A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density.

The growth rates and buoyant densities of a Salmonella typhimurium mutant, TL126 (proB74A+), with enhanced osmotolerance caused by proline overproduction were measured and compared with the growth rates and buoyant densities of an isogenic (wild-type) strain, TL128 (proB+ A+), with normal control of proline production. Growth rates were determined in a rich medium (Luria broth) with added NaCl to produce various osmotic strengths ranging from 300 to 2,000 mosM. At low concentrations of NaCl, there was little variation in doubling times between the two strains. However, as the osmotic strength of the medium approached and exceeded 1,300 mosM, the doubling times of TL126 (osmotolerant) were 1.5 to 2 times faster than those of TL128 (wild type), confirming the osmotolerance of TL126. Buoyant densities were determined by equilibrium sedimentation in a Percoll gradient of osmotic strength equal to that of the growth medium. The osmolarity of the Percoll gradient was adjusted by the addition of NaCl. At low osmolarities (300 to 500 mosM), the buoyant density of TL126 (osmotolerant) was slightly but consistently lower than that of TL128 (wild type). As the osmotic strength was increased, the buoyant density of TL126 (osmotolerant) increased in proportion to the osmotic strength. In contrast, the buoyant density of strain TL128 (wild type) did not increase as much. At high osmolarities (1,600 to 2,000 mosM), the buoyant density of TL126 (osmotolerant) was consistently higher than that of TL128 (wild type). These results suggest that the intracellular accumulation of proline by TL126, the osmotolerant strain, increases both the growth rates and buoyant densities at osmolarities of 1,300 mosM and above.     

Increases in guinea pig small intestinal transepithelial resistance induced by osmotic loads are accompanied by rapid alterations in absorptive-cell tight-junction structure

In some epithelia, mucosal exposure to osmotic loads produces an increase in transepithelial resistance that is presumed to relate to the collapse of the paracellular spaces. Since proximal small intestinal epithelium may transiently encounter osmotic loads during normal digestion, we examined the short-term effect of osmotic loads on resistance and on epithelial structure of mucosal sheets prepared from guinea pig jejunum using Ussing-chamber, thin-section electron- microscopic, and freeze-fracture techniques. After equilibration of mucosal sheets in chambers, mucosal buffer tonicity was increased to 600 mosM with mannitol. This resulted in a 64% increase in resistance within 20 min. Concomitantly, 600 mosM produced a decrease in tight- junction cation selectivity as judged from dilution potentials, collapse of paracellular spaces, decreased cytoplasmic electron density in 10-40% of absorptive cells, and focal absorptive-cell subjunctional lateral-membrane evaginations often associated with microfilament arrays. Freeze-fracture replicas of absorptive-cell tight junctions revealed significant increases in both strand count and depth. Preincubation with 5 micrograms/ml cytochalasin D reduced the 600 mosM resistance increase caused by 600 mosM exposure by 48% but did not prevent the collapse of paracellular spaces. Lowered temperatures that produced morphologic evidence consistent with a gel-phase transition of absorptive-cell lateral membranes prevented both the resistance response and the alterations in tight-junction structure. In conclusion, transient osmotic loads produce an increase in resistance in jejunal epithelium and alter both absorptive-cell tight-junction charge selectivity and structure. These responses, which may have physiologic implications, can be reduced by cytoskeletal inhibitors and ablated by conditions that restrict mobility of absorptive-cell lateral- membrane molecules.     

The maximum and minimum water content and cell volume of human erythrocytes in vitro

The maximum and minimum water contents of human erythrocytes were measured after exposure to various osmotic pressures. Within a range of osmolarities, at which no haemolysis occurred, the water content reached its maximum, 78.1%, at 180 mosM and its minimum, 54.8%, at 800 mosM. Simultaneously, the mean cell volume increased to 98.5 microns 3 at 180 mosM and decreased to 77.2 microns 3 at 800 mosM.     

