Helen L. White

Diversion of prostaglandin endoperoxide metabolism by selective inhibition of thromboxane A2 biosynthesis in lung, spleen or platelets

Infusion of arachidonic acid through the guinea pig lung or the cat spleen causes a release of thromboxane A2 and prostaglandins, as measured by bioassay. After incubation of human platelets with arachidonate similar metabolites are formed, as demonstrated chromatographically. Infusion of imidazole (50-75 microgram/ml) through the lung or spleen specifically inhibits thromboxane A2 production and diverts the pathway to the prostaglandins, mainly prostaglandin F2alpha. In human platelets imidazole causes a dose-dependent inhibition of thromboxane A2 formation (ID50 5.5 X 10(-4) M). This inhibition is accompanied by a dose-dependent increase in prostaglandin F2alpha. Since thromboxane A2 induces platelet aggregation and is a potent vasoconstrictor, diversion of pathways to prostaglandins with opposite or less potent action might be of relevance in the treatment of cardiovascular diseases.

MULTIPLE BINDING SITES OF HUMAN BRAIN AND LIVER MONOAMINE OXIDASE: SUBSTRATE SPECIFICITIES, SELECTIVE INHIBITIONS, AND ATTEMPTS TO SEPARATE ENZYME FORMS

Abstract— MAO of human brain and liver mitochondria was solubilized by a procedure that preserved the substrate and inhibitor selectivities of the original mitochondrial preparation. Techniques that are designed to separate proteins on the basis of molecular size or net surface charge did not yield a physical separation of enzymically active A and B forms, even in the presence of ionic detergents or with limited proteolysis. However, sulfhydryl inhibitors, inorganic salts, ionic detergents, heat treatment, and sonication all tended to cause selective inactivation of serotonin‐metabolizing activity in solubilized preparations. Experiments with selectively inhibited (membrane‐bound or solubilized) MAO supported the concept of at least two independent kinds of substrate binding site, only one of which metabolizes serotonin (A type) and another (B type) which has a very strong affinity for β‐phenethylamine. l‐Norepinephrine, tryptamine, dopamine, and tyramine could be classified as common substrates. The lowest Km values were found for tryptamine at A sites and for β‐phenethylamine at B sites. Results of this study suggest that the different MAO sites could be part of the same large molecular complex, which may normally be embedded in the outer mitochondrial membrane so that A sites are more dependent on their lipid environment within this membrane.

KINETICS OF CHOLINE ACETYLTRANSFERASES (EC 2.3.1.6) FROM HUMAN AND OTHER MAMMALIAN CENTRAL AND PERIPHERAL NERVOUS TISSUES

Abstract— Choline acetyltransferase (EC 2.3.1.6) was partially purified from human caudate nucleus and putamen, human sciatic nerve, rabbit and rat brain, and rabbit sciatic nerve. Kinetic constants were determined under the same conditions for all six extracts. Extrapolated Km values were between 6.6 and 18 μM for acetyl‐CoA and between 0.4 and 1.2 mM for choline. Product inhibition patterns indicated that ChAc from both central and peripheral nervous tissues of man and the rabbit obeys a Theorell‐Chance mechanism. Kinetic parameters suggest a possible influence on ACh synthesis of the in vivo concentration ratio, CoA/acetyl‐CoA.

Acetyl-L-carnitine as a precursor of acetylcholine.

Synthesis of [3H]acetylcholine from [3H]acetyl-l-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [3H] and [14C] labeled acetylcholine were produced when [3H]acetyl-l-carnitine andd-[U-14C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-l-carnitine to acetylcholine was dependent on concentration of acetyl-l-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-l-carnitine in the treatment of age-related cholinergic deficits.

Extracts of Ginkgo biloba leaves inhibit monoamine oxidase.

Extracts of Ginkgo biloba leaves produce reversible inhibition of rat brain monoamine (MAO). Both MAO-A and -B types were inhibited to a similar extent. The MAO inhibitory compound(s) were present in dried or fresh Ginkgo biloba leaves as well as in commercially available capsules of Ginkgo biloba and appear to be heat stable with relatively low molecular weight. MAO inhibition by Ginkgo biloba may be a mechanism underlying reported anti-stress and anxiolytic activities of this natural product.

