Cyrus R. Creveling

A radioisotopic method for measuring the formation of adenosine 3',5'-cyclic monophosphate in incubated slices of brain.

Abstract— A simple and sensitive method for measuring the effect of neurohormones and other chemical agents on the formation of adenosine 3′,5′‐cyclic monophosphate (cyclic 3′,5′‐AMP) in incubated slices of brain was developed. The principle of the method depends on pulse‐labelling of adenosine‐5′‐triphosphate in slices of brain with [8‐14C]adenine, followed by incubation in a medium containing the test substance, separation by thin‐layer chromatography of the cyclic nucleotide formed in the slices, and radioassay. The purity of the cyclic nucleotide was confirmed by chromatography in a variety of systems and by hydrolysis with a specific, bovine‐heart phosphodiesterase. The method was used to study the effect of histamine, norepinephrine, and adenosine on the accumulation of adenosine 3′,5′‐cyclic monophosphate in incubated slices of brain.

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 10−5M. The therapeutic and clinical implications of this observation are pointed out.

Biological Methylation and Drug Design

This paper reviews the work available to date suggesting that elements of the one-carbon cycle may be involved in psychiatric illnesses. Two enzymes of the one-carbon cycle (in erythrocytes) methionine adenosyltransferase (MAT) and serine hydroxymethyltransferase (SHMT) have been reported by our group to be underactive in schizophrenia and depression and overactive in mania. This correlates with the reported anti-depressant effects of S-adenosylmethionine in humans. SHMT has also been found by another group to be underactive in serum of schizophrenics. This defect appears to be linked to abnormalities in the distribution of phospholipids in the membrane, with a relative deficiency of phosphatidylcholine and a relative excess of phosphatidylserine. Evidence showing that medications do not appear to be responsible for these findings will be presented. A review is then made of the role of transmethylation systems for lipids and proteins in cellular control systems. Lipid transmethylation is related in many systems to receptor-final messenger coupling and so to information transfer across the membrane. Protein methylation is involved in many cellular systems including chemotaxis in bacteria and leucocytes, neurotransmitter release, sperm motility and calmodulin function.

Biochemistry of S-Adenosylmethionine and Related Compounds

Immunocytochemical localization of catechol-O-methyltransfersase (COMT) by the peroxidase-antiperoxidase (PAP) technique has demonstrated the presence of the enzyme in normal and cancerous breast tissues of rodents and humans. Glandular epithelial, ductual epithelial, myoepithelial, capillary endothelial and fibroblast cells all exhibi ted reaction product indicative of the presence of COMT. Normal tissues were least reactive, and poorly differentiated, highly malignant tumors were most reactive. Staining intensities of lactating tissues or benign tumors were between the two extremes. Recently, two groups of investigators have reported biochemical evidence of a correlation between COMT activities and metastatic potential (degree of differentiation) of human breast tumors (Assicot et al., 1977; Hoffman et al., 1979). The reasons for such an ·assoc;atlon are not clear;-DUl:there has been speculation about the possible antiestrogenic effects of catecholestrogens or their O-methylated metabolites and what effects such metabol i tes might have on ti ssues normally under estrogen hormonal control (Ball et al., 1971; Gelbke et al., 1977). Havi ng demonstrated the presence of COMT in various ti ssues by immunocytochemical means (Lowe and Creveling, 1979; Inoue et al., 1977), we decided to examine normal and cancerous tissues frOOl mice, rats and humans in order to determine what cell s contain the enzyme and whether or not there appears to be more COMT in cancerous tissues. All tissues were immersion fixed in 95% ethanol 5% glacial

The combined use of α-methyltyrosine and threo-dihydroxyphenylserine—selective reduction of dopamine levels in the central nervous system

