Inhibition by glucocorticoids of phosphate transport in primary cultured renal cells

The regulation by glucocorticoids of phosphate transport in primary cultured chick renal cells was examined. Dexamethasone inhibited the Na+-dependent phosphate uptake system. Na+-independent phosphate uptake and Na+-dependent uptakes of alpha-methylglucoside and L-proline were unaffected. The mineralocorticoid aldosterone did not alter phosphate uptake. The inhibition of Na+-dependent phosphate uptake by dexamethasone was concentration-dependent, exhibited an induction period, was blocked by inhibitors of RNA and protein synthesis, and was rapidly reversed when the steroid was removed. Following reversal, the cells could respond a second time to the glucocorticoid. However, this time the response was rapid, could be evoked at least for 24 h after glucocorticoid withdrawal, and might be prevented by actinomycin D and cycloheximide. These findings demonstrate that glucocorticoids act on renal cells to modulate phosphate transport and suggest that the renal cell system provides an attractive model to examine the mechanism by which glucocorticoids control gene expression and regulate plasma membrane transport function.     

Arachidonic acid inhibits phosphate transport by cultured renal cells

Because arachidonic acid and its metabolites are reported to be intracellular messengers of various exogenous stimuli, we studied whether arachidonic acid influences phosphate transport by cultured mouse renal epithelial cells. Arachidonic acid, at 10(-7)-10(-4)M, inhibited phosphate transport without influencing cyclic adenosine 3':5'-monophosphate production. Nordihydroguaiaretic acid and indomethacin, inhibitors of arachidonic acid metabolism, did not cancel the arachidonic acid-induced inhibition of phosphate transport. Furthermore, unsaturated fatty acids other than arachidonic acid also inhibited phosphate transport and their inhibitory effect increased as the number of double bond increased. These data demonstrate that arachidonic acid inhibits the phosphate transport by the cultured renal epithelial cells, probably not via conversion to its metabolites.     

Stimulation of phosphate transport in rat-liver mitochondria by thyroid hormones

The effect of hyperthyroidism on the transport of phosphate in rat-liver mitochondria has been examined. Thyroid hormones administered in vivo increased carrier mediated (mersalyl-sensitive) phosphate transport. Kinetic analysis of the phosphate transport showed that the thyroid hormone affects the Vmax of this process, while having no effect on the Km values. The higher activity of the phosphate carrier was found not to be due to a change in the endogenous content of phosphate nor to a change in the transmembrane delta pH value. Inhibitor titrations with mersalyl showed that mitochondria from both control and hyperthyroid rats required the same concentrations of inhibitor to produce total inhibition of phosphate transport, thus suggesting that the amount of functional translocase present is unaffected. The level of cardiolipin was significantly higher in mitochondrial membranes from hyperthyroid rats as compared to the control rats. The thyroid hormone induced change in the activity of the phosphate carrier appears to be due to a more favorable lipid microenvironment (cardiolipin content) surrounding the carrier molecule in the mitochondrial membrane.     

Stimulation of glucose utilization and lactate production in cultured human fibroblasts by thyroid hormone

Human dermal fibroblasts were obtained by harvesting outgrowing cells from the dermal tissue explants and cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum. After the cells reached confluency, culture was continued in the medium containing calf serum which was deprived of thyroid hormone by the treatment with activated charcoal. These fibroblasts were responsive to exogeneously added thyroid hormone (triiodothyronine) at physiological concentrations, resulting in enhanced utilization of glucose and production of lactate. This stimulation by thyroid hormone was dependent upon the length of exposure to the hormone and its concentration.The hormone did not show any effects on cellular DNA and protein content. The experimental system described above seems to be easy to reconstitute and should be useful for the elucidation of the mechanism of thyroid hormone action.     

