Frederick J. Ehlert

The binding of [3H]nitrendipine to receptors for calcium channel antagonists in the heart, cerebral cortex, and ileum of rats.

The binding properties of the calcium channel antagonist, [3H]nitrendipine, were investigated in homogenates of the rat cerebral cortex, heart and ileum. The specific component of [3H]nitrendipine binding was consistent with mass-action behavior and was characterized by a high affinity dissociation constant in the range of 0.1-0.3 nM. A variety of other calcium channel antagonists inhibited the binding of [3H]nitrendipine with Kis that agree generally with the ability of these drugs to block contractions of cardiac and smooth muscle. The inhibition of [3H]nitrendipine binding by other dihydropyridines was consistent with competitive antagonism whereas the inhibition caused by verapamil and D600 resembled negative heterotropic cooperativity. Consistent with this latter postulate was the observation that the kinetics of [3H]nitrendipine binding are altered by verapamil, with both the association rate and the dissociation rate being increased. La+3 and several divalent cations caused an inhibition of [3H]nitrendipine with the rank order of potency being Cd+2 greater than La+3 greater than Ni+2 greater than Co+2 = Mn+2 greater than Mg+2 = Ba+2 greater than Ca+2.

An allosteric model for benzodiazepine receptor function.

FREDERICK J. EHLERT*, WILLIAM R. ROESKE, KELVIN W. GEE and HENRY I. YAMAMURA# Departments of Pharmacology, Biochemistry, Psychiatry and Internal Medicine, and the Arizona Research Laboratories, University of Arizona Health Sciences Center, Tucson, AZ 85724, U.S.A. The assignment of specific 3H-labeled ligand binding to a pharmacologically relevant receptor is not a trivial matter. Nevertheless, evidence has accumu- lated during the past few years that benzodiazepines mediate their pharmacological effects by interacting with neuronally localized receptors within the brain. An intriguing question is how the recognition of a benzodiazepine by its receptor initiates a chain of events leading to the pharmacological response, e.g. prevention of seizures, tranquility and sedation. The conventional approach has been to rationalize the initial recognition step within the context of a lock-and-key model. This hypothesis can readily account for the pharmacological specificity and stereospecificity of receptors as being a consequence of their geometry. It is with this recognition process that the implications of receptor binding data have their most immediate application. In addition, ligand binding methods can yield some insight into the activation of the receptor-effector system. It has been pointed out that part of the intrinsic binding energy of an agonist is used to induce a conforma- tional change in the receptor [1]; in other words, the observed affinity of a drug for a receptor depends on both the bimolecular association of the ligand with the receptor and the subsequent conformational change of the ligand-receptor complex. Although this idea is not a new one, the importance of this phenomenon in regard to interpreting binding data is not generally recognized. It is useful to consider how the binding properties of drugs for other neurotransmitter receptors are affected by the intrinsic activity of the ligand. With regard to beta-adrenergic and muscarinic receptors, there are striking differences in the way agonists and antagonists bind to these receptors [2-4]. In general, agonists tend to display heterogeneous binding properties [5]. The magnitude of this heterogeneity can be estimated by computing the relative differ- ences in the affinities of a drug for subclasses of the receptor or, in some instances, by estimating the relative densities of the high and low affinity states of the receptor. Strong correlations between the * Present address: Department of Pharmacology, School of Medicine, University of California, Los Angeles, CA 90024, U.S.A. t Address correspondence to: Henry I. Yamamura, Ph.D., Department of Pharmacology, Arizona Health Sci- ences Center, Tucson, AZ 85724, U.S.A. heterogeneity of binding and the efficacy ligand have been noted for muscarinic and beta- adrenergic receptors [2-4]. Also, the negatively cooperative effects of guanine nucleotides on binding properties show a strong correlation with efficacy [5, 6]. For opiate receptors, a correlation between the influence of Na + on binding and efficacy opiate analogues has been noted [7]. It is not our intent to assess the models that have been proposed to account for these phenomena but merely to emphasize the fundamental principle providing basis for these relationships: the intrinsic activity of an agonist is related to its ability to induce a confor- mational change in the receptor. During the course of our studies of the benzodi- azepine receptor binding activity of the beta-car- boline compounds which are structurally unrelated to benzodiazepines (see Fig. 1), we noticed certain correlations between the influence of gamma-ami- nobutyric acid (GABA) on binding and the intrinsic activity of the drug [8, 9]. These differences have been rationalized in terms of an allosteric model for benzodiazepine receptor function which views the benzodiazepine receptor as a heterotropic site on the GABA receptor-chloride ionophore complex [10], The model is an attempt to define the rela- tionships between the behavioral effects of benzo- diazepines and the influence of GABA on binding. Presently, it is our goal to describe the model in greater detail and to demonstrate that it can account for most of the pharmacological effects of ligands that interact specifically with the recognition site of the benzodiazepine receptor. We shall begin our discussion by reviewing some of the complex binding characteristics of the benzodiazepine receptor. Benzodiazepine receptor heterogeneity Initially, it seemed as if benzodiazepine receptors were homogeneous [11-13]. This assumption was based on the results of numerous binding studies which have shown that the equilibrium binding iso- therms for [3H]diazepam and [3H]flunitrazepam ([3H]FLU) in the range of concentrations studied were consistent with the simple Langmuir isotherm [11-15]. Also, when the specific binding of [3H]FLU or [3H]diazepam in low concentrations is inhibited by other nonlabeled benzodiazepines, the competi- tion curves usually have Hill coefficients of approx- imately 1. However, evidence soon emerged which suggested that benzodiazepine receptors are not sim- ply a homogeneous class of binding sites. In 1979, 2375

