sr-142801 and senktide

Substance P (SP) is a neuropeptide widely expressed throughout the fear-processing pathways of the brain. SP is cleaved by several proteolytic enzymes in amino (N-) and carboxy (C-) terminal sequences, which can have biological activities per se. We have previously shown that the anxiogenic-like effects elicited by SP6-11(C-terminal), a specific metabolite of SP, are mediated via NK1 and NK2 receptors. Nevertheless, there are evidences that C-terminal fragments may have a greater affinity for NK3 receptors.. The aim of the present study was to further investigate the possible involvement of NK3 receptors in the anxiogenic-like effects induced by SP6-11(C-terminal).. Adult male Wistar rats were intracerebroventricularly (i.c.v.) treated with SR142801 (NK3 receptors antagonist) or vehicle one minute to prior SP6-11(C-terminal) or vehicle. Other experimental groups received SP6-11(C-terminal) or vehicle i.c.v. one minute prior to senktide (NK3 receptors agonist) or vehicle. After five minutes, the animals were behaviorally evaluated in the elevated plus-maze test (EPM).. SR142801 (100 pmol) or SP6-11(C-terminal) (10 pmol) reduced all the parameters of open-arms exploration and increased the number of protected stretch-attend postures in the EPM, indicating an anxiogenic-like effect. Senktide (10 pmol) promoted an opposite effect on these behavioral parameters, characterizing an anxiolytic-like profile. Pretreatment with SR142801, in an ineffective dose, potentiated the SP6-11-induced anxiety, especially in the unprotected head-dipping and protected stretch-attend postures behaviors. Moreover, the anxiolytic-like effect induced by senktide (1 pmol) was prevented by SP6-11.. Our results give support to the involvement of NK3 receptors in the anxiogenic-like actions of SP6-11(C-terminal), where this metabolite seems to behave as an antagonist, in a way similar to SR142801.

Topics: Animals; Anxiety; Male; Peptide Fragments; Piperidines; Pyrrolidonecarboxylic Acid; Rats; Rats, Wistar; Receptors, Neurokinin-3; Substance P

The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3.

Topics: Amino Acid Sequence; Animals; Antipsychotic Agents; Binding Sites; Carbamates; Central Nervous System Agents; Dopaminergic Neurons; Female; Gerbillinae; Guinea Pigs; HEK293 Cells; Humans; Locomotion; Male; Models, Molecular; Molecular Sequence Data; Mutation; Neurotransmitter Agents; Pars Compacta; Peptide Fragments; Piperidines; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Substance P; Tissue Culture Techniques

Kisspeptin neurons located in the arcuate nucleus (ARN) coexpress dynorphin and neurokinin B (NKB) and may interact to influence gonadotropin secretion. Using a kisspeptin-green fluorescent protein mouse model, the present study examined whether the neuropeptides kisspeptin, dynorphin, and NKB modulate the electrical activity of ARN kisspeptin neurons in the adult male mouse. Cell-attached recordings showed that kisspeptin itself had no effect on kisspeptin neuron firing. Dynorphin and the κ-opioid receptor agonist U50-488 evoked a potent suppression of all ARN kisspeptin neuron firing that was blocked completely by the κ-opioid receptor antagonist nor-Binaltorphimine. Both NKB and Senktide, a neurokinin 3 receptor agonist, exerted a potent stimulatory action on ∼95% of ARN kisspeptin neurons. Although the selective neurokinin 3 receptor antagonists SB222200 and SR142801 blocked the effects of Senktide on kisspeptin neurons, they surprisingly had no effect on NKB activation of firing. Studies with selective neurokinin 1 receptor (SDZ-NKT343) and neurokinin 2 receptor (GR94800) antagonists revealed that the activation of kisspeptin neurons by NKB was only blocked completely by a cocktail of antagonists against all 3 tachykinin receptors. Whole-cell recordings revealed that individual kisspeptin neurons were activated directly by all 3 tachykinins substance, P, neurokinin A, and NKB. These experiments show that dynorphin and NKB have opposing actions on the electrical activity of kisspeptin neurons supporting the existence of an interconnected network of kisspeptin neurons in the ARN. However, the effects of NKB result from an unexpected activation of multiple tachykinin receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Arcuate Nucleus of Hypothalamus; Dynorphins; Green Fluorescent Proteins; Kisspeptins; Male; Membrane Potentials; Mice; Mice, Transgenic; Neurokinin B; Neurons; Oligopeptides; Patch-Clamp Techniques; Peptide Fragments; Piperidines; Quinolines; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Opioid, kappa; Receptors, Tachykinin; Substance P

This in vitro study investigates and compares the effects of NK3 receptor ligands on the firing rate of rat and guinea pig midbrain dopamine neurons. The findings are discussed in the light of choosing suitable animal models for investigating pharmacological properties of NK3 receptor antagonists, which have been proposed to possess therapeutic activity in neuropsychiatric diseases like e.g. schizophrenia. In vitro midbrain slice preparations of both species were used to record (extracellularly) the firing rates of dopamine neurons located in the substantia nigra (SN) and ventral tegmental area (VTA). Furthermore, the effect of the D2 receptor agonist quinpirole on guinea pig SN and VTA dopamine neurons was investigated. The efficacy of quinpirole in inhibiting guinea pig dopamine neuron firing activity was much less as compared to that of rat dopamine neurons, suggesting a lower dopamine D2 autoreceptor density on the guinea pig neurons. The NK3 receptor agonist senktide induced in subpopulations of rat SN (55%) and VTA (79%) and guinea pig SN (50%) and VTA (21%) dopamine neurons an increase in firing rate. In responsive neurons this effect was concentration-dependent with EC₅₀ values of 3-5 nM (for both species). The selective NK3 receptor antagonist osanetant (100 nM) was able to partly block the senktide-induced increase in firing rates of dopamine neurons and shifted the concentration-response relation curves for senktide to the right (pA₂ values were ~7.5). The fractional block of the senktide responses by osanetant appeared to be larger in guinea pig dopamine neurons, indicating that osanetant is a more potent blocker of NK3 receptor-mediated responses with noncompetitive properties in the guinea pig.

Topics: Action Potentials; Animals; Antipsychotic Agents; Dopamine; Dopamine Agonists; Electrophysiology; Guinea Pigs; Male; Mesencephalon; Neurons; Organ Culture Techniques; Peptide Fragments; Piperidines; Rats; Rats, Wistar; Receptors, Neurokinin-3; Species Specificity; Substance P

Serotonin (5-HT) is a key modulator of neuronal excitability in the central and peripheral nervous system. In the enteric nervous system, 5-HT causes a slow depolarization in the intrinsic sensory neurons, but the receptor responsible for this has not been correlated with known gene products. The aim of this study was to determine whether the newly characterized 5-HT7 receptor may participate in the 5-HT-mediated depolarization of, and synaptic transmission to, the intrinsic sensory neurons of the guinea-pig ileum. Intracellular electrophysiological recordings were made from intrinsic sensory neurons identified as myenteric AH neurons from guinea-pig ileum. 5-HT (5 microM) applied to the cell body evoked both a fast depolarization (5-HT3 mediated) and/or a slow depolarization (5-HT1P-like). The 5-HT1/5/7 receptor agonist 5-carboxamidotryptamine (5-CT) (5 microM) evoked only a slow depolarization. When the fast depolarization evoked by 5-HT was blocked with granisetron (1 microM, 5-HT3 receptor antagonist), only a slow depolarization remained; this was abolished by the 5-HT7 receptor antagonist SB 269970 (1 microM, control: 14+/-2 mV, granisetron+SB 269970: -1+/-2 mV). The slow depolarization evoked by 5-CT was also significantly reduced by SB 269970 (control: 14+/-1 mV, SB 269970: 5+/-2 mV) suggesting a 5-HT7 receptor was activated by exogenous application of 5-CT and 5-HT. Slow excitatory postsynaptic potentials evoked by stimulating descending neural pathways (containing serotonergic fibers) were reduced by SB 269970 (control: 8+/-3 mV, SB 269970: 3+/-1 mV). However, SB 269970 had no effect on slow excitatory postsynaptic potentials evoked by stimulation of circumferential (tachykinergic) pathways (control: 7+/-1 mV, SB 269970: 6+/-1 mV). These data are consistent with the presence on enteric AH neurons of functional 5-HT7 receptors that participate in slow synaptic transmission.

