piperidines and septide

Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum.. Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA.. Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.

Topics: Action Potentials; Analgesics; Animals; Antipsychotic Agents; Atropine; Electric Stimulation; Ileum; Indoles; Isoindoles; Mice; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neurons; Nitroarginine; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P

In the gastrointestinal tract, tachykinin NK1 receptors are widely distributed in a number of neuronal and nonneuronal cells involved in the control of gut motor activity. In particular, in the rabbit isolated distal colon, which is a suitable model system to investigate the contribution of tachykinins as noncholinergic excitatory transmitters, the influence of NK1 receptors in the regulation of peristalsis is not known. The selective NK1-receptor antagonists SR-140333 (0.3 and 1 nM) and MEN-10930 (0.3-10 nM) significantly enhanced the velocity of rabbit colonic propulsion to submaximal stimulation. The prokinetic effect of SR-140333 was prevented by N(omega)-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor, indicating that NK1 receptors located on nitrergic innervation exert a functional inhibitory restraint on the circular muscle and probably on descending excitatory and inhibitory pathways during propulsion. Conversely, the selective NK1-receptor agonist septide (3-10 nM) significantly inhibited colonic propulsion. In the presence of L-NNA, the inhibitory effect of septide was reverted into a prokinetic effect, which is probably mediated by the activation of postjunctional excitatory NK1 receptors.

Topics: Alanine; Animals; Atropine; Colon; Drug Interactions; Enzyme Inhibitors; Indoles; Kinetics; Male; Neurokinin-1 Receptor Antagonists; Nitric Oxide Synthase; Nitroarginine; Peptide Fragments; Peristalsis; Piperidines; Pyrrolidonecarboxylic Acid; Quinuclidines; Rabbits; Receptors, Neurokinin-1; Substance P

The present experiments investigated the role of neurokinin-1 (NK(1)) and neurokinin-3 (NK(3)) receptors on the activity of the locus coeruleus (LC)-noradrenergic system by using a dual probe microdialysis technique in anesthetized guinea pigs. The local application in the LC of the selective NK(1) receptor agonists [SAR(9),Met(O(2))(11)]-SP (10 microM) and septide (1 microM) as well as the selective NK(3) receptor agonist senktide (1 microM), enhanced the extracellular norepinephrine (NE) levels in the prefrontal cortex. The enhancing effect of [SAR(9),Met(O(2))(11)]-SP was completely blocked by the peripheral administration of the selective non peptide NK(1) and NK(3) receptor antagonists, GR 205171 (1 mg/kg, i.p.) and SR 142801 (0.1 mg/kg, i.p.), respectively, whereas SR 142806 (0.1 mg/kg, i.p.) the inactive enantiomer of SR 142801 had no effect. Moreover, the [SAR(9),Met(O(2))(11)]-SP-induced increase in LC DOPAC concentrations, is only antagonized by GR 205171. In contrast, only SR 142801 (0.3 mg/kg, i.p.) could block stereoselectively the senktide-evoked increase in NE levels. Both [SAR(9),Met(O(2))(11)]-SP and senktide effects were blocked by local infusion into the LC of SR 142801 (10(-9) M). These results demonstrate that stimulation of NK(1) and NK(3) receptors located in the LC area modulates the activity of the LC-NE system, and that the excitatory effects of NK(1) receptor agonists require NKB/NK(3) receptor activation in the LC.

Topics: 3,4-Dihydroxyphenylacetic Acid; Animals; Antiemetics; Dose-Response Relationship, Drug; Guinea Pigs; Locus Coeruleus; Male; Neural Pathways; Neurokinin-1 Receptor Antagonists; Neurons; Norepinephrine; Peptide Fragments; Piperidines; Prefrontal Cortex; Pyrrolidonecarboxylic Acid; Receptors, Neurokinin-1; Receptors, Neurokinin-3; Substance P; Tetrazoles

Using a microsuperfusion method in vitro, the effects of the NK1, NK2, and NK3 tachykinin receptor antagonists SR140333, SR48968, and SR142801, respectively, on the NMDA-evoked release of [3H]-acetylcholine were investigated after both acute and chronic suppression of dopamine transmission in striosomes and matrix of the rat striatum. NMDA (1 mm) alone or with D-serine (10 microm) in the presence of alpha-methyl-p-tyrosine (100 microm) markedly enhanced the release of [3H]-acetylcholine through a dopamine-independent inhibitory process. In both conditions, as well as after chronic 6-OHDA-induced denervation of striatal dopaminergic fibers, SR140333, SR48968, or SR142801 (0.1 microm each) reduced the NMDA-evoked release of [3H]-acetylcholine in the matrix but not in striosome-enriched areas. These responses were selectively abolished by coapplication with NMDA of the respective tachykinin agonists, septide, [Lys5,MeLeu9,Nle10]NKA(4-10), or senktide. Distinct mechanisms are involved in the effects of the tachykinin antagonists because the inhibitory response of SR140333 was additive with that of either SR48968 or SR142801. In addition, the SR140333-evoked response remained unchanged, whereas those of SR48968 and SR142801 were abolished in the presence of N(G)-monomethyl-l-arginine (nitric oxide synthase inhibitor). Therefore, in the matrix but not in striosomes, the acute or chronic suppression of dopamine transmission unmasked the facilitatory effects of endogenously released substance P, neurokinin A, and neurokinin B on the NMDA-evoked release of [3H]-acetylcholine. Whereas substance P and neurokinin A are colocalized in same efferent neurons, their responses involve distinct circuits because the substance P response seems to be mediated by NK1 receptors located on cholinergic interneurons, while those of neurokinin A and neurokinin B are nitric oxide-dependent.

Topics: Acetylcholine; Animals; Benzamides; Corpus Striatum; Dopamine; Dopamine Antagonists; Enzyme Inhibitors; In Vitro Techniques; Male; N-Methylaspartate; Neural Inhibition; Neurons; Oxidopamine; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Quinuclidines; Rats; Rats, Sprague-Dawley; Receptors, Tachykinin; Substance P; Synaptic Transmission; Tachykinins; Time Factors

Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations.

Topics: Animals; Binding Sites; CHO Cells; Cricetinae; Humans; Indoles; Iodine Radioisotopes; Isoindoles; Mice; Neurokinin A; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Protein Isoforms; Pyrrolidonecarboxylic Acid; Radioligand Assay; Rats; Receptors, Neurokinin-1; Substance P; Tetrazoles; Tumor Cells, Cultured

The tachykinin NK1 receptor agonist substance P methyl ester (SPOME) impedes intestinal peristalsis by releasing nitric oxide (NO) from inhibitory motor neurones. Since NK1 receptor agonists differ in their receptor interaction, we set out to compare a range of NK1 receptor agonists including SPOME, septide and GR-73 632 in their effects on propulsive peristalsis and circular muscle activity in the guinea-pig isolated small intestine. SPOME (100-300 nM) inhibited peristalsis by a rise of the pressure threshold at which peristaltic waves were triggered, whereas septide and GR-73 632 (30-300 nM) interrupted peristalsis by causing circular muscle spasms. Separate experiments showed that all three NK1 receptor agonists caused contraction of the circular muscle, which was enhanced by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (300 mM) and the P2X purinoceptor antagonist suramin (300 mM). In contrast, tetrodotoxin (300 nM) augmented the contractile effect of septide and GR-73 632 but not that of SPOME. It is concluded that the motor response to NK1 receptor agonists involves release of NO and adenosine triphosphate from inhibitory motor neurones. However, the NK1 receptor agonists differ in the mechanism by which they cause inhibitory transmitter release, which corresponds to differences in their antiperistaltic action.

Topics: Adenosine Triphosphate; Animals; Enzyme Inhibitors; Female; Guinea Pigs; Intestine, Small; Male; Motor Neurons; Muscle Contraction; Muscle, Smooth; Neurokinin A; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Peptide Fragments; Peristalsis; Piperidines; Purinergic P2 Receptor Antagonists; Pyrrolidonecarboxylic Acid; Quinuclidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Sincalide; Spasm; Substance P; Suramin; Tetrodotoxin

