Rauwolscine Binding Research Articles

Anoxia at Birth Induces Behaviorally-Relevant Changes in alpha2-Noradrenergic Receptor Binding in the Adult Rat

Evidence suggests that Caesarean section birth in the rat, with or without an additional period of anoxia, results in long-term changes in brain catecholamine levels as well as reactivity to stress. The purpose of the present investigation was to determine whether Caesarean birth plus anoxia alters (alpha)2-noradrenergic receptor binding and sensitivity to the (alpha)2 receptor agonist, clonidine, in the Porsolt forced swim test. Sprague Dawley rat dams were decapitated and the uteruses were removed by Caesarean section. Pups were then delivered either immediately (Caesarean Only group), or were immersed in a saline bath for approximately 15 minutes (Caesarean plus Anoxia group) before delivery of the pups. A third group of animals born vaginally served as controls (Vaginally-born group). Four to five months postnatally, the expression of (alpha)2 receptors was measured by receptor autoradiography using [3H]-Rauwolscine binding. Receptor binding was increased in the area of the ventral hypothalamus and decreased in the CA1 region of the hippocampus in animals subjected to Caesarean plus Anoxia at birth. These animals also displayed a subsensitive response to the immobilizing effects of clonidine (100 micrograms/kg, i.p.) in the Porsolt forced swim test. Specifically these data show that Caesarean birth produces long-term changes in (alpha)2 receptor density and that, in animals subjected to Caesarean plus anoxia, these changes are reflected in a behavioral subsensitivity to the (alpha)2 agonist, clonidine. The findings reported here provide further experimental support for the hypothesis that birth complications may contribute to the pathophysiology of disorders such as schizophrenia that involve central catecholamine dysfunction.

Knockdown of α2C-adrenoceptors in the occipital cortex rescued long-term potentiation in hidden prenatally malnourished rats

Moderate reduction in the protein content of the mother’s diet calorically compensated by carbohydrates (the so-called “hidden” prenatal malnutrition) leads to increased neocortical expression of the α2C-adrenoceptor subtype, together with decreased cortical release of noradrenaline and impaired long-term potentiation (LTP) and visuospatial memory performance during the rat postnatal life. Ín order to study whether overexpression of the α2C-adrenoceptor subtype is causally related to the decreased indices of neocortical plasticity found in prenatally malnourished rats, we evaluated the effect of intracortical (occipital cortex) administration of an antisense oligodeoxynucleotide (ODN) raised against the α2C-adrenoceptor mRNA on the LTP elicited in vivo in the occipital cortex of hidden prenatally malnourished rats. In addition, we compare the effect of the antisense ODN to that produced by systemical administration of the subtype-nonselective α2-adrenoceptor antagonist atipamezole. Prenatal protein malnutrition led to impaired occipital cortex LTP together with increased expression of α2C-adrenoceptors (about twice Bmax) in the same cortical region. [3H]-rauwolscine binding assay showed that a 7-day intracortical antisense ODN treatment in the malnourished rats resulted in 50% knockdown of α2C-adrenoceptor expression and, in addition, completely rescued the ability of the occipital cortex to develop and maintain long-term potentiation. Atipamezole (0.3mg/kg i.p.) also led to full recovery of neocortical LTP in malnourished rats. The present results argue in favor of our original hypothesis that the deleterious effect of prenatal malnutrition on neocortical plasticity in the adult progeny is in part consequence of increased neocortical α2C-adrenoceptor expression. This receptor subtype is known to be involved in the presynaptic control of noradrenaline release from central neurons, a neurotransmitter that critically influences LTP and memory formation.

Trazodone effects on [3H]-paroxetine and α2-adrenoreceptors in platelets of patients with major depression

