Abstract
Oscillations in cytoplasmic Ca(2+) concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca(2+) oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca(2+)-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca(2+) oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li(+), an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li(+)-treated cells, cytoplasmic Ca(2+) signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca(2+) oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca(2+) oscillations, and these could not be rescued by inositol or PI4P. In Li(+)-treated cells, recovery of the cytoplasmic Ca(2+) oscillations in the presence of inositol or PI4P was suppressed when Ca(2+) influx through store-operated Ca(2+) channels was inhibited. After rundown of the Ca(2+) signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca(2+) release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca(2+) entry is needed to sustain cytoplasmic Ca(2+) signaling following leukotriene receptor activation both by refilling the Ca(2+) stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.
Highlights
Phosphatidylinositol 4,5-bisphosphate levels need to be replenished during calcium signaling, but how this is achieved is unclear
Oscillations in [Ca2ϩ]i Are Sustained by Ca2ϩ Entry through CRAC Channels in RBL-2H3 Cells—Activation of cysteinyl leukotriene type I receptors with LTC4 in RBL-1 cells evokes a series of oscillations in [Ca2ϩ]i that are supported by Ca2ϩ entry through CRAC channels [9]
Application of Phosphatidylinositol 4-Phosphate Rescues Oscillations in [Ca2ϩ]i Evoked by LTC4 in Liϩ-treated Cells— The results described above with wortmannin suggest that phosphatidylinositol 4-phosphate (PI4P) production is important for sustaining oscillations in [Ca2ϩ]i evoked by LTC4
Summary
Phosphatidylinositol 4,5-bisphosphate levels need to be replenished during calcium signaling, but how this is achieved is unclear. Sustained cytoplasmic Ca2؉ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca2؉-releasing second messenger inositol trisphosphate. Our findings show that storeoperated Ca2؉ entry is needed to sustain cytoplasmic Ca2؉ signaling following leukotriene receptor activation both by refilling the Ca2؉ stores and by helping to replenish the PIP2 pool. We explored the possibility that Ca2ϩ influx through CRAC channels, in addition to refilling the stores, regulates replenishment of the agonist-sensitive PIP2 pool during oscillations in [Ca2ϩ]i. We find that the step converting PI4P to PIP2 is dependent on Ca2ϩ entry through the channels, providing an autoregulatory mechanism through which CRAC channels maintain their own activity by ensuring sufficient PIP2 levels in the plasma membrane for store depletion via InsP3 production
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