Short-course subcutaneous treatment with PQ Grass strongly improves symptom and medication scores in grass allergy.

BACKGROUND: A modified grass allergen subcutaneous immunotherapy (SCIT) product with MicroCrystalline Tyrosine and monophosphoryl lipid-A as an adjuvant system (Grass MATA MPL [PQ Grass]) is being developed as short-course treatment of grass-pollen allergic rhinitis (SAR) and/or rhinoconjunctivitis. We sought to evaluate the combined symptom and medication score (CSMS) of the optimized cumulative dose of 27,600 standardized units (SU) PQ Grass in a field setting prior to embarking on a pivotal Phase III trial. METHODS: In this exploratory, randomized, double-blind, placebo-controlled trial subjects were enrolled across 14 sites (Germany and the United States of America). Six pre-seasonal subcutaneous injections of PQ Grass (using conventional or extended regimens) or placebo were administered to 119 subjects (aged 18-65 years) with moderate-to-severe SAR with or without asthma that was well-controlled. The primary efficacy endpoint was CSMS during peak grass pollen season (GPS). Secondary endpoints included Rhinoconjunctivitis Quality of Life Questionnaire standardized (RQLQ-S) and allergen-specific IgG4 response. RESULTS: The mean CSMS compared to placebo was 33.1% (p = .0325) and 39.5% (p = .0112) for the conventional and extended regimens, respectively. An increase in IgG4 was shown for both regimens (p < .01) as well as an improvement in total RQLQ-S for the extended regimen (mean change -0.72, p = .02). Both regimens were well-tolerated. CONCLUSIONS: This trial demonstrated a clinically relevant and statistically significant efficacy response to PQ Grass. Unprecedented effect sizes were reached for grass allergy of up to ≈40% compared to placebo for CSMS after only six PQ Grass injections. Both PQ Grass regimens were considered equally safe and well-tolerated. Based on enhanced efficacy profile extended regime will be progressed to the pivotal Phase III trial.

Perampanel, an antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, for the treatment of epilepsy: studies in human epileptic brain and nonepileptic brain and in rodent models.

Perampanel [Fycompa, 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile hydrate 4:3; Eisai Inc., Woodcliff Lake, NJ] is an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist used as an adjunctive treatment of partial-onset seizures. We asked whether perampanel has AMPA receptor antagonist activity in both the cerebral cortex and hippocampus associated with antiepileptic efficacy and also in the cerebellum associated with motor side effects in rodent and human brains. We also asked whether epileptic or nonepileptic human cortex is similarly responsive to AMPA receptor antagonism by perampanel. In rodent models, perampanel decreased epileptic-like activity in multiple seizure models. However, doses of perampanel that had anticonvulsant effects were within the same range as those engendering motor side effects. Perampanel inhibited native rat and human AMPA receptors from the hippocampus as well as the cerebellum that were reconstituted into Xenopus oocytes. In addition, with the same technique, we found that perampanel inhibited AMPA receptors from hippocampal tissue that had been removed from a patient who underwent surgical resection for refractory epilepsy. Perampanel inhibited AMPA receptor-mediated ion currents from all the tissues investigated with similar potency (IC50 values ranging from 2.6 to 7.0 µM). Cortical slices from the left temporal lobe derived from the same patient were studied in a 60-microelectrode array. Large field potentials were evoked on at least 45 channels of the array, and 10 µM perampanel decreased their amplitude and firing rate. Perampanel also produced a 33% reduction in the branching parameter, demonstrating the effects of perampanel at the network level. These data suggest that perampanel blocks AMPA receptors globally across the brain to account for both its antiepileptic and side-effect profile in rodents and epileptic patients.

