Increased angiotensin II coupled with decreased Adra1a expression enhances cardiac hypertrophy in pregnancy-associated hypertensive mice.

Cardiac hypertrophy is a crucial risk factor for hypertensive disorders during pregnancy, but its progression during pregnancy remains unclear. We previously showed cardiac hypertrophy in a pregnancy-associated hypertensive (PAH) mouse model, in which an increase in angiotensin II (Ang II) levels was induced by human renin and human angiotensinogen, depending on pregnancy conditions. Here, to elucidate the factors involved in the progression of cardiac hypertrophy, we performed a comprehensive analysis of changes in gene expression in the hearts of PAH mice and compared them with those in control mice. We found that alpha-1A adrenergic receptor (Adra1a) mRNA levels in the heart were significantly reduced under PAH conditions, whereas the renin-angiotensin system was upregulated. Furthermore, we found that Adra1a-deficient PAH mice exhibited more severe cardiac hypertrophy than PAH mice. Our study suggests that Adra1a levels are regulated by renin-angiotensin system and that changes in Adra1a expression are involved in progressive cardiac hypertrophy in PAH mice.

Dual-omics reveals temporal differences in acute sympathetic stress-induced cardiac inflammation following α1 and ß-adrenergic receptors activation.

Sympathetic stress is prevalent in cardiovascular diseases. Sympathetic overactivation under strong acute stresses triggers acute cardiovascular events including myocardial infarction (MI), sudden cardiac death, and stress cardiomyopathy. α1-ARs and ß-ARs, two dominant subtypes of adrenergic receptors in the heart, play a significant role in the physiological and pathologic regulation of these processes. However, little is known about the functional similarities and differences between α1- and ß-ARs activated temporal responses in stress-induced cardiac pathology. In this work, we systematically compared the cardiac temporal genome-wide profiles of acute α1-AR and ß-AR activation in the mice model by integrating transcriptome and proteome. We found that α1- and ß-AR activations induced sustained and transient inflammatory gene expression, respectively. Particularly, the overactivation of α1-AR but not ß-AR led to neutrophil infiltration at one day, which was closely associated with the up-regulation of chemokines, activation of NF-κB pathway, and sustained inflammatory response. Furthermore, there are more metabolic disorders under α1-AR overactivation compared with ß-AR overactivation. These findings provide a new therapeutic strategy that, besides using ß-blocker as soon as possible, blocking α1-AR within one day should also be considered in the treatment of acute stress-associated cardiovascular diseases.

Heteromerization between α1B -adrenoceptor and chemokine (C-C motif) receptor 2 biases α1B -adrenoceptor signaling: Implications for vascular function.

Previously, we reported that chemokine (C-C motif) receptor 2 (CCR2) heteromerizes with α1B -adrenoceptor (α1B -AR) in leukocytes, through which α1B -AR controls CCR2. Whether such heteromers are expressed in human vascular smooth muscle cells (hVSMCs) is unknown. Bioluminescence resonance energy transfer confirmed formation of recombinant CCR2:α1b -AR heteromers. Proximity ligation assays detected CCR2:α1B -AR heteromers in hVSMCs and human mesenteric arteries. CCR2:α1B -AR heteromerization per se enhanced α1B -AR-mediated Gαq -coupling. Chemokine (C-C motif) ligand 2 (CCL2) binding to CCR2 inhibited Gαq activation via α1B -AR, cross-recruited ß-arrestin to and induced internalization of α1B -AR in recombinant systems and in hVSMCs. Our findings suggest that CCR2 within CCR2:α1B -AR heteromers biases α1B -AR signaling and provide a mechanism for previous observations suggesting a role for CCL2/CCR2 in the regulation of cardiovascular function.

Vascular Reactions of the Diving Reflex in Men and Women Carrying Different ADRA1A Genotypes.