Trazodone is an antidepressant which behaves as a selective 5-HT(2) antagonist and 5-HT reuptake inhibitor. The lack of information on its effects in vivo prompted us to evaluate alpha(2)-adrenoceptors by means of the specific binding of [(3)H]-rauwolscine, and the 5-HT transporter (SERT) by means of the binding of [(3)H]-paroxetine ([(3)H]-Par), in platelets of depressed patients, before and after one month of treatment with trazodone (75-300 mg/day). Twenty-five outpatients of both sexes with a diagnosis of major depression, as assessed by the Structured Clinical Interview for DSM IV, were included in the study. Depressive symptoms were evaluated by means of the Hamilton Rating Scale for Depression: the total score (mean +/- SD) was 20 +/- 6 at baseline (t(0)) and 7 +/- 4 after one month of treatment (t(1)). Platelet membranes, [(3)H]- rauwolscine and [(3)H]-Par bindings were carried out according to standardized protocols. The results showed that the B(max) values of [(3)H]-Par were statistically lower at t(1) than at t(0) (733 +/- 30 vs 1471 +/- 99, P < 0.001), while the K(d) and the [(3)H]-rauwolscine binding parameters remained unchanged. The findings of this study suggest that in vivo trazodone modifies the number of the SERT proteins and that, perhaps, most of its antidepressant properties are related to this activity.

Role of α 2-adrenoceptors in enhancement of antinociceptive effect in diabetic mice

The present studies investigated behavioral and neurochemical aspects of the noradrenergic and serotonergic nervous systems in streptozotocin-induced diabetic mice. We previously reported that intrathecal (i.t.) injection of norepinephrine significantly potentiated antinociception in diabetic mice compared to that in non-diabetic mice, and that antinociception due to norepinephrine injection was completely abolished by pretreatment with yohimbine, an α 2-adrenoceptor antagonist. The present studies demonstrated that i.t. injection of clonidine also showed more-potent antinociceptive activity in diabetic mice than in non-diabetic mice, but that i.t. methoxamine injection did not affect diabetic or non-diabetic mice. The antinociceptive potency due to i.t. injection of 5-HT was significantly lower in diabetic than in non-diabetic mice. In a neurochemical study, we found that the density of [ 3H]-rauwolscine binding sites in spinal α 2-adrenoceptors was significantly higher in diabetic than in non-diabetic mice, but that the binding affinity was unchanged. Spinal norepinephrine turnover was determined by measuring the decline in tissue norepinephrine concentration at 3 h after injection of the tyrosine hydroxylase inhibitor α-methyl- p-tyrosine. The spinal norepinephrine concentration decreased to 43.7% from the baseline in non-diabetic mice, while it was 21.0% in diabetic mice. These results suggest that, based on the decrease of norepinephrine release in the spinal cord, up-regulation of spinal α 2-adrenoceptors caused the increase of antinociception due to i.t. injection of an α 2-adrenoceptor agonist in streptozotocin-induced diabetic mice, and it seemed that the stimulation of α 2-adrenoceptors potentiated the antinociceptive effect. Thus, the spinal noradrenergic systems play an important moderating role in diabetes-induced neuropathic pain.

Contribution of α 2-adrenoceptors to the mitogenic effect of catecholestrogen in human breast cancer MCF-7 cells

Catecholestrogens are estrogen metabolites formed by hydroxylation of 17β-estradiol and estrone at either the C-2 or C-4 position, rivaling the parent estrogens in concentration. The objective of the present work was to assess if their catechol group could make them induce proliferation of human breast cancer cells via α 2-adrenoceptors. In competition studies in human breast cancer MCF-7 cells, high concentrations of 2-hydroxy-estradiol (2-OH-E 2), 2-hydroxy-estrone (2-OH-E 1) and 4-hydroxy-estrone (4-OH-E 1) competed for [ 3H]-rauwolscine binding, whereas 4-hydroxy-estradiol (4-OH-E 2) did not. The contribution of α 2-adrenoceptors and estrogen receptors (ERs) in proliferation enhancement was analyzed with specific antagonists. The specific α 2-adrenergic antagonist yohimbine partially reversed the effect of catecholestrogens except 4-OH-E 2. The selective ER downregulator ICI-182780 or fulvestrant partially or totally reversed the effect of all hydroxylated catecholestrogens. When analyzing the effect of the combination of both antagonists in MCF-7, the contribution of the α 2-adrenoceptors and ERs for 2-OH-E 2, 2-OH-E 1 and 4-OH-E 1 was mixed, whereas for 4-OH-E 2, the only receptor implied was an ER. In MDA-MB-231 cells (ER-α negative) the proliferation stimulation by these three catecholestrogens and reversal by the adrenergic antagonist was also observed. It can be concluded that α 2-adrenoceptors contribute at least in part to the mitogenic effect of 2-OH-E 2, 2-OH-E 1 and 4-OH-E 1.

