Determination Of Intracellular pH Research Articles

Use of fluorescence ratio imaging for intracellular pH determination of individual bacterial cells in mixed cultures.

The development of a rapid method for measuring intracellular pH (pHi) in single bacterial cells is described. Lactobacillus delbrueckii subsp. bulgaricus and Listeria innocua were used as test organisms. The method is based upon fluorescence microscopy and ratio imaging of cells stained with carboxyfluorescein succinimidyl ester. After staining, the bacteria were immobilized on a membrane filter and transferred to a closed perfusion chamber, allowing control of the cell environment during analysis. The set-up was optimized with regard to the use of neutral-density filters and background subtraction, for determining the excitation ratio 490 nm/435 nm (R490/435) independent of the excitation light intensity, and to reduce photobleaching. This allowed for time-lapse studies with multiple exposures. To study the pHi of Lb. delbrueckii subsp. bulgaricus and L. innocua in response to different extracellular pH (pHex) values, an in vivo calibration curve was constructed in the pHi range 5.0-8.5. Distinct differences between the two cultures were observed. L. innocua maintained a near-neutral pHi almost independently of pHex (5.0-8.0), whereas the pHi of Lb. delbrueckii subsp. bulgaricus decreased with decreasing pHex. In pure and mixed cultures at pHex 5.0, the pHi values of Lb. delbrueckii subsp. bulgaricus and L. innocua were 6.1 +/- 0.2 and 7.5 +/- 0.2, respectively. This difference in pHi may explain how Lb. delbrueckii subsp. bulgaricus obtains a competitive advantage over L. innocua at low pHex.

Hepatocellular defence against acidosis is preserved after cold storage.

Primary non-function of liver allografts is related to preservation time, during which hypoxia leads to intracellular accumulation of acid. Preservation-induced failure of hepatocellular pH regulation may play a role in the pathogenesis of primary graft non-function. Using cultured/suspended rat hepatocytes and fluorimetric determination of intracellular pH, we determined whether preservation in University of Wisconsin solution (4 degrees C) impairs hepatocellular defence mechanisms against acidosis. In non-preserved, 24-h-preserved and 48-h-preserved hepatocytes acidified to pH 6.7-6.8, initial Na+/H+ antiport-mediated H+ fluxes averaged 12 +/- 5, 9 +/- 5 and 12 +/- 5 nmol microL-1 min-1 and initial Na+/HCO3- symport-mediated HCO3- fluxes 7 +/- 2, 7 +/- 3 and 6 +/- 2 nmol microL-1 min respectively (P = NS). Preservation did not affect the inverse relationship between Na+/H+ antiport activity and intracellular pH. Thus, hepatocellular defence against intracellular acidosis is maintained during up to 48 h in University of Wisconsin solution. Altered pHi homeostasis is unlikely to play a role in the pathogenesis of primary non-function of liver allografts.

Acrosome reaction inactivation in sea urchin sperm

Acrosome reaction inactivation (ARI) is a process that renders sperm irreversibly refractory to the egg jelly (the natural inducer of the acrosome reaction, AR). This process triggered by the egg jelly, is associated with an increase in [Ca 2+] i. However, we show here that a rise in [Ca 2+] i alone is not sufficient to induce ARI, since artificially increasing [Ca 2+] i with either an ionophore or rising external pH, does not trigger ARI. Contrary to the AR which strictly requires Ca 2+, ARI can be triggered almost equally well by Sr 2+. On the other hand, Mn 2+ inhibits ARI and, as we showed earlier, does not affect AR. These observations indicate that the mechanisms involved in ARI differ from those leading to AR. In addition, we report here that high external pH (a non-physiological inducer of AR) triggers the AR in previously inactivated sperm by opening the same Ca 2+ channels activated by the egg jelly. Considering that the opening of Ca 2+ channels is one of the earliest responses triggered by the egg jelly and that ARI requires the egg jelly receptor to be activated, we have concluded that ARI involves the uncoupling between the egg jelly receptor and Ca 2+ channels. Furthermore, intracellular pH (pH i) determinations, in the presence or absence of ionomycin to substitute for the uncoupled Ca 2+ channels, indicate that pH i regulation is also impaired in inactivated sperm. In conclusion, ARI is a manifestation of the uncoupling of the egg jelly receptor from the different ion transport systems required for the acrosome reaction.

