Guanosine 5'-O-thiotriphosphate and NaF stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in bovine corneal epithelium.
The role of a GTP-binding protein in activation of phospholipase C in bovine corneal epithelium was determined by investigating the effects of non-hydrolyzable GTP analog, GTP-gamma-S, and NaF on breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) in this tissue. GTP-gamma-S (2-50 microM), when introduced into the permeabilized corneal epithelial cells labeled with myo-[3H]inositol, dose-dependently increased the formation of myoinositol trisphosphate (IP3). Other guanine nucleotides and ATP were ineffective. Incubation of 32P-prelabeled corneal epithelium with NaF (2-50 mM) resulted in increased breakdown of PIP2 and increased synthesis of phosphatidic acid. In myo-[3H]inositol-labeled tissue, NaF dose-dependently increased the accumulation of IP3. Microsomal membrane fraction from corneal epithelium was found to contain phospholipase C activity towards endogenous phosphatidylinositol 4-phosphate and PIP2. The enzyme activity was stimulated by Ca2+ (100 microM). Addition of GTP-gamma-S to microsomal fraction containing phosphoinositides which were radiolabeled with 32Pi in situ or with [gamma-32P]ATP in vitro caused a dose dependent hydrolysis of PIP2. These data, taken collectively, suggest that a GTP-binding protein is involved in activation of phospholipase C towards PIP2 in bovine corneal epithelium, and that this guanine nucleotide regulatory protein may serve to couple norepinephrine and 5-hydroxytryptamine receptors to phospholipase C during transmembrane signalling.
Akhtar
R A
RA
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-3331.
D000818:Animals; D002417:Cattle; D003315:Cornea / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D010539:Permeability; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology*
D006150:Guanine Nucleotides; D007295:Inositol Phosphates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013873:Thionucleotides; C034882:guanosine 5'-O-(1-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012969:Sodium Fluoride