Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag.

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.

Involvement of the Hippocampal Alpha2A-Adrenoceptors in Anxiety-Related Behaviors Elicited by Intermittent REM Sleep Deprivation-Induced Stress in Mice.

Attention deficit/hyperactivity disorder (AD/HD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, and impulsivity. In patients with AD/HD, a decrease in the total and rapid eye movement (REM) sleep times has been observed. We have previously reported that mice with REM sleep deprivation-induced stress (REMSD) may show the hyperactivity- and inattention-like symptoms of AD/HD. However, in this model, impulsivity has not yet been investigated. Impulsivity and anxiety-related behaviors are evaluated by the elevated plus maze test (EPM). In this study, we investigated whether REMSD causes changes in the EPM and expression of alpha2A-adrenoceptors in the hippocampus and frontal cortex in a mouse model. Mice were deprived of REM sleep intermittently using the small-platform method (20 h/d) for 3 d. The time spent in the open arm and the expression levels of alpha2A-adrenoceptor in the hippocampus were significantly increased and decreased, respectively, by the REMSD. The time spent in the open arm was significantly limited by oxymetazoline (an alpha2A-adrenoceptor agonist), methylphenidate, and atomoxetine, which are clinically used to treat AD/HD. Moreover, the positive effects of oxymetazoline were attenuated by yohimbine and BRL44408, which are selective alpha2- and alpha2A-adrenoceptor antagonists, respectively. These results suggest that the increase in the time spent in the open arm induced by REMSD may serve as a model of impulsivity in AD/HD. Furthermore, the REMSD eliciting impulsivity-like behavior and the low-levels of anxiety may be linked to alpha2A-adrenoceptor signaling, as indicated by a decrease in alpha2A-adrenoceptor signaling, particularly in the mouse hippocampus.

Single-Cell Transcriptomic Map of the Human and Mouse Bladders.

BACKGROUND: Having a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer. METHODS: We used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions. RESULTS: We created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A+ and HRH2+ interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1+ epithelial cells that may be involved with bladder emptying. We verify these TNNT1+ epithelial cells also occur in rat and mouse bladders. CONCLUSIONS: This transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.

Prognostic significance of α- and ß2-adrenoceptor gene expression in breast cancer patients.

AIMS: Breast cancer is the most frequently diagnosed and leading cause of cancer death among women worldwide. It was classified within molecular intrinsic subtypes: luminal A, luminal B, human epidermal growth factor receptor 2-enriched and basal-like. Epinephrine and norepinephrine, released during stress, bind to adrenoceptors. α2 -adrenoceptors are encoded by the ADRA2A, ADRA2B and ADRA2C genes and ß2 by ADRB2. METHODS: We compiled several publicly available Affymetrix gene expression datasets, obtaining a large cohort of 1924 patients with distant metastasis-free survival (DMFS) data and evaluated the association between adrenoceptor expression, clinicopathological markers and outcome. RESULTS: ADRA2A high expressing tumours also expressed hormone receptors and presented diminished tumour size, grade and not compromised lymph nodes. ADRB2 high expression was found in smaller, low grade, oestrogen receptor-positive tumours. Both were significantly associated with the absence of metastasis. High expression of ADRA2C was positively associated with increased tumour size and metastatic relapse. We observed a significant increase in DMFS of patients with high ADRA2A (hazard ratio 0.54, 95% CI 0.45-0.65, P < .001) and ADRB2 (0.77, 0.64-0.93, P = .006) expression and a decrease with ADRA2C high expression (1.45, 1.16-1.81, P = .001). For patients with luminal tumours, ADRA2A was the only factor that retained its significance as an independent predictor of DMFS while ADRA2C expression was an independent predictor for worse prognosis in basal-like tumours. CONCLUSIONS: We herein provide new insight for a potential role of ADRA2A and ADRA2C in breast cancer. In low- and medium-income countries, their incorporation to routine immunohistochemistry analysis of biopsies or tumour samples, could provide additional low-cost prognostic factors.

Quantitative determination of α(2B)-adrenoceptor-evoked myosin light chain phosphorylation in vascular smooth muscle cells.

