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Cardiac natriuretic peptides promote adipose 'browning' through mTOR complex-1.
Liu D, Ceddia RP, Collins S
(2018) Mol Metab 9: 192-198
MeSH Terms: Adipose Tissue, Brown, Animals, Atrial Natriuretic Factor, Cells, Cultured, Cyclic GMP-Dependent Protein Kinases, Female, HEK293 Cells, Humans, Male, Mechanistic Target of Rapamycin Complex 1, Mice, Mice, Inbred C57BL, Mitochondria, Signal Transduction, Uncoupling Protein 1
Show Abstract · Added September 25, 2018
OBJECTIVE - Activation of thermogenesis in brown adipose tissue (BAT) and the ability to increase uncoupling protein 1 (UCP1) levels and mitochondrial biogenesis in white fat (termed 'browning'), has great therapeutic potential to treat obesity and its comorbidities because of the net increase in energy expenditure. β-adrenergic-cAMP-PKA signaling has long been known to regulate these processes. Recently PKA-dependent activation of mammalian target of rapamycin complex 1 (mTORC1) was shown to be necessary for adipose 'browning' as well as proper development of the interscapular BAT. In addition to cAMP-PKA signaling pathways, cGMP-PKG signaling also promotes this browning process; however, it is unclear whether or not mTORC1 is also necessary for cGMP-PKG induced browning.
METHOD - Activation of mTORC1 by natriuretic peptides (NP), which bind to and activate the membrane-bound guanylyl cyclase, NP receptor A (NPRA), was assessed in mouse and human adipocytes in vitro and mouse adipose tissue in vivo.
RESULTS - Activation of mTORC1 by NP-cGMP signaling was observed in both mouse and human adipocytes. We show that NP-NPRA-PKG signaling activate mTORC1 by direct PKG phosphorylation of Raptor at Serine 791. Administration of B-type natriuretic peptide (BNP) to mice induced Ucp1 expression in inguinal adipose tissue in vivo, which was completely blocked by the mTORC1 inhibitor rapamycin.
CONCLUSION - Our results demonstrate that NP-cGMP signaling activates mTORC1 via PKG, which is a component in the mechanism of adipose browning.
Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.
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15 MeSH Terms
Second messenger signaling mechanisms of the brown adipocyte thermogenic program: an integrative perspective.
Shi F, Collins S
(2017) Horm Mol Biol Clin Investig 31:
MeSH Terms: Adipocytes, Beige, Adipocytes, Brown, Animals, Cyclic AMP-Dependent Protein Kinases, Cyclic GMP-Dependent Protein Kinases, Energy Metabolism, Gene Expression Regulation, Humans, Intracellular Space, Mechanistic Target of Rapamycin Complex 1, MicroRNAs, Natriuretic Agents, RNA, Long Noncoding, Receptors, Adrenergic, beta, Second Messenger Systems, Signal Transduction, Thermogenesis, Uncoupling Protein 1
Show Abstract · Added September 26, 2018
β-adrenergic receptors (βARs) are well established for conveying the signal from catecholamines to adipocytes. Acting through the second messenger cyclic adenosine monophosphate (cAMP) they stimulate lipolysis and also increase the activity of brown adipocytes and the 'browning' of adipocytes within white fat depots (so-called 'brite' or 'beige' adipocytes). Brown adipose tissue mitochondria are enriched with uncoupling protein 1 (UCP1), which is a regulated proton channel that allows the dissipation of chemical energy in the form of heat. The discovery of functional brown adipocytes in humans and inducible brown-like ('beige' or 'brite') adipocytes in rodents have suggested that recruitment and activation of these thermogenic adipocytes could be a promising strategy to increase energy expenditure for obesity therapy. More recently, the cardiac natriuretic peptides and their second messenger cyclic guanosine monophosphate (cGMP) have gained attention as a parallel signaling pathway in adipocytes, with some unique features. In this review, we begin with some important historical work that touches upon the regulation of brown adipocyte development and physiology. We then provide a synopsis of some recent advances in the signaling cascades from β-adrenergic agonists and natriuretic peptides to drive thermogenic gene expression in the adipocytes and how these two pathways converge at a number of unexpected points. Finally, moving from the physiologic hormonal signaling, we discuss yet another level of control downstream of these signals: the growing appreciation of the emerging roles of non-coding RNAs as important regulators of brown adipocyte formation and function. In this review, we discuss new developments in our understanding of the signaling mechanisms and factors including new secreted proteins and novel non-coding RNAs that control the function as well as the plasticity of the brown/beige adipose tissue as it responds to the energy needs and environmental conditions of the organism.
