CL 218872 antagonism of diazepam induced loss of righting reflex: evidence for partial agonistic activity at the benzodiazepine receptor

A recent hypothesis suggests that the "selective anxiolytic" activity of the triazolopyridazine, CL 218872, is a reflection of this compounds high affinity for a benzodiazepine (BZD) receptor subtype. Subsequent to this proposal, the observation was made that CL 218872 does not effectively discriminate BZD receptor subtypes in vitro at physiological temperatures (37 degrees C). Based upon this observation, a selective effect in vivo related to the high affinity of CL 218872 for a BZD receptor subtype appears unlikely. The present study provides evidence for an alternative hypothesis to explain the unique pharmacological properties of CL 218872. The ability of CL 218872 to antagonize diazepam induced loss of righting reflex and enhance the anticonvulsant effect of diazepam in mice suggests that this triazolopyridazine may act as a partial agonist at the BZD receptor. Compared to the pharmacologically active BZDs, the unique actions of CL 218872 may be related to the lower intrinsic activity of this compound.     

The effect of temperature on CL 218872 and propyl beta-carboline-3-carboxylate inhibition of [3H]-flunitrazepam binding in rat brain

The competitive inhibition of [³H]-flunitrazepam binding by CL 218872 and propyl beta-carboline-3-carboxylate (PCC), non-benzodiazepine compounds that show differential affinities for benzodiazepine (BZD) receptor subtypes, was studied in the rat cerebral cortex and hippocampus at different temperatures of incubation. The potency of both inhibitors was significantly greater at 0° than at 37°C. The magnitude of temperature induced enhancement of potency may correlate with the pharmacological efficacy of compounds that interact with BZD receptors. Hill slopes for CL 218872 shifted from 0.52 to 0.97 in the cerebral cortex when incubations were performed at 0° and 37°C, respectively. Hill values for PCC changed from 0.68 to 0.93 under similar temperature conditions. These observations suggest the presence of a homogenous population of benzodiazepine receptors at physiological temperatures or the inability of CL 218872 and PCC to distinguish between receptor subtypes at 37°C.     

Heterogeneity in the Allosteric Interaction Between the γ-Aminobutyric Acid (GABA) Binding Site and Three Different Benzodiazepine Binding Sites of the GABAA/Benzodiazepine Receptor Complex in the Rat Nervous System

In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.     

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.     

Enhancement of γ-Aminobutyric Acid Binding by Quazepam, a Benzodiazepine Derivative with Preferential Affinity for Type I Benzodiazepine Receptors

We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H] gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4 degrees C in the absence and presence of quazepam or its metabolites. Addition of 10(-6) M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 X 10(-6) M Ro15-1788.     

Type I and Type II γ-Aminobutyric Acid/Benzodiazepine Receptors: Purification and Analysis of Novel Receptor Complex from Neonatal Cortex

The gamma-aminobutyric acid (GABA) type A receptor was purified several thousandfold by affinity chromatography from rat cerebellum, adult cortex, and neonatal cortex. Competition for the benzodiazepine binding site by CL 218872 indicated that cerebellar receptors were predominantly type I, adult cortical receptors were a mixture of subtypes, and neonatal cortex was enriched in type II receptor. The receptor purified from neonatal cortex contained predominantly a 54-kilodalton (kDa), beta-subunit-like protein, whereas receptors from cerebellum and adult cortex contained nearly equal amounts of a 50-kDa, alpha-subunit-like protein and a 54-kDa polypeptide. Peptide maps of trypsin-digested 54-kDa subunits from cerebellum, adult cortex, and neonatal cortex exhibited very similar profiles, a result indicating considerable homology between these proteins in the receptor subtypes. A 59-kDa subunit protein was detected in the receptor complex purified from neonatal cortex. Like the 50-kDa, alpha-subunit of the type I receptor, this protein was photolabeled with [3H]flunitrazepam. The photolabeled peptide fragments, produced by trypsin digestion of these alpha 50- and alpha 59-subunits, exhibited the same retention times on reverse-phase HPLC. A less highly purified GABAA receptor preparation from adult rat spinal cord possessed characteristics that were very similar to those of the receptors purified from neonatal cortex.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [³H]diazepam binding was lost in photolabeled membranes, specific [³H]propyl β-carboline-3-carboxylate ([³H]PCC) binding was spared. In this study, the binding of the BZD antagonists [³H]PCC, [³H]Ro15 1788 and [³H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent K_D and small reductions in the B_max of ³H antagonist binding were observed. However, in the same membranes, up to 89% of specific [³H]FLU binding was lost. When [³H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [³H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[³H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Triazolam, an Anomalous Benzodiazepine Receptor Ligand: In Vitro Characterization of Alprazolam and Triazolam Binding

