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This meta-analytical review examines whether a deletion variant in ADRA2B, a gene that encodes α 2B adrenoceptor in the regulation of norepinephrine availability, influences cognitive processing of emotional information in human observers. Using a multilevel modeling approach, this meta-analysis of 16 published studies with a total of 2752 participants showed that ADRA2B deletion variant was significantly associated with enhanced perceptual and cognitive task performance for emotional stimuli. In contrast, this genetic effect did not manifest in overall task performance when non-emotional content was used. Furthermore, various study-level factors, such as targeted cognitive processes (memory vs. attention/perception) and task procedures (recall vs. recognition), could moderate the size of this genetic effect. Overall, with increased statistical power and standardized analytical procedures, this meta-analysis has established the contributions of ADRA2B to the interactions between emotion and cognition, adding to the growing literature on individual differences in attention, perception, and memory for emotional information in the general population., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cardiomyopathy is a common complication associated with increased mortality in sepsis, but lacks specific therapy. Here, using genetic and pharmacological approaches, we explored the therapeutic effect of α 2A -adrenergic receptor (AR) blockade on septic cardiomyopathy. CLP-induced septic rats were treated with BRL44408 (α 2A -AR antagonist), prazosin (α 1 -AR antagonist) and/or reserpine. CLP-induced cardiomyopathy, indicated by reduced dP/dt and increased cardiac troponin I phosphorylation, was attenuated by BRL44408, this was associated with reduced cardiac TNF-α and endothelial VCAM-1 expression, cardiomyocyte apoptosis and related signal molecule phosphorylation. BRL44408 increased cardiac norepinephrine (NE) concentration in CLP rats. Pretreatment with reserpine that exhausts cardiac NE without affecting the circulating NE concentration or with prazosin partially abolished the cardioprotection of BRL44408 and reversed its inhibitory effects on myocardial TNF-α, apoptosis and related signal molecule phosphorylation, but not on VCAM-1 expression in septic rats. These effects of BRL44408 were confirmed by α 2A -AR gene deletion in septic mice. Furthermore, α 2 -AR agonist not only enhanced LPS-induced TNF-α and VCAM-1 expression in cardiac endothelial cells that express α 2A -AR, but also enhanced LPS-induced cardiac dysfunction in isolated rat hearts. Our data indicate that α 2A -AR blockade attenuates septic cardiomyopathy by promoting cardiac NE release that activates myocardial α 1 -AR and suppressing cardiac endothelial activation.

Noradrenergic signaling, through the α 2A and α 2C adrenergic receptors modulates the cognitive and behavioral symptoms of disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), and addiction. However, it is unknown whether the α 2B receptor has any significant role in CNS function. The present study elucidates the potential role of the α 2B receptor in CNS function via the discovery and use of the first subtype-selective α 2B antagonist (AGN-209419), and behavioral analyses of α-receptor knockout (KO) mice. Using AGN-209419 as radioligand, α 2B receptor binding sites were identified within the olfactory bulb, cortex, thalamus, cerebellum, and striatum. Based on the observed expression patterns of α 2 subtypes in the brain, we compared α 2B KO, α 2A KO and α 2C KO mice behavioral phenotypes with their respective wild-type lines in anxiety (plus maze), compulsive (marble burying), and sensorimotor (prepulse inhibition) tasks. α 2B KO mice exhibited increased marble burying and α 2C KO mice exhibited an increased startle response to a pulse stimulus, but otherwise intact prepulse inhibition. To further explore compulsive behavior, we evaluated novelty-induced locomotor hyperactivity and found that α 2B KO and α 2C KO mice exhibited increased locomotion in the open field. Interestingly, when challenged with amphetamine, α 2C KO mice increased activity at lower doses relative to either α 2A KO or WT mice. However, α 2B KO mice exhibited stereotypy at doses of amphetamine that were only locomotor stimulatory to all other genotypes. Following co-administration of AGN-209419 with low-dose amphetamine in WT mice, stereotypy was observed, mimicking the α 2B KO phenotype. These findings suggest that the α 2B receptor is involved in CNS behaviors associated with sensorimotor gating and compulsivity, and may be therapeutically relevant for disorders such as schizophrenia, ADHD, post-traumatic stress disorder, addiction, and obsessive compulsive disorder., (Copyright © 2016. Published by Elsevier Ltd.)

It has been hypothesized that α2-adrenoceptors (α2-ARs) may be involved in the pathomechanism of colitis; however, the results are conflicting because both aggravation and amelioration of colonic inflammation have been described in response to α2-AR agonists. Therefore, we aimed to analyze the role of α2-ARs in acute murine colitis. The experiments were carried out in wild-type, α2A-, α2B-, and α2C-AR knockout (KO) C57BL/6 mice. Colitis was induced by dextran sulfate sodium (DSS, 2%); alpha2-AR ligands were injected i.p. The severity of colitis was determined both macroscopically and histologically. Colonic myeloperoxidase (MPO) and cytokine levels were measured by enzyme-linked immunosorbent assay and proteome profiler array, respectively. The nonselective α2-AR agonist clonidine induced a modest aggravation of DSS-induced colitis. It accelerated the disease development and markedly enhanced the weight loss of animals, but did not influence the colon shortening, tissue MPO levels, or histologic score. Clonidine induced similar changes in α2B- and α2C-AR KO mice, whereas it failed to affect the disease activity index scores and caused only minor weight loss in α2A-AR KO animals. In contrast, selective inhibition of α2A-ARs by BRL 44408 significantly delayed the development of colitis; reduced the colonic levels of MPO and chemokine (C-C motif) ligand 3, chemokine (C-X-C motif) ligand 2 (CXCL2), CXCL13, and granulocyte-colony stimulating factor; and elevated that of tissue inhibitor of metalloproteinases-1. In this work, we report that activation of α2-ARs aggravates murine colitis, an effect mediated by the α2A-AR subtype, and selective inhibition of these receptors reduces the severity of gut inflammation., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Epinephrine is known as a weak, but important, agonist for platelet activation. It has been reported that the responsiveness of platelets to epinephrine was markedly impaired in 6% of Caucasians and in 16% of Japanese. The purpose of this study was to screen and characterize this abnormality in healthy Taiwanese Chinese volunteers. We used aggregometry, flow cytometry and platelet function analyzer (PFA)-100 system to assess in 50 healthy male volunteers the responsiveness of platelets to epinephrine stimulation. Using α2A adrenoceptor antagonist BRL44408 maleate competition and a [(3)H]yohimbin binding assay, we evaluated α2A adrenoceptors on platelets. The aggregation of platelets after stimulation with 10 μM of epinephrine indicated two distinct groups of study participants: 24 (48.0%) good- and 26 (52.0%) impaired-responders to epinephrine. Flow cytometric analysis of platelets after stimulated with 1 μM epinephrine showed that glycoprotein (GP) IIb/IIIa and P-selectin expression of epinephrine good- and impaired-responders were 27.1 ± 11.0% vs. 9.9 ± 5.4% (p = 0.003) and 12.2 ± 6.2% vs. 3.6 ± 3.5% (p < 0.001), respectively. The PFA-100 system showed that epinephrine-impaired-responders had a longer collagen-epinephrine induced closure time. Good-responder platelets incubated with BRL44408 maleate had an impaired response to epinephrine stimulation. [(3)H]yohimbine binding studies showed fewer α2A adrenoreceptors on the platelets of epinephrine-impaired-responders than on those of good-responders. The prevalence of impaired responsiveness to epinephrine was high and probably due to α2A adrenoreceptor deficiency in male Taiwanese Chinese.

Alpha-2A adrenergic receptor (AR) subtype plays an important role in the analgesic effect of α2-AR agonists. Here, we examined the effects of α2-AR agonists, dexmedetomidine and xylazine, on spinal synaptic transmission in newborn C57BL/6J and α2A-AR mutant mice. Spinal reflex potentials, the monosynaptic reflex potential (MSR) and the slow ventral root potential (sVRP), were measured in isolated spinal cords. The compound action potential was measured in isolated lumbar nerve. Dexmedetomidine and xylazine suppressed both the MSR and sVRP in a concentration-dependent manner. In α2A-AR mutant mice, sVRP suppression by dexmedetomidine was greatly weakened, while that by xylazine (30-100μM) showed only slight attenuation. A high concentration (300μM) of xylazine completely suppressed the sVRP, even in α2A-AR mutant mice spinal cords, and also suppressed the compound action potential. MSR suppression by these α2-AR agonists had no difference between wild-type and α2A-AR mutant mice. These results suggest that sVRP suppression by dexmedetomidine and xylazine is mainly mediated by α2A-AR. In addition, a high concentration of xylazine inhibits conduction of the action potential, which is not mediated by α2A-AR. α2-AR is not responsible for the dexmedetomidine- and xylazine-mediated inhibition of the MSR., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Rationale: Noradrenaline interacts with stress hormones in the amygdala and hippocampus to enhance emotional memory consolidation, but the noradrenergic-glucocorticoid interaction at retrieval, where stress impairs memory, is less understood., Objectives: We used a genetic neuroimaging approach to investigate whether a genetic variation of the noradrenergic system impacts stress-induced neural activity in amygdala and hippocampus during recognition of emotional memory., Methods: This study is based on genotype-dependent reanalysis of data from our previous publication (Li et al. Brain Imaging Behav 2014). Twenty-two healthy male volunteers were genotyped for the ADRA2B gene encoding the α2B-adrenergic receptor. Ten deletion carriers and 12 noncarriers performed an emotional face recognition task, while their brain activity was measured with fMRI. During encoding, 50 fearful and 50 neutral faces were presented. One hour later, they underwent either an acute stress (Trier Social Stress Test) or a control procedure which was followed immediately by the retrieval session, where participants had to discriminate between 100 old and 50 new faces., Results: A genotype-dependent modulation of neural activity at retrieval was found in the bilateral amygdala and right hippocampus. Deletion carriers showed decreased neural activity in the amygdala when recognizing emotional faces in control condition and increased amygdala activity under stress. Noncarriers showed no differences in emotional modulated amygdala activation under stress or control. Instead, stress-induced increases during recognition of emotional faces were present in the right hippocampus., Conclusion: The genotype-dependent effects of acute stress on neural activity in amygdala and hippocampus provide evidence for noradrenergic-glucocorticoid interaction in emotional memory retrieval.

