Complex Inhibition of [3H]Imipramine Binding by Serotonin and Nontricyclic Serotonin Uptake Blockers

Tricyclic antidepressant drugs inhibit [3H]imipramine binding to the rat brain cortex in a competitive manner, giving linear Hofstee plots and Hill coefficients of approximately 1.0. Serotonin, the only neurotransmitter to inhibit [3H]imipramine binding, does so in a complex manner, exhibiting a Hill coefficient of 0.40-0.50. Nontricyclic inhibitors of serotonin uptake such as fluoxetine, paroxetine, norzimelidine, and citalopram inhibit [3H]imipramine binding in the same complex manner as serotonin. These results are interpreted as suggesting that [3H]imipramine binds to a site associated with the serotonin uptake system but different from either the substrate recognition site for serotonin or the site of action of the nontricyclic inhibitors of neuronal uptake of serotonin.     

Distinct Thermodynamic Parameters of Serotonin 5-HT3 Agonists and Antagonists to Displace [3H]Granisetron Binding

Abstract: Specific binding of [³H]granisetron was examined to serotonin 5-HT₃ receptors in synaptosomal membranes of rat cerebral cortex between 1 and 37°C. Displacing potencies were determined for 5-HT₃ antagonists (granisetron, ondansetron, tropisetron, and d -tubocurarine) and agonists (5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, phenylbiguanide, m -chlorophenylbiguanide, and SR 57227A). Displacing potencies of the agonists decreased with decreasing temperature. In contrast, displacing potencies of all antagonists increased with decreasing temperature, whereas those of tropisetron and d -tubocurarine passed a maximum. Scatchard analysis of [³H]granisetron binding resulted in K _D values lower than the IC₅₀ values of granisetron and a decreasing number of binding sites at higher temperatures. It can be reconciled with temperature-dependent agonist and antagonist states of 5-HT₃ receptors. A semiquantitative thermodynamic analysis was based on displacing potencies. The distinct patterns for the signs of entropy, enthalpy, and heat capacity changes on binding can be reconciled with ionic interactions for agonists and hydrophobic interactions for antagonists. The distinctive differences in these thermodynamic parameters exceed those for GABA_A and glycine receptor-ionophore complexes.     

Characterization of Serotonin Transporter in Goldfish Retina by the Binding of [3H]Paroxetine and the Uptake of [3H]Serotonin: Modulation by Light

Abstract: The serotonin (5-HT) uptake system of goldfish retina was evaluated by the binding of [³H]paroxetine to membrane preparations and the uptake of [³H]5-HT into isolated cells from goldfish retina. The order of potency of inhibitors of [³H]paroxetine binding was imipramine > 5-methoxy- N,N -dimethyltryptamine > desipramine > fluoxetine > citalopram > 5-HT. The saturation experiments indicated a high-affinity binding site, and positive cooperativity with Hill coefficient higher than unity. The association reached equilibrium at about one hour of incubation and was efficiently displaced by imipramine. The equilibrium dissociation constants calculated by the antilog of the log concentration of ligand giving 50% of occupation, and by the ratio of dissociation/association constants, were similar: 5.84 and 2.34 n M , respectively. The binding was not significantly reduced by decreasing the temperature of incubation and was sodium dependent. The lesion with 5,7-dihydroxytryptamine reduced the binding to 60%. The uptake of [³H]5-HT into isolated cells also showed positive cooperativity. The order of potency of inhibitors was similar to the one obtained for the binding of [³H]paroxetine. Darkness increased the uptake of 5-HT. The allosteric regulation of the 5-HT transporter and the modulation by light could be related to the physiological role of the monoamine, as a neurotransmitter and as a precursor of melatonin synthesis in the retina.     

Low [3H]Cytochalasin B Binding in the Cerebral Cortex of Newborn Rat

The concentrations of glucose transporter in the cerebral cortex and brainstem of neonatal (4–7 days old) and adult rats were measured using [³H]cytochalasin B binding. There was significantly lower binding in neonatal cortex (1.9 ± 0.7 pmol/mg protein) compared to adult (8.9 ± 2.5 pmol/mg protein). Scatchard analysis indicates this difference is due to a lower B_max (neonate, 9.7 pmol/mg protein; adult, 18.6 ± 1.3 pmol/mg protein). Measurement of [³H]cytochalasin B binding in microvessels prepared from cortex of adult (28.1 ± 3.5 pmol/mg protein) and neonate (12.8 ± 1.9 pmol/mg protein) indicates a lower binding in the microvasculature of neonates, whereas no such difference was seen in the binding in microvessels prepared from adult and neonatal brainstem (adult, 11.8 ± 2.3 pmol/mg protein; neonate, 9.4 ± 2.7 pmol/mg protein). In both adult and neonate brain, there is an enrichment of glucose transporters in the microvasculature.     

Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of (R)-[3H] Tomoxetine and (R/S)-[3H] Nisoxetine Binding in Rat Brain

Abstract: ( R )-[³H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[³H]tomoxetine with those of ( R/S )-[³H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[³H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[³H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[³H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[³H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [³H]citalopram. Therefore, ( R )-[³H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]5-Hydroxytryptamine Binding Sites: Species and Tissue Variation

Abstract: The characteristics of spiperone inhibition of [³H]5-hydroxytryptamine ([³H]5-HT; [³H]serotonin) binding were examined in dorsal (DH) and ventral (VH) hippocampus, corpus striatum (CS) or caudate nucleus (CN), and frontal cortex (FC) in the rabbit, guinea pig, and cat. Some of the properties of spiperone inhibition of [³H]5-HT binding in these species were similar to the properties previously found in the rat. Spiperone was significantly more potent in DH, VH, and FC than in CS or CN. It produced shallow or biphasic inhibition curves, resulting in Hill slopes of less than 1.0. Nonlinear regression analysis of the data showed that the inhibition curves fit a two-site binding model significantly better than a one-site model in each brain region. The dissociation constants of spiperone for the high-affinity binding site ( K _H) for all the tissues and species, except cat FC and rabbit DH, were very close to those previously found in the rat (2-13 n M ). However, the dissociation constants for the low-affinity binding site ( K _L) were different from those in the rat in all species and tissues examined, except cat FC and CS. The present data are consistent with the concept of multiple 5-HT₁ binding sites and suggest the presence of at least two, and perhaps as many as three, groups of sites in the mammalian brain.     

Binding of [3H]Imipramine to Mouse Cerebrocortical Membranes and to Glass Fiber Filters

It was suggested in a recent report by Phillips et al. [J. Neurochem. 43, 479-486 (1984)] that the low-affinity binding of [3H]imipramine in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 microM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low-affinity component, in addition to the high-affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data). (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.     

[3H]Metergoline: A New Ligand of Serotonin Receptors in the Rat Brain

Abstract: A specific binding site for [³H]metergoline characterized by a K_D of 0.5–1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7-dihydroxy-tryptamine- and kainic acid-induced lesions indicated that this specific binding site was localized post-synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [³H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and norad-renaline for displacing bound [³H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [³H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [³H]metergoline and [³H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7-dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [³H]metergoline might well be the appropriate ligand for studying the characteristics of the 'antagonist form' of serotonin receptors postulated by Bennett and Snyder.     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Brain astroglial cells, whether from a bulk isolated preparation or in culture, have been shown to take up serotonin actively. [3H]imipramine has been proposed as a specific label for serotonin uptake sites in brain. We therefore studied the binding of [3H]imipramine to C6 astroglial cells in culture to determine if some of the binding of this radioligand in brain homogenates is actually to serotonin transporting sites on glia. [3H]Imipramine binds saturably (Bmax = 202 fmol/mg protein) and with high affinity (KD = 1.72 nM) to C6 cells. This binding is competitively inhibited by other tricyclic antidepressants. The C6 cells actively transport [3H]serotonin with a Km of 2 microM and a Vmax of 1080 fmol/10(6) cells/min. However, the pharmacological profile for inhibition of serotonin uptake does not correlate with the pharmacological profile for inhibition of [3H]imipramine binding. These results suggest that the binding of [3H]imipramine to astroglial cells is not related to their capacity for active uptake of serotonin. Further, in brain homogenates, some of the binding of [3H]imipramine may not be to neuronal uptake sites but rather may be to sites on astroglial cells.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

A Thermodynamic Study of 5-[3H]Hydroxytryptamine Binding to Human Cortex Membranes

Kinetic and equilibrium measurements of [3H]-serotonin (5-hydroxytryptamine) binding to human frontal cortex membranes have been made between 4 and 30 degrees C. The effects of spiperone and ascorbate on binding have also been determined. Under the conditions used, binding was saturable and reversible. Affinity constants derived from kinetic and equilibrium data were comparable. Serotonin binding to several sites had substantial enthalpic as well as entropic components.     

