Mosé Da Prada

Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range

A radiometric-enzymatic assay for measuring simultaneously femtomole quantities of adrenaline, noradrenaline and dopamine has been developed. The three catecholamines are first converted to their O-methylated analogues by catechol-O-methyltransferase in the presence of S-adenosyl-methionine-3H and thereafter extracted following addition of sodium tetraphenylborate. This extraction, together with an improved quick chromatographic separation and the oxidation of the adrenaline and noradrenaline derivatives to vanillin, yields an extremely high sensitivity and specificity of the method. The present assay allows the determination of adrenaline, noradrenaline and dopamine in tissue samples with a protein content of 100 μg or less and in plasma volumes of 20 – 100 μl. The amine content of 40 – 50 samples can be determined in two days by one person. Due to the high sensitivity achieved, this method promises to be a valid alternative to the gas chromatography-mass spectrometry technique.

Circadian rhythm in rat retinal dopamine

Rat retinal dopamine concentration and synthetic rate exhibit a circadian rhythm that persists in the absence of time cues. Although the population of retinal amacrine cells containing dopamine is small, retinal dopamine neurones may lie on the pathway mediating light information to the circadian system.

Effects of diethyl ether, halothane, ketamine and urethane on sympathetic activity in the rat

The present paper describes the effects of different general anaesthetics on plasma catecholamine (CA) concentrations taken as biochemical index of peripheral sympathetic activity. In chronically catheterized rats, diethyl ether, ketamine and urethane increased plasma adrenaline (A) and noradrenaline (NA) concentrations, indicating that these drugs stimulate both neurosympathetic and adrenomedullary functions. These effects appear to be centrally mediated, since ganglionic blockade or spinal transection completely counteracted the diethyl ether- and ketamine-induced increases in plasma CA levels. Halothane induced a transient decrease in circulating A and an increase in NA. These results support the concept that general anaesthetics may have different effects on sympathetic function. Arterial blood pressure and heart rate were also measured to look for possible correlations with peripheral sympathetic activity. The enhanced release of peripheral CAs seemed to be the determining factor for increasing blood pressure and heart rate with ketamine only. In the other instances the activation of the peripheral sympathetic system appeared to maintain homeostasis by counterbalancing the various depressive effects of anaesthetics on the cardiovascular system.

Serotonin transport and serotonin transporter-mediated antidepressant recognition are controlled by 5-HT2B receptor signaling in serotonergic neuronal cells

The plasma membrane 5‐HT transporter (SERT) is the major protagonist in regulating extracellular 5‐HT concentration and constitutes the target of drugs used to treat a host of metabolic and psychiatric disorders. The exact mechanisms sustaining SERT function still remain elusive. The present work exploits the properties of the 1C11 neuroectodermal progenitor, which acquires, upon 4 days of differentiation, a functional SERT within an integrated serotonergic phenotype to investigate regulatory mechanisms involved in SERT onset and functions. We show that poly(A) addition precedes SERT mRNA translation on day 2 of the serotonergic program. The newly translated transporter molecules immediately bind cocaine. Day 4 must be awaited to monitor antidepressant recognition and 5‐HT uptake. Because external 5‐HT reduces both 5‐HT transport and SERT antidepressant binding, we identify 5‐HT2B receptors as key players in controlling the overall 5‐HT transport system. In the absence of external 5‐HT, 5‐HT2B receptor coupling to NO production ensures SERT phosphorylation to basal level and maximal 5‐HT uptake. In the presence of 5‐HT, the 5‐HT2B receptor‐PKC coupling promotes additional phosphorylations of both SERT and Na+,K+‐ATPase α‐subunit, impairing the electrochemical gradient necessary to 5‐HT uptake. SERT hyperphosphorylation also affects antidepressant recognition. Finally, such 5‐HT2B receptor‐mediated control of SERT activity operates in primary neurons from raphe nuclei. Altogether, our data shed new light on the 5‐HT‐driven post‐translational modifications involved in the control of SERT activity.—Launay, J‐M., Schneider, B., Loric, S., Da Prada, M., Kellermann, O. Serotonin transport and serotonin transporter‐mediated antidepressant recognition are controlled by 5‐HT2B receptor signaling in serotonergic neuronal cells. FASEB J. 20, 1843–1854 (2006)

