Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1–2 in hypothalamic and pituitary cells

Hypothalamic and pituitary cells express G protein-coupled adenosine and P2Y receptors and cation-conducting P2X receptor-channels, suggesting that extracellular ATP and other nucleotides may function as autocrine and/or paracrine signaling factors in these cells. Consistent with this hypothesis, we show that cultured normal and immortalized pituitary and hypothalamic cells release ATP under resting conditions. RT-PCR analysis also revealed the presence of transcripts for ecto-nucleotidase eNTPDase 1–2 in these cells. These enzymes were functional as documented by degradation of endogenously released and exogenously added ATP. Blocking the activity of eNTPDases by ARL67156 led to an increase in ATP release in perifused pituitary cells and inhibition of degradation of extracellularly added ATP. Furthermore, the addition of apyrase, a soluble ecto-nucleotidase, and the expression of recombinant mouse eNTPDase-2, enhanced degradation of both endogenously released and exogenously added ATP. The released ATP by resting hypothalamic cells was sufficient to activate and desensitize high-affinity recombinant P2X receptors, whereas facilitation of ATP metabolism by the addition of apyrase protected their desensitization. These results indicate that colocalization of ATP release sites and ecto-nucleotidase activity at the plasma membrane of hypothalamic and pituitary cells provides an effective mechanism for the operation of nucleotides as extracellular signaling molecules

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating

Constitutive PRL (a) and ATP (b) release by perifused hypothalamic and pituitary cell lines

<p><b>Copyright information:</b></p><p>Taken from "Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1–2 in hypothalamic and pituitary cells"</p><p></p><p>Purinergic Signalling 2005;1(2):135-144.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096527.</p><p></p> Prior to experiments, cells (5 × 10) attached on beads were transferred into 0.5-ml chamber and perifused with ATP-free KrebsYRinger medium for 2.5 h to establish a stable baseline. During experiments, samples were collected every minute and immediately tested for ATP and PRL concentrations. Chambers loaded with beads without cells were used as controls. In this and following figures with perifused pituitary cells, data points are means ± SEM from three independent experiments

Desensitization of recombinant rat P2XRs expressed in GT1-7 cells by endogenous ATP

<p><b>Copyright information:</b></p><p>Taken from "Release and extracellular metabolism of ATP by ecto-nucleotidase eNTPDase 1–2 in hypothalamic and pituitary cells"</p><p></p><p>Purinergic Signalling 2005;1(2):135-144.</p><p>Published online Jan 2005</p><p>PMCID:PMC2096527.</p><p></p> : The lack of effects of exogenously added ATP on activation of P2XR (a) and chimeric receptors (b and c), but not P2XRs (d and e), in cells cultured in medium without apyrase, a soluble ecto-ATPase, for 60 min. : Patterns of ATP-induced [Ca] signaling by parental receptors and their chimeras in cells incubated in medium without apyrase for 15 min. : Patterns of ATP-induced [Ca] signaling by parental and chimeric receptors in cells incubated in medium containing apyrase. In dishes with parental receptors the enzyme was present for 1 h, whereas in those with cells expressing chimeras apyrase was present at least 4 h. Traces shown are mean values from at least 10 EGFP-positive cells in one from three to five dishes. For details on expression of P2XRs, see Materials and methods

Effects of Insulin-Like Growth Factors I and II and Insulin on the Immortalized Hypothalamic GTI-7 Cell Line

Insulin and insulin-like growth factor I (IGF-I) participate in energy metabolism, regulate cellular growth and differentiation, and are thought to act locally in a paracrine manner through specific receptors. Systemic levels of these peptides in humans and primates are directly associated with levels of activity of the reproductive axis. To date, it is unclear whether these peptides participate in reproductive function by acting at the level of the GnRH neuron. In this study we examined the effects of IGF-I, IGF-II and insulin on immortalized GnRH-secreting neurons, the GTI-7 cell line. The GTI-7 cells expressed all three members of the insulin receptor family as determined by analysis of 125I-IGF-1,125I-IGF-II and 125I-insulin binding sites. Insulin receptors bound insulin, IGF-II and IGF-I with a ratio of potency of 1:5:20. IGF-I and IGF-II receptors bound both IGF-I and IGF-II. The ratio of potency of IGF-I/IGF-II was 1:5 for the IGF-I receptor and 100:1 for the IGF-II receptor. The binding characteristics of the growth factors at 22 °C suggested the possibility that these cells may secrete IGF binding proteins. To ensure that changes in GnRH levels in the media were due to secretion and not to changes in cell number, the mitogenic effect of these peptides on GTI cells was evaluated. Both insulin and IGF-I were strong mitogens (48-hour incubation), restoring cell number to that of serum-replete cultures at a dose of 0.1 ng/ml. A 100-fold higher dose of IGF-II was required to produce a similar level of mitogenicity, implicating an action through the IGF-I and/or insulin receptor. Due to these mitogenic effects, the effect of insulin, IGF-I and IGF-II on GnRH secretion was studied after short-term exposure. Insulin and IGF-I did not affect GnRH secretion, but IGF-II had a biphasic effect on GnRH release after 2 h of incubation (a maximum stimulatory effect occurred with a 0.1 ng/ml dose). In order to examine the signal transduction mechanism, the role of cytoplasmic calcium mobilization in IGF-II-induced GnRH secretion was examined in single cells using calcium imaging. The effect of IGF-II on GnRH secretion appeared to operate via a calcium-independent mechanism. The studies document an insulin/IGF system in the GTI-7 neuronal cell line and show that insulin and IGFs can exert direct effects on the immortalized GnRH neurons. The ability of these agents to stimulate mitogenesis and/or secretion suggests that these peptides may play some role in the early development or maturation of GnRH neurons in vivo

