3H-clonidine Binding Research Articles

Effect of glucocorticoids on alpha-2 adrenoceptors in vas deferens of reserpinized rat in organ culture

The changes in alpha adrenoceptors in vasa deferentia of reserpinized rats in organ culture were examined by binding studies with 3H-clonidine, 3H-yohimbine and 3H-prazosin. On organ culture for 1 day, the 3H-clonidine binding sites decreased by 1.07 pmol/g tissue and after 2 days no binding sites were detectable. 3H-Yohimbine binding sites also decreased during culture, but in smaller extent than 3H-clonidine binding sites. On the other hand, 3H-prazosin binding sites showed no apparent change in amount during culture. Addition of 0.1 mM hydrocortisone or dexamethasone to the culture medium inhibited the decrease in 3H-clonidine binding sites (1.7 pmol/g tissue), and the effect of glucocorticoid was blocked by the inhibitors of protein synthesis, cycloheximide and puromycin. However, hydrocortisone showed no effect on the 3H-yohimbine and the 3H-prazosin binding sites. An anti-endocytotic agent, an anti-microtubular agent and protease inhibitors had no effects on the decrease of 3H-clonidine binding sites during culture. These results suggest that the amount of alpha-2 adrenoceptors can change rapidly while alpha-1 adrenoceptors are stable and that glucocorticoids are important in regulation of conformation of alpha-2 adrenoceptor through synthesis of certain protein(s).

Platelet alpha 2 adrenoreceptors are decreased in number after antidepressant therapy

1. 1. Specific binding of 3H-clonidine to alpha 2 adrenoreceptors upon human blood platelet membranes is increased in patients with major depressive disorder (endogenous depression). 2. 2. Specific binding of 3H-yohimbine to the platelet adrenoreceptor is not altered in endogenously depressed patients. 3. 3. Other psychiatric disorders are not associated with alterations in the specific binding of either 3H-clonidine or 3H-yohimbine. In patients with severe congestive heart failure or with symptomatic coronary artery disease the number of platelet alpha 2 adrenoreceptors is actually decreased. 4. 4. Treatment of endogenously depressed patients with tricyclic antidepressants, lithium salts or electroconvulsive therapy results in a decrease in the number of alpha 2 adrenoreceptors on blood platelet membranes. 5. 5. These studies suggest that a supersensitivity of the alpha 2 adrenoreceptor might exist in patients with endogenous depression and that effective forms of therapy lead to a decrease in the number of neural alpha 2 adrenoreceptors which is reflected by a decrease in the number of these receptors upon blood platelet membranes.

Characteristics of alpha-2 receptors in rat vas deferens

The changes of 3H-yohimoine and 3H-clonidine binding sites in rat vas deferens on treatments with 2-chloroadenosine(2CA) and reserpine were examined with binding experiments. The results demonstrated that treatment with 2CA increased 3H-clonidine binding sites, whereas the treatment did not have effects on 3H-yohimbine binding sites. On the other hand, reserpine treatment increased both 3H-clonidine and 3H-yohimbine Dinding sites and also decreased the IC50 values of Clonidine on 3H-yohimbine binding sites. GTP and islet-activating protein inhibited the effects of 2CA on 3H-clonidine binding. The results imply that treatment of 2CA induced a qualitative change of alpha-2 adrenoceptors, while treatment with reserpine caused Doth qualitative and quantitative changes of the receptors. It is suggested that the qualitative change was mediated through a GTP-induced procès s.

3H-clonidine and 3H-rauwolscine binding to membranes from rat cerebral cortex and kidney.

3H-clonidine and 3H-rauwolscine binding to membranes from rat cerebral cortex and kidney.

Changes in alpha 2 adrenoreceptors in various areas of the rat brain after long-term administration of “mu” and “kappa” opiate agonists

