Biomedical subjects

F G Toback

Publications and source records attributed to F G Toback.

At least 55 records · Page 3Linked to original sources

PubMed1988

Sequence of a human kidney cDNA clone encoding thymosin beta 10.

We have isolated a cDNA clone encoding the 43 amino acid protein, thymosin beta 10, from an adult human kidney cDNA library. A comparison of human and rat thymosin beta 10 cDNA sequences revealed 100% identity for the deduced amino acid sequence and 95% nucleotide identity for the coding region. The thymosin beta 10 cDNA clone was used to probe RNA isolated from monkey kidney epithelial cells of the BSC-1 line. The probe recognized 850 bp and 2.4 kb transcripts in quiescent cells which indicates that the gene is constitutively expressed. The abundance of the 2.4 kb transcript was markedly increased one hour after serum stimulation and declined progressively to a basal value at 24 hours.

Adult↗

PubMed1988

Aberrant responses to growth-regulatory signals by variant kidney epithelial cells.

Cultures that achieved a higher cell density than expected were noted during study of growth regulation in monkey kidney epithelial cells of the BSC-1 line. Multiplication of the variant cells was accelerated, compared with parental cells, as the cultures approached confluence. Cytogenetic analysis, immunofluorescence antibody reactions with specific monkey serum, isoenzyme analysis, microbiological studies, and lack of growth in soft agar indicated that the variant cells were not a contaminating cell type, lacked new isoenzymes, were free of microbial contamination, and were not transformed. Confluent variant cultures did not respond to a purified growth inhibitor protein produced by BSC-1 cells that inhibits multiplication and reduces cell Na content in subconfluent variant and parental cells. Vasopressin, which is a mitogen for parental cells, was a potent growth inhibitor for confluent cultures of variant cells. Low-K or high-Na media, which stimulate proliferation of parental cells, had no effect on growth of the variant cell line. These results suggest that enhanced multiplication of the variant cells is mediated by altered signal transduction pathways and/or receptors for growth-regulatory molecules.

Animals↗

PubMed1988

Kidney epithelial cells express c-sis protooncogene and secrete PDGF-like protein.

Nontransformed monkey kidney cells (BSC-1 line), used as a model for renal epithelium, were assayed for release of platelet-derived growth factor (PDGF)-like proteins. BSC-1 cells continuously released a mitogenic activity for fibroblasts and a chemoattractant activity for smooth muscle cells, each of which was inhibited 80-90% by an antibody to human PDGF. A cDNA probe for the PDGF B-chain gene (c-sis), but not for the A-chain gene, hybridized to mRNA obtained from growing and quiescent cells. c-sis gene expression and PDGF-like protein secretion were studied in the presence of known growth-regulatory molecules. A secreted BSC-1 cell protein identical to transforming growth factor beta 2 inhibited DNA synthesis in growing cultures and induced marked accumulation of c-sis mRNA without a corresponding increase in the release of PDGF-like activity. Adenosine diphosphate stimulated DNA synthesis in quiescent cultures and enhanced both c-sis expression and release of PDGF-like activity. However, growing and quiescent cells did not express the PDGF receptor gene or exhibit a mitogenic response to authentic PDGF. Thus the PDGF-like protein released by these kidney epithelial cells could contribute to growth control by a paracrine mechanism.

Animals↗

PubMed1988

Kinetics of a novel cytosolic protein during the onset of renal epithelial cell growth.

Exposure of monkey kidney epithelial cells (BSC-1 line) to medium with a reduced K concentration (3.2 mM) stimulated growth and transiently activated glyceraldehyde-3-phosphate dehydrogenase (G3PD). The increase in enzyme activity was mediated by a cytosolic modifier protein that was purified using affinity and size-exclusion chromatography, and anion-exchange high-performance liquid chromatography. The apparent molecular mass of the protein was 62 kDa. A monospecific antibody to the protein was prepared from rabbit antiserum and used as an immunoprobe. Immunocytochemical staining and Western blotting revealed that the protein was a normal constituent of the cytosol and that it accumulated in cells exposed to low-K medium. A quantitative enzyme-linked immunosorbent assay showed that the amount of modifier protein increased progressively for up to 2 h in cells exposed to low-K medium, and then returned to the control value, a kinetic profile similar to that observed for G3PD activity. These results indicate that the modifier protein is a constituent of renal epithelial cells and accumulates transiently in the cytosol where it could regulate G3PD activity during the onset of growth induced by the low-K mitogenic signal.

Animals↗

PubMed1987

ADP activates protooncogene expression in renal epithelial cells.

Purine nucleotides, particularly adenosine diphosphate (ADP), are the most potent mitogens known for monkey kidney epithelial cells of the BSC-1 line. To explore the molecular mechanisms by which ADP exerts its mitogenic effect, we tested the hypothesis that stimulation of DNA synthesis in these renal cells is mediated by activation of protooncogenes. Transcripts of the c-Ha-ras protooncogene were identified in quiescent, high density cells. Addition of ADP to the culture medium stimulated protooncogene expression fourfold. Maximal expression of c-ras was observed by 12 h after exposure to ADP, and preceded the initiation of DNA synthesis. Expression of the c-myc protooncogene was not detected in unstimulated cells, but accumulated maximally after 1 h of exposure to ADP. As with ADP-stimulated expression of the c-ras protooncogene, transcripts of the transferrin receptor gene reached a maximal value at 12 h, whereas the abundance of gamma-actin mRNA was not altered for up to 24 h. The results indicate that exogenous ADP stimulates protooncogene expression before initiation of DNA synthesis in renal epithelial cells in culture. These findings suggest that some physiological effects of this adenine nucleotide could be mediated by proteins specified by protooncogenes.

Actins↗

PubMed1986

Lowering extracellular Na+ concentration releases autocrine growth factors from renal epithelial cells.

Sodium influx is an important early signal during the onset of mitogenesis in many types of cells. From this observation, one would predict that a decrease in extracellular Na+ concentration might retard cell proliferation. We tested this prediction by exposing sets of cultures of monkey kidney epithelial cells (BSC-1 line) to medium with progressively reduced concentrations of Na+, and we measured the effect on cell multiplication. Unexpectedly, a reduction of the Na+ concentration from 155 mM (control) to 130 mM stimulated proliferation of epithelial cells but not of fibroblasts. Exposure of BSC-1 cells to low Na+ medium for 5 min was sufficient to commit them to accelerated growth. Further study revealed that the cells released two growth factors during this period: anionic proteins with apparent molecular weights of 6200 and 9000 whose properties differ from those of other known growth factors. Thus, a reduction in extracellular Na+ concentration apparently signaled the rapid release of autocrine growth factors that stimulate renal epithelial cell multiplication.

Animals↗

PubMed1985

Extracellular potassium modifies the structure of kidney epithelial cells in culture.

In animals fed a K-deficient diet, alterations in kidney cell structure and function occur in association with changes in the ionic composition of the extracellular fluid. The hypothesis that the extracellular K concentration mediates these changes in renal tissue was tested in cultures of monkey kidney epithelial cells (BSC-1 line) by reducing the K concentration of the culture medium from the control value of 5.4 to 3.2 mM. Exposure of BSC-1 cells to low-K medium raised the maximal rate of uptake for L-glutamic acid by 39% without a change in apparent Km. To determine whether this alteration in plasma membrane function had a structural correlate, studies of the cell surface were performed using scanning and transmission electron microscopy. Morphometric analysis of scanning electron micrographs revealed that the number of microvilli per cell per unit surface area was 45% greater in cells exposed to low-K medium for 3 min than those exposed to control medium. This observation was confirmed by transmission electron microscopy. The results indicate that an alteration in the extracellular K concentration per se can modify specific structural and functional characteristics of kidney epithelial cells.

Animals↗

PubMed1985

Regulation of glyceraldehyde-3-phosphate dehydrogenase by a cytosolic protein.

Stimulation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity and accelerated growth occur in cultures of monkey kidney epithelial cells (BSC-1 line) that are exposed to medium with a reduced K concentration (3.2 mM). We recently found that this activation of G3PD was mediated by the appearance of a new cytosolic protein with an apparent molecular weight of 62,000. G3PD and this modifier protein were isolated from BSC-1 cells, and the interaction between them was characterized to define the mechanism(s) of enzyme activation. The enzyme protein was purified from cells grown in control medium (5.4 mM K). The enzyme, in the presence of modifier, exhibited an increase in maximal rate of enzyme reaction and a decrease in the apparent Km for NAD+. Analysis using Dixon plots revealed that the presence of modifier increased the Ki for NADH by two- to threefold. Inhibition by NADH was competitive with respect to NAD+, glyceraldehyde-3-phosphate, and inorganic phosphate. ATP also inhibited enzyme activity in a competitive manner with respect to NAD+; however, the Ki for ATP was similar both in the presence and absence of modifier. These results suggest that one mechanism by which the cytosolic modifier protein stimulates G3PD activity is to decrease product inhibition by NADH.

