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Monoamine oxidase B (MAO-B) has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Increased MAO-B expression in astroglia has been observed adjacent to amyloid plaques in AD patient brains. This phenomenon is hypothesized to lead to increased production of hydrogen peroxide and reactive oxygen species (ROS), thereby contributing to AD pathology. Therefore, reduction of ROS-induced oxidative stress via inhibition of MAO-B activity may delay the progression of the disease. In the present study we report the pharmacological properties of sembragiline, a novel selective MAO-B inhibitor specifically developed for the treatment of AD, and on its effect on ROS-mediated neuronal injury and astrogliosis in MAO-B transgenic animals. Sembragiline showed potent and long-lasting MAO-B-selective inhibition and did not inhibit MAO-A at doses where full inhibition of MAO-B was observed. Such selectivity should translate into a favorable clinical safety profile. Indeed, sembragiline neither induced the serotonin syndrome when administered together with the serotonin precursor l-5-hydroxytryptophan in combination with antidepressants such as fluoxetine, nor potentiated the pressor effect of tyramine. Additionally, in experiments using a transgenic animal model conditionally overexpressing MAO-B in astroglia, sembragiline protected against neuronal loss and reduced both ROS formation and reactive astrogliosis. Taken together, these findings warrant further investigation of the potential therapeutic benefit of MAO-B inhibitors in patients with AD and other neurologic disorders.

Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon-gamma (IFN-gamma) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN-gamma induced the expression of indoleamine 2,3-dioxygenase (IDO) activity. Whereas tumor necrosis factor-alpha did not affect enzyme induction by IFN-gamma, lipopolysaccharide modulated IDO activity differently in the two IFN-gamma-activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3-hydroxylase activity was stimulated by IFN-gamma. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN-gamma markedly stimulated the activity of this enzyme only in MT2 cells. IFN-gamma-treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3-hydroxyanthranilic and quinolinic acids from L-[5-3H] tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.

The development of 8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-ones 3 starting from (RS)-8-acenaphten-1-yl-1-phenyl-1,3, 8-triazaspiro[4.5]decan-4-one 1 is reported. The synthesis and the binding affinities at human OFQ and opioid (micro, kappa, delta) receptors of the stereoisomers 3a-f are described. In vitro the most selective compound, (1S,3aS)-8-(2,3,3a,4,5, 6-hexahydro-1H-phenalen1-yl)-1-phenyl-1,3,8-triaza-spiro[4. 5]decan-4-one 3c, was found to act as a full agonist at the OFQ receptor in the GTPgamma(35)S binding test. It turned out to be selective versus a variety of other neurotransmitter systems. When tested in vivo following intraperitoneal injection, compound 3c was found to decrease neophobia in a novel environment and to exhibit dose-dependent anxiolytic-like effects in the elevated plus-maze procedure, thus confirming the effects observed following intracerebroventricular infusion of the OFQ peptide in rat.

We have investigated 8-cycloalkyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-o nes as ligands for the ORL1 receptor. These unsophisticated, achiral compounds show remarkable affinity for the ORL1 receptor. Optimizing for selectivity we show that the maximum of affinity and selectivity versus the other opioid receptors is achieved for 8-cyclodecyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-o ne 2e and 8-(cis-4-isopropyl-cyclohexyl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one 2q. The identified compounds (2e, 2q) are more or less equipotent to the natural ligand itself, both in the binding assay and in the functional GTPgammaS assay.

A series of 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan+ ++-4-one derivatives 1 was studied with respect to the binding affinity for the orphanin FQ (OFQ) and opioid (mu, kappa, delta) receptors. The influence of stereochemistry as well as the substitution pattern of the phenyl-ring in position 1 on the affinity for the orphanin FQ receptor and selectivity to opioid (mu, kappa, delta) receptors is discussed. The most interesting compound 1c was tested for its anxiolytic-like properties in vivo.

