Hydroxylation Of Dopamine Research Articles

In vitro Species Different Metabolism and CYP Phenotyping of Zamicastast

BackgroundZamicastat (1) is an inhibitor of dopamine‐β‐hydroxylase, enzyme that catalyzes the hydroxylation of dopamine to norepinephrine in sympathetic nerves and adrenal medulla, and it was shown to increase the survival rate in the monocrotaline rat model of pulmonary arterial hypertension.ObjectivesThe purpose of this study was to evaluate zamicastat clearance and metabolic profile in hepatocytes from different species (human, rat, dog, mouse and monkey) and to identify the CYP450 enzymes isoforms that are involved in zamicastat metabolism. Methods. Metabolite profiling of zamicastat (10 μM) was conducted in human, rat, dog, mouse and monkey cryopreserved hepatocytes. Samples were harvested up to 120 min and analyzed by High Resolution Mass Spectrometry (HR‐MS). The Intrinsic Clearance (CLint) values for zamicastat were assessed in all species. For CYP reaction phenotyping, the study was conducted on human CYP450 recombinant enzymes incubated with zamicastat (5 μM) and NADPH, with samples being harvested up to 45 min and analyzed by LC‐MS/MS.ResultsThe CLint for zamicastat was estimated at 40.3, 32.6, 13.6, 44.2 and 6.29 μl/min/106 cells in mouse, rat, dog, monkey and human hepatocytes, respectively. A total of 21 different metabolites were found where 11 were detected in human, 14 in rat, 7 in dog and 13 in mouse and monkey. In general, the metabolites observed in hepatocytes across species were formed as a result of multiple biotransformation processes, including oxidations, desaturations, acetylations, loss of sulfur, thioureas to ureas and glucuronidations. A potential biotransformation that includes S‐Glutathione conjugation (M21) was only detected in rat species whereas oxidation plus desaturation (M5), loss of sulfur (M7 and M8) and glucuronidation (M20) were detected in all species. Under these experimental conditions, zamicastat was mainly metabolized by CYP2D6. The extent of metabolism, in decreasing order was CYP2D6 > CYP1A2 > CYP2C19 > CYP2C8 > CYP3A4. No zamicastat metabolism was detected when incubated with CYP2B6 or CYP2C9 recombinant enzymes.ConclusionAll the metabolites detected in human hepatocytes were also detected in the pre‐clinical species suggesting that, under the conditions tested no human specific metabolite was observed. The major cytochromes P450 involved in zamicastast metabolism are CYP2D6, CYP1A2 and 2C19.

Effects of zamicastat treatment in a genetic model of salt-sensitive hypertension and heart failure

Hyperactivity of sympathetic nervous system plays an important role in the development and progression of cardiovascular diseases. An approach to mitigate the enhanced sympathetic nervous system drive is restricting the biosynthesis of noradrenaline via inhibition of the enzyme dopamine β-hydroxylase (DβH), that catalyzes the hydroxylation of dopamine to noradrenaline in sympathetic nerves. The aim of the present study was to evaluate the effects of zamicastat, a novel DβH inhibitor that decreases noradrenaline and increases dopamine levels in peripheral sympathetically innervated tissues, on the hemodynamic and cardiometabolic parameters in salt-induced hypertension and heart failure in the Dahl salt-sensitive (SS) rat. Zamicastat (10, 30 and 100 mg/kg body weight) was tested acutely against salt-induced hypertension in the Dahl SS rat. Chronic zamicastat treatment (30 mg/kg/day) was evaluated against salt-induced cardiac hypertrophy and biomarkers of cardiometabolic risk and inflammation in Dahl SS rats and upon the survival rate in aged Dahl SS rats fed a high-salt diet.The reduction in the sympathetic tone attained with zamicastat shaped a dose- and time-dependent effect on blood pressure. Prolonged treatment with zamicastat ameliorated end-organ damage, metabolic syndrome and inflammation hallmarks in hypertensive Dahl SS rats. Survival rate of Dahl SS rats fed a high-salt diet demonstrated that zamicastat increased median survival of Dahl SS rats fed a high-salt diet. The use of DβH inhibitors, like zamicastat, is a promising approach to treat hypertension, heart failure and cardiovascular diseases where a reduction in the sympathetic tone has beneficial effects.

