Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods.

Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media.

Process parameters within a 750,000 litre anaerobic digester during a year of disturbed fermenter performance.

A 750,000litre fermenter was studied throughout one entire year by investigating the concentrations of volatile fatty acids (acetic, butyric, i-butyric, propionic, valeric and i-valeric acids), pH, concentrations of total C, N, S and NH(4)(+)-N, amounts of chemical and biological oxygen demand, and abundance of acetogenic microorganisms. Additionally several process parameters such as temperature, retention time, dry weight and input of substrate and liquids, and the concentrations and amounts of CH(4), H(2), CO(2) and H(2)S within the biogas were monitored continuously. Various volatile fatty acids and the ratio of acetic to propionic acid were shown to allow a rough indication on the fermentation but were not sufficiently precise to describe the fermenter performance. Nutrient compounds and special fractions, such as easily extractable carbohydrates or the concentration of total fats were more strongly correlated to the gas production of the fermenter. Results of an MPN-method for the determination of acetogenic microorganisms point to an important role of these microorganisms during the phase of restoration of the fermenter performance.

Effect of seasonal changes in quantities of biowaste on full scale anaerobic digester performance.

A 750,000l digester located in Roppen/Austria was studied over a 2-year period. The concentrations and amounts of CH4, H2, CO2 and H2S and several other process parameters like temperature, retention time, dry weight and input of substrate were registered continuously. On a weekly scale the pH and the concentrations of NH4+ -N and volatile fatty acids (acetic, butyric, iso-butyric, propionic, valeric and iso-valeric acid) were measured. The data show a similar pattern of seasonal gas production over 2 years of monitoring. The consumption of VFA and not the hydrogenotrophic CH4 production appeared to be the limiting factor for the investigated digestion process. Whereas the changes in pH and the concentrations of most VFA did not correspond with changes in biogas production, the ratio of acetic to propionic acid and the concentration of H2 appeared to be useful indicators for reactor performance. However, the most influential factors for the anaerobic digestion process were the amount and the quality of input material, which distinctly changed throughout the year.

The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture.

Fetal bovine serum (FBS) is a common supplement to in vitro culture media. A workshop was organized to discuss whether or not fetuses might suffer when blood is withdrawn, and to discuss serum replacement methods. When bovine fetuses are exposed after slaughter of the dam, they can suffer only if they inflate their lungs with air and increase their blood oxygen to levels compatible with awareness. Preventing fetuses from breathing air or killing them by an efficient method, according to clearly defined safeguards, ensures that fetal blood collection is humane. Since serum is a supplement of unknown composition, which could be contaminated with unwanted factors, there are scientific and safety reasons for omitting FBS from culture media. Several media have been developed in which minimal or no animal derived components are present. Also, different cell types have been adapted to serum-free media. As yet, no standard serum free media are present, and each cell type requires its own medium composition. Among other recommendations, the establishment of a public database with information on cell types and their serum-free medium composition is proposed.

pH regulation of renal gene expression.

The increase in intracellular pH (pHi) associated with various tumour cells triggers changes in gene expression. Similar adaptations also occur as part of the physiological response to changes in acid base balance. For example, during metabolic acidosis, increased renal ammoniagenesis and bicarbonate synthesis are sustained by the increased expression of various transport proteins and key enzymes of glutamine metabolism. In rat kidney, increased expression of the mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) results from stabilization of their respective mRNAs. The 3'-untranslated region (UTR) of the GA mRNA contains a direct repeat of an 8-base AU sequence that functions as a pH-response element. This sequence exhibits a high affinity and specificity for z-crystallin. The same protein binds to two separate, but homologous, 8-base AU sequences within the 3'-UTR of the GDH mRNA. The apparent binding activity of z-crystallin is increased significantly during onset of metabolic acidosis. Thus, increased binding of z-crystallin may initiate the pH-responsive stabilization of the two mRNAs. In contrast, induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene occurs at the transcriptional level. In LLC-PK1-FBPase+ kidney cells, a decrease in pHi leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of ATF-2. This transcription factor binds to the CRE-1 element within the promoter of the PEPCK gene to enhance transcription. Similar mechanisms may contribute to altered gene expression in tumour cells.