Chronic hypobaric hypoxia induces tolerance to acute hypoxia and up-regulation in alpha-2 adrenoceptor in rat locus coeruleus

Hypoxia preconditioning has been shown to produce tolerance against brain injuries. The hypothesis of this study is that chronic hypobaric hypoxia may also induce acute hypoxia tolerance. We used intracellular recording in slices from rats exposed to chronic hypobaric hypoxia (exposed) and control to investigate the effects of chronic hypobaric hypoxia on the physiology of locus coeruleus (LC) including neuronal excitability. The results showed 35.7% reduced spontaneous firing rate and no change for membrane potential and input resistance in exposed neurons. In response to the alpha-2 adrenoceptor (A2R) agonist clonidine, both the hyperpolarizing potency and efficacy were increased indicated by a decreased EC50 (control: 30.9 nM and exposed: 19.7 nM) and a 50.5% increase in maximum hyperpolarized potential, respectively. A2R binding sites were also increased 21% in exposed neurons measured by radioligand [3H]rauwolscine binding assay. When treated with acute N2-hypoxia, the cell survival time (ST) was longer in exposed neurons, suggesting that a tolerance was induced. In addition, the ST for both groups of LC neurons was decreased by the A2R antagonist yohimbine and increased by the glutamate receptor antagonist kynurenic acid but not by MK-801; the decreased percentage of ST by yohimbine was larger and the increased percentage by kynurenic acid was smaller in exposed neurons. The results suggested that up-regulation of A2R and altered non-NMDA glutamate receptor function induced by chronic hypobaric hypoxia may underlie, in part, the decreased LC neuronal excitability and acute hypoxia tolerance.

Human breast cell lines exhibit functional α2-adrenoceptors

Adrenergic compounds (epinephrine and norepinephrine) are the most important hormones released during stress. Several different receptors are associated with their action in different tissues. However, alpha(2)-adrenoceptors have not yet been described in either normal or tumour human breast tissue. The aim of this work was to describe and characterize these receptors in several tumour and non-tumour human cell lines. The expression of alpha(2)-adrenoceptors was analyzed at the RNA (RT-PCR) and protein ([(3)H]-rauwolscine binding and immunocytochemistry) levels in different human breast cell lines, and the biological activity assessed by [(3)H]-thymidine incorporation. The cancer IBH-6, IBH-7 and MCF-7 and the non-tumour HBL-100 cells line, expressed both alpha(2B)- and alpha(2C)-adrenoceptor-subtypes. A single subtype was expressed in malignant HS-578T (alpha(2A)) and MDA-MB-231 and non-tumour MCF-10A cells (alpha(2B)). All cell lines exhibited significant binding for the specific antagonist [(3)H]-rauwolscine. The alpha-, alpha(2)-, and the alpha(1)-compounds with known affinity for alpha(2)-adrenoceptors, including epinephrine, norepinephrine, yohimbine, clonidine, rauwolscine and prazosin, competed significantly with binding in MCF-7 cells. In addition, IBH-6, IBH-7 and MCF-7 cells showed significant staining with specific antibodies against alpha(2B)- and alpha(2C)-adrenoceptor-subtypes, when tested by immunocytochemistry. In all cell lines, the specific agonist clonidine or oxymetazoline stimulated [(3)H]-thymidine incorporation. EC(50) values were in the range of 20-50 fM for IBH-6, IBH-7, and HS-578T; 0.14 pM for MCF-7; 2-82 pM for HBL-100 and MCF-10A cells, and a biphasic behaviour with a maximum value at 38.0 pM, was observed for MDA-MB-231 cells. The specific alpha(2)-adrenergic antagonist rauwolscine always reversed this stimulation at 0.1 nM. In conclusion, this study describes for the first time, the presence of alpha(2)-adrenoceptors in human epithelial breast cell lines. Moreover, activation of these receptors was associated with an enhancement of cell proliferation.

