Dianne M. Perez

α1-Adrenergic Receptor Subtypes Molecular Structure, Function, and Signaling

The α1ARs are important mediators of sympathetic nervous system responses, particularly those involved in cardiovascular homeostasis, such as arteriolar smooth muscle constriction and cardiac contraction.1 2 In addition, α1ARs have more recently been implicated in the pathogenesis of cardiac hypertrophy, in ischemia-induced cardiac arrhythmias, and in ischemic preconditioning.1 3 Like other ARs, α1ARs are activated by the catecholamines, norepinephrine and epinephrine. They are intrinsic membrane glycoproteins and are members of the GPCR superfamily. Over the past 10 to 15 years, data initially based on functional, radioligand, and biochemical studies have accumulated, indicating that the α1ARs are a heterogeneous group of distinct but related proteins. This conclusion has been confirmed with the molecular cloning of three distinct α1-receptor subtypes, although until recently discrepancies between the properties of the cloned expressed receptors and those characterized pharmacologically and biochemically have led to confusion in the classification of α1-receptor subtypes and their coupled effector responses. As detailed in the present review, much of this confusion has now been clarified for the three cloned α1ARs. These and other recent insights into the molecular structure, function, and signaling of α1ARs, the control of α1AR-gene expression, and pharmacological evidence for additional α1AR subtypes will be reviewed here. For additional information, the reader is also referred to several previous reviews of α1ARs.4 5 6 7 Functional studies of AR responses, particularly from the laboratories of McGrath8 and Ruffolo,9 provided the initial evidence that there may be subtypes of α1ARs. These studies indicated that postjunctional responses mediated by α1ARs could not be explained adequately on the basis of a single population of receptors. This concept was further advanced …

Multiple Signaling States of G-Protein-Coupled Receptors

Studies have been amassed in the past several years indicating that an agonist can conform a receptor into an activation state that is dependent upon an intrinsic property of the agonist usually based upon its chemical composition. Theoretically, each different agonist could impart its own unique activation state. Evidence for multiple signaling states for the G-protein-coupled receptors will be reviewed and is derived from many different pharmacological behaviors: efficacy, kinetics, protean agonism, differential desensitization and internalization, inverse agonism, and fusion chimeras. A recent extension of the ternary complex model is suggested by evidence that the different processes that govern deactivation, such as desensitization and internalization, is also regulated by conformers specific to the agonist. Rhodopsin may serve as a primer for the study of multiple activation states. Therapeutic implications that utilize multiple signaling states hold vast promise in the rationale design of drugs.

Unique microRNA profile in end-stage heart failure indicates alterations in specific cardiovascular signaling networks.

It is well established that gene expression patterns are substantially altered in cardiac hypertrophy and heart failure, but the reasons for such differences are not clear. MicroRNAs (miRNAs) are short noncoding RNAs that provide a novel mechanism for gene regulation. The goal of this study was to comprehensively test for alterations in miRNA expression using human heart failure samples with an aim to build signaling pathway networks using predicted targets for the miRNAs and to identify nodal molecules that control these networks. Genome-wide profiling of miRNAs was performed using custom-designed miRNA microarray followed by validation on an independent set of samples. Eight miRNAs are significantly altered in heart failure of which we have identified two novel miRNAs that are yet to be implicated in cardiac pathophysiology. To gain an unbiased global perspective on regulation by altered miRNAs, predicted targets of eight miRNAs were analyzed using the Ingenuity Pathways Analysis network algorithm to build signaling networks and identify nodal molecules. The majority of nodal molecules identified in our analysis are targets of altered miRNAs and are known regulators of cardiovascular signaling. A heart failure gene expression data base was used to analyze changes in expression patterns for these target nodal molecules. Indeed, expression of nodal molecules was altered in heart failure and inversely correlated to miRNA changes validating our analysis. Importantly, using network analysis we have identified a limited number of key functional targets that may regulate expression of the myriad proteins in heart failure and could be potential therapeutic targets.

Activation of the α1b-Adrenergic Receptor Is Initiated by Disruption of an Interhelical Salt Bridge Constraint

Rhodopsin receptor activation involves the disruption of a salt bridge constraint between glutamic acid 113 on transmembrane 3 and a lysine 296 in transmembrane 7, which forms a Schiffs base with retinal. Light-induced isomerization of cis-retinal to the all trans form breaks this rhodopsin salt bridge leading to receptor activation. The analogous residues in α1b-adrenergic receptors, aspartic acid 125 and lysine 331, also have the potential of forming a constraining salt bridge holding the receptor to an inactive protein configuration. This α1b-adrenergic receptor salt bridge constraint is then released upon binding by the receptor agonist. To test this hypothesis, site-directed mutagenesis was used to eliminate the positive charge at position 331 by substitution of an alanine. The expressed α1b-adrenergic receptor mutant demonstrated a 6-fold increased epinephrine binding affinity with no alterations of affinity values for selective adrenergic receptor antagonists. Furthermore, an increased epinephrine potency for total soluble inositol phosphate production along with an elevated basal inositol triphosphate level was observed in COS-1 cells transfected with mutant versus wild-type α1b-adrenergic receptors. Similar results were obtained for a lysine to a glutamic acid α1b-adrenergic receptor mutation. In addition, increased basal inositol triphosphate levels were also observed for two aspartic acid 125 α1b-adrenergic receptor mutations, consistent with this residues role as the counterion of the salt bridge. Taken together, these α1b-adrenergic receptor mutations suggest a molecular mechanism by which the positively charged lysine 331 stabilizes the negatively charged aspartic acid 125 via a salt bridge constraint until bound by the receptor agonist.

