Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Sensitivity to ethanol across development in rats: comparison to [3H]zolpidem binding

Previous research has suggested that rats tested at 28 to 30 days of age show a marked subsensitivity to the sedative effects of ethanol. In the present study, rats of different ages were tested for aerial righting following acute ethanol (3 g/kg) treatment. These results were compared with the effects of the atypical benzodiazepine zolpidem (3 and 5 mg/kg) and pentobarbital (10 and 15 mg/kg). Animals tested at 25, 28, or 35 days of age were significantly less impaired by ethanol than preweanling rats (age 20 days) or older rats (age 65 to 75 days), whereas animals tested at 25 or 28 days of age were less impaired by the higher dose of zolpidem. With pentobarbital, the most distinct age-related trend was greater impairment in 20-day-old rats. Because ethanol may be active at the same type I GABA(A) receptor site selectively labeled by [3H]zolpidem, levels of [3H]zolpidem binding were determined for rats of different ages. Although some brain regions showed progressive increases in binding of [3H]zolpidem across development, other regions demonstrated increased binding from day 12 or 17 to day 20, then a plateau of binding levels across days 20, 25, and 28, with further increases occurring by day 36 or day 60. This pattern was observed in the cingulate cortex, medial septal nucleus, globus pallidus, inferior colliculus, red nucleus, and cerebellum. Overall, the results indicate that the period of subsensitivity to the sedative effects of ethanol is coincident with a change in the developmental pattern of GABA(A) receptor sites targeted by [3H]zolpidem.     

Characterization of [3H]clozapine binding sites in rat brain

We examined the characteristics of [3H]clozapine binding sites in four rat brain regions (frontal cortex, limbic area, hippocampus and striatum) in order to elucidate the pharmacological profile of this unique atypical antipsychotic drug. The specific [3H]clozapine binding was found to be saturable and reversible in all these brain regions. Scatchard analysis of the saturation data indicated that the specific binding consisted of high- and low-affinity components. Displacement experiments showed that the muscarinic cholinergic receptor represented about 50% of [3H]clozapine binding in each brain area. Serotonin 5-HT2 and dopamine D4 receptor binding sites could also be detected by displacement experiments using ketanserin and nemonapride, respectively, in frontal cortex and limbic area, but not in hippocampus or striatum. Alpha-1, alpha-2, histamine H1, dopamine D1, D2, or D3 receptor components could not be determined within the high-affinity [3H]clozapine binding sites in any brain region. It is possible that the atypical property of clozapine may depend on the modulatory effect on dopaminergic function via 5-HT2 receptor blockade and/or may be mediated via D4 receptor blockade in the mesocortical and mesolimbic area.     

Characterization of [3H]idazoxan binding proteins in solubilized membranes from rabbit and human liver

This study sought to determine the potential role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA)-stimulated efflux of [14C] gamma-aminobutyric acid (GABA) and [3H]acetylcholine from striatal slices in vitro. In Mg2+-free buffer, NMDA-stimulated [14C]GABA and [3H]acetylcholine release were inhibited by the guanylate cyclase inhibitor, 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and, to a lesser extent, by the nitric oxide synthase inhibitor, nitroarginine (N-Arg). Since reversal of catecholamine transporters previously has been implicated in the mechanism underlying NO-induced catecholamine release, we used the GABA transport inhibitor, 1-(2-(((diphenylmethylene)imino)oxy)ethyl)-1,2,5,6-tetrahydro-3-py ridine-carboxylic acid hydrochloride (NNC-711), to address the role of GABA transport in NArg-sensitive NMDA-induced release. NNC-711 inhibited NMDA-stimulated [14C]GABA efflux by 50%, confirming our previous report that NMDA-stimulated GABA release is partially dependent on reversal of the transporter. The effect of N-Arg in the presence of NNC-711 was similar to its effect in the absence of the transport inhibitor, suggesting that reversal of the transporter is not involved in the NO component of NMDA-stimulated [14C]GABA release. These data suggest that glutamatergic transmission through striatal NMDA receptors is partially mediated through activation of the NO/guanylate cyclase pathway and that this mechanism may contribute to the tetrodotoxin sensitivity of NMDA-induced release of GABA and acetylcholine in the striatum.     

