Uptake and release ofmeta-tyramine,para-tyramine,and dopamine in rat striatal slices

The uptakes of high-affinity concentrations (10^–8 M) ofmeta-tyramine (m-TA),para-tyramine (p-TA), and dopamine (DA) into rat striatal slices have been shown to be inhibited by DNP and ouabain. We now demonstrate that cocaine (5×10^–6 M) and low concentrations of sodium ion (26×10^–3 M) also reduced these uptakes. The spontaneous efflux and the release [induced by an elevated concentration of potassium ion (5×10^–2 M)] of each of the previously accumulated amines were studied in the presence and absence of added calcium ions. The spontaneous efflux of each amine (especially the tyramines) was enhanced by the absence of calcium ions. Part of this enhancement seemed to be due to an inhibition of a calcium-dependent reuptake. The elevated concentration of potassium ion proved to be an effective releaser of each amine; and for DA, such release was decreased by the removal of calcium. Form- andp-TA, however, the removal of calcium either did not reduce or completely abolished the releases depending upon the duration of the calcium removal. The significance of these findings is discussed.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Transglutaminase activity in pancreatic islets

1. Pancreatic islet homogenates catalyze, in a Ca²⁺-dependent fashion, the incorporation of [2,5-³H]histamine, [1,4-¹⁴C]putrescine, [1,2-³H]agmatine, [¹⁴C]methylamine, L-[U-¹⁴C]lysine in N,N-dimethylcasein. 2. Using [2,5-³H]histamine as the amine donor, the K_m for Ca²⁺ and histamine amounts to 90μM and 0.7 mM, respectively. 3. The incorporation of [2,5-³H]histamine into N,N-dimethylcasein is inhibited by monodansylcadaverine, N-p-tosyl glycine, bacitracin and methylamine, the relative extent of inhibition depending on the respective concentrations of Ca²⁺, inhibitor and amine donor. 4. Bacitracin and methylamine, but not N-p-tosyl glycine, cause a dose-related inhibition of glucose-stimulated insulin release. 5. It is concluded that, in pancreatic islets, the Ca²⁺-responsive transglutaminase activity plays a critical role in the process of glucose-induced insulin release.     

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma.

Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [³H] tRNA extracted from $\text{E. coli}$ B grown in the presence of [5,6-³H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [³H] tRNA. Neither [5-³H]-uridine, [5,6-³H]-UTP, nor [5,6-³H]-poly U released tritium in the present assay conditions.     

The use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-[3-3H]trimethyl-DL-lysine

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.     

Striatal Transporter for Dopamine: Catechol Structure-Activity Studies and Susceptibility to Chemical Modification

Abstract: The apparent second-order association rate constant of dopamine binding to the striatal transporter (～1 ± 10⁶M^?1 s^?1) as well as the transporter turnover number (～1.5 s^?1) was estimated using rotating disk electrode voltammetry to monitor apparent zero trans entry of dopamine into striatal suspensions. The substrate specificity of the transporter was also assessed using catechol derivatives. Dopamine and norepinephrine were transported, whereas epinephrine and the acidic metabolites of dopamine were not transported. The metabolite, 3-meth-oxytyramine, was transported with a K_m seven times greater than and a V_max close to that for dopamine. 4-Methoxytyramine was transported more facilely than the 3-methoxy derivative. N-Alkylation of the amine side chain of dopamine reduced transport dramatically. 4-Ethylcatechol and 3,4-dihydroxybenzylamine were transported with velocities 79 and 91 % less than that for dopamine, respectively. The rigid analogue 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene was transported with a greater velocity than the 5,7-dihydroxy derivative. Finally, the apparent K_mvalues for 4-ethylcatechol, 1-amino-2-phenylethane, tyramine, and m-tyramine as cosubstrates with dopamine were 1.1, 11, 17, and 2.6 μM, respectively. Pretreatments of striatal suspensions with chloroethylnorapomorphine, N-ethylmaleimide, Hg²⁺, 4,5-dihydroxy-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (a redox modulator of receptors in neuronal as well as other tissues), and neuraminidase reduced the velocity of transport of dopamine, whereas N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline had no effect. Thus, the dopamine transporter requires an intact catechol with a primary ethylamine side chain for optimal activity relative to shorter side chain derivatives (side chains longer than two carbons were not tested), the 3-hydroxyl group of dopamine is the more critical hydroxyl group, and the β rotamer of the extended conformation of dopamine is transported preferentially. The catechol appears to mediate the recognition of the substrate, whereas the amine side chain apparently facilitates the conformational change of the transporter that results in movement of dopamine into or across the membrane. The transporter distinguishes between agents known to block dopamine recognition sites on dopamine receptors? appears to possess a reduction/oxidation modulatory site, and requires sulfhydryl groups and external glycosylation for optimal function.     

