Binding of [3H]idazoxan and of its methoxy derivative [3H] RX821002 in human fat cells: [3H]idazoxan but not [3H] RX821002 labels additional non-alpha 2-adrenergic binding sites

Binding studies were carried out in human fat cell membranes with two alpha 2-adrenergic antagonists, [3H]idazoxan and its methoxy derivative [3H]RX821002. Inhibition studies with epinephrine enantiomers indicate that [3H]RX821002 only binds to alpha 2-adrenoceptors, whereas [3H]idazoxan labels alpha 2-adrenoceptors and additional nonadrenergic sites (NAIBS). NAIBS and alpha 2-adrenoceptors display different affinities towards drugs from various chemical families. Imidazoline and some guanidine derivatives exhibit a high affinity for NAIBS. Pharmacological studies of human NAIBS indicate that they are slightly different from those previously reported in the rabbit, suggesting the existence of several subtypes of NAIBS. Furthermore, NAIBS are different from the previously described "imidazoline-preferring sites." [3H]idazoxan and [3H]RX821002 saturation analyses were performed in human adipocytes from different anatomical locations, in order to compare the number of NAIBS and alpha 2-adrenoceptors. Although there was an important variation in NAIBS and alpha 2-adrenoceptor numbers in the studied samples, a very poor correlation was obtained between the Bmax values of the two sites. Moreover, alkylation of alpha 2-adrenoceptors by phenoxybenzamine produces a 90% reduction in accessible [3H]RX821002 binding sites, without modification of [3H]idazoxan binding. These data show that NAIBS are not closely related to the alpha 2-adrenergic molecule. In addition, benextramine appears to be a reversible competitor at NAIBS. [3H]idazoxan binding, but not [3H]RX821002 binding, is sensitive to K+, suggesting that the domains involved in the ligand-NAIBS interaction are different from those involved in the ligand-alpha 2-adrenoceptor interaction.     

Effect of some antiarrhythmics on [3H]clonidine binding to alpha 2-adrenergic receptors

The effects of the antiarrhythmics quinidine, propranolol, procainamide and lidocaine were determined on the specific binding of [3H]clonidine to the alpha 2-adrenergic receptor. Quinidine is effective in decreasing specific [3H]clonidine binding to the alpha 2 receptor within the range of therapeutic blood levels (Ki = 6 x 10 (-8) M). The effectiveness of all compounds tested, with the exception of quinidine and procainamide, correlate with the membrane/buffer partition coefficient, suggesting a relationship with the local anesthetic activity.     

Characterization of alpha 2-adrenergic receptors of calf retina membranes by [3H]-rauwolscine and [3H]-RX 781094 binding

Alpha 2-adrenergic receptors were identified in calf retina membranes by binding of the radiolabelled antagonists [3H]-RX 781094 and [3H]-rauwolscine. When 10 microM phentolamine was used to determine the non-specific binding, both radioligands labelled a single class of non-cooperative sites: Bmax = 1051 +/- 252 fmol/mg protein, Kd = 5.1 +/- 1.5 nM for [3H]-RX 78104 and Bmax = 1167 +/- 449 fmol/mg protein, Kd = 21.0 +/- 4.1 nM for [3H]-rauwolscine. Competition binding experiments showed the typical pharmacological potency order of alpha 2-adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin. Agonist competition binding curves revealed the presence of two receptor populations, having respectively high affinity (70% of the total receptor population) and low affinity for agonists, but with the same affinity for the antagonists. The high affinity sites could be converted into low affinity sites by guanine nucleotides. The non-specific binding of [3H]-RX 781094 was the same if 0.1 mM (-)-epinephrine was used instead of phentolamine. In contrast, the non-specific binding of [3H]-rauwolscine was markedly lower with (-)-epinephrine than with phentolamine. Under this condition, the Scatchard plot of [3H]-rauwolscine saturation binding was curvilinear, indicating the presence of low affinity sites for the radioligand in addition to alpha 2-adrenergic receptors. Competition binding experiments revealed that these low affinity sites were distinct from adrenergic receptors: the catecholamine agonists (-)- and (-)-epinephrine, (-)-norepinephrine, (-)-isoproterenol and dopamine competed with similar Ki values (microM range) whereas clonidine did not interact. Furthermore, these sites bound reserpine and the alpha 2-adrenergic antagonists yohimbine and rauwolscine but not phentolamine.     

