Fluorescent pH Indicator Research Articles

PH regulation in apoplastic and cytoplasmic cell compartments of leaves.

The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO2, HCl or NH3 differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO2 either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO2 shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO2 was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO2-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO2 under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO2 stress increased in the presence of oxygen with the length of previous exposure to high CO2. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO2 was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO2, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO2 stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m2 leaf area)(-1) s(-1) or less. Proton export under acid stress into the vacuole was about 1200 nmol m(-2) s(-1). The kinetics of pH responses to the addition or withdrawal of CO2 indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast.

The Fluorescent pH-Indicator BCECF: An Inhibitor of the Vacuolar ATPase from Chenopodium rubrum

Summary Selective vacuolar accumulation of the fluorescent pH-indicator 2′, 7′-bis (2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF) may allow to study vacuolar acidification in vivo (Brauer et ai., 1995), which in plant cells is driven by two distinct proton pumps: the V-ATPase (EC 3.6.1.3) and the V-PPase (EC 3.6.1.1). The use of BCECF may help to investigate these proton pumps in vivo . A necessary precondition for such a study is that both enzymes are not affected by the indicator. This was investigated using tonoplast enriched membrane vesicles from Chenopodium rubrum L. suspension cells. We found that BCECF was a strong inhibitor of the V-ATPase. Half-maximal inhibition of the V-ATPase was observed with 10 μmol/L BCECF. The Hill constant (n H = 1.3) for inhibition of the V-ATPase insdicates the presence of two binding sites for BCECF. BCECF was less inhibitory to the vacuolar PPase than to the vacuolar ATPase; half-maximal inhibition was obtained with 500 μmol/L BCECF (n H = 0.5). Moreover, we found that BCECF (2 μmol/L) increased the affinity of the V-ATPase for its specific inhibitor bafilomycin, i.e. it decreased the concentration for half-maximal inhibition from 2 nmol/L to 0.17 nmol/L bafilomycin. We suggest positive cooperation between binding of BCECF and bafilomycin to the V-ATPase. Fluorescein acid and its derivatives 5-carboxyfluorescein, 6-carboxyfluorescein, BCECF and BCECF-AM (acetoxymethyl ester of BCECF) gave similar inhibition of the V-ATPase, indicating that the chromophore fluorescein interacts with the enzyme. Finally, living cells were incubated for 1 h in BCECF-AM (5 μmol/L), a cell-permeable ester derivative of BCECF that is hydrolyzed by cytosolic esterases to yield intracellularly trapped BCECF. Tonoplast vesicles were isolated by sucrose density gradient centrifugation from cells washed free from the loading solution. V-ATPase and V-PPase activity of these tonoplast vesicles showed a decrease of 18 % and 45 %, respectively. Together, these data indicate that the widely used in vivo pH-indicator BCECF alters V-ATPase activity and properties and can therefore interfere with the pH of cellular compartments acidified or alkalized by the V-ATPase, whereas BCECF-AM added to living cells inhibits the V-PPase by an interaction with cellular mechanisms regulating its activity.

Sodium overload through voltage-dependent Na + channels induces necrosis and apoptosis of rat superior cervical ganglion cells in vitro

Using the failure to exclude trypan blue as a criterion for cell death, we found that veratridine, the voltage-dependent Na + channel activator, exerted its toxicity to cultured sympathetic neurons in a dose-dependent manner (half-maximal toxicity occurred at 2 μM). The co-presence of tetrodotoxin completely reversed the toxicity only at concentrations of veratridine <20μM. Veratridine neurotoxicity was due to the influx of Na +; a medium low in Na + (36 mM) completely abolished its neurotoxicity, whereas a Ca 2+-free medium did not attenuate its neurotoxicity. Furthermore, the buffering action of 1,2-Bis-(2-aminophenoxy)ethane-N,N,N′,N′,-tetraacetate (BAPTA) on veratridine-induced increase in intracellular Ca 2+ levels neither blocked veratridine neurotoxicity in normal medium, nor attenuated the low Na + effect. Elevated K + effectively blocked veratridine neurotoxicity in a Ca 2+-dependent manner. Cytoplasmic pH measurements using a fluorescent pH indicator demonstrated that cellular acidification (from pH 7.0 to pH 6.5) occurred upon treatment with veratridine. Both veratridine-induced acidification and cell death were ameliorated by 5-(N-ethyl-N-isopropyl)amiloride, the specific inhibitor of the Na +/H + exchanger (IC 50 = 0.5 μM). Finally, necrosis occurred predominantly in veratridine neurotoxicity, but both staining with bis-benzimide and TUNEL analysis showed nuclear features of apoptosis in sympathetic neurons undergoing cell death.

