Non-adrenergic binding of [3H]atipamezole in rat kidney--regional distribution and comparison to alpha2-adrenoceptors

1 Atipamezole (4-(2-ethyl-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) was first introduced as a potent and specific alpha2-adrenoceptor antagonist, but in some tissues [3H]atipamezole identifies an additional population of binding sites, distinct from both classical alpha2-adrenoceptors and I1- and I2-imidazoline receptors identified with [3H]para-aminoclonidine or [3H]idazoxan. 2 In the present study we have characterized [3H]atipamezole binding sites in rat kidney by receptor autoradiography and membrane binding assays and determined whether they are pharmacologically identical with the previously described binding sites for [3H]para-aminoclonidine and [3H]idazoxan. [3H]RX821002 and [3H]rauwolscine were used to compare the regional distribution of alpha2-adrenoceptors to that of non-adrenergic binding sites of [3H]atipamezole. 3 Comparative autoradiographic experiments demonstrated the differential localisation of [3H]atipamezole, [3H]RX821002 and [3H]rauwolscine binding sites in rat kidney. The pattern of distribution of non-adrenergic [3H]atipamezole binding sites is clearly distinct from that of alpha2-adrenoceptors. 4 The non-adrenergic binding of [3H]atipamezole in rat kidney does not fall into any of the previously identified three classes of imidazoline receptors studied with [3H]para-aminoclonidine, [3H]idazoxan and [3H]RX821002. 5 Atipamezole had no inhibitory effect on MAO-A or MAO-B activity in renal membranes, which speaks against the involvement of MAOs in the observed radioligand binding.     

Antagonistic effect of N-methyltyramine on alpha2-adrenoceptor in mice

We examined the effect of N-methyltyramine (NMT) on alpha2-adrenoceptor. NMT (10(-8)-10(-3) M) inhibited the binding of [3H]p-aminoclonidine to alpha2-adrenoceptor dose-dependently. However, the IC50 value for NMT (5.53 x 10(-6) M) was higher than that for RX821002, an alpha2-adrenoceptor antagonist (1.07 x 10(-8) M). RX821002 (5 mg/kg, i.p.) inhibited hypermotility induced by scopolamine (8 mg/kg, s.c.) in male ddY mice. NMT (20 or 100 mg/kg, i.p.) was found to have a dose-dependent inhibitory effect similar to that of RX821002. These findings indicate that NMT has the properties of an alpha2-adrenoceptor antagonist. However, the affinity of NMT for alpha2-adrenoceptor is weaker than that of RX821002.     

alpha2A-adrenoceptor antagonism increases insulin secretion and synergistically augments the insulinotropic effect of glibenclamide in mice

BACKGROUND AND PURPOSE: The imidazoline-type alpha2-adrenoceptor antagonists (+/-)-efaroxan and phentolamine increase insulin secretion and reduce blood glucose levels. It is not known whether they act by antagonizing pancreatic beta-cell alpha2-adrenoceptors or by alpha2-adrenoceptor-independent mechanisms. Many imidazolines inhibit the pancreatic beta-cell KATP channel, which is the molecular target of sulphonylurea drugs used in the treatment of type II diabetes. To investigate the mechanisms of action of (+/-)-efaroxan and phentolamine, alpha2A-adrenoceptor knockout (alpha2A-KO) mice were used. EXPERIMENTAL APPROACH: Effects of (+/-)-efaroxan, 5 mg kg(-1), and phentolamine, 1 mg kg(-1), on blood glucose and insulin levels were compared with those of the non-imidazoline alpha2-adrenoceptor antagonist [8aR,12aS,13aS]-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine (RS79948-197), 1 mg kg(-1), and the sulphonylurea glibenclamide, in alpha2A-KO and control (wild type (WT)) mice.Key results:In fed WT mice, (+/-)-efaroxan, phentolamine and RS79948-197 reduced blood glucose and increased insulin levels. Fasting abolished these effects. In fed alpha2A-KO mice, (+/-)-efaroxan, phentolamine and RS79948-197 did not alter blood glucose or insulin levels, and in fasted alpha2A-KO mice, blood glucose levels were increased. Glibenclamide, at a dose only moderately efficacious in WT mice (5 mg kg(-1)), caused severe hyperinsulinaemia and hypoglycaemia in alpha2A-KO mice. This was mimicked in WT mice by co-administration of RS79948-197 with glibenclamide. CONCLUSIONS AND IMPLICATIONS: These results suggest that (+/-)-efaroxan and phentolamine increase insulin secretion by inhibition of beta-cell alpha2A-adrenoceptors, and demonstrate a critical role for alpha2A-adrenoceptors in limiting sulphonylurea-induced hyperinsulinaemia and hypoglycaemia.     

