Non-adrenergic Sites Research Articles

Evidence for nonadrenoceptor responses to imidazoline derivatives in the porcine isolated rectal artery.

High concentrations of phentolamine, efaroxan, and idazoxan were found to produce nonadrenoceptor contractions of the porcine isolated rectal artery previously exposed to U46619 and forskolin. These responses were insensitive to the putative imidazoline I(3) receptor antagonist KU-14R, unlike those previously reported in this preparation for oxymetazoline. The pharmacologic nature of this response and the obligate requirement for preconstriction suggests that these imidazoline derivatives modulate ion channel function through a novel nonadrenergic site.

Autoradiographic Comparison of [3H]-Clonidine Binding to Non-Adrenergic Sites and α2-Adrenergic Receptors in Human Brain

Clonidine is a partial agonist at brain α2-adrenoceptors (α2AR), but also has high affinity (KD = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and α2AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 μM norepinephrine (NE; α2AR agonist) or 12.5 μM methoxy-idazoxan (MIDX; selective α2AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 μM cirazoline. Levels of [3H]-clonidine binding to α2AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r2 = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: α2AR (>4:1). Quantitative transepts drawn across hippocampal images revealed α2AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus—areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I1 and I2 subtypes. The rank-order of affinities were cirazoline > harmane > BDF6143 > idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 ± 12 nM). In conclusion: 1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from α2AR; and 2) the affinities of these sites was distinct from α1AR, α2AR, I1 or I2 sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.

Expression of I 2-imidazoline sites in rat prostate : Effect of castration and aging

Clonidine, idazoxan, and related imidazoline adrenergic drugs bind to non-adrenergic sites in brain and several peripheral tissues. These sites, termed imidazoline receptors, appear to exist in two major subclasses, I 1 sites labeled by clonidine and I 2 sites labeled by idazoxan. In this study, we investigated whether rat prostate expresses imidazoline receptors and, if so, whether their expression can be regulated by circulating testosterone. Studies in rat ventral prostate membrane revealed that [ 3H]idazoxan, but not [ 3H]p- aminoclonidine , bound to non-adrenergic sites. The binding of [ 3H]idazoxan was saturable ( B max: 941 ± 105 fmol/mg protein) and high affinity ( K D : 16.4 ± 2.3 nM). The rank order of the inhibition of binding by imidazoline ligands was cirazoline > clonidine > UK 14,304 > guanabenz, indicating an I 2 subclass of imidazoline receptors. Bilateral orchiectomy increased the number of binding sites ( B max) for [ 3H]idazoxan without changing the affinity ( K D ). Testosterone replacement, while completely restoring the plasma testosterone levels, only partially reversed the increase in B max. In contrast, the binding of [ 3H] idazoxan to prostate membranes of rats in different age groups (4, 7, and 16 months) revealed a progressive decrease in the B max without any change in K D . We conclude that the rat prostate expresses the I 2 subclass of imidazoline receptors and that the expression is regulated by circulating testosterone.

Differential characteristics and localisation of [ 3H]oxazoline and [ 3H]imidazoline binding sites in rat kidney

Comparative autoradiography revealed that the imidazolines [ 3H] p-aminoclonidine and [ 3H]idazoxan labelled high densities of α 2A-adrenoceptor sites in the inner medulla and inner stripe of the outer medulla of rat kidney. In contrast, the oxazoline [ 3H]rilmenidine labelled a high density of non-adrenergic sites in the cortex and outer stripe of the outer medulla, a lower density in the inner stripe and inner medulla and a low density of α 2A-adrenoceptor sites in inner medulla. The existence of novel, non-adrenergic oxazoline sites of potential functional importance in rat kidney has important implications for the classification of imidazoline receptors.

Clonidine and cirazoline inhibit activation of nicotinic channels in PC-12 cells.

