Diazepam-potentiated [H]phenytoin binding is associated with peripheral-type benzodiazepine receptors and not with voltage-dependent sodium channels

In the presence of diazepam, [³H]phenytoin binds with high affinity to brain membranes. The present experiments examined whether this high affinity [³H]phenytoin-binding site co-localized with the standard [³H]phenytoin-binding site on the voltage-dependent sodium channel (VDSC). Veratridine, a pharmacological activator of the voltage-dependent sodium channel, that inhibits standard [³H]phenytoin binding, failed to affect the high affinity diazepam-potentiated [³H]phenytoin binding in brain membranes, suggesting that the potentiated binding interaction resides at a site distinct from the voltage-dependent sodium channel. This possibility was confirmed by anion exchange chromatography of digitonin-solubilized rat brain membranes, as diazepam-potentiated high affinity [³H]phenytoin binding eluted in column fractions that were distinct from [³H]saxitoxin-defined voltage-dependent sodium channels. To examine whether diazepam-potentiated [³H]phenytoin binding might be associated with other ‘classic’ benzodiazepine receptor sites, we tested whether specific ligands for benzodiazepine receptors would either produce or block potentiated [³H]phenytoin binding. Neither agonists, nor antagonists, of the high affinity central-type benzodiazepine receptor affected potentiated [³H]phenytoin binding, suggesting that the high affinity potentiated binding site is not likely associated with central benzodiazepine receptors. Peripheral-type benzodiazepine receptor agonists, however, did potentiate [³H]phenytoin binding, and a specific receptor antagonist (PK11195) attenuated the potentiation seen with diazepam. Overall, these data illustrate that [³H]phenytoin interacts with a novel site in brain membranes that is distinct from the voltage-dependent sodium channel and is allosterically revealed by peripheral-type, but not central-type, benzodiazepine receptor agonists.     

Evidence for multiple [3H]prazosin binding sites in canine brain membranes

Two classes of alpha 1 adrenoceptors were identified in canine brain and liver using conventional radioligand binding methods. Scatchard plots of specific [3H]prazosin binding to brain and liver membranes prepared from 100-150-day-old Doberman pinscher dogs were consistently curvilinear and best fit a two-site binding model (frontal cortex, Kd1 = 57.7 +/- 10.0 pM, Bmax1 = 64.6 +/- 17.1 fmol/mg protein, Kd2 = 1.5 +/- 0.5 nM, Bmax2 = 159 +/- 37.6 fmol/mg protein; liver, Kd1 = 82.6 +/- 36 pM, Bmax1 = 7.0 +/- 5.1 fmol/mg protein, Kd2 = 0.8 +/- 0.2 nM, Bmax2 = 62.1 +/- 8.7 fmol/mg protein). Kinetically derived affinity constants from association and dissociation experiments agreed with those obtained by Scatchard analyses of equilibrium binding data. Binding sites were saturable, heat labile, bound ligand reversibly, and appeared to be appropriately distributed in relation to endogenous catecholamine. [3H]Prazosin also bound with high affinity to two classes of binding site in porcine and bovine brain membrane but [3H]prazosin binding in monkey and rat brain was best described by a single-site binding model. Affinities obtained were in between values obtained for high and low affinity Kds in the other species. Competitions for [3H]prazosin binding sites in canine frontal cortex were conducted with the following antagonists: WB-4101, corynanthine, phentolamine, benoxathian, phenoxybenzamine, chlorethylclonidine, thymoxamine, prazosin, yohimbine and agonists: methoxamine, (-)-norepinephrine, and clonidine. All ligands but prazosin, norepinephrine and clonidine competed for specific [3H]prazosin binding in a statistically significant biphasic manner. Benoxathian and WB-4101 displayed the highest affinities (benoxathian: Ki1 = 0.26 nM, WB-4101: Ki1 = 0.20 nM) and selectivity (high affinity/low affinity: benoxathian = 1640, WB-4101 = 13204) for the high affinity [3H]prazosin binding site; chlorethylclonidine had highest affinity (Ki2 = 91 nM) and selectivity (low affinity/high affinity = 405) for the lower affinity [3H]prazosin binding site. As defined, the two sites were similar to the alpha 1a and alpha 1b recently described in the rat and rabbit. A noticeable difference was that the subtypes described in dog brain had a 30-fold difference in affinity for prazosin.     

