Demonstration of alpha 1-adrenoceptors in guinea pig lung using 3H-prazosin.

³H-prazosin, a new radioligand of high specific radioactivity (33 Ci/mmol) was used to characterise postsynaptic (α₁) adrenoceptors in guinea pig lung membranes. Binding was saturable and of high affinity (K_D 0.24 nM) with a Bmax of 54 fmol/mg protein. Adrenergic agonists competed for binding in the order (?)-epinephrine > (?)-norepinephrine ? (?)-phenyl-ephrine > (?)-isoproterenol. (+)-norepinephrine was 100x less potent than (?)-norepinephrine. α-Adrenergic antagonists competed in the order prazosin > WB 4101 > indoramin > phentolamine > haloperidol > chlorpromazine ? piperoxan > yohimbine, indicating that ³H-prazosin binding is probably to α₁-adrenoceptors. Propranolol, methysergide and sulpiride inhibited binding only at high concentrations. Binding of (?)-³H-dihydroalprenolol under identical experimental conditions gave a K_D of 0.93 nM and a Bmax of 870 fmol/mg protein, giving a ratio of beta : α-adrenoceptor binding sites of 16 : 1 in this lung membrane preparation. ³H-prazosin appears to be a useful ligand in studying α₁-adrenoceptors.     

The characteristics of adrenoceptors in homogenates of human cerebral cortex labelled by (3H)-rauwolscine

The alpha-2-adrenoceptor antagonist (³H)-rauwolscine has been used to label adrenoreptors in membranes from human cerebral cortex. The radioligand binds with high affinity (K_D 2.08 nM) to a single population of sites with a density of 135 fmoles/mg protein. Adrenoceptor antagonists displaced binding in a simple monomolecular fashion with an order of affinity rauwolscine > yohimbine > phentolamine > corynanthine > prazosin indicating binding to alpha-2-adrenoceptors. Agonists displaced with an order of affinity clonidine > (-) adrenaline > (-) noradrenaline > dopamine > (-) isoprenaline but all displayed apparent Hill coefficients less than unity indicating heterogeneity of binding. The relatively high affinity of the alpha-1 antagonist prazosin for (³H)-rauwolscine binding sites in rat cerebral cortex was not observed in the human tissue which had pharmacological properties similar to those described previously in human platelet.     

Alpha and beta adrenergic receptors of canine lung tissue identification and characterization of alpha adrenergic receptors by two different ligands

Adrenergic receptors of canine peripheral lung tissues were measured by direct binding techniques using [³H]dihydroergocryptine ([³H]DHE), [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA). All three ligands bound to canine lung tissue with saturability, stereospecificity and reversibility. Adrenergic agonists competed for binding of [³H]DHE and [³H]prazosin in the order: 1-epinephrine > 1-norepinephrine > d-epinephrine > d-norepinephrine > 1-isoproterenol. Adrenergic antagonists competed for binding of [³H]prazosin in the order: prazosin > phentolamine > yohimbine. Inhibition curves of [³H]DHE by prazosin or yohimbine were biphasic suggesting two subtypes of binding sites. Maximum binding capacities of [³H]DHE ranged from 30.6 to 42.7 fmol/mg protein. [³H]prazosin from 18.3 to 26.9 fmol/mg protein and [³H]DHA from 135.2 to 359.4 fmol/mg protein. When both [³H]DHE and [³H]prazosin were used in the same membrane preparation, specific binding of [³H]DHE was always more than that of [³H]prazosin. Since [³H]prazosin is considered to bind to alpha₁ adrenergic receptors specifically and [³H]DHE is considered to bind alpha₂ adrenergic receptors nonselectively, the difference between the numbers of the specific binding sites of these two ligands should represent alpha₂ adrenergic receptors. Alpha₂ adrenergic receptor density ranged from 9.5 to 21.1 fmol/mg protein. Our results suggest the existence of both alpha₁ and alpha₂ adrenergic receptors in canine peripheral lung tissue. Approximately 40% of alpha adrenergic receptors were alpha₂. The ratio of alpha/beta adrenrgic receptors ranged from 1:3.3 to 1:10.4. The ratio of alpha₁/be ta adrenergic receptors ranged from 1:6.7 to 1:21.1.     

