Changes in Na influx precede post-natal increase in Na, K-ATPase activity in rat renal proximal tubular cells.
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Animals↗
Biomedical subjects
C Lechene
Publications and source records attributed to C Lechene.
Alanine stimulation of passive potassium efflux in hepatocytes is independent of Na(+)-K+ pump activity.
We have studied the effects of alanine on electrolyte content and ion transport in rat hepatocytes in primary culture. Application of 10 mM alanine is followed by 1) an increase in the rate of sodium entry; 2) an increase in intracellular sodium content; 3) an increase in ouabain-inhibitable rubidium uptake, a measure of Na(+)-K+ pump rate; 4) an increase in unidirectional potassium efflux, whether or not the Na(+)-K+ pump was inhibited; and 5) an increase in the initial rate of potassium loss after Na(+)-K+ pump inhibition. This increase occurred even when alanine was presented in Ringer made hypertonic by the addition of sucrose. Application of hypotonic solution led to a significant net loss of potassium, but no net loss of potassium was observed after alanine application. Thus alanine stimulates the Na(+)-K+ pump by increasing intracellular sodium secondary to an increase in the rate of sodium entry. Passive potassium efflux is stimulated by a mechanism that is independent of the stimulation of the Na(+)-K+ pump. The stimulated potassium efflux does not appear to be a response to cell swelling.
Alanine↗
Proximal tubule volume regulation in hypo-osmotic media: intracellular K+, Na+, and Cl-.
This study sought to measure the net loss of intracellular K+, Na+, and Cl- that accompanied isosmotic cell volume regulation in hypotonic media and to determine if electrolyte loss depended on the rate at which the extracellular osmolality was reduced. Isolated nonperfused proximal S2 segments from rabbit kidney cortex were studied in vitro. Gradual lowering of osmolality from 295 to 150 mOsm/kg at a rate of 2 mOsm/kg/min did not cause an increase in tubule cell volume until the medium osmolality decreased below 190 mOsm/kg. By contrast, tubules rapidly bathed in low osmolality media exhibited classical osmometric swelling followed by incomplete volume regulatory decrease. Volume regulation associated with gradual and rapid lowering of osmolality was accompanied by the net loss of intracellular K+, Na+, and Cl- (measured by electron probe); however, the temporal pattern of electrolyte loss depended on the rate of osmotic change. With gradual lowering of osmolality, cell K+ content did not decrease significantly until osmolality was lowered below 200 mOsm/kg, whereas Cl- was lost at the 200 mOsm/kg level and below. With rapid lowering of osmolality, cell K+ content was strikingly decreased at the 200 mOsm/kg level, but Cl- did not change appreciably until osmolality was decreased to 150 mOsm/kg. Cell Na+ content decreased in hypo-osmotic media, but the magnitude was relatively small. During volume regulation that accompanied either gradual or rapid lowering of medium osmolality from 295 to 150 mOsm/kg, intracellular osmolal gap, the difference between medium osmolality and the sum of intracellular concentrations of K+, Na+, and Cl- decreased 87 and 58 mOsm/kg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Studies of terminal differentiation of electrolyte transport in the renal proximal tubule using short-term primary cultures.
There are several lines of indirect evidence suggesting that the renal tubule cells have not yet reached terminal differentiation at birth. Methods used in cell biology can now be applied to study renal ontogeny. This review describes how primary cultures of proximal tubule cells from rats can be used to investigate developmental changes in Na permeability and Na-K-ATPase-mediated transport.
Animals↗
Effect of cell spreading on cytoplasmic pH in normal and transformed fibroblasts.
Growth of normal fibroblasts requires an adhesive substratum on which to spread, whereas transformed cells can grow in suspension. Since an alkaline cytoplasm has been shown to be required for growth, we measured cytoplasmic pH in individual cells as a function of spreading. The degree of spreading was controlled by coating tissue culture plastic with varying amounts of the nonadhesive polymer polyHEMA. Completely round BALB/c 3T3 cells were 0.15 pH unit more acidic than spread cells. In short-term experiments, cells were treated with the peptide Gly-Arg-Gly-Asp-Ser-Pro to induce rounding or by plating on fibronectin to induce spreading. When cells were induced to change shape, pH changed rapidly and reversibly. All of the anchorage-dependent cell lines tested behaved similarly, but 3T3 cells transformed by the plasma membrane oncogene src or ras were able to maintain a relatively alkaline pH even when completely round. Cells transformed by the nuclear oncogene myc behaved like normal cells. Our results suggest that the requirement for spreading may in part be mediated by cytoplasmic pH. Anchorage-independent growth due to oncogenes that localize to the plasma membrane is associated with loss of this control mechanism.
