Brain neurotransmitter changes in three patients who had a fatal hyperthermia syndrome.

The authors examined the autopsied brains from three patients who had a fatal hyperthermia syndrome. There was marked hypothalamic noradrenaline depletion in all three patients, severe brain choline acetyltransferase deficiency with nucleus basalis cell loss in two patients, and mild to moderate brain choline acetyltransferase loss in one patient. Striatal dopamine metabolite/dopamine ratio was below normal in two patients and not elevated, as would be expected after short-term neuroleptic administration, in the third. This suggests that reduced capability (aggravated by the cholinergic deficit) of the nigrostriatal dopamine system to respond adequately to stress and/or neuroleptic-induced receptor blockade may be important in the development and course of fatal hyperthermia syndrome.

Cloning and expression of human and rat D1 dopamine receptors.

The importance of the dopaminergic system in brain function has been emphasized by its association with neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. On the basis of their biochemical and pharmacological characteristics, dopamine receptors are classified into D1 and D2 subtypes. As the most abundant dopamine receptor in the central nervous system, D1 receptors seem to mediate some behavioural responses, modulate activity of D2 dopamine receptors, and regulate neuron growth and differentiation. The D dopamine receptor has been cloned by low-stringency screening. We report here the cloning of human and rat D1 dopamine receptors by applying an approach based on the polymerase chain reaction. The cloned human D1 dopamine receptor has been characterized on the basis of four criteria: the deduced amino-acid sequence, which reveals that it is a G protein-coupled receptor; the tissue distribution of its messenger RNA, which is compatible with that of the D1 dopamine receptor; its pharmacological profile when transfected into COS-7 cells; and its ability to stimulate the accumulation of cyclic AMP in human 293 cells.

Molecular cloning and expression of the gene for a human D1 dopamine receptor.

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.

The quantitative analysis of D2-dopamine receptors in baboon striatum in vivo with 3-N-[2'-18F]fluoroethylspiperone using positron emission tomography.

We used the ligand 3-N-[2'-18F]fluoroethylspiperone (FESP), which binds to D2-dopamine receptors in the striatum, and positron emission tomography (PET) to quantify striatal D2-dopamine densities (Bmax) and binding kinetics in baboon brain in vivo. Sequential PET scans were obtained for 4 h post injection. Various similar models based on a nonlinear kinetic four-compartment model that takes into account the effect of ligand specific activity were used. We investigated the effect of exact model configuration on the reliability of Bmax and other kinetic transfer coefficients. We found that with the ligand FESP and dynamic PET studies, the estimated values of Bmax and other model parameters are sensitive to the choice of model configuration, ligand specific activity, and data analysis technique. The limitations of the reliability of parameter estimates in a complex kinetic model for receptor ligands were studied in simulation calculations. Results showed that the accuracy of estimated values of Bmax is affected by both the ligand binding properties and the injected dose of ligand. The estimated average value of kinetic model parameters was as follows: ligand-receptor dissociation constant k4 = 0.0080 min-1; the product of ligand-receptor association constant and fraction of ligand available to bind to specific receptors f2ka = 0.0052 (min nM)-1; and D2-dopamine receptor density Bmax = 37.5 pmol g-1.

Glycoprotein nature of the A2-adenosine receptor binding subunit.

Mammalian A2-adenosine receptor binding subunits (A2AR) can be visualized by covalent labeling with the photoaffinity crosslinking ligand 125I-2-[4-[2-[2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine or directly with the azide derivative described in this paper. The protein comprising the A2-adenosine receptor binding subunit migrates with a Mr of 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the glycoproteins representing the radiolabeled A1- and A2-adenosine receptor binding subunit from bovine brain were compared by partial peptide maps and following treatment with exo- and endoglycosidases. Peptide maps using two separate proteases reveal that the A1- and A2-adenosine receptor binding subunits share no common peptide fragments by two-dimensional gel electrophoresis. Endoglycosidase F treatment of labeled A2AR results in a single labeled peptide of Mr 38,000 without intermediate peptides, suggesting a single N-linked carbohydrate chain. The labeled A2AR demonstrates a sensitivity to neuraminidase, as evidenced by an increased mobility on gel electrophoresis, suggesting the receptors contain a glycan component containing terminal sialic acid. Treatment of the labeled A2AR with alpha-mannosidase reveals two distinct populations of A2ARs, one of which is sensitive and the other resistant to the enzyme. The nonadditivity of sequential treatments with the two exoglycosidases suggests, a heterogeneous population of A2AR containing either complex- or high mannose-type carbohydrate chains. These data suggest the A2AR is a Mr 45,000 glycoprotein with a single carbohydrate chain of either the complex or high mannose type. In addition, the A1- and A2ARs are distinct glycoproteins, as evidenced by their differing molecular weights (before and after deglycosylation) and distinct peptide maps.

