A. M. Cesura

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.

Binding of [3H]Ro 16-6491, a Reversible Inhibitor of Monoamine Oxidase Type B, to Human Brain Mitochondria and Platelet Membranes

Abstract: The reversible inhibitor of monoamine oxidase type B (MAO‐B) [3H]Ro 16‐6491 binds specifically and with high affinity to a single population of binding sites in human frontal cortex crude mitochondria and platelet membranes. In both tissues binding equilibrium was reached after l h incubation at 20°C. Dissociation of bound radioactivity was relatively fast at 20°C (t1/2= 90–120 min) whereas at 0°C [3H]Ro 16–6491 showed the characteristics of a slowly dissociating ligand. Inhibitors and substrates of MAO‐B inhibited binding of [3H]Ro 16‐6491, whereas MAO‐A blockers were much less potent. Ro 16‐6491 was also a substrate for MAO‐B and a stable unidentified intermediate of the oxidation of Ro 16‐6491 possessing high affinity for the enzyme may account for the marked MAO‐B inhibitory effect of the drug. According to this hypothesis Ro 16‐6491 would behave as a mechanism‐based reversible inhibitor. In conclusion, [3H]Ro 16‐6491 binds selectively to MAO‐B and represents an excellent new radioligand probe for studying the regional tissue distribution of this enzyme in normal and pathological conditions.

[3H]Ro 16–6491, a Selective Probe for Affinity Labelling of Monoamine Oxidase Type B in Human Brain and Platelet Membranes

Abstract: [3H]Ro 16–6491 [N‐(2‐aminoethyl)‐p‐chloroben‐zamide HCl], a reversible “mechanism‐based” inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO‐B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16–6491 is fully reversible. However, [3H]Ro 16–6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO‐B inhibitor /‐deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16–6491. The selective inactivation of MAO‐B, e.g., by /‐deprenyl prevented the covalent incorporation of [3H]Ro 16–6491 whereas selective inhibition of the MAO‐A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16–6491 and Ro 19–6327 (a selective and reversible MAO‐B inhibitor closely related to Ro 16–6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO‐B activity whereas MAO‐A activity was unaffected. Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16–6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16–6491 corresponds to one of the two MAO‐B subunits. Therefore, [3H]Ro 16–6491 represents a selective probe for affinity labelling of MAO‐B and for the investigation of the structural composition of the active site of the enzyme. Whether the reduction with NaBH3CN at pH 4.5 of the [3H]Ro 16–6491‐MAO‐B complex results in the formation of a stable adduct with the amino acid chain of the MAO‐B or with its prosthetic group, FAD, remains to be elucidated.

Plasma catecholamines in rats exposed to cold: Effects of ganglionic and adrenoreceptor blockade

Exposure to cold (4 degrees C) of catheterized rats acclimated to 20 degrees C resulted in a progressive increase in plasma noradrenaline (NA) concentrations which reached values consistently more than twice the basal ones (20 degrees C) by about 30 min. No further increase in plasma NA levels were detected when the cold exposure was continued for 24 h. Plasma adrenaline (A) and dopamine levels did not change at any time studied. Adrenalectomized rats exposed to cold exhibited percent rises in plasma NA similar to those in intact rats. An increase in plasma A levels concomitant with that of NA was observed following exposure to cold of rats in which either basal catecholamine release was impaired by chlorisondamine or the vasoconstrictor response was impeded by phentolamine. Propranolol did not modify the acute neurosympathetic response to cold. Exposure to cold (4 degrees C) for short periods of time combined with the measurement of plasma catecholamines is proposed as a useful and reproducible method for studying a pure neurosympathetic response in the rat.

