However, both Mg2+ and Ca2+ increased 5′-AMP hydrolysis
by CP-690550 price about 42% (Fig. 3a). The optimum pH for C. parapsilosis ecto-5′-nucleotidase activity was in the acidic range, with its maximum activity at a pH of 4.5. The enzyme activity decreased with increases in pH (Fig. 3b). In the pH range between 4.5 and 8.5, the rate of 5′-AMP hydrolysis observed from the supernatant was <15–20% of those observed in intact cells (data not shown). In addition to the existence of ecto-ATPase activity (Kiffer-Moreira et al., 2010) on the surface of C. parapsilosis, our group has described the presence of a membrane-bound acid phosphatase activity (Kiffer-Moreira et al., 2007a), which could contribute to AMP hydrolysis. To Temozolomide rule out the influence of acid phosphatase on AMP hydrolysis, we evaluated the influence of a well-known inhibitor of phosphatase activities, sodium orthovanadate (de Almeida-Amaral et al., 2006; Kiffer-Moreira et al., 2007a; Amazonas et al., 2009; Dick et al., 2010). As shown in Fig. 4a, different concentrations of sodium orthovanadate (0.1 and 1.0 mM) inhibited ectophosphatase
activity. Nevertheless, as expected, it did not have an effect on C. parapsilosis ecto-5′-nucleotidase activity (Fig. 4b). On the other hand, ammonium molybdate, a classical 5′-nucleotidase inhibitor (Gottlieb & Dwyer, 1983; Borges et al., 2007), inhibited ecto-5′-nucleotidase in a dose-dependent manner, with maximal inhibition at a concentration of 0.5 mM (Fig. 5). Adenosine has been implicated in many aspects to contribute for pathogens escaping from host immune responses (Bhardwaj & Skelly, 2009; Thammavongsa et al., PTK6 2009). To verify whether adenosine and
5′-AMP would prevent macrophage to phagocyte C. parapsilosis, we perform an in vitro interaction with peritoneal macrophage and C. parapsilosis in the presence of a low concentration of adenosine and 5′-AMP (100 μM). As can be seen in Fig. 6a and b, the addition of adenosine to the interaction medium showed a significant reduction in the percentage of infected macrophages, whereas 5′-AMP at the same concentration did not have an effect, comparing with control. Interestingly, the addition of 5′-AMP, at 1 mM, caused a decrease in the percentage of infected macrophages (Fig. 6a and b), indicating that C. parapsilosis ecto-5′-nucleotidase could have a role in generating extracellular adenosine, to further modulate the macrophage response. On the other hand, no significant differences were observed in the mean number of yeasts per infected macrophage among all system tested (Fig. 6c). In this condition in the presence of 1 mM AMP, C. parapsilosis produced 1.52 ± 0.07 nmol Pi h−1 10−6 cells from AMP hydrolysis. In the same condition, macrophages were also able to promote AMP hydrolysis producing 1.04 ± 0.13 nmol Pi h−1 10−5 cells.