Na(+)-H+ antiport activity in skin fibroblasts from blacks and whites

The predisposition of black people to salt (NaCl)-sensitive essential hypertension may relate to racial differences in cellular Na+ metabolism. This tenet was investigated by examining the Na(+)-H+ antiport in serially passed skin fibroblasts from blacks and whites. Na(+)-dependent stimulation of the Na(+)-H+ antiport by cellular acidification resulted in a greater maximal velocity (Vmax) (mean +/- SEM) of this transport system in quiescent fibroblasts from blacks than fibroblasts from whites; the Vmax for recovery from cellular pH (pHi) of 6.6 was 5.84 +/- 0.50 versus 4.39 +/- 0.34 mmol H+/l X 20 seconds for blacks and whites, respectively (p less than 0.05). Although the Na+ concentration producing 50% stimulation of the Na(+)-H+ antiport for blacks (35.1 +/- 5.7 mM) was greater than for whites (24.1 +/- 3.5 mM), this difference was not statistically significant. No racial differences were observed in the Hill coefficient (n, 1.35 +/- 0.21 for blacks and 1.46 +/- 0.28 for whites). Compared with whites, cells from blacks exhibited a greater response to cytoplasmic acidification over the range of pHi values 6.20-6.60, as exhibited by an augmented rate of recovery in the pHi. These differences were not due to different basal pHi values or cellular buffering capacities, which were similar for blacks and whites. Na(+)-H+ antiport activity was not correlated with family history of hypertension. Increased activity of the Na(+)-H+ antiport in fibroblasts from blacks was confirmed without cellular acidification by stimulating quiescent cells with 10% human serum. This study demonstrates innate racial differences in cellular membrane Na(+)-H+ antiport activity.     

Probucol enhances cholesterol efflux from cultured human skin fibroblasts

To explore whether the reported xanthoma-reducing effects of probucol are related to tissue-specific mechanisms of the drug, the influence of probucol on cholesterol efflux from cultured human skin fibroblasts was investigated. Incubation of cells with probucol led to a 2-fold enhancement of high-density lipoprotein-mediated cholesterol efflux. This result raises the possibility that probucol may reduce cholesterol accumulation in tissues through a cholesterol-mobilizing action.     

Aromatase activity in cultured human genital skin fibroblasts

Aromatase activity of human genital skin fibroblasts grown in cell culture was studied using both [1,2,6,7-3H] androstenedione (A) and [1-3H]A as substrates. With the former substrate the generation of [3H]estrogens was determined, whereas with the latter substrate, the formation of [3H]H2O was measured. Our results showed that the release of [3H]H2O from [1-3H]A provides an accurate and sensitive method for determining aromatase activity in cultured human skin fibroblasts. Because genital skin fibroblasts also possess marked 5 alpha-reductase activity, we found that addition of an alternate substrate for 5 alpha-reductase was necessary to prevent shunting of A from the aromatase pathway. Hence, all aromatase assays were carried out in the presence of 5 microM progesterone. Under these experimental conditions, no correlation was found between levels of 5 alpha-reductase and aromatase activities. The Michaelis-Menten constant (Km) of the aromatase in cultured genital skin fibroblasts measured in the presence of A and added progesterone ranged between 10 and 39 nM, and the maximum velocity (Vmax) ranged between 0.14 and 1.46 pmol product/mg protein/h. These values are in good agreement with those previously described for adipose tissue stromal-vascular cells, suggesting that the aromatase complexes are similar in skin and adipose tissue. We conclude that skin may be an important site for aromatization of androgens to estrogens in men.     

