The Journal of pharmacology and experimental therapeutics, 1996
The present experiments explored the role of endogenous opioids in the behavioral response to a f... more The present experiments explored the role of endogenous opioids in the behavioral response to a formalin-induced nociceptive stimulus in the rat. Flinching was taken as a measure of the intensity of the nociceptive stimulus after the administration of formalin into the dorsal surface of the paw of control animals, or in animals receiving i.p. administration of receptor-selective doses of opioid antagonists including naloxone, naltrindole (delta opioid antagonist), nor-binaltorphimine (kappa opioid antagonist) or beta-funaltrexamine (mu opioid antagonist). Additionally, antisera to [Leu5]enkephalin, [Met5]enkephalin and dynorphin A (1-13) (dynorphin) were administered intrathecally before formalin to explore the contribution of endogenous opioids in modulation of the flinching response. Formalin-induced flinching was increased significantly by naloxone, and receptor selective doses of naltrindole and nor-binaltorphimine, but not beta-funaltrexamine. Additionally, antisera to [Leu5]en...
The TPA (12-O-tetradecanoyl-phorbol-13-acetate) responsive element (TRE) is recognized by the ind... more The TPA (12-O-tetradecanoyl-phorbol-13-acetate) responsive element (TRE) is recognized by the inducible transcription factor AP1, a heterodimeric complex of Fos- and Jun-protein subunits, which each contain a specific structure known as the leucine zipper through which they interact. Studies using site-directed mutagenesis have shown that a basic region adjacent to the leucine zipper in Fos is crucial for the interaction of the Fos-Jun complex with the TRE, and probably represents a site of interaction with DNA. The functionally crucial amino acids in this region are almost completely conserved between Fos and Jun (refs 6, 7 and 11; M.N. and R.M., unpublished results), indicating the formation of a nearly symmetrical DNA-binding site in the Fos-Jun complex. Whereas Jun can form a homodimeric protein complex which binds to the TRE, Fos is unable to do so. The Fos-Jun heterodimer, however, possesses at least a 30-fold-higher affinity for the TRE than does the Jun-Jun homodimer, indica...
This paper outlines the synthesis of selected acid mimics of the non-peptide CCK-B selective anta... more This paper outlines the synthesis of selected acid mimics of the non-peptide CCK-B selective antagonist CI-988, 1. CCK-B and CCK-A binding affinities of these analogues are described and their CCK-B affinity and selectivity rationalized by consideration of the pK(a) values, charge distribution, and geometry of the respective acid mimics. Several of the compounds have CCK-B binding affinities similar to the parent carboxylic acid 1 (CCK-B, IC50 = 1.7 nM; pK(a) = 5.6) and span a pK(a) range of less than 1 (sulfonic acid 27) to greater than 9.5 (5-thio-1,2,4-triazole 24). Among the more active compounds synthesized are tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[2-[[2-[[(3-hydroxy-5-isoxazolyl)acetyl]-amino]-2- phenylethyl]amino]-1-(1H-indol-3-ylmethyl)-1-methyl-2-oxoethyl+ ++]carbamate (15), tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[1-(1H-indol-3-ylmethyl)-1-methyl-2-oxo-2-[[2-[(1-oxo- 3-sulfopropyl)amino]-2-phenylethyl]amino]-ethyl]carbamate, monosodium salt (27), and tricyclo[3.3....
Intracellular calcium concentration ([Ca2+]i) dynamics were simultaneously monitored in multiple ... more Intracellular calcium concentration ([Ca2+]i) dynamics were simultaneously monitored in multiple cultured rat neurons loaded with Fluo-3 and continuously stimulated with glutamate (GLU). Three response types were observed: 10 microM GLU caused an initial transient increase in [Ca2+]i; 20 microM a biphasic response characterized by a 150-350 s 'calcium trough' between peaks; and 40 microM an initial sustained increase in [Ca2+]i. Neurons in calcium-free medium treated with 40 microM GLU showed only an initial transient increase in [Ca2+]i, demonstrating the dependence of sustained secondary increases in [Ca2+]i on extracellular calcium sources. We observed synchronized responses of multiple neurons within a given culture well, after GLU treatment, supporting the hypothesis that sustained influx of extracellular calcium may be stimulated by depletion of intracellular calcium and/or the release of endogenous excitatory amino acids.
Ion channels form a diverse and sophisticated collection of membrane-bound proteins. They are inf... more Ion channels form a diverse and sophisticated collection of membrane-bound proteins. They are influenced by many endogenous compounds and physiological stimuli and modulate neuronal activity. It is thus unsurprising that they provide attractive targets for the design of novel therapeutics. In this article, recent ion channel research and its relevance to modulation of sensory transmission is assessed. In pain research, specific blockade or activation of ion channels has long been considered a desired route for identification of analgesics. Historically, this has proven difficult to attain due to the incidence of side-effects associated with most ion-channel modulators. The recent discovery of several novel classes of ion channels, each of which has a specific distribution and role in sensory processing and nociception, has provided a plethora of targets for pharmaceutical intervention with the promise of an improved therapeutic index.
