We continue investigations on receptors and channels expressed in the anterior pituitary gland and their roles in signaling, gene expression and hormone secretion. In a recent study, we examined the expression of major anterior pituitary genes in five secretory cell types of developing males and females. Corticotrophs show comparable proopiomelanocortin (Pomc) profiles in both sexes, with the highest expression occurring during the infantile period. Somatotrophs and lactotrophs also exhibit no difference in growth hormone (Gh) and prolactin (Prl) profiles during embryonic-to-juvenile age but show the amplification of Prl expression in females and Gh expression in males during peripubertal and postpubertal ages. Gonadotrophs exhibit highly synchronized luteinizing hormone beta (Lhb), follicle-stimulating hormone beta (Fshb) gonadotroph/lactotroph-specific alpha (Cga), and gonadotropin-releasing hormone receptor (Gnrhr) gene expression in both sexes, but the peak of expression occurs during the infantile period in females and at the end of the juvenile period in males. Thyrotrophs also show different developmental thyroid-stimulating hormone beta (Tshb) profiles, which are synchronized with the expression of gonadotroph genes in males but not in females. These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes. In further study on this topic, we examined the in vivo and in vitro expression pattern of three genes that are operative in the thyrotroph subpopulation from postpubertal animals: Cga, Tshb, and thyrotropin-releasing hormone receptor (Trhr). In vivo, the expression of Cga and Tshb was robust, whereas the expression of Trhr was low. In cultured pituitary cells, there was a progressive decline in the expression of Cga, Tshb and Trhr. The expression of Tshb could not be reversed via pulsatile or continuous TRH application in variable concentrations and treatment duration, or by the removal of thyroid and steroid hormones from the sera. In parallel, the expression of CGA and TSHB proteins declined progressively in pituitary cells from both sexes. The lack of the effect of TRH on Tshb expression was not related to the age of pituitary cultures and the presence of functional TRHR. In pituitary fragments, there was also a rapid decline in expression of these genes but TRH was able to induce transient Tshb expression. These observations suggest that the lack of influence of anterior pituitary architecture and/or intrapituitary factors probably accounts for the loss of basal and TRH-stimulated Tshb expression in dispersed pituitary cells. The other focus in this topic was on pituitary lactotrophs, which secrete high levels of PRL in the absence of any hormone action in vitro. We have previously shown that such hyperprolactinemia (HPRL) is driven by spontaneous electrical activity and the accompanying calcium influx. HPRL is a common adverse in vivo effect of antipsychotic medications that are used in the treatment of patients with schizophrenia. We compared the effects of two atypical antipsychotics, paliperidone and aripiprazole, on cAMP/calcium signaling and PRL release in female rat lactotrophs in vitro. Dopamine inhibits spontaneous cAMP/calcium signaling and PRL release. In the presence of dopamine, paliperidone rescues cAMP/calcium signaling and PRL release in a concentration-dependent manner, whereas aripiprazole is only partially effective. In the absence of dopamine, paliperidone stimulates cAMP/calcium signaling and PRL release, whereas aripiprazole inhibits signaling and secretion more potently but less effectively than dopamine. Forskolin-stimulated cAMP production is facilitated by paliperidone and inhibited by aripiprazole, although the latter is not as effective as dopamine. None of the compounds affects Prl transcript activity, intracellular PRL accumulation, or GH secretion. These data indicate that paliperidone has dual HPRL actions in lactotrophs i) by preserving the coupling of spontaneous electrical activity and PRL secretion in the presence of dopamine and ii) by inhibiting intrinsic dopamine receptor activity in the absence of dopamine, leading to enhanced calcium signaling and secretion. In contrast, aripiprazole acts on PRL secretion by attenuating, but not abolishing, calcium-secretion coupling. The other main focus in our investigations is on structural and functional characterization of two ATP-gated P2X receptors, P2X2 and P2X4 expressed in gonadotrophs and lactotrophs, respectively. Activation of P2X2 is characterized by a rapid current growth accompanied with a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using, rat, mouse and human receptors, we show recently that two processes contribute to receptor desensitization: bath calcium-independent and -dependent. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in ATP- and bath calcium-concentration dependent manner. Experiments with NMDG, a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential further indicates that calcium influx through the channels pore at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording show development of calcium-dependent desensitization without increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings. We also studied the influence of allostery on P2X4 pore dilation, using ivermectin (IVM), an established positive allosteric regulator of this channel. In the absence of IVM, this channel activates and deactivates rapidly, does not show transition from open to dilated states, desensitizes completely with a moderate rate, and recovers only fractionally during washout. IVM treatment increases the efficacy of ATP to activate the channel and slows receptor desensitization during sustained ATP application and receptor deactivation after ATP washout. The rescue of the receptor from desensitization temporally coincides with pore dilation, and the dilated channel can be reactivated after washout of ATP. Experiments with vestibular and transmembrane domain receptor mutants further established that IVM has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude and the latter correlating with changes in the EC50 and kinetics of receptor deactivation. The corresponding kinetic (Markov state) model indicates that the IVM-dependent transition from open to dilated state is coupled to receptor sensitization, which rescues the receptor from desensitization and subsequent internalization. Allosterically-induced sensitization of P2X4R thus provides sustained signaling during prolonged and repetitive ATP stimulation.
| 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 |
| Zemková, 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 |
| 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 |
| Jovanovic, Saša; Radulovic, Tamara; Coddou, Claudio et al. (2017) Tonotopic action potential tuning of maturing auditory neurons through endogenous ATP. J Physiol 595:1315-1337 |
Showing the most recent 10 out of 74 publications
