ACTIVATORS OF PROTEIN-KINASE-A DECREASE THE LEVELS OF FREE ARACHIDONIC-ACID IN OSTEOBLASTS VIA STIMULATION OF PHOSPHATIDYLCHOLINE AND PHOSPHATIDYLETHANOLAMINE SYNTHESIS

In order to examine the role of protein kinase A (PKA) in the regulati on of arachidonic acid availability, the interaction between cAMP agon ists and the G protein activator AlF4- in their effects on phospholipi d metabolism were measured in MC3T3-E1 osteoblasts. We show that forsk olin and 8-brcAMP, activators of PKA, amplify the AlF4--induced stimul ation of phosphatidylinositol-specific phospholipase C (phosphatidylin ositol inositolphosphohydrolase; EC 3.1.4.3), measured by the formatio n of [H-3]inositol phosphates in prelabeled cells. However, the AlF4-- stimulated production of 1,2-diacylglycerols and the release of [H-3] arachidonic acid ([H-3]AA) were inhibited 50-75% by forskolin and 8-br omocAMP. Furthermore, pretreatment with PKA activators prevented much of the AlF4--induced loss of [H-3]AA from phosphatidylcholine and phos phatidylethanolamine in prelabeled osteoblasts. In addition, in the ab sence of AlF4-, forskolin was found to stimulate the incorporation of [H-3]AA and [P-32]orthophosphoric acid selectively into these two majo r phospholipids and selectively increased their mass. The effects of f orskolin and 8-BrcAMP on the levels of free [H-3]AA were completely re versed by pretreatment with the PKA inhibitor H-89. Therefore, our fin dings suggest that the activation of cAMP-dependent protein kinase can reduce the availability of free arachidonic acid for prostaglandin sy nthesis in osteoblast cells by stimulating its reesterification via ph ospholipid resynthesis.