Mechanism of increased renal gene expression during metabolic acidosis.

Increased renal catabolism of plasma glutamine during metabolic acidosis generates two ammonium ions that are predominantly excreted in the urine. They function as expendable cations that facilitate the excretion of acids. Further catabolism of alpha-ketoglutarate yields two bicarbonate ions that are transported into the venous blood to partially compensate for the acidosis. In rat kidney, this adaptation is sustained, in part, by the induction of multiple enzymes and various transport systems. The pH-responsive increases in glutaminase (GA) and phosphoenolpyruvate carboxykinase (PEPCK) mRNAs are reproduced in LLC-PK(1)-fructose 1,6-bisphosphatase (FBPase) cells. The increase in GA activity results from stabilization of the GA mRNA. The 3'-untranslated region of the GA mRNA contains a direct repeat of an eight-base AU sequence that functions as a pH-response element. This sequence binds zeta-crystallin/NADPH:quinone reductase with high affinity and specificity. Increased binding of this protein during acidosis may initiate the pH-responsive stabilization of the GA mRNA. In contrast, induction of PEPCK occurs at the transcriptional level. In LLC-PK(1)-FBPase(+) kidney cells, a decrease in intracellular pH leads to activation of the p38 stress-activated protein kinase and subsequent phosphorylation of transcription factor ATF-2. This transcription factor binds to cAMP-response element 1 within the PEPCK promoter and may enhance its transcription during metabolic acidosis.

[Good cell culture practice (GCCP)--an initiative for standardization and quality control of in vitro studies. The establishment of an ECVAM Task Force on GCCP]. / Good Cell Culture Practice (GCCP)--eine Initiative zur Standardisierung und Qualitätssicherung von in vitro Arbeiten. Die Etablierung einer ECVAM Task Force on GCCP.

Cultured human and animal cells are increasingly used as the basis for simplified, direct test systems that have the potential to be more controllable and more reproducible than in vivo test systems. However, if a biological test system is simplified to fundamental levels then it is paramount that the essential components of such a reduced systems are closely defined and reproducible. Thus, minimal requirements for quality standards in cell and tissue culture have to be defined. It is the aim of this GCCP initiative to establish principles for standardisation, rationalisation, and international harmonisation of cell and tissue culture laboratory practices. Therefore, in analogy to Good Laboratory Practice (GLP), a Good Cell Culture Practice (GCCP) was initiated at the 3rd World Congress on Alternatives and Animal Use in the Life Sciences, Bologna, 29. August-2. September 1999. This "Bologna Statement on Good Cell Culture Practice" was presented, discussed, and refined in a Workshop, and a final version was approved at the closing ceremony of the Congress by the scientific audience. Based on the Bologna Statement, an ECVAM Task Force on GCCP was initiated, that is chaired by Thomas Hartung and Sandra Ceocke, in which experts in the field should elaborate minimal requirements for quality standards in cell culture. It is the intention of the GCCP Guidelines to encourage consensus among all concerned with the use of in vitro systems, in order to establish and maintain best laboratory practices, to promote effective quality control systems, to facilitate education and training, to support journal editors, and to help any authorities who need to interpret and apply conclusions based on in vitro data.

Differential expression and acid-base regulation of glutaminase mRNAs in gluconeogenic LLC-PK(1)-FBPase(+) cells.