Association between a functional polymorphism on the dopamine-β-hydroxylase gene and reward dependence in two independent samples

Cloninger's biosocial personality theory postulates a biological basis for the basic temperaments. With respect to reward dependence (RD) characterized by a tendency to sustain rewarding behaviors and to prefer behaviors previously associated with reward, he hypothesized low noradrenaline (NA) levels. Previous endocrine and genetic association studies have supported this hypothesis. The aim of the present study was to test for an association between the functional single nucleotide polymorphism rs1611115 (C-970T) on the dopamine-β-hydroxylase (DBH) gene and RD. DBH is an enzyme crucial for the synthesis of NA by catalyzing the oxidative hydroxylation of dopamine to NA.In two independent samples (N=1144 and N=826) of healthy participants we found significant genotype effects (p≤0.0001 and p≤0.05). Carriers of the C allele had significant higher RD scores than carriers homozygous for the T allele.Although the general RD–NA hypothesis was corroborated, the directionality of the observed effect was antithetical. Based on previous functionality studies reporting low enzymatic activity related to the TT genotype, present results indicate high NA levels in subjects scoring high on RD (in contrast to Cloninger's thesis). Future studies have to analyze how DBH activity and NA levels are dependent on the availability of dopamine.

Simple Photometric Assay of Dopamine-β-Hydroxylase Activity in Human Blood: Useful in Clinical Chemistry

Featured Article: Nagatsu T and Udenfriend S. Photometric assay of dopamine-β-hydroxylase in human blood. Clin Chem 1972;18:980–3.1 This report described a simple assay for measuring the activity of dopamine-β-hydroxylase (DBH2 ) in human blood. DBH is a copper-containing, ascorbate-requiring glycoprotein enzyme that catalyzes the hydroxylation of dopamine to norepinephrine (NE), the precursor of epinephrine (E). DBH is localized in the synaptic vesicles in the central NE and E neurons and peripheral sympathetic NE neurons and in the chromaffin granules in E and NE cells of the adrenal medulla; DBH is released by exocytosis together with NE or E, and appears in both blood and cerebrospinal fluid (CSF). Since I was a medical student at Nagoya University, Japan, I have been interested in the chemical mechanisms of neuropsychiatric diseases and, for that reason, in the biochemistry of catecholamine neurotransmitters that are assumed to be closely related to various brain diseases. I first worked with Sidney Udenfriend in 1964 at NIH, where we discovered tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of catecholamines. Studies …

Catalytic Mechanism of Dopamine β-Monooxygenase Mediated by Cu(III)−Oxo

Mechanisms of dopamine hydroxylation by the Cu(II)-superoxo species and the Cu(III)-oxo species of dopamine beta-monooxygenase (DBM) are discussed using QM/MM calculations for a whole-enzyme model of 4700 atoms. A calculated activation barrier for the hydrogen-atom abstraction by the Cu(II)-superoxo species is 23.1 kcal/mol, while that of the Cu(III)-oxo, which can be viewed as Cu(II)-O*, is 5.4 kcal/mol. Energies of the optimized radical intermediate in the superoxo- and oxo-mediated pathways are 18.4 and -14.2 kcal/mol, relative to the corresponding reactant complexes, respectively. These results demonstrate that the Cu(III)-oxo species can better mediate dopamine hydroxylation in the protein environment of DBM. The side chains of three amino acid residues (His415, His417, and Met490) coordinate to the Cu(B) atom, one of the copper sites in the catalytic core that plays a role for the catalytic function. The hydrogen-bonding network between dopamine and the three amino acid residues (Glu268, Glu369, and Tyr494) plays an essential role in substrate binding and the stereospecific hydroxylation of dopamine to norepinephrine. The dopamine hydroxylation by the Cu(III)-oxo species is a downhill and lower-barrier process toward the product direction with the aid of the protein environment of DBM. This enzyme is likely to use the high reactivity of the Cu(III)-oxo species to activate the benzylic C-H bond of dopamine; the enzymatic reaction can be explained by the so-called oxygen rebound mechanism.

