Prostaglandin f_2αstimulates inositol 1, 4, 5- trisphosphate and inositol 1, 3, 4, 5-tetrakisphosphate formation in bovine luteal cells

The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F_2α(PGF_2α) -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 PGF_2αwere 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_2α. Increases in inositol bisphosphate (InsP₂) were also apparent within 5 sec. Ins₁and InsP₄were observed after a short (5-sec) lag period. HPLC revealed that PGF_2αprovoked 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_2α-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_2α-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_2α-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.