Dopamine receptors in the guinea-pig heart. A binding study

The binding of dopaminergic agonists and antagonists to guinea-pig myocardial membrane preparations was studied using 3H-dopamine and 3H-spiperone as radioligand. 3H-Dopamine bound specifically to heart membranes while 3H-spiperone did not. A Scatchard analysis of 3H-dopamine binding showed a curvilinear plot indicating the presence of two dopamine receptor populations that we have termed high- (Kd = 1.2 nM, Bmx = 52.9 fmol/mg prot.) and low- (Kd = 11.8 nM, Bmx = 267.3 fmol/mg prot.) affinity binding sites, respectively. The characterisation of the high-affinity component of 3H-dopamine binding indicated that the binding is rapid, saturable, stereospecific, pH- and temperature-dependent, and displaced by dopaminergic agonists and antagonists known to act similarly in vivo. The finding that pretreatment with dibenamine (which has been described as an alpha-adrenoceptor irreversible blocker) did not affect the binding of dopamine to cardiac membrane preparations suggests that alpha-adrenoceptors and dopamine receptors have separate recognition sites in the heart. We conclude that 3H-dopamine binds to specific dopamine receptors in the heart of guinea-pigs.     

[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D-1 and D-2 Receptor Sites

[3H]Fluphenazine was used to label both D-1 and D-2 dopamine receptors in mouse striatal membranes. The D-1 and D-2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH-23390 (D-1 selective) and spiperone (D-2 selective). Saturation analyses of these two sites yielded a D-1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D-2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D-2 site was slightly greater than for the D-1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine-specific (apomorphine-sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.     

Temperature-sensitive high affinity [3H]serotonin binding: Characterization and effects of antidepressant treatment

Characterization of temperature-sensitive [³H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S₁ and S₂ receptors. $\text{In vivo}$ pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [³H]5-HT site, although [³H]ketanserin (S₂) densities were decreased by 50%. This suggested that possible S₂ components of [³H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC₅₀ = 3 nM) for a portion of the 1 nM Kd [³H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [³H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after $\text{in vitro}$ incubation. The complex inhibition data may therefore represent indirect interactions through another site.     

Characterization of sulpiride-displaceable 3H-YM-09151-2 binding sites in rat frontal cortex and the effects of subchronic treatment with haloperidol on cortical D-2 dopamine receptors

We investigated the pharmacological properties of the sulpiride-displaceable binding sites labeled by 3H-YM-09151-2 in rat frontal cortex, compared to those in striatum. The IC50 value of ketanserin was 486 nM, which was apparently different from its affinity for the 5HT-2 receptor. Various dopamine antagonists showed almost the same inhibitory effects for binding site in frontal cortex and striatum. Sulpiride-displaceable 3H-YM-09151-2 binding sites were considered to be D-2 dopamine receptors. After subchronic treatment with haloperidol, the D-2 receptor density of frontal cortex (0.55 fmol/mg tissue) increased to the same extent (about 25%) as striatum without significant change in apparent affinity.     

Different Endothelin Receptor Affinities in Dog Tissues

Abstract

Endothelin (ET) is a long-lasting potent vasoconstrictor-peptide. Here we report different binding affinities of endothelin-1 (ET-1) to ET-receptors of various dog tissues. Crude microsomal fractions were prepared after homogenisation of dog tissues in 50 mM Tris/HCl, 20 mM MnCl₂, 1 mM EDTA, p^H 7.4 by differential centrifugation. Aliquots of microsomal fractions (70 u.g of protein) were incubated at 25°C for 180 min in the presence of 20 p^M125I-ET-1 and various concentrations of cold ET-1. Four different ET-1 receptor binding affinities were found: adrenals, cerebrum, liver, heart, skeletal muscle and stomach microsomal membranes contained high affinity binding sites (Kd 50 – 80 p^M, Bmax 60 – 250 fmol/mg). In cerebellum and spleen medium affinity ET-1 receptors (Kd 350 p^M, Bmax 880 and 1200 fmol/mg respectively) were present. In comparison lung and kidney microsomes contained a low affinity ET-1 receptor (Kd 800 and 880 p^M, Bmax 1600 and 350 fmol/mg). Receptors of even lower affinity were present in heart, intestine and liver microsomes with Kd values of 3 – 6 nM.     

