Differences between OK and LLC-PK1 cells: cystine handling

1991 ◽  
Vol 261 (1) ◽  
pp. C8-C16 ◽  
Author(s):  
B. States ◽  
D. Harris ◽  
S. Segal
Keyword(s):  
Cell Lines ◽  
Apical Membrane ◽  
Reduced Glutathione ◽  
Basolateral Membrane ◽  
Growth Period ◽  
Transport Systems ◽  
Amino Acid Transport System ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Dibasic Amino Acid

Cultured opossum kidney (OK) and porcine kidney (LLC-PK1) cells were compared for biochemical characteristics and cystine transport systems. The cell lines differ in amount of protein per cell, with OK cells having approximately one-half the amount found in LLC-PK1. Both cell lines contain 19 micrograms DNA/10(6) cells. As cells reach confluence, cystine uptake increases in OK and decreases in LLC-PK1 cells. Throughout the growth period, only lysine inhibits cystine uptake in OK, whereas glutamate is the inhibitor in LLC-PK1. The predominant site of cystine transport in OK cells is across the apical membrane, and the basolateral membrane is the corresponding site of transport in LLC-PK1 cells. Although the intracellular reduced glutathione pool is the same, the cysteine pool in OK cells is approximately one-fourth that found in LLC-PK1 cells. The ability of OK cells to reflect the shared cystine-dibasic amino acid transport system and LLC-PK1 to exhibit the cystine-glutamate antiporter system makes available two models for investigation of the development and structure of cystine transport systems.

Intracellular compartmentation of organic anions within renal cells

1993 ◽  
Vol 264 (5) ◽  
pp. R882-R890 ◽  
Author(s):  
D. S. Miller ◽  
D. E. Stewart ◽  
J. B. Pritchard
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Organic Anions ◽  
Epifluorescence Microscopy ◽  
Renal Cells ◽  
Bladder Epithelium ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Intracellular Compartments ◽  
Energy Dependent

Epifluorescence microscopy and video-image analysis were used to measure the distribution of the monovalent organic anion fluorescein (FL) within the cells of three organic anion-secreting renal epithelia: crab urinary bladder (a proximal tubule analogue), opossum kidney (OK) cells in culture, and intact teleost proximal tubules. In all three preparations the intracellular FL distribution was nonuniform. Two distinct intracellular compartments were detected, one being diffuse and cytoplasmic and the other punctate. With low FL concentrations in the medium (1 microM and below) dye accumulation in the punctate compartment exceeded that of the cytoplasm. In crab bladder epithelium FL uptake into both compartments was inhibited by external probenecid, p-aminohippurate (PAH), and LiCl and stimulated by 10-50 microM external glutarate, suggesting that the punctate compartment loaded by a two-step mechanism: transport into the cytoplasm at the basolateral membrane, followed by accumulation at specific intracellular sites. Experiments in which FL was microinjected into OK cells directly demonstrated movement of FL from the cytoplasmic to the punctate compartment. Accumulation in the latter was specific, i.e., inhibitable by coinjected PAH and probenecid, and energy dependent. Together, these findings indicate that during secretion organic anions are sequestered within renal cells. The role of sequestration in overall transport remains to be determined.

Functional characterization of albumin binding to the apical membrane of OK cells

1996 ◽  
Vol 271 (2) ◽  
pp. F286-F291 ◽  
Author(s):  
M. Gekle ◽  
S. Mildenberger ◽  
R. Freudinger ◽  
S. Silbernagl
Keyword(s):  
Binding Site ◽  
Apical Membrane ◽  
Binding Capacity ◽  
Specific Binding ◽  
Functional Characterization ◽  
Cellular Protein ◽  
Albumin Binding ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Maximal Binding

We characterized binding of albumin to the apical membrane of opossum kidney (OK) cells using fluorescein isothiocyanate (FITC)-albumin (i.e., bovine serum albumin, BSA) as substrate. Functional analysis of binding data showed one specific binding site characterized by half-maximal binding (Michaelis constant, (Km) at 20 mg/l (300 nmol/l) and maximal binding capacity (Bmax) of 0.61 microgram/mg cellular protein. Excess of unlabeled albumin (BSA) inhibited binding at low concentrations of FITC-albumin completely but only partially at high concentrations. FITC-albumin binding was reversible and pH dependent. Km increased about sixfold when pH decreased from 7.4 to 5.0. The inhibitory effects of conalbumin, alpha-lactalbumin, and transferrin were significantly smaller compared with BSA. We conclude that OK cells express a high-affinity binding site for albumin on the apical membrane. This binding site is pH sensitive, binds albumin in the physiological range, and could be responsible for the effective receptor-mediated reabsorption of albumin in the proximal tubule.

Is leucine an allosteric modulator of the lysine transporter in the intestinal basolateral membrane?

1987 ◽  
Vol 253 (5) ◽  
pp. G637-G642 ◽  
Author(s):  
K. Lawless ◽  
D. Maenz ◽  
C. Cheeseman
Keyword(s):  
Amino Acid ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Low Concentrations ◽  
Carrier Mediated Transport ◽  
Order Of Magnitude ◽  
Dibasic Amino Acid ◽  
Voltage Clamping ◽  
Stimulation Of

The transport of the dibasic amino acid L-lysine was investigated using basolateral membrane vesicles prepared from rat jejunal mucosal scrapings. The majority of the carrier-mediated transport was unaffected by the presence of sodium in the incubation medium, but voltage clamping of the vesicles did increase lysine uptake, indicating an associated movement of charge. Kinetic analysis of lysine influx and efflux showed the system to be symmetrical, but although the Vmax was comparable to other amino acid transport systems in this membrane, the dissociation constant for the overall reaction (KT) was an order of magnitude larger. This low affinity for lysine would explain the relatively slow rate of transport of this amino acid across the basolateral membrane. Competition experiments indicated that this system has a relatively narrow specificity carrying only lysine, arginine, ornithine, and histidine. In contrast the presence of L-leucine caused a marked stimulation of lysine efflux and influx across the vesicles. This effect was observed with leucine concentrations as low as 0.1 microM. It is concluded that although the lysine transport system in the basolateral membrane is slow in its basal state it can be rapidly turned on by the presence of L-leucine. The remarkably low concentrations required to do this suggest a possible allosteric interaction between the transporter and this neutral amino acid.

Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells

1986 ◽  
Vol 251 (1) ◽  
pp. C23-C31 ◽  
Author(s):  
K. Malmstrom ◽  
H. Murer
Keyword(s):  
Parathyroid Hormone ◽  
Cell Lines ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Continuous Cell Lines ◽  
Ok Cells ◽  
Regulatory Cascade ◽  
Regulatory Event ◽  
Apical Membrane Vesicles

Na+-dependent phosphate transport and its response to parathyroid hormone (PTH) has been investigated in three continuous cell lines of renal epithelial origin (LLC-PK1, JTC-12.P3, and OK). The apparent Km for phosphate was similar, but the maximal transport rate (Vmax) was markedly different in the three cell lines. PTH and forskolin produced an increase of cellular adenosine 3',5'-cyclic monophosphate (cAMP) in all cell lines, but Na+-dependent phosphate transport was inhibited exclusively in the OK cells (a threefold reduction of influx after 4 h of exposure to 10(-10) M PTH). The change in phosphate transport is accounted for by a lowered Vmax (30.8 +/- 5.3 vs. 10.2 +/- 1.1 pmol X mg-1 X 3 min-1). The reduction in phosphate transport was reversible, such that 5 h after removal of PTH the Vmax had increased threefold over the inhibited state. Addition of PTH did not alter Na+-dependent L-alanine influx in the OK cells. Experiments with apical membrane vesicles showed that the change in Vmax occurred at the membrane level. It is concluded that the regulatory event responsible for PTH-reduced phosphate transport is beyond cAMP. Of the cell lines studied, only OK cells have a complete regulatory cascade.

Interaction of Cl- and other halogens with Cl- transport systems in rabbit cortical collecting duct

1992 ◽  
Vol 263 (5) ◽  
pp. F870-F877 ◽  
Author(s):  
S. Muto ◽  
M. Imai ◽  
Y. Asano
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cell Types ◽  
Transport Systems ◽  
Cortical Collecting Duct ◽  
Cl Transport ◽  
Distinct Cell ◽  
Cl Conductance ◽  
Basolateral Membrane Potential

