Monitoring Of Intracellular pH Research Articles

Silicon Nanowire-Based Surface-Enhanced Raman Spectroscopy Endoscope for Intracellular pH Detection

Very recently, one-dimensional nanowire (NW) sensors have attracted great attention as smart optical endoscopes to probe and manipulate intracellular biological processes. However, NWs often have limited optical response to the intracellular environment changes. In this work, a near-infrared nanowire optical endoscope for high-resolution intracellular pH detection was developed by integrating the advantages of silicon nanowires and surface-enhanced Raman spectroscopy (SERS). This optical endoscope has a high-resolution, sensitive response to local pH changes over the wide range of pH 4.0-9.0, an important range for most biological processes in cells, with high reproducibility, good reversibility, and at least one-week stability in an aqueous environment. The results indicate the great potential of a single SiNW SERS endoscope for intracellular pH monitoring.

Hypoxia-induced invadopodia formation involves activation of NHE-1 by the p90 ribosomal S6 kinase (p90RSK).

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na+/H+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na+/H+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.

Deletion of the anion exchanger Slc26a4 (pendrin) decreases apical Cl−/HCO3− exchanger activity and impairs bicarbonate secretion in kidney collecting duct

The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl(-)/HCO(3)(-) exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization, Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO(3)(-) concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively; P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl(-)/HCO(3)(-) exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH(4)(+) (ammonium) excretion increased significantly, consistent with increased trapping of NH(3) in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl(-)/HCO(3)(-) exchanger(s) is (are) present in the CCD.

Intracellular pH monitoring as a tool for the study of hybridoma cell behavior in batch and continuous bioreactor cultures.

We show in this paper the results obtained when studying the behavior of hybridoma cells by monitoring of the pHi during batch and continuous bioreactor cultures. A first set of experiments, consisting of a batch culture and a continuous culture at variable dilution rate, was set up under normal physiological operating conditions. Significant pHi variations were measured during these cultures. For the batch culture, maximal pHi values around 7.60 were associated with the middle of the growth phase, while lower values were found in the culture beginning and during the decay phase, respectively 7.47 and 7.40. For the continuous culture, pHi increased with increasing dilution rate, ranging between 7.40 and 7. 60 for dilution rates between 0.010 and 0.040 h-1. These pHi variations were found for both cultures to be linked to variations of the specific growth rate of the cells. The observed link between pHi and cell growth provided us a general framework for the study of the effect of suboptimal operating conditions on cell behavior, all having a particular interest in animal cell culture technology. First, our results indicate that a decrease of the medium pH of its normal value of 7.00 to 6.70 did not necessarily result in cytoplasmic acidification, at least not after prolonged exposition times in continuous culture, and this despite a pronounced growth inhibitory effect. This effect can therefore rather be explained by the combination of the increased demand for maintenance energy associated with the higher DeltapH gradient maintained across the cell membrane and the decreased supply of energy, in particular via the glucose metabolism. Second, our results indicate that also increased ammonium ion concentrations do not lower pHi. This observation, together with the results of batch cultures carried out at a more alkaline pH, indicates that the form NH3, and not the form NH4+, has a negative effect on cell growth of our hybridoma cell line. Third, in the case of an osmolality increase, a significant pHi increase was observed, with mean values of 7.35 at the lower osmolalities 335 and 370 mOsm/kg and 7.45 at the higher osmolalities of 400, 425, and 450 mOsm/kg. This higher pHi might account at least partially for the increased monoclonal antibody production observed at hyperosmolality and coincided furthermore with a faster glutamine consumption rate.

Effect of sodium and potassium ions on intracellular pH and proton excretion in glycolyzing cells of Streptococcus mutans NCTC 10449 under strictly anaerobic conditions.

The effect of sodium and potassium ions on intracellular acid production and acid excretion by glycolyzing cells of Streptococcus mutans was examined. S. mutans NCTC 10449 grown under glucose-limited and strictly anaerobic conditions in a continuous culture system was loaded with bis(carboxyethyl)-carboxyfluorescein, a pH-sensitive fluorescent dye, washed and suspended in 0.00-0.30 M NaCl/KCl solution. The dye allowed for the continuous monitoring of intracellular pH while proton excretion was measured simultaneously with a pH-stat. Sodium ions inhibited and potassium ions, at low pH, accelerated the amount of measurable acid excreted extracellularly. In the presence of both NaCl and KCl, proton excretion following the addition of glucose was slightly higher or similar to that observed in the presence of 0.15 M KCl alone. Sodium and potassium ions did not affect the proton-ATPase enzyme or the intracellular level of ATP, suggesting that these ions did not directly effect proton pumping activity itself. The inhibition of proton excretion by sodium ions was considered to have probably occurred as a result of an indirect inhibition of proton-ATPase activity by the low intracellular pH induced by sodium ions.