Identification of alpha 2-adrenergic receptors in human fat cell membranes by [3H]chlonidine binding

3H-clonidine bound to membrane sites of human fat cells, which have the characteristics of alpha 2-adrenoceptors. Specific binding was rapid, reversible and saturable. [3H]Clonidine binding was of high affinity with a KD of 3.9 nM and with a maximal occupancy of 348 fmol/mg protein. The correlation between alpha-adrenergic agonist or antagonist affinities for the membrane [3H]clonidine binding site with their physiological potencies demonstrates the usefulness of the human fat cell as a model for investigating postsynaptic alpha 2-adrenoceptor properties and regulation.     

Autoradiographic comparison of [3H]-clonidine binding to non-adrenergic sites and alpha(2)-adrenergic receptors in human brain.

Clonidine is a partial agonist at brain alpha(2)-adrenoceptors (alpha(2)AR), but also has high affinity (K(D) = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and alpha(2)AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 microM norepinephrine (NE; alpha(2)AR agonist) or 12.5 microM methoxy-idazoxan (MIDX; selective alpha(2)AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 microM cirazoline. Levels of [3H]-clonidine binding to alpha(2)AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r(2) = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: alpha(2)AR (>4:1). Quantitative transepts drawn across hippocampal images revealed alpha(2)AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus-areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I(1) and I(2) subtypes. The rank-order of affinities were cirazoline > harmane > BDF6143 > idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 +/- 12 nM). In conclusion: 1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from alpha(2)AR; and 2) the affinities of these sites was distinct from alpha(1)AR, alpha(2)AR, I(1) or I(2) sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.     

Evidence for non-adrenergic binding sites for [3H]idazoxan in the smooth muscle of rabbit urethra

The binding characteristics of [3H]idazoxan and [3H]rauwolscine, two potent alpha 2-adrenoceptor antagonists, were compared in the rabbit urethral smooth muscle. The maximal binding capacity was 6 times higher for [3H]idazoxan than for [3H]rauwolscine in male rabbits. No difference was observed for these radioligands in female rabbits. There were marked differences in the ability of alpha 2-adrenergic compounds to inhibit [3H]idazoxan and [3H]rauwolscine binding. These results were consistent with the existence of non-alpha 2-adrenoceptor sites for [3H]idazoxan in the rabbit urethral smooth muscle.     

Non-adrenergic binding sites for the "alpha 2-antagonist" [3H]idazoxan in the rabbit urethral smooth muscle. Pharmacological and biochemical characterization.

In the present study, pharmacological and biochemical binding characteristics of [3H]idazoxan, an originally thought alpha 2-adrenoceptor antagonist, have been determined in smooth muscle of rabbit urethra. It is shown that [3H]idazoxan labels with high affinity non-adrenergic binding sites. Specific binding of [3H]idazoxan is inhibited by compounds possessing an imidazoline or a guanidinium moiety whereas phenylethanolamines and classical alpha 2-antagonists are ineffective competitors which suggests an imidazoline-preferring binding site. However, imidazolidines such as clonidine and paminoclonidine are poorly effective, which differs considerably from pharmacological characteristics of imidazoline binding sites previously reported in the central nervous system. In addition, it is shown that K+ and Mn2+ inhibit [3H]idazoxan binding in a competitive and non-competitive manner, respectively. Other cations such as Na+, Li+ and Mg2+ have no significant effect. It is shown that K+ accelerates the dissociation of [3H]idazoxan binding while Mn2+ does not produce any modification. These results suggest that K+ may bind to an allosteric site, while Mn2+ may bind with a membrane component susceptible to alter [3H]idazoxan binding sites.     

