Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts.

The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.     

Uptake and degradation of virus-associated fluorescent sulfatide by skin fibroblasts

A fluorescent derivative of cerebroside sulfate, pyrene-dodecanoyl sphingosylgalactosylsulfate (P12-CS) was incorporated into the envelope of vesicular stomatitis virus (VSV). When the P12-CS-containing virus was incubated for 24 h with skin fibroblasts, up to 40% of the sulfatide was located in the cells--a value at least 10 fold greater than that observed using sulfatide suspensions without virus. In a similar experiment, in which the 24 h 'pulse' was followed by a 48 h 'chase', about 15-20% of the virus-associated fluorescence was recovered in the skin fibroblasts. Of the latter, about one-third was present as desulfated degradation products of P12-CS. The high uptake and degradation of the virus-associated sulfatide by intact skin fibroblasts suggested that enveloped viruses could be used for introducing other lipids into cells. This could be utilized for studying lipid catabolism and diagnosing lipid storage disorders in intact living cells.     

Uptake of retinol by cultured fibroblasts

Cultured fibroblasts of adult rats were used to determine whether they could take in retinol administered to the culture medium at physiological concentration. After the administration of retinol, cells were observed with a phase-contrast fluorescence light microscope (LM) and a transmission electron microscope (TEM). Retinol and retinyl fatty acyl esters (RFAE) stored in the cells were analyzed with high-performance liquid chromatography (HPLC). It was revealed that these fibroblasts could take in retinol in the medium at a concentration of 1 x 10(-7) M and store it in lipid droplets in the cytoplasm as retinyl palmitate and other RFAE.     

Inactivation by chloroquine of alpha-galactosidase in cultured human skin fibroblasts

When human skin fibroblasts are cultured in the presence of chloroquine or NH₄Cl there is a decrease in the intracellular level of lysosomal hydrolases and a concomitant increase in the extracellular activity as compared with cells grown in the absence of a base (cf [18]). In a medium with 25 μM chloroquine or 5 mM NH₄Cl, the decrease in the intracellular activity of β-hexosaminidase, arylsulphatase and β-glucuronidase is 10–40% after 1 day. A similar decrease in α-galactosidase activity is observed in cells grown in the presence of 5 mM NH₄Cl. However, in the presence of 25 μM chloroquine, the intracellular activity of α-galactosidase decreases by 80–90% within 6 h. The inactivation is irreversible. After removal of the chloroquine and further culture of the cells in chloroquine-free medium, α-galactosidase activity gradually increases due to de novo synthesis. The turnover time of α-galactosidase was calculated to be 1.9 days. Inactivation of α-galactosidase also occurs when homogenates are incubated with chloroquine, but the concentration of the base required for maximum inactivation is at least three orders of magnitude higher than that which must be present in the medium of intact cells to obtain the same effect.     

Uptake of 2,3,7,8-tetrachlorodibenzo-p-dioxin from plasma lipoproteins by cultured human fibroblasts

Tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to human plasma in vitro associated with the plasma lipoproteins. The effects of plasma and lipoproteins on cellular uptake of dioxin were studied using normal human skin fibroblasts and mutant fibroblasts from a patient with homozygous familial hypercholesterolemia. The latter cells lack the normal cell membrane receptor for low density lipoprotein (LDL). The time- and temperature-dependent cellular uptake of [3H]dioxin was greatest from LDL, intermediate from high density lipoprotein (HDL) and least from serum. A significantly greater uptake from LDL by the normal cells compared to the mutant cells indicated the involvement of the LDL receptor-mediated pathway. Concentration-dependent studies indicated that the cellular uptake at 37 degrees C of [3H]dioxin varied linearly with dioxin concentration at constant LDL concentration. Thin-layer chromatography (TLC) showed that conversion to more polar compounds may have occurred after 24-h incubation with cells. [3H]Dioxin could be removed from cells efficiently by incubation with 20% serum greater than HDL greater than LDL. Since the vehicle of delivery may influence subsequent location and metabolism of this compound in cells, it is concluded that the physiologic vehicles (either serum- or LDL-associated dioxin), rather than organic solvents, should be used in experiments with cultured cells or perfused organs.     

