Species-selective binding of [3H]-idazoxan to alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in the central nervous system.

1. We have used the imidazoline derivative [3H]-idazoxan to define alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in cerebral cortex membranes of calf, mouse, rat, guinea-pig and man. 2. Competition experiments using the selective alpha-adrenoceptor drugs, rauwolscine and corynanthine, indicated that [3H]-idazoxan bound to a single population of sites in the calf and mouse membranes. However, [3H]-idazoxan also labelled non-adrenoceptor, imidazoline binding sites in the rat (15%), guinea-pig (30%) and human (40%) cerebral cortex membranes. 3. Competition experiments with adrenaline and cirazoline in the guinea-pig cortex, verified [3H]-idazoxan binding to both alpha 2-adrenoceptors and to non-adrenoceptor, imidazoline binding sites. 4. It has been postulated by several groups that [3H]-idazoxan may possess partial agonist activity. To investigate this further, saturation experiments were performed in the cerebral cortex membranes of all five species in the absence and presence of 300 microM guanosine triphosphate (GTP). GTP had no effect on [3H]-idazoxan binding in guinea-pig cerebral cortex; in both rat and mouse membranes 300 microM GTP increased the dissociation constant for [3H]-idazoxan by 2-3 fold without significantly affecting the Bmax. GTP reduced the Bmax by approximately 30% and 60% in calf and human cerebral cortex membranes, respectively, without significantly altering the Kd. 5. Saturation experiments were performed in the calf cerebral cortex membranes in the absence and presence of 300 microM GTP with the selective alpha 2-adrenoceptor agonist [3H]-clonidine and the selective muscarinic antagonist [3H]-quinuclidinyl benzilate (QNB).(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

[3H]-idazoxan binding to rabbit cerebral cortex recognises multiple imidazoline I2-type receptors: pharmacological characterization and relationship to monoamine oxidase.

1. In rabbit cerebral cortical homogenates, saturation analysis of [3H]-idazoxan, an alpha 2-adrenoceptor antagonist, revealed high affinity binding to a single site with high density. Competition experiments demonstrated that the [3H]-idazoxan recognition site was insensitive to the catecholamines, adrenaline and noradrenaline and possessed a low affinity for the alpha 2- and alpha 1-adrenoceptor antagonists, rauwolscine, yohimbine and prazosin, suggesting that the site was not an adrenoceptor. Mapping [3H]-idazoxan binding sites in the forebrain of rabbits by autoradiography, showed high densities of I2 sites in the medial preoptic area and in the stria terminalis. Moderate binding was found in caudate nucleus, putamen, cerebral cortex and hippocampus. 2. The imidazolines cirazoline, naphazoline, guanabenz and BRL44408 along with amiloride, which is structurally related to the imidazolines, all had high affinity for the [3H]-idazoxan site, suggesting that the site was related to the I2 imidazoline-recognition site described by other groups. However, the imidazolines, clonidine and UK-14,304 and the structurally related rilmenidine all had a low affinity for the binding site, showing that [3H]-idazoxan was not binding to the I1 imidazoline-recognition site found in rat, bovine and human medulla oblongata. 3. Naphazoline, guanabenz, clonidine and amiloride competition studies had Hill slopes which were significantly different from unity (P < 0.01) and computer analysis showed that the [3H]-idazoxan binding data could be best fitted to a model which considers binding to two sites (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

The relationship between density of alpha-adrenoceptor binding sites and contractile responses in several porcine isolated blood vessels.