We have reported that in the rabbit cortical collecting duct (CCD) we can identify electrophysiologically three distinct cell types; the collecting duct (CD) cell and the alpha- and beta-intercalated (IC) cell. To further characterize the Cl- transport properties of each cell type, we examined the interaction between Cl- and other halogens or SCN- in the isolated and perfused CCD by intracellular microelectrode impalement. The rapid depolarization of the basolateral membrane potential (VB) caused by replacement of bath Cl- with each anion revealed that the sequences of apparent halogen selectivity for the basolateral Cl- conductance were similar in all three cell types. The ranking of Cl- > Br- > F- > I- corresponds to the sequence 5 of Eisenman's series, indicating “strong” interaction of the anions with the selectivity site. The basolateral Cl- conductance of these three cell types may share common characteristics, although I- permeability is less in IC cells than in CD cells. Hyperpolarization of the basolateral membrane of the beta-IC cell upon reduction of luminal Cl- reflects alterations in either Cl- entry across the apical membrane, or Cl- exit across the basolateral membrane, or both. Luminal Cl- replacement with each anion showed that the sequence of the hyperpolarization of the basolateral membrane was I- >> cyclamate = SCN- > F- > Br-, suggesting that I-inhibits either apical Cl- entry or basolateral Cl- exit. On the other hand, in the CD cell reduction of the perfusate Cl- by replacement with each anion caused the basolateral membrane to hyperpolarize with a different ranking: cyclamate = F- > I- = SCN- > Br-.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual action of phosphonoformic acid on Na(+)-phosphate cotransport in opossum kidney cells

1992 ◽  
Vol 263 (2) ◽  
pp. F301-F310 ◽  
Author(s):  
M. Loghman-Adham ◽  
T. P. Dousa
Keyword(s):  
Prolonged Exposure ◽  
Apical Membrane ◽  
Competitive Inhibition ◽  
Membrane Vesicles ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Phosphonoformic Acid ◽  
Opossum Kidney Cells ◽  
Dependent Inhibition ◽  
Dual Action

Phosphonoformic acid (PFA, foscarnet) was found to exert both an inhibitory and a stimulatory effect on Na(+)-dependent Pi transport in opossum kidney (OK) cells. When added in the uptake media, PFA produced a dose-dependent inhibition of Na(+)-Pi cotransport. PFA had no effect on the Na(+)-dependent transports of methyl-alpha-D-glucopyranoside (AMG) or L-alanine or on amiloride-sensitive Na(+)-H+ antiport. The inhibition of Na(+)-Pi cotransport was competitive [inhibitory constant (Ki) = 6.0 mM], reversible by dilution, and solute specific. When OK cells were incubated with PFA for longer time periods (1–15 h), the Na(+)-Pi uptake measured after removal of PFA was significantly increased, i.e., “upregulated.” The extent of Na(+)-Pi cotransport upregulation was dependent on time (greater than or equal to 30 min) and dose of PFA (2–10 mM). The increase in Na(+)-Pi cotransport by upregulation with PFA was due to higher apparent Vmax with no change in apparent Michaelis constant (Km) for Pi and was solute specific: uptakes of AMG or L-proline were not changed. Removal of PFA from culture medium resulted in a fast reversal of upregulation. Upregulation was not inhibited by cycloheximide, actinomycin D, or cordycepin. Solute-specific increase of Na(+)-Pi cotransport was also found when measured in apical membrane vesicles isolated from OK cells. Thus PFA exerts a dual action on the Na(+)-Pi cotransporter of OK cells: 1) acute, competitive inhibition and 2) after prolonged exposure it increases Na(+)-Pi uptake, probably by insertion of Na(+)-Pi cotransporters into apical membrane.

Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in OK cells

2005 ◽  
Vol 289 (1) ◽  
pp. F154-F165 ◽  
Author(s):  
Sophia Y. Breusegem ◽  
Nabil Halaihel ◽  
Makoto Inoue ◽  
Hubert Zajicek ◽  
Eleanor Lederer ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Cholesterol Depletion ◽  
Moderate Increase ◽  
Protein Abundance ◽  
Lipid Microdomains ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Per Se

We previously showed an inverse correlation between membrane cholesterol content and Na-Pi cotransport activity during the aging process and adaptation to alterations in dietary Pi in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85–F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-Pi cotransport activity and apical membrane Na-Pi protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with β-methyl cyclodextrin (β-MCD) resulted in a significant increase in Na-Pi cotransport activity accompanied by a moderate increase in apical membrane Na-Pi protein abundance and no alteration of total cellular Na-Pi protein abundance. Conversely, acute cholesterol enrichment achieved with β-MCD/cholesterol resulted in a significant decrease in Na-Pi cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-Pi protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-Pi cotransport activity and apical membrane and total cellular Na-Pi protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-Pi cotransport activity by diverse mechanisms that also include significant interactions of Na-Pi protein with lipid microdomains.

