GPR88 in A2A receptor-expressing neurons modulates locomotor response to dopamine agonists but not sensorimotor gating.

The orphan receptor, GPR88, is emerging as a key player in the pathophysiology of several neuropsychiatric diseases, including psychotic disorders. Knockout (KO) mice lacking GPR88 throughout the brain exhibit many abnormalities relevant to schizophrenia including locomotor hyperactivity, behavioural hypersensitivity to dopaminergic psychostimulants and deficient sensorimotor gating. Here, we used conditional knockout (cKO) mice lacking GPR88 selectively in striatal medium spiny neurons expressing A2A receptor to determine neuronal circuits underlying these phenotypes. We first studied locomotor responses of A2A R-Gpr88 KO mice and their control littermates to psychotomimetic, amphetamine, and to selective D1 and D2 receptor agonists, SKF-81297 and quinpirole, respectively. To assess sensorimotor gating performance, mice were submitted to acoustic and visual prepulse inhibition (PPI) paradigms. Total knockout GPR88 mice were also studied for comparison. Like total GPR88 KO mice, A2A R-Gpr88 KO mice displayed a heightened sensitivity to locomotor stimulant effects of amphetamine and SKF-81297. They also exhibited enhanced locomotor activity to quinpirole, which tended to suppress locomotion in control mice. By contrast, they had normal acoustic and visual PPI, unlike total GPR88 KO mice that show impairments across different sensory modalities. Finally, none of the genetic manipulations altered central auditory temporal processing assessed by gap-PPI. Together, these findings support the role of GPR88 in the pathophysiology of schizophrenia and show that GPR88 in A2A receptor-expressing neurons modulates psychomotor behaviour but not sensorimotor gating.

Prevention of neutrophil extravasation by α2-adrenoceptor-mediated endothelial stabilization.

Adrenergic receptors are expressed on the surface of inflammation-mediating cells, but their potential role in the regulation of the inflammatory response is still poorly understood. The objectives of this work were to study the effects of α2-adrenergic agonists on the inflammatory response in vivo and to determine their mechanism of action. In two mouse models of inflammation, zymosan air pouch and thioglycolate-induced peritonitis models, the i.m. treatment with xylazine or UK14304, two α2-adrenergic agonists, reduced neutrophil migration by 60%. The α2-adrenergic antagonist RX821002 abrogated this effect. In flow cytometry experiments, the basal surface expression of L-selectin and CD11b was modified neither in murine nor in human neutrophils upon α2-agonist treatment. Similar experiments in HUVEC showed that UK14304 prevented the activation-dependent upregulation of ICAM-1. In contrast, UK14304 augmented electrical resistance and reduced macromolecular transport through a confluent HUVEC monolayer. In flow chamber experiments, under postcapillary venule-like flow conditions, the pretreatment of HUVECs, but not neutrophils, with α2-agonists decreased transendothelial migration, without affecting neutrophil rolling. Interestingly, α2-agonists prevented the TNF-α-mediated decrease in expression of the adherens junctional molecules, VE-cadherin, ß-catenin, and plakoglobin, and reduced the ICAM-1-mediated phosphorylation of VE-cadherin by immunofluorescence and confocal analysis and Western blot analysis, respectively. These findings indicate that α2-adrenoceptors trigger signals that protect the integrity of endothelial adherens junctions during the inflammatory response, thus pointing at the vascular endothelium as a therapeutic target for the management of inflammatory processes in humans.

Sex, pain, and opioids: interdependent influences of sex and pain modality on dynorphin-mediated antinociception in rats.

