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Parkinson's disease (PD) is an aging-related movement disorder caused by a deficiency of the neurotransmitter dopamine (DA) in the striatum of the brain as a result of selective degeneration of nigrostriatal DA neurons. The molecular basis of the cell death of DA neurons is unknown, but one hypothesis is the presence of some amine-related neurotoxins that kill specifically nigrostriatal DA neurons over a long period of time. This neurotoxin hypothesis of PD started in the 1980s when 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was discovered to produce acutely PD-like symptoms. Two groups of natural MPTP-like and amine-related neurotoxins have been investigated as endogenous candidate compounds: isoquinolines (IQs) and beta-carbolines. These neurotoxins are speculated to cause oxidative stress, mitochondrial dysfunction, apoptotic cell death, and PD symptoms. However, since PD is a neurodegenerative disorder that progresses slowly over a period of many years, a long-term study may be required to elucidate the neurotoxicity of such neurotoxins in relation to PD.

The (R)- and (S)-enantiomers of salsolinol, the dopamine-derived tetrahydroisoquinolines, were found to inhibit the activity of tryptophan hydroxylase (TPH), prepared from serotonin-producing murine mastocytoma P-815 cells. Inhibition of TPH by salsolinols was found to be non-competitive with the substrate L-tryptophan. Tryptophan hydroxylase is composed of two elements with different kinetic properties in terms of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin and these two elements were inhibited by salsolinols in competitive and uncompetitive ways, respectively. Stereoselectivity of salsolinol was not observed, concerning the potency and the type of inhibition on PTH. These data indicate that salsolinols might be naturally occurring inhibitors of indoleamine metabolism.

The activity of DPP II was higher in gingiva from patients with periodontitis, but the activity of DPP IV, post-proline cleaving enzyme and collagenase-like peptidase was not significantly higher than that of the control group. As DPP II activity is known to be altered in immunological diseases, these findings may suggest some role for DPP II in the pathogenesis of chronic marginal periodontitis.

Effects of a series of heterocyclic amines on type A and B monoamine oxidases were studied using human brain synaptosomal mitochondria as an enzyme source. 3-Amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was the most potent inhibitor of the heterocyclic amines examined. It inhibited type A monoamine oxidase in competition with the substrate and the K(i) value toward type A was 0.84 +/- 0.06 ?M, which was about 1/50 of its K(m) value toward an amine substrate, kynuramine; 46.4 +/- 11.4 ?M. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was also a potent inhibitor. 3- or 2-Aminopyridoindoles or -imidazoles inhibited both type A and B monoamine oxidase competitively to substrate, but inhibition of type B monoamine oxidase by amines with 2-amino-3-methylimidazol ring was non-competitive. In general, type A monoamine oxidase was more sensitive to these heterocyclic amines than type B; the K(i) values toward type A were much smaller than those of type B. Inhibition of type A monoamine oxidase by Trp-P-2 proved to be reversible; the enzyme activity inhibited by Trp-P-2 could be fully recovered by removal of Trp-P-2 by column chromatography with Sephadex G-25.

l-erythro-5,6,7,8-Tetrahydrobiopterin (BH(4)), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH(4) exists as natural (6R)- and unnatural (6S)-isomers. In our previous reports, only (6R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6R)- and unnatural (6S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH(4). Thus, TH activity was observed only in the presence of exogenous BH(4). We compared the difference in in situ DOPA formation by TH activity in the presence of (6R)- or (6S)-BH(4) in the human TH-transfected cells. The effect of exogenous BH(4) was also compared between (6R)- and (6S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 ?M endogenous (6R)-BH(4). The rate of uptake of both BH(4) isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 ?M but was nearly saturated at 1 mM BH(4). TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6R)- or (6S)-BH(4) dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6R)-BH(4) was approximately three times as active as (6S)-BH(4). In contrast, in PC12h cells which contained endogenous (6R)-BH(4) (approximately 100 ?M), exogenous (6R)-BH(4) activated DOPA formation maximally at 500 ?M about 10-fold, while (6S)-BH(4) activated it only slightly, about 2.5-fold. These results suggest that (6S)-isomer has lower cofactor activity with TH in the cells than (6R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell.

The less active form of human tyrosine hydroxylase has been previously reported but its physiological role is unknown. We partially purified the less active form of tyrosine hydroxylase from human adrenals, and examined differences in the properties of the active and less active forms. We succeeded in activation of the less active form of human tyrosine hydroxylase by addition of 100 ?M Fe(2+). Fe(2+) decreased the K(max) for pteridine cofactor in both the active and less active forms, but increased the V(max) only in the less active form. Fe(2+) changed the V(max) but not the K(m) of the less active form for tyrosine. These results suggest that Fe(2+) may regulate tyrosine hydroxylase activity in vivo as a result of activation of its less active form.