1. The aim of this study was to investigate constrictor alpha-adrenoceptors in three isolated blood vessels of the pig, the thoracic aorta (TA), the splenic artery (SA) and marginal ear vein (MEV) and then compare the functional response with the densities of alpha 1- and alpha 2-adrenoceptor binding sites in these and several other porcine vascular tissues, palmar common digital artery (PCDA), palmar lateral vein (PLV) and ear artery (EA). 2. Noradrenaline (NA), phenylephrine (PE) and UK14304 (all at 0.03-10 microM) elicited concentration-dependent contractions in the TA and MEV, with a rank order of potency of UK14304 > NA > PE. UK14304 produced maximal responses which were 58% (TA) and 65% (MEV) of that of NA. In the SA, UK14304 and PE produced maximal responses which were less than 10% and 50% of the NA-induced maximal response respectively, with an order of potency of NA > PE. In the SA, NA-induced contractions were competitively antagonized by prazosin (pA2 = 8.60 +/- 0.15). Further, rauwolscine (1-10 microM) antagonized NA-induced contractions with an apparent pKB of 6.09 +/- 0.11 (n = 6), indicating an action at alpha 1-adrenoceptors. The combination of the two antagonists at concentrations selective for alpha 1- (0.1 microM) and alpha 2-adrenoceptors (1 microM) had no greater effect than either antagonist alone. This suggests that the SA expresses only post-junctional alpha 1-adrenoceptors. 3. In the TA, prazosin produced non-parallel shifts in the NA-induced CRC and this was also observed with rauwolscine, where reductions in the maximal responses were also observed. In the MEV, prazosin was largely inactive in antagonizing NA-induced contractions. In both these vessels a combination of these two antagonists had a greater effect than either alone, indicating the presence of functional alpha 1- and alpha 2-adrenoceptors. The post-junctional alpha 2-adrenoceptors in all of these vessels were resistant to prazosin, suggesting the alpha 2-adrenoceptor to be of the alpha 2A/2D subtype. The expression of functional alpha 2-adrenoceptors was MEV > TA > PLV > PCDA > SA. 4. In radioligand binding studies using TA P2 pellet membranes, [3H]-prazosin and [3H]-RX821002 ([1,4-[6,7(n)-3H] benzodioxan-2-methoxy-2-yl)-2-imidazole) labelled different high affinity sites, and in competition studies using identical membranes corynanthine displaced [3H]-prazosin with 10 fold higher affinity than rauwolscine, indicating that [3H]-prazosin was selectively binding to alpha 1-adrenoceptor sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that the population of postjunctional-adrenoceptors mediating contraction of smooth muscle in the rabbit isolated ear vein is predominantly alpha 2.

1. Noradrenaline (NA), phenylephrine and UK-14304 elicited concentration-dependent contractions of the rabbit isolated ear vein of similar maximal magnitude. The rank order of potency, UK-14304 greater than noradrenaline greater than phenylephrine, is consistent with that of an effect mediated through an alpha 2-subtype. 2. The potent and highly selective alpha 1-adrenoceptor antagonists prazosin and YM-12617, at concentrations as high as 1 microM, produced less than a 4 fold rightward displacement of the NA concentration-response curve. 3. The selective alpha 2-adrenoceptor antagonists rauwolscine, Wy-26703 and CH-38083 antagonized responses to noradrenaline in a competitive manner. For all three antagonists, the pA2 values were consistent with an effect at alpha 2-adrenoceptors. However, 0.1 microM YM-12617 increased the potency of rauwolscine 2 fold indicating the presence of a small population of postjunctional alpha 1-adrenoceptors. 4. The relative antagonist potency of the yohimbine diastereoisomers rauwolscine and corynanthine against noradrenaline (rauwolscine 30 fold greater than corynanthine) is also consistent with an effect at alpha 2-adrenoceptors. 5. Contractions elicited by noradrenaline in the rabbit isolated ear vein appear to be mediated predominantly by postjunctional alpha 2-adrenoceptors.     

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.     

[3H]-idazoxan binds with high affinity to two sites on hamster adipocytes: an alpha 2-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.     

Alpha-adrenoceptors in dog mesenteric vessels--subcellular distribution and number of [3H]prazosin and [3H]rauwolscine binding sites.

Binding of the alpha-adrenergic antagonists [3H]prazosin and [3H]rauwolscine to well-characterized subcellular membrane fractions isolated from dog mesenteric arteries and veins was studied. Binding of both ligands was saturable with Kd values of 0.5 +/- 0.1 nM for [3H]prazosin and 5.85 +/- 0.85 nM for [3H]rauwolscine in arteries, and 0.87 +/- 0.4 nM for [3H]prazosin and 6.6 +/- 1.5 nM for [3H]rauwolscine in veins. In veins, the maximum number of binding sites for [3H]rauwolscine was higher than that for [3H]prazosin, whereas in arteries the maximum number of binding sites for each ligand was similar. In microsomes from dog aorta, the maximum number of bindings sites for [3H]prazosin was higher than that for [3H]rauwolscine. Neuronal membrane contamination in these studies was minimized by dissection procedures and evaluated by the comparison of [3H]saxitoxin binding in various preparations. Only mesenteric veins responded functionally to agonists acting on alpha 2 adrenoceptors. This study thus identified two distinct populations of [3H]prazosin and [3H]rauwolscine binding sites in the plasma membranes of dog mesenteric vessels and suggests that a much higher density of alpha 2-compared to alpha 1-adrenoceptor binding sites is required for a contractile response.     