Evidence for anti-inflammatory and putative analgesic effects of a monoclonal antibody to calcitonin gene-related peptide.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a powerful pro-inflammatory mediator thought to play a significant role in the development of inflammation and pain. We investigated the role of CGRP in trigeminal inflammatory pain by determining the ability of a monoclonal antibody to CGRP to modify central Fos expression in response to stimulation of the inflamed ferret tooth pulp. We also assessed the effect of the antibody on pulpal inflammation. METHODS: Ten adult ferrets were prepared under anaesthesia to allow stimulation of the upper and lower left canine pulps, recording from the digastric muscle and intravenous injections at subsequent experiments. In all animals, pulpal inflammation was induced by introducing human caries into a deep buccal cavity. Four days later animals were treated intravenously with either CGRP antibody (n=5) or vehicle (n=5). After a further 2 days animals were re-anaesthetised and the tooth pulps stimulated at 10 times jaw-opening reflex threshold. Brainstems and tooth pulps were processed immunohistochemically for Fos and the common leucocyte marker CD45, respectively. RESULTS: Fos was expressed in ipsilateral trigeminal subnuclei caudalis (Vc) and oralis (Vo). Significantly fewer Fos-positive nuclei were present within Vc of CGRP antibody-treated animals (p=0.003 vs vehicle-treated). Mean percentage area of staining for CD45 was significantly less in antibody-treated animals (p=0.04 vs vehicle-treated). CONCLUSIONS: This is the first direct evidence that sequestration of CGRP has anti-inflammatory and putative analgesic effects. Previous studies using this Fos model have demonstrated that it is able to predict clinical analgesic efficacy. Thus these data indicate that this antibody may have analgesic effects in dental pain and other types of inflammatory-mediated transmission, and suggest that this is in part due to peripheral anti-inflammatory effects.

Precise quantification of haemoglobin in erythroid precursors and plasma.

INTRODUCTION: Haemoglobin (Hb) quantification in whole blood is possible by various spectrophotometric methods. However, determination of low-level Hb in erythroid precursors or haemolytic plasma is inaccurate because of contribution from light scatter and/or nonhaemoglobin components with overlapping absorbance. Therefore, this study developed a sole method allowing accurate spectrophotometric quantification of Hb at a low concentration range. METHODS: Advantage was taken of the unique absorption spectra of carbon monoxide-Hb complex (COHb) as compared to oxyHb. The visible absorption spectra of samples were recorded prior and following carbon monoxide exposure. A difference extinction coefficient at the maximal difference absorption was used to calculate Hb concentrations. RESULTS: Known amounts of Hb were added to mouse erythroleukaemia (MEL) cells lysate or plasma to yield known 'theoretical' concentrations. The concentrations were measured by the current and known methods. The current method was found much more accurate compared with previous methods specifically at low concentrations. CONCLUSION: The method is valid for accurate quantification of Hb at a wide concentration range (>0.1 µm/L) in erythroid precursors or plasma and is optional for other biological fluids.

Physiological roles of neuronal nicotinic receptor subtypes: new insights on the nicotinic modulation of neurotransmitter release, synaptic transmission and plasticity.

Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the mammalian central nervous system (CNS). Despite this, very little was known, until recently, about their physiological role. In the periphery, nicotinic receptors mediate vital excitatory fast synaptic cholinergic transmission at both the neuromuscular junction and ganglia. In the brain, this role has been mainly "delegated" to glutamate receptors. The very broad cholinergic innervations of most brain areas, including the cortex, have implicated this system, and brain nicotinic receptors in particular, in a unique "modulatory" role of other transmitters systems. Recent evidence confirms, on one hand, that brain nicotinic receptors have a dominant "presynaptic" modulatory function, controlling the release of both acetylcholine (auto-receptors) and other neurotransmitters (hetero-receptors). On the other hand, more experimental data support the idea that a variable component of fast synaptic transmission in the brain can also be mediated by "postynaptic" nicotinic receptors, which, in turn, can control cell excitability. A challenging goal is to identify which one of the plethora of nicotinic receptor subtypes is mediating each effect in different brain areas, and which of these receptors and functions are lost or affected in different human neuro-psychiatric disorders. Needless to say, a better understanding of the physiological role of brain nicotinic receptors will drive our quest for more selective and efficacious nicotinic receptor targeted therapeutic agents.