P 2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and ... more P 2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and following peripheral nerve injury using immunohistochemical methods. Specific antiserum was raised in rabbits and characterized by Western blot, absorption assays and labeling of recombinant receptors. In naive animals, P 2X3 immunoreactivity was present predominantly in a subpopulation of small-diameter sensory neurons in dorsal root ganglia. In the spinal cord, immunoreactivity was observed in the superficial laminae of the dorsal horn. Following a chronic constriction injury to the sciatic nerve, the number of P 2X3 positive small and medium diameter neurons increased in dorsal root ganglia when compared with sham-operated animals. In addition, the spinal cord immunoreactivity increased in magnitude on the side ipsilateral to the ligated nerve, consistent with up-regulation of receptors in presynaptic terminals of the primary sensory neurons.
The novel radioligand [3H]PD140376 was used to label receptors that bind cholecystokinin (CCK) an... more The novel radioligand [3H]PD140376 was used to label receptors that bind cholecystokinin (CCK) and related peptides in membranes prepared from guinea-pig brain and gastric glands. Under control conditions, measurements of the apparent affinity of 11 agonist and 16 antagonist ligands in both tis,;ues revealed a strong positive relationship between the affinity of a compound in either tissue (slope of the regression line = 0.89, r 2 = 0.908). Agonists consistently showed higher affinity for sites in gastric glands compared to brain. If agonists were excluded from the analysis, the degree of correspondence between affinities measured in each tissue was almost perfect (slope = 0.93, r 2 = 0.986). I:a the presence of the guanyl nucleotide 5'-guanylimidodiphosphate (GppNHp), agonist affinity in gastric glands, but not brain, was reduced such that there was a direct relationship between binding affinity in each tissue. These data are consistent with the notion that the receptor sites in brain and gastric glands, which recognise CCK and gastrin related compounds, are the same and of the CCK-B/gastrin subtype. The receptors in the two respective tissues, however, do appear to differ in the degree of post-receptor coupling. These findings may explain previously reported differences between gastrin and CCK-B receptors that were based upon binding studies using agonist ligands. 8s, which are thought to represent the receptors through which these peptides exert their influence, are distributed throughout the brain and the gastrointestinal tract . These receptor binding sites have been differentiated into subtypes on the basis of their affinity for a range of related peptide agonists and non-peptide antagonists. In the case of receptor binding sites for which the endogenous ligand can be considered as being CCK-8s, there are two main subtypes; CCK-A and CCK-B sites . CCK-A receptors discriminate well between sulphated and unsulphated forms of CCK octapeptide and bind the non-peptide antagonist devazepide (L-364,718, MK 329) with picomolar affinity, whilst displaying much lower binding affinity for the peptoid antagonist CI-988 and the benzodiazepine L-365,260. CCK-A receptors are found in high concentrations in the pancreas, gall bladder and also in discrete regions of the brain . CCK-B receptors are ubiquitous throughout the brain and may be defined by 0167-0115/96/$15.00
The possible role of spinal prostanoids in the tactile allodynia and thermal hyperalgesia associa... more The possible role of spinal prostanoids in the tactile allodynia and thermal hyperalgesia associated with an experimental model of neuropathic pain was investigated. Neuropathic pain was induced by tight ligation of the L5 and L6 spinal nerves. Tactile allodynia was assessed 7 days after the surgery by measuring hindpaw withdrawal threshold to probing with von Frey filaments. Thermal hyperalgesia and nociception were determined by the 52°C warm-water tail-flick test and by applying radiant heat to the plantar aspect of the hindpaw ipsilateral to the ligation. Minimal antiallodynic effect was produced by intrathecal (i.th.) administration of ketorolac or morphine up to the highest testable dose (100 mg) or by the (R)-or (S)-enantiomers of ketorolac (up to 6 mg) when administered alone. However, i.th. administration of a fixed ratio (1:1) of morphine plus racemic ketorolac or of morphine plus the (S)-enantiomer of ketorolac (S-ketorolac) produced a dose-and time-related antiallodynic effect: ED 50 114 ± 35.9 mg (total dose) for morphine plus ketorolac and 70.5 ± 21.0 mg (total dose) for morphine plus S-ketorolac. The combination of i.th. morphine plus the (R)-enantiomer of ketorolac (R-ketorolac) (up to 200 mg total dose) was without effect. Similar antiallodynic activity was obtained for the co-administration of i.th. morphine and intravenous (i.v.) racemic ketorolac. In order to investigate the role of cyclooxygenase (COX) isozymes, relatively selective COX 1 (piroxicam) and COX 2 N- [2-cyclohexyloxy-4-nitrophenyl] metanesulfonamide (NS-398) inhibitors were administered i.th. (60 mg) alone or together with i.th. morphine. Piroxicam, NS-398, morphine and vehicle (90% DMSO) were without significant antiallodynic effect when administered alone, but moderate antiallodynic effects were produced by i.th. administration of fixed ratio (1:1) combinations of morphine with 60 mg each (highest soluble dose) of piroxicam (%MPE = 40.8 ± 10.2) or NS-398 (%MPE = 32.4 ± 9.5). Further, the combined i.th. administration of morphine, piroxicam and NS-398 in fixed 1:1:1 ratio (60 mg each) resulted in a supraadditive antiallodynic effect (%MPE = 70.4 ± 10.8). Finally, morphine, but not ketorolac, given i.th. produced dose-dependent anti nociception in either the tailflick or the paw-flick tests. However, there was no synergy between morphine and ketorolac against thermal nociception in either of the tests. These findings suggest that spinal prostanoids produced via both COX