In vivo tonic modulation of the noradrenaline release in the rat cortex by locus coeruleus somatodendritic alpha(2)-adrenoceptors

The regulation of noradrenaline release in the rat cingulate cortex by somatodendritic alpha(2)-adrenoceptors placed in the locus coeruleus was evaluated by dual-probe microdialysis. The alpha(2)-adrenoceptor antagonists BRL44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole), RS79948 ((8,12,13)-decahydro-3methoxy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]-naphthyridine) and RX821002 (2-methoxyidazoxan) administered by reverse dialysis into the locus coeruleus increased concentration-dependently (0.01-100 microM) noradrenaline release in the cortex (maximal effects 170+/-30%, 543+/-17%, 195+/-26%, respectively). Administration of the alpha(2)-adrenoceptor antagonist idazoxan increased at lower (0.1-10 microM) but decreased at the highest dose (100 microM) noradrenaline in the cortex. These data demonstrate that somatodendritic alpha(2)-adrenoceptors in the locus coeruleus exert an inhibitory tonic modulation on noradrenaline release in noradrenergic terminal areas.     

Imidazolinic radioligands for the identification of hamster adipocyte alpha 2-adrenoceptors

Imidazolinic radioligands ([3H]UK 14304, [3H]idazoxan and [3H]RX 821002) were used for the identification of alpha 2-adrenoceptors on hamster fat cell membranes since there are limitations to the use of [3H]yohimbine and [3H]clonidine, which suggest alpha 2-adrenoceptor heterogeneity. Biological assays (lipolysis measurements) were performed on isolated fat cells and binding studies were carried out on fat cell membranes. The imidazolinic derivative, UK 14304, was a full agonist as compared to clonidine. Idazoxan and RX 821002 (2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline), a recently developed alpha 2-antagonist, were more potent alpha 2-antagonists than yohimbine in this fat cell model. [3H]UK 14304 was the most suitable agent for the quantification of the 'high-affinity state' alpha 2-adrenoceptors in binding studies since it did not exhibit the sensitivity to the composition of the buffer shown by [3H]clonidine. Although it is a potent alpha 2-antagonist, [3H]idazoxan had major limitations for use in the identification of alpha 2-adrenoceptors in this cell model since it also bound to 'non-adrenaline displaceable' binding sites which were revealed when imidazolinic derivatives (phentolamine) were used instead of adrenaline to determine the non-specific binding. We demonstrated that [3H]RX 821002 was a more suitable radioligand than [3H]yohimbine for labelling hamster fat cell alpha 2-adrenoceptors (KD = 1.0 +/- 0.1 nM, Bmax = 776 +/- 60 fmol/mg protein). Moreover, since it exhibited low affinity for 'imidazoline-preferring sites', it represents a valuable ligand even in tissues possessing such binding sites. We suggest that [3H]RX 821002 can be used to identify alpha 2-adrenoceptors in various tissues when these sites cannot be labelled with [3H]yohimbine.     

