Contribution of ionic channels in dehydration-elicited rehydration: lysis curves and osmotic fragility data acquisition.
Osmotic fragility curves (OFCs) are extensively used to characterize the hydration state of RBCs in normal, pathological and experimental conditions (Lew et al. 1995; Raftos et al. 1996; Lew et al. 2003; Lew et al. 2005; Tiffert et al. 2005; Baunbaek and Bennekou 2008). With the advent of microwell plates and microplate readers new methodologies were developed (Lew et al. 1995; Raftos et al. 1996) to increase the throughput of OFC data, enabling OFCs to become an important tool for measuring dynamic changes in RBC hydration states, with important applications in research on the condition of intact RBCs in health and disease.
Preparation of red blood cells.
On day one, about 50 ml of blood from healthy volunteers was drawn into heparinized vacutainers after obtaining informed written consent. The blood was spun at 1800 g for 10 min at 10oC, the plasma removed, and the RBCs washed twice by centrifugation and resuspension in solution B and twice more in solution A, with removal of the buffy coat after each spin. The cells were then suspended in RPMI medium (Annex #5) at 50% Hct and stored at 4oC for use in experiments on days 2 and 4.
The biphasic OFC protocol.
RBCs contained in about 7 ml of the suspension in RPMI were washed twice in solution A and twice more in solution C before final suspension at 10% Hct in solution C, 12 ml.
This suspension was divided in two 6 ml aliquots, one control and one test condition in each experiment, both suspensions kept at 37oC. The test condition for each experiment is detailed in Results and in the legends of the figures. The protocol for the biphasic OFC control was as follows. After about 10 min for temperature equilibration, CaCl2 was added from concentrated stock (200 mM) to a final concentration of 50 μM in the suspension. A 0.5 ml sample was taken for duplicate initial OFCs. At t = 0 min, the ionophore A23187 was added to a final concentration of 10 μM in the suspension.
Further 0.5 ml samples for OFCs were taken at t = 10, 30, 60, 120, 180 and 240 min post-ionophore addition. To investigate the extent to which the biphasic OFC pattern of Ca2+ loaded RBCs could be attributed to salt and water shifts as previously assumed (Lew et al. 2007), the cell water content was measured in parallel aliquots of the same samples used for OFCs during the biphasic OFC protocol in all control and test conditions. Osmotic fragility was measured by the profile migration method, as previously described (Lew et al. 1995; Raftos et al. 1996). For water content determinations 0.5 ml aliquots of the cell suspension were distributed in polyethylene micro test tubes and centrifuged at 20 000 g for 10 min at 5°C. After ce ntrifugation the packed cells were separated from the supernatant by slicing the tube with a razor blade above and below the top of the red cell column. After weighing, the packed cells were dried to constant weight for 24 h at 90°C and re-weighed. The measure d water content in gram per gram of cell solids was then divided by 0.34 to express cell water content in units of litre cell water per 340 g of Hb, or its equivalent, litre cell water per litre original packed RBCs (lcw/loc, as reported in the ordinate of the corresponding figures).
Measurement of the volume-fraction of RBC cytosol lost by Ca2+- induced exovesiculation*.
The method followed guidelines derived from early work by Allan and Thomas (Allan et al. 1980). Washed RBCs were suspended at 10 % Hct in solution A or AK supplemented with CaCl2, 0.15 mM, and incubated at 37oC. Samples of this suspension were taken before and 4h after addition of the ionophore A23187, placed in 15 ml centrifuge tubes and gently spun at 500g for 10 min to separate the intact RBCs from the supernatant which contained lysed cells and vesicles. The supernatant was then distributed in 0.5 ml aliquots in microfuge tubes and spun at 16000g for 30 min at 20oC to pellet the vesicles and ghosts. Pellets were recovered from the 4h samples. Microscopic observation of unfixed pellet material showed a dense grainy background and white ghosts. Pellets were pooled together as required for measuring the Hb content of the vesicles, and in preparation for transmission and scanning electronmicroscopy. Hb was measured by resuspending the vesicle pellet in distilled water with 0.01% Triton X-100.
Processing of vesicle samples for transmission and scanning electronmicroscopy.
