John L. Farber

The role of calcium in cell death

Abstract Cell death is frequently encountered in human disease. Ischemia, chemicals, viruses, radiation and toxins are among its varied causes. The resulting pathology, however, is very uniform. The common pattern of altered morphology, coagulative necrosis, implies that, at some point, the diverse causes share common mechanisms. Recent evidence suggests that coagulative necrosis may ultimately reflect an alteration in the control of intracellular calcium homeostasis. Studies in intact animals and in cell culture suggest than an ultimate influx of calcium ions across injured plasma membranes and along a steep concentrations gradient converts potentially reversible alterations into the irreversible injury of cell death. The structural alterations in cellular constituents that characterize coagulative necrosis are themselves very likely the direct result of the action of an elevated calcium concentration on cellular macromolecules. The continuing need to identify the membrane alterations induced by the various causes of cell death in order to assess their potential reversibility in the absence of irreversible calcium accumulation is emphasized.

Accelerated phospholipid degradation in anoxic rat hepatocytes

Abstract Accelerated degradation of membrane phospholipids characterizes the reaction of rat liver and myocardial cells to ischemia. A similar disturbance in phospholipid metabolism was sought in anoxic hepatocytes. Primary cultures of adult rat hepatocytes were made anoxic by evacuation of the CO 2 O 2 atmosphere with N 2 . The resulting loss of ATP was reversible upon reoxygenation after periods of anoxia up to 2 h. With 3–4 h of anoxia, the cells were incapable of regenerating ATP levels. Loss of viability was also indicated by the inability of over 90% of the cells after 3–4 h to exclude trypan blue. The baseline rate of turnover of [ 14 C]-ethanolamine or glycerol prelabeled phospholipids was then established. A constant rate of turnover was found for, at least, the first 3 days the cells were in culture. No loss of total phospholipid occurred during this time. Anoxia induced very significant differences in the fate of prelabeled phospholipids. With [ 14 C]-ethanolamine there was a 30% loss of total cellular radioactivity within 4 h. Total phospholipids determined as lipid phosphate decreased by 20%. This depletion of cellular phospholipids was paralleled by an accumulation of hydrophilic degradation products in the culture medium. Phosphorylethanolamine accounted for 50% of these, with equal amounts of glycerophosphorylethanolamine and ethanolamine the other 50%. A similar accumulation in the medium occurred with [ 14 C]-glycerol- and [ 14 C]choline-prelabeled phospholipids. The accelerated degradation of phospholipid was accompanied by evidence of membrane dysfunction as shown by the loss of 50% of the glucose 6-phosphatase activity in whole cell homogenates. The results of these studies establish that anoxia induces in cultured rat hepatocytes a similar disturbance to phospholipid metabolism as does ischemia of the same cells in the intact animal. This implies that the deprivation of oxygen per se determines the characteristic reaction of cells to ischemia. This conclusion allows further analysis of the effects of O 2 deprivation on cultured hepatocytes as a new experimental model with which to further explore the effects of ischemia on cells.

Differential effects of cycloheximide on protein and RNA synthesis as a function of dose

Abstract The in vivo dose response of rat liver protein and DNA synthesis to cycloheximide have been determined. Protein synthesis was quite sensitive to relatively low doses of cycloheximide being inhibited by more than 90% with 1.5 mg/kg. Maximal inhibition of 98% was achieved with 5 mg/kg. There was no inhibition of RNA synthesis with this dose of cycloheximide. Larger doses of cycloheximide did lead to quite marked inhibition of RNA synthesis without any change in the already maximally inhibited rate of protein synthesis. This differential effect of cycloheximide on protein and RNA synthesis as a function of dose indicates that the inhibition of RNA synthesis caused by the antibiotic is not a consequence of the inhibition of protein synthesis but related otherwise to the effects of large doses of cycloheximide.

A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization.

The yield of hybrid colonies after fusion of mammalian cells with polyethylene glycol (PEG) is increased if the cells are fused in Ca2+-free medium, and kept in Ca2+-free medium for at least 15 min after fusion. The protective effect of Ca2+-free medium is much more obvious when Baker PEG is used than when fusion is carried out with Koch- Light PEG. The increased yield of hybrid colonies is shown to be due to a reduced toxicity rather than to an increased efficiency of cell fusion. These improvements have been found to apply to a variety of cell lines, and also when cell fusion is carried out in suspension. This technique should be particularly useful in studies on mammalian cell hybridization using cell lines that are particularly sensitive to the toxic effect of PEG.

Microsomal membrane structure and function subsequent to calcium activation of an endogenous phospholipase

Abstract An endogenous system in the membranes of rat liver endoplasmic reticulum is capable upon Ca 2+ activation of considerable disruption of normal structure and function. Phosphatidylethanolamine (PE) and to a lesser extent phosphatidylcholine (PC) are degraded to hydrophilic products. This lipid loss is greater at an alkaline pH, preferentially utilizes millimolar Ca 2+ rather than Mg 2+ ions, and is inhibited by KCl. Diethyl ether has no effect on the rate of loss of PE or PC, and the Ca 2+ ionophore A23187 does not lower the Ca 2+ requirement. Phospholipids are most likely lost from the membranes in a two-step process. Lysophospholipids generated in the first, Ca 2+ -dependent step are removed by an endogenous lysophospholipase demonstrated by the hydrolysis of either added lyso PE or lysophospholipids generated from endogenous substrates by Naja naja phospholipase A 2 . The depletion of microsomal membrane phospholipid is accompanied by a loss of glucose 6-phosphatase and of cytochrome P -450. The latter is not associated with any change in total heme content. Polyacrylamide gel electrophoresis showed no difference between the pattern or relative amounts of solubilized membrane proteins before or after depletion of membrane phospholipid. It is concluded that activation of an endogenous phospholipase by Ca 2+ can result in significant depletion of PE and PC that is accompanied by considerable disruption of membrane function. The significance of this system with respect to the maintenance of cell integrity and its possible role in cell injury are discussed.

