Furosemide stimulates K transport in HCD57 erythroid cells.

We examined the influence of serum and furosemide on K movement and cell volume in HCD57 cells, a murine erythroleukemia cell line, which require erythropoietin (EPO) for survival. We found that maintenance of cell volume depends on the concentration of serum in the culture medium. In isotonic medium containing 20% serum, HCD57 cells maintain their steady-state volume. In contrast, the cells shrink progressively as medium serum content is reduced. In serum-free medium, raising external K to 75 mm prevents cell shrinkage and a further increase in K to 145 mm results in swelling, revealing a role for K permeability in the regulation of cell volume. Of particular interest has been a serendipitous finding with furosemide. Below an external K concentration of 2.1 +/- 0.3 mm in medium containing 2% serum, furosemide inhibits K uptake, probably stemming from its well known inhibitory action on KCl cotransport. However, above that K concentration, furosemide stimulates K uptake in a dose-dependent manner. Moreover, furosemide potentiates cell shrinkage induced by serum withdrawal. These findings suggest that the transport machinery mediating cellular shrinkage, once primed by serum depletion, becomes receptive to a second stimulus.

Presence of immunoreactive alpha 3 subunit isoform of Na,K-ATPase in mitochondria of kidney.

BACKGROUND: Sodium-potassium-adenosinetriphosphatase (Na,K-ATPase) is the primary membrane enzyme responsible for the reabsorption of sodium ions in the kidney. It is known that in the nephron the major subunit isoforms of Na,K-ATPase are alpha 1 and beta 1. Previous reports on the presence of alpha 2 and alpha 3 isoforms in the kidney were mixed and controversial. METHODS: Techniques of ultrathin cryosectioning and immunoelectron microscopy were used to study the distribution of alpha subunit isoforms (alpha 1, alpha 2, alpha 3) and beta subunit (beta 1 isoform) of Na,K-ATPase in renal tubular cells. Western blot analysis was used to show the presence of the alpha 3 isoform in the extract of kidney mitochondria. RESULTS: We were able to confirm the previous finding that the alpha 1 isoform and the beta 1 isoform were the preponderant isoforms of the alpha and beta subunits of Na,K-ATPase in the basolateral membrane. In addition, we unexpectedly found the presence of the alpha 3 isoform in the mitochondria of rat renal tubular cells. The alpha 2 and alpha 3 isoforms were not observed in either the apical or basolateral membrane. CONCLUSIONS: Both immunoelectron microscopy and Western blot analysis of the rat kidney mitochondria confirm the presence of the alpha 3 isoform of Na,K-ATPase in the rat kidney mitochondria. The function of this enzyme in the mitochondria is not clear at this time.

Transient increase in the alpha3-isoform of Na,K-ATPase in rat erythroblastic cells.

Using immunoelectron microscopy and isoform-specific antibodies against Na,K-ATPase to study changes in Na,K-ATPase in rat erythroblastic cells during maturation, we unexpectedly observed numerous antigenic sites against the alpha3-isoform in the cytoplasmic phase. There was an increase in the number of alpha3-isoforms after denucleation of the erythroblast. The increase was transient. As the reticulocyte matured into a red blood cell, the number of alpha3-isoforms was reduced drastically. This alpha3-isoform was distributed in a reticular pattern resembling the double layers of endoplasmic reticulum. Western blot analysis confirms the presence of the alpha3-isoform in these cells. X-ray microanalysis of the erythroid series of cells in the bone marrow shows that sodium concentration in the young reticulocyte is higher than that in the nucleated erythroblast. The reason for the transient increase in this pump protein is not clear. It is possible that the increase in sodium concentration in the reticulocyte plays a role in the increase in pump protein synthesis.

Quantitative X-ray imaging of labelled molecules in tissues and cells.

