Characterization of the Alterations in Purine Nucleotide Metabolism in Hypoxanthine‐Guardne Phosphoribosyltransferase‐Deficient Rat Neuroma Cell Line

Abstract

A rat neuroma cell line (B103 4C), deficient of hypoxanthine‐guanine phosphoribosyltransferase (HGPRT), was utilized as a model tissue in search for the biochemical basis of the Lesch‐Nyhan syndrome (LNS). The HGPRT‐deficient neurons exhibited the following properties: an almost complete absence of uptake of guanine and of hypoxanthine into intact cell nucleotides (0.92% and 0.69% of normal, respectively); a significant increase in the availability of 5′‐phosphoribosyl‐1‐pyrophosphate; a three‐ to fourfold acceleration of the rate of de novo nucleotide synthesis; a normal excretion of xanthine, but 15‐fold increase in the excretion of hypoxanthine into the culture media; a normal cellular purine nucleotide content, including the absence of 5‐amino‐4‐imidazole carboxamide nucleotides (Z‐nucleotides), but enhanced turnover of adenine nucleotides (loss of 86% of the radioactivity of the prelabeled pool in 24 h, in comparison to 73% in the normal line), and an elevated UTP content. The results suggest that, under physiological conditions, guanine salvage does not occur in the normal neurons, but that hypoxanthine salvage is of great importance in the homeostasis of the adenine nucleotide pool. The finding of the normal profile of purine nucleotides in the HGPRT‐deficient neurons indicates that the lack of hypoxanthine salvage is adequately compensated by the enhanced de novo nucleotide synthesis. These results did not furnish evidence in support of the possibility that GTP or ATP depletion, or Z‐nucleotide accumulation, occurs in HGPRT‐deficient neurons and that these are etiological factors causing the neurological abnormalities in LNS. On the other hand, the results point to the possibility that elevated hypoxanthine concentration in the brain may have an etiological role in the pathogenesis of LNS.

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

“…Major advantages of B103 cells are that they are well characterized and broadly used in the neurosciences since the 1970s until now for topics encompassing addiction, cellular ultrastructure, differentiation, tumor cell biology, neurodegeneration, neurotoxicity, receptor function, and signal transduction, and that they grow rapidly and reproducibly (Schubert et al 1974;Cabral et al 1987;Kokoz et al 1999;Kawanabe et al 2001;Han et al 2002;Tanura et al 2006;Crespo-López et al 2007;Han et al 2010;Kato et al 2012). A HPRT-deficient clone of B103 cells was generated by selection of 6-thioguanine-resistant cells (Zoref-Shani et al 1993). 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).…”

“…A HPRT-deficient clone of B103 cells was generated by selection of 6-thioguanine-resistant cells (Zoref-Shani et al 1993). 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).…”

Altered membrane NTPase activity in Lesch–Nyhan disease fibroblasts: comparison with HPRT knockout mice and HPRT‐deficient cell lines

“…These abnormalities include decreases in the intracellular concentrations of the purine nucleotide GTP and increased concentration of ATP (Fairbanks et al . 2002) and, in some instances, compensatory increases in the intracellular concentrations of the pyrimidine nucleotides UTP and CTP (Zoref‐Shani et al . 1993; Pelled et al .…”

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

“…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.…”

“…Given the well-known species-specificity of biochemical changes in HPRT-deficiency [18,2,4,12,11], one has to be cautious with extrapolations to LND. Accordingly, in future studies, human neuronal cell culture cell lines will have to be studied.…”