Abstract
The present report shows that thyrotropin (TSH) regulates all three steps involved in prostaglandin synthesis in FRTL-5 rat thyroid cells, i.e. arachidonic acid release from membrane phospholipids, cyclooxygenase (prostaglandin H synthase) action, and individual prostaglandin formation; however, its action at specific steps may require the presence of, or can be duplicated by, insulin, insulin-like growth factor-I (IGF-I), and/or a serum factor. Thus, TSH releases free arachidonic acid from rat FRTL-5 thyroid cells whose phospholipid fraction is radiolabeled with [3H]arachidonic acid; this action involves a pertussis toxin-sensitive G protein, is not cAMP mediated, and does not require insulin or 5% serum. To quantitate TSH effects on cyclooxygenase activity and on individual prostaglandin formation, a homogenate system and a rapid reversed-phase high pressure liquid chromatography procedure have been developed to measure cyclooxygenase metabolites. TSH increased cyclooxygenase activity in homogenates only if the cells were also exposed to insulin, IGF-I, and/or 5% calf serum; TSH alone had no apparent effect on the activity. Maximal activation, 4-fold over basal/micrograms of DNA, took 36 h to achieve and reflected, at least in part, an increase in cyclooxygenase gene expression. Like cyclooxygenase activity, induction of prostaglandin E2 production required 2 or more factors, i.e. TSH plus insulin/IGF-I or TSH plus insulin/IGF-I plus serum. Increased production of prostaglandin D2, could, however, be detected if cells were treated with TSH alone and the TSH activity could be duplicated by insulin, IGF-I, or calf serum alone.
Highlights
The present report shows thatthyrotropin (TSH) the possibility that PGEzmight be involved in the growth of regulates all three steipnsvolved in prostaglandin syn- FRTL-5 cells and thatfactors which regulate that growth, i.e
The present report explores the action of TSH on all three phase high pressure liquid chromatography procedure steps of prostaglandin production by FRTL-5 cells
The pertussis toxin did not affect the ability of TSH to elevate CAMPlevels in the cells (Table I). These results indicate that TSH can cause the release of free arachidonic acid
Summary
Voi. 266, No 1, Issue of January 5, pp. 440-448.1931 Printed in U.S.A. Regulation of Prostaglandin Synthesis by Thyrotropin, Insulin or Insulin-like Growth Factor-I, and Serum in FRTL-5 Rat ThyroidCells*. The sum of all six cyclooxygenase metabolites (PGF,,,, PGE,, PGD,, HHT,15-HETE,and11-HETE, seebelow)was calculated after separation with RP-HPLC using solvent program I1 (analysis time, 60 min). Activity was dependent on the concentration of added substrate and was saturable (Fig. IC); a Ifmax and K,, 0.46 nmol/min/mg protein and approximately 10 p ~ re,spectively, could be calculated Based on these results, all assays were performed at 30°C for 5 min in a 1-ml volume containing 50 mM sodium phosphate, pH 8.0, 3-5mgof homogenate protein, 20 mM tryptophan, 1 p M hematin, and 100 p~ [3H]arachidonicacid (0.1 pCi/nmol). A clone containing a 1230-bpinsert with an internal EcoRI site (860- and 370-bp fragments) and a 85% amino acid sequence identity to thecorresponding region of the sheep cyclooxy-
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