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

Abstract

Hypoxanthine phosphoribosyl transferase (HPRT) deficiency results in Lesch-Nyhan disease (LND). The link between the HPRT defect and the self-injurious behavior in LND is still unknown. HPRT-deficient rat B103 neuroblastoma cells serve as a model system for LND. In B103 cell membranes, HPRT deficiency is associated with a decrease of basal and guanosine triphosphate-stimulated adenylyl cyclase (AC) activity (Pinto and Seifert, J Neurochem 96:454-459, 2006). Since recombinant AC2 possesses a high basal activity, we tested the hypothesis that AC2 function and expression is impaired in HPRT deficiency. We examined AC regulation in B103 cell membranes, cAMP accumulation in intact B103 cells, AC isoform expression, and performed morphological studies. As most important pharmacological tool, we used 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene forskolin (BODIPY-FS) that inhibits recombinant AC2 but activates ACs 1 and 5 (Erdorf et al., Biochem Pharmacol 82:1673-1681, 2011). In B103 control membranes, BODIPY-FS reduced catalysis, but in HPRT(-) membranes, BODIPY-FS was rather stimulatory. 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-[γ-thio]triphosphates inhibit recombinant ACs 1 and 5 more potently than AC2. In B103 control membranes, MANT-guanosine 5'-[γ-thio]triphosphate inhibited catalysis in control membranes less potently than in HPRT(-) membranes. Quantitative real-time PCR revealed that in HPRT deficiency, AC2 was virtually absent. In contrast, AC5 was up-regulated. Forskolin (FS) and BODIPY-FS induced cell clustering and rounding and neurite extension in B103 cells. The effects of FS and BODIPY-FS were much more prominent in control than in HPRT(-) cells, indicative for a differentiation defect in HPRT deficiency. Neither FS nor BODIPY-FS significantly changed cAMP concentrations in intact B103 cells. Collectively, our data show that HPRT deficiency in B103 cells is associated with impaired AC2 function and expression and reduced sensitivity to differentiation induced by FS and BODIPY-FS. We discuss the pathophysiological implications of our data for LND.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…These data also raise the question about the possible physiologic relevance of the high constitutive activity of AC2. The selectivity of BODIPY-FSK for AC2 inhibition, in conjunction with RT-PCR studies and analysis of MANT-nucleotides, was used to define AC2 as the functionally predominant AC isoform in rat B103 neuroblastoma cells (Kinast et al, 2012). Intriguingly, upon loss of hypoxanthine phosphoribosyl transferase, the causative enzyme defect of Lesch-Nyhan disease (Fu et al, 2014), AC2 function is substantially impaired (Kinast et al, 2012).…”

“…AC2 and AC4 are the predominantly expressed AC isoforms in human airway smooth muscle cells, but AC2 expression is lacking in mouse bronchial smooth muscle cells, making confirmation of its function difficult (Bogard et al, 2011. Development of AC2-selective inhibitors may overcome the difficulties to elucidate the (patho)physiologic functions of AC2 (Pinto et al, 2008;Erdorf et al, 2011;Kinast et al, 2012;Conley et al, 2013). ADCY2 polymorphisms may be related to neuropsychiatric (Mühleisen et al, 2014;Suarez-Rama et al, 2015) and pulmonary diseases (Hardin et al, 2012;Panasevich et al, 2013).…”

“…ADCY2 polymorphisms may be related to neuropsychiatric (Mühleisen et al, 2014;Suarez-Rama et al, 2015) and pulmonary diseases (Hardin et al, 2012;Panasevich et al, 2013). Pharmacological studies point to a role of AC2 in the pathogenesis of Lesch-Nyhan disease, a neuropsychiatric disorder with severe self-injurious behavior (Kinast et al, 2012).…”

Naunyn-Schmiedeberg’s Archives of Pharmacology under new editorship: change and continuity

“…In most cases, publication of my students' papers coincided more or less with the defense of their doctoral theses and, thereby, facilitated their professional careers. Most importantly, I could always openly discuss unresolved and controversial questions and did not have to sell a perfect story (see, e.g., Kinast et al 2012;Bräunig et al 2014). Along these lines, I encourage our authors to be very frank and open on limitations of the data and problems in the field.…”

A door opener for future research: agonist-induced β3-adrenoceptor desensitization in HEK cells but not CHO cells

“…As a first step, the expression pattern of AC isoforms in HEK-and CHO cells has to be studied. Such studies are straightforward at the mRNA level (Kinast et al 2012) but highly problematic at the protein level because of the questionable quality of AC antibodies (Göttle et al 2009). Should there be an AC isoform that is expressed exclusively in HEK cells but not in CHO cells, then the corresponding AC isoform could be knocked down with the siRNA technique.…”