ChemInform Abstract: Synthesis, Acidity, and 19F NMR Characteristics of Imidazoles Bearing 1‐Fluorinated Substituents with Potential Application as Probes for Intracellular pH Determination.

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Synthesis, acidity and 19F NMR characteristics of imidazoles bearing 1-fluorinated substituents with potential application as probes for intracellular pH determination

Abstract A series of 1-substituted imidazoles containing fluorine have been prepared. These compounds were identified as potentially useful probes for intracellular pH determination based on 19 F NMR. Key parameters including pK a , 19 F NMR chemical shift sensitivity, water solubility and low toxicity were identified and included in the target molecule selection process. The pK a and 19 F NMR parameters of the molecules are reported and trends analysed.

Loss of GSH, Oxidative Stress, and Decrease of Intracellular pH as Sequential Steps in Viral Infection

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na+/H+ antiporter, the main system responsible for maintaining unaltered pHi in those cells. At the end of the infection period, a rise in both pHi value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

The effect of BCECF on intracellular pH of human platelets

2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) is frequently used for fluorometric determination of intracellular pH (pH i) and its metabolic changes. Studies of BCECF-loaded platelets have reported different pH i values in the range of 6.98 to 7.35, despite the use of the same probe. It is now shown that intracellular BCECF (BCECF i) content affects pH i, and that its over-loading, leads to significantly lower pH i. Different pH i values can be reproduced by changing BCECF i, as reflected by fluorescence intensity. The major loading factors are: the concentration of the probe parent compound, BCECF acetoxymethyl ester (AM), and whether this ester is partly hydrolyzed externally when applied in plasma. When least affected by BCECF, platelet pH i is 7.34. High BCECF i does not affect ATP content, buffer capacity, activation of Na + H + exchange by protein kinase C (PKC) and basal PKC activity. On the other hand high BCECF i does inhibit the Na + H + exchange rate by over 50%. Since the Na + H + exchange strongly affects platelets pH i, it is proposed that this inhibition accounts, at least partly, for the lowered pH i in BCECF over-loaded platelets.

Regulation of gap junction intercellular Communication by pH in MC3T3-E1 osteoblastic cells

Gap junction intercellular communication (GJIC) may be related to coordinating the function of osteoblasts during bone mineralization. Since an alkaline pH supports mineral deposition while an acidic pH promotes mineral dissolution, it was investigated whether GJIC is altered by changes in extracellular pH (pHo) Functional GJIC was assessed by fluorescent dye transfer after microinjection, and connexin protein abundance was examined by immunoprecipitation and immunoblotting in MC3T3-E1 cells, a model of osteoblast-like cells. The percent of cells coupled by GJIC was found to be 40.7% (24 of 59 injected cells) at pH 6.9, 72.2% (26 of 36) at pH 7.2, and 92.8% (26 of 28) at pH 7.6. A decrease in GJIC was detectable by 30-60 minutes of exposure to a pHo of 6.9. Decreased gap junction communication was also found in cells after 3, 8, and 24 h of incubation in a bicarbonate-CO2 system at an ambient pH of 6.9. Connexin protein abundance experiments showed that at after exposure to a pH of 6.9 for 2.75 h, the specific band(s) at 41-43 kD were fainter compared with these same band(s) at pH 7.2 and 7.6. There was no significant difference in band densities at pH 7.2 and 7.6. Determination of intracellular pH (pHi) showed that it was similar to pHo after 2.75 h of incubation at each ambient pH. When pHi was clamped at 6.9 or 7.2, there was a time-dependent decrease in the gap junction coupling frequency at a pHi of 6.9 when pHo was 7.2. Steady-state mRNA levels were decreased at pHo 6.9 but were unchanged at either pHo 7.2 or 7.6. Our conclusions are that (1) longer incubations ( > or = 2.75 h) at low pHo decrease GJIC which in part may be due to a decrease in connexin protein abundance perhaps as a result of a decrease in connexin steady-state mRNA expression; (2) GJIC inhibition or augmentation found at low and high pHo, respectively, suggests that gating of the GJ channel by pH may also occur; (3) pho-induced alterations in GJIC in the MC3T3-E1 osteoblastic model are related to concomitant changes in pHi.

