Leukotriene D 4 -induced Ca 2+ Mobilization in Ehrlich Ascites Tumor Cells

Abstract

Stimulation of Ehrlich ascites tumor cells with leukotriene D4 (LTD4) within the concentration range 1-100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca2+ concentration, [Ca2+]i. The Ca2+ peak time, i.e., the time between addition of LTD4 and the highest measured [Ca2+]i value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration of LTD4 (100 nm), the highest measured increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-containing medium is 260 +/- 14 nm and the EC50 value for LTD4-induced Ca2+ mobilization is estimated at 10 nM. Neither the peptido-leukotrienes LTC4 and LTE4 nor LTB4 are able to mimic or block the LTD4-induced Ca2+ mobilization, hence the receptor is specific for LTD4. Removal of Ca2+ from the experimental buffer significantly reduces the size of the LTD4-induced increase in [Ca2+]i. Furthermore, depletion of the intracellular Ins(1,4,5)P3-sensitive Ca2+ stores by addition of the ER-Ca2+-ATPase inhibitor thapsigargin also reduces the size of the LTD4-induced increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-containing medium, and completely abolishes the LTD4-induced increase in [Ca2+]i in Ehrlich cells suspended in Ca2+-free medium containing EGTA. Thus, the LTD4-induced increase in [Ca2+]i in Ehrlich cells involves an influx of Ca2+ from the extracellular compartment as well as a release of Ca2+ from intracellular Ins(1,4,5)P3-sensitive stores. The Ca2+ peak times for the LTD4-induced Ca2+ influx and for the LTD4-induced Ca2+ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35 min in six out of eight experiments, respectively. Stimulation with LTD4 also induces a transient increase in Ins(1,4, 5)P3 generation in the Ehrlich cells, and the Ins(1,4,5)P3 peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4, 5)P3 content seems to increase before the LTD4-induced Ca2+ release from the intracellular stores but after the LTD4-induced Ca2+ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD4-induced increase in Ins(1,4,5)P3 as well as the LTD4-induced increase in [Ca2+]i, indicating that a U73122-sensitivity phospholipase C is involved in the LTD4-induced Ca2+ mobilization in Ehrlich cells. The LTD4-induced Ca2+ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD4-activated Ca2+ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion, LTD4 acts in the Ehrlich cells via a specific receptor for LTD4, which upon stimulation initiates an influx of Ca2+, through yet unidentified Ca2+ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P3 formation and finally release of Ca2+ from the intracellular Ins(1,4,5)P3-sensitive stores.

The action of cysteinyl‐leukotrienes on intracellular calcium mobilization in human detrusor myocytes

“…These results are in keeping with previous results using HEK-293 cells expressing the CysLT 1 receptor [9] and human neutrophils [5], which showed that most of the response (>80%) persisted after removing extracellular calcium. Furthermore, in agreement with previous results with DMSO-differentiated HL-60 cells [32] and human neutrophils [5] [32,33], almost exclusive release from intracellular stores [5], or both [34].…”

Intracellular Signalling Involved in Volume Regulatory Decrease

“…In rat hepatocytes, LTD 4 activates both Ca 2+ -dependent and Ca 2+ -independent anion conductances, whereas direct application of 100 nM LTD 4 in neuroblastoma cells [61] or 5 nM LTD 4 in EATC [41,62] failed to elicit efflux/currents. An increased Cl --permeability has previously been described after addition of 100 nM LTD 4 to EATC [63], but since this concentration of LTD 4 is known to increase [Ca 2+ ] i in EATC [64], it is likely that this permeability increase represents activation of I Cl,Ca . LTD 4 also failed to induce activation of a Cl --conductance in several other cell types tested [25,65].…”

“…LTD 4 binds to CysLT receptors, of which the human CysLT 1 [119,120], the human CysLT 2 (Gene Bank accession number BAB03601) and the murine CysLT 1 [121] are cloned. Binding of LTD 4 to the receptor results in Ca 2+ release and Ca 2+ influx in EATC cells [63,64] and in many other cell types (see e.g. [122] [62], and addition of LTD 4 decreases the activation time of I K,vol [62]; (vi) the single-channel conductance of the LTD 4 -activated channel is approximately 5 pS, as estimated from non-stationary noise analysis [126] similar to that found for I K,vol .…”

Cysteinyl leukotriene-dependent [Ca2+]iresponses to angiotensin II in cardiomyocytes

“…In human detrusor smooth muscle cells, it was reported that elevation in [Ca 2ϩ ] i levels evoked by LTD 4 were almost exclusively due to mobilization from intracellular Ca 2ϩ stores (4). In other target cells, the LTD 4 -evoked Ca 2ϩ response was dependent on the release of Ca 2ϩ from intracellular stores and enhanced Ca 2ϩ influx (20,23). The present study demonstrates that both ANG II-and CysLTevoked increases in [Ca 2ϩ ] i levels were significantly attenuated by 2-APB.…”

“…They exert their actions via activation of at least two pharmacologically defined G proteincoupled receptors (CysLT 1 and CysLT 2 ) that are linked to PLC-mediated Ca 2ϩ mobilization (8,16). In addition to their well-known bronchiolar smooth muscle spasmogenic effect, these inflammatory mediators have been shown to enhance contractile responses in smooth muscle preparations or [Ca 2ϩ ] i levels in their target cells (4,10,20,21,23,29,30). Recently, with the use of a variety of techniques including in situ hybridization, Northern blotting, and RT-PCR, the presence of CysLT 1 and/or CysLT 2 transcripts in cardiac tissue has been identified (8,10,16,21).…”