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MeSH Terms
Differential activation of natriuretic peptide receptors modulates cardiomyocyte proliferation during development.
Becker JR, Chatterjee S, Robinson TY, Bennett JS, Panáková D, Galindo CL, Zhong L, Shin JT, Coy SM, Kelly AE, Roden DM, Lim CC, MacRae CA
(2014) Development 141: 335-45
MeSH Terms: Animals, Animals, Genetically Modified, Atrial Natriuretic Factor, Cell Proliferation, Cyclic AMP, Cyclic GMP-Dependent Protein Kinases, Gene Knockdown Techniques, Heart, Myocytes, Cardiac, Natriuretic Peptide, Brain, Receptors, Atrial Natriuretic Factor, Signal Transduction, Zebrafish, Zebrafish Proteins
Show Abstract · Added February 19, 2015
Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways.
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2 Members
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14 MeSH Terms
cGMP-dependent protein kinase Ialpha associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake.
Steiner JA, Carneiro AM, Wright J, Matthies HJ, Prasad HC, Nicki CK, Dostmann WR, Buchanan CC, Corbin JD, Francis SH, Blakely RD
(2009) Mol Brain 2: 26
MeSH Terms: Animals, Antibody Specificity, Antidepressive Agents, Cell Line, Cyclic GMP, Cyclic GMP-Dependent Protein Kinase Type I, Cyclic GMP-Dependent Protein Kinases, Gene Knockdown Techniques, Humans, Protein Binding, Protein Kinase Inhibitors, Protein Transport, RNA, Small Interfering, Rats, Serotonin, Serotonin Plasma Membrane Transport Proteins, Transfection
Show Abstract · Added July 10, 2013
BACKGROUND - The Na(+)/Cl(-)-dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear.
RESULTS - In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIalpha specifically associates with hSERT.
CONCLUSION - Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIalpha that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIalpha supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.
1 Communities
3 Members
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17 MeSH Terms
Enhanced activity of human serotonin transporter variants associated with autism.
Prasad HC, Steiner JA, Sutcliffe JS, Blakely RD
(2009) Philos Trans R Soc Lond B Biol Sci 364: 163-73
MeSH Terms: Alleles, Animals, Autistic Disorder, CHO Cells, Cricetinae, Cricetulus, Cyclic GMP-Dependent Protein Kinases, Gene Expression Regulation, Genetic Variation, Glutamic Acid, HeLa Cells, Humans, Lymphocytes, Serotonin, Serotonin Plasma Membrane Transport Proteins, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added February 20, 2014
Rare, functional, non-synonymous variants in the human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) gene (SLC6A4) have been identified in both autism and obsessive-compulsive disorder (OCD). Within autism, rare hSERT coding variants associate with rigid-compulsive traits, suggesting both phenotypic overlap with OCD and a shared relationship with disrupted 5-HT signalling. Here, we document functional perturbations of three of these variants: Ile425Leu; Phe465Leu; and Leu550Val. In transiently transfected HeLa cells, the three variants confer a gain of 5-HT transport phenotype. Specifically, enhanced SERT activity was also observed in lymphoblastoid lines derived from mutation carriers. In contrast to previously characterized Gly56Ala, where increased transport activity derives from catalytic activation, the three novel variants exhibit elevated surface density as revealed through both surface antagonist-binding and biotinylation studies. Unlike Gly56Ala, mutants Ile425Leu, Phe465Leu and Leu550Val retain a capacity for acute PKG and p38 MAPK regulation. However, both Gly56Ala and Ile425Leu demonstrate markedly reduced sensitivity to PP2A antagonists, suggesting that deficits in trafficking and catalytic modulation may derive from a common basis in perturbed phosphatase regulation. When expressed stably from the same genomic locus in CHO cells, both Gly56Ala and Ile425Leu display catalytic activation, accompanied by a striking loss of SERT protein.
