Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

AXOPLASMIC TRANSPORT OF AROMATIC l-AMINO ACID DECARBOXYLASE (EC 4.1.1.26) AND DOPAMTNE β-HYDROXYLASE (EC 1.14.2.I) IN RAT SCIATIC NERVE

The axoplasmic transport of aromatic l -amino acid decarboxylase and dopamine β-hydroxylase, two enzymes involved in the biosynthesis of catecholamines, was studied in rat sciatic nerve. The two enzymes exhibited markedly different axoplasmic flow characteristics, since dopamine β-hydroxylase activity accumulated on the proximal side of a ligation nearly three times as fast as aromatic l -amino acid decarboxylase activity. Distally dopamine β-hydroxylase activity remained essentially constant for 24 h, whereas aromatic l -amino acid decarboxylase activity fell precipitously. Evidence was obtained to rule out the possibility that differences in the rate of inactivation of the two enzymes could account for the different rates of accumulations observed. The conclusion, that aromatic L-amino acid decarboxylase and dopamine β-hydroxylase are transported in sympathetic nerve at different rates is discussed in relation to the biosynthesis of norepinephrine.     

THE BIOSYNTHESIS OF OCTOPAMINE-CHARACTERIZATION OF LOBSTER TYRAMINE β-HYDROXYLASE

—Tyramine β-hydroxylase catalyzes the biosynthesis of octopamine in the lobster nervous system. This enzyme has been characterized and a rapid microassay, based on the enzymic release of tritiated water from [1,2-(side chain) ³H] tyramine, has been developed. Lobster tyramine β-hydroxylase resembled mammalian dopamine β-hydroxylase. The most conspicuous differences were that the lobster enzyme was inhibited by anions, particularly fumarate, and had a higher affinity for substrates. Tyramine β-hydroxylase activity was present in both particulate and soluble fractions of homogenates of the lobster nervous system. Bound activity, extracted by repeated freezing and thawing, was partially purified. The enzyme had the following properties: (1) The optimum pH for the conversion of tyramine to octopamine was 7·4. (2) The apparent Michaelis constant for tyramine was 0·15 mm and for ascorbic acid was 0·2 mm at pH 6·6. (3) The purified enzyme was inhibited by salts; the degree of inhibition was sensitive to the anion and decreased in the order chloride ? fumarate > sulphate > acetate. (4) Tyramine β-hydroxylase was inhibited by metal chelating agents and by cupric sulphate at concentrations greater than 10^?4m ; N-ethylmaleimide had no significant effect on activity in concentrations up to 3 mm . (5) The purified enzyme also β-hydroxylated dopamine to form norepinephrine, with an apparent Michaelis constant of 0·24 mm . This activity co-purified with tyramine β-hydroxylase, suggesting that a single enzyme catalyzed both reactions.     

ON THE DEVELOPMENT OF SYMPATHETIC NERVE TRUNK VESICLES DURING AXONAL TRANSPORT: DENSITY GRADIENT ANALYSIS OF DOPAMINE β-HYDROXYLASE IN BOVINE SPLENIC NERVE

—Dopamine β-hydroxylase was used as a marker enzyme for sympathetic nerve vesicles which were studied by density gradient technique in bovine splenic nerves. The enzyme analyses were complicated by the occurrence of inhibitors which had to be carefully neutralized with copper. The inhibitor was mainly found in the soluble fraction and no evidence for the occurrence of endogenous inhibitors in the nerve vesicles was obtained. A great variation in density of the dopamine β-hydroxylase containing particles was observed. This was probably mainly due to the variation in vesicle maturation since dopamine β-hydroxylase was distributed more towards the lighter gradient fractions in the proximal nerve segment preparations compared with intrasplenic nerve segment preparations. Noradrenaline/protein and noradrenaline/dopaminc β-hydroxylase ratios were found to be increased about 1·7-fold in the vesicle fraction isolated from the proximal nerve segments to those from the intrasplenic segments. A further increase of the noradrenaline/dopamine β-hydroxylase ratio was observed in a fraction with the same density isolated from the spleen. On the basis of these findings the noradrenaline/protein ratio was calculated to be about 500-600 nmol/mg in the nerve terminal vesicles.     

