Intracellular sorbitol content in isolated rat inner medullary collecting duct cells

In order to study the mechanisms involved in the regulation of renal inner medullary sorbitol content, collecting duct cells were isolated from rat inner medulla and the effect of extracellular osmolarity on sorbitol synthesis and sorbitol content was investigated. Cells isolated at 300 mosmol/l and incubated up to 24 h as primary cultures in 300 mosmol/l media or in media made 600 mosmol/l by the addition of 150 mM NaCl showed no difference in total synthesis. Intracellular sorbitol content was, however, 2.3-fold higher in the cells kept in the higher osmotic medium. Cells isolated at 600 mosmol/l released sorbitol about 8 times faster when transferred into hypoosmotic medium (300 mosmol/l) than when transferred into isoosmotic (600 mosmol/l) media. Cells exposed to hyperosmotic media (900 mosmol/l with NaCl) maintained a higher intracellular sorbitol content than cells incubated in isoosmotic media. Changes of intracellular sorbitol content could not be attributed entirely to cell lysis — as demonstrated by determination of cellular content of lactate and lactate dehydrogenase. The alteration in sorbitol membrane permeability was reversible and was only observed when poorly permeable solutes (such as NaCl and sucrose) were used for the experiments, changes in urea elicited no effect. It is proposed that rapid changes in membrane permeability to sorbitol play an important role in the adjustment of intracellular sorbitol concentration in inner medullary collecting duct cells to changes in extracellular osmolarity.Supported by grant DFG Gr 916/1-1     

Osmotically inactive space during hyperosmotic stress in the perfused submandibular gland of the rat.

Cell volume changes were measured by an impedance method during hyperosmotic stress in the perfused rat submandibular gland. When the perfusate osmolarity was raised to 484 mosmol, the cell volume decreased and remained at a plateau level (79%). The decrease was smaller than expected from the extracellular fluid osmolarity change. Furthermore, the potassium content of the gland increased by 7% during the hyperosmotic stress. These results suggest that the cell volume changes observed during hyperosmotic stress are affected by the existence of an osmotically inactive space and by an increase in the intracellular solute content.     

Control of cell proliferation by cell volume alterations in rat C6 glioma cells

K+ and Cl- channels are involved in regulating the proliferation of a number of cell types. Two main hypotheses have been proposed to explain the mechanism by which these channels influence cell proliferation: regulation of membrane potential and regulation of cell volume. In order to test these hypotheses, we measured, under different experimental conditions, the volume, membrane potential and rate of proliferation of C6 glioma cells. Cells cultured in control medium for 1-4 days were compared with cells cultured for the same period of time in the presence of broad spectrum channel blockers: tetraethylammonium, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and Cs+, in hypertonic media (29% increased osmolarity with NaCl, KCl or sucrose), in hypotonic medium (23% decreased osmolarity with H2O) or in the presence of the specific channel blockers, i.e. mast cell degranulating peptide, charybdotoxin or chlorotoxin. In all of these conditions, we observed a close correspondence between the rate of proliferation and the mean cell volume. The proliferation decreased when volume increased. Moreover, whereas control cells were flattened, spindle-shaped, bipolar or multipolar, cells cultured in media supplemented with NPPB, KCl or CsCl were round with few processes. Of the agents tested, only KCl and Cs+ depolarized the cells. These results show that alterations of the rate of proliferation by K+ and Cl- channel blockers or anisotonia are closely related with changes in cell volume or form but are not correlated with changes in membrane potential.     

Influence of osmolarity on the optical properties of human erythrocytes

Plasma osmolarity influences the volume and shape of red blood cells (RBCs). The volume change is inversely related to the hemoglobin concentration and as a consequence to the complex refractive index within the cell. These morphological changes can be linked to changes in the optical behavior of the cells. The optical parameters, absorption coefficient μa, scattering coefficient μs, and effective scattering phase function of red blood cells are investigated in dependence on osmolarity in the spectral range from 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulations are carried out for osmolarities from 225 to 400 mosmol/L. Osmolarity changes have a significant influence on the optical parameters, which can in part be explained by changes in the complex refractive index, cell shape, and cell volume. Spherical forms of RBCs induced by low osmolarity show reduced scattering effects compared to the normal RBC biconcave disk shape. Spinocytes, which are crenated erythrocytes induced by high osmolarity, show the highest scattering effects. Even only a 10% change in osmolarity has a drastic influence on the optical parameters, which appears to be of the same order as for 10% hematocrit and oxygen saturation changes.     