Effect of anisotonic media on cell volume and electrolyte fluxes in slices of the dogfish (Squalus acanthias) rectal gland

Summary Osmotic responses of slices of dogfish rectal gland to hypotonic (urea-free) and hypertonic media were studied. Transfer of tissue from isotonic (890 mosM) to hypotonic (550 mosM) saline produced an osmotic swelling associated with a slow net uptake of cell K⁺ (and Cl^–) and a slow, two-component efflux of urea. Media made hypertonic (1180 mosM) by addition of urea or mannitol produced osmotic shrinkage with a net loss of KCl. The cell osmotic responses in hypotonic media were lower than predicted for an ideal osmometer. No volume regulatory responses were seen subsequent to the initial osmotic effects. The cation influx in hypotonic media lacked specificity: in the presence of 0.5 mM ouabain or in K⁺-free media a net influx of Na⁺ was found. At steady state, the cell membrane potential evaluated from the Nernst potentials of K⁺ and triphenylmethyl phosphonium⁺, was independent of medium tonicity, suggesting the membrane potential as a determinant in the cellular osmotic response. Zero-time⁸⁶Rb⁺ fluxes were measured:⁸⁶Rb⁺ influx was not affected by hypotonicity, implying an unchanged operation of the Na⁺–K⁺-ATPase. On the other hand,⁸⁶Rb⁺ efflux was significantly reduced at hypotonicity; this effect was transient, the efflux returning to the control value once the new steady state of cell volume had been reached. A controlled efflux system is therefore involved in the cell osmotic response. The absence of the volume regulatory phenomenon suggests that the cells are not equipped with a volume-sensing mechanism.Abbreviations and symbols DW dry weight - E extracellular (polyethylene glycol) space - E Nernst potential - H₂O_e H₂O_i tissue water, extra- and intracellular - TPMP ⁺ triphenyl methyl phosphonium salt - WW wet weight     

Membrane stress causes inhibition of water channels in brush border membrane vesicles from kidney proximal tubule

Brush border membrane vesicles (BBMV) from rabbit kidney proximal tubule cells, prepared with different internal solute concentrations (cellobiose buffer 13, 18 or 85 mosM) developed an hydrostatic pressure difference across the membrane of 18.7 mosM, that causes a membrane tension close to 5 × 10⁻⁵ N cm⁻¹. When subjected to several hypertonic osmotic shocks an initial delay of osmotic shrinkage (a lag time), corresponding to a very small change in initial volume was apparent. This initial osmotic response, which is significantly retarded, was correlated with the initial period of elevated membrane tension, suggesting that the water permeability coefficient is inhibited by membrane stress. We speculate that this inhibition may serve to regulate cell volume in the proximal tubule.     

Chondrocyte intracellular calcium, cytoskeletal organization, and gene expression responses to dynamic osmotic loading

While chondrocytes in articular cartilage experience dynamic stimuli from joint loading activities, few studies have examined the effects of dynamic osmotic loading on their signaling and biosynthetic activities. We hypothesize that dynamic osmotic loading modulates chondrocyte signaling and gene expression differently than static osmotic loading. With the use of a novel microfluidic device developed in our laboratory, dynamic hypotonic loading (–200 mosM) was applied up to 0.1 Hz and chondrocyte calcium signaling, cytoskeleton organization, and gene expression responses were examined. Chondrocytes exhibited decreasing volume and calcium responses with increasing loading frequency. Phalloidin staining showed osmotic loading-induced changes to the actin cytoskeleton in chondrocytes. Real-time PCR analysis revealed a stimulatory effect of dynamic osmotic loading compared with static osmotic loading. These studies illustrate the utility of the microfluidic device in cell signaling investigations, and their potential role in helping to elucidate mechanisms that mediate chondrocyte mechanotransduction to dynamic stimuli. cartilage; calcium signaling; actin cytoskeleton; aggrecan     

Amino acid efflux in response to chemotactic and osmotic signals in Bacillus subtilis.

