Idazoxan Binding Research Articles

Effects of idazoxan on 5-hydroxytryptamine-mediated behaviour in the mouse and rat

The α(2)-adrenoceptor antagonists idazoxan and RX811059 induced reciprocal forepaw treading, a component of the 5-HT-behavioural syndrome in rats. This response is independent of 'non-α(2)-adrenoceptor idazoxan binding sites' (NAIBS) at which RX811059 is inactive. Idazoxan pre-treatment, in rats, enhanced forepaw treading, head weaving and tremor induced by the 5-HT agonist 5-methoxy-N,N dimethyltryptamine (5-MeODMT), increased head twitches (but decreased hindlimb abduction) induced by the 5-HT releaser p- chloroamphetamine (pCA), but did not clearly alter head twitches induced by the 5-HT precursor L-5-hydroxytryptophan in mice. The α(1)-antagonist prazosin did not alter behaviour induced by either 5-MeODMT or pCA in rats. The α( 2)-agonist, guanoxabenz, did not alter 5-MeODMT-induced behaviour in rats. St587, an α(1)-agonist, selectively potentiated tremor induced by 5-MeODMT, but no other behaviour. A possible mechanism for these interactions could be through enhanced, α(2)-adrenoceptor-mediated, 5-HT release in specific brain areas. Other possibilities, e.g. direct action at subtypes of 5-HT receptors and the importance of these NA-5-HT interactions in the treatment of resistant depression, are discussed.

Non-adrenergic binding sites for the “ α2-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 α 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 α 2-antagonists are ineffective competitors which suggests an imidazoline-preferring binding site. However, imidazolidines such as clonidine and p-aminoclonidine 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 Mn + inhibit [ 3H]idazoxan binding in a competitive and non-competitive manner, respectively. Other cations such as Na +, Li + and Mg 2+ have no significant effect. It is shown that K + accelerates the dissociation of [ 3H]idazoxan binding while Mn 2+ does not produce any modification. These results suggest that K + may bind to an allosteric site, while Mn 2+ may bind with a membrane component susceptible to alter [ 3H]idazoxan binding sites.

Subcellular distribution of imidazoline-guanidinium-receptive sites in human and rabbit liver. Major localization to the mitochondrial outer membrane.

Imidazoline-guanidinium-receptive site (IGRS) is a membrane protein that, even if recognized by a series of imidazoline and guanidinium alpha 2-adrenergic compounds, is insensitive to catecholamine and physically distinct from alpha 2 receptors (Parini, A., Coupry, I., Graham, R. M., Uzielli, I., Atlas, D., and Lanier, S. M. (1989) J. Biol. Chem. 264, 11874-11878). In the present report, we defined the subcellular localization of IGRS by performing binding studies with the imidazoline radioligand [3H]idazoxan. Binding studies on subcellular fractions of homogenates from human and rabbit liver showed a significant increase in [3H]idazoxan binding in a membrane fraction enriched in cytochrome oxidase activity, a specific marker for mitochondria. The enrichment in [3H]idazoxan binding sites correlates closely with cytochrome oxidase activity in the nuclear, mitochondrial, plasma membrane, microsomal, and soluble fractions (r = 0.966, p less than 0.002) but not with the specific markers for other cell compartments, suggesting a major localization of IGRS in mitochondria. Separation of inner and outer mitochondrial membranes by digitonin treatment showed that [3H]idazoxan binding correlates positively with monoamine oxidase (r = 0.960) and negatively with cytochrome oxidase (r = -0.950) activities. In addition, in highly purified preparations of outer mitochondrial membranes obtained by hypotonic shock, [3H]idazoxan binding activity was 12.5-fold enriched with respect to intact mitochondria. Taken together, these data show, for the first time, that IGRS in human and rabbit liver are mainly associated with the outer mitochondrial membranes. This demonstration of the major mitochondrial localization of IGRS will facilitate the characterization of its functional activity in liver.