LLC-PK(1)-FBPase(+) cells, which are a gluconeogenic substrain of porcine renal LLC-PK(1) cells, exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase (PDG) activity. On adaptation to acidic medium (pH 6.9, 9 mM HCO(-)(3)), LLC-PK(1)-FBPase(+) cells also exhibit a greater increase in ammonia production and respond with an increase in assayable PDG activity. The changes in PDG mRNA levels were examined by using confluent cells grown on plastic dishes or on permeable membrane inserts. The latter condition increased the state of differentiation of the LLC-PK(1)-FBPase(+) cells. The levels of the primary porcine PDG mRNAs were analyzed by using probes that are specific for the 5.0-kb PDG mRNA (p2400) or that react equally with both the 4.5- and 5.0-kb PDG mRNAs (p930 and r1500). In confluent dish- and filter-grown LLC-PK(1)-FBPase(+) cells, the predominant 4.5-kb PDG mRNA is increased threefold after 18 h in acidic media. However, in filter-grown epithelia, which sustain an imposed pH and HCO(-)(3) gradient, this adaptive increase is observed only when acidic medium is applied to both the apical and the basolateral sides of the epithelia. Half-life experiments established that induction of the 4. 5-kb PDG mRNA was due to its stabilization. An identical pattern of adaptive increases was observed for the cytosolic PEPCK mRNA. In contrast, no adaptive changes were observed in the levels of the 5. 0-kb PDG mRNA in either cell culture system. Furthermore, cultures were incubated in low-potassium (0.7 mM) media for 24-72 h to decrease intracellular pH while maintaining normal extracellular pH. LLC-PK(1)-FBPase(+) cells again responded with increased rates of ammonia production and increased levels of the 4.5-kb PDG and PEPCK mRNAs, suggesting that an intracellular acidosis is the initiator of this adaptive response. Because all of the observed responses closely mimic those characterized in vivo, the LLC-PK(1)-FBPase(+) cells represent a valuable tissue culture model to study the molecular mechanisms that regulate renal gene expression in response to changes in acid-base balance.

Impact of culture conditions, culture media volumes, and glucose content on metabolic properties of renal epithelial cell cultures. Are renal cells in tissue culture hypoxic?

When renal proximal tubular cells are brought into tissue culture, they revert from oxidative metabolism and gluconeogenesis to high rates of glycolysis. Among the factors possibly responsible for this metabolic conversion, limited oxygen availability and/or substrate supply are discussed. In order to study the role of these factors on long-term cultures, the impact of growth conditions, culture media volume, and glucose content on carbohydrate metabolism of the continuous renal cell lines LLC-PK(1) (porcine kidney) and OK (opossum kidney) was investigated. The impact of culture media volumes and glucose content, respectively, was determined by overlaying confluent monolayer cultures of LLC-PK(1) and OK cells (i) with increasing volumes of culture medium and thus increasing amounts of glucose, and (ii) with increasing culture medium volumes at constant absolute amounts of glucose by adding glucose-free medium, in order to increase volume at a constant glucose supply. Alternatively, and in order to improve cell oxygenation, LLC-PK(1) cells were also cultured in roller bottles. Cell carbohydrate metabolism was assessed by measuring rates of glucose consumption and lactate production, respectively, and by determination of specific activities of the key glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH). Mitochondrial phosphate-dependent glutaminase (PDG) was assayed as marker enzyme of oxidative metabolism of glutamine. In LLC-PK(1) and OK cells, rates of glucose consumption were independent of the initial glucose concentrations and/or the culture media volumes used. Glucose was quantitatively converted to lactate, which accumulated in a 1:2 molar ratio. Lactate in culture media reached a maximum content after 24 h, and was reutilized by the cell lines thereafter. Interestingly, the rates of lactate reuptake strictly depended on culture medium volume, indicating a volume-induced stimulation of oxidative lactate metabolism. Marked changes were found for the specific activities of glycolytic enzymes. In LLC-PK(1) cells, increased glucose supply caused increases in HK, PFK, PK and LDH activities, which were superimposed to the stimulatory effects of increased media volumes. Enzyme activity showed a biphasic response, indicating that both glucose supply and culture media volumes covering the cell monolayer are factors determining glycolytic rates of LLC-PK(1) renal cells. Conversely, in OK cells glycolytic enzyme activities decreased with increasing culture media volumes at constant glucose levels. As expected, under conditions of enhanced oxygenation of LLC-PK(1) cells in roller bottle culture, glycolytic enzyme activities decreased, whereas PDG activity increased, which was paralleled by increased rates of ammonia generation. Thus, changes in nutrient supply and oxygenation of renal epithelial cell cultures by altered culture media volumes dramatically influence metabolic rates and levels of enzyme activities, respectively.

PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+ cells.

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+ cells caused a rapid decrease in the level of phosphoenolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM HCO-3) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the alpha-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life (t1/2 = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription (t1/2 = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+ cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+ cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.

Nephrotoxicity testing in vitro--what we know and what we need to know.

