Identification of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster

Using reversed-phase high-performance liquid chromatography with electrochemical detection we have demonstrated the occurrence of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster. The former is the first time that has been detected in vivo. The identification has been based on the retention times, hydrodinamic voltagrams and the differential concentration in three strains of Drosophila melanogaster. Compared to the wild type, the Punch2 mutant has diminished levels of both pteridines, whereas Henna-recessive3 lacks completely tetrahydropterin and has increased levels of tetrahydrobiopterin, as expected according to their biochemical lesions.     

Distinctions in metabolism of serotonin and dopamine in rat brain during latent inhibition

Latent inhibition (LI) is a behavioral phenomenon, in which repeated presenting of a non-reinforced stimulus retards conditioning to this stimulus when it is coupled with a reinforcer. In order to find specific serotonin (5-HT- and dopamine (DA) changes mediating the LI, the 5-HT and DA metabolism was investigated in certain brain regions. Oxidative deamination of 5-HT and DA by monoamine oxidase (MAO) was determined in the prefrontal cortex, striatim, amygdala, and hippocampus at preexposure and testing stages of the LI using the passive avoidance procedure in rats. Preexposed animals demonstrated high MAO activity for 5-HT deamination in the amygdala and striatum and lower MAO activity for DA deamination in the amygdala and hippocampus. After testing the LI, a high level of 5-HT deamination by MAO was revealed in the amygdala, white the lower level of 5-HT deamination by MAO was shown in the prefrontal cortex. At the same time, no changes in DA metabolism were found in all the brain regions studied. Thus, the role of dopaminergic system in the LI effect may be limited by the preexposure stage. The obtained evidence suggests that the enhanced 5-HT activity in the amygdala and striatum induced by the preexposed stimulus is a principal biochemical mechanism underlying the LI.     

Increased uptake sites for serotonin and dopamine with decreased S2 serotonin receptors in microencephalic rat brain

Methylazoxymethanol (MAM)-induced cerebral hypoplasia resulted in a significant increase in densities of both serotonin uptake sites in frontal cortex and dopamine uptake sites in striatum, suggesting serotonergic and dopaminergic axon terminals were compressed in the smaller brain volumes. The density of S2 serotonin receptors in MAM-lesioned frontal cortex was decreased probably due to down-regulation, while densities of D1 and D2 dopamine receptors in striatum were identical between MAM-lesioned rats and control rats.     

The uptake of serotonin and dopamine by homogenates of frozen rat and human brain tissue

A recently described procedure of freezing and thawing, which allows retention of metabolic and functional integrity, has been applied in the study of serotonin and dopamine uptake into frozen rat and post mortem human frozen tissue. TheK _m andV _max for the serotonin uptake into human hypothalamus were estimated to be 0.12 M and 0.03 nmol/g/min respectively. TheK _m andV _max for the dopamine uptake into human putamen were estimated to be 0.28 M and 0.13 nmol/g/min respectively. The results indicate that the freezing procedure does not affect the uptake sites for these transmitters. The storage time before freezing is however of importance for theV _max value. TheK _m value for the uptake, on the other hand, seems to be rather resistant to storage time before freezing.     

Stereoselective effects of ketamine on dopamine,serotonin and noradrenaline release and uptake in rat brain slices

Ketamine (2-(2-chlorophenyl)-(1-methylamino)-cyclohexanone) is a rapid-acting dissociative general anaesthetic whose hallucinogenic properties have made it a popular drug of abuse. Ketamine comprises two optical isomers, with differing pharmacology. In the present study, the effects of (+)- and (-)-ketamine on stimulated efflux and reuptake of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) were compared in isolated superfused slices of the rat caudatoputamen (CPu), ventral bed nucleus of the stria terminalis (BSTV) or dorsal raphe nucleus (DRN), respectively. Monoamine efflux was elicited by local electrical stimulation (20 pulses, 100 Hz trains) at tungsten microelectrodes and measured at adjacent carbon fibre microelectrodes using fast cyclic voltammetry (FCV). In CPu (+)-ketamine increased stimulated DA efflux and slowed DA reuptake in a concentration-dependent manner (25-200 microM). At 100 microM (+)-ketamine increased DA efflux by 109+/-20% (mean+/-S.E.M., n=13) of control values after 30 min (P<0.001 versus control) and prolonged uptake half-time (t(1/2)) by 76+/-38% (n=9, P<0.001) of control. In contrast (-)-ketamine (100 microM) had no effect on DA efflux or uptake. In DRN, both isomers (100 microM) increased stimulated 5-HT efflux. (-)-Ketamine had a larger effect (P<0.001), an 88+/-15% increase in 5-HT efflux (n=9) versus 46+/-10% (n=8) for the (+)-isomer. The isomers had similar effects on 5-HT uptake, increasing t(1/2) by approximately 200%. No evidence of stereospecificity was seen in BSTV: both isomers had small effects (+)- and (-)-ketamine (100 microM) increasing NA efflux by 43+/-10% (n=7, P<0.001) and 29+/-8% (n=7, P<0.001), respectively. The isomers also had identical effects on NA uptake, each increasing uptake t(1/2) by approximately 100%. In summary, our data show that the optical isomers of ketamine have strikingly different stereospecificity for the monoamine systems and one might predict, therefore, a different psychotomimetic potential.     