Activity-Dependent Intracellular Acidification Correlates with the Duration of Seizure Activity

Synchronized neuronal activity (seizures) can appear in the presence or absence of synaptic transmission. Mechanisms of seizure initiation in each of these conditions have been studied, but relatively few studies have addressed seizure termination. In particular, how are seizures terminated in the absence of synaptic activity where there is no loss of excitatory drive or augmentation of inhibitory inputs? We have studied dynamic activity-dependent changes of intracellular pH in the absence of synaptic transmission using the fluorescent pH indicator carboxylseminaphthorhodafluo-1. During epileptiform activity we observed intracellular acidification, whereas between seizures the intracellular pH recovered. Experimental conditions that shortened the epileptiform discharge correlated with more rapid intracellular acidification. On the other hand, experimental manipulation of intracellular pH altered the duration of the seizure discharge, with acidification resulting in early termination of the epileptiform activity. These data show a direct relationship between the level of intracellular acidification and the duration of the seizures, suggesting that an intracellular pH-dependent process can terminate nonsynaptic neuronal synchronization.

Measurement of cellular stimulation through monitoring pH changes by bead injection fluorescence microscopy

This work presents a method for extracellular and intracellular pH measurements in live cells based on a combination of the bead injection (BI) technique and fluorescence microscopy. For extracellular pH measurement, cells are grown on fluorescent beads, packed into a small column by a sequential injection instrument, and fluorescence intensity from the beads stained by the indicator is recorded by a fluorescence microscope. The method is applied to quantifying carbachol stimulation of Chinese hamster ovary (CHO) cells transfected with the m1 muscarinic receptor and is verified by a glucose depletion experiment. The results yield an EC 50 value of 1 μM for carbachol, which is in reasonable agreement with the literature value 3 μM determined by an existing potentiometric technique for measuring acid release. The intracellular measurement utilizes CHO M1 cells growing on non-fluorescent beads. For this method the cells rather than the beads are stained by incubating them in a solution of the fluorescent pH indicator BCECF. The cells are also stimulated with carbachol and the intracellular pH dependent fluorescence from the cells is recorded. The results show dependence between intracellular pH changes and carbachol concentration and yield an EC 50 value of 4 μM.

Carbon Dioxide Permeability of Aquaporin-1 Measured in Erythrocytes and Lung of Aquaporin-1 Null Mice and in Reconstituted Proteoliposomes

Measurements of CO(2) permeability in oocytes and liposomes containing water channel aquaporin-1 (AQP1) have suggested that AQP1 is able to transport both water and CO(2). We studied the physiological consequences of CO(2) transport by AQP1 by comparing CO(2) permeabilities in erythrocytes and intact lung of wild-type and AQP1 null mice. Erythrocytes from wild-type mice strongly expressed AQP1 protein and had 7-fold greater osmotic water permeability than did erythrocytes from null mice. CO(2) permeability was measured from the rate of intracellular acidification in response to addition of CO(2)/HCO(3)(-) in a stopped-flow fluorometer using 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) as a cytoplasmic pH indicator. In erythrocytes from wild-type mice, acidification was rapid (t((1)/(2)), 7.3 +/- 0.4 ms, S.E., n = 11 mice) and blocked by acetazolamide and increasing external pH (to decrease CO(2)/HCO(3)(-) ratio). Apparent CO(2) permeability (P(CO(2))) was not different in erythrocytes from wild-type (0.012 +/- 0.0008 cm/s) versus null (0.011 +/- 0.001 cm/s) mice. Lung CO(2) transport was measured in anesthetized, ventilated mice subjected to a decrease in inspired CO(2) content from 5% to 0%, producing an average decrease in arterial blood pCO(2) from 77 +/- 4 to 39 +/- 3 mm Hg (14 mice) with a t((1)/(2)) of 1.4 min. The pCO(2) values and kinetics of decreasing pCO(2) were not different in wild-type versus null mice. Because AQP1 deletion did not affect CO(2) transport in erythrocytes and lung, we re-examined CO(2) permeability in AQP1-reconstituted liposomes containing carbonic anhydrase (CA) and a fluorescent pH indicator. Whereas osmotic water permeability in AQP1-reconstituted liposomes was >100-fold greater than that in control liposomes, apparent P(CO(2)) (approximately 10(-3) cm/s) did not differ. Measurements using different CA concentrations and HgCl(2) indicated that liposome P(CO(2)) is unstirred layer-limited and that HgCl(2) slows acidification because of inhibition of CA rather than AQP1. These results provide direct evidence against physiologically significant AQP1-mediated CO(2) transport and establish an upper limit to the CO(2) permeability through single AQP1 water channels.

Extracellular carbonic anhydrase facilitates carbon dioxide availability for photosynthesis in the marine dinoflagellate prorocentrum micans

This study investigated inorganic carbon accumulation in relation to photosynthesis in the marine dinoflagellate Prorocentrum micans. Measurement of the internal inorganic carbon pool showed a 10-fold accumulation in relation to external dissolved inorganic carbon (DIC). Dextran-bound sulfonamide (DBS), which inhibited extracellular carbonic anhydrase, caused more than 95% inhibition of DIC accumulation and photosynthesis. We used real-time imaging of living cells with confocal laser scanning microscopy and a fluorescent pH indicator dye to measure transient pH changes in relation to inorganic carbon availability. When steady-state photosynthesizing cells were DIC limited, the chloroplast pH decreased from 8.3 to 6.9 and cytosolic pH decreased from 7.7 to 7.1. Re-addition of HCO3- led to a rapid re-establishment of the steady-state pH values abolished by DBS. The addition of DBS to photosynthesizing cells under steady-state conditions resulted in a transient increase in intracellular pH, with photosynthesis maintained for 6 s, the amount of time needed for depletion of the intracellular inorganic carbon pool. These results demonstrate the key role of extracellular carbonic anhydrase in facilitating the availability of CO2 at the exofacial surface of the plasma membrane necessary to maintain the photosynthetic rate. The need for a CO2-concentrating mechanism at ambient CO2 concentrations may reflect the difference in the specificity factor of ribulose-1,5 bisphosphate carboxylase/oxygenase in dinoflagellates compared with other algal phyla.

Short-Term Effects of Thyroid Hormones on the Na/H Antiport in L-6 Myoblasts: High Molecular Specificity for 3,3',5-Triiodo-L-Thyronine

The thyroid hormones l-T3 and l-T4 were shown to activate the Na/H antiport in L-6 cells from rat skeletal muscle by a rapid, nongenomic mechanism. Under pH equilibrium conditions, a significant rise in the intracellular pH, measured by the fluorescent pH indicator 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein was observed after the addition of physiological concentrations (10−10m) of either l-T3 or l-T4, but with different time courses. l-T3 at all concentrations increased the pH after a delay of 2 min, whereas l-T4 showed a concentration-dependent lag time, going from 11 min at 10−11m down to 5 min for a hormone concentration of 10−6m. The effect of l-T4 was blocked in the presence of the 5′-deiodinase inhibitor 6-n-propyl-2-thiouracil, suggesting that the difference in lag time between l-T3 and l-T4 was due to the 5′-deiodination process that transforms l-T4 into the bioactive l-T3. In short term studies (<5 min), a high molecular specificity for l-T3 was found, as l-T4, rT3, the d-isomer of T3, and the deaminated analogues were ineffective at physiological concentrations. In analogy with the results found at equilibrium, intracellular pH recovery from an acid load and set-point were increased after 2 min for l-T3 (10−9m) and after 10 min for l-T4 (10−9m). The effect of the hormones on the intracellular pH was completely blocked by the specific antiport inhibitor 5-(ethyl-N-isopropyl)amiloride. These findings suggest that thyroid hormones may play an active role in the recovery from muscular acidosis through direct stimulation of the Na/H antiport.

