Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.     

Role of Ca2+ in sorbitol release from rat inner medullary collecting duct (IMCD) cells under hypoosmotic stress

The role of Ca2+ was studied in the release of the organic osmolyte sorbitol from rat IMCD cells in response to hypoosmotic stress. When cells were exposed to hypoosmotic media, sorbitol release was greatly reduced in Ca-free media which, on readmission of Ca2+, returned to control values. Under isoosmotic conditions, the ionophore A23187 stimulated sorbitol release without any effect on cell volume. Addition of trifluoperazine, a calmodulin inhibitor, but not the protein kinase C inhibitor H-7, inhibited the osmotically-activated sorbitol release. These results suggest that sorbitol release is a calmodulin-dependent event, possibly activated by a rise in intracellular calcium as a result of cell swelling.     

An in vitro model of nephrocalcinosis using rabbit inner medullary collecting tubular cell culture

In order to investigate the pathogenesis of medullary nephrocalcinosis, rabbit inner medullary collecting duct cells were grown in media containing different Ca++, PTH and pH levels. It was found that high Ca++ (7.8mM) only reduced growth slightly and that crystalline deposits were found under the cells. This suggests that high Ca++ is not severely toxic to the cells but can lead to deposition of calcium beneath the basement membrane. PTH did not effect cell growth even in the presence of high Ca++ implying that it has an indirect effect on tubular cells in medullary nephrocalcinosis associated with hyperparathyroidism. In renal tubular acidosis these cells are subjected to a persistently high urinary pH and low interstitial pH. Raising the pH reduced the cell growth in normal Ca++ medium whereas lowering the pH increased cell growth in vitro. Our results show that nephrocalcinosis is not due to the direct effect of raised pericellular Ca++ or PTH alone and that persistently alkaline tubular fluid may play a role.     

Properties of a polarized primary culture from rat renal inner medullary collecting duct (IMCD) cells

Summary A primary culture from rat renal IMCD cells was established to investigate the permeability characteristics of the luminal and contraluminal plasma membranes of the papillary collecting duct in vitro. Freshly isolated IMCD cells were grown on filters in a special “epithelial cell” medium. Confluency was proved with an epithelial volt/ohm meter. After 7 d of culture the transepithelial resistance reached more than 1000 Ω×cm². A polarization of the cells with regard to a basolateral localization of a lactate efflux system, and an l-alanine transport system was achieved. The hypotonicity-activated release systems for the organic osmolytes sorbitol and betaine were also located basolaterally, whereas taurine, glycerophosphorylcholine, and myo-inositol left the cells at both cell poles but with different capacity. Morphological observations revealed also that the monolayer was well differentiated. Thus, a model of a renal collecting duct epithelium was established which can be used to analyze polarized and differentiated transport processes across the epithelial cells and their plasma membranes.     

The effect of isoproterenol on fluid and electrolyte transport in the inner medullary collecting duct

In the late distal and cortical collecting tubule, which is the principal regulatory site for potassium (K) excretion, vasopressin stimulates, and epinephrine via beta-adrenergic action, inhibits K secretion. In the inner medullary collecting duct (IMCD) we have shown that vasopressin also stimulates K secretion. The present experiments were designed to determine whether the beta-adrenergic agonist, isoproterenol, would induce K reabsorption in the IMCD, and (or) prevent a secretory response to acute KCl infusion. Two groups of rats, with or without isoproterenol administration (3 micrograms/h), were subjected to retrograde microcatheterization of the IMCD before and during infusion of 0.83 mol/h KCl. Isoproterenol reduced plasma K concentration and urinary K excretion, but the response to acute KCl infusion was qualitatively similar to control. Isoproterenol decreased delivery of potassium, chloride, and fluid to the IMCD, there was no net transport of K along the duct in either group, and KCl infusion did not result in K secretion in either group. The results indicate that isoproterenol may inhibit K secretion in the late distal or cortical collecting tubule. However, there was no statistically significant difference in K transport along the IMCD between isoproterenol and control groups. Reduced sodium excretion, which was found during isoproterenol administration both before and after KCl infusion, was associated with no change in sodium delivery but with increased sodium reabsorption in the IMCD. This increased sodium reabsorption may be a direct effect of isoproterenol, or may be due to reflex cardiovascular adjustments associated with systemic actions of the drug.     