The kidney is affected by many chemicals. Some of the chemicals may even contribute to end-stage renal disease and thus contribute considerably to health care costs. Because of the large functional reserve of the kidney, which masks signs of dysfunction, early diagnosis of renal disease is often difficult. Although numerous studies aimed at understanding the mechanisms underlying chemicals and drugs that target various renal cell types have delivered enough understanding for a reasonable risk assessment, there is still an urgent need to better understand the mechanisms leading to renal cell injury and organ dysfunction. The increasing use of in vitro techniques using isolated renal cells, nephron fragments, or cell cultures derived from specific renal cell types has improved our insight into the molecular mechanisms involved in nephrotoxicity. A short overview is given on the various in vitro systems currently used to clarify mechanistic aspects leading to sublethal or lethal injury of the functionally most important nephron epithelial cells derived from various species. Whereas freshly isolated cells and nephron fragments appear to represent a sufficient basis to study acute effects (hours) of nephrotoxins, e.g., on cell metabolism, primary cultures of these cells are more appropriate to study long-term effects. In contrast to isolated cells and fragments, however, primary cultures tend to first lose several of their in vivo metabolic properties during culture, and second to have only a limited life span (days to weeks). Moreover, establishing such primary cultures is a time-consuming and laborious procedure. For that reason many studies have been carried out on renal cell lines, which are easy to cultivate in large quantities and which have an unlimited life span. Unfortunately, none of the lines display a state of differentiation comparable to that of freshly isolated cells or their primary cultures. Most often they lack expression of key functions (e.g., gluconeogenesis or organic anion transport) of their in vivo correspondents. Therefore, the use of cell lines for assessment of nephrotoxic mechanisms will be limited to those functions the lines express. Upcoming molecular biology approaches such as the transduction of immortalizing genes into primary cultures and the utilization of cells from transgenic animals may in the near future result in the availability of highly differentiated renal cells with markedly extended life spans and near in vivo characteristics that may facilitate the use of renal cell culture for routine screening of nephrotoxins.

In vitro nephrotoxicity of Russell's viper venom.

To assess direct nephrotoxicity of Russell's viper venom (RVV; Daboia russelii siamensis), isolated rat kidneys were perfused in single pass for 120 min. Ten micrograms/ml and 100 micrograms/ml RVV were administered 60 minutes and 80 minutes, respectively, after starting the perfusion. Furthermore, cultured mesangial cells and renal epithelial LLC-PK1 and MDCK cells were exposed to RVV (100 to 1000 micrograms/ml) for 5 minutes up to 48 hours. The IPRK dose-dependently exhibited reductions of renal perfusate flow (RPF, 7.7 +/- 2.4 vs. 16.5 +/- 0.7 ml/min g kidney wt in controls, experimental values given are those determined 10 minutes after termination of 100 micrograms/ml RVV admixture), glomerular filtration rate (GFR 141 +/- 23 vs. 626 +/- 72 microliters/min g kidney wt) and absolute reabsorption of sodium (TNa 8 +/- 1.7 vs. 79 +/- 9 mumol/min g kidney wt), and an increased fractional excretion of sodium (FENa 60 +/- 7 vs. 8 +/- 0.8%) and water (FEH2O 68 +/- 3.2 vs. 13 +/- 1.2%). Urinary flow rate (UFR) showed both oliguric and polyuric phases. Functional alterations of this type are consistent with ARF. Light and electron microscopy of perfusion fixed IPRK revealed an extensive destruction of the glomerular filter and lysis of vascular walls. Various degrees of epithelial injury occurred in all tubular segments. In cell culture studies RVV induced a complete disintegration of confluent mesangial cell layers, beginning at concentrations of 200 micrograms/ml. In epithelial LLC-PK1 and MDCK cell cultures only extremely high doses of RVV (> 600 and 800 micrograms/ml, respectively) led to microscopically discernible damage. These results clearly demonstrate a direct dose dependent toxic effect of RVV on the IPRK, directed primarily against glomerular and vascular structures, and on cultured mesangial cells.

Subcellular localization of PEPCK and metabolism of gluconeogenic substrains of renal cell lines.