Concerning Regioselective Photochemical Intermolecular Proton Transfer from Hypericin

Using epifluorescence microscopy on lipid vesicles containing hypericin or several of its O-alkylated derivatives together with a fluorescence pH indicator it was shown that upon excitation of the respective hypericinate ion an excited state derived proton is transferred to the indicator molecule. In addition, it could be also unequivocally derived that this proton originates from one of the peri-hydroxyl groups of the pigment.

Short-term effects of thyroid hormones on the Na/H antiport in L-6 myoblasts: high molecular specificity for 3,3',5-triiodo-L-thyronine.

The thyroid hormones L-T3 and L-T4 were shown to activate the Na/H antiport in L-6 cells from rat skeletal muscle by a rapid, nongenomic mechanism. Under pH equilibrium conditions, a significant rise in the intracellular pH, measured by the fluorescent pH indicator 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein was observed after the addition of physiological concentrations (10(-10) M) of either L-T3 or L-T4, but with different time courses. L-T3 at all concentrations increased the pH after a delay of 2 min, whereas L-T4 showed a concentration-dependent lag time, going from 11 min at 10(-11) M down to 5 min for a hormone concentration of 10(-6) M. The effect of L-T4 was blocked in the presence of the 5'-deiodinase inhibitor 6-n-propyl-2-thiouracil, suggesting that the difference in lag time between L-T3 and L-T4 was due to the 5'-deiodination process that transforms L-T4 into the bioactive L-T3. In short term studies (<5 min), a high molecular specificity for L-T3 was found, as L-T4, rT3, the D-isomer of T3, and the deaminated analogues were ineffective at physiological concentrations. In analogy with the results found at equilibrium, intracellular pH recovery from an acid load and set-point were increased after 2 min for L-T3 (10(-9) M) and after 10 min for L-T4 (10(-9) M). The effect of the hormones on the intracellular pH was completely blocked by the specific antiport inhibitor 5-(ethyl-N-isopropyl)amiloride. These findings suggest that thyroid hormones may play an active role in the recovery from muscular acidosis through direct stimulation of the Na/H antiport.

Pigmentation and Acid Storage in the Tunic: Protective Functions of the Tunic Cells in the Tropical Ascidian Phallusia nigra

The tunic morphology of the tropical ascidian Phallusia nigra was studied with regard to its protective functions by means of light and electron microscopy. The tunic has a smooth surface free of epibionts. Beneath the tunic surface, phagocytic cells containing pigment granules (pigmentary tunic phagocytes) are densely distributed and form the pigmentary layer. These pigments may protect the body from irradiation. When poorly pigmented specimens growing in a dim location are transplanted to a bright location, pigmentation is induced and the tunic becomes black. In this process, tunic phagocytes probably produce pigment granules and migrate to the pigmentary layer. The tunic under the pigmentary layer is packed with highly vacuolated cells (tunic bladder cells). Vital staining with LysoSensor, a fluorescent pH indicator, demonstrated that the vacuoles contain strong acid. Acidic fluid leaks out when the tunic is injured. Leaking acid could serve to disinfect the injured area, be an irritant to potential predators, and/or be involved in anti-fouling. Additional key words: phagocytosis, pigment granules, light, protection, anti-predation, antifouling An integumentary tissue has a variety of functions to protect the body from the environment. It can be the most important defense system in many sessile organisms. The only sessile chordates are ascidians, whose bodies are wholly covered with a leathery or gelatinous matrix called the tunic. Although the tunic is a kind of extracellular matrix situated outside the epidermis, free cells called tunic cells are distributed within it, and the presence of tunic cells makes it a mesenchyme-like tissue. Many types of tunic cells have been described in ascidians: they are involved in phagocytosis (De Leo et al. 1981; Hirose et al. 1994), conduction of impulses (Mackie & Singla 1987), tunic contraction (Hirose & Ishii 1995), bioluminescence (Chiba et al. 1998), photosynthetic symbiosis (Hirose et al. 1996), and allorecognition between colonies (Hirose et al. 1997). The ascidian tunic is, therefore, an attractive material for investigating the diversity of integumentary functions required for adaptation to the environment. In the tropics, sessile organisms can expand potena E-mail: euichi@ sci.u-ryukyu.ac.jp tial habitat through protective systems against strong sunlight (cf. Jokiel 1980; Bingham & Reyns 1999) as well as other environmental pressures, such as predation, infection, and competition for space. Phallusia (= Ascidia) nigra SAVIGNY 1816 is a large solitary ascidian inhabiting shallow tropical waters. Its tunic and mantle contain black pigment that may protect the body from sunlight. Moreover, P. nigra is known to concentrate vanadium and to have an extremely acidic tunic fluid, and these properties may be effective for anti-fouling as well as anti-predation (Stoecker 1978, 1980a,b). The tunic morphology and chemical analysis have been reported for this species with special reference to anti-fouling function (Stoecker 1978), but ultrastructural studies had not been carried out. I describe here the morphology of the tunic cells in P. nigra and examine their possible protective functions in adaptation to the tropical environment. There are two types of tunic cells: one type contains phagosomes and pigment granules, and the other type stores strongly acidic fluid in large vacuoles. These features may be concerned with protection against infection (or scavenging), light, and predation, respectively. This content downloaded from 157.55.39.248 on Sun, 12 Mar 2017 18:08:46 UTC All use subject to http://about.jstor.org/terms Pigmentation and acid storage in ascidian tunic

