Septal tryptophan-5-hydroxylase: divergent response to raphe lesions and parachlorophenylalanine.

A soluble form of tryptophan-5-hydroxylase activity was found to be present in areas rich in serotonergic terminals (colliculi, hippocampus, septal area, and remaining telencephalon) as well as in brainstem, an area rich in cell bodies. The enzymatic activity in all brain regions, except the septal area, was inhibited to varying degrees following administration of parachlorophenylalanine. Destruction of the raphe nuclei in the midbrain led to a large and comparable decrease in both serotonin content and tryptophan hydroxylase activity of the hippocampus. In contrast, these lesions did not significantly affect the enzymatic activity of the septal area although the serotonin content was decreased by 72 percent. These findings suggest that the major portion of the tryptophan hydroxylase activity of the septal area is uniquely different from that found in other telencephalic areas in that it is not localized in serotonergic nerve terminals nor is it inhibited by parachlorophenylalanine.

Cerebral hydroxylases: stimulation by a new factor.

Cerebral tryptophan 5-hydroxylase and tyrosine hydroxylase are stimulated by a solution of lyophilized, protein-free 30,000g supernatant of brain homogenates (concentrate 1). This preparation can be further purified by passage through Sephadex G-75 and phosphocellulose columns to yield concentrate 2. Unlike concentrate 1, concentrate 2 is unstable after several days of storage even at -27 degrees C. Neither preparation is active without the presence of a reduced pteridine cofactor. The stimulating factor appears to be thermostable, alkali-labile, dialyzable, and light-sensitive.

L-kynurenine: its synthesis and possible regulatory function in brain.

One pathway by which tryptophan is metabolized in the brain as well as in the periphery is through cleavage of the indole ring to formylkynurenine and then kynurenine. Indoleamine-2,3-dioxygenase, the enzyme that catalyzes this cleavage, and kynurenine are distributed all across the different anatomic regions of brain. Approximately 40% of the kynurenine in brain is synthesized there, the remainder having come from plasma. Tryptophan loading, which has been used both experimentally and therapeutically as a means of increasing tryptophan conversion to serotonin, also increases kynurenine formation in the brain and in the periphery. Because of the formation of kynurenine, which competes for cerebral transport and cellular uptake of L-tryptophan, and because of substrate inhibition on tryptophan hydroxylase, excessively high doses of tryptophan may actually decrease the production of cerebral serotonin and 5-hydroxyindoleacetic acid.

Biopterin. VII. Inhibition of synthesis of reduced biopterins and its bearing on the function of cerebral tryptophan-5-hydroxylase in vivo.

Repeated intraventricular injections of 2,4-diamino-6-hydroxypyrimidine (DAO-Pyr), inhibitor of D-erythro-q-dihydroneopterin triphosphate synthetase, inhibited q-BH2 synthesis from GTP, markedly increased accumulation of 2-amino-4-hydroxy-5 (or -6)-formamido-6-triphosphoribosylaminopyrimidine (FPyd-P3) and brought about a 60% decrease in the in vivo of reduced biopterin (BH2 and BH4) pool in the brain. Nevertheless, there was no effect on the rate of hydroxylation of L-tryptophan or on the 5-hydroxytryptamine level in rat brain. These data emphasized the significance of the rate of hydrogen transfer and the limitation of the concept of "unsaturation" (i.e., the absolute amount of the carrier pterin molecule) for the synthesis of neurotransmitters in vivo.

Mechanism of irreversible inactivation of phenylalanine-4- and tryptophan-5-hydroxylases by [4-36Cl, 2-14C]p-chlorophenylalanine: a revision.

Intraperitoneal injection of [4-36Cl, 2-14C]p-chlorophenylalanine (pCPA) (300 mg/kg) in rats revealed absence of chlorine in pure hepatic phenylalanine hydroxyase, while the carbon label appeared a 1--4 moles/mole of [14C]tyrosine in the inactivated phenylalanine and cerebral tryptophan-5-hydroxylase. Crystalline muscle aldolase and tyrosine hydroxylase also revealed the presence of [2-14C]tyrosine from [2-14C]pCPA without inactivating these enzymes. Injection of L-[(U)-14C] tyrosine led to its incorporation into the above enzymes, but to a different degree without altering the enzyme activity. Repeated injections of p-chlorophenylacetic acid had no effect on phenylalanine or tryptophan-hydroxylase, Administration of pCPA did not change the levels of cerebral biopterins. Reexamination of the effect of cycloheximide on reversing enzymic inactivation by pCPA failed to confirm our earlier observation.

Mass-spectrographic evidence of the conversion of p-chloroamphetamine to 3,4-dimethoxyamphetamine.

Intraventricularly injected p-chloroamphetamine (p-CA) was actively metabolized to 3,4-dimethoxyamphetamine (3,4-DMA) in the rat brain. Time-course experiments with intraperitoneally injected p-CA confirmed that the presence of cerebral 3,4-DMA was not due to its "one-pass" entry from the peripheral organs. The identity of 3,4-DMA from brain tissue and urine was established by comparison to authentic 3,4-DMA. The synthetic and biological samples were isographic in all analytical systems. 3,4-DMA from biological samples was verified by mass spectrography.

Biopterin : II. Evidence for cerebral synthesis of 7,8-dihydrobiopterin in vivo and in vitro.

The active synthesis of [(14)C]7,8-dihydrobiopterin (BH2) from intraventricularly administered U-[(14)C]GTP was demonstrated in rat brain. The identity of [(14)C]BH2 isolated from brain was confirmed by mass fragmentography. Evidence is presented that [(14)C]BH2 in brain was not synthesized in the peripheral organs. The rate of cerebral synthesis of [(14)C]BH2 from [(14)C]GTP was maximal at 2 h; it was 0.53 nmol/g per h, which is consistent with the estimated turnover rate of cerebral BH2 (0.43 nmol/g per h). Intraventricularly injected 2,4-diamino-6-hydroxypyrimidine (DAOPyr) and 6-thioguanosine were effective inhibitors of the synthesis. U-[(14)C]dGTP and 8-[(14)C]GTP, when given intraventricularly, did not yield [(14)C]BH2. Simultaneous intraventricular injection of U-[(14)C]GTP and DAOPyr resulted in the accumulation of a compound with properties identical to a formamidopyrimidine derivative isolated from the nonenzymatic hydrolysis of GTP. The data from preliminary experiments demonstrated the synthesis of [(14)C]BH2 from U-[(14)C]GTP incubated with 12,000g supernatants of rat brain homogenates.