Non-Adrenergic Binding Sites for the “α-Antagonist” [3H] Idazoxan in Rabbit Urethral Smooth Muscle

In the present study, we have provided evidence that [3H] rauwolscine and [3H] idazoxan bind to different sites in rabbit urethra. The [3H] idazoxan capacity and affinity was 215 +/- 14 fmol/mg protein and 1.59 +/- 0.16 nM while [3H] rauwolscine binding parameters were 45.9 +/- 3.4 fmol/mg protein and 2.39 +/- 0.27 nM. [3H] idazoxan specific binding was inhibited only by compounds possessing an imidazoli(di)ne or a guanidinium moiety, while [3H] rauwolscine specific binding was inhibited by phenylethanolamines and classical alpha 2-antagonists. [3H] idazoxan was inhibited by KCl in a competitive and by MnCl2 in a non-competitive way, while other cations such as Na+, Li+ and Mg2+ did not inhibit [3H] idazoxan binding. Moreover, we investigated the regional distribution of [3H] idazoxan and [3H] rauwolscine along the rabbit urethra using quantitative autoradiography. Analysis of the films revealed a different distribution of these two binding sites on the urethral sections.

Imidazoline-guanidinium and α2-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 α 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 < 0.05: [ 3H]rauwolscine: 56.4 ± 21.4 vs 25.2 ± 7.3 fmol/mg protein. P < 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 α 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, α 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. However, the differences in ligand-recognition properties between the two species propose a possible heterogeneity of IGRS.

Imidazoline receptors in rat liver cells: A novel receptor or a subtype of α2-adrenoceptors?

An imidazoline/guanidine receptor has been characterized in rat liver cells. Binding of [ 3H]idazoxan, a selective benzodioxan antagonist, to imidazoline receptor on intact fresh hepatocytes (B max = 801 ± 23 fmol/mg protein,K d = 11 ± 0.8 nM) and to liver membranes (B max = 400 ± 38 fmol/mg protein, K d = 10 ± 2 nM) was saturable at 4°C within 3.5 h and at 30°C within 30 min, respectively. Rat lung membranes had more imidazoline sites (B max = 578 ± 30 fmol/mg protein, K d = 14 ± 1.4 nM) than α 2-adrenoceptors (B max = 175.0 ± 20.0 fmol/mg protein,K d = 4.8 ± 2.0 nM). We also screened other tissues for imidazoline sites; labeled with [ 3H]idazoxan displaced by cirazoline was lower in rat lung compared to rat brain and human platelets. The imidazoline receptor has common pharmacological properties with α 2-adrenoceptors, although it is not a subtype of the adrenoceptor, since it bound neither the endogenous agonists norepinephrine and epinephrine, nor the selective α 2-antagonists yohimbine and phentolamine. All guanidine type α 2-adrenoceptor drugs (e.g. guanabenz, guanoxan) and imidazolines (e.g., UK-14,304, naphazoline) competed with high affinity for the liver imidazoline receptor. The lack of effect by Gpp(NH)p, a non-hydrolysable GTP analogue, on the affinity of guanidine- and imidazoline-type ligands for liver imidazoline receptors suggests that the mode of action of these drugs at imidazoline receptors is different than at conventional α 2-adrenoceptors. Ionic changes were considered as a possible mechanism underlying the α 2-adrenoceptor effects in various cells. Opening of K + channels by α 2-adrenoceptors agonists is a pathway which might be shared by imidazoline-type agonists at imidazoline sites. Indeed, 4-aminopyridine, a K + channel blocker, inhibited the specific binding of [ 3H]idazoxan to liver cells with an IC 50 of 0.34 ± 0.07 mM, a concentration which is effective in blocking K + channels in neuronal cells. Similarly, Cs + and NH 4 + effectively interfered with [ 3H]idazoxan binding, suggesting a possible coupling of imidazoline sites to K + gating. The endogenous ligand clonidine-displacing substance (CDS), which was isolated from bovine brain and which binds to α 2-adrenoceptors in brain membranes and human platelets competed with idazoxan at rat liver imidazoline receptors. Although the need of an endogenous ligand for imidazoline sites is obvious, it has yet to be determined whether CDS is the endogenous ligand for imidazoline receptors in general, and for liver cells in particular, and whether K + channels are involved in the mechanism of action of these sites.