The role of dynorphin A (1-17; Dyn) and its associated kappa opioid receptor (KOR) in nociception represents a longstanding scientific conundrum: Dyn and KOR (Dyn/KOR) have variously been reported to inhibit, facilitate, or have no effect on pain. We investigated whether interactions between sex and pain type (which are usually ignored) influenced Dyn/KOR-mediated antinociception. Blockade of the spinal α(2)-noradrenergic receptor (α(2)-NAR) using yohimbine elicited comparable spinal Dyn release in females and males. Nevertheless, the yohimbine-induced antinociception exhibited sexual dimorphism that depended on the pain test used: in the intraperitoneal acetic acid-induced writhing test, yohimbine produced antinociception only in females, whereas in the intraplantar formalin-induced paw flinch test, antinociception was observed only in males. In females and males, both intrathecal Dyn antibodies and spinal KOR blockade eliminated the yohimbine-induced antinociception, indicating that Dyn/KOR mediated it. However, despite the conditional nature of spinal Dyn/KOR-mediated yohimbine antinociception, both intraplantar formalin and intraperitoneal acetic acid activated spinal Dyn neurons that expressed α(2)-NARs. Moreover, Dyn terminals apposed KOR-expressing spinal nociceptive neurons in both sexes. This similar organization suggests that the sexually dimorphic interdependent effects of sex and pain type may result from the presence of nonfunctional (silent) KORs on nociceptive spinal neurons that are responsive to intraplantar formalin (in females) versus intraperitoneal acetic acid (in males). Our findings that spinal Dyn/KOR-mediated antinociception depends on interactions between sex and pain type underscore the importance of using both sexes and multiple pain models when investigating Dyn/KOR antinociception.

The antidepressant desipramine is an arrestin-biased ligand at the α(2A)-adrenergic receptor driving receptor down-regulation in vitro and in vivo.

The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as an inhibitor of norepinephrine reuptake. Available evidence, however, suggests a mechanism more complex than simple reuptake inhibition. In the present study, we have characterized the direct interaction between DMI and the α(2A)-adrenergic receptor (α(2A)AR), a key regulator of noradrenergic neurotransmission with altered expression and function in depression. DMI alone was found to be sufficient to drive receptor internalization acutely and a robust down-regulation of α(2A)AR expression and signaling following prolonged stimulation in vitro. These effects are achieved through arrestin-biased regulation of the receptor, as DMI selectively induces recruitment of arrestin but not activation of heterotrimeric G proteins. Meanwhile, a physiologically relevant concentration of endogenous agonist (norepinephrine) was unable to sustain a down-regulation response. Prolonged in vivo administration of DMI resulted in significant down-regulation of synaptic α(2A)AR expression, a response that was lost in arrestin3-null animals. We contend that direct DMI-driven arrestin-mediated α(2A)AR down-regulation accounts for the therapeutically desirable but mechanistically unexplained adaptive alterations in receptor expression associated with this antidepressant. Our results provide novel insight into both the pharmacology of this antidepressant drug and the targeting of the α(2A)AR in depression.

Regional differences in sexually dimorphic protein expression in the spontaneously hypertensive rat (SHR).

Hypertension is sexually dimorphic and modified by removal of endogenous sex steroids. This study tested the hypothesis that endogenous gonadal hormones exert differential effects on protein expression in the kidney and mesentery of SHR. At ~5 weeks of age male and female SHR underwent sham operation, orchidectomy, or ovariectomy (OVX). At 20-23 weeks of age, mean arterial pressure (MAP) was measured in conscious rats. The mesenteric arterial tree and kidneys were collected, processed for Western blots, and probed for Cu Zn superoxide dismutase (SOD1), soluble epoxide hydrolase (sEH), and Alpha 2A adrenergic receptor (A2AR) expression. MAP was unaffected by ovariectomy (Sham 164 ± 4: Ovariecttomy 159 ± 3 mm Hg). MAP was reduced by orchidectomy (Sham 189 ± 5:Orchidectomy 167 ± 2 mm Hg). In mesenteric artery, SOD1 expression was greater in male versus female SHR. Orchidectomy increased while ovariectomy decreased SOD1 expression. The kidney exhibited a different pattern of response. SOD1 expression was reduced in male compared to female SHR but gonadectomy had no effect. sEH expression was not significantly different among the groups in mesenteric artery. In kidney, sEH expression was greater in males compared to females. Ovariectomy but not orchidectomy increased sEH expression. A2AR expression was greater in female than male SHR in mesentery artery and kidney. Gonadectomy had no effect in either tissue. We conclude that sexually dimorphic hypertension is associated with regionally specific changes in expression of three key proteins involved in blood pressure control. These data suggest that broad spectrum inhibition or stimulation of these systems may not be the best approach for hypertension treatment. Instead regionally targeted manipulation of these systems should be investigated.