Clonidine and cirazoline bind with high affinity to a nonadrenergic site in the brain stem, the so-called imidazoline I1 receptor. Our aim was to determine the mechanism by which these receptors act and their possible linkage to signal-transducing heterotrimeric G-proteins. We examined the effects of clonidine and cirazoline on PC-12 cells, a neuronal cell line that is reported to possess the I1 site and have no alpha 2-adrenoceptors. In undifferentiated PC-12 cells loaded with the Ca2+ indicator dye fura-2, clonidine and cirazoline (10-100 microM) inhibited the increase in [Ca2+]i produced by nicotine (10 microM). This inhibition was not reversed by yohimbine (100 microM), and adrenaline and BHT 920 were ineffective at 100 microM. This effect was not inhibited by pretreatment with pertussis toxin (24 hours, 100 ng/ml) and not modulated by pretreatment with IBMX (100 microM). The nicotine-induced increase in [Ca2+]i is apparently due to Ca2+ entering via the intrinsic ion channel of the nicotinic acetylcholine receptor. Clonidine and cirazoline inhibited the inward current produced by nicotine (10 microM) as measured by the whole cell patch-clamp technique in differentiated PC-12 cells, recorded at a holding potential of -60 mV. In agreement with the results found with fura-2, inhibition of inward current was concentration dependent and not blocked by yohimbine (100 microM) or mimicked by adrenaline (100 microM). Pretreatment of PC-12 cells with pertussis toxin or infusion of GDP-beta-S (2 mM) via the patch pipette did not alter the inhibition of the nicotine-induced inward current by clonidine or cirazoline. Clonidine and cirazoline, but not adrenaline, displayed [3H]phencyclidine from Torpedo electroplaque membranes enriched in nicotinic acetylcholine receptors in a concentration-dependent manner (10-100 microM). Taken together, these results suggest that clonidine and cirazoline inhibit Na+ and Ca2+ entry through the nicotinic acetylcholine receptor via a nonadrenergic mechanism that is independent of G-proteins and cyclic nucleotides, presumably by direct blockade of the intrinsic ion channel of the nicotinic acetylcholine receptor.

Imidazoline receptors, non-adrenergic idazoxan binding sites and α 2-adrenoceptors in the human central nervous system

Both [ 3H]clonidine and [ 3H]idazoxan bind to α 2 adrenoceptors. The former also labels imidazoline receptors, and the latter non-adrenergic idazoxan binding sites. In order to investigate whether the imidazoline receptors and non-adrenergic idazoxan binding sites are identical, we compared the binding characteristics of [ 3H]clonidine and [ 3H]idazoxan to these sites by radioligand binding experiments on ultra-thin slices and homogenates of human striatum. A good correlation was found between the effect of different ions on the binding characteristics of [ 3H]clonidine and [ 3H]idazoxan, and the affinities of most competing drugs. However, clonidine and rilmenidine displayed a 100- and 10-fold lower affinity, respectively, for the idazoxan binding sites than for the imidazoline receptors. Autoradiography with [ 3H]clonidine showed that high densities of imidazoline receptors were present in the striatum, pallidum, gyrus dentatus of the hippocampus, amygdala, and substantia nigra. Moderate densities were found throughout the cerebral cortex, thalamus and several brainstem nuclei including the nucleus olivarius inferior. Low densities were seen in the cerebellum, spinal cord and pituitary gland. As for the non-adrenergic sites labelled by [ 3H]idazoxan, the imidazoline receptors can be found in all major brain areas examined. However, there are some striking differences between the concentrations of imidazoline receptors and non-adrenergic idazoxan binding sites in certain brain regions. To reconcile distribution and pharmacologic data, we propose that imidazoline receptors and non-adrenergic idazoxan binding sites represent different proteins or protein complexes and that at least in the nucleus reticularis lateralis and the striatum, imidazoline receptors and non-adrenergic idazoxan binding sites may be physically associated. The regional distribution of α 2 adrenoceptors within the human CNS was determined by quantitative autoradiography with [ 3H]RX821002. The highest densities of α 2 adrenoceptors were found in the cerebral and cerebellar cortex, and certain regions in the medulla oblongata (floor of the IV ventricle, reticular formation, hypoglossal nucleus and nucleus olivarius inferior). No α 2 adrenoceptors were detected in the pituitary gland. There exists no relationship between the distribution pattern of imidazoline receptors and α 2 adrenoceptors, indicating that these binding sites are independent from each other.