Isolation of rat peritoneal mononuclear and polymorphonuclear leucocytes on discontinuous gradients of Nycodenz

Centrifugation of rat leucocytes from thioglycollate-induced inflammatory peritoneal exudate on a discontinuous gradient of Nycodenz with a density of 1106 g/l and an osmolarity of 400 mosmol/l separated the polymorphonuclear from the mononuclear leucocytes. The cells on the interphase between the buffer and the gradient medium contained 96% mononuclear leucocytes with a recovery of greater than 60%, and the bottom fraction consisted of 98% polymorphonuclear leucocytes with a yield of 91%, when an exudate isolated 20 h after the injection of thioglycollate was fractionated. Leucocytes isolated from non-inflamed rat peritoneum could be enriched in the fraction of nonspecific esterase-positive cells from 86% to 96% with a recovery of 82% on a gradient with a density of 1091 g/l and an osmolarity of 325 mosmol/l. The viability of the isolated cells was greater than 95% (trypan blue exclusion test), and there was no measurable reduction in the fraction of phagocytosing cells (latex and opsonized zymosan) after exposure to the hypertonic gradient material.     

渗透压、细胞容积与鼻咽癌细胞增殖

目的:研究渗透压、细胞容积与鼻咽癌细胞增殖的关系。方法:用MTT法检测在不同渗透压培养条件下低分化鼻咽癌细胞(CNE－2Z)的增殖能力,流式细胞仪测定细胞周期分布,活细胞图像分析测量细胞容积,台盼蓝拒染法检测细胞存活率。结果:高渗(370、440mOsmol/L)培养增大细胞容积和促进细胞增殖,细胞容积分别增大8.7%、27.8%,增殖率分别提高22.2%和33.9%;而低渗(160、230mOsmol/L)培养减小细胞容积和抑制增殖,细胞容积分别减小12.8%和4.1%,增殖率分别降低34.0%和15.6%;细胞容积与细胞增殖率呈正相关。非等渗长期培养条件下,细胞周期各时相分布没有显著差异。低渗培养降低细胞生存率。结论:胞外渗透压、细胞容积与鼻咽癌细胞增殖密切相关,低渗培养可能通过减小细胞容积、促进细胞死亡而抑制细胞增殖。     

Micropuncture studies of the osmoregulation in the nauplius of Artemia salina

The osmoregulation of the nauplius of the brine shrimp, Artemia salina, was investigated using micropuncture and microanalytical techniques. The naupliar body fluid, hemolymph was hyposmotic to and had lower Na concentrations than the suspending medium for the range of medium salinities from 80 to 4,900 mM NaCl. In medium containing 20 mM NaCl, the hemolymph was hyperosmotic to the medium, with osmolarity of 101 +/- 8 mosmol/1 and with [Na] of 49 +/- 11 meq/1. Whereas the maximal observed NaCl concentration gradient between hemolymph and medium was 4,785 mM, during the incubation of nauplii in artificial seawater (osmolarity: 932 mosmol/1; and [Na]: 502 meq/1) the osmolarity and [Na] of the naupliar hemolymph were 161 +/- SD 16 mosmol/1 and 86 +/- 14 meq/1, respectively. The influx and efflux of Na between medium and hemolymph were measured using 22Na. The fluxes of this ion were temperature dependent. The main site of efflux of 22Na was the neck organ as was shown by experiments of differential recovery of 22Na introduced in the hemolymph. These studies demonstrate that the nauplius of A. salina has the ability to osmoregulate not only against high environmental salinities but also against low salinities approaching those of freshwater.     

Regulatory volume response of erythrocytes exposed to a gradual and slow decrease in medium osmolality

 A sudden decrease in external medium osmolality (90 mosmol/kg) causes an immediate swelling of trout erythrocytes, followed by a regulatory volume decrease (RVD) due to activation of both a KCl cotransporter and a taurine transport pathway. Here, we determined how trout red cells respond when they are exposed to a gradual and slow decrease in medium osmolality (80 mosmol/kg at a rate of 0.7 mosmol/kg per min). Erythrocytes were unable to regulate their volume efficiently when swollen gradually and it increased continuously throughout the experimental period (120 min). As long as volume was increased slowly by 15–25%, regulatory pathways remained essentially inactivated, erythrocytes losing no significant amount of intracellular osmotically active solutes. Above this swelling threshold, a response was triggered but the quantity of solutes lost via the regulatory pathways was still not sufficient to counterbalance the continuous entry of water due to the slow and gradual decrease in medium tonicity. Received: 18 January 1999 / Received after revision: 10 February 1999 / Accepted: 11 February 1999     

