RT Journal Article
SR Electronic
T1 Trafficking-Dependent and -Independent Pathways of Neurotransmitter Transporter Regulation Differentially Involving p38 Mitogen-Activated Protein Kinase Revealed in Studies of Insulin Modulation of Norepinephrine Transport in SK-N-SH Cells
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 666
OP 677
VO 299
IS 2
A1 Subbu Apparsundaram
A1 Uhna Sung
A1 Raymond D. Price
A1 Randy D. Blakely
YR 2001
UL http://jpet.aspetjournals.org/content/299/2/666.abstract
AB Presynaptic, cocaine- and antidepressant-sensitive norepinephrine (NE) transporters (NETs) dictate levels of extracellular NE after vesicular release. Recent studies suggest that G protein-coupled receptors linked to protein kinase C (PKC) down-regulate cell surface NET protein levels and diminish NE uptake capacity. We identified distinct phosphatidylinositol 3-OH kinase (PI3K)-linked pathways supporting basal and insulin-triggered NE transport in the human noradrenergic neuroblastoma, SK-N-SH. Acute (0–60 min) insulin treatments produced a time- and concentration-dependent stimulation of NE transport, resolved in kinetic studies as an enhancement of NE transport capacity (Vmax) without an alteration in NEKm. Basal and insulin-modulated NET activities were reduced by the tyrosine kinase inhibitor genistein and the PI3K inhibitors wortmannin and LY-294002, but not by the PKC inhibitor staurosporine. PI3K activation was found to support phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). However, basal and insulin-stimulated NET activities were differentiated by their reliance on p38 MAPK activation. Thus, the p38 MAPK inhibitor SB203580 and SB202190 abolished insulin activation of NE transport yet failed to impact basal NET activity. Moreover, p38 MAPK activation and insulin activation of NETs were found to be sensitive to external Ca2+ depletion, blockade of voltage-sensitive Ca2+ channels, and inhibition of protein phosphatase 2A. Effects of tyrosine kinase and PI3K inhibitors on basal NET uptake appear to arise from a loss of cell surface NET protein, whereas the p38 MAPK-dependent enhancement of NE transport occurs without a detectable enhancement of surface NET. Our findings establish two distinct pathways for regulation of NE uptake involving PI3K, one linked to transporter trafficking and a second linked to Ca2+-dependent, p38 MAPK phosphorylation that promotes activation of cell surface NETs. The American Society for Pharmacology and Experimental Therapeutics