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.

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.

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.

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.

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.

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.

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.

Differences of Transport Activity of Arginine and Regulation on Neuronal Nitric Oxide Synthase and Oxidative Stress in Amyotrophic Lateral Sclerosis Model Cell Lines.

L-Arginine, a semi-essential amino acid, was shown to delay dysfunction of motor neurons and to prolong the lifespan, upon analysis of transgenic mouse models of amyotrophic lateral sclerosis (ALS). We investigated the transport function of arginine and neuronal nitric oxide synthase (nNOS) expression after pretreatment with L-arginine in NSC-34 hSOD1WT (wild-type, WT) and hSOD1G93A (mutant-type, MT) cell lines. [3H]L-Arginine uptake was concentration-dependent, voltage-sensitive, and sodium-independent in both cell lines. Among the cationic amino acid transporters family, including system y+, b0,+, B0,+, and y+L, system y+ is mainly involved in [3H]L-arginine transport in ALS cell lines. System b0,+ accounted for 23% of the transport in both cell lines. System B0,+ was found only in MT, and whereas, system y+L was found only in WT. Lysine competitively inhibited [3H]L-arginine uptake in both cell lines. The nNOS mRNA expression was significantly lower in MT than in WT. Pretreatment with arginine elevated nNOS mRNA levels in MT. Oxidizing stressor, H2O2, significantly decreased their uptake; however, pretreatment with arginine restored the transport activity in both cell lines. In conclusion, arginine transport is associated with system y+, and neuroprotection by L-arginine may provide an edge as a possible therapeutic target in the treatment of ALS.

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.

The Alteration of L-Carnitine Transport and Pretreatment Effect under Glutamate Cytotoxicity on Motor Neuron-Like NSC-34 Lines.

L-Carnitine (LC) is essential for transporting fatty acids to the mitochondria for ß-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.

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.

L-Citrulline Level and Transporter Activity Are Altered in Experimental Models of Amyotrophic Lateral Sclerosis.

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/hSOD1G93A, MT) and a control model (NSC-34/hSOD1wt, WT). The [14C]L-citrulline uptake was significantly reduced in MT cells as compared with that of control. The Michaelis-Menten constant (Km) for MT cells was 0.67 ± 0.05 mM, whereas it was 1.48 ± 0.21 mM for control. On the other hand, the Vmax 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 Km and Vmax values showed the high affinity and low capacity for MT as compared with control. Moreover, the uptake of [14C]L-citrulline was significantly inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) and harmaline which is the inhibitor of the large neutral amino acid transporter1 (LAT1) in NSC-34 cell lines. Furthermore, [14C]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 [14C]L-citrulline uptake. It shows the competitive inhibition for L-dopa in ALS cell lines. On the other hand, [14C]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 [14C]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 [14C]L-citrulline may be partially responsible for the pathological alteration in ALS.

Transport Alteration of 4-Phenyl Butyric Acid Mediated by a Sodium- and Proton-Coupled Monocarboxylic Acid Transporter System in ALS Model Cell Lines (NSC-34) Under Inflammatory States.

4-Phenyl butyric acid (PBA) has histone deacetylase inhibitory and neuroprotective effects. We aimed to examine the transport alteration activity of PBA in control (WT) and disease (MT) model cell lines of an amyotrophic lateral sclerosis (ALS) model. The transport characteristics of PBA were examined uptake rates and mRNA expression levels in NSC-34 cell lines. PBA uptake was pH, sodium, and concentration dependent. The Km and Vmax values for PBA uptake in the MT were more than two-fold higher than those in the WT. The presence of monocarboxylic acids (MA) and inhibitors of MA transporter (MCT) inhibited the uptake of PBA. PBA showed competitive inhibition in the presence of MAs in both cell lines. SiRNA transfection studies showed that PBA can be transported to NSC-34 cell lines through sodium-coupled MCT1. TNF-α and H2O2 increased, but LPS and glutamate reduced the uptake rate after the pretreatment of the MT cell lines. SMCT1 mRNA expression levels, in the presence of oxidative stress inducing agents, showed consistent results with the uptake results. These results demonstrate that PBA can be transported to the ALS model NSC-34 cell lines by sodium- and proton-coupled MCTs, and MA plays a vital role in the prevention of neurodegenerative diseases.

Pretreatment Effect of Inflammatory Stimuli and Characteristics of Tryptophan Transport on Brain Capillary Endothelial (TR-BBB) and Motor Neuron Like (NSC-34) Cell Lines.

Tryptophan plays a key role in several neurological and psychiatric disorders. In this study, we investigated the transport mechanisms of tryptophan in brain capillary endothelial (TR-BBB) cell lines and motor neuron-like (NSC-34) cell lines. The uptake of [3H]l-tryptophan was stereospecific, and concentration- and sodium-dependent in TR-BBB cell lines. Transporter inhibitors and several neuroprotective drugs inhibited [3H]l-tryptophan uptake by TR-BBB cell lines. Gabapentin and baclofen exerted a competitive inhibitory effect on [3H]l-tryptophan uptake. Additionally, l-tryptophan uptake was time- and concentration-dependent in both NSC-34 wild type (WT) and mutant type (MT) cell lines, with a lower transporter affinity and higher capacity in MT than in WT cell lines. Gene knockdown of LAT1 (l-type amino acid transporter 1) and CAT1 (cationic amino acid transporter 1) demonstrated that LAT1 is primarily involved in the transport of [3H]l-tryptophan in both TR-BBB and NSC-34 cell lines. In addition, tryptophan uptake was increased by TR-BBB cell lines but decreased by NSC-34 cell lines after pro-inflammatory cytokine pre-treatment. However, treatment with neuroprotective drugs ameliorated tryptophan uptake by NSC-34 cell lines after inflammatory cytokines pretreatment. The tryptophan transport system may provide a therapeutic target for treating or preventing neurodegenerative diseases.

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.

Involvement of a Novel Organic Cation Transporter in Paeonol Transport Across the Blood-Brain Barrier.

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 (VD) of [3H]paeonol in rat brain was about 6-fold higher than that of [14C]sucrose, the vascular space marker of BBB. The uptake of [3H]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 [3H]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.

Blood-to-Retina Transport of Imperatorin Involves the Carrier-Mediated Transporter System at the Inner Blood-Retinal Barrier.

This study investigated the mechanism of transporting imperatorin across the inner blood-retinal barrier (iBRB). The carotid artery single injection method was used to calculate the retinal uptake index (RUI) of [3H]imperatorin in vivo, whereas the retinal capillary endothelial cell lines were used for the in vitro uptake and mRNA expression assays. RUI value of [3H]imperatorin was greater than that of the reference compound ([14C]n-butanol). [3H]Imperatorin significantly reduced the RUI in the presence of neuroprotective organic cationic drugs at 10 mM. However, tetraethylammonium and p-aminohippuric acid showed no significant effects. [3H]Imperatorin uptake by TR-iBRB2 cells was time-, pH-, energy-, and concentration-dependent with a Km value of 679 ± 130 µM. In addition, the uptake study showed insensitivity to sodium and membrane potential. Various organic cations including pyrilamine, nicotine, and clonidine significantly reduced the uptake of [3H]imperatorin, whereas organic anions and monocarboxylic acids did not. Furthermore, the mRNA expression level dropped markedly with rOCTN1, rOCTN2, rPMAT, and rMATE1 small interfering RNAs in the transfection study. Moreover, [3H]imperatorin uptake remained neutral with small interfering RNA transfections. Our results indicate that imperatorin transport across the iBRB involves carrier-mediated transporter system.

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.

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.

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.