Effects of lysosomotropic agents on lipogenesis

Chloroquine, quinine, and NH4Cl are lysosomotropic agents which inhibit lysosomal function, apparently by raising the intralysosomal pH. We found that preincubation of cultured human skin fibroblasts with these lysosomotropic agents under serum-free conditions induced about a 10-fold stimulation of lipogenesis. A similar stimulatory effect on the incorporation of 3H2O, [14C]acetate, [14C]pyruvate, [14C]palmitate, and [14C]choline into cellular lipids was observed. The effect was both time and dose dependent, and was reversible. The concentrations of chloroquine, quinine, and NH4Cl resulting in half-maximal stimulation were about 3 microM, 30 microM, and 9 mM, respectively. At these concentrations, stimulation of lipogenesis correlated with impairment of lysosomal function. At a concentration of 10 microM chloroquine, the half-time for maximal stimulation was about 4 h. Most of the [14C]acetate was incorporated into phosphatidylcholine and other cellular lipids; less than 10% was found in cholesterol and cholesterol ester. Nevertheless, incorporation of [14C]acetate into cholesterol showed a chloroquine-induced stimulation parallel to that observed for phospholipids, suggesting that stimulation of both lipogenesis and cholesterogenesis occurred. The stimulatory effect of lysosomotropic agents on lipogenesis appeared to depend on active synthesis of cellular proteins. In the presence of cycloheximide, an inhibitor of protein synthesis; the stimulation was completely abolished.     

Lysosome inhibitors enhance the ability of cyclic AMP-elevating agents to induce the LDL receptor in human vascular smooth muscle cells.

In human vascular smooth muscle cells cyclic AMP elevation by forskolin increases synthesis of the LDL receptor by a mechanism which appears independent of sterol control. This increased receptor synthesis is further enhanced by chloroquine. Both forskolin and prostaglandin E1 increase the number of cell surface LDL receptors indicating that prostaglandins could exert physiological control over LDL metabolism. This effect is enhanced synergistically by chloroquine. The stimulation by forskolin of LDL receptor synthesis and expression leads to increased metabolism of apo-B and increased hydrolysis of LDL-borne cholesteryl ester. These effects of cyclic AMP on the activity of the LDL pathway are enhanced more than additively by preincubation with the reversible lysosomal inhibitor NH4Cl. Thus cyclic AMP causes up-regulation of the LDL receptor pathway resulting in increased rates of LDL metabolism but this effect can be damped or masked in cell culture by a cyclic AMP-sensitive lysosomal event, probably the acute stimulation of lysosomal cholesterol ester hydrolase.     

Weak base amines can inhibit class I MHC-restricted antigen presentation

This report describes the effects of NH4Cl, CH3NH2, and chloroquine on class I and II MHC-restricted Ag presentation. OVA-specific T-T hybridomas were used to detect processed OVA in association with class I, H-2Kb, and class II, I-Ad/b, molecules on a B lymphoblastoid APC. OVA, internalized by APC under hypertonic conditions, was presented in association with class I and II MHC molecules. Treating the APC with NH4Cl or CH3NH2 inhibited class I- and II-restricted Ag presentation. In contrast, chloroquine markedly inhibited class II, but not class I-restricted Ag presentation. Controls indicated that drug-treated APC were fully competent to interact with T cells and present processing-independent antigenic peptides in association with both class I and II MHC molecules. NH4Cl and CH3NH2 did not inhibit the uptake of radiolabeled Ag by the APC. After the proteolytic removal of H-2Kb from the surface of APC, NH4Cl and CH3NH2-treated and control APC regenerated identical amounts of surface H-2Kb and this regeneration required de novo protein synthesis. These latter results indicate that NH4Cl and CH3NH2 can inhibit Ag presentation without affecting the synthesis, transport, or surface expression of H-2Kb. Also, NH4Cl did not affect the transport of H-2Db to the surface of mutant RMA-S cells that were cultured with exogenous peptides. Taken together these results strongly suggest that NH4Cl and CH3NH2 but not chloroquine can inhibit a critical and early intracellular step in class I-restricted Ag presentation while simultaneously inhibiting class II-restricted Ag presentation.     

Inhibition of the degradation of receptor-bound human choriogonadotropin by lysosomotropic agents, protease inhibitors, and metabolic inhibitors.