Transport of hypoxanthine by human diploid skin fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase

Transport of purine bases and nucleosides by a variety of mammalian cell lines is generally accomplished by facilitated diffusion, a non-concentrative, saturable process. However, previous investigators have been unable to detect a saturable component for the transport of hypoxanthine by human fibroblasts deficient in hypoxanthine-guanine phosphoribosyltransferase, implying that in normal cells the enzyme actively participates in transport. In the present study we have used the phenomenon of countertransport to demonstrate the existence of a saturable transport mechanism in hypoxanthine-guanine phosphoribosyltransferase-deficient human diploid skin fibroblasts.     

Receptor-dependent uptake of human chylomicron remnants by cultured skin fibroblasts

Human chylomicrons were isolated from plasma from a subject with familial hypertriglyceridemia and converted to chylomicron remnants by incubation with postheparin plasma. The interaction of these apolipoprotein E-containing, cholesterol-rich human chylomicron remnants with cultured skin fibroblasts was studied. Chylomicron remnants were internalized by skin fibroblasts as a unit, mainly via the low density lipoprotein (LDL)-receptor pathway, resulting in increased cell cholesterol content. After entering the fibroblast, chylomicron remnants stimulated cholesterol esterification, suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and down-regulated LDL receptor activity similar to the action of LDL. As a function of increasing lipolysis, remnant particles were progressively more effectively taken up by skin fibroblasts, despite a decrease in the apolipoprotein E content per lipoprotein particle. Remnant particles produced after hydrolysis of 70 to 80% of chylomicron triglyceride increased cell cholesterol content to an amount nearly identical to that observed with LDL when the two lipoproteins were incubated at an equal cholesterol concentration. However, when incubated on the basis of equal particle number, chylomicron remnants were 2 to 3 times more effective than LDL in delivering cholesterol to the cells. These results suggest that chylomicron remnants play a role in the regulation of postabsorptive cholesterol homeostasis in nonhepatic cells, and possibly in the pathogenesis of atherosclerosis.     

Phospholipase C activity in cultured human skin fibroblasts

In this paper we report the detection of phospholipase C activity in cultured human skin fibroblasts by a rapid, sensitive method. Sonicates of fibroblasts were incubated with L-3-phosphatidyl-[U-14C]-inositol and the incubation mixture extracted with chloroform/methanol. The solvent components were then separated into 2 phases by the addition of 2 M KCl. Phospholipase C activity, determined from the amount of [14C] in the aqueous phase, agreed well with the enzyme activity assessed by other methods. The optimum pH for the enzyme was 7.0 and the enzyme was found to be dependent on Ca2+ and deoxycholate for optimal activity. The demonstration of phospholipase C activity by this method in cultured skin fibroblasts provides a useful means with which to study, in human tissues, the physiological control of this enzyme and its derangements in disease states in a controlled fashion.     

Absence of hyaluronidase in cultured human skin fibroblasts.

The in vitro degradation of [³⁵S]chondroitin sulfate was investigated in human fibroblasts and rat liver. In rat liver, preparations of chondroitin sulfate were shown to be degraded by the concerted action of endoglycosidase and exoglycosidases. However, with human skin fibroblast preparations, hyaluronidase activity was not detected and chondroitin sulfate was degraded by exoglycosidase action.     

Sericin enhances attachment of cultured human skin fibroblasts

Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles.     

Stimulation of hexose transport in cultured human skin fibroblasts by insulin.