[3H]Dopamine Labeling of D3 Dopaminergic Sites in Human, Rat, and Calf Brain

Abstract: The binding of [³H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [³H]dopamine binding sites, with a B_max value of 185 fmol/mg protein, whereas rat and human striatum contained one-third this number of sites. The K_D values for [³H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin, and N-propylnorapomorphine) inhibited the binding of [³H]dopamine in all three species, at low concentrations, with IC₅₀ values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC₅₀ values of 200 to 40,000 nM). The [³H]dopamine binding sites were saturable, heat-labile, and detectable only in dopamine-rich brain regions; these sites differed from D₂ dopamine sites (labeled by [³H]butyrophenone neuroleptics), and from D_l dopamine sites (labeled by [³H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. We have, therefore, called these high-affinity [³H]dopamine binding sites D₃ sites. [³H]Apomorphine and [³H]ADTN also appeared to label D₃ sites. These ligands however, were less selective than [³H]dopamine, and labeled sites other than D₃ as well. Assay conditions were important in determining the parameters of [³H]dopamine binding. The optimum conditions for selective labeling of the D₃ dopaminergic sites, using [³H]dopamine, required the presence of EDTA and ascorbate.     

THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA

Abstract— The uptake and release of [³H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10^-7m -[³H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [³H]-dopamine. (2) [³H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis-Menten kinetics. This uptake could be explained by a single ‘high-affinity’ mechanism with a K_m of 2.61 ± 0.41 ± 10^-7m and a V_max of 66 ± 12 ± 10^-12 mol/min/mg protein. (3) [³H]dopamine uptake was temperature-dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca²⁺ or the presence of Co²⁺; however, more than 85, uptake was blocked in the absence of external Na⁺. (5) Neither 1 mm -cyanide nor 5 mm -iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [³H]dopamine uptake. (7) [³H]dopamine in the retina could be released by increasing the external K⁺ concentration. This release was Ca²⁺ -dependent and was blocked by 10mm -Co²⁺ or 2Omm -Mg²⁺. The amount of [³H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.     

A sensitive and specific tritium release assay for dopamine-beta-hydroxylase (DbetaH) in serum.

The release of tritium from [7-³H₂]dopamine was investigated as a possible procedure for the assay for dopamine-β-hydroxylase (DβH) in rat and human serum. The release was found to have the same characteristics as those deseribed previously for DβH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu²⁺ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DβH in serum. Tritium release was blocked by the DβH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of ¹⁴C-labeled dopamine along with [7-³H₂]dopamine revealed that, under the standard assay conditions, the formation of [¹⁴C]norepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DβH activity in serum.     

A novel, convenient assay of lanosterol 14α-demethylase

A rapid and sensitive kinetic assay of lanosterol 14α-demethylation has been developed and analyzed. Three substrates, [32-³H]-24,25-dihydrolanosterol, [32-³H]lanost-8-en-3β,32-diol, and [32-³H]lanost-7-en-3β-32-diol, were studied. In all cases, the rate of tritium released into aqueous solution provided a simple and direct assay of 14α-demethylase activity. The kinetic parameters of K_m and V_max for each substrate have been determined in a reconstituted system from rat liver. The percentage of turnover monitored by the novel tritium release assay was comparable to that observed by conventional GC methods. Separation of unreacted sterol from tritiated formate and water via reverse-phase chromatography permitted several samples to be analyzed at once.     