Microsomal membrane dysfunction in ischemic rat liver cells.

Abstract To examine biochemically the effect of ischemia on cellular membranes, microsomal membrane structure and function in ischemic rat liver cells was studied. One-half hour of ischemia produced little or no evidence of histologic cell death 24 h after the reestablishment of blood flow and produced no detectable changes in five separate microsomal parameters measured in vitro. With 2 h of ischemia, histological evidence of liver cell death was quite marked 24 h after reflow had been established, and there were decreases in both microsomal calcium pump and glucose 6-phosphatase activities which could not be explained by differences in relative purity of the samples. Cytochrome P-450 content, glucuronyl transferase activity, and protein composition as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the 2-h ischemic microsomes were similar to those of 0.5-h ischemic preparations. These results indicate the presence of microsomal membrane dysfunction in ischemic rat liver cells temporally related to the onset of irreversible cellular damage. The possible molecular basis of this dyfunction is discussed.

The induction of fibrillar nucleoli in rat liver cells by d-galactosamine and their subsequent re-formation into normal nucleoli

Changes in the fine structure of rat liver cell nucleoli after a single intraperitoneal injection of d -galactosamine are reported. Galactosamine induces extensive fragmentation of nucleoli within 2 hours. This is followed by disappearance of the fragments and the appearance of small, spherical, fibrillar bodies. The altered nucleoli begin to re-form a normal structure between 12 and 24 hours. The reformation seems to involve the fibrillar nucleoli with the appearance of granular material and the formation of small rod-like nucleolonema. The administration of uridine simulataneously with galactosamine completely protects against the induction of nucleolar alterations. Uridine also reverses the galactosamine-induced lesions when given several hours after the galactosamine. These results support the conclusion that the galactosamine-induced disturbance of RNA metabolism and the accompanying nucleolar fragmentation is mediated by the intracellular deficiency of uridine nucleotides. Fibrillar nucleoli like those induced by galactosamine may represent a common morphological manifestation of the absence of ribosomal RNA synthesis.

Control of cellular proliferation in human diploid fibroblasts

SummaryConfluent monolayers of human diploid fibroblasts can be stimulated to synthesize DNA and divide by a change of medium. In the lag period between the application of the stimulus and the onset of DNA synthesis, there occurs a series of biochemical events which include the very early activation of the cell genome. Gene activation requires protein synthesis but not previous RNA synthesis. Experiments suggest that gene activation is dependent upon specific acidic nuclear proteins, whose synthesis is regulated at the post-transcriptional level.

Intracellular calcium homeostasis in galactosamine-intoxicated rat liver cells. Active sequestration of calcium by microsomes and mitochondria.

Abstract Active transport of Ca 2+ by isolated microsomes and mitochondria from galactosamine-intoxicated rat liver cells was studied. The aim was to determine the respective role of each organelle in the disturbed intracellular Ca 2+ homeostasis induced by this hepatotoxin. Calcium uptake by isolated microsomes is ATP dependent and oxalate augmented with a V of 1.45 nmol of Ca 2+ /mg of microsomal protein/mm at 25 °C and an apparent K m for free Ca 2+ of 2.4 μ m . Concentrations of total Ca 2+ higher than 40 μ m are inhibitory. Two hours after administration of galactosamine (200 or 400 mg/kg), at a time when the total cell Ca 2+ content has increased, microsomes isolated from the treated animals exhibited no impairment in calcium transport. The microsomal preparations from the galactosamine-treated animals also had the same content of cytochrome P -450 and the same specific activity of glucose 6-phosphatase as those from the control animals. Calcium uptake by isolated liver mitochondria is also ATP dependent but virtually completely inhibited by 5 m m sodium azide. The V is higher than that of the microsomes, 20.5 nmol of Ca 2+ /mg of protein/min at 25 °C, but the apparent K m for free Ca 2+ is similar, 5.7 μ m There was no alteration in the Ca 2+ uptake activity of mitochondria isolated from galactosamine-treated animals. These results imply that the initial disturbance in intracellular Ca 2+ homeostasis induced by galactosamine is entirely a consequence of the previously described plasma membrane injury. The potential significance of the observed kinetic properties of microsomes and mitochondria with regard to their respective roles in intracellular Ca 2+ homeostasis is discussed.

Increases in the activity of the solubilized rat liver nuclear RNA polymerases following partial hepatectomy

Abstract Nuclei isolated 18 h following partial hepatectomy in the rat synthesize RNA in vitro at twice the rate of liver nuclei from sham-operated animals. This increased RNA synthesis is accompanied by an increase in the α-amanitin-insensitive proportion of the total activity. Twice as much RNA polymerase activity was solubilized from nuclei from hepatectomized animals as from sham-operated animals. Fractionation of this solubilized RNA polymerase activity on DEAE-Sephadex showed increases in the activity of both polymerase I and polymerase II, with a proportionally greater increase in polymerase I. Such an increase in RNA polymerase activity independent of the endogenous chromatin template was also demonstrated by inhibiting the function of the chromatin template in whole nuclei with actinomycin D and then assaying the polymerase activity with poly[d(A-T)]. It is concluded that increases in the activity of the RNA polymerases themselves can account entirely for the changes in the rates of RNA synthesis 18 h following partial hepatectomy.