Evaluation of peripheral serotonin content and α2-adrenergic receptor function as potential markers for life-long recurrent depressive disorder by using methodological improvements.

OBJECTIVE: We aimed to evaluate the role and the relations between peripheral platelet serotonin content, blood plasma serotonin concentration and the function of platelet α2-adrenergic receptors (α2-AR) as potential state or trait biomarkers for recurrent depressive disorder (RDD). METHODS: 26 drug-free patients with life-long RDD and 31 healthy controls were included in the study. Several methodological improvements in blood collection and platelet isolation were implemented following the present standards in Haematology and Light transmission aggregometry. RESULTS: Our results have shown lower platelet serotonin content, higher plasma serotonin concentration and desensitization of platelet α2-AR in patients with RDD. The variables were found heterogeneous and mainly influenced by the clinical characteristics of the current episode. High amplitude of the α2-AR correlated with severe anxious symptoms and high platelet serotonin content (as well as low plasma serotonin levels) were associated with psychotic symptoms. CONCLUSIONS: The evaluated peripheral markers reflect only state (but not trait) abnormalities in patients with current severe episode of RDD. The observed peripheral α2-AR and serotonin abnormalities are mutually not related and they are probably triggered by different mechanisms.

Influence of adrenal hormones in the occurrence and prevention of stress ulcers.

PURPOSE: The aim of the study was to examine whether endogenous cortisol and adrenalin have a role in the formation of stress ulcers in intact and adrenalectomized rats. METHODS: The study was composed of 4 experiments: ulcerated areas in stomachs of adrenalectomized and intact rats were measured, adrenaline (100 µg/kg) and prednisolone (5 mg/kg) were injected intraperitoneally in adrenalectomized rats, metyrapone (200 mg/kg) and metyrosine (200 mg/kg) were administered to intact rats, and metyrapone (200 mg/kg) and metyrosine (200 mg/kg) were administered orally with yohimbine (10 mg/kg) and yohimbine (10 mg/kg) alone were administered to intact rats. After 24-hour restraint stress, ulcerated areas were measured. RESULTS: In the stomach of intact rats, the degree of stress ulcer was 7.25 times more severe than that noted in adrenalectomized rats. Furthermore, stress ulcers in adrenalectomized rats that received adrenaline or prednisolone only were fewer and less severe than rats receiving both adrenaline and prednisolone. CONCLUSIONS: Simultaneous administration of adrenaline and prednisolone did not prevent the formation of stress ulcers. However, either of these hormones alone (adrenaline or prednisolone), in the absence of the other, repressed the formation of stress ulcers. This antiulcer activity may be related to α2-adrenergic receptor activity.

Polymorphisms of adrenoceptors are not associated with an increased risk of adverse event in heart failure: a MERIT-HF substudy.

BACKGROUND: Enhanced sympathetic activation has a central role in the development of heart failure (HF). We assessed whether the alpha(2C)-adrenoceptor (Del322-325) polymorphism exclusively or in combination with a beta(1)-adrenoceptor (Arg389) polymorphism, each with known independent effects on sympathetic function, were associated with an increased risk of adverse events in HF. METHODS AND RESULTS: A total of 526 patients enrolled in the Metoprolol CR/XL Randomized Intervention Trial in Congestive Heart Failure study were genotyped for both adrenoceptor polymorphisms. The distribution of alpha(2C) genotypes was similar between the event and nonevent groups. However, a reduced prevalence of the Del322-325 allele was found in individuals with ischemic congestive HF (P=.022). Patients possessing both the alpha(2C) Del322-325 and beta(1) Arg389 alleles had no increased risk of events. Adjusting for confounding variables and the beta(1) Arg389Gly polymorphism, the odds ratio of being ins/del + del/del for the alpha(2C) Del322-325 and having an event was 0.89 with 95% CI 0.49-1.63, P=.715. Similarly, adjusting for confounding variables and the alpha(2C) Del322-325 polymorphism the odds ratio of being Arg/Arg or Arg/Gly for the beta(1) Arg389Gly polymorphism and having an event was 1.13 with 95% CI 0.52-2.17, P=.864. CONCLUSIONS: The alpha(2C) Del322-325 polymorphism exclusively or in combination with the beta(1)Arg389 allele is not associated with an increased risk of adverse events in HF.