Formation of 3H2O from [2-3H]- and [4-3H]estradiol by rat uteri in vitro: Possible role of peroxidase

The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of ³H₂O from either [2-³H]- or [4-³H]estradiol. Hydrogen peroxide added to this system increased the yield of ³H₂O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H₂O₂ but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of ³H from [2-³H]- or [2,4,6,7-³H]- but not [4-³H]- or [6,7-³H]estradiol. It is proposed that the formation of ³H₂O from ³H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid.     

Roles for Nitric Oxide as an Intra- and Interneuronal Messenger at NMDA Release-Regulating Receptors: Evidence from Studies of the NMDA-Evoked Release of [3H]Noradrenaline and d-[3H]Aspartate from Rat Hippocampal Slices

Abstract: N-Methyl-d -aspartate (NMDA) receptors regulating the release of [³H]noradrenaline ([³H]NA) and d -[³H]aspartate (d -[³H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg²⁺-free Krebs-Henseleit buffer, the NMDA-evoked release of [³H]NA and d -[³H]Asp was Ca²⁺ dependent and inhibited by the NMDA antagonist (±)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [³H]NA was tetrodotoxin (TTX; 0.1–2 µM) sensitive, whereas that for d -[³H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for d -[³H]Asp appear to be presynaptic, whereas those for [³H]NA are extrasynaptic in location. l -Arginine (100 µM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [³H]NA (100%) and d -[³H]Asp (700%). Exogenous NO donors—sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 µM)—stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [³H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (N^G-nitro-, N^G-methyl-, and N^G-amino-l -arginine, all 100 µM) attenuated (50–80%) the NMDA-stimulated release of [³H]NA and d -[³H]Asp, as did KN-62 (10 µM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.     

Allopregnanolone increase in striatal N-methyl-D-aspartic acid evoked [3H]dopamine release is estrogen and progesterone dependent

1. The neurosteroids are compounds derived from steroid hormones and synthesized in the nervous system. They can modulate different neurotransmitter pathways. In previous work we demonstrated that progesterone modulates dopamine release induced by the glutamatergic agonist N-methyl-D-aspartic acid (NMDA).2. The aim of this work was to evaluate a possible modulatory role of the progesterone metabolite allopregnanolone on NMDA-evoked [³H]dopamine release from corpus striatum slices obtained from cycling and ovariectomized female rats.3. We used a dynamic superfusion method to evaluate the release of [³H]dopamine. Allopregnanolone at 50–600 nM was added to the superfusion buffer (Krebs–Ringer–bicarbonate–glucose, pH 7.4, with constant O₂/CO₂ gassing). The results are expressed as a percentage over basal [³H]dopamine loaded by the tissue.4. Allopregnanolone (50 and 100 nM) increased the NMDA-evoked[³H]dopamine release from estrus rats. The remaining doses did not show significant changes in the pattern of release. This effect was not observed in diestrus rats. The ovariectomy abolished the facilitatory effect of allopregnanolone on NMDA-evoked 2 [³H]dopamine release.5. Subcutaneous administration of exogenous estrogen (25 mg/rat) and progesterone (1 mg/rat) restored the facilitatory effect on dopaminergic input.6. These results suggest that allopregnanolone is a neurosteroid able to modulate dopamine release in an ovarian-hormone-fluctuation-dependent manner and provide further support for a role of allopregnanolone as a modulator of glutamatergic–dopaminergic interaction in the corpus striatum.     

