Mitochondrial dysfunction and cytoskeletal disruption during chemical hypoxia to cultured rat hepatic sinusoidal endothelial cells: The pH paradox and cytoprotection by glucose, acidotic pH, and glycine

✍ Scribed by Yoshiya Nishimura; Lewis H. Romer; Dr. John J. Lemasters

• Book ID: 102241137
• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 295 KB
• Volume: 27
• Category: Article
• ISSN: 0270-9139
• DOI: 10.1002/hep.510270420

✦ Synopsis

We investigated mechanisms underlying death of cultured rat liver sinusoidal endothelial cells exposed to chemical hypoxia with KCN (2.5 mmol/L) to simulate the adenosine triphosphate (ATP) depletion and reductive stress of anoxia. During chemical hypoxia, acidotic pH prevented cell death. Glucose (0.3-10 mmol/L) also prevented cell killing. Cytoprotection by glucose but not acidosis was associated with prevention of ATP depletion. After 4 hours of chemical hypoxia at pH 6.2 (simulated ischemia), rapid cell death occurred when pH was restored to pH 7.4 with or without washout of KCN (simulated reperfusion). This pH-dependent reperfusion injury (pH paradox) was prevented after KCN washout at pH 6.2. Glycine (0.3-3 mmol/L) also prevented the pH paradox, but glucose did not. The initial protection by acidotic pH and glycine during simulated reperfusion was lost when pH was later restored to 7.4 or glycine was subsequently removed. Mitochondria depolarized during chemical hypoxia. After washout of cyanide, mitochondrial membrane potential (⌬⌿) did not recover in cells that subsequently lost viability. Conversely, those cells that repolarized after cyanide washout did not subsequently lose viability. The actin cytoskeleton and focal adhesions became severely disrupted during chemical hypoxia at both pH 6.2 and 7.4 and did not recover after cyanide washout under any condition. Glucose during chemical hypoxia prevented cytoskeletal disruption. In conclusion, endothelial cell damage during simulated ischemia/reperfusion involves mitochondrial dysfunction, ATP depletion, and ATP-dependent cytoskeletal disruption. Glycine and acidotic pH prevented cell killing after reperfusion but did not reverse mitochondrial injury or the profound disruption to the cytoskeleton. (HEPATOLOGY 1998;27:1039-1049.)

Ischemia/reperfusion injury is an important problem in liver surgery and systemic shock. Postoperative graft dysfunction and failure after liver transplantation also involve ischemia/reperfusion injury. [1][2][3] In previous studies, sinusoidal endothelial cells were shown to be a critical target of this reperfusion injury. [4][5][6] However, the underlying mechanisms of reperfusion-induced endothelial injury remain unclear.

During ischemia, tissue pH becomes acidotic due to anaerobic glycolysis and adenosine triphosphate (ATP) hydrolysis. Acidosis protects cells from injury during hypoxia, [7][8][9][10][11][12] as does glycolytic metabolism generating ATP. 10,[13][14][15] However, when extracellular pH recovers to physiological levels after hypoxia, rapid cell death occurs, a phenomenon called the pH paradox. 9,11,12,16,17 Because correction of acidosis occurs rapidly after reperfusion, the pH paradox may be a major cause of reperfusion injury. Glycine also strongly protects against hypoxic and toxic injury to many cell types. 12,[18][19][20][21][22] Previously, we reported that glycine prevented reperfusioninduced endothelial cell killing after liver preservation for tissue transplantation surgery and improved graft function and graft survival after liver transplantation in rats. 23,24 The cytoskeleton regulates cell motility, the intracellular distribution of organelles, and overall cell shape and topography. In vascular endothelium, the cytoskeleton is essential for maintenance of barrier function and the regulation of transendothelial permeability. [25][26][27] During cold ischemic liver storage for transplantation, sinusoidal endothelial cells round up and detach from their underlying attachments. 1,28 Such changes suggest disruption of the cytoskeleton. In other cell types, ATP depletion also causes perturbation of the cytoskeleton. 29,30 However, the relation of cytoskeletal disruption to ATP depletion and cell injury in sinusoidal endothelial cells is not well studied.

Because perturbation of structure and loss of viability of endothelial cells are initial events leading to liver graft failure from preservation injury, our aim was to characterize various protective strategies against cell killing and cytoskeletal disruption using a model of ischemia/reperfusion injury to cultured sinusoidal endothelial cells.

MATERIALS AND METHODS

Isolation of Sinusoidal Endothelial Cells.

Sinusoidal endothelial cells of male Sprague-Dawley rats (270-330 g) were isolated and purified by collagenase perfusion and centrifugal elutriation by modification of the procedures described by Braet 31 and Knook. 32 Under pentobarbital anesthesia, the abdomen was opened, a catheter was inserted into the portal vein, and the liver was perfused with Ca 2ϩ -free buffer Abbreviations: ATP, adenosine triphosphate; GBSS, Gay' s balanced salt solution; KRH, Krebs-Ringer-Hepes buffer; TMRM, tetramethylrhodamine methylester; ⌬⌿, membrane potential.

From the Departments of 1 Cell Biology and Anatomy, 2 Pediatrics, and