Calcium-induced calcium release participates in cell volume regulation of rabbit TALH cells

Changes of cell volume and intracellular calcium concentration ([Ca(2+)](i)) in immortalized thick ascending limb of Henle's loop (TALH) cells were monitored using confocal laser scanning microscopy and fura-2 fluorescence, respectively. Reduction of the extracellular osmolarity from 600 to 300 mosmol/l induced cell swelling followed by regulatory volume decrease (RVD). Simultaneously, the [Ca(2+)](i) increased transiently. The calcium rise was not observed in calcium-free solution or in the presence of nifedipine, indicating that the change was, in the first place, due to the activation of a calcium influx. Application of ATP or caffeine in isotonic solutions increased transiently the [Ca(2+)](i) which revealed the existence of stores in TALH cells sensitive to inositol-1,4,5 trisphosphate (IP(3)) and ryanodine. To examine the possibility that the calcium influx might induce calcium release, manganese quenching experiments were performed. In hypotonic calcium-free solutions, the decay of the calcium-insensitive and calcium-sensitive fluorescence occurred simultaneously. In the presence of extracellular calcium however, the calcium-sensitive wavelength revealed initial calcium influx followed by a calcium release from intracellular stores. Thus, the calcium influx was a prerequisite for the calcium release. We conclude that calcium-induced calcium release participates in global calcium signalling during RVD of TALH cells.     

The role of a swelling-activated taurine transport pathway in the regulation of articular chondrocyte volume

Swelling articular chondrocytes by reducing osmolarity stimulates a taurine transport pathway, which is implicated in regulatory volume decrease (RVD) in various cell types. The present study investigated factors controlling the activity of this pathway in chondrocytes, in particular (1) the effects of the acute (seconds) and chronic (hours) exposure of chondrocytes to anisotonic media, and (2) whether there is a role for metabolites from the arachidonic acid cascade in activating the taurine transport pathway. For in situ and isolated chondrocytes, the point at which swelling-activated [14C]taurine efflux was stimulated (the "set-point") corresponded closely to the osmolarity of the incubation medium (180, 280 or 380 mosmol/l). However, the volume of chondrocytes isolated into these media and measured by confocal microscopy was not different ( congruent with 645 microm3). Activity of the swelling-activated taurine transport pathway was inhibited by REV5901 (an inhibitor of steps of the arachidonic acid cascade; K0.5 8+/-4 microM), NDGA (a general lipoxygenase inhibitor; K0.5 28+/-5 microM), or MK886 (an inhibitor of the 5-lipoxygenase-activating protein; 91% inhibition at 10 microM), but weakly by the more potent 5-lipoxygenase inhibitor REV5901 para (K0.5 350+/-100 microM). Addition of the leukotriene (LT) B4 or D4 receptor antagonists, CP-105,696 and L660,711 respectively, or of the leukotrienes LTB4, LTC4, LTD4 and LTE4 or lipoxins (hepoxylin A3 or B3) had no effect on the activity of the pathway in isotonic or hypotonic media. The role of the pathway in RVD was determined in isolated calcein-loaded chondrocytes using fluorescence imaging. RVD was observed and inhibited by REV5901 (50 microM) and by NDGA (75 microM). The data show that despite chronic exposure of chondrocytes to anisotonic media, the cells maintain a pre-determined volume that is the "set-point" for the activation of the taurine transport pathway following acute hypotonic challenge. This pathway appears to play a role in chondrocyte RVD, but its activation does not involve metabolites of the arachidonic acid cascade.     

Isolation of Human Blood Monocytes with Nycodenz, a New Non-Ionic lodinated Gradient Medium

Monocytes were separated from human blood with Nycodenz, an iodinated gradient medium. Monocytes have a lower average density than lymphocytes, but because of overlapping efficient separation cannot be achieved on the basis of density differences alone. Thus the isolation procedure was based on the assumption that the low-density fraction of lymphocytes increases its density more than monocytes by expelling water when exposed to an increased osmolarity. Thereby they might pass through a density barrier present initially, whereas the monocytes remain at the top of the gradient layer. Separation fluids with densities ranging from 1.061 to 1.096 g/ml were prepared by mixing Nycodenz with Nad solutions of various concentrations. EDTA-blood (3 ml) or a leucocyte suspension (2–6 ml) obtained by dextran sedimentation was loaded on 3 ml of separation fluid and centrifuged for 15 min at 1900 rpm. Then the cells in the interface region were collected. At each density level it was possible to obtain an almost pure monocyte suspension (95–98% by increasing the osmolarity. However, the higher the purity, the lower the monocyte yield. Apparently, the viability of monocytes was not affected, even when subjected to an osmo larity of 600 mosmol. For routine use, it appears that separation fluids with densities from 1.061 to 1.078 g/ml and corresponding osmoiarity in the 300 to 410 mosmol range are suitable.     

