Cocaine induces a mixed lysosomal lipidosis in cultured fibroblasts, by inactivation of acid sphingomyelinase and inhibition of phospholipase A1.
This paper reports that cocaine may induce a lysosomal storage disorder. Indeed, culture of Rat-1 fibroblasts with 250-500 microM cocaine induced after 2-3 days a major accumulation in lysosomes of electron-dense lamellar structures. By subcellular fractionation, this was reflected by a selective decrease of the buoyant density of several lysosomal enzymes, indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane-permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A1. At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in Vmax with identical Km. Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal hydrolases was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis.
Nassogne
Marie-Cécile
MC
Cell Biology Unit, Christian de Duve Institute of Cellular Pathology, Louvain University Medical School, B-1200 Brussels, Belgium.
Lizarraga
Chantal
C
N'Kuli
Francisca
F
Van Bambeke
Françoise
F
Van Binst
Roger
R
Wallemacq
Pierre
P
Tulkens
Paul M
PM
Mingeot-Leclercq
Marie-Paule
MP
Levade
Thierry
T
Courtoy
Pierre J
PJ
D000818:Animals; D002460:Cell Line; D003042:Cocaine / Q000378:metabolism / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000633:toxicity*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D008064:Lipidoses / Q000139:chemically induced / Q000201:enzymology*; D016464:Lysosomal Storage Diseases / Q000139:chemically induced / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054466:Phospholipases A1; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D004791:Enzyme Inhibitors; D010741:Phospholipases A; D054466:Phospholipases A1; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase; D003042:Cocaine