Pharmacological characterization of Ro 63-1908 (1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol), a novel subtype-selective N-methyl-D-aspartate antagonist.

Ro 63-1908, 1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol, is a novel subtype-selective N-methyl-D-aspartate (NMDA) antagonist that has been characterized in vitro and in vivo. Ro 63-1908 inhibited [(3)H]dizocilpine ((3)H-MK-801) binding in a biphasic manner with IC(50) values of 0.002 and 97 microM for the high- and low-affinity sites, respectively. Ro 63-1908 selectively blocked recombinant receptors expressed in Xenopus oocytes containing NR1C + NR2B subunits with an IC(50) of 0.003 microM and those containing NR1C + NR2A subunits with an IC(50) of >100 microM, thus demonstrating greater than 20,000-fold selectivity for the recombinant receptors expressing NR1C + NR2B. Ro 63-1908 blocked these NMDA NR2B-subtype receptors in an activity-dependent manner. Ro 63-1908 was neuroprotective against glutamate-induced toxicity and against oxygen/glucose deprivation-induced toxicity in vitro with IC(50) values of 0.68 and 0.06 microM, respectively. Thus, the in vitro pharmacological characterization demonstrated that Ro 63-1908 was a potent and highly selective antagonist of the NR2B subtype of NMDA receptors. Ro 63-1908 was active against sound-induced seizures (ED(50) = 4.5 mg/kg i.p. when administered 30 min beforehand) in DBA/2 mice. The dose required to give a full anticonvulsant effect did not produce a deficit in the Rotarod test. NMDA-induced seizures were also inhibited by Ro 63-1908 with an ED(50) of 2.31 mg/kg i.v. when administered 15 min before testing. Ro 63-1908 gave a dose-related neuroprotective effect against cortical damage in a model of permanent focal ischemia. Maximum protection of 39% was seen at a plasma concentration of 450 ng/ml. There were, however, no adverse cardiovascular or CNS side-effects seen at this dosing level.

Pharmacology of moclobemide.

Moclobemide is a reversible inhibitor of monoamine oxidase (MAO) with clear preference for the A type (so-called RIMA). The enzyme inhibition shows complex kinetics, and the molecular mechanism of interaction with the enzyme is not yet clear. Moclobemide increases the extracellular concentration of the monoamines in rat brain and decreases the level of their metabolites. Neither a loss nor a cumulation of activity has been observed after chronic treatment. Reversibility of MAO-A inhibition was demonstrated in vitro as well as in vivo. In various animal behavioral models, in particular in a novel model of stress-induced anhedonia, moclobemide was as effective as standard antidepressants. Moclobemide improves cognitive functions that are impaired in experimental situations. A neuroprotective action is seen in rats subjected to transient global ischemia/-hypoxia. Moclobemide lacks anticholinergic and other effects and only slightly increases the pressor effect of orally administered tyramine. Possible links between MAO-A inhibition and the various effects of moclobemide on brain function are discussed.

Biochemistry and pharmacology of moclobemide, a prototype RIMA.

RIMA is a term for reversible inhibitors of monoamine oxidase (MAO) with preference for MAO-A; moclobemide is a prototype of this new class of antidepressants and is a highly selective inhibitor of MAO-A in vitro. This inhibition is reversible by dialysis in vitro, which accounts for the dose-dependent duration of in vivo enzyme inhibition of 12-24 h. Moclobemide increases the content of serotonin, noradrenaline and dopamine in the brain, and decreases that of their deaminated metabolites. Its biochemical, neurological and behavioural effects indicate that it increases the extracellular concentration of the classic monoamine neurotransmitters/neuromodulators - in particular 5-HT. Potentiation of the cardiovascular effects of tyramine is less pronounced after taking moclobemide than after irreversible MAO-A inhibitors. Understanding of the physiological role of MAO and of the events that link inhibition of the enzyme with modulation of neuronal activities in the CNS remains incomplete. A major physiological role of intraneuronal MAO is to keep cytosolic amine concentration very low, to enable the neuronal monoamine carriers to produce a net inward transport of monoamines, and thereby to act as the first step in the termination of action of extracellular monoamines. MAO is likely to have a similar function in non-monoaminergic cells with respect to the monoamine carriers they contain. In addition to the classic monoamines, "trace" amines may become functionally active after MAO inhibition. An alternative role for MAO is that of a scavenger, preventing natural substrates from accumulating in monoaminergic neurons and interacting with storage, release, uptake and receptor function of monoamines.