Animals↗
Renal epithelial cyst formation and enlargement in vitro: dependence on cAMP.
Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.
1-Methyl-3-isobutylxanthine↗
Proximal tubule volume regulation in hyperosmotic media: intracellular K+, Na+, and Cl-.
Nonperfused proximal S2 segments from rabbit kidney cortex have been shown to keep cell volume constant as medium osmolality is slowly raised but to shrink and not exhibit regulatory volume increase (RVI) if medium osmolality is abruptly elevated (J. Lohr and J. Grantham. J. Clin. Invest. 78: 1165-1172, 1986). In the current study, 0.5 mM butyrate in the medium 1) extended the range from 361 to 450 mosmol/kgH2O over which cells maintained volume constant as osmolality was gradually raised and 2) restored RVI after cell shrinkage when osmolality was rapidly raised from 295 to 400 mosmol/kgH2O. Volume regulation was associated with net increases in intracellular Na+ and Cl- but no change in K+ (measured by electron probe). The increments in Na+ and Cl- were insufficient to account for the total addition of osmolytes required for volume maintenance or restoration. The fraction of the expected increase in intracellular osmoles accounted for by the increase in [(K+)i + (Na+)i + (Cl-)i] was 52 and 21% for gradual and rapid osmotic changes, respectively. We conclude that butyrate enhances the capacity of S2 segments to regulate volume in hyperosmotic medium by promoting addition of Na+ and Cl- and by other undermined factors.
Animals↗
[Physiologic role of the sodium pump. Implications for the study of arterial hypertension].
Mammalian cells have the same hydroelectrolytic composition (high K, low Na), highly different from that of their surrounding. Constancy of cellular composition is insured by the balance between ionic leaks and (Na, K)-pump activity. Ionic leaks, specially sodium, are fundamental. They allow cells to perform a majority of their general and special functions (import of aminoacids, glucose, phosphates; export of acids; nerve influx; muscular contraction; glandular secretion; intestinal and renal reabsorption and secretion). (Na, K)-pump is essential to life. It is a kind of general motor that creates and maintains ionic concentrations differences whose potential energy is dissipated by leaks to perform cellular functions. Constancy of hydroelectrolytic intracellular composition hides that leak and pump rates, equivalent between them, are extremely variable among cell types (more than 200 times), and can increase 4 times in less than one minute within a cell type with cellular activity. In a cell, (Na, K)-pump rate is far from maximum velocity. This rate is adjusted nearly instantaneously to balance variations in leak rates; it may undergo short term modulation by endo or exocellular factors; it may undergo long term changes through synthesis of new enzyme molecules. Studies on whole cells of the balance between leaks and pump rates is necessary to understand these cells physiology and pathology. Balance between leaks and (Na, K)-pump activity is altered in renal cells from hypertensive rats, spontaneously (SHR) or genetically selected (Milan Hypertensive Strain: MHS). Strikingly, sodium activation of (Na, K)-pump of inner medullary collecting duct cells of MHS rats is greatly blunted compared to controls.
Adenosine Triphosphatases↗
(Na,K)-pump: cellular role and regulation in nonexcitable cells.
The (Na,K)-pump develops and maintains ionic gradients that are of fundamental importance for proper function of most animal cells. These gradients are utilized in the form of ionic leak pathways by a number of special and general cell processes (e.g., nerve conduction, nutrient transport, pH regulation). As the sodium gradient in particular energizes many vital cell processes, alterations in cell activity will often be manifest as changes in sodium entry. The (Na,K)-pump rate varies accordingly, in order to maintain balance between Na entry and exit thereby maintaining the potential energy of the cell. Acute changes in sodium influx are balanced by increases in activity of existing pump units, with only a small change in intracellular sodium concentration. This is possible because intracellular is normally poised on the steep limb of the concentration versus activity curve for the (Na,K)-pump, at a point well below maximal activity, allowing large increases in (Na,K)-pump rate with only small changes in sodium concentration. If the increase in sodium influx is prolonged, it appears that the cell responds by synthesizing new pumps, allowing intracellular sodium concentration to return to its original values. Though increases in (Na,K)-pump activity must be accompanied by increases in potassium leak rates, in the experiments we have presented, there does not appear to be direct functional coupling between (Na,K)-pump and the K leak pathways. In these situations the matching of active influx and passive efflux of K short-term appears to occur by mechanisms not directly related to (Na,K)-pump activation.