The structural features of the active site of human monoamine oxidase B (MAO-B) were investigated by affinity labeling and site-directed mutagenesis. The pseudosubstrate inhibitor N-[2-aminoethyl]-5-chloro-2-pyridine carboxamide HCl (lazabemide) can be irreversibly linked to MAO-B by reduction of the enzyme-inhibitor complex with NaBH(3)CN. Analysis of the flavin spectrum of [(3)H]lazabemide-labeled human MAO-B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO-B. These results indicate that [(3)H]lazabemide is incorporated into the MAO-B peptide stretch containing the FAD-modified Cys397. The function of putative active-site residues contained in this region was investigated by site-directed mutagenesis and expression of the mutant proteins in HEK-293 cells. Substitution of His382 of MAO-B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO-B catalytic mechanism. Whereas it has been shown that mutation of Cys389 with a Ser residue does not markedly affect the activity of the enzyme [Wu, H.-F., Chen, K. and Shih, J.C. (1993) Mol. Pharmacol. 43, 888-893] the mutant carrying an Ala at this position was virtually inactive. Conversely, substitution of Lys386 (to Met) and Ser394 (to Ala) did not markedly modify the kinetic properties of the enzyme. We also report that mutation of MAO-B Thr158 (to Ala) resulted in a dramatic loss of enzymic activity. [on SciFinder (R)]

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2-terminus of one MAO form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide · HCl (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the kcat values of these chimeras. From the data obtained, two sequences, i.e. 62–103 and 146–220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH2- (up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other.

Technological advances in the field of mass spectrometry (MS) are providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion-spray) MS for the biochemical characterization of recombinant human catechol O-methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352±2Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25775±4Da. To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C-terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C-terminal extension resulted from ribosomal frameshift at the codon of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5Da) is in good agreement with our experimental result. The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S-adenosyl-L-methionine (AdoMet) and MgCl2, was also studied. 5-Iodoacetamido fluorescein (5-IAF) was used as thiol-modifying reagent. Under the conditions used, 5-IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl2 partially protected it from inactivation. The 5-IAF-labeled tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5-IAF. Incorporation of 5-IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.

The locus coeruleus (LC), a major noradrenergic nucleus in the brain, probably has a functional role in the regulation of anxiety states as well as vigilance, attention, learning and memory. LC neurons are under the inhibitory control of gamma-aminobutyric acid (GABA) via ionotropic GABAA receptors. However, to date, little is known of the receptor binding characteristics of these neurons. In the present investigation we therefore examined by receptor radioautography the localization of the binding sites for different components of the GABAA receptor complex in the rat and human LC. Both rat and human LC neurons have a high density of binding sites for the pyridazinyl-GABA derivative [3H]SR 95531 (gabazine, a GABAA receptor antagonist for low affinity GABA recognition sites). However, at the concentrations used, no binding sites in the LC were detectable for the benzodiazepine receptor antagonist [3H]flumazenil, the GABAA receptor agonist (for high affinity sites) [3H]muscimol or the ionophore ligand [35S]t-butyl bicyclophosphorothionate (TBPS). Unexpectedly, the pharmacological specificity of [3H]SR 95531 binding to the LC differed markedly from that to most brain regions (IC50 values for GABA and RU 5135 respectively in the LC were > 10(-2) and 10(-3) M; and, for example, in the dentate gyrus the most labelled structure after the LC, 8 x 10(-7) and 1.8 x 10(-9) M). These differences prompted the further characterization of [3H]SR 95531 binding in the LC, revealing a significant affinity for monoamine oxidase type A (MAO-A), which is highly concentrated in this nucleus. In a competition binding study, a reduction of up to 25% of the [3H]SR 95531 binding was observed with MAO-A but not MAO-B inhibitors, at concentrations which produce maximum but selective enzyme inhibition. Correspondingly, 2 h after the oral administration of supramaximal doses of the MAO-A inhibitors moclobemide and Ro 41-1049 (but not the MAO-B inhibitor lazabemide) the in vitro binding of [3H]SR 95531 was markedly reduced (by 77 and 82% of controls respectively). Moreover, enzyme radioautography with [3H]Ro 41-1049 revealed that SR 95531 has a significant affinity for MAO-A (IC50 values were 10(-5) and 4 x 10(-6) M in the LC and dentate gyrus respectively) but not for MAO-B ([3H]lazabemide binding). Altogether, these findings suggest that the high-affinity binding of [3H]SR 95531 to the LC mainly reflects its affinity for MAO-A, which questions its utility as a selective ligand for low-affinity GABA recognition sites in the CNS.(ABSTRACT TRUNCATED AT 400 WORDS)

Serine racemase is responsible for the synthesis of D-serine, an endogenous co-agonist for N-methyl-D-aspartate receptor-type glutamate receptors (NMDARs). This pyridoxal 5'-phosphate-dependent enzyme is involved both in the reversible conversion of L- to D-serine and serine catabolism by alpha,beta-elimination of water, thereby regulating D-serine levels. Because D-serine affects NMDAR signaling throughout the brain, serine racemase is a promising target for the treatment of disorders related to NMDAR dysfunction. To provide a molecular basis for rational drug design the x-ray crystal structures of human and rat serine racemase were determined at 1.5- and 2.1-A resolution, respectively, and in the presence and absence of the orthosteric inhibitor malonate. The structures revealed a fold typical of beta-family pyridoxal 5'-phosphate enzymes, with both a large domain and a flexible small domain associated into a symmetric dimer, and indicated a ligand-induced rearrangement of the small domain that organizes the active site for specific turnover of the substrate.

Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno-electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10-15-fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (approximately 38 kDa) and the extent of glycosylation of platelet-derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno-electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin-like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle-associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.

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.

In the present paper we describe the use of digitonin as a suitable detergent for the effective solubilization of MAO-B from human platelets and frontal cortex membrane preparations and the characteristics of the solubilized enzyme using the selective MAO-B inhibitor [ 3 H] Ro 16-6491 [N-(2-aminoethyl)-p-chlorobenzamide HCl] as binding probe. In addition, we describe an affinity chromatography method for human platelet MAO-B, using as affinity ligand a derivative of Ro 19-6327 [N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide HCl], a compound in clinical trial belonging to a novel class of reversible and selective MAO-B inhibitors

We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm)Chelerythrine (half-maximal uncoupling at about 2 microm)HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.

Novel hexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrroles that act as potent and selective orphanin FQ/nociceptin (N/OFQ) receptor (NOP) agonists were identified. The best compound, (+)-5a, potently inhibited 3H-N/OFQ binding to the NOP receptor (K(i) = 0.49 nM) but was1000-fold less potent in binding to MOP, KOP, and DOP opiate receptors. Further, (+)-5a potently stimulated GTP gamma S binding to NOP membranes (EC50 = 65 nM) and inhibited forskolin-mediated cAMP accumulation in NOP-expressing cells (EC50 = 9.1 nM) with a potency comparable to that of the natural peptide agonist N/OFQ. These results indicate that (+)-5a is a highly selective and potent small-molecule full agonist of the NOP receptor.

The discovery of 8-(5,8-dichloro-1,2,3,4-tetrahydro-naphthalen-2-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1a, as a high-affinity ligand for the human ORL1 (orphanin FQ/nociceptin) receptor led to the synthesis of a series of optimized ligands. These compounds exhibit high affinity for the human ORL1 receptor, exhibit moderate to good selectivity versus opioid receptors, and behave as full agonists in biochemical assays. In this paper we present the synthesis, structure-activity relationship (SAR), and biochemical characterization of substituted 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones culminating in the discovery of 8-(5-methyl-1,2,3,4-tetrahydro-naphthalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1p, and 8-acenaphten-1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one 1q, two high-affinity, potent ORL1 receptor agonists with good to moderate selectivity versus the other opioid receptors.

The biochemical and behavioral effects of a nonpeptidic, selective, and brain-penetrant agonist at the ORL1 receptor are reported herein. This low molecular weight compound {(1 S ,3a S )-8- (2,3,3a,4,5,6-hexahydro-1 H -phenalen-1-yl)-1-phenyl-1,3,8-triaza- spiro[4.5]decan-4-one} has high affinity for recombinant human ORL1 receptors and has 100-fold selectivity for ORL1 over other members of the opioid receptor family. It is a full agonist at these receptors and elicits dose-dependent anxiolytic-like effects in a set of validated models of distinct types of anxiety states in the rat (i.e., elevated plus-maze, fear-potentiated startle, and operant conflict). When given systemically, the compound has an efficacy and potency comparable to those of a benzodiazepine anxiolytic such as alprazolam or diazepam. However, this compound is differentiated from a classical benzodiazepine anxiolytic by a lack of efficient anti-panic-like activity, absence of anticonvulsant properties, and lack of effects on motor performance and cognitive function at anxiolytic doses (0.3 to 3 mg/kg i.p.). No significant change in intracranial self-stimulation performance and pain reactivity was observed in this dose range. Higher doses of this compound (≥10 mg/kg) induced disruption in rat behavior. These data confirm the notable anxiolytic-like effects observed at low doses with the orphanin FQ/nociceptin neuropeptide given locally into the brain and support a role for orphanin FQ/nociceptin in adaptive behavioral fear responses to stress.

