Alkalization Of pH Research Articles

Bis-benzylisoquinoline alkaloids inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy

Flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV), represent a substantial public health challenge as there are currently no approved treatments available. Here, we investigated the antiviral effects of bis-benzylisoquinoline alkaloids (BBAs) on flavivirus infections. We evaluated five specific BBAs—berbamine, tetrandrine, iso-tetrandrine, fangchinoline, and cepharanthine—and found that they effectively inhibited infections by ZIKV, DENV, or JEV by blocking virus entry and genome replication stages in the flavivirus life cycle. Furthermore, we synthesized a fluorophore-conjugated BBA and showed that BBAs targeted endolysosomes, causing lysosomal pH alkalization. Mechanistic studies on inhibiting ZIKV infection by BBAs revealed that these compounds blocked TRPML channels, leading to lysosomal dysfunction and reducing the expression of NCAM1, a key receptor for the entry of ZIKV into cells, thereby decreasing cells susceptibility to ZIKV infection. Additionally, BBAs inhibited the fusion of autophagosomes and lysosomes, significantly reducing viral RNA replication. Collectively, our results suggest that BBAs inhibit flavivirus entry and replication by compromising endolysosomal trafficking and autophagy, respectively, underscoring the potential of BBAs as therapeutic agents against flavivirus infections.

In-situ pH monitoring of pre-notched X80 pipeline steel using an all-solid-state Pt/RuOx ultra-micro pH sensor

In the notch microenvironment caused by the stress corrosion of pipeline steel, the local pH value changes mediated by anodic-cathodic corrosion reactions played an important role in comprehending the local corrosion behavior. To address this issue, a radius 5 µm all-solid-state Pt/RuOx ultra-micro pH sensor was developed using cyclic voltammetry deposition method, which was combined with scanning electrochemical microscopy (SECM) to achieve in-situ monitoring the local pH distribution and evolution of the pre-notched X80 pipeline steel. The micro-sensor demonstrates a near Nernstian slope of −60.0 ± 3 mV/pH within the pH value range of 2.00–12.00, accompanied by an exceptionally rapid response time of 200 ms. Moreover, it exhibits high accuracy, strong long-term monitoring ability and remarkable resistance to the interference of common ions in seawater and ferrous ions generated by the corrosion of pipeline steel. Through monitoring the local pH distribution in simulated seawater solution at the notch of X80 pipeline steel, it was found that obvious pH alkalization occurs first on both the surface and inside of the notch, followed by gradual acidification, and this phenomenon is more obvious at the front of the notch. Combined with other characterization methods, a possible corrosion reaction mechanism at the notch of stress corrosion was proposed.

In situ assay of the reducing sugars in hydrophilic natural deep eutectic solvents by a modified DNS method

The DNS assay has been already a universally method for the in situ determination of reducing sugars (RS) in aqueous phase solvents. Today, “greener biorefineries” require in situ measurement of reducing sugars in non-aqueous solvents such as natural deep eutectic solvents (NADESs). Therefore, that DNS assay cannot be performed successfully in such eutectic phase solvent becomes a puzzle that must be solved. In this work, an improved DNS method, which we call the DNS'' method, was established and implemented successfully in hydrophilic eutectic phase solvent systems (hiNADES). The tickets to the success of DNS'' method are the inserted two essential steps, water-adding disintegration and pH alkalization. Then we tested two excellent RS solvents (ChCl_Gly and ChCl_LA) from six synthetic ChCl-based hiNADESs. The optimal disintegration conditions for assaying RS in ChCl_LA/NaOH (ChCl_Gly/H2O) were volume ratio 3: 1 (3: 1), molar concentration 2.0 mol/L (0 mol/L). Thereafter, we drew the standard curves of seven RSs in ChCl_Gly and ChCl_LA, which are glucose, xylose, fructose, mannose, fucose, rhamnose, and maltose. We determined that HMF interferes with DNS assay in hiNADES. The principles and standard procedures of DNS'' method were also investigated. Finally, the enzymatic saccharification of microcrystalline cellulose (MCC) by two commercial Trichoderma reesei cellulases (Celluclast® 1.5 L and Cellic® CTec2) in neat hiNADES was tracked using the DNS'' method.