Identification of α 2-adrenoceptors and non-adrenergic idazoxan binding sites in rabbit colon epithelial cells

α 2-Adrenoceptors are possibly involved in the regulation of the hydroelectrolytic flux across the digestive mucosa. As no data are available concerning the existence of these receptors in colon epithelial cells, we aimed to investigate the existence of α 2-adrenoceptors in this tissue using tritiated antagonists. [ 3H]Yohimbine and [ 3H]rauwolscine were not usable to label colonic α 2-adrenoceptors because of their very high level of non-specific binding. In contrast, the methoxy derivative of idazoxan, [ 3H]RX821002, appeared a convenient radioligand for the purpose. [ 3H]RX821002 bound with high affinity (k d = 6.2 ± 0.8 nM) to a single population of non-interacting sites (B max = 193 ± 17 fmol/mg protein). The rank order of potency of catecholanaine enantiomers and adrenergic drugs to inhibit [ 3H]RX821002 binding demonstrated that the labelled sites are α 2-adrenoceptors and that 53% of the receptors are in a high-affinity state sensitive to GTP + NaCl [ 3H]Idazoxan also bound to colocyte membranes, but inhibition by (−)-adrenaline and various imidazoline compounds indicated that this radioligands labels α 2-adrenoceptors and non-adrenergic sites. When experiments were performed under binding conditions impeding the interaction of [ 3H]idazoxan with the α 2-adrenoceptors (i.e. in presence of 10 −4 M (−)-adrenaline), the B max of non-adrenergic idazoxan binding sites was 97 ± 8 fmol/mg protein and the K D was 3.5 + 0.5 nM. The sites were pharmacologically characterized with various imidazoline and non-imidazoune drugs. In order to study the putative relationship between α 2-adrenoceptors and non-adrenergic idazoxan binding sites, the expression of both kinds of sites was investigated along the crypt-to-surface axis. Crypt cells had a higher number of α 2-adrenoceptors than surface cells, whereas the number of non-adrenergic idazoxan binding sites remained constant. The results show that (i) α 2-adrenoceptors coexist with non-adrenergic idazoxan binding sites in rabbit colocytes; (ii) the number of ayadrenoceptors is higher in crypt cells than in surface cells and (iii) α 2-adrenoceptors and non-adrenergic binding sites are different and unrelated.

Non-adrenergic sites for imidazolines are not directly involved in the α 2-adrenergic antilipolytic effect of UK 14304 in rat adipocytes

The binding of the α 2-agonist [ 3pH]UK 14304 on Wistar rat adipocyte membranes was separated in two distinct components: one was displaceable by adrenaline or other α 2-adrenergic agents and possessed the characteristics of α 2-adrenoceptors while the other, non-adrenergic in nature, was only recognized by some imidazoline derivatives. [ 3pH]Idazoxan binding shared the same characteristics. The non-adrenergic sites labeled by both radioligands are similar to those described for [ 3pH]idazoxan on other tissues such as brain cortex, smooth muscle and kidney. Even though they were about 10-fold more numerous than the true α 2-adrenoceptors, the non-adrenergic binding sites were not directly involved in the antilipolytic action of UK 14304 since α 2-antagonists devoid of interaction with these sites (yohimbine, phentolamine) totally blocked the UK 14304 effect. However, the existence of such a type of site impairs direct quantification of α 2-adrenoceptors in rat adipocytes. The use of [ 3pH]RX 821002 (2-(2-methoxy-1, 4-benzodioxan-2yl)imidazoline) allowed an accurate quantification of rat adipocyte α 2-adrenoceptors ( B max = 35 ± 2 fmol mg protein, K d = 2.6 ± 0.6 nM) since it did not interact with non-adrenergic binding sites and exhibited the highest α 2-blocking properties among the various α 2-antagonists tested. [ 3pH]RX 821002 binding analysis revealed that α 2-adrenoceptors are, on rat adipocytes; (i) less numerous than in other species well known for their α 2-adrenergic inhibitory regulation of lipolysis (human, hamster, rabbit); (ii) slightly different in nature from the receptors of these species since they had weaker affinity for clonidine and yohimbine; and however (iii) not of the typical α 2-B subtype since the affinity of prazosin was lower than that of oxymetazoline in displacing [ 3pH]RX 821002 or [ 3pH]yohimbine binding.