To gain insight into the structure of monoamine oxidases (MAO) A and B, we investigated the properties of various chimaeric enzymes, engineered by moving progressively the junction between the NH2- and the COOH-termini of each MAO form. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimaeras with increasing length of the NH2-terminus of MAO-A (up to position 256) showed a marked decrease in affinity towards substrates and inhibitors. Two sequences, spanning position 62 to 103 and 146 to 220, appeared of particular importance in putatively constituting the binding site of MAO-B. Conversely, the catalytic properties and specificity of MAO-A were insensitive to substitution of both the NH2-(up to position 112) and COOH-termini (from residue 395), but further modification of the central sequence of MAO-A was not compatible with activity. None of the engineered chimaeras showed a shift in substrate and inhibitor specificity. Investigation on MAO-B by site-directed mutagenesis revealed that His382 and Thr158 may represent residues relevant for MAO-B catalytic mechanism.

Kynureninase (L-kynurenine hydrolase), a pyridoxal-5'-phosphate-(pyridoxal-P)-dependent enzyme, catalyses the cleavage of L-kynurenine and L-3-hydroxykynurenine into anthranilic and 3-hydroxyanthranilic acids, respectively. In this report, we describe the isolation of a cDNA clone encoding human kynureninase. Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse-transcription PCR of rat kidney RNA. The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G2) cDNA library. Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Saccharomyces cerevisiae gene product putatively ascribed to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal-P-binding site consensus sequence assigned to class-V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including brain, showed the presence of an approximately 2.0-kb mRNA species in all tissues tested. A second mRNA species (approximately 2.6 kb) was also detected in some tissues. After transfection of HEK-293 cells with the cDNA coding for kynureninase, the K(m) values of L-kynurenine and DL-3-hydroxykynurenine for the recombinant enzyme were 671 +/- 37 microM and 13.2 +/- 2.0 microM, respectively.

The action of dopamine (DA) released in the synaptic cleft is mainly terminated by its reuptake and catabolism by the enzymes monoamine oxidase (MAO) and catechol O-methyltransferase (COMT). Preclinical data show that the reduction of the catabolism of DA elicited by MAO and COMT inhibitors leads to an enhancement of DA neurotransmission. Moreover, there is evidence suggesting that MAO-B inhibition might protect DA neurons from oxidative stress. Nevertheless, due to differences in enzyme localization and activity between man and rodents, results obtained in experimental animals might not reflect the actual situation in humans. Today the availability of potent and selective MAO and COMT inhibitors makes it feasible for the clinician to test whether the blockade of catabolic enzymes would result in a symptomatic improvement in Parkinsonian patients, and whether MAO-B inhibition might additionally exert a neuroprotective effect.

Noradrenaline (NA) and serotonin, monoamines that increase neurotransmission at the monoaminergic synapses, remain primary targets for antidepressant drugs. To help elucidate NA's role in the action of antidepressants, a model was set up allowing for a simulation of NA turnover under various conditions examining 3 compartments: newly synthesized vesicular NA, released synaptic NA, and vesicular NA reuptake across neuronal membranes. We compared the influence of the reversible MAO inhibitor, moclobemide, and the NA reuplake inhibitor, desipramine, both useful in regaining baseline synaptic NA concentrations. Moclobemide appears less sensitive to fluctuations either in drug concentrations and/or physiological determinants of NA turnover; we conclude that it may influence brain function in a more regulatory way than reuptake inhibitors.

