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PubMed2003

On the mechanism of fluid transport across corneal endothelium and epithelia in general.

The mechanism by which fluid is transported across epithelial layers is still unclear. The prevalent idea is that fluid traverses these layers transcellularly, driven by local osmotic gradients secondary to electrolyte transport and utilizing the high osmotic permeability of aquaporins. However, recent findings that some aquaporin knockout mice epithelia transport fluid sow doubts on local osmosis. This review discusses recent evidence in corneal endothelium that points instead to electro-osmosis as the mechanism underlying fluid transport. In this concept, a local recirculating electrical current would result in electro-osmotic coupling at the level of the intercellular junctions, dragging fluid via the paracellular route. The text also mentions possible mechanisms for apical bicarbonate exit from endothelial cells, and discusses whether electro-osmosis could be a general mechanism.

Animals↗
PubMed1995

Determination of water permeability coefficient and its activation energy for rabbit spermatozoa.

Critical tonicity (tonicity at which 50% of cells swell beyond their maximum volume-to-surface area ratio and lyse) of rabbit spermatozoa was measured as 45.6 mOsm at 22 degrees C. To determine the temperature effect on critical tonicity, cells were equilibrated to 15, 25, and 35 degrees C and critical tonicity was measured as 52.9, 42.2, and 32.4 mOsm, respectively. The time taken for rabbit sperm to achieve lysis at these temperatures was measured by exposing cells for increasing times to distilled water. From these results values for the permeability coefficient of rabbit spermatozoa to water (Lp) and its activation energy (Ea) were calculated. At 25 degrees C, Lp was 0.63 micron/min/atm and was clearly temperature dependent; Ea was 17.8 kcal/mol. Rabbit spermatozoa appear to have a low Lp and high Ea, the opposite of the situation seen with spermatozoa from all other species examined to date which have high Lp and low Ea. Nevertheless, the values obtained permit modeling of cooling rates for cell survival during cryopreservation in keeping with cooling rates commonly employed.

Animals↗
PubMed1994

An analysis of the interaction between interstitial plasma protein, interstitial flow, and fenestral filtration and its application to synovium.

An analysis of the interstitial flow of a protein solution is presented, based on physical interactions with extracellular matrix biopolymers. Previous models were extended by incorporation of three features--(a) interstitial osmotic reflection coefficients and attendant partial molecular sieving, (b) the effect of solute exclusion on apparent intramatrix fluid viscosity, and (c) the effect of discrete capillary porosity on near-pore oncotic and hydraulic gradients. The principles were applied to a distributed, finite difference model of the synovial lining and its fenestrated capillaries. Numerical solutions were used to assess the significance of some of the factors involved and predictions were compared with experimental measurements of the transsynovial flow of albumin solutions in the steady state. The following physiologically important points emerged. (i) Steric exclusion of protein reduces the effective interstitial fluid viscosity and creates nonzero interstitial reflection coefficients, which impose a slight nonuniformity of interstitial plasma protein distribution. Together these two effects produce nonintuitive deviation of interstitial flow from Darcy's law when interstitial protein concentration is increased experimentally. (ii) Flow across fenestrae with high reflection coefficients is buffered by steep local gradients in interstitial oncotic and hydraulic pressures immediately outside the fenestrae. Buffering is underestimated by the mean interstitial Starling forces and by treatment of the capillary as if it were uniformly permeable to water. (iii) A bidirectional flow pattern can develop across the synovial lining and can contribute to fluid turnover within the joint cavity.

Animals↗
PubMed1990

Ca2+ entry through the apical membrane reduces antidiuretic hormone-induced hydroosmotic response in toad urinary bladder.

