We have cloned five ATP-gated P2X receptor channels (P2XRs) from the pituitary gland: P2X2R, P2X3R, P2X4R, P2X6R, and P2X7R. Our ongoing work is focused on P2X7R, which is expressed in a large variety of cells, including microglia and macrophages. In collaboration with Dr. Heegaard, we analyzed the role of these channels in bone cancer pain. We demonstrated that P2X7R knockout mice were susceptible to bone cancer pain and moreover had an earlier onset of pain-related behaviors compared with cancer-bearing, wild type mice. Furthermore, acute treatment with the selective P2X7 receptor antagonist, A-438079, failed to alleviate pain-related behaviors in models of bone cancer pain with and without astrocyte activation, suggesting that astrocytic P2X7R play a negligible role in bone cancer pain. The results support the hypothesis that bone cancer pain is a separate pain state compared with those of neuropathic and inflammatory pain. The P2X7R is a trimeric channel with three binding sites for ATP, but how the occupancy of these sites affects gating is still not understood. At least two conflicting hypotheses have been postulated to reconcile these findings: 1) The pore-dilation hypothesis suggests that there is a progressive dilation of the cation-conducting pore. 2) The two-pore hypothesis implies the activation of an endogenous P2X7R pore permeable for inorganic cations, accompanied by sustained activation of a distinct channel called pannexin, which is permeable to larger organic cations and fluorescent dyes. Recently, we addressed both hypotheses. Our results indicate that naive receptors activated and deactivated monophasically at low and biphasically at higher agonist concentrations. Both phases of response were abolished by application of Az10606120, a P2X7R-specific antagonist. The slow secondary growth of current in the biphasic response coincided temporally with pore dilation. Repetitive stimulation with the same agonist concentration caused sensitization of receptors, which manifested as a progressive increase in the current amplitude, accompanied by a slower deactivation rate. Once a steady level of the secondary current was reached, responses at high agonist concentrations were no longer biphasic but monophasic. Sensitization of receptors was independent of sodium and calcium influx and about 30 min washout was needed to reestablish the initial gating properties. T15E- and T15K-P2X7 mutants showed increased sensitivity for agonists, responded with monophasic currents at all agonist concentrations, activated immediately with dilated pores, and deactivated slowly. The complex pattern of gating exhibited by wild-type channels can be accounted for by a Markov state model that includes negative cooperativity of agonist binding to unsensitized receptors caused by the occupancy of one or two binding sites, opening of the channel pore to a low conductance state when two sites are bound, and sensitization with pore dilation to a high conductance state when three sites are occupied. Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 did not affected P2X7R gating but enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland. In collaboration with Dr. Startakis group, we also studied dependence of spontaneous and cAMP-facilitated electrical activity of pituitary cells on bath sodium. Our results indicate that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (protein kinase A) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated calcium influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 >>TRPC6 >TRPC4 >TRPC5 >TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium. Finally, in our collaborative work with Dr. Kleins group, we perforated patch clamp recording to study the control of membrane potential (Vm) and spontaneous electrical activity in the rat pinealocyte by norepinephrine. Norepinephrine did not alter spiking frequency. However, it was found to act through alpha1-adrenoreceptors in a concentration-dependent manner to produce a biphasic change in Vm. The initial response was a hyperpolarization due to a transient outward potassium current . This current appears to be triggered by calcium released from intracellular stores, based on the observation that it was also seen in cells bathed in calcium-deficient medium. In addition, pharmacological studies indicate that this current was dependent on phospholipase C (PLC) activation and was in part mediated by bicuculline methiodide and apamin-sensitive calcium-controlled potassium channels. The initial transient hyperpolarization was followed by a sustained depolarization due to an inward current. This response was dependent on PLC-dependent activation of sodium/calcium influx but did not involve nifedipine-sensitive voltage-gated calcium channels. Together, these results indicate for the first time that activation of alpha1 adrenoreceptors initiates a PLC-dependent biphasic change in pinealocyte Vm characterized by an initial transient hyperpolarizzation mediated by a mixture of calcium-activated potassium channels followed by a sustained depolarization.

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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
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
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
Rahamim-Ben Navi, Liat; Tsukerman, Anna; Feldman, Alona et al. (2017) GnRH Induces ERK-Dependent Bleb Formation in Gonadotrope Cells, Involving Recruitment of Members of a GnRH Receptor-Associated Signalosome to the Blebs. Front Endocrinol (Lausanne) 8:113
Coddou, Claudio; Sandoval, Rodrigo; Castro, Patricio et al. (2017) Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372. Pain 158:2155-2168

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