Clonidine, an alpha 2 adrenoreceptor agonist, is used to treat opiate dependent individuals who are experiencing the signs and symptoms of withdrawal. Changes in the apparent number of alpha 2 adrenoreceptors in specific areas of the rat brain have been observed after chronic morphine administration. In the present study, the effects of chronically administered morphine sulfate upon alpha 2 adrenoreceptors were compared to those of UM-1072, (±)-5,9-alpha-dimethyl-2-hydroxy-2-tetrahydro-furfuryl-6,7-benzomorphan HCl, a “kappa” agonist which does not produce typical morphine-like dependence. The maximum number of specific binding sites (B max) and dissociation constants (K D's) for 3H-clonidine were measured with neural membranes isolated from saline or drug-treated rats. Rats were injected with saline, morphine or UM-1072, i.p., every 8 hr for 14 days. Doses of morphine ranged from 10 mg/kg, t.i.d., on the first three days to 100 mg/kg, t.i.d., on the last two days. Doses of UM-1072 covered a similar range. In control experiments, the B max's for specific binding of 3H-clonidine were (in fmoles/mg protein): hypothalamus, 142 ± 7; amygdala, 141 ± 3; brainstem, 70 ± 2; parietal cortex, 130 ± 4; hippocampus, 94 ± 2; and caudate nucleus, 62 ± 3. After chronic morphine treatment, the B max's were decreased significantly in all areas except the hippocampus. After chronic UM-1072 treatment, the B max's were decreased significantly in all areas studied. Neither treatment altered appreciably the K D's for 3H- clonidine. This study suggests that “mu” and “kappa” agonists might have similar actions upon noradrenergic systems in the brain.

CGS 7525A, A new, centrally active alpha 2 adrenoceptor antagonist

CGS 7525A, a new tetracyclic compound, was evaluated for alpha 2 adrenoceptor antagonism in receptor binding assays and in behavioral and electrophysiological tests. 3H-Clonidine, but not 3H-prazosin, binding was potently inhibited in vitro by CGS 7525A. In vivo , CGS 7525A attenuated the suppressant action of clonidine on phenylquinone-induced writhing and on locus coeruleus neuronal firing rate. Mianserin was nearly equipotent with CGS 7525A in the 3H-clonidine binding assay, but considerably less potent in the measures of alpha 2 adrenoceptor antagonism in vivo . Both CGS 7525A and mianserin displaced 3H-spiroperidol binding from frontal cortex 5-HT 2 binding sites. Although yohimbine resembled CGS 7525A in most respects, its activity at 5-HT 2 binding sites was relatively low. CGS 7525A was not associated with any appreciable blockade of norepinephrine or serotonin uptake in vitro . Thus, CGS 7525A appears to be a promising new pharmacological tool for investigating the behavioral function of brain alpha 2 adrenoceptors.

Interaction of catecholamine-derived alkaloids with central neurotransmitter receptors.

Catecholamine-derived alkaloids of the simple tetrahydroisoquinoline, 1-benzyl-tetrahydroisoquinoline and tetrahydroprotoberberine classes have been tested for their ability to inhibit the binding of seven different radioligands to neurotransmitter receptors of brain synaptic membranes. Alkaloids of all three classes were active in inhibiting 3H-clonidine binding to alpha 2-adrenergic receptors. Stereoselectivity of tetrahydropapaveroline in binding to alpha 2-adrenergic receptors was evidenced by the marked activity of the S-(--) isomer (IC50 = 0.65 microM) in comparison to the R-(+) enantiomer (IC50 = 50 microM). The simple tetrahydroisoquinolines (3,4-dihydroxytetrahydroisoquinoline and salsolinol), the four isomeric mono-O-methyl derivatives of 2,3,10,11-tetrahydroxyberbine and tetrahydropapaveroline were the most potent inhibitors of 3H-apomorphine binding to dopaminergic receptor agonist sites. The tetrahydroprotoberberines, as a class, were the most potent inhibitors of 3H-spiroperidol binding to dopaminergic receptor antagonist sites and of 3H-WB-4101 binding to alpha 1-adrenergic receptors. The 1-benzyl-tetrahydroisoquinolines exhibited varying degrees of interaction with beta 1-adrenergic receptors. Tetrahydropapaveroline (IC50 = 0.3 microM) was the most active of the 24 alkaloids tested in inhibiting binding of 3H-dihydroalprenolol to beta 1-adrenergic receptors. None of the alkaloids significantly affected 3H-QNB binding to muscarinic-cholinergic receptors, and selected alkaloids from each class interacted only moderately with serotonergic receptors.

A study of the sensitivity of rat brain alpha 2-adrenoceptors during chronic antidepressant treatments.

Two experimental approaches were used to study the effect of chronic antidepressant treatments on the functioning of central alpha2-adrenoceptors. In one, the effect of antidepressant treatment on the ability of a low dose of clonidine (25 μg/kg) to lower rat brain 3-methoxy-4-hydroxyphenylethyleneglycol sulphate (MHPG-SO4) levels was studied. In the other, potential treatment-induced alterations in rat frontal cortex 3H-clonidine binding were determined. The same cortical tissue was also studied in parallel for 3H-dihydroalprenolol binding.

Effect of neonatal 6-hydroxydopamine treatment on alpha 1- and alpha 2-adrenoceptors in rat cerebral cortex.