1 Communities
2 Members
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16 MeSH Terms
Rapid stimulation of presynaptic serotonin transport by A(3) adenosine receptors.
Zhu CB, Steiner JA, Munn JL, Daws LC, Hewlett WA, Blakely RD
(2007) J Pharmacol Exp Ther 322: 332-40
MeSH Terms: Adenosine, Adenosine-5'-(N-ethylcarboxamide), Animals, Biological Transport, Cyclic GMP-Dependent Protein Kinases, MAP Kinase Signaling System, Male, Mice, Mice, Inbred C57BL, Rats, Rats, Sprague-Dawley, Receptor, Adenosine A3, Serotonin, Serotonin Plasma Membrane Transport Proteins, Synaptosomes, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added July 10, 2013
The inactivation of synaptic serotonin (5-hydroxytryptamine, 5-HT) is largely established through the actions of the presynaptic, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Recent studies have demonstrated post-translational regulation of SERT mediated by multiple Ser/Thr kinases, including protein kinases C and G (PKC and PKG) and p38 mitogen-activated protein kinase (MAPK), as well as the Ser/Thr phosphatase PP2A. Less well studied are specific surface receptors that target these signaling pathways to control SERT surface expression and/or catalytic rates. Using rat basophilic leukemia 2H3 cell line (RBL-2H3), we previously established that activation of A(3) adenosine receptors (A(3)AR) stimulates SERT activity via both PKG and p38 MAPK (Zhu et al., 2004a). Whether A(3)ARs regulate SERT in the central nervous system (CNS) is unknown. Here we report that the A(3)AR agonist N(6)-(3-iodobenzyl)-N-methyl-5'carbamoyladenosine (IB-MECA) rapidly (10 min) and selectively stimulates 5-HT transport in mouse midbrain, hippocampal, and cortical synaptosomes. IB-MECA-induced stimulation of 5-HT uptake is blocked by the selective A(3)AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191) and is absent from synaptosomes prepared from A(3)AR knockout mice. Kinetic analyses demonstrate that IB-MECA induces an increase of 5-HT transport V(max) with no significant change in K(m). As in RBL-2H3 cells, IB-MECA stimulation of synaptosomal 5-HT uptake can be blocked by preincubation with PKG antagonists N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) and DT-2 (YGRKKRRQRRRPPLRK(5)H), as well as by the p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Chronoamperometry studies in the anesthetized rat hippocampus support a role for A(3)ARs in SERT regulation in vivo. Together, these results identify a novel, region-specific action of CNS A(3)ARs in the modulation of SERT-mediated 5-HT transport that may be relevant for the etiology and/or therapy of 5-HT-linked brain disorders.
1 Communities
1 Members
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16 MeSH Terms
Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase.
Prasad HC, Zhu CB, McCauley JL, Samuvel DJ, Ramamoorthy S, Shelton RC, Hewlett WA, Sutcliffe JS, Blakely RD
(2005) Proc Natl Acad Sci U S A 102: 11545-50
MeSH Terms: Cyclic GMP-Dependent Protein Kinases, Gene Expression, Genetic Variation, HeLa Cells, Herpesvirus 4, Human, Humans, Lymphocytes, Phosphorylation, Protein Transport, Serotonin, Signal Transduction, Transfection, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added February 20, 2014
Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.
1 Communities
2 Members
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13 MeSH Terms
Role of the small heat shock proteins in regulating vascular smooth muscle tone.
