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

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 μM 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 Cγ1 (PLCγ1) on tyrosine residues were significantly reduced in compactin-pretreated cells. These results open up the possibility that the activation of PLCγ1 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 PLCγ1 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.