Neonatal 6-hydroxydopamine (6-OHDA) treatment was used to destroy the noradrenergic nerve endings in rat cerebral cortex and thus give some insight into the development and regulation of alpha-adrenoceptor subtypes, which in turn provides information concerning the anatomical localization of alpha 1- and alpha 2-adrenoceptors. In cerebral cortex of rats treated in the neonatal period with 6-OHDA, we have observed an irreversible decrease in noradrenaline levels. Differences in 3H-clonidine and 3H-prazosin binding occurred which varied depending upon the time between denervation and the binding assay. In 7-14 day-old rats we observed a 20% decrease in the number of alpha 2-adrenoceptors and a marked increase in alpha 1-adrenoceptors. In older rats (45-50 day-old) both types of alpha-adrenoceptors were increased. Results of this study indicate that alpha 2-adrenoceptors located on presynaptic noradrenergic terminals represent only a minor fraction of the total alpha 2- adrenoceptors in rat cerebral cortex that are lost after denervation. Conversely, noradrenergic denervation resulted in supersensitivity of the alpha 1-adrenoceptors and nonnoradrenergic terminals located on alpha 2-adrenoceptors.

Modulation of rat brain alpha-adrenoreceptor populations four weeks after stimulation of the nucleus locus coeruleus.

We previously showed that electrical stimulation of the nucleus locus coeruleus was followed 4 weeks later by a greatly improved performance in the acquisition of a food-reinforced operant task. To ascertain whether adrenergic receptors were involved in this long-term behavioral modification, we studied the characteristics of the alpha 1, alpha 2, and beta-adrenoreceptors of the cerebral cortex 4 weeks after stimulation of the locus coeruleus. This stimulation induced a slight (14%) but significant increase in the number of alpha 1-receptor [(3H) WB 4101 binding sites] as well a rise in the number of alpha 2-receptor [(3H) clonidine binding sites]. The latter rise mainly affected high-affinity alpha 2-receptor sites (36%) and the number of low-affinity sites remained unchanged. No significant alteration in the number of beta-receptors [(3H)-dihydroalprenolol binding sites] was observed. To confirm this biochemical result, the effect of very small doses of clonidine (1, 2.5, 5 and 10 micrograms/kg) was tested on locomotor activity in the open-field. In rats stimulated 4 weeks before injection, clonidine induced a biphasic effect, comprising firstly sedation which occurred 30 min after injection, and secondly, long-term hyperactivity which began 24 h injection. For the 5 micrograms/kg dose, this rebound of activity was detectable 8 days after injection. In implanted, control rats, only the sedative effect was observed. These findings are interpreted in relation to the current theories about alpha-adrenoreceptors.

Alpha 1- and alpha 2-adrenoceptors in rat cerebral cortex: effect of frontal lobotomy.

Surgical noradrenergic denervation of the cortex via frontal lobotomy was used to destroy the noradrenergic nerve endings and thus give some insight into the distribution of alpha-adrenoceptors. Frontal lobotomy caused a reduction in noradrenaline content in rat cerebral cortex (2.1 +/- 0.4 ng/mg protein for lesioned side, 6.0 +/- 0.3 mg/mg protein for non-lesioned side), indicating an effective noradrenergic denervation. The differences in 3H-clonidine and 3H-prazosin binding observed following surgery were a significant decrease in the number of alpha 2-adrenoreceptors (115.0 +/- 4.5 to 91.7 +/- 3.2 fmol/mg protein, n = 7, P less than 0.001) and a smaller but significant increase in the number of alpha 1-adrenoceptors (119.7 +/- 2.5 to 131.6 +/- 5.4 fmol/mg protein, n = 7, P less than 0.05) in the lesioned cortex. Results of this study indicate that alpha 2-adrenoceptors located on presynaptic noradrenergic terminals represent only a small proportion of the total alpha 2-adrenoceptors in rat cerebral cortex.

Alpha 1 and alpha 2 Adrenergic receptors in mouse brain astrocytes from primary cultures.