1. Effects of two tachykinin NK1 receptor selective agonists (septide and [Sar9, Met(O2)11]SP) were compared on the increases in mean arterial pressure (MAP), heart rate (HR) and motor behaviour following intracerebroventricular (i.c.v.) administration in unanaesthetized rat, and on the vascular permeability increases to intradermal (i.d.) injection in the anaesthetized rat. Moreover, five tachykinin NK1 receptor selective antagonists (LY303870, LY306740, LY303241, SR140333 and RP67580) were tested against the two agonists to compare their pharmacological profile. 2. [Sar9, Met(O2)11]SP and septide (10-100 pmol per rat, i.c.v.) were equipotent in increasing MAP and HR, yet they had dissimilar time-course. Both agonists increased dose-dependently face washing and sniffing while [Sar9, Met(O2)11]SP was the sole to produce grooming, septide was more potent than [Sar9, Met(O2)11]SP (6.5-650 pmol) in increasing vascular permeability. 3. For most centrally mediated responses, LY303870 and RP67580 were significantly more potent in inhibiting septide than [Sar9, Met(O2)11]SP. In some parameters, greater blockade was achieved when antagonists (particularly LY306740) were given 1 h instead of 10 min prior to i.c.v. septide. 4. All antagonists except LY303241 blocked dose-dependently the increases in vascular permeability to equipotent doses of [Sar9, Met(O2)11]SP and septide. LY303870 and LY306740 were more potent against septide. 5. The antagonism afforded by LY303870, LY306740 and LY303241 was stereoselective and only SR140333 was found to cause central and peripheral non specific effects. 6. The data confirm a distinct pharmacological profile for septide in vivo. RP67580 and LY306740 are currently the most valuable tachykinin NK1 receptor antagonists for in vivo studies in rat.

Topics: Acetamides; Animals; Behavior, Animal; Blood Pressure; Capillary Permeability; Heart Rate; Indoles; Injections, Intraventricular; Male; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Rats; Rats, Wistar; Receptors, Neurokinin-1; Stereoisomerism; Substance P

The novel tachykinin receptor antagonist CGP49823 ((2R,4S)-2-benzyl-1-(3,5-dimethylbenzoyl)-4-(quinolin-4-y lmethylamino)piperidine) has been compared with three other selective non-peptide tachykinin NK1 receptor antagonists. The drugs were tested as antagonists of the depolarization of spinal motoneurones induced by bath application of the selective tachykinin NK1 receptor agonist septide-(6-11) (300 nM) for 120 s at 15 min intervals. The antagonists were bath applied and the depolarizations were recorded from lumbar ventral roots of 7 to 12 day old rat and gerbil hemisected spinal cords in vitro. The gerbil preparation is considered to model the human species variant of the tachykinin NK1 receptor. With the exception of SR140333 ((S)-1-[2-[3-(3,4-dichlorophenyl)-1-[[3-(1-methylethoxy)phenyl]ace tyl]-3-piperidinyl]ethyl]-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride), the antagonists were approximately thirty-fold more potent on gerbil preparations. The respective mean IC50 values from gerbil preparations produced by CP96345 ((2S-cis)-2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicy clo[2.2.2]octan-3-amine), CGP49823, SR140333 and CP99994 ((2S-cis)-N-[(2-methoxyphenyl)methyl]-2-phenyl-3-piperidinamine) were, in microM +/- S.E. (n) 0.10 +/- 0.02 (6), 0.22 +/- 0.03 (6), 0.30 +/- 0.10 (5) and 0.38 +/- 0.02 (5) and the corresponding values from the rat preparations were 3.7 +/- 0.4 (5), 7.8 + 1.3 (5), 1.06 +/- 0.16 (6) and 10.5 +/- 2.2 (7). Dominance of tachykinin NK1 receptor activity in the measured responses was confirmed by low potency of the tachykinin NK2-selective antagonist SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl] benzamide) which yielded an IC50 value of 12.0 +/- 2.8 (5) on gerbil preparations and produced less than 50% depression of septide-induced depolarization of rat motoneurones at the highest concentration (100 microM) tested.

Topics: Animals; Anti-Anxiety Agents; Gerbillinae; In Vitro Techniques; Motor Neurons; Neurokinin-1 Receptor Antagonists; Neuromuscular Depolarizing Agents; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Quinolines; Rats; Rats, Inbred Strains; Receptors, Neurokinin-2; Receptors, Tachykinin; Substance P

