Estimation Of Intracellular pH Research Articles

Flow cytometry and growth-based analysis of the effects of fruit sanitation on the physiology of Escherichia coli in orange juice.

Chlorine‐based solutions are commonly used to sanitize orange fruits prior to juice extraction. We used flow cytometry (FCM) to investigate the physiology of Escherichia coli following its subjection to chlorine‐based solutions and alternative sanitizing agents (H2O2 and organic acids). Green fluorescent protein (GFP)‐generating E. coli K‐12 were washed with 50–200 ppm available chlorine (AC), 1%–5% H2O2, 2%–4% citric acid, 4% acetic acid, or 4% lactic acid, after which they were added to 1.2 μm‐filtered orange juice (OJ). Cell physiology was investigated with FCM during storage at 4°C, and culturability was determined using plate counting. Analysis of GFP fluorescence allowed estimation of intracellular pH (pH i). FCM results demonstrated an inverse relationship between the concentration of AC or H2O2 and cellular health in OJ. Higher concentrations of sanitizer also resulted in a significantly greater number of viable but nonculturable (VBNC) cells. Real‐time FCM showed that supplementation of AC with 2% citric acid, but not with 100 ppm of Tween‐80, led to a significant reduction in pH i of the cells incubated in OJ, and that the majority of the reduction in pH i occurred during the first 2 min of incubation in OJ. Organic acids were found to be more effective than both AC and H2O2 in reducing the pH i, viability, and culturability of the cells in OJ. The results confirmed the hypothesis that consecutive subjection of E. coli to maximum legally permitted concentrations of sanitizers and OJ induces the VBNC state. Furthermore, we demonstrate successful application of FCM for monitoring the efficacy of washing procedures.

Morphological homoplasy in Japanese Plocamium species (Plocamiales, Rhodophyta) inferred from the Rubisco spacer sequence and intracellular acidity

Among five species of the genus Plocamium present in Japan, P. cartilagineum, P. recurvatum and P. telfairiae show similar blade morphology, and intermediates exist between them. We investigated the diversity and evolutionary relationships of these Plocamium species using morphological observations, estimation of intracellular pH and analysis of Rubisco spacer sequences. Although P. recurvatum is characterized by the presence of extremely recurved branchlets, most of the specimens examined also have linear or slightly reflexed branchlets in the same thallus. Plocamium cartilagineum is not clearly distinguished from P. telfairiae by the conventional diagnostic characters such as width of main axes, number of branchlets in alternate series and size of the lowermost branchlet. Intracellular pH of these species is estimated to be 1.2–6.6 and is variable within a species. Three haplotypes in P. cartilagineum, two in P. recurvatum and eight in P. telfairiae are recognized on the basis of the Rubisco spacer sequences, and most of these haplotypes show specific intracellular pH. The molecular phylogenetic analyses demonstrate three major clades in the Japanese Plocamium species: P. cartilagineum, one haplotype of P. recurvatum and four of P. telfairiae in one clade (A); P. ovicorne and P. serratulum in another (clade B); the remaining haplotype of P. recurvatum and four haplotypes of P. telfairiae in clade C. Clade C contains only the haplotypes showing strong acidity, whereas clade A is composed of both strongly acidic and weakly acidic or neutral haplotypes. These results suggest that morphological homoplasy occurred in P. recurvatum and P. telfairiae and that physiological properties such as intracellular pH may be informative and contribute to our understanding of the relationship of the Japanese Plocamium species.

Estimation of intracellular pH in muscle of fishes from different thermal environments

A technique based on homogenisation of rapidly frozen tissue was used to investigate the regulation of intracellular pH (pH i) in freshwater and marine fish from diverse environmental temperatures. The following species were held at ambient temperatures of ca. 1°C ( Notothenia coriiceps; Antarctica), 5°C ( Pleuronectes platessa, Myoxocephalus scorpius; North Sea), and 26°C ( Oreochromis niloticus; African lakes). The effects of seasonal acclimatisation to 4, 11 and 18°C were also examined in rainbow trout in the winter, autumn and summer, respectively. Extracellular (whole blood) pH (pH e) did not follow the constant relative alkalinity relationship, where pH +=pOH − for any particular temperature, over a range of 1–26°C (overall δpH e/δ T=0.009±0.002 U °C −1; P<0.001), apparently being regulated by ionic fluxes and ventilation. Intracellular pH (pH i) was also regulated independently of p N(=0.5 p K water) in all species of fish examined. The inverse relationship between pH i and environmental temperature gave an overall δpH i/δ T of −0.010±0.001 U °C −1 (for both white and red muscle) and −0.004±0.003 U °C −1 (cardiac muscle). However, between 1 and 11°C δpH i/δ T was much higher ( P<0.001), −0.022±0.003 U °C −1 (white muscle) and −0.022±0.004 U °C −1 (red muscle). The possible adaptive roles for these different acid–base responses to environmental temperature variation among tissues and species, and the potential difficulties of estimating pH i, are discussed.

