The phosphodiesterase inhibitor zaprinast induces premature egress in a cGMP-dependent manner.

<p>(A) Zaprinast induces merozoite egress. Mature schizonts (∼25% segmented) were supplemented with fresh RBCs to achieve a parasitaemia of ∼40%, then either processed at once for microscopic examination of Giemsa-stained thin films (Start), or after further culture for 50 min in the presence of zaprinast or vehicle only (DMSO, 0.5% v/v). (B) Dose-response profile of zaprinast-induced egress. Schizont preparations similar to those in (A), but in protein-free medium and without addition of uninfected RBCs, were incubated for 50 min in the presence of various concentrations of zaprinast. Culture supernatants were then examined by Western blot using mAbs reactive with SERA5, PfAMA1 or MSP1. (C) Quantitation by capture ELISA of shed PfAMA1, and its dependency on zaprinast concentration. Samples from cultures treated as in (B) for 50 min were serially diluted and assayed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#s4" target="_blank">Materials and Methods</a>. Data are presented as mean values of triplicate readings ±SEM, with PfAMA1 concentrations in arbitrary units based on comparison with a standard. Dotted lines indicate [PfAMA1] values in the absence of zaprinast treatment (lower line), or 50% maximal levels (upper line), used to derive an ED₅₀ for zaprinast of ∼25 µM. (D) Zaprinast-induced egress is blocked by PfPKG inhibitors. Western blot of supernatants harvested at different time-points from a schizont culture treated with a single concentration of zaprinast (75 µM), with or without the additional presence of C1 or C2. Blots were probed with the anti-SERA5 mAb 24C6.1F1. (E) Quantitation by ELISA of shed PfAMA1 levels in the supernatant samples probed in (D). Data are presented as in (C). (F) Zaprinast treatment increases cGMP levels in schizonts. The 0 min sample was taken just prior to addition of zaprinast ±C1. (G) BAPTA-AM inhibits egress, and this can be reversed by zaprinast treatment. Schizonts in protein-free medium were incubated in the presence of the indicated reagents for 50 min. BAPTA-AM was used at 100 µM, and zaprinast at 75 µM. See also Figure S4 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a> and Movies S3 and S4.