Ca2+ Waves Initiate Antigen-Stimulated Ca2+ Responses in Mast Cells

Abstract

Ca2+ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca2+ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca2+ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca2+ oscillations throughout the cell that usually follow the initiating Ca2+ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca2+ puffs at the region of cell contact. Initiating Ca2+ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca2+ release from endoplasmic reticulum stores and to extracellular Ca2+, but they do not depend on store-operated Ca2+ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca2+ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca2+ response.

Spatiotemporal Resolution of Mast Cell Granule Exocytosis

“…Our previous studies demonstrated that Ca 2+ responses to antigen in RBL mast cells usually initiate as waves that begin at the tips of extended cell protrusions (Cohen et al, 2009), and these results prompted us to investigate whether degranulation is influenced by Ca 2+ waves. To monitor Ca 2+ mobilization and degranulation simultaneously in the same cells, we used Fura Red as the Ca 2+ indicator in cells that were preloaded with FITC-dextran.…”

“…7A, left side, only a small percentage of exocytotic events occurred along protrusions compared with the cell body at this high antigen dose, and the difference in total number of events for cells that initiate Ca 2+ responses as P-waves versus Bwaves was small. These cells were shown to initiate Ca 2+ waves from protrusions with a higher probability at low doses of antigen (Cohen et al, 2009), and we investigated whether a low dose of antigen (1.7 ng/ml) caused a higher probability of exocytosis along these extended protrusions than seen with a high dose of antigen. When this low dose of antigen was used to stimulate degranulation, the average number of exocytotic events per cell was less than at the high antigen dose, and this difference was particularly evident for cells with Ca 2+ waves that initiated in the cell body (Fig.…”

“…We previously found that Ca 2+ entry through TRPC1 is important for Ca 2+ -dependent P-wave initiation, which is particularly manifest at low doses of antigen (Cohen et al, 2009). To determine whether TRPC1 contributes to antigen-stimulated degranulation in RBL cells, we compared the degranulation response of these cells in which TRPC1 is knocked down by expression of a vector containing a TRPC1-interfering small hairpin (sh) RNA sequence with one containing a control shRNA sequence.…”

“…In particular, delineating the relationships between degranulation and Ca 2+ mobilization requires high spatial and temporal resolution of both cytoplasmic Ca 2+ changes and individual exocytotic events. We recently showed that antigenmediated crosslinking of IgE-FceRI initiates Ca 2+ responses in mast cells in the form of a fast Ca 2+ wave that usually begins in the tip of an extended protrusion, then moves down the length of the cell over a time period of several seconds (Cohen et al, 2009). At low doses of antigen, initiation of this wave depends on Ca 2+ influx by the canonical transient receptor potential channel TRPC1.…”

“…The initiating waves are typically followed by periodic Ca 2+ oscillations that depend on Ca 2+ influx through SOCE (Ma and Beaven, 2009;Vig et al, 2008). These oscillations encode temporal information relevant to degranulation events (Kim et al, 1997), but they do not manifest the spatial localization displayed by wave propagation (Cohen et al, 2009). Very little is known about the physiological function of the extended protrusions in mast cells where Ca 2+ waves initiate.…”