title: The phosphatidylinositol 3-kinase inhibitor LY 294002 inhibits GlyT1-mediated glycine uptake.
abstract: The actions of neurotransmitter glycine are regulated by the Na+/Cl(-) dependent high-affinity glycine transporters, GlyT1 and GlyT2. These two members of the SLC6 transport family have been cloned and extensively characterized, however relatively little is known regarding their modulation. In the present study, glycine uptake in primary cultures of rat embryonic cortex has been characterized and the effects of the phosphatidylinositol 3 (PI3) kinase inhibitors LY 294002 and wortmannin on GlyT1- and GlyT2-mediated glycine uptake were investigated. GlyT1 inhibitors ALX 5407 and sarcosine reduced total glycine uptake to 80% whereas the specific GlyT2 inhibitor Org 25543 had no effect. In the presence of alanine, glycine uptake was completely blocked by the GlyT1 inhibitors ALX 5407 and sarcosine, suggesting that the high-affinity glycine uptake occurs predominantly via GlyT1. Kinetic analysis of GlyT1 revealed the Km value of 27+/-1.5 microM and Vmax value of 157+/-14 pmol/mg/min. LY 294002, a PI3 kinase inhibitor, blocked the GlyT1-mediated glycine uptake with an IC50 value of 81+/-2 microM, whereas another inhibitor wortmannin did not show any effect. In human placental choriocarcinoma (JAR) cells, which have been previously shown to predominantly express GlyT1a, LY 294002 showed a similar potency with an IC50 value of 86+/-3 microM. Immunoblots demonstrated that LY 294002 and wortmannin inhibited PI3 kinase-dependent Akt phosphorylation in the primary cultures with IC50 values of 10+/-4 microM and 7+/-1 nM, respectively. These results suggest that the commonly used PI3 kinase blocker LY 294002 may modulate GlyT1 function independent of PI3 kinase inhibition. Kinetic analysis in the presence of LY 294002 demonstrated significant decreases of both Km and Vmax values, suggesting a mechanism of uncompetitive inhibition on GlyT1-mediated glycine uptake. In addition, glycine release was blocked by LY 294002. These results raised a possibility that LY 294002 might interact with GlyT1.
authors.affiliation: CNS Biology, Cephalon, Inc. 145 Brandywine Parkway, West Chester, PA 19380, USA. zwu@cephalon.com
authors.forename: Zhi-Liang
authors.initials: ZL
authors.lastname: Wu
authors.forename: Teresa M
authors.initials: TM
authors.lastname: O'Kane
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Connors
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Marino
authors.forename: Hervé
authors.initials: H
authors.lastname: Schaffhauser
mesh_terms: D000409:Alanine / Q000494:pharmacology; D026921:Amino Acid Transport Systems, Neutral / Q000502:physiology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002478:Cells, Cultured; D002540:Cerebral Cortex; D002867:Chromones / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005998:Glycine / Q000493:pharmacokinetics*; D050487:Glycine Plasma Membrane Transport Proteins / Q000502:physiology*; D007150:Immunohistochemistry; D009025:Morpholines / Q000494:pharmacology*; D009474:Neurons; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D011247:Pregnancy; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000409:Alanine; D026921:Amino Acid Transport Systems, Neutral; D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D005998:Glycine; D050487:Glycine Plasma Membrane Transport Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D012521:Sarcosine; D000077191:Wortmannin; C510369:(R)-(N-(3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl))sarcosine; C493269:Slc6a5 protein, rat; C493266:Slc6a9 protein, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one