Correlation between oxidative stress and G6PD activity in neonatal jaundice.

Fetal distress represents a pathophysiological condition in which oxygen is not available to the fetus in sufficient quantities. In cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency, under conditions of oxidative stress, the residual G6PD and complimentary antioxidant mechanisms may become insufficient to neutralize the large amounts of ROS and to prevent severe hemolysis. Alteration in the oxidant-antioxidant profile is also known to occur in neonatal jaundice. The study group included 22 neonates presented with fetal distress during labor and 24 neonates with no evidence of fetal distress (control group). Umbilical cord blood samples were taken immediately after delivery, and the following blood tests were carried out after birth and at discharge from the hospital: erythrocyte count, total bilirubin, G6PD activity, and parameters presenting oxidative status [thiobarbituric acid reactive substances (TBARS), NO, O2 (-), H2O2, SOD, CAT, O2 (-)/SOD, and H2O2/CAT]. There were no significant differences in TBARS and NO values among neonates with or without fetal distress. However, the values of O2 (-), H2O2, SOD, O2 (-)/SOD, and H2O2/CAT among neonates born after fetal distress were significantly higher than in neonates without fetal distress (p < 0.01). In neonates with fetal distress, the total number of RBCs at delivery was significantly lower, accompanied with higher bilirubin content. Also neonates with fetal distress had lower activity of G6PD and lower CAT activity. Higher values of oxidative stress parameters in newborns delivered after fetal distress do not indicate strictly what occurred first-oxidative stress or basic lower G6PD activity.

Psychotomimetics moderately affect dopamine receptor binding in the rat brain.

The hypothesis that psychotomimetics induce a rapid dopamine receptor regulation that could participate in the expression of the brain dopaminergic overactivation and in the early signs of psychotic-like behaviour, was checked by radioligand binding on rat brain cryosections. For this purpose, subchronic 7-day-d-amphetamine pretreatment was combined with acute amphetamine, phencyclidine or LSD challenge. Acute application of psychotomimetics affected only striatal and accumbens but not nigral and olfactory dopamine receptor binding after 40 min, while subchronic amphetamine expressed no effect, as revealed by two-way ANOVA. Post-hoc statistical analysis showed that only striatal and accumbens[3H]SCH 23390 binding decrease (10-12%) following phencyclidine and striatal [3H]spiperone binding increase (11%) after acute amphetamine were significant. It is assumed that such moderate dopamine receptor binding changes probably reflect the fast receptor regulation responses without important influence on a proposed drug-induced dopaminergic overactivity. The registered alterations of D1 receptor binding after phencyclidine are suggested to be capable to modify the activity of some other neural pathways in the basal ganglia and thus participate in a psychotic-like behaviour.

Quantification of human dopamine D2s receptor interactions with G alpha(i,1,2)- and G alpha(o)-proteins.

A simple and rapid in vitro method for qualitative and quantitative estimation of the G alpha-subunits interaction with the third intracellular loop of human D2s dopamine receptor has been developed. For this purpose, D2s-CL3 was cloned in pGEX-2T vector and expressed in E. coli BL21 DE3 as a fusion protein with glutathione-S-transferase (D2s-CL3-GST). The resulting soluble construct was purified by affinity chromatography on glutathione-Sepharose. G alpha-subunits were expressed and purified as His-tagged proteins. For the assay of G alpha/D2s-CL3-GST interactions, varying concentrations of pure His-tagged G alpha-proteins were immobilized on His-Bind Resin and titrated with D2s-CL3-GST fusion protein. G alpha/D2s-CL3-GST interactions were quantified by GST activity determination assay. It was shown that the fusion protein interacts specifically with different G alpha proteins, especially with G alpha(i) proteins. Based on saturation binding analyses, Kd values were determined revealing the highest affinity of His-G alpha(i,2) binding to the fusion protein. The affinities for G alpha(i)/D2s-CL3-GST protein interactions estimated in this way were in nanomolar range of concentrations.

New substituted 2-methylthiomethyl- and 2-methylsulphinylmethylenebenzimidazoles with D2/5-HT1A activity.

