Functional expression of the organic cation/carnitine transporter 2 in rat astrocytes.
In this study, we sought to identify the transporters that mediate the uptake of L-carnitine and acetyl-L-carnitine in cultured rat cortical astrocytes. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake were both saturable, and mediated by a single Na(+)-dependent transport system. Uptake of both was inhibited by L-carnitine, D-carnitine, acetyl-L-carnitine and various organic cations. Acylcarnitines (acetyl-, butyryl-, hexanoyl-, octanoyl- and palmitoyl-L-carnitine) also interacted with L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine transport. 2-Amino-2-norbornane carboxylic acid, a known inhibitor of amino acid transporter B(0,+) (ATB(0,+)), did not cause any significant inhibition. A highly significant correlation was found between the potencies of acylcarnitines in the inhibition of L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake and the acyl chain length of acylcarnitines. The expression of mRNA for organic cation/carnitine transporters (OCTNs), carnitine transporter 2 (CT2) and ATB(0,+) in astrocytes was investigated by reverse transcription (RT)-PCR. OCTN2 mRNA was expressed in astrocytes, whereas the expression of OCTN1, OCTN3 and CT2 mRNA could not be detected. ATB(0,+) mRNA was expressed at very low levels in astrocytes. Western blotting analysis indicated that anti-OCTN2 polyclonal antibody recognized a band of 70 kDa in both kidney and astrocyte preparations. OCTN2 immunoreactivity was detected in rat astrocytes by immunocytochemical staining. Inhibition of OCTN2 expression by RNA interference significantly inhibited L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake into astrocytes. These results suggest that OCTN2 is functionally expressed in rat astrocytes, and is responsible for L-carnitine and acetyl-L-carnitine uptake in these cells.
Department of Pharmacology, Tokyo Medical University, Japan.
Masato
M
Inazu
Hiroshi
H
Takeda
Katsuyuki
K
Maehara
Kyoji
K
Miyashita
Akio
A
Tomoda
Teruhiko
T
Matsumiya
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D002331:Carnitine / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D027701:Organic Cation Transport Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000074058:Solute Carrier Family 22 Member 5; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics
D027682:Cation Transport Proteins; D005904:Glial Fibrillary Acidic Protein; D027701:Organic Cation Transport Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C115441:Slc22a5 protein, rat; D000074058:Solute Carrier Family 22 Member 5; C116917:acylcarnitine; D014316:Tritium; D002331:Carnitine