title: Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation.
abstract: 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.
authors.affiliation: Department of Psychiatry, Vanderbilt School of Medicine, Nashville, Tennessee 37232-8548, USA.
authors.forename: Chong-Bin
authors.initials: CB
authors.lastname: Zhu
authors.forename: William A
authors.initials: WA
authors.lastname: Hewlett
authors.forename: Igor
authors.initials: I
authors.lastname: Feoktistov
authors.forename: Italo
authors.initials: I
authors.lastname: Biaggioni
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D006224:Cricetinae; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D005260:Female; D006162:Guanylate Cyclase / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011687:Purines; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000502:physiology*; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D000068677:Sildenafil Citrate; D013450:Sulfones; D010738:Type C Phospholipases / Q000378:metabolism; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D018047:Receptors, Purinergic P1; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D013450:Sulfones; D012701:Serotonin; D019830:Adenosine-5'-(N-ethylcarboxamide); D000068677:Sildenafil Citrate; D017869:Cyclic GMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium