This project addresses the cellular signaling cascades in endocrine and neuroendocrine cells and the interactions between plasma membrane electrical events and receptor-controlled pathways. Current emphasis is on release and degradation of ATP in hypothalamic and pituitary cells and on the dependence of ATP-gated purinergic receptor-channel (P2XR) activity on their ectodomain structures containing the ATP binding domain. The focus in other line of investigations is on G protein-coupled receptor-controlled calcium signaling, hormone secretion, and gene expression. ATP must be released by cells to act as a ligand for P2XRs and is then hydrolyzed by ecto-nucleotidases, resulting in the formation of the respective nucleoside and a free phosphate. Our experiments in progress indicate that cultured normal and immortalized pituitary and hypothalamic cells release ATP under resting conditions. These cells also express three subtypes of ecto-nucleotidases, which degrade endogenously released and exogenously added ATP. Blocking the activity of these enzymes led to an increase in ATP concentrations in pituitary perfusates and inhibition of degradation of the extracellularly added ATP. On the other hand, the addition of apyrase, a soluble ecto-nucleotidase, and the expression of recombinant mouse ecto-nucleotidase eNTPDase-2 subtype, enhanced degradation of both endogenously released and exogenously added ATP. The ATP released by resting hypothalamic cells was sufficient to activate and desensitize high-affinity P2XRs, whereas facilitation of ATP metabolism by the addition of apyrase protected their desensitization. These results indicate that colocalization of ATP release sites and ecto-nucelotidase activity in hypothalamic and pituitary cells provides an effective mechanism for the operation of ATP as a ligand for P2XRs. Our work with recombinant P2XRs is performed in cells expressing rat P2X2aR, P2X2bR, and P2X3R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X3R instead of the I66-H192 or I66-Y310 sequences of P2X2aR and P2X2bR. Chimeric receptors inherited the P2X3R ligand-selective profile. Furthermore, P2X2a/V60-F301X3R and P2X2a/V60-R180X3R desensitized in a P2X2aR-specific manner and P2X2b/V60-F301X3R desensitized with rates comparable with those of P2X2bR. In striking contrast to the parental receptors, the rates of decay in P2X2a/V60-F301X3R and P2X2b/V60-F301X3R currents after agonist withdrawal were several-fold slower. For these chimeras, the decays in currents where not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels was inversely correlated with the potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X2a/V60-R180X3R-expressing cells. However, the recovery from desensitization of P2X2a/V60-F301X3R and P2X2a/V60-R180X3R was similar and was substantially delayed compared with that of parental receptors. These results indicate that both P2XR ectodomain halves participate in gating, and that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization. Our ongoing experiments with Gs protein-coupled receptors are directed toward investigation of the mechanism by which these receptors activate soluble guanlylyl cyclase. In general, the stimulatory effects of adenylyl cyclase activators on calcium signaling are in accord with observations that intracellular calcium stimulates nitric oxide (NO) synthase activity, leading to increase in cyclic GMP (cGMP) production. Consistent with this, we recently found that activation of adenylyl cyclase by growth hormone-releasing hormone, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and forskolin increased NO and cGMP levels, and that basal and stimulated cGMP production were abolished by inhibition of NO synthase activity. However, activators of adenylyl cyclase were found to enhance this NO-dependent cGMP production even when NO was held constant at basal levels, as well as in cells bathed in calcium-deficient medium. Receptor-activated cGMP production was mimicked by expression of a constitutive active protein kinase A and was accompanied by phosphorylation of native and recombinant alpha-1 soluble guanylyl cyclase subunits. Addition of a protein kinase A inhibitor, over-expression of a dominant negative mutant of regulatory protein kinase A subunit, and substitution of S107-S108 N-terminal residues of alpha-1 soluble guanylyl cyclase subunit with alanine, abolished adenylyl cyclase-dependent cGMP production without affecting basal and NO-donor stimulated cGMP production. These results indicate that phosphorylation of alpha-1 soluble guanylyl cyclase subunit