Monocarboxylate transporter functions and neuroprotective effects of valproic acid in experimental models of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devasting neurodegenerative disorder for which no successful therapeutics are available. Valproic acid (VPA), a monocarboxylate derivative, is a known antiepileptic drug and a histone deacetylase inhibitor. METHODS: To investigate whether monocarboxylate transporter 1 (MCT1) and sodium-coupled MCT1 (SMCT1) are altered in ALS cell and mouse models, a cellular uptake study, quantitative real time polymerase chain reaction and western blot parameters were used. Similarly, whether VPA provides a neuroprotective effect in the wild-type (WT; hSOD1WT) and ALS mutant-type (MT; hSOD1G93A) NSC-34 motor neuron-like cell lines was determined through the cell viability assay. RESULTS: [3H]VPA uptake was dependent on time, pH, sodium and concentration, and the uptake rate was significantly lower in the MT cell line than the WT cell line. Interestingly, two VPA transport systems were expressed, and the VPA uptake was modulated by SMCT substrates/inhibitors in both cell lines. Furthermore, MCT1 and SMCT1 expression was significantly lower in motor neurons of ALS (G93A) model mice than in those of WT mice. Notably, VPA ameliorated glutamate- and hydrogen peroxide-induced neurotoxicity in both the WT and MT ALS cell lines. CONCLUSIONS: Together, the current findings demonstrate that VPA exhibits a neuroprotective effect regardless of the dysfunction of an MCT in ALS, which could help develop useful therapeutic strategies for ALS.

A novel organic cation transporter involved in paeonol transport across the inner blood-retinal barrier and changes in uptake in high glucose conditions.

Paeonol exerts various pharmacological effects owing to its antiangiogenic, antioxidant, and antidiabetic activities. We aimed to investigate the transport mechanism of paeonol across the inner blood-retinal barrier both in vitro and in vivo. The carotid artery single injection method was used to investigate the retina uptake index of paeonol. The retina uptake index (RUI) value of [³H]paeonol was dependent on both concentration and pH. This value decreased significantly in the presence of imperatorin, tramadol, and pyrilamine when compared to the control. However, para-aminohippuric acid, choline, and taurine had no effect on the RUI value. Conditionally immortalized rat retina capillary endothelial cells (TR-iBRB cell lines) were used as an in vitro model of the inner blood-retinal barrier (iBRB). The uptake of [³H]paeonol by the TR-iBRB cell lines was found to be time-, concentration-, and pH-dependent. However, the uptake was unaffected by the absence of sodium or by membrane potential disruption. Moreover, in vitro structural analog studies revealed that [³H]paeonol uptake was inhibited in the presence of organic cationic compounds including imperatorin, clonidine and tramadol. This is consistent with the results obtained in vivo. In addition, transfections with OCTN1, 2 or plasma membrane monoamine transporter (PMAT) small interfering RNA did not affect paeonol uptake in TR-iBRB cell lines. Upon pre-incubation of these cell lines with high glucose (HG) media, [3H]paeonol uptake decreased and mRNA expression levels of angiogenetic factors, such as hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) increased. However, after the pretreatment of unlabeled paeonol in HG conditions, the mRNA levels of VEGF and HIF-1 were comparatively reduced, and the [3H]paeonol uptake rate was restored. After being exposed to inflammatory conditions induced by glutamate, TNF-α, and LPS, paeonol and propranolol pretreatment significantly increased the uptake of both [3H]paeonol and [3H]propranolol in TR-iBRB cell lines compared to their respective controls. Our results demonstrate that the transport of paeonol to the retina across the iBRB may involve the proton-coupled organic cation antiporter system, and the uptake of paeonol is changed by HG conditions.

The Effect of Drug Pre-treatment on Taurine Transport at the Inner Blood-Retinal Barrier Under Variable Conditions.