Increase of deleted mitochondrial DNA in the striatum in Parkinson's disease and senescence.

A mutant mitochondrial DNA (mtDNA) with a 4,977-bp deletion was detected in the parkinsonian brain by using the polymerase chain reaction. Although the deleted mtDNA was detectable even in the brain of aged controls, the proportion of deleted mtDNA to normal mtDNA in the striatum was higher in the parkinsonian patients than in the controls. In both the parkinsonian patients and the aged controls, the proportion was higher in the striatum than in the cerebral cortex. These results indicate that age-related accumulation of deleted mtDNA is accelerated in the parkinsonian striatum and suggest that the deletion contributes to pathophysiological processes underlying Parkinson's disease.

L-threodops increases extracellular norepinephrine levels in the brain: an in vivo study.

Extracellular norepinephrine levels in the striatum of normal rats increased significantly following the administration of L-threodops. When peripheral decarboxylase activity was inhibited with carbidopa, administration of the same dose of L-threodops induced a 2.5-fold greater increase in norepinephrine levels.

Neuropeptide Y expression in rat brain: effects of adrenalectomy.

Neuropeptide Y (NPY) messenger RNA was measured by hybridization of mRNA from the cortex, hippocampus, hypothalamus and striatum of rat brains. Adrenalectomized rats showed lowered level of NPY message in the striatum. A similar decline was found in the hypothalamus, while the cortex and hippocampus were unchanged. Levels of NPY message per unit total RNA were about the same for hypothalamus, cortex and striatum and about 50% less for hippocampus. Adrenalectomized rats that received replacement corticosterone had levels of NPY message that had returned to the levels found in rats receiving sham operation. Response elements consistent with our findings are reported in the NPY genomic sequence.

Plasma and brain kinetics of large neutral amino acids and of striatum monoamines in rats given aspartame.

Two doses (250 and 1000 mg/kg body weight) of aspartame were administered orally to male rats, and plasma and brain phenylalanine and tyrosine kinetic profiles were studied. In both plasma and brain the maximum increase in phenylalanine and tyrosine levels was reached 60 min after treatment. The changes in brain levels of phenylalanine or tyrosine 0, 60, 120 or 180 min after treatment with 1000 mg AMP/kg were directly correlated with the ratio of the plasma concentration of phenylalanine or tyrosine to the overall plasma concentration of the other large neutral amino acids. The time course of monoamine and metabolite concentrations, in the corpora striatum of the brain, was studied after an oral dose of 500 mg phenylalanine/kg. No significant modifications of monoamine levels were found at any of the times studied, up to 5 hr after dosing.

[Modification by alpha-interferon of ethanol-induced changes in the level of opioid peptides in the rat striatum and hypophysis]. / Modifikatsiia pod vliianiem alpha-interferona vyzvannykh étanolom izmenenii soderzhaniia opioidnykh peptidov v striatume i gipofize krys.

Content of Met-enkephalin in striatum and of beta-endorphin in rat hypophysis were estimated after administration of ethanol and alpha-interferon into the animals. Ethanol decreased Met-enkephalin content in striatum and of beta-endorphin in hypophysis. Preadministration of alpha-interferon into brain ventricles before ethanol administration led to an increase in concentration of Met-enkephalin, while content of beta-endorphin was unaltered. In peripheric administration alpha-interferon normalized content of beta-endorphin in adenohypophysis but did not affect the Met-enkephalin concentration. Effects of alpha-interferon on content of Met-enkephalin and beta-endorphin, related to dissimilar organization of the opiate systems in hypophysis and striatum tissues, are discussed.

Non-specific inhibition of imipramine binding argues against an endogenous ligand.

We have attempted to characterize the product(s) inhibiting [3H]imipramine binding from rat cortical membranes. Fractions inhibiting [3H]imipramine binding were analysed by gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-mass spectrometry (HPLC-MS). Tissue extracts showed marked inhibitory activity (two different peaks); however, extracts containing no brain tissue (blanks) also exhibited inhibitory activity (one peak after HPLC pre-purification). The chemical composition of the compound that inhibited [3H]imipramine binding was identical in both cases (tissue and blank extracts) and consisted of zinc chelates, probably formed by the use of zinc sulfate during the extraction procedure. The second inhibitory peak in the tissue extracts was 5-hydroxytryptamine (serotonin, 5-HT). These results suggest that the inhibitory activity on [3H]imipramine binding attributed to a low-molecular weight putative endacoid is non-specific and due to ions introduced in the sample during purification.

