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The opioid receptor-like 1 receptor is a novel member of the opioid receptor family and its endogenous peptide ligand has been termed nociceptin and orphanin FQ. Activation of the opioid receptor-like 1 receptor by nociceptin/orphanin FQ in vivo produces hyperalgesia when this peptide is given supraspinally but analgesia at the spinal level. Nociceptin/orphanin FQ also reverses stress-induced analgesia, suggesting that the peptide has anti-opioid properties. Nociceptin/orphanin FQ knockout mice show alterations in pain sensitivity and stress responses and display increased morphine dependence, suggesting an interaction of the nociceptin/orphanin FQ system with classical opioid receptor function. To determine if the behavioural phenotype of nociceptin/orphanin FQ knockout mice reflects changes in either opioid receptor-like 1 or classical opioid receptor expression, we have carried out quantitative autoradiography of the opioid receptor-like 1, mu-, delta- and kappa-opioid receptors in the brains of these animals. Receptor density was measured on coronal sections from wild-type, heterozygous and homozygous mice using [(3)H]nociceptin, [(3)H][D-Ala(2)-N-methyl-Phe(4)-Gly(5) ol] enkephalin, [(3)H]deltorphin-I, or [(3)H](-)-N-methyl-N-[7-(1-pyrrodinyl)-1-oxospiro[4,5]dec-8-yl]-4-benzofuranacetamide to label opioid receptor-like 1, mu-, delta- and kappa-receptors, respectively. A region-specific up-regulation of the opioid receptor-like 1 receptor (up to 135%) was seen in brains from homozygous mice. Mu-Receptors also showed significant differences between genotypes whilst changes in delta- and kappa- receptors were minor. In conclusion the region-specific up-regulation of the opioid receptor-like 1 receptor indicates a tonic role for nociceptin/orphanin FQ in some brain structures and may suggest the peptide regulates the receptor expression in these regions. The changes in the opioid receptor-like 1 receptor may relate to the anxiogenic phenotype of these animals but the observed change in mu-receptors does not correlate with altered morphine responses.

Three genes for the opioid receptors ( micro, delta and kappa) and a gene coding for a related receptor (ORL1) have been cloned but pharmacological studies suggest that further subtypes exist that remain poorly understood. To determine if there are other classically defined opioid binding sites we have carried out homogenate binding and section autoradiography with [3H]naloxone in mice that lack all three opioid genes and are hyperalgesic in a thermal nociceptive test. We have also examined [3H]bremazocine labelling in triple knockout brain and spinal cord as this ligand has been proposed to label novel kappa-receptors. No receptor labelling for either ligand was detected in the brains or spinal cord of knockout mice demonstrating that all binding is the product of the three known receptors and that there is no cross-labelling of the ORL1 receptor. Nociceptin (1 micro m) caused marked displacement of [3H]bremazocine in wild-type brains indicating that nociceptin at high concentrations can displace classical opioid binding. As a number of studies have proposed a close association between the classical opioid receptors and the ORL1 system we also hypothesized that loss of all of the classical opioid receptors might lead to compensatory changes in ORL1 receptors. Labelling of the ORL1 receptor with [3H]nociceptin showed region-dependent quantitative increases in triple knockout brains indicating a close relationship between the two systems in specific brain areas.

There are two bradykinin receptor subtypes, designated B1 and B2. Whilst both have been implicated in nociception, it is believed that there is a low level of constitutive expression of B1 receptors and that their expression is induced by inflammation or tissue damage. The present study investigated the role of B1 receptors in spinal nociceptive processing using an in vivo electrophysiological assay in decerebrate, spinalized rabbits, a species that shares close B1 receptor homology with the human receptor. Inflammation was induced in the paw by an injection of complete Freund's adjuvant at least 1 h before recording single motor unit activity of the semitendinous/biceps femoris muscle in response to a noxious pinch of the foot. Control animals received an intraplantar injection of saline. The peptide B1 receptor antagonist B9858 was administered i.v. and caused dose-dependent and complete inhibition of the nociceptive spinal reflex (ID50 = 1 mg·kg–1). In control animals without paw inflammation, B9858 had no effect. These findings are consistent with other evidence that peptide B1 receptor antagonists inhibit spinal nociceptive reflexes only after induction of B1 receptors by inflammation and support the potential therapeutic utility of B1 receptor antagonists as analgesic and anti-inflammatory drugs.Key words: bradykinin B1 receptor, inflammation, spinal reflex, rabbit.

These studies investigated the pharmacology of neurogenic dural vasodilation in anaesthetized guinea-pigs. Following introduction of a closed cranial window the meningeal (dural) blood vessels were visualized using intravital microscopy and the diameter constantly measured using a video dimension analyser. Dural blood vessels were constricted with endothelin-1 (3 μg kg−1, i.v.) prior to dilation of the dural blood vessels with calcitonin gene-related peptide (CGRP; 1 μg kg−1, i.v.) or local electrical stimulation (up to 300 μA) of the dura mater. In guinea-pigs pre-treated with the CGRP receptor antagonist CGRP(8-37) (0.3 mg kg−1, i.v.) the dilator response to electrical stimulation was inhibited by 85% indicating an important role of CGRP in neurogenic dural vasodilation in this species. Neurogenic dural vasodilation was also blocked by the 5-HT1B/1D agonist rizatriptan (100 μg kg−1) with estimated plasma levels commensurate with concentrations required for anti-migraine efficacy in patients. Rizatriptan did not reverse the dural dilation evoked by CGRP indicating an action on presynaptic receptors located on trigeminal sensory fibres innervating dural blood vessels. In addition, neurogenic dural vasodilation was also blocked by the selective 5-HT1D agonist PNU-142633 (100 μg kg−1) but not by the 5-HT1F agonist LY334370 (3 mg kg−1) suggesting that rizatriptan blocks neurogenic vasodilation via an action on 5-HT1D receptors located on perivascular trigeminal nerves to inhibit CGRP release. This mechanism may underlie one of the anti-migraine actions of the triptan class exemplified by rizatriptan and suggests that the guinea-pig is an appropriate species in which to investigate the pharmacology of neurogenic dural vasodilation. British Journal of Pharmacology (2001) 133, 1029–1034; doi:10.1038/sj.bjp.0704162

Calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), amylin and calcitonin (CT) are structurally and functionally related neuropeptides. It has recently been shown that the molecular pharmacology of CGRP and ADM is determined by coexpression of one of three receptor activity-modifying proteins (RAMPs) with calcitonin receptor-like receptor (CRLR). Furthermore, RAMP proteins have also been shown to govern the pharmacology of the calcitonin receptor, which in association with RAMP1 or RAMP3, binds amylin with high affinity. In this study, we have cloned the rat RAMP family and characterized the pharmacology of rat CGRP and ADM receptors. Rat RAMP1, RAMP2 and RAMP3 shared 72%, 69% and 85% homology with their respective human homologues. As expected CRLR-RAMP1 coexpression conferred sensitivity to CGRP, whilst association of RAMP2 or RAMP3 with CRLR conferred high affinity ADM binding. Using specific oligonucleotides we have determined the expression of RAMP1, RAMP2 and RAMP3 mRNAs in the rat central nervous system by in situ hybridization. The localization of RAMP mRNAs was heterogeneous. RAMP1 mRNA was predominantly expressed in cortex, caudate putamen and olfactory tubercles; RAMP2 mRNA was most abundant in hypothalamus; and RAMP3 was restrictively expressed in thalamic nuclei. Interestingly, in specific brain areas only a single RAMP mRNA was often detected, suggesting mutual exclusivity in expression. These data allow predictions to be made of where each RAMP protein may heterodimerize with its partner G-protein-coupled receptor(s) at the cellular level and consequently advance current understanding of cellular sites of action of CGRP, ADM, amylin and CT. Furthermore, these localization data suggest that the RAMP family may associate and modify the behaviour of other, as yet unidentified neurotransmitter receptors.