Taurine is essential for the development and function of the central nervous system, retina, and cardiovascular system. It is a naturally occurring amino acid, abundantly found in the retina. It has been shown to exhibit antioxidant, neuroprotective, and osmoregulatory functions in the retina. We used conditionally immortalized rat retinal capillary endothelial cells (TR-iBRB), in vitro, to investigate the effects of oxidative stress, high glucose (HG) and hypertonic conditions on taurine transport. TR-iBRB cells pre-treated with tumor necrosis factor alpha (TNF-α) showed a significant increase in [3H]taurine uptake rate, which, however, decreased when treated with taurine (50 mM). Addition of paeonol and propranolol to TNF-α pre-treated cells had no significant effect on [3H]taurine uptake, but the addition of 10 mM taurine caused a reduction. The uptake rate decreased under HG conditions, in contrast to that under hypertonic conditions. [3H]Taurine uptake increased with pre-incubation time. Additionally, uptake of [3H]taurine and mRNA expression of taurine transporter (TauT) decreased significantly under hypertonic and HG conditions, following pre-incubation with 10 mM taurine, 1 mM paeonol, and 0.1 mM propranolol. [3H]Taurine uptake was significantly inhibited in the presence of taurine transporters such as taurine and ß-alanine. Results indicate that oxidative stress and hypertonic conditions increased taurine uptake in iBRB cell lines, whereas HG conditions reduced the uptake rate. Taurine may be useful in stabilizing the microenvironment in cells affected by oxidative stress as well as hypertonic and HG conditions. Moreover, taurine may play a key role in maintaining taurine concentrations in the taurine transporter system of retinal cells.

l-Citrulline restores nitric oxide level and cellular uptake at the brain capillary endothelial cell line (TR-BBB cells) with glutamate cytotoxicity.

OBJECTIVE: Glutamate excitotoxicity provokes neuronal cell damage and death, leading to collapse of the blood-brain barrier (BBB). Recently, it has been reported that l-citrulline, a neutral amino acid and a major precursor of l-arginine in the nitric oxide (NO) cycle, can prevent both neuronal cell death and cerebrovascular cell loss in brain ischemia. Therefore, the objective of this study was to investigate the effect of l-citrulline on glutamate cytotoxicity in the BBB using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells) as an in vitro model of the BBB. METHODS: Cell viability was determined using MTT assay. Cellular uptake of [14C] l-citrulline and expression levels of rat large neutral amino acid transporter 1 (rLAT1), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) at mRNA level were performed using quantitative real-time polymerase chain reaction (PCR) analysis. NO production from TR-BBB cells was measured using Griess reagents. All experiments were performed after treatment of TR-BBB cells with glutamate alone or co-treatment with l-citrulline, l-arginine, and/or taurine for 24 h. RESULTS: l-Citrulline treatment increased cell viability, [14C] l-citrulline uptake, and the mRNA levels of LAT1 and eNOS in TR-BBB cells treated with glutamate. However, iNOS mRNA expression was inhibited by l-citrulline. NO production and transcript level of iNOS were markedly increased by glutamate treatment alone. However, co-treatment with l-citrulline, taurine, or both l-citrulline and taurine decreased NO levels and mRNA levels of iNOS in TR-BBB cells treated with glutamate. In co-treatment of TR-BBB cells with l-arginine, a NO donor, and glutamate, NO levels were increased and expression levels of iNOS mRNA were similar compared to those in cells treated with glutamate alone. CONCLUSION: l-Citrulline can restore NO level and its cellular uptake in TR-BBB cells with glutamate cytotoxicity. Supplying l-citrulline at the BBB may provide neuroprotective effect to improve cerebrovascular dysfunction such as a brain ischemia.

The alteration of serine transporter activity in a cell line model of amyotrophic lateral sclerosis (ALS).

The alteration of d-serine levels is associated with the pathogenesis of sporadic ALS and mutant SOD1 (G93A) animal model of ALS. However, the exact mechanism of d-serine transport is not known in ALS. To better understand the distribution of d-serine in ALS, we determined the activity and the expression of serine transporter in a motor neuronal cell line model of ALS (NSC-34/hSOD1G93A cells). The uptake of [3H]d-serine was significantly lower in NSC-34/hSOD1G93A cells than in control NSC-34 and NSC-34/hSOD1wt cells. In contrast, the uptake of [3H]l-serine, precursor of d-serine, was markedly increased in NSC-34/hSOD1G93A cells compared to control NSC-34 and NSC-34/hSOD1wt cells. Both [3H]d-serine and [3H]l-serine uptake were saturable in these cells. The estimated Michaelis-Menten constant, Km, for d-serine uptakes was higher in NSC-34/hSOD1G93A cells than in NSC-34/hSOD1wt cells while the Km for l-serine uptake was 2 fold lower in NSC-34/hSOD1G93A cells than in control cells. [3H]d-serine and [3H]l-serine uptakes took place in a Na+-dependent manner, and both uptakes were significantly inhibited by system ASC (alanine-serine-cysteine) substrates. As a result of small interfering RNA experiments, we found that ASCT2 (SLC1A5) and ASCT1 (SLC1A4) are involved in [3H]d-serine and [3H]l-serine uptake in NSC-34/hSOD1G93A cells, respectively. The level of SLC1A4 mRNA was significantly increased in NSC-34/hSOD1G93A compared to NSC-34 and NSC-34/hSOD1wt cells. In contrast, the level of SLC7A10 mRNA was relatively lower in NSC-34/hSOD1G93A cells than the control cells. Together, these data suggest that the pathological alteration of d- and l-serine uptakes in ALS is driven by the affinity change of d-and l-serine uptake system.

Effects of simvastatin on CAT-1-mediated arginine transport and NO level under high glucose conditions in conditionally immortalized rat inner blood-retinal barrier cell lines (TR-iBRB).

OBJECTIVE: Hyperglycemia causes the breakdown of the blood-retinal barrier by impairing endothelial nitric oxide synthase (eNOS) function. Statins have many pleiotropic effects such as improving endothelial barrier permeability and increasing eNOS mRNA stability. The objective of this study was to determine effect of simvastatin on l-arginine transport and NO production under high-glucose conditions in conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB). METHODS: Changes in l-arginine transport uptake and, expression levels of cationic amino acid transporter 1 (CAT-1) and eNOS mRNA were investigated after pre-treatment with simvastatin and NOS inhibitors (l-NMMA and l-NAME) under high-glucose conditions using TR-iBRB, an in vitro model of iBRB. The NO level released from TR-iBRB cells was examined using Griess reagents. RESULTS: Under high glucose conditions, [3H]l-arginine uptake was decreased in TR-iBRB cells. Simvastatin pretreatment elevated [3H]l-arginine uptake, the expression levels of CAT-1 and eNOS mRNA, and NO production under high-glucose conditions. Moreover, the co-treatment with simvastatin and NOS inhibitors reduced [3H]l-arginine uptake compared to pretreatment with simvastatin alone. CONCLUSION: Our results suggest that, in the presence of high-glucose levels, increased l-arginine uptake due to simvastatin treatment was associated with increased CAT-1 and eNOS mRNA levels, leading to higher NO production in TR-iBRB cells. Thus, simvastatin might be a good modulator for diabetic retinopathy therapy by increasing of the l-arginine uptake and improving endothelial function in retinal capillary endothelial cells.

Characteristics of L-citrulline transport through blood-brain barrier in the brain capillary endothelial cell line (TR-BBB cells).

BACKGROUND: L-Citrulline is a neutral amino acid and a major precursor of L-arginine in the nitric oxide (NO) cycle. Recently it has been reported that L-citrulline prevents neuronal cell death and protects cerebrovascular injury, therefore, L-citrulline may have a neuroprotective effect to improve cerebrovascular dysfunction. Therefore, we aimed to clarify the brain transport mechanism of L-citrulline through blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB. METHODS: The uptake study of [14C] L-citrulline, quantitative real-time polymerase chain reaction (PCR) analysis, and rLAT1, system b0,+, and CAT1 small interfering RNA study were performed in TR-BBB cells. RESULTS: The uptake of [14C] L-citrulline was a time-dependent, but ion-independent manner in TR-BBB cells. The transport process involved two saturable components with a Michaelis-Menten constant of 30.9 ± 1.0 µM (Km1) and 1.69 ± 0.43 mM (Km2). The uptake of [14C] L-citrulline in TR-BBB cells was significantly inhibited by neutral and cationic amino acids, but not by anionic amino acids. In addition, [14C]L-citrulline uptake in the cells was markedly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), which is the inhibitor of the large neutral amino acid transporter 1 (LAT1), B0, B0,+ and harmaline, the inhibitor of system b0,+. Gabapentin and L-dopa as the substrates of LAT1 competitively inhibited the uptake of [14C] L-citrulline. IC50 values for L-dopa, gabapentin, L-phenylalanine and L-arginine were 501 µM, 223 µM, 68.9 µM and 33.4 mM, respectively. The expression of mRNA for LAT1 was predominantly increased 187-fold in comparison with that of system b0,+ in TR-BBB cells. In the studies of LAT1, system b0,+ and CAT1 knockdown via siRNA transfection into TR-BBB cells, the transcript level of LAT1 and [14C] L-citrulline uptake by LAT1 siRNA were significantly reduced compared with those by control siRNA in TR-BBB cells. CONCLUSIONS: Our results suggest that transport of L-citrulline is mainly mediated by LAT1 in TR-BBB cells. Delivery strategy for LAT1-mediated transport and supply of L-citrulline to the brain may serve as therapeutic approaches to improve its neuroprotective effect in patients with cerebrovascular disease.