Using cisplatin as a model system, we have been able to demonstrate the feasibility of studying the cellular and subcellular distribution of a labelled molecule containing a single atom of platinum per molecule in bone marrow. An X-ray imaging system consisting of a microcomputer, a 4pi system and a software package was interfaced with an electron microscope enabling the computer to control the beam movements as well as receive signals from the STEM and EDS X-ray detectors. X-ray imaging is useful for both tissue and samples in which the population of cells is not homogeneous. Imaging permits elemental distributions to be measured throughout the sample and not in just randomly selected areas as previously done in X-ray microanalysis. Images are created for not only the element labelling the molecule of interest but also other specified elements present. Three types of maps for imaging labelled molecules are compared and discussed. When the original (collected) data are mapped, the elements of interest are obscured by the continuum. The maps calculated using an internal standard give a concentration distribution on the basis of volume (mmol L(-1) of packed cells). The maps calculated using the continuum normalization method according to Hall produces concentration distribution on the basis of mass (mmolk(-1) dry weight). By recalculating using the 'Peak' or 'Hall' method the continuum problem is removed yielding quantitative images of the intracellular distribution of labelled molecules present in low concentrations.

X-ray microanalysis and mapping: measuring uptake and intracellular distribution of labeled molecules.

X-ray microanalysis is a useful technique for elemental analysis of tissues and cells. This technique utilizes x-rays generated in ultra-thin cryosections of tissue by the electron beam in an electron microscope. The x-rays are characteristic of the various elements, and can be used to measure quantitatively the elemental distribution at subcellular levels. An important application of this x-ray microanalysis and mapping technique is the study of uptake and intracellular distribution of molecules containing an element not normally present in the tissue. It is useful for tissues and samples in which the cell population is not homogeneous. Mapping permits elemental distributions to be measured throughout the sample, not just at selected sites. Maps created are not only for the element of interest but also for other specified elements present. Using an x-ray imaging system and cisplatin as a model, we have been able to demonstrate the feasibility of studying the cellular and subcellular distribution of cisplatin, a molecule containing platinum atom.

Distribution of cisplatin in bone marrow cells: quantitative X-ray imaging.

The distribution of cisplatin in freeze-dried cryosections of rabbit bone marrow cells was measured with an imaging system. Cisplatin contains a single atom of platinum per molecule which allowed the concentration of the drug to be measured throughout the cryosections. This technique was chosen because it does not require the separation of cells into homogeneous subpopulations. X-ray imaging measured not only the distribution of the element labeling the drug, but also other elements which were used to follow the effect of the drug on membrane ion transport. In samples incubated for 10 hr in cisplatin a high selectivity in the uptake of this drug was observed. Only a few of the large early cells were found to accumulate this drug, while in the smaller more mature cells platinum could not be detected. Most of the platinum accumulated in the cytoplasm and was not evenly distributed. Cells containing high levels of platinum lost their cytoplasmic potassium and gained extracellular sodium and chloride. In some cases cells that contain lower concentrations of platinum with normal levels of potassium were found, indicating that the alteration of the membrane permeability or the inhibition of the K/Na pump did not occur during the initial accumulation of the drug. This membrane permeability or pump change requires the accumulation of high concentrations of the drug, resulting in the loss of potassium and the gain of sodium.

X-ray microanalysis of cultured alveolar macrophages with phospholipidosis.

When administered to humans and animals, the iodine-containing drug amiodarone can cause pulmonary toxicity. As part of the pulmonary response to amiodarone, the drug and its principal metabolite, desethylamiodarone, accumulate in alveolar macrophages. Little is known about the susceptibility of lungs with preexisting damage to amiodarone administration. A number of chemicals can cause pulmonary phospholipidosis in humans and animals. To study the effect of a preexisting phospholipidosis on the intracellular accumulation of amiodarone and desethylamiodarone, rats were treated with chlorphentermine to induce a phospholipidosis in alveolar macrophages. The cells were recovered from the lungs by pulmonary lavage and placed in cell culture. They were then exposed to the same concentration of either amiodarone or desethylamiodarone. The intracellular distribution of each drug was quantified by measuring the associated iodine signal using X-ray microanalysis of freeze-dried cryosections of cells. Both drugs accumulated in lipid-rich amorphous bodies which correspond to lysosomally derived lamellar structures observed in conventional plastic sections. The level of desethylamiodarone exceeded that of amiodarone in the amorphous bodies. With both drugs, a higher concentration of iodine was present at the outer edges of the amorphous bodies compared to that in the center core. This suggests that the drugs are unable to freely penetrate the performed structures. By monitoring the concentrations of sodium and potassium ions within the nucleus, it was determined that chlorphentermine treatment disrupted the ionic distribution in the cells. Exposure to amiodarone, but not desethylamiodarone, resulted in further changes in sodium and potassium levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Surfaces of cryosections: is cryosectioning 'cutting' or 'fracturing'?

