Papers by Christian Le Grimellec
Cornea, 2003
To study the surface of normal corneal endothelium by means of atomic force microscopy (AFM). The... more To study the surface of normal corneal endothelium by means of atomic force microscopy (AFM). The central corneal endothelial posterior surface of New Zealand white rabbits was examined. Specimens were observed in Balanced Salt Solution using the contact mode of the AFM either fresh or after fixation in cacodylate-buffered glutaraldehyde solution. Removal of sialic acid residues and hyaluronic acid was achieved by means of enzymatic treatment with neuraminidase and hyaluronidase. Observation of the fresh specimens revealed the presence of an apical endothelial surface coating material (glycocalyx). Removal of sialic acid residues and hyaluronic acid after enzymatic treatment using neuraminidase and hyaluronidase, respectively, permitted the elucidation of the structure of the nondigested coating material. Fixation of the samples resulted in removal of the surface coating material. The imaging of the fixed endothelium surface revealed the mosaic of polygonal cells with the apical flaps of cell junctions emerging over the cell surface. The cell shape and the other characteristics of the posterior surface fixed endothelium were comparable to those described in the literature using scanning electron microscopy. The scanning of very small ranges has provided high-resolution images at the nanometer level in fixed and fresh corneal endothelial surfaces. The atomic force microscope represents a new powerful imaging tool permitting high-resolution observation of corneal endothelium surface in fresh and minimally prepared fixed specimens.
FEBS Letters, Mar 1, 1978
Pflügers Archiv: European Journal of Physiology, Mar 1, 1972
Journal of Membrane Biology, 1999
Journal of Biological Chemistry, Aug 1, 1988
Apical membranes of renal epithelial cells were shown to be more rigid than other plasma membrane... more Apical membranes of renal epithelial cells were shown to be more rigid than other plasma membranes, due in part to the abundance of sphingomyelin among their constituent phospholipids. Tight junctions play a key role in maintaining differences between the apical and the basolateral domains of the plasma membrane with respect to their lipid composition and fluidity. To evaluate the influence of alterations of membrane fluidity on the activity of two apically located transport systems, we compared the effect of opening of tight junctions, by a preincubation period in calcium-deprived medium and of increasing fluidity, with benzyl alcohol, on Na-dependent uptakes of Pi and a-methyl-D-glucopyranoside (MGP) in intact, confluent LLC-PKI cells and MDCK cells. Benzyl alcohol, at 10 mM, increased the Vmaxof Pi uptake by 55 and 42% in LLC-PKl cells and MDCK cells, respectively, but decreased the Vmax of MGP uptake in LLC-PK1 cells by 23%. Similarly to 10 mM benzyl alcohol, opening of tight junctions also increased the Vm,of Pi uptake by 45 and 46% in LLC-PK1 cells and MDCK cells, respectively, and depressed MGP uptake in LLC-PK1 cells by inducing a 15% decrease of the Vm=. None of the two maneuvers (Le. addition of benzyl alcohol or opening of tight junctions) affected the K, values of the transport systems. From these results it is concluded that (i) the increase in membrane fluidity, achieved either by benzyl alcohol or by opening of tight junctions, affects Na-Pi and Na-glucose cotransports differently, reflecting differences in the lipid environments of the two transport systems, and (ii) membrane fluidity might play a physiological role in the modulation of the activity of transport systems. Modifications of membrane lipid order have been shown to affect the activity of numerous membrane-bound proteins, among which are enzymes and transport systems (1). Increasing the fluidity of renal brush-border membrane vesicles with the local anesthetic drug benzyl alcohol elicited a dramatic impairment of Na-dependent D-glUCOSe uptake, demonstrating that this transport system was exquisitely sensitive to
PubMed, Jun 1, 1995
The cytoplasmic face of ventral cell membranes of Madin-Darby canine kidney (MDCK) cells grown on... more The cytoplasmic face of ventral cell membranes of Madin-Darby canine kidney (MDCK) cells grown on glass coverslips was imaged by atomic force microscopy (AFM) in air and under aqueous medium, in "contact" mode. Micrometer range scans on air-dried samples revealed a heterogeneous structure with some filaments, likely corresponding to actin filaments that abut the inner leaflet of the membrane, and a few semi-organized lattice structures that might correspond to clathrin lattices. Experiments in phosphate-buffered saline confirmed the heterogeneity of the inner membrane surface with the presence of large (> 100 nm) globular structures emerging from the surface. Using sub-micrometer scan ranges, protruding particles, that occupy most of the membrane surface, were imaged in liquid medium and in air. These particles, 8 to 40 nm x-y size, were still present following ethanol dehydration which extracts a large fraction of membrane lipids, indicating their proteic nature. Due, at least partly, to the presence of some peripheral proteins, high magnification images of the inner membrane surface were heterogeneous with regard to particle distribution. These data compare with those previously reported for the external membrane leaflet at the surface of living MDCK cells. They show that details of the cytosolic membrane surface can be resolved by AFM. Finally, the images support the view of a plasma membrane organization where proteins come into close proximity.