Taurine Protects Glutamate Neurotoxicity in Motor Neuron Cells.

Amyotrophic lateral sclerosis (ALS) is a fetal neurodegenerative disease that results in motor dysfunction and death. However, there is no cure or effective therapy for ALS. In our previous results, taurine protects motor neurons by repairing for constitutive oxidative stress in an ALS model. ALS is caused by multiple factors including inflammation, oxidative stress, mitochondrial dysfunction, apoptosis, glutamate excitotoxicity and proteasomal dysfunction. Especially, glutamate excitotoxicity has been well known as a mediator in the disease process, and may occur from changes in the excitability of the neurons being stimulated. D-serine is known to a key factor of determination on glutamate toxicity in ALS. Therefore, in the present study, we investigated neuroprotective effects of taurine from glutamate excitotoxicity using motor neuron cells, mtSOD1 (G93A) transgenic cell line model of ALS (NSC-34/hSOD1G93A cells). We evidenced that taurine protects cultured motor neurons from neurotoxic injury. Our findings indicated that taurine has neuroprotective properties and may be a good candidate for therapeutic trials in ALS.

In Vivo and In Vitro Evidence for Brain Uptake of 4-Phenylbutyrate by the Monocarboxylate Transporter 1 (MCT1).

PURPOSE: 4-Phenylbutyrate (4-PBA) is expected to be a potential therapeutic for several neurodegenerative diseases. These activities require 4-PBA transport into the brain across the blood-brain barrier (BBB). The objective of the present study was to characterize the brain transport mechanism of 4-PBA through the BBB. METHODS: The brain transport of 4-PBA across the BBB was investigated following intravenous (IV) injection and internal carotid artery perfusion (ICAP) in vivo. The mechanism of transport was examined using TR-BBB cells, an in vitro model of the BBB. RESULTS: The volume of distribution (VD) of 4-PBA by rat brain was about 7-fold greater than that of sucrose, a BBB impermeable vascular space marker, suggesting the blood-to-brain transport of 4-PBA through the BBB in the physiological state. [(14)C]4-PBA uptake by TR-BBB cells showed time-, pH- and concentration-dependence with a K m of 13.4 mM at pH 7.4 and 3.22 mM at pH 6.0. The uptake was Na(+) independent, and was significantly inhibited by alpha-cyano-4-hydroxycinnamate (a typical inhibitor for monocarboxylate transport), endogenous monocarboxylate compounds and monocarboxylic drugs. Lactate and valproate competitively inhibited [(14)C]4-PBA uptake with K i value of 13.5 mM and 7.47 mM, respectively. These results indicate the role of monocarboxylate transporters (MCTs) in 4-PBA transport into the brain at the BBB. TR-BBB cells expressed mRNA of rMCT1, 2, and 4, especially, rMCT1 showed high mRNA expression level. In addition, [(14)C]4-PBA uptake was inhibited by rMCT1 specific small interfering RNA. CONCLUSION: The transport mechanism of 4-PBA from blood to brain across the BBB likely involves MCT1.

Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator.

Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.

Changes in 5-hydroxytryptamine and cortisol plasma levels in menopausal women after inhalation of clary sage oil.

