Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L. lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.     

The efflux of a fluorescent probe is catalyzed by an ATP-driven extrusion system in Lactococcus lactis.

Many bacteria, both gram positive and gram negative, extrude in an energy-dependent manner the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5[and -6]-carboxyfluorescein (BCECF) (D. Molenaar, T. Abee, and W. N. Konings, Biochim. Biophys. Acta 1115:75-83, 1991). This efflux was studied in detail in Lactococcus lactis, and several indications that a transport system is involved were found. This transport system is most likely driven by ATP or a related compound. The evidence is that BCECF extrusion (i) occurs against a BCECF gradient, (ii) is strictly correlated with ATP concentration and not with the proton motive force, and (iii) is inhibited by vanadate and to a lesser extent by N,N'-dicyclohexylcarbodiimide. Most convincingly, a UV mutant with a strongly reduced efflux rate was isolated. Such a mutant was isolated from a BCECF-loaded and lactose-energized population by selection of highly fluorescent cells in a flow cytometer-cell sorter. The physiological function of this extrusion system is unknown, but its characteristics classify it among the traffic ATPases.     

Cytoplasmic viscosity near the cell plasma membrane: measurement by evanescent field frequency-domain microfluorimetry.

The purpose of this study was to determine whether the unique physical milieu just beneath the cell plasma membrane influences the rheology of fluid-phase cytoplasm. Cytoplasmic viscosity was evaluated from the picosecond rotation of the small fluorophore 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transform microfluorimetry (Fushimi and Verkman, 1991). Information about viscosity within < 200 nm of cell plasma membranes was obtained by selective excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized laser illumination (488 nm) at a glass-aqueous interface. Measurements of fluorescence lifetime and time-resolved anisotropy were carried out in solutions containing fluorescein or BCECF at known viscosities, and monolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canine kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theoretically and/or experimentally, including variations in lifetime due to fluorophore proximity to the interface, and the use of the s and p polarized excitation. In fluorescein solutions excited with s-plane polarized light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotational correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane. The results establish the methodology for time-resolved microfluorimetric measurement of polarization in the evanescent field and demonstrate that the cell plasma membrane has little effect on the fluid-phase viscosity of adjacent cytoplasm.     

Mapping of fluorescence anisotropy in living cells by ratio imaging. Application to cytoplasmic viscosity.

Steady-state and time-resolved fluorescence properties of probes incorporated into living cells give information about the microenvironment near the probe. We have extended studies of spatially averaged fluorescence anisotropy (r) by using an epifluorescence microscope, equipped with excitation and emission polarizers and an image analysis system, to map r of nonoriented fluorophores incorporated into cultured cells. With this imaging system, r for reflected light or glycogen scattering solutions was greater than 0.98. Measurement of r over the range 0.01-0.35 for fluorophores in bulk solution and in thin capillary tubes placed side-by-side gave values equivalent to r measured by cuvette fluorometry. Cytoplasmic viscosity (eta) in Madin-Darby canine kidney (MDCK) cells and Swiss 3T3 fibroblasts was examined from anisotropy images and time-resolved fluorescence decay of the cytoplasmic probes 2,7-bis-carboxyethyl-5 (and 6)-carboxy-fluorescein (BCECF) and indo-1. Nanosecond lifetimes and anisotropy decay were measured using a pulsed light source and gated detector interfaced to the epifluorescence microscope. Anisotropy images of BCECF in MDCK cells revealed two distinct regions of r: one from the cytoplasm (r = 0.144 +/- 0.008) and a second appearing at late times from the interstitial region (r = 0.08 +/- 0.03), representing BCECF trapped beneath the tight junctions. Anisotropy values, taken together with intracellular life-times and the calibration between r and eta/tau f for water/glycerol mixtures, gave eta values of 10-13 cP at 23 degrees C. These values assume little fluorophore binding to intracellular components and are therefore upper limits to cytoplasmic viscosity. These data establish a new methodology to map anisotropy in intact cells to examine the role of fluidity in cellular physiology.     

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter

(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls.     

Cytoplasmic viscosity near the cell plasma membrane: translational diffusion of a small fluorescent solute measured by total internal reflection-fluorescence photobleaching recovery.