Spinal nerve ligation increases α 2-adrenergic receptor G-protein coupling in the spinal cord

Intrathecal and epidural administration of the α 2-adrenergic receptor agonist clonidine in humans results in analgesia to both acute nociceptive and chronic neuropathic pain. The potency of clonidine increases with hypersensitivity to mechanical stimuli after nerve injury, although the reasons for this change are unknown. In the present study, we tested the hypothesis that peripheral nerve injury alters either spinal α 2-adrenergic receptor-mediated G-protein activity or α 2-adrenergic receptor number. Rats were randomized to left spinal nerve ligation (SNL) or sham surgery. Tactile hypersensitivity in the hindpaw was confirmed and lumbar spinal cords were removed for binding assays. To examine agonist-induced G-protein coupling, [ 35S]GTPγS binding experiments were performed in spinal cord membranes and sections using norepinephrine as an α 2-adrenergic agonist. SNL was associated with an increase in maximal efficacy, but not potency, of norepinephrine-stimulated [ 35S]GTPγS binding in dorsal horn. SNL had no effect on basal [ 35S]GTPγS binding or on muscarinic cholinergic-stimulated [ 35S]GTPγS binding. [ 35S]GTPγS autoradiography showed that this increase in α 2-adrenergic-activated G-proteins occurred both ipsilateral and contralateral to SNL surgery. SNL did not alter total α 2-adrenergic receptor number or affinity to [ 3H]-rauwolscine binding, and displacement studies with the α 2A-adrenergic antagonist BRL44408 revealed that most of the binding was associated with the α 2A-adrenergic subtype. These data suggest that the increased potency of clonidine in neuropathic pain could reflect increased efficiency of G-protein coupling from spinal α 2-adrenergic receptors.

Post-pubertal adrenergic changes in rats with neonatal lesions of the ventral hippocampus

Lesions of the ventral hippocampus (VH) in neonatal rats result in post-pubertal alterations in a number of cognitive, social and motor behaviors that bear some analogy to schizophrenia. Increased sensitivity to stress and psychostimulants and prefrontal functional changes in the lesioned animals suggest an involvement of the mesocorticolimbic dopamine (DA) system. DA and norepinephrine (NE) interact in a number of ways in the medial prefrontal cortex (mPFC) to influence each other’s functions. In order to assess the role of adrenergic system in the behavioral responses of neonatal VH (nVH) lesioned animals, we first examined cortical and subcortical bindings of alpha-1 and alpha-2 adrenergic receptors using [ 3H]-prazosin and [ 3H]-rauwolscine respectively, and the norepinephrine transporter (NET) using [ 3H]-nisoxetine. Sprague-Dawley rat pups, at post-natal day (PD) 7, received bilateral injections of ibotenic acid in the VH and were sacrificed pre (PD35)- and post (PD56)-pubertally. A significant increase in [ 3H]-prazosin binding was observed in the frontal and cingulate cortices of lesioned rats at PD56 without any significant change in the caudate putamen or nucleus accumbens. No significant difference was seen in [ 3H]-rauwolscine binding. A significant upregulation of NET binding was observed in subregions of the PFC and nucleus accumbens of PD56 lesioned rats. The functional relevance of changes in adrenergic markers on amphetamine-induced locomotor activity was examined by pre-treatment of PD56 rats with prazosin, an alpha-1 receptor antagonist. Prazosin at doses of 1.0 or 2.0 mg/kg ip significantly reduced amphetamine-induced locomotion in sham but not in PD56 lesioned animals. Taken together, these results suggest that alterations in prefrontal alpha-1 receptors likely contribute to altered behavioral responses observed in post-pubertal VH lesioned rats.

Regional distribution of α 2C-adrenoceptors in brain and spinal cord of control mice and transgenic mice overexpressing the α 2C-subtype: an autoradiographic study with [ 3H]RX821002 and [ 3H]rauwolscine