Randomized clinical trial on the clinical effects of a toothpaste containing extra virgin olive oil, xylitol, and betaine in gingivitis

To determine the effects on gingival bleeding, dental biofilm, and salivary flow and pH in patients with gingivitis of using toothpaste with extra-virgin olive oil (EVOO), xylitol, and betaine in comparison to a placebo or commercial toothpaste. This controlled, double blinded, and multicenter randomized clinical trial included patients with gingivitis randomly assigned to one of three groups: test group (EVOO, xylitol, and betaine toothpaste), control group 1 (placebo toothpaste), or control group 2 (commercial toothpaste). Percentage supragingival biofilm and gingival bleeding were evaluated at baseline (T0), 2 months (T2), and 4 months (T4), measuring non-stimulated salivary flow and salivary pH. Comparisons were performed between and within groups. The final study sample comprised 20 in the test group, 21 in control group 1, and 20 in control group 2. In comparison to control group 1, the test group showed significantly greater decreases in gingival bleeding between T4 and T0 (p = 0.02) and in biofilm between T2 and T0 (p = 0.02) and between T4 and T0 (p = 0.01). In the test group, salivary flow significantly increased between T2 and T0 (p = 0.01), while pH alkalization was significantly greater between T4 and T0 versus control group 2 (p = 0.01) and close-to-significantly greater versus control group 1 (p = 0.06). The toothpaste with EVOO, xylitol, and betaine obtained the best outcomes in patients with gingivitis, who showed reductions in gingival bleeding and supragingival biofilm and an increase in pH at 4 months in comparison to a commercial toothpaste.

The role of crystallinity of palladium nanocrystals in ROS generation and cytotoxicity induction.

Palladium (Pd) nanocrystals with different crystalline forms exhibit distinct enzyme-like activities in generating reactive oxygen species (ROS). How such crystallinity-dependent catalytic activity regulates potential cytotoxicity remains to be elucidated. In the present work, Pd nanocrystals with four different crystalline forms were synthesized, and the underlying mechanisms involved in ROS-mediated cytotoxicity were systematically revealed. Pd nanocrystals with the {100} (nanocubes) and {111} (nanooctahedrons and nanotetrahedrons) facets triggered cytotoxicity by generating singlet oxygen (1O2) and hydroxyl radicals (OH˙), respectively. Meanwhile, Pd nanoconcave-tetrahedrons, which had both the {110} and {111} facets, induced ROS-mediated cytotoxicity via activating the superoxide (O2˙-) pathway. Consumption of protons and generation of hydroxide during intracellular ROS conversion resulted in pH alkalization, eventually leading to cell death. Our findings emphasize the importance of facet-dependent ROS generation promoted by Pd nanocrystals. Furthermore, alkalization is identified as a new biomarker for analyzing ROS-mediated cytotoxicity.

α-Synuclein Overexpression Induces Lysosomal Dysfunction and Autophagy Impairment in Human Neuroblastoma SH-SY5Y.

Although the etiology of Parkinson's disease (PD) is multifactorial, it has been linked to abnormal accumulation of α-synuclein (α-syn) in dopaminergic neurons, which could lead to dysfunctions on intracellular organelles, with potential neurodegeneration. Patients with familial early-onset PD frequently present mutation in the α-syn gene (SNCA), which encodes mutant α-syn forms, such as A30P and A53T, which potentially regulate Ca2+ unbalance. Here we investigated the effects of overexpression of wild-type α-syn (WT) and the mutant forms A30P and A53T, on modulation of lysosomal Ca2+ stores and further autophagy activation. We found that in α-syn-overexpressing cells, there was a decrease in Ca2+ released from endoplasmic reticulum (ER) which is related to the increase in lysosomal Ca2+ release, coupled to lysosomal pH alkalization. Interestingly, α-syn-overexpressing cells showed lower LAMP1 levels, and a disruption of lysosomal morphology and distribution, affecting autophagy. Interestingly, all these effects were more evident with A53T mutant isoform when compared to A30P and WT α-syn types, indicating that the pathogenic phenotype for PD is potentially related to impairment of α-syn degradation. Taken together, these events directly impact PD-related dysfunctions, being considered possible molecular targets for neuroprotection.

