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.