Behavioral studies on gene-manipulated mice have started to elucidate the neurobiological functions of the α 2C-adrenoceptor (AR) subtype. In this study, we applied quantitative receptor autoradiography to investigate the potential anatomical correlates of the observed functional effects of altered α 2C-AR expression. Labeling of brain and spinal cord sections with the subtype non-selective α 2-AR radioligand [ 3H]RX821002 and the α 2C-AR-preferring ligand [ 3H]rauwolscine revealed distinct binding-site distribution patterns. In control mice, [ 3H]rauwolscine binding was most abundant in the olfactory tubercle, accumbens and caudate putamen nuclei, and in the CA1 field of the hippocampus. A mouse strain with overexpression of α 2C-AR regulated by a gene-specific promoter showed approximately two- to four-fold increased levels of [ 3H]rauwolscine binding in these regions. In addition, dramatic increases in [ 3H]rauwolscine binding were seen in the nerve layer of the olfactory bulb, the molecular layer of the cerebellum, and the ventricular system of α 2C-AR-overexpressing mice, representing “ectopic” α 2C-AR expression. Competition-binding experiments with several α 2-AR ligands confirmed the α 2C-AR identity of these sites. Our results provide quantitative evidence of the predominance of the α 2A-AR subtype in most regions of the mouse CNS, but also disclose the wide distribution of α 2C-AR in the normal mouse brain, although at relatively low density, except in the ventral and dorsal striatum and the hippocampal CA1 area. α 2C-AR are thus present in brain regions involved in the processing of sensory information and in the control of motor and emotion-related activities such as the accumbens and caudate putamen nuclei, the olfactory tubercle, the lateral septum, the hippocampus, the amygdala, and the frontal and somatosensory cortices. The current results may help in specifying an anatomical framework for the functional roles of the α 2A- and α 2C-AR subtypes in the mouse CNS.

Correlation between Platelet α2-Adrenoreceptors and Symptom Severity in Major Depression

Background: Abnormalities in different parameters of the norepinephrine system have been widely described in major depression. The presence of α₂-adrenoreceptors in blood platelets, similar to those in the brain, prompted us to evaluate them in depressed patients, as compared with healthy controls. Methods: Fifteen outpatients affected by major depression, according to DSM IV criteria, and 15 comparable healthy control subjects, were included in the study. The α₂-adrenoreceptors were measured by means of the specific binding of [³H]rauwolscine, a highly selective antagonist for this receptor subtype. The severity of depression was assessed by means of the Hamilton Rating Scale for Depression (HRSD). Results: The results did not show any difference in [³H]rauwolscine binding parameters (B_max and K_d) between patients and controls. However, in the patients, a significant and positive correlation between B_max, which measures the density of the receptors, and HRSD total score was detected. Conclusions: Therefore, although no change in α₂-adrenoreceptors seems to occur in major depression, the density of these receptors would seem to be related to the severity of depressive symptoms.

Food Deprivation Increases α2-Adrenoceptor–Mediated Modulation of Jejunal Epithelial Transport in Young and Adult Rats

This study examined the effect of food deprivation on the jejunal response to alpha(2)-adrenoceptor activation in young (20-d-old) and adult (60-d-old) rats, using short-circuit (I(sc)) measurements in the absence or presence of furosemide (1 mmol/L). The effect of alpha(2)-adrenoceptor stimulation by 5-bromo-N:-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 14,304; 0.3-3000 nmol/L) was a concentration-dependent decrease in I(sc) with similar half-maximal effective concentration (EC(50); 12.3 +/- 1.1 vs. 9.6 +/- 1.1 nmol/L) and maximal effect (E(max); 70.6 +/- 6.9 vs. 80.6 +/- 4.5% of reduction) values in adult food-deprived and fed rats. The effect of UK 14,304 on I(sc) in fed and food-deprived rats was markedly (P: < 0.05) attenuated by furosemide (1 mmol/L). E(max) values for UK 14,304 in 20-d-old food-deprived rats were higher (P: < 0.05) than those observed in fed rats (93.3 +/- 3.3 vs. 67.0 +/- 11.3% of reduction), without differences in EC(50) values. The effect of UK 14,304 on I(sc) in 20-d-old fed rats was completely abolished by furosemide (1 mmol/L). In food-deprived young rats, the effect of UK 14,304 was also markedly (P: < 0.05) antagonized by furosemide, but not completely abolished. Specific [(3)H]-rauwolscine binding in membranes from jejunal epithelial cells revealed the presence of a single class of binding sites, with an apparent K:(D) in the low nmol/L range. In 20-d-old food-deprived rats, specific [(3)H]-rauwolscine binding was markedly increased, and this was reversed by refeeding. Na(+),K(+)-ATPase activity in isolated jejunal epithelial cells from 60-d-old fed rats was twice that in 20-d-old fed rats [117 +/- 14 vs. 52 +/- 5 nmol free inorganic phosphorus/(mg protein.min)]. Food deprivation in adult rats, but not in 20-d-old rats, was accompanied by a significant decrease in Na(+),K(+)-ATPase activity. In both young and adult rats (fed and food-deprived), UK 14,304 did not affect Na(+),K(+)-ATPase activity. In conclusion, food deprivation in 20-d-old rats enhanced the response to alpha(2)-adrenoceptor stimulation. This effect, which depends primarily on the stimulation of a furosemide-sensitive antisecretory mechanism, is suggested to result from increases in the number of jejunal epithelial alpha(2)-adrenoceptors.