Alkalization produced by high-dose carbachol in HSG cell line is independent of Ca2+.

The aim of this investigation was to clarify the mechanism of alkalization induced by carbachol in HSG cells. Cells of the HSG cell line derived from a human submandibular gland adenocarcinoma and those of the A-431 human epidermoid carcinoma cell line were loaded with a fluorescent pH indicator, BCECF/AM, and the change in the intracellular pH of adherent cells and suspended ones were measured following stimulation with various concentrations (10(-7) M to 10(-2) M) of neurotransmitters (carbachol, noradrenaline, and isoproterenol). Isoproterenol did not cause alkalization of either cell type, whereas, noradrenaline and carbachol alkalized both types over the concentration ranges of 10(-6) M to 3 x 10(-3) M (HSG cell by noradrenaline), 10(-7) M to 2 x 10(-4) M (A-431 cell by noradrenaline), and 7 x 10(-5) M to 10(-4) M (A-431 cell by carbachol). On the other hand, alkalization induced by carbachol in the HSG cells was recognized at concentrations higher than 6 x 10(-5) M, and it showed no upper limit in terms of carbachol concentration. This high-dose carbachol alkalization was not eliminated by preincubation with nifedipine (100 microM), a Ca2+ channel blocker, or with thapsigargin (100 microM), a microsomal Ca(2+)-ATPase inhibitor. The alkalization system induced by carbachol in the HSG cell was quite different from that in the A-431 cell, and that induced by high-dose carbachol in HSG cells appeared to be independent of intracellular Ca2+. These findings will be useful to clarify the mechanism of salivary secretion stimulated by neurotransmitters.

Effect of ANG II on pHi, [Ca2+]i, and contraction in rabbit ventricular myocytes from infarcted hearts.

In this study we examined Na+/H+ exchange activity, Ca2+ transients, and contractility in rabbit ventricular myocytes isolated from normal and chronically (8-12 wk) infarcted left ventricles. Myocytes from infarcted hearts (post-MI myocytes) were isolated from the peri-infarcted region of the left ventricle. Intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured with the fluorescent pH indicators seminaphthorhodafluor 1 and fluo 3, respectively, and contractility was assessed from changes in cell shortening during field stimulation. Experiments were performed at extracellular pH 7. 4 in the presence and absence (HEPES buffer) of CO2 and HCO-3. Our findings demonstrate that 1) myocytes after myocardial infarction (post-MI) were significantly larger than normal, 2) post-MI hypertrophy was not accompanied by changes in non-CO2 intracellular buffering power, 3) post-MI hypertrophy did not significantly affect the ability of Na+/H+ exchange to mediate pHi recovery from intracellular acidosis, 4) the stimulatory effect of ANG II (100 nM) on Na+/H+ exchange was significantly reduced in post-MI myocytes, 5) in HCO-3-buffered solutions, ANG II did not significantly stimulate pHi recovery from acidosis in post-MI myocytes, 6) the angiotensin AT1 receptor mediates the stimulatory action of ANG II on Na+/H+ exchange in normal and post-MI myocytes, and 7) the stimulatory effect of ANG II on the Ca2+ transient and contraction was blunted in post-MI myocytes bathed in HEPES-buffered solution. A suppressed ventricular responsiveness to ANG II may be beneficial in the intact myocardium by attenuating ATP consumption and by reducing intracellular Na+ accumulation during ischemia-reperfusion.