GABA-TRANSAMINASES OF HUMAN BRAIN AND PERIPHERAL TISSUES—KINETIC AND MOLECULAR PROPERTIES

Abstract— Kinetic experiments with 4‐aminobutyrate‐2‐ketoglutarate transaminase (GABA‐T), partially purified from human brain tissue, supported a Bi Bi Ping‐Pong type of enzyme mechanism in which the enzyme oscillates between forms bound to pyridoxal phosphate and pyridoxamine phosphate. Extrapolated Km values were 0.31 mm for γ‐aminobutyrate, 0.16 mm for α‐ketoglutarate, and 3.8 μm for pyridoxal phosphate. Very similar kinetic parameters were observed with rat brain enzyme. Apparent molecular weight of human GABA‐T by gel filtration was 70,000 ± 3000. Electrofucusing experiments indicated a single ionic form with isoelectric pH = 5.7. Enzyme activity was inhibited by Tris, halides, cadmium and cupric ions, and known GABA‐T inhibitors.

A simple radiochemical assay for prostaglandin synthetase

Abstract A simple radiochemical procedure for prostaglandin synthetase is described, in which incubation mixtures are applied to small disposable columns of Bio-Sil A (silica gel) resin and eluted with two different solvent mixtures to achieve a separation of unmetabolized substrates from prostaglandin products.

Synthesis of prostaglandin 6-keto F1α by cultured aortic smooth muscle cells and stimulation of its formation in a coupled system with platelet lysates

Lysed aortic smooth muscle cells, when incubated with [14C] arachidonate, synthesized only one radioactive product, which was identified as 6-keto-PGF1alpha. Formation of this product from smooth muscle cell lysates was stimulated when human platelet extracts were added to the system, and further stimulation was observed when imidazole, a selective inhibitor of thromboxane synthesis, was added to this coupled system. These observations indicate that the cyclooxygenase of the smooth muscle cells was rate-limiting, that the prostacyclin synthetase of these cells can utilize endoperoxides produced by platelets, and that blocking of thromboxane synthesis might, under certain conditions, shunt arachidonate metabolism toward prostacyclin formation.

MULTIPLE BINDING SITES OF HUMAN BRAIN MONOAMINE OXIDASE AS INDICATED BY SUBSTRATE COMPETITION

Abstract— Six endogenous substrates of monoamine oxidase (EC 1.4.3.4) (serotonin, l‐norepinephrine, dopamine, tyramine, tryptamine and β‐phenethylamine) were used separately and in pairs with human brain mitochondrial extracts. Apparent K1 values were obtained from experiments in which only 1 of 2 substrates was isotopically labelled, and these values were compared with experimental Km values. β‐Phenethylamine appears to be metabolized at enzyme active sites independent from those which bind serotonin. The substrate l‐norepinephrine competes with serotonin for an enzyme site, but also may be catalysed at an additional site which is independent of serotonin binding. Experiments in which [14C]tryptamine was combined with [3E]serotonin indicated that tryptamine is a much more potent inhibitor of serotonin oxidation than was predicted from Km values. It is suggested that the competition among substrates of MA0 which is observed in uitro may have relevance to in uiuo mechanisms for control of biogenic amine concentrations.

Anorexic effects of ethanolamine-O-sulfate and muscimol in the rat: evidence that GABA inhibits ingestive behavior.

Abstract Intracisternal injections of ethanolamine-O-sulfate (EOS), an irreversible selective inhibitor of GABA-transaminase (GABA-T), resulted in relatively long lasting dose dependent decreases in food consumption and body weight of rats. The anorexic effects of EOS generally corresponded in both time course and magnitude to the elevation of GABA levels and associated decreases in GABA-T activity. Chronic treatment with very high intraperitoneal doses of EOS which were able to cross the blood-brain barrier elevated GABA levels and resulted in weight loss. Muscimol, a GABA receptor agonist also produced anorexia. These findings are consistent with the view that GABA may be involved in mediation of satiety in the rat.