[The increase in the proportion of nervous animals bred for catatonia: the participation of central adrenoreceptors in catatonic reactions].

Using a large amount of breeding material, the idea of D. K. Belyaev on the role of selection in the appearance of new behavioral and neuronal forms was confirmed. Experiments were performed using rats of the GC (genetics + catatonia) strain, which are prone to passive defensive reactions of cataleptic freezing. At the current breeding stage, elevation of the proportion of so-called nervous animals was demonstrated, both with respect to the expression of such reactions and their frequency. At this breeding stage, in the brains of GC rats, the mRNA levels of alpha1A- and alpha2A-adrenoreceptor genes were determined. A decrease of alpha1A-adrenoreceptor gene expression in the midbrain and medulla oblongata, along with elevation of alpha2A-adrenoreceptor gene expression in the frontal cortex was observed. It was suggested that changes in the expression of alpha-adrenoreceptor genes could be caused by an increase in the proportion of nervous animals and could contribute to the akinetic behavioral component in GC rats.

Increased presynaptic and postsynaptic α2-adrenoceptor activity in the spinal dorsal horn in painful diabetic neuropathy.

Diabetic neuropathy is a common cause of chronic pain that is not adequately relieved by conventional analgesics. The α(2)-adrenoceptors are involved in the regulation of glutamatergic input and nociceptive transmission in the spinal dorsal horn, but their functional changes in diabetic neuropathy are not clear. The purpose of the present study was to determine the plasticity of presynaptic and postsynaptic α(2)-adrenoceptors in the control of spinal glutamatergic synaptic transmission in painful diabetic neuropathy. Whole-cell voltage-clamp recordings of lamina II neurons were performed in spinal cord slices from streptozotocin-induced diabetic rats. The amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root and the frequency of spontaneous EPSCs (sEPSCs) were significantly higher in diabetic than vehicle-control rats. The specific α(2)-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14304) (0.1-2 µM) inhibited the frequency of sEPSCs more in diabetic than vehicle-treated rats. UK-14304 also inhibited the amplitude of evoked monosynaptic and polysynaptic EPSCs more in diabetic than control rats. Furthermore, the amplitude of postsynaptic G protein-coupled inwardly rectifying K(+) channel (GIRK) currents elicited by UK-14304 was significantly larger in the diabetic group than in the control group. In addition, intrathecal administration of UK-14304 increased the nociceptive threshold more in diabetic than vehicle-control rats. Our findings suggest that diabetic neuropathy increases the activity of presynaptic and postsynaptic α(2)-adrenoceptors to attenuate glutamatergic transmission in the spinal dorsal horn, which accounts for the potentiated antinociceptive effect of α(2)-adrenoceptor activation in diabetic neuropathic pain.

Antinociceptive effect of intrathecal ginsenosides through alpha-2 adrenoceptors in the formalin test of rats.

BACKGROUND: We defined the nature of the pharmacological interaction after intrathecal co-administration of ginsenosides with clonidine, and clarified the contribution of the α-2 adrenoceptors on the effect of ginsenosides. METHODS: Pain was evoked by injection of a formalin solution (5%, 50 µl) into the hindpaw of male Sprague-Dawley rats. Isobolographic analysis was performed to characterize the drug interaction between ginsenosides and clonidine. The antagonism of ginsenosides-mediated antinociception was determined with α-2A (BRL 44408), α-2B (ARC 239), and α-2C (JP 1302) adrenoceptor antagonists. The expression of α-2 adrenoceptor subtypes was examined by reverse transcriptase-polymerase chain reaction. RESULTS: Intrathecal ginsenosides (n=29) and clonidine (n=31) displayed an antinociceptive effect. The ED(50) values (95% confidence intervals) of ginsenosides and clonidine for phases 1 and 2 were 109.5 (63-190.3) and 110.9 (57.1-215.5), and 11.8 (3.7-37.1) and 4.9 (3.1-6.7) µg, respectively. With an isobolographic study (n=48), the ED(50) values (95% confidence intervals) of ginsenosides in the combination of ginsenosides and clonidine for phases 1 and 2 were 58.2 (38.9-87.3) and 57.2 (46.5-70.3) µg, respectively. Intrathecal BRL 44408 (n=6), ARC 239 (n=5), and JP 1302 (n=5) reversed the antinociception of ginsenosides in both phases (P<0.01, <0.001). The injection of formalin increased the expression of α-2C adrenoceptor in the spinal cord (P<0.05). CONCLUSIONS: Intrathecal ginsenosides additively interacted with clonidine in the formalin test. Furthermore, α-2A, -B, and -C adrenoceptors contributed to the antinociception of intrathecal ginsenosides.