The diving reflex is an oxygen-saving mechanism which is accompanied by apnea, reflex bradycardia development, peripheral vasoconstriction, spleen erythrocyte release, and selective redistribution of blood flow to the organs most vulnerable to lack of oxygen, such as the brain, heart, and lungs. However, this is a poorly studied form of hypoxia, with a knowledge gap on physiological and biochemical adaptation mechanisms. The reflective sympathetic constriction of the resistive vessels is realized via ADRA1A. It has been shown that ADRA1A SNP (p.Arg347Cys; rs1048101) is associated with changes in tonus in vessel walls. Moreover, the Cys347 allele has been shown to regulate systolic blood pressure. The aim of this work was to evaluate whether the ADRA1A polymorphism affected the pulmonary vascular reactions in men and women in response to the diving reflex. Men (n = 52) and women (n = 50) untrained in diving aged 18 to 25 were recruited into the study. The vascular reactions and blood flow were examined by integrated rheography and rheography of the pulmonary artery. Peripheral blood circulation was registered by plethysmography. The ADRA1A gene polymorphism (p.Arg347Cys; rs1048101) was determined by PCR-RFLP. In both men and women, reflective pulmonary vasodilation did occur in response to the diving reflex, but in women this vasodilation was more pronounced and was accompanied by a higher filling of the lungs with blood.. Additionally, ADRA1A SNP (p.Arg347Cys; rs1048101) is associated with sex. Interestingly, women with the Arg347 allele demonstrated the highest vasodilation of the lung vessels. Therefore, our data may help to indicate women with the most prominent adaptive reactions to the diving reflex. Our data also indicate that women and men with the Cys allele of the ADRA1A gene polymorphism have the highest risk of developing lung hypertension in response to the diving reflex. The diving reflex is an oxygen-saving mechanism which is accompanied by apnea, reflex bradycardia development, peripheral vasoconstriction, spleen erythrocyte release, and selective redistribution of blood flow to the organs most vulnerable to lack of oxygen, such as the brain, heart, and lungs. However, this is a poorly studied form of hypoxia, with a knowledge gap on physiological and biochemical adaptation mechanisms.

α1B/D-adrenoceptors regulate chemokine receptor-mediated leukocyte migration via formation of heteromeric receptor complexes.

It is known that catecholamines regulate innate immune functions. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family in recombinant systems and that such heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Ligand binding to α1-ARs inhibited migration toward agonists of the CR heteromerization partners of α1B/D-ARs with high potency and 50 to 77% efficacy but did not affect migration induced by a noninteracting CR. Incomplete siRNA knockdown of α1B/D-ARs in THP-1 cells partially inhibited migration toward agonists of their CR heteromerization partners. Complete α1B-AR knockout via CRISPR-Cas9 gene editing in THP-1 cells (THP-1_ADRA1BKO) resulted in 82% reduction of α1D-AR expression and did not affect CR expression. Migration of THP-1_ADRA1BKO cells toward agonists of CR heteromerization partners of α1B/D-ARs was reduced by 82 to 95%. Our findings indicate that CR:α1B/D-AR heteromers are essential for normal function of CR heteromerization partners, provide a mechanism underlying neuroendocrine control of leukocyte trafficking, and offer opportunities to modulate leukocyte and/or cancer cell trafficking in disease processes.

Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha1-adrenoceptors to early cardiac stress responses.

Hyperactivity of the sympathetic nervous system is a major driver of cardiac remodeling, exerting its effects through both α-, and ß-adrenoceptors (α-, ß-ARs). As the relative contribution of subtype α1-AR to cardiac stress responses remains poorly investigated, we subjected mice to either subcutaneous perfusion with the ß-AR agonist isoprenaline (ISO, 30 mg/kg × day) or to a combination of ISO and the stable α1-AR agonist phenylephrine (ISO/PE, 30 mg/kg × day each). Telemetry analysis revealed similar hemodynamic responses under both ISO and ISO/PE treatment i.e., permanently increased heart rates and only transient decreases in mean blood pressure during the first 24 h. Echocardiography and single cell analysis after 1 week of exposure showed that ISO/PE-, but not ISO-treated animals established α1-AR-mediated inotropic responsiveness to acute adrenergic stimulation. Morphologically, additional PE perfusion limited concentric cardiomyocyte growth and enhanced cardiac collagen deposition during 7 days of treatment. Time-course analysis demonstrated a diverging development in transcriptional patterns at day 4 of treatment i.e., increased expression of selected marker genes Xirp2, Nppa, Tgfb1, Col1a1, Postn under chronic ISO/PE treatment which was either less pronounced or absent in the ISO group. Transcriptome analyses at day 4 via RNA sequencing demonstrated that additional PE treatment caused a marked upregulation of genes allocated to extracellular matrix and fiber organization along with a more pronounced downregulation of genes involved in metabolic processes, muscle adaptation and cardiac electrophysiology. Consistently, transcriptome changes under ISO/PE challenge more effectively recapitulated early transcriptional alterations in pressure overload-induced experimental heart failure and in human hypertrophic cardiomyopathy.

