Establishment and validation of a rabbit model for in vivo pharmacodynamic screening of tachykinin NK2 antagonists.

We attempted to establish and validate an in vivo pharmacodynamic (PD) rabbit model to screen tachykinin NK(2) receptor (NK(2)-R) antagonists using pharmacological and pharmacokinetic (PK)/PD analyses. Under urethane anesthesia, changes in intracolonic pressure associated with intravenous (i.v.) administration of a selective NK(2)-R agonist, ßAla(8)-neurokinin A(4-10) (ßA-NKA), was monitored as a PD marker. The analgesic effects of NK(2)-R antagonists were evaluated by monitoring visceromotor response (VMR) to colorectal distension in a rabbit model of visceral hypersensitivity induced by intracolonic treatment of acetic acid. Intravenous administration of ßA-NKA induced transient colonic contractions dose-dependently, which were inhibited by the selective NK(2)-R antagonists in dose- and/or plasma concentration-dependent manners. The correlation between PD inhibition and plasma concentration normalized with the corresponding in vitro binding affinity was relatively high (r(2) = 0.61). Furthermore, the minimum effective doses on the VMR and ID(50) values calculated in the PD model were highly correlated (r(2) = 0.74). In conclusion, we newly established and validated a rabbit model of agonist-induced colonic contractions as a screening tool for NK(2)-R antagonists. In a drug discovery process, this PD model could enhance the therapeutic candidate selection for irritable bowel syndrome, pharmacologically connecting in vitro affinity for NK(2)-R with in vivo therapeutic efficacy.

Neurokinin-1 receptor mediates stress-exacerbated allergic airway inflammation and airway hyperresponsiveness in mice.

BACKGROUND: A wealth of clinical observation has suggested that stress and asthma morbidity are associated. We have previously established a mouse model of stress-exacerbated allergic airway inflammation, which reflects major clinical findings. OBJECTIVE: The aim of the current study was to investigate the role of the neurokinin- (NK-)1 receptor in the mediation of stress effects in allergic airway inflammation. METHODS: BALB/c mice were systemically sensitized with ovalbumin (OVA) on assay days 1, 14, and 21 and repeatedly challenged with OVA aerosol on days 26 and 27. Sound stress was applied to the animals for 24 hours, starting with the first airway challenge. Additionally, one group of stressed and one group of nonstressed mice received the highly specific NK-1 receptor antagonist RP 67580. Bronchoalveolar lavage fluid was obtained, and cell numbers and differentiation were determined. Airway hyperreactivity was measured in vitro by electrical field stimulation of tracheal smooth-muscle elements. RESULTS: Application of stress in sensitized and challenged animals resulted in a significant increase in leukocyte number in the bronchoalveolar lavage fluid. Furthermore, stressed animals showed enhanced airway reactivity. The increase of inflammatory cells and airway reactivity was blocked by treatment of animals with the NK-1 receptor antagonist. CONCLUSION: These data indicate that the NK-1 receptor plays an important role in mediating stress effects in allergen-induced airway inflammation.

Role of neuropeptide receptor systems in vanilloid VR1 receptor-mediated gastric acid secretion in rat brain.

Previously, we reported that the injection of capsaicin into the lateral cerebroventricle (i.c.v.) stimulated gastric acid secretion via vanilloid VR1 receptors and the vagal cholinergic pathways in anesthetized rats. In the present study, we investigated the involvement of receptor systems for neurokinin A, calcitonin gene-related peptide (CGRP) and glutamate in the vanilloid VR1 receptor-mediated response. The i.c.v. injection of neurokinin A (30 nmol) stimulated gastric acid secretion in the presence of cis-2-(diphenylmethyl)-N-[(2-iodophenyl)methyl]-1-azabicyclo[2.2.2]octan-3-amine oxalate (L-703606, a tachykinin NK1 receptor antagonist, 30 nmol) and the effect was inhibited by cyclo[Gln-Trp-Phe-Gly-Leu-Met] (L-659877, a tachykinin NK2 receptor antagonist, 30 nmol); the values were 145.9 +/- 32.3 and 21.1 +/- 16.6 microEq HCl per 120 min, respectively. The value in the control group was 14.3 +/- 3.8 microEq HCl. The tachykinin NK2 receptor-mediated secretion was inhibited by i.c.v. injections of antagonists of the CGRP1 receptor (human CGRP fragment 8-37, 15 nmol) and non-N-methyl-D-aspartate (non-NMDA)-type glutamate receptor (6-cyano-7-nitroquinoxaline-2,3-dione, 10.9 nmol); the values were 30.8+/-29.8 and 5.7+/-16.9 microEq HCl, respectively. Gastric acid secretion induced by the i.c.v. injection of 30 nmol capsaicin (178.4 +/- 34.0 microEq HCl) was inhibited by antagonists of tachykinin NK2 (23.7 +/- 6.2) and CGRP1 (21.2 +/- 8.5), but not tachykinin NK1 (181.4 +/- 37.0), receptors. The gastric acid secretion induced by capsaicin was decreased by the i.c.v. pre-injection of low doses of neurokinin A or CGRP, which alone had no effect on the secretion. These findings suggest the involvement of tachykinin NK2, CGRP and non-NMDA receptor systems in the vanilloid VR1 receptor-mediated regulation of gastric acid secretion in the rat brain regions close to the lateral cerebroventricle.

Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors.

A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to microM agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.

The anti-gonadotropic effects of cytokines: the role of neuropeptides.

The inhibitory effect of inflammation and endotoxins on the secretion of reproductive hormones from the hypothalamo-pituitary axis is well documented. A comparison of the luteinizing hormone (LH) suppressing effects of several pro-inflammatory cytokines revealed that centrally administered IL-1 beta was the most potent inhibitor of pituitary LH secretion; interleukin (IL)-1 alpha and tumor necrosis factor (TNF) alpha were relatively less effective, whereas IL-6 was ineffective. This order of potency suggested that the anti-gonadotropic effects of an immune challenge are most likely attributable to the action of centrally released IL-1 beta, and this was supported by the demonstration that IL-1 beta suppressed hypothalamic luteinizing hormone releasing hormone (LHRH) release. We used a multifaceted approach to identify the afferent signals in the brain that convey immune messages to hypothalamic LHRH neurons. Pharmacological studies with specific antagonists of opioid receptor subtypes demonstrated that activation of the mu 1 receptor subtype was required to transmit the cytokine signal. Furthermore, icv IL-1 beta upregulated hypothalamic POMC mRNA and increased the concentration and release of beta-endorphin, the primary ligand of mu 1 receptors. We have obtained evidence that IL-1 beta also enhanced the gene expression and concentration of tachykinins, a family of nociceptive neuropeptides in the hypothalamus. Blockade of tachykinergic NK2 receptors attenuated IL-1 beta induced inhibition of LH secretion. Collectively, these results demonstrate that IL-1 beta, generated centrally in response to inflammation, upregulates the opioid and tachykinin peptides in the hypothalamus. These two groups of neuropeptides are critically involved in relaying the cytokine signal to neuroendocrine neurons and causing the suppression of hypothalamic LHRH and pituitary LH release.

Involvement of NK-1 and NK-2 tachykinin receptor mechanisms in jaw muscle activity reflexly evoked by inflammatory irritant application to the rat temporomandibular joint.

An electromyographic (EMG) study was carried out in 51 anesthetized rats to assess if neurokinin, NK-1 and NK-2, receptor mechanisms and tachykinins were involved in the increased jaw muscle activity which can be reflexly evoked by injection of the small-fiber excitant and inflammatory irritant mustard oil (MO) into the temporomandibular joint (TMJ) region. A baseline level of EMG activity was recorded bilaterally for 20 min from digastric (DIG) and masseter (MASS) muscles and then each animal was treated with NK-1 or NK-2 antagonist or vehicle. In one series of experiments either the NK-1 antagonist CP-99,994 (20 microg approximately 54 nmol), the NK-2 antagonist MEN-10,376 (10 microg approximately 9 nmol or 20 microg approximately 18 nmol) or vehicle (control) was administrated into the lateral ventricle (i.c.v.); in another series the NK-1 antagonist (4 mg/kg approximately 3-4 micromol/rat) or vehicle (control) was given intravenously (i.v.). After 10 min, MO (20 microl, 20%) was applied to one TMJ (first injection) and 45 min later, MO was applied to the opposite TMJ (second injection). Pretreatment with neurokinin antagonists had little effect on the incidence of the MO-evoked EMG responses but did significantly reduce the EMG magnitude and duration. In the animals pretreated with NK-1 antagonist only the responses to the second MO injection was significantly affected whereas NK-2 pretreatment reduced the EMG responses to both MO injections to the TMJ. The systematic depression of the MO-evoked EMG responses by the NK-2 antagonist suggests that neurokinin A may be involved in the EMG responses. Since the NK-1 antagonist produced no systematic changes in responses elicited by the first MO injection, substance P does not seem to be associated directly with the initiation or maintenance of the EMG responses but may be involved if a 'central sensitization' has been induced by the first MO injection to the TMJ.

Synthesis and biological activity of tripeptide FR113680 analogues containing unconventional amino acids.

In order to further develop structure-activity relationships and to get information about the biological active conformations we synthetized analogues tripeptide to the FR 113680 [Ac-Thr-D-Trp(CHO)-PheNMeBzl; Ac: acethyl], in which the phenylalanine residue was replaced by unconventional amino acids [1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); (3aS, 7aS)-octahydroindole-2-carboxylic acid (Oic); (S,S,S)-2-azabiciclo[3.3.0]octane-3-carboxylic acid (Aoc); 3-(1'-naphthyl)alanine (Nap); phenylglicine (Phg); thienylalanine (Thi)]. The biological activity of the peptides was performed on guinea pig ileum for neurokinin 1 (NK-1) and on rat colon for neurokinin 2 (NK-2). In particular, the replacement of the Phe3 by the Oic (8a) gave an higher antagonist activity in both NK-1 and NK-2 receptors, but no improvement in selectivity with respect to reference tripeptide (FR113680). The compound (8a) represent the first example of highly potent peptides that do not contain an aromatic amino acid of the third position as had been previously considered essential.