Magnesium deficiency accelerates cellular senescence in cultured human fibroblasts

Magnesium inadequacy affects more than half of the U.S. population and is associated with increased risk for many age-related diseases, yet the underlying mechanisms are unknown. Altered cellular physiology has been demonstrated after acute exposure to severe magnesium deficiency, but few reports have addressed the consequences of long-term exposure to moderate magnesium deficiency in human cells. Therefore, IMR-90 human fibroblasts were continuously cultured in magnesium-deficient conditions to determine the long-term effects on the cells. These fibroblasts did not demonstrate differences in cellular viability or plating efficiency but did exhibit a decreased replicative lifespan in populations cultured in magnesium-deficient compared with standard media conditions, both at ambient (20% O(2)) and physiological (5% O(2)) oxygen tension. The growth rates for immortalized IMR-90 fibroblasts were not affected under the same conditions. IMR-90 fibroblast populations cultured in magnesium-deficient conditions had increased senescence-associated beta-galactosidase activity and increased p16(INK4a) and p21(WAF1) protein expression compared with cultures from standard media conditions. Telomere attrition was also accelerated in cell populations from magnesium-deficient cultures. Thus, the long-term consequence of inadequate magnesium availability in human fibroblast cultures was accelerated cellular senescence, which may be a mechanism through which chronic magnesium inadequacy could promote or exacerbate age-related disease.     

Oestrogen formation in genital and non-genital skin fibroblasts cultured from patients with hypospadias

OBJECTIVE Hypospadias is the most common birth defect in males. in most cases the aetiology is unknown. Since penile development is androgen dependent and oestrogen can modify androgen action, we compared the formation of oestrogen In penile tissue from patients with hypospadias to those with normal penile development. DESIGN AND PATIENTS Oestrogen formation was assessed in fibroblast monolayers grown from biopsies of genital and non-genital skin from 11 males with normal genital development (controls) and 18 males with severe hypospadias utilizing the incorporation of tritium into H₂O resulting from the aromatization of 1β-³H-androstenedione. RESULTS in paired fibroblast strains from genital and non-genital skin of nine males with hypospadias, oestrogen formation was significantly (P < 0·025) lower In non-genital skin. Rates of oestrogen formation were also higher in a subset of foreskins from subjects with hypospadias than In normal controls and the remaining hypospadias subjects. In addition, oestrogen formation in this subset of fibroblast strains from patients with hypospadias was markedly enhanced by incubation of Intact monolayers with either cholera toxin or forskolin, agents known to stimulate CAMP formation. Oestrogen formation in the remaining cell strains (controls and hypospadias) was also enhanced in most instances by cholera toxin and forskolin, although to a much lower degree. Thus, we identified in the hypospadias group a subgroup of fibroblast strains in which unstimulated and stimulated oestrogen formation was markedly higher than in other strains examined. CONCLUSIONS Since oestrogen can modify certain androgen effects within cells and since formation of the male genitalia during embryogenesis is mediated by androgens, elevated oestrogen formation in male genital tissue might be a causative factor of hypospadias in some Instances.     

Glycine transport by cultured skin fibroblasts from a patient with isolated hyperglycinuria

Glycine transport is studied in cultured skin fibroblasts from a patient with isolated hyperglycinuria and from five normal subjects. Fibroblasts from the patient take up glycine less well than do cell lines from controls. Kinetic studies are consistent with a single transport system in the patient's and controls' cell lines. Vmax value in the hyperglycinuric lines is normal, but the apparent affinity is always reduced as opposed to those of four different control lines separately tested. Statistical analysis shows significant difference between Km values.     

Glycine transport by cultured skin fibroblasts from a patient with isolated hyperglycinuria

Glycine transport is studied in cultured skin fibroblasts from a patient with isolated hyperglycinuria and from five normal subjects. Fibroblasts from the patient take up glycine less well than do cell lines from controls. Kinetic studies are consistent with a single transport system in the patient's and controls' cell lines.V _max value in the hyperglycinuric lines is normal, but the apparent affinity is always reduced as opposed to those of four different control lines separately tested. Statistical analysis shows significant difference betweenK _m values. The authors gratefully acknowledge Mrs Couturier and Mrs Polini Tor technical assistance and Dr Boegner for the opportunity of studying the patient. This study was supported by grant 773 from UER de Médecine, Université Paris XI (France).     

