Zaprinast

Krebs–Ringer bicarbonate buffer was used for all perifusions prepared as described previously (Neal et al., 2015 (link)). Antimycin A, glucose, oxamate, KCN, and zaprinast were purchased from Sigma-Aldrich. Gases of varying O₂ levels/5% CO₂ and balance N₂ were purchased from Praxair Distribution Inc (Danbury, CT). Cytodex and Cytopore beads were purchased from GE healthcare and biosciences (cat no. 17-0448-01 and 17-0911-01, respectively).

Physiological saline of the following composition was used (mM): NaCl 120.4, KCl 5.9, MgSO₄ 1.2, CaCl₂ 2, glucose 11.5, and HEPES 11. The Ca²⁺-free solutions contained 2 mM EGTA. [Arg⁸]-Vasopressin acetate salt (AVP), Carbachol (CCh), Caffeine, Zaprinast (ZAP), 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), Rp-8-pCPT-cGMPS sodium salt, 8pCPT-cGMP, S-nitroso-N-acetyl-DL-penicillamine (SNAP) were from Sigma. Fluo-4 acetoxymethyl ester was from Molecular Probes, Life Technologies, UK.
[Arg⁸]-Vasopressin acetate salt, Carbachol, Caffeine, Rp-8-pCPT-cGMPS sodium salt, 8pCPT-cGMP were dissolved in water; S-nitroso-N-acetyl-DL-penicillamine (SNAP), 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), Zaprinast in DMSO.

Toxoplasma strain RH tachyzoites were maintained in human foreskin fibroblasts as previously described (65 (link)). Excretory secretory antigens (ESA) were prepared as described previously, with slight modification (29 (link)). Parasites were harvested from fully egressed cultures and resuspended in extracellular (EC) buffer (5 mM KCl, 142 mM NaCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 5.6 mM d-glucose, 25 mM HEPES [pH 7.4]). Tissue culture-grade 24-well plates were coated with 1% bovine serum albumin (BSA) in EC buffer at 4°C overnight. The next day, the wells were washed with EC buffer to remove BSA just prior to stimulating microneme secretion. For stimulation of ESA secretion, 500 µl of freshly harvested parasites was added to BSA-coated wells and 500 µl of 1 mM zaprinast (Sigma-Aldrich) in EC buffer was added. Parasites were allowed to secrete for 20 min at 37°C. After 20 min, plates were chilled for 5 min, the liquid was collected and centrifuged at 2,700 rpm and 4°C for 5 min, and the supernatant was retained as the ESA fraction. The ESA fraction was buffer exchanged with phosphate-buffered saline (PBS) using Amicon (Millipore Sigma) centrifugal filters (3-kDa cutoff), and the retained fraction was stored at −80°C. The protein concentration was estimated using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher).