Miyuki Wada

Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons

Na+‐coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+‐coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+‐coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+‐dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 µm in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3 : 1 and the concentration of Na+ necessary for half‐maximal transport was 53 mm. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (∼ 1.2 mm) than that of succinate. Unlabeled succinate (2 mm) inhibited Na+‐dependent [14C]succinate (18 µm) and [14C]citrate (4.5 µm) transport completely, whereas unlabeled citrate inhibited Na+‐dependent [14C]succinate uptake more weakly. Interestingly, N‐acetyl‐l‐aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+‐dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N‐acetyl‐l‐aspartate and citrate was 15.9, 155 and 764 µm respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 µm, which was different from that observed in astrocytes, suggesting that different Na+‐dependent citrate transport systems are expressed in neurons and astrocytes. RT–PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.

Transport characteristics of N-acetyl-L-aspartate in rat astrocytes : involvement of sodium-coupled high-affinity carboxylate transporter NaC3/NaDC3-mediated transport system

We investigated in the present study the transport characteristics of N‐acetyl‐l‐aspartate in primary cultures of astrocytes from rat cerebral cortex and the involvement of NA+‐coupled high‐affinity carboxylate transporter NaC3 (formerly known as NaDC3) responsible for N‐acetyl‐l‐aspartate transport. N‐acetyl‐l‐aspartate transport was NA+‐dependent and saturable with a Michaelis–Menten constant (Km) of ∼110 µm. NA+‐activation kinetics revealed that the NA+ to‐N‐acetyl‐l‐aspartate stoichiometry was 3 : 1 and concentration of Na+ necessary for half‐maximal transport (KNAm) was 70 mm. NA+‐dependent N‐acetyl‐l‐aspartate transport was competitively inhibited by succinate with an inhibitory constant (Ki) of 14.7 µm, which was comparable to the Km value of NA+‐dependent succinate transport (29.4 µm). l‐Aspartate also inhibited NA+‐dependent [14C]N‐acetyl‐l‐aspartate transport with relatively low affinity (Ki = 2.2 mm), whereas N‐acetyl‐l‐aspartate was not able to inhibit NA+‐dependent aspartate transport in astrocytes. In addition, Li+ was found to have a significant inhibitory effect on the NA+‐dependent N‐acetyl‐l‐aspartate transport in a concentration‐dependent manner. Furthermore, RT–PCR and western blot analyses revealed that NaC3 is expressed in primary cultures of astrocytes. Taken collectively, these results indicate that NaC3 expressed in rat cerebrocortical astrocytes is responsible for NA+‐dependent N‐acetyl‐l‐aspartate transport. This transporter is likely to be an essential prerequisite for the metabolic role of N‐acetyl‐l‐aspartate in the process of myelination.

Functional characterization of brain peptide transporter in rat cerebral cortex: identification of the high-affinity type H+/peptide transporter PEPT2.

In this report, we studied the functional characteristics of a brain peptide transporter using synaptosomes prepared from rat cerebral cortex. Crude synaptosomes (P(2) fraction) were prepared from cerebral cortices in male Wistar rats. Uptake of [14C]glycylsarcosine (Gly-Sar), a substrate for H(+)/oligopeptide transporters PEPT1 and PEPT2, and [3H]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 degrees C by a rapid filtration technique. The uptake of [14C]Gly-Sar into synaptosomes was stimulated by an inwardly directed H(+)-gradient. The uptake system exhibited a Michaelis-Menten constant (K(t)) of 110+/-20 microM for Gly-Sar. This value is comparable to the K(t) value for Gly-Sar uptake via the high-affinity H(+)/peptide transporter PEPT2. The H(+)-dependent uptake of [14C]Gly-Sar into synaptosomes was inhibited by di- and tripeptides and beta-lactam antibiotics, but was unaffected by amino acids glycine and histidine. In particular, kyotorphin (Tyr-Arg) completely inhibited Gly-Sar uptake with the K(i) value of 29+/-14 microM. These uptake properties of the brain peptide transporter (i.e., the K(t) value for Gly-Sar uptake and the K(i) value of kyotorphin for Gly-Sar uptake) are very similar to those of PEPT2. RT-PCR and Western blotting analyses revealed that PEPT2 is actually expressed in the cerebral cortex in rat. These results indicate that a H(+)-coupled high affinity peptide transport system is functionally expressed in the cerebral cortex and that this transport system is identical to PEPT2.

