Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells - PubMed (original) (raw)

Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells

E A Mellor et al. Proc Natl Acad Sci U S A. 2001.

Abstract

The cysteinyl leukotrienes (cys-LTs) LTC(4), LTD(4), and LTE(4) are a class of peptide-conjugated lipids formed from arachidonic acid and released during activation of mast cells (MCs). We now report that human cord-blood-derived MCs (hMCs) express the CysLT1 receptor, which responds not only to inflammation-derived cys-LTs, but also to a pyrimidinergic ligand, UDP. hMCs express both CysLT1 protein and transcript, and respond to LTC(4), LTD(4), and UDP with concentration-dependent calcium fluxes, each of which is blocked by a competitive CysLT1 receptor antagonist, MK571. Stably transfected Chinese hamster ovary cells expressing the CysLT1 receptor also exhibit MK571-sensitive calcium flux to all three agonists. Both hMCs and CysLT1 transfectants stimulated with UDP are desensitized to LTC(4), but only partially to LTD(4). Priming of hMCs with IL-4 for 5 days enhances their sensitivity to each agonist, but preferentially lowers their threshold for activation by LTC(4) and UDP (approximately 3 log(10)-fold shifts in dose-response for each agonist) over LTD(4) (1.3 log(10)-fold shift), without altering CysLT1 receptor mRNA or surface protein expression, implying the likely induction of a second receptor with CysLT1-like dual ligand specificity. hMCs thus express the CysLT1 receptor, and possibly a closely related IL-4-inducible receptor, which mediate dual activation responses to cys-LTs and UDP, providing an apparent intersection linking the inflammatory and neurogenic elements of bronchial asthma.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Calcium responses of hMCs to stimulation with LTD4 and LTC4, and preferential up-regulation by IL-4 of the response to LTC4. (A) hMCs maintained in SCF alone (100 ng/ml) or primed for 5 days with IL-4 (10 ng/ml) in the continued presence of SCF exhibit dose-dependent calcium responses to LTD4 (Upper) or LTC4 (Lower). The depicted results reflect separate experiments for LTD4 and LTC4 performed with hMCs from different donors. Priming the hMCs with IL-4 shifted the 50% maximal response to LTD4 (1.33 ± 0.41 log-fold increment, mean ± SEM, n = 3), and to LTC4 (mean, 3.83 ± 1.94 log-fold increment, n = 3) in every experiment performed. (B) The sustained phase of the cys-LT-induced calcium signal to each agonist was abolished by EGTA. (C) At 1 μM concentrations, LTD4 completely desensitized the hMCs to LTC4, whereas LTC4 partly desensitized them to LTD4. The depicted results for IL-4-primed hMCs were replicated three times for A and C, and two and three times for B.

Figure 2

Figure 2

Effect of pharmacologic receptor inhibitors on cys-LT-mediated calcium signals of IL-4-primed hMCs. (A) The competitive CysLT1 receptor antagonist MK571 (1 μM) completely blocked the responses of IL-4-primed hMCs to 1 μM LTD4 (Upper) and LTC4 (Lower). (B) BAY-u9773 (5 μM), which blocks the CysLT1 receptor but activates and desensitizes the CysLT2 receptor, yielded a small calcium signal in IL-4-primed hMCs that did not attenuate the subsequent calcium response to LTD4 (Upper), but partly blocked the agonist effect of LTC4 (Lower). The data depicted in A and B were replicated in two additional experiments, each with hMCs of different donors.

Figure 3

Figure 3

Expression of the CysLT1 and P2Y4 receptors by hMC. (A) Homology domains in the amino acid sequences of the CysLT1 receptor, the CysLT2 receptor, and the known existing human P2Y receptors, in the first (EC1) and third (EC3) extracellular loops, and second (TM2) and third (TM3) transmembrane domains. Shaded residues indicate identity to the CysLT1 receptor. (B) RNA blot analysis detecting transcripts encoding the cys-LT and P24 receptors in RNA extracted from 107 hMCs maintained over a 5-day period in the presence of SCF alone (−) or with IL-4 (10 ng/ml) (+). Hybridization signals reflecting steady-state levels of mRNA encoding the CysLT1 and P2Y4 receptors are displayed, whereas the CysLT2 and P2Y6 receptors were not detected and are not shown. (C) Membrane expression of the CysLT1 receptor is not altered by 5 days of priming with IL-4. Bold tracings indicate staining with an anti-CysLT1 polyclonal antibody, while lighter tracings depict staining with a rabbit IgG of irrelevant specificity. The depicted cytofluorographs are representative of two experiments performed.

Figure 4

Figure 4

LTC4 and UDP share receptors on hMC. (A) Effect of IL-4 priming of hMCs for 5 days compared with maintenance in SCF alone on dose-dependent calcium flux in response to UDP (10−11 M to 10−6 M). (B) The calcium signal induced by UDP (1 μM) is completely inhibited by MK571 (1 μM). (C) Cross-desensitization between UDP and LTC4 was complete at 1 μM of each agonist. In contrast, UDP did not desensitize the hMCs to LTD4, although LTD4 did desensitize the cells to UDP. (D) Stably transfected CHO cells expressing the human P2Y4 receptor do not respond to cys-LTs or UDP. CHO cells expressing the long isoform of the mouse CysLT1 receptor respond to LTD4 (10−9 M), LTC4 (10−7 M), and UDP (10−6 M, tracings shown with and without the addition of an equimolar concentration of MK571). The depicted results were replicated two times each for A, B, and C, with hMCs from different donors, and three times for D.

Similar articles

Cited by

References

    1. Chervinsky P, Brandon M, Zhang J, Kundu S, McBurney J, Reiss T F. Eur Resp J. 1998;11:1232–1239. - PubMed
    1. Suissa S, Dennis R, Ernst P, Sheehy O, Wood-Dauphinee S. Ann Intern Med. 1997;126:177–183. - PubMed
    1. Dixon R A, Diehl R E, Opas E, Rands E, Vickers P J, Evans J F, Gillard J W, Miller D K. Nature (London) 1990;343:282–284. - PubMed
    1. Reid G K, Kargman S, Vickers P J, Mancini J A, Leveille C, Ethier D, Miller D K, Gillard J W, Dixon R A, Evans J F. J Biol Chem. 1990;265:19818–19823. - PubMed
    1. Lam B K, Penrose J F, Freedman G J, Austen K F. Proc Natl Acad Sci USA. 1994;91:7663–7669. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources