Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH-SY5Y

Abstract: We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K⁺]_o (100 m M ) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [³H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K⁺-evoked release was unaffected by hypoxia (P o ₂≅ 30–38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca²⁺_o with 1 m M EGTA abolished NA release. K⁺-evoked release was also dramatically reduced in the presence of 200 µ M Cd²⁺ to block voltage-gated Ca²⁺ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd²⁺-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca²⁺ influx through the nAChR pore, or by activating an unidentified Cd²⁺-resistant Ca²⁺-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K _i values for binding inhibition and EC₅₀ values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.     

Potassium- and Carbachol-Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH-SY5Y

The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki = 0.33 nM), hexahydrosiladifenidol (Ki = 18 nM), and pirenzepine (Ki = 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists methoctramine (10(-4) M) and AFDX-116 (10(-4) M), by approximately 14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10(-4) M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Mobilization of Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Stores Supports Bradykinin- and Muscarinic-Evoked Release of [3H]Noradrenaline from SH-SY5Y Cells

Abstract: The human neuroblastoma cell line SH-SY5Y, maintained at confluence for 14 days, released [³H]-noradrenaline ([³H]NA) when stimulated with either the muscarinic receptor agonist methacholine or bradykinin. The major fraction of release was rapid, occurring in <10 s, whereas nicotine-evoked release was slower. When the extracellular [Ca²⁺] ([Ca²⁺]_e) was buffered to ～50–100 nM, release evoked by nicotine was abolished, whereas that in response to methacholine or bradykinin was reduced by ～50% with EC₅₀ values of ?5.46 ± 0.05 M and ?7.46 ± 0.06 M (log₁₀), respectively. Methacholine and bradykinin also produced rapid elevations of both inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular free [Ca²⁺] ([Ca²⁺]_i). These elevations were reduced at low [Ca²⁺]_e and under these conditions the EC₅₀ values for peak elevation of [Ca²⁺]_i were ?6.00 ± 0.14 M for methacholine and ?7.95 ± 0.34 M for bradykinin (n = 3 for all EC₅₀ determinations). At low [Ca²⁺]_e, depletion of nonmitochondrial intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin produced a transient small elevation of [Ca²⁺]_i and a minor release of [³H]NA. At low [Ca²⁺]_e, thapsigargin abolished elevation of [Ca²⁺]_i in response to methacholine and bradykinin and completely inhibited their stimulation of [³H]NA release. It is proposed, therefore, that Ca²⁺ release from Ins(1,4,5)P₃-sensitive stores is a major trigger of methacholine- and bradykinin-evoked [³H]NA release in SH-SY5Y cells.     

Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH-SY5Y

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [³H]noradrenaline ([³H]NA) from the human neuroblastoma clone SH-SY5Y with IC₅₀ values of 10, 15, and 30 μ M , respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μ M ). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [³H]NA.     

Biochemical Characterization of the Nicotinic Cholinergic Receptors in Human Brain: Binding of (—)-[3H]Nicotine

(-)-[3H]Nicotine was found to bind specifically to membranes of human brains obtained at autopsy. The binding was stereospecific, (-)-nicotine being 40 times more potent than (+)-nicotine in displacing labeled (-)-nicotine. Saturation binding studies revealed the presence of two binding sites with dissociation constant (KD) values of 8.1 and 86 nM, and maximum binding capacity (Bmax) values of 36 and 90 fmol/mg protein, respectively. In competition studies, nicotinic agonists were 1,000 times more potent than ganglionic, neuromuscular, and muscarinic blocking drugs in displacing labeled (-)-nicotine. IC50 values for cholinergic drugs of (-)-[3H]nicotine binding were as follows: (-)-nicotine, 0.51 nM; acetylcholine, 12.6 nM; (+)-nicotine, 19.9 nM; cytisine, 27.3 nM; and carbachol, 527 nM. IC50 values of alpha-bungarotoxin, hexamethonium, d-tubocurarine, and atropine were larger than 50 microM. (-)-[3H]Nicotine binding was highest in the nucleus basalis of Meynert and thalamus and lowest in the cerebral cortex and caudate in the brain regions tested. These results suggest that nicotinic cholinergic receptors are present in human brain and that there are regional differences in the density of these receptors.     