Analysis of platelet alpha2-adrenergic receptor activity in stable coronary artery disease patients on dual antiplatelet therapy.

Combined antiplatelet therapy reduces recurrent atherothrombotic events in stable coronary disease patients; however, high residual platelet reactivity measured ex vivo still raises concerns as a condition related to treatment failure. Alpha-2 adrenoceptor enhances platelet reactivity and might contribute to this phenomenon. For the present study, 121 stable angina patients on standard dual antiplatelet therapy (75 mg clopidogrel and 100 mg acetylsalicylic acid) were recruited. Born aggregometry was performed with adenosine diphosphate (ADP), collagen and epinephrine. To verify platelet adrenergic activity, potentiation by low-dose epinephrine and inhibition by selective alpha-2 receptor blocker atipamezole were determined. To assess the P2Y(12)-specific residual activity, cangrelor was used. Plasma norepinephrine, soluble CD40-ligand, high-sensitivity-C-reactive protein (hsCRP) - and in 24 subjects platelet P-selectin positivity were measured. Epinephrine - at very low concentration (10(-9)g/ml) - significantly potentiates (1.25 microM ADP: 26.5% vs. 43%; 5 microM ADP: 53% vs. 64.5%; collagen: 17% vs 42%, p < 0.001) while atipamezole inhibits ADP- and collagen-induced platelet aggregations (1.25 microM ADP: 26.5% vs. 23%; 5 microM ADP: 53% vs. 47%; collagen: 17% vs. 11%, p < 0.001). Patients with high adrenergic activity have significantly increased baseline ADP- and collagen-induced platelet aggregation. Based on cangrelor's efficacy, these patients have significantly more residual P2Y(12) activity as well. HsCRP and soluble CD40-ligand levels were similar. In conclusion, stable coronary heart disease patients with prominent adrenoceptor activity in vitro have significantly increased platelet aggregability and more functional P2Y(12) receptor, indicating poor inhibitory response to thienopyridines. Therefore, platelet adrenergic receptor represents a considerable, dynamic factor of high residual platelet reactivity and might contribute to cardiovascular events indicating failure of antiplatelet therapy.

ADRA2A polymorphism and smoking in a Turkish population.

Adrenoceptors (ARs) consist of nine subtypes, which are involved in a wide spectrum of physiological functions and are the site of action for a considerable percentage of currently prescribed therapeutics. All AR subtypes (except alpha(1D)) can be polymorphic because of the genetic variations in the coding and non-coding regions. Sixteen sequence variations were identified in alpha-adrenergic 2A (ADRA2A) gene. Among them, ADRA2A C1291G polymorphism is one of the most important polymorphisms, which plays a major role in regulating neurotransmitter release, blood pressure, lipolysis, insulin secretion, and platelet aggregation. A C-G transversion results in an MspI restriction fragment length polymorphism located at 1291 bp upstream of the origin of transcription. Because Medline search showed no study showing the allelic frequencies, and no information is available on inter-individual variability of ADRA2A C1291G polymorphism in Turkish population, we genotyped 203 healthy Turkish subjects. Because of large genetic variation of the polymorphism, we aimed to find out the distribution of C1291G polymorphism in Turkish population. Furthermore, we evaluated the possible association between the C1291G polymorphism in the ADRA2A receptor gene and smoking. The frequencies for the 1291C and 1291G alleles were 64% and 36%, respectively. The genotype frequencies for C1291C, C1291G, and G1291G were 35.5%, 57.6%, and 6.9%, respectively, in Turkish population. The allelic frequencies (1291C and 1291G) and G1291G homozygous variant genotype were similar to those reported in different Caucasian populations; however, C1291C and C1291G genotypes were different. We also observed that the frequency of the G allele was slightly higher in smoker subjects and lower among controls. The ADRA2A G allele may play a role in the predisposition to smoking. There is a need for expanding genotype and haplotype studies because of its importance in various physiological disorders and to confirm the association of this polymorphism with smoking.

Platelet alpha2-adrenoreceptors in obsessive-compulsive disorder.

