Cells In Hypertonic Medium Research Articles

Cell Volume Regulation of Endothelial Cells Is Impaired in Keratoconus Cornea

In this work the permeability to water and urea of plasma membranes of endothelial cells of normal corneas and corneas with keratoconus was investigated. The endothelial cells were obtained from surgery material. Measurements of osmotic aqueous permeability (Pf) of endothelial cells in normal and in keratoconus did not reveal significant differences of this parameter in the two studied groups. The control cells and the cells from keratoconus cornea have similar osmotic water permeability (control cells, Pf = 0.53 ± 0.045 cm/s; keratoconus cells, Pf = 0.63 ± 0.041 cm/s; n = 25; p ≥ 0.05). Neither coefficient of urea permeability differed significantly in these groups (control, Pu = 0.049 ± 0.003 cm/s; keratoconus, Pu = 0.056 ± 0.003 cm/s; n = 25; p ≥ 0.05). Analysis of cell volume dynamics based on exponential approximation showed a more pronounced decrease of the cell volume of endothelial cells from keratoconus cornea in hypertonic medium in comparison with the cells from normal cornea. The increase of cell volume caused by isotonic entering of urea into the cells in hypertonic medium also was more pronounced in these cells in comparison with the normal ones. We conclude that there are significant changes in cell volume regulating mechanism in keratoconus cornea endothelial cells.

Chemical Composition, Antioxidant and Antimicrobial Activity of Raspberry, Blackberry and Raspberry-Blackberry Hybrid Leaf Buds.

In our investigation, the chemical composition and bioactive potential of leaf buds of raspberry, blackberry, and a raspberry-blackberry hybrid were determined. Antioxidant and antimicrobial properties were tested in water (W), ethanol-water (EW), and glycerol-water (GW) extracts from the buds. These plant organs contain relatively large amounts of minerals, especially Fe. The total antioxidant capacity (TAC) measured by the ABTS and DPPH methods ranged from 2.86 to 12.19 and 6.75 to 24.26 mmol per 100 g fresh weight (FW) of buds, respectively. TAC values were generally higher in the raspberry than in the case of blackberry and raspberry-blackberry hybrid extracts. The antioxidant properties of the extracts were strongly positively correlated with their content of total phenolic (TP). No such relationship was noted for ascorbic acid (AA), whose concentration in all extracts was at a similarly low level. Antioxidant properties determined in vitro were confirmed for the GW extract from raspberry leaf buds in biological test based on the growth parameters of Δsod1 Saccharomyces cerevisiae mutant cells in hypertonic medium. The extracts also exhibited strong antibacterial properties against Staphylococcus aureus and Enterococcus faecalis and weaker against Enterobacter aerogenes. The studied leaf buds could be therefore an unconventional source of minerals, natural antioxidants and antibacterial compounds with potential applications in the food, pharmaceutical, and cosmetics industries.

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.

Hypertonicity induced apoptosis in HL-60 cells in the presence of intracellular potassium

Cell shrinkage is a hallmark of apoptosis. Potassium efflux, which is involved in cell shrinkage, has been previously described as an essential event of apoptosis. This study was designed to address the importance of potassium efflux in hypertonicity (450 mOsm and 600 mOsm) induced apoptosis. We initiated apoptosis in HL-60 cells in hypertonic medium consisting of either high concentrations of NaCl, mannitol or KCl. Apoptotic activity was evaluated based on the DNA content of the cells, annexin-V staining and calcium content. Apoptosis was initiated in hypertonic conditions consisting of high intracellular K(+). We demonstrate that apoptosis can occur in the presence of high intracellular potassium contrary to previous predictions.

Characterization of Na/H exchange systems in immortalized rat caput epididymal (RCE) cells

