2]octane (SR 140333), (S)-N-methyl-N [4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzam ide (SR 48968) and (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)prop yl)-4-phenylpiperidin-4-yl)-N-methylacetamide (SR 142801), were examined. The relative agonist potencies, i.e., [Lys5, MeLeu9, Nle10] neurokinin A-(4-10) > or = neurokinin A > neurokinin B > or = substance P were similar in preparations from rats in dioestrus/metoestrus and those in proestrus/oestrus. Apparent pK(B) values for SR 48968 versus neurokinin A and [Lys5, MeLeu9, Nle10] neurokinin A-(4-10), were 9.9 and 9.2, respectively, indicating activation of an NK2 receptor. SR 140333 (10 nM) produced only a small rightward shift of the log concentration-response curve to substance P. SR 48968 (3 nM), but not SR 142801 (100-300 nM) reduced the effect of neurokinin B. These data indicate that in the rat tachykinin-induced contractions of the uteri during the oestrous cycle are mediated primarily by tachykinin NK2 receptors, and that fluctuations in ovarian hormonal levels during the oestrous cycle have little influence on the uterine response to tachykinins.     

Role for NK(1) and NK(2) receptors in the motor activity in mouse colon

The present study examined the effects induced by endogenous and exogenous activation of NK₁ and NK₂ receptors on the mechanical activity of mouse proximal colon. Experiments were performed in vitro recording the changes in intraluminal pressure from isolated colonic segments. Electrical field stimulation in the presence of atropine and guanethidine produced a small relaxation, followed by nonadrenergic noncholinergic (NANC) contraction. SR140333, NK₁ receptor antagonist, or SR48968, NK₂ receptor antagonist, significantly reduced the contraction, although SR48968 appeared more efficacious. The co-administration of SR140333 and SR48968 virtually abolished the NANC contraction. [Sar⁹, Met(O₂)¹¹]-substance P, selective NK₁ receptor agonist, induced a concentration-dependent biphasic effect, contraction followed by reduction of the mechanical spontaneous activity. Both effects were antagonized by SR140333, but not by SR48968. [β-Ala⁸]-neurokinin A (4-10), selective NK₂ receptor agonist, evoked concentration-dependent contraction, which was antagonized by SR48968, but not by SR140333. The contraction induced by [Sar⁹, Met(O₂)¹¹]-substance P, but not by [β-Ala⁸]-neurokinin A (4-10), was reduced by tetrodotoxin or atropine, and increased by N_ω-nitro-L-arginine methyl ester (L-NAME), inhibitor of nitric oxide synthase. The inhibitory effects induced by [Sar⁹, Met(O₂)¹¹]-substance P were abolished by tetrodotoxin or L-NAME. The results of the present study suggest that in mouse colon both NK₁ and NK₂ receptors are junctionally activated by endogenous tachykinins to cause an additive response. NK₁ receptors appear to be located on cholinergic and on nitrergic neurons as well as on smooth muscle cells, whereas NK₂ receptors seem to be present exclusively on smooth muscle cells.