Biochemical characterization of ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP, E.C. 3.1.4.1) from rat heart left ventricle.

In the present study we investigate the biochemical properties of the members of NPP family in synaptosomes prepared from rat heart left ventricles. Using p-nitrophenyl-5'-thymidine monophosphate (p-Nph-5'-TMP) as substrate for E-NPPs in rat cardiac synaptosomes, we observed an alkaline pH dependence, divalent cation dependence and the K ( M ) value corresponded to 91.42 +/- 13.97 microM and the maximal velocity (V ( max )) value calculated was 63.79 +/- 3.59 nmol p-nitrophenol released/min/mg of protein (mean +/- SD, n = 4). Levamisole (1 mM), was ineffective as inhibitor of p-Nph-5'-TMP hydrolysis in pH 8.9 (optimum pH for the enzyme characterized). Suramin (0.25 mM) strongly reduced the hydrolysis of p-Nph-5'-TMP by about 46%. Sodium azide (10 and 20 mM) and gadolinium chloride (0.3 and 0.5 mM), E-NTPases inhibitors, had no effects on p-Nph-5'-TMP hydrolysis. RT-PCR analysis of left ventricle demonstrated the expression of NPP2 and NPP3 enzymes, but excluded the presence of NPP1 member. By quantitative real-time PCR we identified the NPP3 as the enzyme with the highest expression in rat left ventricle. The demonstration of the presence of the E-NPP family in cardiac system, suggest that these enzymes could contribute with the fine-tuning control of the nucleotide levels at the nerve terminal endings of left ventricles that are involved in several cardiac pathologies.

The effects of angiotensin II and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes.

The extracellular nucleotides, ATP and ADP, as well as adenosine have been implicated in a great number of physiological functions. ADP is one of the major platelet recruiting factors, whereas ATP is considered to be a competitive inhibitor of ADP-induced platelet aggregation and adenosine is able to induce vasodilatation and to inhibit platelet aggregation. The di- and triphosphate nucleosides can be hydrolyzed by members of several families of ectonucleotidases, including ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) that, together with an ecto-5'-nucleotidase, catalyze adenosine formation. The renin-angiotensin system is the most important regulator of renal and cardiovascular functions and angiotensin II induces, physiologically, platelet activation. The aim of this study was to clarify the effects of ANGII and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes. ANGII, in all tested doses (5, 50, 500 and 5000 pmol), was able to increase ATP (21, 31, 44 and 27%, respectively), ADP (22, 28, 78 and 37%, respectively) and AMP (40, 64, 60 and 64%, respectively) hydrolysis by rat platelets. Furthermore, losartan, a specific antagonist of the AT1 angiotensin-receptor, prevented the nucleotide hydrolysis effects. Additionally, an increase in AMP (about 144%) hydrolysis and a decrease in p-Nph-5'TMP (about 27%) hydrolysis were observed in platelets from spontaneously hypertensive rats (SHR) when compared to Wistar normotensive rats. We, herein, present data to demonstrate interactions between rat platelet angiotensinergic and adenosinergic systems that could contribute to the understanding and treatment of cardiovascular diseases such as hypertension, thrombosis and arteriosclerosis.