Diffusional Water Permeability Research Articles

Water and salt transport properties of zwitterionic poly (arylene ether ketone) for desalination membrane applications

A series of novel poly (arylene ether ketone) with pendant zwitterion groups were designed. Three molecules, 1,4-butane sultone, sodium 2-bromoethanesulphonate and sodium bromoacetate were selected to quaternize with tertiary amine-containing poly (arylene ether ketone) to prepare sulfobetaine poly (arylene ether ketone) (PAEK-SB4 and PAEK-SB2) and carboxylbetaine poly (arylene ether ketone) (PAEK-CB) with controllable content of zwitterion group. The fundamental water and salt transport properties in zwitterionic PAEKs were characterized. Except PAEK-SB2, the water and salt diffusivity and permeability of PAEK-SB4 and PAEK-CB increase with the increase of water sorption, which follows the trend of Yasuda's free volume theory. Salt diffusivity and permeability in PAEK-SB2 were suppressed relative to PAEK-SB4 and PAEK-CB with comparable water sorption, which is due to shorter distance between positively charged group (-N+(CH3)2-) and negatively one (SO3−) in SB2 group leading to stronger self-association interaction between zwitterions. The water/salt permeability selectivity of PAEK-SB2 and PAEK-CB change little with the increase of diffusive water permeability, which break through the trade-off between them. The permeability selectivity of zwitterionic PAEKs is more than one order of magnitude higher than sulfonated polysulfone (BPS), and its orders is PAEK-SB2>PAEK-CB > PAEK-SB4>BPS at the comparable diffusive water permeability. This study suggests that shorter distance between positively charged group and negatively one in zwitterion group benefits water/salt selectivity of zwitterionic PAEKs.

Urea and water are transported through different pathways in the red blood cell membrane.

Several studies of the urea transporter UT-B expressed in Xenopus oocytes and in genetically modified red blood cells (RBC) have concluded that UT-B also transports water. In the present study, we use unmodified RBC to test that conclusion. We find that the permeability of urea, Pu (cm/s), has a 10-fold donor variation, while the diffusional water permeability, Pd (cm/s), remains unchanged. Additionally, we observe that phloretin inhibits Pu but not Pd, and that the time course of maximum p-chloromercuribenzosulfonate inhibition of Pu and Pd differs-Pu inhibition takes <2 min, whereas Pd inhibition requires ≥1 h of incubation. The findings in the present study are in line with a previous comparative study using unmodified RBC from four animals and a solvent drag study using human RBC, and they lead us to reject the conclusion that the UT-B transporter represents a common pathway for both solutes.

Light and Scanning Electron Microscopy of Red Blood Cells From Humans and Animal Species Providing Insights into Molecular Cell Biology.

We reviewed the many discoveries in cell biology, made since the 17th century, which have been based on red blood cells (RBCs). The advances in molecular and structural biology in the past 40 years have enabled the discovery with these cells, most notably, of the first water channel protein (WCP) called today aquaporin1 (AQP1). The main aim of our work reviewed was to examine by light and electron microscopy a very wide range of RBCs from reptiles, birds, monotremes, marsupials and placentals, in order to estimate from these images the RBC cell volume and surface area. The diffusional water permeability of the RBC membrane from these species has further been measured with a nuclear magnetic resonance (NMR) spectroscopy technique. The significance of the observed permeability of RBCs to water and possible influences on the whole body are discussed.

Microfluidic platform for rapid measurement of transepithelial water transport.

Water transport across epithelial monolayers is of central importance in mammalian fluid homeostasis, and epithelial aquaporin (AQP) water channels are potential drug targets. Current methods to measure transepithelial water permeability based on indicator dilution have limited accuracy and can require hours for a single measurement. We report here a microfluidics platform for rapid and accurate measurement of water transport across a conventionally cultured epithelial monolayer on a porous filter requiring only a single image obtained using a standard laboratory fluorescence microscope. The undersurface of a porous polyester filter containing cultured epithelial cells on top is contacted with a perfused microfluidic channel of 100 μm width, 20 μm height and 10 cm length with folded geometry, with in-plane size of 3.2 × 3.2 mm2 for visualization with a 2× objective lens. Osmotic water permeability is measured from the steady-state concentration profile along the length of the channel of a membrane-impermeant fluorescent dye in the perfusate, in which an osmotic gradient is imposed by an anisosmolar solution overlying the epithelial monolayer; diffusional water permeability is measured using a D2O/H2O-sensing fluorescent dye in the perfusate with a D2O-containing isosmolar solution overlying the cell layer. Permeability values are deduced from single fluorescence images. The method, named fluid transport on a chip (FT-on-Chip), was applied to measure transepithelial osmotic and diffusional water permeability in control and AQP4-expressing epithelial cell monolayers. FT-on-Chip allows for rapid, accurate and repeated measurements of transepithelial water permeability, and is generalizable to transport measurements of ions and solutes using suitable indicator dyes.