The need for new therapeutic targets in obsessive-compulsive disorder (OCD) prompted us to investigate the putative involvement of the norepinephrine system by means of platelet alpha(2)-adrenoreceptors in a group of 20 OCD patients and healthy control subjects, matched for sex and age. Platelet membranes were prepared according to standard protocols, and the alpha(2)-adrenoreceptors were measured by means of the specific binding of [(3)H]rauwolscine, a highly selective antagonist for this receptor subtype. The results, which showed no difference between patients and controls in the binding parameters of [(3)H]rauwolscine, suggest that the role of alpha(2)-adrenoreceptors, as reflected by the platelet model, is quite limited in OCD and may, perhaps, be restricted purely to some symptoms or dimensions such as motricity, as suggested by the higher density of alpha(2)-adrenoreceptors found in patients concomitantly affected by motor tics.

Correlation between platelet alpha(2)-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 alpha(2)-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 alpha(2)-adrenoreceptors were measured by means of the specific binding of [(3)H]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 [(3)H]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 alpha(2)-adrenoreceptors seems to occur in major depression, the density of these receptors would seem to be related to the severity of depressive symptoms.

Platelet alpha2-adrenoceptor alterations in patients with essential hypertension are normalized after treatment with doxazosin but not propranolol.

OBJECTIVE: Marked alterations have been demonstrated to occur in the platelet alpha2-adrenoceptors of patients with essential hypertension. The purpose of this study was to determine whether antihypertensive treatment with alpha-adrenergic blocker doxazosin or beta-adrenergic blocker propranolol can affect the affinity and the density of platelet alpha2-adrenoceptors in such patients. SUBJECTS AND METHODS: In two groups of 22 previously untreated, essential hypertensive patients, the mean affinity (Kd) and density (B(max)) of platelet alpha2-adrenoceptors were studied by [3H]-UK 14304 binding assays; the first assays were performed before any medication was begun, the second were performed after treatment for up to 13 weeks with doxazosin or propranolol. A third group of 22 healthy normotensive volunteers matched by age, sex and body mass index was used as control. RESULTS: Blood pressure did not differ significantly in the two hypertensive groups, and treatment with the two drugs resulted in closely similar, normal blood pressure levels. Kd and B(max) values were significantly higher in the two hypertensive groups than in controls. After treatment with propranolol the binding parameters did not change significantly, whereas after treatment with doxazosin Kd and B(max) returned to normotensive values. CONCLUSIONS: In previously untreated, essential hypertensive patients platelet alpha2-adrenoceptors have a lower affinity but a higher density than in normotensive subjects. Despite similar effects on blood pressure, the treatment with the alpha-adrenergic blocker doxazosin is followed by restoration of normal findings in the binding assays of platelet alpha2-adrenoceptors whereas the treatment with the beta-adrenergic blocker propranolol does not alter the Kd and B(max) values.

Changes in platelet alpha-2-adrenoceptors in fibromyalgia: effects of treatment with antidepressants.

The aim of this study was to determine platelet alpha(2)-adrenergic receptor (alpha(2)-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 [(3)H]rauwolscine, a selective alpha(2)-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 [(3)H]rauwolscine binding characteristics both before and after subchronic treatment with sertraline or placebo for 12 weeks. [(3)H]rauwolscine binding K(d) values were significantly higher in fibromyalgia patients than in normal volunteers. There were significant inverse correlations between [(3)H]rauwolscine binding K(d) values and duration of illness, age and lower energy. Significantly higher [(3)H]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 [(3)H]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 alpha(2)-ARs.

Characteristics of norepinephrine and clonidine displacement of [3H]yohimbine binding to platelet alpha2-adrenoreceptors in healthy volunteers.

Clonidine's estimates of platelet alpha2-adrenoreceptor (alpha2AR) density are substantially lower than yohimbine's. This discrepancy could have contributed to inconsistent results from studies on the role of alpha2AR in depression. Furthermore, few studies have investigated the relative distribution of alpha2AR between the high- and low-affinity states or their Gi protein coupling. [3H]yohimbine saturable binding to platelet alpha2AR, its displacement by norepinephrine and clonidine, and the effects of Gpp(NH)p on agonist displacement curves were investigated in 11 healthy volunteers. Clonidine estimates of alpha2AR density were close to norepinephrine estimates, and both were strongly correlated. Clonidine's K(L)/K(H) ratio was lower than norepinephrine's, consistent with its partial agonist nature. Norepinephrine and clonidine displacement curves revealed two affinity states. Gpp(NH)p induced a significant rightward shift to a single low-affinity state. When used in combination with a specific antagonist, clonidine's estimates of alpha2AR density were similar to those of norepinephrine's, and both were higher than previously reported, when clonidine was used alone. Re-evaluation of previous studies on alpha2AR in depression using clonidine is needed. The combined use of antagonist-saturation and agonist-displacement experiments to examine possible dysregulation in alpha2AR coupling to Gi protein in psychiatric disorders is recommended.