Topics: Animals; Drug Interactions; Electric Stimulation; Excitatory Postsynaptic Potentials; Female; Granisetron; Guinea Pigs; Ileum; Male; Myenteric Plexus; Neurons; Peptide Fragments; Phenols; Piperidines; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Substance P; Sulfonamides

Recent studies have shown that tachykinins mediate slow synaptic transmission to myenteric AH (afterhyperpolarising) neurons via neurokinin-3 receptors (NK(3)R). This study investigated a similar role for neurokinin-1 receptors (NK(1)R) and compared the effect of selective receptor antagonists on non-cholinergic slow excitatory post-synaptic potentials (EPSPs) recorded in myenteric AH neurons of the guinea-pig ileum. Slow EPSPs evoked by electrical stimulation of circumferentially oriented presynaptic nerves were mimicked by application of senktide, an NK(3)R agonist. [Sar(9),Met(O(2))(11)]-substance P, an NK(1)R agonist, depolarised a smaller number of neurons. SR142801, a selective NK(3)R antagonist (100 nM), inhibited slow EPSPs and responses to senktide, but had no effect on depolarisations evoked by forskolin, an activator of adenylate cyclase. SR140333, a selective NK(1)R antagonist, inhibited slow EPSPs in a subset of neurons and blocked responses to [Sar(9),Met(O(2))(11)]-substance P, but not to senktide or forskolin. Slow EPSPs that were predominantly mediated by NK(1)R had significantly shorter latencies than those due to activation of NK(3)R. After blockade of slow EPSPs, slow hyperpolarizing responses to presynaptic nerve stimulation were revealed in one-third of neurons. These events, which were associated with a decrease in input resistance and blocked by tetrodotoxin, were equated with slow inhibitory postsynaptic potentials. They were abolished by the 5-hydroxytryptamine(1A) receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine (NAN-190), but unaffected by phentolamine, an alpha-adrenoceptor antagonist. In conclusion, these results provide the first direct evidence that NK(1)R mediate some slow excitatory synaptic input to myenteric AH neurons, and suggest that NK(1)R and NK(3)R activate distinct signal transduction pathways. These results also demonstrate that slow inhibitory synaptic transmission, which may be mediated by 5-hydroxytryptamine, is more prevalent in the myenteric plexus than previously indicated.

Topics: Adrenergic alpha-Antagonists; Animals; Antipsychotic Agents; Excitatory Postsynaptic Potentials; Female; Guinea Pigs; Male; Myenteric Plexus; Neural Inhibition; Neurokinin-1 Receptor Antagonists; Neurons; Peptide Fragments; Phentolamine; Piperazines; Piperidines; Quinuclidines; Reaction Time; Receptors, Neurokinin-1; Receptors, Neurokinin-3; Serotonin 5-HT1 Receptor Antagonists; Serotonin Antagonists; Substance P; Synaptic Transmission

As melanin-concentrating hormone (MCH) neurons express the neurokinin 3 receptor (NK3) in the rat diencephalon, their innervation by tachykininergic fibers, the origin of this innervation and the effect of a NK3 agonist on MCH mRNA expression were researched. The obtained results show that the tachykininergic system develops complex relationships with MCH neurons. Overall, MCH cell bodies appeared targeted by both NKB- and SP-inputs. These afferents have multiple hypothalamic and extra-hypothalamic origins, but a local (intra-lateral hypothalamic area) origin from small interneurons was suspected as well. MCH cell bodies do not express NK1, but around 2.7% of the MCH neurons contained SP after colchicine injection. Senktide, a NK3 agonist, produced an increase of the MCH mRNA expression in cultured hypothalamic slices. This effect was reversed by two NK3 antagonists. Tachykinins enhance MCH mRNA expression, and, thus, may modulate the effect of MCH in functions such as feeding and reproductive behaviors in which this peptide has been experimentally involved.

Topics: Animals; Brain; Colchicine; Feeding Behavior; Hypothalamic Hormones; Hypothalamus; Interneurons; Male; Melanins; Neural Pathways; Neurokinin B; Neurokinin-1 Receptor Antagonists; Neurons; Organ Culture Techniques; Peptide Fragments; Piperidines; Pituitary Hormones; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-3; RNA, Messenger; Sexual Behavior; Substance P; Tachykinins

Neurokinin-containing nerve fibers were localized to guinea pig airway parasympathetic ganglia in control tissues but not in tissues pretreated with capsaicin. The purpose of the present study was to determine whether neurokinins, released during axonal reflexes or after antidromic afferent nerve stimulation, modulate ganglionic synaptic neurotransmission. The neurokinin type 3 (NK(3)) receptor antagonists SB-223412 and SR-142801 inhibited vagally mediated cholinergic contractions of bronchi in vitro at stimulation voltages threshold for preganglionic nerve activation but had no effect on vagally mediated contractions evoked at optimal voltage or field stimulation-induced contractions. Intracellular recordings from the ganglia neurons revealed that capsaicin-sensitive nerve stimulation potentiated subsequent preganglionic nerve-evoked fast excitatory postsynaptic potentials. This effect was mimicked by the NK(3) receptor agonist senktide analog and blocked by SB-223412. In situ, senktide analog markedly increased baseline tracheal cholinergic tone, an effect that was reversed by atropine and prevented by vagotomy or SB-223412. Comparable effects of intravenous senktide analog on pulmonary insufflation pressure were observed. These data highlight the important integrative role played by parasympathetic ganglia and indicate that activation of NK(3) receptors in airway ganglia by endogenous neurokinins facilitates synaptic neurotransmission.

Topics: Animals; Bronchi; Bronchoconstriction; Electric Stimulation; Excitatory Postsynaptic Potentials; Ganglia, Parasympathetic; Guinea Pigs; Immunohistochemistry; In Vitro Techniques; Male; Neurons; Peptide Fragments; Piperidines; Quinolines; Receptors, Neurokinin-3; Substance P; Synaptic Transmission; Trachea; Vagotomy; Vagus Nerve

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

The neurokinin 3 (NK3) receptor is predominantly expressed in the central nervous system (CNS). Species differences in neurokinin 3 (NK3) receptor pharmacology have led to the preferential use of guinea pigs and gerbils in the characterization of non-peptide NK3 antagonists. Little is known about the central localization of NK3 receptors in the CNS of these species. To study this, [(3)H]senktide and [(3)H]SR 142801 were used in autoradiography experiments to visualize the NK3 receptors in the guinea pig and gerbil brain and compared to with the distribution of [(3)H]senktide binding sites in the rat brain. In the three species, the NK3 receptor was similarly distributed within the cerebral cortex, the zona incerta, the medial habenula, the amygdaloid complex, the superior colliculus and the interpeduncular nucleus. Outside of these structures, our study has revealed that each species displayed a specific distribution pattern of central NK3 receptors. The rat was the only species where NK3 receptors could be visualized in the striatum, the supraoptic nucleus and the paraventricular nucleus of the hypothalamus. The guinea pig differed mainly from the two other species by the absence of detectable binding sites in the substantia nigra pars compacta and the ventral tegmental area. A specific localization of NK3 receptors in the anterodorsal and anteroventral thalamic nuclei characterized the gerbil. This last species is also unique by in the higher level of NK3 receptors in the dorsal and median raphe nuclei. All these differences suggest that the NK3 receptor mediates different functions in different species.

Topics: Animals; Autoradiography; Brain; Cerebral Cortex; Gerbillinae; Guinea Pigs; Male; Membranes; Peptide Fragments; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, Neurokinin-3; Species Specificity; Substance P

We have used multiple-labeling immunohistochemistry, intracellular dye-filling, and intracellular microelectrode recordings to characterize the distribution of tachykinin receptors and substance P boutons on subpopulations of neurons within the guinea pig celiac ganglion. Superfusion of substance P (SP, 1 microM for 1 min) depolarized 42% of tonic neurons and inhibited afterhyperpolarizations in 66% of long afterhyperpolarizing (LAH) neurons without significant desensitization. Twenty-one percent of tonic neurons and 24% of LAH neurons responded to the NK(3) agonist senktide but did not respond to SP, indicating SP did not activate NK(3) receptors at this concentration. All effects of SP were abolished by the selective NK(1) receptor antagonist, SR140333, but not by the selective NK(3) receptor antagonist, SR142801, suggesting that exogenous SP activated a receptor with NK(1) pharmacology. No dye-filled LAH neuron and only 50% of tonic neurons responding to SP expressed NK(1) receptor immunoreactivity (NK(1)-IR). All neurons responding to SP had SP immunoreactive fibers within one cell diameter, indicating good spatial matching between SP release sites and target neurons. These results indicate that SP may act via a receptor with NK(1)-like pharmacology that has a C terminus not recognized by antibodies to the intracellular domain of the conventional NK(1) receptor. Inward currents evoked by SP acting on this NK(1)-like receptor or senktide acting through NK(3) receptors had identical current-voltage relationships. In LAH neurons, both agonists suppressed I(sAHP) without reducing I(AHP). Responses evoked by SP and senktide were resistant to PKC inhibitors, suggesting that the transduction mechanisms for the NK(1)-like receptor and the NK(3) receptor may be similar.

Topics: Action Potentials; Animals; Biotin; Coloring Agents; Enzyme Inhibitors; Female; Ganglia, Sympathetic; Guinea Pigs; Image Processing, Computer-Assisted; Immune Sera; Male; Microelectrodes; Nerve Tissue Proteins; Neurons; Patch-Clamp Techniques; Peptide Fragments; Piperidines; Protein Kinase C; Quinuclidines; Receptors, Neurokinin-1; Receptors, Neurokinin-3; Substance P; Sympathetic Fibers, Postganglionic; Tachykinins

The role of NK3 receptors in rabbit colonic propulsion has been investigated in vitro with the selective agonist, senktide, and two selective antagonists, SR142801 and SB222200. Peristalsis was elicited by distending a rubber balloon with 0.3 and 1.0 mL of water leading to a velocity of 2.2 and 2.8 mm s-1, respectively. At concentrations of 1 nM, senktide inhibited propulsion evoked by both distensions (range 25-40%), whereas at 6 and 60 nmol L-1 facilitated 'submaximal' propulsion by 30%. In the presence of Nomega-nitro-L-arginine (L-NNA, 200 micromol L-1), which per se caused a slight prokinetic effect, 1 nmol L-1 senktide markedly accelerated propulsion (range 35-50%). Hexamethonium (200 micromol L-1) had minor effects on propulsion. In its presence, 60 nmol L-1 senktide significantly inhibited propulsion induced by both stimuli (range 20-50%). SR142801 (0.3, 3 nmol L-1) and SB222200 (30, 300 nmol L-1) facilitated 'submaximal' propulsion (range 20-40%). Conversely, higher antagonist concentrations (SR142801: 30, 300 nM; SB222200: 1, 10 micromol L-1) inhibited propulsion to both distensions by 20%. A combination of SR142801 (300 nmol L-1) plus hexamethonium (200 micromol L-1) induced an approximately four-fold greater inhibition of propulsion than that induced by SR142801 alone. In conclusion, in the rabbit-isolated distal colon, a subset of NK3 receptors located on descending pathways mediates an inhibitory effect on propulsion by activating a NO-dependent mechanism. Another subset of NK3 receptors, located on ascending pathways mediates a facilitative effect involving a synergistic interaction with cholinergic nicotinic receptors.

