Phosphorylation and protein kinase activities of chromosomal non histone proteins from chick embryo fibroblasts

Confluent chick embryo fibroblasts were cultured in vitro in (i) medium which prevented the cells from dividing, (ii) medium which stimulated the cells to divide synchronously, (iii) medium without lysine in which the cells were blocked in G₁.Chromosomal non histone proteins (NHP) were extracted from cells pulse labelled with ³²P phosphate, and the radioactivity analyzed by acrylamide gel electrophoresis. Several radioactive peaks were found all along the gel in the NHP from confluent and stimulated cells. The highest phosphorylation was found in the fast moving proteins, but the stimulation of the cells increases the phosphorylation of the slower moving proteins. In the NHP from cells cultured in the medium without lysine only the slow migrating proteins were phosphorylated.NHP were extracted from unlabelled cell cultures in the three different media, incubated with [γ-³²P] ATP and analyzed by acrylamide gel electrophoresis. Highly labelled peaks were observed in the fast moving proteins from stimulated cells and from cells cultured in a medium deprived from lysine.By comparing in vivo and in vitro phosphorylation, it can be concluded that in confluent cells the turnover of bound phosphate is slow. In stimulated cells there is a fast turnover of the phosphate bound to fast migrating proteins and a slow turnover of the phosphate bound to slow migrating proteins. In cells cultured in a medium without lysine there is a very fast turnover of the phosphate bound to a small group of fast migrating proteins and very little turnover of the phosphate bound to slow migrating proteins.The cells were incubated with labelled lysine and NHP analyzed by gel electrophoresis. The radioactivity of individual NHP varied with the culture conditions, but in all cases, there was little radioactivity in the fast moving proteins. The phosphate groups submitted to a fast turnover are bound to stable proteins.Phosvitin and casein kinase activities were measured in the NHP fractions. Nine-ten peaks of activities were observed with each substrate. Some variations were observed which apparently correlate with the culture conditions.     

Physiological responses of Zygosaccharomyces rouxii to osmotic stress

When cell suspensions of Zygosaccharomyces rouxii were subjected to osmotic shock with NaCl, the cell volume decreased sharply and plasmolysis was observed. The cell subsequently recovered and volumes similar to those of cells growing at the respective water activity (a_w) values were found. Cycloheximide prevented cell recovery, indicating the involvement of protein synthesis in the recovery process. The intracellular glycerol concentration of Z. rouxii incubated in the presence of [¹⁴C]glycerol increased from 13 to 96 mmol/l during the initial 20 min after an upshock from 0.998 a_w to 0.96 a_w. All the intracellular glycerol was labelled and therefore derived from the medium. Labelled glycerol was subsequently utilized and replaced by unlabelled glycerol produced by the cell within 90 min. The initial increase in glycerol concentration following the upshock was confirmed by ¹³C-nuclear magnetic resonance (NMR) spectroscopic studies of cell extracts. The combined dihydroxyacetone and dihydroxyacetone phosphate concentrations fluctuated during this period, whereas glycerol-3-phosphate initially increased and then remained constant. This indicates that the production of glycerol is regulated. Decreases in ATP and polyphosphate levels were observed following osmotic upshock and may reflect a greater demand for ATP during the period of adjustment to decreased a_w. The changes in cell volume and in ATP concentration following osmotic upshock may serve as osmoregulatory signals in Z. rouxii, as suggested previously for other microorganisms. Correspondence to: S. G. Kilian     

Mode of orthophosphate uptake and ATP labeling by mammalian cells

Incubation of HeLa cells with [³²P]orthophosphate results in more rapid labeling of the γ-phosphorus of ATP than of the intracellular pool of orthophosphate. The specific radioactivity of ATP equals that of extracellular orthophosphate after 2h of incubation. A similar pattern of labeling is seen with human erythrocytes when incubated at physiological concentrations of orthophosphate (2 mM) and pH 7.4–7.8. At lower pH, 6.8–7.2, the rate of orthophosphate uptake increases and exceeds the rate of labeling of ATP. These data are explained by the existence of a primary system for ATP uptake which involves the mediation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. Phosphate first enters the cell as 1,3-diphosphoglyceric acid, is then transferred to ATP, and then enters the intracellular orthophosphate pool. At lower pH monovalent orthophosphate also enters the erythrocyte by a process not involving glyceraldehyde-3-phosphate dehydrogenase.     

