Characterization of calcium-triggered secretion in permeabilized rat basophilic leukemia cells. Possible role of vectorially acting G proteins

• Published in: The Journal of biological chemistry 1991-06, Vol.266 (16), p.10452-10460
• Main Authors: DE MATTEIS, M. A, DI TULLIO, G, BUCCIONE, R, LUINI, A
• Format: Article
• Language: English
• Subjects: Alkaloids - pharmacology; Animals; Biological and medical sciences; calcium; Calcium - metabolism; Cell physiology; Cyclic AMP - pharmacology; Cytoplasmic Granules - metabolism; Cytoplasmic Granules - ultrastructure; Exocytosis; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - physiology; guanine nucleotide-binding protein; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Hydrogen-Ion Concentration; leukemia; Leukemia, Experimental - metabolism; Leukemia, Experimental - pathology; Microscopy, Electron; Molecular and cellular biology; Protein Kinase C - analysis; Proteoglycans - metabolism; Rats; Secretion. Exocytosis; Serotonin Antagonists - metabolism; Serotonin Antagonists - pharmacology; Staurosporine; Tumor Cells, Cultured - ultrastructure
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)99246-X

Strong, albeit indirect, evidence suggests that a GTP-binding (G) protein(s) can act directly on the secretory machinery by a post-second messenger mechanism. The type and function of this putative Ge (exocytosis) protein were investigated in streptolysin-O-permeabilized rat basophilic leukemia (RBL) cells. The exocytotic response to calcium was first characterized both morphologically and biochemically using the release of preloaded [3H]serotonin as an index of exocytosis. Calcium-induced secretion (EC50 about 3 microM) in RBL cells requires ATP (EC50 about 2.5 mM) and is modulated by pH, the optimal value being 7.2. Another requirement for calcium-induced secretion is an activated G protein, since inactivators of G proteins such as GDP beta S (EC50 about 800 microM) inhibit the secretagogue effect of 10 microM free calcium. Conversely, GTP gamma S (EC50 about 1 microM) and other nonhydrolyzable analogs of GTP, which keep G proteins in a permanently active conformation, potentiate the effect of calcium. GTP gamma S alone is without effect. The effect of GTP gamma S on exocytosis is apparently not mediated by known second messengers, suggesting that a Ge protein is involved. Electron microscopic images show that in resting cells, secretory granules are clustered in the perinuclear area, whereas they become scattered upon calcium stimulation. A paradoxical effect of GTP gamma S is observed when applied during permeabilization; under these conditions, in fact, the nucleotide inhibits the subsequent secretory response to calcium. The scattering of granules is also inhibited. This effect of GTP gamma S is counteracted by coadministration of GTP. These responses to guanine nucleotides are typical of vectorially acting G proteins involved in protein synthesis and in intracellular vesicle transport. Taken together, the data presented suggest that calcium-dependent release requires a vectorially acting G protein controlling the movement of secretory granules. This and alternative models are discussed.