The aetiology and pathogenesis of Alzheimer's 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 (~ 185 μm in diameter) which occupied approximately 12% of the cortical areas examined. In other brain regions (hippocampal formation > caudate-putamen > cerebellum), patches of [ 3 H]lazabemide-enriched binding were less abundant. [ 3 H]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 β-amyloid-immunoreactive senile plaques as well as patches of high density binding of [ 3 H]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 Alzheimer's disease—in analogy to its proposed role in neurodegenerative disorders such as Parkinson's 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 Parkinson's and Alzheimer's diseases. We conclude that enzyme radioautography with [ 3 H]lazabemide is a reliable high resolution assay for plaque-associated astroglioses in Alzheimer's disease. Its clinical diagnostic utility for positron emission tomography or single photon emission computer tomography studies is being investigated.

Increased cerebral levels of the endogenous excitotoxin quinolinic acid (QUIN) have been speculatively linked to neuronal damage following neurological and inflammatory disorders. 3-Hydroxyanthranilic-acid dioxygenase (3-HAO; 3-hydroxyanthranilate 3,4-dioxygenase, EC 1.13.11.6) is the enzyme that catalyzes the synthesis of QUIN from 3-hydroxyanthranilic acid, and evidence suggests that it could play a role in disorders associated with altered tissue levels of QUIN. In this report, we describe the isolation of a full-length cDNA clone encoding human 3-HAO (h3-HAO). Degenerate oligonucleotides were designed from the amino acid sequences of tryptic peptides of rat liver 3-HAO, and they were used as primers for reverse transcription-polymerase chain reaction of rat liver RNA. The resulting rat cDNA product was used to screen a human hepatoma cell line (HepG2) cDNA library and to isolate a human 3-HAO cDNA clone. This clone was found to have an insert of 1276 nucleotides. The deduced primary structure of h3-HAO is composed of 286 amino acid residues with a predicted molecular mass of approximately 32.6 kDa. The human sequence exhibits high similarity (94%) to the rat partial amino acid sequence deduced from the rat reverse transcription-polymerase chain reaction fragment. Insertion of the h3-HAO coding sequence into a eukaryotic expression vector yielded relatively high amounts of the active enzyme in human embryonic kidney HEK-293 cells. The Km value of 3-HANA for recombinant h3-HAO (approximately 2 microM) was in good agreement with that reported for the native enzyme. Immunoblot analysis of recombinant h3-HAO revealed a polypeptide with an apparent molecular mass of 32 kDa, as predicted from the deduced amino acid sequence. RNA blot analysis of human liver and HepG2 cells revealed one major species of h3-HAO mRNA of approximately 1.3 kilobases.

Monoamine oxidase (MAO)-A and MAO-B are FAD-containing mitochondrial enzymes which catabolize biogenic and xenobiotic amines. The N-terminal regions of both forms of MAO contain an ADP-binding consensus sequence found in several dinucleotide-dependent enzymes, but otherwise show remarkable sequence differences. In order to investigate whether the N-terminal region of MAOs participates in the different catalytic properties and inhibitor specificities exhibited by MAO-A and MAO-B, we constructed chimeric A/B forms and expressed them in a human embryonic kidney cell line (293 cells). The MAO-A chimeric form containing the N-terminus (36 amino acids) of MAO-B and the B chimera having the first 45 amino acid sequence of MAO-A were both catalytically active. Compared to the respective wild-type form, they did not show any significant difference in their catalytic properties (Km, kcat) towards the substrates tested or in their sensitivity towards inhibitors. This indicates that the N-terminal region of the two isoenzymes is not involved in the different specificities of MAO-A and MAO-B. Substitution of Cys-397 of MAO-B, i.e. the residue covalently anchoring FAD, with an Ala or a His residue resulted in the total loss of enzymatic activity, suggesting that the covalent coupling of FAD to MAO occurs specifically at the -SH group of cysteine.

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.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.

