Characterization of calcium-triggered secretion in permeabilized rat basophilic leukemia cells. Possible role of vectorially acting G proteins.
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.
De Matteis
M A
MA
Laboratory of Neuroendocrinology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, (Chieti), Italy.
Di Tullio
G
G
Buccione
R
R
Luini
A
A
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007942:Leukemia, Experimental / Q000378:metabolism* / Q000473:pathology; D008854:Microscopy, Electron; D011493:Protein Kinase C / Q000032:analysis; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D012702:Serotonin Antagonists / Q000378:metabolism / Q000494:pharmacology; D019311:Staurosporine; D014407:Tumor Cells, Cultured / Q000648:ultrastructure
D000470:Alkaloids; D011509:Proteoglycans; D012702:Serotonin Antagonists; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D019311:Staurosporine; D002118:Calcium