Topics: Animals; Colon; Complement C6; Dose-Response Relationship, Drug; Gastrointestinal Motility; In Vitro Techniques; Male; Peptide Fragments; Piperidines; Quinolines; Rabbits; Receptors, Neurokinin-3; Substance P

Intracellular recording techniques were used to test whether tachykinins could be mediators of slow excitatory postsynaptic potentials (EPSPs) in guinea pig sphincter of Oddi (SO) ganglia. Application of the tachykinin substance P (SP) onto SO neurons caused a prolonged membrane depolarization that was reminiscent of the slow EPSP in these cells. Pressure ejection of the neurokinin 3 (NK3) receptor-specific agonist senktide caused a similar depolarization; however, no responses were detected on application of NK1 or NK2 receptor agonists. The NK3 receptor antagonist SR-142801 (100 nM) significantly inhibited both SP-induced depolarization and the stimulation-evoked slow EPSP, as did NK3 receptor desensitization with senktide. Capsaicin, which causes the release of SP from small-diameter afferent fibers, induced a depolarization that was similar to the evoked slow EPSP in both amplitude and duration. The capsaicin-induced depolarization was significantly attenuated in the presence of SR-142801. These data indicate that tachykinins, released from extrinsic afferent fibers, act via NK3 receptors to provide slow excitatory synaptic input to SO neurons.

Topics: Animals; Calcium; Capsaicin; Culture Techniques; Excitatory Postsynaptic Potentials; Female; Ganglia; Guinea Pigs; Male; Membrane Potentials; Neurokinin-1 Receptor Antagonists; Neurons; Peptide Fragments; Piperidines; Receptors, Neurokinin-3; Receptors, Tachykinin; Sphincter of Oddi; Substance P; Synaptic Transmission; Tachykinins; Tetrodotoxin

The neurokinin 3 (NK3) receptor antagonists represent a novel class of pharmacological agents, which are currently under evaluation for the treatment of psychiatric disorders. An efficient brain penetration is one of the main prerequisites to further evaluate compounds displaying high potency to bind the NK3 receptor. The present report describes a method for determining the in vivo occupancy of central NK3 receptors after peripheral administration of drugs. An ex vivo measurement of NK3 receptor occupancy by quantitative autoradiography employing [3H]senktide as the radioligand has been developed. The speed of the method, which is usually considered low due to the time dedicated to film exposure (from weeks to months), has been considerably increased by the use of the beta-imager. The high sensitivity of this new radioimager was used to visualize and quantitatively analyze the [3H]senktide binding sites in brain sections within hours. Using this method, we have demonstrated that the reference NK3 antagonist SR142801 dose dependently occupied the NK3 receptors in the gerbil brain after subcutaneous administration with an ED50 of 0.85 mg/kg. The less active enantiomer SR142806 occupied the NK3 receptors only by 25% at the highest used dose of 10 mg/kg. These values are in accordance with the reported behavioral effects of the compounds. Our results indicate that ex vivo receptor occupancy measurements can be dependently used to predict the central activity of NK3 antagonists. More generally, the combination of ex vivo receptor autoradiography with the beta-imager detection constitutes a new and fast method to evaluate the brain penetration of drug candidates.

Topics: Animals; Autoradiography; Beta Particles; Brain; Central Nervous System; Dose-Response Relationship, Drug; Gerbillinae; Male; Peptide Fragments; Piperidines; Radionuclide Imaging; Receptors, Neurokinin-3; Substance P

Tachykinins have been suggested to play a significant role in the mammalian striatum, at least in part by the control of acetylcholine release from cholinergic interneurons. In the present study, we have examined the ability of known tachykinin agonists and antagonists to modulate the activity of these interneurons in mouse striatal slices. Using whole-cell patch-clamp recordings, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide each produced a dose-dependent depolarization of visually identified cholinergic interneurons that was retained under conditions designed to interrupt synaptic transmission. The nature of these neurons and the expression of multiple tachykinin receptors was confirmed using single-cell reverse transcriptase-polymerase chain reaction analysis. Using in vitro superfusion techniques, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide, respectively, each produced a dose-dependent increase in acetylcholine release, the selectivity of which was confirmed using the neurokinin-1, neurokinin-2 and neurokinin-3 receptor antagonists SR140333, GR94800 and SR142801 (100 nM). U73122 (10 microM), a phospholipase C inhibitor, blocked [sar9,Met(O2)11]substance P- and senktide-induced acetylcholine release, but had no effect on [beta-ala8]neurokinin A(4-10)-induced release. The protein kinase C inhibitors chelerythrine and Ro-31-8220 (both 1 microM) significantly inhibited responses induced by all three agonists. These findings indicate that tachykinins modulate the activity of mouse striatal cholinergic interneurons. Furthermore, neurokinin-2 receptors are shown to perform a role in mouse that has not been identified previously in other species.

Topics: 2-Amino-5-phosphonovalerate; Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Choline O-Acetyltransferase; Corpus Striatum; Electrophysiology; Enzyme Inhibitors; Estrenes; Excitatory Amino Acid Antagonists; In Situ Hybridization; Indoles; Interneurons; Membrane Potentials; Mice; Mice, Inbred C57BL; Neurokinin A; Oligopeptides; omega-N-Methylarginine; Peptide Fragments; Phenanthridines; Phosphodiesterase Inhibitors; Piperidines; Pyrrolidinones; Quinoxalines; Quinuclidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Tachykinin; RNA, Messenger; Signal Transduction; Substance P; Tachykinins; Tetrodotoxin; Tritium

Effects of two commonly used tachykinin NK-3 receptor antagonists (SR 142801 and R820) intrathecally (i.t.) administered were assessed in the rat tail-flick test. SR142801 and its (R)-enantiomer SR142806 (1.3, 6.5 and 65 nmol) were found as potent as senktide and [MePhe7]NKB (NK-3 selective agonists) to induce transient antinociceptive effects. Naloxone (10 microg) and R820 (6.5 nmol) blocked reversibly the responses to 6.5 nmol senktide, [MePhe7]NKB, SR142801 and SR142806 when administered i.t. 15 min earlier. However, the antinociceptive responses induced by SR142801 and SR142806 were not affected by i.t. pretreatments with NK-1 (6.5 nmol SR140333) and NK-2 (6.5 nmol SR48968) receptor antagonists. In control experiments, the NK-1 and NK-2 antagonists prevented the hyperalgesic effects to NK-1 ([Sar9,Met(O2)11]SP) and NK-2 ([beta-Ala8] NKA(4-10)) receptor agonists (6.5 nmol i.t.), respectively. R820 had no direct effect on nociceptive threshold and failed to alter angiotensin II-induced antinociception. The data suggest that the antinociceptive effect of SR142801 is due to an agonist effect at NK-3 receptor in the rat spinal cord that involves a local opioid mechanism. These results can be best explained by the existence of inter-species NK-3 receptor subtypes.

Topics: Analgesics; Animals; Drug Interactions; Indoles; Male; Naloxone; Narcotic Antagonists; Neurokinin-1 Receptor Antagonists; Oligopeptides; Pain Measurement; Peptide Fragments; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Spinal Cord; Substance P

The contractile effect of capsaicin in the guinea-pig small intestine involves an activation of enteric cholinergic neurons. Our present data show that the P(2) purinoceptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) significantly reduces the contractile response to capsaicin (2 microM) in the presence, but not in the absence, of the tachykinin receptor antagonists [O-Pro(9), (Spiro-gamma-lactam)Leu(10), Trp(11)]physalaemin (1-11) (GR 82334; 3 microM) and (S)-(N)-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidine-4-yl)-N -methylacetamide (SR 142804: 100 nM) (for blocking tachykinin NK1 and NK3 receptors, respectively). PPADS (30 microM) fails to influence submaximal cholinergic contractions evoked by cholecystokinin octapeptide (CCK-8; 2-3 nM) or senktide (1 nM), or the direct smooth muscle-contracting effect of histamine (100-200 nM). A higher concentration (300 microM) of PPADS is also without effect against the stimulatory action of cholecystokinin octapeptide. This means that PPADS can probably be safely used as a purinoceptor antagonist in intestinal preparations. The putative pituitary adenylate cyclase activating peptide (PACAP) receptor antagonist PACAP-(6-38) (3 microM) significantly reduces the contractile effect of PACAP-(1-38) (10 nM) and abolishes that of vasoactive intestinal polypeptide (VIP; 10 nM). PACAP-(6-38) (3 microM) fails to influence the effect of capsaicin (2 microM) both in the absence and in the presence of tachykinin receptor antagonists. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 100 microM) also fails to inhibit the capsaicin-induced motor response. We conclude that an endogenous ligand of PPADS-sensitive P(2) purinoceptors (possibly ATP), but not a VIP/PACAP-like peptide or NO, is involved in the nontachykininergic activation of cholinergic neurons in the course of the capsaicin-induced contraction.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Capsaicin; Enzyme Inhibitors; Guinea Pigs; Ileum; In Vitro Techniques; Intestine, Small; Muscle Contraction; Muscle, Smooth; Neurokinin-1 Receptor Antagonists; Neuropeptides; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Peptide Fragments; Physalaemin; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Pyridoxal Phosphate; Receptors, Neurokinin-3; Sincalide; Substance P; Vasoactive Intestinal Peptide

In the present study the ability of tachykinin agonists and antagonists to modulate gamma-aminobutyric acid (GABA) release has been correlated with tachykinin receptor expression in the mouse striatum. Significant GABA release was observed when striatal slices were challenged with the NK-3 receptor agonist senktide, the selectivity of which was confirmed using the NK-3 receptor antagonist SR142801. In situ hybridisation revealed co-expression of NK-3 receptors with nitric oxide synthase (NOS)/preprosomatostatin containing GABAergic interneurones. These findings suggest that tachykinins modulate GABA release within the striatum via interaction with NK-3 receptors on somatostatin/NOS interneurones.