RCE cells are primary cultures of rat caput epididymal cells immortalized with SV40 large T antigens. The goals of the present studies were to characterize the endogenous Na/H exchange systems in these cells and to understand the regulation of endogenous Na/H exchangers by hyperosmolarity because epididymal fluids are hypertonic. By Western blotting, RCE cells were shown to have endogenous NHE1 (105kD protein), but not NHE2 and NHE3. For measurement of Na/H exchange with BCECF, RCE cells were grown on collagen coated glass cover slips. These cells exhibited Na dependent alkalinization which was completely inhibited by HOE694 (IC50 = 0.3μM), confirming the presence of NHE1. Kinetic analysis with Na showed a Km of 18±3mM with a Hill coefficient of 1.1±0.2. The acute effect of hyperosmolarity on NHE1 was studied at both steady-state pH and by initial rates of pH recovery after NH4Cl acidification. Addition of 100mM mannitol at steady-state pH of cells caused intracellular alkalization (ΔpH = 0.4±0.1) which was blocked by HOE694. Mannitol (100mM) also increased the initial pH recovery rates of cells. Kinetic analysis suggested that there was no change in Vmax but a change in Km(H) (Km(H): control; 6.51±0.01 vs mannitol; 6.75±0.01). Interestingly, mannitol stimulation of NHE1 was inhibited by BAPTA (50μM) and by the calmodulin (CaM) inhibitor, W13 (45μM), suggesting the involvement of Ca/CaM. The chronic effect of hyperosmolarity on NHE1 was also studied by culturing the cells in the presence of 100mM mannitol for 72h. Under this culturing condition, the amount of NHE1 protein was increased by 2.1 fold as determined by Western blotting. We conclude that RCE cells contain endogenous NHE1 which is stimulated by hyperosmolarity in a Ca/CaM dependent manner. Chronic exposure of RCE cells in hypertonic medium increases the amount of NHE1 protein.

The complement inhibitor, CRIT, undergoes clathrin-dependent endocytosis

Complement C2 receptor inhibitor trispanning (CRIT) is a receptor for the second component of complement and is found in various tissues and hemopoietic cells. On binding to CRIT, C2 cannot be activated to potentially form a variant-C3 convertase as it is rendered non-cleavable by C1s. CRIT thus limits the amount of C3 convertase formed on the cell surface. In this study we have shown, using flow cytometry and immunofluorescence microscopy, that human CRIT undergoes endocytosis from the plasma membrane. The endocytosis, possibly ligand mediated, occurs via clathrin-coated pits as it can be inhibited by prior incubation of cells in hypertonic medium or with chlorpromazine, at 37 degrees C. However, inhibition of endocytosis was not possible after treatment with nystatin, or filipin, inhibitors of caveolae/raft-dependent endocytosis. In the presence of C2 alone, CRIT associates with the adapter protein, beta-arrestin-2, and whether in association with C2 or not, then appears in the perinuclear region, but does not appear to be translocated into the nucleus. Apart from the C3aR and C5aR that internalize the anaphylatoxic peptides, this is the first report of the internalization via the clathrin pathway of a receptor for a complement serum protein.

Kidney cell survival in high tonicity.

The kidney medulla of mammals undergoes large changes in tonicity in parallel with the tonicity of the final urine that emerges from the kidney at the tip of the medulla. When the medulla is hypertonic, its cells accumulate the compatible osmolytes myo-inositol, betaine, taurine, sorbitol and glycerophosphorylcholine. The mechanisms by which the compatible osmolytes are accumulated have been explored extensively in kidney-derived cells in culture. Myo-inositol, betaine and taurine are accumulated by increased activity of specific sodium-coupled transporters, sorbitol by increased synthesis of aldose reductase that catalyses the synthesis of sorbitol from glucose. Glycerophosphorylcholine accumulates primarily because its degradation is reduced in cells in hypertonic medium. cDNAs for the cotransporters and for aldose reductase have been cloned and used to establish that hypertonicity increases the transcription of the genes for the cotransporters for myo-inositol, betaine and for aldose reductase. The region 5' to the promoter of the gene for the betaine cotransporter and for aldose reductase confer osmotic responsiveness to a heterologous promoter. The 12-bp sequence responsible for the transcriptional response to hypertonicity has been identified in the 5' region of the gene for the betaine cotransporter.

Regulation of Na+,K(+)-ATPase and the Na+/K+/Cl- co-transporter in the renal epithelial cell line NBL-1 under osmotic stress.

The long-term adaptation of the Na+,K(+)-ATPase to hypertonicity was studied using the bovine renal epithelial cell line NBL-1. Na+,K(+)-ATPase activity measured in intact cells as the ouabain-sensitive fraction of Rb+ uptake was stimulated (40% above controls) after incubating the cells in hypertonic medium. This stimulation was not correlated with significant changes in the amount of Na+,K(+)-ATPase alpha 1 subunit protein. Nevertheless, the amount of alpha 1 but not beta 1 subunit mRNA progressively increased after hypertonic shock (3-4-fold above basal values). These results suggest that the alpha 1 subunit gene is modulated by medium osmolarity, although this does not necessarily involve enhanced translation of the mRNA into active alpha 1 protein. Indeed, the increase in the biological activity of the Na+,K(+)-ATPase is abolished when the electrochemical Na+ transmembrane gradient is depleted by monensin, which is consistent with a post-translational effect on the activity of the sodium pump. A furosemide-sensitive component of Rb+ uptake, attributable to Na+/K+/Cl- co-transporter activity, was very low when cells were cultured in a regular medium, but was greatly induced after hypertonic shock. This induction could not be blocked by cycloheximide. Colcemide addition slightly reduced the absolute increase in Na+/K+/Cl- co-transporter activity, while cytochalasin B significantly potentiated the effect triggered by hypertonic shock. It is concluded: (i) that in NBL-1 cells the alpha 1 but not the beta 1 subunit of the Na+,K(+)-ATPase is encoded by an osmotically sensitive gene, and (ii) that the Na+/K+/Cl- co-transporter, although an osmotically sensitive carrier, is induced by a mechanism that is independent of protein synthesis but may rely, in an undetermined manner, on the structure of the cytoskeletal network.

Synthesis and secretion of endothelin in a cortical collecting duct cell line.

Previous experiments have shown that epithelial cells in the renal medulla produce endothelin-1 (ET-1) and possess ETB receptors. It has been suggested that medullary ET-1 may affect water and sodium absorption along the collecting ducts in an autocrine fashion. To study possible mechanisms responsible for the regulation of medullary ET-1 production, experiments were performed in M-1 cells and mIMCD-K2 cells, cell lines derived from cortical and inner medullary collecting ducts of SV40 transgenic mice, grown to confluence on collagen-coated filter inserts. Both cell lines were found to express ET-1 mRNA and to secrete ET almost exclusively into the basolateral medium as long as the transepithelial resistance was high. Inhibition of transcription with actinomycin D was followed by a decline in both ET mRNA [halftime (t1/2) = 30 min] and ET secretion (t1/2 = approximately 90 min). The addition of arginine vasopressin (AVP, 10(-8) M; 2- or 4-h exposure) or incubation of M-1 cells in hypertonic media (+50 mM NaCl, 4- or 6-h exposure) did not significantly alter ET secretion or ET-1 mRNA expression. In contrast, simultaneously increasing AVP(10(-8) M in the basolateral medium) and tonicity (+50 mM NaCl) for 4 h increased ET secretion (from 28.9 +/- 3.9 to 41.8 +/- 3.8 pg.h-1.mg protein-1; P = 0.029, n = 10) and ET-1 mRNA (control = 2,138 cpm/microliter, log of 3.33 +/- 0.048, n = 4; AVP + NaCl = 3,548.1 cpm/microliter, log of 3.55 +/- 0.09; P = 0.045, n = 5). Exposure of M-1 cells to hypertonic media (+50 mM NaCl or 100 mM mannitol) for 24 h was associated with a marked reduction of ET secretion (-83.9% with NaCl and -78.4% with mannitol; P < 0.0001). This reduction was attenuated, but not prevented, by the presence of AVP in the basolateral medium (-40%). ET-1 mRNA, in contrast, did not change with 24-h exposure to hypertonic media and increased when AVP was present. Results are compatible with the concept that generation of ET by collecting duct cells may contribute in a complex and time-dependent fashion to the paracrine control of collecting duct cell function.

Low Temperatures and Hypertonicity Do Not Block Cytokine-induced Stimulation of the Sphingomyelin Pathway but Inhibit Nuclear Factor-κB Activation

