Reelin secretion from glutamatergic neurons in culture is independent fromneurotransmitter regulation

Reelin (ReIn) is a glycoprotein that in postnatal and adult mammalian brain is believed to be secreted from telencephalic GABAergic interneurons and c erebellar glutamatergic granule neurons into the extracellular matrix. To a ddress the question of whether Rein neurosecretion occurs via a regulated o r a constitutive process, we exposed postnatal rat cerebellar granule neuro ns (CGNs) maintained in culture for 7-9 days to: (i) 100 mu M N-methyl-D-as partate (NMDA) in a Mg+2-free medium to stimulate NMDA-selective glutamate receptors and Ca2+-dependent neurotransmitter release, (ii) 50 mM KCl to de polarize the cells and elicit Ca2+-dependent exocytosis, (iii) 10-100 mu M nicotine to activate excocytosis by nicotinic receptors present in these ce lls, (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 glutamat e receptors activated by endogenously released glutamate, or (v) EGTA (5 mM ) to virtually eliminate extracellular Ca2+ and block Ca2+-dependent exocyt osis. Although, CGNs express and secrete Rein (measured by quantitative imm unoblotting), none of the above-mentioned conditions that control regulated exocytosis alters the stores or the rate of Rein release. in contrast, app lication of either: (i) a Rein antisense oligonucleotide (5' GCAATCTGCAGGGA AATG-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 secretio n. Because, in subcellular fractionation studies. we have shown that Rein i s not contained in synaptic vesicles, these data suggest that Rein secretio n from CGNs does not require Ca2+-dependent exocytosis, but probably is rel ated to a Rein pool stored in Golgi secretory vesicles mediating a constitu tive secretory pathway.