Interaction of dihydroxy-2-aminotetralin derivatives at sites labelled with [3H]clonidine, [3H]prazosin and [3H]spiperone in rat brain membranes

The interactions of 5,6- and 6,7-dihydroxy derivatives of 2-aminotetralin with [³H]clonidine and [³H]clonidine and [³H]prazosin as well as with [³H]spiperone binding sites in rat cerebral cortex membrane preparations were investigated. The hydroxy derivatives of 2-aminotetralin tested showed significant interaction with [³H]clonidine as well as with [³H]spiperone binding sites while for [³H]prazosin binding site these agents appeared virtually inactive. For interaction with [³H]clonidine binding site 6,7-dihydroxy substitutions impart greater potency that 5,6-dihydroxy substitutions and N-alkyl substitutions either make no difference or reduce the affinity of these compounds. N-alkyl substitutions, however, markedly enhance the affinity of 5,6-dihydroxy derivatives for interactions with [³H]spiperone binding site. The results suggest that some hydroxy derivatives of aminotetralin have significant interaction with both central α₂-adrenoceptor and D₂-dopamine receptor systems.     

Pharmacologic characterization of [3H]dopamine and [3H]spiperone binding in mouse cerebellum

• 1.1. [³H]Dopamine and [³H]spiperone binding to cerebellar homogenates was characterized utilizing dopaminergic agonists, antagonists and non-dopaminergic drugs.
• 2.2. The [³H]DA binding to low affinity binding sites reveals a heterogenous population consisting of dopaminergic as well as serotonergic and noradrenergic sites. However, the high affinity binding of [³H]DA reflects dopaminergic sites, although a small contribution of serotonergic and noradrenergic binding sites cannot be excluded.
• 3.3. [³H]Spiperone also labels a heterogenous population of binding sites which, however, are mainly dopaminergic.

     

Inhibition by arylsulphatase A of Na-independent [3H]-GABA and [3H]-muscimol binding to bovine cerebellar synaptic membranes

Over an identical concentration range, both [³H]-GABA and [³H]-muscimol were bound to bovine cerebellar synaptic membranes through a sodium-independent and high-affinity process. Preincubating the synaptic membrane suspensions with arylsulphatase A inhibited [³H]-GABA and [³H]-muscimol binding. Furthermore, by using 4-nitrocatechol sulphate as a substrate for arylsulphatase A, a direct correlation appeared to exist between the amounts of SO^2?in4 released and the degree of inhibition of [³H]-GABA binding to synaptic membranes. The inhibitory effect of arylsulphatase A was blocked by known inhibitors of arylsulphatase such as AgNO₃. Scatchard analysis of specific [³H]-GABA binding to synaptic membranes showed that arylsulphatase decreased the number of [³H]-GABA binding sites from 2.65 to 1.75 pmol mg proteins without changing appreciably the affinity of [³H]-GABA binding to cerebellar synaptic membranes. These observations are interpreted to indicate that some components of GABA recognition sites may also contain sulpholipid(s) in addition to phospholipid(s).     

Characterization of the [3H]-desipramine binding site of the bovine adrenomedullary plasma membrane

Summary The specific (i.e. nisoxetine-sensitive) binding of [³H]desipramine was studied in membranes prepared from bovine adrenal medullae. (1) [³H]desipramine bound reversibly and with high affinity (K _D = 2.8 nmol/l) to a single class of non-interacting binding sites (Hill coefficient = 0.96); the maximal number of binding sites (B_max) was 2.1 pmol/mg protein. (2) Binding of [³H]desipramine was dependent on [Na⁺] and [Cl^–]. Increasing the concentrations of these ions increased binding. (3) Substrates and inhibitors of the neuronal noradrenaline transport system (uptake,) inhibited binding of [³H]desipramine with a rank order of potency typical for an interaction with the uptake, carrier.The characteristics of [³H]desipramine binding remained essentially unchanged after solubilization of adrenomedullary membranes with the non-ionic detergent digitonin.The results indicate that the plasma membrane of bovine adreno-medulary cells is endowed with the neuronal uptake₁ transporter. Correspondence to: H. Bönisch     

3H]Dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. A reassessment using the selective radioligands [3H]prazosin, [3H]yohimbine, and [3H]rauwolscine