Background and Aims: Insulin secretion is controlled by pancreatic α(2A)-adrenoceptors. Mice lacking α(2A)-adrenoceptors (α(2A)AR(-/-) mice) show hyperinsulinaemia, reduced blood glucose levels and improved glucose tolerance., Methods: In the present study, we used α(2AC)AR(-/-), α(2C)AR(-/-) and α(2A)AR(-/-) mice and a mouse line with adrenergic cell-specific expression of α(2A)-adrenoceptors (lacking these receptors in non-adrenergic cells), and their wild-type (WT) controls, to assess the glucoregulatory role of the α(2C)-adrenoceptor subtype in vivo. Glucose and insulin tolerance tests were performed and blood glucose and serum insulin levels were determined after fasting and glucose stimulation. Plasma catecholamines were also measured. In addition, the effect of pretreatment with (±)-propranolol was determined in α(2C)AR(-/-) mice., Results: α(2AC)AR(-/-) mice had a similar glucose and insulin phenotype as α(2A)AR(-/-) mice and mice with restored α(2A)-autoreceptors, suggesting that only deletion of postsynaptic α(2A)-adrenoceptors has major effects on glucose disposition. However, α(2AC)AR(-/-) mice were more sensitive to the glucose-lowering effect of insulin than WT mice. This was not observed in α(2A)AR(-/-) mice. The α(2C)AR(-/-) mice showed impaired glucose tolerance that was reversed by pretreatment with (±)-propranolol. No difference in insulin secretion was observed in α(2C)AR(-/-) mice compared with WT animals., Conclusion: The results underline that depletion of postsynaptic pancreatic α(2A)-adrenoceptors has major effects on the regulation of glucose homeostasis in α(2AC)AR(-/-) and α(2A)AR(-/-) mice. Deletion of the α(2C) subtype leads to increased adrenaline secretion and has the potential to increase blood glucose levels via enhanced glycogenolysis., (Copyright © 2012 S. Karger AG, Basel.)

α(2)-adrenoceptors are known to mediate gastroprotective effect in both acid-dependent and acid-independent ulcer models. The aim of the present study was to determine, which of the three α(2)-adrenoceptor subtypes (α(2A), α(2B) or α(2C)) is responsible for this protection. Various α(2)-adrenoceptor agonists and antagonists were administered intracerebroventricularly (i.c.v.) to C57BL/6 mice with deletion of genes encoding the different subtypes. The gastric mucosal damage was induced by orally injected acidified ethanol. Both the non-selective α(2)-adrenoceptor agonist clonidine (0.3-2.8 nmol) and the α(2B/C)-adrenoceptor subtype preferring agonist ST-91 (0.5-11.5 nmol) induced dose-dependent gastroprotective effect in wild type, α(2A)-, α(2B)- and α(2C)-KO mice. In contrast, the α(2A)-adrenoceptor subtype agonist oxymetazoline (0.07-84 nmol i.c.v.) reduced only slightly the development of ethanol-induced ulcers. The effect of clonidine was antagonized by the non-selective antagonist yohimbine (25 nmol) and the α(2B/C)-adrenoceptor antagonist ARC 239 (10.4 nmol), but not by the α(2A)-adrenoceptor antagonist BRL 44408 (7.5 nmol). ARC 239 also reversed the effect of clonidine in α(2A)-, α(2B)- and α(2C)-KO mice, while the selective α(2C)-adrenoceptor antagonist JP 1302 (52 nmol) antagonized that only in α(2B)-KO, but not in α(2A)- and α(2C)-KO mice. These results suggest that α(2B)- and α(2C)-adrenoceptor subtypes can equally contribute to the mediation of gastroprotective effect induced by α(2)-adrenoceptor agonists in mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Stress has paradoxical effects on pain, causing stress-induced analgesia but also exacerbating pain via poorly understood mechanisms. Adrenergic neurotransmission is integral in pathways that regulate the response to both pain and stress. Hyperalgesia is often associated with enhanced adrenergic sensitivity of primary afferents, but sympathetic nervous system outflow has not been demonstrated to exacerbate pain perception after stress., Methods: Rats or C57/BL6 wild-type mice treated with α-2 receptor antagonists or α-2A receptor knockout mice were exposed to ultrasonic noise stress or footshock stress and subsequently tested for hotplate paw withdrawal latencies. The sensory sensitivity of α-2A knockout mice to electrical and chemical stimuli was tested neurophysiologically and behaviorally. The effects of sympatholytic treatments were investigated., Results: Noise and footshock stressors induced thermal hyperalgesia in rats pretreated systemically with α-2 antagonists. Wild-type mice pretreated with α-2 antagonists and α-2A knockout mice also exhibited thermal hyperalgesia induced by noise stress. Local spinal or intraplantar injection of an α-2 antagonist counteracted stress-induced analgesia without causing hyperalgesia. The α-2A knockout mice had decreased thresholds for peripheral sensitization with sulprostone and for windup of the dorsal horn neuronal response to repetitive electrical stimuli. Stress-induced hyperalgesia was abolished and the sensitization was attenuated by sympathectomy or systemic administration of an α-1-adrenergic antagonist., Conclusions: Sympathetic postganglionic nerves can enhance pain sensation via a peripheral α-1-adrenoceptor mechanism when sympathetic outflow is disinhibited. The net effect of stress on pain sensation reflects a balance between descending spinal inhibition and sympathetic outflow that can shift toward pain facilitation when central and peripheral α-2-adrenoceptor inhibitory mechanisms are attenuated.

α(2)-Adrenoceptors belong to the family of adrenergic receptors, which regulate the neuronal release of norepinephrine as part of a negative feedback loop. Among the α(2)-adrenoceptors, the α(2B)-subtype may also influence developmental signaling pathways involved in angiogenesis of the placenta. Thus, the aim of the present study was to determine whether α(2B)-adrenoceptors are also involved in other developmental processes beyond placental angiogenesis. Ablation of α(2B)-adrenoceptors led to lethality of mutant mice during the first hours after birth. Despite normal breathing and drinking behavior, mutant mice developed cyanosis, which could be traced back to a defect in lung morphology with significantly reduced alveolar volume and thickened interalveolar septi. In α(2B)-deficient lungs and in isolated alveolar type II cells, expression of sonic hedgehog (SHH) was significantly increased, resulting in mesenchymal proliferation. In vitro α(2B)-adrenoreceptor stimulation suppressed expression of sonic hedgehog and the cell cycle genes cyclin D1 and Ki67. In vivo inhibition of enhanced SHH signaling by the smoothened antagonist cyclopamine partially rescued perinatal lethality, lung morphology, and altered gene expression in mutant mice. Thus, α(2B)-adrenoceptors in lung epithelia play an important role in suppressing sonic hedgehog-mediated proliferation of mesenchymal cells and thus prevent respiratory failure.

The cellular mechanisms of positive effects associated with aerobic exercise training on overall intrinsic skeletal muscle changes in heart failure (HF) remain unclear. We investigated potential Ca2+ abnormalities in skeletal muscles comprising different fiber compositions and investigated whether aerobic exercise training would improve muscle function in a genetic model of sympathetic hyperactivity-induced HF. A cohort of male 5-mo-old wild-type (WT) and congenic alpha2A/alpha2C adrenoceptor knockout (ARKO) mice in a C57BL/6J genetic background were randomly assigned into untrained and trained groups. Exercise training consisted of a 8-wk running session of 60 min, 5 days/wk (from 5 to 7 mo of age). After completion of the exercise training protocol, exercise tolerance was determined by graded treadmill exercise test, muscle function test by Rotarod, ambulation and resistance to inclination tests, cardiac function by echocardiography, and Ca2+ handling-related protein expression by Western blot. alpha2A/alpha2CARKO mice displayed decreased ventricular function, exercise intolerance, and muscle weakness paralleled by decreased expression of sarcoplasmic Ca2+ release-related proteins [alpha1-, alpha2-, and beta1-subunits of dihydropyridine receptor (DHPR) and ryanodine receptor (RyR)] and Ca2+ reuptake-related proteins [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)1/2 and Na+/Ca2+ exchanger (NCX)] in soleus and plantaris. Aerobic exercise training significantly improved exercise tolerance and muscle function and reestablished the expression of proteins involved in sarcoplasmic Ca2+ handling toward WT levels. We provide evidence that Ca2+ handling-related protein expression is decreased in this HF model and that exercise training improves skeletal muscle function associated with changes in the net balance of skeletal muscle Ca2+ handling proteins.

Aims: alpha(2)-Adrenoceptors modulate cardiovascular function by vasoconstriction or dilatation, by central inhibition of sympathetic activity, or by feedback inhibition of norepinephrine release from sympathetic neurons. Despite detailed knowledge about subtype-specific functions of alpha(2)-receptors, the relative contributions of sympathetic vs. non-sympathetic receptors involved in these cardiovascular effects have not been identified. The aim of this study was to define the physiological and pharmacological role of alpha(2A)-adrenoceptors in adrenergic vs. non-adrenergic cells at baseline and during sympathetic stress., Methods and Results: Transgenic mice expressing alpha(2A)-adrenoceptors under control of the dopamine beta-hydroxylase (Dbh) promoter were generated and crossed with mice carrying a constitutive deletion in the alpha(2A)- and alpha(2C)-adrenoceptor genes. alpha(2AC)-deficient mice showed increased norepinephrine plasma levels, cardiac hypertrophy, and fibrosis at baseline. Expression of the Dbh-alpha(2A) transgene in sympathetic neurons prevented these effects. In contrast, Dbh-alpha(2A) receptors mediated only a minor part of the bradycardic and hypotensive effects of the alpha(2)-agonist medetomidine. After chronic pressure overload as induced by transverse aortic constriction in mice, the Dbh-alpha(2A) transgene did not reduce norepinephrine spillover, cardiac dysfunction, hypertrophy, or fibrosis. In isolated wild-type atria, alpha(2)-agonist-induced inhibition of [3H]norepinephrine release was significantly desensitized after pressure overload. In primary sympathetic neurons from Dbh-alpha(2A) transgenic mice, norepinephrine and medetomidine induced endocytosis of alpha(2A)-adrenoceptors into neurite processes., Conclusion: alpha(2A)-Adrenoceptors expressed in adrenergic cells are essential feedback inhibitors of sympathetic norepinephrine release to prevent cardiac hypertrophy and fibrosis at baseline. However, these receptors are desensitized by chronic pressure overload which in turn may contribute to the pathogenesis of this condition.