柯拉斯那沉香的生物活性成分研究

为了解柯拉斯那(Aquilaria crassna)的化学成分,从其所产沉香中分离得到10个化合物,经波谱分析分别鉴定为:6,8-羟基-2-(2-苯乙基)色酮(1),6,8-二羟基-2-[2-(4-甲氧基苯)乙基]色酮(2),rel-(1a R,2R,3R,7b S)-1a,2,3,7b-tetrahydro-2,3-dihydroxy-5-(2-phenylethyl)-7H-oxireno[f][1]benzopyran-7-one(3),rel-(1a R,2R,3R,7b S)-1a,2,3,7b-tetrahydro-2,3-dihydroxy-[2-(4-methoxyphenyl)-ethyl]-7H-oxireno[f][1]benzopyran-7-one(4),rel-(1a R,2R,3R,7b S)-1a,2,3,7b-tetrahydro-2,3-dihydroxy-5-[2-(3-hydroxy-4-methoxyphenyl)-ethyl]-7H-oxireno[f][1]benzopyran-7-one(5),oxidoagarochromone B(6),oxidoagarochromone C(7),(5S,6R,7S,8R)-2-[2-(3′-hydroxy-4′-methoxyphenyl)ethyl]-5,6,7,8-tetrahydroxy-5,6,7,8-tetrahydrochromone(8),6,7-cis-dihydroxy-2-(2-phenylethyl)-5,6,7,8-tetrahydrochromone(9),N-trans-feruloyltyramine(10)。化合物3~5和8~10为首次从柯拉斯那沉香中分离得到。化合物1,3,6,7,9和10对乙酰胆碱酯酶具有一定的抑制活性,化合物4对人慢性髓原白血病细胞株K-562和人胃癌细胞株SGC-7901均具有较小的抑制作用,化合物1和3对人肝癌细胞株BEL-7402也有抑制活性。     

SYNTHESIS OF A NEW 5′-O-TRIPHOSPHATE ANALOG OF 5-METHYL 2′-O-DEOXYCYTIDINE. PRELIMINARY IN VITRO LABELING FOR NON-RADIOACTIVE DETECTION OF DNA

We report the synthesis of the triphosphate of 5-methyl 4-N-[6-(p-bromobenzamido)hex-1-yl]-2′-O-deoxycytidine 3A . We also analyzed the formation of intramolecular H-bonds of 5-methyl 4-N-{n-[6-(p-bromobenzamido) caproyl amino]alk-1-yl}-2′-deoxycytidine compounds, and confirmed their presence by ¹H-NMR studies. In vitro DNA labeling with modified nucleotides is preliminarily evaluated.     

Evidence for D2 Receptor Regulation of Dopamine Release in the Goldfish Retina

The possible existence of a dopamine D₂ receptor-mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [³H]dopamine and superfused with D₂ dopamine receptor agonists or antagonists to determine if there was an effect on [³H]dopamine release. The D₂ receptor antagonist sulpiride increased both baseline [³H]- dopamine release and [³H]dopamine release induced by an increase in extracellular potassium concentration. The D₂ receptor agonists LY-171555 and RU-24213 did not reduce baseline [³H]dopamine release but completely inhibited [³H]dopamine release induced by an increase in [K^±]_o. This action of the D₂ agonists was blocked by sulpiride. These studies demonstrate the existence of D₂ receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.     

Role of External and Internal Calcium on Heterocarrier-Mediated Transmitter Release

Abstract: Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [³H]acetylcholine and [³H]noradrenaline from rat hippocampal synaptosomes and of [³H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca²⁺ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [³H]noradrenaline, leaving unaffected that of [³H]acetylcholine or [³H]dopamine. 1,2-Bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [³H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [³H]dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [³H]noradrenaline but not that of [³H]acetylcholine or [³H]dopamine. It is concluded that (a) the release of [³H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca²⁺]-independent releases of [³H]acetylcholine and [³H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca²⁺, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca²⁺.     