Time-Resolved Fluorescence Microscopy Could Correct for Probe Binding while Estimating Intracellular pH

Estimation of intracellular pH by fluorescence ratiometry overcomes many of the limitations such as variations in the pathlength of observation and concentration of the probe, light scattering, and photobleaching. However, binding of probes to membranes and macromolecules is generally not taken into account. By using time-resolved fluorescence microscopy on a variety of cell types, we have shown that the dual-emission fluorescent pH probe carboxy SNARF-1 binds to cellular components in significant levels. The bound population could be resolved in the timescale since its fluorescence lifetime (∼3 ns) is significantly larger than that of the free probe. Intracellular pH was estimated from the relative amplitudes corresponding to free probes. This procedure was validated in simple model systems where carboxy SNARF-1 was present in solutions of bovine serum albumin. It was shown that the intracellular pH could be overestimated by as much as 1 pH unit in the absence of correction for probe binding.

Estimating intracellular pH in developing rodent embryos using a computer imaging technique: changes in embryonic pH and proliferation rates following maternal treatment with acetazolamide.

Using the transplacental distribution of the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO), a computer assisted imaging technique has been developed to permit the estimation of intracellular pH (pHi) in very specific areas of the developing rodent embryo. The study reported here demonstrates the heterogeneity of radiolabeled DMO distribution in the developing mouse forelimb. The pattern of pHi distribution shifts from one of high pHi values in the proximal core of the mesoderm on day 10 of gestation to one of higher pHi values in the mesoderm just underlying the ectoderm on day 11. Studies [Scott et al. (1990) Toxicol. Appl. Pharmacol. 103:238-254] in which DMO concentration was monitored following treatment with acetazolamide or acetazolamide plus amiloride were done in whole embryo homogenates or pooled limb samples which allow for the calculation of an average pHi but may not reflect the pHi in very specific locations of the limb. Two hours after acetazolamide administration, the pHi pattern was not significantly changed from control. Intracellular pH was raised above control levels but was not significant statistically except in the peripheral mesoderm in the ventral third of the forelimb. Fifteen hours after acetazolamide treatment, there was a significant decrease in pHi values with no change in pattern. However, treatment with acetazolamide plus amiloride for 15 hr produced a marked reduction of pHi values throughout the forelimb bud. Changes in bromodeoxyuridine labeling index (an indication of proliferative activity) following treatment with acetazolamide or acetazolamide plus amiloride are reported. The combination treatment reduced the labeling index by approximately 15% below that of control embryos in the limb region where absence of digit(s) will occur. However, we found no overall correlation of proliferative rate and pHi of limb bud mesoderm in treated embryos. Consequently, we were unable to causally associate reduced pHi with decreased proliferative rate.

Reduced PO 2 alters the behavior of Fura-2 and Indo-1 in bovine pulmonary artery endothelial cells