We observed a large efflux of nonvolatile radioactivity from Bacillus subtilis in response to the addition of 31 mM butyrate or the withdrawal of 0.1 M aspartate in a flow assay. The major nonvolatile components effluxed were methionine, proline, histidine, and lysine. In studies of the release of volatile radioactivity in chemotaxis by B. subtilis cells that had been labeled with [3H]methionine, the breakdown of methionine to methanethiol can contribute substantially to the volatile radioactivity in fractions following addition of 0.1 M aspartate. However, methanol was confirmed to be released after aspartate addition and, in lesser quantities, after aspartate withdrawal. Methanol and methanethiol were positively identified by derivitization with 3,5-dinitro-benzoylchloride. Amino acid efflux but not methanol release was observed in response to 0.1 M aspartate stimulation of a cheR mutant of B. subtilis that lacks the chemotaxis methylesterase. The amino acid efflux could be reproduced by withdrawal of 0.1 M NaCl, 0.2 M sucrose, or 0.2 M xylitol and is probably the result of changes in osmolarity. Chemotaxis to 10 mM alanine or 10 mM proline resulted in methanol release but not efflux of amino acids. In behavioral studies, B. subtilis tumbled for 16 to 18 s in response to a 200 mosM upshift and for 14 s after a 20 mosM downshift in osmolarity when the bacteria were in perfusion buffer (40 mosM). The pattern of methanol release was similar to that observed in chemotaxis. This is consistent with osmotaxis in B. subtilis away from an increase or decrease in the osmolarity of the incubation medium. The release of methanol suggests that osmotaxis is correlated with methylation of a methyl-accepting chemotaxis protein.     

Growth and buoyant density of Escherichia coli at very low osmolarities.

The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored. At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml. As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml. From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities. The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml. Both strains of E. coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9. (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM. To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly. From this study, it was determined that both Ca2+ and Mg2+ could stimulate growth below 40 mosM. With Ca2+ - and Mg2+ -supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9. Strain ML3 would only haltingly grow at 15 mosM. Four attempts were made to grow both ML3 and ML308 at 5 mosM. In three of the experiments, ML308 grew, while strain ML3 grew in one experiment. While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently. Therefore, from this study, we believe that E. coli might be defined as an euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.     

Novel Cl- currents elicited by follicle stimulating hormone and acetylcholine in follicle-enclosed Xenopus oocytes

Voltage-clamp techniques were used to study the membrane currents elicited by follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle-enclosed oocytes of Xenopus laevis (follicles). Both agonists caused complex responses that were more evident when the follicles were in hypotonic Ringer solution (HR; 190.4 mosM). In this medium, currents activated by FSH regularly showed three phases whereas currents activated by ACh displayed three to six phases. At a holding potential of -60 mV, FSH, and ACh responses involved combinations of inward and outward currents. Both FSH and ACh responses included a slow smooth inward component that was associated with an increase in membrane conductance, mainly to Cl- (S(in)). This current was strongly dependent on the osmolarity of the external solution: an increase in osmolarity of the HR solution of 18-20 mosM caused a 50% decrease in S(in). In contrast, a fast and transient Cl- current (F(in)) specifically elicited by ACh was not dependent on osmolarity. Both, F(in) and S(in) currents required the presence of follicular cells, since defolliculation using three different methods abolished all the response to FSH and at least four components of the ACh responses. The membrane channels carrying F(in) and oscillatory Cl- currents elicited by stimulation of ACh or serum receptors, were much more permeable to I- and Br- than Cl-, whereas S(in) channels were equally permeable to these anions. Unlike the oscillatory Cl- currents generated in the oocyte itself, S(in) and F(in) currents in follicle-enclosed oocytes were not abolished by chelation of intracellular Ca2+, either with EGTA or BAPTA, which suggests that intracellular Ca2+ does not play a critical role in the activation of these currents. Our experiments show that S(in) and F(in) currents are quite distinct from the previously characterized oscillatory Cl- responses of oocytes. Moreover, the results strongly suggest that the FSH and ACh receptors, the Cl- channels mediating the F(in) and S(in) currents, together with the necessary elements for their activation, are all located in the follicular cells and not in the oocyte. Many aspects of follicular cell physiology in Xenopus laevis, and other species, are regulated by hormones and neurotransmitters, including FSH and ACh. The follicular Cl- currents described in this paper may play an important role in the follicular cell-oocyte development.     

Osmolality-dependent relocation of penicillin-binding protein PBP2 to the division site in Caulobacter crescentus

The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.     

Kinetics of glutaraldehyde fixation of erythrocytes: size, deformability, form, osmotic and hemolytic properties.