A previous report from this laboratory showed that binding of iodine-labeled human choriogonadotropin to Leydig tumor cells is not a reversible process (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 4892--4899). Most of the cell-bound hormone was found to be degraded to 3'-monoiodotyrosine before being released from the cells, and the degradation process could be inhibited by the lysosomotropic agents NH4Cl, chloroquine, and Triton WR-1339. It is reported herein that the degradation of receptor-bound human choriogonadotropin is an energy-dependent process, which can be inhibited by compounds that interfere with glycolysis or oxidative phosphorylation (e.g. NaF, NaN3, NaCN, and 2-deoxyglucose). Hormone degradation is also inhibited by some protease inhibitors such as the chloromethyl ketones of lysine and phenylalanine, but not by specific trypsin inhibitors (e.g. p-aminobenzamidine and p-tosyl-L-arginine methyl ester). With the exception of NH4Cl, it was found that the compounds which inhibit hormone degradation also inhibit hormone-stimulated steroidogenesis. However, the present results involving dose dependency, and those given in the following paper (Ascoli, M. (1978) J. Biol. Chem. 253, 7839--7843), indicate that these two phenomena are not related.     

Accumulation of amino acids by lysosomes incubated with amino acid methyl esters.

Rat liver lysosomal preparations incubated with 10(-5) M L-[4,5-3H]leucine methyl ester hydrolyzed the methyl ester and accumulated radioactivity within a particulate compartment. The acculated radioactivity was identified as free leucine by thin layer chromatography. Free leucine was not itself taken up by the lysosomal preparations. The capacity to accumulate leucine was identified as a specific property of lysosomes and was thought to result from the trapping of the free amino acid within the lysosome following the hydrolysis of the methyl ester. Lysosomes also accumulated phenylalanine, serine, and alanine when incubated with the corresponding methyl esters. Leucine accumulation was inhibited by submillimolar concentrations of chloroquine, by the protease inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone, and by lowering the pH below 7.0. Efflux of leucine from the lysosomes was highly temperature dependent (activation energy 33 kcal/mol). No evidence was found to suggest that leucine efflux was a carrier-mediated process. The results provide a new methodology for the study of amino acid movements across lysosomal membranes.     

Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339.

The proteolytic degradation of 125I-labeled low density lipoprotein by monolayers of cultured human fibroblasts was prevented by exposure of the cells to chloroquine, an agent that has been reported previously to inhibit lysosomal degradative processes. Chloroquine did not inhibit the binding of low density lipoprotein to its cell surface receptor. However, the two regulatory actions that normally follow low density lipoprotein binding to its receptor, namely suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation, were both prevented when degradation of the lipoprotein was inhibited by chloroquine. Two other agents affecting lysosomal function, Triton WR 1339 and concanavalin A, also inhibited the proteolytic degradation of low density lipoprotein in intact fibroblasts and simultaneously prevented low density lipoprotein-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and stimulation of cholesteryl ester formation. Unlike chloroquine, however, these two agents also affect the binding of low density lipoprotein to the cells. The inhibitory action of chloropuine, concanavalin A, and Triton WR 1339 could each be reversed by removal of the agent from the culture medium. These in vivo culture data, together with the observation that cell-free extracts of fibroblasts maximally degrade 125I-labeled low density lipoprotein at pH 4 and do not form acid-soluble material above pH 6, are consistent with the hypothesis that the proteolytic degradation of low density lipoprotein by monolayers of fibroblasts occurs within lysosomes. The data also suggest that normal lysosomal function is required in order for low density lipoprotein to regulate cholesterol synthesis and cholesteryl ester formation in the fibroblast system.     

The effect of chloroquine on the metabolism of [35S]cystine in normal and cystinotic human skin fibroblasts.

The present study concerns the effect of the lysosomotropic drug chloroquine on the uptake and metabolism of [35S]cystine in vitro by normal human fibroblasts and those from patients suffering from the lysosomal storage disease cystinosis. When the cells were cultured with [35S]cystine for periods in excess of 4 h, it was found that chloroquine considerably increased (up to 30-fold) the labelling of the intracellular cystine pool in cystinotic cells, with no increase or a much smaller increase in normal cells. For this effect chloroquine had an optimum concentration of 20 microM, with a small effect still being noticeable at 1 microM. A quinoline analogue, 4-(dimethylaminoethylamino)-7-iodoquinoline, had a similar effect to chloroquine. However, NH4Cl at concentrations of between 100 microM and 50 mM showed either no effect (at the lower concentrations) or a depression of intracellular cystine labelling (at the higher concentrations). The differences between the effects of the quinolines on cystinotic acid normal cells were not due to differences in total cell uptake of drug.     