Both alprazolam and triazolam displaced clonazepam (but not Ro 5-4864) from rat brain membranes with high affinity, showing them to act at central but not peripheral benzodiazepine receptors. At 0 degrees C, 10 microM gamma-aminobutyric acid (GABA) increased the ability of alprazolam, but not of triazolam, to displace ethyl-beta-carboline-3-carboxylate (beta-CCE) and Ro 15-1788 from these receptors. At 37 degrees C, GABA increased the affinity of the receptors for both drugs, with a +GABA/-GABA ratio of 1.5 for each in promoting Ro 15-1788 binding displacement. As both triazolam and alprazolam act as anxiolytics in vivo, the results at 37 degrees C would be compatible with the hypothesis that GABA causes an increase in affinity of drugs that act in this way, but the results at 0 degrees C would not be compatible. At 37 degrees C, alprazolam had a higher IC50 for the benzodiazepine receptor than at 0 degrees C, whereas triazolam showed the reverse effect. The relative IC50 values in vitro at 37 degrees C correlated better with the potency in vivo than those obtained at 0 degrees C. At 0 degrees C, both drugs showed Hill plots with slopes of 0.9-1 with beta-CCE and Ro 15-1788. At 37 degrees C, the slopes with triazolam were much reduced, indicating that the drug may have a selective action on a subclass of central benzodiazepine receptors. In the studies reported here, alprazolam behaved like other benzodiazepines, whereas triazolam showed several anomalous properties. It would be of interest if these properties could be related either to the drug's use as a hypnotic or to the side effects it sometimes induces.     

The type I and type II gamma-aminobutyric acid/benzodiazepine receptor: 1. Purification and two-dimensional electrophoretic analysis of the receptor from cortex and cerebellum

The gamma-aminobutyric acid/benzodiazepine receptor complex was purified from rat cortex and cerebellum by benzodiazepine affinity chromatography. Receptors purified from cortex and cerebellum showed different relative affinities for Cl 218872, a non-benzodiazepine ligand which discriminates type I and type II receptors. In contrast, no differences in subunit composition could be detected between these two purified receptor preparations when analyzed by two-dimensional gel electrophoresis.     

Two forms of the GABAA receptor distinguished by anion-exchange chromatography

The GABAA receptor complex was solubilized from rat brain membranes in Triton X-100, enriched by 1012-S affinity chromatography, and subjected to DEAE anion-exchange chromatography. Two forms were distinguished by their differential elution during this HPLC with a KCl gradient. They displayed similar [3H]muscimol- and [3H]flunitrazepam-binding characteristics, as well as [3H]flunitrazepam-binding inhibition by CL 218872. Rechromatography of these distinct ionic forms indicated that they were not in dynamic equilibrium during chromatography. Resolution of these two pharmacologically similar populations of GABAA receptor by anion-exchange HPLC suggests that they differ in charge densities, a condition which may reflect differing glycosylation or phosphorylation states of the complex.     

The pharmacology of CGRP-responsive receptors in cultured and transfected cells

Historically, CGRP receptors have been classified as CGRP(1) or CGRP(2) subtypes, chiefly depending on their affinity for the antagonist CGRP(8-37). It has been shown that the complex between calcitonin receptor-like receptor (CRLR or CL) and receptor activity modifying protein (RAMP) 1 provides a molecular correlate for the CGRP(1) receptor; however, this does not explain the range of affinities seen for CGRP(8-37) in isolated tissues. It is suggested that these may largely be explained by a combination of methodological factors and CGRP-responsive receptors generated by CL and RAMP2 or RAMP3 and complexes of RAMPs with the calcitonin receptor.     

Diazepam binding inhibitor (DBI): a peptide with multiple biological actions.

