Stimulation of inositol phosphate formation in ROS 17/2.8 cell membranes by guanine nucleotide, calcium, and parathyroid hormone.
In addition to stimulation of cyclic AMP, parathyroid hormone (PTH) may influence cellular events by utilizing other pathways of hormone action, such as the generation of inositol phosphates (IPs). We sought to examine this potential action of PTH by assessing the formation of inositol phosphates in PTH-sensitive ROS 17/2.8 cells. The polyphosphoinositides were labeled by growing the cells with [3H]inositol following which cell homogenates were prepared. The nonhydrolyzable guanine nucleotide, GTP gamma S, and calcium ion, alone and together, stimulated all three IPs, IP1, IP2, and IP3. IP1 formation was linear over 30 minutes but IP2 and IP3 accumulated more rapidly peaking by 5 minutes for all agonist conditions. The proportion of total P as IP3 was enhanced when the cells were grown with retinoic acid (1 microM) or when the assay was conducted at pH 4.5. In addition, the lower pH was associated with much more enzyme activity. PTH agonists, bPTH-(1-84) and bPTH-(1-34), both caused a small but significant stimulation of IP3 formation. When bPTH-(1-84), and the analog bPTH-(3-34)amide, that inhibits PTH-mediated adenylate cyclase activity were present together, there was additive stimulation of IP3 formation compared with that with either agent alone. The results demonstrate that inositol phosphate formation can be stimulated directly in a membrane preparation of ROS cells by GTP gamma S, calcium ion, and PTH and that the enzyme mediating this activity, phospholipase C, is regulated by a guanine nucleotide binding protein.
Cosman
F
F
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY.
Morrow
B
B
Kopal
M
M
Bilezikian
J P
JP
D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D010281:Parathyroid Hormone / Q000494:pharmacology*; D013403:Sugar Phosphates / Q000378:metabolism*; D014212:Tretinoin / Q000494:pharmacology
D006150:Guanine Nucleotides; D007295:Inositol Phosphates; D010281:Parathyroid Hormone; D013403:Sugar Phosphates; D014212:Tretinoin; D002118:Calcium