The aim of this study was to assess at which extent an even co-release of the tachykinins, substance P (SP) and neurokinin A (NKA), occurs from enteric neurons/nerves of the guinea-pig proximal colon during graded depolarization. In this preparation, a sharply diverging NK1/NK2 receptor pattern of tachykininergic co-transmission has been observed in physiological studies. The experiments were performed in capsaicin-pretreated (10 microM for 15 min) mucosa-free smooth muscle of guinea-pig proximal colon, to exclude the mucosa and the peripheral endings of primary afferent nerves as possible sources of released tachykinins. The content of extractable tachykinins was measured as SP- and NKA-like immunoreactivities (-LI) by radioimmunoassay. Chromatographic characterization of aqueous acetic acid extracts showed one peak of SP-LI corresponding to authentic SP, whereas there were multiple peaks of NKA-LI, the major one co-eluting with authentic NKA. An increased outflow of both SP- and NKA-LI was evenly produced in a concentration-dependent manner when the preparations were superfused with a high potassium (K) medium in which NaCl had been replaced with equimolar amounts (20-100 mM) of KCl. The high K-evoked release of SP- and NKA-LI was dependent upon the presence of extracellular calcium and was inhibited by about 50% in the presence of the N-type voltage-dependent calcium channel blocker, omega-conotoxin GVIA (0.1 microM). Omega-conotoxin MVIIC (1 microM), a non-selective blocker of N-, P- and Q-type voltage-dependent calcium channels, likewise produced about 40% inhibition of evoked release of both peptides. No evidence for a role of L-type channels in tachykinin release was obtained, since the addition of nifedipine (1 microM) or Bay K8644 (1 microM) did not significantly affect the response to high K. Neither NK1 receptor agonist (septide, 0.1 microM) or antagonist (SR 140333, 10 nM) nor NK2 receptor agonists ([betaAla8]NKA(4-10) and GR 64349, 0.1 microM each) or antagonist (SR 48968, 10 nM) did affect the high K-evoked release of tachykinins. We conclude that SP and NKA are evenly co-released in response to graded depolarization of enteric nerves in the guinea-pig colon. Therefore, the specialization of tachykininergic transmission observed in functional studies does not originate at the prejunctional level. The co-release of tachykinins involves the influx of extracellular calcium via N-type but not L-type calcium channels. No evidence for the presence

Topics: Animals; Calcium; Colon; Enteric Nervous System; Guinea Pigs; Neurokinin A; Peptide Fragments; Piperidines; Potassium Chloride; Pyrrolidonecarboxylic Acid; Quinuclidines; Receptors, Tachykinin; Substance P; Tachykinins

1. The role of NK1 and NK3 receptors in synaptic transmission between myenteric neurons during motility reflexes in the guinea-pig ileum was investigated by recording intracellularly the reflex responses of the circular muscle to distension or compression of the mucosal villi. Experiments were performed in a three-chambered organ bath that enabled drugs to be selectively applied to different sites along the reflex pathways. 2. When applied in the recording chamber, an NK1 receptor antagonist, SR140333 (100 nM), reduced by 40-50% the amplitudes of inhibitory junction potentials (i.j.ps) evoked in the circular muscle by activation of descending reflex pathways. This effect was abolished when synaptic transmission in the stimulus region was blocked with physiological saline containing 0.1 mM Ca2+ plus 10 mM Mg2+, leaving only the component of the descending reflex pathway conducted via long anally directed collaterals of intrinsic sensory neurons. 3. SR140333 (100 nM) had no effect on descending reflex i.j.ps when applied to the stimulus region. Ascending reflexes were also unaffected by SR140333 in the stimulus region or between the stimulus and recording sites. 4. Septide (10 nM), an NK1 receptor agonist, enhanced descending reflexes by 30-60% when in the recording chamber. [Sar9,Met(O2)11]substance P had no effect at 10 nM, but potentiated distension-evoked reflexes at 100 nM. 5. A selective NK3 receptor antagonist, SR142801 (100 nM), when applied to the stimulus region, reduced the amplitude of descending reflex responses to compression by 40%, but had no effect on responses to distension. SR142801 (100 nM) had no effect when applied to other regions of the descending reflex pathways. 6. SR142801 (100 nM) only inhibited ascending reflexes when applied at the recording site. However, after nicotinic transmission in the stimulus region was blocked, SR142801 (100 nM) at this site reduced responses to compression. 7. Contractions of the circular muscle of isolated rings of ileum evoked by low concentrations of septide, but not [Sar9,Met(O2)11]substance P, were potentiated by tetrodotoxin (300 nM). 8. Contractile responses evoked by an NK3 receptor agonist, senktide, were non-competitively inhibited by SR142801. After excitatory neuromuscular transmission was blocked, senktide produced inhibitory responses that were also antagonised by SR142801, but to a lesser extent and in an apparently competitive manner. 9. These results indicate that tachykinins acting v