MiR-3682 promotes the progression of hepatocellular carcinoma (HCC) via inactivating AMPK signaling by targeting ADRA1A.

INTRODUCTION AND OBJECTIVES: This study aimed to investigate miR-3682 as a biomarker in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: MiRNA and RNA profiles of 375 HCC tissues and 50 normal liver samples were downloaded from The Cancer Genome Atlas (TCGA) database. Multivariate Cox regression and Kaplan-Meier analyses were applied to examine the prognostic value of factors. Target genes of miR-3682 were analyzed by TargetScan and dual-luciferase reporter assay. Online Database for Annotation, Visualization, and Integrated Discovery (DAVID) to perform KEGG pathway enrichment. Cell counting kit-8, colony formation and migration and invasion assays were performed to analyze biological behaviors of HCC cells. RESULTS: MiR-3682 was identified to be highly expressed in HCC tissues and cell lines. And miR-3682 was negatively and independently associated with the outcome of HCC patients. Inhibition of miR-3682 suppressed HCC cell viability and mobility. ADRA1A, predicted and confirmed as the novel target of miR-3682, was an independent and positive prognostic predictor for HCC. In addition, the knockdown of ADRA1A partially offset the inhibitory effect of miR-3682 inhibitor on the growth and mobility of HCC cells. DAVID enrichment and western blot of key signaling-related proteins analyses revealed that miR-3682 inactivated 5'-AMP-activated protein kinase (AMPK) signaling by negatively regulating ADRA1A. Mechanically, it was partially through suppressing AMPK signaling via targeting ADRA1A that miR-3682 supported the HCC cell malignant phenotype. CONCLUSIONS: This study implicates that miR-3682 plays an oncogenetic role in HCC and can be considered a novel therapeutic target and prognostic indicator of HCC.

The selectivity of α-adrenoceptor agonists for the human α1A, α1B, and α1D-adrenoceptors.

Highly selective drugs offer a way to minimize side-effects. For agonist ligands, this could be through highly selective affinity or highly selective efficacy, but this requires careful measurements of intrinsic efficacy. The α1-adrenoceptors are important clinical targets, and α1-agonists are used to manage hypotension, sedation, attention deficit hypersensitivity disorder (ADHD), and nasal decongestion. With 100 years of drug development, there are many structurally different compounds with which to study agonist selectivity. This study examined 62 α-agonists at the three human α1-adrenoceptor (α1A, α1B, and α1D) stably expressed in CHO cells. Affinity was measured using whole-cell 3 H-prazosin binding, while functional responses were measured for calcium mobilization, ERK1/2-phosphorylation, and cAMP accumulation. Efficacy ratios were used to rank compounds in order of intrinsic efficacy. Adrenaline, noradrenaline, and phenylephrine were highly efficacious α1-agonists at all three receptor subtypes. A61603 was the most selective agonist and its very high α1A-selectivity was due to selective α1A-affinity (>660-fold). There was no evidence of Gq-calcium versus ERK-phosphorylation biased signaling at the α1A, α1B, or α1D-adrenoceptors. There was little evidence for α1A calcium versus cAMP biased signaling, although there were suggestions of calcium versus cAMP bias the α1B-adrenoceptor. Comparisons of the rank order of ligand intrinsic efficacy suggest little evidence for selective intrinsic efficacy between the compounds, with perhaps the exception of dobutamine which may have some α1D-selective efficacy. There seems plenty of scope to develop affinity selective and intrinsic efficacy selective drugs for the α1-adrenoceptors in future.

