The in vitro NADPH-dependent inhibition by CCl sub 4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl sub 3 radical

Journal Article · Fri May 25 04:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6742407

Srivastava, S P; Chen, N Q; Holtzman, J L ^[1]

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1. Univ. of Minnesota, Minneapolis (USA)

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3 radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals and are toxic to cultured hepatocytes. The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4.

• OSTI ID: 6742407
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:15; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550501 -- Metabolism-- Tracer Techniques
560300* -- Chemicals Metabolism & Toxicology
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ACID ANHYDRASES
ALKALINE EARTH METALS
AMINO ACIDS
ANIMALS
ATP-ASE
BIOLOGICAL EFFECTS
BODY
CALCIUM
CARBON 14 COMPOUNDS
CARBON TETRACHLORIDE
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHLORINATED ALIPHATIC HYDROCARBONS
COENZYMES
CYSTEINE
DIGESTIVE SYSTEM
ELEMENTS
ENZYMES
GLANDS
HALOGENATED ALIPHATIC HYDROCARBONS
HYDROLASES
INHIBITION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPIDS
LIVER
MAMMALS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
METABOLISM
METALS
MICROSOMES
NADP
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC CHLORINE COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
PORINS
PROTEINS
RADICALS
RATS
REDOX REACTIONS
RIBOSOMES
RODENTS
THIOLS
TRACER TECHNIQUES
VERTEBRATES