Evidence for a striking increase in acetylcholinesterase activity in cultured human fibroblasts which are trisomic for chromosome two

Acetylcholinesterase (AchE) is reported to have a narrowly restricted distribution among human tissues. Three strains of human fibroblasts which are trisomic for chromosome 2 had an average level of AchE activity over 28 times higher than the average level in 19 control strains of human fibroblasts. In contrast, the mean pseudocholinesterase activity of the trisomy-2 strains did not differ appreciably, or significantly, from the mean for the control strains. The 19 control strains included 10 strains trisomic for autosomes other than 2, and 9 euploid strains. Our estimate of the mean AchE activity in the control strains did not differ significantly from zero and might, in any case, have originated from a minute amount of activity contributed to the cells by an esterase in our culture medium. Despite the striking elevation of AchE activity in fibroblasts trisomic for chromosome 2, extracts of these cells had only about 1.5% of the specific AchE activity (per microgram DNA) present in extracts of human cerebral cortex. None of the 22 strains studied had detectable activity for two other enzymes which, like AchE, have a restricted distribution among human tissues: xanthine oxidase and choline acetyltransferase.     

The receptor for urokinase-type plasminogen activator regulates fibronectin matrix assembly in human skin fibroblasts

Previous studies have indicated that the receptor for urokinase-type plasminogen activator, uPAR, can form functional complexes with integrin receptors thereby modulating integrin activity. In the present study, the role of uPAR in the regulation of alpha5beta1-dependent polymerization of the fibronectin matrix was investigated. Incubation of fibroblast monolayers with the P-25 peptide, a uPAR ligand, resulted in a 12-15-fold increase in the accumulation of exogenous fibronectin in the cell layer. The exogenous fibronectin co-localized in the extracellular matrix with endogenous cell-derived fibronectin, and its deposition into the matrix was inhibited by blocking antibodies against the beta1 integrin receptor. The P-25-dependent increase in fibronectin assembly was associated with a 7-8-fold increase in the expression of matrix assembly sites as well as a 37-fold increase in the rate of transfer of cell surface-bound fibronectin into a detergent-insoluble matrix. The effects of P-25 on the matrix assembly were attenuated by incubating cells with either phospholipase C or with antibodies against uPAR, confirming a role for uPAR in the P-25-dependent increase in matrix assembly. P-25-treated cells exhibited a 10-fold increase in the binding of the 120-kDa cell-binding fragment of fibronectin suggesting an increase in alpha5beta1 affinity for fibronectin. Consistent with this, treatment of cells with P-25 also resulted in a 6-10-fold increase in the binding of two different monoclonal antibodies that recognize the active conformation of the beta1 integrin. These results indicate that P-25 increases matrix assembly by altering the activation state of the alpha5beta1 integrin receptor and suggest that changes in integrin activation affect both the number of matrix assembly sites as well as the rate of transfer of cell-bound fibronectin into a detergent-insoluble matrix. These data provide direct evidence that uPAR and integrin receptors synergistically regulate the levels of fibronectin in the extracellular matrix.     

Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous beta-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly

Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K) of fibronectin (FN) stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD) motifs in four of the nine FN type 1 (FNI) modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3)FNI and (5)FNI; (7)FNI and (9)FNI; or (3)FNI, (5)FNI, (7)FNI, and (9)FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3)FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9)FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.     

Effects of thiol protease inhibitors on intracellular degradation of exogenous β-galactosidase in cultured human skin fibroblasts

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of β-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with β-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous β-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.     

Decrease in cAMP levels and increase in cGMP levels in the small intestine mucosa after treatment with fluoride

Sodium fluoride was studied for its effect on the cAMP and cGMP amount in the small intestine mucosa of rats under conditions of acute and chronic experiments. A significant decrease in the cAMP level and an increase in the cGMP level were revealed in both cases. Changes in the content of cyclic nucleotides were followed by marked changes in their physiologically important ratios.     

Agents which increase cyclic AMP have diverse effects on low-density-lipoprotein-receptor function in human vascular smooth-muscle cells and skin fibroblasts.

Receptor-mediated binding and metabolism of low-density lipoproteins (LDL) in cultured human vascular smooth-muscle cells and skin fibroblasts are altered by increased cellular cyclic AMP concentrations. However, the LDL receptor does not respond to changes in cyclic AMP concentration in a simple manner. The activation of adenylate cyclase with forskolin, or the addition of membrane-permeant cyclic AMP analogues, initially decreases the expression of the LDL receptor, but is followed by a substantial increase in receptor expression after 24 h. This increase does not occur in the presence of inhibitors of RNA or protein synthesis, and is due to doubling of the Bmax. of the LDL receptor, without alteration of its affinity for LDL. By contrast, elevation of cyclic AMP concentration by inhibition of phosphodiesterases results in decreased receptor expression throughout the 24 h period. These two response patterns are reproducible phenomena, consistently observed in low-passaged cells derived from seven unrelated individuals.     

