H. Farghali

31P-NMR spectroscopy of perifused rat hepatocytes immobilized in agarose threads: application to chemical-induced hepatotoxicity

A system consisting of isolated rat hepatocytes immobilized in agarose threads continuously perifused with oxygenated Krebs-Henseleit (KH) solution has been found to maintain cell viability with excellent metabolic activity for more than 6 h. The hepatocytes were monitored by phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy at 4.7 Tesla, by measurement of oxygen consumption and by the leakage of lactate dehydrogenase (LD) and alanine aminotransferase (ALT). The data obtained were comparable to those found for an isolated perfused whole liver in vitro. The effects of allyl alcohol (AA), ethanol, and 4-acetaminophenol (AP) were examined. A solution of 225 microM AA perifused for 90 min caused the disappearance of the beta-phosphate resonance of adenosine triphosphate (ATP) in the 31P-NMR spectra, a 7-fold increase in LD leakage and a 70% reduction in oxygen consumption. Ethanol (1.0 M) perifused for 90 min reduced the beta-ATP signal intensity ratio by 20%, the phosphomonoester (PME) signal by 50% and inorganic phosphate (Pi) by 33% (P less than 0.05). AP (10 mM) caused only mild liver-cell damage. The results demonstrate that perifused immobilized hepatocytes can be used as a liver model to assess the effects of a wide range of chemicals and other xenobiotics by NMR spectroscopy.

Fasting enhances the effects of anoxia on ATP, Ca2+i and cell injury in isolated rat hepatocytes

The effect of fasting and anoxia on the intracellular concentration of ATP, Na+, Ca2+, Mg2+, and H+ was studied in isolated perfused rat hepatocytes. ATP and intracellular Mg2+ were measured by 31P-NMR spectroscopy, cytosolic free calcium was measured with aequorin, intracellular Na+ with SBFI, intracellular pH with BCECF, lactic dehydrogenase by NADH absorbance. In hepatocytes from fasted rats, intracellular ATP was depressed 52% (P < 0.001), Nai+ was increased 70% from 16.9 to 27.7 mM (P < 0.02), and Cai2+ was increased 79% from 137 to 245 nM (P < 0.05) when compared to fed rats. Mgi2+ and pHi were unchanged. During anoxia, ATP and the cell phosphorylation potential decreased 90% to practically the same low levels in both fed and fasted groups. On the other hand, in hepatocytes from fasted animals, Cai2+ increased faster and to significantly higher levels than in hepatocytes from fed rats: Cai2+ reached 2.19 microM in 10 min compared to 1.45 microM in 1 h, respectively (P < 0.05). Cell injury assessed by LDH release and trypan blue exclusion also occurred earlier and was more severe in hepatocytes from fasted rats. Fructose and Ca(2+)-free perfusion reduced the rise in Cai2+, abolished LDH release and significantly improved the cell viability measured by Trypan blue exclusion. The data demonstrate that fasting decreases the hepatocytes energy potential and increases Nai+ and Cai2+ which are inversely related to the cell energy potential. Consequently, in hepatocytes isolated from fasted rats, the increase in Cai2+ and the resulting cell injury evoked by anoxia occur earlier and are more severe than in fed rats. These results suggest that Ca2+ plays a crucial role in the development of anoxic cell injury.

Effects of high and low pH on Ca2+i and on cell injury evoked by anoxia in perfused rat hepatocytes.

The effect of high and low pH on anoxic cell injury was studied in freshly isolated rat hepatocytes cast in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB) saturated with 95% O2 and 5% CO2. Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular pH (pHi) with BCECF, and lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate. A 2 h period of anoxia was induced by perfusing the cells with KHB saturated with 95% N2 and 5% CO2. The extracellular pH (pHo) was maintained at 7.4, 6.8 or 8.0 by varying the bicarbonate concentration. The substrate was either 5 mM glucose, 15 mM glucose or 15 mM fructose. In some experiments, anoxia was performed in Ca(2+)-free media by perfusing the cells with KHB without Ca2+ but with 0.1 mM EGTA. Reducing pHo to 6.8 during anoxia did not reduce the increase in Ca2+i, but but completely abolished LDH release. Under these conditions, pHi decreased to 6.56 +/- 0.3 when glucose was the substrate and to 6.18 +/- 0.25 with 15 mM fructose. Apparently, protection against anoxic injury caused by a low pHo is associated with a low pHi but not with a reduced elevation in Ca2+i. Increasing pHo to 8.0 during anoxia increased pHi above 8.0 +/- 0.01 and doubled LDH release without significantly altering the rise in Ca2+i. When 15 mM fructose was present with a pHo of 8.0, pHi was still 8.0, but there was practically no rise in Ca2+i, and LDH release was again completely abolished. On the other hand, a Ca(2+)-free perfusate with a pHo of 8.0 kept the rise in Ca2+i below 400 nM but did not abolish the massive release of LDH caused by high pH. Since cell injury is caused by the activation of Ca(2+)-sensitive hydrolytic enzymes such as phospholipase A2, these experiments suggest that a low pH (< 6.5) prevents their activation even in the presence of a high Ca2+i. Conversely, a high pH (> 8.0) can activate hydrolytic enzymes and cause injury even in the absence of an elevated Ca2+i. The precise mechanism by which fructose protects hepatocytes against cell injury at pHi 8.0 is unclear.