Functional regulation of Na+‐dependent neutral amino acid transporter ASCT2 by S‐nitrosothiols and nitric oxide in Caco‐2 cells

We describe the regulation mechanisms of the Na+‐dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco‐2. Exposure of Caco‐2 cells to S‐nitrosothiol, such as S‐nitroso‐N‐acetyl‐dl‐penicillamine (SNAP) and S‐nitrosoglutathione, and the NO‐donor, NOC12, concentration‐ and time‐dependently increased Na+‐dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V max) of Na+‐dependent alanine uptake in Caco‐2 cells without affecting the Michaelis–Menten constant (K t). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na+‐dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl 3‐oxide sodium salt (carboxy‐PTIO) and N‐(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT‐PCR revealed that Caco‐2 cells expressed the Na+‐dependent neutral amino acid transporter ASCT2, but not the other Na+‐dependent neutral amino acid transporters ATB0,+ and B0AT1. These results suggested that functional up‐regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.

Functional linkage of H+/peptide transporter PEPT2 and Na+/H+ exchanger in primary cultures of astrocytes from mouse cerebral cortex.

In our previous studies, we demonstrated that the high-affinity type peptide transporter PEPT2 is expressed in rat cerebral cortex using synaptosomal membrane study and that the uptake of dipeptide [14C]glycylsarcosine into synaptosomes was stimulated by an inwardly directed H+ gradient (Fujita et al., Brain Res. 972, 52-61, 2004). However, there is no information available for the driving force of PEPT2 function in the nervous system. In the present study, we investigated functional characteristics of PEPT2 mediated transport of Gly-Sar in primary cultured astrocytes from mouse cerebral cortex and examined the effects of Na+/H+ exchanger (NHE) inhibitor on Gly-Sar uptake in mouse astrocytes. In mouse astrocytes, extracellular H+ influenced only the maximal velocity (Vmax) of Gly-Sar uptake without affecting the apparent affinity (Kt). Interestingly, removal of Na+ from uptake buffer significantly reduced Gly-Sar uptake and Gly-Sar uptake was modulated by NHE inhibitors. The treatment of EIPA, an NHE inhibitor, altered the Vmax value of Gly-Sar uptake but had no effect on its Kt value. RT-PCR revealed that NHE1 and NHE2 mRNA are expressed in mouse cerebrocortical astrocytes. These results demonstrated that NHE activity is required to allow optimal uptake of dipeptides mediated by PEPT2 into the astrocytes. This study represents the first description of the functional co-operation of PEPT2 and NHE1 and/or NHE2 in cerebrocortical astrocytes.

Functional expression of constitutive nitric oxide synthases regulated by voltage-gated Na+ and Ca2+ channels in cultured human astrocytes.

We report the functional characterization of constitutive nitric oxide synthase(s) (NOS) such as neuronal and endothelial NOS in cultured human astrocytes. Exposure of cultured human astrocytes to 1 μM veratridine or 50 mM KCl produced a pronounced increase in a calmodulin‐dependent NOS activity estimated from cGMP formation. The functional expression of voltage‐gated Na+ channel, which is estimated by the response to veratridine, appeared to be earlier (at second day in culture) than that of voltage‐gated Ca2+ channels, which are estimated by the response to the KCl stimulation (at fourth day in culture). The KCl‐evoked NO synthesis was totally reversed by L‐type Ca2+ channel blockers such as nifedipine and verapamil, but not by ω‐conotoxin GVIA, an N‐type Ca2+ channel blocker, or ω‐agatoxin IVA, a P/Q‐type Ca2+ channel blocker. In addition, verapamil abolished the KCl‐induced increase in the intracellular free Ca2+ concentration. RT‐PCR analysis revealed that mRNA for neuronal and endothelial NOS was expressed in human astrocytes. In addition, Western blot analysis and double labeling of NOS and glial fibrillary acidic protein (GFAP) showed that cultured human astrocytes expressed neuronal NOS and endothelial NOS as well as the α1 subunit of Ca2+ channel. These results suggest that human astrocytes express constitutive NOS that are regulated by voltage‐gated L‐type Ca2+ channel as well as Na+ channel.