MAPKs Are Highly Abundant but Do Not Contribute to α1-Adrenergic Contraction of Rat Saphenous Arteries in the Early Postnatal Period.

Previously, the abundance of p42/44 and p38 MAPK proteins had been shown to be higher in arteries of 1- to 2-week-old compared to 2- to 3-month-old rats. However, the role of MAPKs in vascular tone regulation in early ontogenesis remains largely unexplored. We tested the hypothesis that the contribution of p42/44 and p38 MAPKs to the contraction of peripheral arteries is higher in the early postnatal period compared to adulthood. Saphenous arteries of 1- to 2-week-old and 2- to 3-month-old rats were studied using wire myography and western blotting. The α1-adrenoceptor agonist methoxamine did not increase the phosphorylation level of p38 MAPK in either 1- to 2-week-old or 2- to 3-month-old rats. Accordingly, inhibition of p38 MAPK did not affect arterial contraction to methoxamine in either age group. Methoxamine increased the phosphorylation level of p42/44 MAPKs in arteries of 2- to 3-month-old and of p44 MAPK in 1- to 2-week-old rats. Inhibition of p42/44 MAPKs reduced methoxamine-induced contractions in arteries of 2- to 3-month-old, but not 1- to 2-week-old rats. Thus, despite a high abundance in arterial tissue, p38 and p42/44 MAPKs do not regulate contraction of the saphenous artery in the early postnatal period. However, p42/44 MAPK activity contributes to arterial contractions in adult rats.

Role of α- and ß-adrenergic signaling in phenotypic targeting: significance in benign and malignant urologic disease.

The urinary tract is highly innervated by autonomic nerves which are essential in urinary tract development, the production of growth factors, and the control of homeostasis. These neural signals may become dysregulated in several genitourinary (GU) disease states, both benign and malignant. Accordingly, the autonomic nervous system is a therapeutic target for several genitourinary pathologies including cancer, voiding dysfunction, and obstructing nephrolithiasis. Adrenergic receptors (adrenoceptors) are G-Protein coupled-receptors that are distributed throughout the body. The major function of α1-adrenoceptors is signaling smooth muscle contractions through GPCR and intracellular calcium influx. Pharmacologic intervention of α-and ß-adrenoceptors is routinely and successfully implemented in the treatment of benign urologic illnesses, through the use of α-adrenoceptor antagonists. Furthermore, cell-based evidence recently established the antitumor effect of α1-adrenoceptor antagonists in prostate, bladder and renal tumors by reducing neovascularity and impairing growth within the tumor microenvironment via regulation of the phenotypic epithelial-mesenchymal transition (EMT). There has been a significant focus on repurposing the routinely used, Food and Drug Administration-approved α1-adrenoceptor antagonists to inhibit GU tumor growth and angiogenesis in patients with advanced prostate, bladder, and renal cancer. In this review we discuss the current evidence on (a) the signaling events of the autonomic nervous system mediated by its cognate α- and ß-adrenoceptors in regulating the phenotypic landscape (EMT) of genitourinary organs; and (b) the therapeutic significance of targeting this signaling pathway in benign and malignant urologic disease. Video abstract.

Deacetylation as a receptor-regulated direct activation switch for pannexin channels.

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.

Antidepressants Differentially Regulate Intracellular Signaling from α1-Adrenergic Receptor Subtypes In Vitro.

Currently utilized antidepressants have limited effectiveness and frequently incur undesired effects. Most antidepressants are thought to act via the inhibition of monoamine reuptake; however, direct binding to monoaminergic receptors has been proposed to contribute to both their clinical effectiveness and their side effects, or lack thereof. Among the target receptors of antidepressants, α1­adrenergic receptors (ARs) have been implicated in depression etiology, antidepressant action, and side effects. However, differences in the direct effects of antidepressants on signaling from the three subtypes of α1-ARs, namely, α1A-, α1B- and α1D­ARs, have been little explored. We utilized cell lines overexpressing α1A-, α1B- or α1D-ARs to investigate the effects of the antidepressants imipramine (IMI), desipramine (DMI), mianserin (MIA), reboxetine (REB), citalopram (CIT) and fluoxetine (FLU) on noradrenaline-induced second messenger generation by those receptors. We found similar orders of inhibition at α1A-AR (IMI < DMI < CIT < MIA < REB) and α1D­AR (IMI = DMI < CIT < MIA), while the α1B-AR subtype was the least engaged subtype and was inhibited with low potency by three drugs (MIA < IMI = DMI). In contrast to their direct antagonistic effects, prolonged incubation with IMI and DMI increased the maximal response of the α1B-AR subtype, and the CIT of both the α1A- and the α1B-ARs. Our data demonstrate a complex, subtype-specific modulation of α1-ARs by antidepressants of different groups.