α 2A Adrenoceptors and non-adrenergic idazoxan binding sites in calf brain and retina are distinct from those in human brain

α 2 Adrenoceptors in membrane preparations of human and calf frontal cortex and of calf retina can be labelled by the antagonists [ 3H]idazoxan, [ 3H]rauwolscine and [ 3H]RX 821002. Present and previous data indicate that [ 3H]idazoxan possesses the highest affinity for the α 2 adrenoceptors in the calf tissues, whereas [ 3H]rauwolscine displays the highest affinity for those in the human frontal cortex. Competition binding experiments with adrenergic and serotonergic drugs further support the notion that the α 2 adrenoceptors in calf frontal cortex and retina are similar, but distinct from the receptors in human frontal cortex. The α 2 adrenoceptors in the three investigated tissues display low affinity for the antagonist prazosin, which suggests that they all belong to the α 2A subclass. The competition binding curves of the α 2A adrenoceptor subtype-selective agonist oxymetazoline are shallow, but undergo a rightward shift and steepening in the presence of GTP. The shallow curves can therefore be attributed to the coupling of the α 2 adrenoceptors to G proteins. The different binding characteristics of the α 2A adrenoceptors from the investigated human and bovine tissues are likely to reflect species-related differences in protein structure. [ 3H]Idazoxan binds also to non-adrenergic sites in membrane preparations from the three tissues. However, the affinity of [ 3H]idazoxan for these sites in calf cortex and retina is appreciably lower than for those in human cortex. The species-related differences of the non-adrenergic idazoxan binding sites may be due to differences in protein structure or even to differences in gene-product.

Expression of non-adrenergic imidazoline sites in rat cerebral cortical astrocytes.

Clonidine and related imidazoline agents, beside binding to alpha 2-adrenergic receptors, have been shown to bind to a non-adrenergic site (imidazoline sites) in brain and peripheral tissues. However, which cell types in brain, namely neurons or glia, express this binding site and the cellular effects of activation of this site are not known. We investigated the cellular localization of imidazoline binding sites in cultured rat cortical astrocytes and neurons. Membranes prepared from cultured astrocytes showed specific, high affinity binding (KD: 4 nM) for 3H-idazoxan with about tenfold higher number of binding sites than alpha 2-adrenergic sites (Bmax: 220 vs. 20 fmol/mg protein). Displacement studies exhibited the rank order of potency: cirazoline > idazoxan > amiloride > clonidine >>> epinephrine = ruawolscine defining this site as I-2a subtype of imidazoline binding sites. Moreover, the binding was inhibited by K+ but not by Na+, another characteristic of imidazoline binding sites. In contrast, membranes prepared from cultured neurons showed fewer binding sites for 3H-idazoxan that were completely displayed by adrenergic agents. Incubation of astrocytes with idazoxan, but not rauwolscine, resulted in a concentration-dependent increase in the levels of mRNA for the astrocyte specific molecule glial fibrillary acidic protein. We conclude that (a) the non-adrenergic imidazoline binding sites are expressed in astrocytes but not in neurons in rat cerebral cortex and (b) these "receptors" may influence astrocyte physiology by regulating the levels of GFAP.

Further pharmacological characterization of [ 3H]idazoxan binding sites in rat brain: evidence for predominant labeling of α2-adrenergic receptors

In addition to binding to α 2-adrenergic receptors, the antagonist [ 3H]idazoxan has been reported to bind to non-adrenergic sites in a number of tissues and species. In the present study, the pharmacological nature of [ 3H]idazoxan binding sites in rat brain slices has been examined using radioligand binding and autoradiographic technniques. In Na 2KHPO 4 buffer, four drugs with high affinity for α 2-adrenergic binding sites were potent inhibitors of [ 3H]idazoxan binding, with the rank order of potency being RX821002 > phentolamine > yohimbine > (-)epinephrine. Non-linear regression analysis resolved all competition curves into two components, with the high affinity site representing the majority of total [ 3H]idazoxan binding. In autoradiographic studies performed in Na 2KHPO 4 buffer, all α 2-selective ligands displaced >/75% of total [ 3H]idazoxan binding to most brain regions. These findings indicate that the major component of [ 3H]idazoxan binding was to sites that are α 2-adrenergic in nature. [ 3H]Idazoxan binding was also examined in glycylglycine buffer. In contrast to binding in Na 2KHPO 4 buffer, the proportion of low affinity sites was significantly increased in glycylcine buffer. Autoradiographic studies confirmed these findings. These pharmacological data are consistent with our previously reported conclusions that, under appropriate assay conditions. [ 3H]idazoxan predominantly labels α 2-adrenergic binding sites in rat brain. These sites are widely distributed and have pharmacological characteristics consistent with those previously reported for α 2A-adrenergic receptors.

Adrenergic and nonadrenergic effects of imidazoline and related antihypertensive drugs in the brain and periphery.

In radioligand binding studies the alpha 2-adrenoceptor antagonist ligand [3H]yohimbine binds exclusively to adrenergic sites. However, in addition to binding to alpha 2-adrenoceptor sites, two other ligands, [3H]clonidine and [3H]idazoxan, also bind at nonadrenergic imidazoline sites. Many of the compounds with a high affinity for these imidazoline sites are centrally acting antihypertensive drugs and there is some evidence that these sites are involved in blood pressure regulation. With chronic treatment of rabbits with guanabenz, clonidine, and rilmenidine, down regulation of alpha 2 but not imidazoline sites occurred in forebrain and hindbrain with guanabenz treatment, and in hindbrain with clonidine treatment. However, no change in either imidazoline or alpha 2-adrenoceptor binding site number occurred in animals given chronic rilmenidine treatment. This rank order of effect, guanabenz greater than clonidine greater than rilmenidine, is consistent with the alpha 2-adrenoceptor activation by the three drugs in the periphery. In contrast, chronic treatment with guanabenz, clonidine, and rilmenidine had similar effects on blood pressure, heart rate, and responses to intracisternal clonidine. We suggest that stimulation of alpha 2-adrenoceptors cannot account for all the changes in cardiovascular responses observed on chronic treatment and that activation of nonadrenergic imidazoline-preferring sites may contribute to the antihypertensive properties of imidazoline and related compounds.

Do Centrally-Acting Antihypertensive Drugs Act at Non-Adrenergic as well as Alpha-2 Adrenoceptor Sites?

Rabbits were treated with guanabenz, clonidine and rilmenidine for 6 days via osmotic minipumps. Blood pressure, heart rate and responses to intracisternal clonidine were measured after 1 and 6 days treatment. Radioligand binding to forebrain and hindbrain membranes after 6 days treatment was examined using [3H]yohimbine to measure the number of adrenergic binding sites and [3H]clonidine and [3H]idazoxan to assess nonadrenergic imidazoline sites. No change in nonadrenergic imidazoline binding was observed but adrenergic binding was decreased in forebrain and hindbrain by guanabenz and in hindbrain by clonidine treatment. Resting heart rate was decreased after 1 day's treatment with partial recovery by day 6. At this time heart rate significantly reduced in the clonidine and rilmenidine treated groups but not the guanabenz group. No significant change in baseline blood pressure was observed in normotensive rabbits. Both depressor and bradycardia responses to intracisternal clonidine were attenuated after 1 day's dosing but only depressor responses were influenced after 6 days. Blood pressure and heart rate thus appeared to be regulated independently. It is possible that imidazoline receptors predominate in the central control of blood pressure while central alpha-2 adrenoceptors play a greater part in heart rate regulation.

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT 1A receptors in human brain using [ 3H]idazoxan and [ 3H]rauwolscine

The regional distribution of [ 3H]idazoxan and [ 3H]rauwolscine was studied autoradiographically in human brain. [ 3H]Idazoxan binds with high affinity to α 2 adrenoceptors as well as to non-adrenergic sites (NAIBS). [ 3H]Rauwolscine, besides binding to α 2 adrenoceptors, also binds to 5-HT 1A receptors. Both radioligands labelled the same population of α 2 adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of α 2 adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no α 2 adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable α 2 adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT 1A receptors labelled by [ 3H]rauwolscine is in agreement with previous studies using [ 3H]8-OH-DPAT.

