Activation of phospholipase D by guanosine 5'[gamma-thio]triphosphate and AlF4- in bovine corneal epithelial cells.
We have investigated the regulation of phospholipase D (PLD) by guanine nucleotides and AlF4- in bovine corneal epithelial cells (BCEC) prelabeled with [3H]myristic acid. In the presence of ethanol, AlF4- increased the production of [3H]PA and [3H]PET indicating activation of PLD in these cells. The effects of AlF4- were time- and dose-dependent. Addition of guanosine 5[gamma-thio]triphosphate (GTP gamma S), to streptolysin O-permeabilized cells also resulted in increased accumulation of [3H]PA and [3H]PEt. Other guanine and adenine nucleotides were ineffective, and guanosine thiodiphosphate inhibited the GTP gamma S-induced activation of PLD. Direct addition of GTP gamma S to microsomal fraction prepared from [3H]myristate-labeled BCEC resulted in increased formation of [3H]PEt in a time- and dose-dependent manner. The activation of PLD by GTP gamma S in the microsomal fraction was absolutely dependent on the presence of Ca2+ > 0.5 microM. Addition of Ca2+ (10-100 microM) alone dose-dependently stimulated the PLD activity. Treatment of the microsomal fraction with phorbol esters had no effect on the ability of GTP gamma S to stimulate PLD. Addition of isoproterenol to BCEC resulted in several-fold stimulation of cAMP, but it had no effect on basal or PDBu-induced stimulation of PLD. Taken together, the data suggest that a GTP-binding protein is involved in regulation of PLD in BCEC, and that maximal stimulation of PLD probably results from an interaction between Ca2+, PKC and G-protein in BCEC.
Akhtar
R A
RA
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Choi
M W
MW
D017607:Aluminum Compounds / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D005459:Fluorides / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology; D020404:Glycerophospholipids; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D008861:Microsomes / Q000201:enzymology; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*
D017607:Aluminum Compounds; D020404:Glycerophospholipids; D009227:Myristic Acids; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D019814:Myristic Acid; C050992:tetrafluoroaluminate; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D005459:Fluorides