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.     

Cardiac pressure overload hypertrophy is differentially regulated by β-adrenergic receptor subtypes

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

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.     

Alpha(1)-adrenergic receptor subtypes: non-identical triplets with different dancing partners?

Alpha(1)-adrenergic receptors are one of the three subfamilies of G protein coupled receptors activated by epinephrine and norepinephrine to control important functions in many target organs. Three human subtypes (alpha(1A), alpha(1B), alpha(1D)) are derived from separate genes and are highly homologous in their transmembrane domains but not in their amino or carboxyl termini. Recent advances in our understanding of these "non-identical triplets" include development of knockout mice lacking single or multiple subtypes, new insights into subcellular localization and trafficking, identification of allosteric modulators, and increasing evidence for an important role in brain function. Although all three subtypes activate the same G(q/11) signaling pathway, they also appear to interact with different protein binding partners. Recent evidence suggests they may also form dimers, and may initiate independent signals through pathways yet to be clearly elucidated. Thus, this subfamily represents a common phenomenon of a group of similar but non-identical receptor subtypes activated by the same neurotransmitter, whose individual functional roles remain to be clearly established.     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.     

Arylsulfonamide derivatives of (aryloxy)ethyl pyrrolidines and piperidines as α1-adrenergic receptor antagonist with uro-selective activity

A series of arylsulfonamide derivatives of (aryloxy)ethyl pyrrolidines and piperidines was synthesized to develop new α₁-adrenoceptor antagonists with uroselective profile. Biological evaluation for α₁- and α₂-adrenorecepor showed that tested compounds 13–37 displayed high-to-moderate affinity for the α₁-adrenoceptor (K_i = 34–348 nM) and moderate selectivity over α₂-receptor subtype. Compounds with highest affinity and selectivity for α₁-adrenoceptor were evaluated in vitro for their intrinsic activity toward α_1A- and α_1B-adrenoceptor subtypes. All compounds behaved as antagonists at both α₁-adrenoceptor subtypes, displaying 2- to 6-fold functional preference to α_1A-subtype. Among them, N-{1-[2-(2-methoxyphenoxy)ethyl]piperidin-4-yl}isoquinoline-4-sulfonamide (25) and 3-chloro-2-fluoro-N-{[1-(2-(2-isopropoxyphenoxy)ethyl)piperidin-4-yl]methyl}benzene sulfonamide (34) displayed the highest preference to α_1A-adrenoceptor. Finally, compounds 25 and 34 (2–5 mg/kg, iv), in contrast to tamsulosin (1–2 mg/kg, iv), did not significantly decrease systolic and diastolic blood pressure in normotensive anesthetized rats to determine their influence on blood pressure.     

Agonist-induced down-regulation of α_(1B)-adrenergic receptor in HEK293 cells transfected with α_(1B)cDNA

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Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

Bromocriptine inhibits adipogenesis and lipogenesis by agonistic action on α2-adrenergic receptor in 3T3-L1 adipocyte cells

The primary goals of the present study were to investigate the inhibitory effects of bromocriptine (BC) on adipogenesis and lipogenesis in 3T3-L1 adipocyte cells as well as to elucidate its molecular mechanism of action. Adipogenic and lipogenic capacity of BC-treated cells was evaluated by oil red-O staining, triglyceride content assay, real-time RT-PCR and immunoblotting. To determine the mechanism responsible for the anti-obesity effect of BC, we applied two methods. Firstly, we knocked down dopamine D2 receptor (D2R) up to 50 % using siRNA. Secondly, we blocked the activity of α2-adrenergic receptor (α2-AR) by yohimbine treatment and monitored its effects on adipogenic and lipogenic events in 3T3-L1 cells. BC decreased the expression levels of adipogenic activators, including Pparα, Pparγ, and Cebpα, as well as major lipogenic target genes, including Me1, Acc1, 6Pgd, Fasn, and Prkaa1. Moreover, BC markedly reduced intracellular nitric oxide formation in a dose-dependent manner and expression of pro-inflammatory genes, Tnfα and Il6, which reflects attenuated pro-inflammatory responses. Further, upon treatment with BC, D2R-deficient cells displayed a significant decrease in lipogenic activity compared to control cells, whereas yohimbine-treated cells exhibited no reduction in lipogenic activity. BC can effectively attenuate adipogenesis and lipogenesis in 3T3-L1 cells by downregulating the expression of lipogenic genes and proteins. Our current experimental data collectively establish that the anti-obesity effects of BC are not D2R-dependent but result from the action of α2-AR in 3T3-L1 adipocytes.     

Opioid μ receptor subtypes (possibly μ1 and μ2) revealed by morphine-induced antinociception vs endothelin-1 in recombinant inbred CXBK mice

Morphine was administered intracerebroventricularly to normal or recombinant inbred CXBK (μ-opioid receptor deficient) mice and antinociception was determined against two different stimuli. Morphine-induced antinociception agains acetylcholine was strain-dependent, whereas against endothelin-1 it was not. The antinociception was mediated via opioid μ receptors (blocked by β-FNA, but not naltrindole, ICI 174,864 or nor-BNI) through separate pathways, one naloxonazine-sensitive and the other naloxonazine-insensitive. Taken together, these results appear to demonstrate supraspinal morphine-induced antinociception through distinct subtypes of the μ opioid receptor, supporting the possibility of novel subtype-selective therepeutic agents with greater separation between analgesia and side-effects or physical dependence. Furthermore, the methodology described herein provides model systems for the in vivo screening of such agents.     