Imidazole binding sites in rabbit kidney and forebrain membranes

1. The binding of [3H]-clonidine, [3H]-idazoxan and [3H]-yohimbine to rabbit forebrain and kidney membranes was compared. 2. Yohimbine bound exclusively to adrenergic sites, idazoxan to non-adrenergic sites and clonidine to both non-adrenergic and adrenergic sites. 3. Differences were observed between the ligands not only in binding at adrenergic and non-adrenergic sites but also between the non-adrenergic binding of [3H]-clonidine and [3H]-idazoxan. 4. However, no tissue specific differences were found.

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.

Binding of [ 3H] p-aminoclonidine to two sites, α 2-adrenoceptors and imidazoline binding sites: distribution of imidazoline binding sites in rat brain

Binding sites labeled by [ 3H] p-Aminoclonidine ([ 3H]PAC were investigated by the competitive analysis with imidazoline and non-imidazoline derivatives. Phenylethylamine derivatives displaced only the part of specific sites for [ 3H]PAC, which was considered as α 2-adrenoceptor, whereas imidazoline derivatives, such as clonidine and tolazoline, competed for a further specific binding of [ 3H]PAC to the non-adrenergic sites, in addition to the α 2-adrenoceptor. Because the non-adrenergic sites were specific for the imidazoline structure, they were termed imidazoline sites. The imidazoline sites were not distributed uniformly among rat brain regions. In striatum, hippocampus and medulla oblongata, they occupied 39.6, 33.0 and 36.5% of the specific binding of [ 3H]PAC, respectively. Saturation isotherms revealed that K d and B max of imidazoline sites for [ 3H]PAC were 3.09 ± 0.59nM, 27.4 ± 1.7fmol/mg protein and 2.23 ± 0.29nM, 21.0 ± 1.5fmol/mg protein in striatum and hippocampus, respectively. Because imidazoline binding sites also displayed weak affinities for imidazole compounds, such as histamine and cimetidine, the imidazoline site may be a subtype of histamine H 2-receptor.

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.

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.

Characterization and Visualization of Clonidine-sensitive Imidazole Sites in Rat Kidney Which Recognize Clonidine-displacing Substance

In the ventrolateral medulla oblongata, clonidine binds not only to alpha 2-adrenergic receptors but also to a novel class of nonadrenergic sites that are specific for imidazoles. Since clonidine has direct actions on the kidney, we sought to determine whether imidazole binding sites could be detected in renal cell membranes. Adrenergic agents having an imidazole ring, like clonidine, completely displaced the specific binding of the high-affinity clonidine analog 3H-p-aminoclonidine (3H-PAC) to kidney membranes. Nonimidazole adrenergic agents inhibited 3H-PAC binding by only 75%, indicating that the remaining 25% of the sites were nonadrenergic. Cimetidine, an imidazole compound lacking adrenergic potency, showed a high affinity for approximately 25% of the sites. 3H-PAC binding to imidazole sites was high-affinity (KD = 11 +/- 3 nmol/L) and saturable (Bmax = 41 +/- 10 fmol/mg protein). Like clonidine, an endogenous clonidine-displacing substance (CDS) completely inhibited 3H-PAC binding to renal cortex membranes. Quantitative receptor autoradiography revealed that imidazole receptors exhibited a specific regional distribution within the kidney that was unique, and distinct from that of alpha 2-adrenergic receptors. We conclude that clonidine binds to specific imidazole sites in the renal cortex of the rat and that CDS may be the endogenous ligand at these sites. Thus, in addition to their role in central nervous system control of arterial pressure, imidazole receptors may be involved in the regulation of renal function.

Guanabenz, guanochlor, guanoxan and idazoxan bind with high affinity to non-adrenergic sites in pig kidney membranes

[ 3H]Idazoxan is a labelled ligand that is frequently used to study α 2-adrenoceptors in the central nervous system. In pig kidney membranes, [ 3H]idazoxan labelled high-affinity binding sites (K d = 1.5 nM) that were not α 2-adrenoptors and which recognized clonidine with low affinity. This new class of binding sites was recognized by amiloride derivatives; however, it is not likely that these sites are the well-known targets of amiloride in the kidney: the Na +/H + exchanger and the epithelium Na + channel. These binding sites may be the normal target of a series of imidazolidines derivatives (guanabenz, guanochlor, guanoxan), which are known for their antihypertensive properties.