细胞内pH值测定方法的研究进展

很多细胞的活动都对pH的变化十分敏感。pH值的有效控制对维持正常的细胞生理活动十分重要。如何有效监测细胞内pH值是很多细胞生物学的重要研究内容之一,如在研究细胞内转运蛋白、Ca2+离子等的变化活动时均需要测定细胞内的pH值,其相关的研究已有100多年的历史。本文将介绍目前几种细胞内pH值的主要测定方法,包括:弱酸弱碱分布法、核磁共振法、微电极法、荧光探针法等;每一种方法将从相关方法、技术的原理、特点、应用、局限性和注意事项等着手,将各个方法的优缺点进行横向的比较。本文还将重点探讨免疫探针法的最新进展,将报道一种最新的基于碳纳米点技术和荧光染料结合的pH定量测定计,还将介绍SNARF的两种最新衍生染料SNARF-F和SNARF-Cl的性能及其应用。     

Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

pH in the extracellular matrix of bacterial biofilms is of central importance for microbial metabolism. Biofilms possess a complex three-dimensional architecture characterized by chemically different microenvironments in close proximity. For decades, pH measurements in biofilms have been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit the monitoring of horizontal pH gradients in biofilms in real time. Quantitative fluorescence microscopy can overcome these problems, but none of the hitherto employed methods differentiated accurately between extracellular and intracellular microbial pH and visualized extracellular pH in all areas of the biofilms. Here, we developed a method to reliably monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4, choosing dental biofilms as an example. Fluorescent emissions of C-SNARF-4 can be used to calculate extracellular pH irrespective of the dye concentration. We showed that at pH values of <6, C-SNARF-4 stained 15 bacterial species frequently isolated from dental biofilm and visualized the entire bacterial biomass in in vivo-grown dental biofilms with unknown species composition. We then employed digital image analysis to remove the bacterial biomass from the microscopic images and adequately calculate extracellular pH values. As a proof of concept, we monitored the extracellular pH drop in in vivo-grown dental biofilms fermenting glucose. The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows the accurate monitoring of extracellular pH in bacterial biofilms in three dimensions in real time and represents a significant improvement to previously employed methods of biofilm pH measurement.     

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 µm thickness, but is limited to the pH range between 4.5 and 7.0.     

Intracellular pH of deep and superficial muscle fibres

Measurements with pH-sensitive microelectrodes have recently shown that the hydrogen ion appears to be in equilibrium in superficial but not in deep muscle fibres. These results are analysed in the present paper and it is concluded that the apparent equilibrium distribution of the hydrogen ion in superficial fibres is real and that the apparent non-equilibrium value of intracellular pH obtained for deeper fibres may possibly be caused by cell damage.     

ECF pH dynamics within the ventrolateral medulla: a microelectrode study

Using pH-sensitive microelectrodes, we evaluated pH dynamics of extracellular fluid (ECF) within the ventrolateral medulla (VLM) beneath the central chemoceptive areas in anesthetized, spontaneously breathing cats. Static ECF pH was acid in the superficial layers (less than 1 mm), compared with the overlying cerebrospinal fluid pH that became alkaline gradually during the experiments. In the deeper VLM areas (1-3 mm), no systematic gradients of ECF pH were observed. We found various, isolated regions where intravertebral artery injections of CO2-saturated saline evoked acidic shift of ECF pH in the time course analogous to ventilatory augmentation. Those responsive regions were found to be scattered not only in the superficial layers but also in the deeper VLM areas, although many nonresponsive regions were also intermingled among them. Occlusions of the principal vessels supplying the tested VLM regions diminished but failed to abolish the ECF pH responses to the CO2 loadings, suggesting a collateral blood flow by fine pial vessels. The present study suggests a possibility that the pH-dependent central chemoreceptors, if any, would be scattered in the deeper VLM areas as well as the superficial layers.     

The mechanism of inhibition of ureogenesis by acidosis

In perfused rat liver a decrease of cytosol pH, determined with pH-sensitive microelectrodes7 from 7.2 to 6.85 is associated with a 50% fall in ureogenesis from ammonium chloride. In isolated rat hepatocytes the fall in ureogenesis due to acidosis is associated with decrease in the mitochondrial and cytosolic concentration of citrulline. Limitation of carbamoyl phosphate synthesis and thus citrulline supply could be responsible for the inhibition of ureogenesis observed.     

Buffer power and intracellular pH of frog sartorius muscle.