Inverse agonism at alpha(2)-adrenoceptors in native tissue

Several alpha(2)-adrenoceptor antagonists have inverse agonist properties in cell culture systems, usually expressing high levels or a constitutively active form of alpha(2)-adrenoceptors. In characterizing the binding of alpha(2)-adrenoceptor agonists to rat brain tissue sections, we found that conditions known to alter agonist affinity for these receptors, particularly the addition of 100 microM GTP, altered the binding of the alpha(2)-adrenoceptor antagonist, [3H](1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride (RX821002). In further studies, we found that under our conditions [3H]RX821002 demonstrates inverse agonist properties at alpha(2)-adrenoceptors. This is the first demonstration of inverse agonism at alpha(2)-adrenoceptors in native tissue. We found that the alpha(2)-adrenoceptor antagonist, (2S,12bS)1', 3'-dimethylspiro(1,3,4,5',6,6',7,12b-octahydro-2H-benzo(b)fu ro(2, 3-a)quinazoline)-2,4'-pyrimidin-2'-one (MK-912), did not have clearly discernible inverse agonist properties and acted as a neutral antagonist in these studies. On the other hand, the antagonist rauwolscine actually displayed partial agonist properties in our studies. These findings indicate that the inverse agonist properties of alpha(2)-adrenoceptor antagonists can be demonstrated in native tissue, as well as in tissue culture, and they strengthen the idea that inverse agonist properties may be of physiological and pharmacological importance.     

In vitro and in vivo approaches to the characterization of the alpha2-adrenoceptor

1. In order to more fully understand the role of the alpha2-adrenoceptor in brain function, a combination of in vitro and in vivo techniques were utilized including radioligand binding, autoradiography, brain microdialysis and antisense oligonucleotides. 2. Binding studies showed the tritiated form of the selective alpha2-adrenoceptor antagonist, RX821002 (methoxy-idazoxan) labelled an apparent single population of sites in rat brain membranes with high affinity (1 nM), for which prazosin had low affinity (1107 nM). Similar studies in rabbit brain membranes found that prazosin and oxymetazoline were able to displace [3H]-RX821002 in a biphasic manner indicating the presence of subtypes of alpha2-adrenoceptors. 3. Receptor autoradiography revealed a distribution of [3H]-RX821002 binding in rat brain consistent with the labelling of all alpha2-adrenoceptor subtypes, namely alpha(2A/D-), alpha2B and alpha2C. 4. In rat, in vivo brain dialysis experiments demonstrated peripherally administered RX821002 elevated basal noradrenaline in frontal cortex and also, although to a lesser extent, in ventral hippocampus. RX821002 was also able to elevate extracellular dopamine in the striatum. 5. A 7-day i.c.v. infusion of an antisense oligonucleotide targeting the alpha(2A/D)-adrenoceptor, resulted in a significant reduction in the autoradiographic density of [3H]-RX821002 binding in specific brain areas, notably the lateral septal nuclei and anterior hypothalamic area. 6. Several years of research by our group has extended our knowledge of the pharmacology and function of the alpha2-adrenoceptor and has provided evidence of the roles of this receptor in the control of monoamine turnover. The successful use of antisense technology to knockdown expression of the alpha(2A/D) subtype provides future opportunities to explore the physiology of this receptor subtype.     

Effect of a 7-day treatment with idazoxan and its 2-methoxy derivative RX 821002 [correction of RX 821001] on alpha 2-adrenoceptors and non-adrenoceptor idazoxan binding sites in rabbits.

1. The present study investigates the influence of a 7-day treatment with 2 mg kg-1, s.c., twice daily of RX 821002 (an alpha 2-adrenoceptor antagonist which binds only to alpha 2-adrenoceptors) or idazoxan (alpha 2-antagonist which binds to alpha 2-adrenoceptors and also to non-adrenoceptor idazoxan binding sites: NAIBS) on alpha 2-adrenoceptor (labelled with [3H]-RX 821002) and NAIBS (labelled with [3H]-idazoxan) number in three tissues (adipocytes, colocytes and platelets) in the rabbit. 2. Acute administration of RX 821002 or idazoxan increased plasma non-esterified fatty acids (NEFA) and catecholamine levels with no change in plasma glucose levels. 3. The 7-day treatment with RX 821002 or idazoxan failed to influence food intake, total body weight or perirenal adipose tissue weight. 4. RX 821002 and idazoxan increased the number of [3H]-RX 821002 binding sites in adipose tissue with no change in colocytes or platelets. 5. RX 821002 and idazoxan failed to modify [3H]-idazoxan binding sites on adipocytes and colocytes. No significant [3H]-idazoxan binding was detected on rabbit platelets. 6. The results show that a 7-day treatment with alpha 2-antagonists induces an up-regulation in adipocyte alpha 2-adrenoceptors. In contrast, this phenomenon does not involve all the tissues since colocytes and platelets escape the effects of alpha 2-antagonists. The data suggest a differential regulation of alpha 2-adrenoceptors according to their location. 7. The fact that NAIBS did not vary suggests that alpha 2-adrenoceptors and NAIBS are two different entities.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-Isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) an irreversible imidazoline(2) binding site ligand: in vitro and in vivo characterisation in rat brain

