Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

Abstract

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders.

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes

“…However, RT-PCR analysis revealed that GH3 cells failed to express transcripts for Npr3 encoding NPR-C (Fig. 1 d), in keeping with our previous studies (Thompson et al 2014 ). As the inhibition of cAMP production has previously been indicated as evidence of NPR-C activity (Sciarretta et al 2013 ), we examined the potential effect of CNP on forskolin (FSK)-stimulated cAMP accumulation in GH3 cells.…”

“…As shown (Fig. 1 b, c), both CNP and ANP caused an increase in the total concentration of cGMP, indicating the presence of functional GC-B and GC-A receptors, as we and others have described previously (Shimekake et al 1994 ; Fowkes et al 2000 ; Thompson et al 2014 ). To establish whether CNP and ANP could stimulate P-ERK1/2, we stimulated GH3 cells with the same concentration range of both peptides for 15 min, prior to the extraction of total protein and Western blotting for phosphorylated ERK1/2 (P-ERK1/2).…”

“…GH3, αT3–1 and LβT2 cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, Poole, UK) containing penicillin/streptomycin and 10% fetal calf serum (FCS; Invitrogen, Paisley, UK) at 37 °C, 5% (v/v) CO 2 in air as previously described (Thompson et al 2012 , 2014 ). Cells were plated at a density of 1 × 10 6 cells/well for Western blotting and RNA extraction and at 3 × 10 5 cells/well for reporter gene assays and cGMP determinations.…”

“…The anterior pituitary gland expresses high tissue concentrations of the GC-B ligand, CNP, plus all three natriuretic peptide receptors (Fowkes and McArdle 2000 ; Komatsu et al 1991 ; Grandclément et al 1995 ). Our previous studies revealed that CNP potently stimulates cGMP accumulation in rodent pituitary cell lines and primary rat pituitary cultures (Fowkes et al 2000 ; Shimekake et al 1994 ; Thompson et al 2009 , 2014 ) and some studies have indicated a function for GC-B and cGMP in mediating pituitary growth hormone secretion (Shimekake et al 1994 ; Hartt et al 1995 ). Furthermore, we have demonstrated the expression of CNP and GC-B in fetal, normal adult and adenomatous human pituitary tissue, including those from acromegalic patients (Thompson et al 2012 ).…”

Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes

“…Signaling through various other hormones and growth factors also reduces the guanylyl cyclase activity of NPR2 in other cells, for example, vasopressin (Abbey and Potter, 2002), PDGF (Chrisman and Garbers, 1999;Abbey and Potter, 2003), lysophosphatidic acid (Abbey and Potter, 2003;Potthast et al, 2004), sphingosine-1-phosphate (Abbey-Hosch et al, 2005) and thyrotropin-releasing hormone (Thompson et al, 2014). In studies of sphingosine-1-phosphate acting on cultured fibroblasts that overexpress NPR2 (Abbey-Hosch et al, 2005), there is a correlation between dephosphorylation and the decrease in guanylyl cyclase activity.…”