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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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Rapid homologous desensitization of CNP-dependent but not ANP-dependent cGMP accumulation in GH3 cells. Cells were treated with PSS containing either (a) 100 nM CNP or (b) 100 nM ANP for 0–15 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 pmol/ml per milligram protein (n = 3). The bar charts (right) indicate a comparison of the rate of cGMP accumulation between 0 and 5 min and between 5 and 15 min of stimulation (***P < 0.001, significantly different from the rate of cGMP accumulation between 0 and 5 min). c GH3 cells were treated as before with 100 nM CNP from 0 to 15 m in the presence or absence of either the protein phosphatase 2A (PP2A) inhibitor, okadaic acid (OA, 100 nM), the PP2B inhibitor, cypermethrin (1 nM), or the protein tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4, 1 mM) and assayed for total cGMP concentration. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the fold change in the rate of cGMP accumulation between 5 and 15 min (n = 3; **P < 0.01, cGMP accumulation maintained at a significant rate)
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Fig2: Rapid homologous desensitization of CNP-dependent but not ANP-dependent cGMP accumulation in GH3 cells. Cells were treated with PSS containing either (a) 100 nM CNP or (b) 100 nM ANP for 0–15 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 pmol/ml per milligram protein (n = 3). The bar charts (right) indicate a comparison of the rate of cGMP accumulation between 0 and 5 min and between 5 and 15 min of stimulation (***P < 0.001, significantly different from the rate of cGMP accumulation between 0 and 5 min). c GH3 cells were treated as before with 100 nM CNP from 0 to 15 m in the presence or absence of either the protein phosphatase 2A (PP2A) inhibitor, okadaic acid (OA, 100 nM), the PP2B inhibitor, cypermethrin (1 nM), or the protein tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4, 1 mM) and assayed for total cGMP concentration. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the fold change in the rate of cGMP accumulation between 5 and 15 min (n = 3; **P < 0.01, cGMP accumulation maintained at a significant rate)

Mentions: To characterize the activity of these receptors further , a series of experiments was performed to examine mechanisms of receptor desensitization. Total cGMP accumulation was measured to determine the changes in activity of the GC-A and GC-B receptors when treated over a short time-course with their native ligands, namely ANP and CNP, respectively. GH3 cells were plated at 3 × 105 cells/well in a 24-well plate, treated with 100 nM of either ANP or CNP in the presence of the phosphodiesterase inhibitor IBMX (1 mM) for 0 to 15 min and assayed for total cGMP concentration (Fig. 2a, b). From 0–5 min of treatment with 100 nM CNP, a rapid increase in cGMP accumulation was seen from 6.4 ± 0.7 to 82.7 ± 15.5 pmol/ml per milligram protein. In contrast, cGMP accumulation between 5 to 15 min of treatment with 100 nM CNP (82.7 ± 15.5 to 117.4 ± 17.6pmol/ml per milligram protein) was significantly attenuated compared with that seen over the first 5 min (reduced to 22.2 ± 3.9 % of the initial rate of accumulation between 0 to 5 min; ***P < 0.001). This failure to maintain the initial rate of cGMP accumulation suggested that rapid homologous desensitization of GC-B-mediated cGMP signaling had occurred. In another experiment performed with 100 nM ANP instead of CNP, a rapid increase in cGMP accumulation (7.91 ± 0.9 to 164.31 ± 9.1pmol/ml per milligram protein) occurred between 0 and 5 min. In contrast to GC-B, this initial rate of cGMP accumulation was maintained for at least 15 min, suggesting that rapid homologous desensitization of GC-A-mediated cGMP signaling did not occur (Fig. 2b).Fig. 2


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)

Rapid homologous desensitization of CNP-dependent but not ANP-dependent cGMP accumulation in GH3 cells. Cells were treated with PSS containing either (a) 100 nM CNP or (b) 100 nM ANP for 0–15 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 pmol/ml per milligram protein (n = 3). The bar charts (right) indicate a comparison of the rate of cGMP accumulation between 0 and 5 min and between 5 and 15 min of stimulation (***P < 0.001, significantly different from the rate of cGMP accumulation between 0 and 5 min). c GH3 cells were treated as before with 100 nM CNP from 0 to 15 m in the presence or absence of either the protein phosphatase 2A (PP2A) inhibitor, okadaic acid (OA, 100 nM), the PP2B inhibitor, cypermethrin (1 nM), or the protein tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4, 1 mM) and assayed for total cGMP concentration. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the fold change in the rate of cGMP accumulation between 5 and 15 min (n = 3; **P < 0.01, cGMP accumulation maintained at a significant rate)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3921447&req=5

Fig2: Rapid homologous desensitization of CNP-dependent but not ANP-dependent cGMP accumulation in GH3 cells. Cells were treated with PSS containing either (a) 100 nM CNP or (b) 100 nM ANP for 0–15 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 pmol/ml per milligram protein (n = 3). The bar charts (right) indicate a comparison of the rate of cGMP accumulation between 0 and 5 min and between 5 and 15 min of stimulation (***P < 0.001, significantly different from the rate of cGMP accumulation between 0 and 5 min). c GH3 cells were treated as before with 100 nM CNP from 0 to 15 m in the presence or absence of either the protein phosphatase 2A (PP2A) inhibitor, okadaic acid (OA, 100 nM), the PP2B inhibitor, cypermethrin (1 nM), or the protein tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4, 1 mM) and assayed for total cGMP concentration. The data are means ± SEM of three independent experiments, each performed in triplicate and are expressed as the fold change in the rate of cGMP accumulation between 5 and 15 min (n = 3; **P < 0.01, cGMP accumulation maintained at a significant rate)
Mentions: To characterize the activity of these receptors further , a series of experiments was performed to examine mechanisms of receptor desensitization. Total cGMP accumulation was measured to determine the changes in activity of the GC-A and GC-B receptors when treated over a short time-course with their native ligands, namely ANP and CNP, respectively. GH3 cells were plated at 3 × 105 cells/well in a 24-well plate, treated with 100 nM of either ANP or CNP in the presence of the phosphodiesterase inhibitor IBMX (1 mM) for 0 to 15 min and assayed for total cGMP concentration (Fig. 2a, b). From 0–5 min of treatment with 100 nM CNP, a rapid increase in cGMP accumulation was seen from 6.4 ± 0.7 to 82.7 ± 15.5 pmol/ml per milligram protein. In contrast, cGMP accumulation between 5 to 15 min of treatment with 100 nM CNP (82.7 ± 15.5 to 117.4 ± 17.6pmol/ml per milligram protein) was significantly attenuated compared with that seen over the first 5 min (reduced to 22.2 ± 3.9 % of the initial rate of accumulation between 0 to 5 min; ***P < 0.001). This failure to maintain the initial rate of cGMP accumulation suggested that rapid homologous desensitization of GC-B-mediated cGMP signaling had occurred. In another experiment performed with 100 nM ANP instead of CNP, a rapid increase in cGMP accumulation (7.91 ± 0.9 to 164.31 ± 9.1pmol/ml per milligram protein) occurred between 0 and 5 min. In contrast to GC-B, this initial rate of cGMP accumulation was maintained for at least 15 min, suggesting that rapid homologous desensitization of GC-A-mediated cGMP signaling did not occur (Fig. 2b).Fig. 2

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