3H-clonidine Binding Research Articles

Identification and characterization of platelet α2-adrenoceptors and imidazoline receptors in rats, rabbits, cats, dogs, cattle, and horses

This study aimed to pharmacologically identify and characterize α2-adrenoceptors and imidazoline (I) receptors (I1- and I2-subtype) on canine, feline, bovine, equine, murine, and leporine platelet membranes. Saturation binding studies with both 3H-yohimbine and 3H-clonidine showed that α2-adrenoceptors were expressed on canine, leporine, feline, and murine platelets but not on bovine and equine platelets. In competition studies, the rank order of affinity of 6 compounds for canine platelet α2-adrenoceptors was similar to that of potency at α2A-subtype reported in human platelets. Saturation binding studies in the presence of norepinephrine showed that canine, feline, bovine, and equine platelets had I1-receptors defined by 3H-clonidine binding, but neither murine nor leporine platelets had I1-receptors; whereas, platelets of all species had I2-receptors defined by 3H-idazoxan binding. In competition studies, more potent compounds displayed biphasic competition curves with 3H-clonidine. The rank orders of affinity of I1 compounds for high-affinity components of I1-receptors of canine, feline, bovine, and equine platelets and I2-receptors of all species platelets were similar to those of compounds for high-affinity components reported in human I1- and I2-receptors, respectively. Guanine nucleotides inhibited the high-affinity component of naphazoline binding to canine I1-receptors, but not to I2-receptors. Furthermore, guanine nucleotides dose-dependently inhibited 3H-clonidine binding to I1-receptors; whereas, they did not interfere with 3H-idazoxan binding to I2-receptors, supporting the notion that Il-receptors may belong to a G protein-coupled receptor superfamily in canine platelets. Interspecific variations of platelet α2-adrenoceptor and imidazoline receptor expressions may explain different platelet responses to catecholamines and imidazoline α-adrenergic agents.

Effects of testosterone on alpha2A-adrenergic receptor expression in the rat brain.

Androgens are involved in regulation of behaviour through intracellular mechanisms owing to their receptors. Involvement of intercellular messengers such as brain norepinephrine and adrenergic receptors (ARs) is seemed to be necessary to realise hormone-dependent behavioural effects. Castration of adult male rats, which decreases copulatory activity in the animals, was accompanied by a significant increase in 3H-clonidine (alpha2-AR agonist) binding site density in the frontal cortex. The levels of mRNA for the alpha2A-ARs (measured by RT-PCR) were increased in the brainstem of castrated males in parallel to the changes in cortical ARs densities. Testosterone treatment, that activates copulatory behaviour in castrates, down regulated alpha2A-AR mRNA levels in the brainstem and 3H-clonidine binding sites densities in the cortex, where terminals of the brain stem neurones are situated. Unlike in the brainstem, castration caused a decrease in alpha2A-AR mRNA in the cortex and testosterone up-regulated this mRNA in the cortical region. The data suggested that down-regulation of alpha2-ARs densities in the cortex that is induced by testosterone can be preferentially related to alpha2-ARs subpopulation which is expressed by the brainstem neurones and imported into the cortex by axons of these neurones.

Distribution and density of monoamine receptors in the primate visual cortex devoid of retinal input from early embryonic stages

Developmental mechanisms that regulate the areal and laminar distribution of various macromolecules, including neurotransmitter receptors in the cerebral cortex, are not known. In the present study, we examined the development of monoaminergic receptors in the rhesus monkey striate and peristriate visual cortex in the absence of input from the retina. Binocular enucleation was performed between embryonic days E60 and E81, prior to the ingrowth of geniculocortical fibers into the cortical plate and before genesis of the granular and supragranular layers of the visual cortex. The animals were delivered at term (E165) and sacrificed at 2 or 12 months of age, and their brains frozen and the occipital lobes cut at 20 microns in the coronal plane. Cortical binding of 3H-clonidine, 125I-pindolol, 3H-5-HT, 3H-ketanserin, 3H-spiperone, 3H-SCH23390, and 3H-prazosin that label various monoamine receptors were autoradiographically visualized and quantified using a computer imaging system. All radioligands displayed specific laminar patterns in the striate and prestriate areas in both groups of animals. The areal and laminar distribution in the anophthalmic monkeys was similar to that in the controls. Significantly, in all enucleated animals, just as in the controls, a particularly high density of 3H-clonidine and 3H-prazosin was observed in the sublayers of layer IV involved in color vision. The present results show that the monoamine receptors in primate visual cortex can establish and maintain distinct laminar and areal patterns in the absence of activity or molecular cues originated from the retina, and provide new insight into the cortical consequences of secondary congenital anophthalmia.