Stimulation of facilitated [3H]uridine transport by thyroid hormone in GH1 cells. Evidence for regulation by the thyroid hormone nuclear receptor

We have previously shown that 3,5,3'-triiodo-L-thyronine (L-T3) stimulates cell growth and a 4- to 8-fold increase in growth hormone mRNA in GH1 cells. These effects appear to be mediated by a thyroid hormone nuclear receptor with an equilibrium dissociation constant for L-T3 of 0.2 nM and an abundance of about 10,000 receptors per cell nucleus. In this report, we show that L-T3 exerts a pleiotypic effect on GH1 cells to rapidly (within 2 h) stimulate [3H]uridine uptake to a maximal value of 2.5- to 3-fold after 24 h. This results from an increase in the number of functional uridine "transport sites" as shown by studies documenting an increase in the apparent Vmax with no change in the Km, 17 microM. Although the labeling of the cellular uridine pool and pools of all phosphorylated uridine derivatives was increased by L-T3, there was no change in the relative amounts of the individual pools in cells incubated with or without hormone. The intracellular concentration of [3H]uridine was estimated to be similar to that of the medium, suggesting that facilitated transport mediates [3H]uridine uptake. That this increase in [3H]uridine transport was nuclear receptor-mediated is supported by the excellent correspondence of the L-T3 dose-response curve for [3H]uridine uptake and that for L-T3 binding to receptor. Finally, inhibition of protein synthesis by cycloheximide and RNA synthesis by actinomycin D demonstrated that the L-T3 effect required continuing protein and RNA synthesis. These results are consistent with an effect of the L-T3-nuclear receptor complex to increase uridine uptake in GH1 cells by altering the expression of gene(s) essential for the transport process.     

Long term regulation of nucleoside transport by thyroid hormone (T3) in cultured chromaffin cells

The adenosine transport in cultured chromaffin cells was increased by the presence of triiodo-l-thyronine (T₃) throughout the prolonged period studied. The V_max values of this transport obtained in absence and presence of 1 M T₃ were 36.21±2.1 and 44.17±3.5 (means±SD) pmol/10⁶cells/min respectively for 26 hours incubation-time with the hormone. The K_m values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by [³H]nitrobenzylthioinosine (NBTI) binding, was increased by 1 M T₃ for 26 hours incubation-time. The values of binding sites per cell were 33,500±3,000 and 40,153±3,700 in absence and presence of T₃ respectively, without changing the K_d constant. When the transport studies were carried out in presence of cycloheximide, an inhibitor of protein synthesis, the adenosine transport capacity decreased with a half-life values of 23.9±2.8 and 24.3±2.1 hours both in the presence or absence of T₃ respectively. When cells were incubated in the presence of both T₃ and cycloheximide, not only the activatory effect of T₃ was completely abolished but also adenosine transport was decreased to the same extent as with cycloheximide alone. These results indicated that T₃ activation of adenosine transport in chromaffin cells required the protein-synthesizing mechanism.     

Basolateral phosphate transport in renal proximal-tubule-like OK cells

It is generally assumed that phosphate (Pi) effluxes from proximal tubule cells by passive diffusion across the basolateral (BL) membrane. We explored the mechanism of BL Pi efflux in proximal tubule-like OK cells grown on permeable filters and then loaded with 32P. BL efflux of 32P was significantly stimulated (P < 0.05) by exposing the BL side of the monolayer to 12.5 mM Pi, to 10 mM citrate, or by acid-loading the cells, and was inhibited by exposure to 0.05 mM Pi or 25 mM HCO3; by contrast, BL exposure to high (8.4) pH, 40 mM K+, 140 mM Na gluconate (replacing NaCl), 10 mM lactate, 10 mM succinate, or 10 mM glutamate did not affect BL 32P efflux. These data are consistent with BL Pi efflux from proximal tubule-like cells occurring, in part, via an electro-neutral sodium-sensitive anion transporter capable of exchanging two moles of intracellular acidic H2PO4- for each mole of extracellular basic HPO4= or for citrate.     