In order to better understand the significance of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta)-receptor internalization in the sphingomyelin pathway signal transduction, we investigated receptor signaling under conditions in which receptor internalization is blocked. We demonstrate that human recombinant TNF-alpha and IL-1 beta both induced sphingomyelin and phosphatidylcholine hydrolysis at either 4, 14, or 37 degrees C in human skin fibroblasts and U937 monocytic cells. Cytokine-induced sphingomyelin degradation also occurred when endocytosis was inhibited by incubating the cells in hypertonic medium. While internalization was not required for the production of ceramide, activation of the transcription factor NF-kappa B was strongly reduced when cells were stimulated with TNF at low temperature or in hypertonic medium. Under these conditions, activation of NF-kappa B by the cell-permeant C2- ceramide (N-acetylsphingosine), by exogenous sphingomyelinase or by phorbol myristate acetate was also inhibited. These results suggest that low temperature and hypertonicity, two inhibitors of receptor internalization: (i) do not affect the TNF-alpha- or IL-1 beta-induced sphingomyelin hydrolysis, but (ii) do inhibit a step distal to ceramide of the intracellular signaling pathway leading to NF-kappa B activation.

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3 gamma subunit of the TCR. To investigate the role of CD3 gamma phosphorylation in PKC-mediated TCR down-regulation, point mutated CD3 gamma cDNA was transfected into the CD3 gamma-negative T cell line JGN and CD3 gamma transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3 gamma was required for PKC-mediated down-regulation of the TCR. Furthermore, analysis of a series of CD3 gamma truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131 and L132) in the cytoplasmic tail of CD3 gamma was required for PKC-mediated TCR down-regulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKC-mediated TCR down-regulation in non-mutated T cells, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucine- or tyrosine-based motifs is proposed.

Aldose Reductase in Human Retinal Pigment Epithelial Cells

Sugar alcohols have been reported to accumulate in retinal pigment epithelium (RPE) of diabetic animals. This finding has raised interest in the role of RPE in diabetes-associated retinal changes such as cystoid macular edema. To confirm the presence of aldose reductase in this tissue, the NADPH-dependent enzyme was purified to an apparent homogeneity from cultured human RPE cells, characterized, and its biochemical properties investigated. The induction of aldose reductase by hypertonic stress was also examined. The purification of aldose reductase was performed by a series of chromatographic steps which include gel filtration, affinity chromatography and chromatofocusing. Final purity achieved was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The kinetic properties and susceptibility to inhibition of the purified aldose reductase were essentially identical to aldose reductase purified from human placenta and kidney. In addition to aldose reductase, chromatofocusing demonstrated the presence of aldehyde reductase, another NADPH-dependent reductase. However, the amounts of aldehyde reductase present were much smaller than those of aldose reductase and the levels of aldehyde reductase appeared too small to contribute to the polyol production in the RPE cells. Culture of RPE cells in hypertonic medium containing 150 mM sodium chloride (600 mosmol total) increased both reductase activity, monitored with d l -glyceraldehyde as substrate, and immunoblot staining for aldose reductase. Chromatofocusing of RPE cells cultured in hypertonic media resulted in a prominent increase in the peak corresponding to aldose reductase compared to the peak height of cells grown in control medium. No increase in aldehyde reductase from RPE cells cultured in hypertonic medium was observed. These results indicate that human RPE cells contain two NADPH-dependent reductases, aldose reductase and aldehyde reductase with aldose reductase being the predominant reductase. Sugar alcohol formation in this tissue is principally catalysed by aldose reductase while the amounts of sugar alcohols generated by aldehyde reductase appear negligible. Only aldose reductase is induced by hypertonic stress. This supports the hypothesis that the aldose reductase mediated polyol production serves as an osmolyte to osmoregulation.

Regulation by cell volume of Na+-K+-2Cl? cotransport in vascular endothelial cells: role of protein phosphorylation

Na(+)-K(+)-2Cl- cotransport in aortic endothelial cells is activated by cell shrinkage, inhibited by cell swelling, and is responsible for recovery of cell volume. The role of protein phosphorylation in the regulation of cotransport was examined with two inhibitors of protein phosphatases, okadaic acid and calyculin, and a protein kinase inhibitor, K252a. Both phosphatase inhibitors stimulated cotransport in isotonic medium, with calyculin, a more potent inhibitor of protein phosphatase I, being 50-fold more potent. Neither agent stimulated cotransport in hypertonic medium. Stimulation by calyculin was immediate and was complete by 5 min, with no change in cell Na + K content, indicating that the stimulation of cotransport was not secondary to cell shrinkage. The time required for calyculin to activate cotransport was longer in swollen cells than in normal cells, indicating that the phosphorylation step is affected by cell volume. Activation of cotransport when cells in isotonic medium were placed in hypertonic medium was more rapid than the inactivation of cotransport when cells in hypertonic medium were placed in isotonic medium, which is consistent with a shrinkage-activated kinase rather than a shrinkage-inhibited phosphatase. K252a, a nonspecific protein kinase inhibitor, reduced cotransport in both isotonic and hypertonic media. The rate of inactivation was the same in either medium, indicating that dephosphorylation is not regulated by cell volume. These results demonstrate that Na(+)-K(+)-2Cl- cotransport is activated by protein phosphorylation and is inactivated by a Type I protein phosphatase. The regulation of cotransport by volume is due to changes in the rate of phosphorylation rather than dephosphorylation, suggesting the existence of a volume-sensitive protein kinase. Both the kinase and the phosphatase are constitutively active, perhaps to allow for rapid changes in cotransport activity.