Until recently the opioid receptor family was thought to consist of only the mu-, delta- and kappa-receptors. The cloning of opioid receptor like receptor (ORL1) and its endogenous ligand nociceptin/orphanin FQ, which displayed anti-opioid properties, has raised the issue of functional co-operativity of this system with the classical opioid system. ORL1 receptor knockout mice have been successfully developed by homologous recombination to allow the issue of potential heterogeneity of this receptor and also of compensatory changes in mu-, delta- or kappa-receptors in the absence of ORL1 to be addressed. We have carried out quantitative autoradiographic mapping of these receptors in the brains of mice that are wild-type, heterozygous and homozygous for the deletion of the ORL1 receptor. ORL1, mu-, delta- and kappa-receptors were labelled with [(3)H] leucyl-nociceptin (0.4 nM), [(3)H] DAMGO (4 nM), [(3)H] deltorphin-I (7 nM), and [(3)H] CI-977 (2.5 nM) respectively. An approximately 50% decrease in [(3)H] leucyl-nociceptin binding was seen in heterozygous ORL1 mutant mice and there was a complete absence of binding in homozygous brains indicating the single gene encodes for the ORL1 receptor and any putative subtypes. No significant gross changes in the binding to other opioid receptors were seen across genotypes in the ORL1 mutant mice demonstrating a lack of major compensation of classical opioid receptors in the absence of ORL1. There were a small number of region specific changes in the expression of classical opioid receptors that may relate to interdependent function with ORL1.

Migraine headache is thought to be caused by a distension of meningeal blood vessels, the activation of trigeminal sensory neurones and the the development of a central sensitization within the trigeminal nucleus caudalis (TNC). It has been proposed that clinically effective 5-HT(1B/1D) agonists act peripherally to inhibit the release of calcitonin gene-related peptide (CGRP) and neurogenic dural vasodilation, and to attenuate nociceptive neurotransmission within the TNC. Since opioids are also effective anti-migraine agents the present studies investigated the role of opioids within the trigemino-vascular system in anaesthetised rats. Electrical stimulation of the dura mater evoked neurogenic dural vasodilation which was significantly inhibited by morphine (1 mg kg(-1)) the selective mu-opioid agonist DAGO (10 microg kg(-1)) and the mixed agonist/antagonist butorphanol (1 mg kg(-1)) but not by the kappa- and delta-opioid agonists (+/-) U50488H (100 microg kg(-1)) and DPDPE (1 mg kg(-1)). Morphine had no effect on CGRP-evoked dural vasodilation. In electrophysiological studies morphine (1 - 10 mg kg(-1)) significantly attenuated brainstem neuronal activity in response to electrical stimulation of the dura by 65% at 10 mg kg(-1). Morphine (3 mg kg(-1)) also inhibited the TNC neuronal sensitization following CGRP-evoked dilation. The present studies have demonstrated that opioids block the nociceptive neurotransmission within the trigeminal nucleus caudalis and in addition inhibit neurogenic dural vasodilation via an action on mu-opioid receptors located on trigeminal sensory fibres innervating dural blood vessels. These peripheral and central actions are similar to those of the 'triptan' 5-HT(1B/1D) agonists and could account for the anti-migraine actions of opioids.

The pharmacology of neurogenic trigeminovascular vasodilator responses in the dura mater is of interest for understanding the pathophysiology of migraine and to develop new therapies for this disabling common condition. Aminergic mechanisms have been implicated in migraine through direct study of amines in patients, and by inference from the pharmacology of many effective anti-migraine compounds, particularly preventative agents. This study used intravital microscopy to assess the role of aminergic transmission in neurogenic dural vasodilatation (NDV) by measuring directly the diameter of dural arteries in sodium pentobarbitone anaesthetised rats. Electrical stimulation of a closed cranial window produces, by local depolarisation of nerves, dural vessel dilation that is monitored continuously on-line using video-microscopy and a video dimension analyser. This dural vasodilatation was not affected by pre-treatment with an alpha1-adrenoceptor agonist (phenylephrine, 1 and 5 microg/kg), or antagonist (corynanthine, 1 and 2 mg/kg), nor by an alpha2-adrenoceptor agonist (UK14,304, 5 microg/kg) or antagonist (yohimbine, 1 and 3 mg/kg). Similarly, we saw no effect of beta-adrenoceptor blockade (propranolol, 1 and 3 mg/kg). The lack of an inhibitory effect of UK14,304 the model of neurogenic dural vasodilation contrasts with its effect in neurogenic dural plasma protein extravasation model. The lack of inhibition of beta-adrenoceptor antagonists in the neurogenic vasodilatation model contrasts with their usefulness as migraine prophylactics, and suggests that their mechanism of action in migraine is unlikely to be through sensory trigeminal fibre terminals at the neurovascular junction. Moreover, the data indicate that the adrenergic system does not play a significant role in neurogenic dural vasodilation.

Vasodilatation in the dura mater has been implicated in migraine pathogenesis. Anti-migraine triptan drugs block vasodilatation by binding to 5-HT1B/1D receptors localized on the peripheral sensory terminals and dural blood vessel smooth muscles. Previous studies suggest that calcitonin gene-related peptide (CGRP) released from Adelta-fibres plays a more important role than substance P (SP) released from C-fibres in inducing dural vasodilatation and that one of the antimigraine mechanisms of triptan drugs is inhibiting CGRP release. In the present study, the relationship between CGRP and 5-HT1B/1D receptors, and between CGRP and SP in the trigeminal ganglion neurons in rats was examined by double immunohistochemical staining. CGRP, 5-HT1B, 5-HT1D and SP-positive trigeminal ganglion neurons were all predominantly small and medium-sized. In the trigeminal ganglia, approximately 50% of CGRP-positive neurons were 5-HT1B positive. Similarly, approximately 55% of CGRP-positive neurons were 5-HT1D immunoreactive. Approximately 50% of CGRP-positive neurons were SP-positive, while 93% of SP-positive neurons were CGRP-positive, suggesting that nearly all SP-positive neurons also contain CGRP. The fibre types of the 5-HT1B- and 5-HT1D-positive neurons were further investigated with an antibody against the A-fibre marker 200-kDa neurofilaments (NF200). Approximately 46% of the 5-HT1B-positive and 43% of the 5-HT1D-positive trigeminal ganglion neurons were also NF200 positive, indicating that many A-fibre trigeminal neurons express 5-HT1B or 5-HT1D receptors. These results support the hypothesis that one important action of antimigraine drugs is the inhibition of CGRP release and that Adelta-fibres may play an important role in migraine pathogenesis.