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

Abstract

In the present study, we have provided evidence that [³H] rauwolscine and [³H] idazoxan bind to different sites in rabbit urethra. The [³H] idazoxan capacity and affinity was 215 ± 14 fmol/mg protein and 1.59 ± 0.16 nM while [³H] rauwolscine binding parameters were 45.9 ± 3.4 fmol/mg protein and 2.39 ± 0.27 nM. [³H] idazoxan specific binding was inhibited only by compounds possessing an imidazoli(di)ne or a guanidinium moiety, while [³H] rauwolscine specific binding was inhibited by phenylethanolamines and classical α-antagonists. [₃H] idazoxan was inhibited by KCI in a competitive and by MnCI₂ in a non-competitive way, while other cations such as Na⁺, Li⁺ and Mg²⁺ did not inhibit [³H] idazoxan binding. Moreover, we investigated the regional distribution of [³H] idazoxan and [³H] rauwolscine along the rabbit urethra using quantitative autoradiography. Analysis of the films revealed a different distribution of these two binding sites on the urethral sections.     

Effect of Noradrenergic Lesions on Subtypes of α2-Adrenoceptors in Rat Brain

Abstract: The binding of [³H]rauwolscine to α_2A- (also referred to as α_2D-) and α_2C-adrenoceptors in homogenates of rat cerebral cortex was measured by exploiting the selectivity of oxymetazoline for α_2A-adrenoceptors. Inhibition of [³H]rauwolscine binding by oxymetazoline was modeled best assuming binding to two sites (p < 0.001). Competition curves for oxymetazoline were shifted rightward by the addition of GTP (250 µM) but were still fit best by a two-site model (p < 0.001). A concentration of oxymetazoline was calculated that would optimally antagonize [³H]rauwolscine binding (with GTP present) to oxymetazoline-sensitive α_2A-adrenoceptors, minimally inhibiting binding to α_2C-adrenoceptors. Subsequently, [³H]rauwolscine binding to α_2A- and α_2C-adrenoceptors in cortex was examined 3 weeks after destruction of noradrenergic terminals. Binding to α_2C-adrenoceptors was increased significantly after treatment with 6-hydroxydopamine (6-OHDA) compared with vehicle-treated controls, whereas binding to α_2A-adrenoceptors was unchanged. Pretreatment of rats with desipramine before 6-hydroxydopamine, to protect noradrenergic neurons, resulted in no changes in binding to either α_2A- or α_2C-adrenoceptors. Thus, α_2C-adrenoceptors are regulated by changes in synaptic availability of norepinephrine. α_2A-Adrenoceptors are either not regulated by synaptic norepinephrine or are located both post- and presynaptically so that up-regulation of postsynaptic α_2A-adrenoceptors is offset by a loss of presynaptic α_2A-adrenoceptors.     

Binding of the imidazoline UK-14, 304, a putative full α2-adrenoceptor agonist,to rat cerebral cortex membranes

The aryl imidazoline compound UK-14, 304 (5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline) is a potent and selective α₂-adrenoceptor agonist with full intrinsic activity, unlike other imidazolines. We examined the characteristics of high specific activity (84 Ci/mmol) [³H] UK-14, 304 binding to rat cerebral cortex membranes. [³H] UK-14, 304 specific binding was enhanced by Mn²⁺ ion, and associated and dissociated moderately rapidly at 25°C. Norepinephrine-displaceable binding was saturable and monophasic, with a K_D of 1.4 nM, in agreement with rate and competition experiments, and a B_max of 200 fmol/mg protein. Competition studies revealed that binding was α₂-adrenoceptor-specific, with yohimbine being 12 times more potent than prazosin. [³H] UK-14, 304 appeared to label predominantly the R(H) state of the brain α₂-adrenoceptor, as judged by the high affinity of catecholamine and imidazoline agonists (IC₅₀, 1–13 nM), and the relatively low affinity of yohimbine and rauwolscine (IC₅₀, 100–300 nM), at the binding site. [³H] UK-14,304 compares favorably with other α₂-adrenoceptor ligands because of its high affinity and specific activity.     

Alpha-noradrenergic receptor binding in mammalian brain: differential labeling of agonist and antagonist states.