Biochimica Et Biophysica Acta - Biomembranes, Aug 1, 1985
Lipid composition, physical state of major phospholipid classes and transbilayer migration of pho... more Lipid composition, physical state of major phospholipid classes and transbilayer migration of phosphatidylcholine have I~n determined in plasma membranes of the dog kidney. The lipid composition of brush-border memhranes markedly differs from that of antiluminal membranes with respect to: (a) the total phospholipid content; (b) the cholesterol to phospbolipid ratio (C/P); (c) the distribution of the major phospholipid classes. Sphingomyelin present in large amounts in both luminal and antiluminal membranes extracts exhibits a transition of phase between 20 and 44°C approximately. In the range of temperature studied (5-55°C) no phase transitions were detected for the other phospholipid species. Our data suggest that: (1) at physiological temperature the higher C/P ratio of brush-border membranes is in large part responsible for their lower fluidity; (2) both the relatively low cholesterol and high sphingomyelin contents contribute to the thermotropic transitions observed in intact membranes. Finally transbilayer migration of phosphatidylcholine in brush-border membranes is a very slow process with a half time of 6.5 h at 37°C which compares with that of other biological membranes.
Physiology, Dec 1, 1988
The apical region of the plasma membrane of renal epithelial cells is characterized by low fluidi... more The apical region of the plasma membrane of renal epithelial cells is characterized by low fluidity related to its lipid composition. Marked changes in transport processes during physiological, pathological, or pharmacological alternations in membrane lipids strongly suggest that lipid composition of apical membranes plays an important role in the renal tubular function.
American Journal of Physiology-renal Physiology, Mar 1, 1982
The physical state of membrane lipids and relationships with the activity of Na+-K+-ATPase and al... more The physical state of membrane lipids and relationships with the activity of Na+-K+-ATPase and alkaline phosphatase were studied in basolateral and brush border membranes of the dog kidney. Fluorescence polarization and electron spin resonance experiments demonstrate that basolateral membranes are much more fluid than brush border membranes. This can be accounted for by a difference in fluidity of the lipid part of the membranes. Broad (43-17 degrees C) thermotropic transitions are observed in liposomes made from total lipid extracts of brush border and basolateral membranes. Fluorescence data strongly suggest that thermotropic transitions also occur in intact membranes and that a change in membrane physical state may take place around the physiological temperature. A nonlinear Arrhenius plot for the Na+-K+-ATPase activity in basolateral membranes (breakpoint 21 degrees C) provides additional support for the existence of a lipid liquid leads to gel transition in antiluminal plasma membranes. A break in the Arrhenius plot of alkaline phosphatase activity is also observed but at a temperature significantly higher (26 degrees C) than that of the end of the thermotropic transition. "Room temperature" appears as a critical zone for lipid physical state and activities of both enzymes.
