The diffusional and osmotic water permeability of the hindgut cuticle in larval Sarcophaga bullata

The diffusional water permeability (P_D) and the pressure filtration coefficient (L_P) of isolated larval hindgut cuticle of the fleshfly, Sarcophaga bullata, were measured using tracer techniques coupled with a simple mathematical model system based on equations of non-equilibrium thermodynamics. Data obtained from the model system were matched to experimental tracer data by means of a mathematical optimization scheme. The following parameter values were obtained: P_D for tritiated water = 1.02 × 10^?6 cm-sec^?1, and L_P = 9.18 × 10^?11 cm³-dyn^?1-sec^?1. These results are now being used to determine reflection coefficients (σ's) and solute mobilities (ω's) for the cuticle system in an attempt to gain an understanding of the mechanisms controlling solute and water movements across the hindgut wall.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V—Rabbit (Oryctolagus Cuniculus)

• 1.1. The diffusional water permeability (P_d) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS).
• 2.2. The values of P_d were around 6.3 × 10⁻³ cm/sec at 15°C, 7.0 × 10⁻³cm/sec at 20°C, 8.0 × 10⁻³ cm/sec at 25°C, 9.1 × 10⁻³ cm/sec at 30°C and10.7 × 10⁻³ cm/sec at 37°C.
• 3.3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37°C with 0.5 mM PCMBS.
• 4.4. The values of maximal inhibition were around 71–74% at all temperatures.
• 5.5. The basal permeability to water was estimated as 1.6 × 10⁻³cm/sec at 15°C, 2.0 × 10⁻³cm/sec at 20°C, 2.4 × 10⁻³cm/sec at 25°C, 2.6 × 10⁻³cm/sec at 30°C, and 3.1× 10^{−3 cm/secat 37°C}.
• 6.6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.
• 7.7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart.
• 8.8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.
• 9.9. Considerable differences in the electrophoretic patterns of the two sources of RBC membranes appeared in the bands migrating in the band 4.5 region and in front of band 7, where some polypeptides were apparent in higher amounts in the rabbit RBC membrane.

     

Comparative studies of water permeability of red blood cells from humans and over 30 animal species: an overview of 20 years of collaboration with Philip Kuchel

NMR measurements of the diffusional permeability of the human adult red blood cell (RBC) membrane to water (P _d) and of the activation energy (E _a,d) of the process furnished values of P _d ~ 4 × 10^?3 cm/s at 25 °C and ~6.1 × 10^?3 cm/s at 37 °C, and E _a,d ~ 26 kJ/mol. Comparative NMR measurements for other species showed: (1) monotremes (echidna and platypus), chicken, little penguin, and saltwater crocodile have the lowest P _d values; (2) sheep, cow, and elephant have P _d values lower than human P _d values; (3) cat, horse, alpaca, and camel have P _d values close to those of humans; (4) guinea pig, dog, dingo, agile wallaby, red-necked wallaby, Eastern grey kangaroo, and red kangaroo have P _d values higher than those of humans; (5) mouse, rat, rabbit, and “small and medium size” marsupials have the highest values of P _d (>8.0 × 10^?3 cm/s at 25 °C and >10.0 × 10^?3 cm/s at 37 °C). There are peculiarities of E _a,d values for the RBCs from different species. The maximum inhibition of diffusional permeability of RBCs induced by incubation with p-chloromercuribenzene sulfonate varied between 0 % (for the chicken and little penguin) to ~50 % (for human, mouse, cat, sheep, horse, camel, and Indian elephant), and ~60–75 % (for rat, guinea pig, rabbit, dog, alpaca, and all marsupials). These results indicate that no water channel proteins (WCPs) or aquaporins are present in the membrane of RBCs from monotremes (echidna, platypus), chicken, little penguin and saltwater crocodile whereas WCPs from the membranes of RBCs from marsupials have peculiarities.     

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 P _d (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 ～6.3 μm. P _d was measured by using an Mn²⁺‐doping 1H NMR (nuclear magnetic resonance) technique. The P _d (cm/s) values were relatively low: ～2.1×10^?3 at 25°C, 2.5×10^?3 at 30°C, 3.4×10^?3 at 37°C and 4.5 at 42°C for the platypus RBCs and ～2.8×10^?3 at 25°C, 3.2×10^?3 at 30°C, 4.5×10^?3 at 37°C and 5.7×10^?3 at 42°C for the crocodile RBCs. In parallel with the low water permeability, the E _a,d (activation energy of water diffusion) was relatively high, ～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.     