5-Isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) is an irreversible ligand based on the highly selective I(2) binding site ligand 2BFI. In competition binding assays it has been shown to have high affinity and selectivity for the I(2) binding site and to irreversibly inhibit the binding of [(3)H]2BFI. In this present study we have sought to confirm and expand on these findings both in vitro and in vivo. In vitro pre-incubation of rat whole brain membranes with BU99006 (10 microM) was shown to reduce the specific binding of [(3)H]2BFI to 10% of the control values, an effect not seen using 2BFI or BU224. Pre-treatment of rat whole brain membranes by BU99006, or by the alpha(2)-adrenoceptor antagonists RX821002 or rauwolscine had no effect on the specific binding of [(3)H]RX821002. In vivo pre-treatment of rats with BU99006 (15 mg x kg(-1), i.v.) caused a substantial loss of [(3)H]2BFI specific binding in subsequent in vitro saturation analysis and autoradiography; this loss was shown to be dose dependent. These data indicate that BU99006 is selectively and irreversibly affecting I(2) binding sites both in vitro and in vivo and that it represents an invaluable tool in the further understanding of the I(2) binding site.     

The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues

BACKGROUND AND PURPOSE: Rodent alpha(2A)-adrenoceptors bind the classical alpha(2)-antagonists yohimbine and rauwolscine with lower affinity than the human alpha(2A)-adrenoceptor. A serine-cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of alpha(2A)-adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding. EXPERIMENTAL APPROACH: Amino acids facing the binding cavity were identified using molecular models: side chains of residues 5.43 in TM5 and xl2.49 and xl2.51 in XL2 differ between the mouse and human receptors. Reciprocal mutations were made in mouse and human alpha(2A)-adrenoceptors at positions 5.43, xl2.49 and xl2.51, and tested with a set of thirteen chemically diverse ligands in competition binding assays. KEY RESULTS: Reciprocal effects on the binding of yohimbine and rauwolscine in human and mouse alpha(2A)-adrenoceptors were observed for mutations at 5.43, xl2.49 and xl2.51. The binding profile of RS-79948-197 was reversed only by the XL2 substitutions. CONCLUSIONS AND IMPLICATIONS: Positions 5.43, xl2.49 and xl2.51 are major determinants of the species preference for yohimbine and rauwolscine of the human versus mouse alpha(2A)-adrenoceptors. Residues at positions xl2.49 and xl2.51 determine the binding preference of RS-79948-197 for the human alpha(2A)-adrenoceptor. Thus, XL2 is involved in determining the species preferences of alpha(2A)-adrenoceptors of human and mouse for some antagonists.     

Discrimination between alpha 2-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 alpha 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 alpha 2-receptivity by means of lipolysis and binding studies. Lipolysis data showed that UK14304 is a full alpha 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 alpha 2-adrenoceptors, [3H]UK14304 and [3H]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 alpha 2-adrenergic population. Inhibition studies of [3H]RX821002 and [3H]UK14304 binding by various compounds confirmed the alpha 2-adrenergic nature of the sites labelled by both radioligands. The other alpha 2-adrenoceptor radioligand, [3H]idazoxan, labelled binding sites which are insensitive to catecholamines. Competition studies of [3H]idazoxan binding with imidazoline derivatives revealed structure-activity relationships different from those of alpha 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 alpha 2-potency is increased or unchanged.     

