SECTION I1

A brain fraction prepared in the same way as liver methylpherase, was also effective in promoting the methylation of normorphine, and was optimally active at pH 5.5 when S-adenosylmethionine was the methyl donor. With the same enzyme preparation from brain, norcodeine was methylated to codeine, and morphine was also recovered in these incuba- tions, presumably by 03-demethylation from codeine. In these in vitro incubations, the concentration of 8-adenosylmethionine was limiting (0 .2 -3 .5 ,umoles/assay) so that less than 1 % of the norderivative (50 ,umoles/assay) was methylated. However, the reaction could be shifted toward completion by doubling the level of S-adenosylmethionine and decreasing the normorphine level to 1 ,umole/assay. I n this case 15.4% of the normorphine was methylated to morphine in an hour of incubation with 15 mg. of liver methylpherase protein/assay.

The methylation of normorphine in vivo in brain was also demonstrated. In these experiments, methionine-(l4C-methyl) was injected into the cisterna magna of the rat (at a dose of 0.5 pmoles containing 2.5 pciiries of W); 15 minutes later, 0.3 pmole of normorphine was administered by the same route. Since maximal analgesic activity had been found in earlier experiments during the 5 to 15 minute period after the intra- cisternal injection of normorphine, the doubly-injected rats were killed 10 minutes later, and the brains were rinsed in buffer and subjected to the isolation procedures used in the in vitro incubations. Labelled mor- phine was isolated from these brains. Thus, in considering the analgesic activity of normorphine, one cannot exclude re-methylation to morphine as a possible metabolic step.

12

REVERSIBLE INHIBITION OF BRAIN MONOAMINE OXIDASE IN VITRO AND IN VIVO

by

R. F. LONG Research Department, Roche Products Lttl., Welwyn Garden City, England

The action of reversible inhibitors on human brain monoamine oxidase has been studied in vitro. As with preparations from other mammalian tissues, different substrates required very different inhibitor concentra- tions to reduce the rates a t which they were oxidized. Using harmaline, 50% inhibition was caused by lop3 M inhibitor when tyramine was the

27

SECTION I1

substrate; M when phenylethylamine or dopamine was used; 3 x M with tryptamine; and 2 x 10-7 M if noradrenaline or 5- hydroxytryptamine (6-HT) was the substrate, all substrates being a t 2 x M concentration. Similar differences were found when other reversible inhibitors were used. Enzymes obtained from different regions of the brain behaved in the same way in this respect. Differences exist however between enzyme preparations from the brains of different species both in the relative rates of oxidation of different amines and in their relative affinities as indicated by the actions of reversible inhibitors.

Selective inhibition of the destruction of endogenous amines should result in selective alteration of their tissue levels when reversible mono- amine oxidase inhibitors are given to animals. It was found that if harmaline or cx-methyltryptamine was administered to mice in doses sufficient to cause marked rises in brain 5-HT and noradrenaline levels, the level of dopamine was not raised. Factors, such as the relative turn- over rates of amines and the availability of alternative metabolic path- ways for their destruction, can determine the relative rises in endogenous amines after blocking monoamine oxidase action. However, since irrevers- ible inhibitors of the enzyme cause dopamine, 5-HT and noradrenaline levels to rise, it seems probable that the differential increase observed in our experiments was due to the selective inhibition of monoamine oxidase by the reversible inhibitors used.

13

EFFECT OF HYDRAZIDES PYRIDOXAL-5-PHOSPHATE ON

AND HYDRAZONES OF THE CEREBRAL GLUTAMIC

DECARBOXYLASE AND PYRIDOXAL PHOSPHOKINASE

by

PIERRE GONNARD Nouvellc Facult6 de MBdecine, 45 Rue des St. PBres, Paris 6e, Frmre

Studies of the cerebral glutamic acid decarboxylase of the rat, the guinea pig and the rabbit made by measurement of the CO, output according to the Warburg manometric technique, invariably demonstrate a defiency of co-enzyme with reference to the apo-enzyme. The addition of pyridoxal phosphate to the medium greatly enhances the CO, output.

The pre-incubation of pyridoxal, together with ATP and rabbit cerebral pyridoxal phosphokinase, leads to the same result. Bringing the

28