␣ 1 -Adrenergic Receptors Regulate Neurogenesis and Gliogenesis

ABSTRACT The understanding of the function of ␣ 1 -adrenergic receptors in the brain has been limited due to a lack of specific ligands and antibodies. We circumvented this problem by using transgenic mice engineered to overexpress either wild-type receptor tagged with enhanced green fluorescent protein or constitutively active mutant ␣ 1 -adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing ␣ 1A -adrenergic receptor subtype-expressing cells with a migratory morphology in the adult subventricular zone that coexpressed markers of neural stem cell and/or progenitors. Incorporation of 5-bromo-2-deoxyuridine in vivo increased in neurogenic areas in adult ␣ 1A -adrenergic receptor transgenic mice or normal mice given the ␣ 1A -adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of ␣ 1 -adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the ␣ 1B -adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. ␣ 1 -Adrenergic receptor stimulation increased the apoptosis of astrocytes and regulated survival of neonatal neurons through phosphatidylinositol 3-kinase signaling. However, in adult normal neurospheres, ␣ 1 -adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and ␤III tubulin neuronal progenitors colocalized with either ␣ 1 -adrenergic receptor subtype in the olfactory bulb. Our results indicate that ␣ 1 -adrenergic receptors can regulate both neurogenesis and gliogenesis that may be developmentally dependent. Our findings may lead to new therapies to treat neurodegenerative diseases

Defective Resensitization in Human Airway Smooth Muscle Cells Evokes β-Adrenergic Receptor Dysfunction in Severe Asthma

<div><p>β₂-adrenergic receptor (β₂AR) agonists (β₂-agonist) are the most commonly used therapy for acute relief in asthma, but chronic use of these bronchodilators paradoxically exacerbates airway hyper-responsiveness. Activation of βARs by β-agonist leads to desensitization (inactivation) by phosphorylation through G-protein coupled receptor kinases (GRKs) which mediate β-arrestin binding and βAR internalization. Resensitization occurs by dephosphorylation of the endosomal βARs which recycle back to the plasma membrane as agonist-ready receptors. To determine whether the loss in β-agonist response in asthma is due to altered βAR desensitization and/or resensitization, we used primary human airway smooth muscle cells (HASMCs) isolated from the lungs of non-asthmatic and fatal-asthmatic subjects. Asthmatic HASMCs have diminished adenylyl cyclase activity and cAMP response to β-agonist as compared to non-asthmatic HASMCs. Confocal microscopy showed significant accumulation of phosphorylated β₂ARs in asthmatic HASMCs. Systematic analysis of desensitization components including GRKs and β-arrestin showed no appreciable differences between asthmatic and non-asthmatic HASMCs. However, asthmatic HASMC showed significant increase in PI3Kγ activity and was associated with reduction in PP2A activity. Since reduction in PP2A activity could alter receptor resensitization, endosomal fractions were isolated to assess the agonist ready β₂ARs as a measure of resensitization. Despite significant accumulation of β₂ARs in the endosomes of asthmatic HASMCs, endosomal β₂ARs cannot robustly activate adenylyl cyclase. Furthermore, endosomes from asthmatic HASMCs are associated with significant increase in PI3Kγ and reduced PP2A activity that inhibits β₂AR resensitization. Our study shows that resensitization, a process considered to be a homeostasis maintaining passive process is inhibited in asthmatic HASMCs contributing to β₂AR dysfunction which may underlie asthma pathophysiology and loss in asthma control.</p></div

βAR function in primary human airway smooth muscle cells (HASMCs) from lungs of non-asthma (Non- asthmatic ASM) and asthma (Asthmatic ASM) patients.

<p><b>a</b>, Non- asthmatic ASM and asthmatic ASM cells were stimulated with β-agonist albuterol (0, 5, 10 & 20 minutes (min)). The cell were lysed and assessed for the ability to generate cAMP. *p<0.005 vs. non-asthmatic ASM or asthmatic ASM 0 min (untreated), #p<0.05 vs. asthmatic ASM 5, 10, 20 min, (n = 6 per group, 6 non-asthmatic ASM and 6 asthmatic ASM). <b>b</b>, Plasma membranes were isolated from non-asthmatic ASM and asthmatic ASM cells following pre-treatment of cells with albuterol for 0, 5, 10, and 20 min. The cell-free membranes were stimulated with albuterol to measure adenylyl cyclase activity by providing radioactive ^32[P]γ-ATP and measuring cAMP generation. *p<0.01 vs. respective <i>in vitro</i> vehicle stimulation, #p<0.05 vs. ASM <i>in vitro</i> albuterol stimulated 0 and 5 min, (n = 6/group). V, Vehicle; Alb, Albuterol (β-agonist).</p

Assessment of βAR desensitization in non-asthmatic ASM and asthmatic ASM cells.