INTRODUCTION: Phosphorylation of myosin light chains is a biochemical readout of smooth muscle cell contraction. α2-Adrenoceptor agonists and antagonists may have important applications in cardiovascular drug development. To assess α2-adrenoceptor-mediated drug effects on vascular smooth muscle contraction, we developed a cell-based assay for the quantitative determination of myosin light chain phosphorylation (pMLC20) in cultured A7r5 smooth muscle cells from rat aorta, transfected to express the human α2B-adrenoceptor (A7r5-α2B cell line). METHODS: In a 96-well format, confluent and serum-starved cells (+/- inhibitor preincubation) were treated with receptor ligands for 5-120 s and the evoked pMLC20 response was monitored with a quantitative in-cell immunoassay, employing time-resolved fluorescence technology. Western blotting, immunofluorescent labelling and intracellular calcium concentration measurements were used for assay validation. RESULTS: The α2-adrenoceptor agonist dexmedetomidine induced rapid, transient and dose-dependent (EC50 30-65 nM) myosin light chain phosphorylation, peaking at 20-45 s with an Emax value of approximately 60% over vehicle control. The endogenous agonist arginine vasopressin produced responses that were comparable to those evoked by dexmedetomidine. Blockers of α2-adrenoceptors, myosin light chain kinase, Gi-proteins, Gßγ subunits, L-type calcium channels and phospholipase C antagonized the dexmedetomidine-evoked myosin light chain phosphorylation, whereas blockers of protein kinase C and protein kinase A potentiated the response to dexmedetomidine. DISCUSSION: The novel method is suitable as a ligand profiling tool to assess the capacity of ligands to evoke or inhibit vascular smooth muscle cell contraction and for investigating the intracellular pathways involved in this process. The assay now allows the quantitative determination of pMLC20 signal induction or inhibition in vascular smooth muscle cells and is superior to conventional Western blotting due to the reduced number of cells required and the potential for measurement of detailed time curves, multiple treatments and replicates on each plate.

Cannabinoid modulation of alpha2 adrenergic receptor function in rodent medial prefrontal cortex.

Endocannabinoids acting at the cannabinoid type 1 receptor (CB1R) are known to regulate attention, cognition and mood. Previous studies have shown that, in the rat medial prefrontal cortex (mPFC), CB1R agonists increase norepinephrine release, an effect that may be attributed, in part, to CB1Rs localised to noradrenergic axon terminals. The present study was aimed at further characterising functional interactions between CB1R and adrenergic receptor (AR) systems in the mPFC using in vitro intracellular electrophysiology and high-resolution neuroanatomical techniques. Whole-cell patch-clamp recordings of layer V/VI cortical pyramidal neurons in rats revealed that both acute and chronic treatment with the synthetic CB1R agonist WIN 55,212-2 blocked elevations in cortical pyramidal cell excitability and increases in input resistance evoked by the α2-adrenergic receptor (α2-AR) agonist clonidine, suggesting a desensitisation of α2-ARs. These CB1R-α2-AR interactions were further shown to be both action potential- and gamma-aminobutyric acid-independent. To better define sites of cannabinoid-AR interactions, we localised α2A-adrenergic receptors (α2A-ARs) in a genetically modified mouse that expressed a hemoagglutinin (HA) tag downstream of the α2A-AR promoter. Light and electron microscopy indicated that HA-α2A-AR was distributed in axon terminals and somatodendritic processes especially in layer V of the mPFC. Triple-labeling immunocytochemistry revealed that α2A-AR and CB1R were localised to processes that contained dopamine-ß-hydroxylase, a marker of norepinephrine. Furthermore, HA-α2A-AR was localised to processes that were directly apposed to CB1R. These findings suggest multiple sites of interaction between cortical cannabinoid-adrenergic systems that may contribute to understanding the effect of cannabinoids on executive functions and mood.

Increased α2- and ß1-adrenoceptor densities in postmortem brain of subjects with depression: differential effect of antidepressant treatment.