Pharmacological characterization of (10bS)-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) as a new alpha2-adrenoceptor antagonist.

Alpha2-adrenoceptor antagonists, which can enhance synaptic norepinephrine levels by blocking feedback inhibition processes, are potentially useful in the treatment of disease states such as depression, memory impairment, impotence and sexual dysfunction. (10bS)-1,2,3,5,6,10b-Hexahydropyrrolo[2,1-a]isoquinoline oxalate (YSL-3S) was evaluated in several in vitro biological tests to establish its pharmacological profile of activities as an alpha2-adrenoceptor antagonist. Saturation binding assay revealed that [3H]rauwolscine bound to the alpha2-adrenoceptors with a Kd value of 6.3+/-0.5 nM and a Bmax value of 251+/-39 fmol/mg protein in rat cortical synaptic membranes. Competitive binding assay showed that YSL-3S inhibited the binding of [3H]rauwolscine (1 nM) in a concentration-dependent manner with a Ki value of 98.2+/-12.1 nM while it did not inhibit the binding of [3H]cytisine (1.25 nM) to neuronal nicotinic cholinergic receptors. The Ki values of yohimbine, clonidine and norepinephrine for [3H]rauwolscine binding were 15.8+/-1.0, 40.1+/-5.9 and 40.0+/-11.5 nM, respectively. In addition, the binding affinity of YSL-3S for alpha2-adrenoceptors was higher than that of its antipode and the racemic mixture. The functional activity of YSL-3S at the presynaptic alpha2-adrenoceptors was assessed using the prostatic portion of the rat vas deferens. Clonidine inhibited field-stimulated contractions of the vas deference in a dose-dependent manner. The presence of YSL-3S or yohimbine caused a parallel, rightward the dose-response curve of clonidine in a dose-dependent manner, indicating an antagonistic action at the presynaptic alpha2-adrenoceptors. The pA2 values of yohimbine and YSL-3S were 7.66+/-0.13 and 6.64+/-0.18, respectively. The results indicate that YSL-3S acts as a competitive antagonist at presynaptic alpha2-adrenoceptors with a potency approximately ten times lower than yohimbine, but is devoid of binding affinity for neuronal nicotinic cholinergic receptors.

Peripheral markers of serotonergic and noradrenergic function in post-pubertal, caucasian males with autistic disorder.

Some studies have suggested that disorders in the peripheral and central metabolism of serotonin (5-HT) and noradrenaline may play a role in the pathophysiology of autistic disorder. This study examines serotonergic and noradrenergic markers in a study group of 13 male, post-pubertal, caucasian autistic patients (age 12-18 y; I.Q. > 55) and 13 matched volunteers. [3H]-paroxetine binding Kd values were significantly higher in patients with autism than in healthy volunteers. Plasma concentrations of tryptophan, the precursor of 5-HT, were significantly lower in autistic patients than in healthy volunteers. There were no significant differences between autistic and normal children in the serum concentrations of 5-HT, or the 24-hr urinary excretion of 5-hydroxy-indoleacetic acid (5-HIAA), adrenaline, noradrenaline, and dopamine. There were no significant differences in [3H]-rauwolscine binding Bmax or Kd values, or in the serum concentrations of tyrosine, the precursor of noradrenaline, between both study groups. There were highly significant positive correlations between age and 24-hr urinary excretion of 5-HIAA and serum tryptophan. The results suggest that: 1) serotonergic disturbances, such as defects in the 5-HT transporter system and lowered plasma tryptophan, may play a role in the pathophysiology of autism; 2) autism is not associated with alterations in the noradrenergic system; and 3) the metabolism of serotonin in humans undergoes significant changes between the ages of 12 and 18 years.

Synthesis and alpha-adrenergic binding ligand affinities of 2-iminoimidazolidine derivatives.

In order to obtain possible veinotonic drugs acting through alpha2 receptor activation, we prepared clonidine analogues in which the 2-imino-imidazolidine was attached to various aliphatic or aromatic heterocycles. Among them, the two benzopyranic derivatives 16 and 22 exhibited interesting affinities (19 and 95 nM respectively on [3H]rauwolscine binding, compared to 35 nM for clonidine). Their affinity for alpha1 receptors was found to be much lower: 7570 and 5030 nM for 16 and 22 respectively, suggesting 16 to be 400 times more selective for alpha2 than for alpha1-adrenoceptors.

Changes in Platelet Alpha-2-Adrenoceptors in Fibromyalgia: Effects of Treatment with Antidepressants

The aim of this study was to determine platelet α₂-adrenergic receptor (α₂-AR) binding sites in fibromyalgia both before and after treatment with sertraline or placebo. The maximum number of binding sites (B_max) and their affinity (K_d) for [³H]rauwolscine, a selective α₂-AR antagonist, were measured in 13 normal volunteers and 22 fibromyalgia patients. Severity of illness was evaluated by means of the Hamilton Depression Rating Scale (HDRS) and dolorimetric assessments of tenderness at tender points. Fibromyalgia patients had repeated measurements of [³H]rauwolscine binding characteristics both before and after subchronic treatment with sertraline or placebo for 12 weeks. [³H]rauwolscine binding K_d values were significantly higher in fibromyalgia patients than in normal volunteers. There were significant inverse correlations between [³H]rauwolscine binding K_d values and duration of illness, age and lower energy. Significantly higher [³H]rauwolscine binding K_d values were found in fibromyalgia patients in an early phase of illness (<3 years) than in fibromyalgia patients with a protracted illness (>3 years). Repeated administration of sertraline had no significant effects on [³H]rauwolscine binding B_max or K_d values. The results suggest that fibromyalgia and, in particular, fibromyalgia in an early phase of illness, is accompanied by lowered affinity of platelet α₂-ARs.

Decreased platelet alpha-2 adrenoceptor density in major depression: effects of tricyclic antidepressants and fluoxetine

Background: It has been suggested that major depression is accompanied by a subsensitivity of central α2-adrenoceptors (α2-ARs) and, consequently, by an impaired negative feedback on the presynaptic catecholaminergic neuron, which, in turn, may induce a disinhibition of noradrenergic output and norepinephrine release in response to any activation. Methods: The maximum number of platelet binding sites (Bmax) and their affinity for [ 3H]-rauwolscine, a selective α2-AR antagonist, were measured in unmedicated and medicated major depressed patients and in normal volunteers. Specific binding was defined as that inhibited by idazoxan, another α2-AR antagonist. Results: Unmedicated major depressed patients had significantly decreased platelet [ 3H]-rauwolscine binding Bmax values compared to normal volunteers. [ 3H]-rauwolscine binding K d values did not differ significantly between unmedicated major depressed patients and normal controls. [ 3H]-rauwolscine binding K d values were significantly higher in depressed patients treated with tricyclic antidepressants than in unmedicated patients. Subchronic treatment with fluoxetine did not significantly alter either [ 3H]-rauwolscine binding Bmax or K d values. [ 3H]-rauwolscine binding Bmax values were significantly greater in men than in women. Conclusions: The results suggest that i) major depression is accompanied by decreased platelet α2-AR density; and that ii) subchronic treatment with tricyclic antidepressants, but not fluoxetine, results in a decreased affinity of rauwolscine for platelet α2-ARs.