Identification of D3 and σ Receptors in the Rat Striatum and Nucleus Accumbens Using (±)-7-Hydroxy-N,N-Di-n-[3H]Propyl-2-Aminotetralin and Carbetapentane

Abstract: Cross-reactions between dopamine D₃ and σ receptor ligands were investigated using (±)-7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin [(±)-7-OH-[³H]DPAT], a putative D₃-selective radioligand, in conjunction with the unlabeled σ ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(?)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(?)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D₃ receptor, neither DTG nor carbetapentane (0.1 µM) displaced (±)-7-OH-[³H]DPAT binding. R(?)-PPAP (0.1 µM) displaced 39.6 ± 1.0% of total (±)-7-OH-[³H]DPAT binding. In striatal and nucleus accumbens homogenates, (±)-7-OH-[³H]DPAT labeled a single site (15–20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(?)-PPAP identified two sites in equal proportion. Carbetapentane and R(?)-PPAP (0.1 µM) displaced ～20–50% of total (±)-7-OH-[³H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[³H]DPAT binding to σ receptors, saturation analysis was performed in the cerebellum while masking D₃ receptors with 1 µM dopamine. Under these conditions (+)-7-OH-[³H]DPAT labeled σ receptors with an affinity of 24 nM. These results suggest that (a) (±)-7-OH-[³H]DPAT binds D₃ receptors with high affinity in rat brain and (b) a significant proportion of (±)-7-OH-[³H]DPAT binding consists of σ₁ sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.     

Increased GABA Release in the Human Brain Cortex as a Potential Pathogenetic Basis of Hyperosmolar Diabetic Coma

Human cerebral cortical slices preincubated with [³H]GABA, [³H]noradrenaline, or 5-[³H]hydroxytryptamine and superfused with Krebs solution or Mg²⁺-free Krebs solution were used to investigate the influence of increased D-glucose concentrations on the release of these [³H]-neurotransmitters evoked by high K⁺ content or NMDA receptor activation, respectively. An increase in level of D-glucose (normal content, 11.1 mM) by 32, 60, and/or 100 mM (a range characteristic for hyperosmolar diabetic coma) increased the [³H]GABA release and inhibited the [³H]noradrenaline release evoked by both methods of stimulation. The K⁺-induced 5-[³H]hydroxytryptamine release was also inhibited by high D-glucose content. Blockade of GABA_B receptors by p-(3-aminopropyl)-p-diethoxymethylphosphinic acid (CGP 35348) attenuated the inhibitory effect of high D-glucose content on the K⁺-evoked release of [³H]noradrenaline and 5-[³H]hydroxy-tryptamine, suggesting that the effect on monoamine release is, at least to a major part, the result of the increased GABA release and, as a consequence, of an increased GABA concentration at inhibitory GABA_B receptors. The membrane-impermeable sorbitol mimicked the increasing effect of D-glucose on [³H]GABA release and its inhibitory effect on 5-[³H]hydroxytryptamine release. However, dimethyl sulfoxide, which is known to permeate rapidly through biological membranes, had no effect at concentrations equiosmolar to D-glucose. It is concluded that a reduction in brain cell volume caused by increased extracellular, compared with cytoplasmic, osmolarity is crucial for the changes in neuronal function observed at high D- glucose and sorbitol content, In view of the fact that GABA is the main inhibitory neurotransmitter in the brain, the increased GABA release may be assumed to contribute to the pathogenesis of hyperosmolar diabetic coma.     

NMDA and AMPA Receptors Evoke Transmitter Release from Noradrenergic Axon Terminals in the Rat Spinal Cord

N-methyl-D-aspartate (NMDA) stimulated release of [³H]noradrenaline (NA) from prelabelled rat spinal cord slices. The release was partially insensitive to tetrodotoxin (TTX) and was inhibited by the NMDA antagonist MK-801. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) also evoked release of [³H]NA, which was enhanced by blocking AMPA receptor desensitization with cyclothiazide. AMPA-evoked release was inhibited by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX) but was not affected by TTX. NMDA and AMPA showed synergistic effects, indicating co-existence of NMDA and AMPA receptors on noradrenergic terminals. Kainate evoked [³H]NA release only at high concentrations and the release was not potentiated by blocking kainate receptor desensitization with concanavalin A. Thus, the results indicate that there are stimulatory presynaptic NMDA and AMPA receptors on noradrenergic axon terminals in the spinal cord and that they interact synergistically to evoke release of [³H]NA.