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PubMed2005

Osmotic tolerance and membrane permeability characteristics of rhesus monkey (Macaca mulatta) spermatozoa.

Biophysical characteristics of the plasma membrane, such as osmotic sensitivity and water and cryoprotectant permeability are important determinants of the function of spermatozoa after cryopreservation. A series of experiments was conducted with rhesus macaque spermatozoa at 23 degrees C to determine their: (1) cell volume and osmotically inactive fraction of the cell volume; (2) permeability coefficients for water and the cryoprotectants dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol; (3) tolerance to anisosmotic conditions; and (4) motility after a one step addition and removal of the four cryoprotectants. An electronic particle counter and computer aided semen analysis were used to determine the cell volume and permeability coefficients, and motility, respectively. Rhesus spermatozoa isosmotic cell volume was 27.7+/-3.0 microm3 (mean+/-SEM) with an osmotically inactive cell fraction of 51%. Hydraulic conductivity in the presence of dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol was 1.09+/-0.30, 0.912+/-0.27, 1.53+/-0.53, and 1.94+/-0.47 microm/min/atm, respectively. Cryoprotectant permeability was 1.39+/-0.31, 2.21+/-0.32, 3.38+/-0.63, and 6.07+/-1.1 (x10(-3)cm/min), respectively. Rhesus sperm tolerated all hyposmotic exposures. However, greater than 70% motility loss was observed after exposure to solutions of 600 mOsm and higher. A one step addition and removal of all four cryoprotectants did not cause significant motility loss. These data suggest that rhesus sperm are tolerant to hyposmotic conditions, and ethylene glycol may be the most appropriate cryoprotectant for rhesus sperm cryopreservation, as it has the highest permeability coefficient of the tested cryoprotectants.

Animals↗
PubMed2003

Annexin A4 reduces water and proton permeability of model membranes but does not alter aquaporin 2-mediated water transport in isolated endosomes.

Annexin A4 (Anx4) belongs to a ubiquitous family of Ca2+-dependent membrane-binding proteins thought to be involved in membrane trafficking and membrane organization within cells. Anx4 localizes to the apical region in epithelia; however, its physiological role is unclear. We show that Anx4 exhibited binding to liposomes (phosphatidylcholine:phosphatidylserine, 1:1) in the presence of Ca2+ and binding was reversible with EDTA. Anx4 binding resulted in liposome aggregation and a reduction in membrane water permeability of 29% (P < 0.001) at 25 degrees C. These effects were not seen in the presence of Ca2+ or Anx4 alone and were reversible with EDTA. Measurements of membrane fluidity made by monitoring fluorescence anisotropy of 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-HPC) demonstrated that Anx4 binding rigidified the outer leaflet of the bilayer (P < 0.001), thus providing a molecular explanation for the inhibition of water flux. To determine whether Anx4 would produce similar effects on physiological membranes we constructed liposomes which recapitulated the lipid composition of the inner leaflet of the MDCK apical membrane. These membranes exhibited reductions to water permeability upon Anx4 binding (19.5% at 25 degrees C, 31% at 37 degrees C; P < 0.01 and P < 0.001, respectively) and to proton permeability (15% at 25 degrees C, 19.5% at 37 degrees C; P < 0.05). Since our in vitro experiments indicated an effect on membrane permeability, we examined localization of Anx4 in the kidney collecting duct, a region of the nephron responsible for concentrating urine through water reabsorbtion. Anx4 was shown to colocalize apically with aquaporin 2 (AQP2) in collecting duct epithelia. To test for the existence of a functional interaction between Anx4 and AQP2 we isolated AQP2-containing endosomes and exposed them to Anx4/Ca2+. Water flux rates were unchanged, indicating Anx4 does not directly regulate AQP2. We conclude that Anx4 can alter the physical properties of membranes by associating with them and regulate passive membrane permeability to water and protons. These properties represent important new functions for Anx4.

Animals↗
PubMed2002

Evidence against aquaporin-1-dependent CO2 permeability in lung and kidney.