Morphology and water permeability of red blood cells from green sea turtle (Chelonia mydas).

The morphology and diffusional water permeability (P d) of red blood cells (RBCs) from green sea turtle (GST) (Chelonia mydas) are presented for the first time. The RBCs had an ellipsoidal shape with full-axis lengths (diameters): D = 14.4μm; d = 10.2μm; h = 2.8μm. The values of P d (cms(-1)) were 5.1 × 10(-3) at 15°C, 5.7 × 10(-3) at 20°C, 6.3 × 10(-3) at 25°C, 6.8 × 10(-3) at 30°C, and 7.9 × 10(-3) at 37°C (i.e., significantly higher than in human RBCs in which it was measured to be 4.2 × 10(-3) at 25°C, 5.0 × 10(-3) at 30°C, and 6.2 × 10(-3) at 37°C). There was a lack of inhibition of P d of GST RBCs by p-chloromercuribenzoate (PCMB), a well-known inhibitor of the RBC water channel proteins (WCPs). The activation energy of water diffusion (E a,d) in GST RBCs was 15.0 ± 1.6kJmol(-1) which is lower than the E a,d for human RBCs (~25kJmol(-1)). These results indicate that in the membrane of GST RBCs, there were no WCPs that were inhibited by the mercurial reagent, while the lipid bilayer of this membrane is unusually permeable to water. This is likely to be a phylogenetically old trait, like that found in amphibians and even the later birds, all of which have nucleated erythrocytes; and it is also likely to be a result of the animal's adaptation to a herbivorous diet (algae and seagrasses).

Direct visualization and quantitative analysis of water diffusion in complex biological tissues using CARS microscopy

To date, it has not been possible to measure microscopic diffusive water movements in epithelia and in the interstitial space of complex tissues and organs. Diffusive water movements are essential for life because they convey physiologically important small molecules, e.g. nutrients and signaling ligands throughout the extracellular space of complex tissues. Here we report the development of a novel method for the direct observation and quantitative analysis of water diffusion dynamics in a biologically organized tissue using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Using a computer simulation model to analyze the CARS O-H bond vibration data during H2O/D2O exchange in a 3D epithelial cyst, we succeeded in measuring the diffusive water permeability of the individual luminal and basolateral water pathways and also their response to hormonal stimulation. Our technique will be applicable to the measurement of diffusive water movements in other structurally complex and medically important tissues and organs.

Protective effects of stem bark of Harungana madgascariensis on the red blood cell membrane

