M. D. Galva

[3H]Ro 19-6327: A reversible ligand and affinity labelling probe for monoamine oxidase-B

This study demonstrated the existence of specific binding sites for [3H]Ro 19-6327 in human platelet membranes. This compound is a novel, time-dependent inhibitor of monoamine oxidase type B (MAO-B) and is structurally closely related to [3H]Ro 16-6491. The density of the sites labelled with high affinity by [3H]Ro 19-6327 was similar to that observed in previous studies with [3H]Ro 16-6491 as ligand. Binding was reversible at 20 degrees C and showed a relatively slow dissociation (t1/2 = 220 min). The dissociation rate was markedly decreased (t1/2 = greater than 24h) at 0 degrees C. MAO-B, but not MAO-A inhibitors, effectively prevented the binding of [3H]Ro 19-6327. Like [3H]Ro 16-6491, [3H]Ro 19-6327 is recognized as a substrate by MAO-B, being eventually deaminated by the enzyme. Since the deaminated aldehyde derivative of Ro 19-6327 did not inhibit MAO-B, a still unidentified reversible adduct, formed at the MAO-B active site, might explain the high potency and selectivity of [3H]Ro 19-6327. Incubation of the radioligand-enzyme complex from platelet and brain membranes with NaBH3CN and acetic acid (to pH 4.5) caused the irreversible incorporation of the radioactivity into a single polypeptide as shown by SDS-PAGE analysis. This polypeptide had a molecular weight identical to that of the MAO-B subunit, i.e. 58,000. The presence of unlabelled MAO-B inhibitors in the incubation mixture prevented the covalent incorporation of [3H]Ro 19-6327. The irreversible MAO-B inhibitor, [3H] pargyline, labelled a protein with a molecular weight identical to the protein labelled by [3H]Ro 19-6327.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effect of aging on lazabemide binding, monoamine oxidase activity and monoamine metabolites in human frontal cortex

Age-related modifications of monoamine oxidase-A and -B (MAO-A and MAO-B) and amine metabolite concentrations were studied in human frontal cortex taken postmortem from 22 subjects of various ages (21–75 years). Qualitative and quantitative analysis for MAO-B was provided by kinetic studies with a specific radioligand, [3H]lazabemide. The data demonstrated a significant (P < 0.05) positive correlation between the density of [3H]lazabemide binding sites (Bmax) and age of the subject, without showing an apparent modification in the dissociation constant (KD) of the radioligand. In parallel experiments, MAO-B but not MAO-A activity was shown to correlate with age (P < 0.05). The concentrations of the amine metabolites 4-hydroxy-3-methoxyphenylacetic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG) were all devoid of a correlation with age. Neither did the concentrations of these metabolites relate to the respective subjects MAO-B enzymatic activity nor to [3H]lazabemide Bmax. A correlation, though rather weak, was obtained between MAO-A activity and MHPG concentration (P=0.045). The MAO-A and -B enzyme characteristics in subjects who had committed suicide (n=9) did not differ from those of subjects deceased for other causes (n=13). Among the measured monoamine metabolites the concentrations of DOPAC and HVA were higher in the suicide versus control group (P < 0.05). The present data confirm in a direct manner that the increase in MAO-B activity in aging brain is due to an enhancement of the number of active sites of the enzyme and not through modifications of its kinetic characteristics. Furthermore, that neither the characteristics nor the activity of the enzyme are changed in the frontal cortex of suicide victims compared to control subjects.

[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.

Effects of oral chlordemethyldiazepam on plasma adrenaline and noradrenaline and cardiovascular reactivity in preoperative patients

SummaryIn 11 preoperative women, plasma adrenaline (A) concentrations were lower after oral administration of an antianxiety dose (19.25 µg/kg) of chlordemethyldiazepam (Cl-DMDZ) than the predrug values, or those in 12 patients given placebo. No significant differences in supine plasma noradrenaline (NA), blood pressure (BP) and heart rate values were observed. Digital plethysmography showed finger vasoconstriction after placebo and vasodilatation after Cl-DMDZ. A mental arithmetic test caused equivalent rises in plasma A in both groups. Standing caused plasma NA to rise to similar levels in both groups of patients, but the BP decrease was less and there was a markedly lower incidence of orthostatic hypotension in the Cl-DMDZ treated group. It is concluded that the effect of Cl-DMDZ on the release of catecholamines from the peripheral sympathetic system consists essentially of decreasing basal adrenomedullary activity. CL-DMDZ appears to prevent the orthostatic hypotension which occurs when neurosympathetic reflex activation is normal.

