title: Persistent cAMP signaling by TSH receptors revealed by phosphodiesterase inhibition.
abstract: BACKGROUND
  It is controversial whether persistent signaling by the thyrotropin (TSH) receptor (TSHR) is cell-type specific. We reported persistent TSHR signaling in human embryonic kidney 293 (HEK293) cells expressing human TSHRs (HEK-TSHRs), whereas another group reported persistent signaling in mouse thyroid follicles but not in HEK293 cells. Herein, we test this hypothesis directly.
  
  
  METHODS
  We used two methods to measure persistent signaling in HEK-TSHRs and confirm our previous observations. In Method 1, we used a chemiluminescent immunoassay to measure intracellular cAMP accumulation over 30-60 min by adding a phosphodiesterase inhibitor to the incubation medium. In Method 2, we used an intracellular biosensor to record cAMP levels continuously.
  
  
  RESULTS
  Using Method 1, we show that TSHR signals persistently in human thyrocytes and human osteosarcoma U2OS-TSHR cells. Using Method 1 in HEK-TSHRs, we show that after 5 min, the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) increases cAMP to 2.5 pmol/well, TSH increases cAMP to 1.6 pmol/well, but IBMX added 30 min after TSH withdrawal increases cAMP to 105 pmol/well. Using Method 2 in HEK-TSHRs, we confirm that without IBMX, TSH causes a transient increase in cAMP and 30 min after TSH withdrawal, IBMX increases cAMP in cells pretreated with TSH more rapidly and to a higher level than IBMX added to cells not pre-exposed to TSH. Lastly, using Method 2, we show that in HEK-TSHRs phosphodiesterases types 3 and 4 are involved in degrading cAMP as the specific inhibitors Rolipram and Milrinone expose persistent TSHR signaling.
  
  
  CONCLUSIONS
  We conclude that persistent TSHR activation occurs in human thyrocytes, U2OS-TSHR cells and HEK-TSHRs; it is not cell-type specific but is revealed by inhibiting phosphodiesterases.
authors.affiliation: Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases , National Institutes of Health, Bethesda, Maryland.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Geras-Raaka
authors.forename: Susanne
authors.initials: S
authors.lastname: Neumann
authors.forename: Marvin C
authors.initials: MC
authors.lastname: Gershengorn
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000737:chemistry; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay; D049449:Luminescence; D020105:Milrinone / Q000737:chemistry; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D020889:Rolipram / Q000737:chemistry; D015398:Signal Transduction; D013961:Thyroid Gland / Q000378:metabolism; D013972:Thyrotropin / Q000378:metabolism*; D013997:Time Factors
chemical_list: D010726:Phosphodiesterase Inhibitors; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D020105:Milrinone; D020889:Rolipram; D015056:1-Methyl-3-isobutylxanthine