Western blot analysis showed that the human neuroblastoma SH-SY5Y expresses the proteins synaptotagmin I, synaptobrevin, synapsin I, rab3a, syntaxin, SNAP-25, NSF, a-SNAP, and munc-1 8, which have been implicated in the movement, docking, and fusion of vesides during exocytosis from other neuroendocrine cells. The subcellular localization of secretogranins land II, synaptotagmin I, neuropeptide Y, rab3a, synaptobrevin, synaptophysin, and syntaxin was investigated by immunofluorescence microscopy and revealed punctate staining patterns characteristic of secretory vesicles. The comigration of noradrenaline, secretogranin II, and dopamine-/~-hydroxylaseon sucrose-D 20 gradient fractions indicates the presence of a population of noradrenaline-contaming large dense-cored vesicles (LDCVs). In addition, a lighter vesicle population is also present that does not appear to be noradrenergic and contains a 48-kDa synaptophysin antigen absent from the large dense-cored vesides. Immunocytochemical experiments show that not alt of the vesicles that express synaptotagmin I contain Secretogranin II. Thus, our studies suggest that two types of vesicle are present in SH-SY5Y cells, one of which, the LDCVs, contains noradrenaline. These findings confirm our previous studies suggesting that depolarizationevoked release of noradrenaline from SH-SY5Y occurs by LDCV exocytosis. This enhances the value of SH-SY5Y as a cell line in which to study the mechanism by which noradrenaline release is regulated. -like microvesicles; a-SNAP, a-soluble NSF attachment protein; SNAP-25, synaptosomal-associated protein of 25 kDa; SSV, small synaptic vesicleT PA, I 2-O-tetradecanoylphorbol 13-acetate; TRITC, tetramethylrhodamine isothiocyanate.

Abstract:The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 1 2-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-mm TPA treatment caused translocation of the rn-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-c from cytosolic and membraneassociated to cytoskeleton-and membrane-associated. TPA had no effect on the cytosolic location of PKC-~. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-mm TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31 -8220 (10 jaM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-ce (but not -e or -~). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-a. Key Words: Myristoylated alanine-rich C-kinase substrate-Noradrenaline release-Phorbol ester-Protein kinase C-a-SH-SY5Y neuroblastoma cells-Translocation.

The human neuroblastoma SH-SY5Y was found to express annexins I, II, IV, V, and VI by western blot analysis. Calcium-dependent membrane-binding proteins were isolated from SH-SY5Y and analysed by 2-dimensional gel electrophoresis. Proteins with Mr and p1 values similar to those of annexins I, II, Ill, IV, V, and VI were observed. The identity of annexins II and V was confirmed by western blotting. The membrane association of annexins II and V was studied in cells that had been stimulated to release noradrenaline by Kdepolarisation or by treatment with the ionophore A23187. Annexins II and V were both found to associate with membranes in a manner that was resistant to elution with EGTA and required Triton X-100 for their solubilisation. Homogenisation of cells in calcium-containing buffers also resulted in the formation of EGTA-resistant membrane-associated annexins II and V. The results demonstrate calcium-dependent relocation of annexins II and V to membranes in intact cells and suggest that these annexins bind in a calcium-dependent manner to nonphospholipid components of SH-SY5Y membranes. Examination of cells by immunofluorescence microscopy demonstrated that annexin II was homogeneously associated with the plasma membrane before treatment with ionophore and relocated to discrete patches of staining after treatment. Annexin V was found by immunofluorescence to be present in the cytoplasm and in the nucleus. Stimulation of the cells produced no change in the cytoplasmic staining pattern but resulted in a partial relocation of nuclear annexin V to the periphery of the nucleus. The results argue for a general role for both annexins in calcium signalling at discrete intracellular locations. The results are not consistent with the specific involvement proposed previously for annexin II in membrane fusion at sites of vesicle exocytosis.

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