Efeito do óxido nítrico sobre a atividade e expressão da ntpdase1 e da ecto-5'-nucleotidase em linhagens de células vasculares

Abstract

Understanding the mechanisms that act in the vasculature represent a promising path for the development and understanding of future studies about vascular disorders. It is known that extracellular nucleotides (ATP, ADP) and nucleoside adenosine are able to modulate vasorelaxation and vasoconstriction and are involved in the production of the vasodilator nitric oxide (NO). It remains to be investigated whether NO is able to modulate the expression and activity of ectonucleotidases, enzymes that hydrolyze nucleotides controlling their availability in the extracellular environment. NTPDases 1 and 2 hydrolyze ATP and ADP and ecto-5'-nucleotidase metabolizes AMP to adenosine, also a vasodilator. To determine the effect of NO on vascular ectonucleotidases, vascular smooth muscle cells A7r5 and endothelial cells tEnd.1 were treated with NO donor (sodium nitroprusside (SNP)), inhibitor of NO synthesis (L-NAME) and mobilizers of NO (ATP, ADP, bradykinin (BK) and 2-Cl-adenosine). After 24 hours, a 35% decrease in cell viability of A7r5 cells was observed by MTT assay in response to 2-Cl-adenosine (10-4M). Nitrite levels, measured by Griess's reagent, significantly increased in the two cell types treated with SNP, indicating an indirect increase in NO levels. The enzymatic activities were detected colorimetrically by the inorganic phosphate released. The hydrolysis of ATP and ADP was not altered. However, AMP hydrolysis was significantly increased by ATP and 2-Cl-adenosine in A7r5 cells and by ATP and ADP in tEnd.1 cells. The increase in ecto-5'-nucleotidase activity seems to be important to increase levels of circulating adenosine, since it is of great importance to ensure regulation of homeostasis and maintenance of vascular tone. The attempt to evaluate the gene expression of ectonucleotidases in response to treatments in A7r5 cells was not successful. Besides, no changes were found in protein level, as assessed by Western blotting. However, the increase in enzyme activity could be explained by the presence of specific highly phosphorylable sites, especially in serine residues, in the structure of the ecto-5'- nucleotidase. Although the results obtained lead us to believe that the ectonucleotidases are modulated independently of the NO in A7r5, additional experiments are required to confirm the concentration of NO in the culture medium of such cells and the levels of regulation undergone by the enzymes in question. The understanding of the functions and mechanisms of action of NO and other vasoactive molecules on vascular ectonucleotidases are of fundamental relevance for the understanding of vascular disorders.