Red blood cells interact with glutaraldehyde (GA) in a complex kinetic pattern of events. At a given GA concentration in phosphate buffered saline (PBS), the sequence of cell 'volume' response, as measured by resistive pulse spectroscopy (RPS), includes: an immediate response to the overall solution osmolality; a constant volume, latent phase; a rapid swelling phase; an intermediate constant volume phase; and a shrinkage phase to a final steady state volume. The final volume depends on fixative solution osmolality; for GA concentrations between 0.05% and 0.25% w/v, fixative osmolalities of less than 355 mosM, including 'isotonic', or greater than 355 mosM, lead to final cell volumes greater or less than native, respectively. Cell-membrane deformability decreases continuously and monotonically with time, as assessed by RPS. The rate of fixation is a direct function of GA concentration, in accordance with a derived empirical expression. The measured kinetic responses are related to considerations of cell size, deformability, and form, and to mechanisms involved in abrupt osmotic hemolysis.     

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions. The influence of in situ field gradients

The 1H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T2 values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with tau = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25-1.98 G/cm and 0.89-2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.     

Effect of tuftsin and oligotuftsins on chemotaxis and chemotactic selection in Tetrahymena pyriformis

The chemotactic properties of tuftsin (H-TKPR-OH), tuftsin derivatives (H-KPR-OH, H-TKPKG-NH(2), Ac-TKPKG-NH(2)) and TKPKG-based oligotuftsins (T20, T30, T40) were investigated in Tetrahymena pyriformis GL. In contrast to its effects on Mammalia, tuftsin elicited chemorepellent or neutral responses; truncation of the N-terminal part (KPR) led to similar results, though with more neutral effects. The significance of the C-terminal part of the molecule was revealed by the chemoattractant properties of TKPKG, which are nevertheless abolished by acylation. Among the oligotuftsins, T20 and T40 were chemoattractants at higher concentrations (10(-9)-10(-6) M), while T30 had a wide-ranging chemorepellent effect, indicating that chemotaxis is elicited in Tetrahymena only by ligands with optimal physicochemical characters (mass, conformation, etc.). The chemotactic selection data indicated that tuftsin-induced chemotaxis results from fairly short-term signalling in Tetrahymena.     

Relationship of pseudopod extension to chemotactic hormone-induced actin polymerization in amoeboid cells

Aggregation-competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells. Changes in cell behavior were correlated with F-actin content by using an NBD-phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D-sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F-actin assembled by 60 sec is localized in these new pseudopods. These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.     

The chemokinetic and chemotactic behavior of Rhodobacter sphaeroides: two independent responses.

Rhodobacter sphaeroides exhibits two behavioral responses when exposed to some compounds: (i) a chemotactic response that results in accumulation and (ii) a sustained increase in swimming speed. This latter chemokinetic response occurs without any apparent long-term change in the size of the electrochemical proton gradient. The results presented here show that the chemokinetic response is separate from the chemotactic response, although some compounds can induce both responses. Compounds that caused only chemokinesis induced a sustained increase in the rate of flagellar rotation, but chemoeffectors which were also chemotactic caused an additional short-term change in both the stopping frequency and the duration of stops and runs. The response to a change in chemoattractant concentration was a transient increase in the stopping frequency when the concentration was reduced, with adaptation taking between 10 and 60 s. There was also a decrease in the stopping frequency when the concentration was increased, but adaptation took up to 60 min. The nature and duration of both the chemotactic and chemokinetic responses were concentration dependent. Weak organic acids elicited the strongest chemokinetic responses, and although many also caused chemotaxis, there were conditions under which chemokinesis occurred in the absence of chemotaxis. The transportable succinate analog malonate caused chemokinesis but not chemotaxis, as did acetate when added to a mutant able to transport but not grow on acetate. Chemokinesis also occurred after incubation with arsenate, conditions under which chemotaxis was lost, indicating that phosphorylation at some level may have a role in chemotaxis. Aspartate was the only chemoattractant amino acid to cause chemokinesis. Glutamate caused chemotaxis but not chemokinesis. These data suggest that (i) chemotaxis and chemokinesis are separate responses, (ii) metabolism is required for chemotaxis but not chemokinesis, (iii) a reduction in chemoattractant concentration may cause the major chemotactic signal, and (iv) a specific transport pathway(s) may be involved in chemokinetic signalling in R. sphaeroides.     