BackgroundAnemia is a condition that has multiple origins. One such origin is the destruction of red blood cells’ (RBCs) membrane induced by free radicals. Treatment of anemia could therefore be enhanced by the use of free radicals’ scavengers potentially found in some medicinal plants. In this study, the protective effect of Harungana madagascariensis on the RBCs’ membrane physiology was investigated in vitro and in vivo.MethodsIn vitro hemolytic anemia was induced by incubation of fresh human RBCs with carbontetrachloride (CCl4) in Olive oil (Oo). Relaxation times of protons excited at 20 MHz (Carr-Purcell-Meiboom-Gill pulse sequence) in the absence or presence of paramagnetic Mn2+ ions (T2i for “extracellular” water and T2a for “intracellular” water, respectively) were determined at several temperatures (25–37°C) via Nuclear Magnetic Resonance (NMR) on a Bruker Minispec spectrometer. Water exchange times (Te) were consequently calculated using the Conlon-Outhred equation: 1/Te = (1/T2a) – (1/T2i). Morphological characteristics (mean cell volume, V, and cell surface area, A) were determined by photonic microscopy and the RBCs’ diffusional water permeability (Pd) was calculated as Pd = (1/Te)*(Va/A), where Va is the aqueous volume in the RBC and is about 0.7 of the cell volume (V). The activation energy of the diffusional process (Ea) for the respective temperature range was estimated using the Arrhenius modified equation k = A(T/T0)n*e-Ea/RT. Inhibition of the water diffusion induced by incubation with para-chloro-mercuribenzoic acid (PCMB) at 25, 30 and 37°C was calculated as I(%) = [(Pd control – Pd sample)/Pd control]*100.To investigate the protective influence of the extract on the RBC membrane, inhibition of the water permeability was evaluated on membranes pre-incubated with the Harungana madagascariensis extract. Male rats were used in in vivo investigations. Malondialdehyde (MDA) and cholesterol in the RBC membrane were estimated by induction of lipid peroxidation while the antioxidant properties of catalase (CAT) and superoxide dismutase (SOD) on the membrane were evaluated in regard to their antioxidant properties on the membrane.ResultsT2a significantly decreased at each temperature. Te results were higher in both RBCs and RBCs + extract groups incubated with PCMB compared to non-incubated controls, but differences were not statistically significant. A high percentage (73.81 ± 7.22) of RBCs pre-incubated with the extract presented the regular biconcave shape. Inhibition by PCMB of the RBCs’ membrane water permeability was increased at 30°C and decreased in the presence of extract (25°C and 37°C), while Ea decreased from 30.52 ± 1.3 KJ/mol to 25.49 ± 1.84 KJ/mol. Presence of the Harungana madagascariensis extract normalized the SOD and CAT activities as well as the MDA and membrane cholesterol concentrations altered by the CCl4-induced oxidative stress.ConclusionHarungana madagascariensis could protect the RBCs’ membrane through its antioxidative properties.

The permeability of red blood cells to chloride, urea, and water

This study extends permeability (P) data on chloride, urea and water in red blood cells (RBC), and concludes that the urea transporter (UT-B) does not transport water. P of chick, duck, Amphiuma means, dog and human RBC to (36)Cl(-), (14)C-urea and (3)H2O was determined under self-exchange conditions. At 25°C and pH 7.2-7.5, PCl is 0.94 × 10(-4)-2.15 × 10(-4) cm s(-1) for all RBC species at [Cl]=127-150 mmol l(-1). In chick and duck RBC, P(urea) is 0.84 × 10(-6) and 1.65 × 10(-6) cm s(-1), respectively, at [urea]=1-500 mmol l(-1). In Amphiuma, dog and human RBC, P(urea) is concentration dependent (1-1000 mmol l(-1), Michaelis-Menten-like kinetics; K1/2;=127, 173 and 345 mmol l(-1)), and values at [urea]=1 mmol l(-1) are 29.5 × 10(-6), 467 × 10(-6) and 260 × 10(-6) cm s(-1), respectively. Diffusional water permeability, Pd, was 0.84 × 10(-3) (chick), 5.95 × 10(-3) (duck), 0.39 × 10(-3) (Amphiuma), 3.13 × 10(-3) (dog) and 2.35 × 10(-3) cm s(-1) (human). DIDS, DNDS and phloretin inhibit PCl by >99% in all RBC species. PCMBS, PCMB and phloretin inhibit P(urea) by >99% in Amphiuma, dog and human RBC, but not in chick and duck RBC. PCMBS and PCMB inhibit Pd in duck, dog and human RBC, but not in chick and Amphiuma RBC. Temperature dependence, as measured by apparent activation energy, EA, of PCl is 117.8 (duck), 74.9 (Amphiuma) and 89.6 kJ mol(-1) (dog). The EA of P(urea) is 69.6 (duck) and 53.3 kJ mol(-1) (Amphiuma), and that of Pd is 34.9 (duck) and 32.1 kJ mol(-1) (Amphiuma). The present and previous RBC studies indicate that anion (AE1), urea (UT-B) and water (AQP1) transporters only transport chloride (all species), water (duck, dog, human) and urea (Amphiuma, dog, human), respectively. Water does not share UT-B with urea, and the solute transport is not coupled under physiological conditions.

