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In vivo inhibition of transcellular water channels (aquaporin-1) during acute peritoneal dialysis in rats.

During peritoneal dialysis (PD), a major portion of the osmotically induced water transport to the peritoneum can be predicted to occur through endothelial water-selective channels. Aquaporin-1 (AQP-1) has recently been recognized as the molecular correlate to such channels. Aquaporins can be inhibited by mercurials. In the present study, HgCl2 was applied locally to the peritoneal cavity in rats after short-term tissue fixation, used to protect the tissues from HgCl2 damage. Dianeal (3.86%) was employed as dialysis fluid, 125I-albumin as an intraperitoneal volume marker, and 51Cr-EDTA (constantly infused intravenously) to assess peritoneal small-solute permeability characteristics. Immunocytochemistry and immunoelectron microscopy revealed abundant AQP-1 labeling in capillary endothelium in peritoneal tissues, representing sites for HgCl2 inhibition of water transport. HgCl2 treatment reduced water flow and inhibited the sieving of Na+ without causing any untoward changes in microvascular permeability, compared with that of fixed control rats, in which the peritoneal cavity was exposed to tissue fixation alone. In fixed control rats, the mean intraperitoneal volume (IPV) increased from 20.5 +/- 0.15 to 25.0 +/- 0.52 ml in 60 min, whereas in the HgCl2-treated rats, the increment was only from 20.7 +/- 0.23 to 23.5 +/- 0.4 ml. In fixed control rats, the dialysate Na+ fell from 135.3 +/- 0.97 to 131.3 +/- 1.72 mM, whereas in the HgCl2-treated rats the dialysate Na+ concentration remained unchanged between 0 and 40 min, further supporting that water channels had been blocked. Computer simulations of peritoneal transport were compatible with a 66% inhibition of water flow through aquaporins. The observed HgCl2 inhibition of transcellular water channels strongly indicates a critical role of aquaporins in PD and provides evidence that water channels are crucial in transendothelial water transport when driven by crystalloid osmosis.

Animals↗

Cytoplasmic involvement in ADH-mediated osmosis across toad urinary bladder.

Several lines of investigation have suggested that antidiuretic hormone (ADH) may have direct effects on the cytoskeletal organization of granular epithelial cells in the toad urinary bladder. To some extent, these effects are in concert with the well-established action of ADH on the hydraulic permeability of the mucosal plasma membrane, but it appears that other conformational adjustments (largely cytoplasmic) may be of comparable importance. The thrust of this review is that the hormone brings about a general restructuring of the granular cells so that the epithelium as a whole may function efficiently as an osmotic pathway. Details of cytoskeletal changes are far from clear as yet, but interference with or modulation of these particular effects infer that cytoplasmic organization is the seat of feedback control of osmotic flow rate, the basis for viability in the presence of dramatic cytosolic dilution and a major factor in the observed disparity in osmotic and diffusional permeability coefficients. In the interest of stimulating new thoughts and experiments in this area, a number of preliminary findings have been freely cited.

Animals↗

Functional characterization of a putative aquaporin from Encephalitozoon cuniculi, a microsporidia pathogenic to humans.

The microsporidia are a group of obligate intracellular parasitic protists that have been implicated as both human and veterinary pathogens. The infectious process of these organisms is believed to be dependent upon the rapid influx of water into spores, presumably via aquaporins (AQPs), transmembrane channels that facilitate osmosis. An AQP-like sequence of the microsporidium Encephalitozoon cuniculi (EcAQP), when cloned and expressed in oocytes of Xenopus laevis, rendered these oocytes highly permeable to water. No permeability to the solutes glycerol or urea was observed. Pre-treatment of EcAQP-expressing oocytes with HgCl(2) failed to inhibit their osmotic permeability, as predicted from EcAQP's lack of mercury-sensitive cysteine residues near the NPA motifs which line the AQP aqueous pore. EcAQP exhibits sequence identity to AQP A of Dictyostelium discoideum (26%) and human AQP 2 (24%). Further study of AQPs in microsporidia and their potential inhibitors may yield novel therapeutic agents for microsporidian infections.

Amino Acid Sequence↗

The relation between salt and ionic transport coefficients.

The reflection coefficient was originally introduced by Staverman to describe the movement of nonelectrolytes through membranes. When this coefficient is extended to salts, one has a choice of defining this term for the whole salt moving as a single electrically neutral component or for the individual ions of the salt. The latter definition is meaningful only in the absence of an electric field across the permeability barrier. This condition may be achieved with the voltage clamp or short-circuit technique and is especially useful in dealing with biological systems in which one rarely has only a single salt or even equal concentrations of the major anion and cation. The relations between the transport coefficients for the salt and its individual ions are derived. The special conditions which may result in negative osmosis through a charged membrane in the presence of a salt are discussed.

Biological Transport↗

Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis.

Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 A) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.

Animals↗

Trichomonas vaginalis haemolysis: pH regulates a contact-independent mechanism based on pore-forming proteins.