Prostanoid receptor-mediated sensitization of sensory nerve fibres is a key contributor to the ge... more Prostanoid receptor-mediated sensitization of sensory nerve fibres is a key contributor to the generation of hyperalgesia. It is generally thought that prostaglandin (PG) E 2 is the principal pro-inflammatory prostanoid. Consequently, prostanoid EP receptors on sensory neurones have been identified as potential therapeutic targets. However, IP prostanoid receptors are also present on sensory neurones, and recent data from transgenic mice lacking the IP receptor demonstrate its importance in the induction of oedema and pain behaviour. PGI 2 , the primary endogenous agonist for the IP receptor, is rapidly produced following tissue injury or inflammation; thus, it may be of equal, or greater, importance than PGE 2 during episodes of inflammatory pain. In this review, Keith Bley, John Hunter, Richard Eglen and Jacqueline Smith compare the roles of EP and IP receptors in nociception and suggest that the IP receptor constitutes a novel target for anti-nociceptive agents. Chemical names AH6809: 7-isopropoxy-9-oxoxanthene-2-carboxylic acid SC19220: 8-chlorodibenx[B,F][1,4]oxazepine-10(11H)carboxylic acid, 2-acetylhydrazide
This report describes the activity of the antiepileptic agent gabapentin (Neurontin) in animal mo... more This report describes the activity of the antiepileptic agent gabapentin (Neurontin) in animal models predictive of anxiolysis and analgesia. Gabapentin displayed anxiolytic-like action in the rat conflict test, the mouse light/dark box and the rat elevated X-maze with respective minimum effective doses (MEDs) of 3, 10 and 30 mg/kg. Furthermore, gabapentin also induced behavioural changes suggestive of anxiolysis in the marmoset human threat test with a MED of 30 mg/kg. In the rat formalin test of tonic nociception, gabapentin dose-dependently (30-300 mg/kg) and selectively blocked the late phase with a MED of 100 mg/kg. However, it failed to block carrageenan-induced paw oedema. The intracerebroventricular (ICV) administration of the glycine/NMDA receptor agonist D-Serine, dose-dependently (10-100 micrograms/animal) reversed the antinociceptive action of gabapentin (200 mg/kg, SC). D-Serine (30 micrograms/animal, ICV) also reversed the anxiolytic-like effects (in the light/dark box and the rat elevated X-maze) of gabapentin (30 mg/kg). In contrast, L-Serine (100 micrograms, ICV) failed to block the antinociceptive action of gabapentin. The antinociceptive action of (+)-HA-966 (25 mg/kg, SC), a partial agonist at the glycine/NMDA receptor, was reversed by D-Serine (100 micrograms/animal, ICV). However, D-Serine (100 micrograms/animal, ICV) failed to affect the antinociceptive action of a competitive NMDA receptor antagonist CGS 19755 (3 mg/kg, SC). Gabapentin has negligible affinity for the strychnine insensitive [3H]glycine binding site. This indicates that the interaction between gabapentin and D-Serine may not involve the NMDA receptor complex. Gabapentin may represent a novel type of anxiolytic and analgesic agent.
Adenosine and dopamine interact within the striatum to control striatopallidal output and globus ... more Adenosine and dopamine interact within the striatum to control striatopallidal output and globus pallidus GABA release. Manipulating striatal adenosine transmission via blockade of the A2A receptor subtype can compensate for the reduced dopamine activity within the striatum that underlies movement disorders such as antipsychotic-induced extrapyramidal syndrome (EPS) and Parkinson's disease (PD). Preclinical studies in the rat have demonstrated that adenosine A2A receptor antagonists can attenuate behaviors reflecting reduced dopamine activity, such as haloperidol-induced catalepsy and hypoactivity. In the present studies using nonhuman primates, adenosine antagonists were tested against haloperidol-induced EPS in Cebus apella and haloperidol-induced catalepsy in Saimiri sciureus (squirrel monkey). Specifically, the A2A receptor antagonists, SCH 412348 (0.3-30 mg/kg PO) and KW-6002 (3-100 mg/kg PO); the A1/A2A receptor antagonist, caffeine (1-30 mg/kg PO and IM); and the A1 receptor antagonist, DPCPX (3-30 mg/kg PO) were tested in at least one of these models. SCH 412348 (10-30 mg/kg), KW-6002 (57-100 mg/kg), and caffeine (30 mg/kg) significantly increased the time to EPS onset. Additionally, SCH 412348, KW-6002, and caffeine afforded protection from the onset of EPS for at least 6 h in some of the primates. SCH 412348 (10 mg/kg) and caffeine (10 mg/kg) significantly reduced haloperidol-induced catalepsy. DPCPX produced a very slight attenuation of EPS at 30 mg/kg, but had no effect on catalepsy. These findings suggest that adenosine A2A receptor antagonists may represent an effective treatment for the motor impairments associated with both antipsychotic-induced EPS and PD.