Aquaporin Expression Contributes to Human Transurothelial Permeability In Vitro and Is Modulated by NaCl

It is generally considered that the bladder is impervious and stores urine in unmodified form on account of the barrier imposed by the highly-specialised uro-epithelial lining. However, recent evidence, including demonstration of aquaporin (AQP) expression by human urothelium, suggests that urothelium may be able to modify urine content. Here we have we applied functional assays to an in vitro-differentiated normal human urothelial cell culture system and examined both whether AQP expression was responsive to changes in osmolality, and the effects of blocking AQP channels on water and urea transport. AQP3 expression was up-regulated by increased osmolality, but only in response to NaCl. A small but similar effect was seen with AQP9, but not AQP4 or AQP7. Differentiated urothelium revealed significant barrier function (mean TER 3862 Ω.cm2), with mean diffusive water and urea permeability coefficients of 6.33×10−5 and 2.45×10−5 cm/s, respectively. AQP blockade with mercuric chloride resulted in decreased water and urea flux. The diffusive permeability of urothelial cell sheets remained constant following conditioning in hyperosmotic NaCl, but there was a significant increase in water and urea flux across an osmotic gradient. Taken collectively with evidence emerging from studies in other species, our results support an active role for human urothelium in sensing and responding to hypertonic salt concentrations through alterations in AQP protein expression, with AQP channels providing a mechanism for modifying urine composition. These observations challenge the traditional concept of an impermeable bladder epithelium and suggest that the urothelium may play a modulatory role in water and salt homeostasis.

Roles of AQP5/AQP5-G103D in carbamylcholine-induced volume decrease and in reduction of the activation energy for water transport by rat parotid acinar cells

In order to assess the contribution of the water channel aquaporin-5 (AQP5) to water transport by salivary gland acinar cells, we measured the cell volume and activation energy (E (a)) of diffusive water permeability in isolated parotid acinar cells obtained from AQP5-G103D mutant and their wild-type rats. Immunohistochemistry showed that there was no change induced by carbamylcholine (CCh; 1 μM) in the AQP5 detected in the acinar cells in the wild-type rat. Acinar cells from mutant rats, producing low levels of AQP5 in the apical membrane, showed a minimal increase in the AQP5 due to the CCh. In the wild-type rat, CCh caused a transient swelling of the acinus, followed by a rapid agonist-induced cell shrinkage, reaching a plateau at 30 s. In the mutant rat, the acinus did not swell by CCh challenge, and the agonist-induced cell shrinkage was delayed by 8 s, reaching a transient minimum at around 1 min, and recovered spontaneously even though CCh was persistently present. In the unstimulated wild-type acinar cells, E (a) was 3.4 ± 0.6 kcal mol(-1) and showed no detectable change after CCh stimulation. In the unstimulated mutant acinar cells, high E (a) value (5.9 ± 0.1 kcal mol(-1)) was detected and showed a minimal decrease after CCh stimulation (5.0 ± 0.3 kcal mol(-1)). These results suggested that AQP5 was the main pathway for water transport in the acinar cells and that it was responsible for the rapid agonist-induced acinar cell shrinkage and also necessary to keep the acinar cell volume reduced during the steady secretion in the wild-type rat.

Dependences of Water Permeation through Cyclic Octa-peptide Nanotubes on Channel Length and Membrane Thickness

Effects of the channel length and membrane thickness on the water permeation through the transmembrane cyclic octa-peptide nanotubes (octa-PNTs) have been studied by molecular dynamics (MD) simulations. The water osmotic permeability (p(f)) through the PNTs of k × (WL)(4)/POPE (1-palmitoyl-2-oleoyl-glycerophosphoethanolamine; k = 6, 7, 8, 9, and 10) was found to decay with the channel length (L) along the axis (~L(-2.0)). Energetic analysis showed that a series of water binding sites exist in these transmembrane PNTs, with the barriers of ~3k(B)T, which elucidates the tendency of p(f) well. Water diffusion permeability (p(d)) exhibits a relationship of ~L(-1.8), which results from the novel 1-2-1-2 structure of water chain in such confined nanolumens. In the range of simulation accuracy, the ratio (p(f)/p(d)) of the water osmotic and diffusion permeability is approximately a constant. MD simulations of water permeation through the transmembrane PNTs of 8 × (WL)(4)/octane with the different octane membrane thickness revealed that the water osmotic and diffusion permeability (p(f) and p(d)) are both independent of the octane membrane thickness, confirmed by the weak and nearly same interactions between the channel water and octane membranes with the different thickness. The results may be helpful for revealing the permeation mechanisms of biological water channels and designing artificial nanochannels.