The cerebral circulation is innervated by calcitonin gene-related peptide (CGRP) containing fibers originating in the trigeminal ganglion. During a migraine attack, there is a release of CGRP in conjunction with the head pain, and triptan administration abolishes both the CGRP release and the pain at the same time. In the search for a novel treatment of migraine, a non-peptide CGRP antagonist has long been sought. Here, we present data on a human cell line and human and guinea-pig isolated cranial arteries for such an antagonist, Compound 1 (4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide). On SK-N-MC cell membranes, radiolabelled CGRP binding was displaced by both CGRP-(8–37) and Compound 1, yielding pKi values of 8.9 and 7.8, respectively. Functional studies with SK-N-MC cells showed that CGRP-induced cAMP production was antagonised by both CGRP-(8–37) and Compound 1 with pA2 values of 7.8 and 7.7, respectively. Isolated human and guinea pig cerebral arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation in human cerebral arteries which was antagonized by both CGRP-(8–37) and Compound 1 in a competitive manner. In guinea pig basilar arteries, CGRP-(8–37) antagonised the CGRP-induced relaxation while Compound 1 had a weak blocking effect . The clinical studies of non-peptide CGRP antagonists are awaited with great interest.

The present study directly compared the antinociceptive and toxic effects of the neuronal nicotinic receptor agonist ABT-594 ((R)-5-(2-azetidinylmethoxy)-2-chloropyridine) with (-)-nicotine and (+)-epibatidine. Like (-)-nicotine (0.8 and 1.6 mg/kg s.c.) and (+)-epibatidine (0.005 and 0.01 mg/kg s.c.), ABT-594 (0.05 and 0.1 mg/kg s.c.) increased response latencies in the hot-plate test in rats, indicating that it has antinociceptive activity. In contrast to (-)-nicotine and (+)-epibatidine, ABT-594 did not cause rotarod impairment at antinociceptive doses but did cause hypothermia and life-threatening adverse effects including seizures. ABT-594 (0.01 and 0.1 mg/kg i.v.) also produced a dose-dependent increase in blood pressure resembling that observed with (-)-nicotine (0.03, 0.1 and 0. 03 mg/kg i.v.) and (+)-epibatidine (0.001 and 0.003 mg/kg i.v.). Both the antinociceptive and toxic effects (convulsions and hypertension) were abolished by pretreatment with the brain penetrant neuronal nAChR antagonist mecamylamine (1 mg/kg s.c.; i.v. for cardiovascular studies), demonstrating that these actions of ABT-594 were mediated via activation of neuronal nicotinic receptors. Continuous infusion of ABT-594 (0.2 mg/kg per day s.c.) to rats for 7 days followed by challenge with mecamylamine (1 mg/kg i.p.) induced a nicotine-like abstinence syndrome suggesting that ABT-594 has nicotine-like dependence liability. These findings indicate that the acute safety profile of ABT-594 is not significantly improved over other nicotinic analgesics.

We have examined the activity of a range of kinins on recombinant human bradykinin receptors, using a high throughput functional assay which measures intracellular Ca 2+ responses. The most potent agonist for Chinese hamster ovary (CHO) cells stably expressing recombinant human bradykinin B 1 receptors were Des-Arg 9 -bradykinin (EC 50 =7.9 nM) and Des-Arg 10 -kallidin (EC 50 =8.6 nM), while the most potent agonist for CHO cells expressing human bradykinin B 2 receptors was bradykinin (EC 50 =2.0 nM). These findings confirm the validity of the recombinant system and the microtitre plate imaging-based characterization system when compared to known agonist properties of the native receptors. The concentration–response relationship for bradykinin at bradykinin B 2 receptors was potently inhibited by [ d -Arg 0 ,Hyp 3 ,β-(2-thienyl)-Ala 5 , d -Tic 7 ,Oic 8 ]-bradykinin (Hoe140) (IC 50 =71 nM), which was 500-fold more potent against the B 2 -expressing cells than the B 1 cells. Bradykinin B 1 receptor-mediated responses activated by Des-Arg 10 -kallidin were fully antagonized by Des-Arg 9 -[Leu 8 ]bradykinin (IC 50 =59 nM), Des-Arg 10 -Hoe140 (IC 50 =211 nM) and most potently by Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser- d -Igl-Oic (B9858) (IC 50 =14 nM), none of which displayed any activity against the bradykinin B 2 receptor cell line up to 3 μM. None of the antagonists displayed partial agonism activity in these cell lines. All bradykinin B 1 and B 2 receptor antagonists tested acted in an apparently non-competitive manner that is likely to be due in part to their kinetics and to the nature of the functional assay used.

The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK-0869 (L-754,030) 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluor o)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorpholine and its water soluble prodrug, L-758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK-0869 and L-758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK-0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5-HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK-0869 (4-16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK-0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK-0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK-0869 and its prodrug, L-758,298, have good activity against cisplatin-induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy.

Aims Vasoconstriction in human coronary artery can be mediated via activation of both 5-HT2 and 5-HT1B-receptors. Coronary vasoconstriction is a rare, but potential adverse effect of the antimigraine drug sumatriptan. In order to investigate the receptor population involved we compared the vasoconstrictor effects of sumatriptan (a mixed 5-HT1B/1D-receptor agonist) with those of L-775,606 (a selective 5-HT1D-receptor agonist) and 5-HT (the endogenous ligand) in human isolated coronary arteries. Methods Coronary arteries were obtained from human hearts removed prior to transplant surgery. Several endothelium denuded ring segments (4 mm in length) were obtained from each artery and mounted for isometric tension recording. Each segment was first exposed to 45 mm KCl and then to 5-HT (1 nm-100 μm ). Concentration-effect curves to L-775,606 (1-(3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl)-4-(2-(3-fluorophenyl)ethyl)piperazine) and sumatriptan were then performed in a consecutive and random manner. The response to repeated application of 5-HT was obtained in separate segments. Results Twenty-five segments from seven different coronary arteries were studied. Concentration-effect curves were fitted to the data using nonlinear regression analysis. The maximum contraction for L-775,606 was significantly less than that for sumatriptan with Emax values (% relative to 45 mm KCl=100%) of 30.1±4.22 and 41.5±2.7, respectively. L-775,606 was significantly (30-fold) less potent than sumatriptan in causing contraction compared with sumatriptan (EC50 values were 6.0 μm and 0.2 μm, respectively). For comparison the Emax value for 5-HT was 77.2% and the EC50 value was 0.2 μm. Conclusions The selective 5-HT1D-receptor agonist L-775,606 has less propensity towards vasoconstriction in human isolated coronary artery (endothelium-denuded) than was mixed 5-HT1B/1D-receptor agonist sumatriptan. The contractions produced were at concentrations where L-775,606 would be expected to occupy 5-HT1B-receptors.

This study investigated whether the selective 5HT1F receptor agonist LY334370 has other possible antimigraine mechanisms in addition to the proposed inhibition of dural plasma extravasation. LY334370 (up to 10−5 M) had no vasoconstrictor effects on human cerebral arteries in vitro. It had no effect (up to 10 mg kg−1, iv) on neurogenic vasodilation of dural blood vessels produced by electrical stimulation of the dura mater in anesthetized rats. Nor had it any effect (at 3 mg kg−1, iv) on the hyperalgesia produced by injection of carrageenan into the paw of conscious rats or on nociceptive reflex responses in the spinalized, decerebrate rabbit (up to 3 mg kg−1, iv), indicating that it has no general analgesic properties. However, it significantly inhibited activation of second-order neurons in the trigeminal nucleus caudalis produced by electrical stimulation of the dura mater in anesthetised rats at 3 mg kg−1, iv. These results provide evidence to suggest that LY334370 has a central mechanism of action in blocking the transmission of nociceptive impulses within the trigeminal nucleus caudalis and that this may represent a mechanism through which it has its antimigraine effect.

