No abstract

Live cell fluorescence microscopy has been widely used to study physiological processes in the human malarial parasite Plasmodium falciparum, including pH homeostasis, Ca 2؉ signaling and protein targeting. However, the reproducibility of the data is often poor. Controversial statements exist regarding cytosolic and vacuolar baseline pH, as well as regarding the subcellular localization of some of the fluorochromes used. When trying to reproduce published baseline values, we observed an unexpected light sensitivity of P. falciparum, which manifests itself in the form of a strong cytoplasmic acidification. Even short exposure times with moderate to low light intensities caused the parasite cytosol to acidify. We show that this effect arises from the selective disruption of the parasite's acidic food vacuole, brought about by lipid peroxidation initiated by lightinduced generation of hydroxyl radicals. Our data suggest that heme serves as a photosensitizer in this process. Our findings have major implications for the use of live cell microscopy in P. falciparum and add a cautionary note to previous studies where live cell fluorometry has been used to determine physiological parameters in P. falciparum.Progress in understanding the physiology of the human malarial parasite Plasmodium falciparum has been slow, despite the fact that P. falciparum is a major health problem, killing millions of humans every year (1). As a consequence, only a limited number of potential drug targets have been identified in recent years. One reason for this is the obligatory intracellular life style of the parasite, which has complicated access to biochemical and physiological pathways.A powerful method to investigate physiological processes of intact cells, under non-disruptive conditions, is live cell fluorescence microscopy. This technique has recently been applied to the intraerythrocytic stages of P. falciparum for measuring intracellular pH, ion concentrations, Ca 2ϩ signaling, and protein targeting (2-7). The main advantage of live cell fluorescence microscopy is that it allows for the spatial separation of signals originating from the parasite and from the surrounding infected erythrocyte. Even subcellular compartments, such as the parasite's acidic food vacuole, can be visualized in situ using this method (5). However, a number of concerns have recently been raised about using single cell fluorescence microscopy in P. falciparum (8).A major disadvantage is the variability between cells, requiring a relatively large number of determinations to obtain statistically significant measurements. Reproducibility between different laboratories seems to be low as well when data obtained from single cell measurements are compared. For example, although some studies have found differences in the parasite's cytosolic pH between chloroquine-sensitive and chloroquine-resistant P. falciparum strains, other studies could not verify this observation (7, 9 -12). Another example represents the use of acridine orange (AO) 1 in the measurement of the...

To study vacuolar chloride (Cl(-)) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl(-) uptake into isolated tonoplast vesicles was measured using the Cl(-)-sensitive fluorescent dye lucigenin (N,N'-dimethyl-9,9'-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl(-), with a Stern-Volmer constant of 173 m(-1) in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl(-)-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K(m) of 17.2 mm and a V(max) of 4.8 mm min(-1). Vacuolar Cl(-) transport was not affected by sulfate, malate, or nitrate. In the presence of 250 microm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl(-) transport was actually significantly increased by 24%. To determine absolute fluxes of Cl(-) using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 x 10(7) m(-1). After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl(-) transport of 31 nmol m(-2) sec(-1) was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques.

Adenophostin A, the most potent known agonist of inositol 1,4, 5-trisphosphate (InsP(3)) receptors, stimulated (45)Ca(2+) release from the intracellular stores of permeabilized hepatocytes. The concentration of adenophostin A causing the half-maximal effect (EC(50)) was 7.1+/-0.5 nM, whereas the EC(50) for InsP(3) was 177+/-26 nM; both responses were positively co-operative. In rapid superfusion analyses of (45)Ca(2+) release from the intracellular stores of immobilized hepatocytes, maximal concentrations of adenophostin A or InsP(3) evoked indistinguishable patterns of Ca(2+) release. The Ca(2+) release evoked by both agonists peaked at the same maximal rate after about 375 ms and the activity of the receptors then decayed to a stable, partially (60%) inactivated state with a half-time (t(1/2)) of 318+/-29 ms for adenophostin A and 321+/-22 ms for InsP(3). Dissociation rates were measured by recording rates of InsP(3)-receptor channel closure after rapid removal of agonist. The rate of adenophostin A dissociation (t(1/2), 840+/-195 ms) was only 2-fold slower than that of InsP(3) (t(1/2), 436+/-48 ms). We conclude that slow dissociation of adenophostin A from InsP(3) receptors does not underlie either its high-affinity binding or the reported differences in the Ca(2+) signals evoked by InsP(3) and adenophostin A in intact cells.

The functional properties of the only inositol trisphosphate (IP(3)) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP(3) receptors of S2 cells bound (1,4,5)IP(3) with high affinity (K(d)=8.5+/-1.1 nM), mediated positively co-operative Ca(2+) release from a thapsigargin-sensitive Ca(2+) store (EC(50)=75+/-4 nM, Hill coefficient=2.1+/-0.2), and they were recognized by an antiserum to a peptide conserved in all IP(3) receptor subtypes in the same way as mammalian IP(3) receptors. As with mammalian IP(3) receptors, (2,4,5)IP(3) (EC(50)=2.3+/-0.3 microM) and (4,5)IP(2) (EC(50) approx. 10 microM) were approx. 20- and 100-fold less potent than (1,4,5)IP(3). Adenophostin A, which is typically approx. 10-fold more potent than IP(3) at mammalian IP(3) receptors, was 46-fold more potent than IP(3) in S2 cells (EC(50)=1.67+/-0.07 nM). Responses to submaximal concentrations of IP(3) were quantal and IP(3)-evoked Ca(2+) release was biphasically regulated by cytosolic Ca(2+). Using rapid superfusion to examine the kinetics of IP(3)-evoked Ca(2+) release from S2 cells, we established that IP(3) (10 microM) maximally activated Drosophila IP(3) receptors within 400 ms. The activity of the receptors then slowly decayed (t(1/2)=2.03+/-0.07 s) to a stable state which had 47+/-1% of the activity of the maximally active state. We conclude that the single subtype of IP(3) receptor expressed in Drosophila has similar functional properties to mammalian IP(3) receptors and that analyses of IP(3) receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP(3) receptors.