Akathisia is a subset of the larger antipsychotic side effect profile known as extrapyramidal syn... more Akathisia is a subset of the larger antipsychotic side effect profile known as extrapyramidal syndrome (EPS). It is associated with antipsychotic treatment and is characterized as a feeling of inner restlessness that results in a compulsion to move. There are currently no primate models available to assess drug-induced akathisia; the present research was designed to address this shortcoming. We developed a novel rating scale based on both the Barnes Akathisia Rating Scale (BARS) and the Hillside Akathisia Scale (HAS) to measure the objective, observable incidence of antipsychotic-induced akathisia-like behavior in Cebus apella non-human primates (NHPs). To induce akathisia, we administered the atypical antipsychotic aripiprazole (1 mg/kg) or the selective phosphodiesterase 10A (PDE10A) inhibitor MP-10 (1-3 mg/kg). Treatment with both compounds produced significantly greater akathisia scores on the rating scale than vehicle treatment. Characteristic behaviors observed included vocalizations, stereotypies, teeth grinding, restless limb movements, and hyperlocomotion. Adenosine A 2A receptor antagonists have previously been shown to be effective in blocking antipsychotic-induced EPS in primates. The selective A 2A receptor antagonist, SCH 412348 (10-30 mg/kg), effectively reduced or reversed akathisialike behavior induced by both aripiprazole and MP-10. This work represents the first NHP measurement scale of akathisia and demonstrates that NHPs are responsive to akathisia-inducing agents. As such, it provides a useful tool for the preclinical assessment of putative antipsychotics. In addition, these results provide further evidence of the utility of A 2A receptor antagonists for the treatment of antipsychotic-induced movement disorders.
Neuropathic pain is a debilitating chronic syndrome that often arises from injuries to peripheral... more Neuropathic pain is a debilitating chronic syndrome that often arises from injuries to peripheral nerves. Such pain has been hypothesized to be the result of an aberrant expression and function of sodium channels at the site of injury. Here, we show that intrathecal administration of specific antisense oligodeoxynucleotides (ODN) to the peripheral tetrodotoxin (TTX)-resistant sodium channel, NaV1.8, resulted in a timedependent uptake of the ODN by dorsal root ganglion (DRG) neurons, a selective 'knock-down' of the expression of NaV1.8, and a reduction in the slow-inactivating, TTX-resistant sodium current in the DRG cells. The ODN treatment also reversed neuropathic pain induced by spinal nerve injury, without affecting non-noxious sensation or response to acute pain. These data provide direct evidence linking NaV1.8 to neuropathic pain. As NaV1.8 expression is restricted to sensory neurons, this channel offers a highly specific and effective molecular target for the treatment of neuropathic pain.
This study investigated the effects of brain ischemia on sodium channel gene (NaCh) expression in... more This study investigated the effects of brain ischemia on sodium channel gene (NaCh) expression in rats. Using quantitative RT-PCR, our findings demonstrated the expression ratio of NaCh genes in normal rat brain to be Nay1.1 > Nay1.8 > Nay1.3 > Nay1.7 (rBI > PN3 > rBIII > PN1). In contrast, brain injury caused by middle cerebral artery occlusion (MCAo) for 2 h followed by reperfusion significantly down-regulated Nay1.3 and Nay1.7 genes in both injured and contralateral hemispheres; whereas the Na~l.8 gene was down regulated in only the injured hemisphere (though only acutely at 2 or 2-6h post-MCAo). However, the time-course of NaCh gene expression revealed a significant down-regulation of Na~l.1 only in the ischemic hemisphere beginning 6 h post-MCAo and measured out to 48 h post-MCAo. In a separate preliminary study Nay1.2 (rBII) gene was found be expressed at levels greater than that of Nay1.1 in normal rats and was significantly down regulated at 24 h post-MCAo. Our findings document, for the first time, quantitative and relative changes in the expression of various NaCh genes following ischemic brain injury and suggest that the Na~l.1 sodium channel gene may play a key role in ischemic injury/recovery.
The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and... more The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and two related compounds (mexiletine and QX-314), in in vitro models of neurotoxicity. Neurotoxicity was produced in primary cerebellar cultures using hypoxia/hypoglycemia (H/H), veratridine or glutamate where, in vehicle-treated neurons, 65%, 60% and 75% neuronal injury was measured, respectively. Dose-response neuroprotection experiments were carried out using concentrations ranging from 0.1-500 ~tM. All the sodium channel blockers were neuroprotective against H/H-induced injury, with each exhibiting similar potency and efficacy. However, against veratridine-induced neuronal injury only RS-100642-198 and mexiletine were 100% protective, whereas QX-314 neuroprotection was limited (i.e. only 54%). In contrast, RS 100642-198 and mexiletine had no effect against glutamate-induced injury, whereas QX-314 produced a consistent, but very limited (i.e. 25%), neuroprotection. Measurements of intraneuronal calcium 2+ .. 9 ([Ca ]i) moblhzatlon revealed that glutamate caused immediate and sustained increases in [Ca2+]i which were not affected by RS-100642-198 or mexiletine. However, both drugs decreased the initial amplitude and attenuated the sustained rise in [Ca2+]i mobilization produced by veratridine or KC1 depolarization. QX-314 produced similar effects on glutamate-, veratridine-or KCl-induced [Ca2+]i dynamics, effectively decreasing the amplitude and delaying the initial spike in [Ca2+]i, and attenuating the sustained increase in [Ca2+]i mobilization. By using different in vitro models of excitotoxicity, a heterogeneous profile of neuroprotective effects resulting from sodium channel blockade has been described for RS-100642-198 and related drugs, suggesting that selective blockade of neuronal sodium channels in pathological conditions may provide therapeutic neuroprotection against depolarization/excitotoxicity via inhibition of voltage-dependent Na + channels.