Modulating monoamine activity as a therapeutic strategy continues to dominate antidepressant research, with a recent emphasis on agents with multiple targets, including combined serotonin/noradrenaline re-uptake inhibitors and numerous serotonin receptor ligands. An important new development has been the emergence of potential novel mechanisms of action, notably modulation of the activity of neuropeptides substance P and corticotrophin-releasing factor, and the intracellular messenger cyclic adenosine monophosphate. Efforts in this area have recently been rewarded by the demonstration of antidepressant efficacy of the substance P receptor antagonist MK-0869.

Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel alpha subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

Abnormal processing of amyloid precursor protein (APP), in particular the generation of beta-amyloid (Abeta) peptides, has been implicated in the pathogenesis of Alzheimer's disease. This study examined the consequences of deleting the APP gene on hippocampal synaptic plasticity, and upon the biophysical properties of morphologically identified neurones in APP-null mice. The hippocampus of APP-null mice had a characteristic increase in gliosis throughout the CA1 region and a disruption of staining for the dendritic marker MAP2 and the presynaptic marker synaptophysin. The disruption of MAP2 staining was associated with a significant reduction in overall dendritic length and projection depth of biocytin labeled CA1 neurones. In two groups of APP-null mice that were examined at 8-12 months, and 20-24 months of age, there was an impairment in the formation of long-term potentiation (LTP) in the CA1 region compared to isogenic age matched controls. This LTP deficit was not associated with an alteration in the amplitude of EPSPs at low stimulus frequencies (0.033 Hz) or facilitation during a 100 Hz stimulus train, but was associated with a reduction in post-tetanic potentiation. Paired-pulse depression of GABA-mediated inhibitory post-synaptic currents was also attenuated in APP-null mice. These data demonstrate that the impaired synaptic plasticity in APP deficient mice is associated with abnormal neuronal morphology and synaptic function within the hippocampus.

1. Migraine headache pain is thought to result from an abnormal distention of intracranial, extracerebral blood vessels and the consequent activation of the trigeminal nervous system. Migraine is also often accompanied by extracranial sensory disturbances from facial tissues. These experiments investigate whether meningeal dilation produces central sensitization of neurones that receive convergent input from the face. 2. Single unit extracellular activity was recorded from the trigeminal nucleus caudalis of anaesthetized rats in response to either noxious stimulation of the dura mater, innocuous stimulation of the vibrissae or to a transient dilation of the meningeal vascular bed. 3. Rat alpha-CGRP (calcitonin gene-related peptide; 1 microg kg(-1), i.v.) caused a dilation of the middle meningeal artery and facilitated vibrissal responses by 36+/-7%. 4. The 5-HT1B/1D agonist, L-741,604 (3 mg kg(-1), i.v.), inhibited responses to noxious stimulation of the dura mater (16+/-7% of control) and, in a separate group of animals, blocked the CGRP-evoked facilitation of vibrissal responses. 5. L-741,604 (3 mg kg(-1), i.v.) also inhibited responses to innocuous stimulation of the vibrissa (14+/-10% of control) with neurones that received convergent input from the face and from the dura mater, but not with cells that received input only from the face (70+/-12% of control). 6. These data show that dilation of meningeal blood vessels causes a sensitization of central trigeminal neurones and a facilitation of facial sensory processing which was blocked by activation of pre-synaptic 5-HT1B/1D receptors. 7. Sustained dural blood vessel dilation during migraine may cause a sensitization of trigeminal neurones. This may underlie some of the symptoms of migraine, such as the headache pain and the extracranial allodynia. Inhibition of this central sensitization may therefore offer a novel strategy for the development of acute and/or prophylactic anti-migraine therapies.

NMDA-mediated neurotoxicity and oxidative stress have been implicated in the etiology of a number of degenerative diseases including motor neuron disease. The present study examined the effect of chronic administration of the glycine/NMDA receptor antagonist L-701,324 and the monoamine oxidase B inhibitor (r)-deprenyl on the onset and rate of progression of neurological impairment in the motor neuron degeneration (mnd) mouse, a murine model of neurodegeneration. Neurological assessment of mnd mice revealed an onset of motor deficits at 6 months of age as observed by the loss of hindlimb reflex extension. By 7 months, balance was also markedly impaired as measured by deficits in rotarod performance and ability to remain on balancing beams. At 8 months of age mnd mice exhibited gross abnormalities in walking pattern; animals were unable to flex their hindlimbs and tended to walk in small labored movements. Daily administration of L-701,324 (10 mg/kg p.o.) or r(-)-deprenyl (1 mg/kg p.o.) to mnd mice from 4 to 8 months of age failed to delay the onset of symptoms or slow the rate of deterioration of motor performance. These findings suggest that excessive activation of NMDA receptors may not be involved in the pathological process leading to motor neuron dysfunction in mnd mice and do not suggest a protective effect of deprenyl on motor neurons in these mice.

The putative neurotrophic effects of the immunophilin ligand GPI-1046 were evaluated in established experimental systems of neuron survival and axon growth in vitro and in vivo. GPI-1046 marginally increased neurite outgrowth of chick dorsal root ganglia in culture under conditions where a very robust effect of nerve growth factor was seen. GPI-1046 failed to protect dopaminergic neurons from 1-methyl-4-phenylpyridinium in culture or to protect cultured cortical neurons from experimentally induced apoptosis in vitro. In adult rats in vivo, daily administration of GPI-1046 (10 mg/kg, s.c.) for three days enhanced the maximal regeneration distance of both motor and large myelinated sensory axons measured using an electrophysiological assay. However, detailed morphometric analysis of these animals failed to provide evidence for an increase in axon numbers in GPI-1046-treated animals. The ability of GPI-1046 to promote the recovery of dopaminergic function following unilateral 6-hydroxydopamine lesions of the substantia nigra was also tested in rats. In the first study, the duration of amphetamine (3 mg/kg, s.c.)-induced circling, but not the maximal number of rotations, was significantly reduced in animals treated with GPI-1046 for five days (10 mg/kg/day). In a second study, testing the effects of delayed GPI-1046 administration, chronic treatment with GPI-1046 (10 mg/kg/day) for two weeks, beginning one month after surgery, did not alter circling responses. Morphometric analysis failed to reveal any changes in either the density of tyrosine hyroxylase-positive fibres in dopaminergic target areas or in cell numbers in the substantia nigra in both experiments. Thus, while GPI-1046 produced marginal effects on neurite outgrowth in dorsal root ganglia cultures and on functional paramaters of nerve regeneration in vivo, we failed to obtain evidence in support of the notion of a general neuroprotective effect of the compound or for an effect on morphologic nerve regeneration in vivo.