Distribution of the Noradrenaline Innervation and Adrenoceptors in the Macaque Monkey Thalamus.

Noradrenaline (NA) in the thalamus has important roles in physiological, pharmacological, and pathological neuromodulation. In this work, a complete characterization of NA axons and Alpha adrenoceptors distributions is provided. NA axons, revealed by immunohistochemistry against the synthesizing enzyme and the NA transporter, are present in all thalamic nuclei. The most densely innervated ones are the midline nuclei, intralaminar nuclei (paracentral and parafascicular), and the medial sector of the mediodorsal nucleus (MDm). The ventral motor nuclei and most somatosensory relay nuclei receive a moderate NA innervation. The pulvinar complex receives a heterogeneous innervation. The lateral geniculate nucleus (GL) has the lowest NA innervation. Alpha adrenoceptors were analyzed by in vitro quantitative autoradiography. Alpha-1 receptor densities are higher than Alpha-2 densities. Overall, axonal densities and Alpha adrenoceptor densities coincide; although some mismatches were identified. The nuclei with the highest Alpha-1 values are MDm, the parvocellular part of the ventral posterior medial nucleus, medial pulvinar, and midline nuclei. The nucleus with the lowest Alpha-1 receptor density is GL. Alpha-2 receptor densities are highest in the lateral dorsal, centromedian, medial and inferior pulvinar, and midline nuclei. These results suggest a role for NA in modulating thalamic involvement in consciousness, limbic, cognitive, and executive functions.

The insulin receptor family and protein kinase B (Akt) are activated in the heart by alkaline pH and α1-adrenergic receptors.

Insulin and insulin-like growth factor stimulate protein synthesis and cardioprotection in the heart, acting through their receptors (INSRs, IGF1Rs) and signalling via protein kinase B (PKB, also known as Akt). Protein synthesis is increased in hearts perfused at alkaline pHo to the same extent as with insulin. Moreover, α1-adrenergic receptor (α1-AR) agonists (e.g. phenylephrine) increase protein synthesis in cardiomyocytes, activating PKB/Akt. In both cases, the mechanisms are not understood. Our aim was to determine if insulin receptor-related receptors (INSRRs, activated in kidney by alkaline pH) may account for the effects of alkaline pHo on cardiac protein synthesis, and establish if α1-ARs signal through the insulin receptor family. Alkaline pHo activated PKB/Akt signalling to the same degree as insulin in perfused adult male rat hearts. INSRRs were expressed in rat hearts and, by immunoblotting for phosphorylation (activation) of INSRRs/INSRs/IGF1Rs, we established that INSRRs, together with INSRs/IGF1Rs, are activated by alkaline pHo. The INSRR/INSR/IGF1R kinase inhibitor, linsitinib, prevented PKB/Akt activation by alkaline pHo, indicating that INSRRs/INSRs/IGF1Rs are required. Activation of PKB/Akt in cardiomyocytes by α1-AR agonists was also inhibited by linsitinib. Furthermore, linsitinib inhibited cardiomyocyte hypertrophy induced by α1-ARs in cultured cells, reduced the initial cardiac adaptation (24 h) to phenylephrine in vivo (assessed by echocardiography) and increased cardiac fibrosis over 4 days. We conclude that INSRRs are expressed in the heart and, together with INSRs/IGF1Rs, the insulin receptor family provide a potent system for promoting protein synthesis and cardioprotection. Moreover, this system is required for adaptive hypertrophy induced by α1-ARs.

Class A G protein-coupled receptors assemble into functional higher-order hetero-oligomers.

