The Effect of Buffers on Weak Acid Uptake by Vesicles

The assessment of weak acid membrane permeability (Pm) frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpHin, in response to a sudden augmentation of external acid concentration. However, ΔpHin may be primarily governed by non-instantaneous protonation and deprotonation reactions of (i) the acid itself, (ii) the buffer molecules, and (iii) the fluorescent pH reporter dye. Moreover, buffer concentration and acid gradient also serve as determinants of ΔpHin, as we show here. The uniexponential time constant (τ) of ΔpHin(t) is an invalid measure of Pm as Arrhenius plots of Pm and τ reveal different activation energies for acid influx. We calculate Pm by fitting a mathematical model to experimental stopped-flow traces. The model takes into account not only the time course of total internal buffer capacity but also (i) water self-dissociation, (ii) volume changes due to acid induced osmotic water flow, and (iii) the spontaneous membrane proton leak. It allows extracting a Pm of 30.8±3.5 µms for formic acid for 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles.

Blood Flow Does Not Limit Peritoneal Transport

Objective We investigated the assumption that blood flow to the microvessels underlying the peritoneum does not limit solute or water exchange between the blood and the dialysis fluid. Design Small plastic chambers were affixed to the serosal side of the liver, cecum, stomach, and abdominal wall of anesthetized rats. Solutions that contained labeled solutes or that were made hypertonic were placed into the chambers, which restricted the area of transfer across the tissue to the base of the chamber and which permitted calculation of mass or water transfer rates on the basis of area. The local blood flow was monitored continuously with a laser Doppler flowmeter during three periods of observation: control, after 50% -70% reduction of the blood flow, and postmortem. Results Urea transfer across all serosa, except for the liver, showed no difference in mean mass transfer coefficient (cm/min) between control (0.0038 0.0046) and after 70% flow reduction (0.0037 -0.0040), but demonstrated a significant decrease with blood flow equal to zero (0.0020). These tissues demonstrated small but insignificant decreases in osmotic water flow into the chamber (0.7 -0.9 μL/min/cm2 under control conditions versus 0.4 -0.7 μL/min/cm2 with reduced blood flow). The liver demonstrated limitations in water and solute transport with a 70% decrease in blood flow. Conclusion Because the liver makes up a small part of the peritoneal area, we conclude that large drops in blood flow do not limit overall solute or water transfer across the peritoneum during dialysis, and therefore acute peritoneal dialysis may be an appropriate modality for ICU patients in shock and renal failure.

Osmotic water flow pathways across Necturus gallbladder: role of the tight junction

To explore the quantitative significance of passive water flow through tight junctions of leaky epithelia, transepithelial water flow rates were measured in Necturus gallbladder mounted in chambers. Osmotic flows generated by raffinose gradients were asymmetrical with the greater flow in the mucosal-to-serosal direction. In tissue fixed in situ, intercellular spaces were dilated during mucosal-to-serosal flow and closed during serosal-to-mucosal flow. Tight junctions were focally separated (blistered), which correlated with the magnitude of mucosal-to-serosal flow. Blisters were not observed during serosal-to-mucosal flow or in nontransporting gallbladders. In freeze-fracture replicas, blisters appeared as pockets between intramembranous strands. Protamine, which decreases electrical conductance and increases depth and complexity of the tight junction, reduced osmotic water flow by approximately 30% in the mucosal-to-serosal direction (100 mosmol/kg gradient) without altering serosal-to-mucosal flow. We suggest that in the steady state, at least 30% of osmotically driven water passes transjunctionally in the mucosal-to-serosal direction, but flow is transcellular in the serosal-to-mucosal direction. Directionally divergent pathways may account for flow asymmetry.

Polyclonal antibodies in study of ADH-induced water channels in frog urinary bladder

It is now generally accepted that changes in water permeability in anti-diuretic hormone (ADH)-responsive target epithelial cells result from the insertion in the plasma apical membrane of new components that contain channels for water. The specificity of these channels suggests that they are formed by intrinsic proteins having access to both facies and spanning the whole membrane. We have previously shown that Triton X-100 apical extracts from ADH-stimulated frog urinary bladder contain some proteins inserted under hormonal stimulation. In the present study we have developed polyclonal antibodies using Triton X-100 extract as an immunogen. After considering the inhibitory effect exerted by the whole immune serum on the osmotic water flow, we used different adsorption steps to select, from the immune serum, antibodies to apical membrane proteins inserted in response to the hormone. Immunoblot analysis of these selected antibodies shows that they recognize seven to eight proteins, of which 55-, 35-, 26-, and 17-kDa proteins are always present. Antibodies to these four proteins, affinity purified on nitrocellulose sheets, inhibited ADH-induced osmotic water flow. Altogether these results strongly suggest that proteins of 55, 35, 26, and 17 kDa (or at least one of them) are likely to be involved in the mechanism of water transport.