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Ascorbic acid transport by 3T6 mouse skin fibroblasts has been characterized using radiometric technique with L-[1-14C]ascorbic acid under the conditions in which oxidation of ascorbic acid was prevented by addition of 1 mM thiourea. The ascorbate transport is temperature-dependent with the energy of activation E and Q10 of 13.3 kcal/mol and 2.0, respectively. The transport requires energy and exhibits Michaelis-Menten kinetics with an apparent Km of 112 microM and Vmax of 158 pmol/min per mg protein, when the extracellular Na+ concentration is 150 mM. The ascorbate transport requires presence of extracellular Na+ and can be inhibited by ouabain treatment. At 40 and 200 microM ascorbate concentrations, respectively, 1.4 and 1.0 moles of Na+ bound the transporter molecule per each mole of ascorbate transported. Increased Na+ binding to the transporter at lower ascorbate concentration may signify multiple Na+-binding sites or ascorbate concentration dependent conformational changes in the transporter molecule. Increasing Na+ concentration decreases Km without affecting Vmax, suggesting that Na+ increases affinity of ascorbate for the transporter molecule without affecting translocation process. An increase in ascorbate concentration reduces the number of Na+ bound to the transporter from 1.4 to 1.0. The ascorbate transport is stimulated by Ca2+ and other divalent cations. The mechanism of stimulation by Ca2+ is not clear. Calcium increases both the Km and Vmax. The data presented support the hypothesis that the ascorbate transport by 3T6 fibroblasts is an energy and temperature-dependent active process driven by the Na+ electrochemical gradient. A potent inhibitor of ascorbate transport is also demonstrated in human serum.

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.

The role of cell density and pH on calcium influx was studied in normal and endotoxin-challenged cultured 3T6 fibroblasts. In normal fibroblasts, at low cell densities, there was no marked difference in calcium influx at pH 6.6, 7.4, and 7.8, whereas at high cell densities, the calcium influx was markedly higher at pH 6.6 as compared to that at pH 7.8. Endotoxin treatment for 4 hr at low cell density and in alkaline pH (7.4-7.8) increased calcium influx in a dose-dependent manner. In contrast, at high cell density and low pH (6.6), endotoxin treatment markedly decreased calcium influx in a dose- and time-dependent manner. These endotoxin-induced changes in calcium influx were not fully compensated by altered calcium efflux because total calcium content of the cells was found to be altered. The efficacy of the endotoxin varied depending on the bacterial source of the endotoxin and the method of purification. There was a relationship between the effect of different endotoxins on the increase in calcium influx and the inhibition of cell proliferation. Endotoxin did not decrease, but slightly increased cell proliferation when added to high cell density cultures even at a concentration of 200 micrograms/ml.

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.

Human gingival fibroblasts were used to study the in vitro attachment of cells to the root surface of periodontally-involved teeth. The portion of the root exposed to the disease process had little or no cell attachment; on the remainder of the root, the cells attached normally. Prior extraction of the roots with phenol-water or the mechanical removal of diseased cementum allowed the cells to attach normally. All things being equal, the extrapolation of these data to an in vivo situation dictates that a clinical success would depend upon complete removal of toxic materials from diseased cementum or the removal of the cementum itself.

Perfusion of isolated intestinal loops has been utilized in the management of uremia, although the technique has been limited by an inability to adequately extract creatinine. In a recent study of uremic rats, the addition of sodium deoxycholate (NaDC) to the perfusate increased clearance of sodium and creatinine sevenfold. This investigation was designed to confirm this effect in canines and to determine if increased creatinine excretion can be maintained in the absence of net water flux. Isolated jejunal loops of 38 to 75 cm in length were created in conditioned canines. Following a 2-week recovery period, loops were perfused with control solutions (Na 60 meq/liter, K 8 meq/liter, Cl 65 meq/liter, polyethylene glycol 5 g/liter, adjusted osmolarity 360 mosm/liter), and experimental solutions of comparable composition and osmolarity including 2 mM NaDC. NaDC perfusion increased loop creatinine excretion four- to sevenfold allowing clearances of up to 79 mg of creatinine/hr. This increase was statistically significant (P < 0.01) when compared to control perfusions (control 6.00 ± 5.16 mg/hr vs NaDC 37.54 ± 16.61 mg/hr). No net water flux and minimal electrolyte flux were noted. Longer loops required higher perfusion rates to maximize creatinine excretion; optimal perfusion rates approximated 40 ml/hr/cm. Bile salts significantly increased creatinine secretion from isolated jejunal loops without obligatory electrolyte or water movement. Addition of such local agents may improve the efficiency of intestinal dialysis and allow reconsideration of clinical use.