Development of an HPLC method for determining the alpha 2-adrenergic receptor agonist brimonidine in blood serum and aqueous humor of the eye.

A procedure for determining brimonidine [5-bromo-6-(2-imidazolidinylideneamino) quinoxaline] in biological samples using a reversed-phase isocratic HPLC method is described. The application in blood serum and eye aqueous humor of patients treated with the Alphagan ophthalmic solution was carried out by enrichment of samples in brimonidine with solid-phase liquid extraction. Brimonidine reached maximum levels in aqueous humor and serum within 2-2.5 h, whereafter a declining pattern was obtained. An approximate 50% level of brimonidine was identified in serum at 12 h after ocular administration, whereas in aqueous humor this percentage was determined after a period of 4-5 h.

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 alpha 2-adrenoceptors (alpha 2-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 alpha 2-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 alpha 2-AR antagonist. RESULTS: Unmedicated major depressed patients had significantly decreased platelet [3H]-rauwolscine binding Bmax values compared to normal volunteers. [3H]-rauwolscine binding Kd values did not differ significantly between unmedicated major depressed patients and normal controls. [3H]-rauwolscine binding Kd 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 Kd 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 alpha 2-AR density; and that ii) subchronic treatment with tricyclic antidepressants, but not fluoxetine, results in a decreased affinity of rauwolscine for platelet alpha 2-ARs.

Platelet alpha2-adrenergic receptor coupling efficiency to Gi protein in subjects with post-traumatic stress disorder and normal controls.

Low platelet membrane alpha2-adrenergic receptor (alpha2AR) density and low basal and forskolin-stimulated cyclic adenosine monophosphate responses, which have been reported in post-traumatic stress disorder (PTSD), suggest either abnormal alpha2AR coupling to G(i) protein or dysregulation in post-receptor signal transduction mechanisms. alpha2AR density in the high- and low-conformational states, agonist affinity in both states and coupling to G(i) protein were investigated in 23 drug-free combat PTSD patients and 25 normal controls. alpha2AR coupling measures were not different between PTSD patients and controls. Total alpha2AR density was higher in PTSD patients than controls, due to a higher density of the receptor in the high-conformational state. There were no differences in agonist affinity to the receptor in either conformational state. Results rule out dysregulation in alpha2AR coupling to G(i) protein. Studies of post-receptor signal transduction mechanisms are warranted.

Adrenergic receptor function in panic disorder. I. Platelet alpha 2 receptors: Gi protein coupling, effects of imipramine, and relationship to treatment outcome.

Various studies suggest alpha 2-adrenergic receptor (alpha 2AR) dysregulation in panic disorder (PD). Platelet alpha 2-AR exist in high- and low-conformational states as a function of their coupling to Gi protein. alpha 2AR coupling is important in signal transduction and is modulated by antidepressants. alpha 2AR density in the high- and low-conformational states, agonist affinity, and coupling efficiency were investigated in 21 healthy controls, 21 drug-free PD patients, and eight imipramine-treated patients using norepinephrine displacement of 3H-yohimbine binding. Percentage of receptors in the high-conformational state (%RH) and the ratio of the agonist dissociation constant to the receptor in the low-/high-conformational state (KL/KH), calculated from displacement experiments, were used as coupling indices. Patients had high alpha 2AR density in both conformational states. %RH and KL/KH ratio were significantly different, particularly in patients with Hamilton scale for depression (HAMD) scores > or = 15. Imipramine treatment (29 weeks) had no effect on alpha 2AR density or coupling, despite improvement in anxiety ratings. High pretreatment alpha 2AR density and coupling predicted low severity of anxiety after treatment. Increased alpha 2AR density and abnormal coupling may represent an adaptive mechanism or trait marker in PD.

