Prostaglandin fstimulates inositol 1, 4, 5- trisphosphate and inositol 1, 3, 4, 5-tetrakisphosphate formation in bovine luteal cells

Robert A. Duncan, John S Davis

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F(PGF) -stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGFwere analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. Ins1and InsP4were observed after a short (5-sec) lag period. HPLC revealed that PGFprovoked rapid (5 sec) increases in inositol 1, 4, 5-tris- phosphate (Ins 1, 4, 5-P3), which was rapidly converted to inositol 1, 3, 4, 5-tetrakisphosphate (Ins 1, 3, 4, 5-P4) and inositol 1, 3, 4-tris- phosphate (Ins 1, 3, 4-P3). The primary inositol bisphosphate isomer present in PGF-stimulated bovine luteal cells was inositol 1, 4-bisphosphate (Ins 1, 4-P2), with lesser amounts of Ins 1, 3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1, 4, 5-P3was followed temporally in saponin- permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1, 4, 5-P3to form Ins 1, 3, 4, 5- P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphos- phorylated inositol phosphates in PGF-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1, 4, 5-P3during agonist-mediated hydrolysis of phosphatidylinositol 4, 5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.

Original languageEnglish (US)
Pages (from-to)1519-1526
Number of pages8
JournalEndocrinology
Volume128
Issue number3
DOIs
StatePublished - Jan 1 1991
Externally publishedYes

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Inositol 1,4,5-trisphosphate 3-kinase
Luteal Cells
Inositol 1,4,5-Trisphosphate
Inositol
Prostaglandins
Dinoprost
Inositol Phosphates
Calcium
Corpus Luteum
Calmodulin
Phosphatidylinositols
calmidazolium
Hydrolysis
Phosphates
High Pressure Liquid Chromatography
Calcium-Calmodulin-Dependent Protein Kinases
inositol-1,3,4,5-tetrakisphosphate
Saponins
Ion Exchange Chromatography
Collagenases

ASJC Scopus subject areas

  • Endocrinology

Cite this

Prostaglandin fstimulates inositol 1, 4, 5- trisphosphate and inositol 1, 3, 4, 5-tetrakisphosphate formation in bovine luteal cells. / Duncan, Robert A.; Davis, John S.

In: Endocrinology, Vol. 128, No. 3, 01.01.1991, p. 1519-1526.

Research output: Contribution to journalArticle

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title = "Prostaglandin f2αstimulates inositol 1, 4, 5- trisphosphate and inositol 1, 3, 4, 5-tetrakisphosphate formation in bovine luteal cells",
abstract = "The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2α(PGF2α) -stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2αwere analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2α. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. Ins1and InsP4were observed after a short (5-sec) lag period. HPLC revealed that PGF2αprovoked rapid (5 sec) increases in inositol 1, 4, 5-tris- phosphate (Ins 1, 4, 5-P3), which was rapidly converted to inositol 1, 3, 4, 5-tetrakisphosphate (Ins 1, 3, 4, 5-P4) and inositol 1, 3, 4-tris- phosphate (Ins 1, 3, 4-P3). The primary inositol bisphosphate isomer present in PGF2α-stimulated bovine luteal cells was inositol 1, 4-bisphosphate (Ins 1, 4-P2), with lesser amounts of Ins 1, 3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1, 4, 5-P3was followed temporally in saponin- permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1, 4, 5-P3to form Ins 1, 3, 4, 5- P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphos- phorylated inositol phosphates in PGF2α-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1, 4, 5-P3during agonist-mediated hydrolysis of phosphatidylinositol 4, 5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2α-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.",
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N2 - The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2α(PGF2α) -stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2αwere analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2α. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. Ins1and InsP4were observed after a short (5-sec) lag period. HPLC revealed that PGF2αprovoked rapid (5 sec) increases in inositol 1, 4, 5-tris- phosphate (Ins 1, 4, 5-P3), which was rapidly converted to inositol 1, 3, 4, 5-tetrakisphosphate (Ins 1, 3, 4, 5-P4) and inositol 1, 3, 4-tris- phosphate (Ins 1, 3, 4-P3). The primary inositol bisphosphate isomer present in PGF2α-stimulated bovine luteal cells was inositol 1, 4-bisphosphate (Ins 1, 4-P2), with lesser amounts of Ins 1, 3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1, 4, 5-P3was followed temporally in saponin- permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1, 4, 5-P3to form Ins 1, 3, 4, 5- P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphos- phorylated inositol phosphates in PGF2α-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1, 4, 5-P3during agonist-mediated hydrolysis of phosphatidylinositol 4, 5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2α-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.

AB - The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2α(PGF2α) -stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2αwere analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2α. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. Ins1and InsP4were observed after a short (5-sec) lag period. HPLC revealed that PGF2αprovoked rapid (5 sec) increases in inositol 1, 4, 5-tris- phosphate (Ins 1, 4, 5-P3), which was rapidly converted to inositol 1, 3, 4, 5-tetrakisphosphate (Ins 1, 3, 4, 5-P4) and inositol 1, 3, 4-tris- phosphate (Ins 1, 3, 4-P3). The primary inositol bisphosphate isomer present in PGF2α-stimulated bovine luteal cells was inositol 1, 4-bisphosphate (Ins 1, 4-P2), with lesser amounts of Ins 1, 3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1, 4, 5-P3was followed temporally in saponin- permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1, 4, 5-P3to form Ins 1, 3, 4, 5- P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphos- phorylated inositol phosphates in PGF2α-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1, 4, 5-P3during agonist-mediated hydrolysis of phosphatidylinositol 4, 5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2α-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.

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