The two gluconeogenic substrains of renal epithelial cells, LLC-PK1-FBPase+ and OKGNG+, have been shown to differ markedly in their metabolism of lactate and pyruvate. OKGNG+ cells consumed lactate as well as pyruvate at high rates in contrast to LLC-PK1-FBPase+ cells, which failed to take up or utilize lactate. (Aminooxy)acetate (AOA), an inhibitor of transamination reactions, was used to further delineate these differences. Lactate consumption of OKGNG+ cells was significantly inhibited by AOA, whereas pyruvate consumption by LLC-PK1-FBPase+ cells was slightly stimulated. Growth of OKGNG+ cultures, however, could be achieved on lactate in the presence of AOA. From these results it was concluded that the cell strains might differ in the subcellular distribution of phosphoenolpyruvate carboxykinase (PEPCK). LLC-PK1-FBPase+ cells may express both mitochondrial and cytosolic PEPCK isoenzymes, whereas OKGNG+ cells express only the mitochondrial isoenzyme. This was tested by directly assaying PEPCK activity in subcellular fractions of the cells. In OKGNG+ cells PEPCK activity fractionated with the mitochondrial marker glutamate dehydrogenase; however, in LLC-PK1-FBPase+ cells two-thirds of PEPCK activity was found in the cytosol. In LLC-PK1-FBPase+ cells, PEPCK activity increased twofold on incubation in acidic culture medium (pH 6.9) for 18 h, in contrast to the PEPCK activity in OKGNG+ cells. Northern blot analysis using cDNA probes specific for the mitochondrial and cytosolic PEPCK mRNAs confirmed the enzyme activity data. In LLC-PK1-FBPase+ cells strong expression of cytosolic PEPCK mRNA was observed, whereas in OKGNG+ cells only very low levels could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity.

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were used as a model system for assessment of nephrotoxic side effects of three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime. Toxic effects of these xenobiotics were monitored on confluent monolayers by light and electron microscopy and by the release of cellular marker enzyme activities into the culture medium. In addition, LLC-PK1 cells were grown on microporous supports, and cephalosporin-induced alteration of epithelial functional integrity was monitored by a novel electrophysiologic approach. For this purpose, an Ussing chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditions in which defined fractions of the apical culture medium NaCl contents were replaced iso-osmotically by mannitol. This method of determining the functional intactness of the epithelial barrier by measuring dilution potentials was found to be far more sensitive than monitoring cell injury by means of morphology or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption of monolayer integrity was detected with all three methodologies applied. Cephaloridine was found the most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of cephaloridine was needed to produce cell injury. Measurement of transepithelial dilution potentials was more sensitive as compared to the release of the apical plasma membrane marker enzyme activities alkaline phosphatase and gamma-glutamyltranspeptidase, the cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase. The data were compared to the effects of the aminoglycoside antibiotic gentamicin, which at least with respect to its effects on LLC-PK1 morphology and enzyme release, but not transepithelial electrical properties, was already investigated.

Selective release of apical membrane enzymes from cultured renal epithelia by phosphatidylinositol-specific phospholipase C.

Membrane proteins can be attached to the plasma membrane in several ways. Recently, a mechanism has been described, by which a number of cell surface proteins are anchored to the exoplasmic side of the plasma membrane by covalent linkage to glycosyl-phosphatidylinositol (GPI). The growth properties of renal epithelial cells in tissue culture enable free access to apical cell surface and brush border membrane proteins. To study the nature of membrane anchoring of apical plasma membrane enzymes in cultured renal epithelial cells, confluent LLC-PK1, OK, NRK, and MDCK epithelia were treated in tissue culture dishes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and the PI-PLC-specific release into the tissue culture medium of the apical membrane enzymes alkaline phosphatase (AP), gamma-glutamyl transpeptidase, leucine aminopeptidase, trehalase, and maltase was determined. Of the five enzymes tested, AP and trehalase, already described as GPI-anchored membrane proteins, were specifically released by PI-PLC from intact cell monolayers. Of the four cell lines investigated, LLC-PK1 cells express AP and trehalase which were released by PI-PLC. In OK cells, which lack AP activity, only trehalase was found to have PI-PLC-releaseable enzyme activity. MDCK cells, on the other hand, express AP activity, releaseable by PI-PLC, but no trehalase activity. In studies on the time course of synthesis and reinsertion of AP into the apical membrane of LLC-PK1 cells after removal by PI-PLC, a 60% recovery of AP activity was obtained only after 7 days. Analysis of protein release by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of culture supernatants after surface labeling with biotin and subsequent Western blotting with streptavidin revealed four protein bands at approximately 130, 90, 30, and 20 kD in LLC-PK1 cells and five GPI-anchored proteins at 110, 85, 65, 40, and 26 kD in OK cultures. The finding of a PI-PLC-specific release of apical membrane enzymes from renal tubular cell lines of different species (pig, opossum, rat, and dog) and of different nephron origin indicates a high conservation of the GPI anchor of renal brush border membrane proteins and further proves the high degree of differentiation retained by the cell lines in tissue culture. In addition, this method may provide a possible tool for isolating GPI-anchored apical membrane proteins from intact epithelial monolayer cultures.