Evidence for the presence of a non-catecholamine, clonidine-displacing substance in crude, methanolic extracts of bovine brain and lung

In the present study we have prepared crude, methanolic extracts of bovine lung and bovine brain and, using radioligand binding assays in conjunction with a number of simple chromatographic techniques, provided evidence for the presence of a non-catecholamine ’clonidine-displacing substance‘ (CDS). The level of CDS in lung extracts (9?units/g wet weight n=11) is approximately 3 times that in the brain extracts. Furthermore, the effect of the crude, methanolic extracts are selective for non-adrenoceptor, imidazoline (labelled by [ ³H]-idazoxan) and a ₂-adrenoceptor binding sites (labelled by [ ³H]-clonidine); both extracts are 5–10-fold more potent displacers of ligand binding to a ₂-adrenoceptors compared with binding to opiate receptors (labelled by [ ³H]-etorphine) and practically inactive against a ₁-adrenoceptor and muscarinic binding sites (labelled by [ ³H]- prazosin and [ ³H]-quinuclidinyl benzilate, respectively). With the exception of the non-adrenoceptor, imidazoline binding assay, which used rat kidney membranes labelled by [ ³H]-idazoxan in the presence of the a ₂-adrenoceptor antagonist RS-15385-197, all radioreceptor assays involved bovine cerebral cortex membranes. Although the extracts contain catecholamines (brain only), histamine (lung only) and monovalent cations (both), which have the potential to interfere with the radioligand binding assays, their concentrations were too low to account for the effects observed. Preliminary attempts at purification of the extracts revealed that CDS activities from the two tissues are similar, i.e., practically insoluble in organic solvents at room temperature, not affected by either Sep-Pak C ₁₈ column or anion exchange resins but retained (along with the monovalent cations) by cation exchange resin. However, following chromatographic separation on a Biogel P2 column, the CDS-containing eluates are cation-free and exhibit qualitatively similar elution profiles. Future experiments will involve further purification of ’clonidine-displacing substance‘ to characterize its interaction with a ₂-adrenoceptor binding sites in greater detail and establish whether it has biological activity consistent with the properties implied by its effects in radioligand binding assays.     

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.     

An examination of the postjunctional alpha-adrenoceptor subtypes for (-)-noradrenaline in several isolated blood vessels from the rabbit.

1. Postjunctional alpha-adrenoceptors in several isolated blood vessels from the rabbit have been characterized on the basis of the relative potency of the agonists noradrenaline (NA, non-selective), phenylephrine (alpha 1-selective) and UK-14304 (alpha 2-selective), and the potency of antagonists rauwolscine (alpha 2-selective) and corynanthine (alpha 1-selective) against contractions elicited by NA. In addition, the potency of prazosin against NA was also assessed in the venous preparations. 2. The thoracic aorta, ear artery and left renal vein appear to possess alpha 1-adrenoceptors since the agonist potency order was NA greater than phenylephrine greater than UK-14304, while corynanthine was 3-10 fold more potent than rauwolscine. 3. The ear vein appears to possess alpha 2-adrenoceptors. The rank order of agonist potency was UK-14304 greater than NA much greater than phenylephrine and all three agonists elicited responses of similar magnitude. Furthermore, rauwolscine was 30 fold more potent than corynanthine while prazosin failed to produce a concentration-dependent inhibition. 4. The saphenous vein and the plantaris vein appear to possess a mixture of both subtypes since the rank order of agonist potency was UK-14304 greater than NA much greater than phenylephrine, while responses elicited by UK-14304 were smaller than those to the other agonists. However, although rauwolscine was 20 to 100 fold more potent than corynanthine in both preparations, suggestive of predominantly alpha 2-adrenoceptors, prazosin was either potent (saphenous vein) or relatively inactive (plantaris vein). 5. The characteristics of postjunctional alpha 1- and alpha 2-adrenoceptors on isolated blood vessels from the rabbit are discussed in relation to the value of both the agonists, particularly NA, and the antagonists used in this study.     

Characterization of the alpha-adrenoceptors in the female rabbit urethra.