Correlations between the hypothalamic density of 3H-clonidine-binding sites and plasma testosterone levels in mice.

Correlations between the specific binding of radiolabelled adrenoligands in frontal cortex and hypothalamus, and plasma testosterone levels were investigated in inbred mouse strains. Binding values were structure and genotype dependent. There were significant negative intrastrain and interstrain correlations between hypothalamic binding of 3H-clonidine, an imidazoline with alpha 2-adrenoceptor agonist specificity, and resting testosterone levels. Neither binding of the alpha 1-adrenoceptor antagonist 3H-prazosin nor of the beta-adrenoceptor antagonist 3H-dihydroalprenolol (DHA) correlated significantly with hormone concentrations. The inverse correlation between hypothalamic 3H-clonidine-binding and plasma testosterone levels suggests a possible role of alpha 2-adrenoceptors in the negative regulation of testosterone plasma levels.

Agmatine recognizes ?2-adrenoceptor binding sites but neither activates nor inhibits ?2-adrenoceptors

It has been suggested that agmatine (decarboxylated arginine) is an endogenous clonidine-displacing substance (CDS) which recognizes alpha 2-adrenoceptor and non-adrenoceptor, imidazoline binding sites. We have examined the effect of agmatine at alpha 2-adrenoceptor binding sites and pre- and postjunctional alpha 2-adrenoceptors. Agmatine produced a concentration-dependent inhibition of 1 nmol/l 3H-clonidine binding to both rat (pKi-5.10 +/- 0.05) and bovine (pKi-4.77 +/- 0.38) cerebral cortex membranes. However, agmatine (0.1-100 microM) failed to activate pre-junctional alpha 2-adrenoceptors regulating transmitter release in the guinea-pig isolated ileum and rat isolated vas deferens, nor did it activate postjunctional alpha 2-adrenoceptors of the porcine isolated palmar lateral vein which mediate contraction or inhibition of forskolin-stimulated cyclic AMP formation. High concentrations of agmatine (10-30-fold the pKi at alpha 2-adrenoceptor binding sites) failed to influence alpha 2-adrenoceptor activation by either clonidine or UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate) in any of the peripheral preparations examined. Moreover, even in a preparation where an interaction with alpha 2-adrenoceptor binding sites on cell membranes can be demonstrated, the rat cerebral cortex, agmatine failed to inhibit forskolin-stimulated cyclic AMP in the intact tissue or affect the inhibition produced by the selective alpha 2-adrenoceptor agonist UK-14304. Agmatine was also devoid of agonist activity in two preparations, the rat isolated thoracic aorta and the rat isolated gastric fundus, in which CDS has been reported to produce non-adrenoceptor effects. Thus, we have confirmed that agmatine recognizes alpha 2-adrenoceptor binding sites and, therefore, is a CDS.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in α2-Adrenoceptors on Vascular Smooth Muscle and Neural Membranes following Hypertension Induced by Renal Ischemia