Population Shift between the Open and Closed States Changes the Water Permeability of an Aquaporin Z Mutant

Aquaporins are tetrameric transmembrane channels permeable to water and other small solutes. Wild-type (WT) and mutant Aquaporin Z (AqpZ) have been widely studied and multiple factors have been found to affect their water permeability. In this study, molecular dynamics simulations have been performed for the tetrameric AqpZ F43W/H174G/T183F mutant. It displayed ∼10% average water permeability compared to WT AqpZ, which had been attributed to the increased channel lumen hydrophobicity. Our simulations, however, show a ring stacking between W43 and F183 acting as a secondary steric gate in the triple mutant with R189 as the primary steric gate in both mutant and WT AqpZ. The double gates (R189 and W43-F183) result in a high population of the closed conformation in the mutant. Occasionally an open state, with diffusive water permeability very close to that of WT AqpZ, was observed. Taken together, our results show that the double-gate mechanism is sufficient to explain the reduced water permeability in the mutant without invoking effects arising from increased hydrophobicity of the channel lumen. Our findings provide insights into how aquaporin-mediated water transport can be modulated and may further point to how aquaporin function can be optimized for biomimetic membrane applications.

Analysis of Aquaporin-Mediated Diffusional Water Permeability by Coherent Anti-Stokes Raman Scattering Microscopy

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (Pf), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (Pd). Here, we report an accurate and instantaneous method for measuring the Pd of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H2O/D2O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τCARS) for the external solution H2O/D2O exchange was 16.1 ms, whereas the intracellular H2O/D2O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τCARS for the intracellular H2O/D2O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate Pd. The average Pd values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10−3 and 8.3 ± 2.6 × 10−4 cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.

Comparative Permeabilities of the Paracellular and Transcellular Pathways of Corneal Endothelial Layers

Layers of rabbit corneal endothelial cells were cultured on permeable inserts. We characterized the diffusional permeability of the cell layer to nonelectrolyte and charged molecules and compared the diffusional and filtration permeabilities of the paracellular and transcellular pathways. We determined the rates of diffusion of (3)H- and (14)C-labeled nonelectrolyte test molecules and estimated the equivalent pore radius of the tight junction. Negatively charged molecules permeate slower than neutral molecules, while positively charged molecules permeate faster. Palmitoyl-DL-carnitine, which opens tight junctions, caused an increase of permeability and equivalent pore radius. Diffusional water permeability was determined with (3)H-labeled water; the permeabilities of the tight junction and lateral intercellular space were calculated using tissue geometry and the Renkin equation. The diffusional permeability (P(d)) of the paracellular pathway to water is 0.57 μm s(-1) and that of the transcellular path is 2.52 μm s(-1). From the P(d) data we calculated the filtration permeabilities (P(f)) for the paracellular and transcellular pathways as 41.3 and 30.2 μm s(-1), respectively. In conclusion, the movement of hydrophilic molecules through tight junctions corresponds to diffusion through negatively charged pores (r = 2.1 ± 0.35 nm). The paracellular water permeability represents 58% of the filtration permeability of the layer, which points to that route as the site of sizable water transport. In addition, we calculated for NaCl a reflection coefficient of 0.16 ≤ σ(NaCl) ≤ 0.33, which militates against osmosis through the junctions and, hence, indirectly supports the electro-osmosis hypothesis.

Aquaporins at a glance

With a concentration of 55,000 mM, water is by far the most prevalent molecule in biological systems. For many years it was assumed that biological membranes are freely water permeable, so that the existence of a family of water channels would not have been predicted. However, biophysical studies in

Influence of sodium nitroprusside on human erythrocyte membrane water permeability: an NMR study

Sodium nitroprusside (SNP) is a nitric oxide (•NO) donor in vitro and in vivo. In this paper the time variation of the intracellular water proton nuclear magnetic resonance (NMR) effective relaxation time T'(2a) in SNP-treated human erythrocyte suspensions, containing 10 mM membrane impermeable paramagnetic MnCl2, has been measured. The observed T'(2a) time-course was analyzed in terms of the two mechanisms by which released •NO affects T'(2a). These are, respectively, enhancement of the intracellular water proton intrinsic NMR relaxation rate 1/T(2a) by paramagnetism of •NO subsequently bonded to iron atoms of intracellular deoxyhemoglobin, and suppression of diffusional water permeability P(d) as a consequence of nitrosylation of aquaporin-1 (AQP1) channel Cys189, either by direct reaction with •NO or with one of the •NO oxidation products, such as N2O3. The bound •NO on the Cys189 thiol residue appears to impose a less efficient barrier to water permeation through AQP1 than the larger carboxyphenylmercuryl residue from p-chloromercuribenzoate. The effect of •NO on P(d) is discussed in terms of NO-induced vasodilation.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVIII platypus (Ornithorhynchus anatinus) and saltwater crocodile (Crocodylus porosus)

