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P2Y purinergic regulation of the glycine neurotransmitter transporters.

ABSTRACT: The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5'-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y(1) and P2Y(13) because the effects are partially reversed by the specific antagonists N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate and pyridoxal-5'-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y(12) receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca(2+) mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y(1) receptor. Sensitivity to 2-methylthioadenosine 5'-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.

SUBMITTER: Jimenez E

PROVIDER: S-EPMC3060522 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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P2Y purinergic regulation of the glycine neurotransmitter transporters.

Jiménez Esperanza E Zafra Francisco F Pérez-Sen Raquel R Delicado Esmerilda G EG Miras-Portugal Maria Teresa MT Aragón Carmen C López-Corcuera Beatriz B

The Journal of biological chemistry 20110118 12

The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5'-diphosphate in rat brainstem/spina ...[more]

PMID: 21245148

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Project description:Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y2 receptor (P2Y2R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y2Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y2R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y2R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10-100 μM, but inhibiting at 300 μM ATP. On the other hand, 1-300 μM UTP, a P2Y2R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y2R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer.

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P2Y purinergic regulation of the glycine neurotransmitter transporters.

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P2Y purinergic regulation of the glycine neurotransmitter transporters.

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