Voltage-operated calcium channel heterogeneity in pancreatic beta cells: physiopathological implications.

Voltage-operated calcium channels play crucial roles in stimulus-secretion coupling in pancreatic beta cells. A growing body of evidence indicates that these channels in beta cells are heterogeneous. In particular, not all the high-threshold calcium channels expressed belong to the best known L-type. In rat insulinoma cells, for example, L, N, and P/Q-type channels are present, while in human beta cells L-type and P/Q-type dominate. Where present, N-type and P/Q-type channels participate, alongside with the dominant L-type, in the control of sugar- or depolarization-induced hormone release. Distinct biophysical properties and selective modulation of the channel subtypes are likely to play important physiological roles. T-type channels are involved in beta cell apoptosis, while calcium channel autoantibodies recognizing high-threshold channels in beta cells, have been described both in neurological and diabetic patients. Subtype-selective calcium channel drugs have the potential for being beneficial in beta cell pathological states.

5-Hydroxyindole potentiates human alpha 7 nicotinic receptor-mediated responses and enhances acetylcholine-induced glutamate release in cerebellar slices.

The effects of 5-hydroxyindole (5-HI) have been investigated on human alpha 7 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes and GH4 cells, on native alpha 7 nAChRs expressed by IMR-32 cells and on alpha 7 nAChR-mediated events in mossy fibre-granule cell synapses in rat cerebellar slices. In oocytes expressing alpha 7 nAChRs, 5-HI potentiated sub-maximal, 60 micro M ACh-induced ion currents in a concentration-dependent manner, the threshold effective concentration being 30 micro M. 5-HI itself did not act as an agonist on alpha 7 nAChRs. A maximum potentiation of 12 times the control was observed at 20 mM 5-HI. The effect of 1 mM 5-HI on the concentration-response curve for ACh revealed that 5-HI increased the potency as well as the efficacy of ACh on alpha 7 nAChRs. 5-HI also potentiated alpha 7-mediated increases in intracellular free calcium levels in both mammalian cells heterologously expressing human alpha 7 nAChRs and in human IMR-32 neuroblastoma cells expressing native alpha 7 nAChRs. At mossy fibre-granule cell synapses, application of 1 mM ACh induced glutamate-evoked excitatory post-synaptic currents (EPSCs). Co-application of 1 mM 5-HI with 1 mM ACh further increased the frequency of the EPSCs. The ACh-induced release, as well as the 5-HI-induced enhancement of release, were blocked by 1-10 nM methyllycaconitine or 200 nM alpha-bungarotoxin, demonstrating that both effects were mediated by presynaptic alpha 7 nAChRs. The results demonstrate that responses mediated by alpha 7 nAChRs are strongly potentiated by 5-HI.

Calcium channel subtypes contributing to acetylcholine release from normal, 4-aminopyridine-treated and myasthenic syndrome auto-antibodies-affected neuromuscular junctions.

1 Acetylcholine release at the neuromuscular junction relies on rapid, local and transient calcium increase at presynaptic active zones, triggered by the ion influx through voltage-dependent calcium channels (VDCCs) clustered on the presynaptic membrane. Pharmacological investigation of the role of different VDCC subtypes (L-, N-, P/Q- and R-type) in spontaneous and evoked acetylcholine (ACh) release was carried out in adult mouse neuromuscular junctions (NMJs) under normal and pathological conditions. 2 omega-Agatoxin IVA (500 nM), a specific P/Q-type VDCC blocker, abolished end plate potentials (EPPs) in normal NMJs. However, when neurotransmitter release was potentiated by the presence of the K(+) channel blocker 4-aminopyridine (4-AP), an omega-agatoxin IVA- and omega-conotoxin MVIIC-resistant component was detected. This resistant component was only partially sensitive to 1 micro M omega-conotoxin GVIA (N-type VDCC blocker), but insensitive to any other known VDCC blockers. Spontaneous release was dependent only on P/Q-type VDCC in normal NMJs. However, in the presence of 4-AP, it relied on L-type VDCCs too. 3 ACh release from normal NMJs was compared with that of NMJs of mice passively injected with IgGs obtained from patients with Lambert-Eaton myasthenic syndrome (LEMS), a disorder characterized by a compromised neurotransmitter release. Differently from normal NMJs, in LEMS IgGs-treated NMJs an omega-agatoxin IVA-resistant EPP component was detected, which was only partially blocked by calciseptine (1 micro M), a specific L-type VDCC blocker. 4 Altogether, these data demonstrate that multiple VDCC subtypes are present at the mouse NMJ and that a resistant component can be identified under 'pharmacological' and/or 'pathological' conditions.

