Incubation In Hypertonic Medium Research Articles

Homologous desensitization of human histamine H3 receptors expressed in CHO-K1 cells

Histamine H3 receptors (H3Rs) modulate the function of the nervous system at the pre- and post-synaptic levels. In this work we aimed to determine whether, as other G protein-coupled receptors (GPCRs), H3Rs desensitize in response to agonist exposure. By using CHO-K1 cells stably transfected with the human H3R (hH3R) we show that functional responses (inhibition of forskolin-induced cAMP accumulation in intact cells and stimulation of [35S]-GTPγS binding to cell membranes) were markedly reduced after agonist exposure. For cAMP accumulation assays the effect was significant at 60 min with a maximum at 90 min. Agonist exposure resulted in decreased binding sites for the radioligand [3H]-N-methyl-histamine ([3H]-NMHA) to intact cells and modified the sub-cellular distribution of H3Rs, as detected by sucrose density gradients and [3H]-NMHA binding to cell membranes, suggesting receptor internalization. The reduction in the inhibition of forskolin-stimulated cAMP formation observed after agonist pre-incubation was prevented by incubation in hypertonic medium or in ice-cold medium. Agonist-induced loss in binding sites was also prevented by hypertonic medium or incubation at 4 °C, but not by filipin III, indicating clathrin-dependent endocytosis. Immunodetection showed that CHO-K1 cells express GPCR kinases (GRKs) 2/3, and both the GRK general inhibitor ZnCl2 and a small interfering RNA against GRK-2 reduced receptor desensitization. Taken together these results indicate that hH3Rs experience homologous desensitization upon prolonged exposure to agonists, and that this process involves the action of GRK-2 and internalization via clathrin-coated vesicles.

Differential Effects of Hyperosmolality on Na-K-ATPase and Vasopressin-Dependent cAMP Generation in the Medullary Thick Ascending Limb and Outer Medullary Collecting Duct

Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of alpha-1, beta-1, and beta-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased alpha-1, beta-1, and beta-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of beta-1, but not that of alpha-1 or beta-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.

Inositol transport in mouse embryonic stem cells

The uptake of myo-inositol by mouse embryonic stem (ES) cells was measured using [2-(3)H]myo-inositol. Uptake of myo-inositol by ES cells occurred in a mainly saturable, sodium-, time- and temperature-dependent manner, which was inhibited by glucose, phloridzin and ouabain. Self inhibition by inositol was much greater than inhibition by glucose indicating that transport was not occurring via a sodium-dependent glucose transporter. Uptake rate was much greater than efflux rate indicating a mainly unidirectional transport mechanism. Estimated kinetics parameters for sodium-dependent inositol uptake were a K m of 65.1 +/- 11.8 micromol L(-1) and a V max of 5.0 +/- 0.59 pmol microg protein(-1) h(-1). Inositol uptake was also sensitive to osmolality; uptake increased in response to incubation in hypertonic medium indicating a possible role for inositol as an osmolyte in ES cells. These characteristics indicate that myo-inositol transport in mouse ES cells occurs by a sodium-dependent myo-inositol transporter protein.

Vesicle Trafficking and Cell Surface Membrane Patchiness

Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited.

The sgk, an aldosterone-induced gene in mineralocorticoid target cells, regulates the epithelial sodium channel

The sgk, an aldosterone-induced gene in mineralocorticoid target cells, regulates the epithelial sodium channel. Aldosterone increases sodium reabsorption in tight epithelia. The early phase of this stimulatory effect is thought to involve activation of apical sodium channels. To identify immediate-early genes that initiate this effect, we used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques. This review summarizes our recent findings. Aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum- and glucocorticoid-regulated kinase), in the native mineralocorticoid target cells, that is, in cortical collecting duct (CCD) cells. The induction of sgk mRNA occurs within 30 minutes of the addition of aldosterone and does not require de novo protein synthesis, indicating that sgk is an immediate/early aldosterone-induced gene. Induction of sgk by aldosterone is mediated through mineralocorticoid receptors (MRs), since it is prevented by ZK91857, an MR antagonist, but not by RU486, a glucocorticoid antagonist. In addition to aldosterone, RU28362, a pure glucocorticoid receptor agonist, also induced sgk mRNA, both in primary cultures of rabbit CCD cells and in the M-1 mouse CCD cell line. Sgk mRNA levels are also influenced by changes in the osmolality of the medium. In M-1 cells, incubation of cells for one hour in a mildly hypotonic medium decreased sgk mRNA levels, whereas incubation in hypertonic medium brought about opposite changes. To determine whether sgk is involved in the regulation of the epithelial sodium channel (ENaC), we coexpressed the full-length sgk cRNA in Xenopus oocytes with the three ENaC subunits. Expression of sgk resulted in a significant increase in the amiloride-sensitive Na current, suggesting that this protein kinase plays an important role in the early phase of aldosterone-stimulated Na transport. These results indicate that sgk is an aldosterone-induced immediate/early gene in native MR target cells, and is involved in the regulation of ion transport and possibly cell volume.