Molecular mechanisms of homologous desensitization and internalization of muscarinic receptors in primary cultures of neonatal corticostriatal neurons.

Homologous desensitization of muscarinic acetylcholine receptors (mAChR) was studied using primary cultures of corticostriatal neurons from neonatal rats. Prolonged incubation with carbachol attenuated phospholipase C responsiveness to muscarinic agonists and decreased the number of cell surface mAChR, as measured by binding of N-[3H] methylscopolamine to neuronal monolayers. When neurons were exposed to carbachol for 15 min, 40% of the mAChR lost from the membrane domain was recovered in the cytosol; a decrease of the total neuronal receptors was detected following an incubation with the agonist lasting longer than 15 min. Both 8-Br-cyclic AMP and forskolin neither affected N-[3H]methylscopolamine binding to cell monolayers or did they prevent the agonist-mediated mAChR desensitization. 8-Br-cyclic GMP also failed to decrease mAChR number. Pertussis toxin failed to prevent the homologous desensitization of mAChR under conditions that blocked the agonist-mediated inhibition of forskolin-stimulated cyclic AMP formation. The phorbol ester 12-O-tetradecanoyl-phorbol-12, 13-acetate induced a concentration-dependent decrease of N-[3H]methylscopolamine binding to neuronal monolayers. However, the protein kinase C inhibitors sphingosine and the ganglioside monosialosyl-gangliotetraglicosylceramide inhibited the 12-O-tetradecanoyl-phorbol-12,13-acetate-induced but not the agonist-induced desensitization of mAChRs. Furthermore, incubation with muscarinic agonists failed to translocate protein kinase C from cytosol to plasma membranes, as measured by binding of the phorbol ester [3H]-4-beta-phorbol-12,13-dibutyrate to neuronal monolayers. In corticostriatal neurons the agonist-induced desensitization and internalization of mAChR involves neither protein kinase C and protein kinase A activation nor changes in cyclic GMP and cyclic AMP content.

Cross-linking of human [125I]beta-endorphin to opioid receptors in rat striatal membranes: biochemical evidence for the existence of a mu/delta opioid receptor complex.

In a modified Krebs buffer at 37 degrees C, the selective mu agonist [3H] D-Ala2,MePhe4,Gly-ol5]enkephalin [( 3H]DAMGO) and the nonselective mu/delta agonist human [125I]beta-endorphin [( 125I]beta-endH) bound to rat striatal membranes with a Kd of about 7 and 5 nM and a Bmax of about 95 and 260 fmol/mg of protein, respectively, consistent with labeling of mu receptors by the former ligand and labeling of both mu and delta receptors by the latter. The binding of 2 nM [125I]beta-endH was displaced by unlabeled DAMGO (IC50 30 nM), [D-Ala2-D-Leu5]enkephalin (IC50 60 nM) as well as by the selective delta agonists [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (DSTBULET, IC50 500 nM) and Tyr-Ala-Phe-Asp-Val-Val-Gly-NH2 (IC50 700 nM) in a monophasic manner within 2 to 3 log concentration units, suggesting an allosteric interaction between mu and delta sites labeled by [125I]beta-endH under these conditions. Accordingly, 500 nM DSTBULET caused almost 40% inhibition of the apparent Bmax without changing the apparent Kd of [3H] DAMGO. The kappa agonist U 50,488 was ineffective as competing ligand even at a concentration of 10 microM. Upon affinity cross-linking of [125I]beta-endH (2 nM) to rat striatal mu- and delta-opioid receptors, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized tissue under reducing conditions followed by autoradiography of the dried gels revealed a major broad band of covalently labeled protein with an apparent molecular weight of 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of chronic dopamine receptor blockade on D2 dopamine receptor mRNA level.

The chronic treatment of rats with antipsychotic drugs which increase striatal D2 dopamine receptors did not measurably increase the level of D2 receptor mRNA indicating that the observed increase in receptors was not primarily due to transcriptional regulation. These and other data suggest that the molecular mechanisms involved in receptor regulation in response to denervation or neurotransmitter depletion may be different from those which occur in response to chronic receptor blockade by antagonist drugs.

Cloning of two additional catecholamine receptors from rat brain.