Abnormal collagen metabolism in cultured skin fibroblasts from patients with Duchenne muscular dystrophy.

Total collagen synthesis is decreased by about 29% (P less than 0.01) in skin fibroblasts established in vitro from male patients with Duchenne muscular dystrophy (DMD) as compared with that in normal male skin fibroblasts in vitro. The reduction in collagen synthesis is associated with an approximately 2-fold increase in collagen degradation in DMD fibroblasts. Correlated to these alterations in the metabolism of collagen, DMD fibroblasts express a significantly higher hydroxyproline/proline ratio (DMD: 1.36-1.45; P less than 0.01) than do normal fibroblasts (controls: 0.86-0.89). The increased hydroxylation of proline residues of collagen (composed of type I and type III) could be the cause for the enhanced degradation of collagen in DMD fibroblasts.     

Copper deficiency in the mitochondria of cultured skin fibroblasts from patients with menkes syndrome

Summary The mitochondrial copper concentrations and cytochrome C oxidase activity of the fibroblasts from the patients with Menkes syndrome were investigated. Both the mitochondrial copper concentrations and cytochrome C oxidase activity of fibroblasts from patients with Menkes syndrome were lower than those of the control fibroblasts. These data indicate that the mitochondria of fibroblasts from patients with Menkes syndrome are in a state of copper deficiency. The activity decline of cytochrome C oxidase, a mitochondrial cuproenzyme, seems to be caused by copper deficiency in the mitochondria.     

Defective organization of actin in cultured skin fibroblasts from patients with inherited adenocarcinoma.

In the cytoplasm of well-spread cultured normal fibroblasts, actin is organized into a network of cables that run the length of the cell just inside the adherent cell membrane. A diffuse matrix replaces the cables in fibroblasts that have become tumorigenic as a result of oncogenic transformation. We have found a similar disruption in actin organization in cultured skin fibroblasts (passage 6-10) obtained by biopsy from patients with the inherited colonic cancer, adenomatosis of the colon and rectum (ACR). Because ACR is inherited as an autosomal dominant trait, about half the children of ACR patients will develop colon cancer, but they typically remain asymptomatic until at least the second decade of life. Actin distribution within cultured cells from children of ACR patients was identical either to that seen in cultured cells from normal persons or to that seen in cultured cells from ACR patients. The two different patterns were independent of age, sex, drug treatment, or infections of the donors. Apparently, this class of colonic carcinoma is accompanied by a systemic aberration in the organization of fibroblast cytoplasm, and this aberration can be detected by immunofluorescent localization of actin within cultured skin fibroblasts, prior to manifestation of any colonic symptoms.     

Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts.

An abnormal regulation of chloride permeability has been described for epithelial cells from patients with cystic fibrosis (CF). To learn more about the biochemical basis of this inherited disease, we have studied chloride metabolism in cultured CF fibroblasts by comparing the efflux of 36Cl- from matched pairs of CF and normal fibroblasts. The rate constants describing 36Cl- efflux did not differ between the two cell types, but in each of the four pairs tested the amount of 36Cl- contained within CF cells was consistently reduced, by 25-30%, relative to normal cells. Comparisons of cell water content and 22Na+ efflux showed no differences between the two cell types, suggesting that overall intracellular chloride concentration is lower than normal in CF fibroblasts. Such data suggest that the CF gene defect is expressed in skin fibroblasts and that this defect may alter the regulation of intracellular Cl- concentration, perhaps through changes in Cl- permeability.     