Mouse brain astrocytes from primary cultures were found to contain both alpha 1 and alpha 2 adrenergic receptors. 3H WB 4101 labeled one category of binding site (KD = 1.5 +/- 0.39 nM, Bmax = 64 +/- 7.9 fmoles/mg protein) with typical alpha 1 adrenergic specificity (WB 4101 greater than prazosin greater than yohimbine). The density of alpha 1 adrenergic receptors was 2-3 times higher in mouse cerebral cortex than in glial cells. Like rat brain [U'Pritchard et al, 1979; Rouot et al, 1980], mouse glial cells were found to contain two categories of 3H clonidine binding sites: high affinity sites, which were identical to the high but not to the low affinity sites found in rat brain, since 1) they displayed the same affinity for 3H clonidine (KD = 1.2 +/- 0.13 nM, n = 4) and the same typical alpha 2 adrenergic specificity (yohimbine greater than WB 4101 greater than prazosin); 2) the dissociation rate constant for clonidine binding was equal to 0.06 min-1, a value close to that found previously for the high affinity 3H clonidine binding sites in rat brain (0.05 min-1); and 3) divalent cations augmented and guanyl nucleotides reduced 3H clonidine binding as in rat brain. Na+ decreased 3H clonidine binding in a complex manner. The number of high affinity sites in glial cells (52 +/- 9.4 fmoles/mg protein, n = 4) was half the number found in mouse cerebral cortex (98 fmoles/mg protein). Low affinity 3H clonidine binding sites (KD = 81 +/- 18 nM, Bmax = 96 +/- 5.8 fmoles/mg protein, n = 3) were not fully characterized. In conclusion, glial cells contained the same alpha adrenergic receptors as those described in brain, but their physiological function is not yet known.

Presynaptically acting catecholamines bind to α2-adrenoceptors labelled by 3H-clonidine

It is known that certain catecholamine congeners can decelerate the heart rate by inhibiting the prejunctional sympathetic neurones to the atrium. The present study was done to determine whether this presynaptic action might be associated with either dopamine receptors or α-adrenergic receptors. The effects of 10 catecholamine congeners were tested on the specific binding of 0.2 nM 3H-WB-4101 and 0.2 nM 3H-clonidine to calf frontal cortex homogenates, and that of 2 nM 3H-apomorphine to calf caudate nucleus homogenates. The drugs tested were apomorphine, the aminotetralin M-7, 3 dialkylated dopamine congeners, and 5 congeners of octahydrobenzo (f)-quinoline. The IC 50 values (concentrations for 50% inhibition of binding) ranged from 400 to 28 000 nM for 3H-WB-4101, from 3 to 270 nM for 3H-apomorphine, and from 9 to 1000 nM for 3H-clonidine. Only the IC 50 values for 3H-clonidine binding correlated with the in vitro IC 50 values for inhibiting atrial acceleration (data from Long et al., 1975, 1979). These findings suggest that 3H-clonidine appears to bind to the same site in brain ( α 2-adrenoceptor) on which catecholaminergic drugs act to produce cardiodeceleration.

Antagonist/agonist-preferring α-adrenoceptors or [formula omitted

Yohimbine and some stereoisomeric alkaloids inhibited the binding of 3H-clonidine and 3H-WB-4101 to rat cerebral cortex membranes. Rauwolscine and yohimbine had much higher affinity to the 3H-clonidine than to the 3H-WB-4101 site, whereas the reverse was true for corynanthine. The results indicate that the 3H-clonidine site is an α 2-adrenoceptor and not an agonist-selective site whereas the 3H-WB-4101 site is an α 1-adrenoceptor and not an antagonist-selective site.

3H-Prazosin binds specifically to 'alpha 1'-adrenoceptors in rat brain.

Prazosin is know to block postsynaptic alpha-adrenoceptors. In this study 3H-prazosin has been used to label biochemically central alpha-adrenoceptors. In rat brain membranes 3H-prazosin bound specifically in a rapid, reversible and saturable manner to a single class of high affinity sites. The relative order of potencies for inhibition of 3H-prazosin binding was WB4101 greater than ARC 239 greater than phentolamine greater than piperoxane greater than yohimbine which is a characteristic of the alpha 1 type of adrenoceptors. In contrast, the relative order of potencies for inhibition of 3H-clonidine binding was yohimbine greater than piperoxane greater than WB4101 greater than ARC239 greater than prazosin which is a characteristic of the alpha 2 type of adrenoceptors. These results indicate that 3H-prazosin binds to central 'alpha 1'-receptors and 3H-clonidine to 'alpha 2'-receptors and confirm the presence of two classes of alpha-adrenoceptors in rat brain membranes.