The purpose of this study was to examine the antidepressant-like effects of clary sage oil on human beings by comparing the neurotransmitter level change in plasma. The voluntary participants were 22 menopausal women in 50's. Subjects were classified into normal and depression tendency groups using each of Korean version of Beck Depression Inventory-I (KBDI-I), KBDI-II, and Korean version of Self-rating Depression Scale. Then, the changes in neurotransmitter concentrations were compared between two groups. After inhalation of clary sage oil, cortisol levels were significantly decreased while 5-hydroxytryptamine (5-HT) concentration was significantly increased. Thyroid stimulating hormone was also reduced in all groups but not statistically significantly. The different change rate of 5-HT concentration between normal and depression tendency groups was variable according to the depression measurement inventory. When using KBDI-I and KBDI-II, 5-HT increased by 341% and 828% for the normal group and 484% and 257% for the depression tendency group, respectively. The change rate of cortisol was greater in depression tendency groups compared with normal groups, and this difference was statistically significant when using KBDI-II (31% vs. 16% reduction) and Self-rating Depression Scale inventory (36% vs. 8.3% reduction). Among three inventories, only KBDI-II differentiated normal and depression tendency groups with significantly different cortisol level. Finally, clary sage oil has antidepressant-like effect, and KBDI-II inventory may be the most sensitive and valid tool in screening for depression status or severity.

Effect of Various Pathological Conditions on Nitric Oxide Level and L-Citrulline Uptake in Motor Neuron-Like (NSC-34) Cell Lines.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a non-essential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [14C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [14C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [14C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [14C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

Factors Predicting Subjective Satisfaction for Successful Hearing Aid Adaptation.

(1) Background: For successful hearing aid (HA) use during daily life, an objective parameter reflecting the subjective satisfaction is required. We explored the aided hearing status, hearing in noise test (HINT) scores, and subjective outcomes to predict performance improvements in everyday living. (2) Methods: A total of 406 patients with hearing loss (HL) who were prescribed HAs were included and were divided into two groups according to the symmetricity of HL. The relationship between audiometric data and subjective questionnaires under unaided and aided (3 months) conditions were investigated. (3) Results: Patients with symmetric HL showed a significant HINT signal-to-noise ratio (SNR) change and significant increase in their subjective satisfaction questionnaire score under the bilateral HA condition. On the other hand, the HINT SNR change and subjective questionnaire score showed various significances according to the side of HA (better or worse hearing) in asymmetric HL HINT SNR and was significantly correlated with the subjective questionnaire score in symmetric HL patients and AHL patients with unilateral HA in their better ear. (4) Conclusions: The HINT SNR improvement after long-term HA use could be an effective tool for predicting the subjective satisfaction of HA use and HA validation.

The effects of bisphosphonates on taurine transport in retinal capillary endothelial cells under high glucose conditions.

Diabetic retinopathy (DR) is a major cause of blindness in diabetic patients. Elevated glucose and vascular endothelial growth factor (VEGF) in retina can trigger many of the retinal vascular changes caused by diabetes and DR. Recently, bisphosphonates, antiosteoporosis drugs, have been reported to have anti-angiogenic effect by decreasing VEGF. Taurine has several biological processes such as osmoregulation and antioxidation in retina. Therefore, the purpose of this study is to clarify the regulation of taurine transport activity by high glucose concentration and the effect of inhibitors for VEGF function, bisphosphonates, on taurine transport under high glucose condition using TR-iBRB cell lines as an in vitro model of inner blood-retinal barrier (iBRB). As a result, by exposing TR-iBRB cells to high glucose for 48 h, [(3)H]taurine uptake was decreased continuously. [(3)H]Taurine uptake was increased significantly by pretreatment of alendronate and pamidronate compared with the values for high glucose. Increased [(3)H]taurine uptake by pretreatment of alendronate and pamidronate was significantly reduced by mevalonate pathway intermediates, geranylgeraniol (GGOH). In conclusion, taurine transport through the iBRB under high glucose condition can be regulated by bisphosphonates via mevalonate pathway. Therefore, we suggest that bisphosphonates could have the beneficial effects on DR by regulation of taurine contents in retina.

Differences in Neurotransmitters Level as Biomarker on Sleep Effects in Dementia Patients with Insomnia after Essential Oils Treatment.

