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

Presynaptic, cocaine- and antidepressant-sensitive norepinephrine (NE) tran sporters (NETs) dictate levels of extracellular NE after vesicular release. Recent studies suggest that G protein-coupled receptors linked to protein kinase C (PKC) downregulate 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 i n the human noradrenergic neuroblastoma, SK-N-SH. Acute (0-60 min) insulin treatments produced a time- and concentration-dependent stimulation of NE t ransport, resolved in kinetic studies as an enhancement of NE transport cap acity (V-max) without an alteration in NE K-m. 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-sti mulated NET activities were differentiated by their reliance on p38 MAPK ac tivation. Thus, the p38 MAPK inhibitor SB203580 and SB202190 abolished insu lin activation of NE transport yet failed to impact basal NET activity. Mor eover, p38 MAPK activation and insulin activation of NETS were found to be sensitive to external Ca2+ depletion, blockade of voltage-sensitive Ca2+ ch annels, and inhibition of protein phosphatase 2A. Effects of tyrosine kinas e and PI3K inhibitors on basal NET uptake appear to arise from a loss of ce ll surface NET protein, whereas the p38 MAPK-dependent enhancement of NE tr ansport occurs without a detectable enhancement of surface NET. Our finding s establish two distinct pathways for regulation of NE uptake involving PI3 K, one linked to transporter trafficking and a second linked to Ca2+-depend ent, p38 MAPK phosphorylation that promotes activation of cell surface NETs .