Pyrimidine biosynthesis in rat brain

—Studies on the incorporation of [¹⁴C]NaHCO₃ into both orotic acid and RNA in tissue slices reveal the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in the rat brain. A comparison of the rates of incorporation of bicarbonate into orotic acid and RNA in tissue slices of brain and liver indicate the brain to be one-fourth to one-half as active as the liver in the de novo biosynthesis of pyrimidines. The results of this study favor the proposal that the adult rat brain can meet its needs for pyrimidines through de novo synthesis and is not dependent upon salvage activity and an extraneural supply of pyrimidines.     

Lipid biosynthesis in anoxic-ischaemic rat brain

The uptake of [U-¹⁴C]glucose and [2-¹⁴C]acetate into lipids was measured in brain slices from anoxic, unilaterally ischaemic, unilaterally anoxic-ischaemic, and control rats. The rate of incorporation was significantly decreased in the brain slices from the treated animals except for the contralateral hemisphere of the unilaterally ischaemic animals. Also, there was no significant difference between the anoxic and the anoxic-ischaemic cerebral hemispheres of the anoxic-ischaemic animals. Fractionation of the total lipid extract demonstrated that the decrease in incorporation was general and not due to any particular class of lipid.     

Calcium as a regulator of juvenile hormone biosynthesis and release in the cockroach Diploptera punctata

The role of calcium in the modulation of juvenile hormone (JH) biosynthesis and release by the corpora allata (CA) of Diploptera punctata was examined using an in vitro radiochemical assay. JH production showed a dose dependence on extracellular calcium in the incubation medium. Rates of JH release were maximal between calcium concentrations of 3–5 mM and were almost totally inhibited in its absence. Upon return to medium containing 5 mM calcium, CA exhibited a rapid increase in JH release, although rates of release remained slightly below normal.Blockers of voltage-dependent calcium channels (verapamil, nifedipine), at physiological doses, were able to modulate JH production whereas non-specific calcium channel blockers such as lanthanum effectively inhibited JH release. The calcium ionophore A23187 caused a rapid and irreversible decline in JH release. The calcium dose-response for A23187 showed 50% inhibition of JH release at about 1 mM calcium and maximal inhibition (93%) at 6 mM calcium. Treatment with lanthanum or A23187 did not result in an accumulation within the CA of either JH or methyl farnesoate and accordingly, these compounds appeared to reduce overall JH biosynthesis rather than inhibiting release. Inhibition of JH release by A23187 was dramatically attenuated by coincubation with cobalt, although cobalt alone was found to stimulate JH release significantly. Intracellular calcium levels thus appear to be important in the regulation of JH biosynthesis and release.     

Sequence and significance of dopamine metabolism in the rat brain

This commentary is a critical evaluation of research on the significance of dopamine (DA) metabolism in the striatum of the rat. The possible sequence of DA metabolism is discussed. Special reference was given to a possible differentiation between “intra-neuronal” and “extra-neuronal” formation of DA. In addition a possible relation between drug-induced changes in DA metabolite levels to processes as release and metabolism of the transmitters, was investigated. It is concluded that our understanding of DA metabolism is still far from clear. The only consistent finding at the present time is the fact that decreased DA release is reflected by decreased 3-MT levels. It is emphasized that one should investigate complete changes in the “fingerprint” of changes of DA metabolism rather than to rely on one or two metabolites, when the influence of a drug on dopaminergic transmission is to be established.     

L-tryptophan and rat fetal brain serotonin.

Rat fetal brain and body tryptophan, and brain serotonin were measured at 15, 17, and 19 days postconception, and on the day of birth. Body tryptophan and brain serotonin increased with age during the last trimester of pregnancy; brain tryptophan increased only slightly during this time period. L-tryptophan injected into the mother or into neonates increased fetal and neonatal body and brain tryptophan, and brain serotonin at all ages studied. The dose- and time-relationships of 1-tryptophan-induced changes in brain tryptophan and serotonin were evaluated in 19 day old fetuses. The systemic administration of 1-tryptophan directly to the 19 day old fetus also increased brain serotonin. Thus, fetal brain serotonin neurons appear to have the capacity to synthesize the neurotransmitter from exogenously administered tryptophan, even though these neurons appear to be relatively immature.     

Effect of low doses of gamma-hydroxybutyric acid on serotonin,noradrenaline, and dopamine concentrations in rat brain areas

The effects of intraperitoneal administration of gamma-hydroxybutyric acid (GHB) on biogenic amine levels in hemispheres, hypothalamus, midbrain, and medulla-pons, and on tryptophan in serum and brain, were studied. One hour after GHB administration (50 and 100 mg/kg) significant increases of dopamine concentration were observed in the hemispheres with both doses and in the hypothalamus with the higher dose, but a significant decrease of noradrenaline in the hypothalamus. No significant changes of serotonin metabolism were observed. These results indicate that low doses of GHB selectively affect the catecholaminergic neuronal activity.     

