David C. U'Prichard

Modulation of rat brain α- and β-adrenergic receptor populations by lesions of the dorsal noradrenergic bundle

Bilateral lesion of the ascending noradrenergic fibers in the dorsal bundle of adult Wistar rats with 4 μg 6-hydroxydopamine caused extensive depletion of norepinephrine in all forebrain areas, but led to a 54% increase in norepinephrine levels in the cerebellum. β-Adrenergic receptor binding of [3Hdihydroalprenolol was significantly increased in all forebrain areas depleted of norepinephrine except hypothalamus. The increase in [3Hdihydroalprenolol binding was due to 62% and 34% increases in the number of β-receptor sites in the frontal cerebral cortex and hippocampus respectively. Binding of [3HWB-4101 toα1-adrenergic receptors after dorsal bundle lesion was augmented generally to a lesser extent than β-receptor binding, with significantly increased numbers of sites only in the frontal cortex (74%), thalamus (20%) and septum. Bothα1-andβ-receptor binding sites were reduced in number by 25–28% in the cerebellum of dorsal bundle-lesioned rats, whereas intraventricular administration of 6-hydroxydopamine to adult rats, which depletes norepinephrine in the cerebellum by 96%, increased cerebellarα1-andβ-receptor binding by 33–40%. Binding of [3Hclonidine to forebrainα2-adrenergic receptors was significantly elevated in the frontal cortex, but reduced in the amygdala and septum, after dorsal bundle lesion.

Differential effects of antidepressant treatment on brain monoaminergic receptors

Chronic (21 days) antidepressant administration to rats results in a decrease in both serotonin and beta-adrenergic, but not cholinergic muscarinic, receptor binding in selected brain regions, with the frontal cortex appearing to be somewhat more sensitive to this effect. Neither nisoxetine nor fluoxetine, potent and specific inhibitors of norepinephrine and serotonin uptake respectively, caused receptor binding changes after chronic administration, suggesting that inhibition of transmitter uptake, in itself, is insufficient to cause receptor subsensitivity. In vitro experiments indicated that antidepressants are relatively weak alpha 2-receptor blocking agents, but some are potent on the alpha 1-receptor system indicating that the norepinephrine releasing potency of some antidepressants may not be mediated by blockade of presynaptic autoreceptors.

[3H]rauwolscine (α-yohimbine): A specific antagonist radioligand for brain α2-adrenergic receptors

[3H]Rauwolscine, a specific and potent alpha 2-antagonist radioligand, was used to characterize alpha 2-receptor binding in bovine cerebral cortex. [3H]Rauwolscine binding was reversible, stereospecific, and saturable. Association, dissociation, and saturation studies revealed one site interactions (k -1/k+1 = 1.2 nM, KD = 2.5 nM, Bmax = 160 fmol/mg protein) and competition studies indicated that [3H]rauwolscine labeled the alpha 2-receptor. Agonists inhibited [3H]rauwolscine binding in a shallow, GTP-sensitive manner. These results suggest that [3H]rauwolscine specifically labels both the high and low affinity states of the alpha 2-receptor in brain membranes.

Human platelet α2-adrenergic receptors: Labeling with 3H-yohimbine, a selective antagonist ligand

Abstract 3 H-Yohimbine, a potent and selective pharmacological antagonist of α 2 -adrenergic receptors, labeled human platelet membrane α 2 -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 Mg 2+ concentrations increased the K D for 3 H-yohimbine without altering the B max . The 3 H-yohimbine site exhibited α 2 -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 α 1 -antagonist prazosin was 340 times less potent than yohimbine. Catecholamine agonists exhibited shallow curves in inhibiting 3 H-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 3 H-prazosin to platelet membranes was observed, indicating the absence of α 1 -receptors. 3 H-Yohimbine labeled fewer platelet sites than did 3 H-dihydroergocryptine under identical conditions (80 vs 130 receptors/ cell), and may be a more specific and useful antagonist probe of platelet α 2 -receptors than 3 H-dihydroergocryptine.

α2-adrenergic receptors in platelet membranes of depressed patients: No change in number of 3H-yohimbine affinity

Abstract The α 2 -adrenergic receptor density in platelet membranes from normal controls and depressed patients was studied using 3 H-yohimbine, a potent α 2 -adrenergic antagonist, as a radioligand. The KD values of 3 H-yohimbine in control and depressed patient samples were 0.92±0.07 nM and 0.97±0.06 nM, respectively. The Bmax values of controls and depressed patients were 240±19 fmoles/mg protein (128±13 receptor platelet, R/PL) and 204±20 fmoles/mg protein (139±14 R/L), respectively. There were no significant differences between the KD and Bmax values of the two groups.

