Tryptamine, a Substrate for the Serotonin Transporter in Human Platelets, Modifies the Dissociation Kinetics of [3H]Imipramine Binding: Possible Allosteric Interaction

Tricyclic antidepressants and nontricyclic serotonin (5-hydroxytryptamine) uptake blockers monophasically inhibit [3H]imipramine binding in human platelets. Similarly, serotonin and tryptamine inhibit the binding of [3H]imipramine in the low micromolar range and with a pseudo-Hill coefficient near unity. Dissociation of the [3H]imipramine receptor complex in the presence of uptake inhibitors follows first-order kinetics with a half-life of approximately 60 min. Although serotonin and tryptamine do not decrease [3H]imipramine binding when added under equilibrium conditions, simultaneous addition of serotonin or tryptamine with serotonin uptake inhibitors decreases the rate of ligand-receptor dissociation in a concentration-dependent manner. These data suggest a common site of action for serotonin, which is the substrate of the transporter system, and of tryptamine, its nonhydroxylated analog. This hypothesis is supported by the identification of a high-affinity (Km = 0.55 microM), saturable, and temperature-dependent uptake of [3H]tryptamine in human platelets. Uptake of [3H]tryptamine was inhibited potently by imipramine and nontricyclic serotonin uptake inhibitors with a potency similar to that observed for [3H]serotonin uptake. These data support the hypothesis that in platelets, [3H]imipramine, tricyclic, and nontricyclic serotonin uptake inhibitors bind to a common recognition site that is associated with the serotonin transporter but that differs from the substrate recognition site of the carrier through which serotonin and tryptamine exert a heterotropic allosteric modulation on [3H]imipramine binding.     

The 5-HT1A receptor probe [3H]8-OH-DPAT labels the 5-HT transporter in human platelets

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using [3H]8-OH-DPAT as the radioligand. [3H]8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average KD of 43 nM and Bmax of 1078 fmol/mg protein. Determinations of IC50 values for various serotonergic characterizing agents in platelets for displacement of [3H]8-OH-DPAT were performed. For example, 8-OH-DPAT 5HT1A had an IC50 of 117 nM; TFMPP 5HT1B (2.3 microM0 and PAPP 1A + 5HT2 (9 microM); ipsapirone 5HT1A (21.1 microM) and buspirone 5HT1A (greater than 100 microM); ketanserin 5HT2 (greater than 100 microM); 5-HT uptake inhibitors: paroxetine (13 nM); chlorimipramine (73 nM) and fluoxetine (653 nM). The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit [3H]imipramine binding, however, it does inhibit [3H]5-HT uptake in human platelets near 5-HT's Km value (IC50 = 2-4 microM). These results suggest that the human platelet site labeled by [3H]8-OH-DPAT is pharmacologically different from the neuronal site and probably is a component of the 5-HT transporter.     

5-Methoxytryptoline, a Competitive Endocoid Acting at [3H]Imipramine Recognition Sites in Human Platelets

5-Methoxytryptoline potently inhibits [3H]imipramine binding to membranes from the cerebral cortex and platelets. Since 5-methoxytryptoline, which appears to occur endogenously with particularly high levels in the human pineal gland, also inhibits 5-hydroxytryptamine (5-HT, serotonin) uptake, it should be considered as a putative endogenous ligand modulating 5-HT transport. As the 5-HT transporter complex comprises the imipramine and the substrate recognition sites, which interact allosterically, it was essential to define the mechanism of inhibition of [3H]imipramine binding by 5-methoxytryptoline. Human platelets show an active and saturable uptake of 5-HT and tryptamine. The uptake of both substrates appears to be mediated by the same carrier and it is inhibited by 5-methoxytryptoline at submicromolar concentrations. 5-HT and tryptamine inhibit [3H]imipramine binding in human platelets with a Hill slope for inhibition close to unity and IC50 values of 3,265 and 3,475 nM, respectively. This inhibition is, however, not competitive because both 5-HT and tryptamine significantly decrease the rate of [3H]imipramine-receptor dissociation. Although 5-methoxytryptoline potently inhibits [3H]imipramine binding (IC50 = 44 nM) in human platelets with a Hill slope of unity, it does not affect the receptor-ligand dissociation rate of [3H]imipramine even at concentrations up to 100 microM. The present experiments show that 5-methoxytryptoline, in spite of its chemical similarity to the indoleamine transporter substrates, interacts with the imipramine receptor through a mechanism of competitive inhibition. This conclusion is supported by a selective effect of 5-methoxytryptoline on the Kd of [3H]imipramine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

p-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.     

Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets

The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.     

[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.     

Partial purification of the 5-hydroxytryptamine-reuptake system from human blood platelets using a citalopram-derived affinity resin [corrected

This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific [3H]imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 microM citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after 125I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of [3H]imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and [3H]imipramine binding activity.     

Two Affinity States for [3H]Imipramine Binding to the Human Platelet 5-Hydroxytryptamine Carrier: An Explanation for the Allosteric Interaction Between 5-Hydroxytryptamine and Imipramine

5-Hydroxytryptamine (5-HT) showed a biphasic effect on the dissociation rate of [3H]imipramine from human platelet membranes: At low concentrations (EC50, approximately 2.5 microM), 5-HT stimulated the rate, as expected for mutually exclusive binding of 5-HT and imipramine; at higher concentrations (EC50, approximately 40 microM), 5-HT reduced this stimulated rate, a result consistent with 5-HT binding at a site that is physically distinct from both the imipramine binding site and the 5-HT transport recognition site of the 5-HT carrier. This modulatory effect could be mimicked by tryptamine, was saturable and independent of Na+ concentration, and could also be demonstrated for detergent-solubilized carriers. Monophasic association kinetics for [3H]imipramine binding were found. Heat stability experiments showed biphasic thermal inactivation curves. These results are consistent with [3H]imipramine binding to two classes of binding sites at the 5-HT carrier on human platelet membranes, with affinities three- to fivefold different. 5-HT can convert the lower-affinity state into the higher-affinity state.     

2-Nitroimipramine: A selective irreversible inhibitor of [3H] serotonin uptake and [3H] imipramine binding in platelets

The effect(s) of a new imipramine analogue, 2-nitroimipramine, on high affinity [³H] imipramine binding and [³H] serotonin uptake in human platelets were studied. 2-Nitroimipramine was found not only to be a very potent inhibitor of [³H] imipramine binding and [³H] serotonin uptake but was found to irreversibly inhibit binding and uptake simultaneously. This finding supports previous observations from our laboratory and others that high affinity imipramine binding labels serotonin uptake or transport sites. 2-Nitroimipramine should prove an important tool for subsequent studies of the molecular mechanism(s) involved in the transport of serotonin and the binding of imipramine to platelet and brain membranes.     

Mechanism of imipramine inhibition of platelet 5-hydroxytryptamine transport.

Plasma membrane vesicles isolated from porcine blood platelets take up approximately 8 to 15 pmol of [3H]imipramine per mg of membrane protein. This apparent binding requires Na+ in the external medium and is reversed by 5-hydroxytryptamine and fluoxetine. The apparent KD for imipramine uptake is 23 nM, which agrees well with the KI for competitive inhibition of 5-hydroxytryptamine transport by imipramine. In contrast to 5-hydroxytryptamine transport, imipramine uptake is not dependent on transmembrane Na+ and K+ gradients and is insensitive to ionophores such as nigericin and gramicidin which dissipate these gradients. Although 5-hydroxytryptamine rapidly and competitively displaces imipramine from membrane vesicles, imipramine does not cause 5-hydroxytryptamine efflux and inhibits 5-hydroxytryptamine exchange. These results are consistent with the proposal that imipramine binds to the substrate site of the 5-hydroxytryptamine transporter but cannot be transported.     

High affinity binding of amiloride analogs at an internal site in renal microvillus membrane vesicles.

Amiloride analogs with hydrophobic substitutions on the 5-amino nitrogen atom are relatively high affinity inhibitors of the plasma membrane Na(+)-H+ exchanger. We demonstrated that a high affinity-binding site for [3H]5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) (Kd = 6.3 nM, Bmax = 1.2 pmol/mg of protein) is present in microvillus membrane vesicles but not in basolateral membrane vesicles isolated from rabbit renal cortex, in accord with the known membrane localization of the Na(+)-H+ exchanger in this tissue. The rank order potency for inhibition of microvillus membrane [3H]MIA binding by amiloride analogs was: MIA (I50 approximately 10 nM) greater than amiloride (I50 approximately 200 nM) greater than benzamil (I50 approximately 1200 nM). This correlated with a qualitatively similar rank order potency for inhibition of Na(+)-H+ exchange: MIA (I50 approximately 4 microM) greater than amiloride (I50 approximately 15 microM) greater than benzamil (I50 approximately 100 microM), but did not correlate with the rank order potency for inhibition of the organic cation-H+ exchanger in microvillus membrane vesicles: MIA approximately benzamil (I50 approximately 0.5 microM) greater than amiloride (I50 approximately 10 microM). However, tetraphenylammonium, an inhibitor of organic cation-H+ exchange, inhibited the rate of [3H]MIA binding without an effect on equilibrium [3H]MIA binding; the dissociation of bound [3H]MIA was inhibited by preloading the membrane vesicles with tetraphenylammonium. These findings indicated that high affinity [3H]MIA binding to renal microvillus membrane vesicles takes place at an internal site to which access is rate-limited by the tetraphenylammonium-sensitive organic cation transporter. Equilibrium [3H]MIA binding was inhibited by H+ but was unaffected by concentrations of Na+ or Li+ that saturate the external transport site of the Na(+)-H+ exchanger. Binding of MIA to its high affinity binding site had no effect on the rate of Na(+)-H+ exchange. This study suggests that the renal Na(+)-H+ exchanger has a high affinity internal binding site for amiloride analogs that is distinct from the external amiloride inhibitory site.     

Characterization of [3H]Paroxetine Binding in Rat Brain

The binding of the 5-hydroxytryptamine (5-HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 microliter, the binding was optimized with an apparent dissociation constant (KD) of 0.03-0.05 nM. Competition experiments with 5-HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single-site binding model. Fluoxetine and imipramine revealed, in addition to a high-affinity (nanomolar) site, also a low-affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5-HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5-HT- and fluoxetine-sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5-HT (100 microM) sensitive [3H]paroxetine binding were also consistent with a single-site binding model, and the binding was competitively inhibited by 5-HT and imipramine. The number of binding sites (Bmax) for 5-HT-sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p-chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a greater than 60% reduction in the striatum and hypothalamus, indicating a selective localization on 5-HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5-HT uptake in rat brain.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Uptake, Release, and Subcellular Localization of l-Methyl-4-Phenylpyridinium in Blood Platelets

The neurotoxic compound 1-[methyl-3H]-4-phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 microM) was 50 times higher than that for serotonin [5-hydroxytryptamine (5-HT]). The uptake of [3H]MPP+ by human platelets was inhibited by selective 5-HT uptake blockers [cianopramine, (-)-paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5-HT organelles (reserpine, mepacrine, and Ro 4-1284). Impairment of the transmembrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5-HT organelle fraction. MPP+ competitively inhibited [14C]5-HT uptake by human platelets and reduced the endogenous 5-HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5-HT carrier and is accumulated predominantly in the subcellular organelles that store 5-HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.     

High affinity [3H]imipramine binding and serotonin uptake to platelets of adolescent females suffering from anorexia nervosa

High affinity [3H]imipramine binding and [3H]serotonin uptake to platelets were investigated in 17 anorexic females aged 15-18 years as compared to 15 healthy females of similar ages. A significant decrease in the density of [3H]imipramine binding sites was observed in anorexics as compared to controls (368 +/- 40 vs 517 +/- 38 fmoles/mg protein, p less than 0.01). No alteration in Kd values or in the kinetic parameters of serotonin uptake (Vmax, Km) were noted. The fact that the decrease in imipramine binding is not accompanied by a parallel reduction in serotonin uptake might indicate that anorexia nervosa is not ultimately related to major depression and that the imipramine binding site is not identical to the serotonin uptake site.     

2-Nitroimipramine: a photoaffinity probe for the serotonin uptake/tricyclic binding site complex.

