Involvement of tachykinin NK1 receptors in nociceptin-induced hyperalgesia in mice.

Intrathecal (i.t.) injection of nociceptin at small doses (3.0 and 30.0 fmol) produced a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of nociceptin (3.0 fmol) and returned to control level within 30 min. Hyperalgesia elicited by nociceptin was inhibited dose-dependently by i.t. co-administration of tachykinin NK1 receptor antagonists, CP-99,994 and sendide. A significant antagonistic effect of [D-Phe7, D-His9] substance P (6-11), a selective antagonist for substance P, was observed against the nociceptin-induced hyperalgesia. Pretreatment with i.t. substance P antiserum and i.t. capsaicin resulted in a complete block of the reduced threshold produced by nociceptin. The NK2 receptor antagonist, MEN-10,376 and pretreatment with neurokinin A antiserum did not alter the behavioural effect of nociceptin. The N-methyl-D-aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and D(-)-2-amino-5-phosphonovaleric acid (D-APV), and L-NG-nitro arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, failed to inhibit nociceptin-induced hyperalgesia. The results obtained suggest that the hyperalgesic effect of nociceptin may be mediated through tachykinin NK1 receptors in the spinal cord.     

An NK1 Receptor-dependent Component of the Slow Excitation Recorded Intracellularly from Rat Motoneurons Following Dorsal Root Stimulation

Intracellular recording from lumbar motoneurons of the neonatal rat spinal cord in vitro was used to study how recently developed non-peptide antagonists such as SR-140333 and SR-48698, known to block distinct subtypes of tachykinin receptors peripherally, might affect synaptic transmission elicited by electrical stimulation of dorsal root fibres. SR-140333 (1 μM) preferentially antagonized responses mediated by an exogenously applied agonist acting on the NK₁ receptor subclass, while SR-48968 (0.5 μM) preferentially reduced responses mediated by an exogenously applied agonist acting on the NK₂ receptor subclass. SR-48968 did not affect fast or slow excitatory postsynaptic potentials (EPSPs) or ‘wind-up’responses induced by repetitive, low-frequency stimulation (mimicking certain types of nociceptive input); binding studies using this radiolabelled ligand disclosed specific binding activity (21 fmol/mg protein) selectively displaced by an NK₂ receptor agonist. SR-140333 reduced the late component of fast and slow EPSPs, and of wind-up. Pharmacological block of ionotropic glutamate receptors abolished all dorsal root-evoked EPSPs. In comparison to glutamate receptor blockers, SR-140333 was a weaker antagonist of slow synaptic responses, though it displayed preferential antagonism towards some components of the wind-up phenomenon. The present results provide evidence obtained with a novel NK₁ antagonist that a neuropeptide (presumably substance P), although not directly released by primary afferents onto motoneurons, is a neurotransmitter (acting via NK₁ receptors) in the pathway mediating slow synaptic responses of motoneurons, and is presumably involved in signalling nociceptive inputs from the periphery.     

Excitatory actions of substance P in the rat lateral posterior nucleus

The lateral posterior nucleus (LP) receives inputs from both neocortex and superior colliculus (SC), and is involved with integration and processing of higher‐level visual information. Relay neurons in LP contain tachykinin receptors and are innervated by substance P (SP)‐containing SC neurons and by layer V neurons of the visual cortex. In this study, we investigated the actions of SP on LP relay neurons using whole‐cell recording techniques. SP produced a graded depolarizing response in LP neurons along the rostro‐caudal extent of the lateral subdivision of LP nuclei (LPl), with a significantly larger response in rostral LPl neurons compared with caudal LPl neurons. In rostral LPl, SP (5–2000 nm ) depolarized nearly all relay neurons tested (> 98%) in a concentration‐dependent manner. Voltage‐clamp experiments revealed that SP produced an inward current associated with a decreased conductance. The inward current was mediated primarily by neurokinin receptor (NK)₁ tachykinin receptors, although significantly smaller inward currents were produced by specific NK₂ and NK₃ receptor agonists. The selective NK₁ receptor antagonist RP67580 attenuated the SP‐mediated response by 71.5% and was significantly larger than the attenuation of the SP response obtained by NK₂ and NK₃ receptor antagonists, GR159897 and SB222200, respectively. The SP‐mediated response showed voltage characteristics consistent with a K⁺ conductance, and was attenuated by Cs⁺, a K⁺ channel blocker. Our data suggest that SP may modulate visual information that is being processed and integrated in the LPl with inputs from collicular sources.     