A comparison of Ca2+ channel blocking mode between gabapentin and verapamil: implication for protection against hypoxic injury in rat cerebrocortical slices

The mode of Ca2+ channel blocking by gabapentin [1‐(aminomethyl)cyclohexane acetic acid] was compared to those of other Ca2+ channel blockers, and the potential role of Ca2+ channel antagonists in providing protection against hypoxic injury was subsequently investigated in rat cerebrocortical slices. mRNA for the α2δ subunits of Ca2+ channels was found in rat cerebral cortex. Nitric oxide (NO) synthesis estimated from cGMP formation was enhanced by KCl stimulation, which was mediated primarily by the activation of N‐ and P/Q‐type Ca2+ channels. Gabapentin blocked both types of Ca2+ channels, and preferentially reversed the response to 30 mM K+ stimulation compared with 50 mM K+ stimulation. In contrast, verapamil preferentially inhibited the response to depolarization by the higher concentration (50 mM) of K+. Gabapentin inhibited KCl‐induced elevation of intracellular Ca2+ in primary neuronal culture. Hypoxic injury was induced in cerebrocortical slices by oxygen deprivation in the absence (severe injury) or presence of 3 mM glucose (mild injury). Gabapentin preferentially inhibited mild injury, while verapamil suppressed only severe injury. ω‐Conotoxin GVIA (ω‐CTX) and ω‐agatoxin IVA (ω‐Aga) were effective in both models. NO synthesis was enhanced in a manner dependent on the severity of hypoxic insults. Gabapentin reversed the NO synthesis induced by mild insults, while verapamil inhibited that elicited by severe insults. ω‐CTX and ω‐Aga were effective in both the cases. Therefore, the data suggest that gabapentin and verapamil cause activity‐dependent Ca2+ channel blocking by different mechanisms, which are associated with their cerebroprotective actions and are dependent on the severity of hypoxic insults.

Functional characterization of Zn2+-sensitive GABA transporter expressed in primary cultures of astrocytes from rat cerebral cortex

The extracellular levels of gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the mammalian cerebral cortex, are regulated by specific high-affinity Na(+)/Cl(-) dependent transporters (GATs). GAT1 mainly expressed in cerebrocortical neurons is thought to play an important role for clearance of GABA in the extracellular fluid, whereas there is a little information available for pharmacological importance for astrocytic GABA transporters. In the present study, we therefore described the functional characterization of GABA transport in primary cultures of astrocytes from rat cerebral cortex and the identification of GABA transporter subtype(s). GABA transport was Na(+) and Cl(-) dependent and saturable with a Michaelis constant (K(t)) of 9.3+/-2.8 microM. Na(+)- and Cl(-)- activation kinetics revealed that the Na(+)-Cl(-)-to-GABA stoichiometry was 2:1:1 and concentrations of Na(+) and Cl(-) necessary for half-maximal transport (K(0.5)(Na) and K(0.5)(Cl)) were 78+/-28 mM and 9.6+/-2.6 mM, respectively. Na(+)-dependent GABA transport was competitively inhibited by various GABA transport inhibitors, especially GAT2- or GAT3-selective inhibitor. In addition, Zn(2+), which has been reported to be a potent inhibitor of GAT3, was found to have a significantly but partially inhibitory effect on the Na(+)-dependent GABA transport in a concentration-dependent manner. Furthermore, reverse transcription-PCR and Western blot analyses revealed that GAT2 and GAT3 are expressed in primary cultures of astrocytes. These results clearly showed that zinc is a useful reagent for separating GAT3 activity from GAT1- and GAT2-activities in CNS. To our knowledge, the present study represents the first report on the inhibitory effect of zinc on the Na(+)-dependent GABA transport in rat cerebrocortical astrocytes.