L'acide indolyl-3-lactique et son métabolisme chez Rhizobium

Summary Among the indole compounds formed when tryptophan 2-¹⁴C is metabolized by Rhizobium, indole-3-lactic acid (ILA) is specially studied. In the course of experiments carried out in the culture medium of growing Rhizobium and in suspensions of washed bacterial cells the amount of ILA formed is compared with that of indole-3-acetic acid (IAA) occurring simulataneously. The formation of ILA and that of IAA directly depend on a transamination reaction. A large quantity of ILA is present in suspensions of washed bacterial cells.When ILA alone, as precursor, is incubated with Rhizobium, several products are identified: IAA, indole-3-acetaldehyde and tryptophol. Tryptophan is also detected in the aqueous fraction and is labelled when ILA 2-¹⁴C is used. The pathway of this metabolism are discussed and a general scheme is suggested.     

Photoassimilation of acetate and metabolism of carbohydrate in Chlorobium thiosulfatophilum

1. Washed cell suspensions of Chlorobium thiosulfatophilum form large amounts of a polyglucose in the light. Addition of acetate to the cells increases the formation of polysaccharide considerably. During incubation in the dark, polysaccharide decreases with time, and organic acids such as succinic and propionic acid are excreted into the medium. 2. Glucose isolated from cells which had photoassimilated 1-, 2-, and U-¹⁴C-acetate had a specific activity which lay between 1 and 2 times that of the acetate substrates. 3. To analyse the distribution of radioactivity in the glucose units formed during photoassimilation of ¹⁴C-acetate, 2 microbial degradations, with bakers' yeast and Zymomonas mobilis respectively, were used. The results show that acetate gives rise to carbon atoms 1+2 and 5+6 of glucose, whereas carbon atomes 3+4 are not labelled. Further, the results indicate that glucose is not formed via the reductive pentose phosphate cycle when acetate is present.     

Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G-3H] hypoxanthine (Hy) into [3H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [3H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.     

INFLUX OF GLUTAMIC ACID IN PERIPHERAL NERVE—CHARACTERISTICS OF INFLUX1

Abstract— —The influx of glutamic acid in frog sciatic nerve has been studied by monitoring the disappearance of ¹⁴C labelled compound from the bathing medium. After 5hr of incubation in 10 ⁻⁶m non-labelled l -glutamic acid and 0·01, μc/ml labelled isotope, the intracellular concentration of labelled glutamic acid is about 15 times the concentration in the bathing medium; however, there appears to be a net loss of non-labelled compound with incubation. Uptake of L,-glutamic acid is accompanied by conversion of significant amounts of labelled E-glutamic acid to carbon dioxide and glutamine; small amounts of γ-aminobutyric acid and aspartic acid are also formed. The rate of disappearance of labelled l -glutamic acid decreases with increasing concentration of non-labelled isotope in the bathing medium. Construction of a Lineweaver-Burk plot from initial velocities of influx yields an average V_m of 4·02 × 10⁻⁹ moles/g/min and an average K_m. of 3·23 × 10 ⁻⁵ moles/l. The influx of glutamic acid is highly specific with regard to molecular structure; of the compounds tested, only l -glutamine, l -glutamic acid, GABA, l -lysine, and l -aspartic acid are taken up, and only l -aspartic acid will compete with l -glutamic acid for uptake.     

Membrane-bound phosphate as driving force for ATP synthesis in chromatophores ofRhodospirillum rubrum

Chromatophores ofRhodospirillum rubrum preilluminated in the presence of unlabelled phosphate form labelled ATP in the dark after being separated from the preincubation mixture by gel-filtration and incubated with ADP and³²P_i. The driving force for the synthesis of this labelled ATP was previously shown to be ATP firmly bound to the membrane. The amount of labelled ATP produced is determined by measuring the incorporated³²P_i and extrapolation of the values towards zero time incubation and is shown to correlate with the phosphorylation activity of the chromatophores used. 2.5 nmoles ATP correspond to a phosphorylation activity of 100 moles ATP/mg Bchl. h. The results were compared with those from assays in which chromatophores were preincubated with pyrophosphate in the dark. The results strongly support the hypothesis that the XP formed in the light does not transfer its P to ADP in solution and therefore give further evidence to a double sited coupling factor as proposed previously.     

Contact Inhibition of Transformed Cells Incompletely Restored by Dibutyryl Cyclic AMP

INCREASED levels of cyclic AMP have been found in normal cells as compared with malignant cells^1,2. Several types of malignant cells become morphologically similar to untransformed cells when incubated in media containing cyclic AMP or its derivative dibutyryl adenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP)^3,4. Sheppard reported that 3T3 mouse fibroblasts, transformed by polyoma virus, grew to low saturation density and became less agglutinable with wheat germ agglutinin if theophylline and dibutyryl cyclic AMP were added to the medium⁵.     