Spinally-mediated behavioural responses evoked by intrathecal high-dose morphine: possible involvement of substance P in the mouse spinal cord

Intrathecal (i.t.) administration of morphine in the spinal subarachnoid space of mice produced a severe hindlimb scratching followed by biting and licking. The onset of the scratching behaviour was observed 60–70 s after i.t. injection of morphine (60 and 90 nmol), and had a duration of 3–4 min. The morphine-induced behaviour was increased additively by i.t. co-administration of substance P (SP). This characteristic behavioural response was inhibited dose-dependently by i.t. co-administration of the tachykinin NK-1 receptor antagonists, sendide and CP-96,345. Significant antagonistic effects of SP (1–7), a putative antagonist for NK-1 receptors and [d-Phe⁷,d-His⁹jSP (6–11), a selective antagonist for SP receptors, were observed against the morphine-induced behaviour. Pretreatment with i.t. SP antiserum and i.t. capsaicin resulted in reduction of the response to morphine. I.t. administration of somatostatin (SOM) antiserum, cysteamine, a relatively selective depletor of SOM and cyclo-SOM, a SOM receptor antagonist, produced no inhibitory effect on the morphine-induced behaviour. These results demonstrate that a spinal system of neurones containing SP may be involved in elicitation of the behavioural episode following i.t. injection of morphine in mice.     

Effect of NMDA receptor antagonists on prostaglandin E2-induced hyperalgesia in conscious mice

Intrathecal (i.t.) injection of prostaglandin E₂ (PGE₂) to conscious mice produced a hyperalgesic action over a wide range of dosages with two apparent peaks at 100 pg and 10 ng per mouse, which may be mediated through EP₃ and EP₂ subtypes of the PGE receptor. In the present study, the effects of NMDA receptor antagonists on hyperalgesia induced by PGE₂ were evaluated by the hot plate test at 30 min after i.t. injection. Hyperalgesia induced by a higher dose of PGE₂ (10 ng/mouse) was relieved byd-AP5 (a competitive antagonist), 7-Cl-KynA (a glycine site antagonist), and ketamine and MK801 (non-competitive channel blockers). Intrathecal injection of butaprost (10 ng/mouse), an EP₂ agonist, induced hyperalgesia, and this hyperalgesia was blocked byd-AP5, 7-Cl-KynA, ketamine, and MK801, similar to that induced by 10 ng of PGE₂. On the other hand, hyperalgesia induced by a lower dose of PGE₂ (100 pg/mouse) was blocked byd-AP5 and 7-Cl-KynA, but not by ketamine and MK801. Intrathecal injection of sulprostone (100 pg/mouse), an EP₁ and EP₃ agonist, induced hyperalgesia, and this hyperalgesia was blocked byd-AP5 and 7-Cl-KynA, but not by ketamine and MK801, similar to that induced by 100 pg of PGE₂. These results first demonstrate that the NMDA receptor is involved in the PGE₂-induced hyperalgesia and suggest that the hyperalgesic action by lower and higher doses of PGE₂ may be mediated through EP₃ and EP₂ subtypes, respectively.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

The tachykinin NK1 receptor. Part I: Ligands and mechanisms of cellular activation

