scholarly journals Parathyroid hormone transport effects and hormonal processing in primary cultured rat proximal tubular cells

1993 ◽  
Vol 293 (2) ◽  
pp. 377-380 ◽  
Author(s):  
R M O'Donovan ◽  
C C Widnell ◽  
T C Chen ◽  
J B Puschett
Keyword(s):  
Parathyroid Hormone ◽  
Specific Binding ◽  
Primary Cultures ◽  
Subcellular Fractionation ◽  
Phosphate Transport ◽  
Surface Binding ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Culture Models ◽  
Proximal Tubular

The development of satisfactory cell culture models for the study of parathyroid hormone (PTH)-induced inhibition of Pi transport has proven difficult. Using subcellular fractionation techniques we investigated the response of primary cultures of rat proximal tubular cells to PTH-(1-34). Specific binding of 125I-bPTH-(1-34) occurred at 2 degrees C. After 5 min of rewarming, trypsin-releasable radioactivity decreased from 90 to 50%, indicating internalization of the ligand. Cell disruption, followed by density centrifugation with 17% Percoll either directly after binding at 2 degrees C or post-rewarming for 20 min, showed a shift of 125I label from the plasma membrane (5′-nucleotidase) to lysosomal fractions (beta-D-glucosaminidase), confirming the sequential occurrence of cell surface binding, internalization and transport to lysosomes of 125I-bPTH-(1-34). Reculture at 37 degrees C revealed steady accumulation of trichloroacetic acid-soluble radioactivity in the medium, indicating degradation of 125I-bPTH-(1-34). Phosphate transport in the absence of sodium was minimal. Incubation of the cells with bPTH-(1-34) resulted in up to 50% inhibition of sodium-dependent phosphate transport. Prior phosphate depletion abrogated the response to PTH.

Carbohydrate metabolism by primary cultures of rabbit proximal tubules

1989 ◽  
Vol 256 (3) ◽  
pp. C532-C539 ◽  
Author(s):  
M. J. Tang ◽  
K. R. Suresh ◽  
R. L. Tannen
Keyword(s):  
Pyruvate Kinase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Glycolytic Metabolism ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular

Renal proximal tubular epithelia were used to assess the factors responsible for the induction of glycolysis in cultured cells. Primary cultures of rabbit proximal tubules, which achieved confluency at 6 days, exhibited hormonal responsiveness and brush-border characteristics typical of proximal tubular cells. Beginning at day 4, these cultured cells exhibited increased glycolytic metabolism reflected by enhanced glucose uptake and lactate production, along with parallel increases in activity of the glycolytic enzymes, pyruvate kinase and lactate dehydrogenase. The gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and fructose-1,6-bisphosphatase (FDP), were downregulated, and the cultured cells exhibited lower oxygen consumption rates than fresh tubules. Cells grown on a rocker, to mitigate hypoxia, exhibited a metabolic and enzymatic profile similar to cells grown under still conditions. ATP levels in cultured cells were higher than in fresh tubules. Furthermore, pyruvate kinase activity was higher in cells grown in media containing 0.5 as contrasted with 25 mM glucose. The enhanced glycolytic metabolism exhibited by cultured proximal tubular cells appears to be a characteristic of proliferation and is not a response to hypoxia, the Pasteur effect, or environmental glucose.

Biochemical and morphological effects of gentamicin in human proximal tubular cells: effects on lipid and lipoprotein metabolism

1987 ◽  
Vol 65 (12) ◽  
pp. 1049-1056
Author(s):  
Subroto Chatterjee ◽  
Anna L. Trifillis ◽  
Annette L. Regec
Keyword(s):  
Lipid Metabolism ◽  
Lipoprotein Metabolism ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Surface Binding ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Cell Surface Binding ◽  
Proximal Tubular ◽  
And Control

The effects of gentamicin, an antibiotic used extensively for antimicrobial therapy on the ultrastructure, binding, internalization, degradation, and cholesterol esterification of low-density lipoproteins, were investigated in cultured human proximal tubular cells. Cells were incubated with 0.3 mM gentamicin for 21 days with the following observations. Cells treated with gentamicin contained numerous "myeloid bodies." The binding, internalization, and degradation of 125I-labeled low-density lipoproteins ([125I]LDL) in cells treated with gentamicin was twofold lower than control cells. Pulse–chase experiments demonstrated that gentamicin did not impair the internalization of receptor-bound LDL and their subsequent transport to the lysosome. The relative amounts of [125I]LDL displaced by increasing concentrations of unlabeled LDL were the same in both gentamicin-treated and control cells. This pattern was reflected in the cell surface binding, internalization, and degradation of [125I]LDL. Gentamicin did not alter the degradation of [125I]LDL in cell homogenates at 4.0. The data suggest that gentamicin decreases me receptor-mediated endocytosis of LDL and subsequent lipid metabolism.

Effect of Ifosfamide Metabolites on Sodium-Dependent Phosphate Transport in a Model of Proximal Tubular Cells (LLC-PK1) in Culture

1993 ◽  
Vol 16 (6) ◽  
pp. 285-298 ◽  
Author(s):  
M. Mohrmann ◽  
A. Pauli ◽  
H. Walkenhorst ◽  
B. Schönfeld ◽  
M. Brandis
Keyword(s):  
Phosphate Transport ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Sodium Dependent ◽  
Proximal Tubular

Cytoplasmic calcium in individual proximal tubular cells in culture

1986 ◽  
Vol 251 (5) ◽  
pp. F938-F944 ◽  
Author(s):  
M. S. Goligorsky ◽  
D. J. Loftus ◽  
K. A. Hruska
Keyword(s):  
Parathyroid Hormone ◽  
Calcium Concentration ◽  
Complete Recovery ◽  
Calcium Transients ◽  
Cytoplasmic Calcium ◽  
Free Medium ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Detailed Technique

The development of high quantal yield Ca2+-sensitive fluorescent indicators has made microspectrofluorometric monitoring of cytoplasmic calcium feasible. The detailed technique to monitor cytoplasmic calcium concentration in individual proximal tubular cells grown on glass cover slips is described. Manipulations of cytoplasmic calcium concentration by means of a Ca2+ ionophore or Ca2+-free medium resulted in corresponding changes of fura-2 fluorescence. Parathyroid hormone elicited a fivefold increase in cytoplasmic calcium concentration, with the subsequent complete recovery of this parameter in 3 min. This effect of parathyroid hormone was abolished by perfusion of the cells with Ca2+-free medium. Repeated pulses of parathyroid hormone spaced at an interval of 20 min, caused refractoriness of adenosine 3',5'-cyclic monophosphate response, whereas cytoplasmic calcium transients remained unaltered. When the frequency of the sequential pulses with parathyroid hormone was increased (5 min intervals), the amplitude of calcium transients was diminished, and the recovery of basal level of cytoplasmic calcium was incomplete and protracted. These observations may have application to disordered renal cell calcium metabolism in hyperparathyroid states.

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