BACKGROUND: Brain α2- and ß-adrenoceptor alterations have been suggested in suicide and major depressive disorder. METHODS: The densities of α2-, ß1- and ß2-adrenoceptors in postmortem prefrontal cortex of 26 subjects with depression were compared with those of age-, gender- and postmortem delay-matched controls. The effect of antidepressant treatment on α2- and ß-adrenoceptor densities was also evaluated. α2- and ß-adrenoceptor densities were measured by saturation experiments with respective radioligands [(3)H]UK14304 and [(3)H]CGP12177. ß1- and ß2-adrenoceptor subtype densities were dissected by means of ß1-adrenoceptor selective antagonist CGP20712A. RESULTS: Both, α2- and ß1-adrenoceptors densities were higher in antidepressant-free depressed subjects (n=14) than those in matched controls (Δ~24%, p=0.013 and Δ~20%, p=0.044, respectively). In antidepressant-treated subjects (n=12), α2-adrenoceptor density remained increased over that in controls (Δ~20%), suggesting a resistance of α2-adrenoceptors to the down-regulatory effect of antidepressants. By contrast, ß1-adrenoceptor density in antidepressant-treated depressed subjects was not different from controls, suggesting a possible down-regulation by antidepressants. The down-regulation of ß1-adrenoceptor density in antidepressant-treated depressed subjects differs from the unaltered ß1-adrenoceptor density observed in citalopram-treated rats and in a group of non-depressed subjects also treated with antidepressants (n=6). ß2-adrenoceptor density was not altered in depressed subjects independently of treatment. LIMITATIONS: Antidepressant-treated subjects had been treated with a heterogeneous variety of antidepressant drugs. The results should be understood in the context of suicide victims with depression. CONCLUSIONS: These results show the up-regulation of brain α2- and ß1-adrenoceptors in depression and suggest that the regulation induced by chronic antidepressant treatment would be altered in these subjects.

Systematic and quantitative analysis of G protein-coupled receptor trafficking motifs.

Plasma membrane expression of G protein-coupled receptors (GPCRs) is a dynamic process balancing anterograde and retrograde trafficking. Multiple interrelated cellular processes determine the final level of cell surface expression, including endoplasmic reticulum (ER) export/retention, receptor internalization, recycling, and degradation. These processes are highly regulated to achieve specific localization to subcellular domains (e.g., dendrites or basolateral membranes) and to affect receptor signaling. Analysis of potential ER trafficking motifs within GPCRs requires careful consideration of intracellular dynamics, such as protein folding, ER export and retention, and glycosylation. This chapter presents an approach and methods for qualitative and quantitative assessment of these processes to aid in accurate identification of GPCR trafficking motifs, utilizing the analysis of a hydrophobic extracellular trafficking motif in α2C adrenergic receptors as a model system.

Synthesis and in vivo evaluation of [11C]MPTQ: a potential PET tracer for alpha2A-adrenergic receptors.

Radiosynthesis and in vivo evaluation of [N-methyl-(11)C] 5-methyl-3-[4-(3-phenylallyl)-piperazin-1-ylmethyl]-3,3a,4,5-tetrahydroisoxazolo[4,3-c]quinoline (1), a potential PET tracer for alpha2-adrenergic receptors is described. Syntheses of nonradioactive standard 1 and corresponding desmethyl precursor 2 were achieved from 2-aminobenzaldehyde in 40% and 65% yields, respectively. Methylation using [(11)C]CH(3)I in presence of aqueous potassium hydroxide in DMSO afforded [(11)C]1 in 25% yield (EOS) with >99% chemical and radiochemical purities with a specific activity ranged from 3-4 Ci/micromol (n=6). The total synthesis time was 30 min from EOB. PET studies in anesthetized baboon show that [(11)C]1 penetrates BBB and accumulates in alpha2A-AR enriched brain areas.

Coiled-coil tag--probe system for quick labeling of membrane receptors in living cell.

The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeting the tag has been craved as an alternative to widely used larger fluorescent proteins. We describe a rapid method with a small tag (21 amino acids) for the fluorescence labeling of cell-surface receptors using a high affinity coiled-coil formation without metals or enzymes. The peptide probes K3 (KIAALKE)3 and K4 (KIAALKE)4 labeled with a fluorophore specifically stained the surface-exposed tag sequence E3 (EIAALEK)3 attached to the N-terminus of the mouse-derived prostaglandin EP3 receptor in living cells (Kd = 64 and 6 nM for K3 and K4, respectively). The labeling was quick (<1 min), nontoxic, and available even in culture medium without affecting receptor function. As an application of this tractable method, the agonist-induced internalization of the human-derived 2-adrenergic receptor and epidermal growth factor receptor was successfully visualized.

Endoplasmic reticulum export of adrenergic and angiotensin II receptors is differentially regulated by Sar1 GTPase.

The molecular mechanism underlying the export of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) remains largely unknown. In this manuscript, we investigated the role of Sar1 GTPase, which coordinates the assembly and budding of COPII-coated vesicles, in the cell-surface targeting, signaling and ER export of alpha(2B)-adrenergic (alpha(2B)-AR), beta(2)-AR and angiotensin II type 1 receptors (AT1R). The cell-surface expression of alpha(2B)-AR, beta(2)-AR and AT1R, and receptor-mediated ERK1/2 activation were significantly attenuated by the GTP-bound mutant Sar1H79G, suggesting that export from the ER of these receptors is mediated through the Sar1-dependent COPII-coated vesicles. Interestingly, subcellular distribution analyses showed that alpha(2B)-AR and AT1R were highly concentrated at discrete locations near the nucleus in cells expressing Sar1H79G, whereas beta(2)-AR exhibited an ER distribution. These data indicate that Sar1-catalyzed efficient GTP hydrolysis differentially regulates ER export of adrenergic and angiotensin II receptors. These data provide the first evidence indicating distinct mechanisms for the recruitment of different GPCRs into the COPII vesicles on the ER membrane.

