Radioligand Binding Data Research Articles

Heterogeneity of vascular muscarinic receptors

Muscarinic receptors mediate diverse effects on the vasculature. Recently, a consensus has been arrived at with regard to muscarinic receptor classification (Levine & Birdsall, 1989). As a result, it may now be possible to clarify the role of each subtype in the responses of vascular tissues to muscarinic agonists. It is apparent that vascular muscarinic receptors form a heterogeneous population. M1 receptors contract canine venous tissue, whilst M3 receptors contract porcine and bovine coronary arteries. M3 receptors also mediate EDRF-dependent relaxant responses in the majority of tissues studied to date. M2 receptors elicit relaxations by a decrease in sympathetic outflow in canine femoral vein, rabbit ear artery and rat portal vein. These conclusions are primarily derived from functional estimations of equilibrium dissociation constants, since comparable radioligand binding data are both scarce and contradictory. It is concluded that all three major subtypes of receptors are present in the vasculature. However, the limited selectivity of the available antagonists, the lack of extensive use of such compounds and the unavailability of selective agonists clearly indicate the need for more definitive studies to be undertaken.

Further Characterization of the Guinea Pig Cerebral Cortex Idazoxan Receptor: Solubilization, Distinction from the Imidazole Site, and Demonstration of Cirazoline as an Idazoxan Receptor‐Selective Drug

We have demonstrated previously that [3H]idazoxan, besides being able to bind to alpha 2-adrenergic receptors, may also bind to a nonadrenergic idazoxan-receptor site with high affinity. The idazoxan receptor is tightly bound to cellular membranes, and we have now developed a method to solubilize it from the guinea pig cerebral cortex by using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The CHAPS-solubilized receptor retains its binding properties for drugs: the membrane-bound, as well as the solubilized, idazoxan receptor shows high affinities for a number of imidazolines (cirazoline, idazoxan, tolazoline, naphazoline, tramazoline, clonidine, and oxymetazoline), some imidazoles (medetomidine, detomidine), and guanfacine. By contrast, catecholamines (adrenaline, noradrenaline, isoprenaline, and dopamine) and a number of other neurotransmitters and neuromodulators (serotonin, histamine, glutamic acid, gamma-aminobutyric acid, glycine, and adenosine) show negligible affinities for the idazoxan receptor. Moreover, the idazoxan receptor shows grossly different binding properties for histamine, cimetidine, and imidazole-4-acetic acid compared to what has been described for the nonadrenergic imidazole site labeled by p-[3H]amino-clonidine, indicating that the two receptor sites are distinct. Radioligand binding data further indicate that cirazoline is an idazoxan receptor-selective drug (KD = 1 nM) showing a 50-210-fold selectivity for binding to the idazoxan receptor when compared to alpha 2-adrenergic receptors and an about 500-fold selectivity when compared to alpha 1-adrenergic receptors. We have also reviewed the literature for possible nonadrenergic actions of idazoxan and cirazoline, and we suggest that idazoxan receptors might be involved in the control of prolactin release from the pituitary.

1,4-Dihydropyridine antagonist activities at the calcium channel: a quantitative structure-activity relationship approach.

The effect of 46 1,4-dihydropyridine-type calcium channel antagonists on the tonic contractile response of longitudinal muscle strips of guinea pig ileum was determined. 2,6-Dimethyl-3,5-dicarbomethoxy-4-phenyl-1,4-dihydropyridine (13) and 13 ortho-, 15 meta-, and seven para-monosubstituted and 10 polysubstituted aromatic derivatives of 13 were studied. The pharmacological activities of the monosubstituted derivatives were best correlated by eq 10, log 1/C = 0.68 pi + 2.50 sigma m -0.47Lmeta -3.40B1para + 11.31, which had a correlation coefficient of 0.89. The full data set was best correlated by eq 11, log 1/C = 0.62 pi + 1.96 sigma m -0.44Lmeta -3.26B1para -1.51Lmeta' + 14.23, which had a correlation coefficient of 0.90. Equations of similar form but involving an ortho steric term were found to correlate the radioligand binding data for this class of compounds.

