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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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Effect of chronic exposure to CNP and ANP on GC-B- and GC-A-dependent cGMP accumulation in GH3 cells. Cells were initially treated with (a) 100 nM CNP or (b) 100 nM ANP in PSS without IBMX for 0 to 6 h. The medium was removed and replaced with 100 nM CNP (a) or 100 nM ANP (b) for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). ***P < 0.001, *P < 0.05, significantly different from the control. c, d Cells were initially treated with 0, 100 nM CNP, or 100 nM ANP for 6 h as before. All pre-treatments were removed and replaced either with (c) 0 or 100 nM CNP or with (d) 0 or 100 nM ANP, for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). *P < 0.05, significantly different from the control
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Fig3: Effect of chronic exposure to CNP and ANP on GC-B- and GC-A-dependent cGMP accumulation in GH3 cells. Cells were initially treated with (a) 100 nM CNP or (b) 100 nM ANP in PSS without IBMX for 0 to 6 h. The medium was removed and replaced with 100 nM CNP (a) or 100 nM ANP (b) for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). ***P < 0.001, *P < 0.05, significantly different from the control. c, d Cells were initially treated with 0, 100 nM CNP, or 100 nM ANP for 6 h as before. All pre-treatments were removed and replaced either with (c) 0 or 100 nM CNP or with (d) 0 or 100 nM ANP, for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). *P < 0.05, significantly different from the control

Mentions: Having established that GC-B signaling but not that of GC-A receptors, underwent rapid homologous desensitization, we next examined the effect of prolonged exposure of each receptor to its specific ligand. GH3 cells pre-treated with either ANP or CNP for 0 (control) to 6 h reduced the response to a subsequent 15-min treatment of ANP or CNP (with IBMX). GH3 cells pre-treated with 100 nM CNP demonstrated a significant reduction in cGMP produced compared with the control response to CNP (no CNP pre-treatment). The amount of cGMP measured from the 15-min pre-treatment sample was reduced by 39.6 ± 0.002 % (***P < 0.001). This effect was maintained up to the 6-h pre-treatment with CNP, which was reduced by 37.9 ± 7.1 % compared with the control (***P < 0.001). The reduction in cGMP accumulation compared with the control did not significantly differ between the 15-min to 6-h pre-treatments (not significant [ns], P > 0.05; Fig. 3a), suggesting that maximal desensitization of GC-B signaling occurred within 15 min of pre-treatment.Fig. 3


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)

Effect of chronic exposure to CNP and ANP on GC-B- and GC-A-dependent cGMP accumulation in GH3 cells. Cells were initially treated with (a) 100 nM CNP or (b) 100 nM ANP in PSS without IBMX for 0 to 6 h. The medium was removed and replaced with 100 nM CNP (a) or 100 nM ANP (b) for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). ***P < 0.001, *P < 0.05, significantly different from the control. c, d Cells were initially treated with 0, 100 nM CNP, or 100 nM ANP for 6 h as before. All pre-treatments were removed and replaced either with (c) 0 or 100 nM CNP or with (d) 0 or 100 nM ANP, for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). *P < 0.05, significantly different from the control
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Fig3: Effect of chronic exposure to CNP and ANP on GC-B- and GC-A-dependent cGMP accumulation in GH3 cells. Cells were initially treated with (a) 100 nM CNP or (b) 100 nM ANP in PSS without IBMX for 0 to 6 h. The medium was removed and replaced with 100 nM CNP (a) or 100 nM ANP (b) for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). ***P < 0.001, *P < 0.05, significantly different from the control. c, d Cells were initially treated with 0, 100 nM CNP, or 100 nM ANP for 6 h as before. All pre-treatments were removed and replaced either with (c) 0 or 100 nM CNP or with (d) 0 or 100 nM ANP, for 15 min in the presence of 1 mM IBMX. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the percentage of the control (n = 3). *P < 0.05, significantly different from the control
Mentions: Having established that GC-B signaling but not that of GC-A receptors, underwent rapid homologous desensitization, we next examined the effect of prolonged exposure of each receptor to its specific ligand. GH3 cells pre-treated with either ANP or CNP for 0 (control) to 6 h reduced the response to a subsequent 15-min treatment of ANP or CNP (with IBMX). GH3 cells pre-treated with 100 nM CNP demonstrated a significant reduction in cGMP produced compared with the control response to CNP (no CNP pre-treatment). The amount of cGMP measured from the 15-min pre-treatment sample was reduced by 39.6 ± 0.002 % (***P < 0.001). This effect was maintained up to the 6-h pre-treatment with CNP, which was reduced by 37.9 ± 7.1 % compared with the control (***P < 0.001). The reduction in cGMP accumulation compared with the control did not significantly differ between the 15-min to 6-h pre-treatments (not significant [ns], P > 0.05; Fig. 3a), suggesting that maximal desensitization of GC-B signaling occurred within 15 min of pre-treatment.Fig. 3

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