Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation

Mol Pharmacol. 2004 Jun;65(6):1462-74. doi: 10.1124/mol.65.6.1462.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine-5'-(N-ethylcarboxamide) / pharmacology
  • Animals
  • Biological Transport
  • CHO Cells
  • Calcium / metabolism
  • Carrier Proteins / metabolism*
  • Cells, Cultured
  • Cricetinae
  • Cyclic GMP / metabolism
  • Cyclic GMP-Dependent Protein Kinases / physiology*
  • Female
  • Guanylate Cyclase / metabolism
  • Membrane Glycoproteins / metabolism*
  • Membrane Transport Proteins*
  • Mitogen-Activated Protein Kinases / physiology*
  • Nerve Tissue Proteins / metabolism*
  • Phosphodiesterase Inhibitors / pharmacology
  • Piperazines / pharmacology
  • Purines
  • Rats
  • Receptors, Purinergic P1 / physiology*
  • Serotonin / metabolism*
  • Serotonin Plasma Membrane Transport Proteins
  • Sildenafil Citrate
  • Sulfones
  • Type C Phospholipases / metabolism
  • Up-Regulation
  • p38 Mitogen-Activated Protein Kinases

Substances

  • Carrier Proteins
  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Phosphodiesterase Inhibitors
  • Piperazines
  • Purines
  • Receptors, Purinergic P1
  • Serotonin Plasma Membrane Transport Proteins
  • Slc6a4 protein, rat
  • Sulfones
  • Serotonin
  • Adenosine-5'-(N-ethylcarboxamide)
  • Sildenafil Citrate
  • Cyclic GMP-Dependent Protein Kinases
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases
  • Type C Phospholipases
  • Guanylate Cyclase
  • Cyclic GMP
  • Calcium