HAL (Le Centre pour la Communication Scientifique Directe), 2007
American Journal of Physiology-renal Physiology, Jul 1, 1988
Fluorescence anisotropy experiments have been done to estimate, in situ, the lipid order of the p... more Fluorescence anisotropy experiments have been done to estimate, in situ, the lipid order of the plasma membrane of polarized Madin-Darby Canine Kidney cells (MDCK) grown on glass cover slips and labeled by 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific marker of the plasma membrane of living cells. Fluorescence microscopy, back-exchange, and quenching experiments indicated that TMA-DPH labeled the highly ordered (r greater than or equal to 0.32, 37 degrees C) apical domain of the plasma membrane of confluent monolayers. Opening of tight junctions or addition of the probe to cell suspensions resulted in a homogeneous distribution of TMA-DPH over the cell surface and in a marked decrease in anisotropy (0.27 less than or equal to r less than or equal to 0.29) that was due neither to a direct effect of Ca2+ on the probe nor to a change in fluorescence lifetime. Our data indicate that the apical domain, likely the external leaflet, of the plasma membrane of polarized MDCK cells is much more ordered than its basolateral counterpart.
Biochimica Et Biophysica Acta - Biomembranes, Jul 1, 1980
The temperature<lependent relationship between K ÷ active influx, Mg 2÷-ATPase activity, transmem... more The temperature<lependent relationship between K ÷ active influx, Mg 2÷-ATPase activity, transmembrane potential (A~) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 ~g/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (-), E). Arrhenius plots of 42K÷ active influx gave a linear relationship for (chol (+), O + P) cells (EA =-9 kcal). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30°C, which is associated with a large increase in the apparent activation energy of the process (t > 30°C, EA =-24 kcal; t < 30°C, EA =-40 kcal). Finally, a biphasic response with a break at approx. 23°C (EA =-7 kcal, t > 23°C; EA =-44 kcal, t <: 23°C) is observed for (chol (-), E) organisms. From the lack of correspondence between these effects on the K ÷ influx and the temperature dependence of both the Mg2+-ATPase activity and A~, it is suggested that changes in the membrane lipid composition affect the K ÷ transport at the level of the K ÷ carrier itself. Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane Abbreviation: SDS, sodium dodecyl sulfate.
Biochimica Et Biophysica Acta - Biomembranes, Jun 1, 1979
Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these... more Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K ÷ concentration (Ki: 200-300 mM) which greatly exceeds that of the growth medium, and a low Na ÷ concentration (Na~: 20 mM). Unlike Na~, K[ varies with cell aging. The K ÷ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K ÷ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, K~i decreases and is partially compensated by a gain in Na ÷. This substitution completely reverses when metabolic substrate is added (K ÷ reaccumulation process). Kinetic analysis of K ÷ movement in cells with steady K ÷ level shows that most of K ÷ influx is mediated by an autologous K÷-K ÷ exchange mechanism. On the other hand, during K ÷ reaccumulation by K+-depleted cells, a different mechanism (a K ÷ uptake mechanism) with higher transport capacity and affinity drives the net K ÷ influx. Both mechanisms are energy-dependent. Ouabain and anoxia have no effect on K ÷ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2÷-dependent ATPase activity. Abbreviation: DCCD, dicyclohexylcarbodiimide.
Journal of Bacteriology, Apr 1, 1981
Adaptation of Mycoplasma gallisepticum, a sterol-requiring Mycoplasma sp., to growth in a serum-f... more Adaptation of Mycoplasma gallisepticum, a sterol-requiring Mycoplasma sp., to growth in a serum-free medium supplemented with cholesterol in decreasing concentrations and with various saturated or unsaturated fatty acids enabled us to control both the cholesterol levels and the membrane fatty acid composition. An estimate of the membrane physical state from fluorescence polarization of 1,6diphenyl-1,3,5-hexatriene indicated that the membrane lipids of native M. gallisepticum were highly ordered. Elongation of the saturated fatty acid chains from 14 to 18 carbon atoms caused only a small increase in the membrane lipid ordering, whereas the introduction of a cis double bond reduced it significantly. Lipid-phase transitions were observed in low-cholesterol-adapted organisms, whose membrane lipids were still highly ordered at the growth temperature. Most species of Mycoplasma are not able to synthesize the cholesterol and long-chain fatty acids necessary for their growth (2, 39, 40, 41). Consequently, variations in the lipid composition of a medium can affect the membrane composition, the growth conditions, the morphology, and the osmotic and mechanical fragility properties of these organisms (22, 27, 28). Control of the exogenous lipids supplied to a medium allows more reproducible growth conditions and makes it possible to study the effects of these lipid components on the functioning of the organisms.
