Sulfate transport in normal and cystic fibrosis fibroblasts.

The glycoconjugate component of cystic fibrosis (CF) epithelial secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation (delta F508/delta F508) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [35S]SO4(2-) uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts.     

The calcium abnormality in cystic fibrosis mitochondria: Relative role of respiration and ATP hydrolysis

Calcium uptake by mitochondria isolated from skin fibroblasts of patients with cystic fibrosis and controls was studied in the presence and absence of inhibitors. Since mitochondrial calcium accumulation may be supported by ATP hydrolysis or respiration, inhibitors of each were used to characterize the basis of previously described alterations in calcium uptake by mitochondria from patients with cystic fibrosis. Calcium uptake measurements under the influence of oligomycin and antimycin A suggest that the increased calcium uptake by mitochondria from patients with cystic fibrosis is related to altered respiratory system activity. Binding constants of calcium to the carrier system in mitochondria were not different between genotypes.     

Taurine uptake by normal and cystic fibrosis fibroblasts

Taurine deficiency recently has been proposed to be clinically significant in cystic fibrosis (CF). Uptake of [14C]taurine by four cystic fibrosis (CF) and three control fibroblast lines was examined to determine whether a generalized defect in taurine transport could contribute to the deficiency. The time course of uptake was linear up to 20 h and was similar in both CF and control fibroblasts. Taurine was avidly retained after uptake, and the effect of metabolic (chlorpromazine) and competitive (hypotaurine, L-leucine) inhibitors was similar in both CF and control cells. In contrast, while taurine uptake in a calcium-free medium was impaired in both CF and control fibroblasts, the impairment was significantly less in CF cells. The findings suggest that a generalized abnormality in taurine transport is unlikely to be responsible for the taurine deficiency in CF.     

Expression of rat liver canalicular sulfate carrier in Xenopus laevis oocytes

Poly(A)+ RNA (mRNA)extracted from rat liver was injected into Xenopus laevis oocytes and the expression of sulfate transport was determined by measuring [35S] sulfate uptake. Compared to water-injected oocytes, which exhibited virtually no sulfate uptake, injection of rat liver mRNA resulted in a time- and dose-dependent increase in uptake of sulfate. Depending on the method used for the isolation of the mRNA, sulfate uptake was stimulated after injection (40 ng after 6 days) between 8- and 72-fold compared to water-injected oocytes. Sulfate uptake of oocytes injected with mRNA was found to be sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC50 less than 20 microM) and could also be inhibited by thiosulfate. Sulfate uptake of injected oocytes showed Michaelis-Menten kinetics (apparent Km, 0.31 mM) which is similar to the Km of the sulfate/bicarbonate antiporter of rat liver canalicular plasma membranes. After fractionation by a sucrose density gradient, the mRNA encoding for the expressed rat liver sulfate carrier was found in fractions containing messages of 3.5-4.0 kilobases in length.     

Sulfate transport by rat liver lysosomes

Sulfate transport was examined using membrane vesicles (pH 7.0 inside) prepared from rat liver lysosomes. Sulfate uptake was dependent upon external pH with increased uptake at lower buffer pH. The Km for uptake was 160 microM at pH 5.0 while at pH 7.0, a lower affinity system with a Km of 1.4 mM was present. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased uptake at pH 5.0 while valinomycin/KCl had no effect. In contrast, at pH 7.0, valinomycin-induced changes in membrane potential stimulated uptake. Countertransport of sulfate at pH 7.0 was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid, and a variety of anions: SO4(2-) greater than MoO4(2-) greater than Cl- greater than HPO4- greater than HCO3-. Trans-stimulation of sulfate uptake at pH 7.0 was observed with MoO4(2-) and, to a lesser extent, with S2O3(2-) while Cl-, HPO4-, and HCO3- had little effect. However, chloride loading of vesicles resulted in marked stimulation of sulfate uptake at pH 5.0. It appears that sulfate and protons exit lysosomes in exchange for chloride by a specific, pH-regulated anion transport system.     

alpha 2-Macroglobulin-proteinase complexes in cystic fibrosis serum. Analysis by monoclonal antibodies and receptor-mediated endocytosis by normal human fibroblasts

alpha 2-Macroglobulin complexed to proteinases activated during clotting of cystic fibrosis and control sera was quantitated with the complex-specific monoclonal antibody F2B2 . Similar amounts of alpha 2-macroglobulin complexes (between 40 and 90 micrograms/ml) were generated in cystic fibrosis and control sera. Endocytosis of the complexes by normal human fibroblasts was compared to the amount of complexes detected by the F2B2 -radioimmunoassay. Normal uptake was observed with 13 out of 14 cystic fibrosis sera. One cystic fibrosis serum showed strongly reduced endocytosis of the complexes. Complexes isolated from this serum on immobilized F2B2 failed to inhibit binding of purified alpha 2-macroglobulin-trypsin to its receptor, demonstrating deficient receptor-binding of these complexes. The low uptake complexes could not be distinguished from complexes isolated from control or other cystic fibrosis sera by isoelectric focusing, rate electrophoresis or SDS-polyacrylamide gel electrophoresis.     