Characterization of alpha 2-adrenergic receptors in the human colon adenocarcinoma cell line HT 29 in culture by [3H]yohimbine binding

The presence of specific binding sites for [3H]yohimbine, a labelled alpha 2-adrenergic agent, on crude membranes of HT 29 cells established in culture from a human colon adenocarcinoma, is reported. The estimated affinity and number of sites (KD = 6.3 +/- 0.9 nM; Bmax = 224 +/- 28 fmol/mg protein) as well as the relative potencies of adrenergic agonists (clonidine greater than phenylephrine greater than amidephrine) and adrenergic antagonists (yohimbine greater than dihydroergotamine much greater than prazosin) to displace [3H]yohimbine binding indicate that the yohimbine sites of these cancer cells have similar characteristics to the alpha 2-adrenergic receptors described in other tissues.     

3H]idazoxan and some other alpha 2-adrenergic drugs also bind with high affinity to a nonadrenergic site

We compared the pharmacological properties of the alpha 2-adrenergic radioligand [3H]idazoxan with those of [3H]rauwolscine in rat and [3H]yohimbine in human renal cortical membranes. The density of "specific" [3H]idazoxan binding sites (defined by 100 microM tolazoline) was twice as high as that of [3H]rauwolscine in rat kidney and four times as high as that of [3H]yohimbine in human kidney. A variety of structurally different drugs fully competed for specific [3H]rauwolscine and [3H]yohimbine binding, with affinities appropriate for the interaction with alpha 2-adrenergic receptors. Specific [3H]idazoxan binding, however, was only partially competed for by the catecholamines epinephrine and norepinephrine in both tissues. Thus, [3H]idazoxan labels both alpha 2-adrenergic receptors and a nonadrenergic site. Clonidine, B-HT 920, moxonidine, phentolamine, prazosin, yohimbine, dopamine, and serotonin also could not compete for this site. However, UK 14,304, guanabenz, indanidine, tolazoline, oxymetazoline, and SK&F 104,078 competed for the additional [3H]idazoxan sites with affinities similar to those at alpha 2-adrenergic receptors. [3H]idazoxan binding substantially in excess of [3H]rauwolscine or [3H]yohimbine binding was also found in human platelets, myometrium, and erythroleukemia (HEL) cells but not in three cell lines lacking alpha 2-receptors (MDCK, BC3H1, and Jurkat cells). Although we have been unsuccessful thus far in defining the precise nature of the additional [3H]idazoxan binding sites, we hypothesize that these sites may be closely affiliated with alpha 2-adrenergic receptors but clearly distinct from the catecholamine binding site of the receptor. The results indicate that care must be taken in the use of [3H]idazoxan or drugs that are recognized at its nonadrenergic site when studying alpha 2-adrenergic effects and receptor subtypes.     

3H]yohimbine and [3H]idazoxan bind to different sites on rabbit forebrain and kidney membranes

The binding of the alpha 2-adrenoceptor ligands [3H]yohimbine and [3H]idazoxan to rabbit kidney and forebrain membranes was compared. The maximum number of [3H]yohimbine binding sites was higher than the number of [3H]idazoxan binding sites in forebrain and lower in kidney. Large differences were observed in the ability of noradrenaline, adrenaline, idazoxan, rauwolscine, yohimbine and WY 26392 to displace [3H]yohimbine and [3H]idazoxan from their binding sites. These data suggest that [3H]idazoxan and [3H]yohimbine bind to different sites on rabbit tissue membranes.     

3H]clonidine and [3H]yohimbine binding to solubilized alpha 2-adrenoceptors from rat cerebral cortex

Alpha 2-adrenoceptors were solubilized from rat cerebral cortex using the zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The CHAPS extract retained binding activity for [3H]clonidine and [3H]yohimbine. Treatment of membranes with 10 mM CHAPS solubilized about 30% of the [3H]clonidine binding sites in the starting membranes. A Scatchard plot of [3H]clonidine binding to the CHAPS extract showed a non-linear curve, indicating the existence of the two distinct binding components. The effects of GTP and cations on alpha 2-agonist and antagonist binding to the CHAPS extract were similar to the effects in membrane preparations. Sepharose CL-4B column chromatography showed the alpha 2-agonist binding complex to be a larger molecule, with a Stokes radius of 85 A, than the alpha 2-antagonist binding complex with a radius of 71 A. These results indicate that the complexes between the alpha 2-adrenoceptors and GTP binding regulatory proteins remain intact throughout the CHAPS solubilization procedure.     

