Studies on the use of cultured cells in a bioassay for parathyroid hormone

1994 ◽  
Vol 143 (2) ◽  
pp. 261-268
Author(s):  
A E Armston ◽  
P J Wood
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Cell Line ◽  
Cultured Cells ◽  
Biologically Active ◽  
Kidney Cell Line ◽  
Opossum Kidney ◽  
Pre Treatment ◽  
Set Up ◽  
Insufficient Sensitivity

Abstract Measurement of parathyroid hormone (PTH) is important for diagnosing hyper- and hypoparathyroidism. The move to two-site immunometric assays that detect the whole molecule has improved the discrimination of these conditions but these assays may be too restrictive because some PTH fragments that are biologically active may not be detected. In addition, PTH-like peptide of malignancy, an important cause of malignancy-associated hypercalcaemia, is not detected by the two-site assays. Experiments were performed to set up a simple, robust and inexpensive bioassay for PTH, exploiting a kidney cell line and using cyclic AMP or an eluted stain assay as the end point. Of the 12 cell lines tested, an opossum kidney (WOK) cell line showed the most promise. Despite optimization of the procedure to include pre-treatment with dexamethasone, insulin and PTH, followed by incubation in the presence of 5′ -guanylimidodiphosphate, isobutyl-1-methylxanthine and forskolin, the WOK cells showed insufficient sensitivity for use in a cultured cell bioassay for PTH in human serum. In addition, the cells were less sensitive to PTH-like peptide precluding their use for an assay for this molecule. Journal of Endocrinology (1994) 143, 261–268

Structure-activity relationships of parathyroid hormone analogs in the opossum kidney cell line

2009 ◽  
Vol 4 (5) ◽  
pp. 723-730 ◽  
Author(s):  
David L. Carnes ◽  
Judith A. Cole ◽  
Leonard R. Forte ◽  
Richard E. Poelling ◽  
Pamela K. Thorne ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Cell Line ◽  
Kidney Cell ◽  
Kidney Cell Line ◽  
Opossum Kidney ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Opossum Kidney Cell ◽  
Hormone Analogs

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone, cyclic AMP and phorbol esters

1990 ◽  
Vol 415 (4) ◽  
pp. 461-470 ◽  
Author(s):  
Corinna Helmle-Kolb ◽  
Marshall H. Montrose ◽  
Gerti Stange ◽  
Heini Murer
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Phorbol Esters ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells

Parathyroid hormone degradation by opossum kidney cells via receptor-mediated endocytosis and lysosomal hydrolysis

1992 ◽  
Vol 127 (3) ◽  
pp. 267-270 ◽  
Author(s):  
Toru Yamaguchi ◽  
Makoto Arao ◽  
Masaaki Fukase
Keyword(s):  
Parathyroid Hormone ◽  
Biologically Active ◽  
Main Role ◽  
Kidney Cells ◽  
Binding Studies ◽  
Opossum Kidney ◽  
Receptor Mediated Endocytosis ◽  
Opossum Kidney Cell ◽  
Opossum Kidney Cells

The mechanisms involved in parathyroid hormone (PTH) degradation by proximal renal tubule cells were studied using an opossum kidney cell line possessing PTH receptors as an in vitro model system. One hour incubation of 5 nmol/l human (h) PTH-(1-84) with intact opossum kidney cells (4.0× 106 cells) resulted in about 70% degradation and disappearance of hPTH-(1-84) from the medium, as determined by a two-site immunoradiometric assay. Preincubation with 100 nmol/l h[Nle8, Nle18, Tyr34]PTH-(1-34)amide for 6, 24, 48 and 72 h caused a 26, 47, 62 and 73% decrease, respectively, in PTH degradation by opossum kidney cells. Binding studies with 125I-labeled h[Nle8, Nle18, Tyr34]PTH-(1-34)amide as a radioligand showed that PTH receptor binding decreased with the time of pretreatment with the agonist. Pretreatments of the cells with monensin, an inhibitor of endocytosis, and the lysosomotropic agents such as chloroquine, ammonium chloride and leupeptin, inhibited degradation of hPTH-(1-84) by 87, 71, 76 and 72%, respectively. Concentrations of 5 nmol/l hPTH-(39-84) and hPTH-(39-68), which are known not to bind to PTH receptors appreciably, were not degraded by opossum kidney cells during 1 h incubations. Thus intact, biologically active PTH, but not its inactive fragments, is degraded by opossum kidney cells, by receptor-mediated endocytosis and lysosomal hydrolysis. A mechanism resembling the peritubular uptake of intact PTH by perfused kidneys reported previously appears to play a main role in PTH metabolism by cultured renal cells.

scholarly journals Relative sensitivity to inhibition by cimetidine and clonidine differentiates between the two types of Na+-H+ exchangers in cultured cells

1991 ◽  
Vol 280 (2) ◽  
pp. 317-322 ◽  
Author(s):  
S Ramamoorthy ◽  
C Tiruppathi ◽  
C N Nair ◽  
V B Mahesh ◽  
F H Leibach ◽  
...  
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Membrane Vesicles ◽  
Type A ◽  
Relative Sensitivity ◽  
Type B ◽  
Opossum Kidney ◽  
Cell Line Hela ◽  
Inhibition Studies

Available evidence indicates that there are two types of Na(+)-H+ exchangers, type A (housekeeping type) and type B (epithelial or apical type), in mammalian cells. We have recently reported, using isolated membrane vesicles, that these two types can be differentiated by their relative sensitivities to inhibition by clonidine and cimetidine [Kulanthaivel, Leibach, Mahesh, Cragoe & Ganapathy (1990) J. Biol. Chem. 264, 1249-1252]. The present study was undertaken to determine whether this approach is also effective in cultured cells. The JAR human placental choriocarcinoma cell line and the opossum kidney (OK) cell line, when grown as confluent monolayer cultures on an impermeable plastic support, express Na(+)-H+ exchanger activity which is measurable by determining Na+ uptake into the cells from the culture medium. The JAR cell Na(+)-H+ exchanger was found to be about 100 times more sensitive to inhibition by dimethylamiloride than the OK cell Na(+)-H+ exchanger. Inhibition studies with clonidine and cimetidine were able to differentiate between these two exchangers very clearly. Cimetidine was 18 times more potent than clonidine in inhibiting the JAR cell Na(+)-H+ exchanger. In contrast, clonidine was at least 8 times more potent than cimetidine in inhibiting the Na(+)-H+ exchanger of the OK cell. The results show that the JAR cell expresses the type A Na(+)-H+ exchanger, whereas the OK cell expresses the type B Na(+)-H+ exchanger. This approach also proved to be very effective in correctly identifying the type of Na(+)-H+ exchanger in a third cell line (HeLa). It is concluded that the relative susceptibility to inhibition by clonidine and cimetidine offers an easy and efficient means of differentiating between the two types of Na(+)-H+ exchangers in cultured cells.

Nuclear transplantation with melanophores, ciliated epidermal cells, and the established cell-line A-8 in Xenopus laevis

Development ◽  
1973 ◽  
Vol 29 (3) ◽  
pp. 539-547
Author(s):  
H. R. Kobel ◽  
R. B. Brun ◽  
M. Fischberg
Keyword(s):  
Cell Line ◽  
Cultured Cells ◽  
Important Change ◽  
Epidermal Cells ◽  
Nuclear Transplantation ◽  
Established Cell Line ◽  
Donor Nucleus ◽  
Established Cell ◽  
Developmental Programme ◽  
Set Up

The developmental potencies of melanophores, ciliated epidermal cells and cells from an established line were tested by means of nuclear transplantation into enucleated Xenopus eggs. Donor nuclei from ciliated epidermal cells of hatching tadpoles never gave rise, after transplantation, to development beyond the blastula stage; nuclei from non-ciliated cells of the same larvae, on the other hand, could fully replace a zygote nucleus. Melanophore nuclei taken from the tailfin of advanced larvae (stage 56) gave rise to blastulae and, in one case, an incipient invagination was observed. Melanophore nuclei taken from tissue cultures, set up from hatching tadpoles, proved to be competent for leading development as far as the heartbeat stage, (stage 33/34), the embryo showing well-elaborated eye Anlagen, muscles, notochord, cement gland, etc. Nuclei from the highly aneuploid established cell-line A-8 were able to give rise to hatching-stage embryos. These results suggest that some condition, provided by culturing, influences the differentiated state so as to improve the chances of the donor nucleus co-operating with the egg's developmental programme. The important change might be the state of proliferation attained in cultured cells.

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