Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells

Abstract

Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)-nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10 μM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y or adenosine A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2 h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2 h cell re-oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re-oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end-products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.

Uncovering the Signaling Pathway behind Extracellular Guanine-Induced Activation of NO System: New Perspectives in Memory-Related Disorders

“…This suggested that the levels of extracellular guanine-based purines depended both on the balance between nucleotide release and nucleoside uptake and on the presence, in the culture medium, of extracellular purine-converting enzymes. Indeed, by using Immunoblot analysis and HPLC for the measurement of the enzyme activities ( Giuliani et al, 2017 ), we confirmed the presence in the culture medium of PNP and GDA ( Figure 1C ). Both the enzymes tended to accumulate in the culture medium over the time (from 2 up to 12 h) following a similar trend even though at different levels (PNP amount was about 7–10 fold higher than GDA).…”

“…According to the method previously described ( Giuliani et al, 2012 , 2017 ), purines were measured by an Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara, CA, United States), by using, for the separation of the compounds, a reverse phase analytical column (LiChrospher 100 RP-18 5 μm in LiChroCART 125-4, Merck) and a 15-min linear gradient [from 100% of buffer A (60 mM KH 2 PO 4 and 5 mM tetrabutylammonium phosphate, pH 6.0) to 100% solvent B (30% methanol plus 70% buffer A)] at a flow rate of 1.5 mL/min. The detection of unlabeled compounds was achieved using a Diode Array Detector (Agilent Technologies, Santa Clara, CA, United States) with wavelength set at 254 nm for all the substances except Uric Acid (UAc), which was 290 nm.…”

“…Intracellular GUA derives from guanosine triphosphate (GTP) breakdown and represents the starting point of reactions deputed to maintain intracellular levels of GTP (purine salvage pathway). When intracellular levels of GUA are excessive, it may be transported outside the cells by specific transmembrane nucleobases transporters, although most of the extracellular GUA derives from the breakdown of the released GTP and it is generated by GUO in a reaction catalyzed by the purine nucleoside phosphorylase (PNP) ( Rathbone et al, 2008 ; Giuliani et al, 2016 , 2017 ; Peña-Altamira et al, 2017 ). On the contrary, GUA degradation to xanthine (Xan) is mediated by Guanine deaminase (GDA) or cypin ( Miyamoto et al, 1982 ), which has been regarded as one of the “intrinsic factors” that regulate dendrite morphology together with the small GTPases RhoA, Rac1, the β-catenin ( Yu and Malenka, 2004 ), PSD-95 ( Charych et al, 2006 ) and the calcium/calmodulin-dependent protein kinase II (CAMKII) ( Fink et al, 2003 ).…”

Purinergic Signalling: Therapeutic Developments

“…P2X7R antagonists blocked the cell cytotoxicity caused by irradiation for glioma ( Gehring et al, 2015 ). Purine nucleoside phosphorylase is released from rat C6 glioma cells, contributing to the purinergic system homeostasis and exhibiting a pathophysiological role ( Giuliani P. et al, 2017 ).…”

Presentation1.pdf

“…Samples containing PNP were obtained as described by Giuliani et al (2017). Briefly, SH-SY5Y cells were incubated in serum-free medium without or with 100 µM GUO.…”