Multiple benzodiazepine receptors and their regulation by γ-aminobutyric

Abstract The interaction of propyl β-carboline-3-carboxylate (PCC) with benzodiazepine receptors in the cerebral cortex of the rat was investigated by direct measurements of [3H]PCC binding and by competitive inhibition of [3H]flunitrazepam (FLU) binding. Initial experiments showed that [3H]PCC binding exhibited characteristics of saturability, stereospecificity and a pharmacological specificity remarkably similar to that of [3H]FLU binding. Analysis of [3H]PCC binding isotherms and PCC/[3H]PCC competition curves revealed the presence of a small population of super high affinity PCC binding sites (KSH = 30–100 pM) which represents approximately 3–6% of the total sites. When measured by competitive inhibition of [3H]FLU binding, receptor occupancy by PCC was generally consistent with that determined by direct measurements of [3H]PCC binding. Analysis of the PCC/[3H]FLU competition curve revealed the presence of two major populations of high and low affinity PCC binding sites with dissociation constants of 0.54 and 10 nM and relative abundances of 52 and 45%, respectively. Collectively, the results of the [3H]PCC binding isotherm, PCC/[3H]PCC competition curve and PCC/[3H]FLU competition curve are internally consistent when rationalized in terms of three populations of benzodiazepine receptors - super high, high, and low affinity - each having different affinities for PCC and equal affinity for FLU. The effects of γ-aminobutyric acid (GABA) on PCC and FLU binding were investigated, and it was observed that GABA enhanced the binding of FLU to the various receptor subtypes whereas no significant effect of GABA on the binding of PCC was detected.

The influence of guanyl-5′-yl imidodiphosphate and sodium on muscarinic receptor binding in the rat brain and longitudinal muscle of the rat ileum

Abstract The effects of guanyl-5′-yl imidodiphosphate (Gpp(NH)p) and sodium on muscarinic receptor binding in the rat brain and longitudinal muscle of the rat ileum were investigated using the specific muscarinic affinity label [ 3 H](−) quinuclidinyl benzilate ([ 3 H](−)QNB). When measured by competitive inhibition of [ 3 H](−)QNB binding to homogenates of the longitudinal muscle of the ileum, the IC 50 ′s of oxotremorine and carbachol increased by a factor of 3 in the presence of 30 μM Gpp(NH)p. In contrast Gpp(NH)p only produced small increases in agonist IC 50 values when binding experiments were done on various rat brain regions. The agonist inhibition curves deviated from the law of mass action and were consistant with a two binding site model. Nonlinear regression analysis showed variation in the percentage of high affinity agonist sites in the cerebellum (68%), brainstem (68%), longitudinal muscle of the ileum (44–50%) and forebrain (21–30%). In the ileum and forebrain, the predominant effect of Gpp(NH)p on agonist binding was a reduction in the affinity of the high affinity agonist binding site. In the cerebellum and brainstem Gpp(NH)p had no effect. Furthermore, Gpp(NH)p had no significant effect on antagonist binding in any tissue. The influence of sodium (200 mM NaCl) on muscarinic receptor binding was investigated in homogenates of the forebrain and longitudinal muscle of the ileum, and a preferential reduction of agonist affinity by sodium was observed.