alpha 2-Adrenoceptors were characterized on neural and vascular membranes from 2-kidney-1-clip renal hypertensive (RHT) and normotensive (NT) rats. Rats were sacrificed 6 weeks after induction of renal ischemia, and the specific binding of 3H-clonidine to smooth muscle membranes form tail arteries and neural membranes from various brain regions was examined. Additionally, isometric contractions of helically cut tail artery strips produced by various alpha-adrenoceptor agonists were measured. Scatchard analysis indicated an increased number of high-affinity binding sites on the smooth muscle membranes from RHT rats (Bmax = 43.5 +/- 1.4 fmol/mg protein) compared to that from the NT rats (25.4 +/- 3.8 fmol/mg protein). An increased contractile sensitivity to clonidine was also observed in tail artery strips from RHT rats (EC50 for RHT = 3.04 x 10(-8) M; NT = 1.52 x 10(-7) M). In neural tissue, the number of alpha 2-adrenoceptor-binding sites was significantly increased in the locus coeruleus from RHT rats, but not in the amygdala, hypothalamus, parietal cortex, hippocampus or lower brain stem. These results demonstrate that renal ischemia produces changes in both peripheral and neural alpha 2-adrenoceptor density. The increase in smooth muscle alpha 2-adrenoceptors might also provide a partial explanation for the supersensitivity to adrenergic agonists in this model of hypertension.

Pharmacological profile of the novel .ALPHA.-adrenoceptor antagonist KT-611(naftopidil).

The pharmacological profile of a new alpha-adrenoceptor antagonist, KT-611 (naftopidil), was studied in vitro. In the dog mesenteric and carotid arteries and in the rabbit, guinea pig and rat thoracic aortae, KT-611 competitively inhibited alpha 1-adrenoceptor-mediated contractions induced by noradrenaline with pA2 values ranging from 6.73 to 8.15. KT-611 also inhibited the postjunctional alpha 2-adrenoceptor-mediated contractions in the dog saphenous vein (pA2 = 6.77) or dog basilar artery. However, the responses mediated through prejunctional alpha 2-adrenoceptors (rat vas deferens), beta-adrenoceptors (rat atria), muscarinic receptors (guinea pig ileum) and 5-HT2 receptors (dog mesenteric artery) were little affected by KT-611. KT-611 also inhibited the sympathetic adrenergic contraction evoked by electrical transmural stimulation in the dog mesenteric artery, and the inhibition was not relieved upon repetitive washing for 1 hour with the drug-free solution. 3H-prazosin and 3H-clonidine binding to the rat cortex membranes was inhibited by KT-611 with pKi values of 7.69 and 5.75, respectively. These results suggest that KT-611 is an alpha 1-adrenoceptor antagonist with a weak antagonistic activity to postjunctional alpha 2-adrenoceptors.

Pharmacological Profile of the Novel α-Adrenoceptor Antagonist KT-611 (Naftopidil)

The pharmacological profile of a new alpha-adrenoceptor antagonist, KT-611 (naftopidil), was studied in vitro. In the dog mesenteric and carotid arteries and in the rabbit, guinea pig and rat thoracic aortae, KT-611 competitively inhibited α1-adrenoceptor-mediated contractions induced by noradrenaline with pA2 values ranging from 6.73 to 8.15. KT-611 also inhibited the postjunctional α2-adrenoceptor-mediated contractions in the dog saphenous vein (pA2 = 6.77) or dog basilar artery. However, the responses mediated through prejunctional α2-adrcnoccptors (rat vas deferens), β-adrenoceptors (rat atria), muscarinic receptors (guinea pig ileum) and 5-HT2 receptors (dog mesenteric artery) were little affected by KT-611. KT-611 also inhibited the sympathetic adrenergic contraction evoked by electrical transmural stimulation in the dog mesenteric artery, and the inhibition was not relieved upon repetitive washing for 1 hour with the drug-free solution. 3H-prazosin and 3H-clonidine binding to the rat cortex membranes was inhibited by KT-611 with pKi values of 7.69 and 5.75, respectively. These results suggest that KT-611 is an α1-adrenoceptor antagonist with a weak antagonistic activity to postjunctional α2-adrenoceptors.

Comorbidity of panic disorder and major depressive disorder: effects on platelet alpha2 adrenergic receptors

Adrenergic receptor dysregulation has been described as occurring in both major depressive disorder (MDD) and panic disorder. Measurements of platelet alpha 2 adrenergic receptors in these patients may be confounded by the coexistence or comorbidity of both diagnoses in the same patient. To explore this possibility, we measured platelet alpha 2 adrenergic receptors (3H-clonidine and 3H-yohimbine binding) in 3 groups of patients (MDD only, panic disorder only, and those showing comorbidity of MDD and panic) and normal controls. Patients with comorbidity of MDD and panic disorder had significantly lower agonist binding (3H-clonidine).

