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ABSTRACT: The role of α(1)-adrenergic receptors (AR) in the regulation of cardiac hypertrophy is still unclear, as transgenic mice have demonstrated hypertrophy or the lack of it despite high receptor overexpression. To further address the role of the α(1)-ARs in cardiac hypertrophy we analyzed unique transgenic mice that overexpress CAM α(1A)-ARs or CAM α(1B)-ARs under the regulation of large fragments of their native promoters. These constitutively active receptors are expressed in all tissues that endogenously express their WT counterparts as opposed to only myocyte-targeted transgenic mice. In this study, we discovered that CAM α(1A)-AR mice in vivo have cardiac hypertrophy independent of changes in blood pressure, corroborating earlier studies, but in contrast to the myocyte-targeted α(1A)-AR mice. We also found cardiac hypertrophy in CAM α(1B)-AR mice, in agreement with previous studies, but hypertrophy only developed in older mice. We also discovered unique α(1)-AR-mediated hypertrophic signaling that was AR subtype-specific with CAM α(1A)-AR mice secreting ANF and IL-6, while CAM α(1B)-AR mice expressed activated NF-κB. These particular hypertrophic signals were blocked when the other AR subtype was co-activated. We also discovered that crossbreeding the two CAM models (double CAM α(1A/B)-AR) inhibited the development of hypertrophy and was reversible with single receptor activation, suggesting co-activation of the receptors can lead to novel antagonistic signal transduction. This was confirmed by demonstrating antagonistic signals that were even lower than normal controls in the double CAM α(1A/B)-AR mice for p-38, NF-κB and the IL-6/gp130/STAT3 pathway. As α(1A/B) double knockout mice fail to develop hypertrophy in response to IL-6, our results suggest that IL-6 is a major mediator of α(1A)-AR cardiac hypertrophy.
Molecular pharmacology 02/2013; · 4.53 Impact Factor
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ABSTRACT: 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 PKCϵ 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 PKCϵ/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.
Journal of Receptor and Signal Transduction Research 01/2012; 32(2):76-86. · 1.59 Impact Factor
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Van A Doze, Robert S Papay,
Brianna L Goldenstein,
Manveen K Gupta,
Katie M Collette,
Brian W Nelson,
Mariaha J Lyons,
Bethany A Davis,
Elizabeth J Luger,
Sarah G Wood,
James R Haselton,
Paul C Simpson,
Dianne M Perez
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ABSTRACT: The role of α(1)-adrenergic receptors (α(1)ARs) in cognition and mood is controversial, probably as a result of past use of nonselective agents. α(1A)AR activation was recently shown to increase neurogenesis, which is linked to cognition and mood. We studied the effects of long-term α(1A)AR stimulation using transgenic mice engineered to express a constitutively active mutant (CAM) form of the α(1A)AR. CAM-α(1A)AR mice showed enhancements in several behavioral models of learning and memory. In contrast, mice that have the α(1A)AR gene knocked out displayed poor cognitive function. Hippocampal brain slices from CAM-α(1A)AR mice demonstrated increased basal synaptic transmission, paired-pulse facilitation, and long-term potentiation compared with wild-type (WT) mice. WT mice treated with the α(1A)AR-selective agonist cirazoline also showed enhanced cognitive functions. In addition, CAM-α(1A)AR mice exhibited antidepressant and less anxious phenotypes in several behavioral tests compared with WT mice. Furthermore, the lifespan of CAM-α(1A)AR mice was 10% longer than that of WT mice. Our results suggest that long-term α(1A)AR stimulation improves synaptic plasticity, cognitive function, mood, and longevity. This may afford a potential therapeutic target for counteracting the decline in cognitive function and mood associated with aging and neurological disorders.
Molecular pharmacology 07/2011; 80(4):747-58. · 4.53 Impact Factor
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ABSTRACT: The understanding of the function of alpha(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 alpha(1)-adrenergic receptor subtypes in tissues in which they are normally expressed. We identified intriguing alpha(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 alpha(1A)-adrenergic receptor transgenic mice or normal mice given the alpha(1A)-adrenergic receptor-selective agonist, cirazoline. Neonatal neurospheres isolated from normal mice expressed a mixture of alpha(1)-adrenergic receptor subtypes, and stimulation of these receptors resulted in increased expression of the alpha(1B)-adrenergic receptor subtype, proneural basic helix-loop-helix transcription factors, and the differentiation and migration of neuronal progenitors for catecholaminergic neurons and interneurons. alpha(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, alpha(1)-adrenergic receptor stimulation increased the expression of glial markers at the expense of neuronal differentiation. In vivo, S100-positive glial and betaIII tubulin neuronal progenitors colocalized with either alpha(1)-adrenergic receptor subtype in the olfactory bulb. Our results indicate that alpha(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.