Overexpression of the α 1B -adrenergic receptor causes apoptotic neurodegeneration: Multiple system atrophy

Progress toward elucidating the function of α1B-adrenergic receptors (α1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate α1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active α1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in α1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the α1AR antagonist terazosin, indicating that α1AR signaling participated directly in the pathology. Our results indicate that overstimulation of α1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.

Hypotension, Autonomic Failure, and Cardiac Hypertrophy in Transgenic Mice Overexpressing the α1B-Adrenergic Receptor

α1-Adrenergic receptors (α1A, α1B, and α1D) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the α1A and α1D subtypes is thought to be mainly responsible for this activity, the role of the α1B-adrenergic receptor (α1BAR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active α1BARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by α1BAR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of α1ARs that is important in systemic regulation of blood pressure.

Localization of the mouse α1A‐adrenergic receptor (AR) in the brain: α1AAR is expressed in neurons, GABAergic interneurons, and NG2 oligodendrocyte progenitors

α1‐Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human α1A‐AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse α1A‐AR promoter. We confirm our transgenic model against the α1A‐AR knockout mouse, which expresses the LacZ gene in place of the coding region for the α1A‐AR. By using these models, we have now determined cellular localization of the α1A‐AR in the brain, at the protein level. The α1A‐AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the α1A‐AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma‐aminobutyric acid (GABA), and N‐methyl‐D‐aspartate (NMDA) receptors. Recordings from α1A‐AR EGFP‐expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the α1A‐AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the α1A‐AR in oligodendrocyte progenitors. We conclude that the α1A‐AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons. J. Comp. Neurol. 497:209–222, 2006.

β1-Adrenergic Receptor Autoantibodies Mediate Dilated Cardiomyopathy by Agonistically Inducing Cardiomyocyte Apoptosis

Background— Antibodies to the &bgr;1-adrenergic receptor (&bgr;1AR) are detected in a substantial number of patients with idiopathic dilated cardiomyopathy (DCM). The mechanism whereby these autoantibodies exert their pathogenic effect is unknown. Here, we define a causal mechanism whereby &bgr;1AR-specific autoantibodies mediate noninflammatory cardiomyocyte cell death during murine DCM. Methods and Results— We used the &bgr;1AR protein as an immunogen in SWXJ mice and generated a polyclonal battery of autoantibodies that showed selective binding to the &bgr;1AR. After transfer into naive male hosts, &bgr;1AR antibodies elicited fulminant DCM at high frequency. DCM was attenuated after immunoadsorption of &bgr;1AR IgG before transfer and by selective pharmacological antagonism of host &bgr;1AR but not &bgr;2AR. We found that &bgr;1AR autoantibodies shifted the &bgr;1AR into the agonist-coupled high-affinity state and activated the canonical cAMP-dependent protein kinase A signaling pathway in cardiomyocytes. These events led to functional alterations in intracellular calcium handling and contractile function. Sustained agonism by &bgr;1AR autoantibodies elicited caspase-3 activation, cardiomyocyte apoptosis, and DCM in vivo, and these processes were prevented by in vivo treatment with the pan-caspase inhibitor Z-VAD-FMK. Conclusions— Our data show how &bgr;1AR-specific autoantibodies elicit DCM by agonistically inducing cardiomyocyte apoptosis.

Recent advances in the molecular pharmacology of the α1-adrenergic receptors

Abstract This review is intended to discuss recent developments in the molecular pharmacology of the α 1 -adrenergic receptor ( α 1 -AR) subtypes. After a brief historical development will focus on the more contemporary issues having to do with this receptor family. Emphasis will be put on recent data regarding the cloning, nomenclature, signalling mechanisms, and genomic organization of the α 1 -AR subtypes. We will also highlight recent mutational studies that identify key amino acid residues involved in ligand binding, as well as the role of the α 1 -AR subtypes in regulating physiologic processes.

α1-Adrenergic Receptor Stimulates Interleukin-6 Expression and Secretion through Both mRNA Stability and Transcriptional Regulation: Involvement of p38 Mitogen-Activated Protein Kinase and Nuclear Factor-κB

Our previous studies have demonstrated that activation of α1-adrenergic receptors (ARs) increased interleukin-6 (IL-6) mRNA expression and protein secretion, which is probably an important yet unknown mechanism contributing to the regulation of cardiac function. Using Rat-1 fibroblasts stably transfected with the α1A-AR subtype and primary mouse neonatal cardiomyocytes, we elucidated the basic molecular mechanisms responsible for the α1-AR modulation of IL-6 expression. IL-6 mRNA production mediated by α1-AR peaked at 1 to 2 h. Studies of the mRNA decay rate indicated that α1-AR activation enhanced IL-6 mRNA stability. Analysis of IL-6 promoter activity using a series of deletion constructs indicated that α1-ARs enhanced IL-6 transcription through several transcriptional elements, including nuclear factor κB (NF-κB). Inhibition of α1-AR mediated IL-6 production and secretion by actinomycin D and the increase of intracellular IL-6 levels by α1-AR activation suggest that α1-AR mediated IL-6 secretion through de novo synthesis. Both IL-6 transcription and protein secretion mediated by α1-ARs were significantly reduced by chemical inhibitors for p38 mitogen-activated protein kinase (MAPK) and NF-κB and by a dominant-negative construct of p38 MAPK. Serum level of IL-6 was elevated in transgenic mice expressing a constitutively active mutant of the α1A-AR subtype but not in a similar mouse model expressing the α1B-AR subtype. Our results indicate that α1-ARs stimulated IL-6 expression and secretion through regulating both mRNA transcription and stability, involving p38 MAPK and NF-κB pathways.