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The purpose of this study was to determine the receptor subtype(s) that mediates tachykinin-induced neurogenic plasma extravasation in the hamster cheek pouch. Changes in microvascular clearance were quantified by counting the number of leaky sites and calculating the clearance of fluorescein isothiocyanate-dextran [mol wt 70,000 (Dextran 70)] during suffusion of the cheek pouch with substance P, neurokinin A, neurokinin B, and capsaicin. Suffusion of substance P, capsaicin, and neurokinin A, but not neurokinin B, was associated with a significant concentration-dependent increase in leaky site formation and clearance of fluorescein isothiocyanate-Dextran 70 (P < 0.05). However, the responses to substance P and capsaicin were significantly greater than those to neurokinin A. Pretreatment with the selective, nonpeptide NK1 receptor antagonist, CP-96,345, significantly attenuated substance P- and capsaicin-induced but not neurokinin A-induced responses (P < 0.05). These effects were specific, since the 2R,3R enantiomer, CP-96,344, was inactive, and CP-96,345 had no significant effect on adenosine-induced responses. We conclude that, in the hamster cheek pouch, NK1 receptors are the predominant receptors that mediate neurogenic plasma extravasation.

The increase in tracheal vascular permeability evoked by hypertonic saline depends on capsaicin-sensitive sensory nerves, which contain substance P and other neuropeptides. The present study was performed to determine whether a novel, nonpeptide, selective antagonist of the NK1 tachykinin receptor CP-99,994, [(+)-(2S-3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine], can prevent the effect of substance P, capsaicin and hypertonic saline on tracheal vascular permeability. CP-99,994 was also tested against a nonpeptide inflammatory mediator, platelet-activating factor (PAF), to assess the selectivity of its action. Anesthetized F-344 rats were injected with either substance P (5 micrograms/kg i.v.), capsaicin (100 micrograms/kg i.v.) or PAF (10 micrograms/kg i.v.), or were exposed to ultrasonically nebulized 3.6% NaCl. In each group, some of the rats were pretreated with CP-99,994 (1 to 4 mg/kg i.v.), and some with its vehicle (0.9% NaCl). Groups of rats injected with substance P or exposed to hypertonic saline were pretreated with the (2R, 3R)-enantiomer CP-100,263, [(-)-(2R-3R)-3-(2-methoxybenzylamino)-2-phenylpiperidine] (2 or 4 mg/kg i.v.). The magnitude of the increase in tracheal vascular permeability was measured by quantifying the extravasation of Evans blue dye. CP-99,994 prevented the increase in tracheal vascular permeability produced by inhalation of hypertonic saline, by substance P and by capsaicin, but did not prevent the effect of PAF. CP-100,263 did not affect substance P- and hypertonic saline-induced increase in vascular permeability. These results indicate that the NK1 receptor antagonist CP-99,994 produces stereoselective inhibition of neurogenic plasma extravasation evoked by inhalation of hypertonic saline.

The molecular mechanism of action for two chemically distinct and highly selective, nonpeptide antagonists, CP-96,345 and SR-48,968, was studied by development of a series of chimeric constructs between their respective target receptors, the NK1 (substance P) and NK2 (neurokinin A) receptors. The binding affinities of the natural peptide ligands, substance P and neurokinin A, were not affected by exchanging almost the entire C-terminal half of the NK1 receptor with the corresponding segment of the NK2 receptor. In contrast, it was found that transfer from the NK2 to the NK1 receptor of a segment corresponding to transmembrane segment VI, the amino-terminal half of transmembrane segment VII, and the connecting extracellular loop 3 completely switched the susceptibility for the nonpeptide antagonists. This chimeric exchange, corresponding to 17 nonconserved residues, conveyed full susceptibility for the NK2-specific compound SR-48,968 to the previously unresponsive NK1 receptor--i.e., the Ki value for inhibition of binding of 125I-labeled substance P decreased from > 10,000 to 0.97 nM. At the same time the affinity for the NK1-selective compound CP-96,345 decreased > 30-fold. The actual binding site for SR-48,968 was localized to this region of the NK2 receptor by use of [3H]SR-48,968, which did not bind to the NK1 receptor but bound with similar high affinities to the wild-type NK2 receptor and to the chimeric NK1 receptor with the NK2 receptor segment incorporated around transmembrane segments VI and VII, Kd = 1.5 nM and 1.0 nM, respectively. Our data indicate that two chemically very different nonpeptide antagonists, CP-96,345 and SR-48,968, act through epitopes located around transmembrane segment VI on their respective target receptors and that at least the nonconserved residues in these epitopes are not important for the binding of the natural peptide ligands, substance P and neurokinin A.

We studied the effect of neurogenic inflammation on airway blood flow in anesthetized F-344 rats. Three successive determinations of blood flow were made by injecting radionuclide-labeled microspheres suspended in 70% dextrose into the left ventricle. A selective agonist of the tachykinin receptor neurokinin 1 (NK1) increased airway blood flow, but NK2- and NK3-selective agonists were without effect. The natural agonist of NK1 receptors, substance P (1 micrograms/kg), increased airway blood flow, an effect that was abolished by the selective NK1 receptor antagonist CP-99,994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] but not by the (2R,3R)-enantiomer CP-100,263. Capsaicin (25 micrograms/kg), a drug that releases tachykinins and calcitonin gene-related peptide from sensory nerves, increased airway blood flow, and again this effect was abolished by CP-99,994. We also studied the effect of a selective inhibitor (captopril, 2.5 mg/kg) of the tachykinin-degrading enzyme kininase II [or angiotensin-converting enzyme (ACE)] on substance P-induced airway vasodilation. Captopril potentiated and prolonged the vasodilator effect of substance P. We conclude that neurogenic vasodilation in rat airways is due to the release of substance P, acts via NK1 receptors, and may be modulated by ACE.

