Imidazoline-guanidinium and alpha 2-adrenergic binding sites in basolateral membranes from human kidney.

In the present study, we used [3H]idazoxan and [3H]rauwolscine to characterize the imidazoline-guanidinium receptive site (IGRS) and alpha 2-adrenoceptors in the human renal proximal tubule, respectively. In purified basolateral membranes, 11-fold enriched in Na(+)-K+ ATPase. [3H]idazoxan and [3H]rauwolscine binding was twofold higher than in homogenates ([3H]idazoxan: 87 +/- 19 vs. 45 +/- 23.3 fmol/mg protein, P less than 0.05; [3H]rauwolscine: 56.4 +/- 21.4 vs. 25.2 +/- 7.3 fmol/mg protein, P less than 0.01). In competition studies performed at saturating concentration of [3H]idazoxan (15 NM), specific binding was competed for by epinephrine and rauwolscine only by 10-15% but was completely inhibited by imidazoline and guanidinium compounds. Thus, in human renal proximal tubule. [3H]idazoxan mainly binds to an IGRS. The highest density of alpha 2-adrenoceptors in basolateral membranes and of IGRS in partially purified membrane preparations, suggests that these two binding sites have a different subcellular localization. When compared to the rabbit renal IGRS, the human [3H]idazoxan binding site displays different affinities for guanabenz, rilmenidine, clonidine, amiloride and its derivatives that persist after membrane solubilization. In contrast, the human and rabbit renal IGRS share similar regulatory properties such as the sensitivity to K+ and the insensitivity to Na+, divalent cations and 5'-guanylylimidodiphosphate (Gpp(NH)p). In conclusion, we demonstrated that, in the human renal proximal tubule, alpha 2-adrenoceptors are mainly located in basolateral membranes while IGRS appear to be associated with another cell compartment. As indicated by their common interaction with imidazoline and guanidinium derivatives and by similar regulatory properties, human and rabbit IGRS belong to the same family of membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of rat liver alpha 1-adrenergic receptors. Agonist specific alteration in receptor binding affinity

An improved method for the solubilization of the alpha 1-adrenergic receptors in rat liver, utilizing digitonin, glycerol and sonication, is described. The yield of solubilized receptors was approximately 20%. The soluble receptors showed characteristics similar to the membrane-bound alpha 1 receptors. However, upon solubilization, the affinity for the agonists (-)norepinephrine and (-)epinephrine increased 35- to 66-fold when compared to the affinity in the membranes. The affinity for antagonists remained unchanged. A number of synthetic partial agonists showed a less marked (5- to 10-fold) increase in affinity upon solubilization. These data are consistent with the notion that these receptors might be capable of existing in two distinct conformational states with the high affinity state for agonists being favored by solubilization.     

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.     

Selective interaction of beta 2- and alpha 2-adrenergic receptors with stimulatory and inhibitory guanine nucleotide-binding proteins.

In Chinese hamster ovary cells expressing recombinant beta 2-adrenergic receptors, isoproterenol enhanced cholera toxin-catalyzed ADP-ribosylation of the large form of G5 alpha. The effect was stereoselectively blocked by the enantiomers of propranolol, indicating receptor mediation. The ADP-ribosylated form of Gs alpha-subunit was resolved into a triplet in gradient gels. beta 2-Adrenergic receptors increased both the labelling and the apparent mass of the slower migrating forms of large Gs alpha, as determined by autoradiography and immunoblotting, suggesting that Gs alpha, can incorporate more than one ADP-ribose per molecule. In cells coexpressing similar amounts of beta 2-adrenergic, alpha 2-adrenergic, and m1 muscarinic receptors, beta 2 receptors stimulated the ADP-ribosylation of only large Gs and alpha 2 receptors that of only Gi; muscarinic receptors had no apparent effect. Thus, in native membranes there appears to be a selectivity for the interaction between adrenergic receptor subtypes and Gs alpha or Gi alpha subunits.     