Recovery of cell volume and electrolytes of A6 cells after re-establishing isotonicity following hypotonic stress

Cellular element concentrations and dry weight contents in A6 cells were determined using electron microprobe analysis to establish whether these cells exhibit a regulatory volume increase (post-RVD-RVI) when re-establishing isotonicity following a hypotonically induced regulatory volume decrease (RVD). Hypotonic stress was induced by reducing basolateral [NaCl], and hence, osmolarity fell from 260 to 140 mosmol/l. The alterations in cell volume after re-establishing isotonicity, calculated from the cellular dry weight changes, indicate within the first 2 min cell shrinkage from 120 to 76% of control, compatible with almost ideal osmometric behaviour of A6 cells, and thereafter a post-RVD-RVI to 94%. The cellular uptake of osmolytes necessary to explain the post-RVD-RVI could be accounted for solely by a gain in cellular K and Cl. The involvement of a Na-K-2Cl cotransporter in most of the KCl uptake seems plausible since basolateral bumetanide blocked KCl uptake and post-RVD-RVI. The net uptake of cations (K uptake of 185.2, Na loss of 8.2 mmol/kg dry wt) during the isotonic period exceeded the Cl uptake by 38.2 mmol/kg dry wt, suggesting the uptake of another anion and/or the alteration of cellular buffer capacity. The relatively low Na concentration maintained during the isotonic period (13.3 vs. 20.4 mmol/kg wet wt under control conditions) might favour electrolyte uptake via the Na-K-2Cl cotransporter.     

The actions of altered osmolarity on guinea-pig detrusor smooth muscle contractility and intracellular calcium

The study measured the effects in vitro of changing extracellular osmolarity on the contractility of detrusor smooth muscle strips. The data were interpreted in the context of separate measurements from isolated cells of alterations to the intracellular [Ca2+], [Ca2+]i. Increased osmolarity (300-700 mosmol l-1) reduced phasic contractions but increased resting tension regardless of whether sucrose, LiCl or NaCl were used as osmolytes. [Ca2+]i was decreased slightly only when NaCl increased osmolarity, otherwise it was unchanged. The contractile effects may be explained by tissue shrinkage and reduction of detrusor excitability. Lowered osmolarity (300-64 mosmol l-1) decreased phasic contractions but increased resting tension and [Ca2+]i. The raised resting tension was due solely to low osmolarity and was independent of changes to [Na], [Cl] or ionic strength. The rise of [Ca2+]i was due partly to Ca2+ influx through Na(+)-Ca2+ exchange but a fraction was independent of extracellular Ca, unaffected by Gd3+, and persisted in the presence of caffeine. By contrast, reduction of phasic tension was due mainly to the reduced ionic strength, not osmolarity. The results do not support the presence of functional stretch-activated channels and suggest only a minor role for Na(+)-Ca2+ exchange under these conditions. However, they do suggest an intracellular source of Ca2+, which is independent of the sarcoplasmic reticulum.     

Induced cell clustering enhances islet beta cell formation from human cultures enriched for pancreatic ductal epithelial cells

A better understanding of the culture conditions that stimulate in vitro beta-cell differentiation from islet precursors would be useful for optimizing the production of tissue-engineered islets. In this study, high- and low-adherent substrates and high- and low-serum media were used to control the clustering of human pancreatic ductal epithelial cells and to determine its effect on their transdifferentiation to beta cells. While the initial epithelial cell cultures were devoid of any beta cells as assessed by dithizone staining, dithizone+ cells were generated during the next 3 weeks under all culture conditions. Although the rate of transdifferentiation was low, a approximately 4-fold greater number and percentage of dithizone+ cells were generated following 23-24 days of culture in the least adherent conditions (low-serum medium, low-adherent substrate), which stimulated cell clustering to the highest degree. Insulin immunohistochemistry data correlated well with the dithizone data (r(2) = 0.99), evidence that dithizone is a reliable measure of insulin+ cells. The preferential distribution of the dithizone+ cells to regions of cell aggregation and the increased efficiency of transdifferentiation in conditions that promote cell clustering suggest that cell-cell interactions and/or cell shape changes are important to the transdifferentiation of adult pancreatic ductal epithelial cells to beta cells in vitro.     