Presynaptic Nicotinic Cholinergic Receptors Labeled by [3H]Acetylcholine on Catecholamine and Serotonin Axons in Brain

Nicotinic cholinergic receptor binding sites labeled by [3H]acetylcholine were measured in the cerebral cortices, thalami, striata, and hypothalami of rats lesioned by intraventricular injection of either 6-hydroxydopamine or 5, 7-dihydroxytryptamine. In addition, [3H]acetylcholine binding sites were measured in the cerebral cortices of rats lesioned by injection of ibotenic acid into the nucleus basalis magnocellularis. [3H]Acetylcholine binding was significantly decreased in the striata and hypothalami of both 6-hydroxydopamine- and 5,7-dihydroxytryptamine-lesioned rats. There was no change in binding in the cortex or thalamus by either lesion. Ibotenic acid lesions of the nucleus basalis magnocellularis, which projects cholinergic axons to the cortex, did not alter [3H]acetylcholine binding. These results provide evidence for a presynaptic location of nicotinic cholinergic binding sites on catecholamine and serotonin axons in the striatum and hypothalamus.     

Ontogeny of Receptor Binding Sites for [3H]Glutamic Acid and [3H]Kainic Acid in the Rat Cerebellum

The development of the specific binding sites for L-[3H]glutamic acid (KD = 370 nM) and for [3H]kainic acid (KD = 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.     

Relationship Between [3H]Mazindol Binding to Dopamine Uptake Sites and [3H]Dopamine Uptake in Rat Striatum During Aging

Certain drugs exhibit a remarkable correlation between their ability to inhibit synaptosomal uptake of dopamine and the binding of [3H]mazindol to striatal membranes. To investigate the role of mazindol binding sites in the dopamine uptake process and the fate of these sites (labeling dopaminergic neurons) during aging, we have examined the properties of mazindol binding and dopamine uptake in individual young and old rats. There was a 48% decrease (p = 0.0001) in the Bmax of mazindol binding and a 23% decrease (p = 0.0166) in the Vmax of dopamine uptake with no apparent change in their affinities with age. Regression analysis of the relationship between Bmax and Vmax exhibited a significant correlation in old (p = 0.0156) but not young rats (p = 0.1398). These data suggest that the number of mazindol binding sites decreases with age and that the number of sites on the dopamine transporter complex far exceeds the number required to elicit maximal dopamine uptake.     

The Intact Human Neuroblastoma Cell (SH-SY5Y) Exhibits High-Affinity [3H]Pirenzepine Binding Associated with Hydrolysis of Phosphatidylinositols

The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of [3H](-)-quinuclidinyl benzilate and its quaternary derivative [3H](-)-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.     

Comparison of the In Vitro Receptor Binding Properties of N-[3H]Methylspiperone and [3H]Raclopride to Rat and Human Brain Membranes

The aim of the present investigation was to study and compare the in vitro binding properties of the two radioligands N-[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug-naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5-hydroxytryptamine 2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10-fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half-life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The Ki values were, however, two- to four-fold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.     

Comparison of the Binding of [3H]Spiperone and [3H]Domperidone in Homogenates of Mammalian Retina and Caudate Nucleus

Abstract— The specific binding of [³H]spiperone and [³H]domperidone, as defined by 1 μ m -(+)butaclamol, was compared in homogenates of bovine retina and caudate nucleus. Scatchard analyses of saturation data for [³H]spiperone binding yielded dissociation constants ( K _d) of 0.35 n m in the retina and 0.64 n m in the caudate nucleus. Comparison of the maximum number of binding sites (B_max) present in each tissue indicated that the density of sites in bovine caudate nucleus (270 fmol/mg protein) was approximately three times higher than in bovine retina (92 fmol/mg protein). This difference was even more marked in guinea pig tissues, with a ratio of 7:1 between corpus striatum and retina. The pharmacological analysis of [³H]spiperone binding in both the bovine retina and caudate nucleus indicated an interaction with dopaminergic rather than serotonergic sites. However, inhibition curves obtained to dopaminergic agonists in the bovine retina were significantly steeper than those observed in the bovine caudate nucleus, as reflected in the greater Hill coefficients obtained for these agents in the retina. Furthermore, only a small amount of specific [³H]domperidone binding was observed in either the bovine caudate nucleus or the guinea pig striatum, whilst no specific [³H]domperidone binding was detectable in homogenates of either bovine or guinea pig retina. These data suggest that the retina possesses only a small population of dopaminergic D2 sites and that these binding sites may differ from those present in the caudate nucleus.     