A radioligand binding technique was used to evaluate the proportions of alpha 1- and alpha 2-adrenoceptors in crude membrane preparations obtained from the female rabbit bladder base and urethra. In addition, urethral rings were studied in vitro in an attempt to determine if alpha 1- and/or alpha 2-adrenoceptors are located postjunctionally in the urethral smooth muscle. Studies of the inhibition of [3H]-dihydroergocryptine binding by the selective alpha 1-adrenoceptor antagonist prazosin or the selective alpha 2-adrenoceptor antagonist rauwolscine revealed the alpha-adrenoceptor population to consist of approximately 25% alpha 1-adrenoceptors and 75% alpha 2-adrenoceptors. These proportions were confirmed in saturation studies with [3H]-prazosin and [3H]-rauwolscine. The sum of alpha 1- and alpha 2-adrenoceptors labelled by these selective alpha 1- and alpha 2-adrenoceptor antagonists was about equal to the number labelled by the non-selective alpha-adrenoceptor antagonist [3H]-dihydroergocryptine. Noradrenaline, as well as the selective alpha 1-adrenoceptor agonist phenylephrine and the selective alpha 2-adrenoceptor agonist clonidine, induced contractions of urethral ring preparations. Prazosin blocked contractions induced by phenylephrine to a greater extent than contractions induced by clonidine. The opposite was true for the inhibitory effect of rauwolscine. In addition to showing that both alpha 1- and alpha 2-adrenoceptor binding sites exist in membrane preparations of the rabbit bladder base and urethra, the results reveal the presence of both adrenoceptor subtypes postjunctionally in the rabbit urethra; and both mediate contraction of the smooth muscle.     

3H]-RS-15385-197, a selective and high affinity radioligand for alpha 2-adrenoceptors: implications for receptor classification.

1. RS-15385-197 is the most potent and selective alpha 2-adrenoceptor antagonist available. We have used [3H]-RS-15385-197 to define alpha 2-adrenoceptor subtypes. The binding of [3H]-RS-15385-197 to membranes of rat cerebral cortex, rat neonatal lung and human platelets was reversible, saturable and of high affinity. Saturation experiments indicated that [3H]-RS-15385-197 bound to a single population of sites in all 3 tissues with high affinity (0.08-0.14 nM). The density of sites labelled by [3H]-RS-15385-197 was greater in the cortex (275 fmol mg-1 protein) than in the neonate lung (174 fmol mg-1 protein) and human platelet (170 fmol mg-1 protein). The density of sites labelled with [3H]-RS-15385-197 in the cortex was significantly greater than that labelled with [3H]-yohimbine (121 fmol mg-1 protein). 2. The selective alpha 2-adrenoceptor antagonists, idazoxan, yohimbine, rauwolscine and WY 26703 displaced [3H]-RS-15385-197 binding to rat cerebral cortex in a simple manner with Hill slopes close to unity. The affinities derived for these antagonists against [3H]-RS-15385-197 were similar to the values obtained for the displacement of [3H]-yohimbine indicating the alpha 2-adrenoceptor nature of the binding site. 3. alpha 2A-Adrenoceptor selective compounds, oxymetazoline and BRL 44409, showed high affinity for [3H]-RS-15385-197 binding in the human platelet and lower affinity in the neonate lung, while the alpha 2B-selective compounds, prazosin and imiloxan, showed high affinity for [3H]-RS-15385-197 binding in the neonate lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat spinal cord alpha 2-adrenoceptors are of the alpha 2A-subtype: comparison with alpha 2A- and alpha 2B-adrenoceptors in rat spleen, cerebral cortex and kidney using 3H-RX821002 ligand binding.