Chloride transport in control and cystic fibrosis human skin fibroblast membrane vesicles.

Plasma membrane vesicles were isolated from either cystic fibrosis (CF) or non-CF cultured fibroblasts derived from skin biopsies of either foetus, child or adolescent human donors. The total membrane yield was essentially identical for either CF or control membranes. By using a rapid filtration technique, 36Cl uptake by these vesicles was quantitated in the absence and presence of alkali-metal ion-, electrical- and/or pH gradients. In the absence of a pH gradient (pHout = pHin = 7.5), Cl uptake took place downhill in both cases. Either cis K+, cis Na+ or an equimolar mixture of cis Na+ plus K+ caused Cl uptake activation. In the presence of an alkaline-inside pH gradient (pHout/pHin = 5.5/7.5), Cl uptake exhibited an apparent overshoot independently of the presence or absence of any metal-ion gradient. The observed potassium-, sodium- and proton-dependent Cl influx rates were all unaffected by voltage clamping, indicating the existence in these vesicles of electroneutral symport systems of the type Cl-/H+, Cl-/K+ and/or Cl-/Na+; but not 2 Cl-/Na+/K+. In the presence of an inward-directed K+ gradient, valinomycin further increased Cl uptake, both in the presence and in the absence of a pH gradient, indicating the presence of a rheogenic Cl uniport. In absolute quantitative terms, the two different modes (rheogenic and electroneutral) of Cl transport evinced in these vesicles were about 45% lower in CF than in control skin fibroblasts. However, qualitatively, there was no difference between normal and CF cells. The evidence obtained indicates that the CF defect, which is expressed in fibroblast plasma membranes, does not affect specifically either the rheogenic or the electroneutral Cl transport systems. Rather, the CF cells appear to give a smaller yield of closed, functional vesicles, reflected by a significantly smaller apparent intravesicular volume. Because it also affects the transport of D-glucose and L-alanine, this anomaly could be the consequence of a generalized membrane defect characterizing CF fibroblasts.     

Membrane function in cystic fibrosis. I. Putrescine transport in normal and cystic fibrosis fibroblasts

Putrescine transport was examined in normal and cystic fibrosis fibroblasts. No differences were observed in accumulation pattern, kinetics of uptake, or efflux between CF and normal cells. In both growing and growth-arrested CF and normal fibroblasts, exogenously supplied putrescine remained unchanged for at least 60 min. Some differences were observed in the response of CF and normal cells to environmental (media) changes.This research was supported by a grant from the Cystic Fibrosis Foundation and by a grant from the National Institutes of Health, Training Grant (GM01316 11 GNC).     

Identification of a defective cAMP-stimulated Cl- channel in cystic fibrosis fibroblasts

Cl- efflux from normal human fibroblasts is stimulated by elevation of cAMP and by elevation of intracellular free Ca2+. In both cases the stimulated Cl- transport occurs via electrically conductive pathways. In six lines of normal human fibroblasts, dibutyryl cAMP increased total Cl- efflux by an average of 13%. In six lines of fibroblasts from patients with cystic fibrosis, dibutyryl cAMP was without effect. The electrically conductive component of Cl- transport was increased an average of 30% by dibutyryl cAMP in normal cells and was unaffected by dibutyryl cAMP in cystic fibrosis cells. Stimulation of the Ca2+-sensitive Cl- channel by addition of A23187 increased Cl- efflux by an average of 30% in normal and 30% in cystic fibrosis fibroblasts. The data indicate that there is a defect in a cAMP-activated Cl- channel in cystic fibrosis fibroblasts.     

Localization of the sulfate/anion exchanger in the rat liver

Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.     

