Carbachol in the presence of guanosine 5'-O-(3-thiotriphosphate) stimulates the breakdown of exogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol by rat brain membranes

E. Claro, M. A. Wallace, H. M. Lee, J. N. Fain

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Abstract

The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTPγS) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTPγS (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 μM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 μM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTPγS was greatest on a percentage basis at the lowest free Ca2+ concentration. Above 1 μM free Ca2+, the stimulatory effect was lost, whereas 10 μM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 μM. The best stimulation of PI breakdown due to carbachol plus GTPγS was seen with 0.3 μM free Ca2+ and 100 μM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 μM GTPγS alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 μM GTPγS. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTPγS in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.

Original languageEnglish
Pages (from-to)18288-18295
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number31
Publication statusPublished - 1989
Externally publishedYes

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Guanosine 5'-O-(3-Thiotriphosphate)
Carbachol
Phosphatidylinositols
Rats
Brain
Membranes
Type C Phospholipases
Inositol
phosphatidylinositol 4-phosphate
Adenosine Triphosphate
Chemical activation
Degradation
Muscarinic Agonists
Deoxycholic Acid
Inositol Phosphates
Guanine Nucleotides
Guanosine
Diglycerides
Atropine
Phosphoric Monoester Hydrolases

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{bfb31db748af45e8be1fc67b1ae17fad,
title = "Carbachol in the presence of guanosine 5'-O-(3-thiotriphosphate) stimulates the breakdown of exogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol by rat brain membranes",
abstract = "The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTPγS) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTPγS (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 μM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 μM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTPγS was greatest on a percentage basis at the lowest free Ca2+ concentration. Above 1 μM free Ca2+, the stimulatory effect was lost, whereas 10 μM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 μM. The best stimulation of PI breakdown due to carbachol plus GTPγS was seen with 0.3 μM free Ca2+ and 100 μM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 μM GTPγS alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 μM GTPγS. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTPγS in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.",
author = "E. Claro and Wallace, {M. A.} and Lee, {H. M.} and Fain, {J. N.}",
year = "1989",
language = "English",
volume = "264",
pages = "18288--18295",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "31",

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TY - JOUR

T1 - Carbachol in the presence of guanosine 5'-O-(3-thiotriphosphate) stimulates the breakdown of exogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol by rat brain membranes

AU - Claro, E.

AU - Wallace, M. A.

AU - Lee, H. M.

AU - Fain, J. N.

PY - 1989

Y1 - 1989

N2 - The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTPγS) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTPγS (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 μM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 μM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTPγS was greatest on a percentage basis at the lowest free Ca2+ concentration. Above 1 μM free Ca2+, the stimulatory effect was lost, whereas 10 μM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 μM. The best stimulation of PI breakdown due to carbachol plus GTPγS was seen with 0.3 μM free Ca2+ and 100 μM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 μM GTPγS alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 μM GTPγS. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTPγS in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.

AB - The breakdown of exogenously added [3H]inositol-labeled phosphoinositides by rat brain cortical membranes was stimulated by the muscarinic cholinergic agonist carbachol. The stimulation required the presence of guanine nucleotide. Optimal conditions were similar to those described for guanosine 5'-O-(3-thiotriphosphate) (GTPγS) + carbachol stimulation of phosphoinositide breakdown in [3H]inositol-prelabeled brain membranes (Claro, E., Garcia, A., and Picatoste, F. (1989) Biochem J. 261, 29-35). Carbachol stimulated [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was inhibited by atropine and guanosine 5'-O-(2-thiobisphosphate). The magnitude of the stimulation of exogenous PIP2 breakdown by carbachol and GTPγS (2- to 3-fold) was little affected over a PIP2 concentration range of 0.03-100 μM. Phosphatidylinositol 4-phosphate (PIP) was as good a substrate at all concentrations as PIP2 for carbachol stimulation of phospholipase C activity. There was appreciable phosphomonoesterase degradation of PIP to phosphatidylinositol (PI) over 10 min. There was also some conversion of added PIP to PIP2 in the presence of added ATP. The effect of calcium on PIP breakdown was similar to that on PIP2 breakdown, with an apparent EC50 for Ca2+ stimulation of 0.74 and 0.72 μM, respectively, under basal conditions. The stimulation of PIP2 and PIP breakdown by carbachol in the presence of GTPγS was greatest on a percentage basis at the lowest free Ca2+ concentration. Above 1 μM free Ca2+, the stimulatory effect was lost, whereas 10 μM free Ca2+ gave a maximal stimulation of basal phospholipase C activity. Degradation of added PI was also stimulated by carbachol in the absence of ATP. PI breakdown had an EC50 for Ca2+ stimulation of 1.07 μM. The best stimulation of PI breakdown due to carbachol plus GTPγS was seen with 0.3 μM free Ca2+ and 100 μM PI. Maximal activation of PI breakdown was seen at 1 mM deoxycholate as was true for PIP2 and PIP breakdown. There was little effect, even of 30 μM GTPγS alone or of carbachol alone, on PI breakdown. Half-maximal activation of the carbachol response required only 0.2 μM GTPγS. These results indicate that the phospholipase C enzyme(s) activated by carbachol in the presence of GTPγS in rat brain cortical membranes can degrade PIP2, PIP, and PI to inositol phosphates and diacylglycerol.

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EP - 18295

JO - Journal of Biological Chemistry

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