Binding of the alpha 2-adrenoceptor antagonist radioligand 3H-RX821002 was investigated in membranes from rat spinal cord, spleen, cerebral cortex and kidney. The ligand was found to bind to saturable binding sites with apparent uniform affinities within each tissue. Seven compounds, some of which have previously been reported to be selective for either alpha 2A- or alpha 2B-adrenoceptors, were used in competition with 3H-RX821002. By using computer modelling, competition curves generated for three of these compounds (ARC 239, prazosin and oxymetazoline) could be resolved into two site fits in the kidney, Kd's of the drugs being compatible with the notion that these sites corresponded to alpha 2A- and alpha 2B-adrenoceptors. Moreover, rauwolscine and yohimbine were found to be about 14 and 9-fold selective for alpha 2B-adrenoceptors in the kidney. In all other tissues studied drug competition curves were uniphasic and computer modelled into one site fits, drug Kd's being well correlated to those for the alpha 2A-adrenoceptor. In rat spinal cord 26 further drugs, which showed wide variation in structure, were evaluated in competition with 3H-RX821002. Of these compounds, competion curves of the agonists UK-14,304, (-) and (+) adrenaline were modelled into two site fits whereas those of the remaining compounds could be modelled only into one site fits. Since the high affinity site for UK-14,304, (-) and (+) adrenaline was eliminated when EDTA, Gpp(NH)p and 140 mM NaCl was present in the assay the heterogeniety observed in spinal cord was considered to be due to formation of high and low affinity conformations of the alpha 2-adrenoceptor for agonists. It is concluded that 3H-RX821002 is useful to label both alpha 2A- and alpha 2B-adrenoceptors in the rat. Moreover, the binding sites labelled by 3H-RX821002 in the spinal cord appear to consist of a single population of alpha 2-adrenoceptors of the alpha 2A-type.     

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.     

Alpha 1-adrenoceptors: the ability of various agonists and antagonists to discriminate between two distinct [3H]prazosin binding sites

Recently, it has been demonstrated that two distinct alpha 1-adrenoceptor binding sites showing high and low affinity for WB-4101 (2-(2,6-dimethoxyphenoxy)ethyl-aminomethyl-1,4-benzodioxane) and 5-methyl-urapidil can be distinguished. In the present study we examined the ability of several agonists and antagonists to discriminate between these alpha 1-adrenoceptor binding sites. [3H]Prazosin binding to membranes of rat liver, heart, cerebral cortex and hippocampus was inhibited monophasically by butanserine, I-BE 2254 (2-(3-(4-hydroxy-3-iodophenyl)ethylaminomethyl)tetralone-hydrochloride), prazosin, rauwolscine and verapamil. In contrast, competition curves of adrenaline, oxymetazoline, amidephrine and YM-12617 (5-[2-[[2-(o-ethoxy-phenoxy)ethyl]-amino]propyl]-2- methoxybenzenesulfonamide HCl) were best described by a model of two binding sites. Chloroethylclonidine (CEC), a compound shown to irreversibly eliminate binding sites with low affinity for WB-4101, increased the proportion of high affinity binding sites for oxymetazoline and amidephrine, whereas the binding data for prazosin and adrenaline remained unchanged. These results indicate that amidephrine, oxymetazoline and YM-12617, but not the other drugs tested discriminate between different alpha 1-adrenoceptor recognition sites labelled by [3H]prazosin.     

Alpha 2-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.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Alpha adrenoceptor sites in vascular smooth muscle. Differentiation by selective antagonist binding

The properties of alpha adrenoceptors in rat-tail artery membranes were studied using tritiated ligands that are selective for the alpha 1 and alpha 2 subtypes. High-affinity saturable binding was obtained for the alpha 1 antagonist prazosin yielding a Bmax of 144 +/- 31.6 fmol/mg protein (mean +/- SEM, N = 3) and a Kd of 0.17 +/- 0.04 nM, and also for the alpha 2 antagonist rauwolscine which yielded a Bmax of 141.3 +/- 19.3 fmol/mg protein and a Kd of 1.57 +/- 0.32 nM. The [3H]prazosin-labelled sites displayed a pharmacological profile characteristic of an alpha 1 adrenoceptor, whereas the [3H]rauwolscine-labelled sites exhibited the expected alpha 2 adrenoceptor profile. Agonist affinity for [3H]rauwolscine sites was reduced by Gpp(NH)p and Na+, and the effects appeared synergistic for adrenaline, but non-interactive for UK-14304. Agonist interaction with [3H]prazosin sites in the rat-tail artery was also regulated by Gpp(NH)p and Na+, although clearly in a qualitatively and quantitatively different manner from the [3H]rauwolscine sites. These results suggest that distinct binding sites for [3H]prazosin and [3H]rauwolscine could be differentiated with antagonist ligands. These distinct antagonist recognition sites demonstrate the pharmacological profile expected for alpha 1 and alpha 2 adrenoceptors, and the quantitatively differing abilities of Na+ and Gpp(NH)p to regulate agonist interactions with these sites are suggestive, but do not necessarily prove, that different G proteins may be involved in this regulation.