Topics: Acetylcholine; Animals; gamma-Aminobutyric Acid; In Situ Hybridization; Interneurons; Mice; Mice, Inbred C57BL; Neostriatum; Nitric Oxide; Nitric Oxide Synthase; Peptide Fragments; Piperidines; Protein Precursors; Receptors, Neurokinin-3; RNA, Messenger; Somatostatin; Substance P; Tachykinins

1. The aim of this study was to determine whether neurokinin B (NKB) or specific agonists of tachykinin NK(3) receptors, [MePhe(7)]NKB and senktide, were able to induce airway hyperresponsiveness in guinea-pigs. The effects of these compounds were compared to those of substance P (SP), neurokinin A (NKA) and the preferential tachykinin NK(1) ([Sar(9), Met(0(2))(11)]SP) or NK(2) ([betaAla(8)]NKA (4-10)) receptor agonists. 2. In guinea-pigs pretreated with phosphoramidon (10(-4) M aerosol for 10 min) and salbutamol (8.7x10(-3) M for 10 min), all tachykinins administrated by aerosol (3x10(-7) to 10(-4) M) induced airway hyperresponsiveness 24 h later, displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine (i.v.). The rank order of potency was: [betaAla(8)]NKA (4-10)>NKA=NKB=senktide=[MePhe(7)]NKB=[Sar(9),Met(0(2))(11)]SP>SP. 3. Airway hyperresponsiveness induced by [MePhe(7)]NKB was prevented by the tachykinin NK(3) (SR 142801) and NK(2) (SR 48968) receptor antagonists. 4. Bronchoconstriction induced by tachykinins administered by aerosol was also determined. SP, NKA, NKB and the tachykinin NK(1) and NK(2) receptor agonist induced bronchoconstriction. The rank order of potency was: NKA=[betaAla(8)]NKA (4-10)>NKB=SP=[Sar(9), Met(0(2))(11)]SP. Under similar conditions, and for concentrations which induce airway hyperresponsiveness, senktide and [MePhe(7)]NKB failed to induce bronchoconstriction. 5. It is concluded that tachykinin NK(3)-receptor stimulation can induce airway hyperresponsiveness and that this effect is not related to the ability of tachykinins to induce bronchoconstriction.

Topics: Acetylcholine; Animals; Bronchi; Bronchoconstrictor Agents; Female; Guinea Pigs; Male; Neurokinin B; Peptide Fragments; Piperidines; Receptors, Neurokinin-3; Substance P; Tachykinins; Vasodilator Agents

Regulated uterine contractions are important in many reproductive functions such as sperm transport and embryo positioning during implantation. The role of classical neurotransmitters including acetylcholine and norepinephrine in regulating myometrial contractility has been well studied; however, the peripheral role of sensory neurotransmitters such as the neurokinins is less clear. The major neurokinins are substance P, neurokinin A, and neurokinin B, which predominantly activate neurokinin receptors (NK-Rs) 1, 2, and 3, respectively. This study utilized selective receptor agonists to examine the role of NK-Rs in uterine contractility. Uterine tissues, obtained from the major stages of the rat estrous cycle, were stimulated with selective NK-R agonists. Addition of each agonist resulted in a significant contractile response. However, the magnitude and nature of the response were dependent upon the stage of the estrous cycle, with responses to all agonists being significantly decreased in tissue from proestrus and estrus. Furthermore, the nature of NK3-R-mediated contraction was different in tissue from proestrus and estrus compared to metestrus and diestrus. The hormonal dependence of NK-R-mediated contractility was then examined in the ovariectomized estrogen-supplemented rat model. These studies confirmed that the magnitude and nature of uterine contractility in response to NK-R activation depend upon the hormonal environment.

Topics: Animals; Diestrus; Estrogens; Estrus; Female; Metestrus; Neurokinin A; Ovariectomy; Peptide Fragments; Piperidines; Proestrus; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Substance P; Uterine Contraction

Although intrathecally administered senktide, an agonist at the neurokinin3 receptor, attenuates withdrawal responses to noxious stimuli in the restrained animal, senktide increases motor neuron activity in spinal cords of neonatal rats and facilitates the electrically-evoked nociceptive flexor reflex in the adult rat. The present study examined the effects of intrathecal administration of senktide on withdrawal responses to noxious thermal and mechanical stimuli in awake, unrestrained, adult rats. Intrathecal administration of senktide (10 nmol) in chronically catheterized rats did not alter the responses elicited by a noxious mechanical stimulus (508 mN, von Frey monofilament). Conversely, intrathecal senktide (10 nmol) induced thermal hyperalgesia, indicated by decreased withdrawal latency to radiant heat. Thermal hyperalgesia peaked 20-26 min following drug injection and returned to normal within 30 min. SR 142801 (60 nmol), a non-peptide neurokinin3 receptor antagonist, inhibited the senktide-induced hyperalgesia, providing further support that the effect of senktide is mediated by neurokinin3 receptors. Pretreatment with N(G)-nitro-L-arginine methyl ester (30 nmol), a nitric oxide synthase inhibitor, blocked the effect of senktide, indicating that senktide-induced thermal hyperalgesia is also mediated by the production of nitric oxide. Intrathecal senktide produced vasodilation and increased skin temperature in the hind paw. Intravenous hexamethonium, a ganglionic nicotinic receptor antagonist, similarly increased paw temperature without decreasing withdrawal latency to radiant heat. Thus, the increased skin temperature associated with intrathecal senktide was insufficient to account for the thermal hyperalgesia observed. Collectively, the present work demonstrates that NK3 receptors mediate thermal but not mechanical hyperalgesia through a pathway that involves the production of NO.

Topics: Animals; Animals, Newborn; Body Temperature; Electric Stimulation; Enzyme Inhibitors; Hot Temperature; Hyperalgesia; Male; Motor Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Peptide Fragments; Physical Stimulation; Piperidines; Postural Balance; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-3; Spinal Cord; Substance P

Tachykinins contribute to the control of gastrointestinal motility and modulation of somatic and visceral pain. The role of neurokinin (NK) B and NK3 receptors in visceral pain and gastrointestinal disorders has not been determined.. Using electromyographic recordings of both abdominal and colonic muscle and electrophysiological recordings of pelvic nerve afferent fibers, we studied drug effects on responses to colorectal distention.. In awake rats, intraperitoneal administration of the NK3-receptor antagonist SR 142,801 reduced, whereas the NK3-receptor agonist senktide increased, both the rectocolonic inhibitory reflex and abdominal contractions produced by colorectal distention. In contrast, intracerebroventricular administration of SR 142,801 increased the number of abdominal contractions without affecting the rectocolonic inhibitory reflex produced by colorectal distention. In a similar manner, intracerebroventricular injection of senktide diminished the number of abdominal contractions. In electrophysiological experiments, SR 142,801 decreased responses of pelvic nerve afferent fibers to colorectal distention. Responses of pelvic nerve fibers to urinary bladder distention, however, were unaffected by SR 142,801.. These results suggest that peripheral NK3 receptors are involved in the mediation of both visceral nociception and gastrointestinal disorders. Also, central NK3 receptors seem to play a role in the modulation of visceral nociception.

Topics: Animals; Colon; Electromyography; Gastrointestinal Motility; Injections, Intraperitoneal; Injections, Intraventricular; Male; Muscle Contraction; Pain Measurement; Peptide Fragments; Piperidines; Pressure; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Neurokinin-3; Rectum; Sensory Thresholds; Substance P; Visceral Afferents

This study investigates the effects of intracerebroventricular injection of selective agonists and antagonists of tachykinin NK(3)receptor on performance of mice in the elevated plus-maze test. Mice were treated with either vehicle or 1, 10, 100 or 500 pmol of neurokinin B or senktide ([succinil-Asp(6), MePhe(8)]substance P(6-11), a natural and synthetic selective NK(3)receptor agonists, respectively. Other mice received similar doses of [Trp(7)beta-Ala(8)]NKA(4-10)or SR 142801 ((S)-N-(1-(3-(1-benzoyl-3-(3, 4-dichlorophenyl)-piperidin-3-yl)propyl)-4-phenyl-piperidin- 4-yl)-N-m ethylacetamide) tachykinin NK(3)receptor selective peptide and non-peptide antagonists, respectively. Senktide significantly increased the frequency of entries and the time spent in the open arms, which is compatible with an anxiolytic action. Neurokinin B treatment did not alter the plus-maze parameters in a significant way. Conversely, the NK(3)peptide antagonist [Trp(7)beta-Ala(8)]NKA(4-10), but not SR142801 non-peptide antagonist, showed a reverse effect, i.e. an anxiogenic profile of action, reducing the frequency and the time spent in the open arms. Co-injection of either senktide plus [Trp(7)beta-Ala(8)]NKA((4-10)), or senktide plus SR 142801, blocked the effects promoted by senktide, indicating that centrally-administered NK(3)receptor agonists and antagonists can modulate experimental anxiety.