Enhanced effects of insulin and angiotensin II on intracellular pH and free cytosolic calcium in fibroblasts from microalbuminuric patients with non-insulin-dependent diabetes mellitus.

1. Whether an alteration in cell membrane cation transport after exposure to insulin and angiotensin II (two important growth promoters that have been shown to be involved in the pathogenesis of atherosclerosis and hypertension) is present in cells from non-insulin-dependent diabetes patients with microalbuminuria, a known risk factor for cardiovascular and renal disease, is unknown. We therefore examined intracellular pH and calcium changes after acute exposure to insulin and angiotensin II in cultured skin fibroblasts from eight non-insulin-dependent diabetes patients with and eight others without microalbuminuria and from a group of seven matched, normal control subjects. 2. Cultured fibroblasts were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester or fura 2-acetoxymethyl ester for continuous monitoring of intracellular pH and free calcium concentrations respectively. 3. In quiescent growth-arrested cells, both intracellular pH and free calcium concentrations were similar in the three groups of subjects. Acutely, insulin induced a gradual alkalinization in all groups of patients. The pH increase was significantly greater in non-insulin-dependent diabetes mellitus patients with microalbuminuria (delta pH +0.24 +/- 0.04 pH units) than in normoalbuminuric patients with non-insulin-dependent diabetes mellitus (0.08 +/- 0.02; P < 0.01) and normal control subjects (0.05 +/- 0.01; P < 0.01). Although the alkalinizing effect of angiotensin II was smaller than that obtained by insulin, intracellular pH increase after angiotensin addition was more pronounced in non-insulin-dependent diabetes mellitus patients with microalbuminuria (delta pH +0.14 +/- 0.04 pH units) than in those without (0.08 +/- 0.02; P < 0.01) and in normal control subjects (0.02 +/- 0.02; P < 0.01). That the increase in intracellular pH was mediated by the sodium-hydrogen antiport was demonstrated by its dependence on the presence of sodium in the medium and its inhibition by amiloride. Whereas insulin addition did not evoke any significant increase in intracellular free calcium levels in fibroblasts from the three groups studied, angiotensin II evoked a fast and transient rise in intracellular free calcium that was higher in fibroblasts from microalbuminuric patients with non-insulin-dependent diabetes mellitus than in cells from normoalbuminuric patients with non-insulin-dependent diabetes mellitus and control subjects. In the whole population of patients with non-insulin-dependent diabetes mellitus, the increase in intracellular pH after exposure to angiotensin II was positively correlated with intracellular free calcium increase (r = 0.53; P < 0.05), suggesting a possible role of intracellular free calcium levels in the activation of the sodium-hydrogen antiport. 4. In conclusion, we have described an association between increased agonist-induced responsiveness of sodium-hydrogen antiport activity and the presence of microalbuminuria in patients with non-insulin-dependent diabetes mellitus. This increased responsiveness, persisting in cultured fibroblasts after several passages in vitro, suggests that in vitro phenotypic characteristics of fibroblasts are likely to be genetically determined and to be, at least in part, independent of the degree of metabolic control in vivo.

Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones.