Alkylation of the bovine anterior pituitary D 2-dopamine receptor: Inconsistency with predictions of the ternary complex model

The ability of a high affinity, N-chloroethyl derivative to irreversibly occupy dopamine receptors has been used to probe the nature of the interaction of the bovine anterior pituitary D2-dopamine receptor with a putative G-protein. Conditions were developed that resulted in a significant 40-60% decrease in the total D2-dopamine receptor concentration without affecting the affinity of the receptor for either antagonists or agonists. Computer analyses of radioligand binding data for the pituitary D2-dopamine receptor have indicated that consistent with the decrease in total receptor concentration following alkylation there was a comparable decrease in the apparent agonist high-affinity state of the receptor as modelled according to independent classes of binding sites. These data were not consistent with the predictions of the ternary complex model. These results indicate that the present form of the model is not sufficient to account fully for the mechanism of ligand interactions with the pituitary D2-dopamine receptor and suggest, furthermore, that the receptor and the G-protein might be considered as a complex where ligand binding to either component regulates ligand interactions of the other, rather than enhancing the degree of association of the components.

A specific and saturable spermine-binding component present in bovine testis membranes.

To determine if polyamine (PA)-binding components were present in testis membranes, a membrane fraction derived from homogenates of immature bovine testes was utilized in a radioligand assay. [14C]Spermine ([14C] Sp) bound to a spermine-binding component (SpBC) in a saturable manner and radioligand-binding data were analyzed and fit to a one-site model. Estimates of the binding constant (Kd = 7.2 +/- 1.2 X 10(-6) M) and of the concentration of sites (11.6 +/- 1.6 X 10(-6) M) were determined assuming a one-to-one stoichiometry. Metal cations (Ca+2, Mg+2, Mn+2 and Na+) as chloride salts had no effect on binding of [14C]Sp to SpBC when tested at concentrations up to 10 mM. Not all polyamines tested were effective competitors for [14C]Sp binding, which had an ID50 of 21.0 +/- 2.0 microM. The inhibitory potencies for putrescine and spermidine relative to spermine were 3.1 +/- 0.43 X 10(-4) and 0.1 +/- 0.01 (nmol/nmol) respectively, illustrating the specificity of SpBC binding. In addition, acetylation of polyamines, which is generally associated with degradation and interconversion of polyamines, resulted in a reduction of potency of 25.2 +/- 2.0-fold of spermine and 31.1 +/- 10.6-fold of spermidine in the SpBC radioligand assay. Binding of SpBC was decreased by tryptic digestion of the membrane fraction, suggesting that protein may be a part of the SpBC. Neuraminidase treatment had no effect on [14C]Sp binding but phospholipase-C digestion increased [14C]Sp binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of radioligand binding experiments: A collection of computer programs for the IBM PC

It is now well accepted that graphic methods are a poor choice of analytical technique for analyzing radioligand binding data given the complexity of some radioligand-binding site interactions; this is true not only for studies performed at equilibrium but also for those examining kinetic properties of the radioligand. As a consequence the computer plays an important role in all forms of radioligand binding experiments because it reduces the labor-intensive calculations associated with such experiments and it allows the use of more appropriate weighted nonlinear curve-fitting techniques that more accurately describe the radioligand-binding site interaction. This paper describes a collection of four programs ( klnetic, ebda, ligad, and lowry) that can be used to analyze most forms of radioligand binding experiments. The programs have been adapted for use on an IBM PC, which is a popular, inexpensive microcomputer and which is well suited to the analysis of radioligand binding data.

Functional interactions between organic calcium channel antagonists in smooth muscle

The Ca2+ channel antagonists D-600, diltiazem, and nifedipine are competitive antagonists of Ca2+ responses in K+-depolarized guinea pig taenia coli and rat mesenteric artery preparations. pA2 values for D-600, diltiazem, and nifedipine in taenia coli were 8.28, 7.44, and 9.27, respectively and in mesenteric artery, 9.6, 7.83, and 10.4, respectively. The combination of nifedipine plus diltiazem gave in both tissues antagonism greater than that calculated on the basis of additivity. This suggests, consistent with published 3H-labelled radioligand binding data, that diltiazem and nifedipine interact at distinct sites. However, the combination nifedipine plus D-600 yielded antagonism consistent with additivity of response.

