Human neuroblastoma cells acquire regulated secretory properties and different sensitivity to Ca2+ and alpha-latrotoxin after exposure to differentiating agents.
IMR-32 human neuroblastoma cells are unable to release [3H]dopamine in response to secretagogues. However, they express a normal complement of membrane receptors and ion channels which are efficiently coupled to second messenger production. In the present study we took advantage of the ability of this cell line to differentiate in vitro in the presence of either dibutyrryl-cAMP or 5-bromodeoxyuridine, to analyze any developmentally regulated changes in its secretory properties. Uptake, storage, and release of [3H]dopamine were studied biochemically and by autoradiography. The calcium ionophore ionomycin, phorbol 12-myristate 13-acetate and the presynaptic acting neurotoxin alpha-latrotoxin were used in both control and differentiated cells as secretagogue agents. The presence of secretory organelles was investigated by electron microscopy; the expression of secretory organelle markers, such as chromogranin/secretogranin proteins (secretory proteins) and synaptophysin (membrane protein), was detected by Western blotting and immunofluorescence. The results obtained indicate that IMR-32 cells acquire regulated secretory properties after in vitro drug-induced differentiation: (a) they assemble "de novo" secretory organelles, as revealed by electron microscopy and detection of secretory organelle markers, and (b) they are able to store [3H]dopamine and to release the neurotransmitter in response to secretagogue stimuli. Furthermore, secretagogue sensitivity was found to be different, depending on the differentiating agent. In fact, dibutyrryl-cAMP treated cells release [3H]dopamine in response to alpha-latrotoxin, but not in response to ionomycin, whereas 5-bromodeoxyuridine treated cells release the neurotransmitter in response to both secretagogues. All together these results suggest that IMR-32 cells represent an adequate model for studying the development of the secretory apparatus in cultured human neurons.
Sher
E
E
CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, Italy.
Denis-Donini
S
S
Zanini
A
A
Bisiani
C
C
Clementi
F
F
D001692:Biological Transport; D001973:Bromodeoxyuridine / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002451:Cell Compartmentation; D002454:Cell Differentiation; D002470:Cell Survival; D002837:Chromaffin Granules / Q000648:ultrastructure; D004298:Dopamine / Q000378:metabolism*; D004987:Ethers / Q000494:pharmacology; D006801:Humans; D015759:Ionomycin; D007700:Kinetics; D009447:Neuroblastoma / Q000378:metabolism* / Q000648:ultrastructure; D011493:Protein Kinase C / Q000502:physiology; D013111:Spider Venoms / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004987:Ethers; D013111:Spider Venoms; D015759:Ionomycin; D003994:Bucladesine; C020763:alpha-latrotoxin; D011493:Protein Kinase C; D001973:Bromodeoxyuridine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D004298:Dopamine