Some basic aspects of reversible inhibitors of monoamine oxidase-A.

A novel class of antidepressants is emerging with considerable therapeutic potential: reversible inhibitors of monoamine oxidase type A (RIMA). Moclobemide (Aurorix) is a representative RIMA. It is a fully and rapidly reversible inhibitor of MAO-A with a correspondingly intermediate duration of action in vivo. It is free of hepatotoxicity and produces a much weaker potentiation of the tyramine pressor effect than the classical irreversible MAO inhibitors. Interaction of MAO inhibitors and monoamine reuptake inhibitors with tyramine is discussed on the basis of experiments in conscious rats. The issue of tyramine content of foods and beverages has been reinvestigated and its relevance for treatment with RIMA antidepressants is discussed. Recently observed antihypoxic (neuroprotective) effects of moclobemide suggest new indications for this compound.

Ro 15-4513: partial inverse agonism at the BZR and interaction with ethanol.

The imidazobenzodiazepinone derivative Ro 15-4513 has the activity profile of a partial inverse (low efficacy) agonist at the benzodiazepine receptor (BZR). It reverses central nervous depressant effects of diazepam, and, in part, of phenobarbitone and ethanol in mice, rats and cats in behavioural, electrophysiological, and neurochemical paradigms. The interaction of Ro 15-4513 with barbiturates and ethanol is due to its inverse agonistic (negative allosteric modulatory) property at the BZR, as it was reversed by the selective BZR blocker flumazenil (Ro 15-1788). In the present experiment situations, other BZR partial inverse agonists in subconvulsant or overt convulsant doses were less effective against ethanol effects than Ro 15-4513. Possible mechanisms for this differential activity of BZR inverse agonists are discussed.

Effect of bilateral decortication on nerve growth factor content in basal nucleus and neostriatum of adult rat brain.

The content of nerve growth factor (NGF) was measured by a sensitive two-site enzyme-linked immunosorbent assay in the basal nucleus and neostriatum at various times following bilateral suction lesions of the cerebral neocortex in adult rats. At 14 and 29 postlesion days, NGF levels in basal nucleus were significantly elevated by 270 and 126%, respectively. In addition, 29 days after cortical lesions, NGF content in the neostriatum was increased 145% above controls. Thus, enhanced NGF production occurs in both basal nucleus and neostriatum of adult rats in response to antero- and/or retrograde neuronal degeneration.

Cholinergic but not monoaminergic denervation increases nerve growth factor content in the adult rat hippocampus and cerebral cortex.

We have investigated whether degeneration of basal forebrain cholinergic neurons is a potential trigger for increased NGF production in the adult rat brain. Electrolytic lesions of cholinergic neurons in the septum-diagonal band and in the nucleus basalis of Meynert induced a transient increase in NGF in the ventral hippocampus (+70%) and cerebral cortex (+125%), respectively. In contrast, selective aminergic denervation of the forebrain by electrolytic lesion of the medial forebrain bundle, did not increase NGF levels in hippocampus and cerebral cortex. Thus, a cholinergic mechanism appears to regulate NGF production in adult rat basal forebrain.

[Hermansky-Pudlak syndrome in a Valais village]. / Syndrome d'Hermansky-Pudlak dans un village valaisan.

Amongst 13 subjects with albinism observed in a village in the Valais, 7 present the clinical and laboratory findings of the Hermansky-Pudlak syndrome. Because of the close family links and the numerous consanguine marriages present in the pedigree, it is probable that the 5 subjects who could not be examined also suffer from the same syndrome. It has an autosomal recessive mode of inheritance. In this isolate the gene and heterozygote frequencies are, respectively, 1:12 and 1:6. Clinically the hemorrhagic diathesis is moderate, the visual and cutaneous symptoms predominate. The platelet dysfunction results from a lack of storage of components (Ca++, nucleotides, amines) normally present in the dense granules. The lack of dense granules is demonstrated by electron and fluorescence microscopy. This structural anomaly is present in the megacaryocyte before individualization of the platelets. Giant melanosomes were found in skin melanocytes.