We studied the role of different tachykinin receptors in mediating neurogenic vasodilation in the nasal mucosa of anesthetized pathogen-free rats. Three successive determinations of blood flow were made by injecting radionuclide-labeled microspheres suspended in 70% dextrose into the left ventricle. A selective agonist of the tachykinin NK1 receptor increased nasal blood flow, but neurokinin NK2- and NK3-selective agonists were without effect. The natural agonist of NK1 receptors, substance P (1 microgram/kg), increased nasal blood flow, an effect that was abolished by the selective NK1 receptor antagonist (+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP-99,994). Capsaicin (25 micrograms/kg), a drug that releases tachykinins from sensory nerves, increased nasal blood flow, and this effect was significantly reduced by CP-99,994. We conclude that a significant component of neurogenic vasodilation in rat nasal mucosa is due to the stimulation of NK1 tachykinin receptors.

Non-peptide ligands for peptide receptors have been discovered in several systems through file screening programs, but the mechanism of action for these candidate drugs is obscure as they do not chemically resemble the native peptides. The compound CP 96345 is a high-affinity, non-peptide antagonist of the substance P (NK1) receptor, which is important in pain perception and neurogenic inflammation. Here we identify epitopes on the NK1 receptor responsible for the specific binding of CP 96345 by systematic exchange of corresponding segments between the NK1 receptor and the homologous NK3 (neurokinin B) receptor, which does not bind the non-peptide ligand. Non-conserved residues, in two epitopes around the top of transmembrane segment V and in one epitope at the top of transmembrane segment VI, are essential for the specific action of CP 96345 on the NK1 receptor, but are surprisingly not important for the binding of the natural peptide ligand, substance P. Susceptibility to the non-peptide antagonists can be conveyed to the previously unresponsive NK3 receptor by mutational transfer of this discontinuous epitope from the NK1 receptor.

The non-peptide substance P (SP) antagonist CP-96,345 has a 90-fold selectivity for the human neurokinin-1 (NK-1) or SP receptor over the rat NK-1 receptor, while the agonist SP shows no such selectivity. The cloned NK-1 receptors from these two species have primary protein structures that differ in only 22 of 407 residues. Wild type, chimeric, and point-mutated NK-1 receptors have been created and functionally expressed to understand the structural basis of this species selectivity. Residue 290 in the seventh putative membrane-spanning region is responsible for 20-fold of the affinity difference seen for CP-96,345 between the rat and human NK-1 receptors. Residues in the second extracellular loop and in membrane-spanning region six contribute the additional 3-5-fold of the species difference. The direct or indirect action of these residues with this nonpeptidic antagonist is discussed. This study serves to emphasize the need for species-appropriate in vitro models for the screening of antagonists in the search for therapeutics, and provides useful information to model nonpeptidic antagonist interactions with peptide hormone receptors.

CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl) methyl]-1-azabicyclo[2.2.2]octan-3-amine) antagonism of substance P-stimulated salivation was investigated in pentobarbital-anesthetized rats. Administered either intraperitoneally or orally, CP-96,345 produced dose-dependent inhibition of the sialogogic response elicited by substance P, with a median effective dose of 12-24 mumol/kg (5-10 mg/kg) of body weight, but had no effect on acetylcholine-stimulated salivation. CP-96,345 produced concentration-dependent inhibition of [3H]substance P binding to rat submaxillary gland membranes, with a median effective concentration of 34 +/- 3.6 nM. These biological activities were confined to CP-96,345 in that the 2R,3R enantiomer (CP-96,344) was without effect.

CP-96,345 [(2S, 3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)- methyl]-1-azabicyclo[2.2.2]octan-3-amine] is a potent nonpeptide antagonist of the substance P (NK1) receptor. CP-96,345 inhibited 3H-labeled substance P binding and was a classical competitive antagonist in the NK1 monoreceptor dog carotid artery preparation. CP-96,345 inhibited substance P-induced salivation in the rat, a classical in vivo bioassay, but did not inhibit NK2, NK3, or numerous other receptors; it is thus a selective NK1 antagonist. This compound may prove to be a powerful tool for investigation of the physiological properties of substance P and exploration of its role in diseases.

A clone of murine neuroblastoma (N1E-115) was shown to have functional receptors for the nonapeptide bradykinin. These receptors mediated a large, rapid (about 1 min to peak) and calcium-dependent increase in cyclic GMP. The median effective concentration (EC50) averaged 1.4 nM. In addition, this event was inhibited by quinacrine, 5,8,11,14-eicosatetraynoic acid, and nordi-hydroguaiaretic acid, suggesting involvement of phospholipase A2 with subsequent formation of lipoxygenase metabolities of arachidonic acid. [3H]Bradykinin binding to intact cells, investigated under conditions nearly identical to those used in the cyclic GMP assay, yielded binding sites with KDS of 0.83 pM, 1.0 nM, and 4.9 nM with respective Bmax values of 12, 160, and 250 fmol/10(6) cells. Apparently, the cyclic GMP response was associated with the binding site in which the KD = 1.0 nM. Peptide analogs of bradykinin stimulated cyclic GMP with EC50S nearly identical to their respective KDS determined in binding assays with [3H]bradykinin, thus providing evidence for receptor specificity of this response. This finding of a biochemical response of bradykinin promises to make N1E-115 cells a convenient model system for study of neuronal bradykinin receptors.