Insulin stimulates phosphate transport in opossum kidney epithelial cells

1990 ◽  
Vol 258 (6) ◽  
pp. F1592-F1598 ◽  
Author(s):  
M. I. Abraham ◽  
J. A. McAteer ◽  
S. A. Kempson
Keyword(s):  
Insulin Action ◽  
Phosphate Uptake ◽  
Phosphate Transport ◽  
In Vivo Studies ◽  
Epithelial Cell Line ◽  
Protein Synthesis Inhibitors ◽  
Renal Epithelial Cell ◽  
Opossum Kidney ◽  
Ok Cells

Insulin is antiphosphaturic in vivo and this effect is due, in part, to increased Na(+)-dependent phosphate uptake across the luminal brush-border membrane of the proximal tubule. The intracellular mechanism is not understood. The present study shows that the stimulatory effect of insulin on phosphate transport can be reproduced in opossum kidney (OK) cells, suggesting that this established renal epithelial cell line may be a good model for further studies on insulin action on renal phosphate transport. The stimulation by insulin was dose related when insulin was used at concentrations within the range of 10(-14) to 10(-8) M. At 10(-8) M, insulin had no effect on Na(+)-independent uptake of phosphate or on the Na(+)-dependent uptakes of methyl-alpha-D-glucopyranoside and glutamate. The onset of insulin action on phosphate uptake was detected within 15 min, and the stimulation was reversed completely within 30 min after removal of insulin from the medium. Insulin action was not blocked by protein synthesis inhibitors and was not altered by bacitracin, an inhibitor of intracellular degradation of insulin. Pretreatment with the calcium-channel blockers, nifedipine and verapamil (10(-4) M), produced significant increases in the stimulatory effect of insulin, suggesting indirectly that insulin action on phosphate uptake may be influenced by Ca2+. In contrast to in vivo studies, there was no evidence that insulin interfered with parathyroid hormone action on OK cells.

scholarly journals Locally formed 5-hydroxytryptamine stimulates phosphate transport in cultured opossum kidney cells and in rat kidney

1996 ◽  
Vol 320 (2) ◽  
pp. 615-621 ◽  
Author(s):  
Zakia HAFDI ◽  
Sylvianne COUETTE ◽  
Etienne COMOY ◽  
Dominique PRIE ◽  
Claude AMIEL ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Mrna Level ◽  
Phosphate Transport ◽  
Inhibitory Effect ◽  
Acid Decarboxylase ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Opossum Kidney ◽  
Amino Acid Decarboxylase ◽  
Ok Cells

Renal proximal tubular cells have been shown to express aromatic l-amino acid decarboxylase (l-AAAD), which converts l-dopa into dopamine and 5-hydroxytryptophan [(OH)Trp] into 5-hydroxytryptamine (5-HT; serotonin). Because 5-HT receptors have been demonstrated in proximal cells, we hypothesized that 5-HT may act as an autocrine/paracrine modulator of proximal transport. We evaluated this possibility in opossum kidney (OK) cells, a renal epithelial cell line with a proximal phenotype expressing 5-HT1B receptors, and in intact anaesthetized rats. 5-HT synthesis by OK cells increased with incubation time and (OH)Trp concentration, and was abolished by benserazide, an l-AAAD inhibitor. 5-HT reversed parathyroid hormone (PTH)-induced cAMP accumulation in a pertussis toxin-sensitive manner and reduced the PTH inhibition of Pi uptake without affecting the NaPi-4 mRNA level. The effects of 5-HT on cAMP generation and Na–Pi co-transport were reproduced by (OH)Trp, except in the presence of benserazide, and by l-propranolol and dihydroergotamine, two 5-HT1B receptor agonists. In rats, (OH)Trp and dihydroergotamine decreased fractional Pi excretion. Benserazide abolished the effect of (OH)Trp but not that of dihydroergotamine. In conclusion: (i) locally generated 5-HT blunts the inhibitory effect of PTH on Na–Pi co-transport in OK cells; (ii) endogenous 5-HT decreases Pi excretion in rats; and (iii) 5-HT is a paracrine modulator involved in the physiological regulation of renal Pi transport.

scholarly journals Thyroid hormone stimulates the renal Na/H exchanger NHE3 by transcriptional activation

1999 ◽  
Vol 276 (1) ◽  
pp. C102-C108 ◽  
Author(s):  
Adriana Cano ◽  
Michel Baum ◽  
Orson W. Moe
Keyword(s):  
Thyroid Hormone ◽  
Transcriptional Activation ◽  
Transcript Abundance ◽  
Epithelial Cell Line ◽  
Protein Abundance ◽  
Renal Epithelial Cell ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Transcript Stability ◽  
Renal Epithelial Cell Line

