Monolayers Of Human Skin Fibroblasts Research Articles

Kinetic assessment of persistent halogenated xenobiotics in cell culture models. Comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3 h to 14C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 μM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (= low accumulation compounds). DBP and DDE showed much higher cellular accumulations (= high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin

The fibronectin matrix contains cryptic sites which are thought to modulate cellular biological responses. One of these sites, located in fibronectin's first type III repeat (III1c), influences signaling pathways that are relevant to cytoskeletal organization and cell cycle progression. The purpose of this study was to identify possible mechanisms responsible for the effects of III1c on cell behavior. Recombinant peptides representing various type III repeats of fibronectin were compared for their effects on fibronectin matrix organization and activation of intracellular signaling pathways. III1c and III13 but not III11c or III10 bound to monolayers of human skin fibroblasts in a dose- and time-dependent manner and were localized to the extracellular matrix. Binding of III13, but not III1c, to matrix was sensitive to heparitinase, suggesting that the association of III1c with the matrix was not dependent on heparan sulfate proteoglycans. Quantitative and morphological assessment indicated that, in contrast to previously published reports, the binding of III1c to cell layers did not result in the loss or disruption of matrix fibronectin. Binding of III1c but not III13 to the extracellular matrix did result in the loss of a conformationally sensitive epitope present within the EDA type III module of cellular fibronectin. III1c-induced loss of the EDA epitope did not require the presence of cells, occurred within 1 hour and was associated with the activation of p38 mitogen-activated protein kinase (MAPK) followed by the formation of filopodia. Maximal phosphorylation of p38 MAPK occurred within 1 hour, whereas cytoskeletal changes did not appear until 12 hours later. These findings are consistent with a model in which the binding of III1c to the extracellular matrix results in a conformational remodeling of the fibronectin matrix, which has both short- and long-term effects on cell physiology.

Study of a (trimethylenecarbonate-co- ε-caprolactone) polymer—Part 2: in vitro cytocompatibility analysis and in vivo ED1 cell response of a new nerve guide

Future surgical strategies to restore neurological function in peripheral nerve loss may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. Random copolymers of trimethylene carbonate and ε caprolactone (P( εCL-TMC), 50 : 50) have been synthesized by ring opening polymerization using rare earth alkoxides as initiator. Their potential use as nerve guide repairs has been assessed through indirect and direct in vitro biocompatibility tests and in vivo soft tissue response to ED1 subclass macrophages. In vitro, we exposed monolayers of human skin fibroblasts and an established continuous cell line (Hela) to liquid extracts (either pure or diluted in the culture medium) of εCL-TMC copolymer including positive (phenol) and negative controls. Then, colorimetric assays (Neutral red and MTT) were performed. The extracts of εCL-TMC induced no significant cytotoxic effect. We also exposed in vitro Schwann cells to pieces of P( εCL-TMC) and P(LA-GA) copolymers. We evaluated cell attachment at 1 and 3 h by measuring the activity of the lysosomal enzyme ( N-acetyl- β-hexosaminidase) and cell proliferation at 1, 3, 6 and 9 days by measuring the cell metabolic activity (MTT assay). Values for attachment slightly decreased between 1 and 3 h but were significantly higher than on agars (negative control). Cells plated on εCL-TMC showed a rate of proliferation comparable with that of normalized controls and higher than on PGA-PLA at day 9. Finally, we evaluated in vivo the soft tissue response after implantation of cylindrical tubes of P( εCL-TMC) and P(LA-GA) copolymers with an immunohistochemistry staining procedure for the newly recruited ED1 macrophages. An image analysis system automatically measured the optical density of labelled positive ED1 cells at 9, 21 and 60 days after implantation. εCL-TMC copolymer showed a mild soft tissue reaction with no adverse chronic inflammatory reaction. These data allowed us to consider this conduit as a potential effective substitute in nerve repair.

Effect of low-power red light laser irradiation on the viability of human skin fibroblast.

Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm2). The energy densities were 2 J/cm2 and 12 J/cm2, respectively, and the irradiation was carried out at a temperature of 22 degrees C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37 degrees C, in the presence of 5% CO2, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation.

Differential growth characteristics and streptomycin susceptibility of virulent and avirulent Mycobacterium tuberculosis strains in a novel fibroblast-mycobacterium microcolony assay.

The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 microgram/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 microgram/ml. Growth of all strains was similarly inhibited by 1.0 microgram of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of M. tuberculosis.

Modifications of proteoglycans extracted from monolayer cultures of young and senescent human skin fibroblasts

Proteoglycans (PGs) were extracted from culture monolayers of human skin fibroblasts (HFs) at early and late passages. Total PGs from senescent cells had markedly reduced abilities to bind type I collagen and hyaluronic acid, but retained normal binding properties with fibronectin and laminin. The constituent polysaccharides of PGs were comparatively characterised. PGs recovered from young and senescent HF cultures had equivalent total polyanionic charges and similar size distributions of the glycosaminoglycan chains. This applied to both types of polysaccharide chains found in PGs, namely the galactosaminoglycuronans (GalN-GAGs) and the glucosaminoglycuronans (GlcN-GAGs). However, senescent HFs produced a greater proportion of PGs containing GlcN-GAG chains and increased the sulphation of the remainding PG fraction with GalN-GAG moieties, yielding a major gain of C6-sulphate groups in the galactosamine residues.

αvβ5 Integrin Receptor-mediated Endocytosis of Vitronectin Is Protein Kinase C-dependent

Previous studies have demonstrated that the alpha v beta 5 integrin receptor functions in the endocytosis and degradation of matrix-bound vitronectin by human skin fibroblasts (Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11988-11993; Panetti, T. S., and McKeown-Longo, P. J. (1993) J. Biol. Chem. 268, 11492-11495). These earlier studies demonstrated that vitronectin degradation was inhibited by either antibodies to the beta 5 integrin or exogenous heparin, suggesting that both integrin receptors and cell surface heparan sulfate proteoglycans are involved in the endocytosis and degradation of vitronectin. The present study was done to define intracellular signaling pathways involved in endocytosis of vitronectin and to evaluate the relative contribution of cell surface heparan sulfate proteoglycans and the alpha v beta 5 integrin in the activation of these signaling pathways. The addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to monolayers of human skin fibroblasts, increased vitronectin degradation. Staurosporine and calphostin C, inhibitors of protein kinase C, blocked internalization and subsequent degradation of vitronectin, while KT5720, an inhibitor of protein kinase A, had no effect on the degradation of vitronectin. PMA was also able to reverse the inhibition of vitronectin degradation seen when cells were pretreated with heparinase or incubated with exogenous heparin. In contrast, the inhibitory effect of either RGD peptides or anti-alpha v beta 5 antibodies on vitronectin degradation were not overcome by the addition of PMA. These data suggest that the internalization of vitronectin from the matrix is mediated by the alpha v beta 5 integrin following activation of protein kinase C.

Sperm functional changes and fertilization in vitro in co-culture with human skin fibroblasts

This study was undertaken to evaluate the effects of human skin fibroblast monolayers on human sperm function and fertilization in vitro. Sperm function was evaluated using the hamster oocyte penetration assay (HOPA) and zona binding assay (ZBA) in medium alone and in co-culture with human skin fibroblast monolayers and suspensions. The ZBA was also studied in fibroblast conditioned medium and in bovine oviduct cell monolayers and suspensions. Fertilization was measured both in in-vitro fertilization (IVF) couples with a normal semen analysis (first study; randomized) and in IVF couples with subnormal semen analysis (second study; each patient served as its own control). The HOPA results were not significantly different with or without fibroblasts. In all co-culture situations and in conditioned medium the ZBA scored significantly lower than medium alone. No significant differences with respect to IVF were observed between the co-culture and the control group in either study. The mean fertilization rate per patient was approximately 60% in the group with normal semen analysis and approximately 25% in the group with abnormal semen analysis. From this study we conclude that although co-culture with human skin fibroblasts and epithelial cells influences the results of some sperm function tests, it does not influence fertilization in vitro.

The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts.

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.

