ATP- and EGF-stimulated phosphatidylinositol synthesis by two different pathways, phospholipase D and diacylglycerol kinase, in A-431 epidermoid carcinoma cells

Abstract

The [3H]inositol incorporation into the membrane fraction of A-431 human epidermoid carcinoma cells was markedly increased by stimulation of the cells with either epidermal growth factor (EGF), ATP, bradykinin, or a calcium ionophore A23187 in the presence of 1 mM extracellular calcium ions; most incorporated [3H]inositol was found to have accumulated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI synthesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Pretreatment of cells with pertussis toxin (IAP, islet-activating protein) inhibited the PI synthesis, [Ca2+]ielevation, and inositol trisphosphate (IP3) production by ATP, suggesting that the phospholipase C (PLC) system coupled with IAP-sensitive G protein is involved in the ATP-stimulated PI synthesis. On the other hand, the ATP stimulation increased the release of [3H]choline and [32P]phosphatidic acid (PA) from radiolabeled cells, and such release was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, the ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP3production and [Ca2+]ielevation, this fact suggests that the IAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P2-purinergic receptors appears to activate the IAP-sensitive PLC system and IAP-insensitive PLD system, both of which are essential for the stimulation of PI synthesis. The present results imply the general prospect that ligand stimulation, which mobilizes second messengers and consumes their precursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.Key words: P2-purinergic receptors, phosphatidylinositol, EGF receptors, phosphatidic acid, A-431 cells.