162 research outputs found
Reductive chain separation of botulinum A toxin — a prerequisite to its inhibitory action on exocytosis in chromaffin cells
Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action
on exocytotic release ofcatecholamines from permeabi1ized chromaffin cells [(1989) FEBS Lett. 242, 245-248; (1989) J.
Neurochern., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide
bond. The actions ofboth neurotoxins on exocytosis were presently compared using streptolysin O-permeabilized bovine
adrenal chromaffin cells. Botulinum A toxin inhibited Ca2 +-stimulated catecholamine release from these cells. Addition
of dithiothreitollowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus
toxin were decreased by a factor of five. This indicates that the interchain S-S bond of botulinum A toxin must
also be split before the neurotoxin can exert its effect on exocytosis
Ca2+-Stimulated Catecholamine Release from alpha-Toxin Permeabilized PC12 Cells
Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC 12 cells
to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can
be activated in a-toxin-permeabilized cells with micromolar levels of free Ca2+. Catecholamine metabolites
formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither
formed nor released from the cells under these conditions. However, when vesicular catecholamines were
discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released
into the medium independent of Ca2+. Both types of experiments provide direct evidence for the operation
of Ca2+-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca2+ dependence
of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines
using the high-performance liquid chromatography technique, exhibits two different phases. One is already
activated below 1 pM free Ca2+ and plateaus at 1-5 pM free Ca2+, while a second occurs in the presence
of larger amounts of free Ca2+ (10-100 pM). Ca2+-induced catecholamine release from the permeabilized
cells can be modulated in different ways: It is enhanced by the phorbol ester 12-0-tetradecanoylphorbol
13-acetate and the diacylglycerol 1 -oleyl-2-acetylglycerol provided Mg*+/ATP is present, and it is inhibited
by guanosine 5’-0-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with
pertussis toxin but not by cholera toxin. Thus, it appears that Ca2+-induced exocytosis can be modulated
via the protein kinase C system, as well as via GTP binding proteins
Minimal requirements for exocytosis
The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular 86Rb+ and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis
Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin
The intracellular action on exocytosis of botulinim A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 μM free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis. It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain
Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin
The intracellular action on exocytosis of botulinim A toxin and constituent chains was studied using permeabilized isolated nerve endings from the rat neural lobe. The release of the neuropeptide vasopressin was measured by radioimmunoassay. In the presence of the reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 μM free calcium. Half maximal inhibition was obtained with 15 nM botulinum A toxin. In the absence of the heavy chain the light chain of the toxin strongly inhibited exocytosis with a half maximal effect of 2.5 nM. The inhibitory effects on secretion could be prevented by incubating the light chain with an immune serum against botulinum A toxin. The heavy chain of botulinum A toxin did not affect vasopressin release. However, it prevented the inhibitory effects of the light chain on stimulated exocytosis. It is concluded that botulinum A toxin inhibits the calcium-dependent step leading to exocytosis by interfering with a target present in the isolated and permeabilized nerve terminals. The functional domain of this neurotoxin, which is responsible for the inhibition of vasopressin release, is present in its light chain
The light chain but not the heavy chain of botulinum A toxin inhibits exocytosis from permeabilized adrenal chromaffin cells
The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography. Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O. Purified light chain inhibited the Ca2+-stimulated [3H]noradrenaline release with a half-maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis
Characterization of hormone and protein release from alpha-toxin- permeabilized chromaffin cells in primary culture
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
- …