[³H]Clonidine, a α-noradrenergic agonist, and [³H]WB-4101, a benzodioxan derivative α-antagonist, bind with high affinity and selectivity to membranes of rat brain in a fashion indicating that they label postsynaptic α-noradrenergic receptors. Binding for both ligands is saturable with K_D values of 5 nM and 0.6 nM respectively for clonidine and WB-4101. The relative affinities of a series of phenylethylamines for binding sites corresponds well with their relative influences at α-receptors. Binding of both [³H]-ligands is stereoselective with about a 50 fold preference for (-)-norepinephrine. Of a series of ergot alkaloids, only those with known α-receptor activity have high affinities for the binding sites. Binding does not involve pre-synaptic norepinephrine nerve endings, because after an 80% depletion of endogenous norepinephrine by treatment with 6-hydroxydopamine, no decrease can be detected in [³H]clonidine and [³H]WB-4101 binding. α-Agonists have much higher affinities for [³H]clonidine than [³H]WB-4101 sites, while the reverse holds true for α-antagonists. Mixed agonist-antagonist ergots have similar affinities for binding of the two [³H]ligands. These data suggest that [³H]clonidine and [³H]WB-4101 respectively label distinct agonist and antagonist states of the α-receptor.     

Human platelet alpha 2-adrenergic receptors: labeling with 3H-yohimbine, a selective antagonist ligand

³H-Yohimbine, a potent and selective pharmacological antagonist of α₂-adrenergic receptors, labeled human platelet membrane α₂-receptors with high affinity. Binding was rapid and reversible at 25°C. Both saturation and kinetic experiments indicated a single order of binding sites, with an equilibrium K_D value of 1.0–1.5 nM. Low Mg²⁺ concentrations increased the K_D for ³H-yohimbine without altering the B_max. The ³H-yohimbine site exhibited α₂-receptor specificity: (?)-norepinephrine and (?)-isoproterenol were 4.8 and 330 times less potent than (?)-epinephrine; (?)-catecholamines were 17–35 times more potent than corresponding (+)-catecholamines; the selective α₁-antagonist prazosin was 340 times less potent than yohimbine. Catecholamine agonists exhibited shallow curves in inhibiting ³H-yohimbine binding, with pseudo-Hill coefficients (n_H) of less than 1.0, whereas the n_H of antagonists was 1.0. No specific binding of ³H-prazosin to platelet membranes was observed, indicating the absence of α₁-receptors. ³H-Yohimbine labeled fewer platelet sites than did ³H-dihydroergocryptine under identical conditions (80 vs 130 receptors/ cell), and may be a more specific and useful antagonist probe of platelet α₂-receptors than ³H-dihydroergocryptine.     

Catecholamine binding to the α-adrenergic receptors of hamster adipocytes evidence that guanine nucleotides regulate this binding to the α2-receptor subtype

The binding characteristics of the α-component of (?)-[³H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, reaching equilibrium in 20 min at 25°C. Dissociation of specific binding by 10 μM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [³H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity (_D = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity (K_D = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands of competed with [³H]norepinephrine binding in the following order of potency: (?)-norepinephrine>(?)-epinephrine>>(+)-norepinephrine>(?)-isoproterenol. Displacement by the selective α-adrenergic drugs prazosin, clonidine and yohimbine yielded biphasic curves consistent with binding of [³H]norepinephrine to both α₁- (14–22%) and α₂- (78–86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [³H]dihydroergocryptine, it severely reduced the binding affinity of (?)-epinephrine, (?)-norepinephrine and the selective α₂-agonist, clonidine. The inhibitory effects of clonidine and of the α-component of (?)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [³H]norepinephrine and [³H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the α-inhibitory effect of norepinephrine on cyclic AMP production. These data led to concluded that [³H]norepinephrine can be successfully used to study the entire α-adrenergic receptor population of hamster fat cells and that the predominant α₂ -receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.     

3H]Para-amino-clonidine: a novel ligand which binds with high affinity to alpha-adrenergic receptors.