Several 2-methylthiomethylbenzimidazoles (3a-e) and the corresponding sulphinyl derivatives 4a-e were synthesized and evaluated measuring their in vitro binding affinity at the D1 and D2 dopamine (source: synaptosomal membranes of the bovine nucleus caudatus) and 5-HT1A serotonin (source: synaptosomal membranes of the bovine hippocampus) receptors. [3H]SCH 23390, [3H]spiperone, and [3H]-8-OH-DPAT were employed as specific radioligands for the D1, D2 and 5-HT1A receptors, respectively. None of the compounds except for 3b acting as a moderate [3H]SCH 23390, competitor, expressed binding affinity at the D1 receptor. Compounds 4a and 4e were inactive displacers of both [3H]spiperone and [3H]-8-OH-DPAT. Ligands 4b, 3d and 4d acted as weak to moderate [3H]spiperone competitors and 3a was a weak [3H]-8-OH-DPAT displacer. The remaining ligands expressed binding affinity at the corresponding receptors in a nanomolar concentration range. Among them, compound 3b with Ki of 14.2 nM and 8.4 nM in [3H]spiperone and [3H]-8-OH-DPAT binding assay, respectively, was the most potent mixed dopaminergic/serotonergic ligand. Although sterically similar, the two classes of ligands differ with regard to electronic properties of substituents in position 2 of the benzimidazole ring. Oxidation of 2-(methylthiomethyl)benzimidazoles afforded ligands devoid of binding affinity at the 5-HT1A receptor and significantly reduced binding affinity at the D2 receptor. This points to the importance of electronic properties of substituents in position 2 of benzimidazole ring for the D2/5-HT1A affinity ratio of this type of ligands.

Adenosine contributes to the amine oxidase-mediated spermine cytotoxicity to human myelogenous leukemia K562 cells.

The effects of adenosine, aminophylline, dipyridamole and salbutamol on the amine oxidase-mediated spermine cytotoxicity to KS62 human myelogenous leukemia cells without Ph-chromosome, spontaneously enriched with mildly adherent cells, were studied. In the absence of spermine, adenosine expressed very mild inhibitory action on K562 cell survival, while in combination with the polyamine an almost additive increase in spermine-FBS cytotoxicity was observed. Aminophylline and salbutamol attenuated both spermine-FBS and spermine-FBS-adenosine suppression of cell survival and viability when equimolar concentration of these agents and the adenosine were applied. Pre-treatment of the cells with higher adenosine levels, in the presence of either aminophylline or dipyridamole, or salbutamol, was associated with decreased K562 cell survival, with the appearance of morphological changes in 10-20% of cells. Additional spermine-FBS cytotoxic effect was not observed in cells pre-treated with adenosine-aminophylline, or adenosine-salbutamole, but morphological changes in 10-20% of cells, even in the presence of spermine, was observed again. Dipyridamole alone suppressed very weakly K562 cell survival. In cells pretreated with dipyridamole, in the presence of spermine-FBS, an additive decrease in cell survival was observed. Pre-treatment of cells with dipyridamole and adenosine in presence of spermine-FBS did not result in a decrease of cell survival compared to the one obtained in dipyridamole-spermine-FBS treated cells.

Mixed dopaminergic/serotonergic properties of several 2-substituted 4-[2-(5-benzimidazole)ethyl]-1-arylpiperazines.

A series of substituted 4-[2-(5-benzimidazole)ethyl]-arylpiperazines was synthesized by introducing different substituents into position 2 of benzimidazole ring of 4-[2-(N,N-di-n-propyl-amino)ethyl]-1, 2-diaminobenzenes. They were evaluated for in vitro binding affinity at the D1 and D2 dopamine and 5-HT1A serotonin receptors using synaptosomal membranes of the bovine caudate nuclei and hippocampi, respectively. Tritiated SCH 23390 (D1 receptor-selective), spiperone (D2 receptor selective) and 8-OH-DPAT (5-HT1A receptor selective) were employed as the radioligands. Only compound 6 expressed a moderate binding affinity at the dopamine D1 receptor, while the remaining ligands were inefficient or weak competitors of [3H]SCH 23390. Compound 12 was an absolutely inactive competitor of all three radioligands. Also, compound 7 was an inefficient displacer of [3H]-8-OH-DPAT. Compound 19 with a Ki value of 3.5 nM was the most potent competitor of [3H]spiperone and compound 13 (Ki = 3.3 nM) was the most efficient in displacing [3H]-8-OH-DPAT from the 5-HT1A serotonin receptor. Ligands 5,6,8-11, and 13-20 expressed mixed dopaminergic/serotonergic activity in nanomolar range of concentrations with varying affinity ratios which strongly depended on the properties of the substituents introduced into position 2 of benzimidazole ring of the parent compounds.

The mechanism of 8-Cl-cAMP action.

