Proc. Nati. Acad. Sci. USA Vol. 87, pp. 5912-5915, August 1990 Neurobiology
Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system (adrenal chromaffin cells/phospholipase A2/arachidonic acid)
URS 0. KARLI, THEO SCHAFER, AND MAX M. BURGER Friedrich Miescher-Institut, P.O. Box 2543, 4002 Basel, Switzerland
Communicated by E. P. Kennedy, May 21, 1990
ABSTRACT In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2+dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2+-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 300C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2+-dependent and -independent process: first, a Ca2+-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2+-independent processing to the terminal fusogen and subsequent Ca2+-independent fusion of the CGs with the PM.
vesicles with the PM. Secretion of catecholamines from brain synaptosomes has been shown to be tightly coupled to stimulation of PLA2 (14, 15). In accordance with this, brain synaptic vesicles have been shown to aggregate and possibly to fuse due to an endogenous PLA2 activity in a cell-free system (16). Based on the data obtained from chromaffin cells and from these cell-free experiments, we have applied the octadecyl rhodamine B (R18) method to dissect the Ca2+ dependence of the exocytotic fusion process of CGs and PM by partial reconstitution in vitro. Vesicle fusion is measured by the elegant method of fluorescence dequenching of the lipid marker R18, as developed by Hoekstra et al. (17). The probe can be incorporated into the membranes of vesicles at a concentration that quenches its fluorescence signal. Fusion of these vesicles with unlabeled target vesicles is measured as a fluorescence signal increase due to dilution of the marker. The validity of the R18 method was confirmed in a number of control experiments (18-20). The kinetics, pH profile, and temperature dependence of fusion were similar when measured for two fluorescent events simultaneously by using R18 for monitoring lipid mixing and a water-soluble fluorescent dye [N-(7-nitrobenzofurazan-4-yl)taurine] for monitoring cytoplasmic continuity (19). Using the R18 technique, we have set up a cell-free system to study the events associated with fusion of CGs with PMs.
A crucial role for Ca2+ in stimulus-secretion coupling has long been recognized, but its precise function in exocytosis remains unknown (1). Neurotransmitter release is mediated by an increase in intracellular Ca2l concentration, which has been postulated to activate intracellular enzymes involved in secretion (2). Adrenal chromaffin cells, derivatives of the neural crest, are a well-characterized model system for studying the synthesis, storage, and secretion of neurotransmitters and hormones (3). Stimulation of adrenal chromaffin cells brings about an increase in intracellular Ca2+ concentration and free arachidonic acid (AA) (4, 5), which trigger the fusion of secretory vesicles, the chromaffin granules (CGs), with the plasma membrane (PM), resulting in the release of the stored catecholamines. Inhibitors of phospholipase A2 (PLA2), an enzyme liberating fatty acids from phospholipids (6), have been shown to inhibit the liberation of AA as well as catecholamine secretion (7). PLA2 activity was postulated to be involved in the release process in other secretory cells, such as mast cells (8) and neutrophils (9). These results are of particular interest, since AA and some of its metabolites have recently been suggested to play an important role in signal transduction (10-13). Cell-free in vitro systems have been established to study the molecular mechanisms promoting fusion of secretory
MATERIALS AND METHODS Purified CGs (21) were lysed in 5 mM phosphate buffer (pH 8.0) and labeled with the fluorescent probe R18 (Molecular Probes) at a molar lipid ratio of 5% in sodium glutamate buffer (20 mM Pipes/139 mM sodium glutamate/5 mM glucose/5 mM EGTA, pH 7.2). Excess R18 was removed by repeated centrifugation at 15,000 x g at 40C. A postnuclear supernatant of bovine adrenal medulla homogenate was centrifuged at 25,000 x g for 20 min. The membrane pellet was layered on a 1.6 M sucrose cushion and centrifuged at 100,000 x g for 2 hr. The resulting interface of light membranes, virtually devoid of CGs, was used for target PM vesicles. PM vesicles were resuspended either in sodium glutamate buffer or in sodium glutamate/Ca2+ buffer (sodium glutamate/Ca2+ buffer also contained 5 mM CaC12, which resulted in -20 1AM free Ca2+). PLA2 (total proteolytic activity,
Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system (adrenal chromaffin cells/phospholipase A2/arachidonic acid)
URS 0. KARLI, THEO SCHAFER, AND MAX M. BURGER Friedrich Miescher-Institut, P.O. Box 2543, 4002 Basel, Switzerland
Communicated by E. P. Kennedy, May 21, 1990
ABSTRACT In adrenal chromaffin cells, stimulation of Ca2+ influx leads to the secretion of neurotransmitters. The intracellular Ca2+ target involved in the fusion of secretory vesicles with the plasma membrane (PM) is still not known. We have reconstituted this fusion in vitro by using chromaffin granules (CGs) and target membrane vesicles and a Ca2+dependent phospholipase A2 (PLA2). Vesicle fusion is measured by a fluorescence dequenching assay with octadecyl rhodamine B used as the marker. CGs fuse with PM vesicles only in the presence of active PLA2. The kinetics of this fusion process depend on the amount of target PM added. Once fusion competence of PM vesicles is achieved by exposure to PLA2 (primed PM vesicles), it is conserved after removal of the PLA2 even in Ca2+-free buffer. The kinetics of fusion between these primed PM vesicles and CGs depend on the amount of PM and on the temperature. Further incubation of the PLA2-treated PM vesicles at 300C in the absence of calcium results in an enhanced fusion competence. During this incubation, the amount of free arachidonic acid liberated by PLA2 decreases, suggesting that during a second process arachidonic acid may be processed to the terminal fusogen. The final steps of secretion can thus be subdivided into a Ca2+-dependent and -independent process: first, a Ca2+-dependent activation of PLA2 liberating fatty acids from phospholipids and second, a Ca2+-independent processing to the terminal fusogen and subsequent Ca2+-independent fusion of the CGs with the PM.