The role of Ca2+ in the regulation of antidiuretic hormone(ADH)-induced water permeability of the apical membrane of the toad urinary bladder was examined. The effects of modifying Ca2+ entry through the apical membrane of toad urinary bladders on the hydroosmotic water flow (phi H2O) and short circuit current (Isc) were measured. In most experiments the bladders were treated with small amounts of Ag+ (10(-7) mol/l) on the apical side. This treatment was used because previous experiments indicate that it markedly increases alkali-earth cation fluxes through an amiloride-insensitive cation channel in the apical membrane of the urinary bladder. Moreover, when Ca2+ is the major cation in the apical solution of these Ag(+)-treated bladders, Isc is mostly due to Ca2+ entry through the apical membrane. Ag+ increased Isc and simultaneously inhibited phi H2O in bladders perfused with Ca2+ solutions on the apical side. Addition of La3+ to the apical solution reversed the stimulation of Isc and the inhibition of phi H2O produced by Ag+. When bladders were perfused with Ca2(+)-free solutions on the apical side, addition of Ag+ did not inhibit phi H2O while the stimulation of cation movements through the amiloride-insensitive cation channel persisted. In bladders perfused with apical Ca2+ solutions and treated with chlorophenyl thio-cyclic adenosine monophosphate (ClPheS-cAMP) the addition of Ag+ did not inhibit phi H2O while it still increased Isc. Finally, addition of Ca2+ to the apical solution of bladders not treated with Ag+ reduced phi H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1980

Quantitative analysis of exocytosis and endocytosis in the hydroosmotic response of toad bladder.

This study concerns the timing and magnitude of exocytosis and endocytosis in the granular cells of toad bladder during the hydroosmotic response to antidiuretic hormone. Granule exocytosis at the luminal cell surface is extensive within 5 min of the administration of a physiological dose of hormone. Hydroosmosis becomes detectable during this time period. The amount of membrane added to the luminal surface by exocytosis during 60 min of exposure to hormone can be of the same order of magnitude as the extent of the luminal plasma membrane. Endocytosis, demonstrated by peroxidase uptake from the luminal surface, becomes extensive during the period 15-45 min after hormone administration. Thus, maximal endocytic activity occurs later than the period of most extensive exocytosis and seems to correlate with the onset of the decline in water movement. The amount of membrane retrieved from the luminal surface by endocytosis during 60 min of stimulation is at least three quarters of that added by exocytosis. The bulk membrane movement in ADH stimulated preparations does not require the presence of an osmotic gradient. Colchicine inhibits the hydroosmotic response, the exocytosis of granules, and endocytosis at the luminal surface. These results strengthen our view that the bulk circulation of membrane at the cell surface, via exocytosis and endocytosis, is closely related to the permeability changes occurring at the surface.

Animals↗
PubMed1989

Volume-activated Na/H exchange activity in fetal and adult pig red cells: inhibition by cyclic AMP.

Hyposmotic swelling of pig red cells leads to a selective increase in K permeability, whereas hyperosmotic cell shrinkage augments the Na permeability. In this regard, the ouabain-resistant (OR) Na flux of red cells of newborn and adult pigs is characterized in detail. A reduction in cell volume by approximately 18% leads to an increase in the OR Na efflux of fetal and adult cells by 15- and fourfold, respectively. The OR Na influx in both cell types is equally influenced by cell shrinkage. Depletion of cellular K does not influence the volume-activated OR Na efflux. Nor does OR Na influx require external K. Both OR Na efflux and influx activated by shrinkage are inhibited by the diuretics furosemide and amiloride. The rank order of decreasing anion sensitivity for diuretic-sensitive Na efflux was acetate greater than chloride greater than gluconate greater than nitrate. Cell shrinkage induced by the addition of hypertonic salts results in an acidification of the unbuffered and CO2-free media, provided that both Na and DIDS are present. The acidification process can be reversed by either of the diuretic agents. These findings suggest that the shrinkage-activated OR Na flux is primarily mediated by a Na/H exchanger rather than by a Na/K/Cl cotransporter. Once loaded with either cAMP or cGMP, cell swelling can no longer activate the Na/H exchanger. The Na/H exchanger activity is detectable in the fetal cells of normal volume but quiescent in adult cells, indicating that the exchanger undergoes a developmental change during the transition from the fetal to adult stage.

Amiloride↗
PubMed2006

A mechano-chemical model for the passive swelling response of an isolated chondron under osmotic loading.