Animals↗
Noninvasive measurement of glucose uptake by two populations of murine embryos.
Glucose uptake was measured by a noninvasive fluorescence technique on a total of 165 morula- and blastocyst-stage murine embryos in two different culture media. Eighty-four embryos were tested in M2 medium, and the remaining 81 embryos were tested in M16. Embryos assayed in M2 took up significantly less glucose over the 4-h assay period than did embryos assayed in M16. The lower uptake of glucose by embryos in M2 corresponded with a decrease in the quality of embryos cultured overnight in M2 as judged by morphological criteria. Embryos that were judged to be degenerate or had gross abnormalities took up significantly less glucose than did normal embryos. Glucose uptake in both populations of embryos covered a wide range of values and was normally distributed. A significant effect between mothers was noted in glucose uptake for embryos assayed in both M16 and M2 media. The possible uses of noninvasive measures of glucose uptake as a test of embryo viability or for optimizing culture conditions are discussed.
Analysis of Variance↗
Studies on terminal differentiation of rat renal proximal tubular cells in culture: ouabain-sensitive K and Na transport.
We have studied the ontogeny of Na-K ATPase-mediated Na and K transport in rat renal proximal tubular cells using electron probe analysis. The cells were cultured from kidneys of 10-day-old, young (Y), and 40-day-old, adult (A) rats. Before an experiment cells were Na-loaded and K-depleted by incubation in K-free medium. The maximum rate of ouabain-sensitive Na and K transport was measured after reactivating the Na-K pump by transferring the cells from K-free medium to medium containing 5 mM K. In cells cultured for 2 days, ouabain-sensitive Na and K net initial transport rates were significantly higher in A than in Y cells. Between 2 and 4 days in culture there was a significant decrease in ouabain-sensitive Na and K transport rates in both Y and A cells. From 2 to 4 days of culture there was, in Y but not in A cells, a significant decrease in K/Na ratio. The decrease in K/Na ratio was due to a significant increase in Na content. After incubation in K-free medium, net intracellular solute accumulation was observed in A and Y cells cultured for 4 days but not in A and Y cells cultured for 2 days. In conclusion, maximal Na- and K-pump-mediated transport increases during terminal differentiation. This increase can be measured in cells cultured for 2 days. With longer time in culture, Na-K pump activity decreases and the difference between A and Y cells is not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)
Age Factors↗
Ouabain-resistant, amiloride-sensitive Na+-K+ pumping activity and morphological changes are inducible.
The functional expression of a ouabain-resistance gene has been studied in a transfected cell line, OR-6 cells. These cells possess a ouabain-inhibitable, amiloride-resistant Na+-K+ pump under control conditions. They can be induced by brief culture in the presence of low concentrations of ouabain to express a ouabain-resistant, amiloride-sensitive Na+-K+ pumping activity. They revert to the uninduced phenotype shortly after removal of ouabain from the culture medium. When placed in low-potassium, low-serum, or dibutyryl adenosine 3'-5'-cyclic monophosphate-containing media, these cells produce long cellular extensions. Thus a single inducible gene appears to regulate sensitivity of the Na+-K+ pumping activity to cardiac glycosides and to amiloride, and it may be related to important characteristics of cellular morphology.
Amiloride↗
Use of experimental models to study the development of renal function.
By use of animal experiments it has been demonstrated that renal cells are immature at birth. Membrane transport is quantitatively and qualitatively different. The control of fluid and electrolyte balance is therefore different in infancy. The increased filtered load at birth as well as hormones will induce tubule maturation. The use of animal experiments will in the future give an insight into how external factors influence growth and maturation.
Animals↗
Acute renal failure with selective medullary injury in the rat.
Since human acute renal failure (ARF) is frequently the result of multiple rather than single insults, we used a combination of treatments to induce ARF in rats. Uninephrectomized, salt-depleted rats injected with indomethacin developed ARF after administration of radiocontrast. After 24 h, the plasma creatine rose from 103 +/- 3 to 211 +/- 22 mumol/liter (mean +/- SE) and the creatinine clearance dropped from 0.7 +/- 0.1 to 0.2 +/- 0.04 ml/min (P less than 0.001). Severe injury was confined to the outer medulla and comprised necrosis of medullary thick ascending limbs (mTALs), tubular collapse, and casts. Other nephron segments were free of damage except for the proximal convoluted tubules which showed vacuole formation originating from lateral limiting membranes that resembled changes reported in human contrast nephropathy. Cell damage to mTALs included mitochondrial swelling, nuclear pyknosis, and cytoplasmic disruption with superimposed calcification; these changes were most severe in the deepest areas of the outer medulla, away from vasa recta in zones remote from oxygen supply. The fraction of mTALs with severe damage was 30 +/- 7% (range 2-68) and the extent of injury was correlated with a rise in plasma creatinine (r = 0.8, P less than 0.001). Thus, the nature of mTAL injury was similar to the selective lesions observed in isolated kidneys perfused with cell-free medium and was shown to derive from an imbalance between high oxygen demand by actively transporting mTALs and the meager oxygen supply to the renal medulla. Combined multiple renal insults in the rat produce ARF that resembles the clinical syndrome of contrast nephropathy and is characterized by selective mTAL injury conditioned by medullary hypoxia.
