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Article L-Type Amino Acid Transporter 1 (LAT1/Lat1)-Utilizing Prodru...

Primary astrocytes from cortex and hippocampi were isolated from 2-days-old wild type and APP/PS1 transgenic mice as previously described58,

Lat1 functionality and ability of compounds to bind to Lat1

All the uptake studies were done by the same way with each cell type. The cells were carefully washed with pre-warmed HBSS (Hank’s balance salt solution) containing 125 mM choline chloride, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.3 mM CaCl2, 5.6 mM glucose, and 25 mM HEPES (pH 7.4 adjusted with 1 M NaOH) after removal of the culture medium. Pre-incubation was done with 500 μL of pre-warmed HBSS at 37 °C for 10 min before adding substrates (250 μL in HBSS) for the uptake experiments. The ability of brain cell to carry a known LAT1/Lat1 substrate, the cells were incubated with [14C]-l-leucine at 37 °C for 2.5 min in uptake buffer (HBSS, 250 μL) containing 0.76–75 μM (0.25 mCi/ml) of [14C]-l-leucine (PerkinElmer, Waltham, MA, USA). After incubation the reaction was stopped by adding 500 µL of ice-cold HBSS and the cells were washed two times with ice-cold HBSS. The cells were then lysed with 500 μL of 0.1 M NaOH (60 min) and the lysate was mixed with 3.5 mL of Emulsifier safe cocktail (Ultima Gold, PerkinElmer, Waltham, MA, USA). The radioactivity in the cells was measured by liquid scintillation counting (MicroBeta2 counter, PerkinElmer Waltham, MA, USA).

The uptake of [14C]-l-leucine (0.76 μM) was also studied in the presence of 100 µM LAT1/Lat1-inhibitor (KMH-233) to confirm the Lat1-mediated uptake. The cells were pre-incubated with LAT1/Lat1 inhibitor for 10 min and the incubation mixture was removed before adding [14C]-l-leucine and LAT1/Lat1-inhibitor on the cells. The competitive uptake (5 min) and analysis was carried out in a manner similar as described. The inhibition was calculated by comparing the counts in the absence and presence of LAT1/Lat1-inhibitor.

Article Targeting of Perforin Inhibitor into the Brain Parenchyma Vi...

Fig. 8

Inhibitory effects of 1 or 10 μM perforin inhibitor (PFI) and its LAT1-utilizing prodrug (PFI-PD) as well as 0.25 μM BACE1 inhibitor (KTEEISEVN-Sta-VAEF-NH2) [38]

According to the results emerging from this study, it is apparent that the conversion of a perforin inhibitor (PFI) into a LAT1-utilizing prodrug (PFI-PD) can increase the cellular uptake and bioavailability in mouse primary astrocytes. Moreover, we have previously shown that the cellular uptake into the primary astrocytes is LAT1-mediated since the uptake was inhibited by over 70% in the presence of a specific LAT1 inhibitor [26]. The ability of PFI-PD to bind LAT1, measured as the inhibition of the [14C]-L-leucine uptake, was relatively high, i.e., the IC50 value was 4 μM (Table 1)