Preconditioning of Bioactive Glasses before Introduction to Static Cell Culture: What Is Really Necessary?

Due to their high bioreactivity, the in-vitro analysis of bioactive glasses (BGs) can be challenging when it comes to maintaining a physiological pH. To improve BG biocompatibility, a heterogenic spectrum of preconditioning approaches, such as “passivation” of the BGs by incubation in cell culture medium, are used but have never been directly compared. In this study, the effect of passivation periods of up to 72 h on pH alkalization and viability of human bone marrow-derived mesenchymal stromal cells was evaluated to determine a time-efficient passivation protocol using granules based on the 45S5-BG composition (in wt%: 45.0 SiO2, 24.5 Na2O, 24.5 CaO, 6.0 P2O5) in different concentrations. pH alkalization was most reduced after passivation of 24 h. Cell viability continuously improved with increasing passivation time being significantly higher after passivation of at least 24 h compared to non-passivated 45S5-BG and the necessary passivation time increased with increasing BG concentrations. In this setting, a passivation period of 24 h presented as an effective approach to provide a biocompatible cell culture setting. In conclusion, before introduction of BGs in cell culture, different passivation periods should be evaluated in order to meet the respective experimental settings, e.g., by following the experimental protocols used in this study.

Edible Oils Attenuate Button Battery-Induced Injury in Porcine Esophageal Segments.

Objective: The objective of the study is to test whether the use of edible oil might be an early treatment strategy for reducing button battery-induced esophageal injury.Methods: A button battery was inserted into esophageal segments collected from pigs. The esophageal segments were randomly allotted to one of the following six treatments: (1) untreated (nothing injected), (2) lemon juice, (3) orange juice, (4) colza oil, (5) peanut oil, and (6) olive oil. Every hour, the battery discharge and the pH value were measured in the esophageal tissue. After treatment for 6 h, the residual voltage of the battery was measured and the esophageal tissue was processed with H&E staining.Results: In esophageal segments of the untreated group, a large area of the mucous membrane was severely eroded. Partial erosion was observed in esophageal tissues treated with either lemon juice or orange juice. Furthermore, the esophageal tissues were basically intact, had little damage when treated with oils. The highest extra-esophageal discharge voltage was recorded in the untreated group, a medium amount of discharge voltage was recorded in the lemon juice and orange juice groups, and the lowest discharge voltage was recorded in all the edible oils groups.Conclusions: Edible oils immersed the battery, reduced the surrounding electrolysis, and thus attenuated battery discharge. As a result, treatment with edible oils attenuated the pH alkalization and tissue damage in button battery injury of pig esophageal segments. These results indicate that edible oils might be used in the treatment of button battery ingestion.

Structural Transformations of Hydrolysates Obtained from Ti-, Zr-, and Ti, Zr-Solutions Used for Clay Pillaring: Towards Understanding of the Mixed Pillars Nature.

Structural characteristics of hydrolysates formed from the aqueous Ti-, Zr-, and Ti, Zr-pillaring solutions prepared from inorganic precursors (TiCl4 and ZrOCl2), was investigated and compared with that of precipitates obtained from the same solutions after a slight alkalization of pH to the values reported for the conditions of clay pillaring. The materials were recovered by lyophilization and subsequently subjected to calcination at 500, 800 and 1000 °C. Of special interest was the effect of pH on the possible formation of mixed Ti, Zr-oxide species. Powder X-ray diffraction (XRD), Raman spectroscopy and scanning electron microscope/energy dispersive spectrometer (SEM/EDS) analysis showed that even a relatively moderate alteration of pH in Ti-, Zr-, or Ti, Zr-precursor solutions caused substantial changes in the outcome of hydrolytic transformations, manifested by different phase and/or chemical composition of the resulting hydrolysates. Analysis of thermal evolution of hydrolysates showed that alkalization facilitated the transformation of anatase into rutile in materials obtained from Ti-pillaring solution, but retarded tetragonal to monoclinic zirconia conversion in samples derived from Zr-pillaring agent. The most striking effect was observed for the mixed Ti, Zr-pillaring solution, where an increase of pH enabled the formation of zirconium titanate as the only crystalline phase, rather than a multiphase mixture of anatase, monoclinic zirconia and zirconium titanate obtained from the more acidic precursor. The finding supports the model of mixed Ti-O-Zr network in props generated in Ti, Zr-pillared montmorillonites.

