Bis(carboxyethyl)-5,6-carboxyfluorescein Research Articles

Serum depletion, multidrug resistance and fluorescent probes: Methodological implications in free radicals evaluation

Intracellular probes used for oxidative burst evaluation could be a substrate of the multidrug resistance proteins (MDR), which may cause misinterpretation of experimental data. We aimed to study the effect of the culture condition and of the MDR-interfering antioxidant quercetin on free radical measurement in serum depleted HCT-8 cells and chick embryo hepatocytes. Serum depletion and/or quercetin affected the traffic of 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and calcein. The comparative results obtained with 2′,7′-dichlorofluorescein (DCF) and with the plasma-membrane probe C11-BODIPY 581/591 [4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid], suggest caution in the use of intracellular probes to evaluate oxidative stress in vitro.

Measuring Interstitial pH and pO2 in Mouse Tumors

This protocol outlines methods to measure two extravascular parameters, interstitial pH and partial pressure of oxygen (pO2), in mouse tumors. The method for measuring interstitial pH uses fluorescence ratio imaging microscopy (FRIM) of the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The method for measuring interstitial pO2 is based on the oxygen-dependent quenching of the phosphorescence of albumin-bound palladium meso-tetra(4-carboxyphenyl)porphyrin, and can be used to measure microvascular as well as interstitial pO2. In addition, the two methods can be used sequentially to measure both pH and pO2 in the same tissues.

Intracellular pH and calcium signaling as molecular targets of diclofenac-induced apoptosis against colon cancer

The role of intracellular pH and Ca2+ and their association with mitochondrial dysfunction and intracellular reactive oxygen species (ROS) are explored in the chemoprevention of colon cancer. 1,2-dimethylhydrazine dihydrochloride (DMH), a potent procarcinogen with selectivity for the colon, at a dose of 30 mg/kg body weight was used to induce initial stages of colon cancer when administered for 6 weeks in male Sprague-Dawley rats. Diclofenac, a preferential cyclooxygenase-2 inhibitor, was used at the anti-inflammatory dose (8 mg/kg body weight) for chemoprevention. The control group was administered vehicles for both DMH and diclofenac. A diclofenac-alone group with the same dose was also run simultaneously. Intracellular pH values as determined by biscarboxyethyl carboxyfluorescein fluorescence assay showed an alkaline pH in colonocytes from the DMH-treated group as compared with the control group. Moreover, the level of intracellular Ca2+ was also found to be decreased with DMH treatment, as shown by the fura-2 acetoxymethyl study and chlortetracycline assay. Apoptosis was studied by comet assay and Apaf-1 immunofluorescent expression and was found to be markedly decreased in this group, indicating that disturbances in pH and Ca2+ homeostasis promoted proliferation in colon and inhibited apoptosis. Changes in mitochondrial membrane potential and ROS levels were analyzed in isolated colonocytes by rhodamine 123 and 2,7-dichlorofluorescein diacetate labeling, respectively. DMH treatment promoted a higher mitochondrial membrane potential while reducing ROS levels. These parameters are known to be associated with pH and Ca2+ changes intracellularly and hence can be suggested to be linked with them in this study also. Diclofenac promoted apoptosis in colonocytes when coadministered with DMH and also ameliorated the changes observed in the above parameters, confirming these mechanisms as early events for the onset of apoptosis in cancer cells.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

Sodium induces simultaneous changes in cytosolic calcium and pH in salt-tolerant quince protoplasts

Previous experiments with salt-resistant quince BA29 (Cydonia oblonga cv. Mill.) have shown that this cultivar takes up sodium transiently into the cytosol of shoot protoplasts only in the absence of calcium chloride, or at <1mM calcium chloride. Addition of NaCl > or =100mM to single protoplasts from in vitro-cultivated quince in the presence of 1.0mM calcium induced instant changes in the cytosolic concentrations of calcium and protons. These changes were investigated by use of tetra [acetoxymethyl] esters of the fluorescent stilbene chromophores Fura 2 and bis-carboxyethyl-carboxyfluorescein (BCECF), respectively. The cytosolic Ca(2+) dynamics in the protoplasts were dependent on the concentration of NaCl added. The changes in calcium differed in amplitude and final concentration and were correlated in time mainly with changes in pH. Addition of 100-400mM NaCl to the protoplasts caused an oscillating increase in the cytosolic level of calcium, and then a decrease. Addition of mannitol, of equiosmolar concentration to NaCl, did not increase the cytosolic calcium concentration. Moreover, there was no increase in cytosolic calcium when NaCl was added in the presence of calcium binding ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA), or lantan or verapamil, two inhibitors of plasma membrane calcium channels. Therefore, we conclude that, in salt-resistant quince, sodium induces an influx of calcium into the cytosol by plasma membrane calcium channels, and a simultaneous increase in cytosolic pH. Because these changes were obtained in the presence of 1mM calcium in the medium, they were not due to sodium uptake into the cytosol.