Presence of dopamine D-2 receptors in human tumoral cell lines

[125I] Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, [125I] iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.     

In vitro binding properties and autoradiographic imaging of 3-iodobenzamide ([125I]-IBZM): a potential imaging ligand for D-2 dopamine receptors in SPECT

The in vitro binding properties of the [125I] labeled benzamide (S(-)-N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-hydroxy-3-iodo-6-methoxy- benzamide, IBZM) were determined in bovine and mouse caudate membrane homogenates and by autoradiography of mouse brain slices. [125I]-IBZM binding is saturable and reversible with a Bmax of 373 +/- 51 fmol/mg protein and a Kd of 3.1 +/- 0.62 nM (mean +/- SD, Scatchard analyses) and 0.56 nM as calculated by association and dissociation time constants. In competition experiments, Ki values for the D-2 antagonists YM-09151-2 and spiperone are 4 orders of magnitude lower than the Ki value for the D-1 antagonist SCH-23390 and S(-)-IBZM is ten-fold more potent than R(+)-IBZM. [125I]-IBZM has a low affinity for serotonin S-2 and for alpha receptors. Therefore, it is a highly selective ligand for dopamine D-2 receptors. Autoradiographic images of brain sections incubated with [125I]-IBZM show the dopamine D-2 receptors of the striatum, nucleus accumbens and olfactory tubercle with a high ratio of specific to nonspecific binding. Thus, S(-)-IBZM, when labeled with [123I], may be useful for in vivo imaging of dopamine D-2 receptors by single photon emission computerized tomography (SPECT).     

[3H]WIN 35,065–2: A Ligand for Cocaine Receptors in Striatum

[3H]WIN 35,065-2 binding to striatal membranes was characterized, primarily by centrifugation assay. Like [3H]cocaine, [3H]WIN 35,065-2 binds to both high- and low-affinity sites. [3H]WIN 35,065-2, however, exhibits consistently higher affinities than [3H]cocaine. Saturation experiments indicate a low-affinity binding site with an apparent KD of approximately 160 nM and a Bmax of 135 fmol/mg of tissue. A high-affinity site has also been identified with an apparent KD of 5.6 nM and a Bmax of 5.2 fmol/mg of tissue. The specific-to-nonspecific binding ratios with [3H]WIN 35,065-2 were higher than with [3H]cocaine in both centrifugation and filtration assays. Pharmacological characterization suggests that [3H]WIN 35,065-2 binds to the dopamine transporter. Mazindol, GBR 12909, nomifensine, and (-)-cocaine are potent inhibitors of [3H]WIN 35,065-2 binding. In contrast, the norepinephrine transporter ligand desipramine is a weak inhibitor, and the serotonin transporter ligand citalopram does not inhibit binding. The effect of sodium on binding was examined under conditions in which (a) the low-affinity site was primarily (87%) occupied and (b) approximately 50% of both sites were occupied. The results indicate that both sites are sodium dependent. Injection of 6-hydroxydopamine into the striatum results in a significant loss of both high- and low-affinity sites, a finding suggesting that both sites are on dopaminergic nerve terminals. Taken together, these data are consistent with the presence of multiple cocaine binding sites associated with the dopamine transporter.     