Effects of clonidine on the rate of noradrenaline turnover in discrete areas of the rat central nervous system.

Turnover of noradrenaline in various regions of the rat brain was estimated by the decrease in noradrenaline content and/or formaldehyde-induced catecholamine fluorescence after inhibition of noradrenaline biosynthesis with alpha-methyl-p-tyrosine. Clonidine (0.1 and 0.3 mg/kg p.o.) decelerated the decrease in noradrenaline content of the locus coeruleus, the nucleus of the solitary tract, the intermediolateral cell column and the ventral horn of the thoracic spinal cord, as measured in tissue punches of the respective regions with a sensitive radioenzymatic method. In all these central regions the clonidine-induced decrease in noradrenaline turnover was antagonized by yohimbine, but not by phenoxybenzamine, indicating mediation through central alpha 2-adrenoceptors, similar to the cardiovascular effects of clonidine. When given alone, both yohimbine and phenoxybenzamine accelerated the disappearance of noradrenaline after inhibition of its biosynthesis. The combined results of radioenzymatic assay and fluorescence histochemistry determinations demonstrated that clonidine markedly reduced noradrenaline turnover in central noradrenaline-containing nerve terminals, but had no effect on the cell bodies of the A1 and A2 cell groups. Noradrenaline turnover was, however, decreased in projection areas of the A1 and A2 cell groups, namely the intermediolateral cell column of the spinal cord and nucleus of the solitary tract, respectively. This observation argues against the existence of a neuronal feedback loop running from the projection areas to the cell bodies of the A1 and A2 cell groups and mediating inhibition of noradrenaline turnover. The effect of clonidine on noradrenaline turnover is, therefore, most likely the result of a local feedback inhibition through presynaptic alpha-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor antagonizes the neurotoxic action of capsaicin on primary sensory neurones.

The protein nerve growth factor (NGF) is a naturally occurring trophic substance for sympathetic neurones and for at least those primary sensory neurones containing substance P (refs 4-6). Thus retrogradely transported NGF increased substance P and protein content in corresponding dorsal root ganglia. Moreover, anti-NGF antibodies administered to newborn rats decreased substance P and somatostatin levels in dorsal root ganglia and dorsal spinal cord, suggesting an important role for NGF in the postnatal development of peptidergic sensory neurones. These neurones appear to be selectively affected by the neurotoxin capsaicin (8-methyl-N-vanillyl-6-nonenamide). Treatment of newborn rats with capsaicin led to degeneration of primary sensory neurones containing substance P, somatostatin, vasoactive intestinal polypeptide and cholecystokinin. The mechanism by which capsaicin evokes its neurotoxic effect is unknown. We report here that in newborn rats concomitant administration of NGF partially antagonized the deleterious effect of capsaicin on substance P-containing neurones in dorsal root ganglia as assessed by morphological and biochemical criteria. We conclude that capsaicin destroys the perikarya of primary sensory peptidergic neurones by interfering with the action of NGF, probably by blocking its retrograde axonal transport.

Nerve growth factor increases substance P, cholecystokinin and vasoactive intestinal polypeptide immunoreactivities in primary sensory neurones of newborn rats.

Immunoreactive substance P, somatostatin, gastrin/cholecystokinin and vasoactive intestinal polypeptide were studied in lumbar dorsal root ganglia of 14-day-old rats treated from day 2 to 11 of life with nerve growth factor. Increased staining intensity of neuronal cell bodies and processes for substance P, gastrin/cholecystokinin and vasoactive intestinal polypeptide was observed by immunohistochemistry indicating increased neuronal peptide concentrations. These results were supported by radioimmunoassays showing increased ganglionic levels of substance P and vasoactive intestinal polypeptide. Both techniques, however, failed to show a significant increase of somatostatin levels.

Supra-ependymal serotoninergic nerves in mammalian brain: morphological, pharmacological and functional studies.

Supra-ependymal nerves in mammals (mainly rats) have been shown to contain serotonin (5-hydroxytryptamine, 5-HT) by combined Falck-Hillarp fluorescence histochemistry, ultrastructural monoamine cytochemistry and pharmacology as well as by immunohistochemistry and autoradiography. Supra-ependymal 5-HT cells do not occur. At least in rats, virtually all supra-ependymal nerves contain 5-HT and in our opinion the occasionally described non-5-HT supra-ependymal nerve cells and their processes contribute little to the supra-ependymal nerve plexus (with the possible exception of those cells above the median eminence). The cells of origin of the supra-ependymal 5-HT nerves are situated in raphe nuclei. The axons and terminals (varicosities) contain small and large dense core vesicles in both of which 5-HT is stored. A co-transmitter has not been found among the candidates investigated so far (leu- and met-enkephalin, substance P and gamma-aminobutyric acid (GABA)). The nerves possess uptake mechanisms specific for 5-HT and possibly GABA. Occasionally desmosome-like junctions are observed between 5-HT nerve terminals and ependymal cells but no true synapses. The function of these nerves is not known. They do not appear to regulate ciliary movement, but might influence the shape of ependymal cells.

Substance P-, met-enkephalin- and somatostatin-like immunoreactivity distribution in the frog spinal cord.

The distribution of substance P-, Met-enkephalin- and somatostatin-like immunoreactivity was studied in the thoracic spinal cord of the frog using immunohistochemical techniques. In fibres, probably nerve terminals, immunoreactivity was greatest in the grey matter (mainly dorsal horn), but it was also present in white matter regions. While substance P- and, perhaps, somatostatin-like immunoreactivity appeared to be contained in primary afferents, the presence of all 3 peptides in neuronal cells of the grey matter indicates the existence of a propriospinal peptidergic system.

Demonstration of supra-ependymal 5-HT nerve fibres in human brain and their immunohistochemical identification in rat brain.

When samples of the ventricle surfaces of human post-mortem brain were examined by scanning- and transmission electron microscopy, varicose nerve fibres could be seen traversing among the cilia and microvilli of ependymal cells. The varicosities contained numerous small electron-lucent vesicles and frequent large electron-dense vesicles, and were usually nonsynaptic but occasionally anchored to the surface by desmosome-like junctions. Supra-ependymal nerve fibres were observed in the lateral ventricles (e.g., n. caudatus), foramen of Monro (stria medullaris), third ventricle (habenula) and floor of the fourth ventricle in brains of the five cases examined. However, only in one of these was a yellow formaldehyde-induced fluorescence observed (on the fourth ventricle floor). Its discrete granular appearance, rapid fading and colour were typical of supra-ependymal 5-HT nerve fibres observed in rat brain. Very recent investigations on serial cryostat sections of rat brain ventricle regions revealed the absence of an immunohistochemical reaction with antisera to substance P, leu- and met-enkephalin and glutamic acid decarboxylase, but the presence of a reaction with 5-HT antiserum. The target for impulse-released 5-HT from this nonsynaptic 5-HT nerve plexus, bathed in cerebrospinal fluid, is not yet known.

Storage pool disease: comparative fluorescence microscopical, cytochemical and biochemical studies on amine-storing organelles of human blood platelets.

The platelet content of ATP, ADP, serotonin (5-HT), dopamine, noradrenaline and adrenaline as well as the net uptake of radiolabelled 5-HT and mepacrine were subnormal in six patients with Storage Pool Disease (SPD). Fewer amine-storing organelles were found by fluorescence microscopy with the fluorescent probe mepacrine and by electron microscopy with a cytochemical (uranaffin) reaction specific for 5'-phosphonucleotides. Both methods showed that SPD platelets have atypical organelles with a reduced capacity to store amines, 5'-phosphonucleotides and mepacrine. The changes were most marked in platelets of the two patients who also had oculocutaneous albinism.

Absence of dopamine sensitive adenylate cyclase in the A10 region, the origin of mesolimbic dopamine neurones.

Dopamine (DA) failed to stimulate the adenylate cyclase of the mesolimbic A10 DA nerve cell body area, in contrast to tis activating effect in the nigrostriatal A9 DA cell body area. The enzyme was stimulated by GMPPNP (a GTP analog) and NaF. This indicates the absence in the A 10 cell area of DA receptors with functional coupling on adenylate cyclase, in contrast to the A9 cell area where such DA receptors are believed to be located on afferent axon terminals.