As part of a programme of comparative measurements of Pd (diffusional water permeability) the RBCs (red blood cells) from an aquatic monotreme, platypus (Ornithorhynchus anatinus), and an aquatic reptile, saltwater crocodile (Crocodylus porosus) were studied. The mean diameter of platypus RBCs was estimated by light microscopy and found to be approximately 6.3 microm. Pd was measured by using an Mn2+-doping 1H NMR (nuclear magnetic resonance) technique. The Pd (cm/s) values were relatively low: approximately 2.1 x 10(-3) at 25 degrees C, 2.5 x 10(-3) at 30 degrees C, 3.4 x 10(-3) at 37 degrees C and 4.5 at 42 degrees C for the platypus RBCs and approximately 2.8 x 10(-3) at 25 degrees C, 3.2 x 10(-3) at 30 degrees C, 4.5 x 10(-3) at 37 degrees C and 5.7 x 10(-3) at 42 degrees C for the crocodile RBCs. In parallel with the low water permeability, the Ea,d (activation energy of water diffusion) was relatively high, approximately 35 kJ/mol. These results suggest that "conventional" WCPs (water channel proteins), or AQPs (aquaporins), are probably absent from the plasma membranes of RBCs from both the platypus and the saltwater crocodile.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVI Dingo (Canis familiaris dingo) and dog (Canis familiaris)

As part of a programme of comparative measurements of Pd (diffusional water permeability) the RBCs (red blood cells) from dingo (Canis familiaris dingo) and greyhound dog (Canis familiaris) were studied. The morphologies of the dingo and greyhound RBCs [examined by light and SEM (scanning electron microscopy)] were found to be very similar, with regard to aspect ratio and size; the mean diameters were estimated to be the same (approximately 7.2 microm) for both dingo and greyhound RBCs. The water diffusional permeability was monitored by using an Mn2+-doping 1H NMR technique at 400 MHz. The Pd (cm/s) values of dingo and greyhound RBCs were similar: 6.5 x 10(-3) at 25 degrees C, 7.5 x 10(-3) at 30 degrees C, 10 x 10(-3) at 37 degrees C and 11.5 x 10(-3) at 42 degrees C. The inhibitory effect of a mercury-containing SH (sulfhydryl)-modifying reagent PCMBS (p-chloromercuribenzene sulfonate) was investigated. The maximal inhibition of dingo and greyhound RBCs was reached in 15-30 min at 37 degrees C with 2 mmol/l PCMBS. The values of maximal inhibition were in the range 72-74% when measured at 25 degrees C and 30 degrees C, and approximately 66% at 37 degrees C. The lowest value of Pd (corresponding to the basal permeability to water) was approximately 2-3 x 10(-3) cm/s in the temperature range 25-37 degrees C. The Ea,d (activation energy of water diffusion) was 25 kJ/mol for dingo RBC and 23 kJ/mol for greyhound RBCs. After incubation with PCMBS, the values of Ea,d increased, reaching 46-48 kJ/mol in the condition of maximal inhibition of water exchange. The electrophoretograms of membrane polypeptides of the dingo and greyhound RBCs were compared and seen to be very similar. We postulate that the RBC parameters reported in the present study are characteristic of all canine species and, in particular in the two cases presented here, these parameters have not been changed by the peculiar Australian habitat over the millennia (as in the case of the dingo) or over shorter time periods, decades or centuries (as in the case of the domestic greyhound).