This study aimed to examine the effects of several essential oils on insomnia in dementia patients following transdermal treatment (aromatherapy). The mean change rates (%) of sleep biomarkers were compared between the single essential and jojoba (vehicle) oil massage groups in this study. The lavender (L) essential oil massage group demonstrated a significant decrease in the mean change rate (%) of 24-h urinary free cortisol, whereas the valerian (V) essential oil massage group demonstrated a significant increase in the mean change rate (%) of serum 5-hydroxytryptamine. In addition, a significant increase in the mean change rate (%) of 24-h urinary norepinephrine was observed in the chamomile (C) essential oil massage group only. Based on these results, valerian, lavender, and chamomile oils were mixed in different ratios to produce blending oils A (L:C:V=2:2:1), B (L:C:V=3:1:1) and C (L:C:V=1:3:1). The highest level of serum 5-hydroxytryptamine was observed after administering blending oil A. These results suggest that blending oil A might possess therapeutic effects against insomnia. Overall, it is hypothesized that the optimally blended essential oil will produce synergic effects when combined with hypnotic drugs.

The acetylcholinesterase inhibitors competitively inhibited an acetyl L-carnitine transport through the blood-brain barrier.

We investigated the interaction of acetylcholinesterase (AChE) inhibitors with acetyl-L-carnitine (ALCAR) transporter at the blood-brain barrier (BBB). ALCAR uptake by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB cells), as an in vitro model of BBB, were characterized by cellular uptake study using [(3)H]ALCAR. In vivo brain uptake of [(3)H]ALCAR was determined by brain uptake index after carotid artery injection in rats. In results, the transport properties for [(3)H]ALCAR by TR-BBB cell were consistent with those of ALCAR transport by the organic cation/carnitine transporter 2 (OCTN2). Also, OCTN2 was confirmed to be expressed in the cells. The uptake of [(3)H]ALCAR by TR-BBB cells was inhibited by AChE inhibitors such as donepezil, tacrine, galantamine and rivastigmine, which IC(50) values are 45.3, 74.0, 459 and 800 µM, respectively. Especially, donepezil and galantamine inhibited the uptake of [(3)H]ALCAR competitively, but tacrine and rivastigmine inhibited noncompetitively. Furthermore, [(3)H]ALCAR uptake by the rat brain was found to be significantly decreased by quinidine, donepezil and galantamine. Our results suggest that transport of AChE inhibitors such as donepezil and galantamine through the BBB is at least partly mediated by OCTN2 which is involved in transport of ALCAR.

Blood-Brain Barrier Solute Carrier Transporters and Motor Neuron Disease.

Defective solute carrier (SLC) transporters are responsible for neurotransmitter dysregulation, resulting in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). We provided the role and kinetic parameters of transporters such as ASCTs, Taut, LAT1, CAT1, MCTs, OCTNs, CHT, and CTL1, which are mainly responsible for the transport of essential nutrients, acidic, and basic drugs in blood-brain barrier (BBB) and motor neuron disease. The affinity for LAT1 was higher in the BBB than in the ALS model cell line, whereas the capacity was higher in the NSC-34 cell lines than in the BBB. Affinity for MCTs was lower in the BBB than in the NSC-34 cell lines. CHT in BBB showed two affinity sites, whereas no expression was observed in ALS cell lines. CTL1 was the main transporter for choline in ALS cell lines. The half maximal inhibitory concentration (IC50) analysis of [3H]choline uptake indicated that choline is sensitive in TR-BBB cells, whereas amiloride is most sensitive in ALS cell lines. Knowledge of the transport systems in the BBB and motor neurons will help to deliver drugs to the brain and develop the therapeutic strategy for treating CNS and neurological diseases.

Protective Effects of Choline against Inflammatory Cytokines and Characterization of Transport in Motor Neuron-like Cell Lines (NSC-34).

