Exocytosis in single chromaffin cells: regulation by a secretory granule-associated Go protein.
ABSTRACT 1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, G alpha o has been found associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells. 2. Microinjection of GTP gamma S and mastoparan strongly inhibits the amperometric response to either nicotine or high K+. 3. The presence of mastoparan in the cell incubation medium had no effect on K(+)-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane. 4. Microinjection of anti-G alpha o antibodies potentiates by more than 50% the K(+)-evoked secretion, whereas anti-G alpha i1/2 antibodies have no effect. 5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolic protein GAP-43 stimulates G alpha o in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin, cells by stimulating a granule-associated Go protein.
- Annual Review of Biochemistry 02/1994; 63:949-90. · 27.68 Impact Factor
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ABSTRACT: Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.Journal of Biological Chemistry 06/1986; 261(13):5777-83. · 4.65 Impact Factor
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ABSTRACT: We have used the caged calcium compound DM-nitrophen to investigate the kinetics of calcium-dependent secretion in bovine chromaffin cells. Perfusion with partially calcium-loaded nitrophen often caused a loading transient--slow secretion for up to 1 min due to displacement of Ca2+ by cytoplasmic Mg2+. Flash photolysis elicited 100 microM [Ca2+]i steps that evoked intense secretion, lasting a few seconds. In cells experiencing a loading transient, [Ca2+]i steps evoked an especially fast secretion. A persistent, slow secretion often followed these fast phases. Distinct kinetic components may reflect secretion from pools that are differentially capable of release. Both secretion and movement of vesicles between pools appear to be [Ca2+]i sensitive. Later [Ca2+]i steps sometimes evoked a rapid capacitance decrease, indicating a fast, [Ca2+]i-dependent phase of endocytosis.Neuron 02/1993; 10(1):21-30. · 15.77 Impact Factor