Inverse Agonist Binding Research Articles

Na +-dependent high affinity binding of [ 3H]LY515300, a 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine opioid receptor inverse agonist

Analogues of 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines are high affinity inverse agonists for μ-, δ- and κ-opioid receptors. To characterize inverse agonist binding, we synthesized a high specific activity radioligand from this series, [ 3H]LY515300 (3-[1-((3-cyclohexyl-[3,4- 3H 2])-3( R, S)-hydroxypropyl)-3( R),4( R)-dimethylpiperidin-4-yl]phenol). In membranes expressing cloned human opioid receptors, [ 3H]LY515300 binding was saturable and exhibited low nonspecific binding. [ 3H]LY515300 bound with high affinity to the μ- ( K d=0.07 nM), δ- ( K d=0.92 nM) and κ-( K d=0.45 nM) opioid receptors. High affinity [ 3H]LY515300 binding to all opioid receptors was Na +-dependent, a characteristic of inverse agonists. Displacement by standard opioid compounds yielded K i values consistent with their known opioid receptor affinities. Autoradiographic localization of specific [ 3H]LY515300 binding in rat and guinea pig brain was high in areas known to express high levels of opioid (particularly μ-opioid receptor) binding sites including the caudate, nucleus accumbens, and nucleus tractus solitarius. Thus, [ 3H]LY515300 is the first radiolabeled opioid receptor inverse agonist useful for the study of opioid receptors in cell lines and native tissues.

Binding of agonists, antagonists and inverse agonists to the human delta-opioid receptor produces distinctly different conformational states distinguishable by plasmon-waveguide resonance spectroscopy.

Structural changes induced by the binding of agonists, antagonists and inverse agonists to the cloned delta-opioid receptor from human brain immobilized in a solid-supported lipid bilayer were monitored using plasmon-waveguide resonance (PWR) spectroscopy. Agonist (e.g. deltorphin II) binding causes an increase in membrane thickness because of receptor elongation, a mass density increase due to an influx of lipid molecules into the bilayer, and an increase in refractive index anisotropy due to transmembrane helix and fatty acyl chain ordering. In contrast, antagonist (e.g. TIPPpsi) binding produces no measurable change in either membrane thickness or mass density, and a significantly larger increase in refractive index anisotropy, the latter thought to be due to a greater extent of helix and acyl chain ordering within the membrane interior. These results are closely similar to those reported earlier for another agonist (DPDPE) and antagonist (naltrindol) [Salamon et al. (2000) Biophys. J.79, 2463-2474]. In addition, we now find that an inverse agonist (TMT-Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists. These results shed new light on the mechanisms of ligand-induced G-protein-coupled receptor functioning. The potential utilization of this new biophysical method to examine structural changes both parallel and perpendicular to the membrane normal for GPCRs is emphasized.

Altered GABAA Receptor Subunit and Splice Variant Expression in Rats Treated With Chronic Intermittent Ethanol

Background: Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in γ-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. Methods: GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase–polymerase chain reaction (RT-PCR) and in situ hybridization. Results: CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the α6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in α6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the γ2 subunit was detected in the hippocampus, especially the CA1 region. Conclusions: Changes in GABAA receptors were found in rats given CIE. Increased α6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa α6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar α6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in γ2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Altered GABAA Receptor Subunit and Splice Variant Expression in Rats Treated With Chronic Intermittent Ethanol

Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Inverse agonist binding of peripheral benzodiazepine receptors in anxiety disorder.

The binding characteristics of the pBR (peripheral benzodiazepine receptor) inverse agonist, [3H]-Ro 5-4864, were examined in patients diagnosed as generalized anxiety disorder. As compared to normal healthy controls, the anxious subjects demonstrated a statistically significant (p < 0.001) increase in the density of pBR in platelets. The enhanced pBR binding correlated significantly with the severity of global anxiety symptom of the Hamilton Anxiety Rating Scale (HAR-S, p < 0.001). The Psychic component, but not the Somatic component, of the HAR-S, correlated significantly (p < 0.001) with the enhanced pBR binding in platelets. The results provide evidence for the hypothesis of dysregulation of peripheral benzodiazepine receptors in generalized anxiety disorder.