The tachykinin NK₁ receptor is widely expressed in the mammalian central and peripheral nervous system. Powerful pharmacological tools (agonists and antagonists) are now available to elucidate the physiological role of NK₁ receptors at these levels, as well as to understand their role in diseases and establish the possible therapeutic usefulness of NK₁ receptor antagonists for treatment of human diseases. The structure-activity studies that have led to the development of potent peptide and non-peptide ligands for the tachykinin NK₁ receptor are here reviewed. Among the peptide agonists and antagonists, linear and cyclic sequences have been developed. The non peptide antagonists belong to different chemical classes, i.e. steroids, perhydroisoindolones, quinuclidines, piperidines and tryptophane derivatives. The first non peptide antagonists for NK₁ receptors have been obtained by random screening of chemical compounds large collections. The resulting leads were optimized with ‘classic’ structure activity approaches, aiming at identifying ‘common’ motifs for interaction with the receptor by ligands of different chemical classes. The results derived from the recent application of molecular biology techniques were useful to drive the design of new ligands toward a precise structural definition of ligand-receptor bi-molecular interactions. Studies on mutant receptors have established that the sites of interaction of peptide agonists and non peptide antagonists with the tachykinin NK₁ receptor are largely non overlapping. Moreover, data obtained from mutagenesis of the NK₁ receptor further indicate that some amino acid residues in the NK₁ receptor sequence are critical for determining the binding affinity of some but not all ligands. Therefore, different antagonists discovered from random screening may not possess common points of interaction or common structural and conformational characteristics for their interaction with the tachykinin NK₁ receptor.The tachykinin NK₁ receptor couples with G-proteins to determine its biological effects in target cells. Several G-proteins both sensitive (Go, Gi) and insensitive (Gq, G11) to pertussis toxin can mediate the action of NK₁ receptors. Moreover, several second messanger signalling systems (elevation of intracellular calcium, stimulation of phosphoinositol turnover, arachidonic acid mobilization, cAMP accumulation) have to be activated following NK₁ receptor signalling. Also a direct modulation of certain ion channels at membrane level has been proposed. The NK₁ receptor undergoes prompt and significant tachyphylaxis upon exposure to the agonist: this has been shown to be linked with receptor internalization which also occurs physiologically when the NK₁ receptor is stimulated by endogenous tachykinins.     

In vivo characterization of the effects of human hemokinin-1 and human hemokinin-1(4-11), mammalian tachykinin peptides, on the modulation of pain in mice

Human hemokinin-1 (h HK-1) and its truncated form h HK-1(4-11) are mammalian tachykinin peptides encoded by the recently identified TAC4 gene in human, and the biological functions of these peptides have not been well investigated. In the present study, an attempt has been made to investigate the effects and mechanisms of action of h HK-1 and h HK-1(4-11) in pain modulation at the supraspinal level in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of h HK-1 (0.3, 1, 3 and 6 nmol/mouse) produced a dose- and time-related antinociceptive effect. This effect was significantly antagonized by the NK₁ receptor antagonist SR140333, but not by the NK₂ receptor antagonist SR48968, indicating that the analgesic effect induced by i.c.v. h HK-1 is mediated through the activation of NK₁ receptors. Interestingly, naloxone, β-funaltrexamine and naloxonazine, but not naltrindole and nor-binaltorphimine, could also block the analgesic effect markedly, suggesting that this effect is related to descending μ opioidergic neurons (primary μ₁ subtype). Human HK-1(4-11) could also induce a dose- and time-dependent analgesic effect after i.c.v. administration, however, the potency of analgesia was less than h HK-1. Surprisingly, SR140333 could not modify this analgesic effect, suggesting that this effect is not mediated through the NK₁ receptors like h HK-1. SR48968 could modestly enhance the analgesic effect induced by h HK-1(4-11), indicating that a small amount of h HK-1(4-11) may bind to NK₂ receptors. Furthermore, none of the opioid receptor (OR) antagonists could markedly block the analgesia of h HK-1(4-11), suggesting that the analgesic effect is not mediated through the descending opioidergic neurons. Blocking of δ ORs significantly enhanced the analgesia, indicating that δ OR is a negatively modulatory factor in the analgesic effect of h HK-1(4-11). It is striking that bicuculline (a competitive antagonist at GABA_A receptors) effectively blocked the analgesia induced by h HK-1(4-11), suggesting that this analgesic effect is mediated through the descending inhibitory GABAergic neurons. The novel mechanism involved in the analgesic effect of h HK-1(4-11), which is different from that of h HK-1, may pave the way for a new strategy for the investigation and control of pain.     