Muscarinic receptor binding in rat brain using the agonist, [3H]cis methyldioxolane

Abstract Muscarinic receptor binding was measured in rat forebrain preparations using the muscarinic agonist, [ 3 H]cis methyldioxolane ([ 3 H]CD). The results of equilibrium binding studies using [ 3 H]CD concentrations between 0.5–64 nM showed that [ 3 H]CD binding did not saturate in this concentration range, although the binding isotherm was concave downward. Nonlinear regression analysis of the binding data revealed the presence of two populations of muscarinic receptors having dissociation constants of 1.83 and 123 nM and binding capacities of 85 and 1320 fmol/mg protein, respectively. Competitive inhibition experiments showed that [ 3 H]CD binding was readily displaced by several muscarinic agonists and antagonists. The stereospecificity of [ 3 H]CD binding was demonstrated in competitive inhibition experiments using the stereoisomers of benzetimide and acetyl-β-methylcholine. Dexetimide was 10,000 times more potent than levetimide and l-acetyl-β-methylcholine was 520 times more potent than d-acetyl-β-methylcholine. A variety of nonmuscarinic cholinergic drugs were not effective at inhibiting [ 3 H]CD binding at a concentration of 10 μM.

Modulation of benzodiazepine receptor binding: insight into pharmacological efficacy.

The effects of GABA on the binding of analogues of benzodiazepines, triazolopyridazines, beta-carbolines and imidazodiazepines were examined in ligand/[3H] flunitrazepam competition experiments. GABA increased the potency of anxiolytics, like flunitrazepam, whereas the potency of benzodiazepine antagonists, like Ro15-1788, was largely insensitive to the influence of GABA. Several other agents including pyrazolopyridines, barbiturates and etomidate caused a chloride dependent enhancement of [3H] flunitrazepam binding but not an enhancement of [3H] propyl-beta-carboline-3-carboxylate binding.

Striatal muscarinic receptors: regulation by dopaminergic agonists.

Abstract The effects of apomorphine on the binding properties of striatal muscarinic receptors were investigated using the specific muscarinic antagonist, [ 3 H](−)3-quinuclidinyl benzilate ([ 3 H](−)QNB). When binding measurements were made in 50 mM sodium/HEPES buffer, pH 7.4, containing Mg +2 , the binding of [ 3 H](−)QNB was consistent with the presence of two binding sites; 57% of the sites had a high affinity dissociation constant of 0.030 nM whereas the remaining sites had a low affinity dissociation constant of 0.64 nM. Apomorphine (1.0 μM) enhanced the binding of [ 3 H](−)QNB by an apparent conversion of low to high affinity sites. A variety of other agents were screened for their ability to enhance [ 3 H](−)QNB binding, and a pattern generally consistent with a dopaminergic effect was observed although some evidence for a β-adrenergic effect was demonstrable. The potent neuroleptics haloperidol, spiperone and sulpiride failed to antagonize the apomorphine enhancement of [ 3 H](−)QNB binding as well as some adrenergic antagonists. However, the potent inhibitors of the dopamine-sensitive adenylate cyclase, α-flupenthixol and fluphenazine, specifically blocked the apomorphine enhancement of [ 3 H](−)QNB binding with K i values of approximately 0.1 μM.

The Nature of Muscarinic Receptor Binding

During the last decade, the availability of muscarinic cholinergic drugs of high specific activity has enabled a direct characterization of the way in which acetylcholine and other drugs interact with the muscarinic receptor. As might be anticipated, the advent of a new experimental method of scientific inquiry is often followed by a surge of information, and such is the relationship between the development of receptor binding techniques and muscarinic receptor Pharmacology. In general, the recent knowledge derived from studies of the binding of muscarinic agonists and antagonists is consistent with classical theories of agonist-receptor interaction and competitive inhibition which were developed primarily from studies of the interaction of drugs with isolated smooth muscle preparations such as the guinea pig ileum. However, additional complicated ligand-receptor interactions have been detected by receptor binding methods which could not have been realized from the results of classical Pharmacological assays on whole tissue preparations.

A simple and rapid radio-receptor assay for the estimation of acetylcholine

The high potency with which acetylcholine (ACh) inhibits the binding of the specific muscarinic agonist, [3H]cis methyldioxolane ([3H]CD), has provided the basis for the development of a radioreceptor assay for estimation of ACh. A synaptosomal preparation of the rat cerebral cortex was used as a source of muscarinic receptors. When binding assays were run at 0 degree C, the IC50 value of ACh was approximately 5 x 10(-9)M, which corresponds to 2.5 - 10 pmoles of ACh, depending upon the assay volume. The ACh content of the rat cerebral cortex and corpus striatum was measured following fast microwave irradiation. By measuring the displacement of [3H]CD binding caused by aliquots of the supernatant from tissue homogenates and comparing the displacement values with an ACh standard curve, the ACh content of the cerebral cortex and corpus striatum was calculated to be 19 and 55 nmoles/g wet tissue weight, respectively.

Heterogeneity of Benzodiazepine Receptors

Evidence has accumulated from almost a decade of study of neurotransmitter and putative neurotransmitter receptors to support the conclusion that most neurotransmitters interact with a heterogeneous population of receptors. The existence of receptor subtypes for various neurotransmitters provides a means by which the functional capabilities of a neurotransmitter may be extended.