Identification of a noradrenergic [ 3H]-idazoxan binding site in rabbit and human liver

Identification of a noradrenergic [ 3H]-idazoxan binding site in rabbit and human liver

Effect of imidazolines on Na + transport and intracellular pH in renal proximal tubule cells

Recently, we characterized an imidazoline-guanidinium receptive site (IGRS) in the renal proximal tubule of rabbit kidney. Although recognized by a series of imidazoline and guanidinium alpha-2 adrenergic compounds, IGRS is insensitive to catecholamines and can be physically separated from alpha-2 adrenergic receptors after solubilization. In the present study, we investigated the effect of imidazoline derivatives on 22Na + uptake and intracellular pH in isolated cells from rabbit renal proximal tubule. After 5 min of preincubation, idazoxan inhibited the total 22Na + influx (−30%) in a dose-dependent manner, with a maximum effect at 10 −5 M. The effect of idazoxan was not competitive as shown by the decrease of the maximal velocity of 22Na + entry (control: 3.80 ± 0.4; idazoxan 10 −5 M: 3.23 ± 0.33 nmol/30 s per mg protein, P < 0.01). A series of imidazoline derivatives inhibited 22Na + entry with an order of potency similar to that previously found for inhibition of [ 3H]idazoxan binding to IGRS (irazoline > idazoxan > UK 14304 > rilmenidine ⪢ cimetidine). The inhibition of 22Na + uptake by these compounds does not appear to be related to interaction with alpha-adrenergic receptors since it was observed in the presence of saturating concentrations of the adrenergic antagonists rauwolscine (alpha-2) or prazosin (alpha-1). When tested on the regulation of intracellular pH by fluorimetric techniques, 10 −5 M cirazoline or idazoxan inhibited by 20% the velocity of the sodium-dependent H + efflux in acidified cells ( P < 0.02). The concomitant inhibition of 22Na + entry and of cell realkalinization suggests that imidazoline derivatives inhibit Na +/H +-exchanger. This effect could be mediated via the renal IGRS and intracellular second messengers that are not yet known.

α2‐Adrenoceptor subtypes and imidazoline‐like binding sites in the rat brain

1. The binding of [3H]-yohimbine and [3H]-idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]-yohimbine in the cortex (Bmax 121 +/- 10 fmol mg-1, protein; Kd 5.2 +/- 0.9 nM) and hippocampus (Bmax 72 +/- 6 fmol mg-1 protein; Kd 5.8 +/- 0.7 nM). [3H]-idazoxan labels one site in the cortex (Bmax 87 +/- 8 fmol mg-1 protein; Kd 4.1 +/- 0.9 nM) and hippocampus (Bmax 30 +/- 6 fmol mg-1 protein; Kd 3.5 +/- 0.5 nM), when 3 microM phentolamine is used to define non-specific binding. A second distinct [3H]-idazoxan binding site (Bmax 110 +/- 21 fmol mg-1 protein; Kd 3.6 +/- 0.07 nM) is identified in rat cortex if 0.3 microM cirazoline is used to define non-specific binding and 3 microM yohimbine is included to prevent binding to alpha 2-adrenoceptors. 2. Displacement studies indicate that the alpha 1-adrenoceptor antagonist prazosin and the 5-HT1 ligands 8-OH-DPAT, RU 24969 and methysergide differentiate [3H]-yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]-idazoxan by each ligand was monophasic. 3. The affinities of 8-OH-DPAT, RU 24969 and methysergide determined against [3H]-idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated alpha 2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated alpha 2B. 4. [3H]-idazoxan, in the presence of 3 microM yohimbine, labels a site that displays high affinity towards cirazoline, naphazoline and guanabenz, but low affinity towards clonidine, p-aminoclonidine, adrenaline, noradrenaline and the alpha 2-adrenoceptor antagonists yohimbine, rauwolscine, WY 26703 and BDF 6143. 5. The results of this study indicate that [3H]-yohimbine labels two sites; the alpha 2A- and alpha 2B-adrenoceptors whereas [3H]-idazoxan labels an alpha 2-adrenoceptor with a profile consistent with the alpha 2A-adrenoceptor subtype. In addition, [3H]-idazoxan labels an imidazoline binding site in the rat cortex that is pharmacologically distinct from alpha 2-adrenoceptors. The low affinity of clonidine and p-aminoclonidine indicates that the imidazoline-like binding site in rat cortex is different from the site labelled by [3H]-clonidine and [3H]-p-aminoclonidine in human, rat and bovine brain stem, providing evidence of potential heterogeneity within this class of binding sites.