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

alpha 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 alpha 2-adrenoceptors in this tissue using tritiated antagonists. [3H]Yohimbine and [3H]rauwolscine were not usable to label colonic alpha 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 (KD = 6.2 +/- 0.8 nM) to a single population of non-interacting sites (Bmax = 193 +/- 17 fmol/mg protein). The rank order of potency of catecholamine enantiomers and adrenergic drugs to inhibit [3H]RX821002 binding demonstrated that the labelled sites are alpha 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 alpha 2-adrenoceptors and non-adrenergic sites. When experiments were performed under binding conditions impeding the interaction of [3H]idazoxan with the alpha 2-adrenoceptors (i.e. in presence of 10(-4) M (-)-adrenaline), the Bmax of non-adrenergic idazoxan binding sites was 97 +/- 8 fmol/mg protein and the KD was 3.5 +/- 0.5 nM. The sites were pharmacologically characterized with various imidazoline and non-imidazoline drugs. In order to study the putative relationship between alpha 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 alpha 2-adrenoceptors than surface cells, whereas the number of non-adrenergic idazoxan binding sites remained constant. The results show that (i) alpha 2-adrenoceptors coexist with non-adrenergic idazoxan binding sites in rabbit colocytes; (ii) the number of alpha 2-adrenoceptors is higher in crypt cells than in surface cells and (iii) alpha 2-adrenoceptors and non-adrenergic binding sites are different and unrelated.     

Pharmacological characterisation of novel alpha 2-adrenoceptor antagonists as potential brain imaging agents

Development of suitable imaging ligands to facilitate in vivo characterisation of alpha(2)-adrenoceptors has been limited in its success. In the present study, a series of iodinated derivatives and a fluorinated derivative of the classical alpha(2)-adrenoceptor antagonist, idazoxan, have been evaluated as potential imaging ligands. These compounds are based on the structure of idazoxan but more closely resemble the selective alpha(2)-adrenoceptor antagonists 2-methoxy-idazoxan (RX821002) and 2-ethoxy-idazoxan (RX811059). Preliminary studies, investigating their affinities at alpha(2)-adrenoceptors, using brain membranes prepared from a variety of species, and their ability to antagonise UK14, 304-induced inhibition of twitch in mouse vas deferens highlighted 2-iodopropoxy-idazoxan and 2-fluoroethoxy-idazoxan as the most promising candidates. Further characterisation of these two compounds showed they had a good selectivity for alpha(2)-adrenoceptors compared with imidazoline(2)-binding sites and beta-adrenoceptors. Additional functional studies also showed a lack of intrinsic activity at alpha(2)-adrenoceptors. Following intravenous injection, both compounds were able to cross the blood brain barrier when tested using an ex vivo binding assay. These data show that both 2-iodopropoxy-idazoxan and 2-fluoroethoxy-idazoxan have binding and functional properties suitable for imaging ligands. Further studies using radiolabelled forms of these ligands and a more extensive characterisation of their binding profiles are necessary but these initial evaluations demonstrate their potential.     

Comparison of the interaction of agmatine and crude methanolic extracts of bovine lung and brain with alpha 2-adrenoceptor binding sites.

1. In the present study we have evaluated whether alpha 2-adrenoceptor binding sites on bovine cerebral cortex membranes labelled by [3H]-clonidine, [3H]-idazoxan and [3H]-RX-821002 can distinguish between known agonists and antagonists. This model has then been used to compare the binding profiles of the putative non-catecholamine, clonidine-displacing substance (CDS), agmatine and crude methanolic extracts of bovine lung and brain. 2. Saturation studies carried out in the presence and absence of noradrenaline, 10 mumol 1(-1), revealed that the maximum number of binding sites on bovine cerebral cortex membranes for [3H]-idazoxan and [3H]-RX-821002 were approximately 60-80% greater than those for [3H]-clonidine (62.6 fmol mg-1 protein). Rauwolscine, the selective alpha 2-adrenoceptor antagonist, was approximately 100 fold more potent against each of the ligands than the selective alpha 1-adrenoceptor diastereoisomer, corynanthine. Also, the pKi value for the selective alpha 1-adrenoceptor prazosin against each ligand was less than 6. 3. Adrenaline, UK-14034, rauwolscine, corynanthine, RX-811059 and prazosin produced concentration-dependent inhibition of binding of all three 3H-ligands. The agonists, adrenaline and UK-14304, were approximately 5 and 10 fold less potent against [3H]-idazoxan and [3H]-RX-821002, respectively, than against [3H]-clonidine. In marked contrast, the antagonists, rauwolscine, corynanthine, RX-811059 and prazosin exhibited a different profile, being approximately 2-3 fold more potent against sites labelled by [3H]-RX-821002 and [3H]-idazoxan compared to sites labelled by [3H]-clonidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of human platelet alpha 2-adrenoceptors with a new antagonist [3H]-RX821002, a 2-methoxy derivative of idazoxan.

1. The binding of a new alpha 2-adrenoceptor antagonist, [3H]-RX821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), was investigated in human platelet membranes and compared with [3H]-yohimbine binding parameters. 2. Analysis of kinetic data revealed association and dissociation time courses consistent with a simple biomolecular reaction. Saturation isotherms showed that [3H]-RX821002 labelled a higher total number of alpha 2-binding sites (224 +/- 31 vs 168 +/- 24 fmol mg-1 protein) than [3H]-yohimbine and with higher affinity (Kd: 0.92 +/- 0.06 vs 1.51 +/- 0.08 nM). Moreover [3H]-RX821002 exhibited a lower percentage of nonspecific binding 3. The difference in total binding is due to a better labelling of the alpha 2-adrenoceptors in the low affinity state by [3H]-RX821002 since the labelled receptors number in high affinity state was identical with the two radioligands. 4. [3H]-RX821002 binding displayed a specificity similar to that obtained with [3H]-yohimbine. The potency of various compounds acting on adrenoceptors was: yohimbine greater than oxymetazoline greater than UK14304 greater than (-)-adrenaline greater than prazosin greater than or equal to (+)-adrenaline greater than isoprenaline. This order of potency is classical for an alpha 2A-adrenoceptor. 5. RX821002 is a more potent alpha 2-adrenoceptor antagonist than yohimbine on adrenaline-induced platelet aggregation. 6. These results indicate that [3H]-RX821002 is a suitable ligand for the identification of human platelet alpha 2-adrenoceptors.     

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.     

Covariation of alpha 2-adrenoceptor density and function following irreversible antagonism with EEDQ.

1. Administration of the irreversible antagonist, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, (EEDQ, 2 mg kg-1, i.p.) to mice reduced binding of [3H]-RX 821002 (2-methoxy-idazoxan) to alpha 2-adrenoceptors in whole mouse brain by 75% 24 h later. The receptor binding returned over time only being reduced by 25% by 16 days post administration; the time taken for binding to return to 50% of control levels was estimated to be 5.25 days. 2. EEDQ administration also resulted in the loss of the sedative effect of the alpha 2-adrenoceptor agonist, medetomidine, measured by the holeboard test of directed exploration and locomotor activity. Agonist-induced sedation returned to control values by 8 days post EEDQ administration. 3. EEDQ administration also resulted in the loss of the hypothermic response to medetomidine (0.1 mg kg-1, i.p.). Medetomidine-induced hypothermia returned to control values by 12 days post EEDQ administration. 4. Pretreatment with the selective alpha 2-adrenoceptor antagonist, RX 821002 (0.1-3.0 mg kg-1, i.p.) 45 min before EEDQ prevented the loss of alpha 2-adrenoceptors as well as the blockade of medetomide-induced sedation and hypothermia by EEDQ. 5. The results of these experiments indicate that there is significant receptor reserve for alpha 2-adrenoceptor-mediated behavioural and physiological responses.     