1. We have previously shown that weaning at day 21 increases delta-opioid receptor binding in the brain at day 25, which might be due to stimulation of the development of a delta-opioid receptor subtype or activation of G-protein coupling processes. 2. We have addressed the possibility that weaning stimulates coupling of the delta-receptor by homogenate binding studies with four agonist and one antagonist radioligand in the presence of a GTP analogue and Na+ in brain tissue from weaned and non-weaned animals. 3. Saturation studies with three agonist ligands ([3H]-deltorphin I, [3H]-S-Atc-Ile(5,6)deltorphin I and [3H]-R-Atc-Ile(5,6)deltorphin II) showed higher levels of maximal binding in brains from 25-day weaned than in brains from non-weaned rats. The magnitude of the effects of GMPPNP and Na+ in decreasing this binding was ligand dependent and in each case was significantly more marked in brains from weaned animals. GMPPNP and Na+ were completely without effect on Bmax for, [3H]-S-Atc-Ile(5,6)deltorphin I and [3H]-R-Atc-Ile(5,6)deltorphin II in brains from non-weaned rats. 4. [3H]-Ile(5,6)deltorphin II and [3H]-naltrindole showed no differences in labelling between weaned and non-weaned groups and both groups responded similarly to the effects of GMPPNP and Na+ treatment. 5. GMPPNP and Na+ had small effects on binding affinity (K(D)) for some of the agonist radioligands which were similar in both weaned and non-weaned groups. 6. Weaning induced increases in binding of delta-receptors in 25-day rats can be explained in terms of the way delta-agonist radioligands recognize the receptor environment.

The localization of substance P in brain regions that coordinate stress responses and receive convergent monoaminergic innervation suggested that substance P antagonists might have psychotherapeutic properties. Like clinically used antidepressant and anxiolytic drugs, substance P antagonists suppressed isolation-induced vocalizations in guinea pigs. In a placebo-controlled trial in patients with moderate to severe major depression, robust antidepressant effects of the substance P antagonist MK-869 were consistently observed. In preclinical studies, substance P antagonists did not interact with monoamine systems in the manner seen with established antidepressant drugs. These findings suggest that substance P may play an important role in psychiatric disorders.

N-methyl-D-aspartic acid (NMDA) receptors are known to play a key role in the induction phase of long-term potentiation (LTP) at certain hippocampal synapses and to represent some component of spatial learning in animals. The ability of NMDA receptor antagonists (or gene knockout) to impair LTP has led to the suggestion that the therapeutic use of such antagonists may impair cognitive function in humans. The present study compares the effects on LTP of NMDA receptor ion channel block by MK-801 and glycine-site antagonism by 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414). In vitro experiments using rat cortical slices revealed L-687,414 to be ∼3.6 fold more potent than its parent analogue, R(+)HA-966 at antagonizing NMDA-evoked population depolarizations (apparent Kbs: 15 μM and 55 μM, respectively). Whole-cell voltage-clamp experiments using rat cultured cortical neurones revealed L-687,414 to shift to the right the concentration-response relationship for NMDA-evoked inward current responses (pKb=6.2±0.12). L-687,414 affinity for the glycine site on the NMDA receptor complex was also determined from concentration-inhibition curves, pKi=6.1±0.09. In the latter experiments, L-687,414 and R(+)HA-966 were unable to completely abolish inward current responses suggesting each compound to be a low efficacy partial agonist (estimated intrinsic activity=∼10 and 20% of glycine, respectively). L-687,414 and MK-801 were compared for their effects on NMDA receptor-dependent LTP in the dentate gyrus of anaethestized rats following high frequency stimulation of the medial perforant path (mPP) afferents. Control rats, administered saline (0.4 ml kg−1 followed by 0.0298 ml min−1), showed a robust augmentation of the population e.p.s.p. risetime (LTP) recorded in the dentate hilus following tetanic stimulation of the mPP. LTP was effectively abolished in a separate group of rats treated with an MK-801 dosing regimen (0.12 mg kg−1 i.v. followed by 1.8 μg kg−1 h−1) known to produce maximal neuroprotection in a rat stroke model but, by contrast, remained largely intact in a third group of animals given a similarly neuroprotective L-687,414 treatment (28 mg kg−1 i.v. followed by 28 mg kg−1 h−1). These experiments suggest that a low level of intrinsic activity at the glycine site may be sufficient to support NMDA receptor-dependent LTP but in circumstances where there is likely to be an excessive NMDA receptor activation the agonism associated with a low efficacy partial agonist, such as L-687,414, is dominated by the antagonist properties. Thus, an NMDA receptor partial agonist profile may offer a therapeutic advantage over neutral antagonists by permitting an acceptable level of `normal' synaptic transmission whilst curtailing excessive receptor activation. British Journal of Pharmacology (1998) 124, 1767–1773; doi:10.1038/sj.bjp.0702010

Recent cloning studies have isolated receptors which confer specific responsiveness to calcitonin gene related peptide (CGRP) and the related peptide adrenomedullin. Using in situ hybridisation, we demonstrate the heterogenous distribution of the mRNAs of two proposed CGRP1 receptors (RDC-1 and calcitonin receptor-like receptor, CRLR) in the rat brain. Adrenomedullin receptor mRNA was weakly expressed, principally in the cerebellum. These findings may assist in the determination of the function of these largely uncharacterised receptors.

These studies in anaesthetised rats showed, using intravital microscopy, that the novel anti-migraine agent, rizatriptan, significantly reduced electrically stimulated dural vasodilation but had no effect on increases in dural vessel diameter produced by exogenous substance P or calcitonin gene-related peptide (CGRP). Rizatriptan also significantly inhibited dural plasma protein extravasation produced by high intensity electrical stimulation of the trigeminal ganglion. We suggest that rizatriptan inhibits the release of sensory neuropeptides from perivascular trigeminal nerves to prevent neurogenic vasodilation and extravasation in the dura mater. These prejunctional inhibitory effects may be involved in the anti-migraine action of rizatriptan.

This study used intravital microscopy to measure the diameter of dural arteries in anaesthetized rats. Electrical stimulation of the surface of a closed cranial window produced increases in dural vessel diameter which were blocked by the CGRP receptor antagonist human-CGRP(8–37) but unaffected by the NK1 receptor antagonist RP67580. Sumatriptan (3 and 0 mg kg-1, iv) significantly reduced the response to electrical stimulation. In contrast, sumatriptan (3 mg kg-1) had no effects on the response to exogenously administered CGRP. These results indicate that neurokinins play no role in neurogenic vasodilation in this preparation and that neurogenic vasodilation in rat dural vessels is mediated predominantly by CGRP. Furthermore, the data indicate that sumatriptan attenuates neurogenic vasodilation, probably by inhibiting the release of CGRP from perivascular trigeminal nerve endings innervating the dura. These experimental data parallel the clinical findings that CGRP levels are elevated in migraine and normalized, concomitantly with headache relief, by sumatriptan.