McLemore EC, Tessier DJ, Thresher J, Komalavilas P, Brophy CM
(2005) J Am Coll Surg 201: 30-6
MeSH Terms: Animals, Blotting, Western, Coronary Vessels, Cyclic GMP-Dependent Protein Kinases, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, HSP20 Heat-Shock Proteins, HSP27 Heat-Shock Proteins, Heat-Shock Proteins, Humans, Muscle Tonus, Muscle, Smooth, Vascular, Nitroprusside, Phosphoproteins, Phosphorylation, Saphenous Vein, Swine, Vasoconstriction, Vasodilation, Vasodilator Agents
Show Abstract · Added March 9, 2015
BACKGROUND - Vasospasm occurs in conduits used for vascular reconstructions. The small heat shock proteins, HSP20 and HSP27, coordinately regulate vascular smooth muscle tone. Phosphorylated HSP20 is associated with vasorelaxation, and phosphorylated HSP27 inhibits the phosphorylation of HSP20 and relaxation. We hypothesized that the relationship between the phosphorylated states of these two proteins might dictate the tone of a vessel and may contribute to vasospasm.
STUDY DESIGN - Sodium nitroprusside relaxation of vascular smooth muscle was recorded using pig coronary artery and human saphenous vein. Segments were frozen and homogenized, and extracted proteins were separated by one- and two-dimensional gel electrophoresis, transferred to Immobilon (Millipore), and probed with anti-cGMP-dependent protein kinase (anti-PKG), -HSP20, -HSP27, and -phosphoHSP27 antibodies. Band intensity was estimated using densitometry.
RESULTS - Pig coronary artery completely relaxed (100%) with SNP (10(-7)M), but human saphenous vein only partially relaxed (20%). The levels of cGMP-dependent protein kinase and HSP20 were similar in the two tissue types. Human saphenous vein had significantly higher levels of HSP27 versus pig coronary artery (30.14 +/- 0.8 versus 6.62 +/- 0.2 pixels/mg; p < or = 0.001) and phosphoHSP27 (8.29 +/- 3.43 versus 0.012 +/- 0.008 pixels/mg; p < or = 0.001).
CONCLUSIONS - Human saphenous vein contained significantly higher levels of HSP27 and pHSP27. Increased levels of phosphorylated HSP27 might contribute to vasospasm in human saphenous vein.
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20 MeSH Terms
cGMP catabolism by phosphodiesterase 5A regulates cardiac adrenergic stimulation by NOS3-dependent mechanism.
Takimoto E, Champion HC, Belardi D, Moslehi J, Mongillo M, Mergia E, Montrose DC, Isoda T, Aufiero K, Zaccolo M, Dostmann WR, Smith CJ, Kass DA
(2005) Circ Res 96: 100-9
MeSH Terms: 3',5'-Cyclic-GMP Phosphodiesterases, Adenoviridae, Adrenergic beta-Agonists, Animals, Carbolines, Cyclic GMP, Cyclic GMP-Dependent Protein Kinases, Cyclic Nucleotide Phosphodiesterases, Type 5, Fluorescence Resonance Energy Transfer, Genetic Vectors, Guanylate Cyclase, Isoproterenol, Mice, Mice, Inbred C57BL, Mice, Knockout, Myocardial Contraction, NG-Nitroarginine Methyl Ester, Nitric Oxide, Nitric Oxide Synthase, Nitric Oxide Synthase Type II, Nitric Oxide Synthase Type III, Phosphodiesterase Inhibitors, Piperazines, Purines, Rats, Rats, Sprague-Dawley, Receptors, Adrenergic, beta, Receptors, Cytoplasmic and Nuclear, Recombinant Fusion Proteins, Second Messenger Systems, Sildenafil Citrate, Soluble Guanylyl Cyclase, Sulfones, Tadalafil
Show Abstract · Added March 4, 2015
Beta-adrenergic agonists stimulate cardiac contractility and simultaneously blunt this response by coactivating NO synthase (NOS3) to enhance cGMP synthesis and activate protein kinase G (PKG-1). cGMP is also catabolically regulated by phosphodiesterase 5A (PDE5A). PDE5A inhibition by sildenafil (Viagra) increases cGMP and is used widely to treat erectile dysfunction; however, its role in the heart and its interaction with beta-adrenergic and NOS3/cGMP stimulation is largely unknown. In nontransgenic (control) murine in vivo hearts and isolated myocytes, PDE5A inhibition (sildenafil) minimally altered rest function. However, when the hearts or isolated myocytes were stimulated with isoproterenol, PDE5A inhibition was associated with a suppression of contractility that was coupled to elevated cGMP and increased PKG-1 activity. In contrast, NOS3-null hearts or controls with NOS inhibited by N(G)-nitro-L-arginine methyl ester, or soluble guanylate cyclase (sGC) inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one, showed no effect of PDE5A inhibition on beta-stimulated contractility or PKG-1 activation. This lack of response was not attributable to altered PDE5A gene or protein expression or in vitro PDE5A activity, but rather to an absence of sGC-generated cGMP specifically targeted to PDE5A catabolism and to a loss of PDE5A localization to z-bands. Re-expression of active NOS3 in NOS3-null hearts by adenoviral gene transfer restored PDE5A z-band localization and the antiadrenergic efficacy of PDE5A inhibition. These data support a novel regulatory role of PDE5A in hearts under adrenergic stimulation and highlight specific coupling of PDE5A catabolic regulation with NOS3-derived cGMP attributable to protein subcellular localization and targeted synthetic/catabolic coupling.