by protein kinase A enhances the NO-dependent sGC activity, and that this is probably the major pathway by which adenylyl cyclase-coupled receptors stimulate cGMP production. In another line of these investigations, we addressed the role of calcium influx in sustained and repetitive calcium signaling induced by Gq-coupled GnRH receptors. In cells bathed in calcium/sodium-containing medium, GnRH-induced baseline calcium oscillations recovered without refractory period and with a time constant of about 20 s, whereas the recovery of spike responses occurred after 25-35 s refractory period and with a time constant of about 30 s. During repetitive GnRH stimulation, removal of calcium had only a minor effect on baseline oscillations but abolished the spike response, whereas removal of sodium slightly extended the duration of baseline oscillations and considerably prolonged the spike response. These results indicate that two calcium handling mechanisms are operative in gonadotrophs: redistribution of calcium within Ins(1,4,5)-trisphosphate-sensitive and -insensitive pools, and sodium-dependent calcium efflux followed by calcium influx. Redistribution of calcium within the cell leads to rapid recovery of the Ins(1,4,5)-trisphosphate-dependent pool, whereas the sodium-dependent calcium efflux pathway is activated by the spike response and limits the time of exposure to elevated cytosolic calcium concentrations. Finally, we investigated the effects of growth hormone on its secretion at the pituitary level. We identified GH receptors in somatotroph fraction of cells and found that their activation by exogenous GH increased steady-state GH-mRNA levels and GH production. Removal of endogenous GH by immunoneutralization with GH antiserum inhibited basal as well as stimulated GH mRNA expression. Cytosolic mature GH mRNA levels were elevated by GH treatment and reduced by GH antiserum, whereas nuclear GH primary transcripts were almost undetectable after GH immunoneutralization. Inhibition of Janus kinase-2, phosphoinositide 3-kinase, and MAPK also abolished GH-induced steady-state GH-mRNA levels. GH immunoneutralization in pituitary cells pretreated with actinomycin D caused a marked decrease in the half-life of GH mRNA, indicating that the clearance of GH transcripts could be enhanced after removing endogenous GH. These results provide evidence that GH can serve as an intrapituitary autocrine/paracrine factor maintaining GH gene expression in somatotrophs, and that this action is mediated by Janus kinase-2/MAPK and Janus kinase-2/phosphoinositide 3-kinase cascades coupled to GH receptors.

Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2004
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
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Country
United States
Zip Code
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
Constantin, Stephanie; Caligioni, Claudia Simone; Stojilkovic, Stanko et al. (2009) Kisspeptin-10 facilitates a plasma membrane-driven calcium oscillator in gonadotropin-releasing hormone-1 neurons. Endocrinology 150:1400-12
Korzeniowski, Marek K; Popovic, Marko A; Szentpetery, Zsofia et al. (2009) Dependence of STIM1/Orai1-mediated calcium entry on plasma membrane phosphoinositides. J Biol Chem 284:21027-35
Stojilkovic, Stanko S (2009) Purinergic regulation of hypothalamopituitary functions. Trends Endocrinol Metab 20:460-8
Jorgacevski, Jernej; Stenovec, Matjaz; Kreft, Marko et al. (2008) Hypotonicity and peptide discharge from a single vesicle. Am J Physiol Cell Physiol 295:C624-31
Cokic, Vladan P; Andric, Silvana A; Stojilkovic, Stanko S et al. (2008) Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells. Blood 111:1117-23
Zemkova, Hana W; Bjelobaba, Ivana; Tomic, Melanija et al. (2008) Molecular, pharmacological and functional properties of GABA(A) receptors in anterior pituitary cells. J Physiol 586:3097-111
Gonzalez-Iglesias, Arturo E; Murano, Takayo; Li, Shuo et al. (2008) Dopamine inhibits basal prolactin release in pituitary lactotrophs through pertussis toxin-sensitive and -insensitive signaling pathways. Endocrinology 149:1470-9
Jelinkova, Irena; Vavra, Vojtech; Jindrichova, Marie et al. (2008) Identification of P2X(4) receptor transmembrane residues contributing to channel gating and interaction with ivermectin. Pflugers Arch 456:939-50
Hatae, Noriyuki; Aksentijevich, Nadia; Zemkova, Hana W et al. (2007) Cloning and functional identification of novel endothelin receptor type A isoforms in pituitary. Mol Endocrinol 21:1192-204

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