Reelin secretion from glutamatergic neurons in culture is independent from neurotransmitter regulation

Reelin (ReIn) is a glycoprotein that in postnatal and adult mammalian brain is believed to be secreted from telencephalic GABAergic interneurons and cerebellar glutamatergic granule neurons into the extracellular matrix. To address the question of whether Rein neurosecretion occurs via a regulated or a constitutive process, we exposed postnatal rat cerebellar granule neurons (CGNs) maintained in culture for 7-9 days to: (i) 100 mu M N-methyl-D-aspartate (NMDA) in a Mg+2-free medium to stimulate NMDA-selective glutamate receptors and Ca2+-dependent neurotransmitter release, (ii) 50 mM KCl to depolarize the cells and elicit Ca2+-dependent exocytosis, (iii) 10-100 mu M nicotine to activate excocytosis by nicotinic receptors present in these cells, (iv) 10 mu M 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7- sulfonamide in combination with 10 mu M dizocilpine to block alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and NMDA-preferring glutamate receptors activated by endogenously released glutamate, or (v) EGTA (5 mM) to virtually eliminate extracellular Ca2+ and block Ca2+-dependent exocytosis. Although, CGNs express and secrete Rein (measured by quantitative immunoblotting), none of the above-mentioned conditions that control regulated exocytosis alters the stores or the rate of Rein release. in contrast, application of either: (i) a Rein antisense oligonucleotide (5' GCAATCTGCAGGGAAATG-3') (10 mu M) that reduces Rein biosynthesis or (ii) brefeldin A (5 x 10(-5) M), an inhibitor of the traffic of proteins between the endoplasmic: reticulum and the Golgi network, sharply curtail the rate of Rein secretion. Because, in subcellular fractionation studies. we have shown that Rein is not contained in synaptic vesicles, these data suggest that Rein secretion from CGNs does not require Ca2+-dependent exocytosis, but probably is related to a Rein pool stored in Golgi secretory vesicles mediating a constitutive secretory pathway.