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EFFECTS OF EXTRACEI
4LULARATP Di HEPATOCVTES
G705
Tabla 3. Effect of extracellular 2ÉL inAIF, isolatedhepatocytes
thapsigargin,
RHQ, azul A-23187 on[Ca
Fíoore sca oce
ity (as a parcentaga of incubations with no additiens,
n = 3) were 73 ±4% (incubations with 1 pM PMA?1,
73 t 3% (incubations with 1 pM PMA plus 10 pM
BHQ), 72 5% (incubatiens with 1 pM PMA plus 0.1
Additions tú tlie Incuhatione
Intensity, %
Nona 100
1iM thapsigargin), and 75 t 5% (incubatiens with 1 piM
PMAplus 2 pMA-23187).
100pMATP
0.1 pM thapsigargin
0.1 iM thapsigargin±100 pMATP
iO
4MBHQ
1OpMBHQ+100pMATP
2 gMA-23187
2jMA-23187 + 100 p.MATP
296
304
371
297
355
230
Afta indo 1 ¡oadirxg, hapatocytas wera jncubated wjth tha additiona
indicatad at 37’C, aad tha intracallular indo 1-emjttad fluorascanee
light was detarmined by flow cvtúmatry. Data are % of
jncubatjons with no additjons (% of control) and reprasent marimuin
value of fluorescenca intensjty at 395 12.5 ma relativa tú that at
488 5 nm. ISaac peak valuas 2~-mebihzjng ware routinaly agente. obtajnad flata correspoad 45—75 a after tú a
representativa addition offha djffereat experjment Ca that was rapeatad 3 times with similar
resulta. BHQ, 2,5~di.(t-buty¡)-1,4-benzohYdrOqmnOaC.
both ACC activity (Fig. 2) arid [Ca2’]t (Tabla 3) was
markedly potentiated by extracellular ATE
Extracallular ATPwas unable te deprasa CPT-I actiyity
arid Rateganasis frem palmitate in Ca2’-dapletad
hapatocytas (Tabla 2).Although this may indicate that
Ca2’ should be involvad in thasa affects, hepatocyta
incubation with thapsigargin, BHQ, er A-23187 had no
effect en CPT-I activity (Fig. 3). Qn the othar hand,
PMA decreased CPT-I activity in a nonadditive and
quantitativaly similar marinar with respact te axtracellular
ATP (Fig. 3). Moreover, bisindolylnialaimida abelished
the extraeeilularAl?-ínediated inhibition of CPT-I
activity (Fig. 3). Bisindelylmaleimide also antagenizad
tha extracallular ATP-depandent inhibition of CPT-I
whan thapsiga.rgin, BHQ, or A-23187 waa presant in
the incubation medium (resulta net shown). The dependency
of the antilcetogenie affact ofextracellularATP en
Ca2~ (Tabla 2) may thus reside in a step prior te PKC
activatien. Interestingly, tite PMA-induced inhibition
of CPT-I activity waa not avidant in Ca2’-depleted
hepatecytas (results not shown). Moreovar, tite PMAinduced
inhibition of CPT-I activity observad in Ca2’contaiing
hepatocytas (Fig. 3) was not potentiatad by
BHQ, titapaigargin, er A-23 187. Values of CPT-I activ-
u
ce
o
-¡00
90
80
70
383
DISCUSSION
o ~I~sc RHO AfllS7 PMA SIM
In tha praaant atudy we show that axtracellularATP
markadly affecta hapatic fatty acid matabolism. Tha
parallel inhibitien of ACC arid fatty acid syntheaia de
nove by extracallular ATP supperts tite general view
that ACC is a key regulatory point of tha fatty acidsynthasizing
proceas (13). Malonyl-CeA, tite preduct of
tite reactien catalyzad byACC, isa phyaielogical inhibiter
of CPT-I and playa an essential reía in tite ceerdinata
control of fatty acid synthasis and exidatien in tite
livar (4, 13, 20, 30). Tha extracellular ATP-induced
dapressien of itepatie ACC activity lad te a parallel
decrease in malonyl-CoA centent. Bacause CPT-I actívity
and palmitata oxidation were inhibitad by extracellular
ATP, titis would indicate that tha intracallular
cencentration of malonyl-CoA is net tha factor raspensible
for the ragulation of hapatie leng-chain fatty acid
oxidation under theaa conditiona. It should also be
pointad eut that te measure CPT-I activity we haya
permeabilized tite plasma membrane, arad titis has
causad tite cytesol te leak out of tite calí, laachng te a
larga dilutien of cytoaolic componanta, including malonyl-CoA
(11, 12). In titis respect it is netewortity that
tite shoñ-term modulatien of CPT-I by extracailular
ATP (rasults not shown), hepatocyta awalling (15), er
tite piteapitatasa inhibiter ekadaic acid (14) la very
stable, since thay survive hepatocyte permeabihzation,
extensive waahing of the permeabilizad calla, arad sub-
sequent incubation of tha parmeabilizad celís at 37 0C
for at leaat 10 mm. Thus, although inhibition of CPT-I
by malenyl-CoA la a wall-described property of tite
anzyme (4, 13, 20, 30), ether types e!ragulatery machanisma
may be invelvad in tite shert-tertn control of
hapatic CPT-I by cellular effactors (cf. Ref. 14).
Tha malonyl-CeA-indepandant inltbition of hapatic
CPT-I activity by extracalluilar ATP is accempaniad by a
dual effect en mitochondrial fatty acid oxidation, sinca
Fig. 3. Medulation ofcifacte of extracallular
ATP on CPT-I activity by conpounds
that changa [Ca2’]
1 and PKC
actjvity. Hepatocytes were preeneabatod
fer 5 mii, with no additiona (—br
with (ja pM) 0.1 TSG, 10 BHQ, 2
A-23187, 1 PMA, or 2 BlM. Hepatocytes
wera further incubated for 1 mm
ja abseace (opea bara) or prasence
(hatehad bara) of100 pM ATP, and ten
CPT-I activity was determinad. Results
are % of activity ra¡ative tú incubationa
with no additions and correspond tú
3—4 differant animala. 100% Value of
CPT-I actjvity ng 177 : 0.35 mno¡
preduct.mia< -mg celular pretein’.
Note ecale en y-axis. Significantly differant
5P < fron 0.01. jacubationa with no additions: