L-Carnitine (LC) is essential for transporting fatty acids to the mitochondria for β-oxidation. This study was performed to examine the alteration of the LC transport system in wild type (WT, NSC-34/hSOD1WT) and mutant type (MT, NSC-34/hSOD1G93A)] amyotrophic lateral sclerosis (ALS) models. The uptake of [3H]L-carnitine was dependent on time, temperature, concentration, sodium, pH, and energy in both cell lines. The Michaelis–Menten constant (Km) value as well as maximum transport velocity (Vmax) indicated that the MT cell lines showed the higher affinity and lower capacity transport system, compared to that of the WT cell lines. Additionally, LC uptake was inhibited by organic cationic compounds but unaffected by organic anions. OCTN1/slc22a4 and OCTN2/ slc22a5 siRNA transfection study revealed both transporters are involved in LC transport in NSC-34 cell lines. Additionally, slc22a4 and slc22a5 was significantly decreased in mouse MT models compared with that in ALS WT littermate models in the immune-reactivity study. [3H]L-Carnitine uptake and mRNA expression pattern showed the pretreatment of LC and acetyl L-carnitine (ALC) attenuated glutamate induced neurotoxicity in NSC-34 cell lines. These findings indicate that LC and ALC supplementation can prevent the neurotoxicity and neuro-inflammation induced by glutamate in motor neurons.
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease caused by the death of the neurons regulating the voluntary muscles which leads to the progressive paralysis. We investigated the difference of transport function of L-citrulline in ALS disease model (NSC-34/hSOD1 G93A , MT) and a control model (NSC-34/hSOD1 wt , WT). The [ 14 C]L-citrulline uptake was significantly reduced in MT cells as compared with that of control. The Michaelis-Menten constant (K m ) for MT cells was 0.67 ± 0.05 mM, whereas it was 1.48 ± 0.21 mM for control. On the other hand, the V max values for MT and control were 10.9 ± 0.8 nmol/mg protein/ min and 18.3 ± 2.9 nmol/mg protein/min, respectively. The K m and V max values showed the high affinity and low capacity for MT as compared with control. Moreover, the uptake of [ 14 C]L-citrulline was significantly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2carboxylic acid (BCH) and harmaline which is the inhibitor of the large neutral amino acid transporter1 (LAT1) in NSC-34 cell lines. Furthermore, [ 14 C]L-citrulline uptakes took place in Na + -independent manner. It was also inhibited by the neutral amino acids such as citrulline and phenylalanine. Likewise, L-dopa, gabapentin, and riluzole significantly inhibited the [ 14 C]L-citrulline uptake. It shows the competitive inhibition for L-dopa in ALS cell lines. On the other hand, [ 14 C]L-citrulline uptake in the presence of riluzole showed competitive inhibition in WT cells, whereas it was uncompetitive for MT cells. The small interfering RNA experiments showed that LAT1 is involved in the [ 14 C]L-citrulline uptake in NSC-34 cell lines. On the other hand, in the examination of the alteration in the expression level of LAT1, it was significantly lower in MT cells as compared with that of control. Similarly, in the spinal cord of ALS, transgenic mice revealed a slight but significant decrease in LAT1 immunoreactivity in motor neurons of ALS mice compared with control. However, the LAT1 immunoreactivity in non-motor neurons and in astrocytes was relatively increased in the spinal cord gray matter of ALS mice. The experimental evidences of our results suggest that the change of transport activity of [ 14 C]L-citrulline may be partially responsible for the pathological alteration in ALS. Keywords Amyotrophic lateral sclerosis (ALS) . L-citrulline . Carrier mediated transporter . Motor neuron disease . NSC-34 cells . LAT1 (large neutral amino acid transporter 1) Abbreviations ALS Amyotrophic lateral sclerosis NSC-34 cells Motor neuron-like cells BCH 2-aminobicyclo-(2,2,1)-heptane-2carboxylic acid ECF Extracellular fluid MeAIB α-(methylamino) isobutyric acid siRNA Small interfering RNA mRNA Messenger RNA MT Mutant type WT Wild type ECF Extracellular fluid * Young-Sook Kang
Paeonol has neuroprotective function, which could be useful for improving central nervous system disorder. The purpose of this study was to characterize the functional mechanism involved in brain transport of paeonol through blood-brain barrier (BBB). Brain transport of paeonol was characterized by internal carotid artery perfusion (ICAP), carotid artery single injection technique (brain uptake index, BUI) and intravenous (IV) injection technique in vivo . The transport mechanism of paeonol was examined using conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) as an in vitro model of BBB. Brain volume of distribution (V D ) of [ 3 H]paeonol in rat brain was about 6-fold higher than that of [ 14 C]sucrose, the vascular space marker of BBB. The uptake of [ 3 H]paeonol was concentration-dependent. Brain volume of distribution of paeonol and BUI as in vivo and inhibition of analog as in vitro studies presented significant reduction effect in the presence of unlabeled lipophilic compounds such as paeonol, imperatorin, diphenhydramine, pyrilamine, tramadol and ALC during the uptake of [ 3 H]paeonol. In addition, the uptake significantly decreased and increased at the acidic and alkaline pH in both extracellular and intracellular study, respectively. In the presence of metabolic inhibitor, the uptake reduced significantly but not affected by sodium free or membrane potential disruption. Similarly, paeonol uptake was not affected on OCTN2 or rPMAT siRNA transfection BBB cells. Interestingly. Paeonol is actively transported from the blood to brain across the BBB by a carrier mediated transporter system.
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