Para-amino-clonidine (PAC) is an α-adrenergic agonist with extraordinarily high potency in some peripheral tissues. We have demonstrated the labeling of α-adrenergic binding sites in central and peripheral tissues with [³H]PAC and compared properties of this binding to those of [³H]clonidine. [³H]PAC binds saturably with a dissociation constant (K_D) of about 0.9 nM to rat cerebral cortex membranes. It has about 2–3 times the affinity of [³H]clonidine for α-receptor binding sites. The greater affinity is attributable mainly to a slower dissociation of [³H]PAC than [³H]clonidine from binding sites. The relative and absolute potencies of various adrenergic agonists and antagonists in competing for [³H]PAC and [³H]clonidine binding are essentially the same. [³H]PAC can also be utilized to label α-adrenergic binding sites in the kidney and spleen where the relative potencies of PAC and clonidine are the same as in the brain.     

Characterization of IBE 2254 Binding to Alpha - Adrenergic Receptors on Intact DDT Smooth Muscle Cells: Comparison with Membrane1 Binding and Correlation with Phosphoinositides Breakdown

Abstract

The binding of the antagonist IBE 2254 (IBE) to α -adrenergic receptors was characterized on intact DDT smooth muscle cells. IBE binding was rapid, reversible, stable and saturable: Bmax = 113000±13000 recetors/cell, K = 110±13 pM (n = 25). Saturation and competition experiments analysed by non linear curve fitting indicated a single population of binding sites with a pharmacological profile typical for α-adrenergic receptors. Antagonists competed for IBE binding sites in the following order: prazosin > phentolamine = phenoxybenzamine > yohimbine. The rank order for agonists was clonidine > epinephrine > norepinephrine > phenylephrine. There was a significant correlation between IBE binding to intact cells, DDT₁ membranes and rat cortex membranes. Neither agonists nor antagonists showed noticeable changes in their affinity for IBE binding on either system. There was also a good correlation between IBE binding and breakdown of phosphoinositides (PI) measured in intact cells.     

Molecular exploration of the α1A-adrenoceptor orthosteric site: Binding site definition for epinephrine,HEAT and prazosin

Despite the physiological and pharmacological importance of the α_1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of ¹²⁵I-HEAT, ³H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist.     

3H]epinephrine and [3H]norepinephrine binding to alpha-noradrenergic.

(±)-[³H]Epinephrine and (?)-[³H]norepinephrine bind saturably to calf cerebral cortex membranes under appropriate incubation conditions in a fashion indicating that they label α-noradrenergic receptors. Binding of the two [³H]catecholamines is saturable with dissociation constants of 20–30 nM. Binding is stereoselective with (?)-norepinephrine displaying about twenty times greater affinity than (+)-norepinephrine. The relative potencies of catecholamines in competing for these binding sites parallels their relative pharmacologic effects at α-noradrenergic receptors in numerous tissues. Thus, (?)-epinephrine is 2–3 times more potent than (?)-norepinephrine and 500 times more potent than (?)-isoproterenol. Binding is inhibited by low concentrations of the α-antagonists phentolamine and phenoxybenzamine but not by the β-antagonist propranolol.     

Characterization of CRF1 receptor antagonists with differential peripheral vs central actions in CRF challenge in rats

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF₁ receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF-induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF₁ receptor binding affinities (15 and 10 nM) and functional activities in reporter gene assay (15 and 9.5 nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10 mg/kg inhibited [¹²⁵I]-CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [¹²⁵I]-CRF binding, indicating that only Compound A inhibited central [¹²⁵I]-CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge-induced anxiety behavior and c-fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF₁ receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders.     

Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

Differential Effects of the Alkylating Agent N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline on Brain α2-Adrenoceptors and I2-Imidazoline Sites In Vitro and In Vivo