Three distinct binding sites for [3H]-prazosin in the rat cerebral cortex.

1. The putative alpha 1-adrenoceptor subtypes of rat cerebral cortex membranes were characterized in binding. 2. Specific binding of [3H]-prazosin was saturable between 20-5000 pm. Scatchard plots of the binding data were non-linear, indicating the presence of two distinct affinity sites for prazosin (pKD, high = 10.18, Rhigh = 308 fmol mg-1 protein; pKD, low = 8.96, Rlow = 221 fmol mg-1 protein). 3. In the membranes pretreated with chlorethylclonidine (CEC) two affinity sites for prazosin were also observed: the affinities were similar to those without CEC pretreatment, but the maximum numbers of binding sites were reduced by CEC pretreatment to 23 and 62% for prazosin-high (Rhigh) and low affinity sites (Rlow), respectively. 4. The prazosin-high affinity sites were further subdivided into two subclasses by WB4101(2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane) and phentolamine; the low affinity sites for WB4101 and phentolamine were more potently inactivated by CEC as compared with the high affinity sites. On the other hand, prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)- amino )-propyl)benzeneacetonitrile fumarate) and yohimbine inhibited [3H]-prazosin binding to prazosin-high affinity sites monophasically. 5. In addition to the high affinity sites, the prazosin-low affinity sites were labelled at high concentrations of [3H]-prazosin. Thus, prazosin and WB4101 showed shallow displacement curves. On the other hand, HV723 and yohimbine did not discriminate between prazosin-high and low affinity sites. 6. Two distinct alpha 1-adrenoceptor subclassifications have been recently proposed (alpha 1A, alpha 1B subtypes and alpha 1H, alpha 1L, alpha 1N subtypes).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of binding sites for [3H]spiroperidol

Experiments were designed to investigate the biochemical properties of binding sites for [3H]spiroperidol ([3H]SPD) solubilized from canine caudate and to define the effect of detergent on the binding of the radioligand. Extraction of canine caudate with 0.75-1.0% digitonin was found to generate the maximum yield of binding sites for [3H]SPD while minimizing extraction of membrane proteins. Although binding sites were solubilized with 1.0% digitonin, a 10-fold reduction in detergent concentration was necessary to achieve maximal binding of [3H]SPD. The rank order of affinity for agonists and antagonists was consistent with the pharmacologic properties of the D2 subtype of the dopamine receptor. However, the binding of antagonists was found to be complex. Studies with some preparations of pooled canine caudate resulted in competition curves for the D2-selective antagonists domperidone and sulpiride that best fit a single-site model. Other preparations exhibited biphasic inhibition curves with these antagonists. The class of binding sites for [3H]SPD with low affinity for D2-selective antagonists constituted as much as 30-40% of the binding sites. Enrichment of solubilized binding sites for [3H]SPD was achieved by size exclusion HPLC followed by adsorption to DEAE-Sephadex and elution with buffer of increasing ionic strength. Enrichment of binding sites was accompanied by a decrease in the affinity of solubilized sites for [3H]SPD.     

3H]idazoxan binding at non-alpha 2-adrenoceptors in rabbit adipocyte membranes

The imidazoline ligand, [3H]idazoxan, labels a large population of high-affinity binding sites in rabbit fat cell membranes (Bmax = 1370 +/- 91 fmol/mg protein; KD = 1.6 +/- 0.6 nM) when imidazoline derivatives are used for definition of non-specific binding. [3H]Idazoxan sites are not alpha 2-adrenoceptors as assessed by competition studies which showed that epinephrine, norepinephrine and yohimbine do not inhibit [3H]idazoxan binding. Naphazoline, tramazoline and the Na+/H+ exchange inhibitor, amiloride, completely inhibited [3H]idazoxan binding. The Ki values were 9, 27 and 48 nM, respectively.     