The effect of bacterial endotoxin on the ascorbic acid uptake by 3T6 fibroblasts was studied. Endotoxin inhibited ascorbic acid uptake by fibroblasts in a dose dependent manner. The inhibition by endotoxin takes place only in the presence of unheated serum; decomplementing serum by heat inactivation at 56 degrees C for 30 minutes eliminates endotoxin's inhibitory effect on ascorbic acid uptake. The effect of endotoxin appears to be instantaneous since the inhibition seen in the cells without any preexposure was similar to the cells preexposed to endotoxin for up to 6 hours. Polymyxin B sulfate which is known to bind the lipid A portion of endotoxin did not reverse the inhibition of ascorbic acid uptake caused by endotoxin.

The effect of sodium salicylate on the metabolism of 3T6 fibroblasts treated with the lathyrogen β-aminopropionitrile was studied. A 5 mM concentration of the lathyrogen and/or 0.5 mM sodium salicylate was used to study proliferation, collagen synthesis, and mucopolysaccharide synthesis as measured by 35SO4 incorporation. Sodium salicylate was able to reverse all lathyrogen-mediated changes to control or near control values. The diversity of these biosynthetic activities suggests that the influence of sodium salicylate on lathyrogen-treated cells is a result of a general metabolic effect rather than a specific one; the exact mechanism of action remains unknown.

Endotoxin was found to be nonselective in its cytotoxicity of cellular organelles. All organelles studied displayed some degree of alteration which became more severe as the concentration of endotoxin was increased. The data suggest that the metabolism of the cell may be compromised at concentrations of endotoxin which do not affect cell viability. The problem involved in attempting to determine the actual concentration of endotoxin at the interface between the culture medium and the cell monolayer, in vitro, and the corresponding situation, in vivo, are also discussed.

The lathyrogen, beta-aminopropionitrile (BAPN), in ascorbate-free medium increased the synthesis of both collagenous and non-collagenous proteins in 3T6 fibroblasts. Cell layer collagen was underhydroxylated and more easily extracted with neutral salt in treated cultures. The ratios of proline: hydroxyproline in sequentially extracted collagen in the cell layer of both BAPN and control cultures showed virtually no difference in the acid soluble and insoluble fractions; however, the BAPN salt-soluble fraction was definitely underhydroxylated when compared to control cultures. The data suggest that the salt-soluble macromolecule represents, in addition to a cross-linking defect, an underhydroxylated intracellular collagen precursor.

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.

SummaryThe exposure of 3T6 fibroblasts to the lathyrogen β-aminopropionitrile fumarate caused a reduction in proliferation of the cells but had no effect upon viability. Chemical analysis for the production of collagen as measured by free, peptide, and protein-bound hydroxyproline showed that the lathyrogen exerted a stimulative effect. The lathyrogen-treated cells produced approximately twice the amount of hydroxyproline as did the control cells. The data also indicate that the initial stages of collagen production are not altered and that the depression in proliferation signifies a change in overall metabolism rather than a direct toxic effect.

The purpose of this investigation was to compare the relative cytotoxic effects of methyl 2-cyanoacrylate, isobutyl 2-cyanoacrylate, and n-octyl 2 cyanoacrylate. Filter paper disks were saturated with these monomers and placed on monolayers of L-929 fibroblasts. Plates containing the cells and disks were incubated and observed at various intervals. zones of cytotoxicity appeared first around the methyl-saturated disks. The cytotoxic response was greatest to the methyl-saturated disks and less severe to the isobutyl and octyl disks. This is in agreement with previous in vivo investigations.