Guanosine 5'-O-(thiotriphosphate)-dependent inositol trisphosphate formation in membranes is inhibited by phorbol ester and protein kinase C

Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with [3H]inositol. GTP gamma S stimulated the formation of [3H]inositol mono-, bis-, and trisphosphate ([3H]InsP, [3H]InsP2, and [3H]InsP3) with a half-maximal effect on [3H]InsP formation...

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Bibliographic Details
Published in:The Journal of biological chemistry 1987-02, Vol.262 (4), p.1638-1643
Main Authors: Orellana, S, Solski, P A, Brown, J H
Format: Article
Language:English
Subjects:
astrocytoma
Biological and medical sciences
Bradykinin - pharmacology
Carbachol - pharmacology
Cell physiology
Fundamental and applied biological sciences. Psychology
guanine nucleotide regulatory protein
Guanosine 5'-O-(3-Thiotriphosphate)
Guanosine Triphosphate - analogs & derivatives
Guanosine Triphosphate - metabolism
Guanosine Triphosphate - pharmacology
Guanylyl Imidodiphosphate - pharmacology
Histamine - pharmacology
Hormonal regulation
Inositol 1,4,5-Trisphosphate
Inositol Phosphates - biosynthesis
Kinetics
Membranes - metabolism
Molecular and cellular biology
Phorbol Esters - pharmacology
phosphoinositides
protein kinase C
Protein Kinase C - metabolism
Quinuclidinyl Benzilate - metabolism
Receptors, Muscarinic - metabolism
Sugar Phosphates - biosynthesis
Tetradecanoylphorbol Acetate - pharmacology
Thionucleotides - metabolism
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Summary:Phosphoinositide hydrolysis was studied in a washed membrane preparation of 1321N1 astrocytoma cells prelabeled with [3H]inositol. GTP gamma S stimulated the formation of [3H]inositol mono-, bis-, and trisphosphate ([3H]InsP, [3H]InsP2, and [3H]InsP3) with a half-maximal effect on [3H]InsP formation at 5 microM. Carbachol increased the accumulation of [3H]inositol phosphates only in the presence of added guanine nucleotide. Calcium increased [3H]InsP3 accumulation over a range of concentrations (10 nM-3 mM free calcium). When 1321N1 cells were treated with phorbol ester (100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)) prior to preparation of the membranes, the maximal [3H]InsP formation induced by GTP gamma S or GTP gamma S plus carbachol was decreased by 50-75%. In contrast, the response to a maximal calcium concentration presumed to activate phospholipase C directly was minimally inhibited (approximately 15%). PMA treatment did not affect muscarinic receptor affinity for carbachol or the effect of GTP on agonist binding. PMA treatment was also without effect on the breakdown of exogenous [3H]InsP3 in homogenates, permeabilized cells, and membranes, indicating that the InsP3-phosphatase was not the site of phorbol ester action. PMA treatment inhibited [3H] InsP3 formation only in membranes and not in cytosol prepared from the same cells, suggesting a membrane site of PMA action. Membranes were also required to demonstrate GTP gamma S-stimulated [3H]InsP3 formation although calcium-stimulated [3H]InsP3 formation was demonstrable in both membranes and cytosol. The addition of purified protein kinase C to the membranes mimicked the effect of PMA treatment to decrease GTP gamma S-stimulated [3H]InsP3 production. These data indicate that the effect of PMA on phosphoinositide metabolism is demonstrable in a cell-free system and that it can be mimicked by protein kinase C. We suggest that the ability of PMA to block GTP gamma S-stimulated formation of [3H]InsP3 results from inhibition of the G protein interaction with phospholipase C.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)75684-1