Thyroid hormone stimulates renal proximal tubule NaCl and NaHCO3 absorption in part by activating the apical membrane Na/H exchanger NHE3. We used a renal epithelial cell line, the opossum kidney (OK) cell, to define the mechanism by which 3,5,3′-triiodothyronine (T3) increases NHE3 activity. T3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or translation. The increase in activity was associated with increases in steady-state cell surface and total cellular NHE3 protein and NHE3 transcript abundance. T3stimulated transcription of the NHE3 gene and had no effect on NHE3 transcript stability. The transcriptional activity of the 5′-flanking region of the rat NHE3 gene was stimulated by T3 when expressed in OK cells. When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells, T3 has no effect on rat NHE3 protein abundance, suggesting the absence of regulation of NHE3 protein stability or translation. These studies demonstrate that T3 stimulates NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance.

Brefeldin A inhibits phosphate transport in opossum kidney cells

1993 ◽  
Vol 264 (1) ◽  
pp. C40-C47 ◽  
Author(s):  
A. W. Capparelli ◽  
M. C. Heng ◽  
L. Li ◽  
O. D. Jo ◽  
N. Yanagawa
Keyword(s):  
Protein Synthesis ◽  
Golgi Apparatus ◽  
Phosphate Uptake ◽  
Brefeldin A ◽  
Phosphate Transport ◽  
Transport Processes ◽  
Free Medium ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Phosphate Deprivation

Brefeldin A (BFA) is a fungal metabolite that blocks the transport processes between the endoplasmic reticulum and the Golgi apparatus. In the present study, we have tested the effect of BFA on phosphate transport in a kidney epithelial cell line, opossum kidney (OK) cells. Electron microscopy showed that exposure of OK cells to BFA caused a rapid and reversible disorganization of Golgi apparatus. Addition of BFA also caused a time (2-8 h)- and dose (1-10 micrograms/ml)-dependent inhibition of Na(+)-dependent cell phosphate uptake. The inhibition of cell phosphate uptake by BFA was reversible and was associated with a decrease in the maximum velocity of phosphate transport. Both the inhibition and the stimulation of cell phosphate uptake by parathyroid hormone and insulin, respectively, were not affected by BFA. BFA at 1 microgram/ml concentration did not affect protein synthesis as determined by [3H]leucine incorporation but diminished the adaptive increase in cell phosphate uptake in response to 2 or 8 h of incubation in nominally phosphate-free medium. On the other hand, inhibition of protein synthesis by cycloheximide (5 microM) abolished the adaptive increase in cell phosphate uptake in response to 8 but not 2 h of incubation in nominally phosphate-free medium, indicating the existence of an early response to phosphate deprivation, which does not require new protein synthesis but is sensitive to the effect of BFA. In summary, results of these studies show that, in OK cells, BFA inhibits phosphate uptake and curtails the adaptive response to phosphate deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

Electrophysiological Measurements of a Toad Renal Epithelial Cell Line (A6) as an Assay for Predicting Ocular Eye Irritancy

1992 ◽  
Vol 20 (2) ◽  
pp. 218-221
Author(s):  
Henning F. Bjerregaard
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
South African ◽  
Mdck Cells ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Electrophysiological Measurements ◽  
Renal Epithelial Cell Line ◽  
Darby Canine Kidney

An established epithelial cell line (A6) from a South African clawed toad (Xenopus laevis) kidney was used as a model for the corneal epithelium of the eye in order to determine ocular irritancy. When grown on Millipore filter inserts, A6 cells form a monolayer epithelium of high electrical resistance and generate a trans-epithelial potential difference. These two easily-measured electrophysiological endpoints showed a dose-related decrease after exposure for 24 hours to seven selected chemicals of different ocular irritancy potential. It was demonstrated that both trans-epithelial resistance and potential ranked closely with in vivo eye irritancy data and correlated well (r = 0.96) with loss of trans-epithelial impermeability of Madin-Darby canine kidney (MDCK) cells, detected by use of a fluorescein leakage assay.

