Biochemical characterization of soluble nucleotide pyrophosphatase/phosphodiesterase activity in rat serum

Biochemical properties of nucleotide pyrophosphatase/phosphodiesterase (NPP) in rat serum have been described by assessing its nucleotide phosphodiesterase activity, using p-nitrophenyl-5-thymidine monophosphate (p-Nph-5-TMP) as a substrate. It was demonstrated that NPP activity shares some typical characteristics described for other soluble NPP, such as divalent cation dependence, strong alkaline pH optimum (pH 10.5), inhibition by glycosaminoglycans, and K (m) for p-Nph-5-TMP hydrolysis of 61.8 +/- A 5.2 mu M. In order to characterize the relation between phosphodiesterase and pyrophosphatase activities of NPP, we have analyzed the effects of different natural nucleotides and nucleotide analogs. ATP, ADP, and AMP competitively inhibited p-Nph-5-TMP hydrolysis with K (i) values ranging 13-43 mu M. Nucleotide analogs, alpha,beta-metATP, BzATP, 2-MeSATP, and dialATP behaved as competitive inhibitors, whereas alpha,beta-metADP induced mixed inhibition, with K (i) ranging from 2 to 20 mu M. Chromatographic analysis revealed that alpha,beta-metATP, BzATP, and 2-MeSATP were catalytically degraded in the serum, whereas dialATP and alpha,beta-metADP resisted hydrolysis, implying that the former act as substrates and the latter as true competitive inhibitors of serum NPP activity. Since NPP activity is involved in generation, breakdown, and recycling of extracellular adenine nucleotides in the vascular compartment, the results suggest that both hydrolyzable and non-hydrolyzable nucleotide analogs could alter the amplitude and direction of ATP actions and could have potential therapeutic application

Long-term intermittent feeding restores impaired GR signaling in the hippocampus of aged rat

Diminished glucocorticoid signaling is associated with an age-related decline in hippocampal functioning. In this study we demonstrate the effect of intermittent, every other day (EOD) feeding on the glucocorticoid hormone/glucocorticoid receptor (GR) system in the hippocampus of middle-aged (18-month-old) and aged (24-month-old) Wistar rats. In aged ad libitum-fed rats, a decrease in the level of total GR and GR phosphorylated at Ser(232) (pGR) was detected. Conversely, aged rats subjected to EOD feeding, starting from 6 months of age, showed an increase in GR and pGR levels and a higher content of hippocampal corticosterone. Furthermore, prominent nuclear staining of pGR was observed in CM pyramidal and DG granule neurons of aged EOD-fed rats. These changes were accompanied by increased Sglc-1 and decreased GFAP transcription, pointing to upregulated transcriptional activity of GR. EOD feeding also induced an increase in the expression of the mineralocorticoid receptor. Our results reveal that intermittent feeding restores impaired GR signaling in the hippocampus of aged animals by inducing rather than by stabilizing GR signaling during aging. (C) 2015 Elsevier Ltd. All rights reserved.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}ON173056, ON173008]; NIH {[}R03AG046216