Characterization of alpha 1-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]WB4104 and [3H]prazosin binding

[3H]Prazosin and [3H]WB4101 [2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4 benzodioxane] have both been proposed to label alpha 1-adrenergic receptors in the rat central nervous system. As many discrepancies between the binding of these two ligands have arisen, we conducted these studies in order to reevaluate their binding characteristics and resolve the similarities and differences in the pharmacological characteristics of their respective binding sites. [3H]Prazosin binding is characterized by a monophasic saturation isotherm. Prazosin, indoramine, and dihydroergocryptine competitions with [3H]prazosin are steep and monophasic, and model best to a single binding site. In contrast, phentolamine and WB4101 competition curves are shallow in rat cortex, exhibiting Hill coefficients significantly less than 1.0, and model to two binding sites of approximately equal proportions. The higher and lower affinity components are defined as alpha 1A and alpha 1B, respectively. [3H]WB4101 also labels two binding sites in rat cortex and hippocampus with picomolar and nanomolar affinity, respectively. However, the nanomolar binding site is serotonergic and not adrenergic. The picomolar site (KD = 150 pm) has characteristics of an alpha 1-receptor binding site: prazosin, WB4101, and phentolamine affinities for this [3H]WB4101 binding site correlate with their affinities for the highest affinity component (alpha 1A) of [3H]prazosin binding. In addition, the Bmax of this [3H] WB4101-labeled site is equal to one-half of the total [3H]prazosin Bmax. Agonist competitions with [3H]prazosin binding are multiphasic with pseudo-Hill slopes less than 1.0 and with a rank order of affinity of epinephrine greater than norepinephrine greater than phenylephrine. When binding to the alpha 1A component is blocked by a 30 nM phentolamine mask, the same rank order of agonist affinities is preserved. Although the affinities of epinephrine and norepinephrine at the two subtypes are identical, phenylephrine is weaker at the alpha 1B site. The ratio of the potency of phentolamine versus prazosin is about 4 at the alpha 1A component but about 80 at the alpha 1B binding site. We discuss these data in relation to the reported potencies of these antagonists in blocking alpha 1-receptor-mediated responses which may correlate with our designation of alpha 1A or alpha 1B binding sites.     

Distinctions between ligand-binding sites for [3H]dopamine and D2 dopaminergic receptors characterized with [3H]spiroperidol

The binding of [3H]Hspiroperidol to D2 dopaminergic receptors in rat striatum was compared to the binding of [3H]dopamine to its binding sites. Both radioligands labeled apparently homogeneous populations of high affinity, stereoselective, saturable sites, determined from analysis of saturation isotherms. [3H]Spiroperidol bound to more than twice as many sites as [3H]dopamine, and antagonist/[3H]spiroperidol competition data were consistent with a single population of receptors. Antipsychotic drugs competed with both high and low affinities for two fractions of [3H]dopamine-binding sites, but for most drugs, their potencies at even the high affinity component were significantly less than their affinities at D2 receptors. The [3H]dopamine-binding sites were altered by kainic acid lesions of the striatum, phenoxybenzamine treatment of tissue homogenates, or reserpine pretreatment of rats. These changes were different from previous reports of alterations in either D2 or D1 dopaminergic receptors. These and other differences in binding properties suggest that [3H]dopamine binds to sites distinct from either D1 or D2 receptors.     

Binding characteristics of the selective alpha 2-adrenoceptor antagonist [3H]idazoxan to rat olfactory cortex membranes

[3H]Idazoxan binding to membranes prepared from rat olfactory cortex obeyed saturation kinetics and was to a single population of sites. Although the density of sites was dependent on the incubation medium, binding was of high affinity (KD approximately 5.5 nM) with a Hill coefficient close to unity. Competition studies with a range of adrenoceptor agonists and antagonists confirmed that [3H]idazoxan binding was to alpha 2-adrenoceptors. Neither chemical lesions with the neurotoxin kainic acid nor chronic unilateral bulbectomy significantly altered any of the [3H]idazoxan binding parameters. These findings suggest that alpha 2-adrenoceptors are not located on the lateral olfactory tract terminals or pyramidal cells of the olfactory cortex.