vesicles with the PM. Secretion of catecholamines from brain synaptosomes has been shown to be tightly coupled to stimulation of PLA2 (14, 15). In accordance with this, brain synaptic vesicles have been shown to aggregate and possibly to fuse due to an endogenous PLA2 activity in a cell-free system (16). Based on the data obtained from chromaffin cells and from these cell-free experiments, we have applied the octadecyl rhodamine B (R18) method to dissect the Ca2+ dependence of the exocytotic fusion process of CGs and PM by partial reconstitution in vitro. Vesicle fusion is measured by the elegant method of fluorescence dequenching of the lipid marker R18, as developed by Hoekstra et al. (17). The probe can be incorporated into the membranes of vesicles at a concentration that quenches its fluorescence signal. Fusion of these vesicles with unlabeled target vesicles is measured as a fluorescence signal increase due to dilution of the marker. The validity of the R18 method was confirmed in a number of control experiments (18-20). The kinetics, pH profile, and temperature dependence of fusion were similar when measured for two fluorescent events simultaneously by using R18 for monitoring lipid mixing and a water-soluble fluorescent dye [N-(7-nitrobenzofurazan-4-yl)taurine] for monitoring cytoplasmic continuity (19). Using the R18 technique, we have set up a cell-free system to study the events associated with fusion of CGs with PMs.
A crucial role for Ca2+ in stimulus-secretion coupling has long been recognized, but its precise function in exocytosis remains unknown (1). Neurotransmitter release is mediated by an increase in intracellular Ca2l concentration, which has been postulated to activate intracellular enzymes involved in secretion (2). Adrenal chromaffin cells, derivatives of the neural crest, are a well-characterized model system for studying the synthesis, storage, and secretion of neurotransmitters and hormones (3). Stimulation of adrenal chromaffin cells brings about an increase in intracellular Ca2+ concentration and free arachidonic acid (AA) (4, 5), which trigger the fusion of secretory vesicles, the chromaffin granules (CGs), with the plasma membrane (PM), resulting in the release of the stored catecholamines. Inhibitors of phospholipase A2 (PLA2), an enzyme liberating fatty acids from phospholipids (6), have been shown to inhibit the liberation of AA as well as catecholamine secretion (7). PLA2 activity was postulated to be involved in the release process in other secretory cells, such as mast cells (8) and neutrophils (9). These results are of particular interest, since AA and some of its metabolites have recently been suggested to play an important role in signal transduction (10-13). Cell-free in vitro systems have been established to study the molecular mechanisms promoting fusion of secretory
MATERIALS AND METHODS Purified CGs (21) were lysed in 5 mM phosphate buffer (pH 8.0) and labeled with the fluorescent probe R18 (Molecular Probes) at a molar lipid ratio of 5% in sodium glutamate buffer (20 mM Pipes/139 mM sodium glutamate/5 mM glucose/5 mM EGTA, pH 7.2). Excess R18 was removed by repeated centrifugation at 15,000 x g at 40C. A postnuclear supernatant of bovine adrenal medulla homogenate was centrifuged at 25,000 x g for 20 min. The membrane pellet was layered on a 1.6 M sucrose cushion and centrifuged at 100,000 x g for 2 hr. The resulting interface of light membranes, virtually devoid of CGs, was used for target PM vesicles. PM vesicles were resuspended either in sodium glutamate buffer or in sodium glutamate/Ca2+ buffer (sodium glutamate/Ca2+ buffer also contained 5 mM CaC12, which resulted in -20 1AM free Ca2+). PLA2 (total proteolytic activity,
