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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