Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Ahnert‐Hilger, Gudrun; Bader, Marie‐France; Bhakdi, Sucharit; Gratzl, Manfred

doi:10.1111/j.1471-4159.1989.tb07253.x

Cited by 109 publications

(37 citation statements)

References 38 publications

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“…3 and 4A). Based on the above results, we conclude that mature components of the ␥-secretase are soluble in Triton X-100, but remain insoluble in Lubrol WX-resistant cholesterol-rich membranes consistent with their recruitment into biochemically distinct lipid rafts, as it has been previously described for microvilli resident protein prominin, copper-transporting P-type ATPase ATP7B, and multidrug resistance protein 1 (47,58).…”

Section: Ps1 and Mature Nicastrin Are Present In Lubrol Wx-insoluble mentioning

confidence: 81%

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes

Vetrivel¹,

Cheng²,

Lin

et al. 2004

Journal of Biological Chemistry

394

369

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Alzheimer's disease-associated ␤-amyloid peptides (A␤) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by ␤-and ␥-secretases. There is growing evidence that cholesterol-and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major ␤-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the ␥-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1 ؊/؊ /PS2 ؊/؊ and NCT ؊/؊ fibroblasts, ␥-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires ␥-secretase complex assembly. Biochemical evidence shows that subunits of the ␥-secretase complex and three TGN/endosomeresident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1-or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of ␥-secretase in lipid raft microdomains of postGolgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.Alzheimer's disease, a neurodegenerative dementing disorder, is pathologically characterized by the cerebral deposition of 39 -42 amino acid peptides termed ␤-amyloid (A␤) 1 peptides.

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Section: Ps1 and Mature Nicastrin Are Present In Lubrol Wx-insoluble mentioning

confidence: 81%

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes

Vetrivel¹,

Cheng²,

Lin

et al. 2004

Journal of Biological Chemistry

394

369

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“…Fractions were collected every 2 min and immediately frozen until radioimmunoassay for released arginine vasopressin (A VP) was performed. 1 The Ca2+ evoked hormone release was calcula ted by subtracting the mean basal release determined in the fractions preceding the onset of the Ca 2 + stimulus from the amount of hormone found in each of the following eight fractions. 8 Free Ca2+ concentrations were calculated using the stability constants listed 18 and measured with a Cal+-sensitive electrode 4 as described recently.11…”

Section: Superfusion Of the Isolated Nerve Endingsmentioning

confidence: 99%

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

et al. 1990

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Abstract-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 ofthe reducing agent dithiothreitol, the two-chain form of botulinum A toxin inhibited vasopressin release induced by 10 Jl M 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.Botulinum A toxin belongs to a family of cIosely reIated neurotoxins. The 150,000 mol. wt protein consists of a heavy chain (100,000 mol. wt) and a light chain (50,000 mol. wt) covalently linked by a disulfide bond. It inhibits neuro transmission at the peripheral cholinergic nerve endings by inhibiting neurotransmitter release. A three-step model is thought to produce the paralytic effects. It consists of (i) extracellular binding, (ii) internalization and (iii) intracellular poisoning (cf. Refs 12 and 19). The toxins' effects are restricted to the nervous tissue when given extracellularly. However, an extracellular application does not allow the differentiation of the intracellular processes involved in the final inhibition of transmitter release and the role played by the individual chains of the toxin.Recently, using cultured adrenal chromaffin cells permeabilized with streptolysin 0, it has been shown that intracellularly applied botulinum A toxin is more effective in the presence of a reducing agent. Indeed, the light chain of botulinum A toxin alone inhibited Ca 2 + -stimulated catecholamine release. 5 ,20,21 In contrast, both the heavy and the light chain must be tPresent address:

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“…Using intact cell preparations, the intracellular action of clostridial neurotoxins could not directly be investigated. The direct intracellular application of the toxins and their fragments in nor~ mal or neoplastic chromaffin cells via a patch clamp pipette [8] or by pemleabilization of tbe plasma membrane [9,10,13,[26][27][28][29] allowed to demonstrate that toxicity is confined to the light chains of both, TeTx and BoNT/A and that the toxins act at a 5tep downstream of the action of Ca 2 + during exocytosis.…”

Section: Discussionmentioning

confidence: 99%

“…Intact TeT~ was less effective in penneabilized synaptosomes than the isolated light chain. This may indicate that chain separation, which presumably is an important process during TeTx and BoNT/A activation [9,10,12,26,31), by endogenous systems [32], is probably not completed within the 5 minutes of iilcubation.…”

Section: Febs Leiters November 1992mentioning

confidence: 99%

“…Also, in permeabilized adrenal chromaffin cells, pheochromocytoma cells (PCI2 cells) [9][10][11] and neurosecretosomes from the posterior pituitary gland [12), TeTx turned out to be a potent inhibitor of noradrenaline (NA) and vasopressin release. In addition it has been shown that TeTx is more effective in these cells in the presence of a reducing agent, wh ich causes chain separation [9,10,12]. Indeed, the ability of TeTx to inhibit release of catecholamines [10,13] and vasopressin [12) [14,15).…”

Section: Introduction Tetanus Toxin (Tetx) Is a Hishly Nellrotoxic Pmentioning

confidence: 99%

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Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin

et al. 1992

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Noradrenaline release frorn rat brain cortical synaptosom,,'S permeabilized with streptolysin 0 can be triggered by,uM conccntrations of free Ca 2 +. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the elfect of tetanus toxin on NA release frem purified synaptosomes is caused by thc intraterminal action of its light chain.

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Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Cited by 109 publications

References 38 publications

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin

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