GABA binding and bicuculline in spinal cord and cortical membranes from adult rat and from mouse neurons in cell culture

Sodium-independent [³H]GABA and [³H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [³H]GABA bound to two sites (K_d=8nM,B_max=0.62pmol/mg protein; K_d=390nM,B_max=3.9pmol/mg protein) whereas the GABA agonist, [³H]muscimol, bound only to a high affinity site (K_d=5.6nM,B_max=1.9pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [³H]GABA (K_d=340nM,B_max=9.8pmol/mg protein) and only a high affinity site was seen with [³H]muscimol (K_d=5.6nM,B_max=0.25pmol/mg protein). The inability to measure a high affinity [³H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cel; culture, [³H]GABA bound to two sites on cortical neurons (K_d=9nM,B_max=0.24pmol/mg protein; K_d=510nM,B_max=1.3pmol/mg protein) and spinal cord neurons (K_d=13nM,B_max=0.12pmol/mg protein; K_d=640nM,B_max=3.2pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1).The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [³H]GABA binding was determined. Bicuculline appeared to inhibit binding to both sites competitively but theK_i for inhibiting the high affinity site was 5 μM and for inhibiting the low affinity site was 115 μM. Bicuculline inhibited [³H]muscimol binding in both brain and spinal cord competitively withK_is of 4μM and 10 μM respectively. Bicuculline inhibition of [³H]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes.The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA receptor. The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments²⁸ supports this hypothesis.     

Stereospecific binding of D-lysergic acid diethylamide (LSD) to brain membranes: relationship to serotonin receptors.

D-[3H]LSD binds saturably, reversibly, and with a high affinity (KD = 10 nM) to rat brain membranes. The association and dissociation rates of binding are temperature dependent and fastest at 37 degrees C. Binding is enriched in crude microsomal (P3) membranes. D-[3H]LSD binding is stereospecific as L-LSD, the psychotropically inactive enatiomer, is 1000 times weaker than D-LSD as a displacing agent. The potencies of other LSD analogues parallel their psychotropic activity with the exception of 2-bromo-LSD (psychotropically inactive) which is as potent as D-LSD in displacing bound D-[3H]LSD. Serotonin is the only putative neurotransmitter with affinity (ED50 = 3 muM) for the LSD binding site, and psychotropically active alkylindoleamines are also potent displacing agents. Destruction of presynaptic serotonin neuronal elements by lesioning the midbrain raphe nuclei does not chang the affinity or maximum number of detectable in vitro D-[3H]LSD bindind sites. The regional distribution in monkey brain of D-[3H]LSD binding and high affinity [3h]serotonin uptake, a marker for pre-synaptic serotonin nerve terminal density, shows some correlation. The most notable exceptions are cerebral cortical areas which are highest in D-[3H]LSD binding and only intermediate in [3h]serotonin uptake. Our evidence suggests that D-[3H]LSD binds to post-synaptic serotonin receptors.     

3H]1-[2-(2-thienyl)cyclohexyl]piperidine labels two high-affinity binding sites in human cortex: further evidence for phencyclidine binding sites associated with the biogenic amine reuptake complex

Previous work demonstrated two high-affinity PCP binding sites in guinea pig brain labeled by [3H]TCP (1-(1-[2-thienyl]cyclohexyl)piperidine): site 1 (N-methyl-D-aspartate [NMDA]-associated) and site 2 (dopamine-reuptake complex associated). The present study examined brain membranes prepared from various species, including human, for the presence of site 2, defined as binding in the presence of (+)-5-methyl-10,11-dihydro-5H-dibenzo [a, d]cyclohepten-5,10-imine maleate ((+)-MK801) minus binding in the presence of 10 microM TCP (nonspecific binding). Studies were conducted in absence of sodium which was found to be inhibitory to [3H]TCP binding. The results demonstrated detectable levels of site 2 in brain membranes of guinea pig, rabbit, pig, mouse, sheep, and human but not in the rat or chicken. Using human cortical membranes, site 2 was the predominant binding site. Detailed studies conducted with human cortical tissue showed that high-affinity dopamine (1-[2- [bis(4-fluorophenyl)-methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909)], [1,2]benzo(b)thiophenylcyclo-hexylpiperidine (BTCP), and serotonin (fluoxetine) uptake inhibitors produced a wash-resistant inhibition of [3H]TCP binding to site 2, but not site 1. Preincubation of guinea pig brain membranes with BTCP was shown to produce an increase in the dissociation rate of [3H]TCP from PCP site 2. Structure activity studies with various uptake inhibitors showed that GBR12909, benztropine, fluoxetine, and BTCP have higher affinity for site 2 than for site 1. (+)-MK801, ketamine, and tiletamine were very selective for site 1, whereas dexoxadrol and TCP were moderately selective for site 1. These results suggest that human cortex possesses high-affinity PCP binding sites associated with biogenic reuptake binding sites, and that guinea pig brain, but not rat brain, may be an appropriate animal model for studying PCP site 2 in human brain.     