Evidence for the presence of dopaminergic receptors in vas deferens

Specific dopaminergic recognition sites were identified in membranes prepared from rat vas deferens with the ligand (³H)-haloperidol. Specific binding, defined as the difference of (³H)-haloperidol binding in the presence or absence of an excess of unlabelled haloperidol (100 μM), was saturable and a Scatchard analysis of the data revealed a Kd = 21 nM and a Bmax = 74 fmol/mg prot. (+)-Butaclamol was several times more active in displacing (³H)-haloperidol from binding sites than its pharmacologically inactive enantiomer, (?)-butaclamol, demonstrating stereospecificity of binding. Dopamine displaced 50% of (³H)-haloperidol binding at a concentration of approximately 10 μM, while norepinephrine, epinephrine and serotonin were practically ineffective at this concentration. Our results support the notion that there are dopaminergic receptors in the rat vas deferens. We speculate that some of the known effects of dopamine and dopaminergic drugs on sexual behavior may be mediated peripherally and not solely via the CNS as is usually assumed.     

Binding of [3H]SKF 38393 to Dopamine D-l Receptors in Rat Striatum In Vitro; Estimation of Receptor Molecular Size by Radiation Inactivation

[3H]SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine) binds with high affinity to 3,4-dihydroxyphenylethylamine (dopamine) D-1 receptors in rat striatum in vitro (KD = 7 and 14 nM in nonfrozen and frozen striatum, respectively). The number of binding sites (Bmax) was approximately 80.0 pmol/g of original tissue, a value similar to the Bmax for the dopamine D-1 antagonist SCH 23390. Nondisplaceable [3H]SKF 38393 binding was approximately 45% of total binding. Irradiation (0-4 Mrad) of frozen whole striata decreased the number of [3H]SKF 38393 binding sites monoexponentially without changing the binding affinity. The functional molecular mass for the agonist dopamine D-1 binding site was 132,800 daltons, which is higher than the functional molecular mass of the antagonist dopamine D-1 binding site (approximately 80,000 daltons).     

Binding of a novel dopaminergic agonist radioligand [3H]-fenoldopam (SKF 82526) to D-1 receptors in rat striatum

Fenoldopam (SKF 82526), a dopamine agonist which exhibits D-1 receptor subtype selectivity, was evaluated as a radioligand for this receptor subtype. In saturation studies in rat striatal membrane preparations, [3H]-fenoldopam appeared to label a single binding site with a KD of 2.3 +/- 0.1 nM and a Bmax of 590 +/- 40 fmoles/mg protein. In competition binding experiments, binding was shown to be stereoselective, and rank ordering of affinities of dopaminergic and non-dopaminergic compounds closely correlated with potencies of these compounds in stimulating or inhibiting dopamine-sensitive adenylate cyclase (D-1) and in binding to D-1 sites labelled with the antagonist [3H]-cis-flupenthixol. The most potent competitors were the recently identified D-1 selective antagonists, SCH 23390 and SKF R-83566. [3H]-Fenoldopam was also used to assess agonist/D-1 receptor interactions. The results suggest that [3H]-fenoldopam is a useful and selective agonist radioligand for the D-1 receptor.     

125I-Spiperone: a novel ligand for D2 dopamine receptors

125I-Spiperone binds with high affinity (KD 0.3 nM) to a single specific site (Bmax 34 pmol/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of 125I-spiperone binding (Ki's 1-10 nM). Selective dopamine antagonists such as sulpiride (Ki 50 nM) and dopamine agonists such as apomorphine (Ki 200 nM) are also potent inhibitors. The drug specificity of 125I-spiperone binding correlates well with that of 3H-spiperone binding, providing good evidence that 125I-spiperone labels D2 dopamine receptors in striatal membranes. 125I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D2 dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.     

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.     

3H-dopamine binding to rat striatal D-2 and D-3 sites: Enhancement by magnesium and inhibition by guanine nucleotides and sodium

Previous studies have demonstrated high affinity ³H-dopamine binding sites on mammalian striatal membranes. These putative dopamine receptors of unknown physiological significance have been termed D-3 sites. Such studies have failed, however, to demonstrate high affinity ³H-dopamine binding to D-2 sites, which can be labeled by ³H-butyrophenones, and which represent the putative dopamine receptors most stronly implicated in the behavioral correlates of dopaminergic CNS activity. We now report that preincubation of membrane homogenates with Mg⁺⁺ and inclusion of Mg⁺⁺ (1–10mM) or other divalent metal cations during binding allows high affinity D-2 specific ³H-dopamine binding in rat striatal membranes, and that these ions also increase the Bmax of D-3 specific ³H-dopamine binding. GTP, GDP, and GppNHp can completely abolish all D-2 specific ³H-dopamine binding, while only a magnesium-dependent portion of D-3 sites appears to be GTP sensitive. These data are consistent with the hypothesis that the striatal D-2 receptor exists in two agonist affinity states whose interconversion is effected by guanine nucleotides and divalent metal cations. The GTP sensitive/magnesium dependent nature of ³H-dopamine binding to so-called D-3 sites suggests that some such sites may in fact represent a high agonist-affinity state of the D-1 adenylate cyclase stimulating dopamine receptor also found in this tissue.     

In vitro evaluation of radioiodinated butyrophenones as radiotracer for dopamine receptor study

Radioiodinated butyrophenone compounds are attracting the interest of those working on dopamine receptor studies; structure-activity relationship study has revealed the ortho position of the p-fluorobutyrophenone moiety as a very plausible iodination site. Various synthesized butyrophenones iodinated at the ortho position of p-fluorobutyrophenone moiety, 2′- iodohaloperidol (2′-IHP), 2′-iodotrifluperidol (2′-ITP) and 2′-iodospiperone (2′-ISP) were tested for their abilities to inhibit ³H-spiperone (SP) binding for the dopamine (D-2) receptor, together with reference compounds (SP, haloperidol (HP) and 4-iodospiperone (4- ISP)). The order of binding affinity of the tested compounds was SP > 2′-ISP > HP > 4-ISP > 2′-IHP > 2′- ITP. Whereas, the serotonin (S-2) receptor binding affinity of SP and its iodinated analogues were in the order of SP > > 4-ISP > 2′-ISP. Furthermore, in the saturation binding study using the striatal membrane preparations, the 2′-ISP displayed a K_D of 0.25 nM with maximum number of binding site B_max of 210 fmol/mg protein. These data indicated the 2′-ISP as holding high affinity for dopamine receptors and a low affinity for serotonin receptors. Thus, the ¹²⁵I-2′-ISP was a very potent radioligand for in vitro dopamine (D-2) receptor studies, and ¹²³I-2′-ISP holds very promising characteristics as for in vivo dopamine receptor studies, as well.     

Solubilization of the D-1 dopamine receptor from rat striatum

The D-1 dopamine receptor was extracted from rat striatal membranes with 0.7% sodium cholate and 1 M NaCl. Pretreatment of the membranes with a D-1 specific agonist, inclusion of crude phospholipids in the solubilization buffer, and subsequent removal of the detergent led to a maximal extraction of 48% of the receptor binding sites. The D-1 antagonist, [125I]SCH 23982, bound to single class of sites with a Kd of 1.8 nM and a Bmax of 1.65 pmol/mg protein. The solubilized receptors retained the ability to discriminate between active and inactive enantiomers of agonists and antagonists selective for the D-1 receptor.     

Photoaffinity labeling of D-2 dopamine binding subunits from rat striatum, anterior pituitary and olfactory bulb with a new probe, [3H]azidosulpride

A novel photoaffinity probe [3H] azidosulpride has been developed for biochemical studies of D-2 dopamine receptors. This ligand binds to the receptors with high affinity (Kd = 3.1 +/- 0.2 nM) and, upon photoactivation, about 20% of the radioactivity bound to membranes becomes covalently incorporated. More than 90% of this irreversible binding is protectable by dopaminergic agents including D-2 selective compounds, whereas D-1 selective and non-dopaminergic compounds are ineffective. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals a single band at Mr = 85 kDa for labeled receptors in striatum, anterior pituitary or olfactory bulb, where pharmacologically distinct binding sites have been previously detected.     

