Abstract
31P nuclear magnetic resonance (NMR) is a unique technique to monitor noninvasively the energetics of living systems at real time through the detection of a variety of phosphorylated metabolites. Using adequately designed α-aminophosphonates as external probes, we have shown earlier that 31P NMR can also give access simultaneously to the accurate pH of cytosolic and acidic compartments in normal and stressed cultured cells or isolated perfused organs, a feature that was not possible using endogenous inorganic phosphate as the probe. More recently, we obtained a series of derivatives of these new pH probes that incorporate a triphenylphosphonium cation as a specific vector to the mitochondrion. Here, we describe the synthesis, 31P NMR pH titrating properties in buffers, and application in cultures of the green alga Chlamydomonas reinhardtii of two of these mitochondria-targeted pH probes in comparison with one nonvectorized, yet still informative α-aminophosphonate.
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References
Lutz NW, Franks SE, Frank MH et al (2005) Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays. MAGMA 18:144–161
Morse DL, Raghunand N, Sadarangani P et al (2007) Response of choline metabolites to docetaxel therapy is quantified in vivo by localized 31P MRS of human breast cancer xenografts and in vitro by high-resolution 31P NMR spectroscopy of cell extracts. Magn Reson Med 58:270–280
Calmettes G, Deschodt-Arsac V, Gouspillou G et al (2010) Improved energy supply regulation in chronic hypoxic mouse counteracts hypoxia-induced altered cardiac energetics. PLoS One 5:e9306
Durand T, Delmas-Beauvieux MC, Canioni P et al (1999) Role of intracellular buffering power on the mitochondria-cytosol pH gradient in the rat liver perfused at 4 °C. Cryobiology 38:68–80
Llopis J, McCaffery JM, Miyawaki A et al (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci U S A 95:6803–6808
Ogata M, Awaji T, Iwasaki N et al (2012) A new mitochondrial pH biosensor for quantitative assessment of pancreatic ß-cell function. Biochem Biophys Res Commun 421:20–26
Bolshakov AP, Mikhailova MM, Szabadkai G et al (2008) Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca2+ deregulation. Cell Calcium 43:602–614
Orij R, Postmus J, Ter Beek A et al (2009) In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth. Microbiology 155:268–278
Pietri S, Miollan M, Martel S et al (2000) α- and β- phosphorylated amines and pyrrolidines, a new class of low toxic highly sensitive 31P NMR pH indicators. Modeling of pKa and chemical shift values as a function of substrates. J Biol Chem 2754:19505–19512
Pietri S, Martel S, Culcasi M et al (2001) Use of diethyl(2-methylpyrrolidin-2-yl)phosphonate as a highly sensitive extra- and intracellular 31P NMR pH indicator in isolated organs. Direct NMR evidence of acidic compartments in the ischemic and reperfused rat liver. J Biol Chem 276:1750–1758
Martel S, Clément JL, Muller A et al (2002) Synthesis and 31P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies. Bioorg Med Chem 10:1451–1458
Gosset G, Satre M, Blaive B et al (2008) Investigation of subcellular acidic compartments using α-aminophosphonate 31P nuclear magnetic resonance probes. Anal Biochem 380: 184–194
Murphy MP, Smith RAJ (2000) Drug delivery to mitochondria: the key to mitochondrial medicine. Adv Drug Deliv Rev 41:235–250
Culcasi M, Casano G, Lucchesi C et al (2013) Synthesis and biological characterization of new aminophosphonates for mitochondrial pH determination by 31P NMR spectroscopy. J Med Chem 56:2487–2499
Hentrich S, Hebeler M, Grimme LH et al (1993) P-31 NMR saturation transfer experiments in Chlamydomonas reinhardtii: evidence for the NMR visibility of chloroplastidic Pi. Eur Biophys J 22:31–39
Sianoudis J, Küsel AC, Mayer A et al (1987) The cytoplasmic pH in photosynthesizing cells of the green algae Chlorella fusca, measured by P-31 NMR spectroscopy. Arch Microbiol 147:25–29
Gorman DS, Levine RP (1965) Cytochrome F and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas Reinhardtii. Proc Natl Acad Sci U S A 54:1665–1669
Hutner SH, Provasoli L, Schatz A et al (1950) Some approaches to the study of the role of metals in the metabolism and microorganisms. Proc Am Philos Soc 94:152–170
Hutson SM, Berkich D, Williams GD et al (1989) 31P NMR visibility and characterization of rat liver mitochondrial matrix adenine nucleotides. Biochemistry 28:4325–4332
Harris EH (1989) The Chlamydomonas sourcebook. A comprehensive guide to biology and laboratory use. Academic, San Diego
Gahan PB (1984) Plant histochemistry and cytochemistry. Academic, London
Acknowledgements
This work was supported by grants from the CNRS and Aix-Marseille Université, and by ANR grants (ANR–09-BLAN-005-03). The authors thank A. Krieger-Liszkay for discussion and G. Herbette (Spectropole, Aix-Marseille Université) for his help in NMR recording.
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Culcasi, M., Thétiot-Laurent, S., Atteia, A., Pietri, S. (2015). Mitochondrial, Acidic, and Cytosolic pHs Determination by 31P NMR Spectroscopy: Design of New Sensitive Targeted pH Probes. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1265. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2288-8_11
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DOI: https://doi.org/10.1007/978-1-4939-2288-8_11
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