Isoosmotic shrinkage by self-stimulated outward Na-K-Cl cotransport in quail erythrocytes

In mammalian erythrocytes, outward fluxes by the Na-K-Cl cotransporter NKCC have been clearly characterized, but NKCC fluxes are small and their physiological role, if any, is poorly understood. Avian erythrocytes are nucleated cells, in which a physiologically relevant NKCC acts as a cell volume regulator. Therefore, we further investigated outward cotransport and its relation to cell volume by using quail erythrocytes. Unlike human or rat erythrocytes, quail erythrocytes exhibit outward cotransport fluxes: (1) of high magnitude [maximal rate of bumetanide-sensitive Li⁺ efflux=12.3±1.1 mmol (l cells·h)^–1, mean ±SEM, n=23] and (2) strongly stimulated by hyperosmotic media (by 100–200% in 500 mosmol/l media). Na⁺- or Li⁺-loaded quail erythrocytes exhibited rapid cell shrinkage when incubated in K⁺-free media. Thus, cell volume remained stationary up to 5–10 min and then started to shrink. Shrinkage was first slow, but progressively accelerated, finally reaching a new stationary state where cell volume had decreased by about 20%. Such rapid cell shrinkage was fully inhibited by bumetanide and was associated with outward cotransport stimulation (self-stimulated or an auto-catalytic process, i.e. a reaction stimulated by its product). External K⁺ reduced all these phenomena, but significant cell shrinkage was still observed at an external K⁺ concentration of 2.8 mM. K⁺ removal failed to stimulate outward cotransport in hypotonic media (250 mosmol/l). Finally, reincubation of shrunken erythrocytes in physiological saline revealed that inward cotransport was stimulated more than outward cotransport. In conclusion, isoosmotic hypokalaemia drives a rapid shrinkage of quail erythrocytes, due to auto-catalytic net outward cotransport stimulation. Whether this is an experimental curiosity or indicates that outward cotransport can have some physiological role deserves further investigation.     

Evidence for several mechanisms of volume regulation in neuroblastoma x glioma hybrid NG108-15 cells

Volumes of neuroblastoma x glioma hybrid NG 108-15 cells were electronically measured in order to characterize the mechanisms involved in volume regulation in isosmotic and anisosmotic conditions. The cells behave as perfect osmometers when tonicity was changed at constant chloride concentration by adding sucrose or replacing NaCl with CaCl2 or MgCl2. In contrast, the cell volume was poorly dependent on tonicity when the Cl- concentration was changed by adding NaCl or H2O. Cell shrinkage was induced by cell stirring or after a hypotonicity-induced swelling. These volume decreases were abolished by caffeine but not by ryanodine or EGTA. Shrinkage was also induced by the Ca2+ ionophore ionomycin. The ionomycin-induced volume decrease was abolished by EGTA. Cell swelling induced an outwardly rectifying Cl- current which was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyloxy-alkanoic acid. When the tonicity was reduced at constant Cl- concentration by replacing NaCl with CaCl2 or MgCl2, the volume increased and then slowly decreased towards its control value. This regulatory volume decrease was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and dihydroindenyl-oxy-alkanoic acid. Long-term (hours-days) cell shrinkage was induced by a reduction of the culture medium osmolarity. Long-term cell swelling was induced by an increase of the culture medium osmolarity. These volume changes were abolished by the protein translation inhibitor cycloheximide. The results suggest that NG 108-15 cell volume is regulated by at least four interacting mechanisms controlled, respectively, by intracellular Ca2+, extracellular NaCl, cell volume and intracellular ionic strength. The speculative nature of ionic systems responsible for these volume regulating mechanisms is discussed.     

