The role of α1-adrenergic receptors in regulating metabolism: increased glucose tolerance,leptin secretion and lipid oxidation

The role of α₁-adrenergic receptors (α₁-ARs) and their subtypes in metabolism is not well known. Most previous studies were performed before the advent of transgenic mouse models and utilized transformed cell lines and poorly selective antagonists. We have now studied the metabolic regulation of the α_1A- and α_1B-AR subtypes in vivo using knock-out (KO) and transgenic mice that express a constitutively active mutant (CAM) form of the receptor, assessing subtype-selective functions. CAM mice increased glucose tolerance while KO mice display impaired glucose tolerance. CAM mice increased while KO decreased glucose uptake into white fat tissue and skeletal muscle with the CAM α_1A-AR showing selective glucose uptake into the heart. Using indirect calorimetry, both CAM mice demonstrated increased whole body fatty acid oxidation, while KO mice preferentially oxidized carbohydrate. CAM α_1A-AR mice displayed significantly decreased fasting plasma triglycerides and glucose levels while α_1A-AR KO displayed increased levels of triglycerides and glucose. Both CAM mice displayed increased plasma levels of leptin while KO mice decreased leptin levels. Most metabolic effects were more efficacious with the α_1A-AR subtype. Our results suggest that stimulation of α₁-ARs results in a favorable metabolic profile of increased glucose tolerance, cardiac glucose uptake, leptin secretion and increased whole body lipid metabolism that may contribute to its previously recognized cardioprotective and neuroprotective benefits.     

Human α1-adrenoceptor subtype selectivity of substituted homobivalent 4-aminoquinolines

A series of ring-substituted ethyl- and heptyl-linked 4-aminoquinoline dimers were synthesized and evaluated for their affinities at the 3 human α₁-adrenoceptor (α₁-AR) subtypes and the human serotonin 5-HT_1A-receptor (5-HT_1A-R). We find that the structure-specificity profiles are different for the two series at the α₁-AR subtypes, which suggests that homobivalent 4-aminoquinolines can be developed with α₁-AR subtype selectivity. The 8-methyl (8-Me) ethyl-linked analogue has the highest affinity for the α_1A-AR, 7 nM, and the greatest capacity for discriminating between α_1A-AR and α_1B-AR (6-fold), α_1D-AR (68-fold), and the 5-HT_1A-R (168-fold). α_1B-AR selectivity was observed with the 6-methyl (6-Me) derivative of the ethyl- and heptyl-linked 4-aminoquinoline dimers and the 7-methoxy (7-OMe) derivative of the heptyl-linked analogue. These substitutions result in 4- to 80-fold selectivity for α_1B-AR over α_1A-AR, α_1D-AR, and 5-HT_1A-R. In contrast, 4-aminoquinoline dimers with selectivity for α_1D-AR are more elusive, since none studied to date has greater affinity for the α_1D-AR over the other two α₁-ARs. The selectivity of the 8-Me ethyl-linked 4-aminoquinoline dimer for the α_1A-AR, and 6-Me ethyl-linked, and the 6-Me and 7-OMe heptyl-linked 4-aminoquinoline dimers for the α_1B-AR, makes them promising leads for drug development of α_1A-AR or α_1B-AR subtype selective ligands with reduced 5-HT_1A-R affinity.     

The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α₁-adrenoceptor (α₁-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α_1A- and α_1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α_1A-AR, but not α_1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α_1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α_1D antagonist BMY7378. Following activation by phenylephrine, α_1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α_1D-AR subtype was not observed via CypHer5 technology. The three α₁-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α_1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization.     

Dominance of the α1B-Adrenergic Receptor and its Subcellular Localization in Human and TRAMP Prostate Cancer Cell Lines

The function and distribution of α₁-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the α₁-AR subtype and suggested that the α_{1 A}-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of α₁-ARs was determined by saturation binding and the distribution of the different α₁-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major α₁-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the α_1B-AR. DU145 cells contained 100% of the α_1B-AR subtype, whereas PC3 cells were composed of 21% α_{1 A}-AR and 79% α_1B-AR. TRAMP cell lines contained between 66% and 79% of the α_1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing α _1B-AR and increasing α_{1 A}- and α_1D-AR densities. Transfection with EGFP-tagged α_1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of α ₁-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that α_1B-ARs are the major α₁-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.     

Syntrophin isoforms play specific functional roles in the α1D-adrenergic receptor/DAPC signalosome

α_1D-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α_1D-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, β₁ and β₂) interact with α_1D-ARs and our previous studies suggest multiple isoforms are required for proper α_1D-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α_1D-AR function. Radioligand binding experiments reveal α-syntrophin enhances α_1D-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate β₂-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α_1D-AR/DAPC signalosome significantly alter the pharmacological properties of α₁-AR ligands in vitro.     