Characterization of purinergic P2X4 receptor channels expressed in anterior pituitary cells

Anterior pituitary cells express cation-conducting P2X receptor channels (P2XRs), but their molecular identity, electrophysiological properties, cell-specific expression pattern, and physiological roles have been only partially characterized. In this study, we show by quantitative RT-PCR that mRNA transcripts for the P2X4 subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X4R protein expression by Western blot analysis. Single-cell patch-clamp recordings show that extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X4R. The channels were activated and desensitized in a dose-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltage-insensitive Ca2+ influx. In the presence of ivermectin, a specific allosteric modulator of P2X4Rs, there was an approximately fourfold increase in the maximum amplitude of the ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. These results indicate that thyrotropin-releasing hormone-responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X4Rs, which facilitate Ca2+ influx and hormone secretion

Connexin 26 regulates epidermal barrier and wound remodeling and promotes psoriasiform response

Inflammatory skin disorders result in significant epidermal changes, including keratinocyte hyperproliferation, incomplete differentiation, and impaired barrier. Here we test whether, conversely, an impaired epidermal barrier can promote an inflammatory response. Mice lacking the transcription factor Kruppel-like factor 4 (Klf4) have a severe defect in epidermal barrier acquisition. Transcription profiling of Klf4(–/–) newborn skin revealed similar changes in gene expression to involved psoriatic plaques, including a significant upregulation of the gap junction protein connexin 26 (Cx26). Ectopic expression of Cx26 from the epidermis-specific involucrin (INV) promoter (INV-Cx26) demonstrated that downregulation of Cx26 is required for barrier acquisition during development. In juvenile and adult mice, persistent Cx26 expression kept wounded epidermis in a hyperproliferative state, blocked the transition to remodeling, and led to an infiltration of immune cells. Mechanistically, ectopic expression of Cx26 in keratinocytes resulted in increased ATP release, which delayed epidermal barrier recovery and promoted an inflammatory response in resident immune cells. These results provide a molecular link between barrier acquisition in utero and epidermal remodeling after wounding. More generally, these studies suggest that the most effective treatments for inflammatory skin disorders might concomitantly suppress the immune response and enhance epidermal differentiation to restore the barrier

Stable GPR101 over-Expressing Cell Lines As an Invaluable Tool for Functional Studies, Ligand Screening, and the Identification of Deregulated Genes/Pathways in Patients with X-Linked Acrogigantism

Background: GPR101 is an orphan G protein-coupled receptor (GPCR) that is duplicated in patients with X-linked acrogigantism (X-LAG) and over-expressed in their GH- and PRL-secreting tumors. GPR101 is a constitutively active GPCR that strongly activates the cAMP pathway. To elucidate the mechanisms through which GPR101 causes GH over-secretion we generated HEK293 and GH/PRL-secreting (GH3) cells with stable GPR101 expression. Methods: Both cell lines were created via direct integration of a human GPR101-coding sequence into their genome. In HEK293 cells this was achieved by transient transfection of a GPR101-expressing plasmid, while GH3 were transduced with GPR101 lentiviral particles. Cells were selected with appropriate antibiotics and the surviving clones expanded. GPR101 expression was quantified by RT-qPCR and immunofluorescence/western blotting. Cell proliferation (MTT assay), cAMP levels (125I-labeled cAMP tracer), and calcium signaling (FURA 2 AM) were determined. RNA was extracted from both cell lines and subjected to RNA-seq. Differential gene expression between control and GPR101-expressing cells and pathway analysis was carried out with the Stirplate and MetaCore softwares, respectively. De-regulated genes were validated by RT-qPCR. Results: High GPR101 expression was achieved in both cell lines and confirmed at the mRNA and protein level. GPR101-expressing cells proliferated at different rates from the respective controls: GPR101-HEK293 cells were slow-dividing, while GPR101-GH3 divided faster. cAMP production was enhanced in GPR101-GH3 and accompained by increased excitability of cells. Differential expression analysis in HEK293 cells revealed several up-regulated and few down-regulated genes. Among the genes with high expression, several were linked to the cAMP pathway: CGA, PCK1, LINC00473 and PDE3A. Enrichment analysis ranked cytoskeleton remodeling and cell cycle regulation (inhibition of G1/S transition) as the most relevant pathways. In GH3 cells most of the genes with a significantly different expression encoded for membrane-localized proteins, among which were ion channels (Trpm8, Kcnj1), GPCRs (Trhr), and calcium sensors (Syt4, Anxa1). Biological processes associated with these genes are: vesicle transport and fusion, cytoskeleton organization, and energy homeostasis. Conclusions: These results show that the intrinsic activity of GPR101 strongly stimulates cAMP production and this in turn facilitates voltage-gated calcium influx. Changes in cAMP/calcium signaling are accompanied with faster/slower cell division depending on the cell type. Accordingly, several genes associated with these and related pathways are differentially expressed. The establishment of these cell lines will be of paramount importance to validate putative GPR101 ligands and to conduct functional studies