Use of a new fluorescent probe, seminaphthofluorescein-calcein, for determination of intracellular pH by simultaneous dual-emission imaging laser scanning confocal microscopy.

A new pH indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual-excitation and dual-emission (FITC/Texas Red) filter set. SNAFL-calcein excitation maxima are approximately 492/540 nm (acid/base) and emission maxima are approximately 535/625 nm (acid/base) with a pKa value at approximately 7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dual-excitation (488/568 nm) and dual-emission (525-614 nm/> or = 615 nm) wavelengths (lambda s). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution approximately 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22 degrees C and 37 degrees C) and constant pH, and at the same temperature (22 degrees C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37 degree C than that at 22 degrees C. The basic form of the dye (lambda Em approximately 625 nm) has a slightly higher bleaching rate than the acidic form (lambda Em approximately 535 nm) in standard culture medium (pH 7.3) at either 22 degree C or 37 degrees C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 +/- 0.03 (mean +/- SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL-calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.

Imidazol-1-ylalkanoic acids as extrinsic 1H NMR probes for the determination of intracellular pH, extracellular pH and cell volume

The synthesis and biological evaluation of a novel series of extrinsic probes for intracellular pH (pH i), extracellular pH (pH o) and cell volume determination by 1H NMR is described. Imidazol-1-ylacetate, malonate, 3-glutarate and 2-succinate esters were synthesized by reaction of imidazole with α-bromo esters or with α,β-unsaturated esters. The corresponding acids were prepared by hydrolysis. Rat erythrocytes were readily permeable to methyl imidazol-1-ylacetate, moderately permeable to diethyl 2-imidazol-1-ylsuccinate and impermeable to diethyl 3-imidazol-1-yl-glutarate esters. Imidazol-1-ylacetic acid was the only acid derivative which penetrated the erythrocyte interior when added directly to the incubation medium. Transport of the permeable compounds to the erythrocyte interior was non-saturable up to 200 mM added compound. Addition of methyl imidazol-1-ylacetate or diethyl 2-imidazol-1-ylsuccinate esters to erythrocyte suspensions, resulted in hydrolysis to imidazol-1-ylacetic acid and 2-imidazol-1-ylsuccinic acid mono-ethyl ester in the intracellular and extracellular spaces, respectively. pH i and pH o were determined from the different chemical shifts of the H-2 proton of the acid derivatives in the intracellular (H-2 i) and extracellular (H-2 o) compartments. In addition, the relative intracellular and extracellular volumes could be calculated from the areas of the intracellular and extracellular H-2 resonances.

19F-NMR study of primary human T lymphocyte activation: effects of mitogen on intracellular pH.

Intracellular pH of purified human T lymphocytes was studied using nuclear magnetic resonance (NMR) spectroscopy under physiological conditions. In this paper we introduce a new improved 19F-NMR pH probe, 2-amino-3,3'-difluoroisobutyric acid (vic-difluoro-alpha-methylalanine), which has a pKa of approximately 7.0, sensitivity of 0.83 ppm/pH, is noncytotoxic, and provides better signal-to-noise ratio for intracellular pH determinations. Quiescent and stimulated lymphocytes display different homeostatic intracellular pH values. Quiescent cells maintain intracellular pH of 7.04 +/- 0.03 at extracellular pH values between 6.9 and 7.3, and stimulated cells maintain intracellular pH of 7.25 +/- 0.05 at extracellular pH values between 7.0 and 7.5. Stimulation with ionomycin plus phorbol 12-myristate 13-acetate leads to intracellular alkalinization within 90 min, reaching the more alkaline steady-state value of 7.25 within 7-10 h. Proliferation, but not viability, of lymphocytes is dependent on extracellular pH in the range of 6.4-8.0, and this dependence is not due to limiting interleukin-2 elaboration. The mechanisms of pH regulation and the possible implications of a permissive pH for T lymphocyte proliferation are discussed.