Intracellular pH (pHi) and buffer power of frog muscle were measured using pH-sensitive microelectrodes under conditions used previously in energy balance experiments because pH strongly influences the molar enthalpy change for phosphocreatine splitting, the major net reaction during brief contractions. The extracellular pH (pHe) of HEPES buffered Ringer's solution influenced pHi, but change in pHi developed slowly. Addition or removal of CO2 or NH3 from the extracellular solution caused a rapid change in pHi. The mean buffer power measured with CO2 was 38.4 mmol.l-1.pH unit-1 (+/- SEM 2.1, n = 49) and with NH3 was 36.2 (+/- SEM 5.5, n = 4) at 20-22 degrees C. At 5 degrees C, in experiments with CO2 the mean buffer power was 40.3 (+/- SEM 2.6, n = 3). For pHi values above approximately 7.0, the observed buffer power was greater than that expected from the values in the literature for the histidine content of intracellular proteins, carnosine and inorganic phosphate in the sarcoplasm. The measured pHi values were similar to those assumed in energy balance calculations, but the high measured buffer power suggests that other buffering reactions occur in addition to those included in energy balance calculations.     

The Correlation of Profiles of Surface pH and Elongation Growth in Maize Roots

High-resolution profiles of surface pH and growth along vertically growing maize (Zea mays) primary root tips were determined simultaneously by pH-sensitive microelectrodes and marking experiments. Methodological tests were carried out that proved the reliability of our kinematic growth analysis, while questioning the validity of an alternative technique employed previously. A distal acidic zone around the meristematic region and a proximal one around the elongation zone proper were detected. This pattern as such persisted irrespective of the bulk pH value. The proximal acidic region coincided with maximum relative elemental growth rates (REGR), and both characters reacted in a correlated manner to auxin and cyanide. The distal acidic band was unrelated to growth, but was abolished by cyanide treatment. We conclude that: (a) the pattern of surface pH as such is a regulated feature of growing root tips; (b) the correlation of extracellular pH and growth rate suggests a functional relationship only along proximal portions of the growing root tip; and (c) the distal acidic band is not caused by pH buffering by root cap mucilage, as suggested previously, but rather is controlled by cellular activity.     

pH-sensitive glass microelectrodes and intracellular pH measurements.

1. Some properties of the open-tipped, uninsulated, pH-sensitive glass microelectrode were examined in several electrical experiments. 2. Based on these observations, technical and theoretical problems were considered for application to the pH measurement in small cells. 3. The intracellular pH, (pH)i, of the epithelial cell in rat duodenum measured was approximately 7.0. A reduction in (pH)i was apparent (about 0.3) with the addition of 20 mM-glucose to the bathing fluid. 4. It was concluded that with certain limitations such uninsulated, open-tipped microelectrodes may be successfully utilized for intracellular pH measurements.     

Fused cells of frog proximal tubule: II. Voltage-dependent intracellular pH

Experiments were performed in intact proximal tubules of the doubly perfused kidney and in fused proximal tubule cells of Rana esculenta to evaluate the dependence of intracellular pH (pHi) on cell membrane potential applying pH-sensitive and conventional microelectrodes. In proximal tubules an increase of the K+ concentration in the peritubular perfusate from 3 to 15 mmol/liter decreased the peritubular cell membrane potential from -55 +/- 2 to -38 +/- 1 mV paralleled by an increase of pHi from 7.54 +/- 0.02 to 7.66 +/- 0.02. The stilbene derivative DIDS hyperpolarized the cell membrane potential from -57 +/- 2 to -71 +/- 4 mV and led to a significant increase of the K+-induced cell membrane depolarization, but prevented the K+-induced intracellular alkalinization. Fused proximal tubule cells were impaled by three microelectrodes simultaneously and cell voltage was clamped stepwise while pHi changes were monitored. Cell membrane hyperpolarization acidified the cell cytoplasm in a linear relationship. This voltage-induced intracellular acidification was reduced to about one-third when HCO-3 ions were omitted from the extracellular medium. We conclude that in proximal tubule cells pHi depends on cell voltage due to the rheogenicity of the HCO-3 transport system.     

Steady-state function of the ubiquitous mammalian Na/H exchanger (NHE1) in relation to dimer coupling models with 2Na/2H stoichiometry

