We continue investigations on receptors and channels expressed in the anterior pituitary gland and their roles in signaling, gene We continue investigations on receptors and channels expressed in the anterior pituitary gland and their roles in signaling, gene transcription, and hormone secretion. Our recent work with corticotrophs was focused on three subjects: developmental pattern of pro-opiomelanocortin gene (Pomc) expression, rapid glucocorticoid effects on corticotrophs function, and electrophysiological characterization of spontaneous and corticotropin-releasing hormone (CRH)-stimulated cells. The qRT-PCR analysis using pre-designed TaqMan Gene Expression Assays, which is a sensitive and reliable method to study gene expression in both male and female pituitaries in vivo and in vitro, revealed that corticotrophs show comparable expression profiles of Pomc in both sexes, with the highest expression occurring during the infantile period. Collaborative experiments with Dr. Greti Aguilera, using cultured pituitary cells, hypothalamic-derived 4B cells, and AtT-20 cells, revealed that the nongenomic/membrane effects of classical glucocorticoid receptor mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation. In contrast to gonadotrophs, lactotrophs, and somatotrophs, the ion channels and mechanisms controlling corticotroph and thyrotrophs excitability are still not well understood. Development of transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the POMC genes regulatory elements are a useful model for studying excitability of corticotrophs and melanotrophs. In the initial stage of investigation done in collaboration with Dr. Greti Aguilera, we established a protocol to distinguish between these two cell types. Next, we characterized the resting membrane potential (RMP), spontaneous electrical activity, and calcium signaling in corticotrophs. These cells were either quiescent or electrically active, with a 22-mV difference. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in spontaneously firing cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory action of CRH was mimicked by forskolin and cell permeable cAMP analog. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling. Mellon and co-workers developed TlphaT1 cells, which represent differentiated thyrotrophs and provide a useful model for studies on signaling and gene expression. Recently, we have characterized for the first time calcium-signaling pathways in these cells. They are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH). Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium concentration, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. Finally, we found that the thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TlphaT1 cells are capable of responding to natively feed-forward TRH signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Our work with pituitary gonadotrophs was focused on signaling pathways accounting for basal and regulated expression of gonadotropin-releasing hormone receptor (GnRHR) mRNA. Basal Gnrhr transcription, the signaling pathways accounting for it have not been systematically investigated, whereas it is well established that hypothalamic GnRH together with gonadal steroids and activins/inhibin regulate its receptor gene (Gnrhr) expression in vivo, which leads to crucial changes in GnRHR numbers on the plasma membrane. This is accompanied by alterations in the gonadotroph sensitivity and responsiveness during physiologically relevant situations. Our in vitro investigations revealed that the Gnrhr expression was progressively reduced but not completely abolished in pituitary cells from adult animals cultured in the absence of GnRH and steroid hormones. The basal Gnrhr expression was also operative in LetaT2 immortalized gonadotrophs never exposed to GnRH. In both cell types, basal transcription was sufficient for the expression of functional GnRHRs. Continuous application of GnRH transiently elevated the Gnrhr expression in cultured pituitary cells followed by a sustained fall without affecting basal transcription. Both basal and regulated Gnrhr transcriptions were dependent on the protein kinase C signaling pathway. The GnRH-regulated Gnrhr expression was not operative in embryonal pituitary and LetaT2 cells and was established neonatally, the sex-specific response patterns were formed at the juvenile-peripubertal stage and there was a strong correlation between basal and regulated gene expression during development. Thus, the age-dependent basal and regulated Gnrhr transcription could account for the initial blockade and subsequent activation of the reproductive system during development.

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U.S. National Inst/Child Hlth/Human Dev
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Alvarez-Prats, Alejandro; Bjelobaba, Ivana; Aldworth, Zane et al. (2018) Schwann-Cell-Specific Deletion of Phosphatidylinositol 4-Kinase Alpha Causes Aberrant Myelination. Cell Rep 23:2881-2890
Zemkov√°, Hana; Stojilkovic, Stanko S (2018) Neurotransmitter receptors as signaling platforms in anterior pituitary cells. Mol Cell Endocrinol 463:49-64
Rokic, Milos B; Castro, Patricio; Leiva-Salcedo, Elias et al. (2018) Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel. Int J Mol Sci 19:
Bjelobaba, Ivana; Stojilkovic, Stanko S; Naor, Zvi (2018) Editorial: Gonadotropin-Releasing Hormone Receptor Signaling and Functions. Front Endocrinol (Lausanne) 9:143
Stojilkovic, Stanko S (2018) Signaling pathways regulating pituitary functions. Mol Cell Endocrinol 463:1-3
Fletcher, Patrick A; Sherman, Arthur; Stojilkovic, Stanko S (2018) Common and diverse elements of ion channels and receptors underlying electrical activity in endocrine pituitary cells. Mol Cell Endocrinol 463:23-36
Li, Shuo; Bjelobaba, Ivana; Stojilkovic, Stanko S (2018) Interactions of Pannexin1 channels with purinergic and NMDA receptor channels. Biochim Biophys Acta Biomembr 1860:166-173
Janjic, Marija M; Stojilkovic, Stanko S; Bjelobaba, Ivana (2017) Intrinsic and Regulated Gonadotropin-Releasing Hormone Receptor Gene Transcription in Mammalian Pituitary Gonadotrophs. Front Endocrinol (Lausanne) 8:221
Mackay, Laurent; Zemkova, Hana; Stojilkovic, Stanko S et al. (2017) Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models. PLoS Comput Biol 13:e1005643
Fletcher, Patrick A; Zemkova, Hana; Stojilkovic, Stanko S et al. (2017) Modeling the diversity of spontaneous and agonist-induced electrical activity in anterior pituitary corticotrophs. J Neurophysiol 117:2298-2311

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