The Journal of pharmacology and experimental therapeutics, 1996
The present experiments explored the role of endogenous opioids in the behavioral response to a f... more The present experiments explored the role of endogenous opioids in the behavioral response to a formalin-induced nociceptive stimulus in the rat. Flinching was taken as a measure of the intensity of the nociceptive stimulus after the administration of formalin into the dorsal surface of the paw of control animals, or in animals receiving i.p. administration of receptor-selective doses of opioid antagonists including naloxone, naltrindole (delta opioid antagonist), nor-binaltorphimine (kappa opioid antagonist) or beta-funaltrexamine (mu opioid antagonist). Additionally, antisera to [Leu5]enkephalin, [Met5]enkephalin and dynorphin A (1-13) (dynorphin) were administered intrathecally before formalin to explore the contribution of endogenous opioids in modulation of the flinching response. Formalin-induced flinching was increased significantly by naloxone, and receptor selective doses of naltrindole and nor-binaltorphimine, but not beta-funaltrexamine. Additionally, antisera to [Leu5]en...
The TPA (12-O-tetradecanoyl-phorbol-13-acetate) responsive element (TRE) is recognized by the ind... more The TPA (12-O-tetradecanoyl-phorbol-13-acetate) responsive element (TRE) is recognized by the inducible transcription factor AP1, a heterodimeric complex of Fos- and Jun-protein subunits, which each contain a specific structure known as the leucine zipper through which they interact. Studies using site-directed mutagenesis have shown that a basic region adjacent to the leucine zipper in Fos is crucial for the interaction of the Fos-Jun complex with the TRE, and probably represents a site of interaction with DNA. The functionally crucial amino acids in this region are almost completely conserved between Fos and Jun (refs 6, 7 and 11; M.N. and R.M., unpublished results), indicating the formation of a nearly symmetrical DNA-binding site in the Fos-Jun complex. Whereas Jun can form a homodimeric protein complex which binds to the TRE, Fos is unable to do so. The Fos-Jun heterodimer, however, possesses at least a 30-fold-higher affinity for the TRE than does the Jun-Jun homodimer, indica...
This paper outlines the synthesis of selected acid mimics of the non-peptide CCK-B selective anta... more This paper outlines the synthesis of selected acid mimics of the non-peptide CCK-B selective antagonist CI-988, 1. CCK-B and CCK-A binding affinities of these analogues are described and their CCK-B affinity and selectivity rationalized by consideration of the pK(a) values, charge distribution, and geometry of the respective acid mimics. Several of the compounds have CCK-B binding affinities similar to the parent carboxylic acid 1 (CCK-B, IC50 = 1.7 nM; pK(a) = 5.6) and span a pK(a) range of less than 1 (sulfonic acid 27) to greater than 9.5 (5-thio-1,2,4-triazole 24). Among the more active compounds synthesized are tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[2-[[2-[[(3-hydroxy-5-isoxazolyl)acetyl]-amino]-2- phenylethyl]amino]-1-(1H-indol-3-ylmethyl)-1-methyl-2-oxoethyl+ ++]carbamate (15), tricyclo[3.3.1.1(3,7)]dec-2-yl [R-(R*,R*)]-[1-(1H-indol-3-ylmethyl)-1-methyl-2-oxo-2-[[2-[(1-oxo- 3-sulfopropyl)amino]-2-phenylethyl]amino]-ethyl]carbamate, monosodium salt (27), and tricyclo[3.3....
Intracellular calcium concentration ([Ca2+]i) dynamics were simultaneously monitored in multiple ... more Intracellular calcium concentration ([Ca2+]i) dynamics were simultaneously monitored in multiple cultured rat neurons loaded with Fluo-3 and continuously stimulated with glutamate (GLU). Three response types were observed: 10 microM GLU caused an initial transient increase in [Ca2+]i; 20 microM a biphasic response characterized by a 150-350 s 'calcium trough' between peaks; and 40 microM an initial sustained increase in [Ca2+]i. Neurons in calcium-free medium treated with 40 microM GLU showed only an initial transient increase in [Ca2+]i, demonstrating the dependence of sustained secondary increases in [Ca2+]i on extracellular calcium sources. We observed synchronized responses of multiple neurons within a given culture well, after GLU treatment, supporting the hypothesis that sustained influx of extracellular calcium may be stimulated by depletion of intracellular calcium and/or the release of endogenous excitatory amino acids.
Ion channels form a diverse and sophisticated collection of membrane-bound proteins. They are inf... more Ion channels form a diverse and sophisticated collection of membrane-bound proteins. They are influenced by many endogenous compounds and physiological stimuli and modulate neuronal activity. It is thus unsurprising that they provide attractive targets for the design of novel therapeutics. In this article, recent ion channel research and its relevance to modulation of sensory transmission is assessed. In pain research, specific blockade or activation of ion channels has long been considered a desired route for identification of analgesics. Historically, this has proven difficult to attain due to the incidence of side-effects associated with most ion-channel modulators. The recent discovery of several novel classes of ion channels, each of which has a specific distribution and role in sensory processing and nociception, has provided a plethora of targets for pharmaceutical intervention with the promise of an improved therapeutic index.