Biochemical and Biological Studies on Dopastin,an Inhibitor of Dopamine β-Hydroxylase

Dopastin produced by a pseudomonas is a potent inhibitor of dopamine β-hydroxylase. Kinetic studies with the purified enzyme indicated that inhibition by dopastin was of the uncompetitive type to the substrate and of the competitive to the cofactor, ascorbic acid. The nitrosohydroxylamino group of dopastin was found to be essential in inhibition of dopamine β-hydroxylase. Both racemic and natural dopastins showed the same activity. Dopastin showed significant hypotensive effect to spontaneously hypertensive rats and phytotoxicity to barley germination.     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

Mutant cell lines resistant to azetidine-2-carboxylic acid: alterations in the synthesis of proline from glutamic acid

Two mutant Chinese hamster lung fibroblast lines have been isolated that are resistant to the toxic proline analog L-azetidine-2-carboxylic acid. The line designated AZCA-1 has 30-fold elevated activity of pyrroline-5-carboxylate synthase and a large increase in the rate of proline production and release compared to controls. Pyrroline-5-carboxylate synthase activity is not elevated in the resistant line designated AZCA-4, but the enzyme is less sensitive to inhibition by ornithine and proline than control enzyme. Intracellular proline is elevated in AZCA-4 cells, with no change in the rate of release of proline synthesized from glutamate. Resistance to azetidine carboxylic acid in both mutant lines is attributed to the expanded intracellular proline pool that results from alterations in pyrroline-5-carboxylate synthase. These results indicate that intracellular proline levels are determined at least in part by the regulated activity of pyrroline-5-carboxylate synthase.     

A time-resolved assay of dopamine β-hydroxylase activity utilizing high-pressure liquid chromatography

A time-resolved assay of dopamine β-hydroxylase (EC 1.14.17.1) activity utilizing high-pressure liquid chromatography is described. The conversion of tyramine to octopamine by the enzyme was used as a standard reaction. The analytical separation of the assay substrate and product employed a reversed-phase ion-pair chromatographic system, with ultraviolet absorbance detection of eluents at 280 nm. Aliquots of the assay solution were injected directly onto the high-pressure liquid chromatography column and were separated in 6 min total elapsed time, thus permitting time-resolved determination of the produet. Quantities of octopamine as small as 20 pmol could be measured. This facile method is more straightforward, convenient, and sensitive than previously published physical and spectroscopic methods of determining dopamine β-hydroxylase activity.     

Muscarinic and nicotinic receptor-mediated Ca2+ dynamics in rat adrenal chromaffin cells during development

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine β-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca²⁺ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca²⁺, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine β-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca²⁺ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca²⁺ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca²⁺ signaling pathway precedes the muscarinic receptor-mediated one during development.     

β-endorphin-induced increase in striatal dopamine turnover

Human β-endorphin administered intracisternally in a dose of 15 μg per rat increased striatal concentrations of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as well as producing catalepsy. These effects were inhibited by naloxone. Pargyline-induced decreases in striatal DOPAC and HVA were greater in endorphin-treated than in saline-treated animals, supporting the concept that β-endorphin increases striatal dopamine turnover. β-endorphin increased the rate of decline in striatal dopamine concentration following synthesis inhibition with α-methyltyrosine, further suggesting that endorphin increases striatal dopamine turnover. β-endorphin and probenecid interacted competitively to decrease the effects of each other to increase striatal HVA. Naloxone prevented the effect of endorphin to decrease the HVA response to probenecid. Thus, probenecid cannot be used to assess the effects of endorphin on striatal dopamine turnover. If β-endorphin acts presynaptically to decrease dopamine release in striatum, the increases in striatal DOPAC and HVA probably represent a compensatory attempt to increase dopamine synthesis. Although turnover of dopamine to its metabolites is increased, dopamine release may be suppressed by β-endorphin.     

A role for synaptotagmin (p65) in regulated exocytosis

Proteins that are specifically localized to synaptic vesicles in the nervous system have been proposed to mediate aspects of synaptic transmission. Antibodies raised against the cytoplasmic domains of five of these proteins, vamp, rab3A, synaptophysin, synaptotagmin, and SV2, were used to investigate their function. Microinjection of monoclonal and polyclonal antibodies raised against synaptotagmin (p65), but not the other vesicle proteins, decreases K⁺/Ca²⁺-mediated dopamine β-hydroxylase surface staining, a measure of regulated secretion in PC12 cells. Microinjection of a soluble fragment of synaptotagmin encompassing one of the domains homologous to the C2 regulatory region of protein kinase C, but lacking the membrane anchor, also inhibits evoked dopamine β-hydroxylase surface staining. These results provide support for the hypothesis that synaptotagmin, a Ca²⁺- and phospholipid-binding protein, is important for regulated exocytosis in neurons.     