Sympathetic hyperactivity (SH) is a hallmark of heart failure (HF), and several lines of evidence suggest that SH contributes to HF-induced skeletal myopathy. However, little is known about the influence of SH on skeletal muscle morphology and metabolism in a setting of developing HF, taking into consideration muscles with different fiber compositions. The contribution of SH on exercise tolerance and skeletal muscle morphology and biochemistry was investigated in 3- and 7-mo-old mice lacking both alpha(2A)- and alpha(2C)-adrenergic receptor subtypes (alpha(2A)/alpha(2C)ARKO mice) that present SH with evidence of HF by 7 mo. To verify whether exercise training (ET) would prevent skeletal muscle myopathy in advanced-stage HF, alpha(2A)/alpha(2C)ARKO mice were exercised from 5 to 7 mo of age. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF and preserved exercise tolerance and muscular norepinephrine with no changes in soleus morphology. In contrast, plantaris muscle of alpha(2A)/alpha(2C)ARKO mice displayed hypertrophy and fiber type shift (IIA --> IIX) paralleled by capillary rarefaction, increased hexokinase activity, and oxidative stress. At 7 mo, alpha(2A)/alpha(2C)ARKO mice displayed exercise intolerance and increased muscular norepinephrine, muscular atrophy, capillary rarefaction, and increased oxidative stress. ET reestablished alpha(2A)/alpha(2C)ARKO mouse exercise tolerance to 7-mo-old wild-type levels and prevented muscular atrophy and capillary rarefaction associated with reduced oxidative stress. Collectively, these data provide direct evidence that SH is a major factor contributing to skeletal muscle morphological changes in a setting of developing HF. ET prevented skeletal muscle myopathy in alpha(2A)/alpha(2C)ARKO mice, which highlights its importance as a therapeutic tool for HF.

Background: Meperidine proved to be more effective in treatment of shivering than equianalgesic doses of other opioids, especially pure mu-agonists. Further, meperidine has well known nonopioid actions including agonistic effects at alpha2-adrenoceptors in vitro. Accordingly, the authors investigated nonopioid receptor-mediated effects of meperidine on thermoregulation using a mice model of nonshivering thermogenesis. To differentiate conceivable alpha2-adrenoceptor subtype specific interactions the authors analyzed wild-type mice and knock-out mice with deletion of the alpha2A-, alpha2B-, or alpha2C-adrenoceptor., Methods: Ten mice per group (n = 60) were injected with saline, meperidine (20 mg/kg), saline plus naloxone (125 microg/kg), meperidine plus naloxone, fentanyl (50 microg/kg) plus naloxone, or meperidine plus atipamezole (2 mg/kg) intraperitoneally. Each mouse was subjected to the six different treatments. Then they were positioned into a plexiglas chamber where rectal temperature and mixed expired carbon dioxide were measured while whole body cooling was performed. Maximum response intensity and thermoregulatory threshold temperature of nonshivering thermogenesis were analyzed., Results: Meperidine decreased the thermoregulatory threshold temperature in wild-type mice and alpha2B- and alpha2C-adrenoceptor knock-out mice. This effect ended after injection of the alpha2-adrenoceptor antagonist atipamezole. In wild-type and alpha2B-adrenoceptor knock-out mice, the decrease of thermoregulatory threshold was not reversible by administration of the opioid receptor antagonist naloxone. In contrast, in alpha2A-adrenoceptor knock-out mice, no decline of thermoregulatory threshold following meperidine injection was detectable. Maximum response intensity of nonshivering thermogenesis was comparable in all groups., Conclusions: The authors' results suggest a major role of alpha2-adrenoceptors, especially the alpha2A subtype, in the mediation of thermoregulatory effects caused by meperidine in mice.

Alpha(2)-Adrenoceptors regulate insulin secretion and sympathetic output. In the present study, alpha(2A)-adrenoceptor knockout (alpha(2A)-KO) mice and their C57BL/6J wild-type (WT) controls were used to assess the glucoregulatory role of the alpha(2A)-adrenoceptor subtype in vivo. Fasting and glucose-stimulated blood glucose and plasma insulin levels were determined with or without (+/-)-propranolol (5 mg/kg) or atropine (10 mg/kg) pre-treatment. Intraperitoneal glucose (1 g/kg) and insulin (0.5 and 1.0 IU/kg) tolerance tests were performed. Fasting plasma glucagon and corticosterone levels were measured. Blood glucose levels (mean+/-S.E.M.) were lower in alpha(2A)-KO males (7.2+/-0.6 mM) and females (7.2+/-0.2 mM) than in WT males (9.8+/-0.3 mM) and females (9.1+/-0.3 mM). Plasma insulin levels were higher in alpha(2A)-KO males (2.2+/-0.5 microg/l) and females (1.7+/-0.3 microg/l) than in WT males (0.7+/-0.1 microg/l) and females (0.8+/-0.2 microg/l). These differences remained after pharmacological beta-adrenoceptor and muscarinic acetylcholine receptor inhibition. In spite of a tendency for slightly decreased insulin sensitivity in alpha(2A)-KO mice, glucose tolerance in alpha(2A)-KO mice was significantly better than in WT mice. However, glucose-stimulated insulin secretion was not increased in alpha(2A)-KO mice compared to WT controls. Plasma glucagon levels, but not corticosterone levels, were elevated in alpha(2A)-KO mice. These results suggest that lack of inhibitory pancreatic beta-cell alpha(2A)-adrenoceptor function results in hyperinsulinaemia, reduced blood glucose levels and improved glucose tolerance in alpha(2A)-KO mice, and demonstrate a key role for the alpha(2A)-adrenoceptor in adrenergic regulation of blood glucose and insulin homeostasis.

Exercise training (ET) is a coadjuvant therapy in preventive cardiology. It delays cardiac dysfunction and exercise intolerance in heart failure (HF); however, the molecular mechanisms underlying its cardioprotection are poorly understood. We tested the hypothesis that ET would prevent Ca(2+) handling abnormalities and ventricular dysfunction in sympathetic hyperactivity-induced HF mice. A cohort of male wild-type (WT) and congenic alpha(2A)/alpha(2C)-adrenoceptor knockout (alpha(2A)/alpha(2C)ARKO) mice with C57BL6/J genetic background (3-5 mo of age) were randomly assigned into untrained and exercise-trained groups. ET consisted of 8-wk swimming session, 60 min, 5 days/wk. Fractional shortening (FS) was assessed by two-dimensional guided M-mode echocardiography. The protein expression of ryanodine receptor (RyR), phospho-Ser(2809)-RyR, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2), Na(+)/Ca(2+) exchanger (NCX), phospholamban (PLN), phospho-Ser(16)-PLN, and phospho-Thr(17)-PLN were analyzed by Western blotting. At 3 mo of age, no significant difference in FS and exercise tolerance was observed between WT and alpha(2A)/alpha(2C)ARKO mice. At 5 mo, when cardiac dysfunction is associated with lung edema and increased plasma norepinephrine levels, alpha(2A)/alpha(2C)ARKO mice presented reduced FS paralleled by decreased SERCA2 (26%) and NCX (34%). Conversely, alpha(2A)/alpha(2C)ARKO mice displayed increased phospho-Ser(16)-PLN (76%) and phospho-Ser(2809)-RyR (49%). ET in alpha(2A)/alpha(2C)ARKO mice prevented exercise intolerance, ventricular dysfunction, and decreased plasma norepinephrine. ET significantly increased the expression of SERCA2 (58%) and phospho-Ser(16)-PLN (30%) while it restored the expression of phospho-Ser(2809)-RyR to WT levels. Collectively, we provide evidence that improved net balance of Ca(2+) handling proteins paralleled by a decreased sympathetic activity on ET are, at least in part, compensatory mechanisms against deteriorating ventricular function in HF.

Alpha2-adrenoceptors are essential presynaptic regulators of norepinephrine release from sympathetic nerves. Previous studies in mice with targeted deletions in the 3 alpha2-adrenoceptor genes have indicated that these receptors are essential for embryonic development. In the present study, we searched for the alpha2-adrenoceptor subtype(s) involved in placental development and its molecular mechanism using mice carrying targeted deletions in alpha2-adrenoceptor genes. Congenic alpha2B-adrenoceptor-deficient mice (Adra2b-/-) developed a defect in fetal and maternal vessel formation in the placenta labyrinth at embryonic day 10.5. This defect was accompanied by reduced endothelial cell proliferation and decreased extracellular signal-regulated kinase 1/2 phosphorylation levels in Adra2b-/- as compared with Adra2b+/+ placentae. Microarray analysis of wild-type and mutant placentae (maternal genotype Adra2b+/-) revealed 179 genes, which were significantly up- or downregulated >1.5-fold in alpha2B-deficient placentae. The type 1 receptor for vascular endothelial growth factor (Flt1), which is coexpressed with alpha2B-adrenoceptors in spongiotrophoblast and giant cells of the placenta, was found to be 2.3-fold upregulated in alpha2B-deficient placentae. Neutralization of Flt1 and its soluble splice variant sFlt1 by a specific antibody in vivo prevented the vascular defect in alpha2B-deficient placentae at embryonic day 10.5. Thus, alpha2B-adrenoceptors are essential to suppress antiangiogenic (s)Flt1 in spongiotrophoblasts to control the coordinated formation of a vascular labyrinth of fetal and maternal blood vessels in the murine placenta during development.