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K(m) and V(max) values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 ?M and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 ?M and 71 pmol/min/g wet weight, respectively.

The effect of the N-methylisoquinolinium ion (NMIQ(+)) on the activity of enzymes related to metabolism of dopamine was studied using a rat clonal pheochromocytoma PC12h cell line. The activities of tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC) and monoamine oxidase (MAO) were inhibited by NMIQ(+), but the mechanism of inhibition of these enzymes differed from each other. TH activity in the cells was inhibited by NMIQ(+) with an IC(50) of about 75 ?M. Aromatic l-amino acid decarboxylase (AADC) was also inhibited by NMIQ(+) but in competition with a co-factor, pyridoxal-5-phosphate, and the K(i) value was 90 ?M. MAO was inhibited by NMIQ(+) in competition with a substrate, kynuramine, and the K(i) value was 20 ?M. In vivo effects of NMIQ(+) on these enzymes in PC12h cells were examined by culture of the cells in the presence of 100 nM-1 mM NMIQ(+) for 6 days. After 6 days culture, TH activity was reduced in cells cultured with NMIQ(+) at concentrations higher than 10 ?M, but the activities of AADC and MAO were reduced only in cells cultured with 1 mM NMIQ(+). In addition, NMIQ(+) was transported into the cells by a transport system specific for dopamine. These data suggest that NMIQ(+) may perturb the catecholamine metabolism of a dopaminergic system in the brain, as a naturally-occurring compound.

The activities of three pterin-requiring monooxygenases, phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase, are regulated by the level of the pterin cofactor, (6R)- l -erythro- tetrahydrobiopterin , which is synthesized from guanosine triphosphate (GTP). Since tyrosine hydroxylase or tryptophan hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines (dopamine, norepinephrine and epinephrine) or serotonin in monoaminergic neurons, biosynthesis of tetrahydrobiopterin from GTP may also regulate the tissue level of monoamine transmitters. Recent evidences indicate that biosynthesis of tetrahydrobiopterin and that of biogenic monoamines may be regulated each other.

The ability of polyamines (putrescine, spermidine, and spermine) to modify tyrosine hydroxylase (TH) activity was examined in crude or purified enzyme preparation and in adrenal tissue slices. Polyamines showed biphasic effects on TH activity in vitro at physiological pH 7.0, with an inhibitory effect at low concentrations (<1 mM) and a stimulatory effect at high concentrations. The degree of both inhibition and stimulation produced by polyamines at low and high concentrations, respectively, were proportional to the number of the amino group in the polyamines (putrescine < spermidine < spermine). The degree of inhibition by polyamines was much greater with purified enzyme than with crude enzyme preparations. Tyrosine hydroxylation in situ in adrenal tissue slices was stimulated by polyamines without inhibition at any concentrations tested. This evidence suggests that TH molecules in vivo could interact with polyamines or polyamine-like substances which inhibit the TH activity at physiological concentrations less than 1 mM.

Monoamine oxidase (MAO) activity towards kynuramine as substrate was measured in 6 hybrid cells derived by fusion of neuroblastoma and glioma, liver or brain cells, and was compared with that of parental or non-parental clones. Activities varied from the lowest level of less than 0.15 pmol/min/mg protein in a neuroblastoma clone NB2A to the highest level of 127 pmol/min/mg protein in NCB20 mouse neuroblastoma x Chinese hamster embryo brain hybrid cells. The relative proportions of A and B types of MAO activities were determined in homogenates of each cell line by inhibition curves with clorgyline and deprenyl. Although the A type activity was found in all cell lines measured, MAO A was predominant in 9 clones, except for NCB20 hybrid cells, N4G-B-a neuroblastoma x glioma hybrid cells, and G8-1 myoblast. The ratio of type A/type B activity in NCB20, N4G-B-a and G8-1 cells was 20/80, 75/25 and 95/5, respectively. The results suggest that NCB20 cells are highly enriched in MAO type B, and that the NCB20 cell is an excellent model for studying the type B activity found in the brain in vivo.