1. Developmental changes in electrophysiological properties of pyramidal neurones correlated with the developmental decline in gap junction-dependent dye coupling were investigated in coronal slices of rat prefrontal and sensorimotor cortex. Effects of intracellular acidification induced by application of weak organic acids on neuronal dye coupling, electrotonic parameters as well as synaptic potentials were examined using the patch clamp technique. Optical monitoring of intracellular pH revealed an acidic shift of 0.4-0.5 pH units following sodium propionate application. 2. Dye coupling between layer II-III neurones was prominent during the first two postnatal weeks. During this period, pre-incubation of slices with 30 mM of the sodium salts of weak organic acids reduced the number of cells coupled to the injected neurones by 64%. 3. Between postnatal days 1 and 18, the mean neuronal input resistance decreased significantly (by 81.0%). Both the membrane time constant (tau 0) and the first equalizing time constant (tau 1) also showed a significant developmental decline of 25.8 and 65.8%, respectively. Electrotonic length decreased by 34.9%. The electrophysiological properties of neurones displayed a pronounced intercellular variability which decreased with on-going development. 4. During the first two postnatal weeks, intracellular acidification led to a mean increase in neuronal input resistance of 55.9% and a mean decreae in electrotonic length of 22.2%. The membrane time constant was reduced by approximately 25% in the majority of neurones tested. Significant electrophysiological effects induced by intracellular acidification were not detected in uncoupled neurones from 18-day-old rats. 5. EPSP width at half-maximal amplitude showed a substantial reduction of approximately 50%, while rise times of the non-NMDA receptor-mediated EPSP components displayed no significant change during development. Both weak organic acids, as well as the gap junction blocker 1-octanol, reduced excitatory synaptic transmission independent of developmental age. 6. We conclude that gap junction permeability is regulated by intracellular pH in developing layer II-III pyramidal cells in the rat neocortex. The prominent correlation between pH-induced reduction in dye coupling and changes in electrophysiological cell properties suggests a significant influence of gap junctions on synaptic integration and information transfer in the immature neocortex.

Simultaneous monitoring of intracellular pH and proton excretion during glycolysis by Streptococcus mutans and Streptococcus sanguis: effect of low pH and fluoride

A system was developed by which 2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein could be used to monitor intracellular pH at the same time that proton excretion was being measured. Streptococcal cells were loaded with the dye, and after the addition of glucose protons were excreted and the intracellular pH increased quickly and remained higher than the extracellular pH of 7.0. The excretion of protons stopped and the intracellular pH returned to the original level when glucose was depleted. The intracellular level of ATP remained high during glucose metabolism and decreased with the depletion of glucose. At extracellular pH of 5.5, and 5.0, the intracellular pH of fasting cells was higher than the extracellular pH value. After addition of glucose there were initial lags of proton excretion and of increases in intracellular pH at the acidic extracellular pH values. In the presence of fluoride, a lag in proton excretion and a simultaneous decrease in intracellular pH were observed, indicating a partial and transient inhibition of proton-ATPase activity.

H +-coupled α-methylaminoisobutyric acid transport in human intestinal Caco-2 cells

Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid α-methylaminoisobutyric acid (MeAIB) across confluent monolayers of the human intestinal epithelial cell line Caco-2 are enhanced by a transepithelial pH gradient (apical pH 6.0, basolateral pH 7.4). In Na +-free conditions (apical pH 7.4, basolateral pH 7.4), net absorption (120 ± 58 pmol/cm 2per h, n = 13) and uptake across the apical membrane (cell/medium ratio 0.56 ± 0.06, n = 13) are low. However, in Na +-free conditions with apical pH 6.0, net absorption (685 ± 95 pmol/cm 2 per h, n = 15) and intracellular accumulation (cell/medium ratio 3.63 ± 0.29, n = 14) were marked. Continuous monitoring of intracellular pH (pH i) in BCECF (2′,7′-bis(2-carbozyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers indicated that apical addition of MeAIB (20 mM) was associated with H +-flow across the apical membrane in both Na + and Na +-free conditions. This transport process is rheogenic in Na +-free media, stimulating an inward short-circuit current in voltage-clamped Caco-2 cell monolayers. On the basis of competition for MeAIB accumulation and pH i experiments, l-proline, glycine, l-alanine and β-alanine are also substrates for H +-linked transport at the apical membrane of Caco-2 cells but l-valine, l-leucine and l-phenylalanine are not. These data are consistent with the expression, in the apical brush-border membrane of Caco-2 cells, of a H +-coupled, Na +-independent MeAIB carrier.

Application of antimony microelectrodes to intracellular pH monitoring

Some novel studies of the properties of the antimony microelectrode used for intracellular pH measurements are described. First, it is shown that currents in the picoampere range, such as those encountered as leakage in some electrometers, induce important changes in pH sensitivity. The response time of the electrode has also been measured and indicates that the electrode exhibits a rapid time course which would be very useful for dynamic cytoplasmic pH investigations. An example of internal pH recording during cellular acidification in Xenopus laevis oocyte is also presented.

Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance.

The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.