Ligand: a versatile computerized approach for characterization of ligand-binding systems.

A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor.

We used radioligand binding techniques and measurement of beta-agonist-mediated positive inotropic responses in isolated cardiac tissue to examine beta-adrenergic-receptor subpopulations in nonfailing and failing human left and right ventricular myocardium. In tissue derived from 48 human hearts the receptor subtypes identified in nonfailing ventricle by radioligand binding were beta 1 (77%) and beta 2 (23%), with no evidence of an "atypical" beta-adrenergic receptor. In failing left ventricle the beta 1:beta 2 ratio was markedly different, i.e., 60:38. This decrease in the beta 1 proportion and increase in the beta 2 proportion in the failing ventricles were due to a 62%, "selective" down-regulation of the beta 1 subpopulation, with little or no change in beta 2 receptors. In muscle bath experiments in isolated trabeculae derived from nonfailing and failing right ventricles, both beta 1- and beta 2-adrenergic receptors were coupled to a positive inotropic response. In nonfailing myocardium, beta 1 responses predominated, as the selective beta 1 agonist denopamine produced a response that was 66% of the total contractile response of isoproterenol. In heart failure the beta 1 component was markedly decreased, while the beta 2 component was not significantly diminished. Moreover, in heart failure the beta 2 component increased in prominence, as the contractile response to the selective beta 2 agonist zinterol increased from a minority (39%) to a majority (60%) of the total response generated by isoproterenol. We conclude that failing human ventricular myocardium contains a relatively high proportion of beta 2 receptors, due to selective down-regulation of beta 1 receptors. As a result, in the failing human heart the beta 2-receptor subpopulation is a relatively important mediator of inotropic support in response to nonselective beta-agonist stimulation and is available for inotropic stimulation by selective beta 2 agonists.

Beta 1- and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure.



Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [3H]5-hydroxytryptamine ([3H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [3H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [3H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [3H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [3H]5-HT binding sites having a differential regional distribution in rat brain.

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

The properties of ligand binding to the beta-adrenergic receptor have been studied using a computer modeling technique to analyze data obtained by indirect binding methods. Antagonists are shown to bind to the receptor with one homogeneous state of uniform affinity, while agonists manifest heterogeneous binding. For agonists, two distinct binding states are apparent; one of high and one of low affinity. Affinity states of the receptor are characterized by specific macroscopic dissociation constants, and the proportion of total receptors in each state can be determined. The ability of an agonist to activate adenylate cyclase (intrinsic activity) correlates closely with the amount of high affinity state formed in the presence of the agonist (p

A quantitative analysis of beta-adrenergic receptor interactions: resolution of high and low affinity states of the receptor by computer modeling of ligand binding data

Frog myocardium appears to possess both beta1 and beta2 receptors, based on the potency order of several adrenergic agonists to compete for [3H]dihydroalprenolo1 binding. Selective beta blocking agents are able to distinguish two receptor subtypes in frog myocardium, but only one site in rat ventricle. Computer modeling using a PDP 11/45 indicates that all rat beta receptors are beta1, whereas only 15%-25% of frog ventricular beta receptors are of the beta1 subtype. Computerized curve fitting can provide a more accurate estimate of receptor parameters than currently available graphical methods of analysis.

Quantitative resolution of beta-adrenergic receptor subtypes by selective ligand binding: application of a computerized model fitting technique.

Recent studies of agonist and antagonist binding to the beta -adrenergic receptor and to other receptors have established the notion of agonist specific binding properties unshared by antagonists and reflecting the activation of the effector. However, previous reports on the dissociation kinetics of the widely used high-affinity beta -adrenergic antagonist (±)-[ 125 I]hydroxybenzylpindolol (HYP) have indicated complex binding kinetics which led to the proposal of a receptor isomerization model involving antagonist promoted transitions. We report here that the binding properties of two high-affinity beta -adrenergic antagonists can be fully explained by their racemic nature. (a) Binding data for the association and the dissociation kinetics of (±)-[ 125 I]HYP to frog erythrocyte membranes can be adequately fitted by computer modeling assuming different rate constants for each enantiomer. (b) In contrast, purified (+)-[ 125 I]HYP shows only uniphasic fast-dissociation kinetics. (c) Similarly, the antagonists (-)-[ 3 H]carazolol and (±)-[ 3 H]carazolol show uniphasic slow-dissociation kinetics and biphasic dissociation kinetics, respectively. These results demonstrate that the complex binding kinetics of these high-affinity racemic radioligands are not due to receptor state transitions but rather to simultaneous binding of both isomers.

Alternative Explanation for the Apparent "Two-Step" Binding Kinetics of High-Affinity Racemic Antagonist Radioligands

Rapid regulation of receptor signaling by agonist ligands is widely accepted, whereas short-term adaptation to inverse agonists has been little documented. In the present study, guanosine 5′- O -(3-[ 35 S]thio)triphosphate ([ 35 S]GTPγS) binding and cAMP accumulation assays were used to assess the consequences of 30-min exposure to the inverse agonist N , N -diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174864) (1 μM) on δ-opioid receptor signaling efficacy. ICI174864 pretreatment increased maximal effect (E max ) for the partial agonist Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (TIPP) at the two levels of the signaling cascade, whereas E max values for more efficacious agonists like (+)-4-[(α R )-α-((2 S ,5 R )-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N , N -diethylbenzamide (SNC-80) and bremazocine were increased in [ 35 S]GTPγS binding but not in cAMP accumulation assays. Pre-exposure to ICI174864 also induced a shift to the left in dose-response curves for bremazocine and TIPP. On the other hand, E max for the inverse agonist H -Tyr-TicΨ[CH 2 NH]Cha-Phe-OH was reduced in both assays, but no changes in potency were observed. For the weaker inverse agonist naloxone, E max in [ 35 S]GTPγS binding was drastically modified because the drug turned from inverse agonist to agonist after ICI174864 pretreatment. Likewise, ICI174864 turned from inverse agonist to agonist when tested in cAMP accumulation assays. In both cases, inversion of efficacy was concomitant with marked increase in potency for agonist effects. Together with functional changes, short-term treatment with ICI174864 reduced basal receptor phosphorylation and increased immunoreactivity for Gα i3 in membrane preparations. Functional consequences of ICI174864 pretreatment were simulated in the cubic ternary complex model by increasing receptor/G protein coupling or G protein amount available for interaction with the receptor. Taken together, these data show that inverse agonists may induce rapid regulation in receptor signaling efficacy.

Reciprocal Regulation of Agonist and Inverse Agonist Signaling Efficacy upon Short-Term Treatment of the Human δ-Opioid Receptor with an Inverse Agonist

Andre Delean

Papers

Quantitative resolution of beta-adrenergic receptor subtypes by selective ligand binding: application of a computerized model fitting technique.

Alternative Explanation for the Apparent "Two-Step" Binding Kinetics of High-Affinity Racemic Antagonist Radioligands

Reciprocal Regulation of Agonist and Inverse Agonist Signaling Efficacy upon Short-Term Treatment of the Human δ-Opioid Receptor with an Inverse Agonist