Topics: Animals; Anxiety; Cerebral Ventricles; Injections, Intraventricular; Male; Maze Learning; Mice; Neurokinin A; Neurokinin B; Peptide Fragments; Piperidines; Receptors, Neurokinin-3; 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

The regulation of dopaminergic and cholinergic function by neurokinin-3 (NK3) receptor activation was examined in vivo in urethane-anaesthetized guinea pigs with microdialysis probes. The local application of the NK3 tachykinin receptor agonist senktide in the region of dopamine cell bodies (pars compacta of the substantia nigra and ventral tegmental area) and in the area of cholinergic cell bodies (septal area) markedly enhanced the extracellular dopamine (DA) and acetylcholine (ACh) concentration throughout their respective target areas, i.e. striatum, nucleus accumbens, prefrontal cortex for dopaminergic systems and hippocampus for cholinergic neurons. The enhancing effect of senktide on neurotransmitter release was dose dependently blocked by the selective non-peptide NK3 receptor antagonist SR142801 (0.1-1 mg/kg, i.p.), whereas its inactive S-enantiomer SR142806 (0.3-1 mg/kg, i.p.) did not exert any antagonistic activity on the effect of intranigral or intraseptal application of senktide. These results demonstrate that NK3 receptors can modulate the activity of central DA and ACh systems.

Topics: Acetylcholine; Animals; Brain; Corpus Striatum; Dopamine; Dose-Response Relationship, Drug; Guinea Pigs; Hippocampus; Male; Microdialysis; Nucleus Accumbens; Peptide Fragments; Piperidines; Prefrontal Cortex; Receptors, Neurokinin-3; Substance P; Substantia Nigra; Synaptic Transmission; Time Factors; Ventral Tegmental Area

Effects of a nonpeptide tachykinin NK3 receptor antagonist, SR 142801, were studied in the isolated spinal cord preparation of the neonatal rat. Potential changes were recorded extracellularly from a lumbar ventral root. Bath-application of neurokinin B induced a dose-dependent depolarization of the ventral root. SR 142801 caused rightward shifts of the concentration-response curve for neurokinin B with pA2 of 6.57, but did not affect the depolarizing responses to other agonists. Stimulation of a dorsal root evoked in the ipsilateral ventral root of the same segment monosynaptic and polysynaptic reflexes of fast time course which were followed by a slow depolarization (ipsilateral slow ventral root potential). SR 142801 depressed the ipsilateral slow ventral root potential. The present results indicate that SR 142801 is a specific antagonist for tachykinin NK3 receptors in the spinal cord and suggest that NK3 receptors are involved in primary afferent-evoked nociceptive responses of spinal neurones.

Topics: Animals; Animals, Newborn; Capsaicin; Electrophysiology; Female; In Vitro Techniques; Male; Membrane Potentials; Neurokinin B; Neurons, Afferent; Patch-Clamp Techniques; Peptide Fragments; Piperidines; Rats; Rats, Wistar; Receptors, Neurokinin-3; Spinal Cord; Spinal Nerve Roots; Substance P

The effects of the transglutaminase-synthesized polyamine derivatives of Substance P (SP) have been further characterized by their ability to contract in vitro the rat portal vein strip (RPV), a pharmacological preparation particularly rich in NK-3 receptors. The effects of selective agonists of NK-1, NK-2 and NK-3 receptors [Sar9,Met(O(2))11]SP, beta-Ala8 NKA(4-10), and senktide respectively, were also evaluated by measuring RPV concentration-response curves. Peptide [GR-82334 (NK-1) and MEN-10,376 (NK-2)] and nonpeptide [WIN 51,708 (NK-1) and SR 142801 (NK-3)] NK receptor antagonists were used to confirm the participation of the different NK receptors to contractile response. Our results demonstrated that the spermine derivative of SP (Spm-SP), previously shown to be unable to recognize NK-1 and NK-2 receptors in some bioassays, contracts RPV (EC50 = 588 nM) better than the native neuropeptide (EC50 = 1120 nM). A pretreatment with thiorphan, an inhibitor of neutral endopeptidases, significantly reduced such a difference. While this inhibitor shifts the SP concentration-response curves to the left (EC50 = 720 nM) the action of Spm-SP and [Sar9,Met(O(2))11]SP were completely thiorphan-resistant. In the absence of thiorphan we found the following rank order of potency: senktide > > beta-Ala8 NKA(4-10) > [Sar9,Met(O(2))11]SP = Spm-SP > SP. Among the mentioned NK receptor antagonists, only the selective NK-3 receptor antagonist, SR 142801, shifted to the right Spm-SP and [Sar9,Met(O(2))11]SP concentration-response curve, showing pKB values of 5.84 and 5.88, respectively. Therefore, the reported results suggest that the introduction of a Spm moiety into the SP alters the parent peptide molecule by increasing its affinity for NK-3 receptors and/or by preventing its degradation by some proteolytic enzymes.

Topics: Androstanes; Animals; Benzimidazoles; In Vitro Techniques; Male; Neurokinin A; Peptide Fragments; Physalaemin; Piperidines; Portal Vein; Rats; Rats, Wistar; Receptors, Neurokinin-3; Substance P; Thiorphan; Transglutaminases; Vasoconstriction

1. Tachykinin NK3 receptors were characterized in the rabbit isolated iris sphincter muscle by use of autoradiography and in vitro functional studies. 2. [125I]-[MePhe7]-neurokinin B (NKB) (1nM), a selective NK3 receptor agonist, specifically labelled a population of NK3 receptors that were uniformly distributed throughout the rabbit iris sphincter muscle. This labelling was inhibited by unlabelled [MePhe7]-NKB (1 microM) but not by the NK1 receptor antagonist CP 99994 (1 microM). 3. In the presence of CP 99994 (1 microM), the selective NK3 receptor agonists senktide (n = 14) and [Pro7]-NKB (n = 4), and the natural preferred ligand for the NK3 receptor, NKB (n = 8), were potent contractile agents in the rabbit iris sphincter muscle. They all produced monophasic concentration-effect curves with pD2 values of 9.53 +/- 0.08, 8.56 +/- 0.09 and 9.75 +/- 0.09, and nH values of 0.93 +/- 0.03, 1.53 +/- 0.17 and 0.76 +/- 0.06, respectively. [MePhe7]-NKB (n = 12) was also a potent agonist, but produced shallow concentration-effect curves which appeared biphasic (nH = 0.45 +/- 0.04). 4. Contractile responses to senktide were surmountably antagonized in a concentration-dependent manner by the selective non-peptide NK3 receptor antagonist, SR 142801 (3-30 nM; pA2 = 8.9; slope = 0.99) and the non-peptide NK2/NK3 receptor antagonist, SR 48968 (3-30 microM; pA2 = 6.1; slope = 1.5). These pA2 values were consistent with functional rabbit NK3 receptors more closely resembling guinea-pig and human NK3 receptors, than rat NK3 receptors. SR 142801 (10-100 nM) and SR 48968 (3 and 30 microM) inhibited responses to low (< or = 1 nM) but not higher (> 1 nM) concentrations of [MePhe7]-NKB, and concentration-effect curves to [MePhe7]-NKb became steeper and monophasic in the presence of either antagonist. 5. SR 142801 (3-30 nM) and SR 48968 (3-30 microM) also surmountably antagonized concentration-effect curves to [Pro7]-NKB and NKB, although results were more difficult to interpret, since the relationship between log concentration-ratios and the concentration of antagonist used did not adhere to the Schild equation. However, analysis of data with the lowest concentration of SR 142801 (3 nM) tested against NKB, and SR 48968 (3 microM) tested against [Pro7]-NKB and NKB, yielded apparent pA2 estimates of 9.3, 6.8 and 6.4, respectively, consistent with blockade of NK3 receptors. 6. SR 142801 (100 nM) had no effect on contractions induced by transmural nerve stimulation (2 Hz, 0.3 ms, 2

Topics: Adrenergic alpha-Antagonists; Animals; Autoradiography; Benzamides; Electric Stimulation; In Vitro Techniques; Iris; Male; Muscle Contraction; Muscle, Smooth; Neurokinin B; Peptide Fragments; Phenoxybenzamine; Piperidines; Rabbits; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Substance P

