C-type natriuretic peptide (CNP) and its cognate guanylyl cyclase receptor, the natriuretic peptide receptor B (NPR-B) together constitute a regulatory system that controls cell function via the generation of intracellular cyclic GMP. In this report we have examined the role of cAMP signaling in the regulation of CNP and NPR-B activity in the FRTL-5 rat thyroid follicular cell line. As had been observed earlier with TSH, the cAMP mimetic, dibutyryl cAMP (dbcAMP; 1 mM) induced a significant reduction in CNP-stimulated cGMP generation that was first apparent after 6 h of treatment. The inhibitory effect of dbcAMP on NPR-B was dose dependent, with an EC50 of 0.2 mM. Pretreatment of FRTL-5 cells with either of two protein kinase A (PKA) inhibitors, KT-5720 and H-89, failed to curtail the dbcAMP reduction in NPR-B activity, suggesting that the cAMP pathway leading to inhibition of NPR-B is PKA independent. Whereas either a 30-min or a 24-h treatment with the protein kinase C-activator phorbol myristate acetate failed to alter maximal levels of CNP-stimulated cGMP, a 24-h exposure to the calcium ionophore A23187 reduced CNP-stimulated cGMP to about one-third of control. Pretreatment of FRTL-5 cells with the cell-permeable calcium chelator 1,2 bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, tetraacetoxymethyl ester completely abrogated the cAMP-induced reduction of CNP-stimulated cGMP. Real-time PCR showed no effect of dbcAMP on NPR-B transcript at 3 and 6 h, but indicated a 40% reduction in transcript by dbcAMP at 24 h. In contrast, real-time PCR indicated a 5-fold increase in CNP transcript at 3 h, reaching 15.4-fold above control at 6 h in cells treated with dbcAMP. In addition, immunofluorescence staining of FRTL-5 cells with a specific antibody for CNP-22 showed the presence of cytoplasmic CNP that was up-regulated by incubation with either TSH or dbcAMP. These results suggested that cAMP signaling regulates the natriuretic peptide system in rat thyroid cells by increasing CNP expression, and reducing NPR-B activity. This latter action of cAMP appears to be both PKA independent and calcium dependent, and provides support for a dominant role for calcium in the regulation of NPR-B in the rat thyroid.
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
