High channel-mediated water permeability in rabbit erythrocytes: characterization in native cells and expression in Xenopus oocytes.

Erythrocytes from several mammalian species contain mercurial-sensitive water transporters. By a stopped-flow light scattering technique, osmotic water permeability (Pf) was exceptionally high in rabbit erythrocytes (0.053 +/- 0.002 cm/s) and reversibly inhibited by 98% by p-(chloromercuri)benzenesulfonate (pCMBS). The activation energy (Ea) was 4.6 kcal/mol (15-37 degrees C). pCMBS inhibition was half-maximal at 0.1 mM (60-min incubation); at 1 mM pCMBS, half-maximal inhibition occurred in 8 min. Pf was also inhibited by HgCl2 and pCMB with greater than 90% inhibition in 5 min. There was no inhibition by high concentrations of phloretin, DNDS, cytochalasin B, amiloride, ouabain, furosemide, and several proteases. In defolliculated Xenopus oocytes microinjected with 50 nL of water or unfractionated mRNA (1 mg/mL) from rabbit reticulocytes, oocyte Pf assayed at 10 degrees C after 72-h incubation increased from (4 +/- 1) X 10(-4) cm/s (water injected) to (18 +/- 2) X 10(-4) cm/s (mRNA injected). Pf increased linearly with [mRNA] (0-75 ng/oocyte) and was inhibited slowly and reversibly by pCMBS and immediately by HgCl2 but not by cytochalasin B, phloretin, or DNDS. Ea was 9.6 kcal/mol (water injected) and 2.6 kcal/mol (mRNA injected). These results demonstrate that rabbit erythrocytes have the highest Pf and the greatest percentage inhibition of Pf by mercurials of any mammalian erythrocyte studied. The characteristics of the expressed and native water channels were similar, suggesting that the erythrocyte water channel is a membrane protein suitable for expression cloning.     

Reconstitution of a Regulated Transepithelial Water Pathway in Cells Transfected with AQP2 and an AQP1/AQP2 Hybrid Containing the AQP2-C Terminus

Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9 ± 8.2 vs. 12.5 ± 3.3 cm · sec⁻¹· 10⁻³), but not in cells transfected with AQP1 (15.3 ± 3.6 vs. 13.4 ± 3.6 cm · sec⁻¹· 10⁻³). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly higher than in mock cells at rest (18.1 ± 4.8 vs. 6.7 ± 1.0 cm · sec⁻¹· 10⁻³). The increases in Posm values were not paralleled by increases in ¹⁴C-Mannitol permeability. HgCl₂ inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed, in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder). Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient. These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2. Received: 24 June 1997/Revised: 16 September 1997     

An improved temperature-jump apparatus

A temperature-jump device of the type devised by M. Eigen (1954, Discuss, Faraday Soc.17, 194–210) was built using an 8000- to 40,000-V capacitor discharge to raise the temperature of a conductive sample as much as 25°C within 5 μs. The temperature-jump cell design, which differs from that of previously built cells, consists of cylindrical electrodes fitted into a quartz cylindrical observation cell. This allowed for variable electrode spacing and eliminated high-voltage arcing as well as the need to degas samples to prevent solution cavitation. The temperature of a 0.3- to 3.0-ml sample volume could be adjusted over a ?20 to 60°C temperature range by circulating 20% glycerol in water through the hollow, stainless-steel electrodes. Absorption, fluorescence, and scattering measurements could be made over the wavelength range 220 to 750 nm with signal to noise ratios generally exceeding 1000:1. A battery-driven, high-intensity mercury arc lamp provided stable, ripple-free light. The optical signal was amplified, digitized by a waveform recorder, transferred to a computer, and analyzed numerically to resolve multiple-exponential decay processes. The performance of this apparatus is illustrated by several membrane-substrate interactions.     

Acclimation or stress symptom? An integrated study of intraspecific variation in the clonal plant Aechmea bromeliifolia,a widespread CAM tank‐bromeliad

The clonal tank-bromeliad Aechmea bromeliifolia (Rudge) Baker was found in four different habitats in a restinga (vegetation mosaic on sandy coastal plains), of south-eastern Brazil. These habitats (swamp forest, dry forest, dry shrubland and herbaceous marsh) lie within a few hundred metres of each other along a gradient extending inland from the coast, and differ markedly in terms of light and flood regime. We compared ramet morphology, leaf anatomy and physiology, and population parameters to examine the amplitude of trait variation of this widespread species in the studied restinga. This integrated approach allowed us to examine which variation conferred acclimation and which was merely a stress symptom. A . bromeliifolia showed site-specific differences in abundance, distribution, rosette size and shape, leaf anatomical arrangement and photochemical efficiency (potential quantum yield; F _v/ F _m) during the day. Most of the variation found seemed to be related to the interaction of light and flooding. The lowest number and size of ramets at the exposed, dry shrubland was matched by a marked leaf photoinhibition, which suggested poor acclimation to local levels of light intensity and limited water supply. In the other habitats, the morpho-physiological parameters measured suggested adequate foraging behaviour and site acclimation.  © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society, 2002, 140 , 391-401.     

Mechanism of acridine orange interaction with phospholipids and proteins in renal microvillus vesicles

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.