Signalling properties of inorganic polyphosphate in the mammalian brain

Abstract

Inorganic polyphosphate is known to be present in the mammalian brain at micromolar concentrations. Here we show that polyphosphate may act as a gliotransmitter, mediating communication between astrocytes. It is released by astrocytes in a calcium-dependent manner and signals to neighbouring astrocytes through P2Y(1) purinergic receptors, activation of phospholipase C and release of calcium from the intracellular stores. In primary neuroglial cultures, application of polyP triggers release of endogenous polyphosphate from astrocytes while neurons take it up. In vivo, central actions of polyphosphate at the level of the brainstem include profound increases in key homeostatic physiological activities, such as breathing, central sympathetic outflow and the arterial blood pressure. Together, these results suggest a role for polyphosphate as a mediator of astroglial signal transmission in the mammalian brain.

Figure 1. PolyP induces Ca²⁺ responses in neurons and astrocytes.

Medium polyP (50 μM) induces [Ca²⁺]_c changes in hippocampal astrocytes (a) and neurons (b) as seen by an increase in the ratio of the Ca²⁺ dye Fura-2. KCl (50 mM) was added at the end of the experiment for cellular (neuronal) identification. (c) Removal of external Ca²⁺ (Ca²⁺-free medium with 0.5 mM EDTA) does not abolish polyP-induced Ca²⁺ responses in astrocytes in response to 50 μM M-polyP. (d) Depletion of intracellular Ca²⁺ stores with thapsigargin (1 μM) and ATP (100 μM) abolishes M-polyP (50 μM)-induced Ca²⁺ responses in astrocytes. (e) M-polyP (50 μM)-induced [Ca²⁺]_c responses are inhibited in the presence of the PLC inhibitor U73122 (5 μM). Shown are representative traces from at least three independent experiments. (f) PLC inhibitor U73122, P2 receptor antagonist suramin (500 μM), and specific P2Y₁ receptor antagonist MRS2279 (50 μM) blocked the polyP-induced [Ca²⁺]_c responses, whereas the ATP-degrading enzymes hexokinase and apyrase (each 25 U ml⁻¹) and the specific P2Y₆, P2Y₁₁ receptor inhibitors (MRS2578 (10 μM) and NF340 (10 μM), respectively) had no effect. All experiments were performed using 50 μM M-polyP. Error bars represent s.e.m., n>100 cells for each condition, **P<0.005, N.S., not significant. (g) Knockdown of the P2Y₁ receptor using shRNA suppressed the polyP-induced Ca²⁺ signal (compared with a scrambled control, Scr). Error bars represent s.e.m., n=201 for P2Y₁ and n=51 for Scr, *P<0.05. (h) Pre-incubation of cells with apyrase (25 U ml⁻¹) did not affect the amplitude of M-polyP (50 μM)-induced Ca²⁺ signal. Student’s t-test was used to asses statistical significance.

Figure 2. Uptake and release of polyP in primary neuroglial co-cultures.

PolyP can be detected in vesicle-like structures in astrocytes using DAPI (DAPI-polyP) (a). Application of M-polyP (50 μM) to DAPI-loaded neuroglial cultures induces release of polyP as seen by a decrease in intracellular vesicular DAPI-polyP (a–c). Error bars represent s.e.m. n=93 cells. Shown are representative images from two time points and the mean trace. Scale bar, 10 μm. PolyP released from astrocytes (a) upon stimulation with M-polyP (50 μM) is taken up by neurons (n) as seen by DAPI-polyP (d). (d) Bright-field image combined with DAPI-polyP staining (blue). Scale bar, 20 μm. (e) Representative mean traces from neurons and astrocytes. Error bars represent s.e.m. n=54 neurons and n=61 astrocytes. (f–i) Application of ionomycin (5 μM) induces release of vesicular polyP from cortical astrocytes. Shown are representative images from three time points (0, 60, 250 s) (f–h) and the corresponding traces (i). (j) Loading of neuroglial cultures with DAPI and Lysotracker (LT) simultaneously indicates partial colocalization of DAPI-polyP with lysosomes (arrows), whereas other DAPI-polyP vesicles do not stain with LT (arrow heads). Scale bar, 10 μm. (k) PolyP is released from a proportion of LT and DAPI co-stained vesicles upon addition of 50 μM M-polyP.

Figure 3. Ca²⁺-induced polyP release.

Uncaging of Ca²⁺ by ultraviolet flash (depicted by black arrows and a black square and trace) in a single cultured astrocyte induces Ca²⁺ signal in neighbouring cells in the presence of apyrase (25 U ml⁻¹; red and blue squares and traces) (a,b). Scale bar, 20 μm. (c) Removal of apyrase from the medium had no effect on the amplitude of the induced Ca²⁺ signal (black arrows indicate time of the flash). (d) The transmission of the signal can be blocked by inhibition of the P2Y₁ receptors with MRS2279 (50 μM).

Figure 4. Effects of polyP in vivo.

Application of M-polyP (50 μM) to the ventral surface of the brainstem increases heart rate (HR), arterial blood pressure (ABP), renal sympathetic nerve activity (RSNA) and phrenic nerve activity (PNA) (a). Expanded traces of PNA and RSNA on the right depict the activities before and after application of polyP. Time points are indicated by numbers (1) and (2) on the time-condensed trace on the left. The effects of M-polyP on the ABP (b) and RSNA (c) in vivo are blocked by pre-treatment with P2 receptor antagonists PPADS (100 μM) or MRS2279 (50 μM). Values are means±s.e.m. Numbers in parentheses indicate sample sizes. *P<0.01 compared with the effect of M-polyP applied alone (ANOVA followed by Tukey–Kramer’s post hoc test).