Protein carboxyl methylation controls intracellular pH in human platelets.

Carboxyl methylation is a reversible post-translational event which regulates the function of several cellular proteins. Because the human Na+-H+ antiporter (NHE-1) possesses a C-terminal consensus sequence for carboxyl methylation, we examined the role of protein carboxyl methylation in the regulation of intracellular pH homeostasis. Experiments were conducted using human platelets and N-acetyl-S-trans,trans-farnesyl-L cysteine (AFC), a specific prenylcysteine methyltransferase inhibitor. The effect of AFC on both basal intracellular pH (pHi) and on the kinetic properties of the Na+-H+ antiporter was characterized. pHi was determined in cell suspensions using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein tetraacetoxymethyl ester, a fluorescent pH indicator. The kinetics properties of the Na+-H+ antiporter activity were determined using platelets acidified with nigericin and challenged with varying extracellular concentrations of Na+. AFC (20 micromol/l) decreased basal pHi significantly (7.047 +/- 0.011 versus 7.133 +/- 0.012 for control, P< 0.001). The acidification was dose-dependent and reached steady state 3 min after AFC addition. In the absence of extracellular Na+, the platelets were acidified to the same extent with AFC or with ethanol (control): 6.530 +/- 0.031 versus 6.532 +/- 0.031 (P= 0.97). However, upon addition of Na+, the platelets treated with AFC showed a significant decrease in the maximal value for initial pHi recovery compared with controls: 0.788 +/- 0.041 versus 0.983 +/- 0.047 pH/min (P< 0.02). AFC also increased the Hill coefficient (2.89 +/- 0.22 versus 2.14 +/- 0.16, P < 0.03), and tended to decrease K0.5, the [Na+] corresponding to half-maximal activation (51.3 +/- 1.8 versus 60.5 +/- 3.9 mmol/l, P = 0.06) of the antiporter. Our data indicate that inhibition of carboxyl methylation reduces basal pHi and alters the kinetic properties of the Na+-H+ antiporter in human platelets, suggesting that carboxyl methylation is implicated in the regulation of intracellular pH homeostasis.

A Trace Amount of the Human Pro-apoptotic Factor Bax Induces Bacterial Death Accompanied by Damage of DNA

An amount of human pro-apoptotic Bax as low as 0.01% of total protein was sufficient to cause cell death in Escherichia coli. The bacterial cell death was examined using a viable bacteria-specific fluorescence indicator system and loss of colony formation ability. Co-expression of anti-apoptotic Bcl-xL showed a modest inhibitory effect on the cell death caused by Bax. The trace amount of Bax elongated E. coli and accumulated monounsaturated fatty acids, suggesting an unusual metabolism of redox in the host. In fact, an increase of KCN-dependent O2 consumption accompanied the expression of Bax. At the same time, a fluorescent pH indicator showed the apparent accumulation of protons outside the cell, suggesting that the membrane is intact. Bax increased the level of superoxide anion as measured by the expression of superoxide-dependent promoter. Nicked DNA was significantly generated, and the frequency of mutations resistant to rifampicin was increased by 30-fold, depending upon the expression of Bax. It is proposed that trace amounts of Bax increase oxygen consumption, triggering generation of superoxide, which affects DNA, leading to bacterial death.

Taurine efflux and intracellular pH during astrocyte volume regulation.

