title: Functional expression of P2 purinoceptors in a primary neuroglial cell culture of the rat arcuate nucleus.
abstract: The arcuate nucleus (ARC) plays an important role in the hypothalamic control of energy homeostasis. Expression of various purinoceptor subtypes in the rat ARC and physiological studies suggest a modulatory function of P2 receptors within the neuroglial ARC circuitry. A differentiated mixed neuronal and glial microculture was therefore established from postnatal rat ARC, revealing neuronal expression of ARC-specific transmitters involved in food intake regulation (neuropeptide Y (NPY), proopiomelanocortin (POMC), tyrosine hydroxylase (TH)). Some NPYergic neurons cosynthesized TH, while POMC and TH expression proved to be mutually exclusive. Stimulation with the general purinoceptor agonists 2-methylthioadenosine-5'triphosphate (2-MeSATP) and ATP but not the P2X1/P2X3 receptor subtype agonist α,β-methyleneadenosine-5'triphosphate (α,β-meATP) induced intracellular calcium signals in ARC neurons and astrocytes. Some 5-10% each of 2-MeSATP responsive neurons expressed POMC, NYP or TH. Supporting the calcium imaging data, radioligand binding studies to hypothalamic membranes showed high affinity for 2-MeSATP, ATP but not α,β-meATP to displace [α-(35)S]deoxyadenosine-5'thiotriphosphate ([(35)S]dATPαS) from P2 receptors. Repetitive superfusion with equimolar 2-MeSATP allowed categorization of ARC cells into groups with a high or low (LDD) degree of purinoceptor desensitization, the latter allowing further receptor characterization. Calcium imaging experiments performed at 37°C vs. room temperature showed further reduction of desensitization. Agonist-mediated intracellular calcium signals were suppressed in all LDD neurons but only 25% of astrocytes in the absence of extracellular calcium, suggestive of metabotropic P2Y receptor expression in the majority of ARC astrocytes. The highly P2Y1-selective receptor agonists MRS2365 and 2-methylthioadenosine-5'diphosphate (2-MeSADP) activated 75-85% of all 2-MeSATP-responsive ARC astrocytes. Taking into consideration the high potency to dose-dependently stimulate ARC cells of the LDD group, the high affinity for rat P2X(1-3) and low affinity for rat P2X4, P2X7 and P2Y receptor subtypes except P2Y1 and P2Y13, the agonist 2-MeSATP primarily acted upon P2X2 and P2Y1 purinoceptors to trigger intracellular calcium signaling in ARC neurons and astrocytes.
authors.affiliation: Institut für Veterinär-Physiologie und -Biochemie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Deutschland. Electronic address: eric-pollatzek@gmx.de.
authors.forename: Eric
authors.initials: E
authors.lastname: Pollatzek
authors.affiliation: Institut für Veterinär-Physiologie und -Biochemie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Deutschland. Electronic address: norma.hitzel@idt-biologika.de.
authors.forename: Norma
authors.initials: N
authors.lastname: Hitzel
authors.affiliation: Institut für Veterinär-Physiologie und -Biochemie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Deutschland. Electronic address: daniela.ott@vetmed.uni-giessen.de.
authors.forename: Daniela
authors.initials: D
authors.lastname: Ott
authors.affiliation: Institut für Veterinär-Physiologie und -Biochemie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Deutschland. Electronic address: katrin.raisl@vetmed.uni-giessen.de.
authors.forename: Katrin
authors.initials: K
authors.lastname: Raisl
authors.affiliation: Institut für Veterinär-Physiologie und -Biochemie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Deutschland. Electronic address: baerbel.reuter@physiologie.med.uni-giessen.de.
authors.forename: Bärbel
authors.initials: B
authors.lastname: Reuter
authors.affiliation: Institut für Veterinär-Physiologie und -Biochemie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Deutschland. Electronic address: ruediger.gerstberger@vetmed.uni-giessen.de.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Gerstberger
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D001111:Arcuate Nucleus of Hypothalamus / Q000378:metabolism*; D001253:Astrocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D008297:Male; D009130:Muscle, Smooth / Q000378:metabolism*; D061251:Primary Cell Culture; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism*
chemical_list: D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium
keywords: calcium imaging; hypothalamic arcuate nucleus; primary mixed neuronal and glial microculture; purinoceptor agonist; radioligand binding; receptor desensitization