Previous research suggested that alpha2A and alpha2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however, the basis for these dissimilarities is not completely known. To better understand the observed functional differences between these autoreceptors, we investigated targeting and signaling of endogenously expressed alpha2A and alpha2CARs in cultured sympathetic ganglion neurons (SGN). At Days 1 and 4, alpha2A and alpha2CARs could be readily detected in SGN from wild-type mice. By Day 8, alpha2A ARs were targeted to cell body, as well as axonal and dendritic sites, whereas alpha2C ARs were primarily localized to an intracellular vesicular pool within the cell body and proximal dendritic projections. Expression of synaptic vesicle marker protein SV2 did not differ at Day 8 nor co-localize with either subtype. By Day 16, however, alpha2C ARs had relocated to somatodendritic and axonal sites and, unlike alpha2A ARs, co-localized with SV2 at synaptic contact sites. Consistent with a functional role for alpha2 ARs, we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older, compared to younger, cultures. These results provide direct evidence of distinct developmental patterns of endogenous alpha2A and alpha2C AR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly, the co-localization at Day 16 of alpha2C ARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between alpha2A and alpha2C ARs in SGN.

Alterations in processing of emotionally salient information have been reported in individuals with major depressive disorder (MDD). Evidence suggests a role for noradrenaline in the regulation of a cortico-limbic-striatal circuit that has also been implicated in the pathophysiology of MDD. Herein, we studied the physiological consequences of a common coding polymorphism of the gene for the alpha(2C)-adrenoreceptor (AR) subtype--the deletion of four consecutive amino acids at codons 322-325 of the alpha2C-AR (alpha2CDel322-325-AR) in medication-free, remitted individuals with MDD (rMDD), and healthy control subjects. After injection of 10 mCi of H2(15)O, positron emission tomography (PET) measures of neural activity were acquired while subjects were viewing unmasked sad, happy, and fearful faces. The neural responses to sad facial expressions were increased in the amygdala and decreased in the left ventral striatum in rMDD patients relative to healthy control subjects. Furthermore, we report that rMDD carriers of one or two copies of the alpha2CDel322-325-AR exhibit greater amygdala as well as pregenual and subgenual anterior cingulate gyrus neuronal activity in response to sad faces than healthy alpha2CDel322-325-AR carriers and rMDD noncarriers. These results suggest that the alpha2CDel322-325-AR confers a change in brain function implicating this alpha2-AR subtype into the pathophysiology of MDD.

Platelet activation plays a central role in hemostasis and thrombosis. Many platelet agonists function through G-protein-coupled receptors. Epinephrine activates the alpha(2A)-adrenergic receptor (alpha(2A)) that couples to G(z) in platelets. Although alpha(2A) was originally cloned from platelets, its role in thrombosis and hemostasis is still unclear. Through analysis of alpha(2A)-deficient mice, variable tail bleeding times were observed. In vitro, epinephrine potentiated activation/aggregation responses of wild-type but not alpha(2A)-deficient platelets as determined by flow cytometry and aggregometry, whereas perfusion studies showed no differences in platelet adhesion and thrombus formation on collagen. To test the in vivo relevance of alpha(2A) deficiency, mice were subjected to 3 different thrombosis models. As expected, alpha(2A)-deficient mice were largely protected from lethal pulmonary thromboembolism induced by the infusion of collagen/epinephrine. In a model of FeCl(3)-induced injury in mesenteric arterioles, alpha(2A)(-/-) mice displayed a 2-fold increase in embolus formation, suggesting thrombus instability. In a third model, the aorta was mechanically injured, and blood flow was measured with an ultrasonic flow probe. In wild-type mice, all vessels occluded irreversibly, whereas in 24% of alpha(2A)-deficient mice, the initially formed thrombi embolized and blood flow was reestablished. These results demonstrate that alpha(2A) plays a significant role in thrombus stabilization.

3,4-Methylenedioxymetamphetamine (MDMA) produces complex effects on body temperature, including hypo- and hyperthermic components that vary with ambient temperature and strain of rat. We have previously reported that MDMA is an alpha(2)-adrenoceptor agonist, and alpha(2)-adrenoceptor agonists such as clonidine produce hypothermia. The purpose of this study was to investigate the effects of MDMA on core body temperature measured by radiotelemetry in conscious wild-type (WT) and alpha(2A)-knockout (alpha(2A)-KO) mice. Clonidine (0.1 mg kg(-1), subcutaneously (s.c.)) produced a hypothermic response in WT mice, but did not significantly affect temperature in alpha(2)-KO mice. MDMA (20 mg kg(-1), s.c.) produced a significant hyperthermia in WT mice beginning at approximately 100 min after injection, recovering by 300 min, but produced a biphasic response, hypothermia followed by hyperthermia, in alpha(2)-KO mice. In WT mice, following the alpha(2A)-adrenoceptor antagonist 2-((4,5-dihydro-1H-imidazol-2-yl)methyl)-2,3-dihydro-1-methyl-1H-isoindole (1 mg kg(-1), s.c.), MDMA (20 mg kg(-1)) produced an initial hypothermia. Hence, alpha(2)-adrenoceptor agonist actions of MDMA contribute to its effects on body temperature, but in a surprising way. Although selective alpha(2A)-adrenoceptor agonism produces hypothermia, the alpha(2A)-adrenoceptor actions of MDMA alter the body temperature response to MDMA from biphasic (hypothermia followed by hyperthermia) to monophasic hyperthemia.

The present studies extend recent findings that mice null for the alpha(2A) adrenergic receptor (alpha(2A) AR KO mice) lack suppression of exogenous secretagogue-stimulated insulin secretion in response to alpha(2) AR agonists by evaluating the endogenous secretagogue, glucose, ex vivo, and providing in vivo data that baseline insulin levels are elevated and baseline glucose levels are decreased in alpha(2A) AR KO mice. These latter findings reveal that the alpha(2A) AR subtype regulates glucose-stimulated insulin release in response to endogenous catecholamines in vivo. The changes in alpha(2A) AR responsiveness and resultant changes in insulin/glucose homeostasis encouraged us to utilize proteomics strategies to identify possible alpha(2A) AR downstream signaling molecules or other resultant changes due to perturbation of alpha(2A) AR expression. Although agonist stimulation of islets from wild type (WT) mice did not significantly alter islet protein profiles, several proteins were enriched in islets from alpha(2A) AR KO mice when compared with those from WT mice, including an enzyme participating in insulin protein processing. The present studies document the important role of the alpha(2A) AR subtype in tonic suppression of insulin release in response to endogenous catecholamines as well as exogenous alpha(2) agonists and provide insights into pleiotropic changes that result from loss of alpha(2A) AR expression and tonic suppression of insulin release.

Alpha(2)-adrenoceptor agonists potentiate opioid analgesia and alleviate opioid withdrawal. The effects of two alpha(2)-adrenoceptor agonists, clonidine (2 mg/kg) and dexmedetomidine (20 and 100 microg/kg), and the alpha(1)-adrenoceptor antagonist prazosin (0.5 mg/kg) were tested on morphine analgesia, tolerance, and withdrawal in wild-type and alpha(2A)-adrenoceptor knock-out (KO) mice. Analgesia and tolerance were assessed with the tail-flick test. Withdrawal was precipitated with naloxone. Prazosin potentiated morphine analgesia equally in both genotypes. Clonidine and dexmedetomidine had no analgesic effects in alpha(2A)-adrenoceptor KO mice, but morphine analgesia and tolerance were similar in both genotypes. Alpha(2A)-Adrenoceptor KO mice exhibited 70% fewer naloxone-precipitated jumps than wild-type mice; weight loss was similar in both genotypes. The alpha(2)-adrenoceptor agonists reduced opioid withdrawal signs only in wild-type mice. We conclude that alpha(2A)-adrenoceptors are not directly involved in morphine analgesia and tolerance, and not critical for potentiation of morphine analgesia by prazosin, but that alpha(2A)-adrenoceptors modulate the expression of opioid withdrawal signs in mice.

1. Our objective was to determine whether alpha(2A)-adrenoceptors modulate the baroreceptor reflex. The efficacy of the reflex was evaluated by measuring the spontaneous blood pressure and heart rate variability at rest and the heart rate responses to evoked changes in blood pressure. Experiments were carried out in conscious, unrestrained, and anaesthetized alpha(2A)-adrenoceptor-deficient (alpha(2A)-KO) mice and WT mice. 2. In conscious alpha(2A)-KO mice, the spontaneous blood pressure variability was greater, and the spontaneous heart rate variability was lower than in conscious WT mice. This was also observed in anaesthetized animals. 3. The reflex bradycardia after intravenous injection of phenylephrine was greatly attenuated in conscious alpha(2A)-KO compared to conscious WT mice; the baroreceptor reflex gain (ratio maximal change in heart rate/maximal change in mean arterial pressure) was decreased by 40%. 4. Similar results were obtained when reflex bradycardia was elicited by intra-arterial volume loading of conscious WT and alpha(2A)-KO mice. The baroreceptor reflex gain upon volume loading was also low in anaesthetized alpha(2A)-KO mice. 5. The reflex tachycardia evoked by intravenous sodium nitroprusside injection was also significantly less in alpha(2A)-KO mice as compared to WT, conscious as well as anaesthetized; the baroreceptor reflex gains were decreased by 50 and 65%, respectively. 6. Direct stimulation of cardiac beta-adrenoceptors by the agonist isoprenaline produced similar cardioacceleration in alpha(2A)-KO and WT animals. 7. Our results show that the baroreceptor reflex function is impaired in mice lacking alpha(2A)-adrenoceptors. We conclude that central alpha(2A)-adrenoceptors facilitate the reflex response to both loading and unloading of the arterial baroreceptors.