ADP-ribosylation factors modulate the cell surface transport of G protein-coupled receptors.

ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of alpha(2B)-adrenergic receptor (AR), beta(2)-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that alpha(2B)-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the alpha(2B)-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.

Transcriptional down-regulation of human alpha(2A)-adrenoceptors by IFNgamma and TNFalpha in intestinal cells.

alpha(2A)-adrenoceptors are expressed on intestinal cells and they participate in the control of epithelial functions such as solute and water transport or cell proliferation. In pathological conditions, pro-inflammatory cytokines secreted by lymphocytes are responsible for modification of intestinal cell characteristics including phenotype switch and changes in the expression of pumps and ion channels. Using the HT29 cell line as a model, the present work examined the effect of two inflammatory cytokines, interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), on the expression of the human alpha(2A)-adrenoceptor. Exposure of cells to either IFNgamma or TNFalpha resulted in a concentration- and time-dependent diminution of [(3)H]RX821002 binding sites, which is preceded by a large decrease in the amount of alpha(2A)-adrenoceptor mRNA. The cytokines did not affect the receptor mRNA half-life, but inhibited the activity of a luciferase construct containing the promoter region of alpha(2A)-adrenoceptor gene, indicating that a decrease in the transcription rate is primarily responsible for the diminution of receptor expression. Exposure of cells to either IFNgamma or TNFalpha caused increased production of reactive oxygen species and transient phosphorylation of extracellular signal-regulated kinase (Erk1/2). The effect of cytokines was mimicked by H(2)O(2) but was unaffected by the addition of anti-oxidants. The blockade of Erk1/2 activation by PD98059 blunted the effect of TNFalpha but not of IFNgamma. In conclusion, the present findings demonstrate that IFNgamma and TNFalpha diminish the alpha(2A)-adrenoceptor expression in HT29 cells by decreasing the transcription rate without modifying the stability of mRNA. The transcription inhibition is however triggered via different signalling pathways. The results suggest that cytokine-mediated down-regulation of alpha(2A)-adrenoceptor could contribute to the pathogenesis of inflammatory bowel disease.

[Adrenergic reactivity and course of the wound process in diabetes mellitus condition: diagnostic significance and pathogenetic interpretation].

In order to establish interrelationship between the organism adrenoreactivity and the wound process course in presence of diabetes mellitus the dynamics of effective concentration of alpha2-adrenoreceptors agonist (alpha2-AR), causing aggregation of 50% of thrombocytes (EC50) in patients, suffering diabetes mellitus, complicated by purulent-necrotic affection of lower extremities in admission to the hospital and during the period of treatment, was studied. The cutaneous wounds healing was accompanied by the organism adrenoreactivity normalization, in condition of durable unhealing of the wound the alpha2-AR sensitivity tolerance to the therapy performed was noted. It permits to consider the EC50 index as a criterion of severity of dysregulation changes and the therapy efficacy.

Sex mediates dopamine and adrenergic receptor expression in adult rats exposed prenatally to cocaine.

The extent of catecholaminergic receptor and respective behavioral alterations associated with prenatal cocaine exposure varies according to exogenous factors such as the amount, frequency, and route of maternal exposure, as well as endogenous factors such as specific brain regions under consideration and sex of the species. The goal of the current study was to use autoradiography to delineate possible moderators of dopaminergic and adrenergic receptor expression in adult rat offspring exposed to cocaine in utero. The current study demonstrated sex-dependent D1 receptor, alpha2, and noradrenergic transporter binding alterations in prelimbic, hippocampus, and anterior cingulate regions of adult rat brains exposed to cocaine during gestational days 8-21. Of further interest was the lack of alterations in the nucleus accumbens for nearly all receptors/transporters investigated, as well as the lack of alterations in D3 receptor binding in nearly all of the regions investigated (nucleus accumbens, prelimbic region, hippocampus, and cingulate gyrus). Thus, the current investigation demonstrated persistent receptor and transporter alterations that extend well into adulthood as a result of cocaine exposure in utero. Furthermore, the demonstration that sex played a mediating role in prenatal cocaine-induced, aberrant receptor/transporter expression is of primary importance for future studies that seek to control for sex in either design or analysis.

Dexmedetomidine Attenuates Lipopolysaccharide Induced MCP-1 Expression in Primary Astrocyte.

Background. Neuroinflammation which presents as a possible mechanism of delirium is associated with MCP-1, an important proinflammatory factor which is expressed on astrocytes. It is known that dexmedetomidine (DEX) possesses potent anti-inflammatory properties. This study aimed to investigate the potential effects of DEX on the production of MCP-1 in lipopolysaccharide-stimulated astrocytes. Materials and Methods. Astrocytes were treated with LPS (10 ng/ml, 50 ng/ml, 100 ng/ml, and 1000 ng/ml), DEX (500 ng/mL), LPS (100 ng/ml), and DEX (10, 100, and 500 ng/mL) for a duration of three hours; expression levels of MCP-1 were measured by real-time PCR. The double immunofluorescence staining protocol was utilized to determine the expression of α2-adrenoceptors (α2AR) and glial fibrillary acidic protein (GFAP) on astrocytes. Results. Expressions of MCP-1 mRNA in astrocytes were induced dose-dependently by LPS. Administration of DEX significantly inhibited the expression of MCP-1 mRNA (P < 0.001). Double immunofluorescence assay showed that α2AR colocalize with GFAP, which indicates the expression of α2-adrenoceptors in astrocytes. Conclusions. DEX is a potent suppressor of MCP-1 in astrocytes induced with lipopolysaccharide through α2A-adrenergic receptors, which potentially explains its beneficial effects in the treatment of delirium by attenuating neuroinflammation.

Mild prenatal protein malnutrition increases alpha 2C-adrenoceptor expression in the rat cerebral cortex during postnatal life.

Mild reduction in the protein content in the diet of pregnant rats from 25 to 8% casein, calorically compensated by carbohydrates, does not alter body and brain weights of rat pups at birth, but results in significant changes of the concentration and release of cortical noradrenaline during postnatal life, together with impaired long-term potentiation and memory formation. Since some central noradrenergic receptors are critically involved in neuroplasticity, the present study evaluated, by utilizing immunohistochemical methods, the effect of mild prenatal protein malnutrition on the alpha 2C-adrenoceptor expression in the frontal and occipital cortices of 8- and 60-day-old rats. At day 8 of postnatal age, prenatally malnourished rats exhibited a three-fold increase of alpha 2C-adrenoceptor expression in both the frontal and the occipital cortices, as compared to well-nourished controls. At 60 days of age, prenatally malnourished rats showed normal expression levels scores of alpha 2C-adrenoceptor in the neocortex. Results suggest that overexpression of neocortical alpha 2C-adrenoceptors during early postnatal life, subsequent to mild prenatal protein malnutrition, could in part be responsible for neural and behavioral disturbances showing prenatally malnourished animals during the postnatal life.

Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2.

The molecular mechanisms that control the targeting of newly synthesized G protein-coupled receptors (GPCRs) to the functional destinations remain poorly elucidated. Here, we have determined the role of Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins 1 and 2 (GGA1 and GGA2) in the cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and studied the underlying mechanisms. We demonstrated that knockdown of GGA1 and GGA2 by shRNA and siRNA significantly reduced the cell surface expression of inducibly expressed α2B-AR and arrested the receptor in the perinuclear region. Knockdown of each GGA markedly inhibited the dendritic expression of α2B-AR in primary cortical neurons. Consistently, depleting GGA1 and GGA2 attenuated receptor-mediated signal transduction measured as ERK1/2 activation and cAMP inhibition. Although full length α2B-AR associated with GGA2 but not GGA1, its third intracellular loop was found to directly interact with both GGA1 and GGA2. More interestingly, further mapping of interaction domains showed that the GGA1 hinge region and the GGA2 GAE domain bound to multiple subdomains of the loop. These studies have identified an important function and revealed novel mechanisms of the GGA family proteins in the forward trafficking of a cell surface GPCR.