Computational Exploration of the Catalytic Mechanism of Dopamine β-Monooxygenase: Modeling of Its Mononuclear Copper Active Sites

Dopamine hydroxylation by the copper-superoxo, -hydroperoxo, and -oxo species of dopamine beta-monooxygenase (DBM) is investigated using theoretical calculations to identify the active species in its reaction and to reveal the key functions of the surrounding amino acid residues in substrate binding. A 3D model of rat DBM is constructed by homology modeling using the crystal structure of peptidylglycine alpha-hydroxylating monooxygenase (PHM) with a high sequence identity of 30% as a template. In the constructed 3D model, the CuA site in domain 1 is coordinated by three histidine residues, His265, His266, and His336, while the CuB site in domain 2 is coordinated by two histidine residues, His415 and His417, and by a methionine residue, Met490. The three Glu268, Glu369, and Tyr494 residues are suggested to play an important role in the substrate binding at the active site of DBM to enable the stereospecific hydrogen-atom abstraction. Quantum mechanical/molecular mechanical (QM/MM) calculations are performed to determine the structure of the copper-superoxo, -hydroperoxo, and -oxo species in the whole-enzyme model with about 4700 atoms. The reactivity of the three oxidants is evaluated in terms of density-functional-theory calculations with small models extracted from the QM region of the whole-enzyme model.

Kinetic Evidence for Channeling of Dopamine between Monoamine Transporter and Membranous Dopamine-β-monooxygenase in Chromaffin Granule Ghosts

The nature of coupling between the uptake and dopamine-beta-monooxygenase (DbetaM) catalyzed hydroxylation of dopamine (DA) was studied in bovine chromaffin granule ghosts. Initial rate and transient kinetics of DA uptake and conversion were determined under a variety of conditions. The uptake kinetics of DA, norepinephrine (NE), and epinephrine demonstrate that DA is a better substrate than NE and epinephrine under optimal uptake conditions. The transient kinetics of DA accumulation and NE production under both optimal uptake and uptake and conversion conditions were zero-order with no detectable lag or burst periods. The mathematical analyses of the data show that a normal sequential uptake followed by the conversion process could not explain the observed kinetics, under any condition. On the other hand, all experimental data are in agreement with a mechanism in which DA is efficiently channeled from the vesicular monoamine transporter to membranous DbetaM for hydroxylation, prior to the release into the bulk medium of the ghost interior. The slow accumulation of DA under optimal conversion conditions appears to be caused by the slow leakage of DA from the channeling pathway to the ghost interior. Because DbetaM activity in intact granules is equally distributed between soluble and membranous forms of DbetaM, if an efficient channeling mechanism is operative in vivo, soluble DbetaM may not have access to the substrate, making the catalytic activity of soluble DbetaM physiologically insignificant, which is consistent with the increasing experimental evidence that membranous DbetaM may be the physiologically functional form.

Multiple Pathways in Regulation of Dopamine β-Hydroxylase

Publisher Summary DBH is localized in membrane-bound (77-kDa) and soluble (73-kDa) forms in neurosecretory vesicles of the noradrenergic neurons of the central and peripheral nervous systems and in the chromaffin granules of the adrenal medullary cells. Experiments from several groups reveal that both forms of DBH arise from a single translational product. Dopamine β -hydroxylase (DBH) catalyzes the hydroxylation of dopamine to form norepinephrine. The expression of DBH is elevated in vivo in response to a variety of transsynaptic signals, hormones, growth factors, and stress. There is now a large body of experiments demonstrating that with prolonged conditions that stimulate catecholamine biosynthesis, the expression of DBH also is markedly elevated. In vivo , DBH gene expression has been shown to be elevated by several important physiological and pharmacological stimuli. These include treatment of rats with reserpine, or nicotine or electrical preganglionic stimulation. Exposure to stress markedly activates DBH gene expression. Using electrophoretic mobility shift assays, it has been shown that the transcription factors are not identical to those reported to interact with its human analogue. For example, the human DBH promoter oligonucleotide analogous to DBH-1 has been shown to bind YY1.

