All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells

Abstract

In nonexcitable cells, receptor stimulation evokes Ca2+ release from the endoplasmic reticulum stores followed by Ca2+ influx through store-operated Ca2+ channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca2+ release-activated Ca2+ (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca2+-dependent activation of protein kinase Cα and the mitogen activated protein kinases ERK1/2 and this is required for the subsequent stimulation of the enzymes Ca2+-dependent phospholipase A2 and 5-lipoxygenase, generating the intracellular messenger arachidonic acid and the proinflammatory intercellular messenger leukotriene C4. In cell population studies, ERK activation, arachidonic acid release, and leukotriene C4 secretion were all graded with stimulus intensity. However, at a single cell level, Ca2+ influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist concentration recruited more mast cells but each cell responded by generating all-or-none Ca2+ influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca2+entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation.

Local Ca2+ Influx through Ca2+ Release-activated Ca2+ (CRAC) Channels Stimulates Production of an Intracellular Messenger and an Intercellular Pro-inflammatory Signal

“…Collectively, these results demonstrate that store-operated Ca 2ϩ entry is much more effective in activating cPLA 2 and LTC 4 secretion than a similar bulk cytoplasmic Ca 2ϩ elevation, establishing that the subplasmalemmal Ca 2ϩ rise is key in stimulating these enzymes. Although the present findings have utilized thapsigargin, we have previously reported that Ca 2ϩ release following activation of muscarinic receptors in RBL cells was entirely ineffective in stimulating ERK, arachidonic acid generation, and LTC 4 secretion, whereas subsequent Ca 2ϩ influx robustly activated these processes (19). Similarly, activation of FC⑀RI receptors with antigen triggered ERK activation and LTC 4 secretion through a mechanism indistinguishable from that utilized by thapsigargin (11).…”

Intercellular Ca 2+ wave propagation involving positive feedback between CRAC channels and cysteinyl leukotrienes

“…We have previously shown that individual mast cells respond in an essentially all-or-nothing manner to agonist (15). Raising agonist concentration recruited more cells in the population, but each cell that responded did so maximally.…”

Regulation of Ca2+ Signaling with Particular Focus on Mast Cells

“…Early studies indicated that influx of Ca 2+ from the external medium was absolutely required for degranulation11 and that Ca 2+ oscillations generated by release of Ca 2+ from intracellular stores failed to support degranulation in antigen-stimulated RBL-2H3 cells 301. Instead, sustained Ca 2+ oscillations or global increases of [Ca 2+ ] i maintained by Ca 2+ influx did support degranulation 309,320. A similar tight dependency on Ca 2+ influx was noted for the recruitment of protein kinase C and components of the ERK/PLA 2 /5-lipoxygenase pathway in RBL cells activated with thapsigargin309 or receptor agonists 320.…”

“…Instead, sustained Ca 2+ oscillations or global increases of [Ca 2+ ] i maintained by Ca 2+ influx did support degranulation 309,320. A similar tight dependency on Ca 2+ influx was noted for the recruitment of protein kinase C and components of the ERK/PLA 2 /5-lipoxygenase pathway in RBL cells activated with thapsigargin309 or receptor agonists 320. Some mast cell stimulants such as agonists of the adenosine A 3 receptor321 and purinergic P2Y receptor320 elicit transient production of IP3 and release of stored Ca 2+ without activating influx mechanisms, Ca 2+ -dependent signals, and degranulation.…”