Age-dependent decrease of alpha 2-adrenergic receptor number in human platelets

In 36 healthy subjects of various ages (14-76 years) the number of alpha 2-adrenergic receptors in platelets - as determined by [3H]yohimbine binding - and plasma catecholamine levels were measured. A highly significant negative correlation (r = -0.666, P less than 0.001) between the number of alpha 2-adrenergic receptors and age was found; on the contrary, plasma catecholamine concentrations increased with increasing age. Thus, reduced responses in the elderly to adrenergic stimuli may be due to reduced number of adrenergic receptors.     

Agonist binding to rat brain somatostatin receptors alters the interaction of the receptors with guanine nucleotide-binding regulatory proteins.

To investigate the interaction of guanine nucleotide-binding regulatory proteins (G proteins) with the agonist-bound brain somatostatin (SRIF) receptor, rat brain SRIF receptor/G protein complexes were solubilized and immunoprecipitated with peptide-directed antisera selective for the different subtypes of G protein alpha subunits (G alpha). In the absence of agonist, solubilized SRIF receptor/G proteins complexes could be immunoprecipitated by antiserum 8730, which is directed against the carboxyl-terminal region of Gi alpha and recognizes all Gi alpha subtypes, and by antiserum 3646, which selectively interacts with internal regions of Gi alpha 1. In contrast, antiserum 1521, which is directed against an internal region of Gi alpha 2, and antiserum 9072, which is directed against the carboxyl-terminal region of Go alpha, did not immunoprecipitate the SRIF receptor. After the binding of agonist to solubilized SRIF receptors, antisera 9072 and 1521, as well as antisera 8730 and 3646, were able to immunoprecipitate the agonist-bound SRIF receptor/G protein complexes, indicating that agonist interaction with SRIF receptors maintained receptor association with Gi alpha 1 and promoted receptor association with Go alpha and, to a lesser extent, Gi alpha 2. Antiserum 1518, which is directed against Gi alpha 3, uncoupled SRIF receptors from Gi alpha and did not immunoprecipitate the agonist-bound or agonist-free brain SRIF receptor. These findings indicate that differences exist in the interaction of the agonist-free and agonist-bound SRIF receptors with G proteins. The binding of agonists to SRIF receptors promotes the association of the receptor with Go alpha and, to a lesser extent, Gi alpha 2, indicating that these G proteins, along with Gi alpha 1 and Gi alpha 3, may be involved in coupling SRIF receptors to cellular effector systems.     

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.     

Pharmacological characterization of rat alpha 2-adrenergic receptors.

We described previously the molecular characterization of a rat alpha 2B-adrenergic receptor and have shown also that the rat genome contains three closely related alpha 2-adrenergic receptor genes. To characterize the ligand-binding properties of these receptor gene products, we expressed the DNAs encoding these receptors individually in COS-1 cells and studied their binding to a wide variety of typical and atypical adrenergic ligands. The receptors displayed high affinity binding to the radioligand [3H] rauwolscine, with equilibrium dissociation constants ranging from 1.4 to 2.8 nM. Kinetic analysis of the binding of [3H]rauwolscine to membranes from transfected cells was in very good agreement with data obtained from saturation analysis. We examined the ability of a number of agents to compete for the binding of [3H]rauwolscine to the alpha 2-adrenergic receptor-transfected membranes. Whereas one of these receptors displayed a pharmacological profile typical of an alpha 2A-adrenergic receptor, the other two receptors showed similar pharmacological properties characteristic of an alpha 2B-adrenergic receptor. The two alpha 2B-like adrenergic receptors differed, however, in the ratios of Ki values for oxymetazoline and prazosin, as well as the Ki ratio of prazosin and yohimbine. In addition, the two alpha 2B-like adrenergic receptors had a 9-fold difference in affinity for chlorpromazine. The pharmacological characterization of the three rat alpha 2-adrenergic receptor gene products is consistent with the known pharmacology of alpha 2-adrenergic receptors, as documented using tissues and cell lines.     