NK1 and NK2 receptors are similar in man and rabbit

A series of 15–18 compounds that act on NK₁ or NK₂ receptors as agonists or antagonists have been tested in the monoreceptor systems of the rabbit vena cava (NK₁) and the rabbit pulmonary artery (NK₂) for biological activities and for their ability to displace [³H] [Sar⁹, Met(O₂)¹¹]SP or [¹²⁵I] NKA respectively from NK₁ or NK₂ human binding sites obtained by transfection and functional expression of the cDNAs for these receptor subtypes in CHO-K1 cells. For the two tachykinin receptors studied, positive highly significant correlations have been shown between binding and biological assays. Slopes of correlations are linear and near unity (r = 0.918 and 0.938). For NK₁ and NK₂ receptors the pharmacology in human and rabbit tissues appears to be very similar. The assays of biological activity on rabbit tissues may be therefore used to complement binding studies on human transfected cells to identify new antagonists for human tachykinin receptors.     

Implication of a Nitric Oxide Synthase Mechanism in the Action of Substance P: L-NAME Blocks Thermal Hyperalgesia Induced by Endogenous and Exogenous Substance P in the Rat

The effects of i.p. administration of the nitric oxide synthase inhibitor N^G-nitro-L-arginine methylester (L-NAME) and its inactive isomer, D-NAME, were tested in two nociceptive paradigms in the rat. In the first paradigm, rats were lightly anaesthetized with a mixture of chloral hydrate (120 mg/kg, i.p.) and sodium pentobarbital (20 mg/ kg, i.p.). Tail flick reaction times were monitored and thermal hyperalgesia was induced by immersion of the tail in hot water at 55°C for 1.5 min. In the groups of rats pretreated with saline (n= 5), 100 mg/kg D-NAME (n= 6), 10 (n= 5) or 25 (n= 6) mg/kg L-NAME, this thermal injury induced a transient reduction in the reaction time that was 54–59% of the baseline value. However, in the groups of rats pretreated with 50 (n= 6) or 100 (n= 7) mg/kg L-NAME the reaction times were 73.9 ± 2.7% (P < 0.05) and 102.3 ± 0.9% (P < 0.001) of the baseline values respectively, indicating a block of the hyperalgesic responses seen in the other groups. As this hyperalgesia has been reported to be blocked by NK-1 receptor antagonists, it is suggested that it is due to the action of endogenous substance P. In the second paradigm, tail flick responses were monitored in the awake rat and thermal hyperalgesia was induced by intrathecal administration of substance P (6.5 nmol) via a chronically implanted catheter. In the group of rats pretreated with saline (n= 5) or D-NAME (n= 5; 100 mg/kg), substance P reduced the reaction time to 39.1 ± 9.9 and 45.5 ± 2.1% of the baseline value respectively. However, in the rats pretreated with L-NAME (n= 6; 100 mg/kg), the reaction time following substance P administration was 108.8 ± 8.8% of the baseline value (P < 0.001), indicating a block of the hyperalgesic response induced by substance P. These data indicate that thermal hyperalgesia induced by endogenously released or exogenously administered substance P, are blocked by L-NAME but not by its enantiomer, D-NAME. Therefore an involvement of a nitric oxide synthase mechanism in the hyperalgesic responses to substance P is suggested.     