Characterization of [3H]AMP binding to rat adipose plasma membranes and its substrate specificity

In order to evaluate the acute effects of two different treatments on changes in the American Urological Association symptom score, we divided 23 men with benign prostatic hyperplasia into 2 groups. Group 1 (n = 16) and group 2 (n = 7) were treated with transurethral resection of the prostate and visual laser ablation of the prostate, respectively. Twice before and about 1 week after surgery, patients completed the AUA symptom questionnaire and underwent urodynamic evaluation. The symptom indexes were subcategorized as obstructive and irritative symptoms. All symptom scores were identical in groups 1 and 2 preoperatively. Postoperatively, significant improvement was found in obstructive scores, the total score, maximum and average flow rates only in group 1. This outcome is probably the reflection of an essential dissmilarity in both therapies. Clinically, the obstructive subscore appears reactive to changes in obstruction and seems meaningful in follow-up even in the early postoperative days.     

Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

1. The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. 2. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg-1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. 3. Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors. 4. It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.     

Unchanged [3H]MK-801 binding and increased [3H]flunitrazepam binding in turtle forebrain during anoxia

In order to determine if functional changes in N-methyl-D-aspartate receptors and GABAA receptors play a role in the remarkable anoxia tolerance of freshwater turtle brain, we used autoradiographic techniques to assay [3H]MK-801 and [3H]flunitrazepam binding in turtle forebrain after turtles had been subjected to anoxia for 2 or 6 h. The effects of glutamate, glycine, competitive N-methyl-D-aspartate antagonists, glycine antagonists, polyamines, magnesium, and zinc on [3H]MK-801 binding were the same in anoxic and control turtle forebrains. These results indicate that NMDA receptor regulation plays no role in the adaptive responses to anoxia in turtle brain. In contrast, [3H]flunitrazepam binding was significantly increased in the anoxic dorsal cortex and striatum. The most parsimonious explanation for elevated benzodiazepine receptor binding is that the rise in extracellular GABA levels known to accompany anoxia enhances benzodiazepine receptor affinity. It is possible, however, that GABAA receptor upregulation during anoxia increases the effectiveness of the inhibitory action of released GABA and contributes to the anoxia tolerance of turtles.     

High affinity [3H]imipramine and [3H]paroxetine binding sites in suicide brains

Specific binding of [3H]imipramine and [3H]paroxetine was simultaneously examined in human brains (frontal cortex, temporal cortex, cingulate cortex, hypothalamus, hippocampus and amygdala) from 11 controls and 11 depressed suicide victims. A single saturable high affinity site was obtained for both radioligands. Age was not related to significant changes in [3H]imipramine and [3H]paroxetine binding parameters, which indicates the stability of the brain serotonergic system with increasing age. A major finding of the present study concerns the existence of a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in hippocampus from depressed suicides as compared with the control group, without changes in the binding affinity (Kd). In contrast, when [3H]paroxetine was used as radioligand, no changes in either Bmax or Kd were detected in any of the brain regions studied. These findings suggest that [3H]imipramine may be a better marker than [3H]paroxetine when alterations in the presynaptic serotonergic uptake site are to be detected.     

Isolation of the [3H]gabapentin-binding protein/alpha 2 delta Ca2+ channel subunit from porcine brain: development of a radioligand binding assay for alpha 2 delta subunits using [3H]leucine

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.     

