Morphine treatment alters nucleotidase activities in rat blood serum

Joanna Ripoll Rozisky,1,2,4,* Yasmine Nonose,1,4,* Gabriela Laste,1,2,4 Vinicius Souza dos Santos,1,4 Isabel Cristina de Macedo,1,2,4 Ana Maria Oliveira Battastini,2,3 Wolnei Caumo,2,4 Iraci LS Torres1,2,41Laboratório de Farmacologia da Dor e Neuromodulação: Modelos Animais, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, 2Programa de Pós-Graduação em Medicina, Ciências Médicas, 3Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil; 4Unidade de Experimentação Animal e Grupo de Pesquisa e Pós-Graduação, Hospital de Clínicas de Porto Alegre, Rio Grande do Sul, Brazil *Both authors contributed equally to this workAbstract: Morphine has been widely used in neonatal pain management. However, this treatment may produce adaptive changes in several physiologic systems. Our laboratory has demonstrated that morphine treatment in neonate rats alters nucleoside triphosphate diphosphohydrolase (NTPDase) activity and gene expression in central nervous system structures. Considering the relationship between the opioid and purinergic systems, our aim was to verify whether treatment with morphine from postnatal days 8 (P8) through 14 (P14) at a dose of 5 µg per day alters NTPDase and 5´-nucleotidase activities in rat serum over the short, medium, and long terms. After the in vivo assay, the morphine group showed increased hydrolysis of all nucleotides at P30, and a decrease in adenosine 5´-diphosphate hydrolysis at P60. Moreover, we found that nucleotidase activities change with age; adenosine 5´-triphosphate hydrolysis activity was lower at P16, and adenosine 5'-monophosphate hydrolysis activity was higher at P60. These changes are very important because these enzymes are the main regulators of blood nucleotide levels and, consequently, nucleotide signaling. Our findings showed that in vivo morphine treatment alters nucleotide hydrolysis in rat blood serum, suggesting that purine homeostasis can be influenced by opioid treatment during the neonatal period.Keywords: 5´-nucleotidase, morphine, neonate, NTPDase, rat blood seru

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases