Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator

A new fluorescent intracellular pH indicator is described ("quene 1") which is related to the tetracarboxylate Ca2+ indicator based on the quinoline fluorophor ("quin 2"). Quene 1 has excitation and emission maxima at 390 and 530 nm, respectively, and shows a 30-fold increase in fluorescence between pH 5 and 9 with a pK alpha of 7.3. The fluorescence is insensitive to Ca2+ and Mg2+ at free concentrations up to 10(-4) M and to the proportions of Na+ and K+ at total concentrations of Na+ and K+ from 100 to 200 mM. The indicator is loaded into thymocytes using the tetraacetoxymethyl ester derivative which is hydrolyzed in the cells to give the tetracarboxylate anion. Intracellular pH can be measured at intracellular quene 1 concentrations of approximately 0.1 mM and quene 1 does not perturb glycolysis or the ATP level in resting cells at concentrations up to 0.8 mM. The intracellular pH of mouse thymocytes indicated by quene 1 is 7.15 +/- 0.04 and it is insensitive to the concentration of Ca2+ or Mg2+ in the extracellular medium. The intracellular pH decreased when the pH of the medium was lowered by addition of HCl, but was insensitive to NaOH at extracellular pH values up to 8.0. Rapid transient changes in intracellular pH are induced by NH4Cl, NaCO2CH3, or HCO3-/CO2. The thymocytes showed no early changes (within 30 min) in intracellular pH in response to mitogenic concentrations of lectins or 4 beta-phorbol-12-myristate-13-acetate.     

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.     

Intracellular pH in single motile cells

Cytoplasmic pH in single living specimens of Chaos carolinensis is determined microfluorometrically by measuring the ratio of fluorescence intensity of microinjected fluorescein-thiocarbamyl (FTC)-ovalbumin at two different excitation wavelengths. The probe is evenly distributed throughout, and confined to, the cytoplasm, and the fluorescence intensity ratio depends only upon pH. It is independent of pathlength, concentration of probe, divalent cations, and ionic strength. Ratios are calibrated with a standard curve generated in situ by adjusting internal pH of FTC-ovalbumin-containing amebae with weak acid and weak base or by injection of strong buffers. With this technique, the average cytoplasmic pH of freely moving ameba is found to be 6.75 (SD +/- 0.3). The pH of a given spot relative to the morphology of a moving ameba remains fairly constant (+/- 0.05 U), whereas the pH of two different spots in the same cell may differ by as much as 0.4 U, and average pH in different amebae ranges from 6.3 to 7.4, with a suggestion of clustering about pH 6.5 and 6.8. During wound healing, there is a local, transient drop in pH (as great as 0.35 U) at the wound site upon puncture, proportional in extent to the degree of damage. Comparison of tails and advancing pseudopod tips reveals no significant difference in cytoplasmic pH at this level of spatial (50 microns diameter spot) and temporal (1.3 s) resolution. Fluctuations in intracellular pH and/or intracellular free Ca++ may be involved in regulation of cytoplasmic structure and contractility.     

Intracellular pH in singleSaccharomyces cerevisiae cells

Saccharomyces cerevisiae cells can be stained with the pH dependent fluorochrome BCECF, to monitor shifts in intracellular pH in individual cells using flow cytometry. Cells stained in the presence of ethanol were found to be stained as much as 100 times more intensely as cells without ethanol. Cells that have been starved for the carbon source showed a significant shift in intracellular pH upon refeeding with a metabolizable carbon source within 3 minutes.     

Intracellular pH gradients in migrating cells

Cell polarization along the axis of movement is required for migration. The localization of proteins and regulators of the migratory machinery to either the cell front or its rear results in a spatial asymmetry enabling cells to simultaneously coordinate cell protrusion and retraction. Protons might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of pH(i), the Na(+)/H(+) exchanger NHE1, is essential for cell migration and accumulates at the cell front. Here, we test the hypothesis that the uneven distribution of NHE1 activity creates a pH(i) gradient in migrating cells. Using the pH-sensitive fluorescent dye BCECF, pH(i) was measured in five cell lines (MV3, B16V, NIH3T3, MDCK-F1, EA.hy926) along the axis of movement. Differences in pH(i) between the front and the rear end (ΔpH(i) front-rear) were present in all cell lines, and inhibition of NHE1 either with HOE642 or by absence of extracellular Na(+) caused the pH(i) gradient to flatten or disappear. In conclusion, pH(i) gradients established by NHE1 activity exist along the axis of movement.     

Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Intracellular site of prolactin synthesis in rat pituitary cells in culture

Free and membrane-bound polyribosomes were isolated from control and thyrotropin releasing hormone-treated GH₃ cells. The two polysome fractions were used to direct {³H}leucine incorporation into prolactin in both heterologous and homologous cell-free protein-synthesizing systems. Prolactin was measured by immunoprecipitation and SDS-disc gel electrophoresis of the reaction products. Only membrane-bound polysomes directed incorporation of {³H}leucine into labeled prolactin. In additon, intact cells were pulselabeled with {³H}leucine, free and membrane-bound polysomes were isolated, and newly synthesized prolactin associated with each polysome fraction was measured. In control cells, {³H}prolactin represented about 0.4 and 4.2% of total acid-insoluble radioactivity in free and membrane-bound polysomes, respectively; whereas, in thyrotropin releasing hormone-treated cells, these values were about 1 and 20%, respectively. Added {³H}prolactin did not associate nonspecifically with membrane-bound polysomes. We conclude that prolactin is synthesized predominantly on membrane-bound polysomes in GH₃ cells.     

Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations

The dual emission, Ca2+ sensitive fluorescent dye, Indo-1, offers several potential advantages over its dual excitation analogue, Fura-2. Most notable among these advantages are increased speed of measurement using dual wavelength photometry and the absence of a requirement for special quartz optics. Despite these potential advantages, only a tiny fraction of the microscopic studies of intracellular free calcium ([Ca2+]i) on substrate-attached cells has employed Indo-1. Among the reasons for the infrequent use of Indo-1 are the fact that it exhibits somewhat different spectral properties in the cytosol than it does in extracellular buffers, and the notion that it is much more sensitive to photobleaching than Fura-2. We report here that under our experimental conditions, Indo-1 photobleaching is small and does not noticeably affect the measurement of free Ca2+, even after 30 minutes of continuous illumination. We also report a new method for creating in situ standard curves that is easy, reproducible, and yields values for [Ca2+]i that are identical to those obtained with Fura-2. In addition, we have found that Indo-1 is less subject than Fura-2 to compartmentalization within subcellular organelles. These results provide baseline data to take advantage of the significant improvement afforded by Indo-1 in the measurement of rapid [Ca2+]i responses and the avoidance of compartmentalization artifacts during experiments of long duration.     

Intracellular free magnesium in excitable cells: its measurement and its biologic significance

As a necessary cofactor for hundreds of enzymes, intracellular Mg2+ influences a wide range of cellular functions such as transmembrane transport of other ions, glycolysis, respiration, muscle contraction, and phosphorylation of ion channels. Unlike Ca2+, Mg2+ does not seem to have a "trigger" function. However, the wide range of enzymes requiring Mg2+ to be activated suggests that Mg2+ plays a pivotal role in fine control and coordination of cell activity, determining the "set point" of hundreds of metabolic reactions. In this sense, intracellular Mg2+ might be regarded as a static rather than a dynamic regulator of cell function. Little is known about the mechanisms by which excitable and other cells keep their [Mg2+]i within narrow limits against large electrochemical gradients. Furthermore, the actual basal level of [Mg2+]i has been the subject of recent controversy. In the present paper the roles of intracellular Mg2+ on cell function as well as four current techniques for measuring [Mg2+]i are briefly reviewed. These techniques are (i) metallochromic indicators, (ii) 31P nuclear magnetic resonance, (iii) null point for plasma membrane permeabilization using the ionophore A23187 and, (iv) Mg2+-selective microelectrodes. The relative advantages and disadvantages of each of these techniques are discussed with special emphasis on Mg2+-selective microelectrodes.     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors

A series of fluorescent, long-wavelength, benzo[c]-xanthene dyes has been characterized for pH measurement in both excitation and emission ratio applications. The two general classes of these indicators are seminaphthofluoresceins (SNAFLs) and seminaphthorhodafluors (SNARFs) which are substituted at the 10-position with oxygen or nitrogen, respectively. These probes show separate emissions from the protonated and deprotonated forms of the fluorophores. The dyes may be excited at 488 or 514 nm with argon ion lasers. Most of the indicators have pKa values between 7.6 and 7.9. Detailed photophysical studies were conducted on the carboxy-SNAFL-1 system and excited-state prototropic reactions were compared to structurally related derivatives, such as the umbelliferones. Membrane permeant esters, such as diacetates and acetoxymethyl esters have also been prepared. The indicators are spectrally well resolved from calcium indicators such as fura-2 and indo-1 and should be suitable for simultaneous determination of pH and Ca2+ transients.     

Synthesis and photophysical properties of new SNARF derivatives as dual emission pH sensors

We report the synthesis and properties of two new seminaphthorhodafluor (SNARF) derivatives, SNARF-F and SNARF-Cl. Both these derivatives exhibit typical red shifts of absorption and fluorescence, and higher cell permeability as compared to traditional SNARF, while the pH-dependent dual-emission characteristics are well retained. In particular, the lower pK_a (7.38) of SNARF-F makes it more suitable than traditional SNARF derivatives for intracellular applications.     

Intracellular free calcium in rat anterior pituitary cells monitored by fura-2

Rat anterior pituitary cells, loaded with the calcium indicator dye fura-2 after primary culture, were challenged with prolactin and growth hormone secretagogues and inhibitory hormones. To initially validate the technique, the calcium channel activator maitotoxin effectively increased intracellular free calcium [( Ca++]i). Various concentrations of the secretagogues thyrotropin releasing hormone or angiotensin II induced peak increases in [Ca++]i within 15 sec, followed by a lower and prolonged plateau phase. The inhibitory hormones dopamine and somatostatin maximally reduced [Ca++]i by 15-20 sec, followed by a spontaneous return to baseline over 5-10 min. The receptor antagonists saralacin and spiperone blocked the angiotensin II and dopamine effects, respectively. Thus, fura-2 appears to be an adequate probe for resolving second-to-second changes in [Ca++]i induced by hormone receptor activation in anterior pituitary cells.     

Intracellular pH based controlled cultivation of yeast cells: I. Measurement methodology

A method has been developed to continuously measure the intracellular pH (pH(i)) of cells cultivated in a bioreactor in an on-line fashion over extend time periods. The methods is attractive in its simplicity and involves the use of a fluorescent pH(i) indicator 9-aminoacridine (9A A) which is a week base. An expression has been derived to calculate changes in pH(i) from measured 9AA-fluorescence changes. The indicator 9AA was found t be nontoxic to yeast cells at concentrations used to measure pH(i) (7 muM). The fluorescence of nicotinamide adenine dinucleotide (NADH) molecules did not interfere significantly with the measurement of 9AA-fluorescence. The pH(i) change in yeast cell following the addition of a proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) measured by 9AA compared favorably with that measured by the well-established pH(i), indicator (which is however unsuitable for on-line applications in a bioreactor) bis-carboxyethyl carboxy fluorescein (BCECF). The pH(i) of yeast under substrate starved conditions was 6.4 units. The responses of pH(i) of yeast cells to induced metabolic transitions were studied. Under aerobic condition, pH(i) increased by 0.12 unit following a 100-ppm glucose pulse addition and by 0.25 unit following a 300-ppm ethanol pulse addition. Under anaerobic condition, pH(i) increased by 0.1 unit following a 500-ppm glucose pulse addition. Comparison of pH(i) with other indicators of cellular metabolic state suggests that pH(i) is a cellular metabolic state indicator. (c) 1993 John Wiley & Sons, Inc.     

Quantitative pH assessment of small-volume samples using a universal pH indicator

We developed a hue-based pH determination method to analyze digital images of samples in a 384-well plate after the addition of a universal pH indicator. The standard error of calibration for 69 pH standards was 0.078 pH units, and no sample gave an error greater than 0.23 units. We then used in-solution isoelectric focusing to determine the isoelectric point of Wnt3A protein in conditioned medium and after purification and applied the described method to assess the pH of these small-volume samples. End users may access our standard to assay the pH of their own samples with no additional calibration.