Cell volume dependence of 1H spin-echo NMR signals in human erythrocyte suspensions: The influence of in situ field gradients

The ¹H spin-echo NMR signal amplitudes and intensities of low molecular weight solutes in the cytoplasm and extracellular fluid of suspensions of human erythrocytes were shown to depend on the osmotic pressure of the media. At low osmotic pressure (220 mosM/kg) freeze-thaw lysis of the cells resulted in signal enhancement which was greatest for extracellular molecules, but both intra- and extracellular species were almost equally enhanced at 580 mosM/kg. This effect is due to field gradients formed at cell boundaries as a result of differences in magnetic susceptibility between the medium and the cytoplasm. T₂ values measured using the Carr-Purcell-Meiboom-Gill pulse sequence, with τ = 0.0003 s, depended little on cell volume and absolute changes in volume magnetic susceptibility were also small. The mean field gradients, calculated from data obtained on cell suspensions at different osmotic pressures, were in the range 0.25–1.98 G/cm and 0.89–2.09 G/cm for intra- and extracellular compartments, respectively. The maintenance of isotonicity of the extracellular fluid during metabolic studies of cell suspensions is important in order to avoid artefacts in the determination of metabolite concentrations when using the spin-echo technique. Conversely it may be possible to perform transport measurements using spin-echo NMR to monitor the cell volume changes which occur during the transmembrane migration of molecules.     

Heterogeneous transmural proteoglycan distribution provides a mechanism for regulating residual stresses in the aorta

The arterial wall contains a significant amount of charged proteoglycans, which are inhomogeneously distributed, with the greatest concentrations in the intimal and medial layers. The hypothesis of this study is that the transmural distribution of proteoglycans plays a significant role in regulating residual stresses in the arterial wall. This hypothesis was first tested theoretically, using the framework of mixture theory for charged hydrated tissues, and then verified experimentally by measuring the opening angle of rat aorta in NaCl solutions of various ionic strengths. A three-dimensional finite element model of aortic ring, using realistic values of the solid matrix shear modulus and proteoglycan fixed-charge density, yielded opening angles and changes with osmolarity comparable to values reported in the literature. Experimentally, the mean opening angle in isotonic saline (300 mosM) was 15 +/- 17 degrees and changed to 4 +/- 19 degrees and 73 +/- 18 degrees under hypertonic (2,000 mosM) and hypotonic (0 mosM) conditions, respectively (n = 16). In addition, the opening angle in isotonic (300 mosM) sucrose, an uncharged molecule, was 60 +/- 16 degrees (n = 11), suggesting that the charge effect, not cellular swelling, was the major underlying mechanism for these observations. The extent of changes in opening angle under osmotic challenges suggests that transmural heterogeneity of fixed-charge density plays a crucial role in governing the zero-stress configuration of the aorta. A significant implication of this finding is that arterial wall remodeling in response to altered wall stresses may occur via altered deposition of proteoglycans across the wall thickness, providing a novel mechanism for regulating mechanical homeostasis in vascular tissue.     

Optical changes in unilamellar vesicles experiencing osmotic stress.

Membrane properties that vary as a result of isotropic and transmembrane osmolality variations (osmotic stress) are of considerable relevance to mechanisms such as osmoregulation, in which a biological system "senses" and responds to changes in the osmotic environment. In this paper the light-scattering behavior of a model system consisting of large unilamellar vesicles of dioleoyl phosphatidyl glycerol (DOPG) is examined as a function of their osmotic environment. Osmotic downshifts lead to marked reductions in the scattered intensity, whereas osmotic upshifts lead to strong intensity increases. It is shown that these changes in the scattering intensity involve changes in the refractive index of the membrane bilayer that result from an alteration in the extent of hydration and/or the phospholipid packing density. By considering the energetics of osmotically stressed vesicles, and from explicit analysis of the Rayleigh-Gans-Debye scattering factors for spherical and ellipsoidal shells, we quantitatively demonstrate that although changes in vesicle volume and shape can arise in response to the imposition of osmotic stress, these factors alone cannot account for the observed changes in scattered intensity.     

Osmotic water permeability of human red cells

The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1.     

Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-beta, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-beta activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand-dependent activation of PDGFR-beta and its downstream effectors Akt, MEK1/2, and ERK1/2 is strongly modulated (inhibited) by hyperosmotic cell shrinkage, whereas cell swelling does not seem to affect the activation of the receptor but rather to activate ERK1/2 via a different mechanism. It is thus likely that cell swelling via activation of ERK1/2 and cell shrinkage via activation of the p38 and JNK pathway and inhibition of the PDGFR signaling pathway may act as key players in the regulation of tissue homeostasis.