Calcium-sensitive fluorophores are used to estimate cytosolic free Ca 2+ in many cell types under various conditions. We tested the effect of reduced PO 2 on the behavior of Fura-2 and Indo-1 in cultured bovine pulmonary artery endothelial cells. Reduced PO 2 (PO 2 25–35 mmHg) caused a significant upward shift of in vivo calibration curves for both fluorophores. The in vivo emission spectrum of Fura-2 indicated that the effect was principally due to attenuated emission at the Ca 2+-unbound 380 nm wavelength, with no shift in position of the emission maxima for either Ca 2+-bound or unbound forms of the fluorophore. Reduced PO 2 did not directly alter the behavior of the dyes, as no shift of in vitro calibration curves was seen. Neither decreased photobleaching nor altered autofluorescence accounted for the shift. We investigated several potential indirect effects, including cellular acidification, reduced viscosity, inhibition of oxidative energy production and reductive stress. In contrast to lowered PO 2, acidification in vitro produced a leftward but not an upward shift. Estimation of intracellular pH with SNAFL-calcein under reduced PO 2 showed no apparent acidification in these cells, further strengthening the argument that altered intracellular pH was not causing the shift. Others have shown that decreases in viscosity in vitro may shift the calibration curve for Fura-2 upward, similar to our finding with reduced PO 2. However, for Indo-1 we found that decreased viscosity in vitro attenuated fluorescence emission at the Ca 2+-bound 405 nm wavelength, thus producing the opposite effect on fluorescence ratio and indicating that reduced PO 2 was not acting through changes in cellular microviscosity. Because reduced PO 2 could be acting through either reduced energy production or redox, we tested the effects of the metabolic inhibitor rotenone (100 nM), and the reducing agent glutathione (50 mM), on the calibration curves. Neither intervention changed Fura-2 fluorescence in vivo, suggesting that these mechanisms were not responsible for the shift in in vivo calibration under reduced PO 2. We conclude that in pulmonary artery endothelial cells, reduced PO 2 alters the behavior of Ca 2+-sensitive fluorophores through an unidentified, indirect mechanism, and recommend that investigators utilizing this technique to study oxygen-dependent effects employ PO 2-specific in vivo calibration curves to avoid errors in interpretation.

Response of ammonia metabolism to acute acidosis: insights from cultured renal epithelium.

LLC-PK1 kidney epithelial cell lines cultured under the condition of continuous rocking exhibit both acute and adaptive changes in glutamine-dependent ammonia metabolism in response to acid-base manipulations in the media pH. Pulse-chase studies with 14C-glutamine as well as studies with various metabolic inhibitors of the ammoniagenic pathways suggest that glutamine in LLC-PK1 cells is metabolized via mitochondrial pathway and that intramitochondrial phosphate-dependent glutaminase pathway plays a predominant role in the regulation of ammoniagenesis to acute acidosis. Furthermore, the measurements of intracellular pH under both basal and acute low pH conditions resulted in the estimation of intracellular pH, which paralleled the alterations in media pH. Therefore, a change in intracellular pH appears to act as a direct signal for alterations in ammonia metabolism in LLC-PK1 cells. Thus, LLC-PK1 cultures provide an excellent model system to investigate renal ammoniagenesis and the intracellular signals that modulate this process.

Red cell volume changes monitored using a new 31P NMR procedure

The application of NMR spectroscopy to whole cell systems has provided considerable insight into a range of cellular phenomena. 3’P NMR is used routinely for the estimation of intracellular pH (I, 2) and in assessing the energy status of whole cell samples (3). ‘H NMR has been used to monitor a variety of membrane transport processes (4) as well as a number of intracellular reactions (5). The rates of reactions inside the cell depend upon the concentrations of enzymes, substrates, and effector molecules and these, in turn, are determined by the intracellular volume. Changes in cell volume have, for example, been shown to affect the glycolytic rate in the human erythrocyte, mainly by altering the intracellular concentration of ATP, a regulator of glycolysis (6). Therefore, in any study of intracellular reaction rates, an understanding of how (and why) the cell volume may be changing during a particular experiment is important. We present here results obtained using a method whereby time-dependent changes in red cell volume may be monitored using “P NMR. The method is based on the observed cell volume dependence of the chemical shift of the “P NMR signal to dimethyl methylphosphonate. It is applicable to high hematocrit cell suspensions and was used to monitor the decrease in erythrocyte volume that followed the addition of the potassium ionophore, valinomycin, to cells suspended in a low-potassium medium. Erythrocyte suspensions were prepared from fresh blood obtained from the Red Cross Transfusion Service, New South Wales, Australia. The cells were washed twice in isotonic saline (0.154 M NaCl, 4°C) then three times in Krebs bicarbonate buffer (7) equilibrated beforehand with Carbogen (Oz/COz ; 19: 1) to give a pH of 7.3. All 3LP NMR spectra were recorded at 162 MHz using a Bruker WM 400 spectrometer operated in the Fourier transform mode at 37°C. Broadband proton decoupling was used throughout the experiments and the sample tubes were spun at - 16 Hz. Chemical shifts are quoted relative to that of 85% (v/v) phos- phoric acid. Dimethyl methylphosphonate (DMMP; CH3PO(OCH3)2; Aldrich, Mich.) is a small, neutral molecule, readily miscible with water. The “P NMR spectrum of DMMP in saline solution appeared as a single resonance (6 = 39.4 ppm). The addition of whole cells to the solution caused the signal to split into two. Furthermore, as the average red cell volume was altered by varying the extracellular