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These binding studies employed the ligand [³H]dihydroergocryptine ([³H]DHE), an alpha-adrenergic antagonist that does not select between alpha₁- and alpha₂-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [³H]prazosin (alpha₁-selective), [³H]yohimbine (alpha₂-selective), and [³H]rauwolscine (alpha₂-selective), and we have compared the binding of these selective radioligands with that of [³H]DHE. [³H]Yohimbine and [³H]rauwolscine both bound with high affinity (K_D = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient ～1.0) of sites (～300 per platelet), but [³H]yohimbine yielded lower nonspecific binding than did [³H]rauwolscine. In paired experiments, [³H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [³H]yohimibine or [³H]rauwolscine. Unlabeled vohimbine and epinephrine competed for fewer [³H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha₂-adrenergic receptors identified by [³H]yohimbine and [³H]rauwolscine, [³H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [³H]prazosin did not identify alpha₁-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [³H]yohimbine and [³H]DHE binding at higher concentrations than usually observed for alpha₁-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha₂ subtype, (2) [³H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [³H]yohimbine and [³H]rauwolscine, and (3) [³H]yohimbine is the preferred antagonist radioligand for studying the alpha₂-adrenergic receptors on human platelets.     

High-affinity [3H]desipramine binding in the peripheral and central nervous system: A specific site associated with the neuronal uptake of noradrenaline

The specific binding of [³H]desipramine to various brain regions and peripheral tissues of the rat was of high affinity, rapid and reversible. It was inhibited with high affinity only by tricyclic antidepressants and noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of [³H]desipramine binding and for the inhibition of noradrenaline uptake. Substrates for the noradrenaline uptake system however inhibited the binding of [³H]desipramine only at very high concentrations. Postganglionic sympathetic denervation of the submaxillary gland and the heart both resulted in a pronounced decrease in [³H]desipramine binding sites, which paralleled the reduction in endogenous noradrenaline levels. High-affinity [³H]desipramine binding sites thus appear to be localised on noradrenergic nerve endings and are probably closely associated with the neuronal uptake system for noradrenaline.     

Heterogeneity of Guinea-Pig Lung Muscarinic Receptors Revealed by [3H]4-DAMP-binding

Summary: Muscarinic receptors present in guinea-pig lung were characterized using the M₃ selective radioligand [³H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([³H]4-DAMP). In saturation studies, [³H]4-DAMP identified two populations of binding sites with approximately 4% of the sites displaying high affinity (K_d=0.21 nM and B_max= 10 fmol/mg prot.) while the remaining sites were low affinity ones (K_d=18.11 nM and B_max=269 fmol/mg prot.). In competition studies with [³H]4-DAMP (0.35 nM), methoctramine and hexahydro-siladifenidol (HHSiD) identified 50 and 70% of high affinity binding sites displaying the pharmacological profile of the M₂ and the M₃ receptors, respectively. No evidence was found for high affinity [³H]pirenzepine binding sites in guinea-pig lung. However, pirenzepine/[³H]4-DAMP competition experiments suggested that pirenzepine recognized an equal proportion of [³H]4-DAMP binding sites with intermediate and low affinity binding constants. The intermediate affinity binding constant was inconsistent with the presence of M₁ receptors and reflected more the presence of M₄ or a mixture of M₃ and M₄ receptors. The low affinity pirenzepine binding sites may represent M₂ receptors. These results provide further evidence for the occurrence of M₂ and M₃ receptors and suggest the presence of the M₄ muscarinic receptor subtype in guinea-pig lung.     

Two distinct α1-adrenergic receptor sites in rat liver: Differential binding of (−)-[3H]norepinephrine, [3H]prazosin and [3H]dihydroergocryptine: Effects of guanine nucleotides and proteolysis; implications for a two-site model of α-receptor regulation