P 2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and ... more P 2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and following peripheral nerve injury using immunohistochemical methods. Specific antiserum was raised in rabbits and characterized by Western blot, absorption assays and labeling of recombinant receptors. In naive animals, P 2X3 immunoreactivity was present predominantly in a subpopulation of small-diameter sensory neurons in dorsal root ganglia. In the spinal cord, immunoreactivity was observed in the superficial laminae of the dorsal horn. Following a chronic constriction injury to the sciatic nerve, the number of P 2X3 positive small and medium diameter neurons increased in dorsal root ganglia when compared with sham-operated animals. In addition, the spinal cord immunoreactivity increased in magnitude on the side ipsilateral to the ligated nerve, consistent with up-regulation of receptors in presynaptic terminals of the primary sensory neurons.
The novel radioligand [3H]PD140376 was used to label receptors that bind cholecystokinin (CCK) an... more The novel radioligand [3H]PD140376 was used to label receptors that bind cholecystokinin (CCK) and related peptides in membranes prepared from guinea-pig brain and gastric glands. Under control conditions, measurements of the apparent affinity of 11 agonist and 16 antagonist ligands in both tis,;ues revealed a strong positive relationship between the affinity of a compound in either tissue (slope of the regression line = 0.89, r 2 = 0.908). Agonists consistently showed higher affinity for sites in gastric glands compared to brain. If agonists were excluded from the analysis, the degree of correspondence between affinities measured in each tissue was almost perfect (slope = 0.93, r 2 = 0.986). I:a the presence of the guanyl nucleotide 5'-guanylimidodiphosphate (GppNHp), agonist affinity in gastric glands, but not brain, was reduced such that there was a direct relationship between binding affinity in each tissue. These data are consistent with the notion that the receptor sites in brain and gastric glands, which recognise CCK and gastrin related compounds, are the same and of the CCK-B/gastrin subtype. The receptors in the two respective tissues, however, do appear to differ in the degree of post-receptor coupling. These findings may explain previously reported differences between gastrin and CCK-B receptors that were based upon binding studies using agonist ligands. 8s, which are thought to represent the receptors through which these peptides exert their influence, are distributed throughout the brain and the gastrointestinal tract . These receptor binding sites have been differentiated into subtypes on the basis of their affinity for a range of related peptide agonists and non-peptide antagonists. In the case of receptor binding sites for which the endogenous ligand can be considered as being CCK-8s, there are two main subtypes; CCK-A and CCK-B sites . CCK-A receptors discriminate well between sulphated and unsulphated forms of CCK octapeptide and bind the non-peptide antagonist devazepide (L-364,718, MK 329) with picomolar affinity, whilst displaying much lower binding affinity for the peptoid antagonist CI-988 and the benzodiazepine L-365,260. CCK-A receptors are found in high concentrations in the pancreas, gall bladder and also in discrete regions of the brain . CCK-B receptors are ubiquitous throughout the brain and may be defined by 0167-0115/96/$15.00
The possible role of spinal prostanoids in the tactile allodynia and thermal hyperalgesia associa... more The possible role of spinal prostanoids in the tactile allodynia and thermal hyperalgesia associated with an experimental model of neuropathic pain was investigated. Neuropathic pain was induced by tight ligation of the L5 and L6 spinal nerves. Tactile allodynia was assessed 7 days after the surgery by measuring hindpaw withdrawal threshold to probing with von Frey filaments. Thermal hyperalgesia and nociception were determined by the 52°C warm-water tail-flick test and by applying radiant heat to the plantar aspect of the hindpaw ipsilateral to the ligation. Minimal antiallodynic effect was produced by intrathecal (i.th.) administration of ketorolac or morphine up to the highest testable dose (100 mg) or by the (R)-or (S)-enantiomers of ketorolac (up to 6 mg) when administered alone. However, i.th. administration of a fixed ratio (1:1) of morphine plus racemic ketorolac or of morphine plus the (S)-enantiomer of ketorolac (S-ketorolac) produced a dose-and time-related antiallodynic effect: ED 50 114 ± 35.9 mg (total dose) for morphine plus ketorolac and 70.5 ± 21.0 mg (total dose) for morphine plus S-ketorolac. The combination of i.th. morphine plus the (R)-enantiomer of ketorolac (R-ketorolac) (up to 200 mg total dose) was without effect. Similar antiallodynic activity was obtained for the co-administration of i.th. morphine and intravenous (i.v.) racemic ketorolac. In order to investigate the role of cyclooxygenase (COX) isozymes, relatively selective COX 1 (piroxicam) and COX 2 N- [2-cyclohexyloxy-4-nitrophenyl] metanesulfonamide (NS-398) inhibitors were administered i.th. (60 mg) alone or together with i.th. morphine. Piroxicam, NS-398, morphine and vehicle (90% DMSO) were without significant antiallodynic effect when administered alone, but moderate antiallodynic effects were produced by i.th. administration of fixed ratio (1:1) combinations of morphine with 60 mg each (highest soluble dose) of piroxicam (%MPE = 40.8 ± 10.2) or NS-398 (%MPE = 32.4 ± 9.5). Further, the combined i.th. administration of morphine, piroxicam and NS-398 in fixed 1:1:1 ratio (60 mg each) resulted in a supraadditive antiallodynic effect (%MPE = 70.4 ± 10.8). Finally, morphine, but not ketorolac, given i.th. produced dose-dependent anti nociception in either the tailflick or the paw-flick tests. However, there was no synergy between morphine and ketorolac against thermal nociception in either of the tests. These findings suggest that spinal prostanoids produced via both COX