Central dopamine receptors mediating pergolide-induced elevation of serum corticosterone in rats: Characterization by the use of antagonists

Fourteen dopamine antagonists were compared for their ability to antagonize the elevation of the concentration of serum corticosterone in rats by pergolide, a dopamine agonist. Clozapine did not antagonize the effect of pergolide at the largest dose (10 mg/kg) that could be tested without alteration of basal levels of corticosterone. For the other antagonists, calculation of ED 50 values [dose antagonizing by 50% the elevation of corticosterone caused by a 0.3 mg/kg (i.p.) dose of pergolide mesylate] revealed the following order of potency: spiperone > loxapine > fluphenazine > perphenazine > flupentixol > haloperidol > cyclophenazine > zotepine > flumezapine > molindone > metoclopramide > chlorpromazine > sulpiride. All of these compounds caused increases in the concentration of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the brains of rats. The dose that increased the concentration of DOP AC to 200% of the control value (ED 200) was calculated for each compound. The ratio of the ED 50 value for antagonism of the elevation of corticosterone induced by pergolide to the ED 200 value for the elevation of DOPAC in brain varied, probably related to differing selectivity for pre- versus postsynaptic dopamine receptors between the compounds. Among those antagonists for which radioligand-binding data were available, the ED 50 values for antagonism of the elevation of corticosterone by pergolide correlated well with the affinity of the compounds for the D2 subtype of dopamine receptors as revealed by their IC 50 values for inhibition of binding of tritiated spiperone to brain membranes in vitro and by their ED 50 values for in vivo antagonism of binding of tritiated spiperone to receptors in the striatum in rat. In contrast, there was no correlation with affinity for D 1 dopamine receptors, revealed by the IC 50 values for inhibition of binding of tritiated flupentixol. The relative potency of the antagonists in preventing the elevation of corticosterone induced by pergolide supports the idea that dopamine D 2 receptors mediate this effect, a conclusion agreeing with the earlier finding that a selective D 2 agonist, trans(±)4,4a,5,6,7,8,8a,9-octahydro-6-propyl-2H-pyrazolo[3,4-g]quinoline-di-HCl (LY141865), mimics pergolide in elevating the concentration of serum corticosterone in rats.

Identification of the angiotensin II receptor in rat mesenteric artery.

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

Binding of prostaglandin F 2a and 20α-hydroxysteroid-dehydrogenase activity of immature rat ovaries throughout pseudopregnancy

PGF 2α is involved in the initiation of progesterone decline in the late luteal phase in the pseudopregnant rat. 20α-Hydroxysteroid-dehydrogenase (20α-OH-SDH), an important enzyme in progesterone metabolism, may participate in luteal regression. It was therefore of interest to investigate whether prostaglandin F 2 (PGF 2α) receptor binding in membrane particles and 20α-OH-SDH activity in a cytosolic fraction in superovulated ovaries of immature rats are related. Scatchard analysis of the radioligand binding data revealed two classes of PGF 2α receptors (K D 10 −10mol/l and 10 −8mol/l). The number of binding sites varied from day 1 through day 21 of pseudopregnancy. Maximal binding/mg protein was obtained on day 7 with a gradual decrease until day 21 after HCG administration. 20α-OH-SDH activity was low in 1 to 9 days old corpora lutea and increased markedly during days 11 to 13. Maximal enzyme activity was monitored on days 15 to 21 after administration of HCG. The time dependent increase in 20α-OH-SDH activity was partially suppressed by the treatment of the rats with indomethacin, a potent inhibitor of prostaglandin biosynthesis. Since PGF 2α-receptor content does not coincide with the increase enzyme activity (maximal receptor content preceded maximal enzyme activity by about 6 days), PGF 2α may not be the only direct factor responsible for the induction of ovarian 20α-OH-SDH, to terminate luteal function.

Methods for the determination of the specific radioactivity of radioligands.