alpha 2-Adrenergic receptors in human polymorphonuclear leukocyte membranes

Human polymorphonuclear cell membranes contain alpha 2-adrenergic receptors which are measured by binding of the alpha 2-adrenergic antagonist [3H]yohimbine. The alpha 1-adrenergic antagonist [3H]prazosin showed no specific binding. High and low affinity sites were detected which had Kd values of 2.38 +/- 0.4 and 139 +/- 12 nM, respectively, and which bound maximally 4.82 +/- 0.9 and 81 +/- 9 fmoles of [3H]yohimbine/mg membrane protein. The high and low affinity sites were also detected by competition studies with phentolamine, epinephrine and norepinephrine and by dissociation kinetics of bound [3H]yohimbine. [3H]Yohimbine binding was stereospecifically inhibited by (-)- and (+)-epinephrine and norepinephrine. [3H]Yohimbine binding to intact cells showed about 500 high affinity sites per cell (Kd 0.5 nM) and approximately 4000 lower affinity sites per cell (Kd 3-4 nM). Yohimbine enhanced the (-)-norepinephrine stimulation of cAMP production in intact cells.     

Agonist binding and Gq-stimulating activities of the purified human P2Y1 receptor

The human P2Y1 receptor (P2Y1-R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y1-R antagonist [3H]MRS2279 ([3H]2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. Ki values determined in [3H]MRS2279 binding assays for antagonists with the purified P2Y1-R were in good agreement with the Ki and KB values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y1-R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) > ADP = 2-methylthioATP = adenosine-5'-O-(3-thio)triphosphate = adenosine-5'-O(2-thiodiphosphate) > ATP was obtained. The signaling activity of the purified P2Y1-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y1-R and Galpha(q)beta(1)gamma(2) was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC50 values of agonists for activation of the purified P2Y1-R were similar to their respective Ki values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC50 and Ki values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-beta1 were potent and efficacious GTPase-activating proteins for Galphaq and Galpha11 in P2Y1-R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y1-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.     

Nonadrenergic [3H]idazoxan binding sites are physically distinct from alpha 2-adrenergic receptors

We have recently demonstrated that the alpha 2-adrenergic radioligand [3H]idazoxan also labels additional sites that do not recognize catecholamines but bind with high affinity several chemically distinct drugs previously assumed to be highly selective for alpha 2-adrenergic receptors [Mol. Pharmacol. 35:324-330 (1989)]. We now have used three approaches to distinguish the nonadrenergic [3H]idazoxan sites from alpha 2-adrenergic receptors. (a) No nonadrenergic [3H]idazoxan binding sites were found in COS-7 cells transfected with the genes for the two known alpha 2-adrenergic receptor subtypes. (b) The ratio of alpha 2-adrenergic and nonadrenergic [3H]idazoxan sites in human platelet membranes varied considerably between various donors. (c) Highly purified platelet plasma membranes were enriched for alpha 2-adrenergic receptors but did not contain any nonadrenergic [3H]idazoxan binding sites. We conclude that the nonadrenergic [3H]idazoxan binding sites are not co-expressed with alpha 2-adrenergic receptors and at least in human platelets may be located in an intracellular compartment.     

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.     

