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Distribution and regulation of GPR12 receptor in rat pituitary
Frontiers in Neuroendocrinology Frontiers in Neuroendocrinology 27 (2006) 91–94 www.elsevier.com/locate/yfrne
ICN 2006 Conference Abstracts
Neuroendocrine gene regulation Distribution and regulation of GPR12 receptor in rat pituitary E. Swinnen, C. Denef Katholieke Universiteit Leuven, Leuven, Belgium GPR12 is a phospholipid G-protein-coupled receptor that exhibits constitutive activity on adenylate cyclase and has been implicated in maintenance of meiotic arrest in rat oocytes and in differentiation of the developing murine brain. GPR12 mRNA was detected by RT-PCR in rat pituitary as early as embryonic day 15 and at different postnatal ages in the anterior and neurointermediate lobe. The pituitary cell lines, GH3, AtT20, aT3-1, and LbT2 (representing different pituitary secretory cell subtypes), also expressed GPR12 mRNA, while the CHO fibroblastic cell line was negative. Confocal laser scanning microscopy revealed strong immunoreactivity (ir) in the melanotrophs in the intermediate lobe, while sparsely distributed, less intensely fluorescent cells were detected throughout the anterior lobe. Positive anterior lobe cells did not appear to uniquely belong to a single secretory cell type. No colocalisation with the folliculo-stellate cell marker S100 or stem cell/progenitor marker nestin was found. GPR12 mRNA and GPR12ir remained expressed in rat pituitary aggregate cell cultures. As measured by means of real-time RT-PCR, thyroid and glucocorticoid hormone significantly enhanced, while estradiol significantly lowered GPR12 mRNA levels. Leukemia inhibiting factor also inhibited GPR12 mRNA level while epidermal growth factor, basic fibroblastic growth factor, and nerve growth factor were without effect. In rat adrenal gland, GPR12-ir was seen in elongated cellular extensions in the cortex and the medulla, in conjunction with S100-positive sustentacular cells that line the endocrine chromaffin lobules. The present findings suggest a characteristic distribution and regulation of GPR12 in endocrine tissues. doi:10.1016/j.yfrne.2006.03.205
b-1-Adrenergic receptor (b-AR) expression in rat anterior pituitary (AP) gonadotrophs and in aT3-1 and LbT2 cells K. Janssens, O. Krylyshkina, N. Hersmus, H. Vankelecom, C. Denef Katholieke Universiteit Leuven, Leuven, Belgium doi:10.1016/j.yfrne.2006.03.204
The AP expresses b2-ARs that mediate stimulation of GH release by epinephrine. We here report the presence of b1-AR in rat AP as measured by Taqman RealTime RT-PCR and Western blot. b1-AR mRNA levels were comparable in adult, 1- to 2-week-old and newborn male rats. Expression was maintained at low level in aggregate cell culture and was strongly upregulated in a dose- and time-dependent manner by adding dexamethasone (DEX) (1–80 nM) to the medium. Contrary to what was expected, DEX had no or only a small stimulatory effect on b2-AR mRNA level. As studied in vibratome sections with confocal laser-scanning microscopy b1-AR immunoreactivity was detected in a subpopulation of the LH-b cells but not in somatotrophs, lactotrophs, corticotrophs, and thyrotrophs. Remarkably, b1AR-positive cells were often surrounded by cup-shaped lactotrophs. Moreover, in aggregates prepared from cells of the top fractions of a velocity sedimentation gradient (containing small but few gonadotrophs), DEX caused a vast increase in b1-AR mRNA levels compared to the ones in aggregates from the bottom fraction containing large gonadotrophs. Further evidence for the presence of b1-ARs in a subpopulation of gonadotrophs was given by studying cAMP levels in response to isoproterenol (ISO) in pituitary cell lines. ISO strongly stimulated cAMP levels in GHFT cells (somatotroph precursor cells), aT3-1 cells (gonadotroph precursors), and LbT2 cells (LH gonadotrophs) but not in GH3 cells (lactosomatotrophs). The magnitude of response was largest in aT3-1 cells (80· above control at 10 nM). In GHFT cells the cAMP response to ISO was not affected by the highly selective b1-antagonist CGP20712A but was abolished by the selective b2-antagonist ICI 118,551. In aT3-1 cells and LbT2 cells both the b1- and b2-AR antagonist partially blocked the response but completely abolished it when added in combination. RT-PCR and Western blotting showed that b1-ARs are more prominent in aT3-1 and LbT2 cells compared to GHFT and GH3 cells. The present data indicate that b1-ARs are present in a subpopulation of gonadotrophs that have a special relationship to lactotrophs and are functionally coupled to adenylate cyclase in gonadotroph cell lines, particularly of the progenitor type. doi:10.1016/j.yfrne.2006.03.206