Topics: Animals; Female; Gastrointestinal Motility; Guinea Pigs; Ileum; In Vitro Techniques; Male; Neurokinin-1 Receptor Antagonists; Neurons; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Quinuclidines; Receptors, Neurokinin-1; Receptors, Neurokinin-3; Substance P; Synaptic Transmission

1. Although peptides are important modulators of synapses, their action on synapse-glia interactions remain unclear. The amphibian neuromuscular junction (NMJ) was used to examine the effects of substance P (SP) on perisynaptic Schwann cells (PSCs), glial cells at the frog NMJ, by monitoring changes in intracellular Ca2+. 2. SP induced Ca2+ responses that were mimicked by the neurokinin 1 receptor (NK-1) agonist septide and with a shorter delay by the SP fragment, SP(6-11). SP and SP(6-11) responses were blocked by NK-1 antagonists SR140333 and LY303870. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed but were blocked after pertussis toxin (PTX) treatment, indicating that the receptors were linked to internal stores of Ca2+ via a PTX-sensitive G-protein. 4. The slowly hydrolysable NK-1 agonist [Sar9, Met(O2)11]-SP only induced Ca2+ responses when applied for a long period of time and not during brief, local applications, suggesting the involvement of SP hydrolysis. Acetylcholinesterase (AChE) may not be involved in SP degradation since Ca2+ responses evoked by SP were unchanged in the presence of the cholinesterase inhibitor neostigmine. 5. Ca2+ responses induced by muscarine and nerve stimulations were almost abolished when preceded by SP applications, while those induced by ATP were significantly reduced. The rundown of the nerve-evoked Ca2+ responses in PSCs was attenuated in the presence of SR140333. 6. These results indicate that endogenous SP is involved in the regulation of PSC activity and that SP is an important modulator of glial cell Ca2+ signalling and synapse-glia communication.

Topics: Acetylcholinesterase; Animals; Calcium; GTP-Binding Proteins; Muscarine; Muscle, Skeletal; Neostigmine; Neurokinin-1 Receptor Antagonists; Neuromuscular Junction; Peptide Fragments; Pertussis Toxin; Piperidines; Pyrrolidonecarboxylic Acid; Quinuclidines; Rana pipiens; Receptors, Neurokinin-1; Schwann Cells; Substance P; Synapses; Virulence Factors, Bordetella

1. The presence of tachykinin NK1 receptors have been shown in the epithelium and smooth muscle of guinea-pig airways. Previous data showed that substance P (SP), and the NK1 receptor agonist, [Sar9, Met (O2)11]-SP, relax guinea-pig tracheal tube preparations by stimulation of epithelial NK1 receptors and via nitric oxide (NO) release. However, the selective tachykinin NK1 receptor agonist, septide, was unable to produce this effect. The aim of the present study was to investigate the ability of a series of SP analogues to stimulate NK1 receptors of guinea-pig airway epithelium. 2. Isometric tension was recorded in isolated tracheal tube preparations in which compounds were administered intraluminally in the presence of phosphoramidon, indomethacin (both 1 microM) and the tachykinin NK2 receptor antagonist, SR 48,968 ((S)-N-methyl N-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl)benzam ide) (0.1 microM). Cumulative concentration-response curves were obtained in preparations under resting tone or in preparations precontracted with acetylcholine (ACh, 10 microM). 3. Contractile responses to low concentrations (0.1-10 nM) of substance P (SP) and the selective agonist of NK1 receptors, [Pro9]-SP. in non precontracted tracheae were higher in preparations pretreated with the NO-synthase inhibitor, NG-monomethyl L-arginine (L-NMMA, 100 microM) than in preparations pretreated with its inactive enantiomer D-NMMA (100 microM). Tracheal tube preparations precontracted with ACh and pretreated with D-NMMA were relaxed by low concentrations of SP and [Pro9]-SP (0.1-10 nM). In contrast, after pretreatment with L-NMMA, SP and [Pro9]-SP contracted tracheae at all the concentrations tested. 4. Concentration-response curves to the NK1 receptor agonists, SP methyl ester, [Apa9-10]-SP and [pGlu6] SP (6-11) obtained in non-precontracted tracheae were similar in the presence of either D-NMMA or L-NMMA. SP methyl ester, [Apa9-10]-SP and [pGlu6] SP (6-11) did not produce any relaxation, but instead, cause contractions in tracheal tube preparations precontracted with ACh and pretreated with D-NMMA. Concentration-response curves produced by all these agonists were similar in preparations precontracted with ACh and pretreated with L-NMMA or D-NMMA. 5. In guinea-pig tracheal tube preparations two groups of NK1 receptor agonists can be distinguished: one group, including [Pro9]-SP, stimulator epithelial NK1 receptors, the other group, including SP methyl ester, [Apa9-

