P2Y purinergic regulation of the glycine neurotransmitter transporters.
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.
Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Esperanza
E
Jiménez
Francisco
F
Zafra
Raquel
R
Pérez-Sen
Esmerilda G
EG
Delicado
Maria Teresa
MT
Miras-Portugal
Carmen
C
Aragón
Beatriz
B
López-Corcuera
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001933:Brain Stem / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D009474:Neurons; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010146:Pain / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D050487:Glycine Plasma Membrane Transport Proteins; D018377:Neurotransmitter Agents; C486690:P2ry13 protein, rat; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; C493269:Slc6a5 protein, rat; C493266:Slc6a9 protein, rat; D000244:Adenosine Diphosphate; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D005998:Glycine
Nat Protoc. 2006;1(5):2406-15
17406484
Neuron. 2003 Nov 13;40(4):785-96
14622582
Pflugers Arch. 2006 Aug;452(5):538-51
16691392
J Biol Chem. 2007 Jul 13;282(28):20351-64
17519230
J Neurosci. 1997 Jul 15;17(14):5297-304
9204914
J Med Chem. 2001 Aug 16;44(17):2679-82
11495577
Trends Pharmacol Sci. 2005 Jun;26(6):283-6
15925702
Histochem Cell Biol. 2003 Nov;120(5):415-26
14564529
J Physiol. 2010 Mar 15;588(Pt 6):995-1009
20123783
J Neurochem. 2008 Jul;106(1):347-60
18384646
J Pharmacol Sci. 2010;112(3):352-60
20173309
Int Immunopharmacol. 2008 Feb;8(2):182-7
18182224
Neuropharmacology. 2005 Nov;49(6):935-44
16181645
FEBS Lett. 2000 Mar 17;470(1):51-4
10722844
Prog Neurobiol. 2008 Jan;84(1):40-56
18036717
Front Neurosci. 2009 Sep 15;3(2):175-81
20011139
J Neurochem. 1990 Jun;54(6):1941-6
2338550
Neurochem Res. 2003 Oct;28(10):1597-605
14570406
Nat Protoc. 2006;1(3):1287-96
17406413
Neuropharmacology. 2005 Nov;49(6):922-34
16143353
Cell Mol Life Sci. 2005 Nov;62(21):2508-15
16231090
Trends Pharmacol Sci. 2003 Feb;24(2):52-5
12559763
Purinergic Signal. 2007 Mar;3(1-2):135-44
18404427
Mol Pain. 2009 Jul 20;5:40
19619286
Neuron. 2003 Nov 13;40(4):797-806
14622583
J Biol Chem. 2007 Apr 20;282(16):11639-47
17310063
Pharmacol Rev. 2006 Sep;58(3):281-341
16968944
J Biol Chem. 1993 Oct 25;268(30):22802-8
8226790
Neuropsychopharmacology. 2003 Mar;28(3):435-44
12629523
Physiol Rev. 2007 Apr;87(2):659-797
17429044
J Physiol. 2004 Aug 15;559(Pt 1):169-86
15235081
Eur J Pharmacol. 2003 Oct 31;479(1-3):249-62
14612155
J Biol Chem. 2008 Apr 18;283(16):11050-63
18270199
Mol Cell Biol. 2010 Aug;30(16):4035-44
20547753
Anesth Analg. 2010 Feb 1;110(2):615-21
20081141
Cell. 2009 Oct 16;139(2):267-84
19837031
Br J Pharmacol. 1999 Jun;127(3):779-87
10401570
Neurosci Lett. 2008 Nov 21;445(3):214-9
18793697
Trends Genet. 2008 Sep;24(9):439-47
18707791
Purinergic Signal. 2004 Dec;1(1):3-10
18404394
Anesthesiology. 2008 May;108(5):929-37
18431130
Cereb Cortex. 2005 Apr;15(4):448-59
15749988
Mol Pharmacol. 1995 Aug;48(2):184-8
7544433
Nat Rev Drug Discov. 2008 Jul;7(7):575-90
18591979
Trends Neurosci. 2009 Jun;32(6):339-46
19414201
J Biol Chem. 2004 May 28;279(22):22934-43
15010455
Acta Anaesthesiol Scand. 2009 Oct;53(9):1107-20
19702699
Neuron. 1992 May;8(5):927-35
1534013
Trends Pharmacol Sci. 2006 Mar;27(3):166-76
16487603
J Comp Neurol. 2000 Jun 5;421(3):374-84
10813793
Mol Pain. 2010 Apr 15;6:21
20398327
Biochem Soc Trans. 2001 Nov;29(Pt 6):742-5
11709067
J Auton Nerv Syst. 2000 Jul 3;81(1-3):289-94
10869734
J Neurosci. 1995 May;15(5 Pt 2):3952-69
7751957
Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8485-90
15159536
Neurosci Lett. 2008 Apr 25;435(3):190-3
18343032
J Neurochem. 1993 Feb;60(2):613-9
8419539
Mol Brain. 2009 Aug 05;2:26
19656393
J Pharmacol Exp Ther. 2008 Aug;326(2):633-45
18448867
Biochem J. 2008 Jun 15;412(3):495-506
18341477
J Pharmacol Exp Ther. 2004 Dec;311(3):1038-43
15345752
Mol Psychiatry. 2004 Nov;9(11):984-97, 979
15278097
Science. 2003 Jun 27;300(5628):2094-7
12829784
Purinergic Signal. 2004 Dec;1(1):31-41
18404398
Neuroscience. 2007 Jun 8;146(4):1808-16
17481825
Traffic. 2009 Jul;10(7):829-43
19374720
Chem Rev. 2008 May;108(5):1654-68
18393466
Proc Natl Acad Sci U S A. 2007 May 8;104(19):8119-24
17452640
Pharmacol Rev. 2001 Dec;53(4):553-68
11734618
Mol Pharmacol. 2003 Jul;64(1):104-12
12815166
Purinergic Signal. 2006 Nov;2(4):595-604
18404462
J Biol Chem. 2001 May 18;276(20):17584-90
11278707
Br J Pharmacol. 2010 Mar;159(5):1106-17
20136836
Nitric Oxide. 2009 Jun;20(4):223-30
19298861
Science. 1965 Nov 19;150(3699):971-9
5320816
Eur J Neurosci. 1995 Jun 1;7(6):1342-52
7582108
Pharmacol Ther. 2009 Jul;123(1):54-79
19393690
J Neurochem. 1995 Jan;64(1):61-8
7798951