ICN 2006 Conference Abstracts
Neuroendocrine gene regulation Distribution and regulation of GPR12 receptor in rat pituitary E. Swinnen, C. Denef Katholieke Universiteit Leuven, Leuven, Belgium GPR12 is a phospholipid G-protein-coupled receptor that exhibits constitutive activity on adenylate cyclase and has been implicated in maintenance of meiotic arrest in rat oocytes and in differentiation of the developing murine brain. GPR12 mRNA was detected by RT-PCR in rat pituitary as early as embryonic day 15 and at different postnatal ages in the anterior and neurointermediate lobe. The pituitary cell lines, GH3, AtT20, aT3-1, and LbT2 (representing different pituitary secretory cell subtypes), also expressed GPR12 mRNA, while the CHO fibroblastic cell line was negative. Confocal laser scanning microscopy revealed strong immunoreactivity (ir) in the melanotrophs in the intermediate lobe, while sparsely distributed, less intensely fluorescent cells were detected throughout the anterior lobe. Positive anterior lobe cells did not appear to uniquely belong to a single secretory cell type. No colocalisation with the folliculo-stellate cell marker S100 or stem cell/progenitor marker nestin was found. GPR12 mRNA and GPR12ir remained expressed in rat pituitary aggregate cell cultures. As measured by means of real-time RT-PCR, thyroid and glucocorticoid hormone significantly enhanced, while estradiol significantly lowered GPR12 mRNA levels. Leukemia inhibiting factor also inhibited GPR12 mRNA level while epidermal growth factor, basic fibroblastic growth factor, and nerve growth factor were without effect. In rat adrenal gland, GPR12-ir was seen in elongated cellular extensions in the cortex and the medulla, in conjunction with S100-positive sustentacular cells that line the endocrine chromaffin lobules. The present findings suggest a characteristic distribution and regulation of GPR12 in endocrine tissues. doi:10.1016/j.yfrne.2006.03.205
b-1-Adrenergic receptor (b-AR) expression in rat anterior pituitary (AP) gonadotrophs and in aT3-1 and LbT2 cells K. Janssens, O. Krylyshkina, N. Hersmus, H. Vankelecom, C. Denef Katholieke Universiteit Leuven, Leuven, Belgium doi:10.1016/j.yfrne.2006.03.204
The AP expresses b2-ARs that mediate stimulation of GH release by epinephrine. We here report the presence of b1-AR in rat AP as measured by Taqman RealTime RT-PCR and Western blot. b1-AR mRNA levels were comparable in adult, 1- to 2-week-old and newborn male rats. Expression was maintained at low level in aggregate cell culture and was strongly upregulated in a dose- and time-dependent manner by adding dexamethasone (DEX) (1–80 nM) to the medium. Contrary to what was expected, DEX had no or only a small stimulatory effect on b2-AR mRNA level. As studied in vibratome sections with confocal laser-scanning microscopy b1-AR immunoreactivity was detected in a subpopulation of the LH-b cells but not in somatotrophs, lactotrophs, corticotrophs, and thyrotrophs. Remarkably, b1AR-positive cells were often surrounded by cup-shaped lactotrophs. Moreover, in aggregates prepared from cells of the top fractions of a velocity sedimentation gradient (containing small but few gonadotrophs), DEX caused a vast increase in b1-AR mRNA levels compared to the ones in aggregates from the bottom fraction containing large gonadotrophs. Further evidence for the presence of b1-ARs in a subpopulation of gonadotrophs was given by studying cAMP levels in response to isoproterenol (ISO) in pituitary cell lines. ISO strongly stimulated cAMP levels in GHFT cells (somatotroph precursor cells), aT3-1 cells (gonadotroph precursors), and LbT2 cells (LH gonadotrophs) but not in GH3 cells (lactosomatotrophs). The magnitude of response was largest in aT3-1 cells (80· above control at 10 nM). In GHFT cells the cAMP response to ISO was not affected by the highly selective b1-antagonist CGP20712A but was abolished by the selective b2-antagonist ICI 118,551. In aT3-1 cells and LbT2 cells both the b1- and b2-AR antagonist partially blocked the response but completely abolished it when added in combination. RT-PCR and Western blotting showed that b1-ARs are more prominent in aT3-1 and LbT2 cells compared to GHFT and GH3 cells. The present data indicate that b1-ARs are present in a subpopulation of gonadotrophs that have a special relationship to lactotrophs and are functionally coupled to adenylate cyclase in gonadotroph cell lines, particularly of the progenitor type. doi:10.1016/j.yfrne.2006.03.206