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is a potential new anticancer agent, but its mechanism of action is not clearly defined. In this work we have studied the effect of various heat inactivated and heat untreated human sera in the absence or in the presence of a nonspecific phosphodiesterase (PDE) inhibitor, IBMX, or of nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole (DP), or of inosine-monophosphatedehydrogenase (IMPDH) inhibitor, tiazofurin, (T), on the antiproliferative 8-Cl-cAMP action towards two human malignant cell lines, K562 and HeLa cells, in vitro. Cell survival was determined 72 hrs after the agents action, using MTT assay. The results obtained, indicated the similar inhibitory effect of 8-Cl-cAMP on HeLa cell survival in the presence of four different heat untreated human sera (IC50 = 4-4.8 microM). Serum heat inactivation caused decrease in 8-Cl-cAMP antiproliferative action depending on the blood donor (IC50 = 23 microM, 15 microM, 19 microM, and 9 microM) and suggesting that some thermolabile ingredient(s) present in sera is involved, at least partially, in the induction or permittance of antiproliferative 8-Cl-cAMP action. K562 Cells were not as much resistant to 8-Cl-cAMP as HeLa cells, or mouse melanoma B16 cells; in the presence of heat untreated FBS, IC50 = 16 microM, while for B16 cells IC50 was 8 microM. Different human sera show different effect on 8-Cl-cAMP action on K562 cells: IC50 = 7.5 microM and 16.5 microM. In the presence of heat inactivated human sera 8-Cl-cAMP IC50 concentrations were higher, with relevant mutual differences. The effect of different sera on 8-Cl-cAMP action was only partly abrogated in the presence of a nonspecific PDE inhibitor, IBMX, suggesting that the serum PDE action is one of the various factors contributing to the induction of 8-Cl-cAMP antiproliferative action. Nucleoside transport inhibitor and cGMP-specific PDE inhibitor dipiridamole inhibited the antiproliferative 8-Cl-cAMP action to HeLa and K562 cells. Tiazofurin and 8-Cl-cAMP acted as antagonists on HeLa, but not on K562 cells.

Bioisosteric approach in the design of new dopaminergic/serotonergic ligands.

Dopaminergic and serotonergic ligands are widely applied in the therapy of some severe diseases in humans connected to the malfunctioning of the corresponding membrane receptors within the CNS. However, no pharmaceuticals of this type with an ideal therapeutic index have been synthesized so far and there is a constant need of producing new dopaminergic/serotonergic ligands with improved properties especially with regard to undesirable side effects expressed after a prolonged therapy. Dopaminergic/serotonergic ratio turned out to be important for a fine tuning of pharmacological profile of new ligands. Employing a bioisosteric approach, we have synthesized numerous quinoxalinediones, benztriazoles, benzimidazoles and 2-substituted benzimidazoles as potential dopaminergic and/or mixed dopaminergic/serotonergic compounds. With this purpose, benzimidazole and its derivatives were incorporated into phenylethylamine, 3- and 4-substituted phenylethylpiperidine, 1-substituted 4-arylpiperazine and semirigid 2-aminotetralin frame and the resulting ligands were checked for the binding affinity at the D1 and D2 dopamine and 5-HT1A serotonin receptors in radioligand binding assays in vitro. Synaptosomal membranes prepared from bovine caudate nuclei and hippocampi served as a source of the dopamine and serotonin receptors, respectively. [3H]SCH 23390 (D1 receptor-selective), [3H]spiperone (D2 receptor-selective) and 8 OH [3H]DPAT (5-HT1A receptor-selective) were employed as radioligands in competition binding assays. Properties of substituents introduced into position 2 of benzimidazole ring, as well as the nature of the frame into which benzimidazole pharmacophore was incorporated have been shown to determine ligand binding affinity, mode of action and receptor preference, i.e. dopaminergic/serotonergic affinity ratio. Benzimidazolyl-2-thione and benztriazole derivatives were the most potent dopaminergic/serotonergic ligands. Molecular ab initio calculations of the electronic properties of pharmacophoric entities of the new ligands revealed different electron density distribution around the benzene ring in the active and inactive ligands. It can be assumed that this difference influences the properties of pi-pi interactions in a receptor-ligand complex. The results are discussed in comparison with the data of other authors working on similar topics.

Synthesis and dopaminergic features of six novel 3-arylpiperidines.