A disintegrin and metalloenzyme (ADAM) 17 activation is regulated by α5β1 integrin in kidney mesangial cells

Background

The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation.

Methodology/Principal Findings

We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT.

Conclusions/Significance

Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.     

A dorsomedial subdivision within the nucleus intercollicularis identified in the Japanese quail (Coturnix coturnix japonica) by means of α2-adrenergic receptor autoradiography and estrogen receptor immunohistochemistry

Summary The nucleus intercollicularis is an important site in the control of vocalization in birds. In oscines, a subregion of the nucleus intercollicularis called the dorso-medial intercollicular nucleus appears to play a key role in this process because it receives the majority of the projections from the nucleus robustus archistriatalis and sends most of the projections to the motor nucleus of the hypoglossal nerve. In this paper, we present neurochemical studies of the nucleus intercollicularis in the Japanese quail which suggest the presence of heterogeneity within this structure. One rostral band contains high densities of cholinergic muscarinic receptors identified by quantitative autoradiography using tritiated N-methylscopolamine as the ligand. A caudal dorso-medial region is specifically labeled by estrogen receptors identified using immunocytochemistry and by ₂-adrenergic receptors which were quantified by autoradiography using tritiated para-amino-clonidine. This latter sub-region is possibly equivalent to the dorso-medial intercollicular nucleus of oscines. Additional track-tracing studies should be performed to confirm this homology. The coexistence of estrogen and ₂-adrenergic receptors within the same structure suggests important functional connections between steroid action and catecholaminergic systems in the brain.     

Trans-Golgi Network (TGN) as a regulatory node for β1-adrenergic receptor (β1AR) down-modulation and recycling

Receptor down-modulation is the key mechanism by which G protein-coupled receptors (GPCRs) prevent excessive receptor signaling in response to agonist stimulation. Recently, the trans-Golgi network (TGN) has been implicated as a key checkpoint for receptor endocytosis and degradation. Here, we investigated the involvement of the TGN in down-modulation of β1-adrenergic receptor in response to persistent isoprotenerol stimulation. Immunofluorescent staining showed that ~50% of endocytosed β1AR colocalized with TGN-46 at 5 h. Disruption of the TGN by brefeldin A (BFA) led to the robust accumulation of endocytosed β1AR in Rab11(+) recycling endosomes, inhibited β1AR entry into LAMP1(+) lysosomes, and as a result enhanced β1AR recycling to the plasma membrane. The lysosomotropic agent, chloroquine, arrested the majority of endocytosed β1AR in the TGN by 4 h. Immunoblot analysis showed that either disruption of the TGN or blockage of the lysosome prevented β1AR degradation. Co-expression of GFP-arrestin-3 in β1AR cells increased the endocytosis of β1AR and facilitated its entry to the TGN but inhibited recycling to the plasma membrane. Arrestin-3-induced inhibition of β1AR recycling was reversed by BFA treatment, whereas chloroquine induced the accumulation of arrestin-3 with β1AR in the TGN. These results demonstrate for the first time that the TGN acts as a checkpoint for both the recycling and down-regulation of β1AR and that arrestin-3 not only mediates β1AR endocytosis but also its recycling through the TGN.     

The antidepressant desipramine is an arrestin-biased ligand at the α(2A)-adrenergic receptor driving receptor down-regulation in vitro and in vivo

The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as an inhibitor of norepinephrine reuptake. Available evidence, however, suggests a mechanism more complex than simple reuptake inhibition. In the present study, we have characterized the direct interaction between DMI and the α(2A)-adrenergic receptor (α(2A)AR), a key regulator of noradrenergic neurotransmission with altered expression and function in depression. DMI alone was found to be sufficient to drive receptor internalization acutely and a robust down-regulation of α(2A)AR expression and signaling following prolonged stimulation in vitro. These effects are achieved through arrestin-biased regulation of the receptor, as DMI selectively induces recruitment of arrestin but not activation of heterotrimeric G proteins. Meanwhile, a physiologically relevant concentration of endogenous agonist (norepinephrine) was unable to sustain a down-regulation response. Prolonged in vivo administration of DMI resulted in significant down-regulation of synaptic α(2A)AR expression, a response that was lost in arrestin3-null animals. We contend that direct DMI-driven arrestin-mediated α(2A)AR down-regulation accounts for the therapeutically desirable but mechanistically unexplained adaptive alterations in receptor expression associated with this antidepressant. Our results provide novel insight into both the pharmacology of this antidepressant drug and the targeting of the α(2A)AR in depression.     

Regulation of α(2B)-adrenergic receptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation by ADP-ribosylation factor 1

A number of signaling molecules are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway by G protein-coupled receptors. In this study, we have demonstrated that α(2B)-adrenergic receptor (α(2B)-AR) interacts with ADP-ribosylation factor 1 (ARF1), a small GTPase involved in vesicle-mediated trafficking, in an agonist activation-dependent manner and that the interaction is mediated through a unique double Trp motif in the third intracellular loop of the receptor. Interestingly, mutation of the double Trp motif and siRNA-mediated depletion of ARF1 attenuate α(2B)-AR-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) without altering receptor intracellular trafficking, whereas expression of the constitutively active mutant ARF1Q71L and ARNO, a GDP-GTP exchange factor of ARF1, markedly enhances the activation of Raf1, MEK1, and ERK1/2. These data strongly demonstrate that the small GTPase ARF1 modulates ERK1/2 activation by α(2B)-AR and provide the first evidence indicating a novel function for ARF1 in regulating the MAPK signaling pathway.