Chloroflexus aurantiacus secretes 3-hydroxypropionate,a possible intermediate in the assimilation of CO2 and acetate

Chloroflexus aurantiacus OK-70 fl secreted 3-hydroxypropionate (3HP) during phototrophic growth. The greatest amounts were secreted by cells grown on propionate (0.35 mM 3HP) while the lowest levels were found in autotrophically grown cultures (1.5 M). Large amounts of 2-fluoro,3-hydroxypropionate were formed by autotrophically grown cells exposed to fluoroacetate (FAc). Increased levels of 3HP were observed in these cultures when incubated with acctate. The secretion of 3HP was further stimulated by 0.2 mM KCN, an inhibitor of CO₂ fixation, but only in the presence of acetate. The pathway of 3HP formation was studied by using ¹³C-labelled substrates and NMR. The 3HP formed in the presence of C1-labelled acetate and FAc was labelled at C3 and somewhat less at C2 while with C2-labelled acetate as the tracer 3HP was labelled predominantly at C2. The carboxyl group was derived from CO₂. The 3HP formed by cells grown on propionate and ¹³CO₂ was labelled at all carbon atoms, the label content of C2 and C3 was about 25 and 65% of that of C1 respectively. It is suggested that 3HP is an intermediate in a pathway for acetate assimilation and in a new reductive carboxylic acid cycle for autotrophic CO₂ fixation.Abbreviations 3HP 3-hydroxypropionate - 2F3HP 2,fluoro,3-hydroxypropionate - FAc fluoroacetate - GC gas chromatography - MS mass spectrometry - NMR nuclear magnetic resonance     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

(14C) acetylcholine synthesis by cortex slices of rat brain

Abstract—

• 1 A procedure has been developed to measure ACh synthesis from [¹⁴C]-precursors. As little as 10^?9 moles of ACh were detected as the result of de nova synthesis. Following incubation of cortex slices of rat brain with eserine and a tagged metabolite, ACh carrier was added to the incubation medium and to an extract from the slices. ACh was purified by chromatography on Amberlite CG-50, precipitation and recrystallization of ACh chloroaurate.
• 2 [U^?14C]glucose and [2^?14C]pyruvate formed similar amounts of [¹⁴C]ACh. Hydrolysis of ACh with subsequent chromatography of the resultant acetic acid demonstrated that all of the label was located in the acetyl moiety. [¹⁴C]acetate did not serve as a precursor of the acetyl group of ACh. Equivalent incorporation of carbons 1 and 6 of glucose into ACh indicated that glucose metabolism to ACh occurred via the Embden-Meyerhof pathway.
• 3 The amount of ACh detected by bioassay after incubation of cortex slices with [U^?14C]glucose was approximately the same as that calculated as labelled ACh; this demonstrates that all of the acetyl groups of ACh formed during incubation were derived from glucose.
• 4 [¹⁴C]choline, either methyl or chain labelled, formed [¹⁴C]ACh while labelled ethanolamine, serine and methionine did not. Synthesis from labelled choline did not occur in the absence of glucose.
• 5 When both [U^?14C]glucose and [¹⁴C]choline were incubated with brain slices, the acetyl and choline moieties of ACh were equally labelled; this demonstrates that the entire molecule was formed from added precursors. Slices supported a high rate of ACh synthesis without addition of choline. The addition of 10^?4m -hemicholinium-3 inhibited ACh formation by more than 90 per cent from either [U-¹⁴C]glucose or [Me-¹⁴C]choline.
• 6 Study of the time course of ACh synthesis from glucose demonstrated a rapid formation of [¹⁴C]ACh within the slices which reached a maximum during the first hour of incubation. [¹⁴C]ACh in the incubation medium accumulated at a linear rate for 3 hr. Replacement of a portion of the sodium chloride of the incubation medium by potassium chloride to a final concentration of 31 mm -KCI markedly increased the formation of [¹⁴C]ACh found in the incubation medium. Decreased amounts of [¹⁴C]ACh were extracted from the slices by homogenization or by subsequent heating at pH 4 in the high potassium ion medium.

