We continue investigations on receptors and channels expressed in neuronal 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 single cell RNA sequencing on freshly dispersed cells from adult male and female rats. Our analysis based on over 7000 cells confirmed the expression of six pituitary-specific cell: folliculostellate cells and hormone producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. 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 include the expression of genes encoding numerous voltage-gated, ligand-gated and other channels in hormone-producing but not follicolostellate cells. 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. Single cell RNA sequencing also confirmed that all hormone producing cell types express ionotropic receptors activated by extracellular ATP, termed P2X4 receptors, whereas folliculostellate cells express P2X2 receptor gene. Our ongoing work on P2X4 receptor channel was focused on gating properties. We utilized electrophysiology and recombinant channels to study in details the antagonistic effect of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro3,2-e-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4 receptor and compared its specificity among another rat P2X receptors. We found that 5-BDBD is a potent P2X4 receptor antagonist, with an IC50 in submicromolar concentration range. In contrast, 5-BDBD did not affect the ATP-induced P2X2a, P2X2bR, and P2X7 receptor current amplitude or the pattern of receptor desensitization when applied in high micromolar concentration range. However, it partially inhibited the P2X1 and P2X3 receptor-gated currents. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease long-term potentiation, a response that is believed to be mediated in part by endogenous P2X4 receptors. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4 receptor in the central nervous system and this antagonist can discriminate between this and other P2X receptors, when they are co-expressed in the same tissue. The other focus in our investigations was on investigations of extracellular matrix proteins known as SIBLINGs (Small Integrin-Binding Ligand, N-linked Glycophosphoproteins). Anterior pituitary gland expresses two SIBLING genes, Dmp1 (dentin matrix protein-1) and Spp1 (secreted phosphoprotein-1) encoding DMP1 and osteopontin proteins, respectively, but their expression pattern and roles in pituitary functions have not been clarified. Here we provide novel evidence supporting the conclusion that Spp1/osteopontin, like Dmp1/DMP1, are expressed in gonadotrophs in a sex- and age-specific manner. Other anterior pituitary cell types do not express these genes. In contrast to Dmp1, Spp1 expression is higher in males; in females, the expression reaches the peak during the diestrus phase of estrous cycle. In further contrast to Dmp1 and marker genes for gonadotrophs, the expression of Spp1 is not regulated by gonadotropin-releasing hormone in vivo and in vitro. However, Spp1 expression increases progressively after pituitary cell dispersion in both female and male cultures. We may speculate that gonadotrophs signal to other pituitary cell types about changes in the structure of pituitary cell-matrix network by osteopontin, a function consistent with the role of this secretory protein in postnatal tissue remodeling, extracellular matrix reorganization after injury, and tumorigenesis. We also studied the in vivo and in vitro expression pattern of gonadotroph marker genes during continuous gonadotropin-releasing hormone (GnRH) receptor (GnRHR) agonist application. Our in vitro experiments with continuous agonist treatments revealed the bidirectional effects on folliculo-stimulating hormone (FSH) beta gene (Fshb) expression, a transient stimulatory followed by prolonged blockade of expression. Such a profile in expression is in full agreement with a hypothesis that regulation of Fshb expression by GnRH is a critical point that determines FSH levels in circulation. In the same experimental conditions, the profile of Gnrhr expression was monophasic, the peak in response was delayed for 3 h when compared to Fshb expression, and there was no blockade of basal expression. Difference in the peak responses for Fshb and Gnrhr/Dmp1 could provide a rationale for the observed differences in the GnRH pulse frequency in regulation of expression of these Fshb (low) and Gnrhr (high) genes and motivate further experiments to clarify whether there is difference in intracellular messengers and/or transcriptional factors controlling their expression. The lack of blockade of Gnrhr expression further points to the physiological importance of basal gene expression for gonadotroph function. Finally, we did not observe a consistent stimulatory effect of GnRHR agonist during continuous application of luteinizing hormone beta gene (Lhb) expression and we detected only a minor and transient inhibition in expression of this gene. These findings argue against the hypothesis that blockade of Lhb expression accounts for desensitization of LH secretion and suggest that pulsatile GnRH provides a mechanism for sustained LH release by allowing sufficient time for de novo LH synthesis to replenish the LH secretory pool. Our results also indicate a need for further experiments on the status of Erg1, Nr5a1, and Pitx1 expression, as well as the expression of other regulatory genes, during the pulsatile GnRH application and how the frequency of pulses affects their patterns of expression.
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