ABSTRACT
Defect of hypoxanthine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), but the link between HPRT deficiency and the self-injurious behavior of LND is unknown. In a previous study (Pinto et al., J. Neurochem. 72 (2005) 1579-1586) we reported on a decrease in nucleotidase activity in membranes of several HPRT(-) cell lines and fibroblasts from LND patients. Since nucleotidases are involved in ATP-induced signal transduction, in the present study, we tested the hypothesis that P2X and P2Y receptor-mediated signal transduction is impaired in HPRT deficiency. As model we studied rat B103 neuroblastoma cells. Compared to control cells, in HPRT(-) cells, NTP and NDP-induced Ca(2+) influx across the membrane and Ca(2+) mobilization from intracellular stores were impaired. Both P2X and P2Y receptors were involved in the responses. Quantitative real-time PCR revealed reduced expression of receptors P2X(3), P2X(5), P2Y(2), P2Y(4), P2Y(12), P2Y(13) and P2Y(14) in HPRT deficiency. Collectively, HPRT deficiency is associated with abnormal purinergic signaling, encompassing P2X and P2Y receptors and nucleotidases.
Subject(s)
Hypoxanthine Phosphoribosyltransferase/deficiency , Neurons/physiology , Receptors, Purinergic P2X/physiology , Receptors, Purinergic P2Y/physiology , Signal Transduction/physiology , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cell Line, Tumor/physiology , Lesch-Nyhan Syndrome , Neuroblastoma/pathology , Neurons/drug effects , Nucleotides/pharmacology , Rats , Real-Time Polymerase Chain Reaction , Receptors, Purinergic P2X/biosynthesis , Receptors, Purinergic P2X/drug effects , Receptors, Purinergic P2X/genetics , Receptors, Purinergic P2Y/biosynthesis , Receptors, Purinergic P2Y/drug effects , Receptors, Purinergic P2Y/genetics , Self-Injurious Behavior , Signal Transduction/drug effectsABSTRACT
Mammalian membranous adenylyl cyclases (mACs) play an important role in transmembrane signalling events in almost every cell and represent an interesting drug target. Forskolin (FS) is an invaluable research tool, activating AC isoforms 1-8. However, there is a paucity of AC isoform-selective FS analogs. Therefore, we examined the effects of FS and six FS derivatives on recombinant ACs 1, 2 and 5, representing members of different mAC families. Correlations of the pharmacological properties of the different AC isoforms revealed pronounced differences between ACs 1, 2 and 5. Additionally, potencies and efficacies of FS derivatives changed for any given AC isoform, depending on the metal ion, Mg(2+) or Mn(2+). The most striking effects of Mg(2+) and Mn(2+) on the diterpene profile were observed for AC2 where the large inhibitory effect of BODIPY-FS in the presence of Mg(2+) was considerably reduced in the presence of Mn(2+). Sequence alignment and docking experiments confirmed an exceptional position of AC2 compared to ACs 1 and 5 with respect to the structural environment of the catalytic core and cation-dependent diterpene effects. In conclusion, mAC isoforms 1, 2 and 5 exhibit a distinct pharmacological diterpene profile, depending on the divalent cation present. mAC crystal structures and modelling/docking studies provided an explanation for the pharmacological differences between the AC isoforms. Our study constitutes an important step towards the development of isoform-specific diterpenes exhibiting stimulatory or inhibitory effects.
Subject(s)
Adenylyl Cyclases/metabolism , Colforsin/analogs & derivatives , Colforsin/pharmacology , Magnesium Compounds/pharmacology , Manganese/pharmacology , Amino Acid Sequence , Animals , Cations, Divalent , Cattle , Cell Line , Colforsin/chemistry , Dogs , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Rats , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Polycystic kidney disease (PKD) is the most common life-threatening genetic disorder with bilateral cysts caused by increased level of cyclic adenosine 3',5'-monophosphate (cAMP). Since adenylyl cyclases (ACs) catalyze cAMP formation, pharmacological characterization of renal AC isoforms is essential. Therefore, we analyzed differences in activation, inhibition, and regulation of AC isoforms in rabbit cortex and medulla membranes. Glucagon, [8-arginine]vasopressin (AVP) and catecholamines significantly activated cortical AC. However, in medulla only glucagon and AVP activated AC. Under Mg(2+) conditions the profile of cortical membrane AC enzyme kinetics and the inhibitory profile of 2'(3')-O-(N-methylanthraniloyl) (MANT) nucleotides resembled recombinant AC5. In contrast, the K (i) values of MANT nucleotides for medullary membrane AC and its kinetic properties were similar to those of recombinant AC1. Reverse-transcriptase PCR confirmed the presence of AC1 and AC5 in medulla and cortex, respectively. Cortical AC was sensitive to inhibition by Ca(2+), corroborating the importance of AC5. However, Ca(2+)/CaM dependency specific for AC1 was not found in medulla. In conclusion, according to expression, kinetics and inhibition by MANT nucleotides both parts of the kidney differ in their AC isoforms. Whereas Ca(2+)-inhibitable AC5 was confirmed in renal cortex, the initially assumed AC1 activation in medulla could not be confirmed, pointing to the involvement of another AC isoform with some similarity to AC1. Since PKD is characterized by predominant involvement of the collecting duct and the distal nephrons located in renal cortex, AC5 may be the major AC isoform in this part of the kidney where cAMP increases cyst growth. Thus, potent and selective AC5 inhibitors could constitute a novel approach to treat PKD.