An approach based on the polymerase chain reaction (PCR) was used to isolate additional members of the G-linked receptor family from a rat striatal lambda gtII cDNA library. Priming with one degenerate probe corresponding to highly conserved consensus sequences in the third transmembrane (TM) domain of 15 G-linked receptors and sequences in the phage vector resulted in one clone (G-13) encoding a dopamine D2 receptor variant with a 29 amino acid insert in the third cytoplasmic loop. In addition, the amino acid sequence encoded by clone G-36 contained conserved sequences characteristic of the G-linked class of receptors and displayed sequence homology in TM domains with the beta 2-adrenergic receptor (48%). Two conserved serine residues in TM5 postulated to be part of a ligand binding site in the adrenergic receptor, suggests that G-36 encodes a catecholaminergic receptor. Northern blot analysis confirmed the expression of G-36 in rat brain, but not in kidney, heart and lung. Several strong hybridizing bands to G-36 were obtained in both human and rat genomic DNA. The general PCR strategy employed here should prove to be extremely useful for the isolation of other members of the G-linked receptor family.

Alterations in neurotransmitter amino acid content in the aging rat striatum are subregion dependent.

The topographical distribution of putative neurotransmitter amino acids in both 6- and 20-month-old Fischer 344 rats was studied in eight striatal subregions. Tissue levels of glutamate, aspartate, GABA, and taurine in the 20-month-old rats were elevated in virtually all of the anterior striatal subregions examined. In addition, aspartate levels were higher in all dorsomedial subregions, while glutamate and taurine levels were elevated in all lateral and ventrolateral subregions, respectively. Other amino acids such as glutamate, serine, and alanine did not display any specific subregional changes. These findings demonstrate specific striatal subregion changes in neurotransmitter amino acid content as a function of aging.

The effects of striatal lesion on turning behavior and globus pallidus single unit response to dopamine agonist administration.

In normal rats, globus pallidus neurons are excited by the systemic administration of postsynaptically active doses of apomorphine. The role of the striatum in mediating this phenomenon was examined by investigating the effects of apomorphine on neuronal activity in the globus pallidus and on turning behavior in rats with unilateral quinolinic acid lesions of the striatum. The lesion markedly reduced striatal choline acetyltransferase activity and GABA content and significantly attenuated apomorphine's effect on the activity of pallidal neurons. Both the extent of attenuation of the electrophysiological response of pallidal neurons in lesioned animals and the neurotoxin-induced decreases in choline acetyltransferase activity and GABA content in the caudal striatum were correlated with the degree of apomorphine-induced turning. The data indicate that striatopallidal neurons contribute to apomorphine's excitatory effect on the activity of pallidal neurons in normal animals.

Regional distribution of progesterone and 5 alpha-pregnane-3,20-dione in rat brain during progesterone-induced "anesthesia".

Progesterone and 5 alpha-pregnane-3,20-dione (5 alpha-DHP) concentrations were measured in seven brain areas and in plasma during "anesthesia" induced by progesterone (1-2 mg IV) in female rats. The highest levels of progesterone were detected in the striatum and hypothalamus (23.3 +/- 5.27 and 22.7 +/- 4.30 micrograms/g +/- SEM, respectively); these concentrations were approximately 1000 times higher than those during the post-ovulatory phase. Highest levels of 5 alpha-DHP were detected in the striatum and hippocampus (11.5 +/- 1.74 and 10.4 +/- 3.15 micrograms/g +/- SEM, respectively). The ratio of 5 alpha-DHP to progesterone was approximately 100 times higher in brain tissue than in plasma. We conclude that a conversion of progesterone to 5 alpha-DHP occurs in the brain during the course of progesterone-induced "anesthesia". This metabolic step may be an important contributory factor to the anesthetic potency of progesterone.

Effect of long-lasting diabetes mellitus on rat and human brain monoamines.

Experimental alloxan- or streptozotocin-produced diabetes in rats was accompanied by an increase in the levels of norepinephrine, dopamine, and serotonin, whereas the contents of metabolites, i.e., 5-hydroxyindoleacetic acid and homovanillic acid, in the whole brain gradually decreased with the duration of diabetes. Among the striatum, thalamus, and hypothalamus of alloxan diabetic rats, monoamine alterations were observed only in the hypothalamus; after 1 week an increase of norepinephrine content and after 13 weeks an increase of norepinephrine and dopamine contents were found. Tissues of 11 brain regions of 10 diabetic and 12 control patients post mortem were investigated for monoamine concentrations. Patients were all male, of similar age and interval between death and autopsy. Diabetic patients had an increase in the content of serotonin in the medial and lateral hypothalamus. The content of dopamine increased in the medial hypothalamus, putamen, and medial and lateral pallidus. In diabetic patients, the content of norepinephrine increased in the lateral pallidus and decreased in the nucleus accumbens and claustrum. Thus, it seems that diabetes mellitus in rats, as well as in humans is associated with regionally specific changes in brain monoamines.