Feedback Inhibition of Pantothenate Kinase Regulates Pantothenol Uptake by the Malaria Parasite

To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake.

Effect of Tris-Hydroxymethyl Aminomethane on intracellular pH depends on the extracellular non-bicarbonate buffering capacity

The effect of Tris-Hydroxymethyl Aminomethane (THAM) on intracellular pH (pHi) is unknown. We previously demonstrated that the effect of sodium bicarbonate on pHi depends on the non-bicarbonate buffering system. First, human hepatocytes from hepatocytes cell culture (HepG2) were perfused with an acidotic artificial medium containing 5-mmol/L (H5) or 30-mmol/L (H30) concentrations of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES), a non-bicarbonate buffer. We studied the effect of THAM on the pHi in both conditions. We repeated the same protocol using an acidotic human blood with a 5% or 40% hematocrit. The pHi was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein (BCECF). Gas analysis was performed before and during the alkaline infusion. The results showed that THAM caused an intracellular alkalization that was higher when the non-bicarbonate buffer concentration was low (0.45 +/- 0.21 and 0.22 +/- 0.14 pH units with H5 and H30, respectively). A significant relationship was found between changes in pHi and changes in PCO(2). Similar results were obtained with the human blood. In conclusion, the intracellular alkalizing effect of THAM is caused by the induced decrease of PCO(2) linked to the extracellular non-bicarbonate buffer capacity: The smaller the concentration of extracellular non-bicarbonate buffer, the higher the PCO(2) decrease caused by THAM.

Evaluation of BCECF fluorescence ratio imaging to properly measure gastric intramucosal pH variations in vivo

Our purpose is to evaluate intramucosal gastric pH video imaging by 2('),7(')-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorescence ratio techniques. We use a video endoscopic imaging system and BCECF as the pH fluorescent probe. Systemic in vivo pH variations are studied in 10 pigs: five in the control group and five with respiratory acidosis induced through rebreathing. The intramucosal pH of the gastric wall is measured every 5 s and the results demonstrate a good correlation (pearson correlation=0.832) between blood gases pH measurements and pH measured with the video endocopic imaging system. Our results confirm the feasibility of using BCECF fluorescence pH imaging to measure intramucosal pH in vivo.

Inhibitory effects of cariporide on LPC-induced expression of ICAM-1 and adhesion of monocytes to smooth muscle cells in vitro

To explore the effects of cariporide on the expression of intercellular adhesion molecule-1 (ICAM-1) and the adhesion of monocytes to vascular smooth muscle cells (SMC) in vitro. Monocytes were isolated from human peripheral blood by the Ficoll-Hypaque method. The expression of ICAM-1 in SMC was detected by ELISA. The adhesion of monocytes to SMC was stimulated by lysophosphatidylcholine (LPC). The adhesion ratio of monocytes was assayed by measuring protein contents. The intracellular pH ([pH]i) of SMC was measured with 2' ,7' -bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Preincubation of SMC with LPC alone (5 microg/mL) for 4 h markedly enhanced the expression of ICAM-1 in SMC and the rate of the adhesion of monocytes to SMC in a concentration-dependent and time-related manner. LPC simultaneously also induced an increase of [pH]i value in SMC. Cariporide concentration-dependently reduced the adhesion ratio of monocytes to SMC and the expression of ICAM-1 in SMC induced by LPC. The inhibitory effects of cariporide on the expression of ICAM-1 in SMC and the adhesion of monocytes to SMC also were associated with blocking LPC-induced elevation of the [pH]i value in SMC. LPC-induced monocyte-SMC adhesion may be mediated via activation of the Na+/H+ (NHE) exchanger. The action mechanism of cariporide may be related with inhibition of activation of the Na+/H+ exchanger of plasma membranes and ICAM-1 expression on the surface of SMC induced by LPC.

Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily.

Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events.

Expression of the Na+/H+ exchanger regulatory protein family in genetically hypertensive rats.

To examine a possible involvement of a regulatory protein of Na+/H+ exchanger (NHE) in the increased renal NHE activity in spontaneously hypertensive rats (SHR), we investigated mRNA expression of inhibitory members of the NHE regulatory protein family, NHERF1 and NHERF2, in the kidney. Prehypertensive 4-week-old and hypertensive 11-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were used to determine the changes in NHE activity and NHERF family expression in the kidney. Dahl salt sensitive (DS) and resistant rats were also used to examine whether these changes are specific for SHR. mRNA expression in the kidney was quantified by RNase protection assay. The NHE activity in primary cultured proximal tubular cells was measured as Na-dependent pHi recovery rate by the NH4Cl prepulse technique with 2'7'-bis-(2-carboxyethyl)-5.6-carboxyfluorescein (BCECF). NHERF1 mRNA expression was significantly decreased in both prehypertensive and hypertensive SHR in comparison with age-matched WKY rats, whereas NHERF2 mRNA expression was significantly increased in SHR only in the hypertensive period. Antihypertensive treatment did not abolish these changes seen in control SHR. On the other hand, hypertensive DS rats fed a high-salt diet showed significant decreases in NHE activity and NHE3 mRNA expression compared with normotensive DS rats fed a low-salt diet, without significant changes in NHERF1 and NHERF2 mRNA expression. These results suggest that decreased expression of NHERF1 may be related to the enhanced NHE activity in SHR and that these changes are likely to be genetically determined, whereas the increased NHERF2 expression may be induced as a compensatory mechanism.

Na+/H+ exchanger inhibitor, SM-20220, is protective against excitotoxicity in cultured cortical neurons.

Recently, it has been reported that Na+/H+ exchanger (NHE) inhibitors demonstrated protective effects on ischemia/reperfusion brain injury in animal models. However, the mechanisms by which the neurons were protected against ischemic insult remain unclear. To reveal the cellular mechanism of the NHE inhibitor on the neuronal death, we examined the effects of a selective NHE inhibitor, SM-20220 (N-[aminoiminomethyl]-1-methyl-1H-indole-2-carboxamide methanesulfonate), on glutamate-induced neuronal death in rat cortical culture. Cortical neurons were prepared from 1-day old rats, and cultured on the glass-based dishes. Glutamate-induced neuronal death was assessed by staining the cells with propidium iodide. Morphological changes in the neurons were observed with a video-enhanced contrast-differential interference contrast microscope. The intracellular calcium concentration ([Ca2+]i) and the intracellular pH (pHi) were measured by fluorescence imaging with a confocal laser microscope using fluo-3/acetoxymethylester (AM) and 2', 7'-bis-2-carboxy-ethyl-5(6)-carboxyfluorescein (BCECF)/AM as a fluorescent dye, respectively. SM-20220 (0.3 to 30 nmol/L) dose-dependently attenuated glutamate (300 micromol/L)-induced neuronal death over a period of 6 hours, and inhibited the acute cellular swelling following glutamate (500 micromol/L) exposure. Dual peaks of [Ca2+]i rise were observed at 5 and 12 minutes after glutamate (500 micromol/L) exposure, followed by a persistent rise. SM-20220 suppressed the persistent [Ca2+]i increase. SM-20220 inhibited intracellular acidification following glutamate (500 micromol/L) exposure. All of the events induced by glutamate were also inhibited by the N-methyl-d-aspartate receptor antagonist, MK-801, indicating the death process was excitotoxicity. NHE inhibitor is neuroprotective through inhibition of both persistent [Ca2+]i increase and acidification in excitotoxicity.

