|Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
In this research project, I investigated functional characterization of H^+/oligopeptide transport system in primary cultures of neurons and astrocytes from mouse cerebral cortex. RT-PCR revealed that the low affinity-type H^+/oligopeptide transporter PEPT1 mRNA was not found in both neurons and astrocytes, whereas the high affinity-type H^+/oligopeptide transporter PEPT2 mRNA was found in astrocytes but not neurons. In addition, expression of PEPT2 protein was confirmed by Western blotting using anti-PEPT2 antibody. Uptake of [^3H]glycylsarcosine, a model dipeptide, in mouse astrocytes was pH-dependent and saturable, and a Michaelis-Menten constant (K_t) value was 110 μM, which is comparable to those values in PEPT2-stable transfectant cells and our previous report using synaptosomal membrane prepared from rat cerebral cortex. These results, therefore, indicate that PEPT2 works **tionally in mouse astrocytes. Furthermore, I demonstrated that mouse astrocytes express Na^+/H^+ exchanger
s (NHEs), suggesting that NHE(s) was responsible for generating inward H^+ gradient which is a driving force of PEPT1/2, in mouse astrocytes as well as renal and intestinal epithelial cells.
In parallel, I investigated the functional characteristics of gabapentin transport and its interaction with voltage-gated Ca^<2+> channel (VGCC) in normal human astrocytes. The uptake of leucine (Leu) and phenylalanine (Phe) in these cells was predominantly Na^+-independent, was stimulated by lowering the extracellular pH, and was inhibited by hydrophobic neutral amino acids. These transport processes were saturable with Michaelis constants (K_t) of 51 ± 7 μM and 24 ± 9 μM for Leu and Phe uptake, respectively. GBP inhibited the Na^+-independent uptake of Leu and Phe in a concentration-dependent manner, and its K_i values were 80 μM and 93 μM for Leu and Phe uptake, respectively. These K_i values were consisted with a previous report by Su et al. and were comparable to the K_t value for GBP in rat astrocytes. GBP decreased K^+ (50 mM)-stimulated [Ca^<2+>]_i increase and cyclic GMP formation, which reflects the activation of nNOS, in a concentration-dependent manner (IC_<50>=111 ± 19 μM). BCH, a specific inhibitor of system L transporter, and Leu were found to recover the inhibitory action of GBP on K^+-stimulated [Ca^<2+>]_i in human astrocytes. However, both compounds per se were failed to decrease the K^+-stimulated [Ca^<2+>]_i. On the other hand, lysine, cationic amino acid, and MeAIB, which is a selective substrate for system A, had no effect. Less