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

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

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

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