Binding sites for elastase on cultured human fibroblasts that do not mediate internalization

The proteolytic actions of elastases have been implicated in extracellular matrix damage, which is characteristic of a variety of pathological conditions including emphysema and rheumatoid arthritis. In order to elucidate the molecular events involved in elastase interaction with connective tissue cells, the present study was designed to investigate the association of elastase with human fibroblasts at 4 degrees C. Elastase bound saturably to binding sites that were present on the surface of these cells. Analysis of cell-bound elastase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a high molecular weight complex (Mr 54,000) that was not formed with elastase whose catalytic site serine was derivatized with a diisopropylphosphate group. The complex did not represent elastase bound to either protease nexin or contaminating serum. The cellular component with which elastase formed a complex could not be detected in the cell culture medium. Unexpectedly, elastase that had been pre-bound at 4 degrees C was not internalized after cells were warmed to 37 degrees C. The elastase binding site described in this report is therefore distinct from high affinity binding sites involved in receptor-mediated endocytosis and intracellular degradation.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Ghrelin concentrations and cardiac vagal tone are decreased after pharmacologic and cognitive-behavioral treatment in patients with bulimia nervosa

Patients with bulimia nervosa (BN) have bulimic and depressive symptoms, which have been associated with abnormalities in the neuroendocrine and vagal systems. Subjects included twenty-four female drug-free outpatients with BN that were selected from patients seeking treatment for eating behavior in our hospital along with twenty-five age-matched healthy females who served as controls. We investigated ghrelin and leptin levels, cardiac vagal tone and sympathovagal balance, frequency of sets of binge-eating and vomiting episodes per week and the Profile of Mood States (POMS) depression scale in BN before and after a 16-week administration of the serotonin selective reuptake inhibitor (SSRI) paroxetine combined with cognitive-behavioral therapy. Compared to controls, the BN group had higher ghrelin levels and resting cardiac vagal tone, and lower leptin levels and resting cardiac sympathovagal balance before treatment, although there was a significant difference between the two groups for the body mass index (BMI). The elevated ghrelin levels (301.7 +/- 18.9 pmol/l, mean +/- SEM vs. 202.8 +/- 15.6 pmol/l, P < 0.01), cardiac vagal tone (2246.4 +/- 335.5 ms(2) vs. 1128.5 +/- 193.3 ms(2), P < 0.01), frequency of sets of binge-eating and purging episodes and T scores for the POMS depression scale were all significantly decreased after treatment despite similar BMI, percent body fat and leptin levels. In close association with cardiac vagal function and ghrelin recoveries, abnormal eating behavior and depressive symptoms improved, indicating the usefulness of these indexes in the assessment of clinical condition and therapeutic efficacy in BN.     

Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added together to intact adipocytes

Twenty-five cyclic nucleotide analogs were tested individually to act as lipolytic agents and to activate adipocyte protein kinase. The lipolytic potency of individual analogs correlated better with their Ka for protein kinase and their lipophilicity rather than with either parameter alone. Some of the most potent lipolytic analogs had I50 values for the particulate low Km cAMP phosphodiesterase suggesting that their effect was not due to raising endogenous cAMP levels through inhibition of phosphodiesterase. The most potent lipolytic analogs contained a thio moiety at the C-8 or C-6 position. These analogs exhibited concave upward dose-response curves. At high concentrations, some analogs were as effective as optimal concentrations of epinephrine in stimulating glycerol release. The regulatory subunit of protein kinase has two different intrachain cAMP-binding sites and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1 and analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2 (Rannels, S. R., and Corbin, J. D. (1980) J. Biol. Chem. 255, 7085-7088). Thus, C-8 and C-6 analogs were tested in combination to stimulate lipolysis in intact adipocytes and to activate protein kinase in vitro. Each process was stimulated synergistically by a combination of a C-6 and C-8 analog. Two C-8 analogs or two C-6 analogs added together did not cause synergism of either process. For both lipolysis and protein kinase activation, C-8 thio analogs acted more synergistically than C-8 amino analogs when incubated in combination with C-6 analogs, a characteristic of type II protein kinase. It is concluded that the observed synergism of lipolysis is due to binding of cAMP analogs to both intrachain sites and that it is the type II protein kinase isozyme which is responsible for the lipolytic response.     