Tachykinins cause inward current through NK1 receptors in bullfrog sensory neurons

The effects of tachykinins on primary afferent neurons of bullfrog dorsal root ganglia (DRG) were examined by using whole-cell patch-clamp methods. Neurokinin A (NKA) caused inward current (I_NKA) in a concentration-dependent manner. Concentration-response curve showed that the EC₅₀ for NKA was 6 nM. The I_NKA showed strong tachyphylaxis, when NKA was continuously applied for more than 1 min. Substance P (SP) also produced inward current with potency similar to that of NKA. Neurokinin B (NKB) was less effective in producing the inward current. The order of agonist potency was NKA = SP NKB. Spantide ([D-Arg¹, D-Trp^7,9, Leu¹¹]SP), non-selective peptide antagonist at tachykinin receptors, reduced the tachykinin-induced current. CP-99,994, a selective non-peptide antagonist for neurokinin-1 (NK₁) receptor, inhibited the inward currents produced by NKA and SP. The I_NKA was associated with decrease in K⁺ conductance. NKA suppressed both a voltage-dependent K⁺ current, the M-current (I_M), and a voltage-independent background K⁺ current, I_K(B). Intracellular dialysis with GTPγS (100 nM) or GDPβS (100 μM) depressed the I_NKA. Pre-treatment of DRG neurons with pertussis toxin (PTX) did not prevent the I_NKA. Depletion of intracellular ATP depressed the I_NKA. These results suggest that the tachykinin-induced inward current is mediated through the NK₁ receptor which mainly couples to PTX-insensitive G-protein in bullfrog primary afferent neurons.     

The glycine site of the NMDA receptor contributes to neurokinin1 receptor agonist facilitation of NMDA receptor agonist-evoked activity in rat dorsal horn neurons

We have investigated the role of the glycine recognition site of the N-methyl-d-aspartate receptor (the Gly_NMDA site) in the facilitation of NMDA receptor agonist-evoked activity in rat dorsal horn neurons that is brought about by neurokinin ₁ (NK₁) receptor agonist and the contribution of protein kinase C (PKC) activation to this phenomenon. Ionophoresis of the selective NMDA receptor agonist 1-aminocyclobutane-ci's-1,3-dicarboxylic acid (ACBD) produced a sustained increase in the firing rate of single laminae III-V neurons recorded extracellularly using multibarrelled glass electrodes. The highly selective NK₁ receptor agonist acetyl-[Arg⁶, Sar⁹, Met(O₂)¹¹]-SP_6–11 (Sar⁹-SP) greatly facilitated this response, but under the present conditions had no effect when applied alone or with a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA receptor agonist) at the same current. In the presence of the Gly_NMDA site antagonists 2-carboxy-4,6-dichloro-(1H)-indole-3-propanoic acid (MDL 29951), 7-chloro-3-(cyclopropylcarbonyl)-4-hydroxy-2(1H)-quinoline (L701,252), 5,7-dinitroquinaxoline-2,3-dione (MNQX) or 7-chlorothiokynurenic acid (7-CTK), or the PKC inhibitors, chelery-thrine or GF109203X, the Sar⁹-SP-induced facilitation of ACBD-evoked activity was prevented, generally restoring activity to a level similar to that in the presence of ACBD alone, whilst an AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo (f) quinoxaline-2,3-dione (NBQX) did not inhibit the facilitation. At the same ionophoretic currents these compounds had no effect on ACBD-evoked activity in the absence of Sar⁹-SP but were inhibitory at significantly greater currents. To further substantiate the importance of the Gly_NMDA site in the interaction, the effects of NMDA receptor antagonists selective for alternative recognition sites on the NMDA receptor were investigated. MK-801, a non-competitive NMDA receptor antagonist and arcaine, a competitive inhibitor at the polyamine site, were applied to the facilitated activity seen in the presence of Sar⁹-SP and ACBD, and to ACBD-evoked activity alone. Unlike the Gly_NMDA site antagonists and PKC inhibitors, these compounds reduced both facilitated and ACBD-evoked activity at similar currents. Furthermore, like the NK₁ receptor agonist, a selective Gly_NMDA site agonist 1-aminocyclopropane carboxylic acid (ACPC) caused facilitation of ACBD-evoked activity which was also blocked by currents of L701,252 that did not alter activity evoked by ACBD alone. These data suggest that activation of the Gly_NMDA site (perhaps as a consequence of glycine release or modification of its influence by intracellular signalling cascades) is an essential component of the means by which NK₁ receptor activation results in facilitated responsiveness of dorsal horn neurons towards NMDA receptor agonists.     