Analysis of Fungal Pellets by UV-Visible Spectrum Diffuse Reflectance Spectroscopy

The application of the UV-visible spectrum diffuse reflectance spectroscopy for the determination of intracellular pH in vivo, for determination of cytochrome content, and for the noninvasive in vivo detection of the redox state of fungal mitochondrial cytochromes in filamentous fungi is introduced. The time course of the intracellular pH values, mitochondrial cytochromes, and CO-binding pigments content and the correlations between the actual redox state of cytochrome aa(3) and saturation of growth medium with oxygen in pellets of the basidiomycete Phanerochaete chrysosporium were determined. As the test microorganism, the yeast Saccharomyces cerevisiae was used. UV-visible spectrum diffuse reflectance spectroscopy proved to be a promising method for the quick and simple analysis of light-impermeable biological structures for which the classical transmittance spectrophotometric methods are difficult to implement.

Simultaneous Determination of Intracellular Magnesium and pH from the Three 31P NMR Chemical Shifts of ATP

The simultaneous determination of intracellular [MgT]/[ATPT] and pH from the three 31P NMR chemical shifts of ATP has been demonstrated using two-dimensional calibrations. The resulting pH will more accurately represent that of healthy tissue than by using the standard NMR technique. As a result of other possible complexes of MgATP and uncertainties in intracellular pH, errors in the values of KMgATPD(pH), the MgATP NMR shift limits, and thus intracellular magnesium levels are reduced by using this self-consistent analytical method. Direct determination of free magnesium from the (γ − β) shifts of ATP may be more sensitive in the alkaline pH range than with the commonly used (α − β) shifts. In addition, the calibration data sets allow for a graphical representation of the uncertainty in magnesium due to the uncertainty in the measured chemical shifts and pH. Our results indicate that KMgATPD is consistent with the literature and favors a value near 50 μM at pH 7.2, but that free magnesium levels will be lower than most prior estimates.

Time-series analysis of measurements on living cells illustrated by analysis of particle movement in the cytoplasm of tomato root hairs

Within a given root hair, the velocity of particle movement, here equated with “cytoplasmic streaming”, is usually very variable, making it difficult to distinguish experimentally-induced changes in velocity from those due to natural variability. Time-series analysis has been combined with piece-wise linear regression to provide an objective means of assessing the results of experiments in which the rate of streaming was recorded repeatedly from the same root hair. Ordinarily, regression and analysis of variance assume that the values in a data set are not strongly autocorrelated. These methods are applicable to sets of single observations (thus, they cannot be used with data obtained by repeated sampling of the same cell). By contrast, time-series analysis can take account of autocorrelations within the data and allows trends in the noise structure to be separated from treatment effects. The statistical methods described in the present paper are illustrated using the results from experiments in which the effect of α-naphthalene acetic acid on streaming velocity in tomato root hairs was recorded. The conclusions reached are in accord with published accounts of variation in protoplasmic streaming and the known behaviour of cells in response to auxins. Our results also provide an explanation for the failure of some workers to observe any consistent changes in streaming velocity in response to exogenous auxin. The method described makes use of well documented statistical techniques and could be applied to other investigations in which sequential measurements of quantifiable parameters, such as fluorometric determination of intracellular pH or concentrations of calcium, are made on intact living cells.

NMRES: an artificial intelligence expert system for quantification of cardiac metabolites from 31phosphorus nuclear magnetic resonance spectroscopy.

The application of high-resolution 31Phosphorus Nuclear Magnetic Resonance (31P NMR) Spectroscopy in biology and medicine has provided new insights into biochemical processes and also a unique assessment of metabolites. However, accurate quantification of biological NMR spectra is frequently complicated by: (a) non-Lorentzian form of peak lineshapes, (b) contamination of peak signals by neighboring peaks, (c) presence of broad resonances, (d) low signal-to-noise ratios, and (e) poorly defined sloping baselines. Our objectives were to develop an expert system that captures and formalizes 31P NMR spectroscopists' expert knowledge, and to provide a reliable, efficient, and automated system for the interpretation of biological spectra. The NMR Expert System (NMRES) was written in the C and OPS5 programming languages and implemented on a Unix-based (Ultrix) mainframe system with XWindows bit-map graphics display. Expert knowledge was acquired from NMR spectroscopists and represented as production rules in the knowledge base. A heuristic weights method was employed to determine the confidence levels of potential peaks. Statistical and numerical methods were used to facilitate processing decisions. NMR spectra obtained from studies of ischemic neonatal and immature hearts were used to assess the performance of the expert system. The expert system performed signal extraction, noise treatment, resonance assignment, intracellular pH determination, and metabolite intensity quantitation in about 10 s per 4 KB (kilobyte) spectrum. The peak identification success rate was 98.2%. Peak areas and pH estimated by the expert system compared favorably with those determined by human experts. We conclude that the expert system has provided a framework for reliable and efficient quantification of complex biological 31P NMR spectra.

Intracellular pH determination by 13C-NMR spectroscopy.

A noninvasive method for the determination of pH by the 13C-nuclear magnetic resonance (NMR) chemical shift of the C-3 carbon of sn-glycerol 3-phosphate is described. Nonlinear least-squares analysis of the chemical shift variation of the C-3 resonance of sn-glycerol 3-phosphate with pH at 37 degrees C in solutions and in perchloric acid extracts of tissue yielded a pKa of 6.2, making it a very sensitive indicator of pH in the approximate range of 5-7. Intracellular pH determined by the present 13C-NMR method correlated well with simultaneous measurements of pH by 31P-NMR spectroscopy over a wide range during normal perfusion and ischemic conditions in intact rat hearts. These findings indicate that this approach is particularly suited for quantification of intracellular pH over the physiological range in intact tissues and that observed in ischemic myocardium.

Imidazol-1-ylalkanoate esters and their corresponding acids. A novel series of extrinsic 1H NMR probes for intracellular pH

A novel series of extrinsic probes for intracellular pH (pH i) determination by 1H NMR is described. Imidazol-1-ylacetate, malonate, 3-glutarate and 2-succinate esters were synthesized by reaction of imidazole either with α-bromoesters or with α,β-unsaturated esters. The corresponding acids were prepared by hydrolysis.

Ca2+ and pH determine the interaction of chromaffin cell scinderin with phosphatidylserine and phosphatidylinositol 4,5,-biphosphate and its cellular distribution during nicotinic-receptor stimulation and protein kinase C activation.

