Limits...
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.

Show MeSH

Related in: MedlinePlus

Lack of ligand-dependent internalization of GC-B or GC-A in GH3 somatolactotrope cells. GH3 cells were cultured on glass coverslips for 24 h prior to being treated with either 100 nM CNP for 0 (a), 1 h (b), or 4 h (c), or 100 nM ANP for 0 (d), 1 h (e), or 4 h (f). GC-A and GC-B immunoreactivity was detected by using Alexa488-conjugated secondary antibodies (green), whereas Alexa635-conjugated phalloidin was used to detect the actin cytoskeleton (red). Nuclear localization was detected by using 4,6-diamidino-2-phenylindole (DAPI) stain for orientation (blue). Panels are representative of three independent experiments (n = 3). Magnification ×40
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3921447&req=5

Fig5: Lack of ligand-dependent internalization of GC-B or GC-A in GH3 somatolactotrope cells. GH3 cells were cultured on glass coverslips for 24 h prior to being treated with either 100 nM CNP for 0 (a), 1 h (b), or 4 h (c), or 100 nM ANP for 0 (d), 1 h (e), or 4 h (f). GC-A and GC-B immunoreactivity was detected by using Alexa488-conjugated secondary antibodies (green), whereas Alexa635-conjugated phalloidin was used to detect the actin cytoskeleton (red). Nuclear localization was detected by using 4,6-diamidino-2-phenylindole (DAPI) stain for orientation (blue). Panels are representative of three independent experiments (n = 3). Magnification ×40

Mentions: To determine whether the GC-A or GC-B receptors undergo internalization upon stimulation with their respective ligands, confocal microscopy for GC-A and GC-B was used to visualize this process directly. Representative immunofluorescence for GC-A- and GC-B-labeled GH3 cells are shown (Fig. 5a–f). GH3 cells treated with 100 nM CNP or 100 nM ANP for 0–4 h did not demonstrate any observable GC-B or GC-A internalization, as observed by the overlay of the staining for actin filaments with that for GC-B and GC-A.Fig. 5


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)

Lack of ligand-dependent internalization of GC-B or GC-A in GH3 somatolactotrope cells. GH3 cells were cultured on glass coverslips for 24 h prior to being treated with either 100 nM CNP for 0 (a), 1 h (b), or 4 h (c), or 100 nM ANP for 0 (d), 1 h (e), or 4 h (f). GC-A and GC-B immunoreactivity was detected by using Alexa488-conjugated secondary antibodies (green), whereas Alexa635-conjugated phalloidin was used to detect the actin cytoskeleton (red). Nuclear localization was detected by using 4,6-diamidino-2-phenylindole (DAPI) stain for orientation (blue). Panels are representative of three independent experiments (n = 3). Magnification ×40
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Lack of ligand-dependent internalization of GC-B or GC-A in GH3 somatolactotrope cells. GH3 cells were cultured on glass coverslips for 24 h prior to being treated with either 100 nM CNP for 0 (a), 1 h (b), or 4 h (c), or 100 nM ANP for 0 (d), 1 h (e), or 4 h (f). GC-A and GC-B immunoreactivity was detected by using Alexa488-conjugated secondary antibodies (green), whereas Alexa635-conjugated phalloidin was used to detect the actin cytoskeleton (red). Nuclear localization was detected by using 4,6-diamidino-2-phenylindole (DAPI) stain for orientation (blue). Panels are representative of three independent experiments (n = 3). Magnification ×40
Mentions: To determine whether the GC-A or GC-B receptors undergo internalization upon stimulation with their respective ligands, confocal microscopy for GC-A and GC-B was used to visualize this process directly. Representative immunofluorescence for GC-A- and GC-B-labeled GH3 cells are shown (Fig. 5a–f). GH3 cells treated with 100 nM CNP or 100 nM ANP for 0–4 h did not demonstrate any observable GC-B or GC-A internalization, as observed by the overlay of the staining for actin filaments with that for GC-B and GC-A.Fig. 5

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