Diagnosis of Gaucher's disease in cultured skin fibroblasts and leucocytes

Washing skin fibroblasts or leucocytes in 0.25 mol/l sucrose increases the activity of-glucosidase at acid pH. This effect is primarily due to removal of low levels of sodium chloride, which inhibit acid-glucosidase. A secondary factor for skin fibroblasts is the removal of residual phosphate buffer pH 7.3 used to wash the cells following trypsinization. As the-glucosidase activity of water-lysed leucocytes is higher at acid pH than that of a saline suspension of leucocytes, the former are better for the diagnosis of Gaucher's disease. However, more reliable results still may be obtained by assay of this enzyme in cultured skin fibroblasts.     

Insulin stimulation of glucose oxidation in cultured human skin fibroblasts

Insulin action on glucose oxidation has been examined in cultured human diploid fibroblastic cells. When the fibroblasts were preincubated in the absence of glucose, insulin stimulation of ¹⁴C-6-D-glucose oxidation was small (approximately 120% of basal). Preincubation in glucose containing medium (2.7–11.0 mM) for 18 hr enhanced insulin stimulation of ¹⁴C-6-D-glucose oxidation to a maximum of 240% of control. Stimulation was observed with as little as 10^?10M insulin and reached maximum at 10^?8M insulin. Half maximum stimulation was achieved at approximately 9 × 10^?10M insulin at all glucose concentrations. In order to examine the specificity of insulin action on glucose oxidation, the relative effectiveness of various analogues was examined. The order of potency was porcine insulin > proinsulin > bonito insulin > desoctapeptide insulin. Multiplication stimulating activity also stimulated glucose oxidation with a half maximal stimulation of 3 × 10^?9M. The optimal pH for insulin stimulation of glucose oxidation was 7.4, and insulin action was less at temperatures lower than 37°C. When the influence of growth conditions on insulin stimulation of glucose oxidation was assessed, confluent cells had greater responsiveness than dividing cells, and responsiveness declined in senescent cells. When insulin action on glucose oxidation and the effect of glucose preincubation were examined using ¹⁴C-1-D-glucose, insulin stimulation after glucose deprivation was again approximately 120%, but glucose preincubation resulted in only a small enhancement of insulin action on ¹⁴C-1-D-glucose oxidation (to 140% of control). To investigate the metabolic site of insulin action on glucose oxidation, the effect of insulin on the distribution of incorporated ¹⁴C-glucose was compared in cells preincubated in the presence or absence of glucose. Insulin stimulation of lactate production (glycolysis) was significantly enhanced by glucose preincubation, and basal glucose incorporation into ribose and glycogen was reduced. Thus, insulin action on CO₂ production seemed to occur at some site in the glycolytic pathway; the preferential glucose enhancement of ¹⁴C-6-D-glucose oxidation appeared to be due to an increased proportion of glucose used for glycolysis and also to an enhancement of insulin action in the glycolytic pathway. These studies indicate that under appropriate culture conditions insulin significantly stimulates ¹⁴C-6-D-glucose oxidation in human skin fibroblasts. Since insulin action in the fibroblast appears to have characteristics similar to insulin action in other cell types, this might be a useful system for the further study of insulin action on cell glucose metabolism.     

Regulation of glucose transporter synthesis in cultured human skin fibroblasts

The present study was designed to see the effects of glucose on glucose transporter expression and glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1-44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in approximately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1-44.4 mM) does not affect the glucose transporter expression and glucose transport activity.     

Testosterone lowers aromatase activity in cultured human genital skin fibroblasts