Endocytosis of gentamicin in a proximal tubular renal cell line

The mechanisms by which aminoglycosides are accumulated in renal proximal tubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the glycoprotein 330/ megalin, have been proposed to be involved in gentamicin transport in renal cells. We used the LLC-PK 1 cell line, derived from the pig proximal tubule, to explore further the regulation of gentamicin endocytosis in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly temperature dependent, whereas total uptake (endocytosis plus binding) did not significantly differ at 4 or 37 °C. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PK 1 cells. Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that is active also on fluid phase endocytosis, significantly reduced the intracellular concentrations of the aminoglycoside. Other maneuvers that perturb clathrin dependent endocytosis without affecting clathrin independent pathway, such as acidification of cytosol or incubation in hypertonic medium, were also without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fluoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PK 1 cells is probably clathrin independent, limited by cytochalasin D, which interacts with cytoskeleton, and increased by substances like mastoparan and aluminum fluoride, which activate heterotrimeric G proteins.

Acute and Chronic Effects of Hyperosmolality on mRNA and Protein Expression and the Activity of Na-K-ATPase in the IMCD

We investigated acute and chronic effects of hyperosmolality on mRNA and protein expressions of Na-K-ATPase α and β isoforms and Na-K-ATPase activity in the rat inner medullary collecting duct (IMCD). Incubation of IMCD in hypertonic medium for 30 min reduced the Na-K-ATPase activity by 50%. The Na-K-ATPase activity of dehydrated rats measured in isotonic medium was decreased, and incubation in hypertonic medium did not further decrease the activity. Incubation of IMCD in hypertonic medium for 6 h did not change α₁ mRNA. In contrast, dehydration decreased α₁ subunit mRNA and protein and β₁ protein expressions without changing β₁ mRNA. These data show (1) that acute hyperosmolality decreases Na-K-ATPase activity in IMCD without changing α₁ and β₁ mRNA and (2) that 2 days of dehydration decreased Na-K-ATPase activity by reducing α₁ and β₁ proteins. Thus, the mechanisms for the inhibition of the Na-K-ATPase activity in IMCD is defferent between acute and chronic exposure to hyperosmolality.

Three Unrelated Perturbations Similarly Uncouple Fluid, Bulk-Membrane, and Receptor Endosomal Flow in Rat Fetal Fibroblasts

We have compared the effects of three perturbations (treatment with 2 μM monensin, potassium depletion, and incubation in 0.35 M NaCl) on recycling of internalized fluid-phase, bulk-membrane, and receptor-mediated tracers in rat fetal fibroblasts. Monensin accelerated 2-fold the regurgitation of the fluid-phase tracer horseradish peroxidase (HRP), as previously described in these cells after potassium depletion or upon incubation in hypertonic medium (1), and all treatments severely inhibited transfer of HRP from endosomes to lysosomes. In comparison, recycling of (6[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl glucosylsphingosine (C6-NBD-GlcCer), a fluorescent lipid used as a bulk-membrane probe, was not significantly affected while transferrin recycling was slowed down 2-fold. The striking similarities of these unrelated perturbations in their distinct effects on fluid, bulk-membrane, and receptor addressing point to common targets regulating these mechanisms.

Measuring volume perturbation of proximal tubular cells in primary culture with three different techniques.

Osmotic cell volume perturbations of rabbit proximal tubule (PT) in primary culture were measured using three independent techniques. Automatic cell thickness monitoring of PT monolayers revealed that cell volume rapidly increased by 39 +/- 2% in hypotonic medium (150 mosM), which was followed by partial regulatory volume decrease (RVD). Subsequent incubation in hypertonic medium (500 mosM) rapidly decreased cell volume by 54 +/- 2% not followed by regulatory volume increase (RVI). When cell volume in PT monolayers was derived from concentration changes in the trapped fluorescent dyes, fura 2 or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, osmotically induced cell volume changes appeared much smaller (17 +/- 1 and 22 +/- 2% for similar hypo- and hypertonicity, respectively). However, changes in fluorescence intensity were most often not in agreement with anticipated cell volume changes. With the Coulter counter, a much larger shift in cell volume was observed in PT cell suspensions. In this situation, cell swelling in hypotonic medium amounted to 74 +/- 2% but was still followed by partial RVD. Hypertonicity resulted in a decrease in cell volume of 42 +/- 3% not followed by RVI. In conclusion, our study indicates that automatic cell thickness monitoring of an epithelial cell layer cultured on a permeable support provides more reliable data than monitoring changes in fluorescence intensity of trapped dyes.