CD56-positive cells with or without synaptophysin expression are recognized in the pancreatic duct epithelium: a study with adult and fetal tissues and specimens from chronic pancreatitis

We observed the distribution of CD56+ epithelial cells in the pancreatic duct system using 25 fetal, one infantile, 3 normal adult, 4 diabetic, and 8 chronically inflamed pancreatic tissue samples. In the early stage of gestation (12 to 17 weeks), CD56+ cells were commonly seen in the immature tubular structures. They were often continuous to pancreatic islets, and their distribution was similar to that of synaptophysin (Syn)+ cells, suggesting that they are precursors of islet neogenesis. Their number decreased in proportion to gestational age. Instead, from 24 weeks of gestation, luminal cell clusters that were common in interlobular ducts revealed CD56+. These cell clusters were unrelated to islet neogenesis and Syn expression. Similar CD56+ luminal cell clusters were also observed in cases of chronic pancreatitis, whereas they were scarce in normal adult and diabetic tissues. CD56+ cells were also occasionally seen in intralobular ducts, intercalated ducts, and centroacinar cells in cases of chronic pancreatitis. We conclude that there are two types of CD56+ epithelial cells in the pancreatic duct system: CD56+ endocrine cells are numerous during the early stage of gestation, when islet neogenesis appears, while CD56+ luminal cells may represent developmental and regenerative changes of pancreatic ducts.     

Hydrostatic forces limit swelling of rat ventricular myocardium

To study ventricular cellular volume regulation when cell membranes and ion pumps cannot prevent swelling, rat ventricular sections were incubated in modified Krebs-Henseleit solutions in which 1) potassium was substituted for sodium, ion for ion; or 2) sodium chloride was reduced to decrease osmolarity to 228, 171, or 114 mosM. Ventricular water, [3H]inulin and [3H]mannitol spaces, potassium, sodium, chloride, and protein contents, and resting transmembrane potentials were measured. Increases in ventricular cellular volume were less than 30% in potassium-substituted and extremely dilute media (114 mosM), in contrast to increases of over 100% in identically treated renal cortical slices. In potassium-substituted solution, the fluid gained by ventricular cells during incubation was hypertonic with respect to the bathing medium. In dilute solution (171 and 114 mosM), ventricular, cellular, and extracellular osmolarities equilibrated only after substantial losses of cellular ions had occurred. These findings support the existence of mechanical limitations to ventricular cellular swelling, which may be caused by a unique network of interstitial collagen present in ventricular myocardium.     

Volumetric behaviour and survival of mouse zygotes and embryos in hyperosmotic media

Volume changes and survival of mouse zygotes and preimplantationembryos were studied after their exposure to hyperosmotic media.Transfer of zygotes from culture medium to medium containing1.5 M DMSO led to a rapid volume reduction to 60% of controlvalues. After 10 min the volume was normal again, illustratingthe rapid penetration of DMSO at room temperature. Re-transferof zygotes equilibrated in 1.5 M DMSO to culture medium in onestep doubled the volume. Twelve minutes later normal diameterswere observed again. Volume changes were also recorded aftertransfer to media rendered hypertonk by the addition of NaClor sucrose. A proportional decrease in volume was noticed upto 1500 mOsmol. A further increase in measured osmolarity upto 3100 mOsmol led only to limited further shrinkage of thezygotes (minimal volume observed was 27% of control volume).Addition of 1.5 M DMSO or 1.5 M propanediol had no influenceon the final volume reaction to NaCl and sucrose. Exposure tomedia with a measured osmotic value of 3200 mOsmol (NaCl added)during 10 min did not prevent further development of the embryosto the hatched blastocyst stage. A solution of 4000 mOsmol provedlethal to nearly all embryos. Addition of 1.5 M DMSO to thismedium did not have a protective effect against the osmoticstress at room temperature.     

Independence of extracellular tortuosity and volume fraction during osmotic challenge in rat neocortex

The structural properties of brain extracellular space (ECS) are summarised by the tortuosity (λ) and the volume fraction (α). To determine if these two parameters were independent, we varied the size of the ECS by changing the NaCl content to alter osmolality of bathing media for rat cortical slices. Values of λ and α were extracted from diffusion measurements using the real-time ionophoretic method with tetramethylammonium (TMA⁺). In normal medium (305 mosmol kg⁻¹), the average value of λ was 1.69 and of α was 0.24. Reducing osmolality to 150 mosmol kg⁻¹, increased λ to 1.86 and decreased α to 0.12. Increasing osmolality to 350 mosmol kg⁻¹, reduced λ to about 1.67 where it remained unchanged even when osmolality increased further to 500 mosmol kg⁻¹. In contrast, α increased steadily to 0.42 as osmolality increased. Comparison with previously published experiments employing 3000 M _r dextran to measure λ, showed the same behaviour as for TMA⁺, including the same constant λ in hypertonic media but with a steeper slope in the hypotonic solutions. These data show that λ and α behave differently as the ECS geometry varies. When α decreases, λ increases but when α increases, λ rapidly attains a constant value. A previous model allowing cellular shape to alter during osmotic challenge can account qualitatively for the plateau behaviour of λ.