The chondron is a distinct structure in articular cartilage that consists of the chondrocyte and its pericellular matrix (PCM), a narrow tissue region surrounding the cell that is distinguished by type VI collagen and a high glycosaminoglycan concentration relative to the extracellular matrix. We present a theoretical mechano-chemical model for the passive volumetric response of an isolated chondron under osmotic loading in a simple salt solution at equilibrium. The chondrocyte is modeled as an ideal osmometer and the PCM model is formulated using triphasic mixture theory. A mechano-chemical chondron model is obtained assuming that the chondron boundary is permeable to both water and ions, while the chondrocyte membrane is selectively permeable to only water. For the case of a neo-Hookean PCM constitutive law, the model is used to conduct a parametric analysis of cell and chondron deformation under hyper- and hypo-osmotic loading. In combination with osmotic loading experiments on isolated chondrons, model predictions will aid in determination of pericellular fixed charge density and its relative contribution to PCM mechanical properties.

Biomechanical Phenomena↗
PubMed2005

Trehalose uptake through P2X7 purinergic channels provides dehydration protection.

The tetra-anionic form of ATP (ATP4-) is known to induce monovalent and divalent ion fluxes in cells that express purinergic P2X7 receptors and with sustained application of ATP it has been shown that dyes as large as 831 Da can permeate the cell membrane. The current study explores the kinetics of loading alpha,alpha-trehalose (342 Da) into ATP stimulated J774.A1 cells, which are known to express the purinergic P2X7 receptor. Cells that were incubated at 37 degrees C in a 50 mM phosphate buffer (pH 7.0) containing 225 mM trehalose and 5 mM ATP, were shown to load trehalose linearly over time. Concentrations of approximately 50 mM were reached within 90 min of incubation. Cells incubated in the same solution at 4 degrees C loaded minimally, consistent with the inactivity of the receptor at low temperatures. However, extended incubation at 37 degrees C (>60 min) resulted in zero next-day survival, with adverse effects appearing even with incubation periods as short as 30 min. By using a two-step protocol with a short time period at 37 degrees C to allow pore formation, followed by an extended loading period on ice, cells could be loaded with up to 50 mM trehalose while maintaining good next day recovery (49 +/- 12% by Trypan blue exclusion, 56 +/- 20% by alamarBlue assay). Cells porated by this method and allowed an overnight recovery period exhibited improved dehydration tolerance suggesting a role for ATP poration in the anhydrous preservation of cells.

Adenosine Triphosphate↗
PubMed2001

The effects of the Na(+)/Ca(++) exchange blocker on osmotic blood-brain barrier disruption.

Osmotic disruption of the blood-brain barrier (BBB) by mannitol is currently being used to enhance drug delivery in human brains. Despite clinical and experimental interest, to date the time course in the early phase of disruption has not been accurately identified. The mechanism in barrier closure also remains elusive. We first studied the rapid change in cerebrovascular permeability after BBB disruption in rats, and then demonstrated that the Na(+)/Ca(++) exchange blocker (KB-R7943) prolongs osmotic disruption. Osmotic BBB disruption was attained by using intra-arterial infusion of hypertonic mannitol in Sprague-Dawley (SD) rats. To measure the changes in cerebrovascular permeability, perfusate containing [14C]-sucrose was infused intra-arterially at different time points following osmotic stress. Cerebrovascular permeability was then measured with the in situ brain perfusion technique. This is the first in vivo study demonstrating that osmotic disruption is prolonged by the Na(+)/Ca(++) exchange blocker, which did not affect the peak level of BBB disruption. The exact time course of cerebrovascular reversibility was studied and the earliest BBB disruption was seen to occur 5 min after osmotic stress. Histopathological examination after osmotic disruption with the Na(+)/Ca(++) exchange blocker showed no neuronal damage in rat brains. Our findings represent important experimental information regarding pharmacological manipulation of BBB disruption. The possibility of prolonging the transient opening of the BBB has major clinical implications.

Animals↗
PubMed2002

The effect of temperature on membrane hydraulic conductivity.