Biochemical and 31P-NMR spectroscopic evaluation of immobilized perfused rat Sertoli cells.

Cell immobilization and perfusion are used for physiologic studies of Sertoli cells with phosphorus 31 nuclear magnetic resonance (NMR) spectroscopy and biochemical methods. In this study the 31P NMR spectra of Sertoli cells isolated from 18-to 21-day-old rats and immobilized in agarose threads continuously perfused with oxygenated Dulbeccos modifed Eagle medium were obtained at 81 MHz on an NMR system. Cytosolic Ca2+, intracellular Mg2+, lactate and pyruvate, and oxygen consumption were measured with standard biochemical methods. Perfused Sertoli cells maintain a stable intracellular adenosine triphosphate concentration for more than 10 hours. Sertoli cells placed in cold storage overnight and then subjected to perfusion partially regenerate cellular adenosine triphosphate levels. Sertoli cells consume an average of 4.8 +/- 0.4 nmol O2/min/10(6) cells and maintain average ambient lactate and pyruvate levels of 7.1 +/- 0.8 mg/dl and 0.65 +/- 0.05 mg/dl, respectively, with a lactate/pyruvate ratio in the range 8 to 12. The basal Ca2+(i) of Sertoli cells is 98 +/- 0.7 nmol/L (n = 58), which declines to a level less than 10 nmol/L when the Sertoli cells are perfused with a calcium-free medium. Perfusion of Sertoli cells with a sodium-free medium, with 10(-6) mol/L carbonyl cyanide P-trifluoromethoxy-thenylhydrozone, or with Ca2+ ionophore A23187 at a concentration of 10(-6) mol/L increases the Ca2+(i) to a level of 426 +/- 107 nmol/L, 274 +/- 29 nmol/L, or 282 +/- 57 nmol/L, respectively. A bioreactor for physiologic studies of Sertoli cells in real time with NMR spectroscopy has been developed. These data demonstrate that isolated, immobilized, and perfused Sertoli cells are stable for prolonged periods. In addition, these data suggest that Sertoli cells possess a functional Na+-Ca2+ antiporter and that they sequester extracellular Ca2+ in one or more intracellular compartments.

Phosphorus-31 nuclear magnetic resonance spectroscopy of rat liver during simple storage or continuous hypothermic perfusion

The adenosine triphosphate (ATP) content and intracellular pH (pHi) of isolated rat liver before, during, and after cold preservation in either University of Wisconsin lactobionate solution (UW) (n = 10) or Euro-Collins solution (EC), (n = 8) were monitored with phosphorus 31 nuclear magnetic resonance. The 31P nuclear magnetic resonance spectra were obtained on a 4.7 T system operating at 81 MHz. Fructose metabolism, liver enzyme release, oxygen consumption, and rat survival after liver transplantation were also evaluated. During simple cold storage (SCS) the ATP level declined to undetectable levels with both preservation solutions whereas the pHi declined to approximately 7.0. In contrast, during continuous hypothermic perfusion (CHP), hepatic ATP levels remained measurable during the 24-hour EC preservation and actually increased significantly (p > 0.01) during UW preservation. After reperfusion at 37 degrees C with Krebs lactate, the livers in SCS treated with EC differed significantly from the UW-treated livers in terms of their ATP level and pHi and their response to a fructose challenge. In contrast, livers undergoing CHP demonstrated similar behaviors with both solutions. These results demonstrate an increase in the hepatic ATP content during CHP, which occurs with UW but is not seen with EC. On the other hand, only livers that were simply stored with UW achieved significant survival after transplantation, whereas CHP livers were affected by vascular damage as demonstrated by fatal thrombosis after transplantation. These data suggest that ATP content is not the only determinant of good liver function. A system of hypothermic perfusion might further improve liver preservation efficacy should injury to the vascular endothelium be avoided.