High- and Low-Affinity Binding of [3H]Imipramine in Mouse Cerebral Cortex

Binding of [3H]imipramine in mouse cerebral cortex was found to be nonhomogeneous. Competition experiments, Scatchard analysis, and Hill plots are compatible with the existence of binding with high (nanomolar) and low (micromolar) affinity. Low-affinity binding could be eliminated by the use of low concentrations of imipramine as the competing ligand. In contrast to the high-affinity binding, the low-affinity binding was found to be unrelated to the neuronal uptake system for serotonin.     

L-[3H]Glutamate Binding to Hippocampal Synaptic Membranes: Two Binding Sites Discriminated by Their Differing Affinities for Quisqualate

The excitatory glutamate analogs quisqualate and ibotenate were employed to distinguish multiple binding sites for L-[3H]glutamate on freshly prepared hippocampal synaptic membranes. The fraction of bound radioligand that was displaceable by 5 microM quisqualate was termed GLU A binding. That which persisted in the presence of 5 microM quisqualate, but was displaceable by 100 microM ibotenate, was termed GLU B binding. GLU A binding equilibrated within 5 min and remained unchanged for up to 80 min. GLU B binding appeared to equilibrate at least as rapidly, but incubation with ligand unmasked latent binding sites. Saturation binding curves were best fitted by single exponentials, which yielded KD values of about 200 nM (GLU A) and 1 microM (GLU B). On the average, GLU B binding sites were about twice as abundant in these membranes as were GLU A sites. Rapid freezing of the membranes, followed by storage at -26 degrees C and rapid thawing markedly diminished GLU A binding, but nearly tripled GLU B binding. Both site bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with about 10 times the affinity of L-aspartate and discriminated poorly between L- and D-aspartate. In contrast, the GLU B site bound L-aspartate with an affinity similar to that for L-glutamate, and with an order-of-magnitude greater affinity than D-aspartate. The structural specificities of the GLU A and GLU B binding sites suggest that these sites may correspond to receptors on hippocampal pyramidal cell dendrites that are activated by iontophoretically applied L-glutamate.     

L-[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.     

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Abstract: [³H]Diazepam and [³H]flunitrazepam ([³H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, γ -aminobutyric acid (GABA) and NaCl both increased [³H]diazepam binding more than [³H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [³H]diazepam in the absence of NaCl and GABA, the B_max (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [³H]FNP, which dissociates more slowly, had the same B_max in both assays. [³H]Diazepam association had two components, and was faster than [³H]FNP association. [³H]Diazepam dissociation, which also had two components, was faster than that of [³H]FNP, and also had a greater fraction of rapidly dissociating species. [³H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [³H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.     

Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites

I. Binding of [³H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris-HCl buffer, pH 7.5, containing 1 mM-EDTA and 0.01% ascorbic acid, using a washed total-membrane fraction. The receptor binding was stereospecifically inhibited by (+)-butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [³H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [³H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [³H]spiperone and [³H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high-ionic-strength Tris buffer with salts added and low-ionic-strength Tris buffer with EDTA. Apparent K_D, values of spiperone decreased with decreasing tissue concentrations. K_D, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the ³H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist-antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high-affinity agonist and antagonist binding.     

Ascorbate Modulates 5-[3H]Hydroxytryptamine Binding to Central 5-HT3 Sites in Bovine Frontal Cortex

Ascorbate is present in millimolar concentrations in mammalian brain and can be released from cellular stores by membrane depolarization. We report here that physiologically relevant concentrations of ascorbate modulate 5-[3H]hydroxytryptamine ([3H]5-HT) binding to bovine frontal cortex membranes. Under conditions where [3H]5-HT binding is reversible and saturable, ascorbate causes a concentration-dependent increase in the affinity of [3H]5-HT for central 5-HT3 binding sites. At pH 7.4, increasing ascorbate from 0 to 5.7 mM changes the equilibrium affinity constant (KD) of binding to 5-HT3 sites from 125 nM to 30 nM, without affecting binding site number. These ascorbate-induced effects are pH dependent. At pH 7.1 binding to central 5-HT3 sites is essentially eliminated in the presence of ascorbate. These studies suggest that ascorbate and hydrogen ion concentration interactions may modulate serotonergic function.