Diazepam binding inhibitor (DBI) is a 9-kD polypeptide that was first isolated in 1983 from rat brain by monitoring its ability to displace diazepam from the benzodiazepine (BZD) recognition site located on the extracellular domain of the type A receptor for gamma-aminobutyric acid (GABAA receptor) and from the mitochondrial BZD receptor (MBR) located on the outer mitochondrial membrane. In brain, DBI and its two major processing products [DBI 33-50, or octadecaneuropeptide (ODN) and DBI 17-50, or triakontatetraneuropeptide (TTN)] are unevenly distributed in neurons, with the highest concentrations of DBI (10 to 50 microMs) being present in the hypothalamus, amygdala, cerebellum, and discrete areas of the thalamus, hippocampus, and cortex. DBI is also present in specialized glial cells (astroglia and Bergmann glia) and in peripheral tissues. In the periphery, the highest concentration of DBI occurs in cells of the zona glomerulosa and fasciculata of the adrenal cortex and in Leydig cells of the testis; interestingly, these are the same cell types in which MBRs are highly concentrated. Stimulation of MBRs by appropriate ligands (including DBI and TTN) facilitates cholesterol influx into mitochondria and the subsequent formation of pregnenolone, the parent molecule for endogenous steroid production; this facilitation occurs not only in peripheral steroidogenic tissues, but also in glial cells, the steroidogenic cells of the brain. Some of the steroids (pregnenolone sulfate, dehydroepiandrosterone sulfate, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one) produced in brain (neurosteroids) function as potent (with effects in the nanomolar concentration range) positive or negative allosteric modulators of GABAA receptor function. Thus, accumulating evidence suggests that the various neurobiological actions of DBI and its processing products may be attributable to the ability of these peptides either to bind to BZD recognition sites associated with GABAA receptors or to bind to glial cell MBRs and modulate the rate and quality of neurosteroidogenesis. The neurobiological effects of DBI and its processing products in physiological and pathological conditions (hepatic encephlopaty, depression, panic) concentrations may therefore be explained by interactions with different types of BZD recognition site. In addition, recent reports that DBI and some of its fragments inhibit (in nanomolar concentrations) glucose-induced insulin release from pancreatic islets and bind acyl-coenzyme A with high affinity support the hypothesis that DBI isa precursor of biologically active peptides with multiple actions in the brain and in peripheral tissues.     

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with AF-DX 116 and pirenzepine

In vitro competition binding experiments with the selective muscarinic antagonists AF-DX 116 and pirenzepine (PZ) vs 3H-N-methylscopolamine as radioligand revealed a characteristic distribution of muscarinic receptor subtypes in different regions of rat brain. Based on non linear least squares analysis, the binding data were compatible with the presence of three different subtypes: the M1 receptor (high affinity for PZ), the cardiac M2 receptor (high affinity for AF-DX 116) and the glandular M2 receptor (low affinity for PZ and AF-DX 116). The highest proportion of M1 receptors was found in the hippocampus, whilst the cerebellum and the hypothalamus were the regions with the largest fraction of the cardiac M2 and glandular M2 receptors, respectively. In certain brain areas, depending on the relative proportions of the subtypes, flat binding curves were seen for AF-DX 116 and PZ. Based on these data, an approximate distribution pattern of the subtypes in the various brain regions is presented.     

Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent [3H]N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37 degrees C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system.     

Binding of progestagens to receptor proteins in MCF-7 cells

With the aim of finding an explanation for the biological properties of progestagens currently used for contraceptive purposes, we have assessed their specificity for progesterone, androgen and oestrogen receptors in MCF-7 cells. The specificity of progestagens for the progesterone receptors in the cytosol fraction of MCF-7 cells was similar to that for progesterone receptors in human and rabbit myometrial cytosol but different from that for the progesterone receptor in rat myometrial cytosol. At 37 degrees C the relative affinity of 3-keto-desogestrel, the major metabolite of desogestrel, for the progesterone receptor in intact MCF-7 cells was twice that of levonorgestrel and Org 2058, three times that of medroxy-progesterone acetate (MPA), 4.5 times that of norethisterone and 5 times that of progesterone and cyproterone acetate whereas at 4 degrees C in the cytosol fraction of MCF-7 cells exposed to molybdate (nontransformed receptor complexes) 3-keto-desogestrel and Org 2058 displayed similar affinity. The stronger binding of 3-keto-desogestrel in intact cells was due to the higher stability of its complex with the progesterone receptor. At 37 degrees C the relative affinity of 3-keto-desogestrel for the androgen receptor in intact MCF-7 cells was half that of levonorgestrel, similar to that of norethisterone and medroxyprogesterone acetate (MPA) and at least three times higher than that of progestagens with anti-androgenic activity whereas at 4 degrees C in the cytosol fraction exposed to molybdate there was no clear difference between the relative affinities of progestagens with androgenic and anti-androgenic properties. Of the progestagens tested in this study, only norethinodrel displayed measurable but very low relative affinity for the oestrogen receptor in MCF-7 cells. We conclude that the present results of binding studies with intact MCF-7 cells correlate better with the known hormonal properties of progestagens than those obtained with the cytosol fraction exposed to molybdate at 4 degrees C.     