Intracellular alkalinization induces Ca 2+ influx via non-voltage-operated Ca 2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca 2+ concentration ([Ca 2+] i) is the primary determinant of contraction, and the intracellular pH (pH i) modulates contractility. Using fura-2 and 2′,7′-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH i on [Ca 2+] i. The application of the NH 4Cl induced concentration-dependent increases in both pH i and [Ca 2+] i. The extent of [Ca 2+] i elevation induced by 20 mM NH 4Cl was approximately 50% of that obtained with 100 mM K +-depolarization. The NH 4Cl-induced elevation of [Ca 2+] i was completely abolished by the removal of extracellular Ca 2+ or the addition of extracellular Ni 2+. The 100 mM K +-induced [Ca 2+] i elevation was markedly inhibited by a voltage-operated Ca 2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca 2+ channel blocker, SKF96365. On the other hand, the NH 4Cl-induced [Ca 2+] i elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca 2+ influx via non-voltage-operated Ca 2+ channels and thereby increases [Ca 2+] i in the vascular smooth muscle cells. The alkalinization-induced Ca 2+ influx may therefore contribute to the enhancement of contraction.

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.

Regulation of intracellular pH in rat renal inner medullary thin limbs of Henle's loop.

Regulation of intracellular pH (pHi) was studied in isolated rat renal inner medullary thin limbs of Henle's loop in bicarbonate/phosphate-buffered medium with high pCO2, high osmolality ( congruent with670 mosmol/kg H2O; 270 mM urea; 180 mM NaCl), organic osmolytes, and a pH of 6.8 to approximate the physiological in vivo environment. The pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi. Resting pHi was always acid and significantly more acid in descending thin limb (DTL) cells than in ascending thin limb (ATL) cells from pure or mixed-type thin limbs. Resting pHi was slightly but significantly higher in both DTLs and ATLs in high osmolality ( approximately 670 mosmol/kg H2O) than in low osmolality ( approximately 290 mosmol/kg H2O) medium but not when sucrose replaced urea. In both DTLs and ATLs the rate of recovery of pHi following additional acidification with an NH4Cl pulse was reduced by Na+ removal from the medium and by the addition of 60 microM HOE642 (an inhibitor of the Na+/H+ exchanger, NHE1), 55 microM S1611 (inhibitor of Na+/H+ exchanger, NHE3), 1 microM bafilomycin A1 (an inhibitor of vacuolar H+ -ATPase), or 20 microM Schering 28080 (an inhibitor of H+ -K+ -ATPase) to the medium. Resting pHi was also reduced by 60 microM HOE642, 55 microM S1611, and 20 microM Schering 28080. In both DTLs and ATLs, RT-PCR revealed message for NHE1, NHE3, and vacuolar H+ -ATPase; immunocytochemistry demonstrated the expression of the protein for NHE1 (basolateral membrane), NHE3 (luminal membrane), and H+ -K+ -ATPase (luminal membrane). These data suggest that pHi in rat inner medullary thin limbs is regulated by urea and by basolateral and luminal H+ extrusion via NHE1, NHE3, vacuolar H+ -ATPase, and H+ -K+ -ATPase.

Na+-dependent pH Regulation by the Amitochondriate Protozoan Parasite Giardia intestinalis

Giardia intestinalis is a pathogenic fermentative parasite, which inhabits the gastrointestinal tract of animals and humans. G. intestinalis trophozoites are exposed to acidic fluctuations in vivo and must also cope with acidic metabolic endproducts. In this study, a combination of independent techniques ((31)P NMR spectroscopy, distribution of the weak acid pH marker 5,5-dimethyl-2,4-oxazolidinedione (DMO) and the fluorescent pH indicator 2',7'-bis (carboxyethyl)-5,6-carboxyfluorescein (BCECF)) were used to show that G. intestinalis trophozoites exposed to an extracellular pH range of 6.0--7.5 maintain their cytosolic pH (pH(i)) within the range 6.7--7.1. Maintenance of the resting pH(i) was Na(+)-dependent but unaffected by amiloride (or analogs thereof). Recovery of pH(i) from an intracellular acidosis was also Na(+)-dependent, with the rate of recovery varying with the extracellular Na(+) concentration in a saturable manner (K(m) = 18 mm; V(max) = 10 mm H(+) min(-1)). The recovery of pH(i) from an acid load was inhibited by amiloride but unaffected by a number of its analogs. The postulated involvement of one or more Na(+)/H(+) exchanger(s) in the regulation of pH(i) in G. intestinalis is discussed.

The cultured human gastric cells HGT-1 express the principal transporters involved in acid secretion.

