ABSTRACT
Title
Purine nucleoside phosphorylase (PNP) is released from cultured rat astrocytes and microglial cells by lysosomal exocytosis.
Authors
E. Polazzi1, B. Monti1, I. Mengoni1, P. Giuliani2, S. Buccella2, S. Cicchitti2, F. Caciagli2, A. Poli1
1Department of Biology, University of Bologna; 2Department of Biomedical and Human Movement Sciences, “G.D’Annunzio” University of Chieti-Pescara. Italy
1Department of Biology, University of Bologna; 2Department of Biomedical and Human Movement Sciences, “G.D’Annunzio” University of Chieti-Pescara. Italy
Abstract
Purine nucleoside phosphorylase (PNP) is an important intracellular catalytic enzyme in the purine salvage pathway.
Its deficiency is associated with T-cell lymphopenia, severe immunodeficiency and neurologic symptoms including ataxia, spasticity and delay in the brain development.
On the other hand,it has been observed that PNP activity and expression were up-regulated in periodontal diseases and in oral cancer tissue (Maeda K, et al.; Cancer 1992) as well as in human colon carcinoma where the enhanced PNP activity was associated with a higher biological aggressiveness of tumoral cells (Sanfilipo O. et al.; Biochem Biophys 1994).
Furthermore, it has been reported that Forodesine, a potent and specific PNP inhibitor, induced apoptosis in several malignant T-cell and B-cell lines, and it is currently tested in phase I/II clinical trials for patients with lymphoblastic/lymphocytic leukemia (Bieghs L. et al.; Adv.Hematol.; 2010).
PNP is present in the cytosol and mitochondria of the major part of cells and its histochemical localization in the brain indicated that it was restricted to glial cells whereas neurons are almost negative (Castellano et al, J Hist Cytochem; 1990).
Our recent results under publication demonstrated that PNP is also localized on cell membranes as it was found for other ecto-enzymes involved in the catabolism of extracellular purines. Furthermore, a PNP activity was observed in plasma as well as in the gingival crevicular fluid, even though the source of such enzyme is substantially unknown.
The aim of the study was to assess whether primary cultures of cerebellar neurons, astrocytes and microglia were able to release PNP into the culture medium and to evaluate whether it occurred in a similar manner in all of them.
Western blot analysis of the conditioned media from equivalent numbers of cultured cells and collected at the same time points, demonstrated that PNP is remarkably released from astrocytes and microglia in a time dependent manner and only to a much lower extent from cerebellar neurons. The level of expression of PNP in the different media revealed that microglial cells are more efficient in the enzyme secretion compared to astrocytes.
These results have been confirmed by the evaluation of the enzyme activity in the conditioned media which was measured by an radio-HPLC analysis of the de novo formed labeled guanine derived from the labeled guanosine, added to media samples and used as substrate of the enzyme.
Microglial activation induced by the cell exposure to lipopolysaccharide or interferon-gdid not cause any over-basal secretion of PNP from these cells whereas the stimulation with extracellular potassium (12,5 - 50 mM) or ATP (100 - 1000 µM) caused a dose-dependent increase in the release of PNP from both cultured microglial cells and astrocytes.
The effect induced by high doses of ATP was mimicked by the P2X7 receptor agonist Benzoyl-ATP (BzATP) both in microglia and in astrocytes and it was blocked by cell pre-treatment with the P2X7 receptor antagonist periodate-oxidized ATP (oATP) suggesting that PNP secretion from glial cells is inducible by the activation of this peculiar ionotropic ATP receptor subtype known to be able to stimulate the release of inflammatory cytokines in immune cells and to cause membrane blebbing and changes in cellular morphology which eventually leads to cell death by apoptosis or necrosis.
Extracellular potassium or ATP have been reported to be able to activate the exocytosis from lysosomes, subcellular organelles widely present in astrocytes and mainly in microglial cells (Potokar et al, Glia, 2010). The immunocytochemical analysis of PNP on astrocytes and microglial cells demonstrated that the enzyme was mainly localized into the cytosol, however it was frequently found also a granular staining that co-localized with LAMP-1, a typical lysosomal marker. This co-localization was more evident following cell challenge with high ATP concentrations or with BzATP.
Its deficiency is associated with T-cell lymphopenia, severe immunodeficiency and neurologic symptoms including ataxia, spasticity and delay in the brain development.
On the other hand,it has been observed that PNP activity and expression were up-regulated in periodontal diseases and in oral cancer tissue (Maeda K, et al.; Cancer 1992) as well as in human colon carcinoma where the enhanced PNP activity was associated with a higher biological aggressiveness of tumoral cells (Sanfilipo O. et al.; Biochem Biophys 1994).
Furthermore, it has been reported that Forodesine, a potent and specific PNP inhibitor, induced apoptosis in several malignant T-cell and B-cell lines, and it is currently tested in phase I/II clinical trials for patients with lymphoblastic/lymphocytic leukemia (Bieghs L. et al.; Adv.Hematol.; 2010).
PNP is present in the cytosol and mitochondria of the major part of cells and its histochemical localization in the brain indicated that it was restricted to glial cells whereas neurons are almost negative (Castellano et al, J Hist Cytochem; 1990).
Our recent results under publication demonstrated that PNP is also localized on cell membranes as it was found for other ecto-enzymes involved in the catabolism of extracellular purines. Furthermore, a PNP activity was observed in plasma as well as in the gingival crevicular fluid, even though the source of such enzyme is substantially unknown.
The aim of the study was to assess whether primary cultures of cerebellar neurons, astrocytes and microglia were able to release PNP into the culture medium and to evaluate whether it occurred in a similar manner in all of them.
Western blot analysis of the conditioned media from equivalent numbers of cultured cells and collected at the same time points, demonstrated that PNP is remarkably released from astrocytes and microglia in a time dependent manner and only to a much lower extent from cerebellar neurons. The level of expression of PNP in the different media revealed that microglial cells are more efficient in the enzyme secretion compared to astrocytes.
These results have been confirmed by the evaluation of the enzyme activity in the conditioned media which was measured by an radio-HPLC analysis of the de novo formed labeled guanine derived from the labeled guanosine, added to media samples and used as substrate of the enzyme.
Microglial activation induced by the cell exposure to lipopolysaccharide or interferon-gdid not cause any over-basal secretion of PNP from these cells whereas the stimulation with extracellular potassium (12,5 - 50 mM) or ATP (100 - 1000 µM) caused a dose-dependent increase in the release of PNP from both cultured microglial cells and astrocytes.
The effect induced by high doses of ATP was mimicked by the P2X7 receptor agonist Benzoyl-ATP (BzATP) both in microglia and in astrocytes and it was blocked by cell pre-treatment with the P2X7 receptor antagonist periodate-oxidized ATP (oATP) suggesting that PNP secretion from glial cells is inducible by the activation of this peculiar ionotropic ATP receptor subtype known to be able to stimulate the release of inflammatory cytokines in immune cells and to cause membrane blebbing and changes in cellular morphology which eventually leads to cell death by apoptosis or necrosis.
Extracellular potassium or ATP have been reported to be able to activate the exocytosis from lysosomes, subcellular organelles widely present in astrocytes and mainly in microglial cells (Potokar et al, Glia, 2010). The immunocytochemical analysis of PNP on astrocytes and microglial cells demonstrated that the enzyme was mainly localized into the cytosol, however it was frequently found also a granular staining that co-localized with LAMP-1, a typical lysosomal marker. This co-localization was more evident following cell challenge with high ATP concentrations or with BzATP.