Prior heat stress enhances survival of renal epithelial cells after ATP depletion

1996 ◽  
Vol 270 (6) ◽  
pp. F1057-F1065 ◽  
Author(s):  
Y. H. Wang ◽  
S. C. Borkan
Keyword(s):  
Heat Stress ◽  
Epithelial Cells ◽  
Recovery Period ◽  
Atp Depletion ◽  
Protective Effects ◽  
Atp Content ◽  
Renal Epithelial Cells ◽  
Opossum Kidney ◽  
Ok Cells

The 72-kDa heat stress protein (HSP-72) is an inducible cytoprotectant protein. Although transient renal ischemia in vivo induces HSP-72, it is not known whether prior heat stress protects renal epithelial cells from injury mediated by ATP depletion. To evaluate this hypothesis, opossum kidney (OK) cells were exposed to sodium cyanide and 2-deoxy-D-glucose in the absence of medium glucose, a maneuver that reduced cell ATP content to < 10% of the control value within 10 min and decreased cell survival. One day after 2 h of ATP depletion, OK cells previously exposed to heat stress (to induce accumulation of HSP-72) exhibited marked improvement in survival (a > 4-fold increase in total DNA), less uptake of vital dye, and less release of lactate dehydrogenase (LDH) than cells subjected to ATP depletion alone (23.0 +/- 1.6 vs. 34.1 +/- 1.2% of total LDH, respectively). Enhanced clonogenicity post-heat stress was completely prevented by cycloheximide and positively correlated with the steady-state content of HSP-72. In the recovery period after ATP depletion, cell ATP content, maximum mitochondrial ATP production rate, and total LDH activity were all significantly higher in cells with abundant HSP-72. Although the protective effects associated with heat stress are likely to be multifactoral, preserved cell metabolism and higher ATP content could enhance cellular repair processes after ATP depletion.

A dual mechanism for regulation of kidney phosphate transport by parathyroid hormone

1987 ◽  
Vol 253 (2) ◽  
pp. E221-E227 ◽  
Author(s):  
J. A. Cole ◽  
S. L. Eber ◽  
R. E. Poelling ◽  
P. K. Thorne ◽  
L. R. Forte
Keyword(s):  
Protein Kinase C ◽  
Parathyroid Hormone ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Rank Order ◽  
Phosphate Transport ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Camp Formation

Regulation of phosphate transport by parathyroid hormone (PTH) was investigated in continuous lines of kidney cells. Phosphate transport was reduced by PTH-(1-34) at physiological concentrations (EC50 5 X 10(-11) M), whereas much higher concentrations were required to stimulate cAMP formation (EC50 1 X 10(-8) M) in opossum kidney (OK) cells. The PTH analogue [Nle]PTH-(3-34) also inhibited phosphate transport but did not enhance cAMP formation. Instead, [Nle]PTH-(3-34) was a competitive antagonist of PTH-(1-34) at cyclase-coupled receptors. PTH-(7-34) had no effect on phosphate transport or cAMP formation. Phorbol esters or mezerein were potent inhibitors of phosphate transport but did not affect cAMP synthesis. Their potencies paralleled the rank-order potency of these agents as activators of protein kinase c in other systems. Maximally effective concentrations of PTH-(1-34) and mezerein did not produce additive inhibition of phosphate transport in OK cells. Phorbol esters stimulated phosphate transport in JTC-12 cells, but PTH-(1-34) had no effect. We concluded that PTH regulates OK cell phosphate transport by interacting with two classes of receptors, and transmembrane-signaling mechanisms. Physiological levels of PTH-(1-34) may regulate phosphate transport by activation of protein kinase c, whereas higher concentrations appear to activate adenylate cyclase.

Clonal sublines that are morphologically and functionally distinct from parental OK cells

1989 ◽  
Vol 256 (4) ◽  
pp. F672-F679 ◽  
Author(s):  
J. A. Cole ◽  
L. R. Forte ◽  
W. J. Krause ◽  
P. K. Thorne
Keyword(s):  
Cell Line ◽  
Phosphate Transport ◽  
Parental Line ◽  
Apical Surface ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Sodium Dependent ◽  
Clonal Lines ◽  
P Cells ◽  
Camp Formation

Three clonal subpopulations of opossum kidney (OK) cells were derived from the parental line. The distribution of apical microvilli suggested that the OK cell line was heterogeneous. The clonal OK sublines appeared homogeneous as reflected by microvilli, which were uniformly distributed on the apical surface. Parathyroid hormone (PTH), forskolin (FSK), and prostaglandin E1 (PGE1) increased adenosine 3',5'-cyclic monophosphate (cAMP) formation in OK cells and all of the clones. PTH inhibited sodium-dependent phosphate transport in parental cells and in OK/B and OK/P clones with maximal effects appearing at 4, 2, and 1 h, respectively. PTH had no effect on phosphate transport in OK/H cells. FSK inhibited phosphate transport in parental cells and OK/B and OK/P clones but was relatively ineffective in OK/H cells. PGE1 decreased phosphate transport in OK/B and OK/P cells but was ineffective in the parental line and in OK/H cells. Phorbol 12-myristate 13-acetate, a potent inhibitor of phosphate transport in the parental OK cell line, had little effect in the clonal sublines. These clonal lines have remained phenotypically stable for 10 passages and should prove useful in studying the regulation of phosphate transport by PTH as well as addressing the question of whether PTH receptor subclasses exist which couple to cAMP and/or calcium effector systems in kidney cells.