Insulin induces chloroquine-sensitive recycling of insulin-like growth factor II receptors but not of glucose transporters in rat adipocytes

Incubation of insulin-treated rat adipocytes with chloroquine, in a time- and concentration-dependent manner, was observed to inhibit the insulin-stimulated increase in insulin-like growth factor II (IGF-II) binding activity, whereas no significant change in IGF-II binding was observed in the absence of insulin. The incremental increase of insulin-stimulated IGF-II binding was inhibited 50% by 0.2 mM chloroquine within 15 min and was nearly completely abolished by 60 min. Interestingly, IGF-II binding was never observed to decrease below the binding value in cells without insulin treatment even when incubation was extended to 180 min. Scatchard analysis of IGF-II binding as well as the specific binding of an anti-IGF-II receptor antibody demonstrated that the loss of IGF-II binding in the insulin-stimulated chloroquine-treated adipocytes was due to a decrease in the number of cell-surface IGF-II receptors, whereas the total number of cellular IGF-II receptors was unaltered. The effect of chloroquine was observed to be reversible, temperature-dependent, and sensitive to the metabolic poison KCN. Furthermore, NH4Cl was also observed to inhibit insulin-stimulated increase in IGF-II binding. In contrast, chloroquine or NH4Cl did not inhibit the basal or insulin-stimulated glucose transport activity. Photoaffinity labeling of the glucose transporter with [3H]cytochalasin B also demonstrated that the basal and insulin-stimulated subcellular distribution of the glucose transporters was unaltered by chloroquine treatment. These results suggest that 1) insulin induces a constitutive, acidotropic agent-sensitive recycling of IGF-II receptor and 2) the glucose transporter and IGF-II receptor do not share the same insulin-regulated intracellular trafficking pathways.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes

We have examined the distribution of mannose-6-phosphate (Man6P) receptors (215 kD) for lysosomal enzymes in cultured Clone 9 hepatocytes at various times after the addition or removal of lysosomotropic weak bases (chloroquine or NH4Cl). Our previous studies demonstrated that after treatment with these agents, Man6P receptors are depleted from their sorting site in the Golgi complex and accumulate in dilated vacuoles that could represent either endosomes or lysosomes (Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320-326). We have now investigated the nature of these vacuoles by labeling NH4Cl-treated cells simultaneously with anti-Man6P receptor IgG and lysosomal or endosomal markers. The structures in which the immunolabeled receptors are found were identified as endosomes based on the presence of endocytic tracers (lucifer yellow and cationized ferritin). The lysosomal membrane marker, lgp120, was associated with a separate population of swollen vacuoles that did not contain detectable Man6P receptors. When cells were allowed to recover from weak base treatment, the receptors reappeared in the Golgi cisternae of most cells (approximately 90%) within approximately 20 min, indicating that as the intra-endosomal pH drops and lysosomal enzymes dissociate, the entire population of receptors rapidly recycles to Golgi cisternae. When NH4Cl-treated cells were allowed to endocytose Man6P, a competitive inhibitor of lysosomal enzyme binding, the receptors also recycled to the Golgi cisternae, suggesting that lysosomal enzymes can dissociate from the receptors under these conditions (high pH + presence of competitive inhibitor). From these results it can be concluded that the intracellular itinerary of the 215-kD Man6P receptor involves its cycling via coated vesicles between the Golgi complex and endosomes, ligand dissociation is both necessary and sufficient to trigger the recycling of Man6P receptors to the Golgi complex, and endosomes rather than secondary lysosomes represent the junction where endocytosed material and primary lysosomes carrying receptor-bound lysosomal enzymes meet.(ABSTRACT TRUNCATED AT 400 WORDS)     

Degradation of chylomicron remnant cholesteryl ester by rat hepatocyte monolayers. Inhibition by chloroquine and colchicine

Rat hepatocytes in monolayer cultures take up and degrade cholesteryl ester of isolated chylomicron remnants. The cholesteryl ester of native chylomicrons was metabolized at a slower rate. The uptake of cholesteryl ester was decreased by the presence of serum. The hydrolysis of cholesteryl ester but not the uptake or binding of chylomicron remnants by the cells was inhibited by chloroquine, which is known to inhibit the lysosomal degradation of protein and of low density lipoproteins by fibroblasts. Colchicine, which inhibits the hydrolysis of chylomicron cholesteryl ester after the uptake by the liver in vivo, had the same effect in hepatocyte monolayers.     

