Intracellular Signaling In Endocrine Cells

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We continue investigations on receptors and channels expressed in neuroendocrine and endocrine cells and their roles in signaling, gene transcription, and hormone secretion. To gain better understanding of the cell type-specific expression and role of these and other proteins in anterior pituitary cell functions and related disorders, we performed recently single cell RNA sequencing on freshly dispersed cells from adult male and female rats. Our analysis confirmed the expression of six pituitary-specific cell: folliculostellate cells, corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells. We continue to explore the information reached by these studies in order to address specific questions regarding pituitary cell functions. To compare the relative expression levels of the transcripts by qRT-PCR, the levels were normalized against a reference gene, i.e. an internal reaction control that had sequences different than the target. Our single cell RNAseq analysis helped to clarify the validity of genes used in previous studies as reference genes. Out of 19 genes studied, only eight genes were expressed in over 90% of anterior pituitary cells: Rpl19, Rpl32, Rps18, Gapdh, Rpl4, Rplp0, B2m, and Actb. Among them, only five genes exhibited comparable expression levels in all subpopulations of anterior pituitary cells: Rpl19, Rpl32, Rps18, Gapdh, and Rpl4. For further testing, we selected Rpl19, Rps18, and Gapdh genes. Using these reference genes, we studied divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr gene expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb gene expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr gene expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 gene expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb gene expression. These data suggest that blockade of Fshb gene expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment. In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of our ongoing project is to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G protein-coupled receptor 68 gene (Gpr68) and acid-sensing ion channel (ASIC) genes Asic1, Asic2, and Asic4 in anterior pituitary cells, and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable folliculostellate cells express GPR68 gene and protein, whereas excitable secretory cells express ASIC1 genes/proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification was accompanied by generation of two currents in a concentration-dependent manner: a fast-developing, desensitizing current, with an estimated EC50 value of 6.7, and a slow-developing, non-desensitizing current, which required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride-insensitive and was driven by voltage-sensitive cation conducting channels. Activation of both channels was accompanied increase in excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx. Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here examined the sex-specific patterns of changes in hypothalamic-pituitary-gonadal axis during experimental autoimmune encephalomyelitis, focusing on hypothalamic Gnrh and Kiss1 and pituitary Gnrhr, Fshb, and Lhb genes. During the disease course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b genes in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh (in males only) gene expression. In females, the expression of gonadotroph-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone levels and a transient arrest of the estrous cycle. In contrast, Fshb gene expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr gene expression and basal and stimulated serum luteinizing hormone and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh expression in females and males, respectively, leading to sex-specific changes downstream the axis.