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This calcium increase appears to initiate extranuclear effects in these tissues. Increases in cytosolic calcium are often a consequence of stimulation of inositol phosphate second messenger pathway. Several calcemic hormones act via this signal transduction route. Therefore we investigated the effects of the metabolically active T3 and the inactive analogues 3,5-diiodotyrosine and rT3 on the inositol phosphate pathway in fetal rat limb bone cultures prelabeled with [3H]myoinositol. Labelled inositol and inositol phosphates were separated by HPLC. There was a significant increase in the radioactivity in inositol bis- and trisphosphates after 1 min of exposure to 10-7 mol/l T3. Stimulation was also observed at 10-6 mol/l T3, but not at 10-5 mol/l. Time course studies demonstrated a rapid effect of T3 on inositol phosphates within 30 seconds that lasted through 5 min. After 20 min incubation with T3, no increase was observed in inositol mono- and bisphoshates, and a decrease was seen in inositol trisphosphate. Pretreatment with indomethacin prevented these effects of T3. 3,5-diiodothyrosine and rT3 did not affect inositol phosphate metabolism. These results suggest the existence of plasma membrane-associated receptors for T3 in bone, in addition to the nuclear receptors demonstrated previously. 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