We describe the steady-state function of the ubiquitous mammalian Na/H exchanger (NHE)1 isoform in voltage-clamped Chinese hamster ovary cells, as well as other cells, using oscillating pH-sensitive microelectrodes to quantify proton fluxes via extracellular pH gradients. Giant excised patches could not be used as gigaseal formation disrupts NHE activity within the patch. We first analyzed forward transport at an extracellular pH of 8.2 with no cytoplasmic Na (i.e., nearly zero-trans). The extracellular Na concentration dependence is sigmoidal at a cytoplasmic pH of 6.8 with a Hill coefficient of 1.8. In contrast, at a cytoplasmic pH of 6.0, the Hill coefficient is <1, and Na dependence often appears biphasic. Results are similar for mouse skin fibroblasts and for an opossum kidney cell line that expresses the NHE3 isoform, whereas NHE1^−/− skin fibroblasts generate no proton fluxes in equivalent experiments. As proton flux is decreased by increasing cytoplasmic pH, the half-maximal concentration (K_1/2) of extracellular Na decreases less than expected for simple consecutive ion exchange models. The K_1/2 for cytoplasmic protons decreases with increasing extracellular Na, opposite to predictions of consecutive exchange models. For reverse transport, which is robust at a cytoplasmic pH of 7.6, the K_1/2 for extracellular protons decreases only a factor of 0.4 when maximal activity is decreased fivefold by reducing cytoplasmic Na. With 140 mM of extracellular Na and no cytoplasmic Na, the K_1/2 for cytoplasmic protons is 50 nM (pH 7.3; Hill coefficient, 1.5), and activity decreases only 25% with extracellular acidification from 8.5 to 7.2. Most data can be reconstructed with two very different coupled dimer models. In one model, monomers operate independently at low cytoplasmic pH but couple to translocate two ions in “parallel” at alkaline pH. In the second “serial” model, each monomer transports two ions, and translocation by one monomer allosterically promotes translocation by the paired monomer in opposite direction. We conclude that a large fraction of mammalian Na/H activity may occur with a 2Na/2H stoichiometry.     

Properties of the pH-sensitive site that controls the lambda max of Limulus metarhodopsin

A pH-sensitive site controls the lambda max of Limulus metarhodopsin. The properties of this site were examined using intracellular recordings of the early receptor potential (ERP) as a pigment assay. ERPs recorded over a range of extracellular pHs indicate that the apparent pK of the site is in the range of 8.3-8.6. Several lines of evidence indicate that the site responds directly to changes in extracellular pH (pHo) rather than to changes in intracellular pH(pHi) that follow as a secondary result of changing pHo : (a) the effect of changing pHo was rapid (less than 60 s); (b) when pHo was raised, the simultaneous rise in pHi, as measured with phenol red, was relatively small; (c) raising pHi by intracellular injection of pH 10 glycine buffer did not affect the site; and (d) the effect of changing pH0 could not be blocked by increasing the intracellular pH buffering capacity. It is concluded that the pH-sensitive site on metarhodopsin is on the extracellular surface of the plasma membrane.     

Vacuolar pH in radish cotyledonal mesophyll cells

The vacuolar pH in cotyledonal mesophyll cells from radish (Raphanus sativus L. var. sativus) seedlings was determined from vacuoles, isolated from protoplasts through osmotic shock, by means of measurement of vacuole extracts with a pH meter and the methylamine method, and gave mean pH values of 6.28 and 6.26, respectively. Direct in situ measurements of the vacuolar pH from intact leaf tissue were recorded with pH-sensitive microelectrodes and gave a mean value of 6.0. The results are discussed with respect to possible erroneous pH measurements and the vacuolar location of specific anabolic reactions.     

Control of cytoplasmic pH under anoxic conditions and its implication for plasma membrane proton transport in Medicago sativa root hairs

In root hairs of Medicago sativa, pH-sensitive microelectrodeshave been applied to study cytoplasmic pH-regulation. To inhibitorslike oligomycin, antimycin A, cyanide and the exchange of O₂for N_2, the root hairs respond with a distinct cytoplasmic acidification.Whereas the cytoplasmic pH under aerobic conditions rests at7.28 0.11 SE (n = 168), under conditions of (chemical) anoxiathe cytoplasmic pH is shifted to a stable, well-regulated 6.78 0.08 SE (n = 81). Once this pH is attained in the presenceof one inhibitor, addition of another has no effect. 2-deoxyglucoseand N-acetylglucosamine, both inhibitors of glycolysis at thehexokinase level, increase cytoplasmic pH by about 0.3 pH units,as do glucogenic amino acids. It is suggested that aerobic energymetabolism does not contribute to acidosis of these cells. SincepH-shift and pump deactivation can be separated by using poorrespiratory inhibitors, it is concluded that the switch from‘aerobic’ to ‘anaerobic’ pH is not correlatedwith proton pump activity. Inversely, since cytoplasmic pH neitherresponds to pump activation by FC with alkalinization, nor topump deactivation by cyanide with acidification, it is alsoconcluded that changes in pump activity do not affect cytoplasmicpH. Key words: Cytoplasmic pH regulation, Medicago, pH-sensitive microelectrodes, proton transport, root hairs     

Intracellular pH of frog sartorius muscle.

