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. Inorganic polyphosphate (poly P)2 is found in all living organisms ranging from bacteria to mammals (1). Poly P performs multiple physiological functions, which are distinct and dependent on the type of organism and the subcellular localization of the polymer. In microorganisms, poly P primarily plays a role in transcription. Additionally, poly P serves as an energy store (2) and as a reserve pool of inorganic phosphates (3). However, in mammalian organisms, poly P plays predominantly a regulatory role (4) and has been implicated in the regulation of enzyme activity in cancer cells (5), stimulation of blood coagulation (6), regulation of mitochondrial ion transport (7), and regulation of respiratory chain activity (8).Although a specific enzyme(s) responsible for poly P production in mammals is currently not known (1), poly P synthesis has been detected in intact mammalian cells. Lysis of mammalian cells leads to loss of poly P synthesis activity, suggesting that poly P synthesis in mammalian cells is likely an energy-dependent process linked to membrane transport and integrity (1, 9). Taking into account that the membrane potential generated at the mitochondrial inner membrane is a major energy source for cellular metabolism, we hypothesized that mitochondria may be the likely source of poly P production in mammalian cells.Poly P is found in mammalian cells at significantly lower levels when compared with microorganisms (9); therefore, it is very difficult to adapt poly P measurement methods developed for bacterial studies for the study of mammalian cells. Recently we developed a protocol, which we optimized for suitability for measuring low amounts of poly P using the fluorescent probe 4Ј,6-diamidino-2-phenylindole (DAPI) (10). In our previous study we used this method to confirm poly P hydrolyzing activity of yeast polyphosphatase expressed in mitochondria of mammalian cultured cells (8). Here we take advantag...

Signal transduction in astrocytes: Localization and release of inorganic polyphosphate

“…Simultaneous measurement of mitochondrial (TMRM) and polyP (JC‐D8) signals showed that ∼40% of cellular polyP resides in mitochondria (here and below coefficient of colocalization is a Mander's X; 0.39 ± 0.08, n  = 204 cells; Figure 1a,d). As we reported previously, the level of polyP in mitochondria is dependent on the energy state of mitochondria and can be modified within seconds (Pavlov et al, 2010). …”

“…Accumulation of polyP in cellular organelles and vesicles can indicate a specific signaling function. In this study, we found that ∼39% of the total pool of intracellular polyP in astrocytes is located in mitochondria (Figure 1) where it plays a role in bioenergetics (Angelova, Baev, et al, 2016; Pavlov et al, 2010) and calcium handling (Abramov et al, 2007; Baev et al, 2017; Elustondo et al, 2016). The level of polyP in mitochondria has been previously shown to be dependent on the metabolic state and the age of the cells (Pavlov et al, 2010).…”

“…PolyP in the mitochondria has its functional role; it plays an important role in energy metabolism and calcium handling (Baev, Negoda, & Abramov, 2017; Pavlov et al, 2010). In agreement with our previous report, we found that in cultured astrocytes the specific polyP indicator has higher intensity in the mitochondrial area (Angelova et al, 2014) (Figure 1a).…”

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

“…In yeast, a genome-wide screen revealed the interdependence of polyP with primary metabolism (29). In mammalian cells, alteration of mitochondrial metabolism affects polyP production (36). It has also been suggested that polyP is an activator or even a constituent of the mitochondrial permeability transition pore (37).…”

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription

“…The method of polyP detection using DAPI was first described in the early 1980s. 21 Since that time, this procedure has been used in several studies (some examples are references: 6,8,13,16,[22][23][24][25] ). Emission fluorescence of DAPI shifts to longer wavelengths in the presence of polyP: this change is specific for polyP and is not produced by PPi, DNA, or other anions.…”