(?)-[³H]Norepinephrine, [³H]prazosin and [³H]dihydroergocryptine bind to rat liver plasma membranes in a manner indicating a selective interaction with α₁-adrenergic receptors. All three ligands display monophasic saturation with a single component on Scatchard analysis. The binding capacities of (?)-[³H]norepinephrine, [³H]prazosin and [³H]dihydroergocryptine are 340 ± 70 fmol/mg of protein 760 ± 40 fmol/mg of protein and 1200 ± 300 fmol/mg of protein, respectively. Differential drug potencies in competing for (?)-[³H]norepinephrine and [³H]prazosin binding sites suggest that these two ligands label two distinct binding sites at the α₁-adrenergic receptor, while [³H]dihydroergocryptine labels both sites. Guanine nucleotides lower the apparent affinity of (?)-[³H]norepinephrine for its binding site, without affecting the number of sites or the binding of [³H]prazosin and [³H]dihydroergocryptine. Incubation of membranes with α-chymotrypsin slightly reduces the binding of [³H]prazosin and [³H]dihydroergocryptine, but causes a 2-fold increase in (?)-[³H]norepinephrine binding. Both the number of (?)-[³H]norepinephrine binding sites and the affinity are increased. Following proteolysis, (?)-[³H]norepinephrine binding still occurs to a single class of sites, but is no longer affected by guanine nucleotides. The effect of α-chymotrypsin is abolished by pretreatment of membranes with the irreversible α-adrenergic antagonist phenoxybenzamine. We propose that in rat liver, (?)-[³H]norepinephrine labels the physiologically active form of the α-adrenergic receptor while [³H]prazosin binds to a precursor form of the active receptor, and/or to an α₁-adrenergic binding site not coupled to an effector system.     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

Multiple [3H]-nemonapride binding sites in calf brain

[³H]-Nemonapride has been the ligand of choice to label D₄ dopamine receptors. Its specificity was questioned when it was discovered that sigma (σ) sites were also labeled by [³H]-nemonapride. To further characterize the binding of [³H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [³H]-nemonapride labeled multiple sites. Dopaminergic and σ sites were the most prominent. The σ binding profile was σ-1 like with a K_i binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and σ sites, respectively, revealed additional, not previously characterized binding sites for [³H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [³H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met. Received: 19 September 1996 / Accepted: 12 March 1997     

Subcellular distribution of [3H]-epsilon-aminocaproic acid and its effects on amine storage mechanisms

ε-Aminocaproic acid (EACA) is an amino-acid reported to cause almost complete depletion of cardiac noradrenaline stores. The present report indicates that this compound can be found in both the particulate and supernatant fractions derived from heart homogenates. The [³H]EACA in the supernatant probably represents a mixture of intra- and extraneuronally located drug. Protriptyline pretreatment decreases the uptake of [³H]EACA suggesting that the amino-acid probably utilizes the amine membrane transport system, EACA, like reserpine, can both impair the retention of exogenously administered amines by adrenergic storage particles and cause the release of amines previously stored in such particles.     

Unaltered 5-HT- and desipramine-sensitive [3H]imipramine binding and [3H]5-HT uptake in rat brain after chronic imipramine and norzimeldine treatment

Several reports have shown heterogeneity of [³H]imipramine binding to brain membranes. Recently, a high affinity and 5-HT sensitive [³H]imipramine binding site of protein nature, that was suggested to be identical to the substrate recognition site for 5-HT uptake, was demonstrated. Since most studies on the regulation of the [³H]imipramine binding sites by antidepressants have used desipramine displaceable binding, which is heterogenous in nature and contains binding not related to 5-HT uptake sites, the present report studies the possible effects of chronic (3 weeks) administration of imipramine or norzimeldine (10 mg/kg intraperitoneally twice daily) on 5-HT sensitive [³H]imipramine binding sites. For comparison, desipramine sensitive binding was also studied, as well as the physiological correlate 5-HT uptake. There were no changes in either [³H]imipramine binding or 5-HT uptake after the antidepressant treatment.Supported by the Swedish Medical Research Council Offprint requests to: J. Marcusson at Dept. of Geriatric Medicine     

[3H]-Spiroperidol labels dopamine receptors in membranes from rabbit mesenteric artery

Summary The potent dopamine receptor antagonist [³H]-spiroperidol was used to label binding sites in a membrane fraction derived from rabbit mesenteric artery which had characteristics expected for dopamine receptors. The binding was of high affinity with an equilibrium dissociation constant (K_D) of 13.1 nM; it was saturable with 110 fmol of [³H]-spiroperidol bound/mg protein at maximal occupancy of the sites. Binding at 37° C was rapid and readily reversible with rate constants of 0.0154 nM^–1 min^–1 and 0.114 min^–1 for forward and reverse reaction, respectively. Dopamine receptor antagonists were about 100–200 times more potent than -adrenolytic drugs in competing for the [³H]-spiroperidol binding sites and dopamine was much more potent than (–)-noradrenaline, adrenaline, (–)-isoprenaline, clonidine or serotonin. It is concluded that in a membrane fraction of the rabbit mesenteric artery there exist binding sites for [³H]-spiroperidol indistinguishable from dopamine receptors. Thus the present results support the view that in vascular smooth muscle there exist specific dopamine receptors.This work was supported by the Deutsche Forschungsgemeinschaft     

Binding of [3H]clonidine to I1-imidazoline sites in bovine adrenal medullary membranes

Summary Imidazolines bind with high affinity not only to -adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [³H]clonidine or [³H]idazoxan and termed I₁- and I₂-IBS, respectively. Since bovine adrenal chromaffin cells lack ₂-adrenoceptors, we investigated the pharmacological characteristics of [³H]clonidine binding sites in the bovine adrenal medulla. The binding of [³H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [³H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [³H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (–)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline >- BDF 7579 (4-chloro-2-isoindolinyl guanidine) >-clonidine>- cirazoline >_ BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazolin-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I₁-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5guanylylimidodiphosphate; 100 mol/l) inhibited specific [³H]clonidine binding by about 50%. Equilibrium [³H]clonidine binding was also significantly reduced by K⁺ and Mg²⁺ In conclusion, [³H]clonidine labels non-adrenergic high-affinity sites in plasma membranes of the bovine adrenal medulla; these sites exhibit the pharmacological properties of I₁-IBS, but not of I₂-IBS. Furthermore, the IBS in the adrenal medulla appear to be coupled to a G-protein.Correspondence to G. J. Molderings at the above address     

Characterization of non-adrenergic [3H]clonidine binding sites in rat stomach: high affinity of imidazolines,guanidines and sigma ligands

We have identified and characterized non-adrenergic [³H]clonidine binding sites in rat stomach. The binding of [³H]clonidine was rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 68% specific binding at [³H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [³H]clonidine to rat stomach membranes was concentration-dependently inhibited by various imidazolines and guanidines including the sigma site ligand 1,2-di-(2-tolyl)guanidine (DTG), by the butyrophenone derivative haloperidol and by the piperidine derivative (+)-3-PPP[(R)-3-(3-hydroxyphenyl)-N-propylpiperidine]; the latter two compounds are also known to exhibit affinity for sigma sites. In contrast, rauwolscine, histamine, ranitidine and the non-hydrolysable GTP-analogue Gpp(NH)p (5 guanylylimidodiphosphate) did not, or with negligible affinity, inhibit [³H]clonidine binding. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases for a single detectable site) was as follows: cirazoline>idazoxanDTG>(+)-3-PPP> clonidine>guanabenz>haloperidol. This rank order is not compatible with the pharmacological properties of either I₁- or I₂-imidazoline binding sites. However, the ability of haloperidol, (+)-3-PPP and DTG to displace [³H]clonidine (the latter two with high affinity) suggests that the [³H] clonidine binding sites in rat stomach may be related to sigma-like sites.     

CHARACTERIZATION OF BINDING SITES FOR [3H]-IDAZOXAN, [3H]-P-AMINOCLONIDINE AND [3H]-RAUWOLSCINE IN THE KIDNEY OF THE DOG

1. We characterized the binding of [³H]-rauwolscine, [³H]-p-aminoclonidine and [³H]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the α₂-adrenoceptor- and imidazoline-preferring binding sites in this organ. 2. [³H]-Rauwolscine bound to an apparent single site with an affinity (K_D) of 2.2 nmol/ L and a maximum density (B_max) of 58.5 fmol/mg protein, when 10 μmol/L idazoxan defined non-specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [³H]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [³H]-rauwolscine binding sites had a relatively low affinity for prazosin (K_I= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (K_I= 7.9 nmol/ L). 3. [³H]-p-Aminoclonidine bound to an apparent single site (K_D= 5.2 nmol/L; B_max= 72.4 fmol/mg protein), when 10 μmol/L phentolamine defined non-specific binding. When 1 μmol/L of the potent and selective α₂-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [³H]-p-aminoclonidine binds only to α₂-adrenoceptors in the dog kidney. 4. [³H]-Idazoxan bound to two sites, with a higher (K_D= 0.95 nmol/L; B_max= 43.9 fmol/mg protein) and lower (K_D= 9.1 nmol/L; B_max= 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non-specific binding. When 10 μmol/ L GTPγS was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2-Methoxyidazoxan displaced [³H]-idazoxan in a monophasic manner and with low potency (IG₅₀=11.5 μmol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [³H]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of α₂-adrenoceptors that can be labelled either with [³H]-rauwolscine or [³H]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenoceptor imidazoline-preferring binding sites of the I₂-subtype, that can be labelled with [³H]-idazoxan. The binding site for which [³H]-idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein.     

