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.     

Toxicity of N-(3,5-dichlorophenyl)succinimide and metabolites to rat renal proximal tubules and mitochondria

The acute nephrotoxicity caused by N-(3,5-dichlorophenyl) succinimide (NDPS) has been shown to be due to a metabolite(s) of the parent compound. This study examined the toxicity of NDPS, its known metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), N-(3,5-dichlorophenyl)malonamic acid (DMA), N-(3,5-dichlorophenyl)succinamic acid (NDPSA), and two postulated metabolites N-(3,5-dichlorophenyl)maleamic acid (NDPMA) and N-(3,5-dichlorophenyl)maleimide (NDPM) to suspensions of renal proximal tubules (RPT) prepared from male Fischer 344 rats. Tubule viability and mitochondrial function were not adversely affected by exposure of RPT to either 1 mM NDPS, NDHS, NDHSA, DMA, NDPSA, or NDPMA for 4 h. However, NDPM caused a concentration-(25-100 microM) and time-dependent (0.25-4 h) loss in basal and nystatin stimulated oxygen consumption and tubule viability. Investigations using isolated renal cortical mitochondria (RCM) showed that NDPM was a potent inhibitor of mitochondrial function. Isolated RCM respiring on pyruvate/malate and exposed to NDPM exhibited a concentration (25-100 microM) dependent decrease in state 3 and state 4 respiration. Inhibition of mitochondrial state 3 respiration by NDPM was mediated through site 1 of the respiratory chain. NDPM did not inhibit cytochrome c-cytochrome oxidase or the electron transport chain. These results indicated that NDPS, its known metabolites, and NDPMA were not directly toxic to rat RPT. However, the postulated metabolite NDPM, was a potent tubule cytotoxicant that inhibited mitochondrial function in isolated RCM and RPT and may produce cell death through this mechanism.     

Factors influencing calcium influx in endotoxin-challenged fibroblasts

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.