Kidney aldose reductase gene transcription is osmotically regulated.

Cells generally adapt to long-term hypertonic stress by accumulating organic osmolytes. PAP-HT25 renal medullary cells in hypertonic medium accumulate sorbitol through a reaction catalyzed by aldose reductase and betaine through osmotically regulated transport. Hypertonicity increases aldose reductase protein synthesis rate by elevating its mRNA abundance. To test whether the rise in aldose reductase mRNA is due to enhanced transcription, PAP-HT25 cells adapted to isotonic medium were switched to hypertonic medium, and transcription rate was measured by nuclear run-on. Aldose reductase transcription rate peaked at 17-fold the isotonic level after 12 h of hypertonicity. Then, transcription fell as sorbitol and betaine accumulated. Transcription stabilized at fivefold the isotonic level within days. Aldose reductase mRNA stability was not significantly different between the hypertonic and isotonic steady states. Thus aldose reductase mRNA is osmotically regulated through changes in its transcription. The osmotically induced rise in aldose reductase transcription is blunted by the accumulation of intracellular betaine and is exaggerated and prolonged by preventing the accumulation of both sorbitol (by aldose reductase inhibition) and betaine (by removal from the medium). This study presents the first description of osmoregulated gene transcription in animal cells.

Role of vesicular traffic in the transport of surface transferrin receptor to the Golgi complex in cultured human cells

We have previously shown that transferrin receptor (TfR) recycles from the cell surface through the Golgi complex in K562 human leukemia cells. However, little is known about the transport pathway that carries these receptors to the Golgi complex. To learn more about this transport, we studied the effects of treatments that block specific types of vesicular traffic. K562 cells were cultured in test media and the transport of surface TfR to the Golgi complex was assessed by measuring the entry of asialo-TfR into the sialyltransferase compartment of the Golgi complex. Depletion of cellular potassium, which blocks formation of coated vesicles at the cell surface, stimulated asialo-TfR resialylation by 60% over controls, suggesting that coated vesicle formation is not the ratelimiting step in cell surface-to-Golgi transport. Similarly, culture in sodium-free medium, which blocks transport from endosomes to lysosomes, increased asialo-TfR resialylation by 40%, arguing that lysosomes do not lie on the transport pathway. In contrast, incubation of cells in hypertonic medium, which blocks many vesicular transport steps, inhibited TfR resialylation by 40%, confirming the importance of vesicular traffic in transport of asialo-TfR from the cell surface to the Golgi complex. These results are consistent with two possible pathways for cell surface-to-Golgi transport. Receptor could be transported via an endosomal intermediate, with the rate-limiting step occurring at a post-endosomal site. Alternatively, receptor could be transported directly to the Golgi via a pathway that does not involve endosomes.

Intracellular betaine substitutes for sorbitol in protecting renal medullary cells from hypertonicity

Renal medullary cells are normally exposed to a variably high extracellular NaCl concentration. They compensate by accumulating large amounts of organic osmolytes, including sorbitol and betaine. The sorbitol is synthesized from glucose, catalyzed by aldose reductase. Previously, inhibition of aldose reductase activity was noted to greatly reduce renal medullary cell survival and growth (measured by cloning efficiency) in tissue cultures of renal medullary cells in hypertonic medium. In contrast, inhibition of aldose reductase and renal medullary sorbitol accumulation is not associated with kidney damage in vivo. In the present experiments we find that addition of betaine to the medium, and its resultant uptake by the cells, largely replaces the decrease in sorbitol caused by aldose reductase inhibitors and restores the cloning efficiency. We presume that in vivo uptake of betaine by renal medullary cells similarly protects them from harm when aldose reductase inhibitors lower sorbitol. The results also demonstrate that one organic osmolyte can substitute for another in protecting cells from hypertonicity, consistent with the compatible osmolytes hypothesis.