The binding of somatostatin to rat synaptosomal membranes: effects of detergent solubilisation and of bacitracin

Specific high and low affinity binding sites for somatostatin have been demonstrated in rat brain synaptosomal membranes using 125I [Tyr"] somatostatin tracer. When incubations were carried out in the presence of low concentrations of bacitracin (0.01% w/v), a 66% increase in association constant of the high affinity binding site to Ka = 1.71 X 10(10) M-1 and a 70% increase in the Ka of the low affinity site to 0.034 X 10(10) M-1 were observed; a similar (50%) increase in the binding capacity of the low affinity site but no increase in the binding capacity of the high affinity site was seen. These results may indicate a direct effect of bacitracin on the membrane that is distinct from its effect as an inhibitor of tracer degradation. The somatostatin-binding capacity of the membranes was solubilised by treatment with Triton X-100 and the apparent molecular weight of the receptor-ligand-detergent micelle complex was estimated by gel filtration to be approximately 250,000.     

Evidence that the delta-selective alkylating agent, fit, alters the mu-noncompetitive opiate delta binding site

Considerable evidence supports the notion that the prototypic delta agonist [3H]D-ala2-D-leu5-enkephalin labels two binding sites on brain membranes in vitro. Recent studies have demonstrated that treatment of brain membranes with the delta-selective, site-directed, alkylating agent, FIT (Rice et al., Science 220, 314-316, 1983) results in a membrane preparation devoid of detectable higher affinity [3H]D-ala2-D-leu5-enkephalin binding sites, but contain residual lower affinity binding sites at which mu-ligands are apparent noncompetitive inhibitors (Rothman et al., Neuropeptides 4:210-215, 1984). In this paper we extend these data by showing that although FIT eliminates the higher affinity binding site, it also alters the properties of the residual lower affinity binding sites.     

3H]GBR-12935 binding to dopamine uptake sites in the human brain

The binding of the selective dopamine uptake inhibitor [3H]GBR-12935 to post-mortem human brain membranes was studied. Competition experiments indicated the presence of multiple binding sites, but when a binding fraction that could be discriminated by either 0.3 microM mazindol or 1 mM dopamine was regarded as specific binding, a single-site binding model was obtained. The [3H]GBR-12935 binding was of protein nature since it was abolished after protease treatment and the binding appeared to label the dopamine uptake site. This was supported by the findings that dopamine uptake inhibitors inhibited the binding with high affinity (Ki 30-130 nM), whereas substances active at dopamine D1, D2 or autoreceptor sites revealed much lower affinities (Ki greater than 10 microM or inactive). Moreover, dopamine was the neurotransmitter with the highest affinity for the [3H]GBR-12935 binding site (Ki 30 microM). The dopaminergic nature of the [3H]GBR-12935 binding was further indicated by its regional distribution, which largely corresponds the known distribution of the dopamine system in the rat brain. The highest binding densities were obtained in the caudate nucleus and putamen (Bmax 1500-2000 fmol/mg protein), followed by the olfactory tubercle (Bmax 900 fmol/mg protein) and the substantia nigra (Bmax 300 fmol/mg protein). The apparent binding affinity (Kd) was the same in all brain regions (Kd 1-1.5 nM). Detectable specific [3H]GBR-12935 binding was obtained also in the globus pallidus, amygdala and cortices of orbital/rectus and cingulate gyri. Drug inhibition studies with the addition of low concentrations of dopamine and mazindol produced only alterations in the apparent Kd values, suggesting a competitive inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]desglycinyl remacemide to rat brain membranes: association with the benzomorphan attachment site of the N-methyl-D-aspartic acid receptor channel.

