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Regulation of Natriuretic Peptide Receptors by Thyrotropin in FRTL-5 Rat Thyroid Cells: Evidence for Nonguanylate Cyclase Atrial Natriuretic Factor-Binding Sites in Cells Lacking the Natriuretic Peptide Receptor C

Department of Medicine, Endocrinology and Nephrology Divisions, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799

Address all correspondence and requests for reprints to: Donald F. Sellitti, Ph.D., Medicine Department, Room A 3060, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799. E-mail: dsellitti{at}mxa.usuhs.mil

Natriuretic peptide receptors (NPR) are expressed in thyroid-derived cells, including the rat FRTL-5 thyroid cell line. We have previously demonstrated that atrial natriuretic factor (ANF) binding consistent with the NPR-A receptor is significantly increased in FRTL-5 cells cultured in the presence of TSH. The purpose of the present study was to determine whether TSH treatment, therefore, results in higher levels of ANF-induced intracellular cGMP, and whether TSH elicits similar effects on cGMP signaling through the NPR-B receptor. We now show that contrary to expectation, long term exposure to 1 mIU/ml bovine TSH (6H medium) does not significantly alter maximal ANF-induced cGMP formation. Moreover, TSH treatment decreased C-type natriuretic peptide (CNP)-induced cGMP generation in FRTL-5 cells, suggesting a down-regulation of NPR-B. A similar effect of TSH on ANF- and CNP-induced cGMP was observed in FRTL cells, the precursor of the FRTL-5 cell line. Scatchard analysis of [¹²⁵I]ANF binding in TSH-treated (6H) FRTL-5 cultures indicated a 5.6-fold increase in high affinity ANF-binding sites compared with TSH-deficient (5H) cultures [binding capacity (B_max) of 6H cells, 227.2 ± 33.7 fmol/mg protein; B_max of 5H cells, 40.2 ± 4.7 fmol/mg protein]. The effect of TSH on [¹²⁵I]ANF binding was mimicked by forskolin and (Bu)₂cAMP, indicating receptor up-regulation via a cAMP pathway. High affinity [¹²⁵I]CNP-binding sites were present in much lower abundance (B_max of 5H, 0.80 ± 0.06 fmol/mg protein), and no effect of TSH treatment on them could be demonstrated. However, low affinity [¹²⁵I]CNP binding was increased by TSH. RT-PCR confirmed the presence of both NPR-A and NPR-B transcripts in FRTL-5 cells and showed that TSH treatment significantly decreased NPR-B, but not NPR-A. NPR-C transcript was not detectable by RT-PCR in FRTL-5 cells cultured in high TSH medium, suggesting that the ANF-binding sites increased by TSH are not NPR-C. Both CNP and ANF transcript were also expressed in FRTL-5 cells, and CNP was increased by TSH. Together the data support the down-regulation of functional NPR-B and no change in functional NPR-A by TSH. The vast majority of ANF-binding sites in FRTL-5 cells, therefore, are not coupled to cGMP production and may represent a novel or altered form of NPR that is regulated by TSH independently of NPR-A and NPR-B.

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