Autoradiographic characterization of alpha(2C)-adrenoceptors in the human striatum.

Indirect experimental evidence suggests that drugs acting on the alpha(2C)-adrenoceptor could be useful in the treatment of neuropsychiatric disorders such as depression and schizophrenia. In rodent brain, the highest levels of alpha(2C)-adrenoceptors are found in the striatum, with lower levels in cerebral cortex and hippocampus. In human brain, because of the poor subtype-selectivity of the available alpha(2)-adrenoceptor ligands, the localization of alpha(2C)-adrenoceptors has remained unknown. Recently, a selective alpha(2C)-adrenoceptor antagonist, JP-1302, was characterized, and to assess the presence of alpha(2C)-adrenoceptors in human brain, we performed competition binding in vitro receptor autoradiography with JP-1302 and the alpha(2)-adrenoceptor subtype nonselective antagonist [ethyl-(3)H]RS79948-197 on rat and human postmortem brain sections. In striatum of both species, JP-1302 vs. [ethyl-(3)H]RS79948-197 competition binding was biphasic, identifying high- and low-affinity binding sites, whereas in cortex and cerebellum, only low-affinity binding sites were detected. The results indicate that a significant portion of the alpha(2)-adrenoceptors in striatum is of the alpha(2C) subtype, whereas non-alpha(2C)-adreocneptors predominate in cortex and cerebellum. Because the alpha(2C)-adrenoceptor subtype distribution pattern appears to be conserved between rodents and humans, results obtained from studies on the role of the alpha(2C)-adrenoceptor in rodent models of neuropsychiatric disorders may be relevant also for human diseases.

Differential modulation of bovine epididymal activity by oxytocin and noradrenaline.

Passage of spermatozoa through the epididymis and emission of sperm during ejaculation are based on spontaneous and induced contractions of epididymal peritubular muscle layers. This study deals with the ejaculation-relevant factors noradrenaline (NA) and oxytocin (OT) and their contractile effects in the course of the bovine epididymal duct. Muscle tension recording revealed excitatory effects of NA in all duct regions. A peculiarity was found in a duct section between the mid-cauda and ductus deferens, where the responsiveness to NA was particularly faint in comparison with the adjacent regions. NA-induced contraction was primarily mediated by postjunctional alpha(2)-adrenoceptors (ADRA) in the caput and corpus regions, and by alpha(1)-ADRA in the cauda region. Contrary to NA, OT exerted regionally varying effects. The peptide induced contraction in intact and epithelium-denuded caput as well as in epithelium-denuded corpus segments but had a relaxant net effect in intact corpus and proximal cauda segments. Within the mid-cauda, OT evoked strong contraction, which progressively decreased distally. Receptor specificity of the epididymal OT effects was verified using the selective OT receptor (OTR) agonist [Thr(4),Gly(7)]OT and vasopressin. OTR immunoreactivity was detected in the epididymal peritubular muscle wall and epithelial principal cells. RT-PCR analysis confirmed the presence of OTR in all duct regions. In summary, different contractile responses to OT and NA occur in the course of the epididymal duct, possibly preventing excessive sperm transport through the corpus and serving orthograde emission of sperm during ejaculation.

Molecules to remember.

"Working memory" is used for the transient storage of information in the brain. In this issue of Cell, Wang et al. (2007) now reveal how a series of molecular events involving alpha2A-adrenoceptors and a class of ion channels gated by cAMP tune the responses of neural circuits that function in working memory in mammals.

Alpha2A-adrenoceptors strengthen working memory networks by inhibiting cAMP-HCN channel signaling in prefrontal cortex.