Functional Supersensitivity of Antinociceptive Spinal Cord α2‐Adrenoceptors, Induced by Depletion of Endogenous Noradrenaline, is Associated with an Enhanced Sensitivity for Guanine Nucleotide Regulation of3H‐Clonidine Binding

In spinal cords from normal mice, 3H-clonidine bound to alpha 2-adrenoceptors with an apparant Kd of 4.6 nM and capacity (Bmax) of 430 fmol/mg protein. The non-hydrolyzable GTP analogue Gpp(NH)p was found to dose-dependently reduce the binding of 3H-clonidine the effect of Gpp(NH)p being essentially maximal at 10(-4) M. Gpp(NH)p was found to reduce the apparent affinity as well as the apparent number of binding sites for 3H-clonidine; the Kd of 3H-clonidine being 16 nM and the Bmax 300 fmol/mg protein when 10(-4) M of the nucleotide was present. In mice pretreated with the noradrenaline neurotoxin DSP4 for 14 days prior to assay, the specific binding of nearly saturating concentrations (10-30 nM) of 3H-clonidine was virtually the same as for control mice. However, for the DSP4 treated animals the down-regulation of 3H-clonidine binding induced by 10(-4) M Gpp(NH)p was significantly higher than for control mice. Thus, at a 3H-clonidine concentration of 10 nM the down-regulation induced by Gpp(NH)p was 64.1 +/- 2.6% for the DSP4 versus 58.9 +/- 2.3% for the control mice (P less than 0.05). At a 3H-clonidine concentration of 30 nM the down-regulation was 52.4 +/- 1.4% for DSP4 versus 41.8 +/- 4.1% for control mice (P less than 0.05). DSP4 pretreatment also potentiated the antinociceptive effect of intrathecal clonidine, as assessed by the tail-flick and hot plate tests. It is suggested that the functional supersensitivity induced by DPS4 for clonidine is related to the increased sensitivity for guanine nucleotide regulation of the spinal cord alpha 2-adrenoceptors.

Formamidine pesticides and alpha 2-adrenoceptors: Studies with amitraz and chlordimeform in rats and development of a radioreceptor binding assay

The interaction of the formamidine pesticides chlordimeform (CDM) and amitraz (AMZ) with rat brain alpha 2-adrenoceptors was investigated. Both compounds inhibited the binding of 3H-clonidine and 3H-yohimbine in vitro with IC 50 values of 62–68 μM (CDM) and 95–110 nM (AMZ). In vivo administration of AMZ and CDM caused a dose-dependent inhibition of 3H-clonidine binding in rat forebrain. The inhibition was short-lasting (24 hr) following CDM administration, while after AMZ recovery of 3H-clonidine binding occurred only after 72 hr. Good correlations were found between inhibition of brain 3H-clonidine binding by the formamidines and “plasma equivalents” of these compounds and/or their biologically active metabolites, as measured by a new radioreceptor assay. These results suggest that 1) formamidines can interact in vivo with brain alpha 2-adrenoceptors when administered at doses previously shown to cause toxic effects on the central nervous system: and 2) this effect is reversible, both in vivo and in vitro, and appears to be linked to the presence of the formamidines and/or their active metabolites at the receptor sites.

Platelet 3H-clonidine and 3H-imipramine binding and plasma cortisol level in depression

Platelet 3H-clonidine ( α 2-adrenergic agonist) binding and 3H-imipramine binding were measured and the Dexamethasone Suppression Test performed in 17 normal controls and 14 unmedicated depressed patients in order to clarify the relationship among these three biological markers. Increases in the B max and the K d for 3H-clonidine binding and decreases in the B max for 3H-imipramine binding of the platelets from depressed patients were observed when compared with controls. There was a significant positive correlation among 3H-clonidine B max , the basal (predexamethasone) plasma cortisol levels, and the severity of depression, as indicated by the Hamilton Depression Rating Scale. On the other hand, no significant correlation was observed in 3H-imipramine binding between the B max and the severity of depression or between the B max and the basal plasma cortisol levels. There was no statistically significant correlation between the B max of 3H-clonidine binding and that of 3H-imipramine binding in depression, but there was a trend toward correlation in normal controls .