Tricyclic antidepressants have analgesic and sedative effects in addition to their antidepressive properties. We tested the acute analgesic and locomotor inhibitory effects of the tricyclic antidepressant amitriptyline and the alpha(2)-adrenoceptor agonist clonidine in wild-type control and in alpha(2A)-adrenoceptor knockout mice in hot-plate and tail-flick tests. Amitriptyline-induced analgesia was lost in alpha(2A)-adrenoceptor knockout mice. The locomotor inhibitory effect of amitriptyline was reduced, but not fully abolished in alpha(2A)-adrenoceptor knockout mice. Similar results were obtained with clonidine. We conclude that alpha(2A)-adrenoceptors appear to have a significant role in amitriptyline-induced acute analgesia in mice, and that alpha(2A)-adrenoceptors also participate in the sedative effects of amitriptyline.

The alpha2-adrenoreceptor (AR) is the most investigated noradrenergic receptor with regard to modulation of seizure activity. However, because of the complexity of multiple alpha2-AR subtypes and their distribution, the exact role of this receptor in modulating seizure activity is not clear. alpha2A- and alpha2C-ARs function as both autoreceptors (presynaptic) on noradrenergic neurons, where they regulate norepinephrine (NE) release, and as postsynaptic receptors on neurons that receive noradrenergic innervation, where they regulate the release of other neurotransmitters (heteroreceptor). The nonselective alpha2-AR agonist clonidine produced a proconvulsant effect on seizure susceptibility, while the selective alpha2A-AR agonist guanfacine was anticonvulsant. The effects of both alpha2-AR agonists were absent in alpha2a knockout mice, suggesting that the alpha2A-AR mediates the proconvulsant and anticonvulsant effect of alpha2-AR agonists on seizure susceptibility. To determine whether the alpha2-AR agonists were acting on inhibitory presynaptic autoreceptors to decrease NE release or on postsynaptic receptors on NE target neurons, the effects of clonidine and guanfacine were determined in dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack NE. The anticonvulsant effect of guanfacine persisted in Dbh -/- mice, suggesting that guanfacine may act preferentially on alpha2A-postsynaptic receptors that regulate the action of NE on target neurons. In contrast, the proconvulsant effect of clonidine was lost in Dbh -/- mice, suggesting that clonidine may act on presynaptic autoreceptors to decrease NE release. We hypothesize that the alpha2A-presynaptic autoreceptor is responsible for the proconvulsant effect of alpha2-AR agonists, while the alpha2A-postsynaptic receptor is responsible for the anticonvulsant effect of alpha2-AR agonists. These data help to clarify the inconsistent effects of alpha2-AR agonists on seizure activity.

Postganglionic sympathetic neurons and brain noradrenergic neurons use alpha(2A)- and alpha(2C)-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic alpha(2B)-autoreceptors as well. Pieces of atria, vasa deferentia, the occipito-parietal cortex and the hippocampus were prepared from either wildtype (WT) mice or mice in which both the alpha(2A)- and the alpha(2C)-adrenoceptor gene had been disrupted (alpha(2AC)KO). The pieces were incubated with (3)H-noradrenaline and then superfused and stimulated electrically. In a first series of experiments, single pulses or brief, autoinhibition-poor pulse trains were used for stimulation. The alpha(2)-adrenoceptor agonist UK 14,304 (brimonidine) reduced the evoked overflow of tritium in all four tissues from WT mice but did not change it in any tissue from alpha(2AC)KO mice. A different pattern was obtained with medetomidine as alpha(2 )agonist. Like UK 14,304, medetomidine reduced the evoked overflow of tritium in all four tissues from WT mice and did not affect overflow in brain slices from alpha(2AC)KO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from alpha(2AC)KO mice, although with a lower maximum and potency than in WT preparations. The alpha-adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in alpha(2AC)KO atria and vasa deferentia to the right. The pK(d) values of the five antagonists against medetomidine in alpha(2AC)KO atria and vasa deferentia correlated with pK(d) values at prototypical alpha(2B) radioligand binding sites but not at alpha(2A) or alpha(2C) binding sites. In a second series of experiments, autoinhibition-rich pulse trains were used for stimulation. Under these conditions, rauwolscine and phentolamine increased the evoked overflow of tritium from alpha(2AC)KO atrial and vas deferens pieces but not from alpha(2AC)KO brain slices. The increase was smaller (by 40% in atria and by 70% in the vas deferens) than previously observed in WT preparations (by 200-400%). In a last series of experiments, mRNA for the alpha(2B)-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, alpha(2A)KO, alpha(2C)KO and alpha(2AC)KO mice but not from alpha(2B)KO mice. The results show that brain noradrenergic neurons express only alpha(2A)- and alpha(2C)-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express alpha(2B)-adrenoceptors as presynaptic autoreceptors as well. The alpha(2B)-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-alpha(2)-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.

Background: Patients with a deletion of 4 consecutive amino acids in the gene encoding for the alpha(2C)-adrenergic receptor (alpha(2C)Del322-325) have an increased prevalence of clinical heart failure, worse clinical status, and a lower left ventricular ejection fraction compared with patients without this deletion. We postulated that patients with the alpha(2C)Del322-325 polymorphism would have a compensatory increase in norepinephrine uptake-1 transporter activity as measured by iodine 123 metaiodobenzylguanidine (MIBG)., Methods and Results: Thirty-nine patients with heart failure related to idiopathic dilated cardiomyopathy were studied. Demographic characteristics, left ventricular ejection fraction, maximum oxygen consumption, exercise duration, and plasma norepinephrine levels did not differ between patients with the alpha(2C) receptor polymorphism (n = 9) and those without it (n = 30). Patients with the alpha(2C)Del322-325 polymorphism had significantly greater heart-to-mediastinum ratios of I-123 MIBG at 4 hours after tracer injection (1.60 +/- 0.19 vs 1.41 +/- 0.19, P =.0117) and greater background-corrected heart counts per pixel at 4 hours compared with patients without the polymorphism., Conclusions: Patients with genetic impairment of the alpha(2C)-adrenergic receptor have augmented activity of the norepinephrine uptake-1 transporter as measured by I-123 MIBG. Further studies are needed to clarify the mechanism by which uptake-1 transporter activity is increased in this setting.

We have investigated sensitivity to the conditioned fear procedure of mice is influenced by the genetic deletion of alpha2A adrenoceptors (ARs). We observed a heightened freezing response in the discrete cue memory test in alpha2A AR knockout (alpha2A AR KO) mice and in D79N mice, a transgenic mouse strain with functionally impaired alpha2A ARs. No significant differences in contextual memory were observed between control and alpha2A AR KO or D79N mice suggesting a minimal role for the noradrenergic system in contextual memory. We speculated that the increased freezing response of the alpha2A AR KO and D79N mice in the discrete cue setting was due to increased release of norepinephrine evoked by the unconditioned footshock stimulus. In alpha2A AR KO mice we measured a doubling in the number of noradrenergic neurons in the locus coeruleus (LC) and a large increase in the cell volume of tyrosine hydroxylase positive neurons, likely due to selective preservation of large, multipolar neurons in the subcoeruleus. Hyperplasia of the noradrenergic neurons in the nucleus tractus solitarius, A5 and A7, was also observed. Alpha2A AR KO mice exhibit greater c-Fos expression in the LC compared to wild type mice suggesting that the LC neurons in the alpha2A AR KO mice were spontaneously more active. This study suggests that alpha2A ARs are involved in the development of the central noradrenergic system and raises the possibility that alterations in alpha2A AR expression may contribute to variations in fear and stress responses.

Background: The intravenous anesthetic etomidate exhibits structural similarities to specific alpha2-adrenoceptor agonists of the type such as dexmedetomidine. The current study was performed to elucidate the possible interaction of etomidate with alpha2-adrenoceptors in mice lacking individual alpha2-adrenoceptor subtypes (alpha2-KO)., Methods: Sedative and cardiovascular responses to etomidate and the alpha2-agonist, dexmedetomidine, were determined in mice deficient in alpha2-receptor subtypes. Inhibition of binding of the alpha2-receptor antagonist [3H]RX821002 to recombinant alpha2-receptors by etomidate was tested in human embryonic kidney (HEK293) cells in vitro., Results: In vivo, loss and recovery of the righting reflex required similar times after intraperitoneal injection of etomidate in wild-type and in alpha2A-receptor-deficient mice, indicating that the hypnotic effect of etomidate in mice does not require the alpha2A-receptor subtype. Intravenous injection of etomidate resulted in a transient increase (duration 2.4 +/- 0.2 min) in arterial blood pressure in wild-type mice (17 +/- 3 mmHg). Etomidate did not affect blood pressure in alpha2B-KO or alpha2AB-KO mice. In membranes from HEK293 cells transfected with alpha2-receptors, etomidate inhibited binding of the alpha2-antagonist, [3H]RX821002, with higher potency from alpha2B- and alpha2C-receptors than from alpha2A-receptors (Ki alpha2A 208 microm, alpha2B 26 microm, alpha2C 56 microm). In alpha2B-receptor-expressing HEK293 cells, etomidate rapidly increased phosphorylation of the extracellular signal-related kinases ERK1/2., Conclusions: These results indicate that etomidate acts as an agonist at alpha2-adrenoceptors, which appears in vivo primarily as an alpha2B-receptor-mediated increase in blood pressure. This effect of etomidate may contribute to the cardiovascular stability of patients after induction of anesthesia with etomidate.

