Characterization of the ascorbic acid transport by 3T6 fibroblasts.

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.

Diabetes and periodontal diseases. Possible role of vitamin c deficiency: an hypothesis.

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.

Intracellular localization of bacterial lipopolysaccharide using the avidin biotin complex method at the electron microscopic level.

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.

Possible role of calcium in periodontal disease.

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.

In vitro attachment of human gingival fibroblasts to root surfaces.

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.

Histochemical study of strain L fibroblasts exposed to endotoxin. The effect on cellular organelles.

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.

Stimulation of macromolecular synthesis by endotoxin-treated 3T6 fibroblasts.

The interaction of endotoxin with cultured fibroblasts resulted in a depression of cellular proliferation and an increased synthesis of macromolecules, namely collagenous and non-collagenous proteins. The collagen salt-soluble fraction was increased at the expense of the insoluble fraction, and both the salt-soluble fraction and collagen secreted into the medium was underhydroxylated.

On the possible mechanism of cyanoacrylate histotoxicity.

The loss of tissue fluid as a possible mechanism of cyanoacrylate histotoxicity was studied in vitro. There appears to be no statistical difference in the rates of fluid removal between methyl, isobutyl and N-octyl 2-cyanoacrylates, nor is there any correlation between fluid removed and the observed in vivo tissue damage. It is suggested that tissue fluids play a dual role in initiating polymerization and in polymer degradation; and that tissue damage depends upon the rate of polymer decomposition with the formation of toxic degradation products.

Inhibition of ascorbic acid uptake by endotoxin: evidence of mediation by serum factor(s).

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.

Activation of serum complement leads to inhibition of ascorbic acid transport.

Ascorbic acid is transported into 3T6 fibroblasts by a carrier-mediated, energy-dependent saturable active process with a Km of 112 microM and Vmax of 158 pmole/min/mg protein. The transport is dependent on extracellular Na+ concentration which reduces the Km. 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 179:128-131, 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.

In vitro lipid synthesis by lathyrogen-treated L-929 fibroblasts.

Aside from cholesterol, cholesterol esters and lyso-lecithin, the de novo lipid synthesic mechanisms which operate in cells grown in the presence of beta-amino-propionitrile are largely depressed and suggest that there may be in operation specific metabolic control mechanisms for regulation of cellular lipid composition.

Ascorbic acid transport by 3T6 fibroblasts. Regulation by and purification of human serum complement factor.

It was earlier reported (Padh, H., and Aleo, J. J. (1987) Proc. Soc. Exp. Biol. Med. 185, 153-157) that the activation of serum complement by endotoxin or immunocomplexes inhibited ascorbate transport in 3T6 fibroblasts. We show here that the inhibitor of 3T6 fibroblasts. We show here that the inhibitor of ascorbate transport increased the Km for ascorbate without affecting the Vmax, indicating that the inhibitor reduces the affinity of the ascorbate transporter for ascorbate without affecting the process of translocation. Inhibition by serum and endotoxin was reversible, and the generated inhibitor was no longer heat-labile (at 56 degrees C for 30 min) suggesting that the inhibitor of ascorbate transport is likely to be a small protein molecule. Utilization of complement components suggested that C3 was consumed during formation of the inhibitor of ascorbate transport while C5 and factor B were not consumed. These data along with other results indicate that the inhibitor is generated at C3 step of complement activation. The inhibitor was purified from inulin activated human serum and it had an apparent molecular mass of around 9000 daltons. The inhibitory effect of the purified factor was abolished by antiserum to C3a suggesting that the 9000-dalton factor could be related to this fragment of complement protein. These data raise the possibility that tissue supply of ascorbate may be compromised during infection or autoimmune processes when serum complement is activated.