Total internal reflection-fluorescence recovery after photobleaching (TIR-FRAP) was applied to measure solute translational diffusion in the aqueous phase of membrane-adjacent cytoplasm. TIR fluorescence excitation in aqueous solutions and fluorescently labeled cells was produced by laser illumination at a subcritical angle utilizing a quartz prism; microsecond-resolution FRAP was accomplished by acousto-optic modulators and electronic photomultiplier gating. A mathematical model was developed to determine solute diffusion coefficient from the time course of photobleaching recovery, bleach time, bleach intensity, and evanescent field penetration depth; the model included irreversible and reversible photobleaching processes, with triplet state diffusion. The validity and accuracy of TIR-FRAP measurements were first examined in aqueous fluorophore solutions. Diffusion coefficients for fluorescein isothiocyanate-dextrans (10-2000 kDa) determined by TIR-FRAP (recovery t1/2 0.5-2.2 ms) agreed with values measured by conventional spot photobleaching. Model predictions for the dependence of recovery curve shape on solution viscosity, bleach time, and bleach depth were validated experimentally using aqueous fluorescein solutions. To study solute diffusion in cytosol, MDCK epithelial cells were fluorescently labeled with the small solute 2',7'-bis-2-carboxyethyl-5-carboxyfluorescein-acetoxymethyl-ester (BCECF). A reversible photobleaching process (t1/2 approximately 0.5 ms) was identified that involved triplet-state relaxation and could be eliminated by triplet-state quenching with 100% oxygen. TIR-FRAP t1/2 values for irreversible BCECF bleaching, representing BCECF translational diffusion in the evanescent field, were in the range 2.2-4.8 ms (0.2-1 ms bleach times), yielding a BCECF diffusion coefficient 6-10-fold less than that in water. These results establish the theory and the first experimental application of TIR-FRAP to measure aqueous-phase solute diffusion, and indicate slowed translational diffusion of a small solute in membrane-adjacent cytosol.     

Characterization of Zn(2+) transport in Madin-Darby canine kidney cells

The aim of this study was to characterize the mechanism implicated in Zn(2+) transport in MDCK cells. Trace elements such as Zn(2+), Cd(2+) or Cu(2+) induced MDCK cell depolarization at the micromolar level as demonstrated by bis-oxonol fluorescence and whole-cell patch experiments. This depolarization was inhibited by La(3+) and Gd(3+) and was not related to the activation of Na(+) or Cl(-) channels. Uptake of 65Zn was assessed under initial rate conditions. The kinetic parameters obtained at 37 degrees C were a K(m) of 18.9 microM and a V(max) of 0.48 nmol min(-1) (mg protein(-1)). Intracellular pH measurements using BCECF probe demonstrated that Zn(2+) transport induced a cytoplasmic acidification. The cytoplasmic acidification resulting from Zn(2+) uptake activated Na(+)/H(+) antiporter, which allowed for the recycling of protons. These data suggest that Zn(2+) enters MDCK cells through a proton-coupled metal-ion transporter, the characteristics of which are slightly different from those described for the metal transporter DCT1. This mechanism could be in part responsible of the metal transport evidenced in the distal parts of the renal tubule.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

Inhibition of alpha-aminoisobutyric acid uptake by mastoparan in Madin-Darby Canine Kidney cells

Mastoparan, a wasp venom, was found to inhibit Na(+)-dependent net alpha-aminoisobutyric acid (AIB) uptake in Madin Darby Canine Kidney (MDCK) cells. Mastoparan also produced a significant increase in AIB efflux when compared to controls. Pretreatment of MDCK cells with 2 mM neomycin attenuated mastoparan's inhibition of net AIB uptake and completely suppressed mastoparan-mediated increases in AIB efflux when compared to controls. These data suggest that mastoparan's inhibition of net AIB uptake involves more than a single basic mechanism.     

Activation of basolateral membrane K+ permeability by bradykinin in MDCK cells

We study bradykinin-stimulated K+ efflux in Madin-Darby canine kidney (MDCK) cells using 86Rb as an isotopic tracer. Bradykinin brings about a rapid increase in the permeability of MDCK cells to K+, the effect is dose-dependent with a plateau at 10(-6) M. The effect seems to be mediated by Ca2+-activated K+ channels, localised at the basolateral aspect of the epithelium. Unlike alpha-receptors, which mediate a similar effect of adrenalin in these cells, bradykinin receptors seem to be present at both sides of the epithelium. Bradykinin increases the labelling of IP3, and bradykinin-stimulated K+ efflux persists even in cells which are bathed in Ca2+-free medium, suggesting that the effects seen in the present work are probably due to Ca2+ release from intracellular stores. Some extracellular Ca2+ also might be involved in the bradykinin effect, consistent with the kinin-increasing membrane permeability to Ca2+.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.     