Objective: Adrenaline inhibits insulin secretion through activation of alpha(2)-adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads to inhibition of adenylyl cyclase, inhibition of Ca(2+) channels and activation of K(+) channels. Recently, three distinct subtypes of alpha(2)-AR were described, alpha(2A)-AR, alpha(2B)-AR and alpha(2C)-AR. At present, it is unknown which of these alpha(2)-AR subtype(s) may regulate insulin secretion. We used mice deficient in alpha(2)-ARs to analyze the coupling and role of individual alpha(2)-AR subtypes in insulin-secreting beta cells., Methods: The inhibitory effect of adrenaline on insulin secretion was measured in freshly isolated and cultured wild type (wt) and alpha(2)-AR knockout (KO) mouse islets in order to examine the receptor subtypes which mediate adrenaline-induced inhibition of insulin secretion. Adenylyl cyclase activity was measured in isolated cultured islets. Membrane potential was measured using the amphotericin B permeabilized patch clamp method in isolated and cultured single islet cells., Results: In wt, alpha(2A)- and alpha(2C)-AR KO mouse islets, adrenaline, 1 microM/L, inhibited secretion by 83, 80 and 100% respectively. In contrast, in alpha(2A/2C)-AR double KO mouse islets, adrenaline had no effect on stimulated secretion indicating that both alpha(2A)-AR and alpha(2C)-AR, but not alpha(2B)-AR, are functionally expressed in mouse islets. Surprisingly, glucose (16.7 mM/L)-induced secretion in the presence of 1 microM/L forskolin was greatly impaired in alpha(2A)-AR KO islets. However, when cAMP levels were increased further by the combination of forskolin (5 microM/L) and 3-isobutyl-1-methylxanthine (100 microM/L), secretion was stimulated 2.7-fold (8.5-fold in wt islets). Adrenaline lowered the concentration of cAMP in wt and alpha(2C)-AR KO mouse islets by 74%. Adrenaline also hyperpolarized wt and alpha(2C)-AR KO beta cells. In contrast, adrenaline did not inhibit adenylyl cyclase in islets of alpha(2A)-AR KO mice, nor did it hyperpolarize alpha(2A)-AR KO beta cells., Conclusion: Adrenaline inhibits insulin release through alpha(2A)- and alpha(2C)-ARs via distinct intracellular signaling pathways.

The alpha(2C)-adrenoceptor occurs in high density in the striatum relative to other brain regions, but its biological role in striatal physiology is perplexing because of the paucity of noradrenergic terminals in this region. In this study, mice with a targeted inactivation of the alpha(2C)-adrenoceptor gene (alpha(2C)-KO mice), and genetically related mice (WT mice), were used to study the potential role of the striatal alpha(2C)-adrenoceptor in modulating GABA release. Perfused brain slices were pre-loaded with [(3)H]GABA and were stimulated electrically. In WT mice, the alpha(2)-adrenoceptor agonist, UK14304 (brimonidine), significantly enhanced [(3)H]GABA release from striatal slices, while the alpha(2)-adrenoceptor antagonist, RX821002, alone evoked a significant decrease in [(3)H]GABA release. In alpha(2C)-KO mice, the effect of RX821002 was absent, while UK14304 retained its ability to enhance [(3)H]GABA release. Pharmacological depletion of monoamines in WT mice also abolished the effect of RX821002 on [(3)H]GABA release. In hippocampal slices, RX821002-induced reduction in [(3)H]GABA release was present in WT and alpha(2C)-KO mice. In the presence of tetrodotoxin, RX821002 increased [(3)H]GABA release in striatal slices from both WT and alpha(2C)-KO mice. Together, these data imply that alpha(2A)- and alpha(2C)-adrenoceptors are located on different neurons in the striatum, that alpha(2C)-adrenoceptor-mediated effects on striatal GABA release are mediated by an endogenous catecholamine that could be dopamine, and that the alpha(2C)-adrenoceptor effect of RX821002 does not occur at the GABAergic terminal.

1. In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release-enhancing effect when presynaptic alpha(2)-autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with alpha(2) antagonists. We now studied this operational condition in alpha(2)-adrenoceptor-deficient mice. Release of (3)H-noradrenaline was elicited by electrical stimulation. 2. In tissues from wild-type (WT) mice, angiotensin II and bradykinin increased the overflow of tritium evoked by 120 pulses at 3 Hz. This enhancement did not occur or was much reduced when tissues were stimulated by 120 pulses at 3 Hz in the presence of rauwolscine and phentolamine, or when they were stimulated by 20 pulses at 50 Hz. 3. In tissues from mice lacking the alpha(2A)-adrenoceptor (alpha(2A)KO) or the alpha(2B)-adrenoceptor (alpha(2B)KO), the concentration-response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the alpha(2C)-adrenoceptor (alpha(2C)KO) or both the alpha(2A)- and the alpha(2C)-adrenoceptor (alpha(2AC)KO), the concentration-response curves were shifted to the same extent downwards. 4. As in WT tissues, angiotensin II and bradykinin lost most or all of their effect in alpha(2A)KO and alpha(2AC)KO tissues when rauwolscine and phentolamine were present or trains consisted of 20 pulses at 50 Hz. 5. Rauwolscine and phentolamine increased tritium overflow evoked by 120 pulses at 3 Hz up to seven-fold in WT and alpha(2B)KO tissues, three-fold in alpha(2A)KO and alpha(2C)KO tissues, and two-fold in alpha(2AC)KO tissues. 6. Results confirm that angiotensin II and bradykinin require ongoing alpha(2)-autoinhibition for the full extent of their release-enhancing effect. Specifically, they require ongoing alpha(2C)-autoinhibition. The peptide effects that remain in alpha(2C)-autoreceptor-deficient mice seem to be because of alpha(2B)-autoinhibition. The results hence also suggest that in addition to alpha(2A)- and alpha(2C)- mouse postganglionic sympathetic neurons possess alpha(2B)-autoreceptors.

Alpha2-adrenergic drugs modulate cortical arousal and EEG. However, the role of individual alpha2-adrenoceptor (alpha(2)-AR) subtypes in these functions is not clear. We investigated the role of alpha(2C)-ARs in the modulation of baseline cortical EEG activity and EEG responses to the alpha(2)-AR selective agonist, dexmedetomidine (3-300 microg/kg, s.c.), and antagonist, atipamezole (3-1000 microg/kg, s.c.), by using alpha(2C)-AR knockout (KO) and wildtype (WT) mice. The overall amplitude (1-30 Hz) was not significantly altered in KO mice although the activity of theta band (4-8 Hz) was increased in these mice. The main finding was that dexmedetomidine (30-300 microg/kg) more effectively slowed and atipamezole (30-1000 microg/kg) less effectively increased cortical EEG arousal in KO mice compared to WT controls. Importantly, autoradiographical results showed no compensatory increase in other alpha(2)-AR subtypes in cortical, thalamic or other brain structures of KO mice. Furthermore, there were no differences between the genotypes in the levels of hippocampal choline acetyltransferase, monoamines or their metabolites. Altered baseline cortical EEG activity and EEG responses to alpha(2)-AR selective drugs in KO mice indicate that alpha(2C)-ARs are involved in regulation of cortical arousal. These results suggest that alpha(2C)-ARs may antagonize the sedative effect of alpha(2)-AR agonists mediated by activation of alpha(2A)-ARs.

Background: Elevated plasma norepinephrine levels are associated with increased mortality in patients and in animal models with chronic heart failure. To test which alpha2-adrenoceptor subtypes operate as presynaptic inhibitory receptors to control norepinephrine release in heart failure, we investigated the response of gene-targeted mice lacking alpha2-adrenoceptor subtypes (alpha2-KO) to chronic left ventricular pressure overload. In addition, we determined the functional consequences of genetic variants of alpha2-adrenoceptors in human patients with chronic heart failure., Methods and Results: Cardiac pressure overload was induced by transverse aortic constriction. Three months after aortic banding, survival was dramatically reduced in alpha2A-KO (52%) and alpha2C-KO (47%) mice compared with wild-type and alpha2B-deficient (86%) animals. Excess mortality in alpha2A- and alpha2C-KO strains was attributable to heart failure with enhanced left ventricular hypertrophy and fibrosis and elevated circulating catecholamines. The clinical importance of this finding is emphasized by the fact that heart failure patients with a dysfunctional variant of the alpha2C-adrenoceptor had a worse clinical status and decreased cardiac function as determined by invasive catheterization and by echocardiography., Conclusions: Our results indicate an essential function of alpha2A- and alpha2C-adrenoceptors in the prevention of heart failure progression in mice and human patients. Identification of heart failure patients with genetic alpha2-adrenoceptor variants as well as new alpha2-receptor subtype-selective drugs may represent novel therapeutic strategies in chronic heart failure and other diseases with enhanced sympathetic activation.

Genetic manipulation of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) in mice has revealed the role of this subtype in numerous responses, including agonist-induced hypotension and sedation. Unexpectedly, alpha(2)-agonist treatment of mice heterozygous for the alpha(2A)-AR (alpha(2A)-AR(+/-)) lowers blood pressure without sedation, indicating that more than 50% of alpha(2A)-AR must be activated to evoke sedation. We postulated that partial activation of alpha(2A)-AR in wild-type alpha(2A)-AR(+/+) animals could be achieved with partial agonists, agents with variable ability to couple receptor occupancy to effector activation, and might elicit one versus another pharmacological response. In vitro assays reveal that moxonidine is a partial agonist at alpha(2A)-AR. Although moxonidine was developed to preferentially interact with imidazoline binding sites, it requires the alpha(2A)-AR to lower blood pressure because we observe no hypotensive response to moxonidine in alpha(2A)-AR-null (alpha(2A)-AR(-/-)) mice. Moreover, we observe that moxonidine lowers blood pressure without sedation in wild-type mice, consistent with the above hypothesis regarding partial agonists. Our findings suggest that weak partial agonists can evoke response-selective pathways and might be exploited successfully to achieve alpha(2A)-AR pharmacotherapy where concomitant sedation is undesirable, i.e., in treatment of depression or attention deficit hyperactivity disorder, in suppression of epileptogenesis, or enhancement of cognition. Furthermore, rigorous physiological and behavioral assessment of mice heterozygous for particular receptors provides a general strategy for elucidation of pathways that might be selectively activated by partial agonists, thus achieving response-specific therapy.