Mechanism-based inhibitors of dopamine β-hydroxylase

The copper-containing monooxygenase dopamine β-hydroxylase catalyzes the hydroxylation of dopamine at the benzylic position to form norepinephrine. Mechanismbased inhibitors for dopamine β-hydroxylase have been used as probes of the mechanism of catalysis. The variety of such inhibitors that have been developed for this enzyme can be divided into three groups: (i) those in which the inactivating species is formed by abstraction of a hydrogen atom to form a radical intermediate; (ii) those in which the inactivating species is formed by abstraction of an electron to form an epoxide-like intermediate; and (iii) those in which the product is the inactivating species. A mechanism consistent with inactivation by all three groups of inhibitors which proposes that hydroxylation of dopamine by dopamine β-hydroxylase involves formation of a benzylic radical has been developed. The benzylic radical is formed by abstraction of a hydrogen atom from the substrate by a high-potential copper-oxygen species.

Ascorbic acid regulation of norepinephrine biosynthesis in isolated chromaffin granules from bovine adrenal medulla.

The effect of ascorbic acid on the conversion of dopamine to norepinephrine was investigated in isolated chromaffin granules from bovine adrenal medulla. Ascorbic acid was shown to double the rate of [3H]norepinephrine formation from [3H]dopamine, despite no demonstrable accumulation of ascorbic acid into chromaffin granules. The enhancement of norepinephrine biosynthesis by ascorbic acid was dependent on the external concentrations of dopamine and ascorbate. The apparent Km of the dopamine beta-hydroxylation system for external dopamine was approximately 20 microM in the presence or absence of ascorbic acid. However, the apparent maximum velocity of norepinephrine formation was nearly doubled in the presence of ascorbic acid. By contrast, the apparent Km and Vmax of dopamine uptake into chromaffin granules were not affected by ascorbic acid. Norepinephrine formation was increased by ascorbic acid when the concentration of ascorbate was 200 microM or higher; a concentration of 2 mM appeared to induce the maximal effect under the experimental conditions used here. The effect of ascorbic acid on conversion of dopamine to norepinephrine required Mg-ATP-dependent dopamine uptake into chromaffin granules. In contrast to ascorbic acid, other reducing agents such as NADH, glutathione, and homocysteine were unable to enhance norepinephrine biosynthesis. These data suggest that ascorbic acid provides reducing equivalents for hydroxylation of dopamine despite the lack of ascorbate accumulation into chromaffin granules. These findings imply the functional existence of an electron carrier system in the chromaffin granule which transfers electrons from external ascorbic acid for subsequent intragranular norepinephrine biosynthesis.

Enhancement of norepinephrine biosynthesis by ascorbic acid in cultured bovine chromaffin cells.

Ascorbic acid donates electrons to dopamine beta-monooxygenase during the hydroxylation of dopamine to norepinephrine in vitro. However, the possible role of ascorbic acid in norepinephrine biosynthesis in vivo has not been defined. We therefore investigated the effect of newly accumulated ascorbic acid on catecholamine biosynthesis in cultured bovine adrenal chromaffin cells. Cells supplemented for 3 h with ascorbic acid accumulated 9-fold more ascorbic acid than found in control cells. Under these conditions, the cells loaded with ascorbate were found to double the rate of norepinephrine biosynthesis from [14C]tyrosine compared to control. By contrast, the amounts present of [14C] 3,4-dihydroxyphenylalanine and [14C]dopamine synthesized from [14C]tyrosine were unaffected by the preloading of ascorbic acid. Ascorbate preloaded cells incubated with [3H]dopamine also showed a similar increase in the rate of norepinephrine formation, without any change in dopamine transport into the cells. Thus, these data were consistent with ascorbate action at the dopamine beta-monooxygenase step. In order to determine if ascorbate could interact directly with dopamine beta-monooxygenase localized within chromaffin granules, we studied whether isolated chromaffin granules could accumulate ascorbic acid. Ascorbic acid was not transported into chromaffin granules by an uptake or exchange process, despite coincident [3H]dopamine uptake which was Mg-ATP dependent. These data indicate that ascorbic acid does augment norepinephrine biosynthesis in intact chromaffin cells, but by a mechanism that might enhance the rate of dopamine hydroxylation indirectly.

Evidence for two copper atoms/subunit in dopamine beta-monooxygenase catalysis.