Although class A seven-transmembrane helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC), and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4, atypical chemokine receptor 3, α1a -adrenoceptor, and arginine vasopressin receptor 1A form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function.

GPR55 regulates the responsiveness to, but does not dimerise with, α1A-adrenoceptors.

Emerging evidence suggests that G protein coupled receptor 55 (GPR55) may influence adrenoceptor function/activity in the cardiovascular system. Whether this reflects direct interaction (dimerization) between receptors or signalling crosstalk has not been investigated. This study explored the interaction between GPR55 and the alpha 1A-adrenoceptor (α1A-AR) in the cardiovascular system and the potential to influence function/signalling activities. GPR55 and α1A-AR mediated changes in both cardiac and vascular function was assessed in male wild-type (WT) and GPR55 homozygous knockout (GPR55-/-) mice by pressure volume loop analysis and isolated vessel myography, respectively. Dimerization of GPR55 with the α1A-AR was examined in transfected Chinese hamster ovary-K1 (CHO-K1) cells via Bioluminescence Resonance Energy Transfer (BRET). GPR55 and α1A-AR mediated signalling (extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation) was investigated in neonatal rat ventricular cardiomyocytes using AlphaScreen proximity assays. GPR55-/- mice exhibited both enhanced pressor and inotropic responses to A61603 (α1A-AR agonist), while in isolated vessels, A61603 induced vasoconstriction was attenuated by a GPR55-dependent mechanism. Conversely, GPR55-mediated vasorelaxation was not altered by pharmacological blockade of α1A-ARs with tamsulosin. While cellular studies demonstrated that GPR55 and α1A-AR failed to dimerize, pharmacological blockade of GPR55 altered α1A-AR mediated signalling and reduced ERK1/2 phosphorylation. Taken together, this study provides evidence that GPR55 and α1A-AR do not dimerize to form heteromers, but do interact at the signalling level to modulate the function of α1A-AR in the cardiovascular system.

The C-terminal of the α1b-adreneroceptor is a key determinant for its structure integrity and biological functions.

The C-terminal of G protein-coupled receptors is now recognized as being important for G protein activation and signaling function. To detect the role of C-terminal tail in receptor activation, we used the α1b-AR, which has a long C-terminal of 164 amino acids. We constructed the intramolecular FRET sensors, in which the C-terminal was truncated to 10 (∆C-10), 20 (∆C-20), 30 (∆C-30), 50 (∆C-50), 70 (∆C-70), or 90 (∆C-90). The truncated mutants of ∆C-10, ∆C-20, or ∆C-30 cannot induce FRET signal changes and downstream ERK1/2 phosphorylation. However, the truncated mutants of ∆C-50, ∆C-70, or ∆C-90 induce significant FRET signal changes and downstream ERK1/2 phosphorylation, especially ∆C-90. This is particularly true in the case of the ∆C-90, ∆C-70, or ∆C-50 which retained the potential phosphorylation sites (Ser401, Ser404, Ser408, or Ser410). The ∆C-90 showed an increase in agonist-induced FRET signal changes and ERK1/2 phosphorylation in PKC- or endocytosis-dependent and EGFR-, src-, or ß-arrestin2-independent.

Identification of novel therapeutic targets for contrast induced acute kidney injury (CI-AKI): alpha blockers as a therapeutic strategy for CI-AKI.