American Journal of Physiology-renal Physiology, Oct 1, 1982
To evaluate the effect of hyper- and hypotonicity on proximal convoluted tubule (PCT) cell volume... more To evaluate the effect of hyper- and hypotonicity on proximal convoluted tubule (PCT) cell volume, nonperfused PCT were studied in vitro with hypertonic solutions containing sodium chloride, urea, or mannitol (450 mosmol/kg H2O) and with hypotonic low sodium chloride solutions (160 mosmol/kg H2O). When the tubules were subjected to hypertonic peritubular solutions containing NaCl, cell volume immediately decreased by 15.5% and remained constant throughout the experimental period (60 min). With mannitol, the initial decrease was identical to that with NaCl (17.7%), but the PCT volume increased slightly during the experimental period. With urea, the decrease in cell volume was smaller (7%) and transient. In hypotonicity, the PCT swelled rapidly, but this swelling was followed by a rapid regulatory phase in which PCT volume nearly returned to control values after less than 10 min. With a potassium-free peritubular medium or 10(-3) M ouabain, the regulatory phase of hypotonicity completely disappeared, whereas the cells did not maintain their reduced volume in NaCl-induced hypertonicity. These results suggest that Na-K-ATPase plays an important role in the maintenance of a reduced cellular volume in hypertonicity and in the regulatory phase of hypotonicity, probably by an active extrusion of sodium and water from the cell.
American Journal of Physiology-cell Physiology, Jul 1, 1993
The events accounting for the adaptation of the sodium-dependent phosphate cotransport (Na-Pi) to... more The events accounting for the adaptation of the sodium-dependent phosphate cotransport (Na-Pi) to phosphate deprivation other than genomic regulation remain unknown. The involvement of changes in intracellular calcium concentration was investigated in Madin-Darby canine kidney (MDCK) cells. Calcium concentration was decreased by 15 h of phosphate deprivation (-24 to -35%) or low-calcium medium (calcium deprivation) (-45%), or 8-(N,N&#39;-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB8) (-32%). Calcium deprivation stimulated Na-Pi (2-fold at 1 h and up to 15 h) by increasing the affinity for phosphate. Combined calcium and phosphate deprivation had more than additive effects on phosphate uptake. The effect of a 15-h calcium deprivation, but not of a 2-h one, was dependent on gene transcription and protein synthesis. TMB8 stimulated phosphate uptake similarly to phosphate deprivation (increase in maximum velocity dependent on gene transcription). The ionophore A23187 decreased basal Na-Pi as well as its stimulation by phosphate or calcium deprivation or by TMB8. Calcium deprivation stimulated (3.2-fold increase) the sodium-coupled alanine transport, whereas phosphate deprivation and TMB8 did not. We conclude that 1) phosphate deprivation decreases intracellular calcium concentration, 2) low intracellular calcium concentration is instrumental in the stimulation by prolonged calcium or phosphate deprivation of Na-Pi, and 3) phosphate or calcium deprivation modulates Na-Pi through different cellular pathways.
Nephron, 1969
Summary Electron Probe Analysis (EPA), using a MS 46 microprobe (Cameca, France), has been develo... more Summary Electron Probe Analysis (EPA), using a MS 46 microprobe (Cameca, France), has been developped and applied to the determination of Cl, Na, K, Ca, P and Fe concentrations in 48 micropuncture samples collected from proximal and distal convolutions in three rats perfused with both ferrocyanide and tritiated inulin, and undergoing salt diuresis of various intensities. The technique for EPA,
S. Karger AG eBooks, Apr 15, 2015
Revue de Médecine Interne, Jun 1, 1993
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Papers by Christian Le Grimellec