Constitutive and inducible expression of a transgene directed by heterologous promoters in a trout liver cell line

Activities of heterologous promoters and enhancers in cultured rainbow trout liver cells were examined employing the bacterial chloramphenicol acetyltransferase gene as the reporter. SV40 promoter-enhancer and Rous sarcoma virus long terminal repeat directed constitutive expression at high levels. Moloney murine leukemia virus long terminal repeat and SV40 promoter combined with Adenovirus type 2 enhancer were also constitutively expressed. Drosophila Hsp70 promoter was activated when the transformed cells were cultured at 25 degrees C, a higher temperature than the temperature normally used, in faithful response to heat shock.     

Mitochondrial expression of arginase II in male and female rat inner medullary collecting ducts.

Microdissected rat proximal straight tubules (PST) and inner medullary collecting ducts (IMCD) highly produce urea from l-arginine, supporting the expression of the mitochondrial arginase II. However, IMCD contain a very low density of mitochondria compared with PST. Recently, arginase II has been localized by immunohistochemistry in rat PST but not IMCD. This study was designed to verify whether rat IMCD express arginase II and to identify its subcellular localization. We developed an antibody raised against arginase II that allowed the detection of a band of 38 kDa corresponding to arginase II on immunoblots. In male and female rat kidneys, Western blot analyses revealed that arginase II was highly expressed in the inner medulla (IM), the outer stripe of the outer medulla (osOM), and the deep cortex. Immunocytochemistry demonstrated that arginase II was homogeneously expressed in IMCD. Proteins of the cytosolic and mitochondrial fractions extracted from osOM and IM and analyzed by Western blot showed that 86% of arginase II was associated with mitochondria. The molecular weight of arginase II was similar in the cytosolic and mitochondrial fractions. Immunoelectron microscopy confirmed the presence of arginase II in the mitochondria of IMCD. In conclusion, arginase II is expressed in mitochondria of male and female rat IMCD.     

Scavenger receptor of human monocytic leukemia cell line (THP-1) and murine macrophages for nonenzymatically glycosylated proteins

Long-term incubation of proteins with glucose undergo a series of nonenzymatic reactions to form advanced glycosylation end product (AGE) with fluorescence and brown color. The receptor for AGE-proteins was demonstrated in murine macrophages (Vlassara et al. (1985) Proc. Natl. Acad. Sci. USA 82. 5588). Our recent study with rat macrophages revealed that the receptor also recognized proteins modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, indicating its close identity to a scavenger receptor for aldehyde-modified proteins (Takata, K. et al. (1988) J. Biol. Chem. 263. 14819). This notion was tested in the present study with human monocytic leukemia cell line (THP-1 cells), human monocyte macrophages and murine peritoneal macrophages. Endocytic uptake of AGE-proteins and aldehyde-modified proteins was inhibited in a cross-competitive fashion. The receptor activities of THP-1 cells for AGE-albumin and aldehyde-modified proteins were induced synchronously by phorbol 12-myristate 13-acetate. Furthermore, upon reduction by NaBH4 of the Schiff base formed between proteins and glucose or aldehydes, no ligand activity was generated. However, once the ligand activity was generated, NaBH4 was no longer effective for the ligand activity. Thus, a structure in common between AGE-proteins and aldehyde-modified proteins may be crucial for recognition by the human macrophage receptor.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage

M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.     