Neurokinin-1 projection cells in the rat dorsal horn receive synaptic contacts from axons that possess alpha2C-adrenergic receptors.

Thealpha2C subclass of adrenergic receptor (alpha2C-AR) mediates some of the antinociceptive actions of norepinephrine in the spinal cord. Axon terminals, which possess this receptor, are concentrated in the superficial dorsal horn and originate from spinal interneurons. We performed a series of combined tract-tracing and immunocytochemical studies to determine whether alpha2C-AR-immunoreactive axons target projection neurons that possess the neurokinin-1 (NK-1) receptor because such cells are likely to transmit nociceptive information to the brain. Spinomedullary neurons were labeled by stereotaxic injection of the B-subunit of cholera toxin (CTb) into the caudal ventrolateral medulla of three anesthetized adult rats. After 3 d, the animals were anesthetized again and fixed by perfusion. Sections were cut from midlumbar segments and reacted with antibodies to reveal alpha2C-ARs, CTb, and NK-1 receptors. Retrogradely labeled neurons possessing the NK-1 receptor (n = 45) were examined with confocal microscopy to investigate their relationship with alpha2C-AR-immunoreactive axons. Numerous alpha2C-AR axons were apposed to cell bodies and proximal dendrites of cells in lamina I and also to distal dendrites that originate from labeled cell bodies in lamina III/IV. A combined confocal and electron microscopic method confirmed that these appositions were synaptic. Additional experiments showed that virtually all alpha2C-AR terminals in contact with labeled cells are also immunoreactive for the vesicular glutamate transporter 2 and therefore are glutamatergic. These data suggest that norepinephrine can modulate excitatory synaptic transmission from spinal interneurons to projection cells by acting at alpha2C-ARs. This could be one of the mechanisms that underlie the antinociceptive actions of norepinephrine.

Identification in islets of Langerhans of a new rat alpha 2-adrenergic receptor.

The islets of Langerhans are richly innervated, and an inhibitory effect on insulin secretion, mediated through alpha 2-adrenergic receptors, appears to be an important physiological neural modulator of beta-cell function. An alpha 2-receptor was cloned from isolated newborn rat islets using a polymerase chain reaction (PCR) approach. This receptor was shown by sequencing to be a new rat alpha 2-receptor very similar to the human alpha 2-C2 receptor. No other alpha 2-receptor subtype was identified in normal islets by the PCR using alpha 2-receptor primers. This was also the only alpha 2-receptor subtype present in the exocrine pancreas and liver. In contrast, in the beta-cell line, beta TC3, the alpha 2-C2 receptor was not detected, but the alpha 2-C4 and alpha 2-C10 receptor subtypes were detected. It is suggested that the alpha 2-C2 subtype may be the principal alpha 2-receptor mediating inhibitory autonomic nervous system activity in the gastrointestinal tract. A comparison of the rat islet, pancreas, and liver alpha 2-receptor sequences reported here with previously reported alpha 2-receptor sequences indicates that the rat islet alpha 2-receptor is not the rat alpha 2-C2 homologue previously denoted as RNG alpha 2, but is a new, fourth rat subtype with an even higher similarity to the human alpha 2-C2 receptor.

Imidazoline and alpha(2a)-adrenoceptor binding sites in postmenopausal women before and after estrogen replacement therapy.

BACKGROUND: Platelet alpha(2A)-adrenoceptors (alpha(2A)AR) and imidazoline binding sites (subtype I(1)) have been proposed as peripheral markers of brain stem receptors that mediate sympathetic outflow and are reported to be elevated in major depression. METHODS: In our study, p[(125)I]-iodoclonidine was used to assess platelet alpha(2A)AR and I(1) binding sites in healthy postmenopausal women (n = 34) compared with healthy women of reproductive age (n = 26). Receptor determinations were repeated in 19 postmenopausal women following 59-60 days of estrogen replacement therapy (ERT; 0.1 mg estradiol transdermal patches). RESULTS: I(1) binding sites were twofold higher in platelets of postmenopausal women compared with women of reproduction age but were down-regulated (normalized) after 59-60 days of ERT. All other binding parameters, including platelet alpha(2A)AR density, were not different between groups nor were they changed after ERT. Platelet I(1) densities after 59-60 days of ERT were positively correlated with plasma luteinizing hormone concentrations. CONCLUSIONS: It is suggested that increased imidazoline binding sites might be associated with mood and behavioral changes in postmenopausal women.