Prostanoid receptor-mediated sensitization of sensory nerve fibres is a key contributor to the ge... more Prostanoid receptor-mediated sensitization of sensory nerve fibres is a key contributor to the generation of hyperalgesia. It is generally thought that prostaglandin (PG) E 2 is the principal pro-inflammatory prostanoid. Consequently, prostanoid EP receptors on sensory neurones have been identified as potential therapeutic targets. However, IP prostanoid receptors are also present on sensory neurones, and recent data from transgenic mice lacking the IP receptor demonstrate its importance in the induction of oedema and pain behaviour. PGI 2 , the primary endogenous agonist for the IP receptor, is rapidly produced following tissue injury or inflammation; thus, it may be of equal, or greater, importance than PGE 2 during episodes of inflammatory pain. In this review, Keith Bley, John Hunter, Richard Eglen and Jacqueline Smith compare the roles of EP and IP receptors in nociception and suggest that the IP receptor constitutes a novel target for anti-nociceptive agents. Chemical names AH6809: 7-isopropoxy-9-oxoxanthene-2-carboxylic acid SC19220: 8-chlorodibenx[B,F][1,4]oxazepine-10(11H)carboxylic acid, 2-acetylhydrazide
This report describes the activity of the antiepileptic agent gabapentin (Neurontin) in animal mo... more This report describes the activity of the antiepileptic agent gabapentin (Neurontin) in animal models predictive of anxiolysis and analgesia. Gabapentin displayed anxiolytic-like action in the rat conflict test, the mouse light/dark box and the rat elevated X-maze with respective minimum effective doses (MEDs) of 3, 10 and 30 mg/kg. Furthermore, gabapentin also induced behavioural changes suggestive of anxiolysis in the marmoset human threat test with a MED of 30 mg/kg. In the rat formalin test of tonic nociception, gabapentin dose-dependently (30-300 mg/kg) and selectively blocked the late phase with a MED of 100 mg/kg. However, it failed to block carrageenan-induced paw oedema. The intracerebroventricular (ICV) administration of the glycine/NMDA receptor agonist D-Serine, dose-dependently (10-100 micrograms/animal) reversed the antinociceptive action of gabapentin (200 mg/kg, SC). D-Serine (30 micrograms/animal, ICV) also reversed the anxiolytic-like effects (in the light/dark box and the rat elevated X-maze) of gabapentin (30 mg/kg). In contrast, L-Serine (100 micrograms, ICV) failed to block the antinociceptive action of gabapentin. The antinociceptive action of (+)-HA-966 (25 mg/kg, SC), a partial agonist at the glycine/NMDA receptor, was reversed by D-Serine (100 micrograms/animal, ICV). However, D-Serine (100 micrograms/animal, ICV) failed to affect the antinociceptive action of a competitive NMDA receptor antagonist CGS 19755 (3 mg/kg, SC). Gabapentin has negligible affinity for the strychnine insensitive [3H]glycine binding site. This indicates that the interaction between gabapentin and D-Serine may not involve the NMDA receptor complex. Gabapentin may represent a novel type of anxiolytic and analgesic agent.
Adenosine and dopamine interact within the striatum to control striatopallidal output and globus ... more Adenosine and dopamine interact within the striatum to control striatopallidal output and globus pallidus GABA release. Manipulating striatal adenosine transmission via blockade of the A2A receptor subtype can compensate for the reduced dopamine activity within the striatum that underlies movement disorders such as antipsychotic-induced extrapyramidal syndrome (EPS) and Parkinson's disease (PD). Preclinical studies in the rat have demonstrated that adenosine A2A receptor antagonists can attenuate behaviors reflecting reduced dopamine activity, such as haloperidol-induced catalepsy and hypoactivity. In the present studies using nonhuman primates, adenosine antagonists were tested against haloperidol-induced EPS in Cebus apella and haloperidol-induced catalepsy in Saimiri sciureus (squirrel monkey). Specifically, the A2A receptor antagonists, SCH 412348 (0.3-30 mg/kg PO) and KW-6002 (3-100 mg/kg PO); the A1/A2A receptor antagonist, caffeine (1-30 mg/kg PO and IM); and the A1 receptor antagonist, DPCPX (3-30 mg/kg PO) were tested in at least one of these models. SCH 412348 (10-30 mg/kg), KW-6002 (57-100 mg/kg), and caffeine (30 mg/kg) significantly increased the time to EPS onset. Additionally, SCH 412348, KW-6002, and caffeine afforded protection from the onset of EPS for at least 6 h in some of the primates. SCH 412348 (10 mg/kg) and caffeine (10 mg/kg) significantly reduced haloperidol-induced catalepsy. DPCPX produced a very slight attenuation of EPS at 30 mg/kg, but had no effect on catalepsy. These findings suggest that adenosine A2A receptor antagonists may represent an effective treatment for the motor impairments associated with both antipsychotic-induced EPS and PD.