Regulation of GABA release by nicotinic acetylcholine receptors in the neonatal rat hippocampus.

1. The whole-cell configuration of the patch-clamp technique was used to study the modulation of giant depolarizing potentials (GDPs) by nicotinic acetylcholine receptors (nAChRs) in CA3 hippocampal neurons in slices from postnatal day (P) 2-6 rats. 2. Bath application of nicotine increased GDP frequency in a concentration-dependent manner. For example, nicotine (0.5-1 microM) enhanced GDP frequency from 0.05 +/- 0.04 to 0.17 +/- 0.04 Hz. This effect was prevented by the broad-spectrum nicotinic receptor antagonist dihydro-beta-erythtroidine (DHbetaE, 50 microM) and partially antagonized by methyllycaconitine (MLA, 50 nM) a competitive antagonist of alpha7 nAChRs. GDP frequency was also enhanced by AR-17779 (100 microM), a selective agonist of alpha7 nAChRs. 3. The GABA(A) receptor antagonist bicuculline (10 microM) and the non-NMDA glutamate receptor antagonist DNQX (20 microM) blocked GDPs and prevented the effects of nicotine on GDPs. In the presence of DNQX, nicotine increased GABA-mediated synaptic noise, indicating that this drug may have a direct effect on GABAergic interneurons. 4. Bath application of edrophonium (20 microM), a cholinesterase inhibitor, in the presence of atropine (1 microM), increased GDP frequency, indicating that nAChRs can be activated by ACh released from the septo-hippocampal fibres. This effect was prevented by DHbetaE (50 microM). 5. In the majority of neurons tested, MLA (50 nM) and DHbetaE (50 microM) reduced the frequency of GDPs with different efficacy: a reduction of 98 +/- 11 and 61 +/- 29 % was observed with DHbetaE and MLA, respectively. In a subset of cells (40 % in the case of MLA and 17 % in the case of DHbetaE) these drugs induced a twofold increase in GDP frequency. 6. It is suggested that, during development, nAChRs modulate the release of GABA, assessed as GDPs, through distinct nAChRs. The rise of intracellular calcium via nAChRs would further strengthen GABA-mediated oscillatory activity. This can be crucial for consolidation of synaptic contacts and for the fine-tuning of the developing hippocampus.

Ultrastructural distribution of the alpha7 nicotinic acetylcholine receptor subunit in rat hippocampus.

Acetylcholine (ACh) is an important neurotransmitter in the mammalian brain; it is implicated in arousal, learning, and other cognitive functions. Recent studies indicate that nicotinic receptors contribute to these cholinergic effects, in addition to the established role of muscarinic receptors. In the hippocampus, where cholinergic involvement in learning and memory is particularly well documented, alpha7 nicotinic acetylcholine receptor subunits (alpha7 nAChRs) are highly expressed, but their precise ultrastructural localization has not been determined. Here, we describe the results of immunogold labeling of serial ultrathin sections through stratum radiatum of area CA1 in the rat. Using both anti-alpha7 nAChR immunolabeling and alpha-bungarotoxin binding, we find that alpha7 nAChRs are present at nearly all synapses in CA1 stratum radiatum, with immunolabeling present at both presynaptic and postsynaptic elements. Morphological considerations and double immunolabeling indicate that GABAergic as well as glutamatergic synapses bear alpha7 nAChRs, at densities approaching those observed for glutamate receptors in CA1 stratum radiatum. Postsynaptically, alpha7 nAChRs often are distributed at dendritic spines in a perisynaptic annulus. In the postsynaptic cytoplasm, immunolabeling is associated with spine apparatus and other membranous structures, suggesting that alpha7 nAChRs may undergo dynamic regulation, with insertion into the synapse and subsequent internalization. The widespread and substantial expression of alpha7 nAChRs at synapses in the hippocampus is consistent with an important role in mediating and/or modulating synaptic transmission, plasticity, and neurodegeneration.

