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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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Molecular and pharmacological characterization of natriuretic peptide receptors in GH3 somatolactotropes and primary rat pituitary tissue. a Total RNA was extracted from either GH3 cells or primary rat pituitaries, prior to cDNA synthesis and reverse transcription plus the polymerase chain reaction for the indicated gene targets. Data shown are representative of at least three independent experiments. b, c GH3 cells were stimulated with the indicated concentrations of (b) atrial natriuretic peptide (ANP) or (c) C-type natriuretic peptide (CNP) in physiological saline solution (PSS) containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 15 min, before termination with 100 %(v/v) ice-cold ethanol. Following extraction under vacuum, total cGMP accumulation was measured by enzyme-immunoassay and concentration-response curves were constructed by using pre-existing equations in GraphPad Prism 5.0. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3)
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Fig1: Molecular and pharmacological characterization of natriuretic peptide receptors in GH3 somatolactotropes and primary rat pituitary tissue. a Total RNA was extracted from either GH3 cells or primary rat pituitaries, prior to cDNA synthesis and reverse transcription plus the polymerase chain reaction for the indicated gene targets. Data shown are representative of at least three independent experiments. b, c GH3 cells were stimulated with the indicated concentrations of (b) atrial natriuretic peptide (ANP) or (c) C-type natriuretic peptide (CNP) in physiological saline solution (PSS) containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 15 min, before termination with 100 %(v/v) ice-cold ethanol. Following extraction under vacuum, total cGMP accumulation was measured by enzyme-immunoassay and concentration-response curves were constructed by using pre-existing equations in GraphPad Prism 5.0. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3)

Mentions: Our previous studies have revealed that Npr1, Npr2, Npr3 and Nppc are all expressed in mouse pituitaries and/or in gonadotropes, suggesting a local role in the regulation of reproduction (Thompson et al. 2009). However, Npr2−/− not only are infertile but also have dwarfism and growth hormone deficiencies, suggesting that natriuretic peptides and their receptors also control somatotropes. To establish which guanylyl cyclases the GH3 somatolactotropes express, total RNA was extracted from GH3 cells and normal rat pituitary tissue, before qualitative reverse transcription (RT)-PCR. As shown (Fig. 1a), in GH3 cells, specific transcripts were detected for Npr1 (GC-A), Npr2 (GC-B1, GC-B2) and the somatotrope transcription factor, Pou1f1 (Pit1). Interestingly, no expression of Npr3 (clearance receptor) was detectable. In contrast, specific transcripts were detected for all these genes in primary rat pituitary tissue.Fig. 1


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)

Molecular and pharmacological characterization of natriuretic peptide receptors in GH3 somatolactotropes and primary rat pituitary tissue. a Total RNA was extracted from either GH3 cells or primary rat pituitaries, prior to cDNA synthesis and reverse transcription plus the polymerase chain reaction for the indicated gene targets. Data shown are representative of at least three independent experiments. b, c GH3 cells were stimulated with the indicated concentrations of (b) atrial natriuretic peptide (ANP) or (c) C-type natriuretic peptide (CNP) in physiological saline solution (PSS) containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 15 min, before termination with 100 %(v/v) ice-cold ethanol. Following extraction under vacuum, total cGMP accumulation was measured by enzyme-immunoassay and concentration-response curves were constructed by using pre-existing equations in GraphPad Prism 5.0. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Molecular and pharmacological characterization of natriuretic peptide receptors in GH3 somatolactotropes and primary rat pituitary tissue. a Total RNA was extracted from either GH3 cells or primary rat pituitaries, prior to cDNA synthesis and reverse transcription plus the polymerase chain reaction for the indicated gene targets. Data shown are representative of at least three independent experiments. b, c GH3 cells were stimulated with the indicated concentrations of (b) atrial natriuretic peptide (ANP) or (c) C-type natriuretic peptide (CNP) in physiological saline solution (PSS) containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) for 15 min, before termination with 100 %(v/v) ice-cold ethanol. Following extraction under vacuum, total cGMP accumulation was measured by enzyme-immunoassay and concentration-response curves were constructed by using pre-existing equations in GraphPad Prism 5.0. The data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as pmol/mg protein (n = 3)
Mentions: Our previous studies have revealed that Npr1, Npr2, Npr3 and Nppc are all expressed in mouse pituitaries and/or in gonadotropes, suggesting a local role in the regulation of reproduction (Thompson et al. 2009). However, Npr2−/− not only are infertile but also have dwarfism and growth hormone deficiencies, suggesting that natriuretic peptides and their receptors also control somatotropes. To establish which guanylyl cyclases the GH3 somatolactotropes express, total RNA was extracted from GH3 cells and normal rat pituitary tissue, before qualitative reverse transcription (RT)-PCR. As shown (Fig. 1a), in GH3 cells, specific transcripts were detected for Npr1 (GC-A), Npr2 (GC-B1, GC-B2) and the somatotrope transcription factor, Pou1f1 (Pit1). Interestingly, no expression of Npr3 (clearance receptor) was detectable. In contrast, specific transcripts were detected for all these genes in primary rat pituitary tissue.Fig. 1

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