Osmotic permeability of Novikoff hepatoma cells

The osmotic permeability coefficient (P_f) for water movement across Novikoff hepatoma cells was found to be 82 ± 3 (S.E.) · 10^?5 cm · s^?1 at 20°C. The corresponding diffusional permeability coefficient for ³HHO (P_d) was 97 ± 10 (S.E.) · 10^?5 cm · s^?1, therefore the ratio $\text{P}{}_{\mathrm{<mtext>f</mtext>}}\text{P}{}_{\mathrm{<mtext>d</mtext>}}$ is close to unity. The apparent activation energy for water filtration was 10.4 ± 0.4 (S.E.) kcal · mol^?1. This value is significantly greater than the activation energy for the self diffusion of water. The product of the hydraulic permeability coefficient and the viscosity coefficient for water was temperature-dependent. However, the product of the hydraulic permeability coefficient and the viscosity coefficient for membrane lipid did not vary with temperature. These data are interpreted as evidence for water movement across a lipid membrane barrier rather than through aqueous channels.     

Water permeability of thin lipid membranes

The osmotic permeability coefficient, P_f, and the tagged water permeability coefficient, P_d, were determined for thin (<100 A) lipid membranes formed from ox brain lipids plus DL-α-tocopherol; their value of approximately 1 x 10^-3 cm/sec is within the range reported for plasma membranes. It was established that P_f = P_d. Other reports that P_f > P_d can be attributed to the presence of unstirred layers in the experimental determination of P_d. Thus, there is no evidence for the existence of aqueous pores in these thin phospholipid membranes. The adsorption onto the membrane of a protein that lowers its electrical resistance by a factor of 10³ was found not to affect its water permeability; however, glucose and sucrose were found to interact with the membrane to modify P_f. Possible mechanisms of water transport across these films are discussed, together with the implications of data obtained on these structures for plasma membranes.     

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 P _d (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 (～7.2 μm) for both dingo and greyhound RBCs. The water diffusional permeability was monitored by using an Mn²⁺‐doping ¹H NMR technique at 400 MHz. The P _d (cm/s) values of dingo and greyhound RBCs were similar: 6.5×10^?3 at 25°C, 7.5×10^?3 at 30°C, 10×10^?3 at 37°C and 11.5×10^?3 at 42°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°C with 2 mmol/l PCMBS. The values of maximal inhibition were in the range 72–74% when measured at 25°C and 30°C, and ～66% at 37°C. The lowest value of P _d (corresponding to the basal permeability to water) was ～2–3×10^?3 cm/s in the temperature range 25–37°C. The E _a,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 E _a,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).     

Investigation of Need of Natural Bioenhancer for a Metabolism Susceptible Drug—Raloxifene,in a Designed Self-Emulsifying Drug Delivery System

Bioenhancers can increase the bioavailability of metabolism susceptible drugs. The present study was designed to understand the impact of bioenhancer on permeability and bioavailability of a biopharmaceutical drug disposition classification system (BDDCS) class II drug raloxifene (RLX). RLX undergoes extensive first pass metabolism by UGT enzymes in gastrointestinal tract (GIT) and has an oral bioavailability of about 2%. Self-emulsifying drug delivery system (SEDDS) of RLX was developed using a designed approach and this formulation was loaded with reported bioenhancers: quercetin and piperine. These formulations were tested for improvement in permeability and bioavailability of the RLX. The apparent permeability using everted gut sac (P _app) for SEDDS (5.26?±?1.10?×?10^?8 cm/s) was found to be similar to that of SEDDS with bioenhancers (5.11?±?1.05?×?10^?8 cm/s). In oral bioavailability study in rat, SEDDS demonstrated a 4-fold and 2.5-fold higher AUC_0–∞ than RLX suspension (control) and marketed product, respectively. No additional improvement in permeability and bioavailability was offered by inclusion of piperine and quercetin (bioenhancers) in the SEDDS.     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