Cytotoxic cerebral edema is characterized by enlarged astroglial cells. In tissue culture, osmotically swollen astrocytes return toward normal volume over a period of 15-30 min in a process termed regulatory volume decrease (RVD). RVD is due, in part, to net efflux of taurine and other amino acids. Our objective in these studies was to examine changes in astrocyte intracellular pH (pHi) which may be related to taurine loss during RVD. We hypothesized net efflux of anionic taurine abandons a proton inside the cell, thus lowering pHi. Primary cultures of cerebral astrocytes were prepared from neonatal rats pups and grown on glass coverslips. Confluent cells were loaded at 37 degrees C with the fluorescent pH indicator BCECF. Fluorescence intensity ratios for excitation wavelengths of 440 nm and 494 nm (530 nm emission) were computed every 2 sec. Intensity ratios were calibrated to pHi at the end of each experiment using 140 mM KCl plus 8.6 microM nigericin at pH 7.4. pHi was measured in isoosmotic Hepes-buffered saline (290 mOsm) and then in hypoosmotic Hepes-buffered saline (200 mOsm) in the presence of 0.5 mM amiloride. Some solutions also contained 150 microM niflumic acid (NA). Cellular taurine content was determined in parallel studies using HPLC. Changes in pHi were compared between groups using Student's t-test with Bonferroni correction. Significance was assumed if p < 0.05. In isoosmotic saline, mean +/- SEM pHi was 7.58 +/- 0.04 and decreased to 7.35 +/- 0.09 after adding amiloride. Hypoosmotic exposure caused a further drop in pHi of 0.29 +/- 0.03 within 15 min. Recovery of pHi in isoosmotic saline was amiloride-sensitive. Subsequent hypoosmotic exposure after recovery in isoosmotic saline produced a change in pHi which was 81 +/- 9% of the change measured during the initial hypoosmotic exposure. Taurine content decreased from 147 +/- 6 nmol/(mg protein) to 116 +/- 7 nmol(mg protein) during the 15 min hypoosmotic exposure in 0.5 mM amiloride. NA significantly reduced the hypoosmotically induced change in pHi to 0.17 +/- 0.02 while completely blocking taurine loss. Assuming an intracellular buffering power of 13 mM, the NA-sensitive intracellular acidification of cells during hypoosmotic exposure in the presence of 0.5 mM amiloride corresponds to 1.6 mequiv/l additional intracellular H+. This increase in intracellular H+ content is equivalent to approximately 32% of the NA-sensitive loss of taurine. The association of changes in pHi with taurine efflux is supported by these data; however, efflux of other weak acids may contribute to intracellular acidification during astrocyte RVD and a significant portion of taurine must leave the cell with a proton.

Changes in Luminal pH Caused by Calcium Release in Sarcoplasmic Reticulum Vesicles

Fast (milliseconds) Ca 2+ release from sarcoplasmic reticulum is an essential step in muscle contraction. To electrically compensate the charge deficit generated by calcium release, concomitant fluxes of other ions are required. In this study we investigated the possible participation of protons as counterions during calcium release. Triad-enriched sarcoplasmic reticulum vesicles, isolated from rabbit fast skeletal muscle, were passively loaded with 1 mM CaCl 2 and release was induced at pCa = 5.0 and pH = 7.0 in a stopped-flow fluorimeter. Accompanying changes in vesicular lumen pH were measured with a trapped fluorescent pH indicator (pyranin). Significant acidification (∼0.2 pH units) of the lumen occurred within the same time scale ( t 1/2 = 0.75 s) as calcium release. Enhancing calcium release with ATP or the ATP analog 5′-adenylylimidodiphosphate (AMPPNP) produced >20-fold faster acidification rates. In contrast, when calcium release induced with calcium with or without AMPPNP was blocked by Mg 2+, no acidification of the lumen was observed. In all cases, rate constants of luminal acidification corresponded with reported values of calcium release rate constants. We conclude that proton fluxes account for part (5–10%) of the necessary charge compensation during calcium release. The possible relevance of these findings to the physiology of muscle cells is discussed.

Na+/H+ exchanger activity does not contribute to protection by ischemic preconditioning in the isolated rat heart.

