| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
|
|---|
| Research Abstract |
In the present research, I have investigated the involvement of Na^+-independent neutral amino acid transport system L in voltage-gated Ca^<2+> channels blocking action by gabapentin in normal human astrocytes and primary cultured mouse cerebrocortical neurons. In particular, there is no information available for amino acid transport system L in neurons that are thought to be a major target site for gabapentin. Therefore, we primarily investigated the functional characteristics of gabapentin transport into the neurons using primary cultures of mouse cerebrocortical neurons. Cerebrocortical neurons were isolated from 15-day-old-fetal ddY mice. The purity of neurons and contamination of glial cells were confirmed by immunocytochemistry using anti-MAP2 (neuronal marker) and anti-GFAP (glial marker) antibodies, respectively.
The uptake of [^3H]gabapentin in neurons was predominantly Na^+-independent, was stimulated by lowering the extracellular pH, and was inhibited by hydrophobic neutral amino acids. The transport process was saturable with a K_t of 66±0.7 μM for gabapentin uptake. RT PCR revealed that mouse cerebrocortical neurons express the system L amino acid transporters, LAT1 and LAT2, and the heavy chain of the cell surface antigen 4F2 (4F2hc/CD98), which is required for the functional expression of LAT1 and LAT2. Structurally-related compounds of gabapentin, such as 1-amino-1-cyclohexanecarboxylic acid (ACHC), 1-amino-1-cyclopentane- carboxylic acid (cycloleucine), and BCH inhibited the Na^+-independent gabapentin transport in a concentration-dependent manner, and their IC_<50> values were±13, 185±24, and 56±21 μM, respectively. In addition, preloading of various neutral amino acids into neurons markedly enhanced Na^+-independent [^3H]gabapentin uptake (trans-stimulation effect).
These results suggest that functional amino acid transport system L is expressed in mouse cerebrocortical neurons.
|