The cytochrome P-450-dependent aromatase pathway utilizes the androgens testosterone (T) and androstenedione, as substrates for estrogen formation. In addition, androgens have been shown to influence the level of aromatase activity in various tissues. In cultured human skin fibroblasts, incubation with T for 14 h resulted in a dose-dependent decline in aromatase activity, the concentration of T producing a half-maximal decline being 6 nM. In the presence of T (50 nM), aromatase activity declined in a time-dependent fashion with maximal reduction occurring by 9 h. When aromatase kinetics were determined after preincubation of cells with T, there was a significant decline in the calculated Vmax with no significant change in the apparent Km, suggesting that incubation of cells with T reduced the number of active enzyme sites. Aromatase activity was unaffected by preincubation of cells with the synthetic androgen methyltrienolone. In addition, the decline in aromatase activity following preincubation with T was observed in cells derived from patients with complete androgen insensitivity demonstrating that the effect of T was not mediated by androgen receptors. Furthermore, new protein synthesis was not necessary for the T-mediated effect as the presence of cycloheximide (50 micrograms/ml) did not prevent it. When cells were incubated at low oxygen tension, the inhibition of aromatase activity by T was diminished. Testosterone is rapidly metabolized in genital skin fibroblasts to dihydrotestosterone, androstanedione, androsterone, 3 alpha-androstanediol, 3 beta-androstanediol and estradiol. To determine if a metabolite of T might be responsible for the repression of aromatase activity, aromatase activity was determined in cells following preincubation with various metabolites of T. Preincubation of cells with androstenedione, androstanedione or 3 alpha-androstanediol produced a small but significant decline in aromatase activity, whereas preincubation of cells with dihydrotestosterone, androsterone, or 3 beta-androstanediol did not have a significant effect. Aromatase activity was also unaffected by preincubation of cells with estradiol or diethylstilbestrol. When aromatase activity was assayed in microsomal preparations from cells preincubated with T, activity was reduced. Although cells preincubated with 50 nM [3H]T contained between 0.25 and 0.51 pmol of residual steroid/mg microsomal protein, the amount of [1-3H]androstenedione and T was insufficient to account for the observed decline in aromatase activity on the basis of competitive inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impaired collagen gel contraction with cultured skin fibroblasts from patients with systemic sclerosis

We investigated the capacity of skin fibroblasts, derived from 9 patients with systemic sclerosis (SSc), to contract collagen lattices in a three-dimensional culture system. In comparison with control fibroblasts (N = 8), more than 30% of SSc fibroblasts exhibited markedly impaired ability to contract collagen lattices. The expression of alpha2beta1 integrins and integrin-mediated signals were not significantly different between normal and SSc fibroblasts. Although the underlying mechanisms remain to be determined, our present data provide evidence that certain aspects of interaction with collagen are impaired in SSc fibroblasts.     

Selective resistance to parathyroid hormone in cultured skin fibroblasts from patients with pseudohypoparathyroidism type Ib

We measured cAMP production in response to agonists in cultured skin fibroblasts from subjects with pseudohypoparathyroidism type Ib (PHP Ib; normal phenotype, resistance to PTH only, normal guanine nucleotide stimulatory coupling protein activity) and skin fibroblasts from normal subjects. There were no significant differences in basal or prostaglandin E1- and forskolin-stimulated cAMP production in PHP Ib vs. normal fibroblasts. Fibroblasts from 7 of 10 subjects with PHP Ib had significantly reduced peak cAMP responses to PTH [3.95 +/- 0.88 vs. 15.9 +/- 4.2 pmol/100 micrograms protein (mean +/- SD); n = 7 for both groups; P less than 0.001]. PTH-stimulated cAMP production was significantly reduced in the 7 subjects with PHP Ib at all concentrations of PTH tested [3-1000 ng/ml human PTH-(1-34)]. In the other 3 subjects with PHP Ib, the cAMP response to PTH was either normal (2 subjects) or above the normal range (1 subject). Thus, skin fibroblasts from many, but not all, subjects with PHP Ib have selective resistance to PTH in terms of cAMP response. Since the defect is hormone specific and persists in culture, we suggest that an intrinsic defect in the PTH receptor may cause PTH resistance in certain subjects with PHP Ib. The cause of PTH resistance in the subjects with a normal cAMP response to PTH is not known, but the data suggest heterogeneity even within the PHP Ib subgroup.     