     

Isolation and photosynthetic characteristics of mesophyll cells from developing leaves of soybean

Mesophyll cells were isolated from developing sink leaves (25 to 30 mm in length) of soybean, Glycine max (L.) Merr. cv. Will. Leaf strips were incubated for two h in a buffered medium containing osmoticum and 0.2% Pectolyase Y-23. Gently stirring the leaf strips released from 7 to 16% of the total leaf mesophyll cells. Other pectinase enzymes, effective in releasing cells from mature source leaves (70 to 75 mm in length), did not release cells from sink leaves. Sink and source cell preparations were about 50 and 95% intact, respectively, based on the exclusion of Evans Blue dye. Intact cells could not be separated from broken cells on Ficoll or metrizamide density gradients. Total protein and catalase, glyceraldehyde-3-phosphate dehydrogenase, glycolate oxidase, phosphoenolpyruvate carboxylase, and ribulose 1,5-bisphosphate carboxylase activities on a chlorophyll basis were about 50% lower in sink mesophyll cells than in sink leaf homogenates indicating that broken sink cells lost soluble protein to the medium. Source cells and source leaf homogenates had comparable amounts of protein and enzymatic activities. Enzymatic activities on a chlorophyll basis were similar in source and sink leaves with the exception of phosphenolpyruvate carboxylase, which was two times higher in sink leaves. This enzyme was also exceptionally low in source and sink cells being only 61 and 23%, respectively, of whole leaf activities. Sink cell rates of ¹⁴CO₂ fixation were only 7% of source cell rates and sink cells did not show light-dependent O₂ evolution. Both cell preparations had photosystem II activiteis which were comparable to rates of ¹⁴CO₂ fixation at satuarating light and CO₂ concentration. It was concluded that the reduced photosynthetic rate of sink cells was limited by the low photochemical capacity rather than a limitation of Calvin cycle enzymes.     

Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines

Methionine dependence is a metabolic defect found thus far only in transformed and malignant cells. The defect is manifested as the inability of cells to grow in media in which methionine (Met) is replaced by its immediate precursor homocysteine (Hcy). We have termed this Met ^? Hcy ⁺ media. We demonstrate here that methionine-dependent cells derived from human tumors, compared to normal methionine-independent cells, have low levels of free Met, low levels of S-adenosylmethionine (AdoMet) and elevated levels of S-adenosylhomocysteine (AdoHcy) when incubated in Met ^? Hcy ⁺ medium. Methionine-independent human tumor cells also have very low levels of free Met compared to normal cells but generally have levels of AdoMet and AdoHcy comparable to normal cells in Met ^? Hcy⁺ medium. All tumor cell types incorporate amounts of Met into protein similar to normal methionine-pindependent human fibroblasts when incubated in Met ^? Hcy⁺ medium, thereby indicating apparently normal levels of Met synthesis in the tumor cells. The methionine-independent tumor cell lines in Met ^? Hcy⁺ medium seem able to regulate their AdoMet/AdoHcy ratios normally despite this defect in having very low levels of free Met. Thus, in a diverse set of human tumor cell lines, all are defective in at least one aspect of Met metabolism, giving rise to the possibility of a general metabolic defect in cancer.     

Transplantation of expanded bone marrow‐derived very small embryonic‐like stem cells (VSEL‐SCs) improves left ventricular function and remodelling after myocardial infarction

Adult bone marrow‐derived very small embryonic‐like stem cells (VSEL‐SCs) exhibit a Sca‐1⁺/Lin^–/CD45^– phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (1 × 10⁶) of freshly isolated, non‐expanded VSEL‐SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL‐SCs in the marrow is very low, we examined whether VSEL‐SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n= 11), 1 × 10⁵ enhanced green fluorescent protein (EGFP)‐labelled expanded untreated VSEL‐SCs (group II, n= 7), or 1 × 10⁵ EGFP‐labelled expanded VSEL‐SCs pre‐incubated in a cardiogenic medium (group III, n= 8). At 35 days after myocardial infarction (MI), mice treated with pre‐incubated VSEL‐SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle‐treated controls. In contrast, transplantation of expanded but untreated VSEL‐SCs did not produce appreciable reparative benefits. Scattered EGFP⁺ cells expressing α‐sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)‐1, or von Willebrand factor were present in VSEL‐SC‐treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL‐SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic growth factors and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre‐incubation confers therapeutic efficacy does not involve differentiation into new cardiac cells. These results support the potential therapeutic utility of VSEL‐SCs for cardiac repair.     

Ammonium ions prevent methylation of uridine to ribothymidine inAzotobacter vinelandii tRNA

It was shown that tRNA fromAzotobacter vinelandii grown in the presence of ammonium chloride lacks ribothymidine while that grown in the absence of the ammonium salt contains this modified nucleoside. [³²P]-Labelled tRNA from this organism grown in a medium containing the ammonium salt was digested with RNase T1 and the pseudouridinecontaining tetranucleotide, common to all tRNAs was isolated and analysed for the nucleoside replacing the ribothymidine. It was found to be uridine. Cells previously labelled with [³²P]-phosphate in the ammonium salt medium were washed and incubated in the ammonium saltfree medium to test whether ribothymidine would be formed upon removal of the ammonium ions. Methylation of the uridine did not take place.     

