M. Da Prada

Immunoregulation mediated by the sympathetic nervous system.

Abstract A postulated immunoregulatory role for the autonomous nervous system was explored utilizing several in vivo and in vitro approaches. Local surgical denervation of the spleen in rats and general chemical sympathectomy by 6-hydroxydopamine combined with adrenalectomy yielded a similar removal of restraint expressed as enhancement in the number of PFC in response to immunization. Noradrenaline and the synthetic α-agonist clonidine which are, respectively, natural and artificial effector molecules of the sympathetic nervous system each strongly suppressed the in vitro induced immune response of murine spleen cells to SRBC. Further, radiometric-enzymatic assay of noradrenaline in the splenic pulp revealed a decrease in the content of this neurotransmitter just preceding the exponential phase of the immune response to SRBC (Days 3 and 4) in this site. Taken together, these findings point to a dynamic immunoregulatory relationship between the immune and sympathetic nervous system.

Increased monoamine oxidase b activity in plaque-associated astrocytes of Alzheimer brains revealed by quantitative enzyme radioautography

The aetiology and pathogenesis of Alzheimers disease are currently poorly understood, but symptomatic disease is associated with amyloid plaques, neurofibrillary tangles, neuronal loss and numerous alterations of neurotransmitter systems in the CNS. Monoamine oxidase type B is known to be increased in Alzheimer diseased brains. The distribution and abundance of catalytic sites for monoamine oxidases A and B in post mortem human brains of 11 Alzheimer disease cases and five age-matched controls were investigated by quantitative enzyme radioautography. Using tritiated monoamine oxidase inhibitors (Ro41-1049 and lazabemide)--as high affinity substrates selective for monoamine oxidases A and B, respectively--it was found that monoamine oxidase B activity increased up to three-fold exclusively in temporal, parietal and frontal cortices of Alzheimer disease cases compared with controls. This increase was restricted to discrete patches (approximately 185 microns in diameter) which occupied approximately 12% of the cortical areas examined. In other brain regions (hippocampal formation >> caudate-putamen > cerebellum), patches of [3H]lazabemide-enriched binding were less abundant. [3H]Ro41-1049 binding (i.e. monoamine oxidase A) was unchanged in all tissues of diseased versus control brains. The monoamine oxidase B-enriched patches in all cortical regions correlated, in their distribution and frequency, with glial fibrillary acidic protein-immunoreactive clusters of astrocytes. Diffuse and mature beta-amyloid-immunoreactive senile plaques as well as patches of high density binding of [3H]PK-11195--a high-affinity ligand for peripheral-type (mitochondrial) benzodiazepine binding sites in microglia/macrophages--were found throughout Alzheimer diseased cortices. The up-regulation of monoamine oxidase B in plaque-associated astrocytes in Alzheimers disease--in analogy to its proposed role in neurodegenerative disorders such as Parkinsons disease--might, indirectly, be a potential source of cytotoxic free radicals. Lazabemide, a selective reversible monoamine oxidase B inhibitor, is currently under clinical evaluation for the treatment of Parkinsons and Alzheimers diseases. We conclude that enzyme radioautography with [3H]lazabemide is a reliable high resolution assay for plaque-associated astroglioses in Alzheimers disease. Its clinical diagnostic utility for positron emission tomography or single photon emission computer tomography studies is being investigated.

Plasma adrenaline, noradrenaline and dopamine in man and different animal species

1. The catecholamines adrenaline (A), noradrenaline (NA) and dopamine (DA) were determined in plasma samples of man and various animal species using a highly sensitive radioenzymatic method.

Platelets as a model for neurones

The multiple biochemical and pharmacological similarities existing between blood platelets and 5-hydroxytryptamine (5-HT)-containing neurones of the CNS point to the platelets as a reliable model for the biochemical characterization of 5-HT releasers and uptake blockers which interfere with the storage and the active carrier mechanism of 5-HT in the neurones, respectively. In addition, the affinity displayed by dopamine and by dopaminergic neurotoxin MPP+ for the platelet 5-HT transport and storage indicates also some similarities between platelets and the dopaminergic system of the CNS. Since human platelets contain almost exclusively monoamine oxidase type B (MAO-B), they can be used as a source for the purification and characterization of this human enzyme. Human platelets thus offer an excellent peripheral model to indirectly assess the degree and duration of MAO-B inhibition occurring in the CNS. To date, knowledge of the many biochemical mechanisms underlying platelet physiology is still fragmentary. In fact, the functional role of binding sites located on the platelet cytoplasmic membrane, i.e. their coupling to a specific transmembrane signalling mechanism, is still in need of a precise biochemical and physiological characterization.