1. Inhibition of NK3 receptor agonist-induced contraction in the rabbit isolated iris sphincter muscle was used to assess the in vitro functional activity of three 2-phenyl-4-quinolinecarboxamides, members of a novel class of potent and selective non-peptide NK3 receptor antagonists. In addition, an in vivo correlate of this in vitro response, namely NK3 receptor agonist-induced miosis in conscious rabbits, was characterized with some of these antagonists. 2. In vitro senktide (succinyl-[Asp9,MePhe8]-substance P (6-11) and [MePhe7]-neurokinin B ([MePhe7]-NKB) were potent contractile agents in the rabbit iris sphincter muscle but exhibited quite different profiles. Senktide produced monophasic log concentration-effect curves with a mean pD2=9.03+/-0.06 and mean nH=1.2+/-0.02 (n=14). In contrast, [MePhe7]-NKB produced shallow log concentration-effect curves which often appeared biphasic (nH=0.54+/-0.04, n=8), preventing the accurate determination of pD2 values. 3. The contractile responses to the NK3 receptor agonist senktide were antagonized in a surmountable and concentration-dependent manner by SB 223412 ((-)-(S)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-ca rboxamide; 3-30 nM, pA2=8.4, slope=1.8+/-0.3, n=4). SB 222200 ((-)-(S)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car box amide; 30-300 nM, pA2=7.9, slope=1.4+/-0.06, n=4) and SB 218795 ((-)-(R)-N-(alpha-methoxycarbonylbenzyl)-2-phenylquinoline-4-carboxamide; 0.3 and 3 microM apparent pKB=7.4+/-0.06, n=6). 4. Contractile responses to the NK3 receptor agonist [MePhe7]-NKB in the rabbit iris sphincter muscle were unaffected by SB 218795 (0.3 and 3 microM, n=8). In contrast, SB 223412 (30 and 300 microM n=4) and SB 222200 (0.3 and 3 microM, n=4) inhibited responses to low concentrations (< or = 1 nM), to a greater extent than higher concentrations (> 1 nM) of [MePhe7]-NKB. Furthermore, log concentration-effect curves to [MePhe7]-NKB became steeper and monophasic in the presence of each antagonist. 5. SB 218795 (3 microM, n=4) had no effect on contractions induced by transmural nerve stimulation (2 Hz) or substance P, exemplifying the selectivity of this class of antagonist for functional NK3 receptors over NK1 receptors in the rabbit. 6. In vivo, senktide (1, 10 and 25 microg i.v., i.e. 1.2, 11.9 and 29.7 nmol, respectively) induced concentration-dependent bilateral miosis in conscious rabbits (maximum pupillary constriction=4.25+/-0.25 mm; basal pupillary diameter 7.75+/-0.48 mm; n=4).

Topics: Animals; In Vitro Techniques; Iris; Male; Miosis; Muscle Contraction; Muscle, Smooth; Neurokinin B; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Quinolines; Rabbits; Receptors, Neurokinin-3; Substance P

The uterotonic potencies of the naturally occurring mammalian tachykinins and the synthetic subtype-selective agonist analogues of these agents [Lys5,MeLeu9,Nlel0]neurokinin A-(4-10) and [Nle10]neurokinin A-(4-10) (tachykinin NK2 receptor-selective), [Sar9,Met(O2)11]substance P (tachykinin NK1 receptor-selective) and senktide (tachykinin NK3 receptor-selective) were determined using preparations from oestradiol-treated rats. The endopeptidase 24.11 inhibitor, N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl-(S)-isoserine+ ++ (SCH 39370), potentiated responses to neurokinin A, neurokinin B and substance P, but not to [Lys5,MeLeu9,Nle10)]neurokinin A-(4-10) or senktide. [Nle10]neurokinin A-(4-10) effects were potentiated by SCH 39370 with amastatin and those to [Sar9,Met(O2)11]substance P were potentiated by SCH 39370 and captopril in combination. In the presence of optimal concentrations of peptidase inhibitors the relative order of agonist potency was: neurokinin A > substance P > neurokinin B for the naturally occurring mammalian tachykinins and [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) > [Nle10]neurokinin A-(4-10) > [Sar9,Met(O2)11]substance P > senktide for the synthetic tachykinin analogues. Thus, while a tachykinin NK2 receptor predominates in the oestrogen-primed uterus, a tachykinin NK1 receptor may also be present. The non-peptide tachykinin NK3 receptor antagonist, SR 142801, did not antagonise the effects of senktide suggesting that tachykinin NK3 receptors do not mediate its relatively minor effect on the uterus of the oestrogen-primed rat.

Topics: Animals; Dipeptides; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Estradiol; Female; Muscle, Smooth; Neurokinin A; Peptide Fragments; Piperidines; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Tachykinin; Substance P; Tachykinins; Uterine Contraction; Uterus

This study characterized tachykinin-evoked secretomotor responses in in vitro submucosal and mucosal-submucosal preparations of the guinea pig ileum using combined intracellular and Ussing chamber recording techniques. Superfusion of endogenous tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B depolarized single submucosal neurons and evoked increased short-circuit current (Isc) responses in Ussing chamber preparations. The NK1-receptor agonist [Sar9,Met(O2)11]SP [50% effective concentration (EC50) = 2 nM] depolarized all submucosal neurons examined. The NK3-receptor agonist senktide (EC50 = 20 nM) depolarized approximately 50% of neurons examined, whereas the NK2-receptor agonist [Ala5,beta-Ala8]NKA-(4-10) had no effect on membrane potential. [Sar9,Met(O2)11]SP and senktide evoked similar increases in Isc that were tetrodotoxin sensitive (91 and 100%, respectively) and were selectively blocked by the NK1 antagonist CP-99,994 and the NK3 antagonist SR-142,801, respectively. Capsaicin-evoked increases in Isc were significantly inhibited (54%, P < 0.05) by CP-99,994 but not by SR-142,801. Neither antagonist inhibited slow excitatory postsynaptic potentials. These findings suggest that tachykinin-evoked secretion in guinea pig ileum is mediated by NK1 and NK3 receptors on submucosal secretomotor neurons and that capsaicin-sensitive nerves release tachykinin(s) that activate the NK1 receptors.

Topics: Animals; Capsaicin; Electric Stimulation; Evoked Potentials; Guinea Pigs; Ileum; In Vitro Techniques; Intestinal Mucosa; Membrane Potentials; Muscle, Smooth; Neurokinin A; Neurokinin B; Neurokinin-1 Receptor Antagonists; Neurons; Peptide Fragments; Piperidines; Receptors, Neurokinin-1; Receptors, Neurokinin-3; Substance P; Tachykinins

1. In the conscious rat, three tachykinin NK3 receptor antagonists, namely SR142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)pro pyl)-4-phenylpiperidin-4-yl)-N-methylacetamide), R820 (3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) and R486 (H-Asp-Ser-Phe-Trp-beta-Ala-Leu-Met-NH2) were assessed against the intracerebroventricular (i.c.v.) effects induced by senktide, a selective NK3 receptor agonist, on mean arterial blood pressure (MAP), heart rate (HR) and motor behaviour. 2. Senktide (10-650 pmol per animal; i.c.v; n = 4-16) at the lowest dose caused a significant fall in MAP (-10 +/- 6 mmHg), while at the highest doses (100 and 650 pmol), senktide caused a rise in MAP (9 +/- 3 and 12 +/- 1 mmHg, respectively) when compared to vehicle. The intermediate doses (25 and 65 pmol) had no effect on MAP. The highest two doses caused a tachycardia of 62 +/- 15 and 88 +/- 8 beats min(-1), respectively. The dose of 65 pmol had a biphasic effect on HR, an initial bradycardia of 47 +/- 12 beats min(-1) followed by a tachycardia of 46 +/- 14 beats min(-1). The lowest doses caused either a rise of 52 +/- 10 beats min(-1) (25 pmol) or no effect (10 pmol) on HR. All doses of senktide caused similar increases in face washing, sniffing and wet dog shakes except at the dose of 100 pmol, when wet dog shakes were more than double those observed with the other doses. 3. The antagonist SR142801 (100 pmol -65 nmol per animal; i.c.v.; n = 6-8) caused increases in MAP at the highest two doses (6.5 and 65 nmol) while HR, dose-dependently, increased (23 +/- 6 to 118 +/- 26 beats min[-1]) and the onset dose-dependently decreased. The (R)-enantiomer, SR142806 (100 pmol - 65 nmol per animal; i.c.v.; n = 6-8) only caused rises in MAP (13 +/- 2 mmHg) and HR (69 +/- 11 beats min[-1]) at the highest dose. These drugs had no apparent effect on behaviour, except for the highest dose of SR142801 which increased sniffing. The antagonist R820 (650 pmol - 6.5 nmol per animal; i.c.v.; n = 6) had no effect on MAP or HR and only increased sniffing behaviour at 6.5 nmol. At 650 pmol (n = 6), R486 had no effect on any variable, but at 3.25 nmol, i.c.v. (n = 4) a delayed tachycardia and a significant increase in all behavioural variables were observed. 4. The cardiovascular responses induced by 6.5 nmol SR142801 and 25 pmol senktide were inhibited by R820 (6.5 nmol, 5 min earlier i.c.v.). In contrast, R820 failed to affect the central cardiovascular and behavioural responses induced by 10

Topics: Animals; Behavior, Animal; Cardiovascular System; Dogs; Indoles; Injections, Intraventricular; Male; Oligopeptides; Peptide Fragments; Piperidines; Rats; Rats, Wistar; Receptors, Neurokinin-3; Substance P

1. The effects of intracerebroventricular (i.c.v.) administration of the NK3 tachykinin receptor agonist, senktide (10 nmol each side), in guinea-pigs pretreated with the selective NK3 tachykinin receptor antagonist, SR142801 (3 mg kg(-1) subcutaneous, s.c., 30 min before senktide), or its less active enantiomer, SR142806 (3 mg kg(-1) s.c. 30 min before senktide), on behaviour and on the distribution of Fos-like immunoreactivity (Fos-LI) in central neurones were investigated. Guinea-pigs were chosen for the study since they possess NK3 tachykinin receptors with pharmacological characteristics similar to those in man. 2. Wet-dog shakes, but not locomotor activity, elicited by senktide i.c.v. were significantly reduced by SR142801 but not by SR142806, confirming the involvement of NK3 tachykinin receptors in wet-dog shake behaviour. 3. Senktide induced increased numbers of Fos-LI neurones in the following brain areas: frontal, parietal and piriform cortex, the lateral septum, the CA1, CA2, subiculum and dentate gyrus of the hippocampus, most areas in the amygdala, thalamus and hypothalamus, medial geniculate nucleus and the ventral cochlear nucleus. Pretreatment with SR142801, but not with SR142806, before administration of senktide inhibited Fos-LI expression in the cingulate cortex, dentate gyrus of the hippocampus, some regions of the thalamus, hypothalamus and amygdala and the ventral cochlear nucleus. 4. The present results are the first demonstration that senktide induces Fos-LI in widespread areas of the guinea-pig brain. It is proposed that NK3 tachykinin receptors may play a more extensive role in the control of diverse brain functions, including cortical processing, learning and memory, neuroendocrine and behavioural regulation, than is currently recognized.