Discrimination between α2-adrenoceptors and [ 3H]idazoxan-labelled non-adrenergic sites in rabbit white fat cells

Previous results indicated that the rabbit could represent a suitable model for investigations on the functional role of α 2-adrenoceptors in fat cells, but the characterization of these receptors was not resolved yet. In the present report, imidazoline compounds were used to attempt a better definition of rabbit adipocyte α 2-receptivity by means of lipolysis and binding studies. Lipolysis data showed that UK14304 is a full α 2-adrenoceptor agonist promoting a strong antilipolysis in rabbit fat cells. Moreover, the methoxy derivative of idazoxan, RX821002, is a more potent antagonist of UK14304-induced antilipolysis than idazoxan or yohimbine. Whereas [ 3H]yohimbine failed to bind at rabbit adipocyte α 2-adrenoceptors, [ 3H]UK14304 and [ 3]RX821002 are valuable tools to study this receptor. Analysis of binding data demonstrated that [ 3H]UK14304 labels the high-affinity-state receptor while [ 3H]RX821002 binds to the whole α 2-adrenergic population. Inhibition studies [ 3H]RX821002 and [ 3H]UK14304 binding by various compounds confirmed the α 2-adrenergic nature of the sitesl labelled by both radioligands. The other α 2-adrenoceptor radioligand, [ 3H]idazoxan, labelled binding sites which are insentitive to catecholamine. Competition studies of [ 3H]idazoxan binding with imidazoline derivatives revealed structure-activity relationships different from those of α 2-adrenoceptors. The most striking observation is that substitutions in the 2-position of idazoxan markedly reduce the affinity for the non-adrenergic sites, whereas the α 2-potency is increased or unchanged.

Transient elevation of amygdala α2 adrenergic receptor binding sites during the early stages of amygdala kindling

Enhanced noradrenergic neurotransmission retards but does not prevent the development of kindling. We previously reported that locus coeruleus (LC) α 2 adrenergic receptor binding sites are transiently elevated during the early stages of kindling development. Since the firing activity of LC noradrenergic neurons is partially regulated via an α 2 receptorsmediated recurrent inhibition, the transient elevation in LC α 2 receptors could decrease LC activity and consequently facilitate the development of kindling. Transient elevation of α 2 receptor binding sites during early stages of kindling may also occur on noradrenergic axon terminals projecting to forebrain sites. Using in vitro neurotransmitter autoradiography techniques, we investigated this hypothesis by measuring specific [ 3H]idazoxan binding in 5 different areas of rat forebrain at 2 different stages of kindling development. After 2 class 1 kindled seizures, specific [ 3H]idazoxan binding was elevated significantly in the amygdala, but not in other forebrain regions. No differences in specific [ 3H]idazoxan binding were observed in any of the 5 brain regions in rats kindled to a single class 5 kindled motor seizure. Saturation of binding experiments indicated that the increase in amygdala [ 3H]idazoxan binding, following 2 class 1 kindled motor seizures, was due to an increase in the total number of α 2 receptor binding sites without a change in the affinity of the binding sites for [ 3H]idazoxan. Thus, the transient increase in α 2 receptors that occurs in the LC in the early stages of kindling also occurs in the forebrain region in which the kindled seizure originates.