A weak base, morpholine, has been labelled with 3H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20 degrees C in a solution of the same ionic strength as frog Ringer was found to be 8.45 +/- 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indcator. Intracellular pH was then measured by distribution of morpholine (6.985 +/- 0.08), nicotine (6.915 +/- 0.03) and the weak acid 5,5'-dimethyl-2,4-oxazolidinedione (7.10 +/- 0.05) and the pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and all ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H+ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Direct measurement of intracellular pH changes in Xenopus eggs at fertilization and cleavage

We have used Thomas-type recessed-tip pH-sensitive microelectrodes to measure the intracellular pH (pHi) in Xenopus eggs during both fertilization and ionophore activation. The average pHi in unfertilized eggs is 7.33 +/- 0.11 (SD; n = 21) with a resting membrane potential of -10.1 +/- 3.5 (SD; n = 38) mV. Within 2 min after the onset of the fertilization potential, there is a slight, transient pHi decrease of 0.03 +/- (SD, n = 8), followed by a distinct, permanent pHi increase of 0.31 +/- 0.11 (SD; n = 7) beginning approximately 10 min after the start of the fertilization potential and becoming complete approximately 1 h later. The pHi remains near this level of 7.67 +/- 0.13 (SD, n = 10) through at least 10 cleavage cycles, but it is possible to discern pHi oscillations with a mean amplitude of 0.03 +/- 0.02 (SD, n = 38). Eggs perfused for at least 2 h in Na+-free solution with 1 mM amiloride exhibited all of these pHi changes, so these changes do not require extracellular Na+. Similar cytoplasmic alkalinizations that accompany the activation of metabolism and the cell cycle in a wide variety of cell types are discussed.     

pH-responsive sulfonamide/PEI system for tumor specific gene delivery: an in vitro study

The feasibility of pH-sensitive polymeric nanoparticles that effectively target the acidic extracellular matrix of tumors is demonstrated. Plasmid DNA was complexed with polyethyleneimine (PEI) and further with a pH-sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine) (PSD)-block-PEG (PSD-b-PEG), to obtain naonparticles. The shielding/deshielding of nanoparticles was tested along with cell viability and transfection efficiency at physiological and tumor pH. The nanoparticles composed of DNA/PEI/PSD-b-PEG were 300 nm in size and showed low cytotoxicity and transfection at pH 7.4 due to shielding of PEI by PSD-b-PEG. The PSD-b-PEG bound to PEI/DNA complex decreased the interaction of PEI positive charges with cells and reduced the cytotoxicity by 60%. At pH 6.6, the nanoparticles demonstrated high cytotoxicity and transfection, indicating PSD-b-PEG detachment from the nanoparticles and permit PEI to interact with cells. PSD-b-PEG is able to discern the small difference in pH between normal and tumor tissues and hence has remarkable potential in drug targeting to tumor areas.     

Intracellular pH of frog sartorius muscle

A weak base, morpholine, has been labelled with ³H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20°C in a solution of the same of ionic strength as frog Ringer was found to be 8.45 ± 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indicator. Intracellular pH was then measured by distribution of morpholine (6.985 ± 0.08), nicotine (6.915 ± 0.03) and the weak acid 5,5′-dimethyl-2,4-oxazolidinedione (7.10 ± 0.05) and with pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H⁺ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pKi values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.     

Effect of locally applied drugs on the pH of luminal fluid in the endolymphatic sac of guinea pig

The aim of the present work was to assess the effect of various drugs applied locally on the pH of the luminal fluid (pH(lum)) in guinea pig endolymphatic sac. pH(lum) and transepithelial potential, when measured in vivo by means of double-barrelled pH-sensitive microelectrodes, were 7.06 +/- 0.08 and +6.1 +/- 0.34 mV (mean +/- SE; n = 84), respectively, which is consistent with a net acid secretion in the luminal fluid of the endolymphatic sac. Bafilomycin and acetazolamide increased and decreased, respectively, pH(lum). Amiloride, ethylisopropylamiloride, ouabain, and Schering 28080 had no effect on pH(lum). Results obtained with inhibitors of anionic transport systems were inconclusive; e.g., DIDS reduced pH(lum), whereas neither SITS nor triflocin had any effect. We conclude that bafilomycin-sensitive H(+)-ATPase activity accounts for the transepithelial acid gradient measured in the endolymphatic sac and that intracellular and membrane-bound carbonic anhydrase probably participates in regulating endolymphatic sac pH(lum). The relationship between acid pH, endolymph volume, and Ménière's disease remains to be further investigated.