Binding, internalization, and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat

Binding, internalization, and degradation of 125I-labeled-rat atrial natriuretic peptide (rANP) were studied in cultured rat aortic vascular smooth muscle cells (VSMC). At 37 degrees C, 125I-labeled-rANP rapidly bound to VSMCs, but the cell-bound radioactivity rapidly decreased upon subsequent incubation, while the binding was slow at 4 degrees C, reaching to an apparent equilibrium after 6 hrs. The cell-bound 125I-labeled-rANP at 37 degrees C is rapidly dissociated from VSMC (t 1/2: approximately 40 min) with the appearance of degradaded product(s) of radioligand in the medium, whereas the degradation was minimal at 4 degrees C. This degradative process was blocked by inhibitors of metabolic energy production (azide, dinitrophenol), inhibitors of lysosomal cathepsins (leupeptin, pepstatin), and lysosomotropic agents (NH4Cl, chloroquine, lidocaine, methylamine, dansylcadaverine), but not by inhibitors of serine or thiol proteases. 125I-labeled-rANP initially bound to the cell-surface was rapidly internalized, and delivered to lysosomal structures, which was confirmed by autoradiographic studies. These data indicate that rANP, after binding to the cell-surface receptors, is rapidly internalized into the cells through receptor-mediated endocytosis, and subsequently degradaded by lysosomal hydrolases.     

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.     

Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.     

Modulation of mannose receptor activity by proteolysis.

The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.     

Modulation of human natural killer cell function by L-leucine methyl ester: monocyte-dependent depletion from human peripheral blood mononuclear cells

L-leucine methyl ester (Leu-OMe) causes lysosomal disruption and death of human monocytes (M phi). In addition, Leu-OMe removed natural killer cell (NK) activity from human peripheral mononuclear cells (PBM). Thus, a brief preincubation of PBM with Leu-OMe (greater than 1 mM) caused irreversible loss of NK function as assessed by the lysis of K562 targets. By contrast, a variety of other amino acid methyl esters, including L-glutamic dimethyl ester, L-valine methyl ester, and L-isoleucine methyl ester caused reversible inhibition of NK activity in a manner that was similar to other lysosomotropic agents such as chloroquine and ammonium chloride, but did not cause irreversible loss of all NK function. Leu-OMe appeared to cause actual removal of NK effector cells from PBM, because K562 target binding cells, Leu-11b+ lymphocytes, and OKM1+ lymphocytes were depleted. If M phi were removed from PBM before the incubation, Leu-OMe caused only reversible inhibition of NK function in a manner similar to that observed with other amino acid methyl esters. Upon the addition of freshly isolated M phi, polymorphonuclear leukocytes, or sonicates of these cells to M phi-depleted lymphocyte populations, irreversible ablation of NK function was again observed as a result of Leu-OMe exposure. After in vitro culture, M phi lost their susceptibility to Leu-OMe toxicity and the ability to mediate the irreversible deletion of NK cells resulting from Leu-OMe incubation. These results indicate that in the absence of M phi, Leu-OMe and a variety of other amino acid methyl esters are reversible inhibitors of NK function. However, Leu-OMe is unique in that it can interact with M phi or granulocytes to effect an irreversible loss of NK activity from human peripheral blood lymphocytes.     

Lysosome mediated Kir2.1 breakdown directly influences inward rectifier current density

The inward rectifier current generated by Kir2.1 ion channel proteins is primarily responsible for the stable resting membrane potential in various excitable cell types, like neurons and myocytes. Tight regulation of Kir2.1 functioning prevents premature action potential formation and ensures optimal repolarization times. While Kir2.1 forward trafficking has been addressed in a number of studies, its degradation pathways are thus far unknown. Using three different lysosomal inhibitors, NH₄Cl, chloroquine and leupeptin, we now demonstrate involvement of the lysosomal degradation pathway in Kir2.1 breakdown. Upon application of the inhibitors, increased steady state protein levels are detectable within few hours coinciding with intracellular granular Kir2.1 accumulation. Treatment for 24 h with either chloroquine or leupeptin results in increased plasmamembrane originating inward rectifier current densities, while current-voltage characteristics remain unaltered. We conclude that the lysosomal degradation pathway contributes to Kir2.1 mediated inward rectifier current regulation.     

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.     

Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin A and ammonium chloride administration.

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14-day-old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.