Differences in the regulation of [ 3H]idazoxan and [ 3H]yohimbine binding sites in the rabbit

In vitro studies suggest that [ 3H]yohimbine binds to α 2-adrenoceptors while [ 3H]idazoxan binds preferentially at a non-adrenergic site. In order to compare in vitro with in vivo effects male New Zealand White rabbits received the following treatments: 5 days idazoxan 1.1 mg/kg per h, 10 days noradrenaline 46 μg/kg per h (intravenous infusion), 21 days amitriptyline 30 mg/kg per day (intraperitoneally) or vehicle. The effect of these treatments on the number of [ 3H]yohimbine and [ 3H]idazoxan binding sites was examined. Ten days noradrenaline infusion and 21 days amitripytyline treatment significantly reduced [ 3H]yohimbine binding in kidney and hindbrain membranes respectively, but had no significant effect on [ 3H]idazoxan binding. Five days idazoxan infusion significantly increased [ 3H]yohimbine binding in the forebrain, while a significant reduction in [ 3H]idazoxan binding sites in the kidney was observed. Thus differential regulation of the two binding sites was observed in vivo. These alterations in binding site number are consistent with the differing affinities of noradrenaline and idazoxan for the [ 3H]yohimbine and [ 3H]idazoxan binding sites previously observed in vitro and support the hypothesis that in the rabbit idazoxan binds preferentially at non-adrenergic sites while yohimbine binds to an α 2-adrenergic site. The idazoxan site may be an imidazoline type of receptor but further work, including functional studies, is required to substantiate this.

3H]‐idazoxan binds with high affinity to two sites on hamster adipocytes: an α2‐adrenoceptor and a non‐adrenoceptor site

1. [3H]-idazoxan labels a single population of high affinity sites (Kd 2.26 +/- 0.02 nM; Bmax 372 +/- 25 fmol mg-1 protein) in hamster adipocyte membranes. In the presence of 1 microM yohimbine to preclude binding to alpha 2-adrenoceptors, the density of [3H]-idazoxan binding sites was reduced (287 +/- 18 fmol mg-1 protein) without an apparent decrease in the affinity (Kd 2.19 +/- 0.24 nM) of the radioligand. 2. Displacement studies indicate that alpha-adrenoceptor ligands with an imidazoline side chain completely inhibit [3H]-idazoxan binding to hamster adipocyte membranes; in contrast, the alpha 2-adrenoceptor antagonists yohimbine, rauwolscine, BDF 6143 and phentolamine inhibited only 20-30% of the specific binding with affinity values consistent with an interaction at alpha 2-adrenoceptors. 3. The low potency of noradrenaline and adrenaline in displacing [3H]-idazoxan binding to the second site on hamster adipocyte membranes indicates that it is unlikely that this site is a type of adrenoceptor. 4. These results suggest that [3H]-idazoxan binds with high affinity to two sites in hamster adipocytes: an alpha 2-adrenoceptor and a non-adrenoceptor imidazoline site.

Characterization of an imidazoline/guanidinium receptive site distinct from the α2-adrenergic receptor