Autocrine/paracrine regulation of renal Na(+)-phosphate cotransport by dopamine

1993 ◽  
Vol 264 (4) ◽  
pp. F618-F622 ◽  
Author(s):  
R. P. Glahn ◽  
M. J. Onsgard ◽  
G. M. Tyce ◽  
S. L. Chinnow ◽  
F. G. Knox ◽  
...  
Keyword(s):  
In Vivo Model ◽  
Acid Decarboxylase ◽  
System Support ◽  
Opossum Kidney ◽  
Amino Acid Decarboxylase ◽  
Ok Cells ◽  
Proximal Tubular ◽  
Phosphate Cotransport

We tested the hypothesis that dopamine (DA) acts as an autocrine/paracrine regulator of Na(+)-Pi symport in proximal tubules, using opossum kidney (OK) cells as an in vivo model. Both DA and parathyroid hormone (PTH) increased adenosine 3',5'-cyclic monophosphate (cAMP) and inhibited Na(+)-gradient-dependent uptake of 32P but not that of L-[3H]-alanine. Incubation of OK cells with L-dopa, a DA precursor, resulted in accumulation of DA (7.4 nM), a ninefold increase of cAMP in the medium, and an inhibition (-10%) of Na(+)-Pi uptake. Carbidopa, an inhibitor of aromatic-L-amino acid decarboxylase, prevented the formation of DA from L-dopa, the increase in cAMP, and the inhibition of Na(+)-Pi cotransport. Pi-replete OK cells produced more DA (+15%) from L-dopa than Pi-deprived cells; however, the endogenous DA inhibited Na(+)-Pi cotransport both in Pi-deprived and in Pi-replete cells. Thus OK cells can synthesize DA from L-dopa in a quantity sufficient to elicit both the maximum DA-stimulated cAMP accumulation and inhibition of Na(+)-Pi cotransport in the same cell population. Our data, obtained on an in vitro system, support the hypothesis proposing that DA generated in proximal tubular cells can modulate, via cAMP, the Na(+)-Pi symport in the same or adjacent cells. If present in the kidney, this pathway might represent an autocrine/paracrine system that can contribute to regulation of renal Pi homeostasis.

scholarly journals Extracts from tumors causing oncogenic osteomalacia inhibit phosphate uptake in opossum kidney cells

2001 ◽  
Vol 169 (3) ◽  
pp. 613-620 ◽  
Author(s):  
KB Jonsson ◽  
M Mannstadt ◽  
A Miyauchi ◽  
IM Yang ◽  
G Stein ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Phosphate Transport ◽  
P Uptake ◽  
Low Molecular Weight ◽  
Kidney Cells ◽  
Camp Accumulation ◽  
Oncogenic Osteomalacia ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Hypophosphatemic Osteomalacia

In oncogenic osteomalacia (OOM), a tumor produces an unknown substance that inhibits phosphate reabsorption in the proximal tubules. This causes urinary phosphate wasting and, as a consequence, hypophosphatemic osteomalacia. To characterize this poorly understood biological tumor activity we generated aqueous extracts from several OOM tumors. Extracts from three of four tumors inhibited, dose- and time-dependently, (32)P-orthophosphate uptake by opossum kidney (OK) cells; maximum inhibition was about 45% of untreated control. Further characterization revealed that the factor is resistant to heat and several proteases, and that it has a low molecular weight. The tumor extracts also stimulated cAMP accumulation in OK cells, but not in osteoblastic ROS 17/2.8 and UMR106 cells, or in LLC-PK1 kidney cells expressing the parathyroid hormone (PTH)/PTH-related peptide receptor or the PTH-2 receptor. HPLC separation of low molecular weight fractions of the tumor extracts revealed that the flow-through of all three positive tumor extracts inhibited (32)P uptake and stimulated cAMP accumulation in OK cells. Additionally, a second peak with inhibitory activity on phosphate transport, but without cAMP stimulatory activity, was identified in the most potent tumor extract. We have concluded that several low molecular weight molecules with the ability to inhibit phosphate transport in OK cells can be found in extracts from OOM tumors. It remains uncertain, however, whether these are related to the long-sought phosphaturic factor responsible for the phosphate wasting seen in OOM patients.

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