The stoichiometry of the copper requirement for the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine has been investigated by rapid chemical-quench techniques. This approach, which employs concentrated samples of enzyme, overcomes ambiguities of interpretation arising from levels of trace copper in excess of enzyme concentrations normally added to steady state kinetic assays. Low turnover numbers are observed when rapid quench kinetic studies are performed under conditions in which enzyme concentrations (2.5-7.1 microM) are in excess over trace copper levels (about 0.7 microM). The addition of exogenous Cu(II) results in full restoration of activity, which is maximal at a stoichiometry of 2 mol of copper/mol of enzyme subunit. From the dependence of catalytic activity on copper levels we conclude that both coppers are required for catalysis. No stimulation of activity was observed upon addition of the following metal ions: Ni(II), Co(II), Mn(II), Fe(III), and Zn(II). In addition, the magnitude of the tritium isotope effect for [2-3H]dopamine hydroxylation is invariant over a large range of enzyme activities accompanying changes in the ratio of copper to enzyme concentration. These results appear to rule out an effector role for the second mole of copper/subunit, implicating both copper atoms in active site redox chemistry.

Tyrosine hydroxylase and dopamine β-hydroxylase inhibiting properties of a new series of pyridazinyl hydrazones

New pyridazinyl hydrazones are described as a novel class of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) inhibitors. A great number of these substances showed potent TH- or DBH-inhibiting capacities. The structure-activity analysis suggested an important role for the N 2 substitution of hydrazine in the inhibiting characteristics of the compounds. β-Substituted pyridazinyl hydrazones showed favourable TH- or DBH-inhibiting activities, whereas the free hydrazines were only slightly effective on tyrosine hydroxylation and noneffective on dopamine hydroxylation. The β-ketoester derivatives of 6-chloro-3-pyridazinyl hydrazones were characterized as potent DBH-inhibiting substances while the cyclohexylidene or the bicycloheptylidenepyridazinyl hydrazones were found to be marked TH- and DBH-blocking agents. Among the above substances, GYKI 11679 {chemically 1-(6-morpholino-3-pyridazinyl)-2-[(1-tertiary-butoxycarbonyl)-2-propylidene] hydrazine} has been found to be the most potent inhibitor of tyrosine hydroxylation both in vitro and in vivo. Its in vitro as well as its in vivo TH-blocking activity was comparable with that of α-methyltyrosine. The most effective representative of this new series on dopamine and tyramine hydroxylations in vitro and in vivo, compound GYKI 11473 {chemically 1-(6-chloro-3-pyridazinyl)-2-[(1-ethoxycarbonyl)-2-propylidene] hydrazine}, has been characterised as a reversible and competitive inhibitor of DBH with dopamine and a noncompetitive inhibitor of DBH with tyramine as substrate at concns of 10 −5–10 −6 M. The compound has also been found to inhibit the neuronal and the vesicular uptake of dopamine in hypothalamic slices. GYKI 11473 showed a greater effect in the heart than in the brain and its in vivo efficiency has been established as being more potent than those of fusaric acid in both tissues. The noradrenaline (NA) determinations suggested similar, but not equal, and exclusive contributions of TH and DBH in the maintenance of NA pools in these tissues.

Dopamine and norepinephrine in the brains of hepatectomized rats

After complete hepatectomy in rats, there was a profound depletion of norepinephrine throughout the brain, whereas dopamine concentrations remained within the normal range. This suggests that in liver failure there is inhibition of norepinephrine synthesis at one of two processes subsequent to the formation of dopamine--that is, either the uptake of dopamine by vesicles or the hydroxylation of dopamine may be inhibited. However, other factors could contribute to the depletion of norepinephrine, including degeneration of its specific storage sites, displacement by false neurotransmitters, or an increased rate of catabolism. The present experiments do not allow a selection among these possibilities.

A possible change in the rate-limiting step for cardiac norepinephrine synthesis in the cardiomyopathic Syrian hamster.