Mitochondrial reactive oxygen species regulate the induction of CD8+ T cells by plasmacytoid dendritic cells

Cross-presentation allows exogenous antigen presentation in association with major histocompatibility complex class I molecules, a process crucial for the priming of CD8+ T-cell responses against viruses and tumors. By contrast to conventional dendritic cells (cDC), which cross-present antigens in the steady state, plasmacytoid dendritic cells (pDC) acquire this ability only after stimulation by Toll-like receptor (TLR) ligands. The intracellular pathways accounting for this functional difference are still unknown. Here we show that the induction of cross-presentation by pDCs is regulated by mitochondria through a reactive oxygen species (ROS)-dependent mechanism, involving pH alkalization and antigen protection. The reduction of mitochondrial ROS production dramatically decreases the cross-presentation capacity of pDCs, leading to a strong reduction of their capacity to trigger CD8+ T-cell responses. Our results demonstrate the importance of mitochondrial metabolism in pDC biology, particularly for the induction of adaptive immune responses.

Silver nanoparticles induce lysosomal-autophagic defects and decreased expression of transcription factor EB in A549 human lung adenocarcinoma cells

Although silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary damage is unclear. AgNPs are incorporated into cells and processed via the autophagy pathway. We examined the effects of AgNP exposure on autophagic flux and expression of transcription factor EB (TFEB) in A549 lung adenocarcinoma cells. In cells exposed to citrate-coated 60-nm AgNPs, confocal laser microscopic examination showed a decrease in the LysoTracker fluorescence signal and an increase in that of Cyto-ID, indicating lysosomal pH alkalization and autophagosome formation, respectively. The proteins p62 and microtubule-associated protein light chain 3B-II (LC3B-II) are both degraded by autophagy, and their levels increased depending on AgNP dose. Furthermore, AgNP-induced increase in LC3B-II was not enhanced by treatment with the autophagic inhibitor bafilomycin A1. TFEB mRNA levels, and protein levels in cytosolic and nuclear fractions, were suppressed by exposure to AgNPs, suggesting transcriptional inhibition of TFEB expression. Overexpression of TFEB did not suppress AgNP-induced LC3B-II accumulation and cellular damage, indicating that impairment of autophagic flux and cellular damage by AgNPs might not be primarily caused by reduced TFEB expression. The present study suggests that AgNP-induced lysosomal dysfunction plays a principal role in the autophagic flux defect.

Ratiometric imaging of mitochondrial pH in living cells with a colorimetric fluorescent probe based on fluorescein derivative

Mitochondrial pH plays a pivotal role in the regulation of physiological process. Developing ratiometric fluorescent probes for real-time detection of mitochondrial pH fluctuation is still highly demanded yet challenging. Herein, we present a novel strategy to design a ratiometric probe (FDI) by broadening absorption spectrum (360–700nm) of fluorescein fluorophore. Unsaturated dialkene/indole quaternary ammonium moiety has broadened absorption and emission spectrum of fluorescein fluorophore to provide ratiometric detection by CLSM in dual excitation/dual emission mode, as well as directionally accumulated in mitochondria in living cells. Superior to commercially mitochondrial tracker, Carboxy-SNARF, probe FDI exhibits excellent and fast cell-membrane permeability and mitochondrial-targetability. It has been demonstrated that FDI could permit real-time monitoring of pH alkalization of mitochondria stimulated by chloroquine. Owing to nondestructive process and reversible ratiometric response to pH, image acquisition can be repeated frequently to trace and monitor the time course of mitochondrial pH responses. It is clearly confirmed that FDI would be a promising probe for real-time tracking of mitochondrial pH changes in the biomedical and biological fields.