Further evidence for an opioid receptor complex

We recently presented evidence that distinct morphine and enkephalin receptors coexist in an opioid receptor complex. These studies used membranes prepared from whole rat brain. In this paper the receptor complex is demonstrated to occur in membranes prepared from rat striatum, cortex, and pooled nonstriatal-noncortical regions of the brain. The observation that morphine masks enkephalin receptors is confirmed using 3H-methionine enkephalin to label the enkephalin receptor. These data further support the hypothesis that populations of morphine and enkephalin receptors coexist in an opioid receptor complex.     

GABAA Receptor Subtypes: Ligand Binding Heterogeneity Demonstrated by Photoaffinity Labeling and Autoradiography

Abstract: Heterogeneity of binding affinities for a variety of ligands was observed for γ-aminobutyric acid type A (GABA_A) receptors in the rat CNS, at both GABA and ben-zodiazepine recognition sites. Photoaffinity labeling by [³H]flunitrazepam and [³H]muscimol to affinity column-purified receptor proteins was examined by gel electropho-resis in sodium dodecyl sulfate. Anesthetic barbiturates (pentobarbital) and steroids (alphaxalone) both differentially stimulated the incorporation of [³H]flunitrazepam more so into the 51-kDa α1 subunit than into the 53-kDa aL2 polypeptide, and incorporation of [³H]muscimol into the 55-kDa β2 subunit more so than the 58-kDaβ3 polypeptide. Binding to these polypeptides was also affected differentially by other allosteric modulators and competitive inhibitors, including the benzodiazepine “type 1” selective ligand CL218.872. Heterogeneity in affinity of this drug for the single 51-kDa α1 polypeptide strongly suggests that type I receptors, like type II, are heterogeneous. In brain sections, the extent of enhancement of [³H]muscimol binding showed significant regional variation, similar for both steroids and barbiturates, and the GABA analogues THlP and taurine inhibited muscimol binding with regional variations in affinity that were almost opposites of each other. Modulation of [³H]flunitrazepam binding by steroids, barbiturates, and THlP significantly varied with regions. Taken together, ligand binding heterogeneity exhibited by photoaffinity labeling and autoradiography demonstrate the existence of multiple pharmacological-binding subtypes resulting from the combination of multiple polypeptide gene products into several oligomeric isoreceptors. Comparison of the regional distribution of binding subtypes with that of different subunit gene products allows the following conclusions about possible subunit compositions of native pharmacological receptor subtypes present in the brain: Benzodiazepine pharmacology of the oligomeric receptor isofotms is dependent on the nature of α and subunits other than α, GABA-benzodiazepine coupling is dependent on the nature of the α subunits, GABA site pharmacology is dependent on the nature of the β sub-units, and several subunits including α and β contribute to the degree of sensitivity to steroids and barbiturates. Finally, the presence of discrete subunits may be necessary but is not sufficient to postulate a defined pharmacological property.     

Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor

125I-Labeled recombinant human interleukin-3 (IL-3) was used to study the characteristics and distribution of receptors for IL-3 on human cells. Receptors were found on primary monocytes, on some strains of KG-1 cells, and on pre-B cell lines. Binding was rapid at 37 degrees C, while requiring several hours to reach equilibrium at 4 degrees C. Equilibrium binding studies indicated that IL-3 bound to a single class of high affinity receptor (less than 500 receptors/cell) with a Ka of approximately 1 x 10(10) M-1. Inhibition studies revealed that human granulocyte/macrophage colony stimulating factor partially inhibited the binding of 125I-IL-3 to human monocytes but not JM-1 cells. Additional analysis showed that on KG-1 cells, both IL-3 and GM-CSF partially competed specific binding of heterologous radiolabeled ligand, with approximately equivalent capacities. This competition occurred at both 37 and 4 degrees C. These results suggest heterogeneity in the binding sites for IL-3 and GM-CSF in which a subset of receptors binds only IL-3, a subset only GM-CSF, and another subset can bind both, all with high affinity. Additional heterogeneity was suggested by equilibrium binding of 125I-IL-3 to KG-1 cells which revealed a biphasic Scatchard plot containing a low affinity component not observed on monocytes and JM-1 cells.     

Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands

gamma-aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha5 subunit were substituted by their corresponding alpha1 residues. Given the high affinity and selectivity of alpha1-containing compared with alpha5-containing GABAA receptors for zolpidem, mutated alpha5 subunits were co-expressed with beta2 and gamma2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha5P162T and alpha5E200G, might alter binding pocket conformation, whereas alpha5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha1 subunit, particularly alpha1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha1-containing receptors, and a model of this binding pocket is presented.