Akathisia is a subset of the larger antipsychotic side effect profile known as extrapyramidal syn... more Akathisia is a subset of the larger antipsychotic side effect profile known as extrapyramidal syndrome (EPS). It is associated with antipsychotic treatment and is characterized as a feeling of inner restlessness that results in a compulsion to move. There are currently no primate models available to assess drug-induced akathisia; the present research was designed to address this shortcoming. We developed a novel rating scale based on both the Barnes Akathisia Rating Scale (BARS) and the Hillside Akathisia Scale (HAS) to measure the objective, observable incidence of antipsychotic-induced akathisia-like behavior in Cebus apella non-human primates (NHPs). To induce akathisia, we administered the atypical antipsychotic aripiprazole (1 mg/kg) or the selective phosphodiesterase 10A (PDE10A) inhibitor MP-10 (1-3 mg/kg). Treatment with both compounds produced significantly greater akathisia scores on the rating scale than vehicle treatment. Characteristic behaviors observed included vocalizations, stereotypies, teeth grinding, restless limb movements, and hyperlocomotion. Adenosine A 2A receptor antagonists have previously been shown to be effective in blocking antipsychotic-induced EPS in primates. The selective A 2A receptor antagonist, SCH 412348 (10-30 mg/kg), effectively reduced or reversed akathisialike behavior induced by both aripiprazole and MP-10. This work represents the first NHP measurement scale of akathisia and demonstrates that NHPs are responsive to akathisia-inducing agents. As such, it provides a useful tool for the preclinical assessment of putative antipsychotics. In addition, these results provide further evidence of the utility of A 2A receptor antagonists for the treatment of antipsychotic-induced movement disorders.
Neuropathic pain is a debilitating chronic syndrome that often arises from injuries to peripheral... more Neuropathic pain is a debilitating chronic syndrome that often arises from injuries to peripheral nerves. Such pain has been hypothesized to be the result of an aberrant expression and function of sodium channels at the site of injury. Here, we show that intrathecal administration of specific antisense oligodeoxynucleotides (ODN) to the peripheral tetrodotoxin (TTX)-resistant sodium channel, NaV1.8, resulted in a timedependent uptake of the ODN by dorsal root ganglion (DRG) neurons, a selective 'knock-down' of the expression of NaV1.8, and a reduction in the slow-inactivating, TTX-resistant sodium current in the DRG cells. The ODN treatment also reversed neuropathic pain induced by spinal nerve injury, without affecting non-noxious sensation or response to acute pain. These data provide direct evidence linking NaV1.8 to neuropathic pain. As NaV1.8 expression is restricted to sensory neurons, this channel offers a highly specific and effective molecular target for the treatment of neuropathic pain.
This study investigated the effects of brain ischemia on sodium channel gene (NaCh) expression in... more This study investigated the effects of brain ischemia on sodium channel gene (NaCh) expression in rats. Using quantitative RT-PCR, our findings demonstrated the expression ratio of NaCh genes in normal rat brain to be Nay1.1 > Nay1.8 > Nay1.3 > Nay1.7 (rBI > PN3 > rBIII > PN1). In contrast, brain injury caused by middle cerebral artery occlusion (MCAo) for 2 h followed by reperfusion significantly down-regulated Nay1.3 and Nay1.7 genes in both injured and contralateral hemispheres; whereas the Na~l.8 gene was down regulated in only the injured hemisphere (though only acutely at 2 or 2-6h post-MCAo). However, the time-course of NaCh gene expression revealed a significant down-regulation of Na~l.1 only in the ischemic hemisphere beginning 6 h post-MCAo and measured out to 48 h post-MCAo. In a separate preliminary study Nay1.2 (rBII) gene was found be expressed at levels greater than that of Nay1.1 in normal rats and was significantly down regulated at 24 h post-MCAo. Our findings document, for the first time, quantitative and relative changes in the expression of various NaCh genes following ischemic brain injury and suggest that the Na~l.1 sodium channel gene may play a key role in ischemic injury/recovery.
The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and... more The present study investigated the effects of RS-100642-198 (a novel sodium channel blocker), and two related compounds (mexiletine and QX-314), in in vitro models of neurotoxicity. Neurotoxicity was produced in primary cerebellar cultures using hypoxia/hypoglycemia (H/H), veratridine or glutamate where, in vehicle-treated neurons, 65%, 60% and 75% neuronal injury was measured, respectively. Dose-response neuroprotection experiments were carried out using concentrations ranging from 0.1-500 ~tM. All the sodium channel blockers were neuroprotective against H/H-induced injury, with each exhibiting similar potency and efficacy. However, against veratridine-induced neuronal injury only RS-100642-198 and mexiletine were 100% protective, whereas QX-314 neuroprotection was limited (i.e. only 54%). In contrast, RS 100642-198 and mexiletine had no effect against glutamate-induced injury, whereas QX-314 produced a consistent, but very limited (i.e. 25%), neuroprotection. Measurements of intraneuronal calcium 2+ .. 9 ([Ca ]i) moblhzatlon revealed that glutamate caused immediate and sustained increases in [Ca2+]i which were not affected by RS-100642-198 or mexiletine. However, both drugs decreased the initial amplitude and attenuated the sustained rise in [Ca2+]i mobilization produced by veratridine or KC1 depolarization. QX-314 produced similar effects on glutamate-, veratridine-or KCl-induced [Ca2+]i dynamics, effectively decreasing the amplitude and delaying the initial spike in [Ca2+]i, and attenuating the sustained increase in [Ca2+]i mobilization. By using different in vitro models of excitotoxicity, a heterogeneous profile of neuroprotective effects resulting from sodium channel blockade has been described for RS-100642-198 and related drugs, suggesting that selective blockade of neuronal sodium channels in pathological conditions may provide therapeutic neuroprotection against depolarization/excitotoxicity via inhibition of voltage-dependent Na + channels.
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