Fluorescence-based viability assay for studies of reactive drug intermediates

Studies of drug toxicity, toxicologic structure-function relationships, screening of idiosyncratic drug reactions, and a variety of cytotoxic events and cellular functions in immunology and cell biology require the sensitive and rapid processing of often large numbers of cell samples. This report describes the development of a high-sensitivity, high-throughput viability assay based on (a) the carboxyfluorescein derivative 2'-7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) as a vital dye, (b) instrumentation capable of processing multiple small (less than 100 cells) samples, and (c) a 96-well unidirectional vacuum filtration plate. Double staining of cultured peripheral blood mononuclear cells with BCECF and propidium iodide (PI) showed no overlap between PI+ (nonviable) and BCECF+ (viable) cells by flow cytometric analysis. Optimal conditions were developed for dye loading and minimizing physical cell damage and fluorescence quench during the assay procedure. The ratio of BCECF fluorescence to internal standard fluorescent particles was linear from 40 to greater than 20,000 cells with a signal:noise ratio of approximately 3 at 40 cells/well. Sulfamethoxazole hydroxylamine (SMX-HA) was used as a model toxic drug metabolite to explore the validity of the BCECF procedure. SMX-HA, but not its parent compound sulfamethoxazole, resulted in a dose dependent loss of cellular fluorescence and the parallel accumulation of PI+ nonviable cells. When compared to the currently used tetrazolium dye reduction viability assay, the BCECF method was 3-fold more sensitive, greater than 10-fold faster, and required 1/10-1/100 the cell numbers.     

High throughput assay to detect compounds that enhance the proton permeability of Candida albicans membranes.

We describe a 96-well microtiter plate format assay to detect changes in proton permeability in membranes of the pathogenic yeast, Candida albicans. Candida albicans cells were incubated with the lipophilic ester of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH-sensitive fluorescein derivative. Inside the cells, BCECF was released and trapped in the vacuole. Compounds that destroyed membrane integrity increased the pH value of the vacuole due to proton leakage into the cytoplasm. This was paralleled by an increase in BCECF fluorescence intensity, which could be quantified. The test assay was validated with amphotericin B, as well as with other membrane-active compounds known to increase membrane permeability. Possible applications and limitations of this assay in the field of antifungal drug discovery are discussed.     

Genomic structure,gene expression,and promoter analysis of human multidrug resistance-associated protein 7

The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5'-flanking region at -1,780-1,287 bp, and at -611 to -208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226% by genotoxic 2-acetylaminofluorene and 347% by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.     

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.     

The role of multidrug resistance protein 1 (MRP1) in transport of fluorescent anions across the human erythrocyte membrane

We employed human red blood cells as a model system to check the affinity of MRP1 (Multidrug Resistance-associated Protein 1) towards fluorescein and a set of its carboxyl derivatives: 5/6-carboxyfluorescein (CF), 2,7-bis-(2-carboxyethyl)-5/6-carboxyfluorescein (BCECF) and calcein (CAL). We found significant differences in the characteristics of transport of the dyes tested across the erythrocyte membrane. Fluorescein is transported mainly in a passive way, while active efflux systems at least partially contribute to the transport of the other compounds. Inside-out vesicle studies revealed that active transport of calcein is masked by another, ATP-independent, transport activity. Inhibitor profiles of CF and BCECF transport are typical for substrates of organic anion transporters. BCECF is transported mainly via MRP1, as proven by the use of QCRL3, a monoclonal antibody known to specifically inhibit MRP1-mediated transport. Lack of effect of QCRL3 on CF uptake excludes the possibility of MRP1 being a transporter of this dye. No inhibition of CF accumulation by cGMP, thioguanine and 6-mercaptopurine suggests also that this fluorescent marker is not a substrate for MRP5, another ABC transporter identified in the human erythrocyte membrane.     

Activation of Na+/H+ exchange and Ca2+ mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization.

