The inhibitory effect of prostaglandin E1 on oxidative stress-induced hepatocyte injury evaluated by calpain-mu activation.
BACKGROUND
Prostaglandin E1 (PGE1) is known to inhibit ischemia-reperfusion injury of the liver. The calcium-dependent neutral proteinase, calpain-mu, is involved in oxidative stress-induced hepatocyte injury. We investigated the mechanisms of cytoprotection by PGE1, focusing on the elevation of intracellular calcium ([Ca2+]i), activation of calpain-mu, and calpain-mu-mediated activation of protein kinase C-alpha (PKC-alpha).


METHODS
Cultured hepatocytes were treated with various amounts of PGE1 (0, 0.1, 1.0, 10, and 100 ng/ml) for 30 min and subsequently with 0.5 mM tert-butyl hydroperoxide (TBHP). Cell injury was evaluated by the release of lactate dehydrogenase. Plasma membrane bleb formation was examined by phase contrast microscopy. Activation of calpain-mu and limited degradation of PKC-alpha was evaluated by Western blotting using antibodies that specifically recognize the amino-terminal regions of calpain-mu and PKC-alpha. [Ca2+]i was measured by confocal microscopy using Fluo-3AM.


RESULTS
LDH release from cells treated with 10 ng/ml PGE1 was significantly lower than from untreated cells (135 +/- 12 vs. 258 +/- 18 IU/L, respectively; P < 0.05). Morphologically, many blebs were observed in untreated cells, but very few were seen in those treated with 10 ng/ml PGE1. Western blotting revealed that the amount of activated calpain-mu and [Ca2+]i increased up to 1,300 nM at 35 min after the addition of TBHP (0.5 mmol/L) in control experiments (without PGE1). PGE1 (10 ng/ml) delayed the rise in [Ca2+]i for about 30 min, but did not suppress it completely. PKC-alpha decreased in experiments using PGE1 (10 ng/ml).


CONCLUSIONS
PGE1 exerts its cytoprotective effect in TBHP-induced hepatocyte injury partly by inhibiting Ca2+-calpain-mu-mediated mechanisms.
Kishimoto
S
S
Second Department of Surgery, Osaka University Medical School, Japan.
Sakon
M
M
Umeshita
K
K
Miyoshi
H
H
Taniguchi
K
K
Meng
W
W
Nagano
H
H
Dono
K
K
Ariyosi
H
H
Nakamori
S
S
Kawasaki
T
T
Gotoh
M
M
Monden
M
M
Imajoh-Ohmi
S
S
D000527:Alprostadil / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D009994:Osmolar Concentration; D018384:Oxidative Stress / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D051381:Rats; D017208:Rats, Wistar
D007527:Isoenzymes; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002154:Calpain; D000527:Alprostadil; D002118:Calcium