Bert Grobben

Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family.

The ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) multigene family contains five members. NPP1-3 are type II transmembrane metalloenzymes characterized by a similar modular structure composed of a short intracellular domain, a single transmembrane domain and an extracellular domain containing a conserved catalytic site. The short intracellular domain of NPP1 has a basolateral membrane-targeting signal while NPP3 is targeted to the apical surface of polarized cells. NPP4-5 detected by database searches have a predicted type I membrane orientation but have not yet been functionally characterized. E-NPPs have been detected in almost all tissues often confined to specific substructures or cell types. In some cell types, NPP1 expression is constitutive or can be induced by TGF-beta and glucocorticoids, but the signal transduction pathways that control expression are poorly documented. NPP1-3 have a broad substrate specificity which may reflect their role in a host of physiological and biochemical processes including bone mineralization, calcification of ligaments and joint capsules, modulation of purinergic receptor signalling, nucleotide recycling, and cell motility. Abnormal NPP expression is involved in pathological mineralization, crystal depositions in joints, invasion and metastasis of cancer cells, and type 2 diabetes. In this review we summarize the present knowledge on the structure and the physiological and biochemical functions of E-NPP and their contribution to the pathogenesis of diseases.

An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma

Abstract : The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the α‐and γ‐phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for ~50‐75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), a membrane‐impermeable inhibitor of the enzyme. PPADS concentration above 20 μM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 ± 5 μM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3′,5′‐bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD‐I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen‐related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD‐I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists.

The effect of ecto‐nucleotide pyrophosphatase (ecto‐NPPase; EC 3.6.1.9) on the ATP‐ and ADP‐mediated receptor activation was studied in rat C6 glioma cells. The P2‐purinoceptor antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC50=12±3 μM) of the latter enzyme. 4,4′‐diisothiocyanatostilbene‐2,2′ disulfonic acid (DIDS), 5′‐phosphoadenosine 3′‐phosphate (PAP) and suramin were less potent inhibitors with an IC50 of 22±4, 36±7 and 72±11 μM respectively. P1‐purinoceptor antagonists CGS 15943, cyclo‐pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto‐NPPase. ATP‐ and ADP‐mediated P2Y1‐like receptor activation inhibited the (−)‐isoproterenol‐induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y‐antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto‐NPPase. We conclude that ecto‐NPPase has a modulatory effect on purinoceptor‐mediated signalling in C6 glioma cell cultures.

Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3‐kinase (PI 3‐K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3‐K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3‐K construct, resulting in a decrease of the enzymatic activity of PI 3‐K, inhibited the cAMP‐dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3‐K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3‐K‐dependent mechanism.

Agonists of the P2YAC-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma

We have previously shown that an ecto‐NPPase modulates the ATP‐ and ADP‐mediated P2YAC‐receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap3A induced no detectable PI turnover and were identified as specific agonists of the P2YAC‐receptor with EC50 values of 250 ± 37 pm and 1 ± 0.5 µm, respectively. P2YAC‐receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2YAC‐purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP3‐independent Ca2+‐influx was observed after P2YAC‐receptor activation. Inhibition of this influx by Ca2+‐chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC‐inhibitors and a specific MEK‐inhibitor inhibited the 2MeSADP‐ and Ap3A‐induced MAP kinase activation. In addition, transfection with dominant negative RhoAAsn19 rendered C6 cells insensitive to P2YAC‐receptor‐mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf‐1 after P2YAC‐receptor activation. We may conclude that P2YAC‐purinoceptor agonists activate MAP kinase through a Gi‐RhoA‐PKC‐raf‐MEK‐dependent, but ras‐ and Ca2+‐independent cascade.