[3H]2-Nitroimipramine ([3H]2-NI), a compound with high affinity for the serotonin uptake system, is shown to be an effective photoaffinity probe which incorporates covalently into membrane homogenates prepared from human platelets, as well as rat brain and liver. In all cases, [3H]2-NI preferentially incorporated into a minor membrane component of 30 kd protein, as determined by SDS-polyacrylamide gel electrophoresis and subsequent fluorography. A number of selective and general serotonin uptake inhibitors quantitatively chased labeling of the 30-kd band at nanomolar concentrations. Pharmacological characterizing agents unrelated to the serotonin uptake system generally had little effect on labeling. In platelet membranes, a broad band of approximately 35-kd protein was also labeled by [3H]2-NI, but this labeling was not inhibited by any of the selective serotonin uptake blockers. Interestingly, serotonin itself increased incorporation into the 30-kd band and selectively decreased labeling of the 35-kd band. Photolytic incorporation into the 30-kd band was of high affinity, saturable, and Scatchard analyses of irreversible labeling were linear. In contrast, Scatchard transformations of [3H]2-NI equilibrium binding saturation isotherms were markedly curvilinear. Cross-linking unlabeled 2-NI to intact platelets, followed by extensive dialysis, decreased the maximal velocity (Vmax) of platelet serotonin uptake, but did not alter the affinity (Km) of serotonin for its transport site. These results are noteworthy since current theories implicate prejunctional allosteric interactions between serotonin and imipramine at serotonergic synapses.(ABSTRACT TRUNCATED AT 250 WORDS)     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

The sites of high affinity binding of L-[3H]glutamic acid in human platelets. A new type of platelet receptor?

The total membrane fraction of human platelets was found to contain high affinity sites of L-[3H]glutamic acid binding (Kd = 100 nM, Bmax = 1.06 pmol/mg protein). The pH optimum for binding is at pH approximately 6.9 Na+ (1-150 mM) inhibit glutamate binding by platelet membranes (IC50 = 12 mM). Ca2+ (50-100 microM) stimulate the binding by 10-20% and inhibit it by 20-30% at concentrations of 1-5 mM. Monoclonal antibodies to the glutamate receptor strongly suppress the L-[3H]glutamate binding by platelet membranes (IC50 = 300 nm). The presence in human platelets of a glutamate-sensitive receptor complex similar to the central nervous system glutamate receptor is postulated.     

"Specific" Binding of [3H]Imipramine to Protease-Sensitive and Protease-Resistant Sites

A number of 5-hydroxytryptamine (5-HT) uptake inhibitors have been shown to displace the binding of [3H]imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 microM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10 microM desipramine. The protease-sensitive [3H]imipramine binding sites were found to be saturable, high-affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5-HT but not [3H]noradrenaline uptake. This was not the case however for the protease-resistant but desipramine-displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 microM) to define "specific binding," these data would suggest that either protease-sensitivity or displacability by 1 microM norzimeldine would give more reliable estimates of the specific binding.     

Interaction of the anxiolytic agent buspirone with serotonin and other synaptic receptors in the human brain

Buspirone and Mj 138-05 (up to 0.1 mM) did not displace specifically bound (3H) tryptamine, (3H) strychnine, (3H) flunitrazepam and (3H) imipramine in human cortical and hippocampal membrane preparations. At the same time both compounds displayed similar to serotonin affinity (IC50 in the range of 2-6 microM) for (125I)-LSD specific binding sites in the human cortex and hippocamp. IC50 of serotonin and buspirone and Mj 138-05 for (3H) LSD (2 nM) specific binding sites in the hippocamp was determined as 0.14 microM, 2.3 microM and 6.1 microM, respectively; and for (3H) serotonin specific binding sites in the hippocamp as 0.005 microM, 3.8 microM and 21 microM, respectively. The affinity for human cortex (3H) LSD binding sites was 10-fold lower in case of serotonin and 4-fold lower in case of buspirone and Mj 138-05 than in the hippocamp. However, the affinity for (3H) serotonin binding sites in the cortex was the same as in the hippocamp in case of serotonin and 12-15-fold lower than in the hippocamp in case of buspirone and Mj 138-05. It is concluded that in human brain buspirone and Mj 138-05 interact with micromolar affinity with 5 HT2 and are capable of binding to a subpopulation of 5 HT1 receptors in the hippocamp.