Inorganic polyphosphate and energy metabolism in mammalian cells

Abstract

Inorganic polyphosphate (poly P) is a polymer made from as few as 10 to several hundred phosphate molecules linked by phosphoanhydride bonds similar to ATP. Poly P is ubiquitous in all mammalian organisms, where it plays multiple physiological roles. The metabolism of poly P in mammalian organisms is not well understood. We have examined the mechanism of poly P production and the role of this polymer in cell energy metabolism. Poly P levels in mitochondria and intact cells were estimated using a fluorescent molecular probe, 4',6-diamidino-2-phenylindole. Poly P levels were dependent on the metabolic state of the mitochondria. Poly P levels were increased by substrates of respiration and in turn reduced by mitochondrial inhibitor (rotenone) or an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Oligomycin, an inhibitor of mitochondrial ATP-synthase, blocked the production of poly P. Enzymatic depletion of poly P from cells significantly altered the rate of ATP metabolism. We propose the existence of a feedback mechanism where poly P production and cell energy metabolism regulate each other.

FIGURE 1.

Inhibition of the activation of mitochondrial metabolism changes the level of DAPI-poly P in the cells. Mitochondrial uncoupler, FCCP (1 μm, A), and an inhibitor of glycolysis, IAA (20 μm, B), decrease poly P-DAPI fluorescence in mitochondrial and cytosolic areas of cortical astrocytes. C, effect of substrates for respiratory complexes I (pyruvate, 5 mm) and II (Me-succinate, 5 mm), inhibitor of complex I (rotenone, 5 μm) and the F₁F₀-ATP synthatase inhibitor (oligomycin, 2 μg/ml) on the level of poly P of cortical astrocytes. D, the rate of changes in the poly P level in response to mitochondrial substrates and inhibitor and inhibitors of glycolysis and oxidative phosphorylation in cortical astrocytes. a.u., arbitrary units.

FIGURE 2.

Oligomycin causes the opposite effect on DAPI-poly P fluorescence in HEK cells and astrocytes. Inhibitor of the F₁F₀-ATP synthase, oligomycin (2 μg/ml), increases the level of poly P in HEK cells (A) and decrease DAPI-poly P fluorescence in cortical astrocytes (B). Note that oligomycin-induced mitochondrial depolarization in HEK cells (mitochondrial membrane potential was measured as tetramethylrhodamine methylester fluorescence, C), but hyperpolarized mitochondria in cortical astrocytes (D). Uncoupler FCCP (1 μm) indicates complete mitochondrial depolarization.

FIGURE 3.

DAPI fluorescence in the presence of purified ATP and poly P. Fluorescence was excited at 405 nm and emission was monitored simultaneously at 475 and 550 nm. Arrows indicate points of addition of 6 μm poly P and 1.25 mm ATP. The change in DAPI fluorescence in response to ATP addition is larger when emission is measured 475 nm as compared with 550 nm. Subsequent addition of poly P substantially increased the 550-nm emission fluorescence while reducing the 475-nm emission fluorescence, consistent with a shift in DAPI-poly P emission spectrum. Addition of hexokinase to convert all the ATP in the cuvette to ADP reduced the fluorescence emission at 475 nm without any measurable effect on the 550 nm emission.

FIGURE 4.

Changes in DAPI fluorescence in isolated rat liver mitochondria in response to activation and inhibition of the respiratory chain. A, the emission fluorescence of DAPI was monitored at 475 and 550 nm after addition of succinate (5 mm) and phosphate (1 mm) in the presence of rotenone. The increase in emission fluorescence at 550 nm with no change in 475 nm confirmed that the measured changes in DAPI fluorescence were due to direct changes in poly P levels. B, changes in DAPI fluorescence measured at 550 nm emission in response to substrates (glutamate, 5 mm, and succinate, 5 mm); inhibitor (rotenone, 5 μm), and uncoupler (CCCP, 1 μm) of the respiratory chain. C, the presence of oligomycin (2 μg/ml) to block mitochondrial ATP synthase prevented the increase in DAPI fluorescence (gray trace) observed with activation of mitochondrial respiration after addition of rotenone (5 μm) and succinate (5 mm, black trace). D, change in poly P length in mitochondria treated with succinate (5 mm) in the absence (S) and presence of CCCP. Average length of poly P standards is as follows: short, 14; middle, 60; long, 130. a.u., arbitrary units.

FIGURE 5.

Fluorescence spectrum of DAPI in the presence of mitochondria. A, emission spectrum of DAPI was first collected after addition of rat liver mitochondria and again from the same sample following stimulation by 5 mm succinate in the presence of 5 μm rotenone. B, emission spectrum of DAPI was first collected after addition of mitochondria and again following the addition of various amounts of synthetic poly P. C, normalized fluorescence along with results of Gaussian fits obtained by subtraction of spectrum shown on panel A of stimulated mitochondria minus control mitochondria (gray lines) and B, mitochondria in the presence of 150 ng of poly P minus control mitochondria (black lines). Samples were excited at 415 nm wavelength.

FIGURE 6.

Role of poly P in ATP metabolism. A shows representative normalized traces of C2C12 cells expressing mitochondrially targeted luciferase (mt-Luc) and mitochondrially targeted GFP (mt-GFP) as control (black trace) or MTS-PPX (gray trace) perfused with 50 μm luciferin and challenged in sequence with 5 mm pyruvate, 10 μm rotenone, and 5 mm Me-succinate (n = 3 different transfections). B, measurements of [Mg²⁺]_i, which increase as ATP is hydrolyzed (see text) in control and MTS-PPX C2C12 cells in response to inhibitors of ATP production, IAA (20 μm) and oligomycin (2 μg/ml). C, effect of poly P on the sarcoplasmic-endoplasmic reticulum Ca²⁺-ATPase activity estimated as the rate of Ca²⁺ uptake into isolated canine cardiac sarcoplasmic reticulum vesicles.