Topics: Animals; Behavior, Animal; Brain; Female; Guinea Pigs; Male; Neurons; Peptide Fragments; Piperidines; Proto-Oncogene Proteins c-fos; Receptors, Neurokinin-3; Substance P; Tyrosine 3-Monooxygenase

Topics: Animals; Binding, Competitive; CHO Cells; Ciliary Body; Cricetinae; Cricetulus; Drug Design; Guinea Pigs; Humans; Muscle Contraction; Peptide Fragments; Piperidines; Quinolines; Rabbits; Receptors, Neurokinin-3; Recombinant Fusion Proteins; Structure-Activity Relationship; Substance P

In urethane-anaesthetized rats, moderate colonic distention (0.5 ml) induced reflex rhythmic contractions (5 mm Hg amplitude and 1.1 cycles/min frequency). Senktide (1-10 nmol/kg, i.v.), a tachykinin NK3 receptor selective agonist, transiently suppressed distension-induced contractions. SR 142,801 (1-10 mumol/kg i.v.), a non-peptide tachykinin NK3 receptor antagonist, had no effect on distension-induced contractions but prevented the inhibitory effect of senktide. Infusion of N-omega-nitro-1-arginine methyl esther hydrochloride (L-NAME, 20 mumol/ml/h, i.v) increased the amplitude of colonic contractions and decreased the inhibitory effect of senktide. Hexamethonium (15 mumol/ml/h, i.v.) or atropine (1 mumol/ml/h, i.v.) inhibited the distension-induced contractions. In hexamethonium- or atropine-treated rats, senktide (10 nmol/kg) transiently and selectively enhanced the amplitude of contractions. Also SR 142,801 (10 mumol/kg), but not its inactive enantiomer SR 142,806, increased both amplitude and frequency of contractions. During continuous infusion of L-NAME and hexamethonium or atropine both frequency and amplitude of distension-induced colonic contractions were higher than when in hexamethonium or atropine only. Senktide (10 nmol/kg) had no effect and SR 142,801 (10 mumol/kg) produced a slight enhancement of colonic contractions. Infusion of sodium nitroprusside (3 mumol/ml/h, i.v.) decreased amplitude and frequency of distension-induced contractions. SR 142,801 had no effect in the presence of the nitric oxide (NO) donor. We conclude that tachykinins acting through NK3 receptors exert at least four different actions on colonic motility activated by distension: 1) a hexamethonium-resistant, NO-dependent, suppressant effect on contractions; 2) a hexamethonium-sensitive, NO-independent inhibitory effect on the amplitude of contractions; 3) a hexamethonium-resistant, NO-independent inhibitory effect on the amplitude of contractions and 4) a hexamethonium resistant and L-NAME-sensitive excitatory effect on amplitude of contractions. The prevalent inhibitory effect evoked in normal conditions along with the excitatory activity induced by SR 142,801 on hexamethonium-resistant colonic motility indicates that tachykinins, acting through neuronal NK3 receptors, activate NO-dependent and NO-independent inhibitory neurotransmission in the rat colon.

Topics: Adrenergic Agents; Animals; Colon; Dose-Response Relationship, Drug; Guanethidine; Hexamethonium; Male; Muscle Contraction; Neurokinin A; NG-Nitroarginine Methyl Ester; Nicotinic Antagonists; Peptide Fragments; Piperidines; Rats; Rats, Wistar; Receptors, Neurokinin-3; Receptors, Tachykinin; Substance P

1. The cardiovascular responses to intravenous (i.v.) injection of natural tachykinins, substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and selective tachykinin (NK) receptor agonists, [Sar9, Met(O2)11]SP, [beta Ala8]NKA(4-10), [MePhe7]NKB and senktide were assessed in conscious, freely moving, guinea-pigs. 2. SP and [Sar9, Met(O2)11]SP (1-1000 pmol kg-1) induced dose-dependent decreases in mean arterial blood pressure (MAP) accompanied by increases in heart rate (HR). NKA evoked only weak hypotensive effects at high doses (3000 pmol kg-1) whereas [beta Ala8]NKA(4-10) (1-3000 pmol kg-1) had no effects. By contrast, NKB [MePhe7]NKB (1-10,000 pmol kg-1) and senktide (1-1000 pmol kg-1), produced dose-related hypertensive effects with the following rank order of potency: senktide > [MePhe7]NKB > NKB. Bradycardia occurred simultaneously with the increases in arterial pressure. 3. The pressor response to intravenous injection of senktide (300 pmol kg-1) was partially reduced by pretreatment with prazosin (0.71 mumol kg-1), or clonidine (0.38 mumol kg-1) and was completely inhibited by the combination of the two compounds. Atropine (1.5 mumol kg-1) suppressed the decrease in HR induced by senktide without altering the blood pressure response. These findings suggest that the blood pressure response to senktide is an indirect effect mediated by noradrenaline released from sympathetic nerve endings, whereas the bradycardia is of vagal reflex origin. 4. SR 142801, ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenyl-piperidin-4-yl)-N-methylacetamide), a potent and specific non-peptide NK3 receptor antagonist dose-dependently (0.46-4.6 mumol kg-1, i.v.; 4.6-46 mumol kg-1, p.o.) inhibited the cardiovascular effects of senktide and displayed a long-lasting inhibitory effect after oral administration. By contrast, SR 142806 (4.6 mumol kg-1, i.v.), the (R)-enantiomer of SR 142801 had no effect on the responses to senktide. SR 142801 at a high dose (15 mumol kg-1, i.v.) was inactive toward the [Sar9, Met(O2)11]SP-induced hypotension. 5. SR 142801 did not modify MAP in conscious guinea-pigs both after i.v. (4.6 and 15 mumol kg-1) and oral (46 and 150 mumol kg-1) administration, showing a lack of agonistic properties. However, a slight reduction in HR was observed only after i.v. injection. 6. In conclusion, these results show evident differences in the functional role of tachykinin receptors in the peripheral control of the cardiovascu

Topics: Analysis of Variance; Animals; Blood Pressure; Dose-Response Relationship, Drug; Guinea Pigs; Heart Rate; Injections, Intravenous; Male; Neurokinin A; Neurokinin B; Peptide Fragments; Piperidines; Substance P

The objective of the present in vitro and in vivo experiments was to examine the involvement of neurokinin NK3 receptors in the regulation of the noradrenergic function in gerbils and guinea-pigs. Application of senktide, a peptide NK3 receptor agonist, on guinea-pig locus coeruleus slices increased the firing rate of presumed noradrenergic neurons (EC50 = 26 nM) in a concentration-dependent manner. Given i.c.v., senktide (0.5-2 micrograms) and (MePhe7)neurokinin B (1-10 micrograms), another NK3 receptor agonist, reduced exploratory behaviour in gerbils in a dose-dependent manner (2 micrograms of senktide producing a 50% reduction of locomotor activity and rearing). In vivo microdialysis experiments in urethane-anaesthetized guinea-pigs showed that senktide (2-8 micrograms i.c.v.) induced a dose-dependent increase in norepinephrine release in the medial prefrontal cortex. The electrophysiological, behavioural and biochemical changes elicited by senktide were concentration- or dose-dependently reduce by SR 142801, the selective non-peptide NK3 receptor antagonist. In the locus coeruleus slice preparation, complete antagonism of senktide (30 nM) was observed with 50 nM of SR 142801, while injected i.p. (0.1-1 mg/kg) it abolished the senktide-induced norepinephrine release in guinea-pigs. In gerbils, SR 142801 (1-10 mg/kg i.p.) reversed the reduction of exploratory behaviour induced by senktide (1 microgram). By contrast, the 100-fold less active enantiomer, SR 142806, did not exert any antagonism in these models. Finally, the reduction of exploratory behaviour in gerbils was found to be reversed by prazosin (0.25-2.56 micrograms/kg i.p.) and to some extent by clonidine, drugs known to depress noradrenergic function. All these experiments strongly support the hypothesis that brain noradrenergic neurons can be activated by stimulation of neurokinin NK3 receptors.