Hypertonic Treatment Does Not Affect the Radiation Yield of Interphase Chromosome Breaks in DNA Double-Strand Break Repair-Deficient xrs-5 Cells

A DNA double-strand break (DSB) repair-deficient CHO cell line, xrs-5, was used to evaluate the effect of hypertonic treatment on the radiation yield of interphase chromosome breaks, as visualized by means of premature chromosome condensation (PCC). For this purpose, interphase chromosome breaks were measured in plateau-phase G1 cells after exposure to 10 Gy X rays using either the standard protocol for PCC induction or a revised protocol that includes a brief (20 min) treatment in hypertonic medium (500 mM NaCl) during chromosome condensation. Experiments at the chromosome level were complemented by experiments at the DNA and the cellular level performed under similar postirradiation conditions. The results obtained were compared to those reported previously for CHO cells. Radiation yields of interphase chromosome breaks, as measured using standard protocols for PCC induction, were higher in xrs-5 than in CHO cells. However, the yields became similar between the two cell lines when a 20-min incubation in hypertonic medium was introduced immediately after fusion with mitotic cells. This equalization was due to the fact that incubation in hypertonic medium did not affect the radiation yield of interphase chromosome breaks in xrs-5 cells but increased it in CHO cells to levels similar to those measured in xrs-5 cells. This result suggests that xrs-5 cells constitutively express as interphase chromosome breaks a subset of DSBs which in repair-proficient CHO cells requires incubation in hypertonic medium for transformation to interphase chromosome breaks. Rejoining of DSBs, when measured in isotonic medium, was incomplete in xrs-5 cells compared to CHO cells. Rejoining of DSBs normally repaired in xrs-5 cells was completely inhibited during treatment in hypertonic medium. Postirradiation treatment in hypertonic medium affected the survival of xrs-5 cells only modestly, suggesting that the sector of PLD (beta-PLD) which required treatment in hypertonic medium for fixation in repair-proficient CHO cells is fixed constitutively in xrs-5 cells. These results are in agreement with a model developed to interpret results obtained in similar experiments with CHO cells (Iliakis et al., Radiat. Res 135, 160-170, 1993). The model postulates that potentiation in CHO cell killing induced by hypertonic treatment is caused by the transformation of a subset of fast-repairing DSBs to irreparable interphase chromosome breaks and assumes that this transformation is equivalent to fixation of beta-PLD.(ABSTRACT TRUNCATED AT 400 WORDS)

Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification.

The effects of methods known to perturb endocytosis from clathrin-coated pits on the localization of clathrin and HA2 adaptors in HEp-2 carcinoma cells have been studied by immunofluorescence and ultrastructural immunogold microscopy, using internalization of transferrin as a functional assay. Potassium depletion, as well as incubation in hypertonic medium, remove membrane-associated clathrin lattices: flat clathrin lattices and coated pits from the plasma membrane, and clathrin-coated vesicles from the cytoplasm, as well as those budding from the TGN. In contrast, immunofluorescence microscopy using antibodies specific for the alpha- and beta-adaptins, respectively, and immunogold labeling of cryosections with anti-alpha-adaptin antibodies shows that under these conditions HA2 adaptors are aggregated at the plasma membrane to the same extent as in control cells. After reconstitution with isotonic K(+)-containing medium, adaptor aggregates and clathrin lattices colocalize at the plasma membrane as normally and internalization of transferrin resumes. Acidification of the cytosol affects neither clathrin nor HA2 adaptors as studied by immunofluorescence microscopy. However, quantitative ultrastructural observations reveal that acidification of the cytosol results in formation of heterogeneously sized and in average smaller clathrin-coated pits at the plasma membrane and buds on the TGN. Collectively, our observations indicate that the methods to perturb formation of clathrin-coated vesicles act by different mechanisms: acidification of the cytosol by affecting clathrin-coated membrane domains in a way that interferes with budding of clathrin-coated vesicles from the plasma membrane as well as from the TGN; potassium depletion and incubation in hypertonic medium by preventing clathrin and adaptors from interacting. Furthermore our observations show that adaptor aggregates can exist at the plasma membrane independent of clathrin lattices and raise the possibility that adaptor aggregates can form nucleation sites for clathrin lattices.

