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

Abstract

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 1Y3 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.Abbreviations: [Ca 2+ ] i -intracellular calcium concentration; eNTPDase -ecto-nucleoside triphosphate diphosphohydrolase;

Expression of P2Y receptors in the rat anterior pituitary

“…They were found in clusters formed by a few positive cells. These P2Y 13 receptor-ir cells were not labeled by ACTH, FSH, PRL, TSH, ED1 or S100 antibodies or GH oligonucleotide probes. P2Y 13 receptor-ir cells were also found in the intermediate lobe of the rat pituitary and these P2Y 13 receptor-ir cells were also labeled by the ACTH antibody (Fig.…”

“…The section slides were washed 3×5 min in PBS, and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton-X-100, 0.4% sodium azide in 0.01 mol/l PBS pH7.2) for 30 min, followed by incubation with two primary antibody dilutions, one a P2Y antibody (P2Y 6 , P2Y 12 , and P2Y 13 ) and the second either a hormonal antibody adrenocorticotropin (ACTH), follicle-stimulating hormone (FSH), prolactin (PRL), and thyroid-stimulating hormone (TSH) or a cell type marker (ED1 [macrophage marker] and S-100 [folliculo-stellate cell marker]) at room temperature, overnight. Subsequently, the sections were incubated at room temperature for 2 h with a mixed secondary antibody solution of Cy3-conjugated donkey anti-rabbit IgG diluted 1:400 for P2Y antibodies and either FITC-conjugated donkey anti-goat IgG diluted 1:200 for hormonal antibodies or FITC-conjugated donkey anti-mouse IgG diluted 1:200 for cell type marker antibodies.…”

“…Using calcium image analysis, sheep and rat anterior pituitary cells were found to express P2Y receptors [10][11][12]. Following RT-PCR analysis, P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 12 receptor transcripts were detected in mixed anterior pituitary cells [13,14]. The identified cells in the anterior pituitary with P2Y receptors were also found in rat pituitary folliculo-stellate cells in primary culture [15], gonadotrophs [16] and lactotrophs [17].…”

“…There are two groups of P2 receptors: P2X and P2Y receptors, which are ligand-gated ion channels and G protein-coupled receptors, respectively. So far, seven P2Y receptors and seven P2X receptors have been cloned and are denoted P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , P2Y 14 , and P2X 1 to P2X 7 receptors [1].…”

“…In this study, we used single-labeling and doublelabeling immunofluorescence, in situ hybridization and Western blot analyses to study the distribution patterns of P2Y 1 , P2Y 2 P2Y 4 , P2Y 6 , P2Y 12 , and P2Y 13 receptors in anterior pituitary cells of rat and found that gonadotrophs and thyrotrophs express P2Y 1 and P2Y 4 receptors, lactotrophs and folliculo-stellate cells express P2Y 2 , and macrophages express P2Y 6 .…”