Topics: Animals; Dose-Response Relationship, Drug; Epithelium; Guinea Pigs; Male; Muscle, Smooth; Neurokinin-1 Receptor Antagonists; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Peptide Fragments; Physalaemin; Piperidines; Pyrrolidonecarboxylic Acid; Quinuclidines; Receptors, Neurokinin-1; Substance P; Tachykinins; Trachea

Three chimeric receptors were constructed by exchanging exons between human neurokinin NK1 and NK3 receptor genes. The N-terminal sequences of these chimeric receptors are encoded by exon 1, exon 1-2, or exon 1-3 of the NK1 receptor gene, whereas the remaining C-terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK3 receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK1 receptor gene may also play an important role for substance P binding. On the contrary, potent NK1 antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK1 agonists, [Sar9, Met(O2)11] substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK1 receptor. These results show that the exon-exchanging technique can be a useful tool for studying structure-function relationships of receptors in which exon-intron junctions are fully conserved among receptor subtypes.

Topics: Amino Acid Sequence; Cloning, Molecular; Exons; Humans; Iodine Radioisotopes; Molecular Sequence Data; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Phosphatidylinositols; Piperidines; Protein Binding; Pyrrolidonecarboxylic Acid; Quinuclidines; Radioligand Assay; Receptors, Neurokinin-1; Recombinant Fusion Proteins; Stereoisomerism; Substance P

Vascular NK-1 and NK-2 tachykinin receptors in the rat and the guinea pig were characterized pharmacologically by using available agonists and antagonists exhibiting varying degrees of selectivity for these receptors. Because the anesthetized guinea pig has unusually low blood pressure, these animals were pithed and vagotomized and infused, throughout the experimental procedure, with norepinephrine (6 micrograms/kg/min). This treatment raised their blood pressure to a level appropriate for the determination of dose-hypotensive response curves. The NK-1 receptor agonists substance P (SP) and septide (0.004 to 1 microgram/kg iv) decreased carotid artery blood pressure in a dose-dependent manner in both species, but they were more potent (13- and 33-fold, respectively) in guinea pigs than in rats. The NK-2 receptor agonist [beta Ala8]-NKA(4-10) (0.06 to 1 microgram/kg) also dose-dependently lowered blood pressure in the pithed guinea pig with noradrenaline-supported blood pressure, although it failed to do so in the same rat preparation. RP 67580, a selective NK-1 antagonist, antagonized the SP- or septide-induced hypotensive response in rats, but not in guinea pigs. Conversely, RPR 100893, a novel NK-1 receptor antagonist chemically related to RP 67580, dose-dependently inhibited hypotension induced by SP, and even more, that induced by septide only in guinea pigs. In the latter species, neither RP 67580 nor RPR 100893 affected decreases in blood pressure induced by [beta Ala8]-NKA(4-10). These decreases were, however, inhibited by the NK-2 receptor antagonist SR 48968. The selectivity of this compound for the latter receptor was confirmed by its failure to affect SP- or septide-induced hypotension in either guinea pigs or rats. These results confirm that the hypotensive responses to SP and septide are mediated by NK-1 receptors in the two species studied. However, functional NK-2 receptors appear to be present in the vascular bed of the guinea pig but not that of the rat, since in the former species the hypotensive responses induced [beta Ala8]-NKA(4-10) were inhibited by SR 48968 but not by the NK-1 receptor antagonists studied. This conclusion is, to our knowledge, drawn here for the first time from clear-cut pharmacological results.

Topics: Animals; Benzamides; Blood Pressure; Guinea Pigs; Hypotension; Indoles; Isoindoles; Male; Neurokinin A; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P

Experiments were performed in the longitudinal muscle strip of the guinea pig ileum to characterize the receptors involved in the contractile response of this preparation to neurokinins. Antagonists for the NK-1 (CP 96345, CP 99994) and NK-2 (SR 48968) receptors, atropine for NK-3 receptors, as well as diphenhydramine (histamine H1 receptor antagonist) and indometacin (cyclooxygenase inhibitor) were used to determine the relative contribution of neurokinin receptors and some endogenous agents to the myotropic effects of substance P (SP) and neurokinin receptor selective agonists. The present findings indicate that the three neurokinin receptor types take part in the contractile activities of SP-related peptides. NK-1 receptors, probably localized in the smooth muscle, are inhibited only by the two CP compounds and not by atropine or the other agents. NK-2 receptors contribute to the contraction by 5-10% and are blocked by SR 48968. NK-3 receptors act indirectly through the release of acetylcholine from the myenteric plexus, since activities of [MePhe7]NKB and senktide are blocked by atropine. Septide behaves as a selective NK-1 receptor agonist and does not show any difference with SP, except for higher sensitivity to CP antagonists. The same is observed with Ac[Arg6,Sar9,Met(O2)11]SP(6-11), another NK-1-selective fragment. Discrepancies between antagonist pA2 values obtained against undeca- and hexapeptide agonists are interpreted as due to a stronger binding affinity of undecapeptide agonists as compared with the hexapeptides. Results of binding assays confirm data from the literature by showing that undecapeptide agonists have higher affinities than hexapeptides, particularly septide,, and such discrepancies (with the biological assays) can also be explained by the reduction or absence of the cationic charge at the N terminal of septide.

Topics: Acetylcholine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzamides; Biphenyl Compounds; Guinea Pigs; Ileum; Male; Myenteric Plexus; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Radioligand Assay; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Stereoisomerism; Structure-Activity Relationship; Substance P

The selective NK2 agonist [Lys5-MeLeu9,Nle10]NKA(4-10) markedly stimulated [3H]inositol monophosphate (PI1) formation in prisms from the rat urinary bladder. This response was blocked by the NK2 antagonist SR 48968. Senktide (NK3 agonist) was inactive. Septide, a short SP analogue, and the NK1 agonists [Pro9]SP and [Sar9,Met(O2)11]SP also stimulated [3H]IP1 formation and several NK1 tachykinin antagonists (RP 67580, CP 96345, GR 82334, and [D-Pro9,t beta-BPr10,Trp11]SP) were more potent in blocking the septide than the [Pro9]SP response. GR 82334 was the most discriminative. SR 48968 (10(-6) M shifted the [Pro9]SP dose-response curve but did not modify the septide dose-response curve. Septide had a low affinity for [3H][Pro9]SP binding sites, suggesting further that septide and NK1 agonists act on different receptors. Finally, both [Pro9]SP and [Sar9,Met(O2)11]SP blocked the septide-evoked response, acting as partial agonists at the septide-sensitive tachykinin receptors.

Topics: Amino Acid Sequence; Animals; Benzamides; Hydrolysis; Indoles; Isoindoles; Male; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neurokinin A; Peptide Fragments; Phosphatidylinositols; Piperidines; Pyrrolidonecarboxylic Acid; Rats; Rats, Sprague-Dawley; Receptors, Tachykinin; Substance P; Urinary Bladder

The effects of a novel nonpeptide NK1 tachykinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with 125I-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with Ki values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar9,Met(O2)11]-substance P, stimulated the formation of inositol phosphates with an EC50 of 3.8 x 10(-9) M. SR 140333 blocked the stimulatory effect of this agonist (10(-7) M) with an IC50 of 1.6 x 10(-9) M, whereas the effect of another NK1 agonist, septide (EC50 = 1.5 x 10(-8) M) was antagonized with an IC50 of 2.1 x 10(-10) M. Enhancement of [3H]taurine release by [Sar9,Met(O2)11]-substance P (EC50 = 7.4 x 10(-9) M) was also inhibited by SR 140333 with an IC50 of 1.8 x 10(-9) M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [3H]taurine release. The calcium mobilization induced by [Sar9,Met(O2)11]-substance P (10(-8) M) was totally prevented by 10(-8) M SR 140333. Patch-clamp experiments showed that SR 140333 depressed the outward current evoked by 5 x 10(-8) M [Sar9, Met(O2)11]-substance P with an IC50 of 1.3 x 10(-9) M. The expression of c-fos was stimulated by [Sar9,Met(O2)11]substance P with an EC50 of 2.5 x 10(-10) M, an effect that was also inhibited by SR 140333 with an IC50 of 1.1 x 10(-9) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Astrocytoma; Binding, Competitive; Calcium; Electric Conductivity; Genes, fos; Humans; Inositol Phosphates; Iodine Radioisotopes; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Potassium; Pyrrolidonecarboxylic Acid; Quinuclidines; Receptors, Neurokinin-1; Stereoisomerism; Substance P; Succinimides; Taurine; Transcription, Genetic; Tumor Cells, Cultured

1. Following the recent proposal that the selective agonist septide, ([pGlu6,Pro9]SP(6-11)), acts on a novel tachykinin receptor distinct from the 'classical' NK1 receptor, the aim of the study was to investigate the possible heterogeneity of tachykinin NK1 receptors in the rat urinary bladder. 2. The synthetic tachykinin receptor agonists, septide (pD2 7.87) and [Sar9]substance P (SP) sulphone (pD2 7.64) produced concentration-dependent contractions of the rat isolated urinary bladder. 3. The NK1 receptor antagonists GR82,334, (+/-)-CP96,345, and RP67,580 competitively antagonized (slopes of Schild plot not significantly different from unity) the response to septide with the rank order of potency (pKB values in parentheses): RP 67,580 (7.57) > GR 82,334 (7.01) > (+/-)-CP 96,345 (6.80). The same antagonists were significantly less potent when tested against [Sar9]SP sulphone, while maintaining the same rank order of potency: RP 67,580 (7.00) > GR 82,334 (5.93) > (+/-)-CP 96,345 (< 6). The antagonists did not affect the concentration-response curve to bombesin. 4. To exclude the involvement of the NK2 receptor, a second series of experiments was performed in the presence of the potent nonpeptide NK2 receptor antagonist, SR 48,968. SR 48,968 (1 microM) produced a rightward shift of the concentration-response curve to the NK2 receptor selective agonist, [beta Ala8]neurokinin A (NKA) (4-10). SR 48,968 did not significantly modify the response to SP, NKA, neurokinin B (NKB), neuropeptide K (NPK), neuropeptide gamma (NP gamma), SP(4-11), SP(6-11), septide or [Sar9]SP sulphone. 5. In the absence or presence of SR 48,968, RP 67,580 antagonized in a competitive manner the response to septide, [Sar9]SP sulphone, SP(4-11) and SP(6-11): pKB values obtained in the absence and presence of SR 48,968 were not significantly different for any of these four agonists.6. RP 67,580 antagonized the response to SP and NKA both in the absence and presence of SR 48,968.In both cases, the slopes of the Schild plots were significantly different from unity. Mean dose-ratios produced by RP 67,580 in the presence of SR 48,968 were larger than those measured without NK2receptor blockade for both SP and NKA.7. RP 67,580 (3 MicroM) did not antagonize the response to NKB in the absence of SR 48,968. In the presence of SR 48,968, RP 67,580 acted as a competitive antagonist of NKB-induced contractions with apKB value (7.63) not significantly different from that measured towards septide. In t

Topics: Animals; Benzamides; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Rats; Rats, Wistar; Receptors, Tachykinin; Substance P; Sulfones; Tachykinins; Urinary Bladder

1. The effects of the NK1-receptor antagonists, (+/-)-CP 96,345 and CP 99,994, on NK1-agonist evoked contractions were compared in isolated rings of guinea-pig tracheal smooth muscle. 2. (+/-)-CP 96,345 and CP 99,994 were similarly effective in antagonizing responses evoked by septide, whereas CP 99,994 was more effective than (+/-)-CP 96,345 in inhibiting responses evoked by [Sar9Met11(O2)] substance P. 3. These results suggest that responses to septide and [Sar9Met11(O2)] substance P may be operated via different populations of NK1-receptors.

Topics: Animals; Biphenyl Compounds; Guinea Pigs; In Vitro Techniques; Indoles; Isoindoles; Male; Muscle Contraction; Muscle, Smooth; Neurokinin A; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Substance P; Trachea