Leukotriene D4 and E4 induce transmembrane signaling in human epithelial cells. Single cell analysis reveals diverse pathways at the G-protein level for the influx and the intracellular mobilization of Ca2+.
Leukotrienes are recognized as important mediators of the inflammatory process. Recently, increasing attention has been paid to the role of noninflammatory cells in the regulation of the inflammatory process. To further increase our knowledge of this matter we have, in the present study, investigated leukotriene-induced Ca2+ signaling, using a single cell technique in a human epithelial cell line, Intestine 407. It was evident that both LTD4 and LTE4, at physiological concentrations (10 nM), triggered rapid and pronounced cytosolic free Ca2+ transients, due to both influx across the plasma membrane and intracellular mobilization. Preincubation with pertussis toxin (1200 ng/ml) decreased the level of agonist-induced Ca2+ transients to an extent similar to that caused by depletion of extracellular Ca2+, suggesting that the toxin affected the influx but not the intracellular mobilization of Ca2+. Indeed, by using the Mn2+ quenching technique, it could be shown that pertussis toxin totally inhibited the influx of Ca2+. The fact that, even after pertussis toxin treatment, direct G-protein activation by AIF4- was still able to trigger a cytosolic free Ca2+ transient, indicates that, in these cells, G-proteins (GTP-binding proteins) that are insensitive to pertussis toxin are capable of mediating a Ca2+ signal. In order to test the idea that such G-proteins regulate mobilization of intracellular Ca2+ induced by LTD4 and LTE4, we electropermeabilized and preincubated the Intestine 407 cells with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), let them reseal, and, after loading with fura2, investigated the effects on agonist-stimulated Ca2+ signaling. Electropermeabiization and resealing alone did not significantly affect the Ca2+ responses triggered by LTD4 or LTE4. Addition of GDP beta S, in the presence of extracellular Ca2+, reduced the Ca2+ responses by approximately 60-70%. In Ca2(+)-depleted medium, GDP beta S also impaired the LTD4-induced response by 65%, however, it had no effect on the Ca2+ response induced by LTE4. In conclusion, LTD4 and LTE4 trigger cytosolic free Ca2+ signaling in a human epithelial cell line by causing both an influx of Ca2+ and mobilization of intracellular Ca2+. The Ca2(+)-signaling mechanism appears to consist of dual pathways, since the influx is regulated by a pertussis toxin-sensitive G-protein, but, the mobilization of Ca2+ is not. Furthermore, our data suggest that the LTD4-induced mobilization is regulated by a pertussis toxin-insensitive G-protein whereas the LTE4-induced mobilization is relatively insensitive to both pertussis toxin and GDP beta S.
Sjölander
A
A
Department of Cell Biology, Faculty of Health Sciences, University of Linköping, Sweden.
Grönroos
E
E
Hammarström
S
S
Andersson
T
T
D000535:Aluminum / Q000494:pharmacology; D017607:Aluminum Compounds; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D005459:Fluorides; D005461:Fluorine / Q000494:pharmacology; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D017999:Leukotriene E4; D037342:Pertussis Toxin; D013189:SRS-A / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D012969:Sodium Fluoride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D017607:Aluminum Compounds; D013189:SRS-A; D010566:Virulence Factors, Bordetella; C050992:tetrafluoroaluminate; D005461:Fluorine; D017999:Leukotriene E4; D012969:Sodium Fluoride; D000535:Aluminum; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D005459:Fluorides; D002118:Calcium; D016257:Fura-2