Dopaminergic 3H-agonist receptors in rat brain. New evidence on localization and pharmacology

Recent methodological advances have allowed the reliable assay of specific dopaminergic 3H-agonist binding sites in rat striatum. Successful assay depends on preincubation of tissue homogenates at 37 degrees C; this results in a guanyl nucleotide-sensitive and dopamine (DA)-dependent increase in the density (Bmax) of 3H-agonist binding. Lesions of DA terminals or drugs which deplete DA levels prevent the preincubation-induced increase in binding, and this effect is completely reversible by preincubation with added DA. In contrast, kainic acid lesions irreversibly reduce 3H-agonist binding. It is concluded that the evidence supporting the existence of presynaptic "D-3" sites is artefactual and that 3H-DA binding sites are more likely related to post-synaptic receptors. 3H-DA binding involves two sites, one of which has pharmacologic properties similar to D-1 receptors, whereas the other resembles D-2 receptors. The affinity of 15 antipsychotic drugs for 3H-haloperidol binding sites was highly correlated (R = 0.94) with their inhibitory potency at a subset of 3H-DA binding sites. However, the inhibition of 3H-DA binding by antipsychotic drugs was noncompetitive. These findings can be explained by an allosteric model, whereby antagonists bind to a site different from but allosterically linked to a high-affinity 3H-DA binding site.     

Effect of long-term L-dopa administration on the dopaminergic and cholinergic (muscarinic) receptors of striatum in 6-hydroxydopamine lesioned rats

L-Dihydroxyphenylalanine (L-Dopa) (200 mg/kg/day) was administered for 30 days to the rats whose nigrostriatal dopamine pathway was lesioned unilaterally with 6-hydroxydopamine and the receptor binding of ³H-spiperone and ³H-quinuclidinyl benzilate (³HQNB) was measured in the dopaminergic and muscarinic cholinergic receptors of the striatum. ³H-spiperone binding increased by 73% and ³HQNB binding decreased by 14% in the lesioned side when compared to the control side of L-Dopa-non-treated rats. ³H-spiperone binding was measured in the lesioned sides of L-Dopa-treated and L-Dopa-non-treated rats and was found to have decreased by 21% in the former. In the control side of the L-Dopa-treated lesioned rats, however, ³H-spiperone binding increased by 27% when compared to the opposite striatum of the same rats. ³HQNB binding in the lesioned side of L-Dopa-treated rats was not significantly different from that of the control side statistically. These results suggest that changes in functional equilibrium between the dopaminergic and cholinergic mechanisms influence the muscarinic cholinergic receptors and that supersensitivity of dopamine receptors after lesion of the nigrostriatal pathway also remains after long-term L-Dopa treatment.     

Pharmacological characterization and autoradiographic localization of substance P receptors in guinea pig brain

[3H]Substance P ([3H]SP) was used to characterize substance P (SP) receptor binding sites in guinea pig brain using membrane preparations and in vitro receptor autoradiography. Curvilinear Scatchard analysis shows that [3H]SP binds to a high affinity site (Kd = 0.5 nM) with a Bmax of 16.4 fmol/mg protein and a low affinity site (Kd = 29.6 nM) with a Bmax of 189.1 fmol/mg protein. Monovalent cations generally inhibit [3H]SP binding while divalent cations substantially increased it. The ligand selectivity pattern is generally similar to the one observed in rat brain membrane preparation with SP being more potent than SP fragments and other tachykinins. However, the potency of various nucleotides is different with GMP-PNP greater than GDP greater than GTP. The autoradiographic distribution of [3H]SP binding sites shows that high amounts of sites are present in the hippocampus, striatum, olfactory bulb, central nucleus of the amygdala, certain thalamic nuclei and superior colliculus. The cortex is moderately enriched in [3H]SP binding sites while the substantia nigra contains only very low amounts of sites. Thus, the autoradiographic distribution of SP binding sites is fairly similar in both rat and guinea pig brain.