This study investigated the properties of a novel piperidine ether-based tachykinin NK1 receptor antagonist L-733,060, ((2S,3S)-3-((3,5-bis(trifluoromethyl)phenyl)methyloxy)-2-phenyl piperidine and its 2R,3R-enantiomer L-733,061 on [Ca2+]i mobilisation in Chinese hamster ovary cells transfected with human tachykinin NK1 receptors, compared to their effects in rodent cardiovascular and neurogenic plasma extravasation assays. Using FURA-2-imaging techniques, L-733,060 inhibited substance P-induced [Ca2+]i mobilisation with an estimated affinity of 0.8 nM whereas L-733,061 (30-300 nM) did not. No significant effects of L-733,060 were observed on mean arterial blood pressure or heart rate in conscious or anaesthetised rats at doses of3000 micrograms kg-1 i.v. L-733,060 also stereoselectively inhibited neurogenic plasma extravasation in rat dura produced by electrical stimulation of trigeminal nerves with an ID50 of 212 +/- 19 micrograms kg-1 i.v. Thus, L-733,060 is a novel antagonist of human tachykinin NK1 receptors which stereoselectively inhibits neurogenic plasma extravasation at doses that do not cause adverse cardiovascular effects.

No significant new drugs nor pharmacological strategies were revealed, whilst all trade stands and symposia stressed the use of existing agents (sometimes in new formulations). The cyclo-oxygenase (COX)-2 inhibitors probably constituted the only real innovation in drug therapy since the last Congress in 1993. However, basic research has been productive, and a number of emerging strategies are likely to lead to novel analgesics. Key areas of interest included: neuropeptides; with bradykinin, substance P (SP), calcitonin gene related peptide (CGRP), galanin, and opioids receiving significant attention. N-Methyl-D-aspartate (NMDA) receptor antagonism was a favoured topic in both preclinical and clinical presentations, and there was much enthusiasm for the idea of an NMDA receptor antagonist with an improved therapeutic index (should this be possible) as an analgesic strategy. The idea that morphine tolerance was in part due to protein kinase C (PKC) induction, leading to NMDA receptor phosphorylation and rem...

This small yet popular meeting attracted its usual enthusiastic group of neuropeptide devotees. The bulk of the material presented had already been published, but the symposium on neuropeptide Y (NPY) saw the first public disclosure of the sequence of the NPY Y5 receptor by Synaptic and heard the news that a non-peptide NPY receptor antagonist from Sanofi was moving into clinical development. Interesting sessions were held on the role of neuropeptides in drug and alcohol dependence and on the neurotrophic actions of peptides. This report is selective and deals with only some of the presentations made, which reflects the interests of the authors and is not a value judgement.

The expression of the messenger RNAs encoding N-methyl- d-aspartate receptor subunits in neurologically normal post-mortem human brain was studied by in situ hybridization. In the caudate, putamen and nucleus accumbens strong hybridization signals were observed for N-methyl- d-aspartate R1−1 messenger RNA but much weaker signals for N-methyl- d-aspartate R1–3 and N-methyl- d-aspartate R1–4. N-Methyl- d-aspartate R1–2 was not detectable. N-methyl- d-aspartate R2B was the only N-methyl- d-aspartate R2 subunit detected in these nuclei. In the hippocampus the messenger RNAs for both N-methyl- d-aspartate R1–1 and N-methyl- d-aspartate R1–4 were strongly expressed in the dentate gyrus, CA3-CA1 pyramidal cells, subiculum, entorhinal cortex and perirhinal cortex. Much lower expression was seen for N-methyl- d-aspartate R1–2 and N-methyl- d-aspartate R1–3. The messenger RNAs for both N-methyl- d-aspartate R2A and N-methyl- d-aspartate R2B, but not N-methyl- d-aspartate R2C, subunits were expressed in the hippocampus. In the temporal cortex all N-methyl- d-aspartate R1 isoforms were expressed (N-methyl- d-aspartate R1–1 and N-methyl- d-aspartate R1–4 being the most abundant) and N-methyl- d-aspartate R2A and N-methyl- d-aspartate R2B but not N-methyl- d-aspartate R2C were also moderately expressed. In the brain stem N-methyl- d-aspartate R1–4 was strongly expressed in various nuclei including the locus coeruleus, nucleus centralis superior and deep pontine nuclei. Only weak expression was seen for N-methyl- d-aspartate R1–1 and N-methyl- d-aspartate R1–3 but not N-methyl- d-aspartate R1–2; of the N-methyl- d-aspartate R2 subunits only N-methyl- d-aspartate R2C was found to be expressed in these nuclei. In the cerebellum all the N-methyl- d-aspartate 1 isoforms were expressed (mostly N-methyl- d-aspartate R1–4) in the Purkinje layer which also expressed N-methyl- d-aspartate R2A and N-methyl- d-aspartate R2C. In the molecular layer cells were found expressing N-methyl- d-aspartate R1–4 and N-methyl- d-aspartate R2B and cells in the granule layer were found to express N-methyl- d-aspartate R1−1, N-methyl- d-aspartate R1–3 and N-methyl- d-aspartate R1–4 and N-methyl- d-aspartate R2C only. Preliminary studies indicated that the messenger RNA for the N-methyl- d-aspartate R2D subunit was not expressed in the above areas of brain. These results give the first demonstration of the distribution of N-methyl- d-aspartate receptor subunit messenger RNAs in the human brain. The region-specific expression of subunit combinations suggests a heterogeneity of N-methyl- d-aspartate receptors with diverse physiological/pathophysiological roles and provides a rationale for the development of discriminatory N-methyl- d-aspartate receptor antagonists to target selective neuronal populations.

1. NMDA receptor antagonists have previously been shown to have antinociceptive effects in behavioural experiments, but controversy remains as to the role of NMDA receptors in mechanical hyperalgesia. We have studied the effects on mechanical nociceptive thresholds in rats with carrageenin-induced paw inflammation of L-687,414, a low efficacy partial agonist which acts as a functional antagonist at the glycine modulatory site of the NMDA receptor and of L-701,324, a structurally novel, highly selective, full antagonist at this site. 2. Mechanical thresholds were measured for both hind paws 1 h before and 3 h after carrageenin or saline was injected into 1 hind paw. Dose-response curves were constructed for each test compound in separate experiments, with test compound or vehicle being given i.p. 1 h before the final test. 3. Both compounds produced selective dose-dependent and statistically significant reversal of mechanical hyperalgesia, with minimum effective doses of 100 mg kg-1 L-687,414 and 3 mg kg-1 L-701,324. Neither L-687,414 nor L-701,324 affected the response threshold of the contralateral non-inflamed paw over the dose-range producing reversal of carrageenin-induced hyperalgesia. Neither compound had any effect on the paw oedema produced by carrageenin injection. 4. These results show that both a full antagonist and a low efficacy partial agonist at the glycine modulatory site of the NMDA receptor complex reverse inflammation-induced mechanical hyperalgesia, thus supporting the argument that maximal activation of the glycine site is required for transmission via NMDA receptors, and showing that NMDA receptor-mediated actions are important in mechanical hyperalgesia induced by inflammation.