Quantitative analysis of radioligand binding data requires the knowledge of an accurate and actual value for the specific radioactivity of the radiolabeled ligand. Several protocols, based on simple radioligand binding experiments, which allow the determination of the molar assay concentration of the radioligand and of the radioligand/receptor complex, are described. Their appropriateness is demonstrated by the use of tritiated and iodinated beta-adrenergic radioligands.

Analysis of dose-response curves and calculation of agonist dissociation constants using a weighted nonlinear curve fitting program

The K A value of a full agonist (Furchgott, 1966) is usually calculated following the transformation of a rectangular hyperbolic relationship between equiactive concentrations into a linear relationship via a double reciprocal plot. Such a transformation will introduce error into the value of K A unless the pre- and postinactivation dose-response curves are also rectangular hyperbolas. Nonlinear curve fitting computer programs, which have been developed to evaluate radioligand binding data, fit data to rectangular hyperbolas and, thus, can be used to fit concentration-response data for pre- and postinactivation curves. Examination of ( − )-isoprenaline concentration-response curves for positive inotropic effects in left atrial preparations from the guinea pig, established before and after treatment with the irreversible antagonist Ro 03-7894, indicated a marked deviation from the theoretical rectangular hyperbola (Hill coefficient greater than 1). This was accounted for by the presence of a threshold effect. The present study reports on the use of the computer program LIGAND to fit a rectangular hyperbola to the pre- and postinactivation curves for an agonist, while simultaneously calculating and allowing for the presence of a threshold phenomenon. Since both curves are fitted to rectangular hyperbolas, the K A values can be calculated with greater precision than that possible from double reciprocal plots constructed from uncorrected concentration-response curves.

Quantitation of radio-ligand binding data using a desk-top calculator in the program mode

Quantitation of specific estrogen-binding capacity was facilitated using an inexpensive procedure for the rapid processing of data from radioligand binding measurements from sucrose gradient analyses. Ligand-binding data from each fraction of a gradient were punched into a paper tape in ASCII code and, later, read by a tape reader into an Olivetti Programma 101. Using the total instructional capacity (120 steps) a program was written in symbolic language which describes: (a) the calculation of counting efficiency of the instrument for each fraction, (b) the conversion of counts/minute to disintegrations/minute, (c) the computation of the concentration of [ 3H]estradiol-17β in each fraction and (d) the summation of the quantity of [ 3H]estradiol-17β bound by specified regions of the gradient. The program can easily be adapted for use with data generated from separation procedures such as centrifugation, electrophoresis or chromatography in which large number of samples are analysed.

Effects of GABA and related amino acids on neurones in Deiter's nucleus

GABA and the GABAA and GABAB receptors play a pivotal role in the coordination of the central vestibular pathways. The commissural inhibition, which exists between the two vestibular nucleus complexes (VNCs) and which is responsible for enhancing the dynamic sensitivity of VNC neurons to head acceleration, is known to be substantially mediated by GABA acting on GABAA and GABAB receptors. After unilateral vestibular deafferentation (UVD), the large asymmetry in spontaneous resting activity between the two VNCs is reinforced and exacerbated by the GABAergic interaction between the ipsilateral and contralateral sides. Although it has been suggested that reduced GABAergic inhibition of the ipsilateral VNC may be partially responsible for the recovery of resting activity that underlies vestibular compensation of the static symptoms of UVD, at present there are few data available to test this hypothesis systematically. There is some evidence that GABA concentrations change in the ipsilateral VNC during the development of compensation; however, it is unclear whether these changes relate to GABA release or to metabolic pools of GABA. Most biochemical studies of GABA receptors have been conducted at the gene expression level. Therefore, it is unclear whether changes in the receptor protein also occur, although the most recent data suggest that changes in GABAA and GABAB receptor density in the VNC are unlikely. The few radioligand binding data relate to GABAA receptors with benzodiazepine binding sites only. A decrease in the sensitivity of ipsilateral VNC neurons from compensated animals to GABA receptor agonists has been reported; however, these studies have employed brainstem slices and therefore the functional identity of the neurons involved has been unclear. Although it seems likely that some changes in central GABAergic systems accompany the recovery of resting activity in the ipsilateral VNC during the development of vestibular compensation, at the present stage there is no compelling evidence that these changes have a causal role in the compensation process.