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

Thompson IR, Mirczuk SM, Smith L, Lessey AJ, Simbi B, Sunters A, Baxter GF, Lipscomb VJ, McGonnell IM, Wheeler-Jones CP, Mukherjee A, Roberson MS, McArdle CA, Fowkes RC - Cell Tissue Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Signaling Group, Comparative Biomedical Sciences, Royal Veterinary College, University of London, Royal College Street, London, NW1 0TU, UK.

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.

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CNP fails to alter expression of Npr2 mRNA or GC-B protein in GH3 somatolactotrope cells. a GH3 cells were stimulated with 0 or 100 nM CNP for 24 h prior to extraction of total RNA, cDNA synthesis, and quantitative PCR for endogenous Npr2 expression. The data shown are means ± SEM of three independent experiments (n = 3). b GH3 cells were stimulated with 100 nM CNP for the indicated time, prior to extraction of total protein and Western blot analysis for GC-B expression. Blots were stripped and re-probed for β-actin. Panels are representative of three independent experiments (n = 3)
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Fig4: CNP fails to alter expression of Npr2 mRNA or GC-B protein in GH3 somatolactotrope cells. a GH3 cells were stimulated with 0 or 100 nM CNP for 24 h prior to extraction of total RNA, cDNA synthesis, and quantitative PCR for endogenous Npr2 expression. The data shown are means ± SEM of three independent experiments (n = 3). b GH3 cells were stimulated with 100 nM CNP for the indicated time, prior to extraction of total protein and Western blot analysis for GC-B expression. Blots were stripped and re-probed for β-actin. Panels are representative of three independent experiments (n = 3)

Mentions: We next examined whether Npr2 or GC-B down-regulation (as determined by a reduction in Npr2 mRNA expression and or GC-B protein expression) mediated the inhibitory effects of sustained CNP exposure. The effect of CNP exposure on the expression of the endogenous Npr2 gene was examined by using qPCR. A 24-h treatment with 100 nM CNP failed to alter Npr2 expression in GH3 cells (Fig. 4a). To establish whether down-regulation occurred at the protein level, total proteins were extracted from GH3 cells incubated for up to 24 h with 100 nM CNP. Western blotting for GC-B protein revealed that CNP failed to alter expression levels (Fig. 4b). Collectively, these data suggest that a loss of Npr2 mRNA or GC-B protein is not responsible for the reduced cGMP accumulation seen in pre-treated GH3 cells.Fig. 4


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

Thompson IR, Mirczuk SM, Smith L, Lessey AJ, Simbi B, Sunters A, Baxter GF, Lipscomb VJ, McGonnell IM, Wheeler-Jones CP, Mukherjee A, Roberson MS, McArdle CA, Fowkes RC - Cell Tissue Res. (2013)

CNP fails to alter expression of Npr2 mRNA or GC-B protein in GH3 somatolactotrope cells. a GH3 cells were stimulated with 0 or 100 nM CNP for 24 h prior to extraction of total RNA, cDNA synthesis, and quantitative PCR for endogenous Npr2 expression. The data shown are means ± SEM of three independent experiments (n = 3). b GH3 cells were stimulated with 100 nM CNP for the indicated time, prior to extraction of total protein and Western blot analysis for GC-B expression. Blots were stripped and re-probed for β-actin. Panels are representative of three independent experiments (n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3921447&req=5

Fig4: CNP fails to alter expression of Npr2 mRNA or GC-B protein in GH3 somatolactotrope cells. a GH3 cells were stimulated with 0 or 100 nM CNP for 24 h prior to extraction of total RNA, cDNA synthesis, and quantitative PCR for endogenous Npr2 expression. The data shown are means ± SEM of three independent experiments (n = 3). b GH3 cells were stimulated with 100 nM CNP for the indicated time, prior to extraction of total protein and Western blot analysis for GC-B expression. Blots were stripped and re-probed for β-actin. Panels are representative of three independent experiments (n = 3)
Mentions: We next examined whether Npr2 or GC-B down-regulation (as determined by a reduction in Npr2 mRNA expression and or GC-B protein expression) mediated the inhibitory effects of sustained CNP exposure. The effect of CNP exposure on the expression of the endogenous Npr2 gene was examined by using qPCR. A 24-h treatment with 100 nM CNP failed to alter Npr2 expression in GH3 cells (Fig. 4a). To establish whether down-regulation occurred at the protein level, total proteins were extracted from GH3 cells incubated for up to 24 h with 100 nM CNP. Western blotting for GC-B protein revealed that CNP failed to alter expression levels (Fig. 4b). Collectively, these data suggest that a loss of Npr2 mRNA or GC-B protein is not responsible for the reduced cGMP accumulation seen in pre-treated GH3 cells.Fig. 4

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Signaling Group, Comparative Biomedical Sciences, Royal Veterinary College, University of London, Royal College Street, London, NW1 0TU, UK.

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.

Show MeSH
Related in: MedlinePlus