Serotonergic and Noradrenergic Markers of Post-Traumatic Stress Disorder with and without Major Depression

Some studies have suggested that disorders in the peripheral and central metabolism of serotonin (5-HT) and noradrenaline (NE) may play roles in the pathophysiology of post-traumatic stress disorder (PTSD). This study examines (1) the availability of plasma total tryptophan, the precursor of 5-HT, and tyrosine, the precursor of NE; and (2) the platelet 5-HT transporter and α2-adrenoceptor (α2-AR) binding sites in patients with PTSD and healthy volunteers. High-performance liquid chromatography (HPLC) was employed to measure plasma tryptophan and tyrosine as well as amino acids known to compete with the same cerebral transport system; that is, valine, leucine, phenylalanine, and isoleucine. The maximum number of binding sites (Bmax) and their affinity (Kd) for binding to [3H]-paroxetine and [3H]-rauwolscine, a selective α2-AR antagonist, were determined. [3H]-paroxetine and [3H]-rauwolscine binding Kd values were significantly higher in patients with PTSD than in healthy volunteers. [3H]-rauwolscine binding Kd values were significantly higher in patients with PTSD and concurrent major depression (MD) than in PTSD patients without MD and healthy volunteers. Plasma tyrosine concentrations and the ratio of tyrosine/valine + leucine + isoleucine + phenylalanine + tryptophan were significantly higher in PTSD patients with MD than in those without MD and healthy volunteers. The results show that PTSD is accompanied by lower affinity of paroxetine binding sites and that PTSD with concurrent MD is accompanied by lower affinity of α2-ARs and increased plasma tyrosine availability to the brain. The results suggest that (1) serotonergic mechanisms, such as defects in the 5-HT transporter system, may play a role in the pathophysiology of PTSD; and (2) that catecholaminergic mechanisms, such as increased precursor availability and lowered affinity of α2-ARs, may play a role in the pathophysiology of PTSD with concurrent MD.

Passive avoidance learning involves alpha2-noradrenergic receptors in a day old chick.

Day-old chicks were trained on one-trial passive avoidance task, using methyl-anthranilate (MeA) as an aversive substance. Bilateral pharmacological manipulation of the intermediate hyperstriatum ventrale was performed by intracerebral application of an alpha2-noradrenergic agonist, clonidine (5 microM), or an antagonist, rauwolscine (300 microM). Only rauwolscine application (pre- or post-training) induced significant memory impairment. Quantitative receptor autoradiography was used to determine the kinetic properties of the binding sites for [3H]clonidine or [3H]rauwolscine in MeA-trained or water-trained (control) chicks, in forebrain areas known to be involved in avoidance learning. Scatchard analysis revealed that MeA-training resulted in a significant bilateral upregulation in the number of [3H]rauwolscine binding sites (Bmax) in the area of hyperstriatum ventrale. These findings suggest the importance of activation of alpha2-noradrenergic receptors in aversive learning in chicks.

The expression of functional postsynaptic alpha2-adrenoceptors in the corpus cavernosum smooth muscle.

1. The purpose of this study was to determine if corpus cavernosum smooth muscle expresses functional postsynaptic alpha2-adrenoceptors (AR). 2. The alpha2-adrenoceptor agonist UK 14,304 elicited concentration-dependent contractions in rabbit corpus cavernosum smooth muscle (CCSM). The half-maximal response occurred at 0.32+/-0.03 microM and the maximum contraction at 10 microM UK 14,304. 3. Pretreatment of CCSM strips with selective alpha2-adrenoceptor antagonists, rauwolscine and RS-15385, produced rightward shifts in the dose-response curves to UK 14,304 (pA2 values 7.1 and 8.5, respectively). In contrast, these antagonists did not alter contraction induced by the alpha1-adrenoceptor agonist phenylephrine (PE) or oxymetazoline. UK 14,304-induced contractions were also inhibited by prazosin (pA2 = 9.08). 4. UK 14,304-induced contractions, unlike those to PE, were highly dependent on the presence of extracellular Ca2+. 5. [3H]-rauwolscine bound to CCSM membranes with high affinity (Kd = 1.5 nM). [3H]-rauwolscine binding was displaced by unlabelled rauwolscine, RS-15385, UK 14,304 and prazosin, but not by PE. 6. UK 14,304 inhibited forskolin and prostaglandin E1 (PGE1)-induced increases in intracellular cyclic AMP concentration in primary cultures of rabbit CCSM cells. 7. These results demonstrate that CCSM expresses Gi-coupled postsynaptic alpha2-adrenoceptors, and activation of these receptors causes contraction of trabecular smooth muscle.