Although an increase in cytosolic pH (pHi) caused by Na+/H+ exchange enhances Ca2+ mobilization in platelets stimulated by low concentrations of thrombin [Siffert & Akkerman (1987) Nature (London) 325, 456-458], studies using fluorescent indicators for pHi (BCECF) and [Ca2+]i (fura2) suggest that Ca2+ is mobilized while the cytosolic pH decreases. Several lines of evidence indicate that the initial fall in BCECF fluorescence is not due to cytosolic acidification but is caused by a platelet shape change. (1) Pulse stimulation of platelets by successive addition of hirudin (4 unit/ml) and thrombin (0.2 unit/ml) induced a shape change of 43 +/- 8% and a fall in BCECF fluorescence, which both remained unchanged when Na+/H+ exchange was inhibited by ethylisopropylamiloride (EIPA, 100 microM). (2) Increasing the thrombin concentration to 0.4 unit/ml doubled the shape change and the fall in BCECF fluorescence, but again EIPA had no effect on these responses. (3) Treating platelets with 2 microM-ADP induced shape change and a decline in BCECF fluorescence that was unaffected by EIPA. (4) A second addition of thrombin to platelets that had already undergone shape change induced an immediate increase in BCECF fluorescence without a prior decrease. (5) Activation of protein kinase C by 1,2-dioctanoyl-sn-glycerol (DiC8) neither induced shape change nor a decline in BCECF fluorescence; in contrast BCECF fluorescence rapidly increased indicating an immediate cytosolic alkalinization. Concurrent analysis of [Ca2+]i under conditions in which shape change did not interfere with BCECF fluorescence showed that cytosolic alkalinization and Ca2+ mobilization started almost simultaneously. These observations suggest that cytosolic alkalinization is not preceded by a fall in pHi and can support Ca2+ mobilization induced by weak agonists.     

Osmotic Swelling Stimulates Ascorbate Efflux from Cerebral Astrocytes

Abstract: Ascorbate (reduced vitamin C) is an important enzyme cofactor, neuromodulator, and antioxidant that is stored at millimolar concentrations in the cytosol of cerebral astrocytes. Because these cells swell during hyponatremia, cerebral ischemia, and trauma, we investigated the effects of osmotic stress on astrocytic transport of ascorbate. Ascorbate efflux from primary cultures of rat astrocytes was rapidly (within 1 min) increased by incubation in hypotonic medium. Efflux also increased when astrocytes, which had been adapted to a hypertonic environment, were swollen by transfer to isotonic medium. Swelling-induced ascorbate efflux was inhibited by the anion-transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). The pathway that mediates ascorbate efflux was found to be selective because a larger anion, 2',7'-bis(carboxyethyl)-5-(or -6)-carboxyfluorescein (BCECF), was retained in the swollen astrocytes. Na⁺-dependent ascorbate uptake into astrocytes was inhibited slightly during the first minute of hypotonic stress, indicating that the sodium ascorbate cotransporter does not mediate swelling-induced efflux. Cell concentration of authentic ascorbate was measured by HPLC with electrochemical detection. When astrocytes were incubated in ascorbate-free medium, hypotonicity decreased cell ascorbate concentration by 50% within 3 min. When astrocytes were incubated in ascorbate-supplemented hypotonic medium, intracellular ascorbate concentration was restored within 10 min because uptake remained effective. Many pathological conditions cause brain cell swelling and formation of reactive oxygen species. Ascorbate release during astrocytic swelling may contribute to cellular osmoregulation in the short-term and the scavenging of reactive oxygen species.     

Simultaneous measurements of intracellular pH in the leech giant glial cell using 2'',7''-bis-(2-carboxyethyl)-5,6-carboxyfluorescein and ion-sensitive microelectrodes.

We have employed two independent techniques to measure the intracellular pH (pHi) in giant glial cells of the leech Hirudo medicinalis, using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and double-barreled neutral-carrier, pH-sensitive microelectrodes, which also record the membrane potential. We have compared two procedures for calibrating the ratio of the BCECF signal, excited at 440 nm and 495 nm: 1) the cell membrane was H(+)-permeabilized with nigericin in high-K+ saline at different external pH (pHo) values, and 2) the pHi of intact cells was perturbed in CO2/HCO3(-) -buffered saline of different pH, and the BCECF ratio was calibrated according to a simultaneous microelectrode pH reading. As indicated by the microelectrode measurements, the pHi did not fully equilibrate to the pHo values in nigericin-containing, high-K+ saline, but deviated by -0.12 +/- 0.02 (mean +/- SEM, n = 37) pH units. In intact cells, the microelectrode readings yielded up to 0.15 pH unit lower values than the calibrated BCECF signal. In addition, larger dye injections into the cells (> 100 microM) caused an irreversible membrane potential loss indicative of some damage to the cells. The amplitude and kinetics of slow pHi changes were equally followed by both sensors, and the dye ratio recorded slightly higher amplitudes during faster pHi shifts as induced by the addition and removal of NH4+.