Adenosine Triphosphate↗

PubMed1985

Purine nucleotides stimulate DNA synthesis in kidney epithelial cells in culture.

Adenine nucleotides infused into animals with acute renal failure appear to enhance recovery of kidney function and structure. To determine whether these compounds could act by a direct effect on renal cell metabolism, their capacity to stimulate DNA synthesis was evaluated in cultures of monkey kidney epithelial cells (BSC-1 line). AMP and ADP enhanced DNA synthesis by threefold more than was previously observed with other mitogens for these cells. Guanosine and inosine and their nucleotides and adenosine and ATP were also mitogenic but to a lesser extent, whereas pyrimidine derivatives were ineffective. In the presence of AMP, autoradiography of [3H]thymidine-labeled cells indicated that a greater number of cells entered the S phase of the cell cycle, and assessment of cell number revealed increased multiplication. The mitogenic effect of adenine nucleotides was not reproduced by agents that raise the cellular content of cAMP and was serum independent. Adenine nucleotides did not alter DNA synthesis when added to cultures of mouse fibroblasts. These results indicate that provision of exogenous purine nucleosides and nucleotides stimulate DNA synthesis in renal epithelial cells in culture.

Adenine Nucleotides↗

PubMed1984

Cell growth and net Na+ flux are inhibited by a protein produced by kidney epithelial cells in culture.

Proliferation of confluent kidney epithelial cell cultures (BSC-1 line) is inhibited by a protein (Mr approximately equal to 24,000) that is secreted by the cells. The mechanism of action of this growth inhibitor was sought by studying its effect on net Na+ flux because increased availability of Na+ in the culture medium had been shown to stimulate cell growth. The increase in cell Na+ content observed during stimulation of the growth after a medium change was attenuated in the presence of the purified inhibitor. Inhibition of both cell Na+ accumulation and growth in the presence of the protein was reversed completely by addition of NaCl to the medium. These results suggest that control of net Na+ flux and growth in kidney epithelial cells could be mediated, at least in part, by a secreted cellular protein.

Animals↗

PubMed1984

Growth of kidney epithelial cells in culture: evidence for autocrine control.

The factors that stimulate kidney growth in K+-deficient animals are unknown. Cultures of renal epithelial cells (BSC-1 line) were used to study this phenomenon because their growth is accelerated in medium containing a reduced K+ concentration. We tested the hypothesis that growth induced by low-K+ medium is mediated by factors produced by the cells; i.e., is subject to autocrine control. Low-K+ (3.2 mM) or control (5.4 mM) medium was conditioned by placing it on confluent cultures of BSC-1 cells for 1 h and was then collected. The K+ concentration of the low-K+ conditioned medium was then adjusted to the control value by addition of KCl. This conditioned medium stimulated growth of fresh cultures of cells to the same extent as did unconditioned low-K+ medium. The appearance of growth-promoting activity was maximal at a K+ concentration of 3.2 mM during conditioning of the medium. Low-K+ conditioned medium, corrected to a K+ concentration of 5.4 mM, required 6 h to commit cells to enhanced proliferation. Growth-stimulating activity in low-K+ conditioned medium was antagonized by a purified growth inhibitor produced by the cells. These observations are consistent with the hypothesis that autocrine products with opposite effects on growth can regulate proliferation of renal epithelial cells.

Animals↗

PubMed1984

Stimulation of DNA synthesis in kidney epithelial cells in culture by potassium.