Actin Filaments Are Involved in the Coupling of V0-V1 Domains of Vacuolar H+-ATPase at the Golgi Complex

We previously reported that actin-depolymerizing agents promote the alkalization of the Golgi stack and thetrans-Golgi network. The main determinant of acidic pH at the Golgi is the vacuolar-type H(+)-translocating ATPase (V-ATPase), whose V1domain subunitsBandCbind actin. We have generated a GFP-tagged subunitB2construct (GFP-B2) that is incorporated into the V1domain, which in turn is coupled to the V0sector. GFP-B2 subunit is enriched at distal Golgi compartments in HeLa cells. Subcellular fractionation, immunoprecipitation, and inversal FRAP experiments show that the actin depolymerization promotes the dissociation of V1-V0domains, which entails subunitB2translocation from Golgi membranes to the cytosol. Moreover, molecular interaction between subunitsB2andC1and actin were detected. In addition, Golgi membrane lipid order disruption byd-ceramide-C6 causes Golgi pH alkalization. We conclude that actin regulates the Golgi pH homeostasis maintaining the coupling of V1-V0domains of V-ATPase through the binding of microfilaments to subunitsBandCand preserving the integrity of detergent-resistant membrane organization. These results establish the Golgi-associated V-ATPase activity as the molecular link between actin and the Golgi pH.

Increased Springtail Abundance in a Garlic Mustard-Invaded Forest

Alliaria petiolata (Garlic Mustard) decreases diversity of native plants and introduces novel litter inputs to forests, but whether Garlic Mustard's presence influences detritivores is unclear. Our goal was to determine if Garlic Mustard alters springtail (detritivore) densities during early invasion. We obtained epigeal (litter, humus, and soil) cores from invaded and uninvaded areas in a Pinus strobus (White Pine) forest and used high-gradient dynamic sampling to extract springtails. Invaded areas contained nearly three times more springtails than uninvaded areas, attributable to two springtail morphospecies from Collembola families Tomoceridae and Entomobryidae. Higher pH in invaded epigeal samples correlated with increased springtail abundance. pH alkalization also correlated with increased proportion of juvenile rosettes in mixed stands. Our results suggest a possible role for pH modulation of springtail abundance in response to Garlic Mustard invasion.

Osmolarity effects on bovine articular chondrocytes during three-dimensional culture in alginate beads

With the development of engineered cartilage, the determination of the appropriate culture conditions is vital in order to maximize extracellular matrix synthesis. As osmolarity could affect the fate of chondrocytes, the purpose of this study was to determine the effects of osmolarity on chondrocytes during relatively long-term culture. Bovine articular chondrocytes were cultured in alginate beads in a biocarbonate free system at 280, 380 and 550 mOsm at pH 7.4 for up to 12 days, respectively. Cell volume, intracellular pH (pH(i)), cell number, glucosaminoglycan (GAG) and collagen retention were measured at day 5 and 12. Cell viability and volume were monitored over the 12 days of culture. By day 5 and 12, compared to the cell volume at 380 mOsm, around 20% (P<0.01) swelling and 15% (P<0.05) shrinkage were observed when the cells were cultured at 280 and 550 mOsm. The pH(i) over the 12 days of culture varied with osmolarity of the culture medium. In comparison with fresh cells, pH(i) became slightly more acidic by 0.15 pH units at 280 mOsm at day 5. However, by day 12, an alkalization of pH(i), by 0.2 pH units, was noted. A higher proliferation rate was seen at 280 mOsm than at other osmolarities while less GAG was produced. Chronic exposure to anisotonic conditions results in cell swelling at 280 mOsm and shrinkage at 550 mOsm. The osmolarity of 280 mOsm appears to encourage proliferation of chondrocytes, but inhibits matrix production.

Gating, permselectivity and pH‐dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to approximately 400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is approximately 90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pH(i) from 7.2 to 7.8 increased gap junctional conductance (g(j)) of approximately 100-fold with pK(a) = 7.41. We show that this g(j) increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca(2+)](o) = approximately 0.3 mm or below, demonstrating that a moderate reduction of [Ca(2+)](o) can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pH(i) with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell-cell coupling in horizontal cell in the retina.

