Joseph J. Aleo

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.

Glucose inhibits cellular ascorbic acid uptake by fibroblasts in vitro

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.

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.

Collagen Synthesis in Cultured Cells The Influence of Beta-Aminopropionitrile

Summary The 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.

Inhibition of Ascorbic Acid Uptake by Endotoxin: Evidence of Mediation by Serum Factor(s)

Abstract 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°C for 30 minutes eliminates endotoxins 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

Abstract Ascorbic acid is transported into 3T6 fibroblasts by a carrier-mediated, energy-dependent saturable active process with a Km of 112 μM and V max 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.

An in vitro bioassay of cyanoacrylate cytotoxicity

Abstract 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.

Factors Influencing Calcium Influx in Endotoxin-Challenged Fibroblasts

Abstract 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 μg/ml.

Collagen Synthesis by Lathyrogen-Treated 3T6 Fibroblasts

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.

Activation of Serum Complement Generates Inhibitor of Ascorbate Transport

Ascorbic acid (vitamin C) is transported into 3T6 fibroblasts, which synthesize and secrete collagen, by an energy-dependent saturable active process with a K,,, of 112 pM and V,,,,, of 158 pmol/min/mg protein. The transport is dependent on the extracellular Na+ concentration, which reduces K,,,.’ While studying the ascorbate transport we observed that serum contained a heat-labile factor which, when interacted with bacterial endotoxin, inhibited ascorbate transport? There is a possibility that the endotoxin activated the complement system in the unheated serum and the activation of complement-generated factor that was observed to be inhibitory for ascorbic acid transport. The basis for this suggestion is the endotoxin’s ability to activate complement pathways and the similar heat lability of complement and the inhibitory factor (56°C for 30 minutes). Therefore, we have examined if it is the activation of serum complement that generated the “factor” inhibitory for ascorbate transport. This was done by activation of complement by other activators like zymosan and immunocomplexes (e.g., albumin and antibodies to albumin). This was further investigated by using serum devoid of component 3 of complement (C3). In addition, the effect of inhibitory factor on the kinetic parameters was also examined. Since our initial studies were done with bovine serum, we examined whether sera from different species also contained the inhibitory factor for ascorbate transport. As shown in TABLE 1, sera from different species showed endotoxin-dependent inhibition of ascorbate transport. Guinea pig serum exhibited the maximum inhibition and was therefore used for further studies. The next thing we examined was to see whether activation of serum complement by immunocomplexes leads to inhibition of ascorbate transport. As shown in FIGURE 1, immunocomplexes in the presence of guinea pig serum inhibited ascorbate transport by 3T6 fibroblasts. Immunocomplexes in the absence of serum or in the presence of heat-inactivated serum were ineffective. Then we examined the effect of endotoxin in guinea pig serum devoid of C3 of complement. As shown in FIGURE 2, guinea pig serum devoid of C3 did not show any inhibition of ascorbate transport by endotoxin, unless reconstituted with purified C3. These results clearly demonstrate that activation of complement by endotoxin and immunocomplexes generates a factor that inhibits ascorbate transport. It is not known at which stage of complement activation the inhibitor is generated. However, from the