Role of NH2 and COOH termini in targeting, stability, and activity of sodium bicarbonate cotransporter 1

2006 ◽  
Vol 291 (3) ◽  
pp. F588-F596 ◽  
Author(s):  
Doris Joy D. Espiritu ◽  
Angelito A. Bernardo ◽  
Jose A. L. Arruda
Keyword(s):  
Cell Membrane ◽  
Sodium Bicarbonate ◽  
Basolateral Membrane ◽  
Membrane Stability ◽  
Hek293 Cells ◽  
Opossum Kidney ◽  
Cell Membrane Stability ◽  
Ok Cells ◽  
Sodium Bicarbonate Cotransporter

Sodium bicarbonate cotransporter 1 (NBC1) mediates 80% of bicarbonate reabsorption by the kidney, but the molecular determinants for activity, targeting, and cell membrane stability are poorly understood. We generated truncation mutants involving the entire NH2 (ΔN424) or the entire COOH (ΔC92) terminus and examined the effects of these truncations on targeting, cell membrane stability, and NBC1 activity. ΔN424 and ΔC92 targeted to the plasma membrane of HEK293 cells or to the basolateral membrane of opossum kidney (OK) cells at 24 h but did not display NBC1 activity. Unlike the NBC1 wild-type and the ΔN424, ΔC92 expression was significantly decreased in the basolateral membrane at 48 h and yet the total ΔC92 expression in the cell was constant. We found that decreased ΔC92 expression in the basolateral membrane was due to increased endocytosis and mistargeting to the apical membrane. Increased endocytosis was prevented when both ΔN424 and ΔC92 were cotransfected together and more stable expression of ΔC92 was observed. Immunoprecipitation studies using NBC1 antibody specific for the COOH epitope were able to detect the COOH truncated NBC1 when probed with NH2 epitope-specific antibody or vice versa. Similar findings were observed with Ni-NTA pull-down assay. Cotransfection of both mutants partially restored NBC1 activity. In summary, NBC1 targets to the basolateral membrane of OK cells by a default mechanism and the COOH terminus plays a role on NBC1 stability in the basolateral membrane.

Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells

1989 ◽  
Vol 257 (6) ◽  
pp. E895-E902
Author(s):  
R. Rabkin ◽  
C. Yagil ◽  
B. Frank
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Insulin Receptors ◽  
Proximal Tubules ◽  
Time Dependent ◽  
Dependent Manner ◽  
Opossum Kidney ◽  
High Insulin ◽  
Receptor Number

In vivo, filtered insulin is absorbed and degraded in proximal tubules after binding to the apical membrane. Peritubular removal also occurs and involves basolateral receptor binding and degradation. Whether basolateral degradation proceeds within the cell or on the cell surface is unknown. Because of the difficulties in addressing this question in vivo, this study was carried out with a cultured opossum kidney epithelium cell line with proximal-like features and insulin receptors. Cells were grown in partitioned wells on polycarbonate filters and, when confluent, the monolayer effectively separated the culture well into apical and basolateral compartments. Apical and basolateral binding, internalization, and degradation were studied separately by incubating monolayers with 125I-insulin added to either the apical or basal compartment. At 37 degrees C insulin associated with either pole in a time-dependent manner. This interaction was specific, for it was competitively inhibited by cold insulin but not by unrelated peptides. Separation of surface-bound from internalized insulin was achieved by lowering extracellular pH. At 4 degrees C, 92% of the radioactivity added to either side of the monolayer was surface-bound, whereas at 37 degrees C and after 1 h, 57% was surface-bound and 43% internalized. Affinity of apical and basolateral receptors were similar (1-2 nM), but basolateral receptor number was greater, for at high insulin concentrations (5 x 10(-8) M) basolateral membrane binding exceeded apical by fivefold (250 +/- 81 vs. 56 +/- 11 fm/10(6) cells). Degradation followed exposure to either pole of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Dibutyryl cAMP activates bumetanide-sensitive electrolyte transport in Malpighian tubules

1991 ◽  
Vol 261 (3) ◽  
pp. C521-C529 ◽  
Author(s):  
J. L. Hegarty ◽  
B. Zhang ◽  
T. L. Pannabecker ◽  
D. H. Petzel ◽  
M. D. Baustian ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Ringer Solution ◽  
Malpighian Tubules ◽  
Membrane Voltage ◽  
Dibutyryl Camp ◽  
K Secretion ◽  
Transepithelial Voltage

The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.

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