Four substituted 3-aryl-1-¿2-[5-(1H-benzimidazole)]ethyl¿piperidines and two 3-aryl-1-¿2-[6-(1,4-dihydroxyquinoxaline-2,3-dione)]ethyl¿ piperidines were synthesized, characterized and evaluated for in vitro binding affinity at the D1 and D2 dopamine and 5-HT1A serotonin receptors using synaptosomal membranes of fresh bovine caudate nuclei and hippocampi as a source of the dopamine and serotonin receptors, respectively, and the corresponding specific radioligands. All six novel compounds expressed no binding affinity at the D1 and 5-HT1A receptors. Derivatives of 1,4-dihydroxyquinoxaline-2,3-dione (compounds 8a and 8b) and of 2-dihalomethylbenzimidazole (ligands 9 and 10) were moderate competitors of [3H]spiperone binding at the D2 dopamine receptors. However, benzimidazole-2-thiones (compounds 7a and 7b) behaved as selective D2 receptor ligands with the binding affinity in the nanomolar range of concentrations.

Synthesis of several substituted phenylpiperazines behaving as mixed D2/5HT1A ligands.

Twenty-two different compounds have been synthesized with the aim of creating new, mixed D2/5HT1A ligands. For this purpose 1-substituted phenylpiperazines attached by the N-4 nitrogen to dopaminergic pharmacophores of the 2-(5-benzimidazole)ethyl-, 2-(5-benztriazole)ethyl-, 2-[5-(benzimidazole-2-thione)]ethyl- and 2-[6-(1,4-dihydroquinoxaline-2,3-dione)]ethyl-type were selected according to known structure-affinity requirements of 1-arylpiperazines. All the new compounds were evaluated for in-vitro binding affinity at the dopamine (D1 and D2) and 5-HT1A receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei and hippocampi were used as a source of dopamine and 5-hydroxytryptamine receptors, respectively. [3H]SCH 23390 (D1 selective), [3H]spiperone (D2 selective) and 8-OH-[3H]DPAT (5-HT1A selective) were employed as the radio-ligands. None of the compounds expressed affinity for binding at D1 dopamine receptors. Compounds 3b and 4b were inactive 8-OH-[3H]DPAT competitors whereas 1b, 2b and 4b were inactive in the [3H]spiperone-binding assay. The other compounds tested showed fair (1c, 1e, 1f, 2c, 2f, 3b, 3c and 4c) to high (1a, 1d, 2a, 1d, 3a, 3d-3f, 4a, and 4d) affinity in the [3H]spiperone-binding assay, the most potent representative being 4-[2-(5-benzimidazole-2-thione)ethyl]-1-(2-methoxyphenyl)piperazine, 3a (Ki = 1.7 nM). In the 8-OH-[3H]DPAT-displacement assay compounds 1b, 1d, 1f, 2b, 2f and 3f behaved as moderate competitors and 1a, 1c, 1e, 2a, 2c, 2d, 3a, 3c-3e, 4a, 4c, 4d and 4f as rather strong competitors; 4-[2-(5-benztriazole)ethyl]-1-(2-methoxyphenyl)piperazine, 2a had the highest binding affinity at the 5-HT1A receptors (Ki = 2.6 nM). Because many antipsychotic and anxiolytic agents behave as mixed dopaminergic and serotonergic ligands, the high affinity of several of these new ligands for binding at both D2 and 5-HT1A receptors make them promising candidates deserving further pharmacological evaluation as antipsychotic or anxiolytic pharmaceuticals.

Structure-affinity relationship studies on D-2/5-HT1A receptor ligands. 4-(2-Heteroarylethyl)-1-arylpiperazines.

With an aim of creating new, mixed D-2/5-HT1A ligands, sixteen different compounds were synthesized. For this purpose 1-arylpiperazines attached through N-4 nitrogen to the dopaminergic pharmacophores of 2-(5-benzimidazole)-ethyl-, 2-(5-benztriazole)-ethyl-, 2-[5-(benzimidazole-2-thione]-ethyl- and 2-[6-(1,4-dihydroquinoxaline-2,3-dione)]-ethyl-type were selected according to their structural diversity (phenyl, substituted phenyl, heteroaryl and naphthyl). All new compounds were checked for in vitro binding affinity at the dopamine (D-1 and D-2) and serotonin (5-HT1A) receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei and hippocampus were used as a source of dopamine and serotonin receptors, respectively, [3H]SCH 23390 (D-1 selective), [3H]spiperone (D-2 selective) and 8-OH-[3H]DPAT (5-HT1A selective) were employed as the radioligands. All compounds were inactive competitors of [3H]SCH 23390, 1c and 2b being inactive in the two other binding assays, as well. Derivatives of 1-(2-pyridyl)piperazine 1b and 2a and of 1-phenylpiperazine 1a expressed moderate to low affinity for both D-2 and 5-HT1A receptors, while 1-(2-pyridyl)-piperazines 3b and 4b and 4-(1-phenylethyl)-1-(1-naphthyl)-piperazine 10 were moderate [3H]spiperone, but potent 8-OH-[3H]DPAT competitors. Among them, derivatives of 1-(1-naphthyl)-piperazine 1e, 2d and 3e and of 1-(2,3-dimethylphenyl)-piperazine 1d, 2c and 3d were the strongest competitors at both D-2 and 5-HT1A receptors.