Tyrosine hydroxylase (TH) was isolated from human brain (caudate nucleus + putamen). The major form of the active enzyme in the cytoplasmic fraction was purified to apparent homogeneity. The molecular weight of the purified enzyme was estimated to be 280 kdalton by gel filtration. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) of the purified enzyme gave a single subunit with mol. wt 60 kdalton, which is similar to the subunit of human adrenal TH. Using a sandwich enzyme immunoassay (EIA), the presence of inactive form(s) of TH in human brain was demonstrated, and the total content of this immunoinactive form(s) was approx. 8 times higher than that of the active form. By the Western blot technique after two-dimensional (2-D) electrophoresis, TH in the crude fraction of the human brain was found to consist of multiple forms with different pI-values and with the same molecular weight. The pl of the major spots ranged from 5.3 to 5.8, and that of the minor spot was 6.0. Because the pl of the purified enzyme preparation was 6.0, this protein with pI at 6.0 may be the active form of TH.

The uptake and accumulation of N-methyl-4-phenylpyridinium ion (MPP(+)), a neurotoxin produced by oxidation of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), into PC12h pheochromocytoma cells were examined. Concentration gradients of MPP(+) were established at its low concentrations of 10 to 100 nM. Uptake of MPP(+) into PC12h cells was mediated by saturable, carrier mediated transport systems with two different kinetic properties; a high-affinity and low-capacity system and a low-affinity and high-capacity system. The apparent K(m) values of these two systems were obtained to be 254.4 +/- 96.5 nM and 23.1 +/- 6.9 ?M, respectively, and the maximal uptake velocity was obtained to be 8.47 +/- 1.72 and 28.6 +/- 5.2 pmol/min/mg protein, respectively. The uptake by a high-affinity system was mediated by a carrier system common to dopamine and noradrenalin and MPTP itself proved to be taken up by this system, which was further confirmed by the inhibition of the MPP(+) uptake by nomifensine and mazindol. The uptake was inhibited by metabolic inhibitors, such as carbonyl cyanide m-chlorophenyl hydrazone, sodium cyanide and 2,4-dinitrophenol, and the uptake was inhibited by ouabain and nigercin. By subcellular fractionation, MPP(+) taken up was found to be localized mainly in cytosol fraction, but a definite amount of MPP(+) was found also in mitochondrial fraction.

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.

Tyrosine hydroxylase (TH) was purified from the soluble fraction of human adrenal glands. The enzyme in human adrenal glands that was purified to apparent homogeneity had an apparent M(r) of about 280,000. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a single band with a M(r) of 60,000 similar to the M(r) of bovine adrenal enzyme. The enzyme is considered to be composed of four identical subunits. The specific activity of the final preparation was approximately 310 nmol 3,4-dihydroxyphenylalanine (DOPA) formed/min/mg protein. The use of the "Western Blot" method showed that human adrenal TH did not aggregate as rapidly as bovine adrenal TH.

The chromosomal gene for human phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) was isolated from a human genomic library using a cloned human PNMT cDNA as a probe, and the nucleotide sequence was determined. PNMT is encoded in a single gene which consists of three exons. We observed newly the presence of minor PNMT mRNA (type B) besides the major mRNA (type A) as reported previously (Kaneda et al., J. Biol. Chem. 263, 7672-7677, 1988) by Northern hybridization. Type B mRNA carries an approximately 700 nucleotide-long untranslated region in the 5? terminus. This suggests that two types of mRNA are produced from a single gene through the use of two alternative promoters. A TATA-like sequence locates 30 base pair upstream from the cap site of type A mRNA. Upstream of the cap site, there are several sequences resembling Spl binding sites and glucocorticoid responsive elements, with the latter also found in the first intron.

By use of in vivo voltammetry technique, in vivo release of dopamine and serotonin in the striatum under stress was found to be more prominent in spontaneously hypertensive rats (SHR) at 4 weeks of age than in control Wistar-Kyoto rats (WKY). Simultaneously, a greater activation of tyrosine hydroxylase in the striatum was demonstrated in SHR than in WKY. These results indicate that SHR is more susceptible to stress in the central monoaminergic neurons than WKY.

Mouse neuroblastoma x rat glioma NG108-15 hybrid cells contain a considerable amount of serotonin, and possess small but significant tryptophan hydroxylase activity. The results suggest that NG108-15 hybrid cells are serotonergic, in addition to the known cholinergic property.

A sandwich enzyme immunoassay (EIA) was established by using purified human serum dopamine-?-hydroxylase (DBH) as a standard protein and a monospecific polyclonal antibody raised against human DBH purified from human pheochromocytoma. The EIA was applied to measuring DBH levels in human CSF from Parkinsonian patients and control patients devoid of neurological diseases. The control group had DBH content of 21.1 +/- 3.1 ng/ml CSF and DBH activity of 24.0 +/- 3.7 ? U/ml CSF, and Parkinsonian group 3.3 ? 0.7 ng/ml CSF (16% of control) and 4.6 +/- 0.7 ?U/ml CSF (19% of control) (mean +/- SEM). Thus, both DBH content and DBH activity in CSF were reduced in Parkinsonian patients to less than 20% of the control values (P $ ? 0.005 ). However, the specific activity (units of enzyme activity/mg of DBH protein) in CSF of Parkinsonian patients was similar to that of control patients. These results suggest that the reduced DBH activity in CSF from Parkinsonian patients is caused by a reduction in DBH protein content, and is not due to production of an inactive form of DBH, for example, by combining with endogenous inhibitor(s). These data support our previous findings that DBH activities in the Parkinsonian brain and CSF are decreased.

A new method was developed to study the unsupplemented tryptophan hydroxylase system in brain tissue slices from the raphe nuclei of the rat by high-performance liquid chromatography (HPLC) with fluorescence detection. Tryptophan hydroxylase activity was measured by determining 5-hydroxytryptophan (5-HTP) accumulation in raphe nuclei slices containing all of the enzyme system (the hydroxylase, tetrahydrobiopterin, and dihydropteridine reductase) in the presence of NSD-1055 (an inhibitor of aromatic l-amino acid decarboxylase). An optimum temperature was observed at 25 degrees C and the reaction progressed linearly for 60 min. The hydroxylation of tryptophan was maximal by the addition of 0.2 mM tryptophan in the medium. A maximum 1.5-fold activation was shown at 0.2 mM 6-methyltetrahydropterin in the presence of 10 mM dithiothreitol. Dithiothreitol alone did not affect the activity. A 1.5-fold activation was observed when incubation was carried out under gas phase of 95% oxygen and 5% CO(2) instead of air. The activity was inhibited by 75% at 10(?4) M p-chlorophenylalanine. Both A-23187, a calcium ionophore, and dibutyryl cyclic AMP (DBc-AMP) stimulated the hydroxylation of tryptophan. The activation by A-23187 plus DBc-AMP was more than additive, suggesting the two activating mechanisms by Ca(2+) and cyclic AMP may be operating synergistically.

By the use of the brain micro-dialysis technique combined with HPLC, the changes in the extracellular levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and a serotonin(5-HT) metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were examined in the rat striatum before and after intracerebral injection of a vehicle or (6R)-l-erythro-tetrahydrobiopterin (6R-BH(4)), the natural form of the cofactor for the tryrosine hydroxylase and tryptophan hydroxylase. No apparent change after the 6R-BH, treatment was found in the levels of DA, DOPAC, HVA and 5-HIAA in the striatal dialysate. In contrast, the levels of total biopterin in both the operated (dialysis probe-implanted) and unoperated striatum of 6R-BH(4)-treated rats increased by 23- and 93-fold, respectively, when compared with those of the control, vehicle-treated rats. The results indicate that increased levels of the tetrahydrobiopterin cofactor may not affect the release of DA and the extracellular level of DA and 5-HT metabolites in the physiologically normal brain.

It was reported that tryptophan hydroxylase was activated in vitro only under calcium-calmodulin-dependent phosphorylating condition, but not under cyclic AMP-dependent phosphorylating condition. This is attributed to the absence of cyclic AMP-dependent protein kinase in serotonergic neurons. Since we had evidence that in tissue slices of the raphe regions of rats both calcium-calmodulin-dependent and cyclic AMP-dependent processes may activate tryptophan hydroxylase in situ, we have investigated possibility of coexistence of calcium-calmodulin-dependent protein kinase and cyclic AMP-dependent protein kinase with tryptophan hydroxylase in rat raphe regions. The activities of cyclic AMP-dependent and calcium-calmodulin-dependent protein kinases were measured in the raphe serotonergic regions of rats treated intracisternally with 5,7-dihydroxytryptamine, which is a specific neurotoxin for serotonergic neurons. At 6 days after treatment with 200 ?g of 5,7-dihydroxytryptamine, activity of tryptophan hydroxylase and that of cyclic AMP-dependent protein kinase decreased by 60 and 40%, respectively, and a significant correlation was observed between the activities of the two enzymes. Calcium-calmodulin-dependent protein kinase activity disappeared in 5,7-dihydroxytryptamine-treated rats. These results suggest that both cyclic AMP-dependent and calcium-calmodulin-dependent protein kinases are present in raphe serotonergic neurons of rats, and may regulate tryptophan hydroxylase in vivo.