The regional distribution of dopamine and serotonin uptake and transmitter concentrations in the human brain

The regional distribution of the dopamine and serotonin uptake sites in human brain have been assessed and compared with the distribution of the transmitters and their metabolites measured in the same brains and also with a limited regional distribution of the uptake sites in rat and sheep brain. The affinity of the uptake sites for both transmitters was determined and found to be c. 0.2 μ M in all 3 species. Most dopamine uptake in all species was in caudate and putamen samples. Many regions of the human brain showed no dopamine uptake and little dopamine uptake was seen in sheep cortex or nigral preparations. Dopamine and metabolite concentrations were highest in the caudate, putamen and substantia nigra. Most serotonin uptake was seen in the hypothalamus in all 3 species; less was observed in the striatal regions; the cortical and nigral preparations of sheep brain showed little serotonin uptake though cortical preparations of rat brain had high levels of uptake. In the human brain, other regions did not show serotonin uptake. Highest concentrations of serotonin were found in the substantia nigra and medulla, intermediate concentrations in the putamen, globus pallidus, hypothalamus, olfactory tubercle and thalamus; very low concentrations of serotonin were found in other regions. The use of the human uptake site for pharmacological studies and as a marker for monoaminergic afferents in human health and disease is discussed.     

IGF-1 in the brain as a regulator of reproductive neuroendocrine function

Given the close relationship among neuroendocrine systems, it is likely that there may be common signals that coordinate the acquisition of adult reproductive function with other homeostatic processes. In this review, we focus on central nervous system insulin-like growth factor-1 (IGF-1) as a signal controlling reproductive function, with possible links to somatic growth, particularly during puberty. In vertebrates, the appropriate neurosecretion of the decapeptide gonadotropin-releasing hormone (GnRH) plays a critical role in the progression of puberty. Gonadotropin-releasing hormone is released in pulses from neuroterminals in the median eminence (ME), and each GnRH pulse triggers the production of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These pituitary hormones in turn stimulate the synthesis and release of sex steroids by the gonads. Any factor that affects GnRH or gonadotropin pulsatility is important for puberty and reproductive function and, among these factors, the neurotrophic factor IGF-1 is a strong candidate. Although IGF-1 is most commonly studied as the tertiary peripheral hormone in the somatotropic axis via its synthesis in the liver, IGF-1 is also synthesized in the brain, within neurons and glia. In neuroendocrine brain regions, central IGF-1 plays roles in the regulation of neuroendocrine functions, including direct actions on GnRH neurons. Moreover, GnRH neurons themselves co-express IGF-1 and the IGF-1 receptor, and this expression is developmentally regulated. Here, we examine the role of IGF-1 acting in the hypothalamus as a critical link between reproductive and other neuroendocrine functions.     

Structural basis for pterin antagonism in nitric-oxide synthase. Development of novel 4-oxo-pteridine antagonists of (6R)-5,6,7,8-tetrahydrobiopterin

Pathological nitric oxide (NO) generation in sepsis, inflammation, and stroke may be therapeutically controlled by inhibiting NO synthases (NOS). Here we targeted the (6R)-5,6,7,8-tetrahydro-l-biopterin (H(4)Bip)-binding site of NOS, which, upon cofactor binding, maximally increases enzyme activity and NO production from substrate l-arginine. The first generation of H(4)Bip-based NOS inhibitors employed a 4-amino pharmacophore of H(4)Bip analogous to antifolates such as methotrexate. We developed a novel series of 4-oxo-pteridine derivatives that were screened for inhibition against neuronal NOS (NOS-I) and a structure-activity relationship was determined. To understand the structural basis for pterin antagonism, selected derivatives were docked into the NOS pterin binding cavity. Using a reduced 4-oxo-pteridine scaffold, derivatives with certain modifications such as electron-rich aromatic phenyl or benzoyl groups at the 5- and 6-positions, were discovered to markedly inhibit NOS-I, possibly due to hydrophobic and electrostatic interactions with Phe(462) and Ser(104), respectively, within the pterin binding pocket. One of the most effective 4-oxo compounds and, for comparisons an active 4-amino derivative, were then co-crystallized with the endothelial NOS (NOS-III) oxygenase domain and this structure solved to confirm the hypothetical binding modes. Collectively, these findings suggest (i) that, unlike the antifolate principle, the 4-amino substituent is not essential for developing pterin-based NOS inhibitors and (ii), provide a steric and electrostatic basis for their rational design.     

The effect of cocaine treatment on the level of dopamine and serotonin regeneration and on the level of acetylcholine in rat brain]

Cocaine treatment (10 or 50 mg/kg, i.m.): 1) did not change significantly the amount of 3H dopamine synthesized from 3H tyrosine in the striatum but reduced the specific activity of 3H tyrosine; 2) delayed the release of cerebral dopamine after inhibition of its biosynthesis by alpha-methylparatyrosine; 3) reduced the amount of 5-hydroxyindolacetic acid accumulated after Probenecid, which reflects an inhibitory effect on serotonin release; 4) did not change significantly the acetylcholine level of the brain.