Influence of follicle-stimulating hormone on glucose transport by cultured Sertoli cells

Transport of 3-O-methyl-D-[14C]glucose by Sertoli cells cultured in plastic dishes, is competitively inhibited by glucose (Ki 4 microM). The glucose analogue was therefore used to study glucose transport in these cells in which it is not metabolized. Addition of follicle-stimulating hormone (FSH) (10 micrograms/ml) or dibutyryl cyclic AMP (1 mM) to the cells, increases transport of methylglucose by Sertoli cells. The increased transport results from increased influx and involves decrease in Km without change in Vmax. These changes in the kinetics of transport are seen with both FSH and dibutyryl cyclic AMP. FSH does not stimulate transport of methylglucose in peritubular fibroblasts nor in germ cells. In view of the importance of lactate as a substrate for spermatids (Mita and Hall, 1982) it is proposed that stimulation of the transport of glucose by Sertoli cells in response to FSH is important in the increased production of lactate by these cells in response to FSH and hence is one mechanism by which the tropic hormone enables the Sertoli cell to promote spermatogenesis.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats

The transport of phosphate by primary cultures of renal cells from young (5-6 weeks) and adult (10-12 months) rats was studied. Renal tubule cells isolated from young and adult groups exhibited typical epithelial morphology and similar growth rates. The Na-dependent phosphate uptake was saturable with a Km of 5-7 microM over a substrate range of 1-500 microM. A decrease in Na-dependent phosphate uptake in adult cells (30%) was found compared to that of young cells. The Na-independent component of phosphate uptake did not vary with age. In addition, the inhibition of phosphate uptake by a variety of compounds (ouabain, gramicidin, 2,4-dinitrophenol, KCN, and arsenate) were similar in both age groups. Kinetic analysis showed that a significant reduction in Vmax (4.4 +/- 0.4 vs. 3.1 +/- 0.2 nmol Pi/mg protein/10 min in young and adult cells, respectively), but not Km, resulted in this decreased uptake of phosphate in adult groups. There was no difference in the efflux of phosphate from both age groups. When cells were preincubated in a phosphate-free medium for 24 hours, the uptake of phosphate was increased to 46% and 24% of their corresponding controls in young and adult cells, respectively. The decreased phosphate uptake and limited adaptation to a phosphate-free medium by the adult renal cells may account for the hypophosphatemia and phosphaturia seen in adult and old animals in vivo.     

Effects of confluence on phosphate transport capacity in cultured renal cell lines

The LLC-PK1 cell line transports phosphate (Pi), glucose, and amino acids using carriers similar to those in proximal tubular cells. Others have reported that when monolayers reach confluence, hexose transport increases and activity of the A-amino acid transporter falls. The present study evaluates Pi uptake by two continuous cell lines derived from renal proximal tubule, and demonstrates that phosphate uptake falls sharply upon reaching confluence in LLC-PK1 cells but not in cultured opossum kidney (OK) cells. The fall in Pi uptake in LLC-PK1 cells at confluence represents a halving in Vmax for Na-dependent phosphate uptake (2.33 vs. 5.00 nmol/mg protein/5 min) without a change in Km (82 vs. 94 microM). Suppression of phosphate transport in confluent monolayers of LLC-PK1 cells is completely reversed by bringing the cells into suspension. As has been shown for the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), exposure of monolayers to serum stimulates phosphate uptake, but unlike phorbol ester, serum does so without stimulating alanine uptake. OK cells differ from LLC-PK1 in that no change occurs in Pi uptake at confluence, although they resemble LLC-PK1 cells in that sugar uptake rises and alanine uptake falls at confluence. The different temporal patterns for Pi uptake in the two cell lines indicates that developmental change in the uptake of Pi is not linked to that of glucose or alanine.     

Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.     