Nicotinic stimulation and high K(+)-depolarization of chromaffin cells cause disassembly of cortical filamentous actin networks and redistribution of scinderin, a Ca(2+)-dependent actin filament-severing protein. These events which are Ca(2+)-dependent precede exocytosis. Activation of scinderin by Ca2+ may cause disassembly of actin filaments leaving cortical areas of low cytoplasmic viscosity which are the sites of exocytosis (Vitale, M. L., A. Rodríguez Del Castillo, L. Tchakarov, and J.-M. Trifaró. 1991. J. Cell. Biol. 113:1057-1067). It has been suggested that protein kinase C (PKC) regulates secretion. Therefore, the possibility that PKC activation might modulate scinderin redistribution was investigated. Here we report that PMA, a PKC activator, caused scinderin redistribution, although with a slower onset than that induced by nicotine. PMA effects were independent of either extra or intracellular Ca2+ as indicated by measurements of Ca2+ transients, and they were likely to be mediated through direct activation of PKC because inhibitors of the enzyme completely blocked the response to PMA. Scinderin was not phosphorylated by the kinase and further experiments using the Na+/H+ antiport inhibitors and intracellular pH determinations, demonstrated that PKC-mediated scinderin redistribution was a consequence of an increase in intracellular pH. Moreover, it was shown that scinderin binds to phosphatidylserine and phosphatidylinositol 4,5-biphosphate liposomes in a Ca(2+)-dependent manner, an effect which was modulated by the pH. The results suggest that under resting conditions, cortical scinderin is bound to plasma membrane phospholipids. The results also show that during nicotinic receptor stimulation both a rise in intracellular Ca2+ and pH are observed. The rise in intracellular pH might be the result of the translocation and activation of PKC produced by Ca2+ entry. This also would explain why scinderin redistribution induced by nicotine is partially (26-40%) inhibited by inhibitors of either PKC or the Na+/H+ antiport. In view of these findings, a model which can explain how scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated conditions is proposed.

Extracellular fluid volume measurements in tissues of the rainbow trout (Oncorhynchus mykiss)in vivo and their effects on intracellular pH and ion calculations

Extracellular fluid volume (ECFV) estimates were determined in various tissues and whole body of resting, chronically cannulated rainbow trout (Oncorhynchus mykiss). Fish were infused with(14)C-inulin,(3)H-polyethylene glycol (PEG, M.W. 4000),(3)H-mannitol, or(14)C-mannitol, and values of ECFV determined from tissue: plasma water distribution ratios after 6 h or 13 h equilibration. Overall,(3)H-PEG provided the most conservative and reliable estimates after 13 h equilibration, with ECFV values in the order: brain < white muscle < red muscle < liver < heart < gill tissue.(14)C-inulin yielded generally similar values to(3)H-PEG at 13 h, but probably overestimated ECFV in liver.(3)H-mannitol and(14)C-mannitol spaces were similar to each other and far greater than(3)H-PEG or(14)C-inulin values in most tissues.(3)H-mannitol values increased significantly between 6 h and 13 h, in contrast to(14)C-inulin. Mannitol clearly overestimated ECFV in liver and gill, and probably also heart and whole body, but may have provided more realistic estimates in brain due to better penetration of the blood-brain barrier. The Cl(-)/K(+) space technique overestimated ECFV in gills, but was satisfactory in white muscle. Measurements and model calculations evaluated sources of error in intracellular pH (by(14)C-DMO) and ion determinations. Trapped red cells in the gills have negligible influence. Errors in ECFV are much more influential in a tissue with a high ECFV (gills) than a low ECFV (white muscle).(3)H-PEG is the marker of choice for intracellular pH determinations. However, even when(3)H-PEG is used, the potential for absolute errors in intracellular ion concentrations remains high.

The Simultaneous Determination of Intracellular pH and Cell Energy Status

The relationship between the apparent equilibrium constant of creatine kinase and intracellular pH was evaluated in CHO and murine FSaII tumor cells. The apparent equilibrium constant, K' = [ATP][Cr]/[ADP][PCr], was determined from acid extracts at variable pH. Intracellular pH (pHi) was determined from the intracellular/extracellular distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione. Over the intracellular pH range of 7.2 to 6.1, K' increased by a factor of approximately 10. Intracellular pH was related to the apparent equilibrium constant by the equation pHi = -log K' + log K, where the value of the constant log (log[K'/H+]) was 8.09. Over the same pH range, the concentration of phosphocreatine decreased with pH. Essentially identical results were obtained in CHO and FSaII tumor cells. The similar apparent equilibrium constants in CHO and FSaII cells suggest that assessment of the creatine kinase metabolites will be useful not only for determination of cell energy status but also for the determination of intracellular pH. This information may be useful for the design of therapeutic strategies which are influenced by pH or energy status such as hyperthermia, and drugs which are weak acids or bases, including hypoxic cell radiosensitizers.