Water channels in membranes

A systematic programme of comparative nuclear magnetic resonance measurements of the membrane permeability for water (Pd) and of activation energy (E_a,d) of this process in red blood cells of various wild, laboratory and domestic animals was carried out here. The RBC from humans, cow, sheep and kangaroos had P_d values around 5·10^?3 cm/s at 25 °C, 7 · 10^?3 cm/s at 37 °C with E_a,d values around 25 kJ/mol. For RBC from other ten marsupial species and from mouse, rat and rabbit, the P_d values were more than twice as high as for human RBC. For mosr RBC a high value of Pd was associated with a low value of E_a,d (range from 15 to 21 kJ/mol), pointing to specialized channels for water diffusion incorporated in membrane proteins. Recently a channel-forming integral protein of 28 kDa (CHIP 28) was identified as a major water channel protein in the RBC membrane. A procedure for quantitating the purified CHIP 28 by densitometry of silver-stained polyacrylmide gel electrophoreograms was developed. The analysis of a purified fraction of CHIP 28 showed that the 28 kDa component represents approximately two-thirds of the sample with the remainder comprising the glycosylated high-molecular-weight component. A correlation between the content in CHIP 28 and the relative water permeability among RBC from different vertebrate species was attempted.     

The permeability of the membranes of experimental secondary cysts of Echinococcus granulosus to [14C]mebendazole

The permeability of secondary E. granulosus cysts to [¹⁴C]mebendazole was studied. The cysts were obtained by transplanting secondary cysts raised in mice into rats. The permeability to [¹⁴C]mebendazole was established by two different experiments: uptake and washout of the drug. The cyst wall permeability to [¹⁴C]mebendazole was found to be 1·33 × 10^?4 cm s^?1, which is of the same order as the diffusion permeability coefficient to water (1·88 × 10^?4 cm s^?1, Rotunno, Kammerer, Perez Esandi & Cereijido, 1974).The drug readily permeates through the cyst wall and experimental data suggest that it moves across the barrier by simple diffusion.     

Cation-dependent light-induced structural changes in visual receptor membranes

A stearamide spin probe was used to study the light-induced structural changes in Rod Outer Segment Membranes in the presence of sodium and calcium ions. The correlation time (τ_C) for the reorientation of the probe was calculated in the dark and light. In the presence of sodium ions, τ_C = 3.3 × 10^?9 sec in the dark, and 2.7 × 10^?9sec in the light while the opposite was noticed in the presence of calcium ions, τ_C = 2.9 × 10^?9 sec in the dark and 3.6 × 10^?9 sec in the light. The correlation times for reorientation of the probe were also calculated in aqueous glycerol solutions of varying viscosities at 20°C. Comparison of the values of τ_C (dark and light) suggests a change in local mobility in the ROS corresponding to a macroscopic viscosity difference of approximately 150 cp. The significance of calcium ion interaction with negatively charged groups and the formation of a Schiff base is emphasized.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

Electron paramagnetic resonance spectroscopy of spin-labelled cuticle of Centruroides sculpturatus (Scorpiones: Buthidae): Correlation with thermal effects on cuticular permeability

Cuticular phase transitions and molecular dynamics have been studied in the buthid scorpion Centruroides sculpturatus using the techniques of thermogravimetric analysis (TGA) and electron paramagnetic resonance (EPR). TGA studies of the cuticular permeability reveal discontinuous changes in thermal dependence of transcuticular water loss rates (transitions). The first transition occurs between 30–40 C and results in a small increase in water loss rate, whereas the second transition begins near 55 C and is accompanied by a large increase in water loss rate. EPR spectra of spin-labelled cuticle indicate that the epicuticular lipids are very mobile at ambient temperature, with the translational diffusion coefficient being about 5 × 10^?6 cm² sec at 22 C. and that the low-temperature transition is associated with an increase in mobility of the hydrocarbon chains of the epicuticular lipids. The high-temperature transition probably results from melting of the epicuticular lipids. The results of this study are discussed with reference to current models of the structure of the arthropod cuticle.     

KINETICS OF AMINO ACID INFLUX INTO NITELLA FLEXILIS1

The uptake of amino acids by Nitella flexilis has been investigated. Influx of glycine, alanine, and valine appears to be a diffusive process. Influx ranged from 0.14 to 0.06 and 0.04 pmoles/(cm)(sec), respectively. Aspartic acid uptake is an active transport mechanism. The V_max is 2.8 pmoles/(cm)(sec); the transport constant (Michaelis constant) K_m, 7.8 × 10^?3 M. The uptake of arginine is apparently due to 2 transport systems, one with a V_max and K_m of 3.1 pmoles/(cm)(sec) and 3.2 × 10^?3M, respectively. The second system has a V_max of 1.4 pmoles/(cm)(sec) and a K_m of 2.1 × 10^?4 M. The possibility that the second system is diffusive has been considered.     