AQP1-dependent CO2 transport has been suggested from the increased CO2 permeability in Xenopus oocytes expressing AQP1. Potential implications of this finding include AQP1-facilitated CO2 exchange in mammalian lung and HCO3-/CO2 transport in kidney proximal tubule. We reported previously that: (a) CO2 permeability in erythrocytes was not affected by AQP1 deletion, (b) CO2 permeability in liposomes was not affected by AQP1 reconstitution despite a 100-fold increased water permeability, and (c) CO2 blow-off by the lung in living mice was not impaired by AQP1 deletion. We extend these observations by direct measurement of CO2 permeabilities in lung and kidney. CO2 transport across the air-space-capillary barrier in isolated perfused lungs was measured from changes in air-space fluid pH in response to addition/removal of HCO3-/CO2 from the pulmonary artery perfusate. The pH was measured by pleural surface fluorescence of a pH indicator (BCECF-dextran) in the air-space fluid. Air-space fluid pH equilibrated rapidly (t(1/2) approximately 6 s) in response to addition/removal of HCO3-/CO2. However, the kinetics of pH change was not different in lungs of mice lacking AQP1, AQP5 or AQP1/AQP5 together, despite an up to 30-fold reduction in water permeability. CO2 transport across BCECF-loaded apical membrane vesicles from kidney proximal tubule was measured from the kinetics of intravesicular acidification in response to rapid mixing with a HCO3-/CO2 solution. Vesicles rapidly acidified (t(1/2) approximately 10 ms) in response to HCO3-/CO2 addition. However the acidification rate was not different in kidney vesicles from AQP1-null mice despite a 20-fold reduction in water permeability. The results provide direct evidence against physiologically significant transport of CO2 by AQP1 in mammalian lung and kidney.

Algorithms↗
PubMed1999

Inhibitory effect of high [Mg2+] on the vasopressin-stimulated hydroosmotic permeability of the isolated perfused cortical collecting duct.

High magnesium concentration inhibits the effect of arginine vasopressin (AVP) on smooth muscle contraction and platelet aggregation and also influences hepatocyte AVP receptor binding. The aim of this study was to determine the role of magnesium concentration [Mg2+] in AVP-stimulated water transport in the kidney collecting duct. The effect of low and high peritubular [Mg2+] on the AVP-stimulated osmotic water permeability coefficient (Pf) was evaluated in the isolated perfused rabbit cortical collecting duct (CCD). Control tubules bathed and perfused with standard Ringer bicarbonate solution containing 1 mM Mg2+ presented a Pf of 223.9 +/- 27.2 microm/s. When Mg2+ was not added to the bathing solution, an increase in the AVP-stimulated Pf to 363.1 +/- 57.2 microm/s (P<0. 05) was observed. An elevation of Mg2+ to 5 mM resulted in a decrease in Pf to 202.9 +/- 12.6 microm/s (P<0.05). This decrease in the AVP-stimulated Pf at 5 mM Mg2+ persisted when the CCDs were returned to 1 mM Mg2+, Pf = 130.2 +/- 20.3 microm/s, and was not normalized by the addition of 8-[4-chlorophenylthio]-adenosine 3', 5'-cyclic monophosphate, a cAMP analogue, to the preparation. These data indicate that magnesium may play a modulatory role in the action of AVP on CCD osmotic water permeability, as observed in other tissues.

Animals↗
PubMed2004

Nanoleakage types and potential implications: evidence from unfilled and filled adhesives with the same resin composition.

PURPOSE: To compare nanoleakage patterns of an unfilled (OS; One-Step), a 6 wt% spherical silica-filled (OSs; One-Step Plus) and a 15 wt% glass-filled (OSg) version of a two-step, acetone-based self-priming adhesive. Permeability of bonded dentin treated with OS and OSs was also examined. METHODS: Deep, coronal dentin from extracted third molars were etched and bonded using these adhesives. One-mm thick sections were immersed in 50 wt% ammoniacal silver nitrate (pH 9.5) for 24 hours. Unstained, undemineralized sections were examined by TEM. The permeability of dentin bonded with OS and OSs were investigated at 20 cm of H2O hydrostatic pressure and compared with the osmotic conductance determined with 4.8 M CaCl2 at zero hydrostatic pressure. Composite-dentin beams bonded with OS, OSs and OSg that were fractured after microtensile bond testing were examined by SEM. RESULTS: Two types of nanoleakage patterns were recognized along the resin-dentin interfaces. The reticular type consisted of discontinuous islands of silver deposits and was exclusively seen in hybrid layers. The spotted type consisted of isolated silver grains and was evident throughout the hybrid and adhesive layers in OS. These two patterns were seen to variable extents in the two filled adhesive versions OSs and OSg and their distribution was independent of one another. OS and OSs bonded dentin were permeable to fluid filtration. However, part of this fluid movement was due to the permeability of the adhesive layer, as demonstrated by osmotic fluid conductance in the absence of hydrostatic pressure. Fractographic analysis revealed denuded collagen fibrils within fractured hybrid layers that were indicative of incomplete resin infiltration.