In order to determine if cryosectioning involves 'fracturing' or 'cutting' we examined the surfaces obtained in cryosectioning by a metal-replicating procedure commonly used in freeze-fracture microscopy. Platinum-carbon replicas were made of the surfaces of both the sections and the complementary surfaces of the sample stubs from which the sections were cut. When samples of frozen red cells were sectioned at -120 degrees C with large knife advancements (1 micron), the chips produced did not resemble sections. Membrane fracture faces, produced by splitting of the lipid bilayer, were found in electron micrographs of replicas of the sample stubs. This demonstrates that a cryomicrotome can be used to produce large intact replicas. When dull knives were used with small knife advancements, both smooth and fractured regions were found. The sections produced with dull knives had a snowflake appearance in the light microscope. When sharp knives were used with small advancements (0.1 microns), replicas of the surfaces were free of fracture faces and the sections had a cellophane-like appearance in the light microscope. Therefore, in cryosectioning a different process other than 'fracturing' is responsible. This 'cutting' process may be micromelting of a superficial layer by the mechanism of melting-point depression from the pressure exerted by the sharp edge of the knife.

Application of X-ray microanalysis to the study of drug uptake in cell culture.

X-ray microanalysis has been used previously to study the accumulation of iodine in alveolar macrophages of rats treated with the iodinated drug, amiodarone. Due to metabolism of the drug in vivo, primarily to desethylamiodarone, it was not possible to identify the source of the iodine signal. In the present study we have utilized primary cell cultures of alveolar macrophages to study the intracellular accumulation of each of these drug species in vitro. Neither drug is metabolized by these cells in culture, permitting characterization of the accumulation of each independent of the other. Cells were incubated with equimolar concentrations of either amiodarone or desethylamiodarone for 42 hr, and X-ray microanalysis of freeze-dried cryosections of cells was used to quantify accumulation by monitoring the iodine signal associated with each drug. For both drug exposures, the highest iodine content was present in amorphous bodies and dense granules, consistent with the pattern following in vivo exposure. Higher levels of desethylamiodarone, compared to amiodarone, were measured in all compartments of the cells. The results of the in vitro investigation further demonstrate the utility of X-ray microanalysis in the study of the cellular response to amiodarone and desethylamiodarone.

Quantitative X-ray microanalysis of alveolar macrophages after long-term treatment with amiodarone.

Treatment with the iodine-containing antiarrhythmic drug, amiodarone, can cause pulmonary toxicity. Alveolar macrophages are particularly susceptible to formation of lipidrich lamellar bodies in amiodarone-treated animals. Amiodarone and several of its metabolites accumulate in the cell. Previously, we have reported that the technique of X-ray microanalysis is useful in monitoring the distribution of iodine in freeze-dried cryosections of alveolar macrophages from Fischer 344 rats 24 hr after a single dose of amiodarone. In the present study, we examine the effects of longer term amiodarone treatment of 1 or 9 weeks. Substantial changes in iodine distribution occur in the cells with increasing length of drug treatment. High concentrations of iodine are found early in the lamellar bodies. The iodine levels in the nuclei slowly increase with the length of treatment, and after 9 weeks of treatment, approach those found in the lamellar bodies. It is possible that this accumulation of iodine in the nuclei is due to the presence of polar metabolites. In addition, the potassium concentration in the cell decreases and the sodium increases with treatment duration. These changes in cations are most likely due to altered ion transport in the macrophages by the inhibition of membrane Na-K-ATPase by the drug and its principal metabolite, desethylamiodarone.