Increased 3H-Clonidine binding in the platelets of patients with depressive and Schizophrenic disorders

To examine whether α 2-adrenergic receptor function is altered in affective and schizophrenic disorders, we determined 3H-clonidine binding in platelets obtained from 33 normal control subjects and from 24 patients with depressive, 22 patients with schizophrenic, 18 with bipolar, and 8 patients with schizoaffective disorders during a drug-free period. The maximum number of binding sites ( B max) and apparent dissociation constant ( K d) for high affinity 3H-clonidine binding was computed by Scatchard analysis. Comparison of the diagnostic groups indicated that the B max in depressed, schizophrenic, and schizoaffective patients was significantly higher than in normal controls, but there were no significant B max differences between bipolar patients and controls. Comparison of the K d among the diagnostic groups indicated no significant differences among the groups or between patient diagnostic groups and normal controls. Baseline B max in schizophrenic patients was significantly correlated with the decrease in Brief Psychiatric Rating Scale (BPRS) scores after treatment, suggesting a relationship between baseline B max and clinical response. Treatment with lithium caused a significant decrease in the baseline B max, whereas treatment with desipramine or trifluoperazine did not cause significant changes in the baseline B max. Our results thus indicate an increase in the number of α 2-adrenergic receptors in depressed and schizophrenic patients as compared to normal controls.

A trial of CI-943 in schizophrenia

350 determined in the platelet membrane obtained from 33 normal control subjects, 24 patients with depressive illness, 22 patients with schizophrenia and 18 with bipolar illness. The maximum number of binding sites (B,,) and apparent dissociation constant (K,) for high affinity 3H-clonidine binding were computed by Scatchard analysis. The mean B,, (fmoles/mg protein) in depressed patients (46.3 2 26.4 SD), schizophrenic patients (53.7 + 27.4 SD) and schizoaffective patients (58.6 + 30.4 SD) was significantly higher than normal controls (3 1.6 ? 23 SD). There were no significant differences in the Kn between any of the diagnostic groups. Treatment with lithium for four to six weeks caused a significant decrease in the B,,, of ‘H-clonidine binding, but treatment with desipramine or trifluoperazine had no significant effect on the B max. Our results thus indicate that increased high affinity alpha*-adrenergic receptor number may be associated with schizophrenia and depression.

Detection and Characterization of 3H-Clonidine Binding Sites in Coated Vesicles Isolated from Bovine Brain

AbstractThe binding of 3H-clonidine to bovine brain coated vesicles was studied. The binding was reversible and saturable. Saturation studies revealed a one-site interaction: Kd was 8.7 nM and Bmax was 24 5 fmol/mg protein. Noradrenaline, yohimbine and phenoxybenzamine displaced 3H-clonidine binding from the binding sites at concentrations of 10-7-10-4 M. The results indicate that the coated vesicles from the bovine cerebral cortex contain 3H-Clonidine binding sites comparable to the low affinity sites of α2-adrenergic receptors in bovine brain membranes

Detection and characterization of 3H-clonidine binding sites in coated vesicles isolated from bovine brain.

The binding of 3H-clonidine to bovine brain coated vesicles was studied. The binding was reversible and saturable. Saturation studies revealed a one-site interaction: Kd was 8.7 nM and Bmax was 24.5 fmol/mg protein. Noradrenaline, yohimbine and phenoxybenzamine displaced 3H-clonidine binding from the binding sites at concentrations of 10(-7)-10(-4) M. The results indicate that the coated vesicles from the bovine cerebral cortex contain 3H-clonidine binding sites comparable to the low affinity sites of alpha 2-adrenergic receptors in bovine brain membranes.