Evidence for nicotinic acetylcholine receptor activation in rat cerebellar slices.

Neuronal nicotinic ACh receptor (nAChR) activation is known to enhance glutamate and GABA release in different brain areas. Moreover, nAChRs play an important role in neuronal differentiation. By using the patch-clamp technique, we have investigated the presence of nAChRs in cerebellar granule cells in slices from P5-P14 rats. Application of ACh (1 mM) could elicit a variety of effects. Some cells did not respond at all. In other cells, a somatic current was activated. In a proportion of cells, postsynaptic currents (PSCs), with or without somatic current, were elicited. Somatic nAChRs are likely to be of the alpha(4)beta(2) subtype, but the presence of other subunit combinations (alpha(7)- or beta(4)-containing receptors) cannot be ruled out. The ACh-induced PSCs were glutamatergic in nature. Thus, in a reasonable proportion of cells, nicotinic receptors are present presynaptically. They are likely to be alpha(7) receptors whose activation elicits Glu release via a TTX-sensitive mechanism. Our experiments are the first electrophysiological evidence showing, in a native cerebellar preparation, the presence of nicotinic receptors at the mossy fibre-granule cell synapse at early developmental stages.

Peptide neurotoxins, small-cell lung carcinoma andneurological paraneoplastic syndromes.

Peptide neurotoxins isolated from the venom of snakes, spiders and snails have represented invaluable tools for the identification and characterisation of membrane ion channels and receptors in vertebrate cells, including human neurons. We here report on the use of these toxins for the characterisation of membrane ion channels and receptors expressed by one of the most aggressive human cancers, small-cell lung carcinoma. This tumour shares many properties with other neuro-endocrine cell types, including the ability of firing action potentials and release hormones in a calcium-dependent manner. Toxins such as alpha-bungarotoxin and omega-conotoxins, among others, have been successfully used to characterise neuronal nicotinic receptors and voltage-dependent calcium channels, respectively, in human small-cell lung carcinoma cells. These receptors and ion channels are not only crucial for the growth of this specific tumour, but also represent autoantigens against which cancer patients build an autoimmune response. Although the aim of this autoimmune response is eventually the destruction of the cancer cells, the circulating antibodies cross-react with similar ion channels and receptors present in normal neurons or other cells, causing a number of different paraneoplastic diseases, the best characterised of which is the Lambert-Eaton myasthenic syndrome. Conotoxin-based radioimmunoassays have become an invaluable tool for the diagnosis and follow up of these paraneoplastic disorders and could represent a step forward in the early diagnosis of small-cell lung carcinoma itself.

Identification and cellular localisation of voltage-operated calcium channels in immature rat testis.

Sertoli cells regulate the spermatogenic process mainly through the secretion of a complex fluid into the lumen of the seminiferous tubules behind the blood-testis barrier, containing many of the essential proteins necessary for maintenance and maturation of male germ cells. Thus, the study of Sertoli cell secretory processes is strictly correlated with the understanding of the regulatory mechanisms of spermatogenesis. In this work the authors have explored the voltage-sensitive calcium channel variety in the immature rat testis, their localisation and distribution within the seminiferous epithelium and peritubular and interstitial tissues as well as the possible role in the control of Sertoli cell secretion. The results reported in this paper, obtained by in situ hybridisation, immunohistology of rat testicular sections and Western blot analysis of Sertoli cell plasma membranes, show that mammalian Sertoli cells express mRNA encoding for several voltage-operated calcium channel subunits and express such proteins on their surface. Experiments performed on Sertoli cell monolayers cultured in the presence of specific toxins indicate that both N and P/Q-type Ca(2+) channels are involved in the regulation of protein secretion.