Acetone↗
PubMed1990

Transport mechanisms in iontophoresis. III. An experimental study of the contributions of electroosmotic flow and permeability change in transport of low and high molecular weight solutes.

The objective of this research was to provide in vitro transport data designed to clarify the relative importance of permeability increase and electroosmotic flow in flux enhancement via iontophoresis. Iontophoretic fluxes were measured with both anode and cathode donor cells, and passive fluxes were measured both before iontophoresis (Passive 1) and after iontophoresis (Passive 2). Data were generated for three uncharged low molecular weight solutes (glycine, glucose, and tyrosine) and two high molecular weight anionic species (carboxy inulin and bovine serum albumin). Flux enhancement is greater for anodic delivery than for cathodic delivery, even for the negatively charged molecules, and anodic flux of glucose decreases as the concentration of NaCl increases. Both observations are consistent with a mass transfer mechanism strongly dependent on electroosmotic flow. Steady-state anodic flux at 0.32 mA/cm2, expressed as equivalent donor solution flux (in microliters/hr cm2), ranged from 6.1 for glycine to about 2 for the large anions. As expected, iontophoretic flux is higher at 3.2 mA/cm2 than at 0.32 mA/cm2, and passive flux measured after iontophoresis is about a factor of 10 greater than the corresponding flux measured before the skin was exposed to electric current. There are two mechanisms for flux enhancement relative to passive flux on "fresh" hairless mouse skin: (1) the effect of the voltage in increasing mass transfer over the passive diffusion level, the effect of electroosmotic flow dominating this contribution in the systems studied in this report; and (2) the effect of prior current flow in increasing the "intrinsic permeability" of the skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Gravitational effects in the passive osmotic flows across polymeric membrane of electrolytic solutions.

Results of experimental study of volume flux in one-membrane system were presented. This system contains horizontal, microporous and symmetrical flat polymeric membrane (Nephrophan), which separate water and electrolyte solution. As binary solutions, aqueous ammonia solutions, which density is lower than water density, were used. As ternary solutions the ammonia with KC1 (0.1 or 0.2 mole.l-1) in aqueous solution were used. The density of ternary solutions was lower, higher or the same as water density. Two configurations of membrane system (A and B) in gravitational field were studied. In configuration A, water was in compartment over the membrane and the solution was under the membrane. In configuration B the succession was reverse. The thermodynamic model of flux graviosmotic effect was elaborated, and the calculations of this effect were performed for A and B configurations of one membrane system. The experimental results are interpreted in terms of gravitational instability that reduces concentration boundary layer dimensions and increases the diffusion permeability coefficient value of the complex: boundary layer/membrane/boundary layer.

Ammonia↗
PubMed2004

Mechanistic evaluation of the glucose-induced reduction in initial burst release of octreotide acetate from poly(D,L-lactide-co-glycolide) microspheres.

One major obstacle for development of injectable biodegradable microspheres for controlled peptide and protein delivery is the high initial burst of drug release occurring over the first day of incubation. We describe here the significant reduction in initial burst release of a highly water-soluble model peptide, octreotide acetate, from poly(D,L-lactide-co-glycolide) microspheres by the co-encapsulation of a small amount of glucose (e.g., 0.2%w/w), i.e., from 30+/-20% burst - glucose to 8+/-3% + glucose (mean+/-SD, n=4). This reduction is unexpected since hydrophilic additives are known to increase porosity of microspheres, causing an increase in permeability to mass transport and a higher burst. Using the double emulsion-solvent evaporation method of encapsulation, the effect of glucose on initial burst in an acetate buffer pH 4 was found to depend on polymer concentration, discontinuous phase/continuous phase ratio, and glucose content. Extensive characterization studies were performed on two microsphere batches, +/-0.2% glucose, to elucidate the mechanism of this effect. However, no significant difference was observed with respect to specific surface area, porosity, internal and external morphology and drug distribution. Continuous monitoring of the first 24-h release of octreotide acetate from these two batches disclosed that even though their starting release rates were close, the microspheres + glucose exhibited a much lower release rate between 0.2 and 24h compared to those - glucose. The microspheres + glucose showed a denser periphery and a reduced water uptake at the end of 24-h release, indicating decreased permeability. However, this effect at times was offset as glucose content was further increased to 1%, causing an increase in surface area and porosity. In summary, we conclude that the effect of glucose on initial burst are determined by two factors: (1) increased initial burst due to increased osmotic pressure during encapsulation and drug release, and (2) decreased initial burst due to decreased permeability of microspheres.

Coated Materials, Biocompatible↗