CONTENT AND SUBCELLULAR LOCALIZATION OF CATECHOLAMINES AND 5‐HYDROXYTRYPTAMINE IN HUMAN AND ANIMAL BLOOD PLATELETS: MONOAMINE DISTRIBUTION BETWEEN PLATELETS AND PLASMA

1 The content of adrenaline (Ad), noradrenaline (NA) and dopamine was measured in human, guinea‐pig, cat, rabbit and rat blood platelets by a highly sensitive and specific radioenzymatic method. 2 In all platelet specimens analyzed, the content of the three catecholamines (CA) was several thousand times lower than that of 5‐hydroxytryptamine (5‐HT). 3 In basal conditions, the NA concentration in platelets and plasma always exceeded that of Ad and dopamine. 4 In rat and rabbit platelets, Ad, NA and dopamine were present only in the free (unconjugated) form. 5 Platelets of rats with storage pool deficiency (Fawn‐hooded) contained much less 5‐HT and CA than normal rat platelets. 6 Following restraint stress, platelets of Fawn‐hooded rats, in contrast to normal rat platelets, did not accumulate CA in spite of a dramatic rise in plasma CA. 7 Reserpine, a monoamine depletor, released CA as well as 5‐HT from rabbit platelets in vivo. 8 Subcellular fractionation experiments with rabbit platelets indicate that both CA and 5‐HT are most concentrated in the fraction consisting of pure 5‐HT organelles. 9 Both in humans and rabbits the concentration gradient between platelets and plasma was much lower for CA than for 5‐HT, indicating that a high affinity transport mechanism operates in vivo for 5‐HT but not for CA. 10 In conclusion, the present data show that both human and animal platelets contain Ad, NA and dopamine. The bulk of the CA seems to be stored as unconjugated amines together with 5‐HT, histamine and p‐octopamine in a multitransmitter storage site, namely the 5‐HT organelle.

Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry

Monoamine oxidases are key enzymes in the metabolism of amine neurotransmitters and neuromodulators and are targets for drug therapy in depression, Parkinsons and Alzheimers diseases. Knowledge of their distribution in the brain is essential to understand their physiological role. To study the regional distribution and abundance of monoamine oxidases A and B in human brain, pituitary and superior cervical ganglion, we used quantitative enzyme radioautography with radioligands [3H]Ro41-1049 and [3H]lazabemide, respectively. Furthermore, 35S-labelled oligonucleotides complementary to isoenzyme messengerRNAs were used to map the cellular location of the respective transcripts in adjacent sections by in situ hybridization histochemistry. A markedly different pattern of distribution of the isoenzymes was observed. Highest levels of monoamine oxidase A were measured in the superior cervical ganglion, locus coeruleus, interpeduncular nucleus and ventromedial hypothalamic nucleus. The corresponding messengerRNA was detected only in the noradrenergic neurons of the superior cervical ganglion and locus coeruleus. In contrast to rat brain, monoamine oxidase B was much more abundant in most human brain regions investigated. Highest levels were measured in the ependyma of ventricles, stria terminalis and in individual hypothalamic neurons. Monoamine oxidase B transcripts were detected in serotoninergic raphe neurons, histaminergic hypothalamic neurons and in dentate gyrus granule cells of the hippocampal formation. We conclude that [3H]Ro41-1049 and [3H]azabemide are extremely useful radioligands for high-resolution analyses of the abundance and distribution of catalytic sites of monoamine oxidases A and B, respectively, in human brain sections. From levels of messenger RNA detected, the cellular sites of synthesis of the isoenzymes are the noradrenergic neurons of the locus coeruleus (for monoamine oxidase A) and the serotoninergic and histaminergic neurons of the raphe and posterior hypothalamus, respectively (for monoamine oxidase B). The combination of quantitative enzyme radioautography with in situ hybridization histochemistry is a useful approach to study, with high resolution, both the physiology and pathophysiology of monoamine oxidases in human brain.