The effect of insulin on hexose transport in cultured human skin fibroblasts. Studies were carried out on cultures of human skin fibroblasts to explore the effect of insulin on hexose transport in serum-starved monolayers. Insulin (100 mU/ml) stimulated 2-deoxy-D-glucose transport (30% above control values) after 30 minutes exposure time, the response being similar up to four hours exposure to insulin. In several experiments (n = 22) employing three cell strains, insulin (100 mU/ml) exposure led to variable stimulation of 2-deoxy-D-glucose transport (an average of 37% above control values, with a range of 0 = 120%). The insulin-induced stimulation of 2-deoxy-D-glucose transport showed a dose dependency with increasing amounts of insulin, the response being maximal at an insulin concentration of 100 mU/ml. Kinetic analysis of 2-deoxy-D-glucose transport showed that insulin addition resulted in a slight change in the transport K_m (3.13 to 4.06 mM) and a 1.8-fold increase in the transport V_max (17.6 nanomoles/mg protein/min to 32.1 nanomoles/mg protein/min). Insulin also stimulated the transport of 3-0-methyl-D-glucose while the hexokinase activity of the cells was not affected. Further, this insulin-induced stimulation of sugar transport was not blocked by cycloheximide. The results indicate that insulin stimulated the stereospecific carrier-mediated of hexose transport in cultured human skin fibroblasts.     

Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts

Trypsin released from the surface of intact human skin fibroblasts β-N-acetylglucosaminidase. The amount of trypsin removable β-N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β-N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β-N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β-N-acetylglucosaminidase, α-N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β-N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β-N-acetylglycosaminidase, α-N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface.     

Glucose transport and metabolism in cultured human skin fibroblasts

Human skin fibroblast cultures, seeded at $\text{10}{}^5\text{cells}\text{5}\text{cm}$ plate and allowed to grow to confluence at approx. $\text{10}{}^6\text{cells}\text{5}\text{cm}$ plate, utilized a glycolytic mode of metabolism where the ratio of glucose utilized to lactate produced wa 0.62±0.05 (Zielke, R.H., Ozand, P.T., Tyldon, J.I., Sevdalian, D.A. and Cornblath, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4110–4114) (mean±S.E.). When the glucose in the medium was exhausted, the lactate produced during the highly glycolytic phase was then reutilized. In monolayer cultures that had been washed with phosphate-buffered saline, rates of glucose utilization were measured at 0.25 and 2 mM glucose by monitoring the appearance of ³H₂O from [5-³H]glucose. Rate of utilization for each concentration of glucose decreased markedly as the cultures became more confluent. This decrease also correlated with a reduced ability to transport glucose as measured by 2-deoxy-[³H]glucose uptake in washed monolayer cultures. In washed confluent culture of fibroblasts, glucose utilization was markedly decreased by the presence of pyruvate and lactate but not by glutamine. The respiratory inhibitors, rotenone and antimycin, did not increase the rate of glucose utilization except when added in combination with pyruvate. We conclude that cultured skin fibroblasts posses a highly glycolytic mode of metabolism but that this mode can become more oxidative in the presence of sufficient quantities of pyruvate and lactate.     

Phytanic acid alpha-oxidation in human cultured skin fibroblasts.

We studied the oxidation of [1-14C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts. The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.     

Hexosaminidase a deficient adults: Presence of α chain precursor in cultured skin fibroblasts

Cultured skin fibroblasts from hexosaminidase A deficient adults synthesize the α and β chain precursors of β-hexosaminidase (EC 3.2.1.30) of the same molecular weight as that synthesized by normal fibroblasts. However, the amount of the α chain precursor is greatly reduced. The α chain precursor in secretions from these fibroblasts consists of 19% of the total β-hexosaminidase secreted compared to about 50% in normal cells. Attempts to increase the amount of detectable cellular α chain precursor by addition of protease inhibitors or by more extensive extraction methods have failed. Mature α chains were not detected. The presence of α chain precursor in fibroblasts from hexosaminidase A deficient adults can be used to distinguish between them and true Tay-Sachs disease homozygotes.