Hyperosmolality regulates endothelin release by Madin-Darby canine kidney cells.

ET release by the renal epithelial Madin-Darby canine kidney (MDCK) cell line was investigated under isosmotic (300 mosmol/kg H2O; pH 7.4) and hyperosmotic (400, 500, or 600 mosmol/kg H2O) culture and assay conditions by the use of a specific and sensitive RIA. During isosmotic incubation, MDCK cells, which may be of collecting duct origin, secreted by far more ET into the cell culture supernatant (495.7 +/- 25.5 fmol.mg of protein-1.24 h-1) than did the proximal tubule-derived LLC-PK1 (2.42 +/- 0.20 fmol.mg of protein-1.24 h-1) and opossum kidney (3.12 +/- 0.47 fmol.mg of protein-1.24 h-1) cells. ET secretion by MDCK monolayers increased progressively within 24 h and then only slightly declined up to 48 h. Phosphoramidon (100 mumol/L) inhibited the constitutive ET synthesis in MDCK cells by 60%, indicating the participation of a phosphoramidon-sensitive ET-converting enzyme in the processing of bigET to ET in these cells. MDCK epithelia grown on filter inserts showed a clear polarity in their ET release. The baseline secretion of ET was 2.5 times higher to the basolateral than to the apical side, which might be in support of a predominantly basolateral action of the peptide. Short-term incubation of MDCK cells in hyperosmotic NaCl media for 24 h dose dependently decreased ET production. When urea was used as the solute to generate hyperosmolality, ET release by MDCK cells significantly increased. In contrast, when raffinose was added to increase osmolality to 500 mosmol/kg H2O, a decrease of ET production in a range similar to the effect of NaCl was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammoniagenesis in renal cell culture. Lack of extracellular ammoniagenesis at the apical surface of LLC-PK1 epithelia.

In renal ammoniagenesis, two major pathways of glutamine metabolism have been described: (i) intracellular metabolism by phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase and (ii) extracellular metabolism by phosphate-independent glutaminase. The latter has been identified as the hydrolytic activity of the apically membrane-bound gamma-glutamyl transpeptidase (gamma-GT). The growth properties of cultured renal epithelia enable the study of in vitro extracellular metabolic properties occurring at the apical epithelial surface in the culture dish. Therefore, confluent epithelia of the LLC-PK1 renal epithelial cell line were used to elucidate the role of extracellular (apical) hydrolysis of glutamine by gamma-GT in LLC-PK1 ammonia production. To distinguish between intra- and extracellular metabolism of glutamine, confluent LLC-PK1 epithelia were incubated with either D-glutamine as substrate, which cannot be metabolized intracellularly by PDG, or with L-glutamine and hippurate to stimulate, and AT-125 (acivicin) to inhibit gamma-GT activity, respectively. In addition, cellular uptake of the glutamate, extracellularly formed by gamma-GT, was inhibited by D-aspartate. D-Glutamine (2 mM) did not increase ammonia formation above endogenous production levels, indicating the negligible role of extracellular hydrolysis of glutamine by gamma-GT. After modulating gamma-GT activity by hippurate or AT-125, almost identical ammonia production rates were found within the various experimental protocols, further confirming that extracellular metabolism of glutamine does not significantly contribute to LLC-PK1 ammoniagenesis.