Effects of combined administration of amphetamine and iprindole on brain adrenergic receptors

Mobley et al. 1° have shown that treatment of rats with amphetamine for 4 days can reduce the sensitivity of the forebrain cyclic AMP generating system to norepinephrine (NE) stimulation. Furthermore, treatment of rats with amphetamine in combination with iprindole, a drug which inhibits the metabolism of amphetamine 4 and increases its brain concentrations in the rat 6 , reduced the time required for this desensitization to 2 days 9. Chronic treatment of rats with antidepressant drugs has also been shown to decrease the sensitivity of the limbic forebrain cAMP generating system to NE stimulation 19, and this effect has been ascribed to a decrease in brain fladrenergic receptor density 1. Therefore, it was of interest to us to determine whether the amphetamine-iprindole effect described above might also be explained by a decrease in brain fl-receptor density, or perhaps an effect on other receptors. Our results reveal that after 3 days of treatment, while amphetamine or iprindole administration alone did not alter cortical fl-adrenergic receptors, the combination of iprindole and amphetamine reduced the density of these receptors. In addition, all 3 drug treatments significantly elevated a2-adrenergic receptor densities but did not affect the properties of al-adrenergic receptors. Groups of 4 Charles River C-D male rats, weighing 230--270 g, were treated twice daily with amphetamine (10 mg/kg i.p.) and once daily with iprindole (25 mg/kg i.p.) for 3 days. Amphetamine and iprindole were administered at 08.30 h and amphetamine again at 16.30 h. Separate groups were treated with each drug alone on the same schedule as in the combination study. A water-injected group served as a control. All rats were sacrificed by decapitation between 08.30 and 09.00 h on the fourth day and the whole brain was rapidly removed and frozen (--20 °C). For receptor binding assays, tissues were prepared in the following manner. The

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 alpha 2-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 degrees C. Norepinephrine-displaceable binding was saturable and monophasic, with a KD of 1.4 nM, in agreement with rate and competition experiments, and a Bmax of 200 fmol/mg protein. Competition studies revealed that binding was alpha 2-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 alpha 2-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 alpha 2-adrenoceptor ligands because of its high affinity and specific activity.

Differential supersensitivity of β-receptor subtypes in rat cortex and cerebellum after central noradrenergic denervation

Abstract Inhibition of 3 H-dihydroalprenolol binding to rat cortex and cerebellum β-receptors by the selective β 1 -antagonist practolol, and the selective β 2 -agonist salbutamol, was shallow and biphasic, with log-logit slopes less than 1.0. The relative affinities of these inhibitors suggested that the predominant β-adrenergic receptor population in cortex and cerebellum was β 1 and β 2 respectively. specific lesion of the ascending dorsal norepinephrine bundle, in addition to increasing β-receptor number in the cortex, significantly increased the affinity of practolol, but did not change the affinity of salbutamol, at cortex β receptor sites. Similar lesions decreased cerebellar β-receptor binding and reduced the affinity of salbutamol but not of practolol for those same sites. Iterative computer analysis of the inhibition data showed mixed populations of β 1 - and β 2 - receptors in both cortex and cerebellum. Dorsal NE bundle lesion doubled the number of cortical β 1 -receptors, but did not alter the number of β 2 -receptors. In contrast, these lesions induced a selective decrease in cerebellar β 2 -receptors. It is concluded that the relevant neuronal β-receptors which are postsynaptic to central NE nerve terminals are β 1 in the cerebral cortex and β 2 in the cerebellum.

In vitro modulation of CNS β-receptor number by antidepressants and β-agonists

In vitro incubation of rat cerebral cortex slices with antidepressant drugs reduced beta-adrenergic receptor binding of 3H-dihydroalprenolol by 30%. The decrease was maximum after 60 min, and was reversible after 120 min. (-)-Isoproterenol incubation caused a rapid and reversible loss (60%) of beta-receptor binding. Both effects were due to a decrease in beta-receptor sites. In vitro beta-receptor subsensitivity due to desipramine and isoproterenol was non-additive. The observed reversible loss of beta-receptor sites may be an initial phase of the beta-receptor down-regulation by antidepressants seen in vivo.

Tricyclic antidepressant radioreceptor assay

A radioreceptor assay for tricyclic antidepressants described here is based on the ability of these drugs to compete with [3H]-3-quinuclidinyl benzilate (3H-QNB) for binding to muscarinic cholinergic receptors in rat brain membranes. The assay is sensitive, in that in can detect, for example, 2 ng/ml nortriptyline in plasma. Seven plasma samples from depressed patients treated with nortriptyline were assayed with the radioreceptor and gas liquid chromatographic methods, and the results from these two methods were almost identical. This assay should be used cautiously, if at all, in patients treated with other drugs that have potent anticholinergic effects.