Desglycinyl remacemide (DGR), a biologically active metabolite of remacemide, was radiolabeled in an attempt to develop a ligand binding assay to identify its site of action. Incubation of the radioligand with membranes obtained from P2 fractions of whole rat brain revealed a single population of specific [3H]-DGR binding sites having a Kd of 290 nM and a Bmax of 1.3 pmole/mg protein. The specific binding of [3H]-DGR is most enriched in the P2 subcellular fraction and is heterogeneously distributed throughout the brain. The binding of [3H]-DGR to rat brain membranes was inhibited most potently by MK-801 and SKF-10,047. In contrast, haloperidol, and other sigma receptor-active agents, were relatively inactive at this site. These data suggest that DGR interacts with a channel blocking site on the NMDA receptor.     

Further pharmacological characterization of [3H]idazoxan binding sites in rat brain: evidence for predominant labeling of alpha 2-adrenergic receptors.

In addition to binding to alpha 2-adrenergic receptors, the antagonist [3H]idazoxan has been reported to bind to non-adrenergic sites in a number of tissues and species. In the present study, the pharmacological nature of [3H]idazoxan binding sites in rat brain slices has been examined using radioligand binding and autoradiographic techniques. In Na2KHPO4 buffer, four drugs with high affinity for alpha 2-adrenergic binding sites were potent inhibitors of [3H]idazoxan binding, with the rank order of potency being RX821002 greater than phentolamine greater than yohimbine greater than (-)epinephrine. Non-linear regression analysis resolved all competition curves into two components, with the high affinity site representing the majority of total [3H]idazoxan binding. In autoradiographic studies performed in Na2KHPO4 buffer, all alpha 2-selective ligands displaced greater than or equal to 75% of total [3H]idazoxan binding to most brain regions. These findings indicate that the major component of [3H]idazoxan binding was to sites that are alpha 2-adrenergic in nature. [3H]Idazoxan binding was also examined in glycylglycine buffer. In contrast to binding in Na2KHPO4 buffer, the proportion of low affinity sites was significantly increased in glycylglycine buffer. Autoradiographic studies confirmed these findings. These pharmacological data are consistent with our previously reported conclusions that, under appropriate assay conditions, [3H]idazoxan predominantly labels alpha 2-adrenergic binding sites in rat brain. These sites are widely distributed and have pharmacological characteristics consistent with those previously reported for alpha 2A-adrenergic receptors.     

Pharmacological characterization of solubilized 5-HT1 serotonin binding sites from bovine brain

This report describes the pharmacologic characterization of [3H]serotonin binding activity solubilized from bovine frontal cortical membranes. The ability of a number of serotonin (5-HT) and lysergic acid diethylamide (LSD) analogs to compete with [3H]serotonin and D-[3H]LSD for binding to membrane and solubilized 5-HT1 sites has been investigated. The results indicate that the solubilized binding site is probably of the 5-HT1B type. Fifteen of the 21 compounds tested exhibit nearly identical affinity for membrane or solubilized 5-HT1 binding sites. However, some important differences were observed, and these may help elucidate the molecular structure of the binding site. In particular, some N-substituted tryptamine analogs show a markedly lower affinity for solubilized 5-HT1 sites compared to their binding to intact membranes. Further, the solubilized site does not distinguish stereoisomers of LSD: both D- and L-LSD bind to solubilized 5-HT1 sites with comparable high affinities, whereas D-LSD has a markedly higher affinity for the membrane 5-HT1 site. Methiothepin, which binds to the 5-HT1 site primarily through its amine groups, has virtually no affinity for the solubilized receptor, whereas it is quite potent at competing for [3H]serotonin binding to membrane sites. These observations lead to the conclusions that in bovine cortical membranes, the 5-HT1 site contains both indole and amine attachment sites. After solubilization, the indole attachment site retains its binding properties, but the amine attachment site has been significantly altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity [3H]paroxetine binding to serotonin uptake sites in human brain tissue

[3H]Paroxetine binding to human brain tissue was characterized. Competition studies in the putamen and frontal cortex revealed single-site binding models for binding sensitive to 5-hydroxytryptamine (5-HT) (Ki 1-3 microM) and citalopram (Ki 0.6 nM), which displaced the same amount of binding. However, desipramine, norzimeldine and fluoxetine displaced additional binding (10-20%) and these competitors fitted two-site binding models with high affinity components in the nanomolar range and low affinity components in the micromolar range. The high affinity components approximated the 5-HT- and citalopram-sensitive binding fraction. Most of the [3H]paroxetine binding sites were protease-sensitive, but the low-affinity (microM) sites appeared to be protease-resistant. Based on these findings, only the [3H]paroxetine binding representing the fraction sensitive to 30 microM 5-HT (or e.g. 0.3 microM norzimeldine), was regarded as specific binding. This binding fraction was saturable with an apparent binding affinity (Kd) of 0.03-0.05 nM throughout the brain. The highest binding densities were obtained in the hypothalamus and substantia nigra (Bmax 500 fmol/mg protein). The basal ganglia reached intermediate densities (Bmax 200 fmol/mg protein), whereas cortical areas had low Bmax values (less than 100 fmol/mg protein). The lowest B max value was noted in cerebellar cortex (30 fmol/mg protein). The [3H]paroxetine binding was competitively inhibited by low concentrations of 5-HT, imipramine and norzimeldine, suggesting that the substrate recognition site for 5-HT uptake was labeled. Compounds active at dopaminergic, noradrenergic, histaminergic, 5-HT1, 5-HT2 and cholinergic muscarinic sites did not affect the binding at 100 microM concentrations. It is concluded that [3H]paroxetine is a marker for the 5-HT uptake site in the human brain, provided that an adequate pharmacological definition of specific binding is performed.     

Developmental Pb2+ exposure alters NMDAR subtypes and reduces CREB phosphorylation in the rat brain

In the present study we show that chronic exposure to low levels of lead (Pb(2+)) during development alters the type of N-methyl-D-aspartate receptor (NMDAR) expressed in the developing and young adult rat brain. Ifenprodil inhibition of [3H]MK-801 binding to the NMDAR channel in cortical and hippocampal neuronal membranes expressed high and low affinity components. Previous studies have shown that the high affinity component is associated with NR1/NR2B receptor complexes while the low affinity component is associated with the appearance and insertion of the NR2A subunit to NMDAR complexes. Pb(2+)-exposed rats express a greater number of [3H]MK-801 binding sites associated with the high affinity and low affinity components of ifenprodil inhibition. Further, [3H]ifenprodil saturation isotherms and Scatchard analysis in cortical and hippocampal membranes showed a higher number of binding sites (B(max)) with no change in binding affinity (K(d)) in Pb(2+)-exposed animals relative to controls. Quantitative [3H]MK-801 autoradiography in response to glutamate and glycine provided evidence that NMDAR complexes in Pb(2+)-exposed rat brain were maximally activated in situ. Higher levels of ifenprodil-sensitive binding sites and increased sensitivity to agonists are properties characteristic of NR1/NR2B recombinant receptors. Thus, our results strongly suggest that a greater proportion of the total number of NMDAR are NR1/NR2B receptors in the Pb(2+)-exposed rat brain. This Pb(2+)-induced change in NMDAR subtypes in the rat brain was associated with reduced CREB phosphorylation in cortical and hippocampal nuclear extracts. These findings demonstrate that chronic exposure to environmentally relevant levels of Pb(2+) altered the subunit composition of NMDAR complexes with subsequent effects on calcium-sensitive signaling pathways involved in CREB phosphorylation.     

The development of alpha 2-adrenoceptors in the rat kidney: correlation with noradrenergic innervation

The development of the renal alpha 2-adrenoceptors in the rat was studied by binding renal membranes of prenatal and postnatal rats with p-[3,5-3H]aminoclonidine (PAC), a selective alpha 2-adrenoceptor agonist. The results demonstrate that prior to birth and on the day of birth [3H]PAC binding is present and reflects a single linear binding affinity (Kd approximately equal to 1.0 nM). In contrast, data from postnatal day 3 demonstrate the addition of a second, lower affinity binding site (Kd approximately equal to 7.0 nM) that develops rapidly during the ensuing weeks of life. Both high and low affinity alpha 2-adrenoceptor binding sites continue to increase in receptor density through 30 weeks of age; however, the binding affinity of each site remains relatively stable. The sympathetic noradrenergic innervation of the rat kidney is present prenatally and rapidly develops during the first 3 weeks of life with peak noradrenaline concentrations being reached at approximately prenatally and rapidly develops during the first 3 weeks of life with peak noradrenaline concentrations being reached at approximately postnatal day 21. Immunohistochemical staining demonstrates that tyrosine hydroxylase-positive axons rapidly develop in the kidney during the same period, indicating that the increase in noradrenaline is related to expansion of renal sympathetic innervation.     

Characterization of [3H] CCK4 binding sites in mouse and rat brain

We have investigated the possible occurrence of distinct CCK8 and CCK4 binding sites in the brain by comparing the binding characteristics of [3H] CCK4 to those of the CCK8 analogue, [3H] Boc (Nle28,31]CCK27-33 (BDNL-CCK7). [3H] CCK4 and [3H] BNDL-CCK7 were shown to interact with mouse brain membranes with very similar maximal binding capacities 31.7 +/- 2.1 fmol/mg prot (KD = 3.78 +/- 0.47 nM) and 38.9 +/- 2.2 fmol/mg prot (KD = 0.26 +/- 0.02 nM) respectively. The apparent affinities of five CCK analogues for the sites labelled by both probes were almost identical. Autoradiographic studies revealed that the distribution of [3H] CCK4 binding sites in rat forebrain was the same as that of [3H] BDNL-CCK7, with high densities of receptors in the cortex, nucleus accumbens, olfactory bulb and the medial striatum, moderate densities in the amygdala, the hippocampus, several nuclei of the thalamus and hypothalamus. However in the interpenduncular nucleus where there was moderate binding of [3H]BDNL-CCK7, no [3H]CCK4 labelling was observed. These studies demonstrated the occurrence of one class of high affinity binding sites for [3H] CCK4 in mouse and rat brain, with characteristics similar to those already reported with CCK33, CCK8 and pentagastrin probes. Nevertheless the presence of a small amount of very high affinity binding sites for [3H]CCK4 cannot be excluded.     

The binding properties and regional ontogeny for [3H]glutamic acid Na+-independent and [3H]kainic acid binding sites in chick brain

The binding kinetics, pharmacological properties and regional ontogeny of L-[3H]glutamic acid Na+-independent and [3H]kainic acid binding sites were studied in preparations of chick brain. One binding component was found for L-[3H]glutamic acid with a Kd value of 176 x 10(9) M. For [3H]kainic acid two binding components were found in the hemispheres, optic lobes and brain stem, one with high affinity and a Kd value of 12.5 x 10(9) M and one with low affinity and a Kd value of 260 x 10(9) M. In cerebellum only one binding site was detected for [3H]kainic acid with a Kd value of 144 x 10(9) M. The ontogeny of L-[3H]glutamic acid and [3H]kainic acid binding sites was studied using membrane preparations (48,000 g pellet) of hemispheres, optic lobes, brain stem and cerebellum. Binding of L-[3H]glutamic acid was already significant in all brain regions by embryonic day 11 but major increases in total receptor number per brain region or per mg of protein were apparent by embryonic day 19 and especially after hatching. Cerebral hemispheres, optic lobes and brain stem showed few [3H]kainic acid binding sites by day 13 in ovo. An increase follows which, in hemispheres and optic lobes, continues at the same rate during the first two weeks after hatching. In cerebellum, by contrast, the kainic acid binding site is almost undetectable until embryonic day 15. The appearance of these binding sites in cerebellum takes place during the restricted period between days 15 in ovo and 5 post-hatching. This pattern of development of [3H]kainic acid binding sites almost parallels the developmental patterns of the molecular layer of chick cerebellum and it is consistent with the results of our autoradiographic study showing that the great majority of kainic acid binding sites are localized in the molecular layer.     

Characterization of adenosine receptors in brain using N cyclohexyl [H]adenosine

The presence of distinct binding sites for adenosine in both the CNS and PNS has been proposed in numerous studies. The recent availability of stable adenosine analogues such as cyclohexyladenosine, 2-chloroadenosine and diethylphenylxanthine has made the characterization of such a receptor feasible. In the present report the binding of N⁶ cyclohexyl [³H]adenosine ([³H]CHA) to rat brain synaptosomal membranes is characterized. [³H]CHA binding is saturable and exhibits a biphasic kinetic saturation profile characteristic of 2 binding sites. The high affinity site has a K_d of 0.7 nM and the low affinity site 2.4 nM. The respective B_max values are 230 and 120 fmol/mg protein in rat forebrain. The highest density of binding sites is found in the hippocampus and subcellular distribution studies indicate that the [³H]CHA site is predominantly synaptosomal. [³H]CHA binding is highly dependent on the presence of adenosine deaminase since only 30% of the binding capacity is observed in synaptosomal membranes not treated with this enzyme. Of the many cations and anions tested only copper and zinc have effects on [³H]CHA binding. Both metals are potent inhibitors of binding with copper having an IC₅₀ of 30 μM and zinc 150 μM. Sulfhydryl reducing and alkylating agents also inhibit binding indicating that the binding site is a sulfhydryl-dependent protein.     