Spatial working memory (WM; i.e., "scratchpad" memory) is constantly updated to guide behavior based on representational knowledge of spatial position. It is maintained by spatially tuned, recurrent excitation within networks of prefrontal cortical (PFC) neurons, evident during delay periods in WM tasks. Stimulation of postsynaptic alpha2A adrenoceptors (alpha2A-ARs) is critical for WM. We report that alpha2A-AR stimulation strengthens WM through inhibition of cAMP, closing Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels and strengthening the functional connectivity of PFC networks. Ultrastructurally, HCN channels and alpha2A-ARs were colocalized in dendritic spines in PFC. In electrophysiological studies, either alpha2A-AR stimulation, cAMP inhibition or HCN channel blockade enhanced spatially tuned delay-related firing of PFC neurons. Conversely, delay-related network firing collapsed under conditions of excessive cAMP. In behavioral studies, either blockade or knockdown of HCN1 channels in PFC improved WM performance. These data reveal a powerful mechanism for rapidly altering the strength of WM networks in PFC.

Differential effects of the tricyclic antidepressant desipramine on the density of adrenergic receptors in juvenile and adult rats.

Although the tricyclic antidepressants, such as desipramine (DMI), are among the most efficacious treatments for adult depression, they are not effective in treating childhood and adolescent depression. Because the adrenergic nervous system is not fully developed until late adolescence, we hypothesized that the mechanisms regulating receptor density may not yet be mature in young mammals. To test this hypothesis, the effects of DMI treatment on cortical alpha-1-, alpha-2-, and beta-adrenergic receptors were compared in juvenile and adult rats. DMI was delivered either by 4 days of twice daily injections to postnatal day 9 to 13 (4 and 7 mg/kg/day) and adult (20 mg/kg/day) rats, or by 2 weeks of continual drug infusion (osmotic minipumps) to postnatal day 21-35 (15 mg/kg/day) and adult (10 mg/kg/day) rats. These delivery paradigms gave juvenile brain concentrations of DMI similar to those in adult rats. The beta-adrenergic receptor was down-regulated with both treatment paradigms in both juvenile and adult rats. By contrast, in the postnatal day 9 to 13 rats, there was a dose-dependent up-regulation of the alpha-1 in the cortex and alpha-2-adrenergic receptor in the prefrontal cortex, whereas there was no change in density in adult rats. These differences in the alpha-adrenergic receptor regulation after DMI treatment suggest that the lack of efficacy of tricyclic antidepressants in treating childhood depression may be related to immature regulatory mechanisms for these receptors.

Possible involvement of alpha-2A adrenergic receptors in attention deficit hyperactivity disorder: radioligand binding and polymorphism studies.

Neuropharmacological and genetic association studies have implicated norepinephrine and adrenergic receptors in the pathogenesis of ADHD. The purpose of this study was to compare genetic association studies of three polymorphisms of the alpha-2A adrenergic receptor gene (ADRA2A) with radioligand binding studies of the alpha-2A adrenergic receptor protein in platelets from a sample of children without or with ADHD. The pediatric subjects ranged from 6 to 18 years of age. A thorough clinical assessment of each child resulted in one of the following DSM-IV ADHD diagnoses: inattentive, hyperactive/impulsive, combined, or no ADHD. No significant linkage was found between the ADRA2A polymorphisms (MspI, HhaI, and DraI) and any of the phenotypes tested. Association analysis, however, did detect significant linkage disequilibrium for the DraI polymorphism. Association was also evaluated considering the three ADRA2A single nucleotide polymorphisms as haplotypes. The HhaI-DraI and the MspI-HhaI-DraI haplotypes were significantly associated with ADHD. The platelet alpha-2 adrenergic receptor density did not differ between children without or with ADHD. The affinity of the receptor for the radioligand however, differed significantly between those without and with ADHD. In addition, there were some significant correlations between binding parameters and severity of ADHD in this well-characterized clinical population, and significant association was found between these measures of receptor function and MspI and DraI polymorphisms. Thus, both the genetic and the binding studies indicate that the alpha-2 adrenergic receptor may play a role in ADHD.

Alpha2C-adrenergic receptors exhibit enhanced surface expression and signaling upon association with beta2-adrenergic receptors.