K dependence of the Na-K pump is abnormal in erythrocytes from patients with cystic fibrosis and obligate heterozygotes

The affinity of the Na-K pump for K was significantly (P less than .001) lower in erythrocytes from patients with cystic fibrosis (Km 4.6 +/- 0.35 mM; n = 26) or from heterozygotes (Km 3.9 +/- 0.57 mM; n = 12) than in controls (Km 2.2 +/- 0.10 mM; n = 20). The affinity of the Na-K pump for K was lower in normal erythrocytes than in normal fibroblasts which may explain the variability in the severity of involvement of different organs in cystic fibrosis. We have now shown in human skin fibroblasts and erythrocytes, that the K affinity of the Na-K pump is lower in patients with cystic fibrosis than in controls. Since the abnormality is also present in erythrocytes from heterozygotes who are clinically normal, it is likely that this abnormality is closely related to the genetic defect in cystic fibrosis.     

The response of chloride transport to cyclic AMP, calcium and hypotonic shock in normal and cystic fibrosis fibroblasts

It has widely been established that Cl- transport is defective in cystic fibrosis fibroblasts. In the present study, the effect of elevation of intracellular concentration of cyclic AMP and calcium on the efflux of Cl- from human fibroblasts has been investigated. Cyclic AMP analogs (8-bromo cAMP and dibutyryl cAMP) and a beta agonist (isoproterenol) induced only a weak stimulation (5-10%) of Cl- efflux. Conversely, elevation of cytoplasmic calcium concentration produced by addition of the Ca2+ ionophore A23187 in the efflux medium, did not affect Cl- efflux. Our data indicate that the response of Cl- efflux to elevation of cAMP and calcium is similar in normal and cystic fibrosis fibroblasts. Exposure to hypotonic medium induced a significant stimulation of Cl- efflux in fibroblasts from both normal and cystic fibrosis individuals. Substitution of Cl- in the medium by gluconate and the subsequent addition of furosemide did not inhibit the effect of hypotonicity, indicating the involvement of a conductive pathway for Cl- transport, which was insensitive to oligomicin C.     

Assimilatory sulfur metabolism in marine microorganisms: characteristics and regulation of sulfate transport in Pseudomonas halodurans and Alteromonas luteo-violaceus.

Sulfate transport capacity was not regulated by cysteine, methionine, or glutathione in Pseudomonas halodurans, but growth on sulfate or thiosulfate suppressed transport. Subsequent sulfur starvation of cultures grown on all sulfur sources except glutathione stimulated uptake. Only methionine failed to regulate sulfate transport in Alteromonas luteo-violaceus, and sulfur starvation of all cultures enhanced transport capacity. During sulfur starvation of sulfate-grown cultures of both bacteria, the increase in transport capacity was mirrored by a decrease in the low-molecular-weight organic sulfur pool. Little metabolism of endogenous inorganic sulfate occurred. Cysteine was probably the major regulatory compound in A. luteo-violaceus, but an intermediate in sulfate reduction, between sulfate and cysteine, controlled sulfate transport in P. halodurans. Kinetic characteristics of sulfate transport in the marine bacteria were similar to those of previously reported nonmarine systems in spite of significant regulatory differences. Sulfate and thiosulfate uptake in P. halodurans responded identically to inhibitors, were coordinately regulated by growth on various sulfur compounds and sulfur starvation, and were mutually competitive inhibitors of transport, suggesting that they were transported by the same mechanism. The affinity of P. halodurans for thiosulfate was much greater than for sulfate.     

Sulfate transport in human placenta: further evidence for a sodium-independent mechanism

Sulfate transport in isolated placental brush-border membrane vesicles has properties consistent with an anion exchange process. To ascertain the relevance of this finding to sulfate accumulation by the fetus and placenta in vivo, we examined sulfate transport in human placental tissue slices, comparing sulfate uptake with that of a non-metabolizable amino acid marker, alpha-aminoisobutyrate (AIB). In contrast to AIB, which was actively concentrated from physiological media, sulfate uptake by the placenta slice was concentrative only in the absence of sodium and at low pH. Uptake of sulfate reached a steady state after 60 min. It was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a specific inhibitor of anion transport, but not by ouabain. We found no evidence for Na(+)-dependent uptake of sulfate in incubated placental tissue. It seems unlikely that Na(+)-dependent sulfate transport by the placenta can be responsible for net sulfate accumulation by the human fetus.     