3H]RX821002: a new tool for the identification of alpha 2A-adrenoceptors

The human adenocarcinoma cell-line HT29 was used as a model to investigate the binding properties of a new antagonist radioligand of the imidazoline series, [3H]RX821002. All aspects of [3H]RX821002 binding conclusively prove that this radioligand is a valuable tool for labelling alpha 2A-adrenoceptors. [3H]RX821002 binding was very rapid and reversible. Computer-assisted analysis of kinetic data revealed association and dissociation time courses consistent with a simple bimolecular reaction. Saturation isotherms indicated that [3H]RX821002 labeled with high affinity a single population of non-interacting sites displaying a KD of 1.7 +/- 0.1 nM. Adrenoceptor agonists and antagonists inhibited [3H]RX821002 and [3H]yohimbine binding with a strictly similar rank order of potency which is characteristic of alpha 2A-adrenoceptors. The binding parameters of [3H]RX821002 were compared with those of other commercially available [3H]antagonists, [3H]yohimbine and [3H]idazoxan. Analysis of the saturation isotherms for the three radioligands showed that (1) [3H]RX821002 was the radioligand exhibiting the lower percentage of non-specific binding and the better affinity, (2) the Bmax of [3H]RX821002 was significantly higher than that of [3H]yohimbine. The difference in Bmax was not due to better labelling of one of the two affinity states of the receptor but was greatly reduced in glycylglycine buffer, suggesting that, in Tris-Mg2+ buffer, [3H]yohimbine does not label the entire alpha 2-adrenoceptor population.     

Characterization and autoradiographical localization of non-adrenoceptor idazoxan binding sites in the rat brain.

1. In rat whole brain homogenates, saturation analysis revealed that both [3H]-idazoxan and [3H]-RX821002, a selective alpha 2-adrenoceptor ligand, bound with high affinity to an apparent single population of sites. However, the Bmax for [3H]-idazoxan was significantly (P less than 0.01) greater than that for [3H]-RX821002. 2. In competition studies, (-)-adrenaline displaced 3 nM [3H]-idazoxan binding with an affinity consistent with [3H]-idazoxan labelling alpha 2-adrenoceptors. However, this displacement was incomplete since 23.68 +/- 1.11% of specific [3H]-idazoxan binding remained in the presence of an excess concentration (100 microM) of (-)-adrenaline. In contrast, unlabelled idazoxan promoted a complete displacement of [3H]-idazoxan binding with a Hill slope close to unity and an affinity comparable with its KD determined in saturation studies. 3. Displacement of [3H]-idazoxan binding by the alpha 2-adrenoceptor antagonists yohimbine, RX821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline) and RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline) was more complex, with Hill slopes considerably less than unity, and best described by a two-site model of interaction comprising a high and low affinity component. The proportion of sites with high affinity for each antagonist was similar (60-80%). 4. The rank order of antagonist potency for the high affinity component in each displacement curve (RX821002 greater than RX811059 greater than yohimbine) is similar to that determined against the binding of [3H]-RX821002 to rat brain, suggesting that these components reflect the inhibition of [3H]-idazoxan binding to alpha 2-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that the novel imidazoline compound FT005 is an alpha(2)-adrenoceptor agonist

1: The aim of this study was to determine whether the hyperglycaemic action of the novel imidazoline compound FT005 could be mediated by activation of alpha(2)-adrenoceptors, using a variety of in vivo and in vitro methods including radioligand binding. 2: FT005 produced a dose-dependent increase in blood glucose levels of CBA/Ca mice (0.125-25 mg kg(-1), i.p.). The time course of this hyperglycaemic effect matched that of adrenaline (1 mg kg(-1)) more closely than glucagon (1 mg kg(-1)) or the K(ATP) channel opener diazoxide (25 mg kg(-1)). The hyperglycaemic effect of FT005 (1 mg kg(-1)) was significantly reduced by the alpha(2)-adrenoceptor antagonist rauwolscine (0.5 mg kg(-1)). 3: FT005 produced a significant reduction in plasma insulin levels of mice 30 min after administration. The hyperglycaemic effect of FT005 (25 mg kg(-1)), although still present, was significantly less in fasted mice in which insulin levels are lower, suggesting that a reduction of insulin secretion contributes to the action of FT005. 4: When studied in the mouse isolated vas deferens preparation, FT005 produced a complete inhibition of neurogenic contractions, which was blocked by rauwolscine. This is consistent with activation of pre-synaptic alpha(2)-adrenoceptors. 5: In radioligand binding studies FT005 completely displaced the alpha(2)-adrenoceptor antagonist [(3)H]-RX821002 from mouse whole brain homogenates. The displacement was best described by a two-site model of interaction comprising high and low affinity components. 6: The results indicate that FT005 is an agonist at alpha(2)-adrenoceptors. A reduction in insulin secretion contributes to the hyperglycaemic action of FT005, although an additional mechanism can not be excluded.