Abstract Dihydroxyphenylserine- α - 14 C (DOPS- α - 14 ) is taken up into mouse brain and decarboxylated to form norepinephrine- 14 C. The combined use of nontracer doses of this amino acid ( threo- or erythro-DOPS ) and the tyrosine hydroxylase inhibitor, α-methyltyrosine ethyl ester, causes a 50 per cent depletion of dopamine in the central nervous system in mice while leaving norepinephrine levels unchanged. Without threo -DOPS, α-methyltyrosine depletes both norepinephrine and dopamine levels to less than one-half their normal values. dl - Threo -DOPS rapidly replenishes norepinephrine levels in reserpine-treated mice, but does not cause the awakening effect shown by l -DOPA under the same conditions. It is suggested that the reversal of the reserpine syndrome by l -DOPA is due to formation of dopamine rather than norepinephrine in the central nervous system.

Accumulations of Cyclic AMP in Adenine‐Labeled Cell‐free Preparations from Guinea Pig Cerebral Cortex: Role of α‐Adrenergic and H1‐Histaminergic Receptors

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine‐labeled nucleotides in particulate fractions from Krebs‐Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac‐like entities, which apparently are associated with a significant portion of the tnembranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine‐labeled cyclic AMP‐generating systems by norepinephrine is by means of α‐adrenergic receptors, while activation by histamine is through H1‐ and H2‐histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via „release” of endogenous adenosine. It is proposed, based on the virtual absence of an α‐adrenergic or H1‐histaminergic response in the presence of a combination of potent adenosine and H2‐histaminergic antagonists, that α‐adrenergic and H1‐histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs‐Ringer particulate fractions, but merely facilitate activation by β‐adrenergic, H2‐histaminergic, or adenosine receptors.

LOCALIZATION OF DOPAMINE‐β‐OXIDASE IN BRAIN

THE sympathetic hormone norepinephrine has been shown to be derived from tyrosine and it is generally recognized that the immediate precursor is 3 :4dihydroxyphenylethylamine (dopamine). Conversion of the latter compound to norepinephrine in isolated peripheral tissues has been demonstrated many times. The presence of both dopamine and norepinephrine in the central nervous system suggested that this conversion also takes place in brain. The data presented in this report indicate that not only does brdn convert dopamine to norepinephrine but that the enzyme responsible for this conversion, dopamine-8-oxidase, is localized in those areas of the brain which are richest in norepinephrine and other catalysts and intermediates involved in its synthesis.

Soluble and Particulate Forms of Rat Catechol‐O‐Methyltransferase Distinguished by Gel Electrophoresis and Immune Fixation

Abstract: Catechol‐O‐methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDSPAGE) of all tissue homogenates examined revealed three major immune‐specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 × g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an “aggregate” of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two‐dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pl of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1–5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is “soluble” with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.

The purification and kinetic properties of liver microsomal-catechol-O-methyltransferase☆

Abstract A membrane-bound form of catechol-O-methyltransferase (COMT) has been solubilized and partially purified from rat liver microsomes. The purified microsomal-COMT was found to be neutralized by antibody to the soluble-COMT. The pH optimum, the kinetic constants for catechol substrates and S-adenosylmethionine, and the sensitivity to inhibitors of this microsomal-COMT were all found to be similar to the soluble-COMT.

The effect of ring-fluorination on the rate of O-methylation of dihydroxyphenylalanine (DOPA) by catechol-O-methyltransferase: significance in the development of 18F-PETT scanning agents.

The three ring-fluorinated analogs of dihydroxyphenylalanine (DOPA) were synthesized and the kinetics of their O-methylation catalyzed by catechol-O-methyltransferase compared to those of DOPA. The affinities (Km) of 2-FDOPA, 5-FDOPA, DOPA, and 6-FDOPA were 20, 23, 55, and 552 microM, respectively, whilst the Vmax values were 4.0, 4.4, 5.0 and 5.4 nmol per min per mg protein. The importance of the low affinity and decreased rate constant of 6-FDOPA for COMT are discussed with regard to the use of 6-FDOPA as an 18-fluorine labeled probe for positron emission transaxial tomography (PETT).