Binding of an imidazolidine (clonidine), an oxazoloazepin (B-HT 933) and a thiazoloazepin (B-HT 920) to rat brain α-adrenoceptors and relation to cardiovascular effects

The specific binding to α-adrenoceptors in crude plasma membrane preparations of the rat brain was studied by means of 3H-clonidine (specific radioactivity 26.7 Ci/mmole). Equilibrium binding of 3H-clonidine could be described adequately according to a two-site model with a minor population of high affinity sites (K D1 = 0.4 nM) and a major population of low affinity sites (K D2 = 6.1 nM). The heterogeneity of 3H-clonidine binding was also indicated by a biphasic association rate in kinetic binding studies. In competition experiments with 3H-clonidine concentrations of 0.5 or 4.0 nM respectively, concentration-dependent displacement was observed with the non-radioactive compounds: clonidine, B-HT 920 (2-amino-6-ally--5,6,7,8-tetrahydro-4H-thiazolo-[5,4-d]-azepin-dihydrochloride) and B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxozolo-[5,4-d]-azepinddihydrochloride). IC 50 values of 3, 21 and 160 nM or 10, 63 and 380 nM respectively were thereby evaluated. Cardiovascular effects were estimated in rats. The blood pressure increase in spinal animals was taken as parameter for α-adrenoceptor stimulation at peripheral vascular sites. The bradycardic effect in vagotomized animals was taken as parameter for central nervous sympathoinhibition. The ranking order of the potency of the three drugs was the same in both in vivo tests and parallels the in vitro binding affinities at both binding sites: clonidine > B-HT 920 > B-HT 933. These results indicate the similarity of the α-adrenoceptor structures in brain membrane preparations, at peripheral vascular sites and at central sympathoinhibitory sites.

Short-term surgical denervation increases 3H-clonidine binding in rat salivary gland

Short-term surgical denervation increases 3H-clonidine binding in rat salivary gland

Binding to α-adrenergic receptors: Differential pharmacological potencies and binding affinities of benzodioxanes

We have compared the influence of a series of benzodioxane α-adrenergic antagonists in 3H-WB-4101 and 3H-clonidine binding to α-receptor sites in the brain and peripheral tissues with their pharmacological properties. The drug specificity of 3H-WB-4101 bidning is quite similar in central and peripheral tissues. Pharmacological potencies of benzodioxanes at postsynaptic α-receptors in the rat vas deferens correlate with potencies at 3H-WB-4101 but not at 3H-clonidine binding sites. These findings suggest pharmacological effects of these drugs are mediated by “α-1 postsynaptic receptors” labeled by 3H-WB-4101. For several benzodioxanes absolute pharmacological potencies at postsynaptic α-receptors of the rat vas deferens are substantially less than theie pontencies at 3H-WB-4101 sites. The potencies of benzodioxane analogues at 3H-clonidine binding sites are similar to their pharmacological potencies at presynaptic autoreceptors in the rat vas deferens.

Negligible binding of 3H-clonidine to histamine H 2 receptor

Negligible binding of 3H-clonidine to histamine H 2 receptor

Binding of 3H-clonidine to an alpha-adrenoceptor in membranes of guinea-pig ileum.

The binding of 3H-clonidine to membrane particles from guinea-pig ileum was investigated. The specific binding, i.e. the binding that could be inhibited by high concentrations of unlabeled clonidine or noradrenaline, was of high affinity, KD3 nM. The number of sites was approximately 25 fmol/mg protein. Rate constants of association and dissociation were 5.3×107 M−1 min−1 and 0.18 min−1, respectively. Affinites of various drugs to the binding site were determined by measuring their effect on the binding of 3H-clonidine. The affinity of adrenergic agonists decreased in the order clonidine = tramazoline > (−)-erythro-α-methylnoradrenaline > (−)-noradrenaline ≫ (−)-phenylephrine. (−)-Noradrenaline had about 20 times more affinity than the (+)-isomer. The affinity of α-adrenoceptor antagonists decreased in the order phentolamine > rauwolscine = yohimbine > WB 4101 > pseudoyohimbine > prazosin = corynanthine. Yohimbine and rauwolscine had about 100 times more affinity than their stereoisomer corynanthine. Serotonin 10 μM and metiamide 10 μM did not affect the binding, and propranolol inhibited it only at high concentrations. — The results indicate that 3H-clonidine labels an β2-adrenoceptor in guinea-pig ileum. The orders of affinity of α-adrenoceptor agonists and antagonists agree well with their orders of potency in functional tests, namely as modulators of cholinergic transmission in the guinea-pig ileum and as modulators of noradrenaline release in the rabbit pulmonary artery. An α-adrenoceptor should be classified as α2 when the affinities of clonidine, tramazoline and α-methylnoradrenaline greatly exceed the affinity of phenylephrine, and when the affinities of rauwolscine and yohimbine exceed those of prazosin and corynanthine.