<p><b>a</b>, To determine whether βAR are differentially phosphorylated in non-asthmatic ASM and asthmatic ASM cells, the cells were plated on cover slips and β₂AR phosphorylation was visualized by confocal microscopy using anti-phospho-S-355/356 β₂AR antibody (green) (Scale-100 μM) (n = 5/group). <b>b</b>, Plasma membranes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to ^[125]I-CYP (cyanopindalol) βAR binding at saturation concentration of 250 pmol. *p<0.005 vs. non-asthmatic ASM, (n = 7/group). <b>c</b>, To investigate changes in desensitization βAR components lysates (100 μg) from non-asthmatic ASM and asthmatic ASM cells were immunoblotted for ubiquitously expressed GRKS, GRK2, 3, 5 or 6. The blots were stripped for each probing. Furthermore, the blots were immunoblotted for β-arrestin 1 and 2. Actin was blotted as loading control (n = 7/group).</p

Measure of βAR resensitization in non-asthmatic ASM and asthmatic ASM cells (βAR function, density and distribution).

<p><b>a</b>, To obtain a measure of resensitization that mediates βAR dephosphorylation leading to generation of agonist ready receptors in the endosomes, endosomes were isolated from non-asthmatic ASM and asthmatic ASM cells, and subjected to various assays. <b>b</b>, Plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to ^[125]I-CYP (cyanopindalol) βAR binding at saturation concentration of 250 pmol. *p<0.005 vs. Non-ASM, (n = 4/group). <b>c</b>, Plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells were subjected to cell-free membrane associated β-agonist (isoproterenol, ISO)-stimulated adenylyl cyclase activity. *p<0.01 vs. respective cell-free vehicle (V) stimulation, #p<0.05 vs. asthmatic ASM ISO, (n = 4/group). <b>d</b>, Upper panel, plasma membranes and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells (100 μg) were immunoblotted using anti-phospho-S-355/356 β₂AR antibody. Lower panel, densitometry for phospho- β₂AR (n = 4/group). *p<0.01 vs. plasma membrane (P) non-asthmatic ASM, #p<0.05 vs. endosomes (E) non-asthmatic ASM. V, Vehicle, ISO, isoproterenol (β-agonist), P, Plasma membrane, E, Endosome.</p

Measure of βAR resensitization in Non-ASM and ASM HASMCs (measure of resensitization components PI3Kγ and PP2A activity).

<p><b>a</b>, Upper panel, PI3Kγ was immunoprecipitated from plasma membrane and endosomes isolated from non-asthmatic ASM and asthmatic ASM cells (120 μg) and the washed immunoprecipitates were subjected to <i>in vitro</i> lipid kinase assays. Lower panel, densitometry for PI3Kγ (n = 4/group). *p<0.01 vs. plasma membrane (P) non-asthmatic ASM, #p<0.0001 vs. endosomes (E) non-asthmatic ASM. <b>b</b>, PP2A was immunoprecipitated from (100 μg) of non-asthmatic ASM and asthmatic ASM cells using anti-PP2A antibodies and the immunoprecipitates were subjected to <i>in vitro</i> phosphatase assay with malachite green as a read for activity, (n = 4/group). *p<0.001 vs. non-asthmatic ASM,). <b>c</b>, Illustration depicting the loss of resensitization as a underlying cause for βAR dysfunction leading to paradoxical loss in β-agonist response. Left Panel (non-asthmatic ASM conditions), βAR activation by β-agonist leads to desensitization by phosphorylation and resensitization by dephosphorylation via PP2A resulting in normal βARs recycling. Right Panel (asthmatic ASM conditions), desensitization is well understood but, little is known of resensitization mechanisms. Since we have shown that βAR resensitization is regulated by PI3Kγ-PP2A axis, assessment in the endosomes shows marked increase in PI3Kγ activity associated with loss in PP2A activity. Based on this observation, we propose that asthma HASMCs are characterized by loss in βAR resensitization wherein, increased PI3Kγ inhibits PP2A activity blocking receptor resensitization resulting in accumulation of βARs in the endosomes.</p