Iodinated contrast is used for imaging and invasive procedures and it can cause contrast induced acute kidney injury (CI-AKI), which is the third leading hospital-acquired health problem. The purpose of the present study was to determine the effect of α-adrenergic receptor-1b (Adra1b) inhibition by using terazosin on change in kidney function, gene, and protein expression in C57BL/6J male mice, 6-8 weeks with chronic kidney disease (CKD). CKD was induced by surgical nephrectomy. Twenty eight days later, 100-µL of iodinated contrast (CI group) or saline (S group) was given via the carotid artery. Whole-transcriptome RNA-sequencing (RNA-Seq) analysis of the kidneys was performed at day 2. Mice received either 50-µL of saline ip or terazosin (2 mg/kg) in 50-µL of saline ip 1 hour before contrast administration which was continued every 12 hours until the animals were euthanized 2 and 7 days later. The kidneys were removed for gene expression, immunohistochemical analysis, and blood serum analyzed for kidney function. Differential gene expression analysis identified 21 upregulated and 436 downregulated genes (fold change >2; P < 0.05) that were common to all sample (n = 3 for both contrast and saline). We identified Adra1b using bioinformatic analysis. Mice treated with terazosin had a significant decrease in serum creatinine, urinary Kim-1 levels, HIF-1α, apoptosis, and downstream Adrab1 genes including Ece1, Edn1, pMAPK14 with increased cell proliferation. Contrast exposure upregulated Adra1b gene expression in HK-2 cells. Inhibition of Adra1b with terazosin abrogated Ece1, Edn1, and contrast-induced Fsp-1, Mmp-2, Mmp-9 expression, and caspase-3/7 activity in HK-2 cells.

α1A-adrenaline receptors in dorsal horn inhibitory neurons have an inhibitory role in the regulation of chloroquine-induced itch in mice.

Our previous study showed the intrinsic ability of descending noradrenergic neurons projecting from the locus coeruleus to the spinal dorsal horn (SDH) to suppress itch-related behaviors. Noradrenaline and α1A-adrenaline receptor (α1A-AR) agonist increase inhibitory synaptic inputs onto SDH interneurons expressing gastrin-releasing peptide receptors, which are essential for itch transmission. However, the contribution of α1A-ARs expressed in SDH inhibitory interneurons to itch-related behavior remains to be determined. In this study, RNAscope in situ hybridization revealed that Adra1a mRNA is expressed in SDH inhibitory interneurons that are positive for Slc32a1 mRNA (known as vesicular GABA transporter). Mice with conditional knock-out of α1A-ARs in inhibitory interneurons (Vgat-Cre;Adra1aflox/flox mice) exhibited an increase in scratching behavior when induced by an intradermal injection of chloroquine, but not compound 48/80, which are known as models of histamine-independent and dependent itch, respectively. Furthermore, knockout of inhibitory neuronal α1A-ARs in the SDH using the CRISPR-Cas9 system also increased the scratching behavior elicited by chloroquine but not compound 48/80. Our findings demonstrated for the first time that α1A-ARs in SDH inhibitory interneurons contribute to the regulation of itch signaling with preference for histamine-independent itch.

Adaptation to exercise-induced stress is not dependent on cardiomyocyte α1A-adrenergic receptors.

The 'fight or flight' response to physiological stress involves sympathetic nervous system activation, catecholamine release and adrenergic receptor stimulation. In the heart, this induces positive inotropy, previously attributed to the ß1-adrenergic receptor subtype. However, the role of the α1A-adrenergic receptor, which has been suggested to be protective in cardiac pathology, has not been investigated in the setting of physiological stress. To explore this, we developed a tamoxifen-inducible, cardiomyocyte-specific α1A-adrenergic receptor knock-down mouse model, challenged mice to four weeks of endurance swim training and assessed cardiac outcomes. With 4-OH tamoxifen treatment, expression of the α1A-adrenergic receptor was knocked down by 80-89%, without any compensatory changes in the expression of other adrenergic receptors, or changes to baseline cardiac structure and function. Swim training caused eccentric hypertrophy, regardless of genotype, demonstrated by an increase in heart weight/tibia length ratio (30% and 22% in vehicle- and tamoxifen-treated animals, respectively) and an increase in left ventricular end diastolic volume (30% and 24% in vehicle- and tamoxifen-treated animals, respectively) without any change in the wall thickness/chamber radius ratio. Consistent with physiological hypertrophy, there was no increase in fetal gene program (Myh7, Nppa, Nppb or Acta1) expression. In response to exercise-induced volume overload, stroke volume (39% and 30% in vehicle- and tamoxifen-treated animals, respectively), cardiac output/tibia length ratio (41% in vehicle-treated animals) and stroke work (61% and 33% in vehicle- and tamoxifen-treated animals, respectively) increased, regardless of genotype. These findings demonstrate that cardiomyocyte α1A-adrenergic receptors are not necessary for cardiac adaptation to endurance exercise stress and their acute ablation is not deleterious.