Retinoic acid inhibition of hyaluronate synthesis in cultured human skin fibroblasts

The effects of all-trans retinoic acid on glycosaminoglycan (GAG) accumulation were determined in cultured primary human skin fibroblasts. Confluent cultures treated with retinoic acid accumulated less [3H]GAG than those without the compound, an effect with an apparent threshold of 10 nM which was dose dependent in the concentration range tested (0-10 microM). At 10 microM, the inhibition was 54%. Greater than 80% of the labeled macromolecular material was streptomyces hyaluronidase digestible in cultures labeled with [3H]acetate. The incorporation of H2[35S]O4 into chondroitin sulfate and dermatan sulfate was unaffected, as was total protein synthesis. Retinol also inhibited accumulation of [3H]GAG, but was far less potent. T3 and dexamethasone can inhibit [3H]hyaluronate synthesis. When retinoic acid was added to cultures treated with either of these hormones at concentrations that maximally inhibit [3H] GAG accumulation, there was a further decrease in the rate of macromolecular accumulation. The retinoic acid effect evolved over 24-48 h after addition to the culture medium. A pulse-chase study failed to demonstrate any effect on [3H]GAG degradation.     

Human milk stimulates DNA synthesis and cellular proliferation in cultured fibroblasts.

Human milk contains a mitogenic factor that stimulates DNA synthesis and cell division in mouse and human fibroblasts in vitro. Milk at a concentration of 1% (vol/vol) is as active in stimulating DNA synthesis as is 5% (vol/vol) human serum and 10% (vol/vol) calf serum. The mitogenic activity of human milk is destroyed by incubation with trypsin and chymotrypsin. However, neither urea, guanidine hydrochloride-dithiothreitol, nor exposure to pH 1 will inactivate the milk-derived growth factor. Gel filtration and isoelectric focusing indicate that the mitogenic activity of human milk has a molecular weight between 14,000 and 18,000 and an isoelectric point between 4.4 and 4.7.     

Thyroid hormone inhibits fibronectin synthesis by cultured human skin fibroblasts

Fibronectin (Fn) is a glycoprotein composed of two different subunits, each with a mol wt of approximately 230K. Fn is important for cell adhesion and for maintaining normal cell morphology. Recent in vivo studies suggested that thyroid hormone affects the plasma Fn level in man. The aim of this study was to determine whether T3 regulates the synthesis of Fn in cultured human skin fibroblasts. Skin fibroblasts obtained from seven normal subjects were grown in Minimum Essential Medium supplemented with 10% fetal calf serum. After reaching confluence, the cells were exposed for 3 days to the same medium in which fetal calf serum was replaced by 10% thyroidectomized bovine serum without or with added T3. At the end of this period, the cultures were incubated with [35S]methionine (95 microCi/dish) for 4 h before harvesting. Cell lysates and corresponding media were combined, and 35S incorporation into total protein and Fn was determined by trichloroacetic acid precipitation and immunoprecipitation with antihuman Fn immunoglobulin, respectively. Analysis of the immunoprecipitated material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the 35S activity migrated as a 230K band which was displaced by excess unlabeled Fn. Addition of T3 did not affect trichloroacetic acid-precipitable 35S activity but decreased the 35S activity precipitated with anti-Fn in a dose-dependent manner. While addition of 10(-10) M T3 to the medium had no effect, 35S incorporation into Fn was inhibited by 22.5 +/- 7.4% (mean +/- SD) with 10(-9) M T3 (free T3, 6 X 10(-12) M) and by 31.3 +/- 5.8% with 10(-7) M T3. To assess whether the inhibition of Fn accumulation by T3 was due to suppression of Fn synthesis or enhanced Fn degradation, cells were labeled for 4 h with [35S]methionine and chased for another 4 h with excess unlabeled methionine. T3 had no effect on the rate of decline of [35S]Fn during the chase. We conclude that physiologic T3 concentrations inhibit the synthesis of Fn in normal human fibroblasts. This effect provides a new method to study the action of thyroid hormone which may prove useful in the tissue diagnosis of resistance to thyroid hormone.