The uptake and distribution of radiolabelled thyroxine by isolated cardiac myocytes.

The uptake and distribution of ¹⁴C and ¹²⁵I-labelled thyroxine was studied in ventricular myocytes, isolated from the hearts of male Sprague-Dawley rats. Equilibrium was established between the radioactivity of the incubation medium and the cells within 15 minutes. At equilibrium the concentration of ¹⁴C-thyroxine in the cells was approximately 50 times the concentration in the incubation medium. Fractionation of the cells revealed that the equilibrium had been attained for all fractions except the nuclear. The radioactivity of the nuclear fraction showed an increase for at least 60 minutes of incubation. At equilibrium the distribution of radioactivity was: Soluble fraction 51.3%, Mitochondria 33.6%, Microsomal 7.0% and Nuclear 7.0%. When the values for these fractions were corrected for mitochondrial contamination the specific activity (CPM/MG protein) of the mitochondrial fraction was by far the highest, exceeding the next highest fraction (the supernatant) by an order of magnitude. The presence of equimolar amounts of triiodothyronine produced little change in the pattern of uptake of the label by any of the cell fractions. The uptake of labelled thyroxine was profoundly affected by the presence of calcium in the media. The uptake of ¹⁴C-thyroxine by cells incubated in media containing 1.25mM calcium was less after 60 minutes than in cells incubated in calcium free buffer. Fractionation of the cells revealed that the amount of label bound to the mitochondria of cells in calcium containing medium was significantly increased while the radioactivity bound to the other cellular fractions was decreased. The data indicate that the cell fraction with the highest specific activity was the mitochondria. The relation of these findings to some of the current theories of thyroid hormone action is discussed.     

An investigation of acid-soluble nuclear proteins of human leucocytes in relation to fraction RP2-L, a component of neoplastic cells

1. The claim that tumour cells contain a specific nuclear protein was investigated. The presence of this component was confirmed in Walker tumour cells by the chromatography on CM-cellulose of nuclear proteins labelled with [¹⁴C]lysine. This protein was studied further in a number of human leucocyte cells. 2. The labelling of leucocyte nuclear proteins with [¹⁴C]lysine was attempted during incubation and culture in vitro. Incorporation of the label into acid-soluble nuclear proteins was highest in normal lymphocytes cultured with phytohaemagglutinin, followed by chronic-myeloid-leukaemic leucocytes and mixed samples of normal leucocytes incubated in plasma. Little incorporation was seen in similar extracts of chronic-lymphatic or normal leucocytes. 3. Lymphocytes were the only cells that gave nuclear extracts with amino acid analysis similar to that of unfractionated histones. 4. Little of the [¹⁴C]lysine in nuclear extracts of incubated leucocytes proved to be of chromosomal origin. No evidence was found of an RP2-L component in the highly labelled nuclear extracts of phytohaemagglutinin-treated lymphocytes until after 6 days of culture with [¹⁴C]lysine. This component was soluble in saline. 5. Evidence is presented that fraction RP2-L is a non-histone protein constituent of cell nuclei whose labelling with [¹⁴C]lysine may be dependent on the metabolic state of the cell. Thus this component is not specific to the neoplastic state.     

Response of cultured chick heart cells to changes in ionic environment

Ventricles from 11-day-old chick embryonic heart were disaggregated by elastase and the component cells cultured on glass in maintenance medium containing 10 μc of P³². After 48 hours incubation at 37°C the medium was removed, the cells rinsed and exposed to a phosphate-free test solution for two hours. During this period samples of the test medium were removed for counting and spectrophotometric analysis. Cells incubated in solutions lacking amino acids or vitamins or serum components lost phosphate at essentially the same rate as in the complete culture medium; furthermore such cells lost very small amounts of nucleotide materials. Cells incubated in 0.16 M NaCl lost phosphate and nucleotides rapidly; the addition of either K⁺ or Ca⁺² or Mg⁺² reduced phosphate and nucleotide loss and cells in balanced saline media containing all four cations, retained phosphate and nucleotides at essentially the same level as in the complete medium. These results show that primary isolated chick heart cells can be maintained for short periods in physiological saline solutions without injury and that saline balance in short term studies is a primary factor in maintaining these cells in an uninjured state.     

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na⁺K⁺ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 μM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na⁺K⁺ ATPase activity in whole cells, Na⁺K⁺ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na⁺K⁺ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na⁺K⁺ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na⁺K⁺ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na⁺K⁺ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.