Hippocampal alpha2a-adrenergic receptors are located predominantly presynaptically but are also found postsynaptically and in selective astrocytes.

Alpha-adrenergic receptor, subtype 2A (alpha2A-AR), activation is one of the primary modes of action for norepinephrine (NE) in the rat hippocampal formation. In this study, alpha2A-AR immunoreactivity (alpha2A-AR-I) was localized by light and electron microscopy in the rat hippocampus and dentate gyrus by using a previously characterized antibody to the rat alpha2A-AR. By light microscopy, dense alpha2A-AR-I was observed in the pyramidal and granule cell layers. Diffuse and slightly granular alpha2A-AR-I was found in the neuropil in all other laminae, notably stratum lacunosum-moleculare. Ultrastructurally, alpha2A-AR-I was found in neuronal cytoplasm associated with large multivesicular-like organelles and with clusters adjacent to endoplasmic reticula and/or plasmalemma. The distribution of alpha2A-AR-I in the strata oriens, radiatum, and lacunosum-moleculare of hippocampal CA1 and CA3 regions and in the molecular layer of the dentate gyrus was remarkably similar (n > 2,000 profiles examined): alpha2A-AR-I was found in axons and terminals (approximately 40%), glia (approximately 30%), dendritic spines (approximately 25%), and dendritic shafts (approximately 5%). This mixed pre- and postsynaptic distribution was not seen in the stratum lucidum of the CA3 region and the dentate hilar region, where most alpha2A-AR-I was found in axons (approximately 60%) and glia (approximately 30%). Alpha-2A-AR-labeled axons were small and unmyelinated; labeled terminals usually formed asymmetric synapses on unlabeled spines; and labeled dendritic spines were morphologically similar to pyramidal or granule cells. Dual labeling studies demonstrated that some axons contained alpha2A-AR-I and tyrosine hydroxylase (TH), the catecholaminergic synthesizing enzyme, and that some TH-labeled terminals were in close proximity to alpha2A-AR-labeled spines and glia. These studies demonstrate that hippocampal alpha2A-AR-I is localized (1) presynaptically in both noncatecholaminergic and catecholaminergic terminals, (2) postsynaptically in the dendritic spines of pyramidal and granule cells near catecholaminergic terminals, and (3) in some glial processes. These results suggest several sites for NE to exert its effects on hippocampal alpha2A-ARs.

Increased mRNA expression of alpha2A-adrenoceptors, serotonin receptors and mu-opioid receptors in the brains of suicide victims.

The development of new therapies for the treatment of psychiatric disorders requires an in-depth knowledge of the molecular bases underlying these pathologies, which remain largely unknown. Alterations in adrenoceptors, serotonin receptors, and other G protein-coupled receptors (GPCRs) have been associated with suicide and depression. However, to date, there is little information about mRNA expression of the GPCRs in the frontal cortex of suicide victims. Our goal was to study the expression in the brain of these receptors. For this purpose, we measured mRNA levels by RT-PCR. We found that the expressions of alpha2A-adrenoceptors, 5-HT1A, 5-HT2A serotonin receptors, and mu-opioid receptors were elevated in the post-mortem brains of these suicide victims with respect to matched controls. Moreover, in the case of alpha2A-adrenoceptors (the only for which these data were available), a significant correlation was observed between the level of mRNA and protein quantified in the brain of the same subjects, indicating that protein synthesis of this receptor was not influenced by post-translational regulatory mechanisms. In addition, the degree of adrenoceptor and 5-HT receptor expressions appeared to be correlated in the brains of suicide victims and control subjects. Alterations in the expression of adrenoceptors, serotonin, and opioid receptors indicate that these signaling proteins might be related to the etiopathology of suicidal and depressive behaviors. Alternatively, such changes may represent adaptive mechanisms to compensate for other as yet unknown alterations. The results also suggest that these receptors could share common regulatory mechanisms.