Insulin and insulin-like growth factors (IGF-I, IGF-II) are closely related polypeptides which are found in the CNS and which promote neuronal survival and neurite outgrowth. They are each associated with specific cell surface receptors and several soluble binding proteins (IGFBPs) which are involved in regulating function and availability. Two analogues of IGF-I were produced by site directed mutagenesis: [Gln3, Ala4, Tyr15, Leu16]IGF-1 (QAYL-IGF) and a B-chain mutant in which the first 16 amino acids of IGF-1 were replaced by the first 17 amino acids of insulin. These analogues have significantly reduced binding affinity for IGFBPs. Using glucose deprivation as a damaging stimulus and assaying lactate dehydrogenase released from cultures as a marker for cell death, we have investigated the effect of IGF analogues on cell death of cerebrocortical and cerebellar granule cell cultures. In the presence of IGF-I, QAYL-IGF or B-chain mutant, the amount of LDH released from cortical and cerebellar granule cell cultures was significantly reduced compared to control (no glucose), indicating that these molecules promote survival. Both QAYL and B-chain mutants, which have reduced affinity for IGFBPs, are as effective as IGF-I in promoting cell survival in conditions of glucose deprivation and their reduced affinity for IGFBPs has no apparent deleterious effect on their neuroprotective function. We also show that the neuroprotective effect of the IGF analogues is due to a direct effect on the neurones in these cultures and is independent of the presence of glia.

1. The ability of bradykinin and its analogues to depolarize rat and mouse superior cervical ganglia was studied by use of in vitro grease-gap recording techniques, and the ability of antagonists selective for bradykinin receptor subtypes to block their effects was examined. 2. Bradykinin (3 microM) depolarized ganglia from both species, although the magnitude of the maximal response was less in mouse (15 +/- 5%, n = 7) than rat tissue (33 +/- 6%, n = 7), relative to muscarine (1 microM). 3. Interleukin 1 beta (30 u ml-1 for 18 h at 37 degrees C) increased the depolarization caused by bradykinin (3 microM) in mouse ganglia from 15% to 54% (P0.001, n = 12). Responses to the B1 receptor agonist, [des-Arg10]-kallidin (3 microM) were similarly potentiated but this was only detected after inhibition of peptidase activity with 10 microM captopril (4% to 35%, n = 5). 4. In ganglia from both species the rank order of agonist potency was bradykinin = [Lys0]-bradykinin[des-Arg10]-kallidin. However, like responses to [des-Arg10]-kallidin in mouse tissue, both the potency of bradykinin and the maximal depolarization achieved (EC50 = 912 nM; 80%, n = 11) was enhanced following inhibition of angiotensin converting enzyme with 10 microM captopril (EC50 = 50 nM; 135%, n = 4). 5. Responses to bradykinin were selectively antagonized by the B2 receptor antagonist, Hoe 140 but not by the B1 antagonist, [Leu8]-bradykinin1-8. From Schild analysis the pA2 value for Hoe 140 in mouse tissue was 9.65, although the slope of the regression line was significantly greater than unity, indicating non-competitive kinetics (slope = 1.88 +/- 0.18, n = 9). The depolarization caused by [Lys0]-bradykinin was also antagonized by Hoe 140 (3 nM).6. Thus the predominant bradykinin receptor in mouse superior cervical ganglia is compatible with a B2 subtype. Furthermore the depolarizations caused by B1 and B2 agonists in this tissue can be increased following exposure to interleukin l beta, and by blocking peptide degradation with captopril.

A time course study in untreated male Sprague-Dawley rats showed that there was no significant difference in the rate of regeneration of motor and sensory axons after a crush injury. Acidic fibroblast growth factor, given topically directly to the site of the crush injury (osmotic minipump for three days) or systemically (i.v. once daily for three days), stimulated the regeneration of motor axons and myelinated sensory axons in the sciatic nerve of the rat. Dose-dependent increases in regeneration distance were seen after 3.6, 36 or 360 ng/day were applied locally and 3 or 10 micrograms/kg per day were given systematically. The greatest effects were achieved with 36 ng/day locally or 10 micrograms/kg per day systematically, when the increase in regeneration distance over three days compared to untreated rats was 47% and 48%, respectively. Administration of heparin vehicle had no significant effect on regeneration. We conclude that since a crush injury has little effect on the endoneurial tubes and supporting cells, the stimulatory effects of acidic fibroblast growth factor on peripheral nerve regeneration seen in this study are likely to be due to a direct acceleration of axonal extension. This is in contrast with the axonal regeneration that occurs across a gap after nerve transection, where axonal extension may be secondary to stimulatory effects on non-neuronal cells providing a supporting "bridge" across the gap. These results suggest that acidic fibroblast growth factor may be clinically useful in the treatment of peripheral neuropathy in man, particularly since systemic treatment for short periods may be effective.

1. Epibatidine is an analgesic substance, isolated from the skin of the poisonous frog Epipedobates tricolor, for which the mechanism of action was previously unknown. 2. The IC50 of synthetic (+)-epibatidine oxalate (the naturally occurring isomer) for [3H]-nicotine binding to rat whole-brain membranes was 0.1 nM. The (-)-isomer also exhibited high affinity (IC50 = 0.2 nM). 3. (+)- and (-)-Epibatidine exhibited much lower affinity for displacement of the muscarinic ligand [3H]-N-methylscopolamine binding to rat cortical membranes (Kapp = 6.9 microM and 16.0 microM respectively). The (+)-enantiomer of epibatidine had an antagonist/agonist (NMS/oxo-M) binding ratio of 4.2 This is consistent with a muscarinic antagonist profile. 4. (+)-Epibatidine oxalate (10 microM) did not cause significant (> 30%) displacement of radioligand binding to opioid, excitatory amino acid, benzodiazepine, 5-HT, dopamine, adrenaline or peptide receptors. 5. (+)- and (-)-Epibatidine (5-20 micrograms kg-1 s.c.) doubled response latency in the mouse hot-plate test. Antinociception and behavioural depression induced by (+)-epibatidine (5 micrograms kg-1) was fully blocked by the nicotinic antagonists mecamylamine (2 mg kg-1 s.c.) or dihydro-beta-erythroidine (2 mg kg-1 s.c.). The muscarinic antagonist scopolamine (0.4 and 10 mg kg-1 s.c.) caused partial reversal of antinociception induced by (+)-epibatidine in mice, but not in rats. 6. These findings demonstrate that (+)-epibatidine oxalate salt is a highly selective and potent nicotinic analgesic agent.