Modulation of thyroid hormone nuclear receptors by cholera toxin in cultured GH1 cells

The cellular actions of the thyroid hormones L-thyroxine and L-triiodothyronine are mediated by the association of hormone with a chromatin-associated receptor. In cultured GH1 cells, a hormone-responsive rat pituitary cell line, thyroid hormone decreases the concentration of its receptor at early incubation times by reducing the accumulation of newly synthesized receptor. In this study, we demonstrate that cholera toxin also reduces the amount of nuclear receptor in GH1 cells in a time- and dose-dependent fashion, without altering the affinity of the receptor for hormone. The reduction of receptor mediated by cholera toxin is not secondary to a generalized inhibition of cell protein synthesis or cell replication rates and this effect can be abolished by pretreatment of the cholera toxin with soluble ganglioside II3-alpha-N- acetylneuraminosylgangliotetraosylceramide . This effect requires an intact cholera toxin molecule and does not occur at similar concentrations of the membrane-binding B subunit of cholera toxin. In order to study the influence of cholera toxin on thyroid hormone receptor turnover, we have used a dense amino acid-labeling technique. The results indicate that cholera toxin does not change the half-life of receptor, but decreases the rate of appearance of newly synthesized receptor. This decreased rate completely accounts for the lowered steady state receptor levels. The extent of cAMP stimulation by cholera toxin does not correlate with the extent of receptor reduction and forskolin, which stimulates cAMP 25- to 500-fold, does not decrease thyroid hormone receptor abundance. These studies suggest that cholera toxin modulates receptor levels by a mechanism(s) that is not mediated by cAMP in GH1 cells.     

Stimulation of glucose transport in osteoblastic cells by parathyroid hormone and insulin-like growth factor I

Insulin and parathyroid hormone (PTH) regulate glucose metabolism in bone cells. In order to differentiate between the effects of these hormones and to compare the potency of insulin with that of insulin-like growth factor (IGF) I, we treated rat bone-derived osteoblastic (PyMS) cells for different time periods and at different concentrations with insulin, IGF I, or PTH, and measured [1-(14)C]-2-deoxy-D-glucose (2DG) uptake and incorporation of D-[U-(14)C] glucose into glycogen. 2DG uptake was Na-independent with an apparent affinity constant (K (M)) of ~2 mmol/l. Expression of the high affinity glucose transporters (GLUT), GLUT1 and GLUT3 but not of GLUT4, was found by Northern and Western analysis. Similar to the findings with primary rat osteoblasts, but distinct from those in rat fibroblasts, 2DG uptake and glycogen synthesis were increased in this cell line after exposure to low concentrations (0.1 nmol/l and above) of PTH. IGF I at low doses (0.3 nmol/l and above) or insulin at higher doses (1 nmol/l and above) stimulated 2DG uptake and [(3)H] thymidine incorporation into DNA. 2DG transport was enhanced already after 30 min of IGF I treatment whereas the effect of PTH became significant after 6 h. It is concluded that IGF I rather than insulin may be a physiological regulator of 2DG transport and glycogen synthesis in osteoblasts.     

Stimulation of iodine organification in porcine thyroid cells by thyroid stimulators

Several Graves' sera were simultaneously assessed in a bioassay based on the ability of porcine thyroid cells to organify 125I and in a radioreceptor assay for TSH receptor binding activity. Both assay systems were sensitive to 1 mcU/ml (final concentration) of unlabelled bovine TSH. Six Graves' sera were studied in detail over a wide (0-1.0 mcl sera) dose response range in repeat determinations. Two sera exhibited parallel binding and stimulating. However, two sera revealed significant inhibition of 125I-TSH binding prior to the demonstration of stimulation and the other two sera showed stimulatory capabilities before significant binding was evident. IgG was prepared from one serum by ammonium sulphate precipitation and chromatography on Sepharose 6B and then subjected to preparative isoelectric focusing. The isoelectric distribution of the two activities were found to be identical with major peaks of activity at pl=9.5 and pl=8.5. In summary: 1) each Graves' sera exhibits different dose-response curves with respect to binding and stimulation, 2) at certain concentrations of sera, only binding or stimulation were evident, 3) neither assay was consistently more sensitive for the presence of Graves' immunoglobulins, 4) for one Graves' sera, binding and stimulation could not be separated by isoelectric focusing. These studies would suggest each Graves' immunoglobulin has inherently different characteristics in its interaction with the TSH receptor.