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0702
EFEECTS OF EXTRACELLULAR ATP Di HEPATOCYTES
[U-
14C]glycerol(0.2 Ci/mol, 0.5 mM final concn). Cellular
triacylglycerols ami phospholipids were isolated by thin-layer
chro¡natography and quantified as described by Tijburg
st aL (25).
For ita determination of ratas of fatty acid oxidation,
reactiona were atañed by the addition to ceil incubationa of
[1-’4C]fatty acid (either palmitate or octanoate, 0.05 CiImol,
0.4 mM final concn) bound te albumin. Oxidation producta
(both acid-soluble producta ami CO
2) were extractad ami
quantiñed exactly as described before (10). Ketone bodies
routinely repreaent 80—90% oftotal acid-solubleproducta (10).
Finzymatic assays. Acetyl-CoA carboxylase (ACC) activity
was determinad ja digitonin-penneabiitad hepatocytes as
the incorporation of radiolabeled acatyl-CoA into fatty acids
la a raaction conpled to the fatty acid synthase (FAS) reaction.
This method avoids the number of interferences inherant
to ita classic bicarbonate-fixation aasay of ACC activity
(5). Te measure enzynie activity, 100 pl of hepatocyte suspensien
was added to 100 pl of prewarmed digitonin-containiflg
aasay medium exactly as describad by Bijleveld andGeelen (5).
FAS activity was monitored in digitonin-permeabilized
hepatocytas as described previously (5).
Carnitine O-palmitoyltransferase 1 (CPT-I) activity was
determinad in digitonin-parmeabilited hepatecytas aB the
tetradecyiglyeidate (TDGA)-sensitive incorporation of radiolabeled
L-carmtine into palxnitoylcarnitine. In brief, hepatocytea
were preincubated for 20 mm in the absence or presente
of5 wM TIJGA, apotent andspeciflcinhibitor ofCPT-I (7, 12).
Aliquota ware removed from both sets of incuhationa to
monitor CFT-1 activity. For that purpose, 100 pl of hapatocyte
suspansien was added to 100 pl of prewarmad digitonincontaining
assay medium exactly as described by Guzmán
and Gealen (12). Peroxisomal CFI? activity was determinad lii
the sanie incubationa as tha TDGA-insensitive, malonyl-CoAsensitiva
hepatoceHular CFI? activity (12).
Lactate dehydrogeaase activity was determinad by a standard
spectrophotometric methed Hepatocytes (29). (2—3 mg of cellular
Determination of [Ca 2~.l~.
protainlml) were incubated witb 6 pM indo 1-acetoxymethyl
ester (indo 1-AM) in bicarbonate-free Krebs-Henseleit medium
suppiemeated with 10 mMN~2.hydroxyethylpiPeraZin5
N’~2-ethanesulfoniC acid-NaOH (pH 7.4). 10 mM glucosa, and
1% (wt/vol) defatted and dialyzed bovina saruin albumin.Ita
incubation was performad at 370C for 45 mm with constant
shalci.ng (85 oscillations/min).
The fluorescence of indo 1-loadad calla wasexcitad by a5-W
laser tunad to 345—365 sun, andthe amitted fluorescance was
simultaneously measurad at 895 t 12.5 nm (for Ca2t~bound
indo 1) and 488 ± 5 nm (for Ca2t-free indo 1) in a FACStar
Plus (Becton Dickinson) Iiow cytometer
Oth-eranalyticalproeedures. Intracellular lavela of malonyl-
CoA ware detarmined in neutralizad perchloric acid celí
extracta by a radieenzymatic mnethed as describad by Baynan
et al. (2). Protein was determinad by the merhod of Lewry at
al. (19). with bovine sarum albuminas standard.
Materíais. [1-’4C]acatyl-CoA (54 CiImol), [1-’4C]acetate (56
CiImol), 3H
4C]palniitic acid (58 Ci/mol),
[1-’4C]octanoic 20 (5 acid CiImol), (32 Ci/moll. [1-’ [U-’4Clglycerol (171 CiImol),
and L-(Me-’4C]carmtlfla (54 Cf/mal) wera suppliad by Aniershani
International (Ainershani, Bucks, UK). Digitonia, collagenase
(type 1), 4~3-phorbol 12r3-myristate iSa-acetata (PMA),
and ATP were purchased from Sigma Chemical (St. Louis,
MO). TIRiA was a gift from Dr. M. J. H. Geelan, Utracht
University, The Natherlands. Bisindolylmalaintide, thapsigargin,
A-23187, ~,5~di.(t.butyl).1,4-banZOhytfrOqrnnOne (BHQ),
and indo 1-AM wara from Calbiocham (San Diego, CA).
Statistical analysi-s. Unlees ctharwise indicated, resulta
are meana ±SD of the number of aniinals indicated in every
case. Ceil incubationa and/or enzyme assays were always
carried out iii triplicate. Statiatical analysis was performed
by Studant’s t-tast.
RESULTS
Ita affeeta of extracallular ATP en fatty acid synthesis
and oxidation were studied in isolated rat hepatocytea.
In addition, digitonin-permeabilizad hapatocytes
were uaed for the study of tha effects exerted by
extracallular ATP en the activity of ACO (a kay regulatory
enzyme of fatty acid syntheais de novo in the liver)
and CPT-I (an important regulatory site ofhepatic fatty
acid oxidation). Exposura of hepatoeytea to the concentration
of digitonin used in our system (—40 pg/mg of
cellular protein) liberatea more than 95% of total
lactate dehydroganase (a cytosolic markar enzyme)
within 15 8(11, 12). The intagrity of the mitochondrial
ontar and inner membranas is preved by the fact that
lasa than 1% of total monoamine oxidase (a mitochondrial
outer membrana marker enzyme) and of total
glutamate dehydrogenase (a mitochondrial matrix
ma.rker enzyme) la raleased from tha permeahilized
celia after 1 mm of exposure to digitonin (11, 12).
Because extracellular ATP acta on hepatic matabolism
in a vary rapid fashion (8, 9), and because it is
actively hydrolyzad by plasma membrana ecto-adanosinetriphoaphataaes
(8, 9, 18), the incubation times usad
in the presant study wera vary ahort. Itese incubation
timea ware observad to allow maximal effects en the
parametera experimental1)’ determinad (resulta not
shown). In the casa of maasurements of ratas of fatty
acid synthesia, esterification, and oxidation, incubationa
wera carried out for np te 5 mm to achieve
adequate incorporation of the radiolabeled subatrates
into producta.
Prolongad exposura of rat hepatocytes te high desea
of ATP has been ahown te produce cytotoxic affects (31).
Howaver we did not observe any cytotoxic affect of thia
compeund in our short-term cali incubationa. Thua
challenge of hepatocytea te 200 pM ATP for 5 mm did
not induce any signiñcant variation of calí viability, as
determinad by both trypan bíne axcluaion and lactate
dehydrogenaae ralease.
Sifects of extracellular A7’P on Upogenesis. Incubation
of hepatocytea with ATP markadly decreased the
rata of fatty acid synthesia de novo when [‘4C]acetate
was usad as a subatrate (Tabla 1). Similarly, extracalínlar
ATP depresaed fatty acid synthesia da novo by
39.3 t 6.4% (n = 3, P