Platelet alpha2A-adrenoceptor function in major depression: Gi coupling, effects of imipramine and relationship to treatment outcome.

Studies suggest alpha2A-adrenoceptors (alpha(2A)AR) dysregulation in major depressive disorder (MDD). Platelet alpha(2A)ARs exist in high- and low-conformational states that are regulated by Gi protein. Although alpha(2A)AR coupling to Gi protein plays an important role in signal transduction and is modulated by antidepressants, it has not been previously investigated. Alpha2AR density in the high- and low-conformational states, agonist affinity and coupling efficiency were investigated in 27 healthy control subjects, 23 drug-free MDD patients and 16 patients after imipramine treatment using [3H]yohimbine saturation and norepinephrine displacement of [3H]yohimbine binding experiments. Coupling measures were derived from NE-displacement experiments. Patients had significantly higher alpha(2A)AR density, particularly in the high-conformational state, than control subjects. Coupling indices were normal in patients. High pre-treatment agonist affinity to the receptor in the high-conformational state and normal coupling predicted positive treatment outcome. Decreased coupling to Gi predicted a negative treatment outcome. Imipramine induced uncoupling (-11%) and redistribution of receptor density in treatment responders only, but had no effect on alpha(2A)AR coupling or density in treatment non-responders. Increased alpha(2A)AR density may represent a trait marker in MDD. The results provide indirect evidence for abnormal protein kinase A (PKA) and protein kinase C (PKC) in MDD which may be pursued in future investigations.

Adrenergic receptors in premenstrual dysphoric disorder: I. Platelet alpha 2 receptors: Gi protein coupling, phase of menstrual cycle, and prediction of luteal phase symptom severity.

BACKGROUND: Abnormal alpha 2-adrenergic receptor (AR) function is implicated in anxiety and depressive disorders. Premenstrual dysphoric disorder (PMDD) is characterized by anxiety and depressive symptoms, which may be associated with changes in alpha 2AR function. Previous studies on alpha 2AR function during phases of the menstrual cycle in controls and PMDD patients are inconsistent. METHODS: alpha 2AR function was examined in 16 PMDD patients and 15 controls during the follicular phase, and in 10 PMDD patients during late luteal phase. Antagonist-measured maximum binding capacity, agonist-measured receptor density in high- and low-conformational states, and agonist affinity to both states were measured. Coupling efficiency to Gi protein was estimated. RESULTS: There were no significant differences in coupling efficiency. PMDD patients had significantly low antagonist affinity; there were no differences in other binding parameters. There were no changes in alpha 2AR binding parameters between phases of menstrual cycle in PMDD women. alpha 2AR density and symptom severity were inversely related during the follicular phase in controls and patients. During luteal phase, alpha 2AR density correlated positively with symptom severity in patients. High follicular alpha 2AR density predicted more severe luteal symptoms in PMDD patients. CONCLUSIONS: These findings are discussed in view of the molecular biology of alpha 2AR, and their role in PMDD, anxiety, and depressive disorders.

Effects of high-altitude chronic hypoxia on platelet alpha 2-receptors in man.

During chronic high-altitude (HA) exposure, basal and exercise-induced noradrenaline (NA) increases do not parallel blood pressure (BP) changes observed; unlike beta-adrenergic receptors, to our knowledge no data are available on alpha-receptors. We studied platelet alpha 2- and leucocyte beta-receptors and basal catecholamine levels in 11 trained climbers before and after they had spent a 15-day period at a height of over 4400 m. In six of the climbers we also evaluated catecholamines after maximal bicycle ergometer exercise. After chronic high-altitude exposure, a significant decrease was found in platelet alpha 2-receptor density and affinity [Bmax from 92.6 +/- 6.7 to 54.6 +/- 4.2 fmol mg-1 protein (P < 0.001) and KD from 1.271 +/- 0.034 to 1.724 +/- 0.077 nmol L-1 (P < 0.05)], although no changes to beta-receptors were observed. No changes were found in basal pre- and post-expedition NA and adrenaline (A), and there was only a slight decrease in post-expedition NA after maximal exercise. Our results suggest that prolonged exposure to hypoxia induces a down-regulation of alpha 2-receptors, which may be a contributory factor in the regulation of the physiological vascular response to acclimatization.