The cellular immediate early genes are involved in the transcriptional events associated with the dopaminergic regulation of neurotransmitter expression within neurons of the neostriatum. To characterize these events in detail, quantitative in situ hybridization histochemistry was used to assess the temporal effects of acute dopamine receptor blockade with eticlopride, a dopamine D2 receptor antagonist, on the messenger RNA expression of the immediate early genes and neurotransmitters/receptors in the caudate-putamen and ventral tegmental area/substantia nigra pars compacta of the rat. Groups of rats were injected with a single dose of either isotonic saline or eticlopride (0.5 mg/kg i.p.) and killed at various time intervals ranging from 5 min to 24 h and frozen brain sections processed by in situ hybridization histochemistry. Using computerized image analysis, the changes in messenger RNA expression for c-fos, c-jun, jun B, jun D, nerve growth factor I-A and nerve growth factor I-B and for neurotensin, glutamate decarboxylase, proenkephalin, the dopamine D1 receptor and the short and long isoforms of the D2 receptor were examined in the caudate-putamen. In the ventral tegmental area and substantia nigra pars compacta, the messenger RNA expression of the above early response genes and that for neurotensin, tyrosine hydroxylase, cholecystokinin and the D2 receptor isoforms were also examined. In the neostriatum, eticlopride caused a rapid increase in c-fos messenger RNA with significantly increased levels at 10 min (P < 0.01). The levels peaked at 30 min and thereafter declined to control levels. A similar profile was observed for jun B messenger RNA, although levels were still significantly (P < 0.01) elevated at 1 h and declined to basal levels thereafter. No significant changes were observed for c-jun, jun D, nerve growth factor I-A and nerve growth factor I-B messenger RNAs. In the dorsolateral neostriatum, there was an increase in proneurotensin messenger RNA 10 min after eticlopride, this increase becoming significant (P < 0.01) at 60 min. Levels were maximal at 2-6 h and decreased after 12 h to basal levels. There were small increases in proenkephalin messenger RNA, but these were not significant (P < 0.05) until 6 h after the injection. Eticlopride did not have any significant effects on the messenger RNA levels for glutamate decarboxylase, the D1 receptor and the short and long isoforms of the D2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Monoclonal antibodies (MAbs) against rat alpha-calcitonin gene-related peptide (alpha CGRP) were produced. Those which bound CGRP in a radioimmunoassay and inhibited the binding of 2-[125I]-iodohistidyl10-CGRP in a receptor binding assay were selected for immunoblockade experiments. 2. The effect of MAbs on CGRP inhibition of electrically stimulated contractions of the rat isolated vas deferens was characterized. Four out of 11 MAbs tested shifted the concentration-response curve of CGRP to the right compared with vehicle or irrelevant MAb control. MAb C4.19 produced equipotent blockade of rat alpha CGRP and rat beta CGRP and was chosen for further studies. MAb C4.19 had no pharmacologically significant effect on the concentration-response relationship of isoprenaline, rat beta-endorphin or somatostatin. 3. We demonstrated that the pharmacological response to CGRP in the presence of MAb C4.19 could be predicted when the dissociation constant and concentration of binding sites of the antibody were known. Comparison of experimental and computer simulated data showed good agreement for EC50 and maximum effect of CGRP in the presence of MAb C4.19. 4. Capsaicin at 1 microM inhibited the electrically stimulated contractions by 60.8% (95% confidence interval 51.8% to 69.9%). This effect was significantly attenuated by MAb C4.19 to 26.0% (95% confidence interval 15.2% to 36.8%; P < 0.003). 5. The immunoblockade of exogenous and endogenous CGRP described here, together with complementary evidence from other studies, strongly suggest that CGRP has a major neurotransmitter role at the neuroeffector junction of the rat vas deferens.

To study whether weaning stimulates the development of a subtype of the delta-opioid receptor we have measured antinociceptive activity of putative delta 1 (DELT I) and delta 2 (DSLET) agonists in weaned and non-weaned 25-day-old rats. In weaned rats, DSLET produced antinociception reversed by the delta 2-antagonist naltriben, but in non-weaned rats DSLET had no effect. Responses to DELT I were evident in both weaned and non-weaned rats and were not antagonised by naltriben. This is the first evidence that delta-receptor subtypes develop independently and suggests that weaning is the stimulus for the expression of the delta 2-subtype.

The 4th International Symposium on CGRP was expertly organized, at a difficult time, by Inger Jansen-Olesen and Lars Edvinsson and held on 28-30 September 2001 at the Royal Danish School of Pharmacy, Copenhagen, Denmark.

1. N-methyl-D-aspartate (NMDA) receptor ion channel antagonists have been reported to cause pronounced increases in cerebral glucose metabolism (CMRglc) and transient reversible vacuolation within pyramidal cortical neurones. The present studies examined in rats the effects of the NMDA receptor antagonist, L-687,414 (R-(+)-cis-4-methyl-3-amino-l-hydroxypyrolid-2-one (+)-cis-4-methyl-HA-966) on regional CMRglc and cortical neuronal morphology. 2. L-687,414 was given as a steady state intravenous infusion for 4 h in a neuroprotective dose regime of 17.5 mg free base kg-1 bolus followed by 225 micrograms kg-1 min-1 (n = 8) or at the higher dose rate of 35 mg kg-1 bolus followed by 440 micrograms kg-1 min-1 (n = 10). Data were compared to a parallel series of experiments in rats given the NMDA receptor ion channel antagonist, dizocilpine for 4 h in the optimum intravenous neuroprotective dose-regime of 0.12 mg kg-1 bolus followed by 1.8 micrograms kg-1 min-1 (n = 8) or at the higher dose rate of 0.4 mg kg-1 bolus followed by 6 micrograms kg-1 min-1 (n = 4; morphology only studied). A saline-infused group of rats (n = 8) were used as controls. 3. CMRglc was studied by use of [14C]-2-deoxyglucose and autoradiography (n = 4 each group) whilst plasma drug levels were in a steady state during the final 45 min of the 4 h drug infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

On behalf of the British Pharmacological Society it is a pleasure, albeit a sad one, to introduce this supplement to the British Journal of Pharmacology which has been put together in order to mark the passing of Jimmy Black and to celebrate his life and work. He was by some margin our most distinguished and celebrated member but he never lost his interest in the practical nuts and bolts of our subject. He had little time for formality and adulation but always seemed happiest talking to young colleagues about their data. His very down to earth persona made it easy to forget that he was responsible for two dramatic therapeutic advances in the introduction of s-adrenoceptor antagonists for the treatment of cardiovascular disease and the H2-antagonists for treating peptic ulcer and associated disorders. His record in drug discovery is unparalleled and it could be argued that all of the honours he accumulated would be more than justified had he made just one of these major therapeutic advances. It is also noteworthy that he made both discoveries whilst working in industrial pharmacology laboratories in the UK, employing small teams of dedicated colleagues and classical in vitro and in vivo pharmacological tools. I hope that those who are unfamiliar with his work will read this and gain an appreciation of why he was so important to our subject and to the BPS. For those who knew him and for whom he was a role model I hope that this will be a memento of a much loved and admired colleague.

The bradykinin B 1 receptor is thought to be induced by tissue injury and inflammation. In the present study, we have investigated whether there is a basal expression of B 1 receptor in dorsal root ganglion (DRG) and trigeminal ganglion neurons in rats. A substantial number of neurons in both DRGs and trigeminal ganglia were found to be B 1 -immunoreactive in rats. Both small and medium-sized DRG neurons were B 1 -immunoreactive, suggesting that they are likely to be Aδ- or C-fibre neurons which are involved in nociceptive transmission. These results support a possible role for B 1 receptors in the modulation of nociceptive sensory transmission.

ATP activates damage-sensing neurons (nociceptors) and can evoke a sensation of pain1. The ATP receptor P2X3 is selectively expressed by nociceptors2, 3 and is one of seven ATP-gated, cation-selective ion channels4, 5, 6. Here we demonstrate that ablation of the P2X3 gene results in the loss of rapidly desensitizing ATP-gated cation currents in dorsal root ganglion neurons, and that the responses of nodose ganglion neurons to ATP show altered kinetics and pharmacology resulting from the loss of expression of P2X2/3 heteromultimers. Null mutants have normal sensorimotor function. Behavioural responses to noxious mechanical and thermal stimuli are also normal, although formalin-induced pain behaviour is reduced. In contrast, deletion of the P2X3 receptor causes enhanced thermal hyperalgesia in chronic inflammation. Notably, although dorsal-horn neuronal responses to mechanical and noxious heat application are normal, P2X3-null mice are unable to code the intensity of non-noxious 'warming' stimuli.

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