Temperature-sensitive high affinity [3H]tryptamine binding sites in rat brain

The influence of prior incubation on [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. A 55 min preincubation of the membranes at 37 degrees C induced an approx. 2.4-fold increase in the specific binding of [3H]ligand to the subsequently washed preparations and this phenomenon was quite temperature-dependent. On the other hand, the proportion of nonspecific binding sites was significantly decreased by 70% of the original sites within 20 min of the start of preincubation. Pargyline, ascorbic acid, EGTA, metal ions (Ca2+, Mg2+, Na+) and guanine nucleotides, included in the preincubation buffer, were all inactive on the stimulation of [3H]tryptamine binding, while the pretreatment of membranes with glutaraldehyde antagonized the augmentation of this binding. Furthermore, it was revealed that the Scatchard plot of the [3H]tryptamine binding preincubated at 0 degree C conformed to a straight line (KD = 33.1 nM, Bmax = 543 fmoles/mg protein), whereas a curvilinear Scatchard plot was obtained at 37 degrees C preincubation. Nonlinear regression analysis of the latter resulted in apparent KD (nM) & Bmax (fmoles/mg protein) values of 0.45 & 102.7 and 33.7 & 603.4 for the high and low affinity sites, respectively. All these observations lead to the inference that the preincubation-induced increase in [3H]tryptamine binding (i.e., nearly high affinity proportion of sites) may occur as a result of temperature-sensitive interconvertible conformational changes.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Demonstration of specific high-affinity binding sites for [3H]5-hydroxytryptamine in the cestode Hymenolepis diminuta

[3H]5-HT (0.16-8.32 nM) exhibited saturable and specific binding in membrane preparations of Hymenolepis diminuta. The saturation data produced a non-linear Scatchard plot which could be resolved into sites having apparent dissociation constants (Kd) of 0.17 and 8.30 nM for the high-affinity and low-affinity components, respectively. Drug displacement studies, using radioligand concentrations of 0.6 and 6 nM, revealed that the two [3H]5-HT binding components are pharmacologically distinct and do not conform to any known class of 5-HT recognition site. The physiological significance of these putative 5-HT receptors and their potential usefulness for the selection of new antiparasitic agents are discussed.     

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.     

Characterization of specific binding sites for [3H]-staurosporine on various protein kinases

Binding of [3H]-staurosporine to different protein kinases was time-dependent, reversible and saturable. Scatchard analysis of saturation isotherms indicated one class of binding sites for [3H]-staurosporine with dissociation constants (KD) of 9.6, 2.0, 3.0 and 7.4 nM for protein kinase C, cAMP-dependent protein kinase, tyrosine protein kinase and calcium/calmodulin-dependent protein kinase respectively. [3H]-staurosporine binding was fully displaced by unlabelled staurosporine or the related compound K-252a whereas other protein kinase inhibitors (H-7, H-8 and W-7) did not compete with [3H]-staurosporine. These data confirm that sataurosporine shows no selectivity for different protein kinases and suggest the putative existence of distinct, specific binding sites for [3H]-staurosporine on these enzymes.     

Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain

High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7-DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline.     

Identification of high and low (GTP-sensitive) affinity [3H]glibenclamide binding sites in cardiac ventricular membranes

Glibenclamide is an antagonist of the ATP-modulated K+ channel in cardiac tissue. This study showed glibenclamide to bind to high (0.2 nM) and low (40 nM) affinity binding sites in canine ventricular membranes. Gpp [NH]p significantly altered the binding characteristics of the low affinity site, while those of the high affinity site were unchanged. This indicates independence of the two sites and suggests the low affinity site may be coupled to a G-binding protein. Although we have identified two [3H]glibenclamide binding sites, the importance of these sites to the cardiac effects of glibenclamide remains to be determined.     

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.     

Comparison of filtration and equilibrium dialysis methods for [3H]imipramine binding to human platelets

Receptor binding of imipramine in human platelets was assessed by filtration through glassfiber filters and by equilibrium dialysis. Both methods yield drug-receptor dissociation constants of similar magnitude (10^?9m) to literature values. However, the density of binding sites (B_max) was fivefold lower by filtration (473 ± 92 fmol/mg protein) compared to equilibrium dialysis (2652 ± 765 fmol/mg protein). Dialysis allows direct assessment of free imipramine and avoids drug loss during the separation step of the filtration assay. Additional advantages were found for computer nonlinear regression analysis of untransformed data to eliminate errors owing to linear transformation in the Scatchard analysis and for simultaneous quantitation of nonspecific and total drug binding.     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Sex-differences and stress: effects on regional high and low affinity [3H]GABA binding

Sex-differences are observed in the GABAergic neurotransmitter system both at rest and following acute stress, yet the brain regions and functional implications of these differences are unknown. We examined sex-differences in the number of low- and high-affinity [3H]GABA binding sites in various brain regions of male and female mice and the effect of stress on such sex-differences. Male (n=6) and female (n=6) QS mice were exposed to a brief swim stress (3 min at 32+/-1 degrees C) either individually or with cage-mates whilst control males (n=6) and females (n=6) remained undisturbed in the home cage. Using quantitative receptor autoradiography, sections of mouse brain were labelled with either 30 or 1000 nM [3H]GABA to label high or low affinity binding sites, respectively. Results indicated that males had more low affinity [3H]GABA binding sites in various forebrain cortical regions but less high affinity binding sites in many of these regions compared with females. Forced swim stress-induced rapid changes in forebrain GABA binding sites in females and group stressed males, suggesting a mechanism for rapid GABAergic adaptations. However the number of functional binding sites for GABA in certain forebrain regions was altered by stress in opposite directions in males and females, such that baseline sex-differences were removed following stress. These results exemplify sex-differences in brain chemical function and stress responses, and are of potential importance for understanding sex-differences in response to GABAergic compounds and disorders with sex and stress as predisposing factors.     

Purification of L-[3H]nicotine eliminates low affinity binding

Some studies of L-[3H]nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicates that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-[3H]nicotine eliminates the low affinity site.     

Modulation of specific [3H]phenytoin binding by benzodiazepines

We have shown that diazepam (ED₅₀ 2.4 M), flunitrazepam (ED₅₀ 10.2 M) and Ro5-4864 (ED₅₀ 5 M) are able to enhance both total and specific [³H]phenytoin binding. Picrotoxin (IC₅₀ 1.43 M) and chloride, either NaCl or KCl (IC₅₀ 42.4 M) inhibit both the increase in total and specific binding of [³H]phenytoin, Ro 15-1788 does not. The optimum time for this enhancement was 3–4 hours. While the ED₅₀'s for the benzodiazepines are high their order of potency suggests that an involvement of both the peripheral type benzodiazepine receptor and the GABA-chloride ionophore complex is likely. Clonazepam (IC₅₀ 23 M), oxazepam (IC₅₀ 12 M) chlordiazepoxide (IC₅₀ 35 M) and Ro8682-10, a convulsant benzodiazepine (IC₅₀ 16 M) all inhibit both total and specific [³H]phenytoin binding. These effects were not blocked by chloride ions, picrotoxin or Ro 15-1788, and reached equilibrium within 45 minutes. This order of potency also parallels that for the peripheral benzodiazepine receptor in rat brain. These data suggest the presence of a micromolar benzodiazepine receptor site which may play a role in the control of CNS excitability. Nitrazepam, medazepam, bromazepam and the tetralobenzodiazepines U38335, U42794, U43434, and U37834 had no effect on total or specific [³H]phenytoin binding nor on the actions of the other benzodiazepines described in concentrations up to 50 M.     

[3H]Serotonin binding sites in goldfish retinal membranes

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [³H]5HT was sensitive to pre-treatment of the membranes at 40° or 60° C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [³H]5HT binding to retinal membranes. The 5HT₂ agonist, 1-(-naphtyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodopheny1-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT_1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B_max 2.45 pmol/mg of protein, k_d 6.86 nM), and a low affinity component (B_max 53.46 pmol/mg of protein, K_d 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (K_d 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existance of 5HT₁ receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a suptype of receptor expressed in glial cells.Abbreviations used B _max maximum binding capacity - CPP, 1 (3 chlorophenyl)piperazine - CLN clonidine - DMI desimipramine - DMT 5-methoxy-N,N-dimethyltryptamine - DOI (+)-1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane - DPAT (+)-8-hydroxy-2-(D1-N-propylamino)tetralin - GppNHp 5-guanylylimidodiphosphate - HAL haloperidol - 5HT serotonin - IC₅₀ concentration of drug producing 50% inhibition of binding - IMI imioramine - K_d equilibrium dissociation constant - MIAN mianserin - NOM nomifensin - NP 1-(1-napthyl)piperazine - PRP propranolol In memory of Dr. Boris Druian who died on Dec. 24, 1991.     

Widespread occurrence of specific, high affinity binding sites for amino acids.

The high affinity binding of several amino acids to various membrane and protein preparations has been measured. Binding of radioactive amino acids suspected of being neurotransmitters and also of leucine and tyrosine to brain, liver and heart muscle membranes was saturable, reversible and stereospecific. Similar characteristics were found using chloroform-methanol extracted brain tissue and heat denatured albumin. Compounds thought to act as blockers of postsynaptic binding such as strychnine, bicuculline and kainic acid did not inhibit binding. Thus, specific high affinity interactions between amino acids and proteins are widespread and largely unrelated to neurotransmission.     

Solubilization and characterization of [3H]Imipramine and [3H]Paroxetine binding sites from calf striatum

The serotonin (5-HT) transporter from calf striatum cerebral membranes was solubilized with digitonin and characterized by gel exclusion chromatography. [³H]Imipramine and [³H]paroxetine were utilized as markers for labeling it.³H-imipramine labels a high- and a low-affinity site on striaturn membranes, whereas it binds to a single high-affinity site on the solubilized fraction. [³H]Paroxetine binds with the same affinity to a single site on both membranes and solubilized preparations. After gel exclusion chromatography of the solubilizate both [³H]imipramine and [³H]paroxetine bind on an identical fraction of 205 kDa molecular weight, with a similar maximum number of binding sites (Bmax). Our results suggest that both³H-imipramine and [³H]paroxetine bind to a common site on the 5-HT transporter.     

Localization of kappa opioid receptor binding sites in human forebrain using [3H]U69,593: Comparison with [3H]bremazocine

1. The autoradiographic distribution of kappa opioid receptor binding sites in human brain was examined using two radiolabeled probes, namely [3H]U69,593 and [3H]bremazocine. 2. [3H]U69,593 binding was performed in the absence of blockers for other sites, while [3H]bremazocine binding was investigated in the presence of saturating concentrations of mu and delta blockers to ensure selective labeling of kappa opioid receptors. 3. Our results show that the autoradiographic distribution of [3H]U69,593 and [3H]bremazocine (plus blockers) binding sites is identical, with high densities of sites found in deep cortical layers and claustrum. 4. This indicates that [3H]U69,593 is a highly selective ligand of the kappa opioid receptor type.