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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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Evidence of sphingosine-mediated but not calcium-mediated, heterologous desensitization of GC-B signaling in GH3 somatolactotrope cells. GH3 cells were pre-treated with PSS containing either (a, b) 10 μM sphingosine-1-phosphate (S1P) or (c, d) 1 μM A23187 (calcium ionophore) for 30 min in the absence of IBMX, before a 15-min stimulation with either 100 nM CNP (a, c) or 100 nM ANP (b, d) in the presence of 1 mM IBMX. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3). **P < 0.01, significantly different from control response to CNP
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Fig6: Evidence of sphingosine-mediated but not calcium-mediated, heterologous desensitization of GC-B signaling in GH3 somatolactotrope cells. GH3 cells were pre-treated with PSS containing either (a, b) 10 μM sphingosine-1-phosphate (S1P) or (c, d) 1 μM A23187 (calcium ionophore) for 30 min in the absence of IBMX, before a 15-min stimulation with either 100 nM CNP (a, c) or 100 nM ANP (b, d) in the presence of 1 mM IBMX. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3). **P < 0.01, significantly different from control response to CNP

Mentions: Sphingolipids and calcium have been implicated in regulating natriuretic peptide signaling (Abbey-Hosch et al. 2004; Abbey and Potter 2002). Therefore, we examined the role of sphingosine-1-phosphate (S1P) on CNP-stimulated cGMP accumulation. Pre-treating GH3 cells with 10 μM S1P for 30 min failed to significantly alter basal cGMP accumulation (Fig. 6a). However, S1P pre-treatment did cause desensitization of CNP-stimulated cGMP accumulation (by 30 ± 2.7 % compared with control, **P < 0.01). Interestingly, pre-treating GH3 cells with 10 μM S1P did not result in desensitization of ANP-stimulated cGMP accumulation, compared with the ANP control samples (Fig. 6b; ns, P > 0.05), suggesting that GC-B and GC-A receptors are differentially regulated in GH3 cells. In similar experiments, cells were pre-treated with either 0 or 10 μM A23187 for 30 min prior to stimulation with either 0, 100 nM CNP, or 100 nM ANP for 15 min; however, pre-treatment with this calcium ionophore failed to alter either CNP- or ANP-stimulated cGMP accumulation (Fig. 6c, d).Fig. 6


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)

Evidence of sphingosine-mediated but not calcium-mediated, heterologous desensitization of GC-B signaling in GH3 somatolactotrope cells. GH3 cells were pre-treated with PSS containing either (a, b) 10 μM sphingosine-1-phosphate (S1P) or (c, d) 1 μM A23187 (calcium ionophore) for 30 min in the absence of IBMX, before a 15-min stimulation with either 100 nM CNP (a, c) or 100 nM ANP (b, d) in the presence of 1 mM IBMX. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3). **P < 0.01, significantly different from control response to CNP
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3921447&req=5

Fig6: Evidence of sphingosine-mediated but not calcium-mediated, heterologous desensitization of GC-B signaling in GH3 somatolactotrope cells. GH3 cells were pre-treated with PSS containing either (a, b) 10 μM sphingosine-1-phosphate (S1P) or (c, d) 1 μM A23187 (calcium ionophore) for 30 min in the absence of IBMX, before a 15-min stimulation with either 100 nM CNP (a, c) or 100 nM ANP (b, d) in the presence of 1 mM IBMX. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3). **P < 0.01, significantly different from control response to CNP
Mentions: Sphingolipids and calcium have been implicated in regulating natriuretic peptide signaling (Abbey-Hosch et al. 2004; Abbey and Potter 2002). Therefore, we examined the role of sphingosine-1-phosphate (S1P) on CNP-stimulated cGMP accumulation. Pre-treating GH3 cells with 10 μM S1P for 30 min failed to significantly alter basal cGMP accumulation (Fig. 6a). However, S1P pre-treatment did cause desensitization of CNP-stimulated cGMP accumulation (by 30 ± 2.7 % compared with control, **P < 0.01). Interestingly, pre-treating GH3 cells with 10 μM S1P did not result in desensitization of ANP-stimulated cGMP accumulation, compared with the ANP control samples (Fig. 6b; ns, P > 0.05), suggesting that GC-B and GC-A receptors are differentially regulated in GH3 cells. In similar experiments, cells were pre-treated with either 0 or 10 μM A23187 for 30 min prior to stimulation with either 0, 100 nM CNP, or 100 nM ANP for 15 min; however, pre-treatment with this calcium ionophore failed to alter either CNP- or ANP-stimulated cGMP accumulation (Fig. 6c, d).Fig. 6

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