P Controls Toxoplasma gondii-purchase 181223-80-3 infection less pronounced in infected cells. In contrast, other nucleotide treatments did not alter ROS production by uninfected or T. gondii-infected macrophages. Necrotic cell death was analyzed by measuring lactate dehydrogenase activity in culture supernatants harvested 4h after nucleotide treatment. Treatment with 0.1% Triton X-100 was used as a positive control for necrosis. Nucleotide treatment did no induce necrosis in uninfected or T. gondii-infected cells. Apoptotic cell death was analyzed by flow cytometry using ethidium bromide, to identify cells with < 2C DNA content. As a positive control for apoptosis induction, cells were treated with 5 M staurosporine for 24 h before analyze. Treatment with nucleotides for 12 h did not induced apoptosis in uninfected or T. gondii-infected macrophages. Data represent mean and SEM of three independent experiments.P2Y receptor agonists induce premature egress of tachyzoites PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748118 from host cells Since the reduction in T. gondii macrophage infection induced by P2Y agonist treatment was not due to increased NO or ROS production, or cell death induction, we hypothesized that tachyzoites might be egressing prematurely from infected cells upon nucleotide treatment. To test this hypothesis, we determined the infection index in cultures fixed immediately after treatment with 100 M UTP or UDP for 30 minutes, and also quantified the number of parasites observed in the culture medium after treatment. We observed that both UTP and UDP treatment reduced the number of parasite inside of cells with an overall reduction in parasite burden of 38% and 33%, respectively. Ca2+ influx into host cells induces T. gondii egress even after a short period of infection. Thus, treatment with the Ca2+ ionophore 4BrA23187 was used as a positive control for early tachyzoite egress from host cells. Similarly to that observed after treatment with both UTP and UDP treatments 4BrA23187, reduced the number of infected macrophages and the infection index,. Although infection index reduction immediately after P2Y agonist treatment suggested that tachyzoites egressed prematurely from host cells, it was important to verify whether tachyzoites were indeed released into the medium after treatment. For these experiments, we also treated cells with the Ca2+ chelator BAPTA-AM prior to treatment with nucleotides or with 4BrA23187, to examine the possibility that early parasite egress after nucleotide treatment was dependent on a Ca2+ influx. Culture supernatants of infected cells treated with UTP, UDP or 4BrA23187 contained a larger number of parasites than untreated cell supernatant. Importantly, this effect was reduced by pre-treatment of cells with BAPTA-AM before UTP treatment. These results suggest that the activation of P2Y receptors on the surface of T. gondii-infected macrophages induced early parasite egress from host cells, in a Ca2+-dependent manner. To examine the mechanisms of nucleotide-mediated parasite egress, we performed UTP treatments in the presence of U73122, a phospholipase C inhibitor expected block the 
P2Y intracellular signaling cascade. We found that the pre-treatment with U73122 completely abolished the UTP-mediated parasite egress, but had no effect on the response to Ca2+ ionophore treatment. These results confirm that the UTP-response depends on PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748480 P2Y receptor signaling, via intracellular calcium mobilization. Videomicroscopy analysis showed tachyzoites egressing from infected macrop