Topics: Adrenergic Fibers; Animals; Brain; Dose-Response Relationship, Drug; Gerbillinae; Guinea Pigs; Locus Coeruleus; Microdialysis; Neuropeptides; Norepinephrine; Peptide Fragments; Piperidines; Prazosin; Receptors, Neurokinin-3; Substance P

Primary cultures of gerbil mesencephalon were used for studying the modulation exerted by tachykinin NK(3) receptor activation on the activity of dopamine (DA) neurons. Dopamine neurons were identified by their ability to take up [(3)H]DA in a nomifensine-dependent manner. Moreover, tyrosine hydroxylase immunohistochemistry revealed that these neurons accounted for 5-7% of the total cell population. The NK(3) receptor agonists, senktide (EC(50) = 0.58 nM) and [MePhe(7)]neurokinin B (EC(50) = 3 nM), increased spontaneous [(3)H]DA release in a concentration-dependent manner. In contrast, tested at a supramaximal concentration (IC(50) = 0.89 nM), neither septide nor substance P were found to affect [(3)H]DA release. The senktide-evoked [(3)H]DA release was not observed when extracellular Ca(2+) was chelated, but was unaffected by nomifensine. This indicates that this increase in [(3)H]DA outflow resulted more from an exocytotic process than from reversal of carrier-mediated DA uptake. Moreover, the senktide effect was unaffected by the Na+ channel blocker tetrodotoxin, a result suggesting a direct action of senktide on DA neurons. The non-peptide NK(3) receptor antagonist, SR 142801, shifted or blocked (IC(50) = 0.89 nM) the senktide-evoked [(3)H]DA release, while its (-)-antipode, SR 142806, was 80-fold less potent, in agreement with binding data. Selective antagonists for Nk1 (SR 140333) or Nk2 (SR 48968) receptors failed to reduce the senktide effect. Light scanning microscopic analysis of mesencephalic cells loaded with the Ca(2+) sensitive dye, fluo-3, showed that senktide induced a rise in cytosolic Ca(2+) in 8-10% of the cell population. The senktide-induced elevation in intracellular Ca(2+) was rapid in onset and transient (at 10-8 M) or more sustained with no further increase in fluorescence intensity (at 10(-7) M). The proportion of senktide-responsive cells was not significantly modified when extracellular Ca(2+) was chelated, but was reduced by 87% in the presence of SR 142801 and by 75% in cultures that were pre-treated with the DA neurotoxin 1-methyl-4-phenylpyridinium. The present study shows that enhancement of spontaneous [(3)H]DA release and intracellular Ca(2+) mobilization may be observed after NK(3) receptor stimulation and that both biochemical events are likely to occur in DA neurons.

Topics: Animals; Calcium; Cells, Cultured; Dopamine; Gerbillinae; Intracellular Membranes; Mesencephalon; Neurons; Peptide Fragments; Piperidines; Receptors, Neurokinin-3; Receptors, Tachykinin; Substance P

The pharmacological profile of the novel tachykinin NK3 receptor antagonist SR 142801, ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide), was studied at tachykinin NK1, NK2 and NK3 receptors, in several in vitro bioassays. In the guinea-pig isolated ileum longitudinal muscle preparation, SR 142801 (10 nM-1 microM) caused an insurmountable antagonism of tachykinin NK3 receptor-mediated contractions produced by senktide (apparent pKB = 9.27). The blockade induced by SR 142801 was essentially irreversible, since it was not removed by washout (up to 2 h) and was increased by prolonging the incubation from 15 to 120 min. SR 142801 showed similar antagonist potency at rat tachykinin NK3 receptors (portal vein) and rabbit tachykinin NK2 receptors (pulmonary artery) (pKB = 7.49 and 7.66, respectively), whereas it was distinctly less potent at hamster tachykinin NK2 receptors (trachea; pKB = 6.84) and inactive at guinea-pig tachykinin NK1 receptors (ileum, longitudinal muscle). In the guinea-pig whole ileum SR 142801 (100 nM) did not affect the contraction produced by capsaicin (1 microM). The combined SR 142801 pretreatment and tachyphylaxis of neuronal CGRP (calcitonin gene-related peptide) receptors produced a slight (about 25%), but significant reduction of the response to capsaicin, suggesting that tachykinin NK3 receptors play a minor role in capsaicin-induced neuronal excitation of afferent nerves in the guinea-pig ileum.

Topics: Analysis of Variance; Animals; Binding, Competitive; Capsaicin; Cricetinae; Guinea Pigs; Ileum; In Vitro Techniques; Male; Mesocricetus; Muscle Contraction; Muscle, Smooth; Muscle, Smooth, Vascular; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Piperidines; Portal Vein; Pulmonary Artery; Rabbits; Rats; Rats, Wistar; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Substance P; Trachea

The aims of this study were: (i) verify the usefulness of the recently described non-peptide antagonist, SR 142801, for blocking tachykinin NK3 receptors in the circular muscle of the guinea-pig colon and (ii) after occlusion of NK3 receptors by SR 142801, test the hypothesis that tachykinins may activate non-adrenergic non-cholinergic inhibitory neurons via non-NK3 receptors. In sucrose gap, we found that SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced atropine (1 microM)-sensitive depolarization, action potentials and contractions of circular muscle of guinea-pig colon without affecting the cholinergic excitatory junction potential and contraction produced by single pulse electrical field stimulation. Likewise, SR 142801 (0.1 microM) time-dependently inhibited the senktide-induced non-adrenergic non-cholinergic hyperpolarization and relaxation of the circular muscle, without affecting the non-adrenergic non-cholinergic inhibitory junction potentials and relaxation produced by single pulse electrical field stimulation. Therefore, SR 142801 is a suitable tool to occlude neuronal NK3 receptors in guinea-pig colon. In the presence of SR 142801 (0.1 microM), atropine (1 microM), guanethidine (3 microM), indomethacin (3 microM) and nifedipine (1 microM) superfusion with neurokinin A (0.3 microM) produced depolarization on which a series of inhibitory junction potentials were superimposed. The incidence, number and amplitude of the inhibitory junction potentials evoked by neurokinin A was partly reduced by pretreatment with either apamin (0.1 microM) or L-nitroarginine (30 microM) and was totally blocked by pretreatment with apamin plus L-nitroarginine or by tetrodotoxin (1 microM). None of these treatments affected the depolarization and contraction produced by neurokinin A. The NK1 receptor selective antagonist, GR 82,334 (3 microM), did not affect the responses to neurokinin A, which were abolished by the NK2 receptor-selective antagonist GR 94,800 (0.1 microM). Substance P (0.3 microM) produced a large depolarization of the membrane but was poorly effective in producing superimposed inhibitory junction potentials. The NK1 receptor-selective agonist [Sar9]substance P sulfone (0.3 microM) produced large depolarization without inducing superimposed inhibitory junction potentials, while the NK2 receptor-selective synthetic agonist [beta-Ala8]neurokinin A(4-10) (0.3 microM) produced depolarization and superimposed inhibitory junction pote

Topics: Animals; Colon; Guinea Pigs; Male; Membrane Potentials; Peptide Fragments; Piperidines; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Substance P

We have determined the ability of the novel nonpeptide tachykinin (TK) NK3 receptor antagonist, SR 142801, [(S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methylaceta mide] in inhibiting the nitric oxide (NO)-independent prejunctional inhibition of cholinergic twitches and the NO-dependent relaxation produced by the NK3 receptor selective agonist, senktide, in the circular muscle of the guinea-pig proximal colon. Under moderate load (10 mN) and isometric recording of mechanical activity, single pulse electrical field stimulation (EFS) produced atropine- and tetrodotoxin-sensitive twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon. In the presence of NK1 and NK2 receptor antagonists (SR 140333 0.01 microM and GR 94800 0.1 microM, respectively) the NK3 receptor selective agonist, senktide (EC50 33 pM) and the NK3 receptor preferring natural TK, neurokinin B (NKB, EC50 13 pM) produced a concentration-dependent slowly developing inhibition of cholinergic twitches. Senktide (1 nM) did not affect the contractile response to acetylcholine (1 microM) indicating that depression of evoked twitches occurs prejunctionally. The inhibitory effect of senktide was unaffected when evoked in the presence of the cyclooxygenase inhibitor (S)-ketoprofen (10 microM), guanethidine (10 microM), naloxone (0.3 microM), the GABAB receptor antagonist 2-hydroxysaclofen (10 microM) or the combined application of the adenosine A1 and A2 receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (10 microM) and 3,7-dimethyl-1-propargylxanthine (30 microM) respectively. In the presence of NK1 and NK2 receptor antagonists, the NO-synthase inhibitor L-nitroarginine (L-NOARG 30-100 microM) did not affect twitch inhibition induced by senktide (EC50 33 pM). The response to NKB (EC50 95 pM) was slightly reduced by L-NOARG, yet the bulk of the inhibitory effect of both agonists on cholinergic twitches was substantially independent of NO generation. SR 142801 (0.1-0.3 microM) produced a moderate rightward shift of the concentration-response curve to senktide without depression of the Emax to the agonist, yielding an apparent pKB value of 7.65. Under low resting tone (3 mN) and isotonic recording of mechanical activity, mucosa-free circular muscle strips from the guinea-pig proximal colon gained a high intrinsic tone suitable for testing the response to relaxant agents. In the presence of atropine (1 microM)

Topics: Animals; Arginine; Colon; Electric Stimulation; Enzyme Inhibitors; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Peptide Fragments; Piperidines; Receptors, Neurokinin-3; Substance P