Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1.
Neurotransmitter transporters are regulated by phosphorylation but little is known about endogenous substances and receptors that regulate this process. Adenosine is an ubiquitous neuromodulator operating G-protein coupled receptors, which affect the activity of several kinases. We therefore evaluated the influence of adenosine upon the GABA transporter 1 (GAT-1) mediated GABA uptake into hippocampal synaptosomes. Removal of endogenous adenosine (adenosine deaminase, 1 U/mL) decreased GABA uptake, an effect mimicked by blockade of A2A receptors (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine, 50 nM) but not A1 or A2B receptors. A2A receptor activation (4-[2-[[6-amino-9-(N-ethyl-beta-d-ribofuranuronamidosyl)-9H-purin-yl]amino]ethyl]benzenepropanoic acid hydrochloride, 3-100 nM) enhanced GABA uptake by increasing the transporter Vmax without change of K(M). This was mimicked by adenylate cyclase activation (forskolin, 10 microM) and prevented by protein kinase A (PKA) inhibition (N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, 1 microM), which per se did not influence GABA transport. Blockade of protein kinase C (PKC) (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide, 1 microM) facilitated GABA transport whereas PKC activation (4-beta-phorbol-didecanoate, 250 nM) inhibited it. PKA blockade did not affect the facilitatory action of the PKC inhibitor or the inhibitory action of the PKC activator. However, when adenylate cyclase was activated neither activation nor inhibition of PKC affected GABA uptake. It is concluded that A2A receptors, through activation of the adenylate cyclase/cAMP/PKA transducing pathway facilitate GAT-1 mediated GABA transport into nerve endings by restraining tonic PKC-mediated inhibition.
Cristóvão-Ferreira
Sofia
S
Institute of Pharmacology and Neurosciences, Institute of Molecular Medicine, University of Lisbon, Portugal.
Vaz
Sandra H
SH
Ribeiro
Joaquim A
JA
Sebastião
Ana M
AM
D000818:Animals; D050485:GABA Plasma Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D058805:GABA Uptake Inhibitors; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D050485:GABA Plasma Membrane Transport Proteins; D058805:GABA Uptake Inhibitors; D007211:Indoles; D008301:Maleimides; D043705:Receptor, Adenosine A2A; D005680:gamma-Aminobutyric Acid; D011493:Protein Kinase C; C070515:bisindolylmaleimide I