Modulation of N-type calcium channels translocation in RINm5F insulinoma cells.

An intracellular pool of N-type voltage-operated calcium channels has recently been described in both IMR32 human neuroblastoma and PC12 rat pheochromocytoma cells. These channels were found to be accumulated in subcellular fractions where the chromogranin B-containing secretory granules were also enriched. Upon exocytosis N-type calcium channels were reversibly inserted in the plasma membrane. We have now extended this study to RINm5F rat insulinoma cells, and characterized the parallelism between the 'regulated' secretion of serotonin and the recruitment of surface calcium channels. Exocytosis was stimulated by different means, such as depolarization with high KCl, high Ba(2+)alone or protein kinase C activation; on the other hand exocytosis was inhibited with the non-selective calcium channel antagonist Cd(2+)or with noradrenaline. Stimulated release was always accompanied, with parallel kinetics, by calcium channel recruitment, while inhibition of secretion blocked calcium channel recruitment too. During repetitive depolarizations we revealed a potentiation of [Ca(2+)]()i transients in single Fura-2 loaded RINm5F cells, that was accompanied by an increase in surface VOCCs, suggesting a physiological role for the newly recruited channels. 2000 Academic Press@p$hr

Inactivation properties of human recombinant class E calcium channels.

The electrophysiological and pharmacological properties of alpha(1E)-containing Ca(2+) channels were investigated by using the patch-clamp technique in the whole cell configuration, in HEK 293 cells stably expressing the human alpha(1E) together with alpha(2b) and beta(1b) accessory subunits. These channels had current-voltage (I-V) characteristics resembling those of high-voltage-activated (HVA) Ca(2+) channels (threshold at -30 mV and peak amplitude at +10 mV in 5 mM Ca(2+)). The currents activated and deactivated with a fast rate, in a time- and voltage-dependent manner. No difference was found in their relative permeability to Ca(2+) and Ba(2+). Inorganic Ca(2+) channel blockers (Cd(2+), Ni(2+)) blocked completely and potently the alpha(1E,)/alpha(2b)delta/beta(1b) mediated currents (IC(50) = 4 and 24.6 microM, respectively). alpha(1E)-mediated currents inactivated rapidly and mainly in a non-Ca(2+)-dependent manner, as evidenced by the fact that 1) decreasing extracellular Ca(2+) from 10 to 2 mM and 2) changing the intracellular concentration of the Ca(2+) chelator 1. 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), did not affect the inactivation characteristics; 3) there was no clear-cut bell-shaped relationship between test potential and inactivation, as would be expected from a Ca(2+)-dependent event. Although Ba(2+) substitution did not affect the inactivation of alpha(1E) channels, Na(+) substitution revealed a small but significant reduction in the extent and rate of inactivation, suggesting that besides the presence of dominant voltage-dependent inactivation, alpha(1E) channels are also affected by a divalent cation-dependent inactivation process. We have analyzed the Ca(2+) currents produced by a range of imposed action potential-like voltage protocols (APVPs). The amplitude and area of the current were dependent on the duration of the waveform employed and were relatively similar to those described for HVA calcium channels. However, the peak latency resembled that obtained for low-voltage-activated (LVA) calcium channels. Short bursts of APVPs applied at 100 Hz produced a depression of the Ca(2+) current amplitude, suggesting an accumulation of inactivation likely to be calcium dependent. The human alpha(1E) gene seems to participate to a Ca(2+) channel type with biophysical and pharmacological properties partly resembling those of LVA and those of HVA channels, with inactivation characteristics more complex than previously believed.