Cerebral microvessels contain a beta 2-adrenergic receptor

Cerebral microvessels isolated from cat forebrain contain a specific β-adrenergic-sensitive adenylate cyclase. Among various compounds tested, the most potent activator of enzyme activity is isoproterenol (k_a = 1.4 × 10^?7M), followed in order by epinephrine (k_a= 1.5 × 10^?6M), norepinephrine (k_a= 1.4 × 10^?5M) and phenylephrine (k_a> 3 × 10^?4M). Isoproterenol-stimulated enzyme activity is blocked by propranolol (k_i= 2.4 × 10^?9M, IPS 339 (k_i= 4 × 10^?9M), H35/25 (k_i = 1.2 × 10^?7M), atenolol (k_i= 5.9 × 10^?6M) and practolol (k_i= 1.8 × 10^?5M). These agonist and antagonist properties are quite similar to those demonstrated by β₂-adrenergic receptors and β₂-stimulated adenylate cyclase present in other tissues and indicate that the majority of adenylate cyclase-associated adrenergic receptors in cerebral microvessels are β₂. The findings are relevant to physiological studies of cerebral blood flow and vascular permeability.     

Probiotic Effect of <Emphasis Type="Italic">Bacillus licheniformis fb11</Emphasis> on the Digestive Efficiency and Growth Performance in Juvenile <Emphasis Type="Italic">Chitala chitala</Emphasis> (Hamilton, 1822)

Five isocaloric (430 kcal 100 g^?1), isonitrogenous (40% CP) experimental diets were formulated with different concentrations of Bacillus licheniformis fb11 probionts (isolated from the gut of Chitala chitala) viz. Control (without probionts), 5 × 10⁴ CFU g^?1 (D₁), 5 × 10⁵ CFU g^?1 (D₂), 5 × 10⁶ CFU g^?1 (D₃), 5 × 10⁷ CFU g^?1 (D₄), 5 × 10⁸ CFU g^?1 (D₅) to evaluate its efficiency in C. chitala juvenile. The best growth performance, feed utilisation, specific α-amylase, total protease and lipase activity were observed with the diet D₃ (P < 0.05). The lowest Presumptive Pseudomonas Count, Motile Aeromonad Count, Total Coliform Count was observed for D₃ (P < 0.05) on 90th day of trial. Two uppermost values were achieved in case of crude protein for D₃ and D₂ (P > 0.05). The highest lipid content (12.12 ± 0.4 g 100 g^?1) was found for D₅ (P < 0.05). The highest gross energy (18.75 ± 0.21 MJ 100 g^?1) of carcass was recorded for D₃. Thus B. licheniformis fb11 at the concentration 5 × 10⁶ CFU g^?1 as probiotic supplement promoted growth, digestion in C. chitala juvenile significantly by modulating intestinal microflora.     

An improved water content model for Periplaneta cuticle: Effects of epidermis removal and cuticle damage

All previously reported integumental permeabilities to water for Periplaneta at 20°C have been shown to be overestimates by at least an order of magnitude. Cuticle damage, apparently due to mechanical contact, is a common occurrence in cockroach cultures. Damage is increased by handling during experimental manipulation. Repair of the damage is readily observed in isolated individuals as cuticle permeability declines over a few days. Repaired cuticle permeability is not humidity dependent, but damage affecting a few per cent of the cuticle area, renders it so. Cuticle-water contents are slightly influenced by sex related size differences. Cuticle-water contents are unaffected by variations in ambient activity. Epidermal removal initiates a drop in cuticle-water content, apparently not involving active processes. Cuticle-water contents can be accurately predicted in any ambient vapour pressure using a two-layered model combining equilibrium water contents and component permeabilities. Incorporating a parallel unprotected endocuticle pathway into the model successfully simulates the observed humidity dependence of damaged permeabilities. Epidermal permeability cannot be less than 2× 10⁻⁵ cm.s⁻¹; 2000 times more permeable than the overlying cuticle.