Pellets of vesicles destined for ultrastructural observation were initially fixed by 500-fold dilution in 4% formaldehyde in 0.1M phosphate buffer for 24 h at 20oC. They were then rinsed in 0.1M PIPES buffer at pH 7.4 and post fixed in 2% glutaraldehyde in 0.1M PIPES buffer for a further 2 h. They were again rinsed in PIPES buffer and treated with 1% osmium tetroxide for one hour, rinsed in deionized water (DIW) and stained with 2% uranyl acetate in 0.05 M Maleate buffer. The pellets were then split into two samples one for TEM and one for SEM. TEM : The pellet was dehydrated in an ascending series of ethanol solutions from 70% to 100%, rinsed twice in acetonitrile and infiltrated with Quetol epoxy resin. The resin was cured at 60°C for 48 hours. Thin sections were cut using a Leica ultracut UCT mounted on 300 mesh copper grids and stained with uranyl acetate and lead citrate. The sections were viewed in an FEI CM100 operated at 80 kV and images were recorded with a Deben AMT 16000 digital camera. SEM : The pellet was re-suspended in 250 μl of DIW and allowed to settle on a 12 mm diameter coverslip coated with poly-L-lysine for 5 min in a humid chamber. The coverslip was dehydrated to 100% ethanol and critical point dried in a Polaron B7010 critical point dryer. The coverslips were mounted on Cambridge SEM stubs, sputter coated with 5 nm of gold with a Quorum Emitech K575X sputter coater and viewed in a FEI XL30 FEGSEM operated at 5 kV.
Table of contents :
LIST OF FIGURES.
LIST OF TABLES.
I. IN BRIEF.
I. 1. Aims.
I. 2. Summary of results.
I. 3. Scientific communication.
I. 3. 1. Publications.
I. 3. 2. Oral communications.
I. 4. IN BRIEF en Fraçais.
I. 5. IN BRIEF po polsku.
II. GENERAL CONTEXT.
II. 1. Oxygen.
II. 2. Haemoglobin.
II. 3. Erythrocytes.
II. 4. Human red blood cell.
II. 5. RBC membrane transporters.
II. 5. 1. Pumps, exchangers, cotransporters.
II. 5 .2. Aquaporins.
II. 5. 3. Ionic channels.
II. 5. 3. 1. Cationic channels.
II. 5. 3. 2. Anionic channels.
II. 5. 3. 3. Physiological role of ionic channels.
III. MATERIALS AND METHODS.
III. 1. Contribution of ionic channels in dehydration-elicited rehydration: lysis curves and osmotic fragility data acquisition.
III. 1. 1. Experimental design.
III. 1. 2. Solutions.
III. 1. 3. Preparation of red blood cells.
III. 1. 4. The biphasic OFC protocol.
III. 1. 5. Measurement of the volume-fraction of RBC cytosol lost by Ca2+-induced exovesiculation*.
III. 1. 5. 1. Processing of vesicle samples for transmission and scanning electronmicroscopy.
III. 2. Electrophysiology.
III. 2. 1. Preparation of cells.
III. 2. 2. Solutions.
III. 2. 3. Current recordings.
III. 3. Chemicals.
III. 4. Red blood cell model.
IV. 1. First question: Are RBC’s membrane channels involved in rehydration elicited by isosmotic dehydration?
IV. 1. 1. Results.
IV. 1. 2. Discussion.
IV. 2. Second question: Does membrane deformation induce channel activity?
IV. 2. 1. Results.
IV. 2. 1. 1. Evidence for spontaneous channel activity following seal formation.
IV. 2. 1. 2. Transient nature of recorded currents.
IV. 2. 1. 3. Identification of Gardos channels.
IV. 2. 1. 4. Simulation by mathematical RBC model.
IV. 2. 1. 5. Evidence for anionic channel activation.
IV. 2. 2. Discussion.
IV. 2. 2. 1. Activation of Gardos channels.
IV. 2. 2. 2. Activation of anionic channels.
IV. 2. 2. 3. Transient nature of Gardos channel activity.
IV. 2. 2. 4. Fast transitions.
IV. 3. Third question: What is the molecular identity of anionic channels present in RBC membrane?
V. GENERAL CONCLUTIONS AND PROSPECTS.