Uptake of glycoproteins into brain nerve terminals was studied, using glycosylated ?-galactosidase and human brain synaptosomes. A series of p-aminophenyl ?- or ?-glycosides were covalently bound to ?-galactosidase purified from Aspergillus oryzae and uptake of the enzyme into human brain synaptosomes was measured. ?-Galactosidase coupled with ?-d-mannoside (?-d-Man ?-gal) was the best to be taken up among glycoconjugates tested. The uptake was dependent on the amount of ?-d-Man ?-gal, the incubation temperature and the incubation time. Kinetic studies showed that the uptake was a saturable and receptor-mediated process, which followed Michaelis-Menten kinetics. The specificity of the uptake was confirmed by competition of the uptake of ?-d-Man ?-gal by ?-methyl mannoside. The uptake was dependent on Ca(2+) and that of ?-galactosidase coupled with ?-l-fucoside (?-l-Fuc ?-gal) was saturated by Ca(2+) at lower concentrations than that Of ?-d-Man ?-gal.Occurrence of two different uptake systems was also suggested by kinetic study; a low-affinity and high-capacity system for ?-d-mannoside and a high-affinity and low-capacity system for ?-l-fucoside. The former system may be mediated by a process coupled with a Ca(2+)/calmodulin-dependent protein kinase, but the later may not be dependent on the protein kinase.

The effects of ?- and ss-adrenergic drugs on the activity of tryptophan hydroxylase were investigated in rat midbrain raphe slices. The tryptophan hydroxylase activity in slices was estimated by measuring the formation of 5-hydroxytryptophan (5-HTP) under inhibition of aromatic l-amino acid decarboxylase using 3-hydroxy-4-bromobenzyloxyamine (NSD 1055). Isoproterenol, a ss-adrenergic stimulant, significantly increased 5-HTP formation to 122% (P < 0.05) of control at 10(?6) M and this effect was prevented by 10(?6) M of propranolol, a ss-adrenergic blocker. 5-(1-Hydroxy-2-isopropylaminobutyl)-8-hydroxycarbostryril hydrochloride hemihydrate (OPC 2009), a ss-adrenergic stimulant which does not contain a catechol group, increased 5-HTP formation to 145% at 10(?6) M. A-23187 at 5 x 10(?7) M further enhanced the isoproterenol-stimulated 5-HTP formation to 156% of control. Dibutyryl cAMP at 10(?2) M, however, did not enhance it. 8-Bromo cAMP did not enhance the OPC 2009-stimulated 5-HTP formation, either. An ?-adrenergic stimulant, clonidine, had no effect on 5-HTP formation. But an ?-adrenergic blocker, yohimbine, reduced 5-HTP formation to 78% at 10(?6) M. These results suggest that the activity of tryptophan hydroxylase can be controlled by a ss-adrenergic receptor coupled with adenylate cyclase via an intracellular cAMP-dependent process.

A highly-sensitive sandwich enzyme immunoassay for serum dipeptidyl aminopeptidase IV (DAP IV) was developed; its level in sera of patients with oral cancer [1.50 +/- 0.35(SD)mg/l serum] was significantly lower than in normal subjects [2.27 +/- 0.77(SD)mg/l serum].

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was systemically administrated into C57BL/6N mice for 8 days. Tyrosine hydroxylase activity and dopamine content in striatum and hypothalamus were reduced markedly, and the reduction was more manifest in the striatum. Cyclosporin A, an immune suppresser, enhanced the neurotoxicity of MPTP, when it was injected to mice in combination with MPTP. Tyrosine hydroxylase activity in the striatum was reduced to 109 +/- 20 from 350 +/- 46 pmol/min/mg protein of control, when mice were injected by MPTP alone. The enzyme activity was further reduced to 68.4 +/- 12.2 pmol/min/mg protein by injection of MPTP in combination to cyclosporin A. In addition, dopamine and biopterin contents in the striatum decreased quite in parallel to the reduction of tyrosine hydroxylase activity. On the other hand, cyclosporin A itself did not effect tyrosine hydroxylase activity and dopamine and biopterin contents in the striatum and hypothalamus. These data suggest cumulative effect of cyclosporin A to the neurotoxicity of MPTP.

Biopterin, the cofactor for tyrosine hydroxylase and tryptophan hydroxylase, was decreased in caudate nucleus, hypothalamus and cerebellum of the rolling mouse. Though there were not significant differences of tyrosine hydroxylase and tryptophan hydroxylase activities between the rolling and normal control mouse in the hypothalamus, the rolling showed significant increase of biopterin concentration and tyrosine hydroxylase activity after administration of thyrotropin releasing hormone (TRH). These results suggest that ataxic gait of the rolling mouse may be partly due to some abnormalities of catecholaminergic neurons, especially noradrenergic neurons, and that TRH may improve the abnormalities of catecholaminergic neurons. The changes of biopterin concentration by TRH administration indicate that biopterin may be a regulatory factor in catecholamine biosynthesis.