Influence of an Uremia-Associated Serum Factor on CNS α2-Adrenoceptors

The action of blood serum from uremic rats and chronically hemodialyzed patients was investigated for effects on alpha 2-adrenoceptors labeled with 3H-clonidine. Compared to blood sera of rats and patients with normal kidney function, uremic serum significantly inhibited specific 3H-clonidine binding. In saturation experiments the density and affinity of alpha 2-adrenoceptors for 3H-clonidine was lowered by uremic serum. Heating, or trypsin or lipase treatment of the serum did not affect this phenomenon. The effect of the patient's serum could likewise be demonstrated after hemodialysis treatment. The presence of an allosteric regulating substance for clonidine binding to adrenoceptors could at least partially explain the altered and attenuated action of this drug in renal insufficiency.

Evidence that high affinity (3H)clonidine binding cooperates with H2-receptors in the canine coronary smooth muscle membrane.

We have investigated the effects of myocardial ischemia and exogenous histamine and 4-methylhistamine on the regulation of membrane bound alpha 2- and beta-adrenoreceptors (ARs) in the canine coronary artery smooth muscle (CAS). The results indicate that exposure of CAS to ischemia and histamine is associated with the stimulation of adenylate cyclase and with a down-regulation of alpha 2-ARs which is accompanied by the sequestration of alpha 2-AR sites into light membrane particles. The increased number of beta-AR sites in CAS represents a c-AMP mediated adaptational pathway in compromised CAS.

Super high affinity 3H-Para-Aminoclonidine binding to platelet adrenoceptors in depression

1. 1. An assay was developed using sucrose gradient purified platelet plasma membranes which allowed detection, for the first time in patients, of both super-high affinity (K D-17 pM) and high affinity (K D-1.7 nM) binding sites. 2. 2. Limited Scatchard plot analyses were performed on platelet membranes from depressed patients and controls using 10 pM-2.5 nM 3H-p-aminoclonidine ( 3 H-PAC). 3. 3. Patients (n = 9) were age-paired with healthy control subjects for simultaneous blood drawing, platelet preparation and analysis. 4. 4. All patients were endogenous depressives with Hamilton-Depression scores ranging from 19 to 30 at the time of pre-treatment. Seven of the nine patients were analyzed again at six weeks of treatment with antidepressant medication. 5. 5. Using 60 pM 3H-PAC (a concentration determined to bind predominantly to the super-high affinity receptor state) pre-treatment patient values were higher then paired controls (p = 0.06). Post-treatment analysis of seven of the patients and paired controls showed no differences (p = 0.5) suggesting a normalization of receptor binding following treatment. 6. 6. No differences were observed in platelet yield or morphology or in the percent of other blood cell contaminants in the platelet preparations between patients at pre-treatment and controls. However, the platelet yield was significantly lower in patients post-treatment (p = 0.06). 7. 7. These results are in agreement with two previous studies showing elevated 3H-clonidine binding to high affinity sites from depressed patients. The data presented herein suggest that there is a modest 1.25-fold elevated super-high affinity platelet adrenoceptor binding in depressed patients pre-treatment. Receptor binding becomes normal post-treatment.

Interactions of heparin with alpha-2-adrenoceptors.

The interactions of the anticoagulant Heparin with the alpha-2-adrenoceptor in rat brain cortex membranes were investigated. Binding experiments with 3H-Clonidine were performed in both the absence and presence of Heparin. 1 uM Na-Heparin caused a significant decrease in the maximal number of binding sites (Bmax) from 129.4 fmol/mg protein to 93.7 fmol/mg protein with an associated decrease in affinity (KD = 0.79 pM vs. KD = 1.53 pM) of these binding sites. Addition of Na+-Heparin to 3H-Clonidine (3.1 nM) labelled membranes inhibited 50% of specific 3H-Clonidine binding (IC50) at a concentration of 0.95 uM. Based on our findings we conclude that the simultaneous long term administration of Na-Heparin and the antihypertensive agonist Clonidine should be regarded under consideration of the inhibitory effect of Na-Heparin to the alpha-2-adrenoceptors.