The hypothesis that the K+ concentration of extracellular fluid is a determinant of renal DNA synthesis was examined in quiescent, high-density cultures of monkey kidney epithelial cells of the BSC-1 line. The addition of KCl to the medium increased the number of cells engaged in DNA synthesis in a concentration-dependent manner. The capacity of K+ to stimulate DNA synthesis in a greater number of cells was additive with exogenous NaCl and calf serum and was associated with an increment in the steady-state cell K+ content. Studies with other monovalent cations indicated that the stimulatory effect of K+ on DNA synthesis was not mediated by increments in the chloride concentration or osmotic pressure of the medium. The addition of K+ to confluent cultures was associated with a concentration-dependent increase in cell multiplication. The commitment of cells to increased multiplication required exposure of the culture to added KCl for longer than 3 but not more than 6 h. Addition of KCl to cultures of mouse fibroblasts did not alter DNA synthesis, multiplication, or cell K+ content. These observations indicate that increased availability of K+ in the extracellular fluid can stimulate DNA synthesis in kidney epithelial cells in culture.

Animals↗

PubMed1984

Na regulates growth of kidney epithelial cells induced by lowering extracellular K concentration.

Accelerated kidney growth and increased tissue Na content have been observed in rats fed a K-deficient diet. These observations suggest that enhanced Na influx could mediate renal growth, a hypothesis that was tested in cultures of kidney epithelial cells of the BSC-1 line. Reduction of the K concentration in the culture medium from 5.4 to 3.2 mM augmented cell growth and induced a transient increase in the cellular content of Na and a decrease in that of K. That low-K-induced growth was Na dependent was shown by decreasing the medium Na concentration from 155 to 150 mM, which abolished the increases in both growth and cell Na content in a concentration-dependent manner. The stimulation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity that occurs in cells exposed to low-K medium for 1 h was similarly prevented by decreasing the medium Na concentration. Thus decreased availability of extracellular Na prevented the increase in cell Na content, stimulation of G3PD activity, and accelerated growth induced by low-K medium. The hypothesis was also tested by adding vasopressin to cultures of BSC-1 cells exposed to low-K medium; the hormone prevented the increments in cell Na content, G3PD activity, and growth to the same extent as did decreased availability of extracellular Na. These results are consistent with the interpretation that transient accumulation of Na is a critical determinant of the initiation of kidney epithelial cell growth.

Animals↗

PubMed1984

Phosphatidylcholine metabolism during renal growth and regeneration.

Phosphatidylcholine, the most abundant phospholipid in renal cellular membranes, is synthesized predominantly via the Kennedy pathway in normal and growing kidney tissue. Augmented biosynthesis of phosphatidylcholine is one of the earliest responses to growth signals in renal cells. During potassium depletion, regeneration after acute tubular necrosis, and compensatory growth after uninephrectomy increased membrane phosphatidylcholine biosynthesis precedes the appearance of new organelles and surface structures and the onset of cell division. The increment in phosphatidylcholine biosynthesis in the growing kidneys of potassium-depleted rats appears to be mediated by enhanced cellular uptake of the precursor choline and activation of the enzyme cytidine diphosphocholine:1,2-diacylglycerol cholinephosphotransferase. Specific amino acids, cations, and polyamines can modify the activity of this microsomal enzyme in normal and growing renal cells. Phospholipase A also plays a regulatory role in phosphatidylcholine metabolism because inhibition of this catabolic enzyme favors phospholipid accretion and kidney growth during potassium depletion, whereas stimulation of the enzyme leads to brisk phospholipid breakdown and a decrease in tissue mass during potassium repletion.

Acute Kidney Injury↗

PubMed1983

Appearance of a cytosolic protein that stimulates glyceraldehyde-3-phosphate dehydrogenase activity during initiation of renal epithelial cell growth.

Rats fed a K-deficient diet exhibit accelerated kidney growth and enhanced activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase, EC 1.2.1.12). Cultures of renal epithelial cells (BSC-1 line) served as a model for this phenomenon because a reduction in the medium K concentration from 5.4 to 3.2 mM resulted in augmented growth and increased G3PD activity. Mixing the soluble supernatant fraction from cells grown in low-K medium (3.2 mM) with that from cells grown in control medium indicated that the cytosol of low-K cells contained a positive modifier of G3PD activity. Appearance of modifier activity that occurred within 1 hr after exposure of cells to low-K medium was blocked by cycloheximide but not by actinomycin D. Modifier activity was also observed in mouse fibroblasts stimulated by low-K medium and in proliferating renal medullary tissue from rats fed a K-deficient diet. A single protein that contained G3PD-stimulating activity was isolated from the soluble supernatant of BSC-1 cells exposed to low-K medium. This protein was not detectable in control cells. The material yielded a single band on NaDodSO4/polyacrylamide gel electrophoresis with an apparent Mr of 62,000. These results suggest that a new protein with the capacity to stimulate G3PD activity appears in the cytosol during the initiation of cell growth.