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34 MeSH Terms
Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation.
Zhu CB, Hewlett WA, Feoktistov I, Biaggioni I, Blakely RD
(2004) Mol Pharmacol 65: 1462-74
MeSH Terms: Adenosine-5'-(N-ethylcarboxamide), Animals, Biological Transport, CHO Cells, Calcium, Carrier Proteins, Cells, Cultured, Cricetinae, Cyclic GMP, Cyclic GMP-Dependent Protein Kinases, Female, Guanylate Cyclase, Membrane Glycoproteins, Membrane Transport Proteins, Mitogen-Activated Protein Kinases, Nerve Tissue Proteins, Phosphodiesterase Inhibitors, Piperazines, Purines, Rats, Receptors, Purinergic P1, Serotonin, Serotonin Plasma Membrane Transport Proteins, Sildenafil Citrate, Sulfones, Type C Phospholipases, Up-Regulation, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added December 10, 2013
Serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) are critical determinants of synaptic 5-HT inactivation and the targets for multiple drugs used to treat psychiatric disorders. In support of prior studies, we found that short-term (5-30 min) application of the adenosine receptor (AR) agonist 5'-N-ethylcarboxamidoadenosine (NECA) induces an increase in 5-HT uptake Vmax in rat basophilic leukemia 2H3 cells that is enhanced by pretreatment with the cGMP phosphodiesterase inhibitor sildenafil. NECA stimulation is blocked by the A3 AR antagonist 3-ethyl-5-benzyl-2-methyl-phenylethynyl-6-phenyl-1,4(+/-)dihydropyridine-3,5-dicarboxylate (MRS1191), by the phospholipase C inhibitor 1-(6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl)-1H-pyrrole-2,5-dione (U73122), by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, and by the guanyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Hydroxylamine, a nitric-oxide donor, and 8-bromo-cGMP, a membrane-permeant analog of cGMP, mimic the effects of NECA on 5-HT uptake, whereas the protein kinase G (PKG) inhibitor N-[2-(methylamino)ethy]-5-isoquinoline-sulfonamide (H8) blocks NECA, hydroxylamine, and 8-bromo-cGMP effects. NECA stimulation activates p38 mitogen-activated protein kinase (MAPK), whereas p38 MAPK inhibitors block NECA stimulation of SERT activity, as does the protein phosphatase 2A (PP2A) inhibitor calyculin A. 5-HT-displaceable [125I]3beta-(4-iodophenyl)-tropane-2beta-carboxylic acid methylester tartrate (RTI-55) whole-cell binding is increased by NECA or sildenafil, and both surface binding and cell surface SERT protein are elevated after NECA or sildenafil stimulation of AR/SERT-cotransfected Chinese hamster ovary cells. Whereas p38 MAPK inhibition blocks NECA stimulation of 5-HT activity, it fails to blunt stimulation of SERT surface density. Moreover, inactivation of existing surface SERTs fails to eliminate NECA stimulation of SERT. Together, these results reveal two PKG-dependent pathways supporting rapid SERT regulation by A3 ARs, one leading to enhanced SERT surface trafficking, and a separate, p38 MAPK-dependent process augmenting SERT intrinsic activity.
1 Communities
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28 MeSH Terms