Characteristics of assimilatory sulfate transport in Rhodobacter sulfidophilus

Abstract Sulfate uptake was investigated with four species of phototrophic sulfur bacteria. Rhodobacter sulfidophilus and Chromatium vinosum took up ³⁵S-labeled sulfate added in micromolar concentrations. Sulfate uptake by C. vinosum was expressed only under sulfate starvation. R. sulfidophilus took up 10 μM sulfate almost completely and accumulated it up to 5300-fold, also when grown with excess sulfate. Sulfite (1 mM) as an intermediate of sulfate assimilation inhibited sulfate uptake completely within 1 min. Moderate inhibition was observed with cysteine (1 mM) and none with sulfide (1 mM). Transport was not dependent on the cations K⁺, Na⁺, Li⁺ or protons, but was sensitive to uncouplers and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). The accumulation of sulfate correlated with the ATP concentration in the cells, indicating an ATP-dependent uptake mechanism.     

Electron paramagnetic resonance spectroscopy as a monitor of the pathway of the thermal unfolding of ribonuclease A.

α-L-Fucosidase activity is elevated in skin fibroblasts from cystic fibrosis patients when compared to controls. The activities of nine other acid hydrolases including neuraminidase are similar in cystic fibrosis and control fibroblasts. The relationship of these results to the recent finding of a decreased activity of α-L-fucosidase in the serum of cystic fibrosis patients is discussed. It is proposed that an abnormal distribution of α-L-fucosidase is involved in the pathogenesis of this disease.     

The Escherichia coli CysZ is a pH dependent sulfate transporter that can be inhibited by sulfite

The Escherichia coli inner membrane protein CysZ mediates the sulfate uptake subsequently utilized for the synthesis of sulfur-containing compounds in cells. Here we report the purification and functional characterization of CysZ. Using Isothermal Titration Calorimetry, we have observed interactions between CysZ and its putative substrate sulfate. Additional sulfur-containing compounds from the cysteine synthesis pathway have also been analyzed for their abilities to interact with CysZ. Our results suggest that CysZ is dedicated to a specific pathway that assimilates sulfate for the synthesis of cysteine. Sulfate uptake via CysZ into E. coli whole cells and proteoliposome offers direct evidence of CysZ being able to mediate sulfate uptake. In addition, the cysteine synthesis pathway intermediate sulfite can interact directly with CysZ with higher affinity than sulfate. The sulfate transport activity is inhibited in the presence of sulfite, suggesting the existence of a feedback inhibition mechanism in which sulfite regulates sulfate uptake by CysZ. Sulfate uptake assays performed at different extracellular pH and in the presence of a proton uncoupler indicate that this uptake is driven by the proton gradient.     

The Km of NADH dehydrogenase is decreased in mitochondria of cystic fibrosis cells

The kinetic properties of the NADH dehydrogenase of the mitochondrial respiratory chain, assayed as NADH-dependent rotenone-sensitive cytochrome c reductase have been studied in mitochondria isolated from mononuclear white blood cells in patients affected by cystic fibrosis. Data reported here show that the apparent Km of the enzyme for NADH is significantly decreased in cystic fibrosis mitochondria. These findings are independent of the age or the clinical state of the disease and have also been obtained with mitochondria isolated from cultured skin fibroblasts. These observations support the notion that cystic fibrosis is possibly accompanied by alterations of intracellular membranes and these are evident also in circulating cells and cultured fibroblasts.     

Insulin-induced insulin resistance of alpha-aminoisobutyric acid transport in cultured human skin fibroblasts.

Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin.     

Surface enzymes in cultured fibroblasts from cystic fibrosis patients.

Membrane function was examined in cultured cells from cystic fibrosis patients by assaying several enzymes on intact skin fibroblasts attached to culture dishes. This technique required few cells and minimized disruption of cellular organization. Comparison of enzyme activities of intact and broken cells showed that 12% of total glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was measurable using intact cells, while all adenosine monophosphatase was measurable using intact cells. Alkaline paranitrophenylphosphatase activity was divided between the cell surface and interior. Substrate competition experiments indicated that substrate specificities for adenosine monophosphatase and paranitrophenylphosphatase activities were different. Adenosine monophosphatase activities of 2 control and 2 cystic fibrosis strains fluctuated similarly during the cell culture cycle. The apparent Km values relative to adenosine monophosphate were similar in all strains. A chromatographic fraction of serum from a cystic fibrosis patient that was inhibitory to oyster ciliary activity had no effect on adenosine monophosphatase activity of normal fibroblasts. Furthermore, fractions of media from cystic fibrosis homozygote and heterozygote fibroblast cultures were not inhibitory to adenosine monophosphatase activities of intact normal fibroblasts or of part iculate fractions prepared from them. In light of previous studies that showed that factors from cystic fibrosis serum of culture medium disrupted specific membrane activities, it is proposed that the cystic fibrosis factor interacts with the plasma membrane, interfering most conspicuously with the protein functions that are sensitive to changes in their membrane environment.