THE STORAGE OF NORADRENALINE, NEUROPEPTIDE-Y AND CHROMOGRANINS IN ANDSTOICHIOMETRIC RELEASE FROM LARGE DENSE CORED VESICLES OF THE UNDIFFERENTIATED HUMAN NEUROBLASTOMA CELL-LINE SH-SY5Y

Sucrose gradient centrifugation combined with electron microscopy reve aled that undifferentiated SH-SY5Y cells contain predominantly one pop ulation of noradrenaline containing vesicles, i.e. large dense cored v esicles. These vesicles have been purified approximately twenty times using sucrose/D2O gradients. Electron microscopy of sucrose/D2O fracti ons confirms that large dense cored vesicles are enriched in the fract ions containing predominantly dopamine-beta-hydroxylase, chromogranin A, noradrenaline and neuropeptide Y. The membranes of these vesicles c ontain the typical large dense cored vesicle markers dopamine-beta-hyd roxylase, synaptotagmin, cytochrome b(561) and rab 3. Stimulation of S H-SY5Y cells with carbachol and KCl shows that noradrenaline and neuro peptide Y are released in the same proportion as stored in the large d ense cored vesicles. The immune-blot pattern and intensity of chromogr anin A and chromogranin B present in large dense cored vesicles and in the released material were definitely the same. This suggests that no radrenaline and the proteins/peptides are released in the same molar s toichiometry as they are stored in large dense cored vesicles. These d ata provide for the first time experimental evidence that the neurobla stoma cell line SH-SY5Y contains functionally active large dense cored vesicles similar to those of sympathetic neurons and indicate that th is cell line is a suitable experimental cell model to study the exocyt otic pathway of large dense cored vesicles.