Baclofen and γ-hydroxybutyrate: similar effects on cerebral dopamine neurones

Abstract Baclofen (20 mg/kg) caused an increase in the content of homovanillic acid (HVA) and dopamine (DA) in rat brain 2–3 h after drug injection without appreciable changes in the level of other monoamines and their main metabolites. Six and eight hours after baclofen, the content of HVA but not that of DA was reduced. Moreover, baclofen initially (20 min after injection) reduced, but later (105 min post drug) enhanced the accumulation of HVA induced by probenecid. The shortlasting (20 min) initial reduction of HVA elevation in probenecid-pretreated animals as well as the longlasting (6–8 h) decrease of HVA levels in rats injected with baclofen alone are interpreted to be due to a decreased release and metabolism of DA, probably as a consequence of the blockade of impulse flow in mesolimbic and nigro-striatal DA neurones. The increase in HVA and DA seen during the first few hours is thought to result from enhanced DA synthesis similar to that known for γ-hydroxybutyrate (GHB). This initial rise in HVA due to synthesis stimulation probably masked a reduction of HVA to be expected immediately after baclofen injection. The similarity between baclofen and GHB is stressed by the finding that baclofen counteracted the increase of HVA occuring after chlorpromazine and D-amphetamine but not that induced by the benzoquinolizine derivative, Ro 4-1284.

Rapid and Sensitive Single‐Step Radiochemical Assay for Catechol‐O‐Methyltransferase

Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol‐O‐methyltransferase activity is described. The method is based on the conversion of catechol to [3H]guaiacol by catechol‐O‐methyltransferase in the presence of Mg2+, adenosine deaminase and S‐adenosyl l‐[methyl‐3H]methionine. Incubation and direct extraction of [3H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol‐O‐methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.

Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line.

Abstract: Human catechol‐O‐methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane‐bound catechol‐O‐methyltransferase (MB‐COMT; amino acids 1‐271) and the soluble catechol‐O‐methyltransferase COMT (S‐COMT; Δ membrane anchor hCOMT; amino acids 27‐271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high‐level synthesis of catalytically active enzymes was obtained. Insertion of the human MB‐COMT‐coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV‐hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol‐O‐methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37°C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 μM for MB‐COMT and 108 μM for S‐COMT, indicating that recombinant MB‐COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of ∼ 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue. Using the polymerase chain reaction (PCR) with primers surrounding the putative membrane anchor region, we have clearly identified a single‐size PCR product generated from hCOMT mRNA of various human tissues. Hence, the two forms of the enzyme cannot be the products of an alternative splicing of transcripts. We suggest that S‐COMT is generated by proteolytic cleavage between the NH2‐terminal membrane anchor and the catalytic domain of the membrane‐bound form. Lack of the N‐terminal fragments reduces the catalytic activity of the enzyme.

Characterisation of wild-type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line

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 (K m, k cat) 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.

Effects of tolcapone, a novel catechol-O-methyltransferase inhibitor, on striatal metabolism of L-DOPA and dopamine in rats

In vivo brain microdialysis was used to assess the effects of tolcapone, a novel central and peripheral inhibitor of catechol-O-methyltransferase on striatal 3,4-dihydroxyphenyl-L-alanine (L-dopa) and dopamine metabolism. The oral administration of 30 mg/kg of tolcapone failed to change dopamine output but elicited a marked and long-lasting decrease of the extracellular levels of homovanillic acid (HVA) and 3-methoxytyramine with a concomitant increase of 3,4-dihydroxyphenylacetic acid (DOPAC). The administration of L-dopa (20 and 60 mg/kg p.o.) + benserazide (15 mg/kg p.o.) resulted in dose-dependent increase of dialysate levels of L-dopa and 3-O-methyl-DOPA. Tolcapone (30 mg/kg p.o.), given as adjunct to both doses of L-dopa, markedly enhanced the elevation or extracellular L-dopa, while it completely prevented the formation of 3-O-methyl-DOPA. In another experiment, the administration of L-dopa + benserazide (30 + 15 mg/kg p.o.) resulted in increased extracellular levels of dopamine, DOPAC, HVA and 3-methoxytyramine. The co-administration of tolcapone (30 mg/kg p.o.) further increased dopamine and DOPAC levels, whereas HVA and 3-methoxytyramine effluxes were reduced. These findings support the notion that tolcapone has the ability to enhance striatal dopamine neurotransmission by increasing L-dopa bioavailability through peripheral and central inhibition of L-dopa O-methylation, as well as by blocking the central conversion of dopamine into 3-methoxytyramine.

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.