Choline, a component of the neurotransmitter acetylcholine, is essential for nervous system functions, brain development, and gene expression. In our study, we investigated the protective effect and transport characteristics of choline in amyotrophic lateral sclerosis (ALS) model cell lines. We used the wild-type (WT) motor neuron-like hybrid cell line (NSC-34/hSOD1WT) as a control and the mutant-type (MT; NSC-34/hSOD1G93A) as a disease model. The uptake of [3H]choline was time-, pH-, and concentration-dependent. [3H]Choline transport was sodium-dependent, and, upon pretreatment with valinomycin, induced membrane depolarization. Gene knockdown of Slc44a1 revealed that choline-like transporter 1 (CTL1) mediates the transport of choline. In NSC-34 cell lines, the specific choline transporter inhibitor, hemicholinium-3 demonstrated significant inhibition. Donepezil and nifedipine caused dose-dependent inhibition of [3H]choline uptake by the MT cell line with minimal half inhibitory concentration (IC50) values of 0.14 mM and 3.06 mM, respectively. Four-day pretreatment with nerve growth factor (NGF) resulted in an inhibitory effect on [3H]choline uptake. Choline exerted protective and compensatory effects against cytokines mediators. Hence, the choline transport system CLT1 may act as a potential target for the delivery of novel pharmacological drugs, and the combination of drugs with choline can help treat symptoms related to ALS.

Antioxidant and Neuroprotective Effects of Paeonol against Oxidative Stress and Altered Carrier-Mediated Transport System on NSC-34 Cell Lines.

Paeonol is a naturally occurring phenolic agent that attenuates neurotoxicity in neurodegenerative diseases. We aimed to investigate the antioxidant and protective effects of paeonol and determine its transport mechanism in wild-type (WT; NSC-34/hSOD1WT) and mutant-type (MT; NSC-34/hSOD1G93A) motor neuron-like amyotrophic lateral sclerosis (ALS) cell lines. Cytotoxicity induced by glutamate, lipopolysaccharides, and H2O2 reduced viability of cell; however, the addition of paeonol improved cell viability against neurotoxicity. The [3H]paeonol uptake was increased in the presence of H2O2 in both cell lines. Paeonol recovered ALS model cell lines by reducing mitochondrial oxidative stress induced by glutamate. The transport of paeonol was time-, concentration-, and pH-dependent in both NSC-34 cell lines. Kinetic parameters showed two transport sites with altered affinity and capacity in the MT cell line compared to the WT cell line. [3H]Paeonol uptake increased in the MT cell line transfected with organic anion transporter1 (Oat1)/Slc22a6 small interfering RNA compared to that in the control. Plasma membrane monoamine transporter (Pmat) was also involved in the uptake of paeonol by ALS model cell lines. Overall, paeonol exhibits neuroprotective activity via a carrier-mediated transport system and may be a beneficial therapy for preventing motor neuronal damage under ALS-like conditions.

Change in Cationic Amino Acid Transport System and Effect of Lysine Pretreatment on Inflammatory State in Amyotrophic Lateral Sclerosis Cell Model.

Amyotrophic lateral sclerosis (ALS) is a lethal neurological disorder characterized by the deterioration of motor neurons. The aim of this study was to investigate alteration of cationic amino acid transporter (CAT-1) activity in the transport of lysine and the pretreatment effect of lysine on pro-inflammatory states in an amyotrophic lateral sclerosis cell line. The mRNA expression of cationic amino acid transporter 1 was lower in NSC-34/hSOD1G93A (MT) than the control cell line (WT), lysine transport is mediated by CAT-1 in NSC-34 cell lines. The uptake of [3H]L-lysine was Na+-independent, voltage-sensitive, and strongly inhibited by inhibitors and substrates of cationic amino acid transporter 1 (system y+). The transport process involved two saturable processes in both cell lines. In the MT cell line, at a high-affinity site, the affinity was 9.4-fold higher and capacity 24-fold lower than that in the WT; at a low-affinity site, the capacity was 2.3-fold lower than that in the WT cell line. Donepezil and verapamil competitively inhibited [3H]L-lysine uptake in the NSC-34 cell lines. Pretreatment with pro-inflammatory cytokines decreased the uptake of [3H]L-lysine and mRNA expression levels in both cell lines; however, the addition of L-lysine restored the transport activity in the MT cell lines. L-Lysine exhibited neuroprotective effects against pro-inflammatory states in the ALS disease model cell lines. In conclusion, studying the alteration in the expression of transporters and characteristics of lysine transport in ALS can lead to the development of new therapies for neurodegenerative diseases.