Serge Thomas

A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum

A recent study on malaria‐infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host‐ or parasite‐derived and their molecular nature have not been resolved. Here we report a comparison of a malaria‐induced anion channel with an endogenous anion channel in Plasmodium falciparum‐infected human RBCs. Ion channel activity was measured using the whole‐cell, cell‐attached and excised inside‐out configurations of the patch‐clamp method. Parasitised RBCs were cultured in vitro, using co‐cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (∼15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I− > Br− > Cl−. In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (∼80 pS, outward conductance) was spontaneously active. The host membrane of malaria‐infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria‐infected RBCs were shown to have a low open‐state probability (Po) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole‐cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up‐regulated by the malaria parasite P. falciparum.

Adaptive respiratory responses of trout to acute hypoxia. I. Effects of water ionic composition on blood acid-base status response and gill morphology

The effects of various levels of hypoxia (PWO2 ranging from 10 to 60 Torr) on arterial blood gases (PaO2 and PaCO2) and acid-base status were investigated in trout at 15 degrees C. The hypoxic responses of two stocks of trout living in natural waters having very different levels of NaCl (1.0 mmol.L-1 and 0.1 mmol.L-1) and carbonate alkalinity (0.4 mmol.L-1 and 2.4 mmol.L-1) were compared. The use of an extracorporeal circulation method made it possible to continuously monitor the pH changes. The different patterns of the acid-base status observed in response to hypoxia depend on the evolution of PaO2. Two critical PaO2 thresholds were defined. Crossing the upper (about 15 Torr) induces metabolic acidosis which is normally followed by pH recovery, while crossing the lower (about 10 Torr) promotes loss of capacity to compensate acidosis. The NaCl concentration of the water drastically modifies the fish sensitivity to hypoxia: fish living in water with a low NaCl concentration have less resistance to hypoxic exposure. This may be explained by the fact that in fish living in low NaCl concentrations, the secondary gill lamellae are surrounded by chloride cells, which considerably reduce the surface area available for gas exchange. Consequently a modest fall in PWO2 induces a drastic reduction of the arterial oxygen tension which crosses the lower critical PaO2 threshold.

Modulation of whole-cell currents in Plasmodium falciparum-infected human red blood cells by holding potential and serum

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum‐infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole‐cell configuration of the patch‐clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole‐cell recordings in malaria‐infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4‐fold and inward currents by approximately 2‐fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.

Red cell investigations: Art and artefacts

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

HCO3− dehydration by the blood of an elasmobranch in the absence of a Haldane effect

We measured in vivo arterial PCO2 and CaCO2 in Scyliorhinus canicula and found them to be very low (approximately 1 Torr and 3 mmol l-1 respectively). In vitro, the Haldane effect was functionally absent, and there was no detectable beta-adrenergic Na+/H+ exchange, in contrast to teleosts. The HCO3- dehydration rate of the blood, measured by a radioisotopic assay (Wood and Perry, J. Exp. Biol. 157:349-366, 1991), was independent of steady-state deoxygenation or oxygenation, unaffected by rapid oxygenation, and insensitive to isoprenaline, amiloride, and removal of urea or TMAO. SITS and acetazolamide reduced the rate; HCO3-/Cl- exchange rather than intracellular carbonic anhydrase (CA) was the rate-limiting factor. The rate was not altered by steady-state plasma [HCO3-], but increased linearly with PCO2 and with RBC concentration, saturating at hematocrits > or = 15%. The rate in separated plasma accounted for approximately 50% of the whole blood rate, was higher than in trout plasma or saline, and was inhibited by acetazolamide. The presence of CA in the normally circulating blood plasma of dogfish may contribute to highly efficient CO2 excretion in vivo.

Volume regulation following hypotonic shock in isolated crypts of mouse distal colon