Acute Kidney Injury↗
Physiological role of the Na-K pump.
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Animals↗
Rescue of a wild-type MDCK cell by a ouabain-resistant mutant.
When wild-type MDCK cells (W-MDCK) were cocultured in mixed monolayers with a ouabain-resistant mutant (R-MDCK), the wild-type cells were protected from the effect of ouabain up to concentrations as high as 100 microM. Rescue depended on the dose of ouabain and on the proportion of each cell type in the coculture. The survival of R-MDCK cells at 1 microM ouabain was not endangered by varying from 1:9 to 9:1 the proportion of W-MDCK cells to be rescued. Ouabain binding revealed two kinds of binding sites in R-MDCK cells, one with high and the other with low affinity. Only the high affinity site was present in W-MDCK cells. Electron probe analysis of individual cells revealed that rescued cells kept a high K and a low Na intracellular contents, similar to control cells. Histograms of intracellular K/Na in cocultured cells treated with ouabain were unimodal. Using microinjection of Lucifer yellow or electrophysiological techniques we estimated that at most 13% of the R-MDCK and W-MDCK cells may be connected at a given time through cell-to-cell junctions. Therefore permanent cell-to-cell communication did not seem to play a central role in the rescue. W-MDCK cells cocultured with R-MDCK cells and subsequently separated, were not rescued. Thus rescue did not seem to depend on the transfer from R-MDCK to W-MDCK cells of either ouabain-resistant Na-K pumps or of information to synthesize them. It is speculated that intercellular communications were sporadic events, so that all cells may become intermittently connected and rescued.
Animals↗
Pancreatic zymogen granules differ markedly in protein composition.
The activities of both chymotrypsin and amylase in individual zymogen granules of rat pancreas were measured by means of micromanipulation and microfluorometric methods. The enzyme content and the ratio of amylase to chymotrypsin varied widely among granules taken from the same animal. These results are compatible with short-term nonparallel bulk secretion of the two enzymes through exocytosis. The distribution of each enzyme activity in a population of granules suggests quantal packaging of amylase and chymotrypsinogen into the granules.
Amylases↗
Ionic transport in individual renal epithelial cells from adult and young rats.
Renal epithelial cells were isolated from the outermost superficial cortex of adult and young rats. The cells, likely of proximal origin, were plated on silicon pieces, and cultured during 1-3 days. Intracellular content and concentrations of K, Na, Cl, and P, and the kinetics of change in intracellular content, after inhibition of Na-K ATPase by incubation with ouabain or in K-free medium, were measured in individual cells in small populations using electron probe analysis. In control medium, concentrations in mM were approximately: K, 130; Na, 15; Cl, 28; P, 140. After 6 h inhibition of Na-K ATPase, cells exchanged all K for Na, and the intracellular Na concentration increased to 139 mM in K-free medium. The Cl concentration increased at most to 46 mM. The sum of intracellular K + Na + Cl did not increase more than 25% after 24 h incubation in K-free medium. There were no differences in intracellular K, Na, and Cl for adult and young rat cells in similar conditions. The half-times of K efflux and Na influx after inhibition of Na-K ATPase measured in adult rat were approximately 16-20 min. In the absence of serum, in K-free medium, the half-times of K efflux and Na influx in young rat cells were approximately 30 min, significantly higher than the half-time in the presence of serum, and with ouabain, being approximately 13 min. Histograms of distributions of K and Na content showed that the cells behaved as a single functional population. Ouabain Ki was estimated to be 10(-4) M. After 24 h preincubation in K-free medium, when returned in 5 mM K-containing medium, adult rat cells recovered rapidly normal intracellular K and Na concentrations. Using this approach, expression of the kinetics ionic transport properties of renal epithelial cells during development, and the hormonal influences on terminal differentiation may be studied.
Age Factors↗