The alpha(2C)-adrenergic receptor (alpha(2C)AR) is known to be poorly trafficked to the cell surface when expressed in a variety of cell types. We tested the hypothesis that the surface expression and signaling of alpha(2C)AR might be enhanced by heterodimerization with other G protein-coupled receptors (GPCRs). Cotransfection of alpha(2C)AR with more than 25 related GPCRs revealed that only coexpression with the beta(2)-adrenergic receptor (beta(2)AR) increased the surface localization of alpha(2C)AR in human embryonic kidney-293 cells. Coimmunoprecipitation of alpha(2C)AR with beta(2)AR confirmed a physical interaction between the two receptors. Confocal microscopy studies demonstrated that alpha(2C)AR expressed alone was mainly intracellular, whereas alpha(2C)AR coexpressed with beta(2)AR was predominantly localized to the plasma membrane. Ligand binding studies revealed a significant increase in alpha(2C)AR binding sites upon coexpression with beta(2)AR, with no apparent change in affinity for alpha(2)AR ligands. Functional assays with the alpha(2)AR-specific agonist brimonidine (UK 14,304) revealed that coexpression of beta(2)AR with alpha(2C)AR enhanced alpha(2C)AR-mediated activation of extracellular signal-regulated kinase 1/2. Furthermore, analyses of agonist-promoted receptor endocytosis demonstrated enhanced alpha(2C)AR internalization in response to alpha(2)AR agonists when alpha(2C)AR and beta(2)AR were coexpressed. In addition, substantial cointernalization of alpha(2C)AR in response to betaAR agonists was observed when alpha(2C)AR was coexpressed with beta(2)AR. These data reveal that alpha(2C)AR can interact with beta(2)AR in cells in a manner that regulates alpha(2C)AR surface expression, internalization, and functionality.

Decreased alpha2-adrenergic receptor in the brain stem and pancreatic islets during pancreatic regeneration in weanling rats.

Sympathetic stimulation inhibits insulin secretion. alpha(2)-Adrenergic receptor is known to have a regulatory role in the sympathetic function. We investigated the changes in the alpha(2)-adrenergic receptors in the brain stem and pancreatic islets using [(3)H]Yohimbine during pancreatic regeneration in weanling rats. Brain stem and pancreatic islets of experimental rats showed a significant decrease (p<0.001) in norepinephrine (NE) content at 72 h after partial pancreatectomy. The epinephrine (EPI) content showed a significant decrease (p<0.001) in pancreatic islets while it was not detected in brain stem at 72 h after partial pancreatectomy. Scatchard analysis of [(3)H]Yohimbine showed a significant decrease (p<0.05) in B(max) and K(d) at 72 h after partial pancreatectomy in the brain stem. In the pancreatic islets, Scatchard analysis of [(3)H]Yohimbine showed a significant decrease (p<0.001) in B(max) and K(d) (p<0.05) at 72 h after partial pancreatectomy. The binding parameters reversed to near sham by 7 days after pancreatectomy both in brain stem and pancreatic islets. This shows that pancreatic insulin secretion is influenced by central nervous system inputs from the brain stem. In vitro studies with yohimbine showed that the alpha(2)-adrenergic receptors are inhibitory to islet DNA synthesis and insulin secretion. Thus our results suggest that decreased alpha(2)-adrenergic receptors during pancreatic regeneration functionally regulate insulin secretion and pancreatic beta-cell proliferation in weanling rats.

Orexin neurons are directly and indirectly regulated by catecholamines in a complex manner.

We reported elsewhere that orexin neurons are directly hyperpolarized by noradrenaline (NA) and dopamine. In the present study, we show that NA, dopamine, and adrenaline all directly hyperpolarized orexin neurons. This response was inhibited by the alpha2 adrenergic receptor (alpha2-AR) antagonist, idazoxan or BRL44408, and was mimicked by the alpha2-AR-selective agonist, UK14304. A low concentration of Ba2+ inhibited NA-induced hyperpolarization, which suggests that activation of G protein coupled inward rectifier potassium channels is involved in the response. In the presence of a high concentration of idazoxan, NA induced depolarization or inward current. This response was inhibited by alpha1-AR antagonist, prazosin, which suggests the existence of alpha1-ARs on the orexin neurons along with alpha2-AR. We also examined the effects of NA on glutamatergic and GABAergic synaptic transmission. NA application dramatically increased the frequency and amplitude of spontaneous inhibitory synaptic currents (sIPSCs) and inhibited excitatory synaptic currents (sEPSCs) in orexin neurons; however, NA decreased the frequency of miniature EPSCs (mEPSCs) and IPSCs and the amplitude of evoked EPSCs and IPSCs through the alpha2-AR, because the NA response on mPSCs was inhibited by idazoxan. These results suggest that the NA-induced increase in sIPSC frequency and amplitude is mediated via alpha1-ARs on the somata of GABAergic neurons that innervate the orexin neurons. Calcium imaging using orexin/YC2.1 transgenic mouse brain revealed that NA-induced inhibition of orexin neurons is not altered by sleep deprivation or circadian time in mice. The evidence presented here revealed that orexin neurons are regulated by catecholamines in a complex manner.