Abstract
Ca2+ entry through store-operated Ca2+ channels drives the production of the pro-inflammatory molecule leukotriene C4 (LTC4) from mast cells through a pathway involving Ca2+-dependent protein kinase C, mitogen-activated protein kinases ERK1/2, phospholipase A2, and 5-lipoxygenase. Here we examine whether local Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane stimulates this signaling pathway. Manipulating the amplitude and spatial extent of Ca2+ entry by altering chemical and electrical gradients for Ca2+ influx or changing the Ca2+ buffering of the cytoplasm all impacted on protein kinase C and ERK activation, generation of arachidonic acid and LTC4 secretion, with little change in the bulk cytoplasmic Ca2+ rise. Similar bulk cytoplasmic Ca2+ concentrations were achieved when CRAC channels were activated in 0.25 mm external Ca2+ versus 2 mm Ca2+ and 100 nm La3+, an inhibitor of CRAC channels. However, despite similar bulk cytoplasmic Ca2+, protein kinase C activation and LTC4 secretion were larger in 2 mm Ca2+ and La3+ than in 0.25 mm Ca2+, consistent with the central involvement of a subplasmalemmal Ca2+ rise. The nonreceptor tyrosine kinase Syk coupled CRAC channel opening to protein kinase C and ERK activation. Recombinant TRPC3 channels also activated protein kinase C, suggesting that subplasmalemmal Ca2+ rather than a microdomain exclusive to CRAC channels is the trigger. Hence a subplasmalemmal Ca2+ increase in mast cells is highly versatile in that it triggers cytoplasmic responses through generation of intracellular messengers as well as long distance changes through increased secretion of paracrine signals.
Highlights
Hormones, neurotransmitters, and other signaling molecules that impinge on cell-surface receptors
We compared the amplitude of the cytoplasmic Ca2ϩ signal arising from readmission of external Ca2ϩ for 4 min to cells treated with thapsigargin in Ca2ϩ-free solution with that obtained after 4 min of stimulation of cells with thapsigargin in Ca2ϩ-free solution but in the presence of La3ϩ (Fig. 2B)
We have shown that a subplasmalemmal Ca2ϩ rise driven by Ca2؉ release-activated Ca2؉ (CRAC) channels in native cells, but not bulk cytoplasmic Ca2ϩ elevation, promotes the generation of the important intracellular messenger arachidonic acid, which is metabolized to form leukotriene C4 (LTC4)
Summary
Hormones, neurotransmitters, and other signaling molecules that impinge on cell-surface receptors. An increase in cytoplasmic Ca2ϩ concentration activates a variety of key cellular responses, including neurotransmitter release, muscle contraction, gene transcription, and cell growth and proliferation [1, 2] The use of such a promiscuous messenger raises the question of specificity. Local Ca2ϩ entry through store-operated channels, which are activated by emptying intracellular Ca2ϩ stores [7], regulates specific Ca2ϩ-dependent enzymes anchored at the plasma membrane, including the Ca2ϩ-ATPase pump [8], adenylate cyclase [9], and endothelial NO synthase [10]. Ca2ϩ release from the stores fails to activate this signaling pathway, despite raising bulk cytoplasmic Ca2ϩ to levels only slightly lower than those attained by Ca2ϩ entry [12] Activation of these enzymes might be critically dependent on the local subplasmalemmal Ca2ϩ rise accompanying CRAC channel opening. By stimulating the secretion of leukotrienes that stimulate cells some distance away, subplasmalemmal Ca2ϩ operating over distances of a few nanometers can result in transferral of information over distances of up to several centimeters
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