Binding characteristics of [3H] guanfacine to rat brain α-adrenoceptors: Comparison with [3H]clonidine

The tritium-labeled α-adrenoceptor agonist and antihypertensive drug guanfacine, N-amidino-2-(2,6-dichlorophenyl)-acetamide (sp. act. 24.2 $\text{Ci}\text{mmole}$) was employed for a direct identification and characterization of α-adrenoceptors in rat brain membranes. Its usefulness as a radioligand was studied in comparison with [³H]clonidine (sp. act. 26.7 $\text{Ci}\text{mmole}$). The nonspecific binding of [³H]guanfacine to rat cerebral membranes was considerably more pronounced than that observed for [³H]clonidine. The specific binding of [³H] guanfacine (0.1–20 nM) and [³H]clonidine (0.1–20 nM) as defined as the excess over blanks containing (?)-norepinephrine (10μM) was saturable. Scatchard analyses of these binding data indicated single populations of binding sites for both ligands. K_D values of 3.9 ([³H]guanfacine) and 3.7 nM ([³H]clonidine) were calculated. Maximal number of specific binding sites amounted to 220 and 195 $\text{fmole}\text{mg}$ protein for [³H]guanfacine and [³H]clonidine, respectively. In case unlabeled guanfacine (1 μM) was used to characterize the specific binding of [³H] guanfacine, K_d value and maximal number of binding sites were about twice as high as determined in the presence of excess (?)-norepinephrine. The rate of association of both radioligands was rapid. Binding reached equilibrium by about 10–15 min of incubation. Half-maximal binding was attained at approximately 1–2 min. The rates of dissociation were biphasic. A rapid and a slow component were identified. The specific binding sites of [³H] guanfacine in rat brain possess the general characteristics of α₂-adrenoceptors. Selective antagonists of α₂-adrenoceptors, like yohimbine and rauwolscine strongly interfered with this binding. However, preferential blocking agents of α₁-adrenoceptors, such as prazosin and corynanthine, were weak competitors. The relative potency of agonists and antagonists in displacing [³H]guanfacine was identical to their effectiveness in competing for [³H]clonidine specific binding sites. It is concluded that [³H]guanfacine labels the same α₂-adrenoceptor population in rat brain as [³H]clonidine. However, [³H]guanfacine seems not as suitable as [³H]clonidine for routine use in the direct identification of α₂-adrenoceptors in view of its relatively high nonspecific binding.     

Solubilization and characterization of [3H]spiroperidol binding sites from subcellular fractions of the calf striatum

Specific [³H]spiroperidol binding sites were obtained from subcellular fractions of the calf striatum in a solubilized form using salt extraction with potassium chloride. Binding activity was assayed by adsorption on Norit SGX to separate unbound radioactive ligand from the macromolecular ligand complex. The solubilized membrane preparation exhibited stereoselectivity for butaclamol with the (+)-enantiomer being the active agent. Neuroleptic drugs were found to have an affinity for the soluble preparation which was identical to that of the native membrane-bound receptor. The effects of various parameters on the binding of [³H]spiroperidol to the soluble extract, including temperature, salt concentration, ionic strength and stabilizing agents, suggest that the binding site is biochemically stable. Electron micrographs of the soluble material reveal the absence of any recognizable membrane structures. Overall, these data demonstrate that a macromolecular component with high affinity and stereospecificity for [³H]spiroperidol can be isolated from calf striatal synaptosomes and microsomes, and suggest that at least one of the receptors for dopamine may be an extrinsic protein.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Characterization of [3H]tryptamine binding sites in brain

[3H]Tryptamine binds with high affinity to sites on rat brain membranes. The sites have the characteristics of tryptamine receptor recognition sites. These sites are widely distributed among rat brain regions with the highest density occurring in the cerebral cortex, striatum and hippocampus. The site is also found in human cerebral cortex. The binding site is localized mainly to the synaptosomal fraction. Drug competition studies indicate that the [3H]tryptamine binding site is distinct from serotonin receptors. Drugs that are potent inhibitors of [3H]tryptamine binding include tetrahydro-beta-carboline, quipazine, phenylethylamine, amphetamine, p-chloroamphetamine and methamphetamine.