Molecular pharmacology 07/2009; 76(2):314-26. · 4.53 Impact Factor
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ABSTRACT: Our previous studies have demonstrated that activation of alpha(1)-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 alpha(1A)-AR subtype and primary mouse neonatal cardiomyocytes, we elucidated the basic molecular mechanisms responsible for the alpha(1)-AR modulation of IL-6 expression. IL-6 mRNA production mediated by alpha(1)-AR peaked at 1 to 2 h. Studies of the mRNA decay rate indicated that alpha(1)-AR activation enhanced IL-6 mRNA stability. Analysis of IL-6 promoter activity using a series of deletion constructs indicated that alpha(1)-ARs enhanced IL-6 transcription through several transcriptional elements, including nuclear factor kappaB (NF-kappaB). Inhibition of alpha(1)-AR mediated IL-6 production and secretion by actinomycin D and the increase of intracellular IL-6 levels by alpha(1)-AR activation suggest that alpha(1)-AR mediated IL-6 secretion through de novo synthesis. Both IL-6 transcription and protein secretion mediated by alpha(1)-ARs were significantly reduced by chemical inhibitors for p38 mitogen-activated protein kinase (MAPK) and NF-kappaB 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 alpha(1A)-AR subtype but not in a similar mouse model expressing the alpha(1B)-AR subtype. Our results indicate that alpha(1)-ARs stimulated IL-6 expression and secretion through regulating both mRNA transcription and stability, involving p38 MAPK and NF-kappaB pathways.
Molecular pharmacology 05/2009; 76(1):144-52. · 4.53 Impact Factor
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ABSTRACT: Adrenergic receptors (ARs) play an important role in the regulation of cardiac function. Cardiac inotropy is primarily regulated by beta(1)-ARs. However, alpha(1)-ARs may play an important role in inotropy during heart failure. Previous work has suggested that the alpha(1B)-AR modulates beta(1)-AR function in the heart. The potential role of the alpha(1A)-AR has not been previously studied. We used transgenic mice that express constitutively active mutant (CAM) forms of the alpha(1A)-AR or alpha(1B)-AR regulated by their endogenous promoters. Expression of the CAM alpha(1A)-AR or CAM alpha(1B)-AR had no effect on basal cardiac function (developed pressure, +dP/dT, -dP/dT, heart rate, flow rate). However, both alpha(1)-AR subtypes significantly decreased isoproterenol-stimulated +dP/dT. Pertussis toxin had no effect on +dP/dT in CAM alpha(1A)-AR hearts but restored +dP/dT to non-transgenic values in CAM alpha(1B)-AR hearts. Radioligand binding indicated a selective decrease in the density of beta(1)-ARs in both CAM mice. However, G-proteins, cAMP, or the percentage of high and low affinity states were unchanged in either transgenic compared with control. These data demonstrate that CAM alpha(1A)- and alpha(1B)-ARs both down regulate beta(1)-AR-mediated inotropy in the mouse heart. However, alpha(1)-AR subtypes are coupled to different beta-AR mediated signaling pathways with the alpha(1B)-AR being pertussis toxin sensitive.
Journal of Molecular and Cellular Cardiology 12/2005; 39(5):777-84. · 5.17 Impact Factor
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ABSTRACT: Brief periods of ischemia stimulate an endogenous mechanism in the heart that protects the myocardium from subsequent ischemic injury. alpha1-Adrenergic receptors (ARs) have been implicated in this process. However, the lack of sufficiently selective antagonists has made it difficult to determine which alpha1-AR subtype protects the heart from ischemic injury. The goal of this study was to identify the alpha1-AR subtype that is involved in ischemic preconditioning.
We developed transgenic mice that express constitutively active mutant (CAM) forms of the alpha1A-AR or the alpha1B-AR regulated by their endogenous promoters. Hearts isolated from transgenic and non-transgenic mice were perfused by the Langendorff method using an ischemic preconditioning perfusion protocol or a non-preconditioning perfusion protocol prior to 30-min ischemia and 40-min reperfusion. Contractile function was continuously monitored through an intraventricular balloon.