Molecular cloning, stable expression and cellular localization of human α1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of α1D -adrenergic receptor

The cDNAs encoding for three subtypes of adrenergic receptors, α_1A-, α_1B- and α_1D-ARs, were cloned and expressed in HEK 293 cells. Expression of α_1A- and α_1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of α_1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of α_1A- and α_1B-ARs was primarily localized on the cell membrane whereas expression of α_1D-AR was␣predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing α_1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of α_1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.Sunil M. Khattar, Roop Singh Bora and Priyanka Priyadarsiny contributed equally to this work.     

Effect of naftopidil,an alpha1D/A-adrenoceptor antagonist,on the urinary bladder in rats with spinal cord injury

AimsAlpha_1D-adrenoceptors (α_1D-ARs) located in the spinal cord are involved in the control of lower urinary tract function. In order to clarify the effect of α_1D-ARs on storage function in the spinal cord, we examined the effect of oral administration and intrathecal injection of the α_1D/A-AR antagonist, naftopidil, on bladder activity, as well as the effect of naftopidil on bladder wall histology, in female rats with spinal cord injury (SCI).Main methodsAdult female Sprague–Dawley rats with Th9–10 spinal cord transection were used. In SCI rats with or without 5 mg/day of naftopidil for 4 weeks, bladder activity was examined via continuous cystometry. In other SCI rats, bladder activity was examined before and after intrathecal injection of naftopidil. In addition, bladder wall histology was compared between SCI rats with or without oral administration of naftopidil for 4 weeks.Key findingsOral administration of naftopidil decreased the number of non-voiding contractions (NVCs). Intrathecal injection of naftopidil prolonged the interval between voiding contractions, decreased the maximum voiding contraction pressure and the number of NVCs, and increased bladder capacity without affecting the residual urine volume. Oral administration of naftopidil also decreased bladder wall fibrosis.SignificanceThe α_1D/A-AR antagonist naftopidil might act on the bladder and spinal cord to improve detrusor hyperreflexia in the storage state in SCI female rats. Naftopidil also suppressed bladder wall fibrosis, suggesting that it may be effective for the treatment of neurogenic lower urinary tract dysfunction after SCI.     

Design,synthesis, crystal structure,biological evaluation and molecular docking studies of carbazole-arylpiperazine derivatives

Subtype-selective α₁-adrenoceptor (AR) antagonists display optimum therapeutic efficacies for the treatment of benign prostatic hyperplasia (BPH). In this study, we designed and synthesized novel carbazole-arylpiperazines derivatives (1 and 2) on the basis of the proposed pharmacophore model for α₁-AR antagonists. Structural properties were investigated using single-crystal X-ray diffraction analysis. Comparison of crystal structures with ligand-based pharmacophore models revealed that the two agents may possess antagonistic effects on α_1D subtype. Tissue functional assay in vitro showed that compound 2 exerted strong antagonistic activity on α_1B-AR (pA₂ 7.13) with a poor selectivity for α_1A and α_1D subtypes. Compound 1 exhibited enhanced antagonistic effect on α_1D subtype (pA₂ 7.06) and excellent selectivity for α_1D over α_1B (α_1D/α_1B ratio = 79.4). To illustrate the relationship between antagonistic activity and chemical structure, molecular docking studies were performed using the homology models of α₁ receptors. Binding mechanism indicated that small hydrophobic substituents attached to the arylpiperazine moiety were essential for rational design of α_1D-selective antagonists.     

α1A-Adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α₁-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α₁-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α_1A-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α₁-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α_1A-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α_1A-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α_1A-AR agonism.     

Cardiac and neuroprotection regulated by α1-adrenergic receptor subtypes

Sympathetic nervous system regulation by the α₁-adrenergic receptor (AR) subtypes (α_1A, α_1B, α_1D) is complex, whereby chronic activity can be either detrimental or protective for both heart and brain function. This review will summarize the evidence that this dual regulation can be mediated through the different α₁-AR subtypes in the context of cardiac hypertrophy, heart failure, apoptosis, ischemic preconditioning, neurogenesis, locomotion, neurodegeneration, cognition, neuroplasticity, depression, anxiety, epilepsy, and mental illness.     

Different phosphorylation patterns regulate α1D-adrenoceptor signaling and desensitization

Human α_1D-adrenoceptors (α_1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α_1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α_1D-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/β, phosphorylate α_1D-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α₁-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.     