Iodine in rat alveolar macrophages following amiodarone treatment: quantitative X-ray microanalysis.

The techniques of cryomicrotomy and X-ray microanalysis were used to quantitatively measure the subcellular distribution of iodine in rat alveolar macrophages following a single administration of the iodine-containing antiarrhythmic drug, amiodarone. When frozen, dried sections were analyzed, small amounts of iodine were found throughout the alveolar macrophages, but the major accumulations were observed in amorphous bodies and dense granules. The highest to lowest accumulation is in the following order: amorphous bodies (90 mmole I/kg dry wt) greater than dense granules (50 mmole I/kg dry wt) greater than nucleus = cytosol (10 mmoles I/kg dry wt). The amorphous bodies can contain high and low levels of iodine and the granules are found to have high and low levels of iron. Granules with the high and low levels of iron and amorphous bodies with the high and low levels of iodine can be found in the same cells. X-ray microanalysis proved useful in describing the intracellular distribution of iodine-labeled species following amiodarone administration.

X-ray microanalysis of Plasmodium falciparum and infected red blood cells: effects of qinghaosu and chloroquine on potassium, sodium, and phosphorus composition.

Cryosections of human red blood cells infected by Plasmodium falciparum were analyzed by energy dispersive x-ray microanalysis to determine the elemental composition of the parasites and their red cell hosts separately. The effects of two antimalarial drugs, qinghaosu and chloroquine, on potassium, sodium, and phosphorus concentrations were studied. Malarial infection causes a decrease in potassium concentration and an increase in sodium concentration in the host red cells. The drastic change in the cation composition, however, occurs only in red cells infected by late stage parasites (late trophozoite and schizont). Red cells infected by early stage parasites (ring stage) show only small changes in sodium concentration. Furthermore, the noninfected red cells in parasitized cultures show no difference in composition from those of normal red cells. Treatment of the parasitized cultures with qinghaosu (10(-6) M) or chloroquine (10(-6) M) for 8 hr causes phosphorus concentration of both early and late parasites to decrease. An 8 hr treatment with qinghaosu also produces a reduction in potassium and an increase in sodium concentrations in early and late parasites. In contrast, 8 hr treatment with chloroquine only causes a change in the sodium and potassium concentrations of the late stage parasites and does not affect the early stage parasites.

Anion transport during maturation of erythroblastic cells.

Bromide uptake was measured in single maturing erythroblastic cells of rabbits by means of X-ray microanalysis. Increase in bromide uptake as the cells matured was observed. The order of cells from low to high bromide uptake was: early erythroblast less than late erythroblast less than marrow red cells less than peripheral red blood cells. The transition from low to high bromide uptake is correlated to the accumulation of iron which begins in the late erythroblast. A decrease in rubidium uptake also occurs as iron accumulates in the cell. These results indicate that the anion and cation transport changes during maturation are parallel in time course but opposite in direction. In addition, the increase in bromide uptake can be accounted for by the increase in surface-to-volume ratios of the cells. Surface-to-volume ratios were estimated by morphometric techniques.

Interrelations among Na and K content, cell volume, and buoyant density in human red blood cell populations.

This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na + K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966. Scand. J. Lab. Invest. 18:167-180) cell water and cell volume was found to vary directly with the summed content of Na + K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na + K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.

Rubidium uptake in single cells.

Rubidium uptake was measured in single erythroid and myeloid cells of rabbit by means of X-ray microanalysis. It was found in the nucleated bone marrow cells that after incubation in rubidium the sums of potassium and rubidium concentrations were similar to the original potassium concentrations, indicating that there was one-to-one replacement of potassium by rubidium. Although the nuclear potassium and rubidium concentrations were higher than those in the cytoplasm, the nuclear and cytoplasmic ratios of K/Rb were similar. This implies that the potassium in both compartments exchanged freely with rubidium. In the erythroid line of cells there was a continuous reduction of potassium transport activity during the maturation process as indicated by the decrease in rubidium uptake rates. The uptake was measured in seven groups of cell types that could be distinguished on the basis of morphology and chemical composition. The order of the groups from high to low rubidium uptake were: esosinophilic myelocyte greater than early erythroblast and thin-rimmed erythroblast greater than late erythroblast greater than early bone marrow red cell greater than late bone marrow red cell greater than peripheral blood red cell. Thus, there is a continuous decrease in rubidium transport as the erythroid cells mature.