Compartmentation of alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells

We have identified alpha 2-adrenergic receptors on human erythroleukemia (HEL) cells, a suspension-grown cell line related to human platelets. properties of receptors were assessed in intact cells by binding of the antagonist [3H]yohimbine and by inhibition of cAMP accumulation. [3H]Yohimbine labeled 5900 +/- 2100 receptors/cell with a Kd of 3.6 +/- 0.9 nM (n = 7). alpha 2-Adrenergic receptors were potently coupled to inhibition of adenylate cyclase, with EC50 values for epinephrine, UK-14,304, and p-aminoclonidine in the low nM range. Treatment of cells with pertussis toxin abolished this response. In radioligand binding studies with membrane preparations [3H]yohimbine and [3H]UK-14,304 bound to the same number of sites (71 versus 69 fmol/mg of protein), and epinephrine competed for [3H]yohimbine binding in a biphasic manner. After addition of GTP, no high affinity [3H]UK-14,304 binding was detected, and epinephrine competed for [3H]yohimbine binding with lower affinity at both 4 degrees and 37 degrees. In studies with intact cells, we detected no specific binding of [3H]UK-14,304 at either 37 degrees or 4 degrees. At 37 degrees, epinephrine competed for all [3H]yohimbine binding sites with a low apparent affinity (Ki = 21 microM), whereas at 4 degrees epinephrine (up to 1 mM) was able to compete for only 59 +/- 13% of [3H]yohimbine-binding sites. The potency of epinephrine in competing for [3H]yohimbine sites in intact cells at 4 degrees was greater than at 37 degrees (Ki = 1 microM) and was similar to that observed with membranes in the presence of GTP. We hypothesize that sites not detectable by epinephrine at 4 degrees are sequestered within the cell. Treatment of HEL cells with pertussis toxin reduced the proportion of receptors on the surface from 51 +/- 12% to 23 +/- 7% (n = 3, p less than 0.05) of the total sites. Treatment of HEL cells with epinephrine (100 microM, 1 hr) reduced the cell surface component to 25 +/- 8% (n = 3) of the total sites. This treatment was not accompanied by significant desensitization of the ability of epinephrine to inhibit cAMP accumulation. We conclude that alpha 2-adrenergic receptors exist in more than one compartment in HEL cells and that interaction of receptors with a guanine nucleotide-binding protein or with agonist may regulate this compartmentation. These cells provide a new model system for the study of expression and metabolism of alpha 2-adrenergic receptors.     

Interaction of purified bovine brain A1-adenosine receptors with guanine nucleotide-binding proteins of human platelet membranes following reconstitution

A1-adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) have been co-purified from bovine cerebral cortex by agonist affinity chromatography [J. Biol. Chem. 264:14853-14859 (1989)]. In this study we have reconstituted purified bovine brain A1 receptors into human platelet membranes that contain A2- but no detectable A1-adenosine receptors. The recovery of reconstituted receptors was assessed from the binding of the antagonist radioligand [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentyl-xanthine and ranged from 32 to 84%. Coupling of reconstituted A1 receptors to platelet G proteins was evaluated by measurement of the high affinity binding of an agonist radioligand, 125I-aminobenzyladenosine, to receptor-G protein complexes and by stereospecific photoaffinity labeling of a 35,000-Da receptor polypeptide with the agonist photoaffinity label 125I-azidobenzyladenosine. Fifty percent of receptors reconstituted into platelet membranes bound agonists with high affinity, indicative of coupling to platelet G proteins. Reconstituted A1 receptors bound various ligands with affinities characteristic of A1 receptors of bovine brain. Although platelets contain both pertussis toxin-sensitive and -insensitive G proteins, reconstituted high affinity agonist binding was almost completely abolished by treatment of platelet membranes with guanosine 5'-3-O-(thio)triphosphate, pertussis toxin, N-ethylmaleimide, or heparin. Following reconstitution, A1 receptors could be resolubilized in complexes with platelet G proteins. The data suggest that marked species differences in the binding affinity of ligands to adenosine receptors result from differences in the receptors rather than membrane structure or G proteins and, further, that A1 receptors couple selectively and tightly to pertussis toxin-sensitive G proteins.     

Selective blockade and recovery of cell surface alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells. Studies with the irreversible antagonist benextramine

alpha 2-Adrenergic receptors are present on human erythroleukemia (HEL) cells, both on the cell surface and in a sequestered compartment. In the current study we show that benextramine, a hydrophilic irreversible antagonist, can be used to investigate alpha 2-adrenergic receptor compartmentation in these cells. In membranes prepared from HEL cells, benextramine competed for all alpha 2-adrenergic receptors ( [3H]yohimbine sites). In intact cells, at 4 degrees, benextramine exhibited a biphasic competition curve for alpha 2-adrenergic receptors, with EC50 values of approximately 10 microM and greater than 1 mM for the high and low affinity components, respectively. We propose that the alpha 2-adrenergic receptors preferentially blocked by benextramine are those on the surface of the cell, whereas those with low affinity are sequestered receptors because: 1) only epinephrine-accessible sites [i.e., cell surface sites; McKernan et al., Mol. Pharmacol. 32:258-265 (1987)] are removed by prior treatment of cells with benextramine, 2) a preparation enriched with surface membranes is also enriched in receptors with a high affinity for benextramine; and 3) after blockade of cell surface receptors (54 +/- 6% of total sites, n = 7) by benextramine, the ability of the alpha 2-adrenergic agonists epinephrine and UK-14,304 to inhibit forskolin-stimulated cAMP accumulation is lost. The latter result implies that only cell surface and not sequestered receptors are functionally coupled to adenylate cyclase. The return of receptors from the sequestered compartment to the cell surface and the recovery of alpha 2-adrenergic receptor function were measured after HEL cells were treated with benextramine (50 microM for 1 hr at 4 degrees). The recovery of receptor binding (t1/2 = 25 min) was somewhat slower than the recovery of function (t1/2 approximately 8 min). This is consistent with the existence of "spare receptors" and also suggests that the sequestered compartment of alpha 2-adrenergic receptors can rapidly exchange with those on the surface. When all alpha 2-adrenergic receptors were blocked by incubation of HEL cells with benextramine for 1 hr at 37 degrees, repopulation of surface and sequestered receptors was much slower (t1/2 = 9 hr for recovery of total receptors). Surface receptors recovered even more slowly than did total cellular receptors consistent with the idea that alpha 2-adrenergic receptors must traverse through intracellular locations before insertion into the cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

Agonist binding at alpha2-adrenoceptors of human platelets using 3H-UK-14,304: regulation by Gpp(NH)p and cations

Summary The agonist/₂-adrenoceptor interactions at human platelet membranes have been examined in radioligand binding studies with the full agonist ligand ³H-UK-14,304 [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline] and the antagonist ligand ³H-yohimbine. From association kinetics of different concentrations of ³H-UK-14,304 (0.75–8.1 nmol/l) a K _D-value of 2.37 nmol/l in agreement with the high-affinity KD-value (K _DH = 1.60 ± 0.15 nmol/1) obtained from equilibrium binding studies was derived. In the presence of Gpp(NH)p about 6% of specific radioligand binding was observed in the association reaction. Addition of Gpp(NH)p at equilibrium resulted in a rapid loss (t _1/2 < 1 min) of 80% of bound radioligand. Dissociation after addition of an excess of phentolamine (10 mol/l) showed a biphasic time course independent of the radioligand concentration with the proportions of /15 of rapidly (t _/12 < 2 min) and /45 of slowly dissociating ligand (k–1 = 0.033±0.004 min^–1). Application of a sequential binding model resulted in K _D-values from this approach also in agreement with K _DH from equilibrium binding studies. The rank order of potency for different agonists and antagonists to compete for binding with ³H-UK-14,304 indicated an ₂-adrenoceptor interaction: (–)adrenaline > clonidine > (–)noradrenaline > (–)isoprenaline and yohimbine = rauwolscine > phentolamine > prazosin >- corynanthine > timolol respectively. The analysis of competition isotherms of UK-14,304 versus ³H-yohimbine (Hill-coefficient = 0.59 ± 0.03) showed that the agonist binds to two affinity states of the ₂-adrenoceptor, with high (K _DH = 1.77 ± 0.50 nmol/l) and low affinity (K _DL = 71.2 ± 11.6 nmol/l) respectively. From these experiments a fraction of 56.9%±2.1% of the total number of ₂-adrenoceptors (B _max = 198.4 ± 8.0 fmol/mg of protein) in the high-affinity state was calculated. Similar results were obtained from ³H-UK-14,304 saturation isotherms according to a two-state binding model (K _DH = 1.60±0.15 nmol/l; K _DL = 66.2±10.7 nmol/l; B _maxH = 57.6% ± 2.3%). Adrenoceptor agonists competed for specific binding of ³H-UK 14,304 and ³H-yohimbine in a manner that suggests that the ³H-UK-14,304 (3.5 nmol/l) labeled sites represent predominantly the agonist induced or stabilized high-affinity state of the ₂-adrenoceptor. Adrenoceptor antagonists had equal affinities irrespective of the receptor states labeled by the agonist or antagonist radioligand. A loss of the high-affinity binding capacity (B _maxH) of the agonist due to the presence of Gpp(NH)p was delineated from ³H-UK-14,304 saturation isotherms. An IC₅₀-value of 0.181 ± 0.007 mol/l for this Gpp(NH)p-effect was calculated. Divalent cations such as magnesium and manganese (10 mmol/l) increased specific binding of ³H-UK-14,304 by a factor of 3, without any influence on binding of the antagonist ³H-yohimbine. In contrast, sodium chloride strikingly decreased high-affinity binding of the agonist radioligand (IC₅₀ = 41.9 ± 3.7 mmol/l). Unlike Gpp(NH)p, sodium chloride (> 30 mmol/l) additionally promoted a marked decrease of the affinity of UK-14,304 at the low-affinity binding component. In contrast to the effects on agonist binding, sodium chloride concentrations of 30 to 300 mmol/l increased the binding affinity of the antagonist ³H-yohimbine about 2-fold. The sodium substitute N-methyl-D-glucamine was without effect on binding of ³H-UK-14,304 indicating that the influence of sodium chloride on binding properties was not due to changes in osmolarity. In conclusion these results suggest that ³H-UK-14,304 labels preferentially the agonist induced or stabilized high-affinity state (_2H) of the platelet ₂-adrenoceptor.Abbreviations UK 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline - Gpp(NH)p guanylyl-imidodiphosphate - EDTA ethylene dinitrilo tetraacetic acid Preliminary data were presented at the Joint Meeting of the Belgian, Dutch and German Pharmacological and Toxicological Societies at Aachen, FRG, September 23–26, 1985 (Schloos et al. 1985)This report is part of the thesis to be presented by J. Schloos in partial fulfillment of the requirements for a Doctor of Natural Science degree Send offprint requests to J. Schloos at the above address     