There is a controversy in literature about involvement of secreted factors in the pathogenetic mechanisms of Trichomonas vaginalis, described mostly as contact-dependent. We found that the protozoan, under triggering conditions, is able to release molecules that lead to lysis without direct contact between parasite and target cells as a prerequisite. In this paper we characterize contact-independent cytotoxicity using the red blood cell as a cellular model. Contact-independent haemolysis is a phenomenon were pH exerts a key role, triggering the secretion of a lytic molecule and regulating its activity. A partial physicochemical characterization of the haemolytic factor suggests that a protein of M(r) > 30 kDa could be the effector responsible for damage. Furthermore, the parasite-induced membrane permeabilization, detected by measuring potassium escape from the target cell, and an effective osmotic protection by carbohydrates allowed us to relate the previously described pore-forming mechanism involved in contact-dependent cytotoxicity with the contact-independent lysis.

Animals↗

Current concepts of amniotic fluid dynamics.

Amniotic fluid at term in the human is the product of numerous exchanges with the fetal and indirectly, the maternal compartments. Our current information on the formation, circulation, and removal of this fluid is limited but provides an interesting concept of the steady-state regulation of this space. Further confirmation of this model comes from data describing drug exchange between mother, fetus, and amniotic fluid.

Amniotic Fluid↗

Thermally defined cryomicroscopy and thermodynamic analysis in lymphocyte freezing.

A cryomicroscope is described which provides the possibility of quantifying the volume loss of cells during freezing, detection of intracellular ice formation during cooling and warming, as well as the determination of viability as function of (constant) cooling rates. The basic mechanisms occurring in cryopreservation have been studied with this system using the human lymphocyte suspended in pure saline as a biological model system; experimentally observed exosmosis during freezing is compared to predictions from a thermodynamic model. Cell volume loss during freezing has been determined experimentally for cooling rates of 2.4, 12, 48, and 120 degrees K/min. Exosmosis also was calculated corresponding to various assumptions regarding the concentration dependence of the hydraulic permeability of the cells. Further calculations of exosmosis are performed for determining the effects of the initial cell volume. The temperatures and transition cooling rate ranges of intracellular ice formation have been determined. On the basis of exosmosis and a lethal level of intracellular salt concentration, a hypothetical relative optimum of the cooling rate is theoretically predicted and compared to the experiments.

Blood Preservation↗

Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures.

Monocytes were isolated from fresh whole human blood and resuspended in Hanks balanced salt solution; a portion of the cells was mixed with an equal volume of 2M dimethyl sulfoxide (DMSO) to form a 1 M solution. Microliter volumes of cell suspension were placed directly onto a computer-controlled cryostage and cooled to a predetermined subzero temperature. Ice was nucleated in the extracellular medium and a continuous video record was made of the subsequent osmotically induced volume changes of individual cells owing to exposure to the concentrated extracellular solutes. Selected micrographs emphasizing the initial transient data were digitized for computer analysis with an interactive boundary tracing algorithm to determine metric parameters of specific cells, and apparent volume changes were measured as a function of elapsed time after nucleation. The Kedem-Katchalsky-coupled transport equations were fit to the data using a network thermodynamic model implemented on a microcomputer to determine values for the permeability properties Lp, omega, and sigma. Experiments were performed over the temperature range from -7 degrees to -10 degrees C. Cells pre-equilibrated with DMSO had a lower Lp and a higher activation energy, delta E, than without additive, although the statistical significance of the difference could not be substantiated. It was found that the movement of DMSO across the plasma membrane in response to extracellular freezing was apparently so much smaller than the water flux that values for omega and sigma could not be determined from the data base.

Cell Membrane Permeability↗

Mercury free operation of the Coulter counter MultiSizer II sampling stand.

Electronic particle counters have gained widespread acceptance as a means to measure osmotic properties of cell membranes. Because most current instruments do not allow for the collection of true volume as a function of time data, investigators use older models such as the MultiSizer II sampling stand. A significant drawback to this and other older models is that they rely on mercury to maintain a constant pressure and to connect electrodes. The presence of mercury is a human health hazard that is exacerbated by the sometimes irregular vacuum pressures that cause mercury spills inside of the machine. To eliminate this hazard, we have determined that the MultiSizer II model can be simply and easily modified to function and collect temporal volume data without the use of mercury.

Animals↗

Electroosmotic characteristics of canine aorta and vena cava wall.

Experiments in which canine aorta and vena cava walls are subjected to electroosmosis in an open system at constant pressure are described. Electroosmosis reveals that the blood vessel walls studied have a negative zeta potential. The calculated zeta potentials are different for aorta and vena cava, -9.0 +/- 5.0 mv compared with -4.7 +/- 1.2 mv, respectively, and again of different magnitude with different bathing solutions. The calculated membrane pore charge per centimeter of effective pore surface in statcoulombs is approximately 6.2 x 10(3) for aorta compared with 3.5 x 10(3) for vena cava. The implications of the negative electroosmotic zeta potential in terms of the surrounding electric double layer, ion transport, and thrombosis are briefly discussed.

Animals↗