Heterogeneity of [H]neurotensin binding: studies with dynorphin, L-156,903 and levocabastine

The binding of [3H]neurotensin (NT) to membranes from rat forebrain was complex, exhibiting 'high' affinity (Kd approximately 0.5 nM) and 'low' affinity (Kd approximately 5.0 nM) binding components. Dynorphin A(1-13) (DYN A(1-13] and L-156,903 (N-oxo-3-(10H-phenothiazine-10-yl)propyl-1- arginyl-1-prolyl-1-phenylalanine) potently inhibited [3H]NT binding to brain with shallow biphasic competition curves. Saturation binding studies conducted in the presence or absence of DYN A(1-13) or L-156,903 indicated that these compounds, like levocabastine, exhibited substantial selectivity for 'low' affinity NT site. Structure-activity studies indicated rigid structural requirements for the NT binding activity of DYN A(1-13) and L-156,903. In contrast to the results using brain tissue, DYN A(1-13), L-156,903 and levocabastine were very weak or inactive to inhibit [3H]NT binding to rat uterus. These studies further characterize the heterogeneity of [3H]NT binding in vitro and demonstrate clear tissue differences in binding within a given species.     

The complex binding of tricyclic antidepressants to rat brain: The case of nortriptyline

[3H]Nortriptyline (NT) binding to rat brain sections was characterized using saturation and competition experiments, quantitative autoradiographic localization and monoaminergic lesions. [3H]NT binding exhibits a saturable component in the nM range (high affinity binding). Final saturation is reached only in micromolar concentrations (low affinity binding). The high affinity binding sites of [3H]NT appear in areas rich in noradrenergic and serotonergic terminals, and are decreased by 6-OHDA and 5,7-DHT lesions, to an extent depending on the brain region and the relative density of noradrenergic and serotonergic terminals there. We conclude that [3H]NT binds with high affinity to both noradrenergic and serotonergic nerve terminals, and with low affinity to other yet uncharacterized entities. This complex binding pattern is compatible with the multiplicity of biochemical, physiological and behavioral effects of NT and tricyclic antidepressants in general.     

Temperature-dependent and -independent apparent binding activities of [3H]glutathione in brain synaptic membranes

An apparent binding activity of [3H]glutathione was examined by using synaptic membrane preparations of the rat brain. The activity was found to be more than two times as high at 30 degrees C as that found at 2 degrees C. At 2 degrees C, the apparent binding sites consisted of a single component with a Kd of 0.77 microM and a Bmax of 5.60 pmol/mg protein. In contrast, two independent separate sites with Kds of 0.56 and 12.6 microM and Bmaxs of 2.50 and 28.5 pmol/mg protein were observed at 30 degrees C. In vitro addition of Triton X-100 significantly inhibited the apparent binding activities detected at both temperatures, whereas pretreatment of the membranes with the detergent did not significantly affect both binding activities. Among 3 constituent amino acids of glutathione, L-cysteine induced a selective and irreversible potentiation of the apparent activities, which occurred independently of the incubation temperature. Scatchard analysis revealed that L-cysteine drastically increased the number of the low affinity sites without significantly altering their affinity. Apparent binding activities determined at both incubation temperatures were unevenly distributed in the central and peripheral structures. Distribution profile of the temperature-dependent activities was found to be closely related to that of the basal binding activity of [3H]L-glutamic acid, a putative central excitatory neurotransmitter. These results suggest that brain synaptic membranes may indeed contain specific binding sites of [3H]glutathione which have an interaction with the glutamate binding sites. Possible presence of two distinctly different apparent binding sites of [3H]glutathione, such as temperature-independent high affinity sites and temperature-dependent low affinity sites, is also suggested.