1 A video‐imaging technique of morphometry was used to measure the diameter as an index of cell volume in intact mouse distal colon crypts submitted to hypotonic shock. 2 Transition from isotonic (310 mosmol l−1) to hypotonic (240 mosmol l−1) saline caused a pronounced increase in crypt diameter immediately followed by regulatory volume decrease (RVD). 3 Exposure of crypts to Cl−‐free hyposmotic medium increased the rapidity of both cell swelling and RVD. Exposure of crypts to Na+‐free hyposmotic medium reduced the total duration of swelling. Return to initial diameter was followed by further shrinkage of the crypt cells. 4 The chloride channel inhibitor NPPB (50 μM) delayed the swelling phase and prevented the subsequent normal decrease in diameter. 5 The K+ channel blockers barium (10 mM), charybdotoxin (10 nM) and TEA (5 mM) inhibited RVD by 51, 44 and 32%, respectively. 6 Intracellular [Ca2+] rose from a baseline of 174 ± 17 nM (n= 8) to 448 ± 45 nM (n= 8) during the initial swelling phase 7 The Ca2+ channel blockers verapamil (50 μM) and nifedipine (10 μM), the chelator of intracellular Ca2+ BAPTA AM (30 μM), or the inhibitor of Ca2+ release TMB‐8 (10 μM), dramatically reduced volume recovery, leading to 51% (n= 9), 25% (n= 7), 37% (n= 6), 32% (n= 8) inhibition of RVD, respectively. TFP (50 μM), an antagonist of the Ca2+‐calmodulin complex, significantly slowed RVD. The Ca2+ ionophore A23187 (2 μM) provoked a dramatic reduction of the duration and amplitude of cell swelling followed by extensive shrinkage. The release of Ca2+ from intracellular stores using bradykinin (1 μM) or blockade of reabsorption with thapsigargin (1 μM) decreased the duration of RVD. 8 Prostaglandin E2 (PGE2, 5 μM) slightly delayed RVD, whereas leukotriene D4 (LTD4, 100 nM) and arachidonic acid (10 μM) reduced the duration of RVD. Blockade of phospholipase A2 by quinacrine (10 μM) inhibited RVD by 53%. Common inhibition of PGE2 and LTD4 synthesis by ETYA (50 μM) or separate blockade of PGE2 synthesis by 1 μM indomethacin reduced the duration of RVD. Blockade of LTD4 synthesis by nordihydroguaiaretic acid (NDGA) did not produce any significant effect on cell swelling or subsequent RVD. 9 Staurosporine (1 μM), an inhibitor of protein kinases, inhibited RVD by 58%. Taken together the experiments demonstrate that the RVD process is under the control of conductive pathways, extra‐ and intracellular Ca2+ ions, protein kinases, prostaglandins and leukotrienes.

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cells diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. Methodology/Principal Findings The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. Conclusions/Significance The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Adaptive respiratory responses of trout to acute hypoxia. II. Blood oxygen carrying properties during hypoxia

The effects of deep and acute hypoxia (PwO2 = 25 Torr) on oxygen transport characteristics (Hill number (n) and P50) were investigated in trout at 15 degrees C. When a fish is submitted to such an acute and deep hypoxia, a metabolic acidosis develops as soon as the arterial oxygen tension drops to about 15 Torr. We first showed that the hemoglobin of blood sampled at the end of the acidification period has an increased oxygen affinity. This improved affinity could be explained by the internal alkalisation of erythrocytes due to the extrusion of protons via a beta-adrenergic stimulation of Na+/H+ exchanges occurring at the onset of hypoxia and responsible for extracellular acidosis. Secondly we observed a significant increase (about 20%) of the number of blood cells per volume of blood during the acidosis. This cell number stays constant afterwards. The dual effects of a higher hemoglobin oxygen affinity and a greater amount of available hemoglobin improving blood oxygen loading at the fish gills appear to be a fast adaptive response to acute hypoxia. Surprisingly, we found that the elevated affinity occurring during acidosis remained constant as long as the fish were maintained in hypoxia, in spite of possible large variations of extracellular pH (pHe). This result is difficult to reconcile with the idea that the increase in affinity is imposed by intracellular pH (pHi), since in red blood cells pHi depends on pHe, thus any modification of pHe would in this case modify oxygen affinity.

Effects of graded exercise on blood gas tensions and acid-base characteristics of rainbow trout

A swim tunnel respirometer and an extracorporeal blood circulation technique allowed continuous collection of data from exercising fish below and near their critical speed. Swimming at speeds below maximum showed no changes in plasma Na+, Cl- and lactate concentrations but increased levels of blood hemoglobin and plasma K+. PaO2 and PaCO2 showed an exponential decrease and increase respectively and related to swimming speed. Increased swimming speed changed the acid-base status toward a mixed respiratory and metabolic acidosis. Upon reaching maximum speed sudden and large increases in plasma Na+, K+ and lactate concentrations occurred associated with a large metabolic acidosis.

Adaptive respiratory responses of trout to acute hypoxia. III: Ion movements and pH changes in the red blood cell

In the preceding paper acute hypoxia was shown to elicit within minutes an increase in the blood O2 affinity. From the present data it appears that this rapid change in blood P50 value can be ascribed to an important alkalization of the red blood cell despite a simultaneous decrease in extracellular pH (pHe). The intracellular alkalization is only partially due to beta-adrenergic stimulation of Na/H exchange, deoxygenation of hemoglobin and the rapid decrease of PaCO2 due to hyperventilation being involved in this process via the chloride shift. This high value of intraerythrocytic pH (pHi) is then maintained practically constant throughout the time the fish is kept in hypoxia despite wide changes of external pH. The blocking of pHi accounted for the constant O2 content observed during hypoxia. The uncoupling of pHi from pHe, which occurs at the onset of hypoxia, is still unexplained: for instance, it is not due to inhibition of the anion exchanger responsible for the passive distribution of H+ across the red cell membrane. A general scheme of all the mechanisms involved in the emergency adaptive response to acute hypoxia is presented.