Localization of the noradrenaline transporter in rat adrenal medulla and PC12 cells: Evidence for its association with secretory granules in PC12 cells

The noradrenaline transporter (NAT) is present in noradrenergic neurons and a few other specialized cells such as adrenal medullary chromaffin cells a nd the rat pheochromocytoma (PC12) cell line. We have raised antibodies to a 49-residue segment (NATM2) of the extracellular region (residues 184-232) of bovine NAT. Affinity-purified NATM2 antibodies specifically recognized an 80-kDa band in PC12 cell membranes by western blotting. Bands of a simil ar size were also detected in membranes from human neuroblastoma (SK-N-SH) cells expressing endogenous NAT and human embryonic kidney (HEK293) cells s tably expressing bovine NAT. Immunocytochemistry of rat adrenal tissue show ed that NAT staining was colocalized with tyrosine hydroxylase in medullary chromaffin cells. Most NAT immunoreactivity in rat adrenal chromaffin and PC12 cells was present in the cytoplasm and had a punctate appearance. Cell surface biotinylation experiments in PC12 cells confirmed that only a mino r fraction of the NAT was present at the cell surface. Subcellular fraction ation of PC12 cells showed that relatively little NAT colocalized with plas ma membrane, synaptic-like microvesicles, recycling endosomes, or trans-Gol gi vesicles. Most of the NAT was associated with [H-3]noradrenaline-contain ing secretory granules. Following nerve growth factor treatment, NAT was lo calized to the growing tip of neurites. This distribution was similar to th e secretory granule marker secretogranin I. We conclude that the majority o f NAT is present intracellularly in secretory granules and suggest that NAT may undergo regulated trafficking in PC12 cells.