Formation of [3H]Inositol Metabolites in Rat Hippocampal Formation Slices Prelabelled with [3H]Inositol and Stimulated with Carbachol

Rat hippocampal formation slices were prelabelled with [3H]inositol and stimulated with carbachol for times between 7 s and 3 min. The [3H]inositol metabolites in an acid extract of the slices were resolved with anion-exchange HPLC. Carbachol dramatically increased the concentration of [3H]inositol monophosphate, [3H]inositol bisphosphate (two isomers), [3H]inositol 1,3,4-trisphosphate, [3H]inositol 1,4,5-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate. The levels of [3H]inositol 1,4,5-trisphosphate rose most rapidly; they were maximally elevated after only 7 s and declined toward control levels in 1 min followed by a more sustained elevation in levels for up to 3 min. When [3H]inositol 1,4,5-trisphosphate was incubated with hippocampal formation homogenates in an ATP-containing buffer it was very rapidly metabolised. After 5 min [3H]inositol 1,4-bisphosphate, [3H]inositol 1,3,4-trisphosphate, and [3H]inositol 1,3,4,5-tetrakisphosphate could be detected in the homogenates. Under similar experimental conditions [3H]inositol 1,3,4,5-tetrakisphosphate is metabolised to [3H]inositol 1,3,4-trisphosphate and an inositol bisphosphate isomer that is not [3H]inositol 1,4-bisphosphate. We conclude that like other tissues the primary event in the hippocampus following carbachol stimulation is the activation of phosphatidylinositol 4,5-bisphosphate selective phospholipase C.     

[3H]N-[1-(2-Thienyl)cyclohexyl]-3,4-Piperidine ([3H]TCP) Binding in Human Frontal Cortex: Decreases in Alzheimer-Type Dementia

We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.     

Comparison of Tryptic Peptides of Benzodiazepine Binding Proteins Photolabeled with [3H]Flunitrazepam or [3H]Ro 15–4513

When rat brain membranes were incubated with the benzodiazepine agonist [3H]flunitrazepam or the partial inverse benzodiazepine agonist [3H]Ro 15-4513 in the presence of ultraviolet light one protein (P51) was specifically and irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. After digestion of the membranes with trypsin, protein P51 was degraded into several peptides. When P51 was photolabeled with [3H]Ro 15-4513, four peptides with apparent molecular weights of 39,000, 29,000, 21,000, and 17,000 were observed. When P51 was labeled with [3H]flunitrazepam, only two peptides with apparent molecular weights of 39,000 and 25,000 were obtained. Protein P55 was only partially degraded by trypsin, and whether it was labeled with [3H]flunitrazepam or [3H]Ro 15-4513 it yielded the same two proteolytic peptides with apparent molecular weights of 42,000 and 45,000. These results support the existence of at least two different benzodiazepine receptor subtypes associated with proteins P51 and P55. The different receptors seem to be differentially protected against treatment with trypsin. In addition, these results indicate that in the benzodiazepine receptor subtype associated with P51 benzodiazepine agonists and partial inverse benzodiazepine agonists irreversibly bind to different parts of the molecule.     

In Vivo Regulation of [3H]Acetylcholine Recognition Sites in Brain by Nicotinic Cholinergic Drugs

The in vivo regulation of [3H]acetylcholine [( 3H]ACh) recognition sites on nicotinic receptors in rat brain was examined by administering drugs that increase stimulation of nicotinic cholinergic receptors, either directly or indirectly. After 10 days of treatment with the cholinesterase inhibitor diisopropyl fluorophosphate, [3H]ACh binding in the cortex, thalamus, striatum, and hypothalamus was decreased. Scatchard analyses indicated that the decrease in binding in the cortex was due to a reduction in the apparent density of [3H]ACh recognition sites. In contrast, after repeated administration of nicotine (5-21 days), the number of [3H]ACh recognition sites was increased in the cortex, thalamus, striatum, and hypothalamus. Similar effects were observed in the cortex and thalamus following repeated administration of the nicotinic agonist cytisin. The nicotinic antagonists mecamylamine and dihydro-beta-erythroidine did not alter [3H]ACh binding following 10-14 days of administration. Further, concurrent treatment with these antagonists and nicotine did not prevent the nicotine-induced increase in these binding sites. The data indicate that [3H]ACh recognition sites on nicotinic receptors are subject to up- and down-regulation, and that repeated administration of nicotine results in a signal for up-regulation, probably through protracted desensitization at the recognition site.     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.     

Inhibition of [3H]Spiroperidol Binding by In Vitro Addition of Ethanol

: The in vitro addition of ethanol at concentrations greater than 0.2% produced an inhibition of [³H]spiroperidol binding. This inhibition was competitive, lowering the affinity without altering the maximum number of sites. Both the rates of association and dissociation were altered such that the ligand spent less time attached to the receptor. Since the binding was competitive, little inhibition was seen at ligand concentrations greater than 300 pm . These results are important for in vitro studies when drugs are dissolved in ethanol, and also for in vivo studies on the effects of ethanol.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)