We continue investigations on receptors and channels expressed in anterior pituitary cells and their roles in pituitary functions. Acetylcholine (ACh) has been established as a paracrine factor in the pituitary gland, but the receptors mediating ACh action and the cell types bearing these receptors have not been identified. Our results showed that the expression of the nicotinic subunits mRNAs followed the order beta2 >beta1 = alpha9 >alpha4 in cultured rat pituitary cells. M4 >M3 muscarinic receptor mRNAs were also identified in pituitary cells. The treatment of cultured pituitary cells with GnRH down-regulated the expression of alpha9 and alpha4 mRNAs, without affecting the expression of M3 and M4 receptor mRNAs, and ACh did not alter the expression of GnRH receptor mRNA. We also performed double immunostaining to show the expression of beta2 subunit and M4 receptor proteins in gonadotrophs. Functional nicotinic channels capable of generating an inward current, facilitation of electrical activity and calcium influx were identified in single cells. The M3 receptor-mediated, phospholipase C-dependent calcium mobilization activated an outward apamin-sensitive potassium current and caused hyperpolarization. The activation of M4 receptors by ACh inhibited cAMP production and GnRH-induced LH release. We concluded that multiple cholinergic receptors are expressed in gonadotrophs and that the main secretory action of ACh is inhibitory through M4-receptor-mediated down-regulation of cAMP production. The expression of nicotinic receptors in vitro compensates for the lack of regular GnRH stimulation of gonadotrophs. cAMP and cGMP are intracellular messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. In pituitary cells, our previous work revealed that the cyclic nucleotides and their kinases play important role in the control of electrical activity of the endocrine pituitary cells and channels involved in this process. More recently, we focused on the role of these messengers in control of hormone secretion downstream on electrical activity in calcium signaling. The hormone release studies show that prolactin (PRL) release from isolated rat lactotrophs stimulated by forskolin, an activator of adenylyl cyclases, and membrane- permeable cAMP analog (dbcAMP), exhibit a biphasic concentration dependency. Although at lower concentrations (2-10 M forskolin and 2.5-5 mM dbcAMP) these agents stimulate PRL release, an inhibition is measured at higher concentrations. By using high-resolution capacitance (Cm) measurements, we recorded discrete increases in Cm, which represent elementary exocytic events. An elevation of cAMP leaves the frequency of full-fusion events unchanged, while increasing the frequency of transient events. These exhibited a wider fusion pore as measured by increased fusion-pore conductance and a prolonged fusion-pore dwell-time. The probability of observing rhythmic reopening of transient fusion pores was elevated by dbcAMP. In conclusion, cAMP-mediated stabilization of wide fusion pores prevents vesicles from proceeding to the full-fusion stage of exocytosis, which hinders vesicle content discharge at high cAMP concentrations. Many endocrine disrupters affect cyclic nucleotide signaling in target cells. Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In a recent study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate PRL release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in these cells together with mRNA for PDE8A. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s.We also progressed in investigation the gating properties of P2X receptor (P2XR) channels cloned from pituitary cells. The P2X7R, natively expressed in lactotrophs, operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we combined electrophysiological, imaging, and mathematical studies using wild type and mutant rat P2X7Rs. Activation of nave receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov state model that included, in addition to nave and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of nave receptors favored a slow transition to desensitized states, whereas the occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions. Activation of P2XR causes an enlargement of the receptor extracellular vestibule, leading to opening of the transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. Our alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. The Y54A and Y54C receptor function was partially rescued by ivermectin, suggesting a decreased potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a beta-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor traffickin, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues cause vestibule widening.

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Zemkova, Hana; Khadra, Anmar; Rokic, Milos B et al. (2015) Allosteric regulation of the P2X4 receptor channel pore dilation. Pflugers Arch 467:713-26
Rokic, Milos B; Stojilkovic, Stanko S; Zemkova, Hana (2014) Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating. Front Cell Neurosci 8:3
Khadra, Anmar; Tomic, Melanija; Yan, Zonghe et al. (2013) Dual gating mechanism and function of P2X7 receptor channels. Biophys J 104:2612-21
Stojilkovic, Stanko S; Zemkova, Hana (2013) P2X receptor channels in endocrine glands. Wiley Interdiscip Rev Membr Transp Signal 2:173-180
Andric, Silvana A; Kojic, Zvezdana; Bjelic, Maja M et al. (2013) The opposite roles of glucocorticoid and ?1-adrenergic receptors in stress triggered apoptosis of rat Leydig cells. Am J Physiol Endocrinol Metab 304:E51-9
Fa, Svetlana; Pogrmic-Majkic, Kristina; Dakic, Vanja et al. (2013) Acute effects of hexabromocyclododecane on Leydig cell cyclic nucleotide signaling and steroidogenesis in vitro. Toxicol Lett 218:81-90
Rokic, Milos B; Stojilkovic, Stanko S; Vavra, Vojtech et al. (2013) Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor. PLoS One 8:e59411
Stojilkovic, Stanko S; Leiva-Salcedo, Elias; Rokic, Milos B et al. (2013) Regulation of ATP-Gated P2X Channels: From Redox Signaling to Interactions with Other Proteins. Antioxid Redox Signal :
Calejo, Ana Isabel; Jorgacevski, Jernej; Kucka, Marek et al. (2013) cAMP-mediated stabilization of fusion pores in cultured rat pituitary lactotrophs. J Neurosci 33:8068-78
Zemkova, Hana; Kucka, Marek; Bjelobaba, Ivana et al. (2013) Multiple cholinergic signaling pathways in pituitary gonadotrophs. Endocrinology 154:421-33

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