Regulation of gill transcellular permeability and renal function during acute hypoxia in the Amazonian oscar (Astronotus ocellatus): new angles to the osmorespiratory compromise

Earlier studies demonstrated that oscars, endemic to ion-poor Amazonian waters, are extremely hypoxia tolerant, and exhibit a marked reduction in active unidirectional Na(+) uptake rate (measured directly) but unchanged net Na(+) balance during acute exposure to low P(O(2)), indicating a comparable reduction in whole body Na(+) efflux rate. However, branchial O(2) transfer factor does not fall. The present study focused on the nature of the efflux reduction in the face of maintained gill O(2) permeability. Direct measurements of (22)Na appearance in the water from bladder-catheterized fish confirmed a rapid 55% fall in unidirectional Na(+) efflux rate across the gills upon acute exposure to hypoxia (P(O(2))=10-20 torr; 1 torr=133.3 Pa), which was quickly reversed upon return to normoxia. An exchange diffusion mechanism for Na(+) is not present, so the reduction in efflux was not directly linked to the reduction in Na(+) influx. A quickly developing bradycardia occurred during hypoxia. Transepithelial potential, which was sensitive to water [Ca(2+)], became markedly less negative during hypoxia and was restored upon return to normoxia. Ammonia excretion, net K(+) loss rates, and (3)H(2)O exchange rates (diffusive water efflux rates) across the gills fell by 55-75% during hypoxia, with recovery during normoxia. Osmotic permeability to water also declined, but the fall (30%) was less than that in diffusive water permeability (70%). In total, these observations indicate a reduction in gill transcellular permeability during hypoxia, a conclusion supported by unchanged branchial efflux rates of the paracellular marker [(3)H]PEG-4000 during hypoxia and normoxic recovery. At the kidney, glomerular filtration rate, urine flow rate, and tubular Na(+) reabsorption rate fell in parallel by 70% during hypoxia, facilitating additional reductions in costs and in urinary Na(+), K(+) and ammonia excretion rates. Scanning electron microscopy of the gill epithelium revealed no remodelling at a macro-level, but pronounced changes in surface morphology. Under normoxia, mitochondria-rich cells were exposed only through small apical crypts, and these decreased in number by 47% and in individual area by 65% during 3 h hypoxia. We suggest that a rapid closure of transcellular channels, perhaps effected by pavement cell coverage of the crypts, allows conservation of ions and reduction of ionoregulatory costs without compromise of O(2) exchange capacity during acute hypoxia, a response very different from the traditional osmorespiratory compromise.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XV. Agile wallaby ( Macropus agilis), red-necked wallaby ( Macropus rufogriseus) and Goodfellow's tree kangaroo ( Dendrolagus goodfellowi)

The water diffusional permeability ( P d) of red blood cells (RBC) from agile wallaby ( Macropus agilis), red-necked wallaby ( Macropus rufogriseus) and Goodfellow's tree kangaroo ( Dendrolagus goodfellowi) was monitored using an Mn 2+-doping 1H nuclear magnetic resonance (NMR) technique at 400 MHz. The P d (cm s − 1 ) values of agile wallaby RBCs were 7.5 × 10 − 3 at 25 °C, 9 × 10 − 3 at 30 °C, 11 × 10 − 3 at 37 °C, and 13 × 10 − 3 at 42 °C. The inhibitory effect of a mercury-containing sulfhydryl (SH)-modifying reagent p-chloromercuribenzoate (PCMB) on agile wallaby RBCs was investigated. The maximal inhibition was reached in 90 min at 37 °C with 2 mmol L − 1 PCMB. The value of maximal inhibition was ~ 63% when measured at 25 °C, ~ 52% at 37 °C and ~ 45% at 42 °C. The lowest value of P d (corresponding to the basal permeability to water) was ~ 3 × 10 − 3 cm s − 1 at 25 °C. For the RBCs from red-necked wallaby ( M. rufogriseus) the values of P d (cm s − 1 ) were 7 × 10 − 3 at 25 °C, 8 × 10 − 3 at 30 °C, 10 × 10 − 3 at 37 °C, and 12 × 10 − 3 at 42 °C. Higher values of P d (cm s − 1 ) were found for the RBCs from Goodfellow's tree kangaroo ( D. goodfellowi): 8.5 × 10 − 3 at 25 °C, 10 × 10 − 3 at 30 °C, 13 × 10 − 3 at 37 °C, and 15 × 10 − 3 at 42 °C. The mean values of the activation energy of water diffusion ( E a,d) were ~ 25 kJ mol − 1 for RBCs from the agile wallaby and tree kangaroo, respectively, and ~ 23 kJ mol − 1 for RBCs from red-necked wallaby. The values of E a,d increased after exposure of agile wallaby RBCs to PCMB, reaching a value of ~ 43–46 kJ mol − 1 when the maximal inhibition of P d was achieved.