Platelet Met-Enkephalin Immunoreactivity and 5-Hydroxytryptamine Concentrations in Migraine Patients: Effects of 5-Hydroxytryptophan, Amitriptyline and Chlorimipramine Treatment

In thirty patients with common migraine the platelet concentrations of met-enkephalin immunoreactivity (ME) (76 9 pg/mg protein) were similar to those in 23 healthy volunteers (77 5), suggesting that there is no alteration in the ME pool in this biochemical compartment in migraine. Chronic treatment (4 weeks) with drugs that interfere with 5–hydroxytryptamine (5–HT) synthesis or uptake induced the expected changes in platelet 5–HT levels, i.e. a rise following administration of the 5–HT precursor 5–hydroxytryptophan (daily dose: 300–500 mg, n = 9) and a decrease after amine uptake inhibition by amitryptyline (30–75 mg, n = 7) and even more by chlorimipramine (30–50 mg, n = 9). Platelet ME concentrations rose by up to ∼90% over the basal values after either 5–hydroxytryptophan (significantly from week 2) or amitriptyline (at week 2) and were unchanged after chlorimipramine, indicating that 5–HT and ME concentrations in platelets can vary independently. The high platelet ME levels following 5–hydroxytryptophan and amitriptyline cannot be explained at present. They might be due either to increased ME synthesis, possibly in the megakaryocyte, or to decreased utilization by platelets or both.

In vitro effects on monoamine uptake and release by the reversible monoamine oxidase-B inhibitors Lazabemide and N-(2-aminoethyl)-p-chlorobenzamide: A comparison with l-deprenyl

To investigate whether the reversible monoamine oxidase-B (MAO-B) inhibitors lazabemide and Ro 16-6491 have any additional effect on monoamine uptake and release, in vitro experiments were performed on rat forebrain synaptosomes and blood platelets. The effects of the two drugs were compared with those of L-deprenyl, the well-known irreversible MAO-B inhibitor which is reported to affect amine uptake. Both lazabemide and Ro 16-6491 behaved as weak inhibitors of [3H]monoamine uptake by synaptosomes, with a similar rank order of potency for amine uptake inhibition (noradrenaline (NA) > or = 5-hydroxytryptamine (5 HT) > dopamine (DA)). The IC50 values for lazabemide and Ro 16-6491, respectively, were: 86 microM and 90 microM for NA uptake; 123 microM and 90 microM for 5HT uptake; > 500 microM and > 1000 microM for DA uptake. L-Deprenyl (rank order of inhibitory potency: NA > DA > 5 HT) was four to 10 times more potent than either compound in inhibiting [3H]catecholamine uptake (IC50 = NA 23 microM, DA 109 microM), and two to three times less potent in inhibiting 5 HT uptake (IC50 233 microM). Lazabemide and Ro 16-6491 also differed from L-deprenyl in their ability to induce release of endogenous monoamines from synaptosomes. Thus, Ro 16-6491 (500 microM) induced a greater 5 HT release than did L-deprenyl, but was less effective than L-deprenyl in releasing DA. On the contrary, lazabemide was almost completely inactive on either 5 HT and DA release. The differential effect of the three MAO-B inhibitors on synaptosome 5 HT uptake and release was confirmed by [14C]5HT uptake and liberation experiments with isolated rat platelets. The data indicate that the reversible MAO-B inhibitors lazabemide and Ro 16-6491 at relatively high concentrations possess amine uptake-inhibiting properties. With regard to the effects examined, lazabemide markedly differs from L-deprenyl since it does not interfere with DA uptake nor induce amine release from synaptosomes.

Alcohol impairs age-dependent adaptation of human lymphocyte beta-adrenergic receptors

Abstract. Lymphocyte beta‐adrenergic receptor function and norepinephrine (NE) plasma concentration have been compared in normal subjects and in ethanoladdicted patients of different ages. Direct measurement of the density and properties of beta‐adrenoceptors in membrane fractions was performed using the radioligand 125I‐Iodocyanopindolol (ICYP). In normal subjects beta‐receptor density decreased and norepinephrine plasma concentration increased with age. There was a statistically significant negative correlation between plasma norepinephrine and beta‐receptor number. In ethanol‐addicted patients the agerelated modification in beta‐receptor number and the correlation between plasma norepinephrine and beta‐receptor density were lost, in spite of the fact that the increase of NE plasma concentration was still present. The ethanol‐induced effects in lymphocyte beta‐receptor may have consequences on immunological function and may be qualitatively similar to alterations in other tissues not routinely accessible in humans.

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).