Mechanisms of inverse agonism of antipsychotic drugs at the D2 dopamine receptor: use of a mutant D2 dopamine receptor that adopts the activated conformation

The antipsychotic drugs have been shown to be inverse agonists at the D(2) dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D(2) dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D(2) dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G-protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non-activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.

Differential modulation by GTPgammaS of agonist and inverse agonist binding to h5-HT(1A) receptors revealed by [3H]-WAY100,635.

1. The interaction of serotonergic ligands at human (h) 5-HT(1A) receptors expressed in Chinese hamster ovary cells was examined with the selective 'neutral' 5-HT(1A) antagonist [(3)H]-WAY100,635. Its binding was saturable (K(D)=0.056 nM) with a B(max) (3.65 pmol mg(-1)) significantly higher than that of two other selective 5-HT(1A) radioligands: the partial agonist, [(3)H]-S15535 (2.77 pmol mg(-1)) and the agonist, [(3)H]-8-OH-DPAT (2.02 pmol mg(-1)). 2. The influence of GTPgammaS (100 microM) on the binding affinity of 15 serotonergic agonists, partial agonists, antagonists and inverse agonists was investigated in competition binding experiments with [(3)H]-WAY100,635. 3. Agonists, including 5-HT, 8-OH-DPAT and buspirone, displayed biphasic isotherms which shifted to the right in the presence of GTPgammaS. In contrast, isotherms of the inverse agonists, methiothepin, (+)butaclamol and spiperone, were shifted to the left in the presence of GTPgammaS. Unlabelled WAY100,635 was the only ligand that was unaffected by GTPgammaS, consistent with 'neutral' antagonist properties. 4. The magnitude of affinity changes induced by GTPgammaS for 13 ligands was highly correlated (r = 0.98) with their efficacy (positive and negative) previously determined by [(35)S]GTPgammaS binding. 5. In contrast, the napthylpiperazine derivative and high efficacy agonist, S14506, displayed only a modest GTPgammaS shift, in accordance with previous indications of 'atypical' binding properties of this ligand. A further full agonist, S14671, which is chemically closely-related to S14506, also displayed a minimal GTPgammaS shift, underpinning this observation. 6. In conclusion, [(3)H]-WAY100,635 constitutes a useful neutral antagonist radioligand for the characterization of drug actions at h5-HT(1A) receptors. GTPgammaS-induced affinity changes of agonist and inverse agonist competition isotherms generally correlate well with ligand efficacy, with the notable exception of two chemically-similar agents, S14506 and S14671, which are efficacious agonists, yet relatively insensitive to h5-HT(1A) receptor/G-protein coupling changes.

Chronic pentobarbital administration alters gamma-aminobutyric acidA receptor alpha 6-subunit mRNA levels and diazepam-insensitive [3H]Ro15-4513 binding.

In order to study the chronic effects of pentobarbital, a positive GABAA receptor modulator, on the inverse agonist binding of the benzodiazepine site, binding of [3H]Ro15-4513 and levels of GABAA receptor alpha 6-subunit mRNA were investigated in the brains of pentobarbital-tolerant/dependent animals, using receptor autoradiography and in situ hybridization histochemistry in consecutive brain sections. Pentobarbital was administered to rats either 60 mg/kg, i.p., once, for acute treatment, or 300 micrograms/10 microliters/h i.c.v. continuously for 6 days via osmotic minipumps to render rats tolerant to pentobarbital. Rats assigned to the dependent group were sacrificed 24 h after discontinuance of pentobarbital infusion, while those assigned to the tolerant group were sacrificed at the end of infusion. The alpha 6 subunit mRNA was increased in the tolerant group only. Diazepam-insensitive [3H]Ro15-4513 binding was increased in the cerebellar granule layer of pentobarbital-tolerant and -dependent rats. No alterations in these parameters were observed in acutely treated animals. These data suggest that chronic pentobarbital treatment induced expression of alpha 6-subunit mRNA. This was in contrast to alpha 1- and gamma 2-subunit mRNA, which in tolerant animals are unchanged, but for which withdrawal triggers a surge in levels. Because the alpha 6-subunit is a major component of the diazepam-insensitive [3H]Ro15-4513 binding site, the increased diazepam-insensitive [3H]Ro15-4513 binding implied de novo synthesis of the receptor subunit protein.

Chronic pentobarbital administration alters γ‐aminobutyric acidA receptor α6‐subunit mRNA levels and diazepam‐insensitive [3H]Ro15‐4513 binding

In order to study the chronic effects of pentobarbital, a positive GABAA receptor modulator, on the inverse agonist binding of the benzodiazepine site, binding of [3H]Ro15-4513 and levels of GABAA receptor α6-subunit mRNA were investigated in the brains of pentobarbital-tolerant/dependent animals, using receptor autoradiography and in situ hybridization histochemistry in consecutive brain sections. Pentobarbital was administered to rats either 60 mg/kg, i.p., once, for acute treatment, or 300 μg/10μl/h i.c.v. continuously for 6 days via osmotic minipumps to render rats tolerant to pentobarbital. Rats assigned to the dependent group were sacrificed 24 h after discontinuance of pentobarbital infusion, while those assigned to the tolerant group were sacrificed at the end of infusion. The α6 subunit mRNA was increased in the tolerant group only. Diazepam-insensitive [3H]Ro15-4513 binding was increased in the cerebellar granule layer of pentobarbital-tolerant and -dependent rats. No alterations in these parameters were observed in acutely treated animals. These data suggest that chronic pentobarbital treatment induced expression of α6-subunit mRNA. This was in contrast to α1- and γ2-subunit mRNA, which in tolerant animals are unchanged, but for which withdrawal triggers a surge in levels. Because the α6-subunit is a major component of the diazepam-insensitive [3H]Ro15-4513 binding site, the increased diazepam-insensitive [3H]Ro15-4513 binding implied de novo synthesis of the receptor subunit protein. © 1996 Wiley-Liss, Inc.

Altered γ-aminobutyric acid/benzodiazepine interaction after chronic diazepam exposure

Binding to components of the GABA/benzodiazepine receptor complex was examined in cortical membranes from rats treated for 3 weeks with continuously releasing diazepam pellet implants. Chronic diazepam treatment resulted in a decrease in the ability of GABA to inhibit benzodiazepine inverse agonist binding. The amount of binding of benzodiazepine agonist, antagonist, and inverse agonist to benzodiazepine recognition sites was unaltered by the chronic treatment, as were the potencies of these benzodiazepine ligands in inhibiting agonist ( 3H-flunitrazepam) binding. Chloride channel binding ( 35S-TBPS) was also unchanged by chronic diazepam treatment. A change in GABA/benzodiazepine coupling and/or a decreased effectiveness of GABA may be responsible for the desensitizalion of GABA/benzodiazepine interaction.

Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors

Binding of the benzodiazepine inverse agonist [3H]methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate [( 3H]DMCM) and the agonist [3H]flunitrazepam [( 3H]FNZ) was compared in rat cortical membranes. Halide ions enhanced [3H]DMCM binding three- to fourfold, increasing both the apparent affinity and the number of binding sites for this radioligand. The effect was present at both 0 and 37 degrees C. In contrast, the magnitude of halide stimulation of [3H]FNZ binding was much smaller, resulting solely from an increase in the apparent affinity for this radioligand, and was not observed at 37 degrees C. The potencies but not the efficacies of a series of anions to stimulate both [3H]DMCM and [3H]FNZ binding to benzodiazepine receptors were highly correlated with their relative permeabilities through gamma-aminobutyric acid (GABA)-gated chloride channels. Two stress paradigms (10 min of immobilization or ambient-temperature swim stress), previously shown to increase significantly the magnitude of halide-stimulated [3H]FNZ binding, did not significantly affect [3H]DMCM binding. Phospholipase A2 treatment of cortical membrane preparations was equipotent in preventing the stimulatory effect of chloride on both [3H]DMCM and [3H]FNZ binding. These data strongly suggest that anions modify the binding of [3H]DMCM and [3H]FNZ by acting at a common anion binding site that is an integral component of the GABA/benzodiazepine receptor chloride channel complex.