Regulation of Na(+)-K(+)-ATPase expression in cultured renal cells by incubation in hypertonic medium.

To determine whether alterations in cell volume affect Na(+)-K(+)-adenosinetriphosphatase (ATPase) expression, a subclone of the Madin-Darby canine kidney (MDCK) cell line was incubated in anisotonic serum-free medium and alpha- and beta-subunit mRNA, Na(+)-K(+)-ATPase activity, and active K+ transport were measured. In medium adjusted to 500 mosmol/kgH2O by adding NaCl, the alpha-subunit mRNA concentration was 2.93 +/- 0.14 (SE) times control and beta-mRNA was 1.93 +/- 0.27 times control. When sucrose was added to increase osmolality, alpha-subunit mRNA increased to 1.85 +/- 0.18 times control. Na(+)-K(+)-ATPase activity of homogenates from cells incubated in 500 mosmol/kgH2O medium for 24 h increased to 2.62 +/- 0.52 times control when NaCl was added and 2.31 +/- 0.34 times control when sucrose was added. Active K+ transport increased between 60 and 90% after cells were incubated in 450 mosmol/kgH2O medium with either NaCl or sucrose added. Stimulation of Na(+)-K(+)-ATPase expression in renal cells facing hypertonic stress may represent a long-term mechanism that allows cells to maintain cation gradients in a hypertonic environment.

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.

Trivalent chromium is neither cytotoxic nor mutagenic in permeabilized hamster fibroblasts

BHK cells became reversibly permeable by a 30-min incubation in hypertonic medium. During permeabilization they were exposed to water-soluble Cr(VI) (K 2Cr 2O 7) and Cr(III) (CrCl 3). Thymidine uptake in the intracellular nucleotide pool, DNA replication, DNA damage and repair and sister-chromatid exchanges (SCE) were examined to detect the cytotoxic and genetic effects of Cr compounds. Cr(III) remained inactive also in permeabilized cells. An apparent induction of DNA damage by Cr(lII), suggested by the Painter's test, was considered unreliable. Cr(VI) cytotoxic and genetic activity was enhanced in permeabilized cells, as demonstrated by increased inhibition of DNA replication and higher frequency of SCE.

Effect of Hypertonicity and X Radiation on DNA Synthesis in Normal and Ataxia-Telangiectasia Cells

Normal human cells and cells from patients with ataxia-telangiectasia (A-T) were exposed to culture medium made hypertonic by raising the NaCl concentration. The rate of DNA synthesis in both types of cells was depressed as a function of increasing hypertonicity. When cells of both types were exposed to X radiation and incubated in hypertonic medium, DNA synthesis appeared to be more radioresistant than in cells incubated in normal medium. Velocity sedimentation analysis showed that this was due to a hypertonicity-induced inhibition of replicon initiation, which is the same process affected by X radiation, indicating that the two treatments were not additive. After a 5-hr incubation in hypertonic medium, there was a new steady state of replicon initiation and elongation similar to that existing in cells grown in normal medium, except that fewer replicons were participating. At this time DNA synthesis in each type of cell had a characteristic response to radiation, i.e., radiosensitive in normal cells and radioresistant in A-T cells. These results suggest that radioresistant DNA synthesis

Arrest of segregation leads to accumulation of highly intertwined catenated dimers: Dissection of the final stages of SV40 DNA replication

When SV40-infected cells are placed into hypertonic medium, newly synthesized DNA accumulates as form C catenated dimers. These molecules consist of two supercoiled monomer circles of SV40 DNA interlocked by one or more topological intertwinings and are seen as transiently labeled intermediates during normal replication. Form C catenated dimers represent pure segregation intermediates, replicative DNA structures in which DNA synthesis is complete but which still require topological separation of the two daughter circles. Hypertonic shock seems to block selectively a type II topoisomerase activity involved in disentangling the two circles. This is reflected in the fact that form C catenated dimers that accumulate during the block are highly intertwined with catenation linkage numbers up to C L = 20. While initiation of replication is also inhibited by hypertonic treatment, ongoing SV40 DNA synthesis is not affected, and replication is free to proceed from the earliest cairns structure through to form C catenated dimers. The block to segregation is rapidly and completely released by shifting the cells back to normal medium. A much slower recovery of DNA segregation takes place on prolonged incubation in hypertonic medium, perhaps because of some cellular homeostatic mechanism. The results of this work lead to a detailed view of the final stages of SV40 DNA replication.