A substantial percentage of human pregnancies are lost as spontaneous abortions after implantation. This is often caused by an inadequately developed placenta. Proper development of the placental vascular system is essential to nutrient and gas exchange between mother and developing embryo. Here we show that alpha(2)-adrenoceptors, which are activated by adrenaline and noradrenaline, are important regulators of placental structure and function. Mice with deletions in the genes encoding alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors died between embryonic days 9.5 and 11.5 from a severe defect in yolk-sac and placenta development. In wildtype placentae, alpha(2)-adrenoceptors are abundantly expressed in giant cells, which secrete angiogenic factors to initiate development of the placental vascular labyrinth. In placentae deficient in alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors, the density of fetal blood vessels in the labyrinth was markedly lower than normal, leading to death of the embryos as a result of reduced oxygen and nutrient supply. Basal phosphorylation of the extracellular signal regulated kinases ERK1 and ERK2 was also lower than normal, suggesting that activation of the mitogen-activated protein kinase (MAP kinase) pathway by alpha(2)-adrenoceptors is required for placenta and yolk-sac vascular development. Thus, alpha(2)-adrenoceptors are essential at the placental interface between mother and embryo to establish the circulatory system of the placenta and thus maintain pregnancy.

The present study investigated the role of alpha2A-adrenoceptor subtype in the regulation of noradrenaline and dopamine release in the medial prefrontal cortex. The effect of local introduction of the alpha2-adrenoceptor agonist dexmedetomidine (10-9-10-8 m) on noradrenaline and dopamine release was investigated in alpha2A-adrenoceptor knockout and control mice by using in vivo microdialysis. Furthermore, to reveal a possible distinction between regulation of baseline and peak release, we sampled the dialysate during both rest and handling-induced mild stress. Baseline noradrenaline and dopamine concentrations did not differ between alpha2A-adrenoceptor knockout and control mice. Dexmedetomidine decreased, in a concentration-dependent manner, noradrenaline and dopamine levels in both genotypes. However, the effect of dexmedetomidine on noradrenaline release was attenuated in the alpha2A-adrenoceptor knockout mice, whereas the effect on dopamine release did not differ between the genotypes. The first handling episode increased noradrenaline and dopamine levels to the same extent in both genotypes. However, in alpha2A-adrenoceptor knockout mice the noradrenaline and dopamine levels remained elevated in the samples following the first handling whilst, in the control mice, transmitter levels returned to baseline levels. In control mice the handling-induced peak noradrenaline and dopamine levels were lower after the administration of dexmedetomidine than during the first handling episode, but in alpha2A-adrenoceptor knockout mice no drug effect on handling-induced peak noradrenaline and dopamine levels was found. Our results suggest that the release of noradrenaline in the medial prefrontal cortex is mainly regulated via alpha2A-adrenoceptors, whilst other alpha-adrenoceptor subtypes play a significant role in the regulation of dopamine release.

Background: Both alpha(2)-adrenergic and non--alpha(2)-adrenergic mechanisms seem to be involved in the hypotensive effect of imidazoline-like drugs. This study aimed at investigating how these 2 mechanisms work together to modify blood pressure (BP)., Methods and Results: LNP 509, which appeared in this study to be devoid of alpha(2A)-adrenergic activity, was administered to anesthetized rabbits and wild-type (WT) mice into the cisterna magna and into the fourth ventricle, respectively. Mean arterial pressure decreased by a maximum of 46 +/- 4% and 16 +/- 2%, respectively. In D79N mice, which lack functional alpha(2A)-adrenergic receptors, LNP 509 also reduced mean arterial pressure by 17 +/- 2%. The hypotension induced by LNP 509 (100 microg/kg intracisternally) was prevented by S23757 (1 mg/kg intracisternally), an antagonist highly selective for I(1)-imidazoline binding sites (I(1)BS). A synergy between LNP 509 and the alpha(2)-adrenergic agonist alpha-methylnoradrenaline (alpha-MNA) was observed in rabbits (cisterna magna injection) and in WT mice (fourth ventricle injection) but not, as expected, in D79N mice. Similar to LNP 509 alone, rilmenidine (fourth ventricle injection), which binds both to alpha(2)-adrenergic receptors and to I(1)BS, decreased BP in D79N mice. In WT animals, rilmenidine had a significantly greater effect. Microinjections performed in rabbits showed that the synergism occurred at least in part in the nucleus reticularis lateralis of the brain stem., Conclusions: These results demonstrate that a central imidazoline-sensitive, but non--alpha(2)-adrenergic, mechanism can modify BP by itself. This mechanism, which may involve I(1)BS, interacts synergistically with an alpha(2)-adrenergic mechanism to decrease BP.

Mammals possess three types of alpha(2)-adrenoceptor, alpha(2A), alpha(2B) and alpha(2C). Our aim was to determine the type of alpha(2)-adrenoceptor involved in the control of gastrointestinal motility. In transmitter overflow experiments, myenteric plexus longitudinal muscle (MPLM) preparations of the ileum were preincubated with [(3)H]-choline and then superfused. The alpha(2)-adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium from preparations taken from wild type but not alpha(2A)-adrenoceptor-knockout mice. In a second series of overflow experiments, MPLM preparations were preincubated with [(3)H]-noradrenaline and then superfused. Again medetomidine reduced the electrically evoked overflow of tritium from wild type but not alpha(2A)-knockout preparations. In organ bath experiments, medetomidine reduced electrically evoked contractions of segments of the ileum from wild type but not alpha(2A)-knockout mice. In each of these three series, phentolamine antagonized the effect of medetomidine in wild-type preparations with greater potency than rauwolscine. In conscious mice, gastrointestinal transit was assessed by means of an intragastric charcoal bolus. In alpha(2A)-knockout mice, the speed of gastrointestinal transit was doubled compared to wild-type. Medetomidine, injected intraperitoneally, slowed gastrointestinal transit in wild type but not alpha(2A)-knockout mice. We conclude that the cholinergic motor neurons of the enteric nervous system of mice possess alpha(2)-heteroreceptors which mediate inhibition of acetylcholine release, of neurogenic contractions and of gastrointestinal transit. The noradrenergic axons innervating the intestine possess alpha(2)-autoreceptors. Both hetero- and autoreceptors are exclusively alpha(2A). It is the alpha(2A)-adrenoceptor which in vivo mediates the inhibition of intestinal motility by the sympathetic nervous system.

alpha(2)-Adrenergic receptors play an essential role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the CNS. However, the role of each of the three highly homologous alpha(2)-adrenergic receptor subtypes (alpha(2A), alpha(2B), alpha(2C)) in this process has not been determined unequivocally. To address this question, the regulation of norepinephrine and dopamine release was studied in mice carrying deletions in the genes encoding the three alpha(2)-adrenergic receptor subtypes. Autoradiography and radioligand binding studies showed that alpha(2)-receptor density in alpha(2A)-deficient brains was decreased to 9 +/- 1% of the respective wild-type value, whereas alpha(2)-receptor levels were reduced to 83 +/- 4% in alpha(2C)-deficient mice. These results indicate that approximately 90% of mouse brain alpha(2)-receptors belong to the alpha(2A) subtype and 10% are alpha(2C)-receptors. In isolated brain cortex slices from wild-type mice a non-subtype-selective alpha(2)-receptor agonist inhibited release of [(3)H]norepinephrine by maximally 96%. Similarly, release of [(3)H]dopamine from isolated basal ganglion slices was inhibited by 76% by an alpha(2)-receptor agonist. In alpha(2A)-receptor-deficient mice, the inhibitory effect of the alpha(2)-receptor agonist on norepinephrine and dopamine release was significantly reduced but not abolished. Only in tissues from mice lacking both alpha(2A)- and alpha(2C)-receptors was no alpha(2)-receptor agonist effect on transmitter release observed. The time course of onset of presynaptic inhibition of norepinephrine release was much faster for the alpha(2A)-receptor than for the alpha(2C)-subtype. After prolonged stimulation with norepinephrine, presynaptic alpha(2C)-adrenergic receptors were desensitized. From these data we suggest that two functionally distinct alpha(2)-adrenergic receptor subtypes, alpha(2A) and alpha(2C), operate as presynaptic inhibitory receptors regulating neurotransmitter release in the mouse CNS.

The stimulation frequency-noradrenaline release relationship was studied in the vas deferens and the cerebral cortex of NMRI mice, mice in which the alpha2A-, the alpha2B-, the alpha2C- or both the alphaCA- and the alpha2C-adrenoceptor gene had been disrupted (alpha2AKO, alpha2BKO, alpha2CKO and alpha2ACKO), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically with a constant number of pulses (30 in vas deferens and 50 in brain cortex) at frequencies between 0.03 and 100 Hz. The frequency-evoked tritium overflow curves ascended monophasically in the vas deferens of wildtype and NMRI mice. Disruption of the alpha2B-adrenoceptor gene caused no change. In the vas deferens of alpha2CKO mice, the overflow evoked by low frequencies (0.3 and 1 Hz) was slightly increased. In the vas deferens of alpha2AKO and alpha2ACKO mice, the evoked overflow was increased to a greater extent. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the vas deferens of NMRI, wildtype, alpha2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the vas deferens of alpha2AKO and alphaC2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine slightly steepened the slope of the vas deferens frequency-overflow curve. In the brain cortex of wildtype and NMRI mice, the frequency-evoked tritium overflow curves were U-shaped. In the brain cortex of alpha2BKO and alpha2CKO mice, the evoked overflow was slightly reduced. In the brain cortex of alpha2AKO and alpha2AcKO mice, in contrast, the evoked overflow was increased. Rauwolscine (1 microM) caused a marked increase of the evoked overflow of tritium from the brain cortex of NMRI, wildtype, Q2BKO and alpha2CKO mice. Rauwolscine also increased the evoked overflow of tritium from the brain cortex of alpha2AKO and alpha2ACKO mice, but to a smaller extent. The gene disruptions and rauwolscine flattened the U shape of the brain cortex frequency-overflow curve. It is concluded that alpha2-autoinhibition is one factor that shapes the frequency-noradrenaline release relationships in the mouse vas deferens and cerebral cortex. The autoreceptors are mainly alpha2A and to a minor extent, and well detectable in the vas deferens only, alpha2C. When both the alpha2A- and the alpha2C-adrenoceptor have been deleted, alpha2B-adrenoceptors may be expressed as autoreceptors in noradrenergic neurons. It seems possible that alpha2C-autoreceptors depress mainly release at low (around 1 Hz) whereas alpha2A-autoreceptors depress mainly release at high (around 10 Hz) frequencies.