The development of heart failure in the cardiomyopathic hamster is associated with a decrease in norepinephrine stores and parallel increases in cardiac sympathetic tone and tyrosine hydroxylase activity. Despite the increase in tyrosine hydroxylase, cardiac norepinephrine synthesis does not increase in heart failure. In this study, we have shown that an accumulation of cardiac dopamine accompanies the decline of cardiac norepinephrine. The abnormal content of norepinephrine and of dopamine in the decompensating hamster heart is restored to normal by peripheral ganglionic blockade. The acute increase in cardiac sympathetic tone induced by immobilization stress in control hamsters mimics the alterations in cardiac catecholamine distribution found in heart failure. Other investigators have demonstrated similar alterations in the catecholamine content of the rat submaxillary gland and adrenal medulla following an increase in sympathetic input to these organs. We conclude that the increase in cardiac sympathetic tone in the late stages of hamster cardiomyopathy appears to lead to a shift in the rate-limiting step for norepinephrine synthesis from the hydroxylation of tyrosine to the hydroxylation of dopamine. There is evidence that this shift which results in an accumulation of dopamine in the noradrenergic nerve terminals of the heart is a general manifestaion of augmented sympathetic nerve traffic rather than a peculiarity of hamster cardiomyopathy.

Differential responsiveness of central noradrenergic and dopaminergic neuron tyrosine hydroxylase to hypophysectomy, ACTH and glucocorticoid administration.

Hypophysectomy leads to a small increase in tyrosine hydroxylase activity of all brain areas containing noradrenergic neurons or tuberoinfundibular dopamine neurons, but nigroneostriatal dopamine neurons are not so affected. ACTH or corticosterone treatment inhibited this effect of hypophysectomy in some noradrenergic neurons and in tuberoinfundibular dopamine neurons. These data showing differential responsiveness of tyrosine hydroxylase in different brain areas are compatible with differences in regulation or molecular form of tyrosine hydroxylase in central noradrenergic and dopaminergic neurons. The disparity between increased hypothalamic tyrosine hydroxylase activity and decreased norepinephrine turnover following hypophysectomy may result from a change in the rate-limiting step to the hydroxylation of dopamine.

Studies of enzyme-mediated reactions. Part VII. Stereospecific syntheses of tritium-labelled (2R)- and (2S)-dopamines: stereochemical course of hydroxylation of dopamine by dopamine β-hydroxylase (E.C. 1.14.17.1)

An efficient synthesis has been developed for the preparation in high configurational purity of dopamines (3,4-di-hydroxyphenethylamines) stereospecifically labelled at C-2 with isotopes of hydrogen. By incubating (2R)-, and (2S)-[2-2H1]dopamines with dopamine β-hydroxylase (E.C. 1.14.17.1) it has been shown that the pro-R-hydrogen atom is lost in the formation of noradrenaline; thus the hydroxylation of the benzylic methylene group occurs with retention of configuration.

Stereochemical course of hydroxylation of dopamine by dopamine-β-hydroxylase (EC1.14.17.1)

(2R)-, and (2S)-[2-3H1]Dopamines are synthesised having high configurational purity; they are used to prove that hydroxylation of dopamine to yield noradrenalin occurs with retention of configuration.

An inherited deficiency in noradrenaline biosynthesis in the brindled mouse.

Abstract—A reduction in central and peripheral synthesis of noradrenaline has been demonstrated in mice hemizygous for the X‐linked brindled (Mobr) mutant in the mouse. The results are consistent with defective hydroxylation of dopamine to NA, arising either as a result of a primary genetic defect in the enzyme dopamine‐β‐hydroxylase or from the presence of a highly specific inhibitor of this enzyme in mutant mice. Associated with this deficiency of noradrenaline are an increase in the activity of tyrosine hydroxylase in the central nervous system and an increase in the active uptake of tyrosine (and other amino acids sharing a common uptake mechanism with tyrosine) across the blood‐brain barrier.

Substrates and inhibitors of dopamine-β-oxidase

The enzyme dopamine-β-oxidase not only effects the hydroxylation of dopamine to norepinephrine, but accepts as substrates a wide vareety of phenethylamine derivatives such as epinine, m-tyramine and their branched α-methyl derivatives, m-methoxytyramine, which is converted to normetanephrine, 3,5-dimethoxytyramine, and to some extent even mescaline. The fact that many well-known sympathomemetic drugs are good substrates forthe enzyme raises the pissibility that compounds such as amphetamine, paredrinol, paradrine and α-methyl- m-tyramine owe some of their activity to the corresponding metabolites which are all derivatives of ephedrine. The non-specificity of dopamine-β-oxidase prompted a search for competitive or specific inhibitors. enzylhydrazine, isosteric with phwnethylamine, inhibited the enzyme strongly at concentrations of 10 −5 M. The therapeutic and clinical implications of this observation are pointed out.