ATP- and EGF-stimulated phosphatidulinositol synthesis by two different pathways, phospholipase D and diacylglycerol kinase, in A-431 epidermoid carcinoma cells.
The [(3)H]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 [(3)H]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, [Ca(2+)]i elevation, and inositol trisphosphate (IP(3)) 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 [(3)H]choline and [(32)P)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 IP(3) production and [Ca(2+)]i elevation, this fact suggests that the lAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P(2)-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.
Hosoi
K
K
Department of Physiology, Tokushima University School of Dentistry, Japan.
Shioda
Y
Y
Ueha
T
T
Atsumi
T
T
Sugita
K
K
Kurihara
K
K
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001920:Bradykinin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology*; D002614:Chelating Agents / Q000494:pharmacology; D002794:Choline / Q000378:metabolism; D019852:Diacylglycerol Kinase; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000187:drug effects / Q000502:physiology*; D037342:Pertussis Toxin; D010712:Phosphatidic Acids / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000096:biosynthesis* / Q000235:genetics; D010739:Phospholipase D / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011744:Pyrimidinones / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000187:drug effects* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D007476:Ionophores; D009363:Neoplasm Proteins; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D011744:Pyrimidinones; D018048:Receptors, Purinergic P2; D013449:Sulfonamides; D013844:Thiazoles; D010566:Virulence Factors, Bordetella; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D000001:Calcimycin; D004533:Egtazic Acid; D004815:Epidermal Growth Factor; C017967:W 7; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; C047505:R 59022; D037342:Pertussis Toxin; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; D066246:ErbB Receptors; D010738:Type C Phospholipases; D010739:Phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D019311:Staurosporine; D002794:Choline; D001920:Bradykinin; D002118:Calcium