III. Conformational shifts at the benzodiazepine receptor related to the binding of agonists antagonists and inverse agonists

III. Conformational shifts at the benzodiazepine receptor related to the binding of agonists antagonists and inverse agonists

I. Demonstration and purification of an endogenous benzodiazepine from the mammalian brain with a monoclonal antibody to benzodiazepines

Four hybridoma lines secreting monoclonal antibodies to benzodiapines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Roll-6896, and Ro5-3438 (the K d values are in the 10 −9 M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (β-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistent. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter γ-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam.

Aging does not alter the sensitivity of benzodiazepine receptors to GABA modulation

The effects of GABA on benzodiazepine receptor binding in cerebral cortical, hippocampal, and cerebellar membranes from 2–3 months old and 28–32 months old rats were studied. GABA modulation of agonist, antagonist, and inverse agonist binding to the receptor was examined using the displacement of 3H-Ro15-1788 by diazepam, Ro15-1788, and β-carboline-3-carboxylate methyl ester, respectively, in the absence and presence of 100 μM GABA and 150 mM sodium chloride. GABA modulation was alike in old and young rats, with respect to the particular ligand and brain region. The results support the hypothesis that, in the brain regions studied, the allosteric modulation of benzodiazepine receptor binding by GABA remains intact as a function of aging.

Demonstration of benzodiazepine-like molecules in the mammalian brain with a monoclonal antibody to benzodiazepines.

An anti-benzodiazepine monoclonal antibody has been used to demonstrate the existence of benzodiazepine-like molecules in the brain. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. Small benzodiazepine-like molecules were isolated from the brain soluble fraction by immunoaffinity chromatography. They block the binding of agonists, inverse agonists, and antagonists to the neuronal-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified endogenous molecules. The results indicate that the immunoaffinity-purified molecules behave like the neuronal-type benzodiazepine receptor agonists. The purified molecules, however, do not inhibit the binding of tritiated Ro 5-4864 to the "peripheral-type" benzodiazepine receptor. The results demonstrate the existence of benzodiazepine-like molecules in the brain that bind to the benzodiazepine receptor. These molecules are different from the endogenous benzodiazepine receptor ligands reported by others.

Avermectin B1a modulation of gamma-aminobutyric acid/benzodiazepine receptor binding in mammalian brain.

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.

Barbiturates allosterically inhibit GABA antagonist and benzodiazepine inverse agonist binding.

Barbiturates and the related depressant drugs, etazolate and etomidate, inhibited both the binding of [3H]bicuculline methochloride (BMC) to gamma-aminobutyric acid (GABA) receptor sites and the binding of [3H] beta-carboline-3-carboxylic acid methyl ester (beta CCM) to benzodiazepine receptor sites in mammalian brain. These concentration-dependent effects were chemically specific and stereospecific in a manner correlating with the activity of barbiturates to enhance GABA responses in neurons and to enhance GABA and benzodiazepine receptor agonist binding in vitro. The barbiturate inhibition of [3H]BMC binding involved a decrease in affinity which at high concentrations of barbiturates results in an effective complete loss of detectable binding. The maximal inhibition of [3H] beta CCM binding involved a more modest decrease in affinity (increase in KD from 1.35 to 1.85 nM). The barbiturate inhibitions of both ligands could be reversed by picrotoxin, suggesting an indirect action at previously defined picrotoxin/barbiturate modulatory sites on the GABA-benzodiazepine receptor/chloride ion channel complex.