Abstract— The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a peptide-coupling agent that is being used to inactivate irreversibly α₂-adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l₂-imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10^?8-10^?5M) markedly decreased (20–90%) the specific binding of the selective antagonist [³H]R821002 to α₂-adrenoceptors without affecting that of [³H]idazoxan (in the presence of adrenaline) to l₂-imidazoline sites. In EEDQ-pretreated membranes (10^?5M, 30 min at 25°c), the density of l₂-imidazoline sites (B_max= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10^?6M (-)-adrenaline (B_max= 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [³H]idazoxan binding sites (B_max= 107 ± 6 fmol/mg of protein) (l₂-imidazoline sites plus a₂-adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 ± 1%) the specific binding of [³H]-R821002 to α₂-adrenoceptors, but also the binding of [³H]idazoxan to l₂-imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced α₂-adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ-induced inactivation of l₂-imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase-A sites labeled by [³H]Ro 41–1049 or that of monoamine oxidase-B sites labeled by [³H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [³H]idazoxan to l₂-imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ-pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l₂-imidazoline sites when using [³H]-imidazoline ligands that also recognize α₂-adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l₂-imidazoline sites probably by an indirect mechanism. Key Words: Brain l₂-imidazoline sites—[³H]-Idazoxan—α₂-Adrenoceptors—[³H] R821002—N -Ethoxycarbonyl-2-ethoxy-li2-dihydroquinoline—Monoamine oxidase-A—[³H]Ro 41–1049—Monoamine oxidase-B—[³H]Ro 19–6327.     

Quantification of in vivo binding of [3H]RX 821002 in rat brain: Evaluation as a radioligand for central α2-adrenoceptors

On the basis of its established in vitro characteristics, [³H]RX 821002 was evaluated in rats as an in vivo radioligand for central α₂-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmental analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the α₂-antagonists, idazoxan or yohimbine, the α₂-agonist, clonidine, or the α₁-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP-4, had no significant effect on [³H]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [³H]RX 821002 can be used experimentally as an in vivo marker for central α₂-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [¹¹C]RX 821002 for clinical PET studies.     

Further Characterization of the Guinea Pig Cerebral Cortex Idazoxan Receptor: Solubilization, Distinction from the Imidazole Site, and Demonstration of Cirazoline as an Idazoxan Receptor-Selective Drug

We have demonstrated previously that [3H]idazoxan, besides being able to bind to alpha 2-adrenergic receptors, may also bind to a nonadrenergic idazoxan-receptor site with high affinity. The idazoxan receptor is tightly bound to cellular membranes, and we have now developed a method to solubilize it from the guinea pig cerebral cortex by using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The CHAPS-solubilized receptor retains its binding properties for drugs: the membrane-bound, as well as the solubilized, idazoxan receptor shows high affinities for a number of imidazolines (cirazoline, idazoxan, tolazoline, naphazoline, tramazoline, clonidine, and oxymetazoline), some imidazoles (medetomidine, detomidine), and guanfacine. By contrast, catecholamines (adrenaline, noradrenaline, isoprenaline, and dopamine) and a number of other neurotransmitters and neuromodulators (serotonin, histamine, glutamic acid, gamma-aminobutyric acid, glycine, and adenosine) show negligible affinities for the idazoxan receptor. Moreover, the idazoxan receptor shows grossly different binding properties for histamine, cimetidine, and imidazole-4-acetic acid compared to what has been described for the nonadrenergic imidazole site labeled by p-[3H]amino-clonidine, indicating that the two receptor sites are distinct. Radioligand binding data further indicate that cirazoline is an idazoxan receptor-selective drug (KD = 1 nM) showing a 50-210-fold selectivity for binding to the idazoxan receptor when compared to alpha 2-adrenergic receptors and an about 500-fold selectivity when compared to alpha 1-adrenergic receptors. We have also reviewed the literature for possible nonadrenergic actions of idazoxan and cirazoline, and we suggest that idazoxan receptors might be involved in the control of prolactin release from the pituitary.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.     

Alterations in central and peripheral adrenergic receptors in deoxycorticosterone/salt hypertensive rats

The treatment of uninephrectomized rats with deoxycorticosterone (DOCA) and salt for 6 weeks caused a significant systolic hypertension and cardiac and renal hypertrophy. There was a significant decrease in the density of cardiac α₁- and β-, and renal α₁-adrenoceptors in DOCA/salt hypertensive rats, as compared to uninephrectomized salt loaded control rats. In contrast, the cerebral cortex, corpus striatum, hippocampus and hypothalamus/thalamus of hypertensive rats showed a significant increase in adrenoceptor binding in these hypertensive rats. In contrast, muscarinic cholinergic receptors and [³H]yohimbine binding sites were not altered in most tissues of the hypertensive rats. The present study suggests an important role for central and peripheral α₁- and β-adrenoceptors in the pathogenesis of hypertension.