Δ1-Pyrroline-5-carboxylate: The product of proline dehydrogenase from Cucurbita moschata cotyledons

The product of oxidation of proline by pumpkin proline dehydrogenase reacted with o-aminobenzaldehyde to give a yellow compound that had an absorption spectrum similar to that obtained from chemically synthesized Δ¹-pyrroline-5-carboxylate. The product of the proline dehydrogenase reaction and synthetic Δ¹-pyrroline-5-carboxylate had identical R_f values. Both authentic Δ¹-pyrroline-5-carboxylate and the product of the enzyme gave a pink colour with acid ninhydrin on paper chromatograms and both had identical elution patterns on Dowex 50(H⁺) columns. Neither synthetic Δ¹-pyrroline-5-carboxylate nor the product of proline-dehydrogenase produced γ-amino butyrate with hydrogen peroxide.     

The inhibition of mammalian d-amino acid oxidase by metabolites and drugs. Inferences concerning physiological function

The inhibition of d-amino acid oxidase (EC 1.4.3.3) by a large number of metabolites and drugs is reported. When the substrate is an adduct (thiazolidine-2-carboxylate) formed from cysteamine and glyoxylate, at least five different mechanisms of inhibition are possible, and examples of each are given. Effective inhibitors include (a) some adenosine containing nucleotides, in particular ADP, AMP, ADP ribose, NADH, NADPH and dephospho-CoA; (b) diuretics, especially furosemide, ethacrynic acid, and mersalyl; (c) anti-inflammatory agents, such as salicylate, indomethacin, phenylbutazone, acetylsalicylate, mefenamic, and flufenamic acids; (d) hypoglycemic, hypocalcemic, and hypolipidemic compounds, including 5-methylpyrazole-3-carboxylate, pyrrole-2-carboxylate, 5-methylthiophene-2-carboxylate, thiophene-2-carboxylate, benzoate, and nicotinate; (e) aldehydes, for example, formaldehyde, acetaldehyde, and succinate semialdehyde; (f) thiols, especially β-aminothiols such as cysteine and penicillamine; (g) tropolone, and (h) hydrogen peroxide. The rate of the reaction is also very sensitive to oxygen presure; at pH 7.4 and 25°C the K_m for O₂ is 1.1 mM.These data, in conjunction with literature information concerning the biological affects of such compounds, are used to suggest possible physiological processes in which the d-amino acid oxidase reaction may be involved. Although such correlations based on circumstantial evidence are not conclusive, they suggest that d-amino acid oxidase may play a major role in the control of metabolism in animals. Some processes in which it may participate include maintenance of ion and water balance in the kidney, inflammatory response, transmission of nerve impulses, sensing of O₂ concentration, control of cell growth, and as part of an intracellular messenger system for some hormones, especially insulin.     

The isolation and characterization of the methyl acceptor protein from adrenal chromaffin granules

A methyl acceptor protein (MAP), which serves as a substrate for adrenal medullary protein carboxymethylase (PCM, E.C. 2.1.1.24), has been isolated from a hypotonic lysate of adrenal chromaffin granules. The isolated MAP was shown to be distinct from the adrenal chromaffin granule protein, dopamine β-hydroxylase (DBH). The properties of MAP, including its amino acid composition, were comparable to those reported for chromogranin A, a major acidic protein found in adrenal chromaffin granules.     

Solubilization of histamine H-1, GABA and benzodiazepine receptors.

Dopamine 3-0-sulfate is present in considerable amounts in mammalian plasma and peripheral tissues. Incubation of dopamine 3-0-sulfate (0.1 μmole) with purified bovine dopamine-β-hydroxylase resulted in the formation of free norepinephrine (7.3 × 10^?3 μmole). The conversion to norepinephrine was inhibited by 0.6 mM of fusaric acid, an inhibitor of dopamine-β-hydroxylase. The reaction of dopamine 3-0-sulfate with dopamine-β-hydroxylase followed Michaelis-Menten kinetics. The calculated K_m was 17 mM, different from the K_m for free dopamine (0.1 mM). The incubation medium does not contain any sulfatase activity.     

