Serum-induced membrane depolarization in quiescent fibroblasts: activation of a chloride conductance through the G protein-coupled LPA receptor.

Abstract

Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin‐bound lysophosphatidic acid (LPA) acting on its own G protein‐coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl‐ efflux. This depolarizing Cl‐ current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume‐regulated Cl‐ currents. Activation of the Cl‐ current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras‐related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein‐coupled receptor agonists evoke membrane depolarization by activating a new type of Cl‐ channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers.

Cloning and Expression of a Complementary DNA Encoding a Molluscan Octopamine Receptor That Couples to Chloride Channels in HEK293 Cells

“…Recently, it was shown that activation of the G-proteincoupled lysophosphatidic acid receptor present on fibroblasts leads to a long lasting depolarization of these cells due to an efflux of chloride ions (20). As was found in our studies, the signal transduction pathway responsible for opening the chloride channel was suggested to be independent of known second messengers.…”

“…All available data suggest that the chloride channel in HEK293 cells that we have found to be G-protein-activated is highly similar to the channel described by Postma et al (20). Our data, however, exclude the option that phosphoinositide hydrolysis is important in the signaling pathway leading from the activated receptor to the opening of the chloride channel, since stimulation of Lym oa 2 does not result in any change in intracellular concentrations of inositol phosphates.…”

“…G-protein-mediated activation of a similar Cl Ϫ current was recently described by Postma et al (20) in rat fibroblasts. Their study also indicated that none of the known second messenger system was involved in the Cl Ϫ channel activation.…”

“…Also, activation of a serotonin receptor that has been shown to be coupled to the activation of PLC does not influence the outward Cl Ϫ current. The G-protein-mediated activation of the chloride channel described by Postma et al in Rat-1 fibroblasts (20) and by ourselves in HEK293 cells must proceed via an as yet unknown signaling pathway. Here we show that protein phosphorylation is important in this pathway but that protein kinase A and protein kinase C are not involved.…”

“…The opening of this particular chloride channel could also be induced by activation of the thrombin receptor, the endothelin receptor, and the neurokinin A receptor. Since these receptors (as well as the lysophosphatidic acid receptor) all couple to PLC, it was suggested that the signaling pathway leading to the increased Cl Ϫ conductance is closely associated with phosphoinositide hydrolysis (20).…”