The function of presynaptic alpha2-autoreceptors was studied in the hippocampus, occipito-parietal cortex, atria and vas deferens of NMRI mice, mice in which the alpha2A/D-, the alpha2B- or alpha2c-adrenoceptor gene had been disrupted (alpha2A/DKO, alpha2BKO and alpha2CKO, respectively), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. The alpha2-adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium in all tissues from all mouse strains (stimulation with single pulses or single high-frequency pulse trains, called POPs, i.e. pulse patterns leading to minimal autoinhibition). The effects of medetomidine did not differ in NMRI, wildtype, alpha2BKO and alpha2CKO mice but were greatly reduced in alpha2A/DKO brain preparations and to a lesser extent in alpha2A/DKO atria and vasa deferentia. Six drugs were tested as antagonists against medetomidine. Their pKd values indicated that the hippocampal and occipito-parietal alpha2-autoreceptors in NMRI and wildtype mice were alpha2D (the rodent variant of the alpha2A/D-adrenoceptor) whereas the atrial and vas deferens alpha2-autoreceptors in NMRI and wildtype mice could not be identified with a single alpha2 subtype. Deletion of the alpha2A/D gene changed the pKd values in all tissues so that they now reflected alpha2C properties, whereas deletion of the alpha2C gene changed the pKd values in atria and vasa deferentia so that they now had alpha2D properties (as they had in NMRI and wildtype brain preparations). Autoinhibition by released noradrenaline was created using trains of up to 64 pulses or up to 4 POPs, and the overflow-enhancing effect of the alpha2 antagonist rauwolscine was determined. Results did not differ, irrespective of whether preparations were obtained from NMRI, wildtype, alpha2BKO or alpha2CKO mice: the overflow of tritium elicited by p pulses or POPs was much smaller than p times the overflow elicited by a single pulse or POP, and rauwolscine greatly increased the evoked overflow. Results differed, however, in tissues taken from alpha2A/DKO mice: in these tissues, the overflow of tritium elicited by p pulses or POPs was close to p times the overflow elicited by a single pulse or POP, and rauwolscine did not increase the evoked overflow of tritiumor increased it only marginally. When a greater degree of autoinhibition was produced in atria and vasa deferentia by stimulation with 120 pulses, both disruption of the alpha2A/D gene and disruption of the alpha2C gene but not disruption of the alpha2B gene attenuated the overflow-enhancing effects of phentolamine and rauwolscine. In NMRI and wildtype atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine at enhancing the evoked overflow were not easily compatible with a single alpha2 subtype. In alpha2A/DKO atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine indicated that the autoinhibition-mediating receptors were alpha2C, whereas in alpha2CKO atria and vasa deferentia the relative potencies indicated that the autoinhibition-mediating receptors were alpha2D. It is concluded that alpha2-autoreceptors function identically in NMRI mice and the wildtype mice from which the receptor-deficient animals had been generated. There is no evidence from the experiments for any contribution of alpha2B-adrenoceptors to autoreceptor function. The main presynaptic alpha2-autoreceptors are alpha2A/D, both as sites of action of exogenous agonists and as sites of action of previously released noradrenaline. However, there are in addition non-alpha2A/D-, probably alpha2C-autoreceptors. They are less prominent in mediating the inhibitory effects of exogenous agonists and the negative feedback effect of released noradrenaline. They operate not only after deletion of the alpha2A/D-adrenoceptors but also in normal (NMRI, wildtype) mice without gene deletion.

The noradrenergic system is involved in the regulation of many physiological and psychological processes, including the modulation of mood. The alpha(2)-adrenergic receptors (alpha(2)-ARs) modulate norepinephrine release, as well as the release of serotonin and other neurotransmitters, and are therefore potential targets for antidepressant and anxiolytic drug development. The current studies were undertaken to examine the role of the alpha(2A) subtype of alpha(2)-AR in mouse behavioral models of depression and anxiety. We have observed that the genetic knock-out of the alpha(2A)-AR makes mice less active in a modified version of Porsolt's forced swim test and insensitive to the antidepressant effects of the tricyclic drug imipramine in this paradigm. Furthermore, alpha(2A)-AR knock-out mice appear more anxious than wild-type C57 Bl/6 mice in the rearing and light-dark models of anxiety after injection stress. These findings suggest that the alpha(2A)-AR may play a protective role in some forms of depression and anxiety and that the antidepressant effects of imipramine may be mediated by the alpha(2A)-AR.

The present study investigated the role of alpha(2C)-adrenoceptors in the regulation of activity and discrimination accuracy in an operant chamber test. We trained food deprived control and alpha(2C)-adrenoceptor knockout mice to collect liquid food rewards in an operant chamber during the light (20 s) period. No food reward was delivered during the dark period (40 s). The alpha(2C)-adrenoceptor knockout mice tended to make fewer total responses and collect less rewards than their controls after saline treatment. However, only response accuracy of alpha(2C)-adrenoceptor knockout mice was significantly lower than that of the control mice. Methylphenidate, a drug blocking dopamine re-uptake and increasing dopamine release, dose-dependently decreased the number of total responses and collected food rewards in control mice but increased those measures in alpha(2C)-adrenoceptor knockout mice. In addition, the effect of methylphenidate on discrimination accuracy differed between knockout and control mice. Our results indicate that alpha(2C)-adrenoceptors may regulate dopamine-mediated functions.

Alpha2-Adrenoceptor-mediated inhibition of [3H]noradrenaline release and alpha2-adrenoceptor-mediated inhibition of voltage-activated Ca2+ currents were compared in cultured thoracolumbar postganglionic sympathetic neurons from newborn wildtype (WT) mice and mice in which the alpha2A/D-adrenoceptor gene had been disrupted (alpha2A/DKO). In cultures prepared from WT mice and preincubated with [3H]noradrenaline, the alpha2-adrenoceptor agonist 5-bromo-6-(2-imidazolidinylidenamino)quinoxaline (UK 14,304) reduced the (autoinhibition-free) release of [3H]noradrenaline elicited by single electrical pulses or trains of 8 pulses at 100 Hz. The maximal inhibition by UK 14,304 amounted to 70%-85%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) and, to a smaller extent, rauwolscine (0.3 microM). Pretreatment of the cultures with pertussis toxin abolished the effect of UK 14,304. Phentolamine and rauwolscine increased the (alpha2-autoinhibited) release of [3H]noradrenaline elicited by 18, 36 or 72 pulses at 3 Hz. In cultures from alpha2A/DKO mice, UK 14,304 failed to reduce the release of [3H]noradrenaline elicited by single pulses and phentolamine and rauwolscine failed to increase the release of [3H]noradrenaline elicited by 18-72 pulses at 3 Hz. In neurons from WT mice examined with the amphotericin B-perforated configuration of the patch clamp method, UK 14,304 reduced depolarisation-evoked Ca2+ currents. The inhibition was voltage-dependent as shown by a decline at strong depolarisation during ramp-like voltage commands and by an attenuation briefly after a conditioning depolarising pulse. The maximal inhibition by UK 14,304 was 39%. Its concentration-response curve was shifted to the right by phentolamine (0.3 microM) but not significantly changed by rauwolscine (0.3 microM) and prazosin (1 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304. In neurons from alpha2A/DKO mice, UK 14,304 also reduced depolarisation-evoked Ca2+ currents, but with a smaller maximal effect, namely 18% inhibition. Its concentration-response curve was shifted to the right by rauwolscine (0.3 microM) and prazosin (1 microM) but not significantly changed by phentolamine (0.3 microM). Pretreatment with pertussis toxin abolished the effect of UK 14,304 also in cultures from alpha2A/DKO mice. It is concluded that the only presynaptic alpha2-autoreceptors that detectably depress transmitter release from cultured thoracolumbar sympathetic neurons taken from newborn mice are alpha2A/D. In contrast, the soma-dendritic alpha2-autoreceptors that inhibit voltage-gated Ca2+ channels are both alpha2A/D and non-alpha2A/D (i.e. alpha2B or alpha2c). Both presynaptic alpha2A/D- and soma-dendritic alpha2A/D- and non-alpha2A/D-autoreceptors operate through pertussis toxin-sensitive G proteins in these neurons.

alpha2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of alpha2-ARs have been cloned (alpha2A, alpha2B, and alpha2C). Here we describe the physiological consequences of disrupting the gene for the alpha2A-AR. Mice lacking functional alpha2A subtypes were compared with wild-type (WT) mice, with animals lacking the alpha2B or alpha2C subtypes, and with mice carrying a point mutation in the alpha2A-AR gene (alpha2AD79N). Deletion of the alpha2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 +/- 21 min-1; WT, 395 +/- 21 min-1), depletion of cardiac tissue norepinephrine concentration (knockout, 676 +/- 31 pg/mg protein; WT, 1178 +/- 98 pg/mg protein), and down-regulation of cardiac beta-ARs (Bmax: knockout, 23 +/- 1 fmol/mg protein; WT, 31 +/- 2 fmol/mg protein). The hypotensive effect of alpha2 agonists was completely absent in alpha2A-deficient mice. Presynaptic alpha2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective alpha2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from alpha2B-AR knockout, alpha2C-AR knockout, alpha2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the alpha2 agonist in alpha2A-AR knockout mice was only 42 +/- 9%. [3H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the alpha2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual alpha2-mediated effect in the alpha2A-AR knockout mice suggests that a second alpha2 subtype (alpha2B or alpha2C) also functions as a presynaptic autoreceptor to inhibit transmitter release.