ATP-STIMULATED AND EGF-STIMULATED PHOSPHATIDYLINOSITOL SYNTHESIS BY 2DIFFERENT PATHWAYS, PHOSPHOLIPASE-D AND DIACYLGLYCEROL KINASE, IN A-431 EPIDERMOID CARCINOMA-CELLS

The [H-3]inositol incorporation into the membrane fraction of A-431 hu man epidermoid carcinoma cells was markedly increased by stimulation o f 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 [H-3]inositol was found to have accumu lated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI syn thesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H- 7), and an intracellular calcium chelator, ,2-bis(o-aminophenoxy)ethan e-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not b y the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesul fonamide hydrochloride (W-7). Pretreatment of cells with pertussis tox in (IAP, islet-activating protein) inhibited the PI synthesis, [Ca2+]( i) elevation, and inositol trisphosphate (IP3) production by ATP, sugg esting that the phospholipase C (PLC) system coupled with LAP-sensitiv e G protein is involved in the ATP-stimulated PI synthesis. On the oth er hand, the ATP stimulation increased the release of [H-3]choline and [P-32]phosphatidic acid (PA) from radiolabeled cells, and such releas e was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, th e ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP3 production and [Ca2+](i) elevation, this fact suggest s that the IAP-insensitive PLD system is involved in the ATP-stimulate d PI synthesis. In A-431 cells, the stimulation of P-2-purinergic rece ptors appears to activate the IAP-sensitive PLC system and IAP-insensi tive 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 pre cursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.