Animals↗

PubMed1983

Kidney epithelial cell growth is stimulated by lowering extracellular potassium concentration.

The factors that induce kidney growth in K+-depleted animals are unknown. To determine if the low extracellular fluid K+ concentration could act as a growth stimulus, cultures of monkey kidney epithelial cells from the BSC-1 line were studied in media with a low-K+ concentration. Growth of confluent cultures was accelerated maximally at a K+ concentration of 3.2 mM, whereas concentrations of 2.9 and 3.5 mM were also stimulatory but to a lesser extent. Because growing renal tissue from K+-depleted rats was previously found to exhibit increased uptake of nutrient molecules, evidence for enhanced uptake was sought in BSC-1 cells after exposure to low-K+ medium. The uptake of 10 different nutrient molecules was enhanced in cells exposed to low-K+ medium for 30 s. These observations indicate that a reduced extracellular K+ concentration per se stimulates proliferation of renal epithelial cells in culture and could be one of the factors that mediate kidney growth in K+-depleted animals.

Amino Acids↗

PubMed1983

Vasopressin stimulates growth of renal epithelial cells in culture.

The hypothesis that arginine vasopressin could regulate kidney epithelial growth by its effect on Na+ transport was examined in cultures of cells from the BSC-1 line. Addition of vasopressin (75 pg/ml) or NaCl (25 mM) to the medium stimulated growth of confluent cultures but retarded growth of sparse cells in the presence of 0.5% calf serum. Thus the capacity of vasopressin or exogenous NaCl to regulate growth of BSC-1 cells was cell density dependent. Vasopressin stimulated growth of confluent cultures only in the narrow concentration range of 50-100 pg/ml (approximately 10(-10)M), whereas concentrations of 10 pg/ml and 125-1,000 pg/ml had no effect. In contrast, vasopressin at or above concentrations of 10 pg/ml raised cell Na+ content to its maximal value, which indicated that the hormone could increase the Na+ content of cells without necessarily stimulating their growth. To determine if vasopressin modulates growth by acting on the plasma membrane, nutrient transport and ligand binding were assessed in high-density quiescent cultures. The hormone augmented uptake of alpha-aminoisobutyric acid and binding of epidermal growth factor, whereas the addition of NaCl (25 mM) did not. Thus growth stimulation by vasopressin was associated with increased cell Na+ content, enhanced uptake of an amino acid, and augmented binding of a growth factor. These observations suggest that the growth-promoting effect of vasopressin is not a simple function of its capacity to alter cell Na+ flux but could be mediated by other actions of the hormone, perhaps at the level of the plasma membrane.

Animals↗

PubMed1983

Amino acid administration enhances renal protein metabolism after acute tubular necrosis.

The capacity of exogenous amino acids to alter renal protein metabolism was studied during renal regeneration after mercuric chloride-induced acute tubular necrosis in the rat. In regenerating cortical tissue, the free leucine concentration was 17% lower than normal, and was decreased further after glucose infusion. The concentration was raised above normal by amino acid infusion thereby ameliorating the deficit of this amino acid. Synthesis and degradation of rapidly-turning over proteins in renal cortical cells was examined in vitro. Renal protein synthesis in cortical slices was assessed by measurements of tissue leucine specific radioactivity and cycloheximide-inhibitable [14C]leucine incorporation into protein. Protein synthesis in regenerating tissue was 52% higher than normal and was not increased further by glucose infusion. In contrast, amino acid infusion increased the rate 47% above that observed after an isocaloric glucose infusion, thereby demonstrating that amino acid enhancement of protein synthesis is superimposed upon the increased synthetic rate observed during renal regeneration. Renal protein degradation remained at the normal rate after amino acid infusion, but was increased in regenerating tissue and after glucose infusion. These results indicate that infused amino acids act on the kidney to enhance protein synthesis and reduce protein degradation in regenerating renal cells after acute tubular necrosis.

Acute Kidney Injury↗