Marked reduction in intramembranous particle clusters in the terminal portion of inner medullary collecting ducts of antidiuretic rats

Summary Antidiuretic hormone (ADH) induces an aggregation of intramembranous particles (IMP) into discrete clusters in the luminal plasma membrane of rat renal papillary collecting duct cells (Harmanci et al. 1978). The correlation between an elevated dose of ADH, increased urine osmolality, and greater IMP cluster frequency has led to speculation that the water permeability of the luminal plasma membrane is reflected by the IMP cluster density (Harmanci et al. 1980). The present study indirectly evaluated this water permeability by quantitating collecting duct IMP cluster frequency from freeze fracture replicas in two regions of the renal papilla, at its base and at its tip, in antidiuretic and in water diuretic rats. During antidiuresis there was a high frequency of IMP clusters (189/100 m² of luminal plasma membrane) in cells from the papilla base but not at the papilla tip (9.0/100 m²). During water diuresis there were few IMP clusters in either cells from the papilla base (5.9/100 m²) or at the papilla tip (1.4/100 m²). Most significantly these results suggest that the water permeability of the terminal portion of the inner medullary collecting duct of antidiuretic rats is significantly lower than that of the collecting duct epithelium higher in the papilla.Preliminary findings of this study were presented at the Second International Congress of Cell Biology, West Berlin 1980     

Gamma-melanocyte stimulating hormone regulates the expression and cellular localization of epithelial sodium channel in inner medullary collecting duct cells

Gamma₂-melanocyte-stimulating hormone (γ₂MSH) is a peptide hormone released by the pituitary gland which is thought to act directly on the renal inner medulla to promote increased sodium excretion into urine (natriuresis). The aim of this study was to determine if a stable analog, [Nle³, D-Phe⁶]-γ₂MSH (NDP-γ₂MSH), of the native peptide regulated the activity, expression and cellular localization of epithelial sodium channel (ENaC) in a murine inner medullary collecting duct (mIMCD-3) cell line. Our results indicate that expression of the γ₂MSH receptor, melanocortin receptor 3 receptor (MC3R), is up-regulated by culturing the cells in media with an increased osmolality (∼400 mOsm/kg). Furthermore, stimulation of cAMP signaling and sodium transport by 1 nM NDP-γ₂MSH occurs only in cells cultured in the high osmolality media. Finally, treatment of mIMCD-3 cells cultured in high osmolality medium for 1 h with 1 nM NDP-γ₂MSH causes a reduction in expression of serum- and glucocorticoid-induced kinase (sgk1) and a reduction in expression and cell surface abundance of the alpha subunit of ENaC. Collectively, this data suggest that γ₂MSH directly regulates both ENaC expression and cellular localization in the inner medulla to exert its natriuretic effect.     

固有无序蛋白研究进展及其对细胞胁迫耐受性的影响

固有无序蛋白(intrinsically disordered proteins,IDPs)是指在生理条件下缺乏有序稳定的高级结构,整体或局部不折叠,但能够参与多种生物学过程、行使特定生物学功能的一类蛋白质.固有无序蛋白决定了其不同于经典蛋白质"序列-结构-功能"的功能范式,丰富了蛋白质"结构-功能"的多样性.固有无序...     

Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.

Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.     

A murine cell line (2E10.4.13) produces five hemopoietic stimulators, and interleukin-2 and interleukin-3

An interleukin-2 (IL-2)-independent murine lymphocyte clone (2E10.4.13) with the Thy1+Lyt1+2-T200+ phenotype was separated from the original IL-2-dependent natural killer (NK) cell line (PEC-1). Erythroid burst-promoting activity (BPA), erythropoietin (Ep), granulocyte/macrophage, megakaryocyte and eosinophil colony-stimulating factors (GM-, MK- and Eo-CSF), IL-2 and Interleukin-3 (IL-3) were produced when these cells were stimulated with phorbol myristate acetate (PMA). When the conditioned medium was run through ion-exchange high-performance liquid chromatography, BPA, Ep, GM-CSF, MK-CSF and Eo-CSF were eluted in the same region as IL-3. In contrast, MK-CSF, much of the GM-CSF and half of the Eo-CSF were eluted in a distinct region where no IL-3 was detected. Chemical analyses of the hemopoietic factors derived from a single T inducer clone indicated that all the hemopoietic activities were associated with IL-3 activity. Some CSF activities (GM-, MK- and Eo-CSF) also could be mediated by the distinct molecules from IL-3, evidence that heterogeneous molecules are responsible for CSF activity.     

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM)

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.