Despite evidence that pharmacological inhibition of the Na+/H+ exchanger (NHE) is cardioprotective, activation of NHE has been proposed as a protective mechanism of ischemic preconditioning (PC). In isolated rat ventricular myocytes (n=8 to 11 per group) loaded with the fluorescent pH indicator C-SNARF-1, we showed that HOE-642 (HOE) was a potent inhibitor of the sarcolemmal NHE (80% inhibition at 1 micromol/L); such inhibition was readily reversible by washout of the drug. We confirmed that 1 micromol/L HOE produces significant and reversible inhibition of NHE activity in isolated rat hearts as well (n=4), and in this model, we tested (n=8 per group) whether the presence of the drug during (1) the prolonged period of ischemia (40 or 60 minutes) or (2) the preceding brief periods of PC ischemia (3 minutes plus 5 minutes) modulates the protective efficacy of PC. In protocol 1, HOE was infused for 5 minutes immediately before the prolonged ischemic period. With 40 minutes of prolonged ischemia, the postischemic recovery of left ventricular developed pressure (LVDP) was 15+/-2% in controls and was improved to 45+/-7% with HOE (P<.05), 55+/-5% with PC (P<.05), and 68+/-2% with PC+HOE (P<.05 versus all groups). When the prolonged ischemic period was extended to 60 minutes, an additive effect of PC and HOE was readily apparent and LVDP recovery with PC+HOE (66+/-2%) was almost double that observed with HOE (37+/-4%) or PC (34+/-5%) alone (P<.05). In protocol 2, HOE was infused for 3 minutes immediately before each episode of PC ischemia and was subsequently washed out before a 40-minute prolonged ischemic period (HOE+PC). LVDP recovery was 34+/-4% in controls and was improved to 57+/-2% with PC (P<.05) and 55+/-3% with HOE+PC (P<.05). Improved recovery of LVDP was matched by reduced creatine kinase leakage in all cases. Because coadministration of HOE (at a concentration sufficient to inhibit NHE activity) did not reduce the efficacy of PC in either protocol, we conclude that NHE activity does not contribute to the cardioprotective actions of PC. On the contrary, NHE inhibition during the prolonged ischemic period may enhance the protection afforded by PC.

Measurement of Carbonic Anhydrase Activity Using a Sensitive Fluorometric Assay

The dehydration reaction of bicarbonate was measured using the fluorescent pH indicator, 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), in combination with stopped-flow spectrofluorometry. The initial rate of bicarbonate dehydration was measured after mixing a pH 6.0 solution with a pH 8.0 solution containing bicarbonate. Addition of carbonic anhydrase to the pH 6.0 solution enabled the measurement of the initial rate of activity at physiological temperatures with resolution times of 2 ms. This assay was used to resolve differences in activity and sensitivity to sulfonamides by comparing mammalian carbonic anhydrase isoforms. The fluorescent technique used in this study is very sensitive, allowing the determination of initial rates with a protein concentration as little as 65 ng/ml. Pyranine can also be loaded into membrane vesicles to follow carbonic anhydrase activity within vesicles. The change in pH within vesicles is dependent on the concentration of externally added bicarbonate and the presence of carbonic anhydrase on either side of the membrane. Therefore, this assay can be used to measure carbon dioxide flux across membranes and to assess the contribution of carbonic anhydrase to this flux.

Flow cytometric determination of intracellular free potassium concentration

A flow cytometric assay has been developed for determination of intracellular free potassium concentration ([K+]i). Investigated cells, loaded with the fluorescent pH indicator 2',7bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF), are incubated in the presence of nigericin, and the intracellular pH is measured. The ionophore maintains the same ratio value [H+]/[K+] on both sides of the cytoplasmic membrane, so [K+]i can be evaluated from the measured intracellular pH (pHi) and the known parameters of the buffer. Application of the method revealed that the intracellular potassium concentration is significantly higher in lymphocytes than in proliferating cells of HUT-78, U266, and JY cell lines. A surprisingly low (60 mM) [K+]i concentration was observed with sperm cells of common carp. This method allows measurements on individual cells, provides data of excellent statistics, and still does not require large amounts of material. These features offer remarkable advantages over other techniques used for intracellular K+ measurements, such as steady-state fluorescence, atom absorption photometry, or energy-dispersive x-ray analysis.