HGT-1 is a human cell line sharing a number of physiological features with gastric parietal cells. HGT-1 cell monolayers were able to secrete H+ when stimulated with histamine (calculated external pH variation, deltapH(e) 0.46+/-0.05) as assessed using the impermeant, pH-sensitive fluorescence dye 8-hydroxypyrene-1,3,6-trisulphonic acid, trisodium salt (HPTS). Treatment with 100 microM omeprazole inhibited the histamine-induced apical acidification by about 60%. Intracellular pH (pH(i)) measurements using the fluorescent pH-sensitive dye 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) demonstrated the expression of a functional, omeprazole-sensitive H+/K+-pump. A monoclonal antibody directed against the alpha subunit of the H+/K+-ATPase immunoprecipitated a 95-kDa protein from HGT-1 cells and human stomach which corresponds to the expected molecular size of the native protein. HGT-1 cells were also positive for the anion exchanger AE2 that is expressed in gastric parietal cells. In addition, we identified a histamine- and pH(i)-sensitive Na+/H+ exchanger in HGT-1 cells, which might correspond to the functional expression of the NHE4 isoform that has been detected in gastric epithelial cells as well as in primary cultured parietal cells. HGT-1 cells therefore display the principal features of parietal cells and might represent an interesting cell culture model for studying the regulatory mechanisms involved in acid secretion.

Intracellular pH in isolated rat renal papillary thin limbs of Henle's loop.

Intracellular pH (pHi) was measured in isolated, nonperfused and perfused rat papillary thin limbs of Henle's loops in N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES)- or HEPES/bicarbonate-buffered medium at pH 7.4 using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Resting pHi was about 6.7 in descending thin limbs (DTL) and about 6.9 in ascending thin limbs (ATL), even with a medium pH of 7.4. These values appeared to reflect the acid pH of the blood in the neighboring vasa recta found in vivo. The resting pHi did not differ whether or not the medium contained bicarbonate although the total buffering capacity of the tubule cells was increased in the presence of bicarbonate. In nonperfused DTL and ATL, pHi was further acidified following an NH4Cl pulse. The rate of recovery of pHi from this level to the resting pHi was reduced by Na+ removal from the bath in both DTL and ATL and by the addition of ethylisopropylamiloride (EIPA) to the bath in the presence of Na+ in DTL. The rate of recovery was not affected by Cl- removal from the bath or K+ (75 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) addition to the bath in either DTL or ATL. These results suggest that the common, amiloride-sensitive, basolateral Na+/H+ exchanger plays a role in the regulation of pHi in rat papillary DTL but that a different basolateral Na+/H+ exchanger or a luminal Na+/H+ exchanger is important in rat papillary ATL.

In Vivo Application of Intestinal pH Measurement Using 2,7‘‐Bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) Fluorescence Imaging

Measurement of gastrointestinal intramucosal pH (pHim) has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolutions. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). This study aimed to set up and validate a fluorescence imaging technique to measure in vivo the intramucosal pH (pHim) of the intestine. The intestine was inserted into an optical chamber placed under a microscope. Animals were injected intravenously with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pHim by constructing a calibration curve. The pHim controls were performed with a pH microelectrode and were correlated with venous blood gas sampling. Results show that pHim is determined with an accuracy of +/- 0.07 pH units and a response time of 1 min. In conclusion pHim mapping of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurements.

Basolateral regulation of pHi in proximal tubules of avian loopless and long-looped nephrons in bicarbonate

In isolated, nonperfused chicken proximal tubules from both loopless reptilian-type and long-looped mammalian-type nephrons, resting intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.1 under control HCO3- conditions [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/5 mM HCO3(-)-buffered medium with pH 7.4 at 37 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this level to the resting level in proximal tubules from both nephron types was (1) significantly reduced by the removal of Na+ or both Na+ and Cl- from the bath, and (2) unaffected by the removal of Cl- from the bath or the presence of a high K+ concentration or Ba2+ in the bath. In proximal tubules from long-looped mammalian-type, but not loopless reptilian-type, nephrons, dpHi/dt was significantly reduced by the addition of either 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) to the bath. These data suggest that a Na+/H+ exchanger and most likely a Na(+)-dependent Cl-/HCO3- exchanger are involved in basolateral regulation of pHi in mammalian-type nephrons whereas none of the commonly identified basolateral acid-base transporters appear to be involved in regulation of pHi in reptilian-type nephrons.