Leukotriene D4-induced mobilization of intracellular Ca2+ in epithelial cells is critically dependent on activation of the small GTP-binding protein Rho

Abstract

We have previously shown that the leukotriene D4 (LTD4)-induced mobilization of intracellular Ca2+ in epithelial cells is mediated by a G-protein that is distinctly different from the pertussis toxin-sensitive G-protein that regulates the subsequent influx of Ca2+. In the present study, we attempted to gain further knowledge about the mechanisms involved in the LTD4-induced mobilization of intracellular Ca2+ in epithelial cells by investigating the effects of compactin, an inhibitor of the isoprenylation pathway, on this signalling event. In cells preincubated with 10 microM compactin for 48 h, the LTD4-induced mobilization of intracellular Ca2+ was reduced by 75% in comparison with control cells. This reduction was reversed by co-administration of mevalonate (1 mM). The effect of compactin occurred regardless of whether or not Ca2+ was present in the extracellular medium, suggesting that isoprenylation must occur before Ca2+ is released from intracellular stores. In accordance with this, we also found that both the LTD4-induced formation of inositol 1,4,5-trisphosphate and the LTD4-induced phosphorylation of phospholipase C gamma 1 (PLC gamma 1) on tyrosine residues were significantly reduced in compactin-pretreated cells. These results open up the possibility that the activation of PLC gamma 1 is related to a molecule that is sensitive to impaired activity of the isoprenylation pathway, such as a small monomeric G-protein. This idea was supported by the observation that Clostridium botulinum C3 exoenzyme-induced inhibition of Rho proteins abolished the LTD4-induced intracellular mobilization of Ca2+. A regulatory role of Rho proteins in the LTD4-induced activation of PLC gamma 1 is unlikely to be indirectly mediated via an effect on the cytoskeleton, since cytochalasin D had no major effect on the LTD4-induced mobilization of Ca2+. Although the mechanism of interaction remains to be elucidated, the present findings indicate an important role of an isoprenylated protein such as Rho in the LTD4-induced Ca2+ signal.

Leukotriene D4 Triggers an Association between Gβγ Subunits and Phospholipase C-γ1 in Intestinal Epithelial Cells

“…Rhee and Bae (21) originally described the PLC-␥ isoform as a calcium signaling enzyme involved in growth factor signal transduction, and these investigators noted that activation of PLC-␥ was associated with increased tyrosine phosphorylation and translocation of this enzyme to the plasma membrane (21). Although we have found that LTD 4 activated the heterotrimeric G␣ i3 -protein (22), we have also observed that LTD 4 stimulated PLC-␥1, detected as an increase in tyrosine phosphorylation and translocation of this isoform of the enzyme to the plasma membrane (23). Moreover, in the latter investigation, we noted that the PLC-␥1-mediated release of intracellular calcium in Int 407 cells included a Rho-dependent event.…”

“…We have previously found that such calcium mobilization is blocked by compactin, an inhibitor of protein isoprenylation (23). This finding can at least partly be due to decreased isoprenylation of G␤␥ subunits and to an inadequate association of these proteins with the plasma membrane (40).…”

“…Gel Electrophoresis-The precipitated proteins were solubilized by boiling at 100°C for 5 min in 62 mM Tris (pH 6.8), 1.0% SDS, 10% glycerol, 15 mg/ml dithiothreitol, and 0.05% bromphenol blue (23). All samples were then subjected to electrophoresis in the presence of SDS on 10% homogeneous polyacrylamide gels.…”

International Union of Pharmacology XXXVII. Nomenclature for Leukotriene and Lipoxin Receptors

“…However, these observations have not been pursued. The initial observations of Gronroos et al (1995Gronroos et al ( , 1996Gronroos et al ( , 1998 have recently received support from several investigators (Hoshino et al, 1998;Thodeti et al, 2000;Massoumi and Sjolander, 2001;Paruchuri et al, 2002) and provided evidence that LTD 4 activation of an epithelial cell line (THP-1) led to an association of a protein other than the heterotrimeric G-proteins and this mobilization was necessary for the mobilization of calcium. Although these investigators observed that LTD 4 activation of THP-1 cells altered mitogen-activated protein kinase via protein kinase C and Raf-1, an effect which was PTX-insensitive, they also reported that the chemotactic response of these cells was PTX-sensitive.…”

International Union of Basic and Clinical Pharmacology. LXXXIV: Leukotriene Receptor Nomenclature, Distribution, and Pathophysiological Functions

“…In further reports, investigators have shown that the Ca 2ϩ modulation may be through two distinct G proteins, one PTX-sensitive and one insensitive, in monocyte/macrophage U937 cells (Pollock and Creba, 1990;Capra et al, 2003) or in a human epithelial cell line (Sjölander et al, 1990;Adolfsson et al, 1996). Subsequently, CysLT 1 R-dependent actin reorganization was shown to be coupled with a PTX-sensitive G protein and to Rho in an intestinal epithelial cell line (Grönroos et al, 1996;Massoumi and Sjölander, 1998;Massoumi et al, 2002) or in bronchial SMCs (Saegusa et al, 2001). Thus, these data confirm CysLT 1 R promiscuity in G protein coupling in constitutive systems, in good agreement with the finding that LTD 4 activates distinct signaling pathways differently coupled to G proteins in MNCs (Saussy et al, 1989;Skoglund and Claesson, 1991;Hoshino et al, 1998;Capra et al, 2003) or intestinal epithelial cells (Grönroos et al, 1998;Paruchuri and Sjölander, 2003;Nielsen et al, 2005b).…”