Isolated 5‐hydroxytryptamine organelles of rabbit blood platelets: physiological properties and drug‐induced changes

1 . In isolated 5‐hydroxytryptamine (5‐HT) organelles of rabbit platelets, the concentrations of 5‐HT, histamine and adenosinetriphosphate (ATP) respectively are about 200 times higher than in intact platelets. Organelles incubated in plasma at 37° C gradually lose endogenous 5‐HT, histamine and ATP and take up 14C‐5‐HT against a considerable concentration gradient. Liberation and uptake of 5‐HT markedly decrease with dimishing incubation temperature. 2 . Exposure to reserpine in vitro strongly counteracts the uptake of 14C‐5‐HT by isolated organelles, whereas the 14C‐5‐HT uptake of intact isolated platelets is less affected by the drug. 5‐HT organelles of platelets from reserpinized rabbits also take up very little 14C‐5‐HT. 3 . Imipramine inhibits the uptake of 14C‐5‐HT in isolated organelles less markedly than in isolated platelets. 4 . It is concluded that in the organelles 5‐HT and possibly histamine may be associated with ATP. Reserpine probably impairs the uptake of 5‐HT at the level of the organelles (possibly by interfering with the association 5‐HT/ATP), whereas imipramine seems to act preferentially on the cell membrane.

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.

Measurement of human cerebral monoamine oxidase type B (MAO-B) activity with positron emission tomography (PET): a dose ranging study with the reversible inhibitor Ro 19-6327

SummaryEight normal subjects (3 females and 5 males) were studied using intravenous L-11C] deprenyl and positron emission tomography. In a single blind study one subject received tracer alone, one subject received an oral pre-dose of 20 mg of L-deprenyl and 6 subjects received oral pre-doses of 10 to 50 mg of a novel reversible MAO-B inhibitor (Ro 19-6327). Dynamic PET scans beginning 12 h after the oral dose were collected over 90 min and arterial blood was continuously sampled. Data analysis was modelled for two tissue compartments and using an iterative curve fitting technique the value of the rate constant for irreversible binding of L-[11C] deprenyl to MAO-B (k3) in whole brain was obtained for each subject.The dose response curves obtained indicated that a dose of at least 0.48 mg·kg−1 of Ro 19-6327 was necessary for >90% decrease in whole brain k3. Inhibition of MAO-B in platelets isolated from blood samples taken at the time of scanning correlated strongly with decrease in whole brain k3 (r=0.949).The results indicate that PET can be used to determine the dose of Ro 19-6327 necessary to inhibit >90% of brain MAO-B. This technique is an attractive alternative to traditional large scale patient-based dose-finding studies. Moreover it is shown that inhibition of platelet MAO-B can be used as a marker for central MAO-B inhibition with Ro 19-6327.

From moclobemide to Ro 19-6327 and Ro 41-1049: the development of a new class of reversible, selective MAO-A and MAO-B inhibitors

This study describes the serendipitous discovery of moclobemide, a short-acting MAO-A inhibitor which is in an advanced stage of clinical development as an antidepressant. The short duration of action of this MAO inhibitor containing a morpholine ring moiety is due to the complete reversibility (probably by metabolism of the inhibitory molecular species) of MAO-A inhibition. Since moclobemide is much more effective in vivo than expected from its in vitro activity, investigations to identify a possible metabolite(s) more active as MAO-A inhibitor than the parent compound were carried out. The study of the MAO inhibitory characteristics of several known and putative moclobemide metabolites did not allow the identification of a potent MAO-A inhibitor but led to the discovery of Ro 16-6491, a potent MAO-B inhibitor of novel chemical structure. Systematic chemical modification of the aromatic ring system of Ro 16-6491 finally provided Ro 19-6327 and Ro 41-1049 which are highly selective and reversible inhibitors of MAO-B and MAO-A, respectively. Tritiated derivatives of Ro 19-6327 and Ro 41-1049 were used in binding studies to elucidate their mechanisms of action and to study their cellular distribution by quantitative enzyme radioautography.