Effects of fasting and food restriction on sympathetic activity in brown adipose tissue in mice

Summary The activity of the sympathetic nervous system in mice that were either fed ad libitum, food restricted or fasted was estimated by measuring the accumulation of dopamine following the inhibition of dopamine -hydroxylase activity. Mice in each group were injected with the dopamine -hydroxylase inhibitor 1-cyclohexyl-2-mercaptoimidazole and were exposed to either 30°C (warm) or 4°C (cold). Mice were killed 1 h after the injection. Both heart and brown adipose tissue were then quickly removed and homogenized in ice-cold perchloric acid. Dopamine and noradrenaline were determined using high performance liquid chromatography. Regardless of whether mice were warm or cold exposed, both content and concentration of brown adipose tissue and dopamine were predictably higher in 1-cyclohexyl-2-mercaptoimidazole-injected mice than in non-injected animals. In mice fed ad libitum, post-injection content and concentration of dopamine in both brown adipose tissue and heart were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice, post-injection concentrations of dopamine in brown adipose tissue were higher in cold-exposed mice than in warm-exposed animals. In food-restricted and fasted mice there was no difference between warm- and cold-exposed animals with respect to post-injection contents and concentrations of dopamine in heart tissue. In fasted mice there was no difference between warm- and cold-exposed animals in post-injection content of dopamine in brown adipose tissue. This study provides further evidence that fasting, in contrast to food restriction, may blunt the tissue sympathetic nervous system response in brown adipose tissue of cold-exposed mice.Abbreviations BAT brown adipose tissue - CHMI 1-cyclohexyl-2-mercaptoimidazole - DA dopamine - DHBA dihydroxybenzylamine - EDTA ethylenediaminetetra-acetic acid - HPLC high performance liquid chromatography - NA noradrenaline - PCA perchloric acid - SNS sympathetic nervous system     

Discovery of β-aminoacyl containing thiazolidine derivatives as potent and selective dipeptidyl peptidase IV inhibitors

A series of β-aminoacyl containing thiazolidine derivatives was synthesized and evaluated for their ability to inhibit DPP-IV. Several thiazolidine derivatives with an acid moiety were found to be potent DPP-IV inhibitors. Among them, compound 2da is the most active in this series with an IC₅₀ value of 1 nM, and it showed excellent selectivity over DPP-IV related enzymes including DPP-2, DPP-8, and DPP-9. Compound 2da is chemically and metabolically stable, and showed no CYP inhibition, hERG binding or cytotoxicity. Compound 2db, an ester prodrug of 2da, showed good in vivo DPP-IV inhibition after oral administration in rat and dog models.     

Dopamine β-hydroxylase of bovine adrenal medullae. A rapid purification procedure

1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca²⁺ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro. 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p-chloromercuribenzoate or Cu²⁺ ions.     

Formation and excretion of pyrrole-2-carboxylic acid. Whole animal and enzyme studies in the rat.

A corrected method for the measurement of pyrrole-2-carboxylate in rat urine was used in studies of its excretion under various experimental conditions. The findings implicated administered hydroxy-L-proline as a relatively efficient source of urinary pyrrole-2-carboxylate and tended to exclude administered L-proline as a significant direct source. Removal of aerobic gut flora had no influence on the excretion of pyrrole-2-carboxylate either endogenously or following hydroxy-L-proline administration. Related studied showed that rat kidney L-amino acid oxidase catalyzes oxidation of hydroxy-L-proline to delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate on acidification of reaction mixtures. All findings were consistent with hydroxy-L-proline as the source of endogenous pyrrole-2-carboxylate excretion. Excretion patterns and labeling patterns were compared after administration of pyrrole-2-carboxylate or of hydroxy-proline epimers. From these data, the true excretion product of hydroxy-L-proline oxidation by L-amino acid oxidase appeared to be the unstable oxidation product, delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate in urine. The capacity of homogenates of guinea pig kidney and human kidney to carry out oxidation of hydroxy-L-proline to pyrrole-2-carboxylate was much less than that of rat kidney, consistent with the lower levels of urinary pyrrole-2-carboxylate in these species. Experiments designed to examine the modest increase of pyrrole-2-carboxylate excretion after proline loads led to new observations on tissue levels of hydroxy-L-proline following proline administration and on the inhibition by L-proline of hydroxy-L-proline oxidase.     

Uncoupling by proteolysis of alpha-adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets

Ethyl 2{5(4-chlorophenyl)pentyl}oxiran-2-carboxylate (POCA) is a new hypoglycaemic compound. The POCA-CoA ester strongly inhibits β-oxidation at carnitine palmitoyltransferase I. Chronic administration of POCA to rats decreases plasma concentrations of cholesterol and triacylglycerol and increases the number of hepatic peroxisomes similarly to hypolipidaemic drugs related to clofibrate. Peroxisomal fractions from rats fed a diet containing 0.2% of POCA for 4 weeks were prepared on self-generated Percoll gradients. POCA induced a 4-fold increase in catalase activity and peroxisomal β-oxidation, agreeing with the morphological data. The increase in peroxisomal β-oxidation caused by POCA feeding does not prevent accumulation of lipid following the inhibition of mitochondrial β-oxidation.