Fluorescent measurement of the intracellular pH during sporulation of Saccharomyces cerevisiae

This work reports the intracellular pH (pH i) dynamics of Saccharomyces cerevisiae cells in sporulation medium. Cells loaded with the pH-sensitive dye carboxy-seminaphthorhodafluor-1 (C.SNARF-1) exhibited an alkalization of the pH i following the extracellular pH during sporulation in the absence of buffer and almost no change in pH i or ΔpH when sporulation was carried out in buffered medium. The results indicate that the pH gradient does not appear to be directly involved in the regulation of acetate uptake during sporulation. However, the alkalization of pH i by eliciting a decrease in metabolic fluxes could account for a lower demand for acetate.

Inorganic carbon uptake for photosynthesis by the symbiotic coral-dinoflagellate association II. Mechanisms for bicarbonate uptake

Mechanisms of HCO 3 − uptake as a source of dissolved inorganic carbon (DIC) for photosynthesis by the intracellular symbiont, Symbiodinium sp. were studied using microcolonies of the coral Galaxea fascicularis, freshly isolated zooxanthellae (FIZ) and cultured zooxanthellae (CZ). For this purpose we used specific inhibitors of anion transport 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid - SITS -, 4,4′-diisothiocyanato-stilbene-2,2'-disulfonic acid - DIDS -, carbonic anhydrase (acetazolamide, ethoxyzolamide), H + -ATPase (N,N′-dicyclohexylcarbodiimide - DCCD -, diethylstilbestrol - DES -, vanadate) or Ca 2+ channels (verapamil). We also tested the effect of ions known to play a role in HCO 3 − transport, like Na + and Ca 2+. Chloride uptake experiments were also performed to determine whether Cl − and HCO 3 − fluxes were coupled in CZ. Furthermore, the presence of carbonic anhydrase was tested using indirect immunoflurescence. Our results suggest that bicarbonate uptake by the animal symbiont is likely to be achieved by two types of DIDS-sensitive HCO 3 − carriers, each sharing 50% of the total uptake. The first is Na +-dependent, while the second is Na +-independent. We suggest the presence of a Na +-independent Cl − HCO 3 − exchange and either a Na +-dependent Cl − HCO 3 − exchange or a Na + HCO 3 − symport. Pharmacological data suggest that the enzyme carbonic anhydrase plays an important role in maintaining the photosynthetic rate. In the intact symbiosis, the major fraction of carbonic anhydrase activity is located in the zooxanthellae. Striking differences in DIC absorption mechanisms were found for FIZ and CZ. In FIZ, H +-ATPase and carbonic anhydrase participate in the carbon supply while in CZ the mechanism of HCO 3 − uptake appears to be strictly Na +-dependent and could be the result of Na + HCO 3 − symport activity. We hypothesize that stimulation of HCO 3 − uptake by the animal host is a consequence of intracellular pH alkalization by zooxanthellae photosynthesis. These results were summarized in a synthetic scheme of DIC absorption by both host cell and isolated zooxanthellae.

Intracellular pH of Candida albicans blastospores as measured by laser microspectrofluorimetry and 31P-NMR

The intracellular pH (pH i) of Candida albicans blastospores harvested from 8 h or 48 h cultures was determined under identical experimental conditions by two different techniques: 31P-NMR and laser microspectrofluorimetry. Time dependence of pH i was monitored by 31P-NMR on the whole cell population. Microspectrofluorimetry, after loading of the cells with SNARF-1, enabled the determination of pH i in isolated cells and its distribution among the cell population. By this method, the vacuolar pH could not be distinguished from the cytoplasmic pH in C. albicans blastospores, but alkalization of pH i was observed at the beginning of germ tubes. The absolute values of pH i determined by 31P-NMR were slightly different from those obtained by laser microspectrofluorimetry. However, the pH distributions in the cell population were converging. For blastospores in exponential phase a gaussian distribution of pH i was observed with both methods, the cells maintained a steady pH i value when the external pH was varied from 5.5 to 8.5. For cells in stationary phase two pools were identified: the combination of the two techniques demonstrated the presence of two different subpopulations. One of these population (with lower pH) was able to commute to the other one with time as shown by 31P-NMR kinetics. This information is reported here for the first time in C. albicans.