title: Botulinum neurotoxin light chain inhibits norepinephrine secretion in PC12 cells at an intracellular membranous or cytoskeletal site.
abstract: Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described "cell cracking" procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell "ghosts" which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca(2+)-activated [3H]norepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150-kDa NT and the 50-kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca(2+)-activated [3H]norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100-kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by "cell cracking." The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]-norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.
authors.affiliation: Food Research Institute, University of Wisconsin, Madison 53706.
authors.forename: R
authors.initials: R
authors.lastname: Lomneth
authors.forename: T F
authors.initials: TF
authors.lastname: Martin
authors.forename: B R
authors.initials: BR
authors.lastname: DasGupta
mesh_terms: D000310:Adrenal Gland Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D003599:Cytoskeleton / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D009498:Neurotoxins / Q000494:pharmacology; D009638:Norepinephrine / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D010673:Pheochromocytoma / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D009498:Neurotoxins; D001905:Botulinum Toxins; D009638:Norepinephrine