Mitogen-induced [Ca2+]i changes in individual human fibroblasts. Image analysis reveals asynchronous responses which are characteristic for different mitogens

Temporal changes in intracellular free Ca2+ concentration ([Ca2+]i) in cultured human foreskin fibroblasts were investigated using image analysis techniques to simultaneously monitor Lys-bradykinin (BK)- or thrombin-induced elevations of [Ca2+]i in each individual cell within a microscopic field. Responses to BK are heterogeneous with respect to the shapes of the [Ca2+]i time courses. Furthermore, the onsets of these responses follow a variable lag period such that the individual cell responses occur asynchronously. The asynchrony and heterogeneity of individual cell responses are not related to cell cycle differences since noncycling cells respond in a similar manner. When cells are ranked according to order of an initial response to BK (the first cell to respond is ranked 1, the second to respond is 2, etc.), restimulation of the same cells with BK elicits a similar order of cell responses, and the shape of the [Ca2+]i time course of an individual cell is similar for both responses to BK. If cells that were stimulated with BK are washed and restimulated with thrombin (which produces [Ca2+]i changes similar to those induced by BK), the response order to thrombin does not correspond to the response order following BK stimulation. These data suggest that the asynchrony of [Ca2+]i changes induced by BK or thrombin is characteristic for each mitogen and may be determined by cell-to-cell variation in receptor number.     

High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

The binding of high specific activity, radioactive Concanavalin A to cultured normal human fibroblasts was investigated. We report the presence of two classes of Concanavalin A binding sites on the plasma membranes of these cells. These classes of binding sites are distinguished by their affinities for the lectin. Scatchard analysis of the binding data indicates the presence of a class of high affinity sites which are saturated at about 0.25 μg/ml of Concanavalin A. The other, lower affinity binding sites are not saturated until 50–100 μg/ml Concanavalin A levels are achieved. At 4°C the K_a for the high affinity sites varies between 1.5 – 5 × 10⁹ M^?1 depending on the method used to label the Concanavalin A. For the lower affinity sites K_a varies between 1 – 4 × 10⁶ M^?1. The average number of high affinity sites per cell is 8 × 10⁵ representing less than 1% of the total receptor sites for the lectin.     

Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: Evidence for intracellular complexes

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azido-benzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37°C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Flg receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4°C with ¹²⁵I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170–180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130–160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with ¹²⁵I-HSAB-FGF-2 at 4°C and then washed, were shifted to 37°C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of ¹²⁵I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex. © 1996 Wiley-Liss, Inc.     

Interactions of methylmalonyl CoA mutase from normal human fibroblasts with adenosylcobalamin and methylmalonyl CoA: evidence for non-equivalent active sites

We have examined interactions between human methylmalonyl CoA mutase and two critical ligands, its cofactor adenosylcobalamin (AdoCbl) and its substrate methylmalonyl CoA, by performing in vitro experiments with preparations of mutase apoenzyme and holoenzyme from normal cultured human fibroblasts. When extracts are prepared from cells grown in medium containing high concentrations of hydroxocobalamin, a precursor of AdoCbl, mutase activity measured in Tris-containing buffers in the absence of added AdoCbl accounts maximally for only 50% of that activity measured in the presence of excess AdoCbl. A similar result is observed when mutase holoenzyme is formed in vitro by incubating cell extracts containing apoenzyme with AdoCbl and removing excess AdoCbl by gel filtration. When such holoenzyme preparations are heated at 45 °C and then assayed for activity, their thermostability is less than that of mutase holoenzyme heated in the presence of excess cofactor, but far greater than that of mutase apoenzyme. Methylmalonyl CoA modulates these enzyme-coenzyme interactions, since mutase holoenzyme formed in Triscontaining buffers is resolved to apoenzyme upon exposure to substrate. Qualitatively different data are obtained when buffers containing cations other than Tris are used. Under these conditions, mutase activity measured in the absence of added AdoCbl accounts for nearly 100% of the activity measured in the presence of excess cofactor, whether holoenzyme is formed in intact cells in culture or in cell extracts in vitro. Furthermore, holoenzyme formed in vitro in potassium phosphate buffer is not resolved to apoenzyme upon exposure to substrate. We suggest that the “holoenzyme” form of mutase obtained and assayed in Tris-containing buffers is that molecular species with only one of its two potential AdoCbl binding sites occupied in a catalytically active fashion, and that other ions can influence markedly the interactions between mutase, AdoCbl, and methylmalonyl CoA. These data are consistent, therefore, with the hypothesis that the dimeric mutase apoenzyme is characterized, under certain conditions, by nonequivalent active sites.