Acetylated low-density lipoprotein as a vehicle for antiinfectious drugs: preparation and antileishmanial activity of Ac-LDL containing ketoconazole-oleate

Previous studies have demonstrated that mouse peritoneal macrophages take up low-density lipoproteins (LDL) which have been chemically modified by the acetylation of lysine residues (Ac-LDL). This uptake is mediated through a specific receptor known as the scavenger receptor. Ac-LDL therefore appear to have excellent potential for antiinfectious drug targeting. We have developed a new method to incorporate ketoconazole-oleate, a lipophilic derivative of ketoconazole, into Ac-LDL. The method involves solubilization of LDL in the presence of Na deoxycholate, the addition of a micellar solution of the drug to be incorporated, and the subsequent removal of the detergent leading to the formation of reconstituted LDL (referred to as LDL-KOL). The LDL-KOL contain 200 drug molecules per LDL particle and compete for the binding of native 125I-LDL on human skin fibroblast monolayers. Furthermore, reconstituted LDL-KOL are indistinguishable from native LDL with regard to lipid composition and electrophoretic mobility. LDL-KOL, when acetylated, are recognized by the scavenger receptor. Acetylated LDL-KOL show antileishmanial activity when tested on infected macrophages. The activity appears to be mediated through the scavenger-cell pathway, as LDL-KOL are inactive under the same conditions. Moreover, acetylated LDL-KOL are selectively accumulated within infected macrophages rather than in normal cells. This may be of value in the treatment of intracellular infections.

Multiple Factors Influence Insulin-Like Growth Factor-I Binding to Human Skin Fibroblasts*

The binding of [125I]insulin-like growth factor-I ([125I]IGF-I) to human skin fibroblasts (HSF) is regulated by multiple factors. In monolayers of HSF, IGF-I binds to both the type I IGF receptor and IGF-binding proteins (BPs) associated with the cell surface. [125I]IGF-I binding to both of these proteins depends markedly on the sodium chloride concentration of the binding buffer. In monolayers of HSF, replacing 120 mM NaCl with isoosmotic concentrations of sucrose increases binding of [125I]IGF-I by 2- to 6-fold. Enhancement of [125I]IGF-I binding in the absence of sodium chloride is also seen in HSF in suspension, in human erythrocytes, in monolayers of HEP G2 cells and FRTL5 cells, and in membranes prepared from human placentae. Kinetic analysis of [125I]IGF-I binding to HSF monolayers reveals that association rates are increased and dissociation rates are decreased in the absence of sodium chloride. The binding of [125I]alpha IR-3, a monoclonal antibody to the human type I IGF receptor, to monolayers and suspensions of HSF also depends on the sodium ion concentration; it is 5- to 7-fold higher in the absence of sodium chloride. Binding of [125I]IGF-I to monolayers of HSF also depends on NaCl under conditions where alpha IR-3 saturates the type I IGF receptor but does not affect IGF-BPs. These findings demonstrate that sodium chloride has a marked effect on the interaction of IGF-I with the type I IGF receptor in the plasma membrane and with BPs associated with the surface of intact HSFs. Since an effect is also evident in membranes prepared from intact tissues (human placenta), occurs at 4 C, and occurs with cells devoid of BPs, a mechanism involving receptor or BP translocation seems unlikely, at least as the sole explanation for these findings. Sodium ions (and other ions) may induce a conformational change in the receptor and BPs and cause decreased availability of both the IGF-I-binding site and the alpha IR-3 epitope on the receptor and the IGF-binding site on the BP.

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s).

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the "apparent" Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column. Uptake of 125I-LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep-G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep-G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver-derived Hep-G2 cells.

Cyclobutane-type pyrimidine photodimer formation and excision in human skin fibroblasts after irradiation with 313-nm ultraviolet light.

The formation and excision of 313-nm light-induced cyclobutane-type pyrimidine photodimers were determined in confluent cultures of human fibroblasts. A new method was developed for the resolution and determination of cytosine-thymine (CT) and thymine-thymine dimers (TT) by using sodium borohydride reduction and high-pressure liquid chromatography. This assay can detect as little as 1.8 TT or 5.6 CT per 10(8) daltons, levels induced in monolayers of human skin fibroblasts by doses of 1 and 2 kJ m-2 of 313-nm light, respectively. CT formation was 20% more efficient than TT formation in the physiological dose range of 2.25-15 k m-2 at 37 degrees C. Normal fibroblasts removed 61% TT within the first 8 h of incubation following a dose of 5.5 kJ m-2. CT was removed approximately twice as efficiently as TT during the same time period following exposure to 10 kJ m-2. The lack of removal of CT as well as TT observed in xeroderma pigmentosum fibroblasts indicates that the repair deficiency in these cells affects the repair of both classes of dimers.

The role of extracellular pH in the uptake of chloroquine and its effect on mucopolysaccharide metabolism in normal and Hurler's-disease fibroblasts

The preceding paper (Danpure & Patel, 1983) demonstrated the different effects of the lysosomotropic drug chloroquine on mucopolysaccharide metabolism in normal human fibroblasts and those from patients suffering from the mucopolysaccharidosis Hurler’s disease (a-L-iduronidase deficiency). It was noted that there was considerable quantitative inter-experiment variability with respect to chloroquine uptake and its effect on 35S-labelled mucopolysaccharide metabolism, especially in normal fibroblasts. Chloroquine is a weak base and concentrates in lysosomes by proton entrapment (de Duve et al., 1974; Wibo & Poole, 1974; Reijngoud & Tager, 1976), and therefore its uptake is highly dependent on the difference in pH between the lysosomes and the extracellular medium. The pH of tissueculture medium is notoriously difficult to control for any length of time in the presence of actively metabolizing cells (Eagle, 1971), and the present study concerns an investigation into the effect of extracellular pH on the uptake of chloroquine and its effect on 3sS-labelled mucopolysaccharide metabolism in normal and Hurler’s-disease fibroblasts. Monolayers of human skin fibroblasts from normal volunteers and patients suffering from Hurler’s disease were grown to confluence in Ham’s F 10 medium (pH 7.2) supplemented with 15% foetal-calf serum. Before the experiments the medium was replaced with Eagle’s minimal essential medium containing 15% foetal-calf serum. The pH was adjusted by adding appropriate amounts of NaHCO, (Taylor, 1962) and gassed with air/CO, (19 : 1). The cells were labelled with Na25S04 (10pCi/ml, 1 mM) for 3 days (the medium being changed every 24 h), again in the presence or absence of chloroquine. For the chloroquine-uptake experiments [ 14Clchloroquine (Amersham International; 13 nCi/ml, 2 0 , ~ ~ ) was used. At the end of the experiments the cells were washed with phosphate-buffered saline (Dulbecco formula Ca2+and Mg2+free) and harvested by scraping with a ‘rubber policeman’ in this medium, sonicated (IOs, 1 O p ~ amplitude) and assayed for protein (Lowry et al., 1951), and for radioactivity by liquidscintillation counting. The effect of chloroquine on the [35Slsulphate labelling of cellular mucopolysaccharides was found to be markedly dependent on the pH of the medium (Fig. 1). As shown previously (Lie et al., 1972, 1973), in the absence of chloroquine normal cells progressively labelled more cellular mucopolysaccharide as the pH was increased from 6.8 to 7.6. In the presence of chloroquine the optimum pH for the increased labelling in normal cells was 7.2. At pH 7.6 chloroquine had no effect on [35Slsulphate uptake. The effect of pH on Hurler cells was quite different. In the absence of chloroquine there was no clear effect of pH. However, chloroquine slightly increased labelling at pH6.8-7.0, but decreased it rapidly at more alkaline pH values, to reach only 30% of the control value at pH 7.6. The changes in I 35Slsulphate uptake with pH were to some extent paralleled by total cellular protein, so that the plots of [35Slsulphate/mg of protein against pH were almost horizontal. The uptake of [ 14Clchloroquine was also highly dependent on the pH of the medium, especially in normal cells (Fig. 2). In normal cells the uptake of chloroquine increased gradually as the pH of the medium increased from 6.8 to 7.4 (see Polet, 1970), but still remained lower than the equivalent Hurler-cell uptake, which showed little change with pH of the medium. However, at more alkaline pH (7.6), the uptake of chloroquine by normal cells increased dramatically, to extents similar to those in Hurler cells (the 5 h uptake point being greater). The pH optimum for the effect of chloroquine on [35S]sulphate metabolism in normal cells was not directly related to chloroquine uptake. The pH-dependence of chloroquine uptake by normal fibroblasts in the present study is at variance with the observations of Ohkuma & Poole (1981), who showed a pH optimum of 7.0. However, they used macrophages and 100pM-chloroquine, which we have found to be toxic in our