Synthesis and dopaminergic properties of 3- and 4-substituted 1-[2-[5-(1H-benzimidazole-2-thione)]ethyl]piperidines and related compounds.

With an aim of creating new, high affinity dopaminergic ligands, six different 3- and 4-substituted 1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]piperidines and nine related heterocyclic congeners were synthesized and evaluated for in vitro binding affinity at D1 and D2 dopamine receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei were used as a source of the dopamine receptors. Only 4-[bis-(4-fluorophenyl)methylene]-piperidines, compounds 9e, 10d, and 11d, expressed moderate affinity for the D1 receptors, while all other compounds were inactive competitors of [3H]SCH 23390. Compounds 9c, 9d, 10c, 11a, and 11c were inactive in the D2 receptor binding assay, as well. Derivatives of 4-phenylpiperidine (9-11b) and 3-phenylpiperidine (10a) expressed a moderate to low affinity for the D2 receptors. However, racemic (+/-)-1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]-3-phenylpiperidine 9a and its enantiomer (+)-9a behaved as selective, high affinity D2 receptor ligands, the latter being some four times more active than the racemate.

Acute amphetamine and/or phencyclidine effects on the dopamine receptor specific binding in the rat brain.

Psychotic-like behaviour was induced in rats with a single i.p. injection of AMPH (20 mg/kg b.w.) and/or PCP (10 mg/kg b.w.). The D1 and D2 dopamine receptor (DAR) specific binding of [3H]SCH 23390 and [3H]spiperone, respectively, during the 120 min period upon the treatment was examined on cryosections using computerized scanning and image analysis. AMPH, alone or in combination with PCP, induced a transient decrease of the D1 receptor specific binding in the striatum (30 min; AMPH, -18%; AMPH+PCP, -31%) and nucleus accumbens (30 min; AMPH, -30%; AMPH+PCP, -40%), which was completely abolished at the 120 min point. Only AMPH persistently elevated nigral D1 receptor specific binding. PCP-induced striatal and accumbal D1 receptor down-regulation was intensive throughout the 120 min period, while in the s. nigra it was non-significant. A significant increase of the D2 receptor specific binding was observed only 30 min after the treatment in striatum (AMPH, 15%; PCP, 16%; AMPH+PCP, 13%) and n. accumbens (AMPH, 16%). These alterations of DAR specific binding may reflect a regulation of the DAR and the changes in nigrostriatal and mesolimbic DA-ergic neurotransmission during an intensive drug-induced psychotic-like behavioral expression.

Synthesis of N-n-propyl-N-(2-arylethyl)-5-(1H-benzimidazol-2-thione)-eth ylamines and related compounds as potential dopaminergic ligands.

Twelve different N-n-propyl-N-(2-arylethyl)-5-(1H-benzimidazol-2-thione)-ethy lamines and 12 related heterocyclic congeners were synthesized with the aim of creating new high affinity dopaminergic ligands. Upon thorough chemical analysis they were evaluated for their affinity towards the D-1 and the D-2 dopamine receptors (DAR) by in vitro binding assay using synaptosomal membranes of the bovine caudate nuclei and [3H]SCH 23390 (R (+)-7-chloro-8-hydroxy-3-methyl-1-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) (D-1 selective) and [3H]spiperone (D-2 selective) as the radioligands. None of the synthesized compounds expressed affinity for the binding to the D-1 DAR Compounds 11e, 11j, 11l, 12b, 12d, 13a, 13d, 14a, and 14d were also inactive competitors of [3H] spiperone binding to the D-2 DAR. However, 1H-benzimidazol-2-thione derivatives 11k, 11h, and 11f and 1, 4-di-hydroquinoxalin-2,3-dione derivative 12c (in this rank order of potencies) acted as strong competitors of [3H] spiperone binding to the D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT(2) receptors. Relationship between the structure and affinities of the new ligands for the binding to the D-2 DAR as well as the demands of the receptor itself for the interaction with the new compounds are discussed.

Synthesis and evaluation of N,N-di-n-propyl-5,6,7,8-tetrahydro-1 H-benz[f]benzimidazol-6-yl-amine and related congeners as dopaminergic ligands.

Four semirigid dopaminergic biosiosteres (N,N-di-n-propyl-5,6,7,8-tetrahydro-1 H-benz[f]benzimidazol-6-yl-amine [sequence: see text] and three related congeners) were synthesized and chemically characterized. The affinity and selectivity of these compounds for D-1 and D-2 classes of the dopamine receptor were determined in competition binding experiments using synaptosomal membranes of the bovine caudate nuclei and [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective) as radioligands. None of the four compounds tested expressed significant affinity for D-1 receptors and all of them competed moderately with [3H]spiperone binding to D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT2 receptors. Some aspects of structure affinity relationship for this class of dopaminergic ligands are discussed.

Design, synthesis and properties of several heterocyclic dopaminergic ligands.

Two series of non-catechol dopamine (DA) bioisosteres with the hydroxyl groups on the benzene ring replaced by N-H groups were synthesized using phenethylamine as a parent molecule. Compounds from the first series (1-9) contained a primary amine group, while those from the second one (10-19) had a N,N-di-propylamino group introduced into the side chain of the phenethylamine. The affinity and selectivity of these compounds for the D-1 and D-2 subtypes of the DA receptor were determined in competition binding assays using synaptosomal membranes from bovine caudate nuclei and [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective) as radioligands. None of the 19 compounds examined expressed significant affinity for D-1 receptors, while compounds 15, 17 and 19, in this order of potency, strongly competed with [3H]spiperone binding to D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT2 receptors. Varying affinities of the compounds examined for the DA receptors can be ascribed to different acidity of the N-H groups introduced at meta- and para-positions of the phenethylamine molecule.

Synthesis and binding properties of several new dopaminergic ligands.

Four different heterocyclic dopamine bioisosteres were synthesized. The affinity and selectivity of these compounds for the D-1 and D-2 classes of the dopamine receptor were determined in competition binding experiments using synaptosomal membranes from bovine caudate nuclei and [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective) as radioligands. None of these compounds expressed significant affinity for the D-1 receptors, while compounds 3, 4, 2 and 1 in this order of potency strongly competed with [3H]spiperone binding to D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT2 receptors (50 nM ketanserin). Compounds 3 and 4 behave as agonists as judged by the data obtained in competition binding experiments in the presence of Gpp(NH)p, the former (3) expressing a very high affinity for D-2 receptors.

To the mechanism of spermine-FBS cytotoxicity toward K562 human myelogenous leukemia cells.

The effects of several compounds acting through adenylate cyclase system and/or influencing prostaglandin biosynthesis on spermine-FBS cytotoxicity to human myelogenous leukemia K562 cells were studied. Salbutamol, a beta 2-adrenoceptor agonist inhibited to a certain extent spermine-FBS cytotoxic action to K562 cells, and propranolol, a beta 2-adrenoceptor antagonist, did not affect this inhibition. Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase, acted suppressing spermine-FBS cytotoxicity to K562 cells. Pretreatment of the cells with dexamethasone did not significantly alter salbutamol-related inhibition of spermine-FBS cytotoxicity. Indomethacin, an inhibitor of cyclooxygenases directly involved in prostaglandin biosynthesis, did not interfere with protective terbutaline effects against spermine-FBS cytotoxicity to K562 cells during the 24-hour period.

Solubilization of dopamine D-1 receptors with a zwitterionic detergent DCHAPS and their reconstitution.

1. Dopamine D-1 receptors of the bovine caudate nucleus were solubilized with different detergents. They were labelled with [3H]SCH 23390 and assayed by filtration through PEI-coated glass fibre filters and Sephadex G-50 columns. 2. DCHAPS was the best solubilizer among all detergents used and at 0.075% DCHAPS, 10 mg/ml protein, 30 min, 4 degrees C, gave the yield of 48.7%. 3. Reconstitution of solubilized receptors was performed using SM-2 Bio-Beads. Phosphatidylcholine did not improve reconstitution suggesting that DCHAPS solubilized sufficient amounts of the membrane phospholipids. 4. Loss of affinity of solubilized receptors to [3H]SCH 23390 binding was reversible. Apparent Kd values of 0.36 +/- 0.02, 21.3 +/- 3.2 and 0.77 +/- 0.05 nM were obtained for membrane-bound, solubilized and reconstituted receptors, respectively.