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The uptake of Ca2+ by endotoxin-challenged 3T6 fibroblasts, in vitro, was studied. In recent years, the role of calcium in cell injury ultimately leading to cell death has attracted a fair amount of interest. The purpose of the study was to determine whether the direct toxic action of endotoxin is related to a disturbance in Ca2+ homeostasis. Increased calcium uptake in endotoxin-challenged cells was found to be directly related to the bacterial source and method of extraction of endotoxin, the cell density of the culture and the pH of the medium. The effect of endotoxin on calcium uptake was completely reversed by polymyxin B which is known to neutralize the endotoxicity of lipopolysaccharides. These results imply that the increased calcium uptake may be one of the mechanisms by which endotoxin causes direct tissue damage. The potential significance of these data to periodontal disease is discussed.

Ascorbic acid is transported into 3T6 fibroblasts by a carrier-mediated, energy-de- pendent saturable active process with a K, of 1 12 pM and V,,, of 158 pmole/min/mg protein. The transport is dependent on extracellular Na' concentration which reduces the K,,,. It was recently observed in this laboratory that bovine serum contained a heat-labile factor which, after interaction with bacterial endotoxin (lipopolysaccharides), inhibited ascorbic acid transport (J. J. Alleo and H. Padh, Proc SOC Exp Biol Med 179: 128-1 3 1, 1985). We report here that the inhibition of ascorbic acid transport by endotoxin is mediated by the activation of serum complement. This was done by examining the activation of complement by other activators like zymosan and immunocomplexes (e.g., albumin and antibodies to albumin). Ascorbate transport was inhibited by the mixture of unheated serum and the activators. No inhibition was observed with serum devoid of C3 (component 3 of the complement). When C3-deficient serum was reconstituted by the addition of purified C3, the endotoxin-induced inhibition of ascorbate transport was restored. The implication of these findings is that in spite of a normal intake and blood level of the vitamin, tissues may not be getting adequate vitamin C during disease states when the complement in serum is activated. In other words, what may be considered an adequate intake of vitamin C under health conditions may not be adequate under disease conditions. o 1987 Society for Experimental Biology and Medicine.

Previous studies have shown that cementum-bound endotoxin or endotoxin-like products from untreated periodontally involved teeth depress the proliferation of, and have direct toxic effects on, cultured fibroblasts. The purpose of this investigation was to study the effect(s) of ascorbate and several other compounds on the toxicity of commercially prepared endotoxin. The 3T6 fibroblasts were grown in Dulbecco—Vogt modification of Eagles' medium containing 10% heat-inactivated calf serum. Replicate cultures were prepared and grown for 5 days. The proliferation of endotoxin-challenged cells was monitored in the presence of either l-ascorbate, d-isoascorbate, dehydroascorbate, carbonylcyanide m-chlorophenylhydrazone, or cycloheximide. Under very specific conditions, ascorbate reversed the endotoxin-mediated depression of cellular proliferation. An uncoupler of oxidative phosphorylation, carbonylcyanide m-chlorophenylhydrazone also effectively reversed the effect of endotoxin while cycloheximide had no...

It has been suggested earlier that the local deficiency of ascorbic acid in tissues could be responsible for development of various angiopathies in diabetes. Hyperglycemia is one of the factors which could contribute considerably to the development of local ascorbic acid deficiency. Therefore, the effect of glucose on uptake of L-[1-14C] ascorbic acid by fibroblasts was studied in vitro. The data clearly show that ascorbic acid uptake is inhibited instantly by glucose in a concentration dependent fashion. The results support the contention that local ascorbic acid deficiency in tissues could be a natural consequence of hyperglycemia of whatever cause. The rate of ascorbic acid uptake under various conditions suggests that additional supplements of ascorbic acid might be helpful to individuals in averting deleterious effects of hyperglycemia on tissue ascorbic acid supply.

Polymyxin B sulfate treatment induced an increase in calcium influx in mouse fibroblasts (3T6) and normal human skin fibroblasts. This increase in calcium influx occurred in a dose- and time-dependent fashion and was dependent on pH but independent of the electrochemical gradient of calcium across the plasma membrane. This effect was prevented when cycloheximide (20 micrograms/ml) was added with polymyxin B sulfate. Addition of actinomycin D (2 micrograms/ml) also remarkably reduced this effect. In view of these findings, it is possible that polymyxin B sulfate therapy-induced side effects, such as neuromuscular blockade and kidney dysfunction, are conditional and may be due to an increase in calcium influx.

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.

The intracellular localization in 3T6 fibroblasts of Escherichia coli lipopolysaccharide (LPS) using the rapid avidin-biotin-immunoperoxidase technique at the electron microscopic level was studied. The role of bacterial endotoxin in the etiology of periodontal disease has been well documented previously. The purpose of the present study was to localize LPS within the cell, thereby determining which organelles concentrate the material and relate this to the cytologic pathophysiology. An increased concentration of LPS was found in the cell nuclei and, specifically, in association with nuclear chromatin and nucleoli. The concentration of LPS in the nucleus was directly related to the time of incubation, with some product appearing in that site within 2 minutes. There was no specific localization of endotoxin in mitochondria, lysosomes, Golgi, endoplasmic reticulum or ribosomes. These results imply that bacterial endotoxin may have a direct effect on nuclear components of fibroblasts. The relationship of these results to the etiologic mechanisms of periodontal disease is discussed.

An hypothesis is proposed relating the possible role of vitamin deficiency as an etiologic factor contributing to periodontal disease in diabetes. The hypothesis is based upon the following: (1) transport of ascorbate across cell membranes may be impaired by glucose, but facilitated by insulin; (2) glucose utilization is significantly accelerated by sublethal concentrations of endotoxin; (3) endotoxin-induced histamine sensitivity of tissue is enhanced by ascorbic deficiency; and (4) ascorbic acid deficiency alters mucosal barrier function. The interrelationship of these factors is discussed.

Enterobacter aerogenes was examined for its ability to utilize a known lathyrogen or chemically related compounds for use in developing an in vitro model system to study experimental lathyrism. Enterobacter aerogenes grew in a minimal medium containing the lathyrogen, beta-aminopropionitrile fumarate. Related compounds or combinations of related compounds which together contained all the constituents of the lathyrogen failed to support growth. The specificity of this compound in supporting growth of this polysaccharide-producing microorganism suggests that this microorganism may be useful in developing an in vitro model system to study polysaccharide metabolism in experimental lathyrism.