The novel reversible and selective inhibitor of monoamine oxidase-A (MAO-A) Ro 41-1049 [N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide HCl] shows inhibition characteristics similar to those of the structurally related reversible MAO-B inhibitors Ro 16-6491 and Ro 19-6327. In the present study, tritiated Ro 41-1049 was used as a high affinity ligand to study the binding characteristics of this inhibitor to MAO-A and its interactions with the enzyme. An homogeneous population of high affinity binding sites for [3H]Ro 41-1049 was found in membrane preparations from human frontal cortex and placenta (Kd = 16.5 +/- 1.4 and 64.4 +/- 19.2 nM, respectively). In frontal cortex the Bmax value for [3H]Ro 41-1049 (2.6 +/- 0.4 pmol/mg of protein) was about one third of the Bmax calculated for the MAO-B-selective ligand [3H]Ro 16-6491. The density of [3H]Ro 41-1049 binding sites in human placenta varied greatly in the different tissue samples investigated, showing an average Bmax of 101.7 +/- 36.5 pmol/mg of protein. Apparent binding equilibrium was reached after 1 hr of incubation at 37 degrees. At this temperature the binding was reversible, with a dissociation t 1/2 of about 35 min. At lower temperatures the radioactivity dissociation was much slower. Among the various drugs tested, only inhibitors of MAO-A were able to effectively prevent [3H]Ro 41-1049 specific binding. As previously reported for the MAO-B ligands [3H]Ro 16-6491 and [3H]Ro 19-6327, the analysis of the membrane-bound radioactivity showed that [3H]Ro 41-1049 was entirely recovered in the form of its aldehyde derivative, indicating that Ro 41-1049 was deaminated by MAO-A. The existence of a Ro 41-1049 adduct reversibly bound to the enzyme active site might explain the inhibition mechanism of this compound. The exposure of the radioligand-enzyme complex to NaBH3CN at pH 4.5 caused the irreversible covalent incorporation of about 70% of the specifically bound radioactivity into a 60-kDa polypeptide. This incorporation was dependent on the pH and on the amount of NaBH3CN added. The presence of MAO-A- but not MAO-B-selective inhibitors prevented the covalent incorporation of [3H]Ro 41-1049. The present results indicate that [3H]Ro 41-1049 is incorporated into a subunit of MAO-A, in the presence of NaBH3CN, and modifies a protein domain that is essential for the enzyme activity.(ABSTRACT TRUNCATED AT 400 WORDS)

The selective, reversible inhibitors of monoamine oxidase (MAO) moclobemide and Ro 41-1049 (selective for MAO-A), as well as of Ro 16-6491 and Ro 19-6327 (selective for MAO-B) inhibited the enzyme with an initial competitive phase, followed by a time-dependent inhibition of MAO. Ro 41-1049, Ro 16-6491 and Ro 19-6327, being activated by MAO into reversible adducts, fit into the classification as mechanism-based inhibitors. Conversely, since no product formation was observed after incubation of tissue homogenates with moclobemide, this drug probably belongs to the class of the "slow-binding" MAO inhibitors.

In rabbits, 3 mg/kg Ro 19-6327 p.o., induced complete monoamine oxidase type B (MAO-B) inhibition in striatum, cerebral cortex, liver and platelets for about 12 h. In healthy human volunteers complete inhibition of platelet MAO was obtained with only 5 mg Ro 19-6327. After 40 and 200 mg Ro 19-6327 the duration of complete MAO-B inhibition was prolonged to 12 and 24 hours, respectively. Changes in brain MAO-B in humans after Ro 19-6327 are likely to be faithfully reflected by platelet MAO activity.

Picogram amounts (50–150 pg/mg protein) of immunoreactive met-enkephalin material (met-enkephalin in 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 in 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 in IR than age matched normotensive Wistar-Kyoto rats.

In three untreated patients with phenylketonuria (PKU), three PKU and six hyperphenylalaninaemic (HPA) patients in good metabolic control, the kinetic constants of platelet in vitro uptake of [14C]serotonin (5HT) did not significantly differ from those in 12 control subjects matched for age. The platelet concentrations of endogenous 5HT and noradrenaline (NA), taken as long-term indices of the amount of these amines circulating in plasma, were lower than normal in PKU and HPA patients, whether or not they were kept on a diet. However, a reduction in plasma NA concentrations at the moment of blood collection was seen only in untreated PKU patients. These data indicate that there may be a chronic inhibition of 5HT and possibly of NA synthesis even in PKU or HPA subjects in good metabolic control, with normal psychomotor development and only slightly raised plasma phenylalanine levels.

MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine) causes selective and irreversible degeneration of the substantia nigra of human and non-human primates. In the central nervous system, the oxidative metabolism of MPTP to 1-methyl-4-phenyl-pyridinium (MPP + ) by monoamine oxidase type B (MAO-B) seems to be a critical feature in the neurotoxic process. We now report that [ 3 H]MPTP is rapidly converted in vitro into [ 3 H]MPP − by human platelet MAO-B. The formation of [ 3 H]MPP + in human platelets is prevented by specific MAO-B but not by MAO-A or by 5-hydroxytryptamine uptake inhibitors.

[3H]Ro 16-6491 [N-(2-aminoethyl)-p-chlorobenzamide 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 l-deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16-6491. The selective inactivation of MAO-B, e.g., by l-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.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The effects of selective deactivation and stimulation of cardiopulmonary receptors on plasma noradrenaline (radioenzymatic method) were studied in nine normotensive subjects by reducing and increasing central venous pressure for 20 min via lower body suction and leg-raising manoeuvres that did not alter arterial blood pressure and heart rate. 2. Deactivation of cardiopulmonary receptors was accompanied by a rise in plasma noradrenaline that achieved a peak within 5 min (91.8 ± 22%, mean ± se) and was then sustained. Stimulation of cardiopulmonary receptors was accompanied by a fall in plasma noradrenaline (−16.6 ± 3.4%) that levelled off at the second minute and was then sustained. 3. On average the increase and the reduction in plasma noradrenaline had a time course and a magnitude similar to the increase (80.5 ± 10.5%) and the reduction (−28.4 ± 5%) in forearm vascular resistance (derived from plethysmographic flow measurement) concomitantly caused by cardiopulmonary receptors. Furthermore, analysis of individual data showed that changes in plasma noradrenaline and forearm vascular resistance were linked by a positive relationship (r = 0.64). 4. Thus the cardiopulmonary receptor reflex can produce rapid, marked and sustained changes in both plasma noradrenaline and forearm vasomotor tone. This is in sharp contrast with the previously observed inability of the carotid baroreflex to alter both these humoral and haemodynamic variables. Taken together these findings support the hypothesis that sympathetic tone to skeletal muscle is an important determinant of the concentration of plasma noradrenaline in blood.

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 1 h incubation at 20 degrees C. Dissociation of bound radioactivity was relatively fast at 20 degrees C (t1/2 = 90-120 min) whereas at 0 degrees 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.

In 8 essential hypertensive patients infusion of 7.5 ml kg-1 b.w. of isotonic saline over 20 min decreased plasma renin activity but did not affect plasma adrenaline or noradrenaline concentrations in suprarenal aortic, renal venous and caval blood. Mean blood pressures and heart rates were unchanged throughout the saline infusion period, whereas the hematocrit was significantly decreased. These data suggest that inhibition of renin secretion after slight volume expansion with saline in hypertensive humans occurs without parallel inhibition of either renal neurosympathetic drive or humoral adrenergic tone to the kidney through circulating catecholamines.

Various procedures for the solubilization of the imipramine-binding protein (IBP) of human platelets were compared. An IBP of a molecular weight of 300 000–400 000, as determined by exclusion chromatography on Sepharose 6B, was obtained with high amounts of digitonin and with lysolecithin. With smaller amounts of digitonin the molecular weight varied between more than 1 million and about 550 000 depending on the batch of detergent. The K D values and the IC 50 values of drugs inhibiting imipramine binding were similar in the soluble preparation and in intact membranes. CHAPS and CHAPSO, even in high amounts, yielded solubilized IBP of high molecular weight (>1 million). Membrane preparations of human platelets solubilized with high amounts of digitonin and with lysolecithin would therefore seem to be the most suitable for further purification of IBP.

Kynurenine 3-monooxygenase, an NADPH-dependent flavin monooxygenase, catalyses the hydroxylation of L-kynurenine to L-3-hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3-monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55,762 Da. Transfection of the human cDNA in HEK-293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of approximately 2.0 kb in liver, placenta and kidney.