Influence of acute potassium loading on renal phosphate transport in the rat kidney.

UNLABELLED: This study examined 1) whether potassium-induced depression of phosphate excretion is a parathyroid hormone-dependent phenomenon, and 2) whether such stimulation of tubular phosphate reabsorption capacity involves increased phosphate reabsorption in the distal tubule. Potassium was infused into intact rats (25 mumol X min-1 X kg-1) during stepwise addition of phosphate to the infusion and led to a significant drop in phosphate excretion; this effect was abolished in thyroparathyroidectomized (TPTX) animals. In intact rats the maximal tubular Pi reabsorption per milliliter of glomerular filtrate (max TRPi/ml GF) was significantly higher in the potassium group (2.54 +/- 0.06 mumol/ml GF) compared with the control group (2.31 +/- 0.06 mumol/ml GF) (means +/- SE). In TPTX rats no difference in max TRPi/ml GF was observed: 3.44 +/- 0.07 and 3.49 +/- 0.07 mumol/ml GF during potassium and sodium infusion, respectively. Free-flow micropuncture was carried out on superficial distal tubules of intact rats and fluid samples were analyzed for [3H]inulin and phosphorus (electron microprobe). Phosphorus delivery into the distal tubule was similar in control and potassium-loaded rats. Whereas net phosphorus reabsorption along the distal tubule was absent in the control group, intravenous potassium administration stimulated distal phosphorus reabsorption. CONCLUSION: potassium stimulates renal phosphate reabsorption capacity, an effect that is abolished after TPTX. The potassium effect on phosphate occurs along the distal tubule.

The distribution of intracellular ions in the avian salt gland.

To investigate the mechanism of salt secretion in the avian salt gland, we used quantitative electron probe microanalysis to measure the intracellular elemental concentrations in dry cryosections of unspecialized and partially specialized secretory epithelial cells from fresh water- and salt water-adapted ducklings, respectively. In conjunction with this, human and duckling erythrocytes were also analyzed, since these provided the experimental basis for using in situ erythrocytes as standards for determining the local water content of epithelia from the analysis of dried cryosections. The microprobe results from both types of erythrocytes compared favorably with chemical determinations of elemental concentrations. The nucleated avian erythrocytes, whose wet-weight elemental concentrations were determined by a compartmental analysis that required neither a peripheral standard nor a measure of the local mass, revealed a marked accumulation of P and K in the nucleus (388 and 190 mmol/kg wet wt, respectively) relative to the cytoplasm (67 and 85 mmol/kg wet wt). In both developmental states of the epithelial cells, the nucleus and apical cytoplasm had essentially similar and unremarkable concentrations of Na (76 and 83 mmol/kg dry wt, respectively, in the adapted cells vs. 72 and 81 mmol/kg dry wt in the control cells) and K (602 and 423 mmol/kg dry wt vs. 451 and 442 mmol/kg dry wt). Chloride, however, which was in general rather high, was significantly depressed in the apical cytoplasm of adapted cells only (164 and 124 mmol/kg dry wt in the nucleus and cytoplasm, respectively, of adapted cells (P less than 0.05) vs. 138 and 157 mmol/kg dry wt for control cells (P less than 0.05). Cation concentrations (Na + K) were elevated approximately 15% in the basal regions of adapted cells as compared with apical cytoplasm. When tissue water variations are accounted for, the results suggest that: (a) an active, energy-requiring process is responsible for chloride accumulation in this cell; (b) the apical membrane is a regulatory site for secretion; and (c) there are regional distinctions in the distribution of ions and water, particularly in the salt water-adapted cell. These conclusions are consistent with active chloride transport as the basis for salt secretion in this tissue.