Decreased GTP‐stimulated adenylyl cyclase activity in HPRT‐deficient human and mouse fibroblast and rat B103 neuroblastoma cell membranes

Abstract

Defect of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT), results in Lesch-Nyhan disease (LND). It is unknown how the metabolic defect translates into the severe neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in GTP, UTP and CTP concentrations in HPRT-deficient cells. Moreover, GTP, ITP, XTP, UTP and CTP differentially support G s -protein-mediated adenylyl cyclase (AC) activation. Based on these findings we hypothesized that abnormal AC regulation may constitute the missing link between HPRT deficiency and the neuropsychiatric symptoms in LND. To test this hypothesis, we studied AC activity in membranes from primary human skin and immortalized mouse skin fibroblasts, mouse Neuro-2a neuroblastoma cells and rat B103 neuroblastoma cells. In B103 control membranes, GTP, ITP, XTP and UTP exhibited profound stimulatory effects on basal AC activity that approached the effects of hydrolysis-resistant nucleotide analogs. In HPRT -membranes, the stimulatory effects of GTP, ITP, XTP and UTP were strongly reduced. Similarly, in human and mouse skin fibroblast membranes we also observed a decrease in GTP-stimulated AC activity in HPRT-deficient cells compared with the respective controls. In mouse Neuro-2a neuroblastoma membranes, AC activity in the presence of GTP was below the detection limit of the assay. We discuss several possibilities to explain the abnormalities in AC regulation in HPRT deficiency that encompass various species and cell types.

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

“…In mouse fibroblasts, NTPDase 3 expression decreased, whereas NTPDase 4-6 expression actually increased. Thus, our present expression data for individual NTPDase isoenzymes confirm the notion that secondary biochemical changes in HPRT-deficiency are cell type-and species-specific [12,11].…”

“…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.…”

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

“…In a previous study, we examined AC regulation in membranes of B103 control and HPRT − cells (Pinto and Seifert 2006). Compared with control membranes, in HPRT − membranes, AC activities under basal conditions and in the presence of guanosine triphosphate (GTP) were substantially decreased.…”

“…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).…”