digitonin and pervanadate

Receptor-mediated phospholipase D activation in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR) apparently involves the small G protein RhoA. Here, activation of RhoA was examined by measuring cytosol-membrane translocation, which is a sign of RhoA activation. RhoA translocation was induced by guanosine 5'-O-(3-thio)triphosphate in digitonin-permeabilized HEK cells, and in intact cells by the agonist-activated mAChR and by direct activation of heterotrimeric G proteins. RhoA translocation was also induced by the phosphotyrosine phosphatase inhibitor pervanadate, while the tyrosine kinase inhibitors tyrphostin 23 and genistein inhibited the mAChR-induced RhoA translocation. These data suggest that translocation and thus activation of RhoA by the G protein-coupled m3 mAChR in HEK cells apparently involves a tyrosine kinase-dependent reaction.

Topics: Atropine; Biological Transport; Carbachol; Catechols; Cell Line; Cell Membrane; Cell Membrane Permeability; Cytosol; Digitonin; Enzyme Inhibitors; Genistein; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Isoflavones; Kidney; Muscarinic Agonists; Muscarinic Antagonists; Nitriles; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Receptor, Muscarinic M3; Receptors, Muscarinic; rhoA GTP-Binding Protein; Tyrphostins; Vanadates