The contractile function of non-transgenic hearts perfused according to the ischemic preconditioning protocol completely recovered from 30-min ischemia. However, non-transgenic hearts perfused according to the non-preconditioning protocol recovered only 60% of their contractile function. The contractile function of CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, completely recovered from 30-min ischemia even though they were perfused according to the non-preconditioning protocol. Thus, CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, were inherently preconditioned against ischemic injury. Staurosporine, but not chelerythrine, completely reversed the preconditioning effect of CAM alpha1A-ARs.
These data demonstrate that alpha1A-ARs protect the heart from ischemic injury through a staurosporine-sensitive signaling pathway that is independent of protein kinase C.
Cardiovascular Research 03/2005; 65(2):436-45. · 6.06 Impact Factor
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ABSTRACT: The alpha1-adrenergic receptors (alpha1ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central alpha1ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each alpha1AR subtype to various central functions is currently unclear. Transcription regulation through alpha1AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the alpha1BAR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of alpha1BAR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N-methyl-d-aspartate (NMDA) receptors and decreased GABAA, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The alpha1BAR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures.
Brain 01/2004; 126(Pt 12):2667-81. · 9.46 Impact Factor
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ABSTRACT: Alpha(1)-adrenoceptor subtypes (alpha(1A)-, alpha(1B)-, alpha(1D)-) are known to couple to similar signaling pathways, although differences among the subtypes do exist. As a more sensitive assay, we used oligonucleotide microarrays to identify gene expression changes in Rat-1 fibroblasts stably expressing each individual subtype. We report the gene expressions that change by at least a factor of 2 or more. Gene expression profiles significantly changed equally among all three subtypes, despite the unequal efficacy of the inositol phosphate response. Gene expressions were clustered into cytokines/growth factors, transcription factors, enzymes, and extracellular matrix proteins. There were also a number of individual subtype-specific changes in gene expression, suggesting a link to independent pathways. In addition, all three alpha(1)-AR subtypes robustly stimulated the transcription of the prohypertrophic cytokine interleukin (IL)-6, but differentially altered members of the IL-6 signaling pathway (gp-130 and STAT3). This was confirmed by measurement of secreted IL-6, activated STAT3, and gp-130 levels. Activation of STAT3 Tyr705 phosphorylation by the alpha(1)-ARs was not through IL-6 activation but was synergistic with IL-6, suggesting direct effects. Interestingly, alpha(1B)-AR stimulation caused the dimerization-dependent phosphorylation of Tyr705 on STAT3 but did not activate the transcriptional-dependent phosphorylation of Ser727. The alpha(1B)-AR also constitutively down-regulated the protein levels of gp-130. These results suggest that the alpha(1B)-AR has differential effects on the phosphorylation status of the STAT3 pathway and may not be as prohypertrophic as the other two subtypes.
Molecular Pharmacology 06/2003; 63(5):1104-16. · 4.88 Impact Factor
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ABSTRACT: Adrenergic receptors (ARs) play an important role in the regulation of cardiac function. Cardiac inotropy is primarily regulated by β1-ARs. However, α1-ARs may play an important role in inotropy during heart failure. Previous work has suggested that the α1B-AR modulates β1-AR function in the heart. The potential role of the α1A-AR has not been previously studied. We used transgenic mice that express constitutively active mutant (CAM) forms of the α1A-AR or α1B-AR regulated by their endogenous promoters. Expression of the CAM α1A-AR or CAM α1B-AR had no effect on basal cardiac function (developed pressure, +dP/dT, –dP/dT, heart rate, flow rate). However, both α1-AR subtypes significantly decreased isoproterenol-stimulated +dP/dT. Pertussis toxin had no effect on +dP/dT in CAM α1A-AR hearts but restored +dP/dT to non-transgenic values in CAM α1B-AR hearts. Radioligand binding indicated a selective decrease in the density of β1-ARs in both CAM mice. However, G-proteins, cAMP, or the percentage of high and low affinity states were unchanged in either transgenic compared with control. These data demonstrate that CAM α1A- and α1B-ARs both downregulate β1-AR-mediated inotropy in the mouse heart. However, α1-AR subtypes are coupled to different β-AR mediated signaling pathways with the α1B-AR being pertussis toxin sensitive.
Journal of Molecular and Cellular Cardiology.
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ABSTRACT: The α<SUB>1</SUB>-adrenergic receptors (α<SUB>1</SUB>ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central α<SUB>1</SUB>ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each α<SUB>1</SUB>AR subtype to various central functions is currently unclear. Transcription regulation through α<SUB>1</SUB>AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the α<SUB>1B</SUB>AR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of α<SUB>1B</SUB>AR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N -methyl-D-aspartate (NMDA) receptors and decreased GABA<SUB>A</SUB>, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The α<SUB>1B</SUB>AR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures.