P2YAC−-receptor agonists enhance the proliferation of rat C6 glioma cells through activation of the p42/44 mitogen-activated protein kinase

Extracellularly added P1,P3‐di(adenosine‐5′) triphosphate (Ap3A), P1,P4‐di(adenosine‐5′) tetraphosphate (Ap4A), ATP, ADP, AMP and adenosine are growth inhibitory for rat C6 glioma cells. Analysis of nucleotide hydrolysis and the use of nucleotidase inhibitors demonstrated that the latter inhibition is due to hydrolysis of the nucleotides to adenosine. Agonists of the P2YAC−‐receptor enhance the growth of C6 cells if their hydrolysis to adenosine is inhibited by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). In these conditions, the potency to stimulate cell growth parallels the ranking of the receptor agonists, i.e. 2‐methylthioadenosine‐5′‐diphosphate (2MeSADP)>Ap3A>Ap4A. ATP and ADP are still hydrolysed in the presence of PPADS and have no proliferative effect on C6 cells. The enhanced growth is due to a P2YAC−‐receptor‐mediated activation of p42/44 mitogen‐activated protein kinase (MAPK) as shown by immunoblotting and protein kinase assays for active MAPK and the use of the MAPK/extracellular signal‐regulated kinase kinase (MEK) inhibitor PD98059. The UTP‐induced enhancement of the growth of C6 cells is due to activation of MAPK by a PPADS sensitive nucleotide receptor. In conclusion, the effect of nucleotides on the growth of C6 cells is determined by ecto‐nucleotidases and by activation of nucleotide receptors. Hydrolysis of nucleotides to adenosine induces growth inhibition while inhibition of the hydrolysis of agonists of the P2YAC−‐receptor enhances cell growth by activation of MAPK.

Reperfusion injury after focal myocardial ischaemia: polymorphonuclear leukocyte activation and its clinical implications

The only way to rescue ischaemic tissue is to re-instate the oxygen supply to the tissue. However reperfusion of the ischaemic area not only oxygenates the tissue but also initiates a cascade of processes, which may in some cases result in temporary dysfunction of the myocardium. In order to devise protective measures, it is essential to understand the mechanisms and the triggers of this reperfusion phenomenon. In this review we will mainly focus on the inflammatory response caused by reperfusion. We will cover the different steps of polymorphonuclear leukocyte activation and will briefly discuss the molecular biology of the receptors involved. The currently used pharmacological medications in acute cardiology will be reviewed and in particular their actions on polymorphonuclear leukocyte activation, adhesion and degranulation. This review is a compilation of the current knowledge in the field and the therapeutic progress in the prevention of reperfusion injury made today.

PROTEIN TYROSINE KINASE-DEPENDENT REGULATION OF ADENYLATE CYCLASE AND PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATES THE EXPRESSION OF GLIAL FIBRILLARY ACIDIC PROTEIN UPON INDUCTION OF DIFFERENTIATION IN RAT C6 GLIOMA

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP‐mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or β‐adrenergic receptor‐mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3‐kinase (PI 3‐K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3‐K activity in C6 glioma cells.

Cyclic AMP-dependent down regulation of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) in rat C6 glioma

In this communication, we demonstrate that an increase in intracellular cAMP by 1) addition of dibutyrylic cAMP (dbcAMP), a membrane-permeable cAMP-analogue, or 2) activation of the β-adrenoceptor with (-)-isoproterenol, down regulates the levels of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) mRNA, NPP1 protein and ecto-NPPase activity in rat C6 glioma cells. DbcAMP and (-)-isoproterenol inhibit NPP1 expression in a time and dose-dependent manner. After 48h of stimulation, 1mM dbcAMP or 5μM (-)-isoproterenol decreases the amount of NPP1 protein by 75±3% and 81±1% respectively. Contrary to down regulation of NPP1, we observe an up regulation of glial fibrillary acidic protein (GFAP), a differentiation marker for astrocytic cells. Using specific inhibitors and activators, we have shown that Ca(2+), PKA, PI 3-K/PKB/GSK-3, Epac/Rap1/PP2A and MAP kinase modules are not involved in the inhibition of NPP1 gene expression. The transcription factor c-jun is significantly reduced while c-fos becomes up regulated after cAMP elevation. However an electrophoretic mobility shift assay with the activator protein-1 motif present in the promoter of the rat NPP1 gene indicates that this motif is not involved in the cAMP-dependent inhibition of NPP1 expression. In conclusion, these results indicate that intracellular cAMP levels regulate the expression of NPP1 in rat C6 glioma cells by a signalling pathway that is different from the GFAP signal transduction pathway.