A high affinity binding site for [3H]-Dofetilide on human leukocytes

Certain Class III anti-arrhythmic agents have been shown to interact with human leukocytes and after antigenic and mitogenic activation. We hypothesized that a binding site for the Class III anti-arrhythmic agent, dofetilide, would exist on human leukocytes. Analysis of binding isotherms defined the presence of a single high affinity binding site on mononuclear cells and neutrophils: Kd 26+/-4 nm, Bmax 61+/-14 fmol/10( 6) cells and Kd 33+/-14 nm, Bmax 163+/-45 fmol/10(6) cells, respectively. Other Class III drugs inhibited [3H]-dofetilide binding at physiologically relevant concentrations, but the IC50 values of E4031 and quinidine were significantly higher for leukocytes than for cardiac myocytes. Interestingly, verapamil inhibited [3H]-dofetilide binding to leukocytes, but not to cardiac myocytes at physiologic concentrations (10 microM). Charybdotoxin and tetraethlyammonium inhibited [3H]-dofetilide binding to leukocytes at microM mm concentrations, respectively, however, apamin did not inhibit binding even at 1 microM concentrations. These data suggest that a Ca2+-activated K+ channel, like K(Ca) mini (apamin-insensitive isoform), is a candidate for the leukocyte [3H]-dofetilide binding site. To assess the functional significance of defetilide binding to leukocyte biology, we evaluated fMLP-stimulated superoxide production in the presence or absence of dofetilide. Dofetilide, at 30 nm suppressed of superoxide production. In conclusion, dofetilide binds to human leukocytes at physiologic concentrations and this binding alters leukocyte function possibly through interaction with a Ca2+-activated K+ channel.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.     

Characterization of 5,7-dichlorokynurenate-insensitive D-[3H]serine binding to synaptosomal fraction isolated from rat brain tissues

To explore target sites for endogenous D-serine that are different from the glycine site of the N-methyl-D-aspartate (NMDA) type glutamate receptor, we have studied the binding of D-[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 microM) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled D-serine. Association, dissociation, and saturation experiments indicated that D-[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K(D) of 614 nM and a Bmax of 2.07 pmol/mg of protein. D-Serine, L-serine, and glycine produced a total inhibition of the specific DCK-insensitive D-[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 microM. The profiles of displacement of the DCK-insensitive D-[3H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive D-[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive D-[3H]serine binding site could be a novel candidate for a target for endogenous D-serine in mammalian brains.     

3H]-BIBP3226 and [3H]-NPY binding to intact SK-N-MC cells and CHO cells expressing the human Y1 receptor

We have studied the binding of [3H]-NPY and the newly developed non-peptide Y1 receptor antagonist [3H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y1 receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [3H]-NPY binding was slow, the binding kinetics of [3H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y1 agonists NPY, PYY and [Leu31-Pro34]-NPY completely and potently displaced [3H]-NPY binding, they could only displace 70 to 80% of the [3H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [3H]-NPY binding sites in CHO-Y1 cells whereas [3H]-BIBP3226 binding parameters remained unchanged.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Changes in apparent in vivo binding of [3H]raclopride and [3H]N-methylspiperone induced by oxotremorine

The effects of muscarinic agonist, oxotremorine (0.3 mg/kg), and antagonist, scopolamine (0.5 mg/kg), on in vivo [3H]raclopride (RAC) and [3H]N-methylspiperone (NMSP) binding were investigated. Following tracer administration to control or pretreated mice, binding potentials, and the rate constants k3 and k4 were determined by kinetic analysis. Oxotremorine resulted in a 70% increase in striatal RAC binding potential compared with controls. RAC and NMSP showed almost identical decreases in k3 (40%), whereas k4 for RAC was unexpectedly decreased by 64%. Scopolamine resulted in no significant changes in RAC or NMSP binding. These results, in combination with previous data obtained in reserpinized mice, show that 1) competition by endogenous ligand may not be the only factor influencing the magnitude of apparent in vivo receptor binding, and 2) interneuronal communication may be partly mediated by changes in the rates of ligand-receptor binding.     

Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.