alpha 2-Adrenergic receptors recognize a number of molecules with diverse chemical structures, including the yohimban diastereoisomers yohimbine and rauwolscine, catecholamines, guanidinium analogs, and imidazolines, such as clonidine. The affinity of the receptor protein for some of these ligands can vary by 10-100-fold among various tissues and species, suggesting a heterogeneous class of binding sites. Certain cellular effects elicited by the compounds possessing an imidazoline or guanidinium moiety may actually be mediated by a membrane receptor distinct from the alpha 2-adrenergic receptor. To determine whether this imidazoline/guanidinium receptive site (IGRS) and the alpha 2-adrenergic receptor represent distinct proteins, we solubilized and partially characterized the two binding sites in rabbit kidney. This tissue expresses both alpha 2-adrenergic receptors and high affinity imidazoline/guanidinium binding sites, the latter which are rauwolscine-insensitive but can be identified with the benzodioxan [3H]idazoxan. The IGRS and alpha 2-adrenergic receptor in rabbit kidney exhibit distinct ligand recognition properties, which are maintained after solubilization and partial purification. In addition, the two receptors can be physically separated by heparin-agarose or lectin affinity chromatography indicating that the two binding sites are distinct entities. [3H]Idazoxan binding is trypsin-sensitive, indicating that the IGRS is a protein rather than a lipid component of the plasma membrane. [3H]Idazoxan binding is not inhibited by endogenous agonists for known neurotransmitter receptors. However, the IGRS does recognize clonidine-displacing substance, a small non-catechol compound isolated from calf brain, suggesting the existence of a previously uncharacterized hormonal/neurotransmitter receptor system.

Coupling between hypothalamic α 2-adrenoceptors and [ 3H]mazindol binding site in response to several hyperglycaemic stimuli in mice

The hypothalamic response to circulating glucose and insulin levels was studied in the mouse by differentially attenuating glucose-homeostasis. The administration of glucose, 2-deoxyglucose or the α 2-adrenoceptor agonist UK 14.304 was accompanied by a persistent hyperglycaemia; however, an increase in insulin levels was obtained with glucose and a decrease with the other two manipulations. Both α 2-adrenoceptors (labeled with [ 3H]idazoxan) and the anorectic recognition site (labeled with [ 3H]mazindol) were upregulated by the three treatments. A good correlation was obtained between circulating glucose levels and either hypothalamic [ 3H]mazindol binding ( r = 0.70, P < 0.001) or [ 3H]idazoxan binding ( r = 0.63, P < 0.001), as well as between the two binding sites ( r = 0.88, P < 0.001). No correlation was obtained between circulating insulin levels and these binding sites ( r = 0.18, r = 0.26, P =n.s. for [ 3H]mazindol and [ 3H]idazoxan binding, respectively). It is suggested the α 2-adrenoceptors and the anorectic binding sites are associated in their response to glucose as part of a hypothalamic center involved in the regulation of feeding mechanisms.

Amiloride interacts with [ 3H]idazoxan and [ 3H]rauwolscine binding sites in rabbit urethra

Amiloride and its analogues (N-ethylisopropylamiloride and banzamil) interact more potently with [ 3H]idazoxan binding sites (nM range) than with [ 3H]rauwolscine binding sites (μM range) in the rabbit urethra. The binding of both radioligands was competitivelly inhibited by amiloride, with increased K D values and no change in their binding capacity. Interestingly, amiloride was a potent inhibitor at [ 3H]idazoxan binding sites in rabbit urethral smooth muscle at concentrations far below those required to inhibit the Na +/H + exchanger or electrogenic pump.

Acute and chronic effects of methylphenidate on cortical adrenoceptors in the rat

The effects of acute and chronic treatments with methylphenidate were examined on cortical adrenoceptors. A single dose of methylphenidate increased the affinity of [ 3H]prazosin and [ 3H]idazoxan binding sites. Acute and chronic methylphenidate treatments caused a down-regulation of α 1-adrenoceptors. There were no changes in cortical β-adrenoceptors measured with [ 3H]dihydroalprenolol nor in endogenous monoamine levels. The alterations in α-adrenoceptors can be attributed to an indirect action of methylphenidate possibly through an accured release of noradrenaline or tyramine.

3H]Idazoxan binding at non- α2-adrenoceptors in rabbit adipocyte membranes

The imidazoline ligand, [ 3H]idazoxan, labels a large population of high-affinity binding sites in rabbit fat cell membranes (B max = 1370 ± 91 fmol/mg protein; K D = 1.6 ± 0.6 nM) when imidazoline derivatives are used for definition of non-specific binding. [ 3H]Idazoxan sites are not α 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 K i values were 9, 27 and 48 nM, respectively.