Uptake, Release, and Subcellular Localization of l‐Methyl‐4‐Phenylpyridinium in Blood Platelets

Abstract: : The neurotoxic compound 1‐[methyl‐3H]‐4‐phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 μM) was 50 times higher than that for serotonin [5‐hydroxytryptamine (5‐HT)]. The uptake of [3H]MPP+ by human platelets was inhibited by selective 5‐HT uptake blockers [cianopramine, (−)‐paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5‐HT organelles (reserpine, mepacrine, and Ro 4–1284). Impairment of the trans‐membrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5‐HT organelle fraction. MPP+ competitively inhibited [14CJ5‐HT uptake by human platelets and reduced the endogenous 5‐HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5‐HT carrier and is accumulated predominantly in the subcellular organelles that store 5‐HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.

Binding of [3H]dihydrotetrabenazine and [125I]azidoiodoketanserin photoaffinity labeling of the monoamine transporter of platelet 5-HT organelles.

The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage organelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodokentanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 +/- 1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values = 1.1 +/- 0.2, 1.2 +/- 0.1 and 0.52 +/- 0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine.

Met-enkephalin immunoreactivity in blood platelets

Picogram amounts (50-150 pg/mg protein) of immunoreactive met-enkephalin material (met-enkephalin IR) were detected by radioimmunoassay in human, rat and rabbit platelets. Characterization of this material by thin-layer chromatography, gel filtration chromatography and high-pressure liquid chromatography indicated that it behaves identically with synthetic met-enkephalin. No high molecular weight met-enkephalin IR could be detected in the platelet extracts, even after trypsin hydrolysis, using two antisera which are able to recognize some of the putative met-enkephalin precursors present in the adrenal gland or striatum. In vitro, thrombin released platelet-met-enkephalin IR concomitantly with 5-hydroxytryptamine (5-HT), suggesting a common subcellular localization, i.e. the 5-HT storing organelles, for met-enkephalin IR and the amine. In vivo, platelet met-enkephalin IR in the Sprague-Dawley rat was affected neither by adrenalectomy nor by hypophysectomy. Thirteen-and 18-week-old spontaneous hypertensive rats (SHR) had lower platelet concentrations of met-enkephalin IR than age matched normotensive Wistar-Kyoto rats.

Free (Unconjugated) Catecholamine Concentrations in Platelets: Biological Significance and Clinical Implications

Mammalian platelets do not contain monoamine synthesizing enzymes, but take up and accumulate in their “dense bodies”, the serotonin (5-HT) organelles, monoamines present in plasma, e.g. 5-HT, catecholamines (CA), normetanephrine and p-octopamine (Da Prada et al., 1980; for review see Da Prada et al., 1981). The limits of sensitivity of the fluorimetric methods used in earlier studies (Weil-Malherbe and Bone, 1975; Markwardt, 1976) for measuring the low concentrations of plasma and platelet CA have recently been overcome by highly sensitive and specific radioenzymatic assays, which allow precise and simultaneous measurements of adrenaline (A), noradrenaline (NA) and dopamine (DA) in minute platelet (from 1 to 2.5 ml of platelet rich plasma) and plasma (<0.1 ml) samples (Da Prada and Picotti, 1979). These methods applied to platelet extracts submitted to acid hydrolysis have also made it possible to establish the presence of substantial amounts of conjugated CA in human but not in animal platelets (Da Prada et al., 1980).

Monoamin-Oxidase-Hemmer

Die Monoamin-Oxidase (MAO, EC 1.4.2.4; Amin: Sauerstoff-Oxidoreduktase [desaminierend]) ist ein relativ unspezifisches mitochondriales Enzym. Es metabolisiert monoaminerge Neurotransmitter (wie Adrenalin, Noradrenalin, Dopamin) und Neuromodulatoren (wie (α-Phenethylamin), andere endogene und exogene Monoamine (wie Tyramin), aber auch tertiare Amine wie das dopaminerge Neurotoxin 1-Methyl-4phenyl-1,2,3,6-tetrahydropyridin (MPTP) entsprechend folgender Nettoreaktionsgleichung (YouDIM et al. 1988, GERLACH und RIEDERER 1993).