Cyclic GMP-dependent activation of Ca2+ release from the ER and digestive vacuole at distinct phases of intraerythrocytic Plasmodium falciparum development.

The malaria parasite Plasmodium falciparum relies on coordinated signaling cascades to control progression through its complex life cycle in the mammalian host and insect vector. Mobilization of cytosolic Ca2+ ([Ca2+]c) is central to many of these processes and is linked to the second messengers cGMP, cyclic AMP and inositol 1,4,5-trisphosphate. During the symptomatic blood stage these signaling pathways play key roles in erythrocyte invasion, maturation and egress of the parasite. Intraerythrocytic P. falciparum expressing the genetically encoded Ca2+ indicator GCaMP3 were used to investigate [Ca2+]c signals elicited by the cGMP phosphodiesterase inhibitor zaprinast during the late trophozoite and schizont stages, using post hoc Hoechst staining to categorize the parasites. Zaprinast elicited two distinct phases of [Ca2+]c increase with the first appearing only in parasites in which DNA replication was already underway and the second arising later in schizogony. Both phases were dependent on activation of protein kinase G and could be mimicked by the cGMP analog 8-Br-cGMP. The first phase occurred as a transient [Ca2+]c increase derived from cyclopiazonic acid-sensitive endoplasmic reticulum Ca2+ stores and was suppressed by the 1,4,5-trisphosphate signaling inhibitor 2-aminoethoxydiphenyl borate. The second phase [Ca2+]c increase induced by zaprinast was kinetically distinct and unaffected by cyclopiazonic acid, but was selectively eliminated by chloroquine, suggesting that it is derived from the digestive vacuole. These findings demonstrate that two separate Ca2+ mobilization pathways under the control of cGMP contribute to the [Ca2+]c signals that become active during schizogony in preparation for egress from the erythrocyte.