The objective of this study was to use the temperature dependence of water permeability to suggest the physical mechanisms of water transport across membranes of osmotically slowly responding cells and to demonstrate that insight into water transport mechanisms in these cells may be gained from easily performed experiments using an electronic particle counter. Osmotic responses of V-79W Chinese hamster fibroblast cells were measured in hypertonic solutions at various temperatures and the membrane hydraulic conductivity was determined. The results were fit with the general Arrhenius equation with two free parameters, and also fit with two specific membrane models each having only one free parameter. Data from the literature including that for human bone marrow stem cells, hamster pancreatic islets, and bovine articular cartilage chondrocytes were also examined. The results indicated that the membrane models could be used in conjunction with measured permeability data at different temperatures to investigate the method of water movement across various cell membranes. This approach for slower responding cells challenges the current concept that the presence of aqueous pores is always accompanied by an osmotic water permeability value, P(f)>0.01 cm/s. The possibility of water transport through aqueous pores in lower-permeability cells is proposed.

Animals↗
PubMed1992

Permeability of rat liver microsomal membrane to glucose 6-phosphate.

Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer revealed the following. (1) The increase in extravesicular osmolality by addition of glucose 6-phosphate or mannose 6-phosphate (25 mM each) caused a rapid shrinking of microsomal vesicles. After shrinkage, a rapid swelling phase (t1/2 approx. 22 s) was present with glucose 6-phosphate but absent with mannose 6-phosphate, indicating that the former had entered microsomal vesicles, but the latter had not. (2) Almost identical results were obtained in the absence of any glucose 6-phosphate hydrolysis, i.e. with microsomes pre-treated with 100 microM-vanadate. (3) The anion-channel blocker 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid (DIDS) suppressed the glucose 6-phosphate-induced swelling phase. (4) The swelling phase was more prolonged as the glucose 6-phosphate concentration increased (t1/2 = 16 +/- 3, 22 +/- 3 and 35 +/- 4 s with 25 mM, 37.5 mM- and 50 mM-glucose 6-phosphate respectively). The behaviour of glucose-6-phosphatase activity of intact and disrupted microsomes measured in the presence of high concentrations (less than 30 mM) of substrate also indicated the saturation of the glucose 6-phosphate permeation system by extravesicular concentrations of glucose 6-phosphate higher than 20-30 mM. Additional experiments showed that vanadate-treated microsomes pre-equilibrated with 0.1 mM- and 1.0 mM-glucose 6-phosphate (and [1-14C]glucose 6-phosphate as a tracer) rapidly (t1/2 less than 20 s) released [1-14C]glucose 6-phosphate when diluted in a glucose 6-phosphate-free medium. The efflux of [1-14C]glucose 6-phosphate was largely prevented by DIDS, allowing an evaluation of the intravesicular space of glucose 6-phosphate of approx. 1.0 microliter/mg of microsomal protein.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2002

Glycophorin A-mediated haemolysis of normal human erythrocytes: evidence for antigen aggregation in the pathogenesis of immune haemolysis.

The inexplicable severity of anti-Pr autoimmune haemolytic anaemia led us to test the hypothesis that the haemolysis was primarily due to a change in the function of glycophorin A, on which the Pr antigen is located. The lectins Maclura pomifera and wheat germ agglutinin that bind to glycophorin A induced the haemolysis of normal erythrocytes in vitro. Lectin binding led to an increase in erythrocyte membrane permeability to sodium and potassium, the former resulting in an influx of water and subsequent haemolysis. The response was glycophorin A specific as Concanavalin A, which binds to band 3, did not cause haemolysis and peanut agglutinin only did so after removal of erythrocyte sialic acid. The lectin-induced cation leak was not mediated by activation of cation channels as the inhibitors, tetrodotoxin, amiloride and 4,4' disothiocyanate stilbene 2,2'disulphonate, had no effect, suggesting that the haemolysis was due to exacerbation of the inherent cation permeability of the erythrocyte membrane. A human IgAK anti-Pr autoantibody and a mouse anti-human glycophorin A antibody increased erythrocyte permeability to sodium. The role of glycophorin A in stabilizing and, upon aggregation, destabilizing the phospholipid bilayer is discussed. Our findings may help explain the severity of anti-Pr autoimmune haemolytic anaemia and other pathophysiological changes in human erythrocytes.

Anemia, Hemolytic, Autoimmune↗