Tight agonist binding may prevent the correct interpretation of agonist competition binding curves for alpha 2-adrenergic receptors

alpha 2-Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated antagonist 3H-RX 781094. Saturation binding occurs to a single class of noncooperative sites. The number of sites amounts to 1070 +/- 243 and 935 +/- 178 fmol/mg of protein, and the equilibrium dissociation constants equal 1.8 +/- 0.4 and 3.8 +/- 0.3 nM at 25 degrees and 37 degrees, respectively. Binding is rapid, equilibrium being reached within 5 min, and is reversible. At both temperatures, (-)-epinephrine competition binding curves are shallow in the presence of magnesium ions. The curves, obtained for incubation periods varying between 5 and 60 min, are superimposable at 37 degrees. Computer-assisted analysis indicates that approximately 75% of the receptors (RH sites) display high agonist affinity for (-)-epinephrine as well as for the other agonists tested: (-)-norepinephrine, clonidine, and UK 14304. However, the (-)-epinephrine competition curves display a time-dependent leftward shift at 25 degrees. This can be attributed to an increase in agonist affinity for the RH sites. Addition of 0.1 mM Gpp(NH)p causes a marked steepening and rightward shift of the curves, at both 25 and 37 degrees. These curves are superimposable for all of the incubation times tested. The nonequilibrium of agonist competition binding at 25 degrees can be attributed to slow dissociation of the agonist (i.e., tight binding) when the receptor is coupled to the regulatory component Ni. This dissociation rate can be measured by preincubation of the membranes with 10 microM (-)-epinephrine, followed by extensive washing and incubation with 3H-RX 781094 for increasing lengths of time. The first order rate of agonist dissociation (i.e., receptor recovery) is appreciably faster at 37 degrees than at 25 degrees: i.e., 0.029 min-1 and 0.0044 min-1, respectively. These findings are confirmed by kinetic experiments using the radiolabeled agonist 3H-UK 14304. Slow agonist dissociating kinetics may prevent the correct evaluation of the agonist binding parameters by computerized analysis of competition binding curves when the incubation time is too short, especially at low temperature.     

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.