Mechanisms of α1-adrenoceptor mediated QT prolongation in the diabetic rat heart

AimsDiabetes mellitus is associated with changes of α₁-adrenoceptor (α₁-AR) on heart electrical function and expression. In this study, we investigated the ionic basis underlying abnormal α₁-AR mediated QT prolongation in the diabetic rat hearts.Main methodsElectrophysiological and biochemical techniques were used in Streptozotocin (STZ)-induced diabetic and control rat hearts.Key findingsIn both control and diabetic rats, the α₁-AR agonist, phenylephrine (PE, 10–100 µM) prolonged the rate-corrected QT intervals (QTc) and action potential durations at 30% (APD₃₀) and 90% (APD₉₀) repolarization levels with the increased QTc and APD₉₀ significantly greater in diabetic rats. PE significantly decreased the transient outward K⁺ current (I_to) and the steady-state K⁺ current (I_ss) in both control and diabetic rats but had no effects on the delayed rectifier K⁺ current (I_k). However, PE induced a greater reduction mainly in the I_ss, but not I_to, in diabetic rats. Furthermore, using RT–PCR and Western blot analyses, we found that α_1A-ARs were over-expressed in the left ventricular tissues of the diabetic rat hearts at both the mRNA and the protein levels.SignificanceThese data suggested that in diabetic hearts, a greater sensitivity of the α_1A-AR mediated the larger suppression of I_ss and resulted in a more prolonged APD₉₀ and QTc. Thus, higher α_1A-AR expression levels in diabetic heart may underlie this type of diabetic cardiomyopathy and suggests that α_1A-AR may serve as a therapeutic target.     

α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake

While α₁-adrenergic receptors (ARs) have been previously shown to limit ischemic cardiac damage, the mechanisms remain unclear. Most previous studies utilized low oxygen conditions in addition to ischemic buffers with glucose deficiencies, but we discovered profound differences if the two conditions are separated. We assessed both mouse neonatal and adult myocytes and HL-1 cells in a series of assays assessing ischemic damage under hypoxic or low glucose conditions. We found that α₁-AR stimulation protected against increased lactate dehydrogenase release or Annexin V⁺ apoptosis under conditions that were due to low glucose concentration not to hypoxia. The α₁-AR antagonist prazosin or nonselective protein kinase C (PKC) inhibitors blocked the protective effect. α₁-AR stimulation increased ³H-deoxyglucose uptake that was blocked with either an inhibitor to glucose transporter 1 or 4 (GLUT1 or GLUT4) or small interfering RNA (siRNA) against PKCδ. GLUT1/4 inhibition also blocked α₁-AR-mediated protection from apoptosis. The PKC inhibitor rottlerin or siRNA against PKCδ blocked α₁-AR stimulated GLUT1 or GLUT4 plasma membrane translocation. α₁-AR stimulation increased plasma membrane concentration of either GLUT1 or GLUT4 in a time-dependent fashion. Transgenic mice overexpressing the α_1A-AR but not α_1B-AR mice displayed increased glucose uptake and increased GLUT1 and GLUT4 plasma membrane translocation in the adult heart while α_1A-AR but not α_1B-AR knockout mice displayed lowered glucose uptake and GLUT translocation. Our results suggest that α₁-AR activation is anti-apoptotic and protective during cardiac ischemia due to glucose deprivation and not hypoxia by enhancing glucose uptake into the heart via PKCδ-mediated GLUT translocation that may be specific to the α_1A-AR subtype.     

Inverse agonism at α2A adrenoceptors augments the hypophagic effect of sibutramine in rats

Because the use of monoamine reuptake inhibitors as weight-reducing agents is limited by adverse effects, novel antiobesity drugs are needed. We studied acute effects of the noradrenaline (NA) and serotonin (5-HT) reuptake inhibitor sibutramine (SIB), alone and after pretreatment with α1- and α2-adrenoceptor (AR), and 5-HT1/2/7, 5-HT1B and 5-HT2C receptor antagonists in order to determine which ARs and 5-HT receptors act downstream of SIB on feeding and locomotion. Acute effects on caloric and water intake, meal microstructure and locomotion were assessed, using an automated weighing system and telemetry in male rats with restricted 18-h access to Western style diet. SIB 3 mg/kg reduced meal size and frequency, which suggests enhanced within- and postmeal satiety. Imiloxan (α2B-AR), WB4101 (α1-AR), SB-224289 (5-HT1B), and modestly BRL 44408 (α2A/D-AR) attenuated SIB's effect on meal size, suggesting that α2B- and α1-ARs and 5-HT1B receptors mediate within-meal satiety, with a modest role for α2A/D-ARs. Only prazosin (α1/2B/2C-AR) counteracted SIB's effect on meal frequency. At 3 mg/kg, SIB modestly increased locomotion. This effect was blocked by metergoline (5-HT1/2/7), WB4101 (α1-AR), and RX821002 (α2-AR). Interestingly, the α2-AR antagonists atipamezole and RX821002 enhanced SIB's effect on caloric intake, probably due to inverse agonistic actions at α2A-autoreceptors that further enhanced release of NA that regulates caloric intake. Thus, an inverse agonist of presynaptic α2A-ARs might beneficially enhance SIB's weight-reducing effect and offer novel treatment for obesity. All in all, the present data supports the ARs and 5-HT receptors involved in the effects of SIB on different aspects of caloric intake and locomotion.