Evaluation of the activity of a novel metabotropic glutamate receptor antagonist (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) in the in vitro neonatal spinal cord and in an in vivo pain model.

The cyclobutylglycine (+/-)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) has been identified as a functionally potent metabotropic glutamate receptor antagonist. It is most potent on the two group I metabotropic glutamate receptors, 1alpha and 5alpha, with IC50 values of 1.0+/-0.4 microM and 1.6+/-1.4 microM, respectively. In this study, LY393053 has also been evaluated electrophysiologically on native group I metabotropic glutamate receptors in an in vitro spinal cord preparation as well as behaviourally, in a mouse model of visceral pain. LY393053 dose-dependently antagonised group I agonist, (RS)-3, 5-dihydroxyphenylglycine, or a broad-spectrum agonist (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation of spinal motoneurons. The apparent Kd values were estimated to be 0.3 microM against (RS)-3, 5-dihydroxyphenylglycine-induced depolarisation and 0.5 microM against (1S,3R)-amino-1,3-cyclopentanedicarboxylic acid-induced depolarisation, respectively. On the other hand, the dorsal root-ventral root potential elicited at 8 x threshold was depressed by LY393053 with IC50 values of 9.0+/-0.7 microM and 12.7+/-1.7 microM on monosynaptic and polysynaptic responses, respectively. When investigated using the mouse acetic acid writhing test, LY393053 showed significant analgesic effects at doses of 1-10 mg/kg intraperitoneally. An ED50 value of 6.0 mg/kg was obtained in this test. By revealing a potent effect of LY393053 in antagonising the native group I metabotropic receptor-mediated responses in the spinal cord in rodents, and an antinociceptive efficacy in a mouse visceral pain model, these results, therefore, provide additional evidence in support of the analgesic potential of metabotropic glutamate receptor antagonists.

Distribution of the voltage-dependent calcium channel alpha1G subunit mRNA and protein throughout the mature rat brain.

The molecular identity of a gene which encodes the pore-forming subunit (alpha1G) of a member of the family of low-voltage-activated, T-type, voltage-dependent calcium channels has been described recently. Although northern mRNA analyses have shown alpha1G to be expressed predominantly in the brain, the detailed cellular distribution of this protein in the central nervous system (CNS) has not yet been reported. The current study describes the preparation of a subunit specific alpha1G riboprobe and antiserum which have been used in parallel in situ mRNA hybridization and immunohistochemical studies to localize alpha1G in the mature rat brain. Both alpha1G mRNA and protein were widely distributed throughout the brain, but variations were observed in the relative level of expression in discrete nuclei. Immunoreactivity for alpha1G was typically localized in both the soma and dendrites of many neurons. Whilst alpha1G protein and mRNA expression were often observed in cells known to exhibit T-type current activity, some was also noted in regions, e.g. cerebellar granule cells, in which T-type activity has not been described. These observations may reflect differences between the subcellular distribution of channels that can be identified by immunohistochemical methods compared with electrophysiological techniques.

Metabolism and trafficking of N-type voltage-operated calcium channels in neurosecretory cells.

The N-type voltage-operated calcium channel has been characterized over the years as a high-threshold channel, with variable inactivation kinetics, and a unique ability to bind with high affinity and specificity omega-conotoxin GVIA and related toxins. This channel is particularly expressed in some neurons and endocrine cells, where it participates in several calcium-dependent processes, including secretion. Omega-conotoxin GVIA was instrumental not only for the biophysical and pharmacological characterization of N-type channels but also for the development of in vitro assays for studying N-type VOCC subcellular localization, biosynthesis, turnover, as well as short-and long-term regulation of its expression. We here summarize our studies on N-type VOCC expression in neurosecretory cells, with a major emphasis on recent data demonstrating the presence of N-type channels in intracellular secretory organelles and their recruitment to the cell surface during regulated exocytosis.