Effects of hypertonic and sodium‐free medium on transport of a membrane glycoprotein along the secretory pathway in cultured mammalian cells

Incubation of cultured cells in hypertonic medium and sodium-free medium have been shown to block transport at two different stages along the endocytic pathway. To determine the effects of these treatments on the exocytic pathway, we studied the transport of the membrane glycoprotein of vesicular stomatitis virus (VSV-G) in cells infected with tsO45 mutant virus. This mutant synthesizes a VSV-G that accumulates in the endoplasmic reticulum (ER) when cells are incubated at 39.5 degrees C. In addition, VSV-G accumulates in the post-ER pre-Golgi compartment when cells are incubated at 15 degrees C and in the trans-Golgi network (TGN) when cells are incubated at 18 degrees C. Upon transfer of cells to 32 degrees C in control medium, VSV-G exits each of these compartments and is transported to the cell surface. Incubation in sodium-free medium at 32 degrees C did not block transport from any of these three compartments. In contrast, incubation in hypertonic medium blocked export from the ER, transport from the pre-Golgi compartment to the Golgi complex, and transport from the TGN to the cell surface. Our results, in combination with previous studies, suggest that hypertonic medium blocks at least five distinct transport steps; the three exocytic steps described here, endocytosis from the cell surface, and transport of cell surface proteins into the Golgi complex. This raises the possibility that vesicular transport in different parts of the cell shares common elements that are inhibited by this treatment.

Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system.

A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a cap-independent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4- to 7-fold increase in total CAT activity. A further approximately 2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for greater than 10% of the total cell protein.

Volume-sensitive K transport in human erythrocytes.

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.

Evidence for Differences among the Sectors of Potentially Lethal Damage Expressed by Hypertonic Treatment in Plateau-Phase V79 Cells after Exposure to Neutrons or γ Rays: The Importance of Distinction between α and β-PLD Forms

Expotentially growing and plateau-phase V79 cells were exposed to various doses of neutrons and plated either immediately or after treatment in hypertonic medium (250-500 mM NaCl) to express radiation-induced potentially lethal damage (PLD). Postirradiation treatment of exponentially growing cells in hypertonic medium (500 mM) resulted in a decrease in both Dq and D0, whereas postirradiation treatment of plateau-phase cells in hypertonic medium (in the range between 200 to 1,500 mM) resulted mainly in a reduction of Dq. This difference in response between exponentially growing and plateau-phase cells may reflect differences in the chromatin structure in cells at various stages of the cell cycle, affecting fixation of radiation-induced damage. Exposure of plateau-phase cells to gamma rays, on the other hand, resulted in a treatment time and salt concentration-dependent decrease in Dq along with a decrease in D0. Repair of neutron-induced, hypertonic treatment-sensitive PLD, measured by delaying treatment for various periods after irradiation, was found to proceed with a t1/2 of about 1 h. This is similar to the repair kinetics obtained by delaying treatment of plateau-phase cells with 150 microM beta-D-arabinofuranosyladenine (araA) after exposure to gamma rays or neutrons and contrasts the repair kinetics observed after exposure of cells to gamma rays. In this case, hypertonic treatment was found to affect a form of PLD repaired with a t1/2 of 10-15 min (beta-PLD) and araA, a different form of PLD, repaired with a t1/2 of about 1 h (alpha-PLD). Based on these results it is hypothesized that the sector of lesions affected by hypertonic treatment and araA coincides after exposure to neutrons (effect on alpha-PLD) but only partly overlaps after exposure to gamma rays (due to the effect on beta-PLD of hypertonic treatment). The results presented, together with previously published observations, suggest a differential induction and/or fixation by hypertonic medium of the alpha- and beta-PLD forms as the LET of the radiation increases. Furthermore, they indicate that direct comparison of the effects of a postirradiation treatment, as well as of the repair kinetics obtained by its delayed application after exposure to radiations of various LET, should be made with caution.