Complex changes in ecto-nucleoside 5′-triphosphate diphosphohydrolase expression in hypoxanthine phosphoribosyl transferase deficiency

Abstract

Lesch-Nyhan disease is caused by a deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT). The link between HPRT deficiency and the neuropsychiatric symptoms is unknown. In rat B103 neuroblastoma cell membranes and mouse Neuro2a neuroblastoma cell membranes, nucleoside 5 -triphosphatase (NTPase) activity is substantially reduced, whereas in fibroblast membranes from HPRT knock-out mice, NTPase activity is increased. Candidate genes for these NTPase activity changes are ecto-nucleoside 5 -triphosphate diphosphohydrolases (NTPDases). Therefore, we studied expression of NTPDases in B103 cells, Neuro2a cells and skin fibroblasts by reverse transcriptase polymerase chain reaction and restriction enzyme digestion of amplified cDNA fragments. In B103 cells, expression of NTPDases 1, 3 and 6 decreased, whereas expression of NTPDases 4 and 5 increased in HPRT deficiency. In Neuro2a cells, expression of NTPDases 3-6 increased in HPRT deficiency. In fibroblasts, NTPDase 3 expression decreased, and expression of NTPDases 4-6 increased in HPRT deficiency. Collectively, there are complex decreases and increases in NTPDase isoform expression in HPRT deficiency that depend on the specific cell type and species studied. These changes in NTPDase expression may reflect an (insufficient) attempt of cells to compensate for the changes in nucleotide metabolism caused by HPRT deficiency. © 2007 Elsevier Ireland Ltd. All rights reserved.Keywords: Lesch-Nyhan disease; Hypoxanthine phosphoribosyl transferase; Ecto-nucleoside 5 -triphosphate diphosphohydrolase; Fibroblasts; Neuroblastoma cells Lesch-Nyhan disease (LND) is caused by a defect of hypoxanthine phosphoribosyl transferase (HPRT) [10,6]. Clinical symptoms of LND are hyperuricemia, gouty arthritis, mental retardation, dystonia, spasticity, delayed motor development and a compulsive form of self-injurious behavior. The link between HPRT deficiency and the neuropsychiatric symptoms is unknown. To study the biochemical basis for the neuropsychiatric symptoms in LND, a HPRT knock-out mouse was generated [15]. Similar to LND patients, there are abnormalities in dopaminergic neurotransmission in the HPRT knock-out mouse [7,16]. In addition, several HPRT-deficient cell lines including primary human skin fibroblasts, immortalized mouse skin fibroblasts, mouse Neuro2a neuroblastoma cells and rat B103 neuroblastoma are used as biochemical models for LND [18,2,4,12,11]. In a recent study [11] we showed that nucleoside 5 -triphosphatase (NTPase) activity in B103-and Neuro2a cell membranes is reduced in HPRT-deficiency. In contrast, in mouse skin fibroblast membranes, NTPase activity is increased in HPRT deficiency. Thus, altered NTPase activity is a hallmark of HPRT deficiency and these changes are cell type-and species-specific. However, it is very difficult to reveal the molecular identity of the NTPase(s) in complex systems by measuring enzyme activity. Specifically, analysis of enzyme activity with various substrates and inhibitors revealed monoph...

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor

“…During the measurement of the ATP affinity of ecAT3.10, continuous bath perfusion conditions were employed in order to continuously replenish ATP and control the extracellular ATP concentration. Physiologically, however, ectonucleotidases contribute to purinergic signaling dynamics by clearing extracellular ATP by hydrolysis[ 27 , 58 ], thereby generating ADP, AMP, and adenosine, which can act independently as purinergic signals. In order to test ecAT3.10’s ability to detect endogenous ectonucleotidase activity[ 58 ], we performed experiments in which a single addition of exogenous ATP was made to cultured Neuro2A cells expressing ecAT3.10 in an imaging dish under static bath conditions without solution exchange.…”

“…Physiologically, however, ectonucleotidases contribute to purinergic signaling dynamics by clearing extracellular ATP by hydrolysis[ 27 , 58 ], thereby generating ADP, AMP, and adenosine, which can act independently as purinergic signals. In order to test ecAT3.10’s ability to detect endogenous ectonucleotidase activity[ 58 ], we performed experiments in which a single addition of exogenous ATP was made to cultured Neuro2A cells expressing ecAT3.10 in an imaging dish under static bath conditions without solution exchange. We initially used Neuro2A cells because they express the ectonucleotidase NTPDase1 (CD39)[ 51 ].…”

Impairment of adenylyl cyclase 2 function and expression in hypoxanthine phosphoribosyltransferase-deficient rat B103 neuroblastoma cells as model for Lesch–Nyhan disease: BODIPY–forskolin as pharmacological tool

“…TaqMan probes and SYBR Green reagents were from Applied Biosystems (Darmstadt, Germany). Sources of other reagents have been described elsewhere (Pinto et al 2005(Pinto et al , 2008Pinto and Seifert 2006;Lorenz et al 2007;Erdorf et al 2011a).…”

“…In HPRT-deficient cells, 6-thioguanine cannot be converted to the cytotoxic 6-thioguanosine 5′-triphosphate and hence, cells survive (Zoref-Shani et al 1993;Hacke et al 2012). HPRTdeficient B103 cells are an established cell culture model for LND and have already been used for two decades to study various aspects of the pathophysiology of LND including alterations in neuronal differentiation, nucleotide metabolism, purinoceptor function, nucleotidase activity, nucleotidase gene expression, and adenylyl cyclase (AC) regulation (Zoref-Shani et al 1993;Ma et al 2001;Connolly 2001;Pinto et al 2005;Pinto and Seifert 2006;Lorenz et al 2007;Erdorf et al 2011a).…”

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase

“…A series of evidences have suggested that these enzymes may be involved in eukaryotic events such as: virulence (Berredo-Pinho et al, 2001), purinergic signaling (Deaglio and Robson, 2011;Knowles, 2011), inflammation (Kannan, 2002), hemostasis (Bernardes et al, 2000;Jin et al, 2005;Marcus et al, 2005), purine salvage (de Souza Leite et al, 2007;Lorenz et al, 2007) and host-pathogen interactions (Bisaggio et al, 2003;Matin and Khan, 2008).…”