The role of nociceptin in cognition

The physiological role of the modulation via the nociceptin receptor is still unclear. Here we report the role of the nociceptin system in learning and memory. Nociceptin-knockout mice possess greater learning ability in the water maze task, show an enhanced latent learning in the water finding task, have better memory in the passive avoidance task, and further, show larger long-term potentiation in the hippocampal CA1 region than wild-type mice. Nociceptin itself induces an impairment of the passive avoidance task in wild-type mice. This impairment is reversed by naloxone benzoylhydrazone (NalBzoH), but not by other opioids in wild-type mice. Further, experiments on cultured cells transfected with nociceptin receptor cDNA show that NalBzoH competes [³H]-nociceptin binding and attenuates the nociceptin-induced inhibition of cAMP accumulation induced by forskolin. These results demonstrate that the nociceptin system seems to play a negative role in learning and memory and that NalBzoH acts as a potent antagonist for the nociceptin receptor. In addition, the antagonists for the nociceptin receptor may be worth testing for alleviating memory disorders.     

[H]substance P binding in the intermediolateral cell column and striatum of the rat

[³H]substance P binding was studied in the intermediolateral cell column and striatum in the rat using slide-mounted sections. The intermediolateral cell column had a single high affinity binding component with a dissociation constant,K_d = 1.45 nM and the number of sites,B_max= 18.1fmol per mg protein. The striatum had aK_d = 0.77nM and aB_max= 23.5fmol per mg protein. The relative potency of various substance P-like tachykinins in displacing [³H]substance P suggested that both these areas may contain a substance P-P (for physalaemin) receptor subtype. (d-Pro⁴,d-Trp^7,9)substance P(4–11), a substance P antagonist, has a relatively low affinity (micromolar range) in both these areas.     

Involvement of spinal neurokinins, excitatory amino acids, proinflammatory cytokines, nitric oxide and prostanoids in pain facilitation induced by Phoneutria nigriventer spider venom

The major local symptom of Phoneutria nigriventer envenomation is an intense pain, which can be controlled by infiltration with local anesthetics or by systemic treatment with opioid analgesics. Previous work showed that intraplantar (i.pl) injection of Phoneutria nigriventer venom in rats induces hyperalgesia, mediated peripherally by tachykinin and glutamate receptors. The present study examined the spinal mechanisms involved in pain-enhancing effect of this venom. Intraplantar injection of venom into rat hind paw induced hyperalgesia. This phenomenon was inhibited by intrathecal (i.t.) injection of tachykinin NK1 (GR 82334) or NK2 (GR 94800) receptor antagonists, a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP8-37) and N-methyl-D-aspartate (NMDA; MK 801 and AP-5), non-NMDA ionotropic (CNQX), or metabotropic (AIDA and MPEP) glutamate receptor antagonists, suggesting the involvement of spinal neurokinins and excitatory amino acids. The role of proinflammatory cytokines, nitric oxide (NO), and prostanoids in spinally mediated pain facilitation was also investigated. Pharmacological blockade of tumour necrosis factor-alpha (TNFalpha) or interleukin-1beta (IL-1beta) reduced the hyperalgesic response to venom. Intrathecal injection of L-N6-(1-iminoethyl)lysine (L-NIL), but not of 7-nitroindazole (7-NI), inhibited hyperalgesia induced by the venom, indicating that NO, generated by the activity of the inducible form of nitric oxide synthase, also mediates this phenomenon. Furthermore, indomethacin, an inhibitor of cyclooxigenases (COX), or celecoxib, a selective inhibitor of COX-2, abolished venom-induced hyperalgesia, suggesting the involvement of spinal prostanoids in this effect. These data indicate that the spinal mechanisms of pain facilitation induced by Phoneutria nigriventer venom involves a plethora of mediators that may cooperate in the genesis of venom-induced central sensitization.     

