Functional characteristics of H+ -dependent nicotinate transport in primary cultures of astrocytes from rat cerebral cortex.
In the present study, we report the characteristics of H(+)-coupled nicotinate transport in primary cultures of astrocytes from rat cerebral cortex. The [(3)H]nicotinate transport in rat astrocytes increased up to a pH 5.5. The nicotinic acid uptake at pH 6.0 was both energy-dependent and saturable with a Michaelis constant (K(t)) of 2.8+/-0.4 mM and the maximal uptake rate (V(max)) of 31+/-3.2 nmol/mg protein/10 min. This process was reduced by a protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, and a typical monocarboxylate transporter (MCT) inhibitor, alpha-cyano-4-hydroxycinnamic acid, suggesting that nicotinate uptake by rat astrocytes is mediated by H(+)-coupled monocarboxylate transport system. [(3)H]Nicotinate transport in rat astrocytes was significantly inhibited by various monocarboxylic acids such as l-lactic acid and pyruvic acid with a relatively low affinity (K(i)>10 mM). On the other hand, the uptake process of l-lactic acid was also saturable with a high-affinity component (K(t)=0.27 mM) and a low-affinity component (K(t)=35.9 mM). Reverse transcription-PCR and Western blot analyses revealed that three MCT subtypes, MCT1/Slc16a1, MCT2/Slc16a7, and MCT4/Slc16a3, were expressed in these cells. Because l-lactate reduced to 67% of the nicotinate uptake even at 10mM, it is unlikely that nicotinate uptake in rat astrocytes is mediated by MCT1 and/or MCT2. These results provide biochemical evidence of a H(+)-coupled and saturable transport system, presumed to be a low-affinity monocarboxylate transporter MCT4 or other unknown H(+)-coupled monocarboxylate transport system, for nicotinate in rat cerebrocortical astrocytes.
Shimada
Ayumi
A
Department of Biochemical Pharmacology, Kyoto Pharmaceutical University, Japan.
Nakagawa
Yoko
Y
Morishige
Hiroyuki
H
Yamamoto
Akira
A
Fujita
Takuya
T
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology*; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000493:pharmacokinetics; D009525:Niacin / Q000378:metabolism* / Q000493:pharmacokinetics; D011522:Protons; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014316:Tritium / Q000493:pharmacokinetics
D004791:Enzyme Inhibitors; D011522:Protons; D012333:RNA, Messenger; D014316:Tritium; D009525:Niacin; D019344:Lactic Acid