1. Plasma noradrenaline and adrenaline concentrations and plasma renin activity were measured in 21 mothers at delivery and in their babies at birth (umbilical cord blood) and on days 1 and 5 of extrauterine life. 2. At birth plasma renin activity was significantly higher in the newborn than in mothers. Plasma renin activity increased further, but not significantly, on day 1 of life and significantly decreased on day 5. On day 5, 10 min head-up tilting caused no change in plasma renin activity. 3. Plasma noradrenaline in the newborn was higher than in mothers at birth and significantly decreased thereafter. Plasma adrenaline levels at birth were similar in the newborn and their mothers and significantly lower in the newborn in subsequent days. Tilting caused no increase in either plasma adrenaline or noradrenaline levels. 4. No correlation was found between plasma noradrenaline and adrenaline levels and plasma renin activity, or between noradrenaline, adrenaline or plasma renin activity and blood pressure.

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 microM) 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 transmembrane 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 [14C]5-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.

To evaluate the role of adrenergic mechanisms in the acute response of renin to furosemide, plasma renin activity (PRA) and plasma catecholamine concentrations were measured for 3 h after i.v. administration of furosemide 1 mg/kg to 8 patients with mild essential hypertension. Furosemide induced a prompt and long-lasting increase in renin, with PRA more than doubled at all times. The increase in PRA within the first 30 min paralleled the peak increases in urinary water and sodium flow rates, and significant decreases in plasma volume and central venous pressure. There was no change in plasma catecholamine concentrations. Plasma noradrenaline was increased significantly at 60 min and adrenaline at 90 min, once furosemide had induced a marked loss of body-fluid and approximately 65% decrease in central venous pressure. Both catecholamines remained elevated until the end of the study, whereas urinary water and sodium flow rates had returned to their pre-treatment values by 150 min. Mean blood pressure was essentially unchanged throughout the study, whereas heart rate increased significantly after 90 min. The findings suggest that in mildly hypertensive patients adrenergic mechanisms are not involved in the initial renin response to furosemide, but they come into play later, probably as a result of reflex sympathetic activation triggered by marked volume depletion.

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.

2-Hydroxy-(1-hydroxy-[(1-methyl-3-phenylpropyl)amino]-2-ethyl)-5-benzamide (labetalol), a new alpha- and beta-adrenergic blocking agent, was employed in 21 patients with severe hypertension by slow (6 patients) and rapid (15 patients) i.v. infusion. A marked and significant fall of blood pressure was observed in both groups, though more gradual in patients treated by slow infusion. A rapid blood pressure fall with cardiac output decrease was observed by passing from supine to standing position in the first hours after infusion. Therefore, it is advisable to keep a supine position for a few hours. Plasma renin activity decreased after labetalol infusion, but basal plasma renin levels were not related to hypotensive effect of labetalol. In slow infusion patients, plasma noradrenaline levels increased and no changes of plasma adrenaline levels were observed during infusion. The most likely explanation of these variations is an increase of sympathetic activity secondary to hypotension, caused by labetalol, whereas a technical interference seems to be excluded. The simultaneous blockade of alpha- and beta-receptors can inhibit, in this case, the pressor effects of the sympathetic reflex.

Plasma catecholamines and cardiovascular responses to acute insulin i.v. injection (0.12 U.I. per Kg of body weight) have been studied in eight patients with mild essential hypertension. The hypoglycaemia test was carried out before and after a week of atenolol treatment. In six patients the test was also repeated after propranolol treatment. Following insulin injection, blood sugar fell abruptly with a nadir at 30 m, diastolic blood pressure decreased, and systolic blood pressure and heart rate rose significantly. Atenolol treatment abolished almost completely these hemodynamic changes. By contrast, propranolol caused an increase in both systolic and diastolic blood pressures, leaving the heart rate unmodified. During hypoglycaemia, plasma adrenaline rose sharply in coincidence with the glycaemic nadir and declined thereafter during the glycaemic recovery. Plasma noradrenaline rose less markedly than adrenaline, but the increments were significant after 30 and 60 m. Neither atenolol, nor propranolol affected significantly these changes in plasma catecholamine concentration. These data indicate that beta-blockers do not alter the hypoglycaemia-induced adrenomedullary release of catecholamines. We conclude therefore that beta-blockers modify the pattern of the hemodynamic response to acute hypoglycaemia by interfering with the peripheral effects of circulating catecholamines in a way which depends on the degree of beta-1 selectivity of the drug used.