An NK1 receptor antagonist affects the circadian regulation of locomotor activity in golden hamsters

Substance P (SP) is a neuromodulator which may participate in the photic regulation of the circadian timing system in mammals. The biological effects of SP are mediated by interaction with specific receptors, designated as NK₁, NK₂, and NK₃. The NK₁ subtype receptor is expressed in the circadian system. Experiment 1 was designed to test whether an NK₁ antagonist mimics the effects of dark pulses. Hamsters were housed in constant lighting conditions, either constant darkness or constant light (around 250 lx), and they received an i.p. injection of either the specific NK₁ receptor antagonist, L-760,735 (5 mg/kg), or saline during the mid-subjective day, a time when dark pulses cause a phase-advance in circadian rhythm of locomotor activity. After treatment with the NK₁ antagonist, significant phase-advances of wheel-running activity rhythm were found in constant light, but not in constant darkness. Experiment 2 was designed to test the ability of the NK₁ antagonist to block the phase-delaying and/or the phase-advancing effects of light in animals kept in constant darkness. Phase-advances of locomotor activity rhythm that can normally be induced by light pulses given during the late subjective night were markedly reduced by pre-treatment with the NK₁ antagonist. By contrast, phase-delays that can be induced by lights pulses given during the early subjective night were unaffected by the NK₁ antagonist. These data support the hypothesis that SP within the circadian system may, by interacting with NK₁ receptors, modulate photic responses of the SCN pacemaker.     

Evidence for Modulation of DoDamine-neuronal Function by Tachykinin NK3 Receptor Stimulation in Gerbil Mesencephalic Cell Cultures

Primary cultures of gerbil mesencephalon were used for studying the modulation exerted by tachykinin NK₃ receptor activation on the activity of dopamine (DA) neurons. Dopamine neurons were identified by their ability to take up [³H]DA in a nomifensine-dependent manner. Moreover, tyrosine hydroxylase immunohistochemistry revealed that these neurons accounted for 5–7% of the total cell population. The NK₃ receptor agonists, senktide (EC₅₀= 0.58 nM) and [MePhe⁷]neurokinin B (EC₅₀= 3 nM), increased spontaneous [³H]DA release in a concentration-dependent manner. In contrast, tested at a supramaximal concentration (10-⁷ M), neither septide nor substance P were found to affect [³H]DA release. The senktide-evoked [³H]DA release was not observed when extracellular Ca²⁺ was chelated, but was unaffected by nomifensine. This indicates that this increase in [³H]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 NK₃ receptor antagonist, SR 142801, shifted or blocked (ICs0 = 0.89 nM) the senktide-evoked [³H]DA release, while its (-)-antipode, SR 142806, was 80-fold less potent, in agreement with binding data. Selective antagonists for NK, (SR 140333) or NK₂ (SR 48968) receptors failed to reduce the senktide effect. Light scanning microscopic analysis of mesencephalic cells loaded with the Ca²⁺ sensitive dye, fluo-3, showed that senktide induced a rise in cytosolic Ca²⁺ in 8-10% of the cell population. The senktide-induced elevation in intracellular Ca²⁺ was rapid in onset and transient (at lo⁴ 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 Ca²⁺ 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 l-methyl-4-phenylpyridinium. The present study shows that enhancement of spontaneous [³H]DA release and intracellular Ca²⁺ mobilization may be observed after NK₃ receptor stimulation and that both biochemical events are likely to occur in DA neurons.     

Effects of a tachykinin NK3 receptor antagonist, SR 142801, studied in isolated neonatal rat spinal cord

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.