The storage of noradrenaline, neuropeptide Y and chromogranins in and stoichiometric release from large dense cored vesicles of the undifferentiated human neuroblastoma cell line SH-SY5Y.

Sucrose gradient centrifugation combined with electron microscopy revealed that undifferentiated SH-SY5Y cells contain predominantly one population of noradrenaline containing vesicles, i.e. large dense cored vesicles. These vesicles have been purified approximately twenty times using sucrose/D2O gradients. Electron microscopy of sucrose/D2O fractions confirms that large dense cored vesicles are enriched in the fractions containing predominantly dopamine- -hydroxylase, chromogranin A, noradrenaline and neuropeptide Y. The membranes of these vesicles contain the typical large dense cored vesicle markers dopamine- -hydroxylase, synaptotagmin, cytochrome b561 and rab 3. Stimulation of SH-SY5Y cells with carbachol and KCl shows that noradrenaline and neuropeptide Y are released in the same proportion as stored in the large dense cored vesicles. The immuno-blot pattern and intensity of chromogranin A and chromogranin B present in large dense cored vesicles and in the released material were definitely the same. This suggests that noradrenaline and the proteins/peptides are released in the same molar stoichiometry as they are stored in large dense cored vesicles. These data provide for the first time experimental evidence that the neuroblastoma cell line SH-SY5Y contains functionally active large dense cored vesicles similar to those of sympathetic neurons and indicate that this cell line is a suitable experimental cell model to study the exocytotic pathway of large dense cored vesicles.

Phenotype plasticity and immunocytochemical evidence for ChAT and D beta H co-localization in fetal pig superior cervical ganglion cells.

The early expression of the cholinergic phenotype in sympathetic neurons was already studied in superior cervical ganglion cells derived from rat, quail and chicken embryo. In the present work, we set up a neuron culture derived from the superior cervical ganglia of fetal pigs. The yield is 1000 times of that of a neonatal rat [17], 100 times of a 10- to 13-day-old chick embryo [26] and 20 times of a 10-day-old quail embryo [3]. This high yield will greatly facilitate further biochemical studies concerning neuronal differentiation. Using these cells as a model, the phenotype plasticity was studied by both biochemical and immunocytochemical methods in normal physiological medium, in a high KCl (30 mM) medium and in a splenocyte co-culture. The phenotype shift occurs in the normal physiological medium and in the splenocyte co-culture, but not in the high KCl medium. Taking into account the species difference, the fetal pig superior cervical ganglion neurons behave in a comparable manner as reported in earlier studies for other animal models. Moreover, for the first time, using immunocytochemical methods, direct evidence for a co-localization of choline-acetyl-transferase and dopamine-beta-hydroxylase in mammalian fetal sympathetic neurons, at least during a certain period, is given.