Harumi Hata

Functional identification of ASCT1 neutral amino acid transporter as the predominant system for the uptake of l-serine in rat neurons in primary culture

The uptake of L-serine, a nonessential amino acid known to be transported by the neutral amino acid transporter system ASC, was studied in primary cultures of rat neurons and astrocytes, and compared with that in human embryonic kidney (HEK293) cells transfected with rat ASCT1 cDNA. We first cloned neutral amino acid transporter ASCT1 from rat neurons in primary culture as a transporter candidate for L-serine uptake in the brain. The predicted amino acid sequence from rat ASCT1 exhibited significant homology with mouse and human ASCT1s. The amino acid sequence of rat ASCT1 was 92 and 84% identical to that of mouse and of human ASCT1, respectively. HEK293 cells expressing the rat ASCT1 cDNA showed a saturable dose-dependent and Na(+)-dependent increase in L-[(3)H] serine uptake by high affinity ( K(m) = 67 microM). The substrate selectivity of rat ASCT1 was the same as those of the mouse and human transporter. Northern blot analysis revealed that ASCT1 mRNA was ubiquitously expressed in the brain, with its highest concentration in the striatum and hippocampus. When the uptake of L -[(3)H] serine into rat primary neurons or astrocytes was compared with that of HEK293 cells expressing rat ASCT1 or rat ASCT2 cDNA, the inhibition profile of amino acids for the rat neurons quite resembled that for HEK293 cells expressing rat ASCT1. In contrast, the profile for rat astrocytes was a mixture of that for HEK293 cells expressing rat ASCT1 and that for the cells expressing rat ASCT2. Furthermore, L-[(3)H] serine uptake in neurons was fully Na(+)-dependent. ASCT1 mRNA was expressed in both primary neurons and astrocytes, whereas ASCT2 mRNA was expressed only in astrocytes, as determined by using RT-PCR with primers specific for the rat ASCT1 or rat ASCT2 transporter. Taken together, these findings indicate that ASCT1 predominantly contributes to the uptake of L-serine in primary neurons.

Association of morphine-induced antinociception with variations in the 5'flanking and 3' untranslated regions of the μ opioid receptor gene in 10 inbred mouse strains

Objective Genetic factors are hypothesized to be involved in interindividual differences in opioid sensitivity. Inbred mouse strains that are genetically different and isogenic within each strain are useful for elucidating the genetic mechanisms underlying the interindividual differences in opioid-induced analgesia. Methods We examined the effects of morphine in 10 inbred mouse strains, including wild-derived strains that have a wide range of genetic diversity, including BLG2, CHD, KJR, MSM, NJL, PGN2, and SWN. We also performed full sequencing of the 5′ flanking region and exons of the mouse μ opioid receptor gene Oprm1 and analyzed the association between genotypes and phenotypes in these mice. Results The effects of morphine on locomotor activation and antinociception varied among the inbred strains. The nucleotide differences that cause amino acid substitutions were not found in the Oprm1 gene in the inbred strains analyzed in this study. In the 5′ flanking region and 3′ untranslated region of the Oprm1 gene, four highly variable regions containing novel short tandem repeat polymorphisms (GA, T, TA, and CA/CT) were identified. The GA, T, and TA repeat numbers were significantly associated with morphine-induced antinociception. Conclusion These results suggest that the short tandem repeats in the 5′ flanking and 3′ untranslated regions of the μ opioid receptor gene are involved in interstrain differences in opioid sensitivity in mice. Wild-derived inbred mouse strains with different numbers of these repeats may be useful models for examining interindividual differences in opioid sensitivity.

Association analysis of δ-opioid receptor gene polymorphisms in methamphetamine dependence/psychosis†

The role of the δ‐opioid receptor (OPRD1) in methamphetamine (MAP) addiction was investigated using association analysis between OPRD1 gene polymorphisms and MAP dependence/psychosis. DNA samples from Japanese patients with MAP dependence/psychosis were analyzed to find polymorphisms in OPRD1 gene exons and exon–intron boundaries. One novel single nucleotide polymorphism (SNP) in intron 1 and two SNPs in exon 3 were identified. The two SNPs in exon 3 were in linkage disequilibrium. No significant difference was observed in either genotypic or allelic frequencies of these SNPs between controls (n = 260) and MAP dependent/psychotic patients (n = 170). Global analyses using the three SNPs and subcategory analyses on clinical parameters also showed no significant differences. These results suggest that the OPRD1 gene variants may not be a factor in vulnerability to MAP dependence/psychosis.

Involvement of the 3′ non‐coding region of the mu opioid receptor gene in morphine‐induced analgesia

Abstract  The mu opioid receptor (MOR) is known to play an essential role in morphine‐induced analgesia. MOR‐1 mRNA, the major MOR transcript, possesses a long 3′ untranslated region (3′UTR) in both mouse and human species. The sequence of the MOR‐1 3′UTR, especially that of its 3′ end region, is conserved between mice and humans. In the MOR‐1 3′UTR, AU‐rich elements (AREs) are densely localized at the 3′ end region, suggesting low stability of this mRNA. Numerous putative transcription factor‐binding motifs are located in the 3′ non‐coding regions of the mouse and human MOR genes. The CXBK mouse strain, known as a MOR‐deficient strain, possesses a decreased amount of MOR‐1 mRNA containing an abnormally long MOR‐1 3′UTR with a long nucleotide insertion. This insert might disrupt the stability of the MOR‐1 mRNA or might reduce the transcription of the MOR‐1 mRNA by separating the transcription factor‐binding motifs in the 3′ non‐coding region of the MOR gene, thereby decreasing MOR‐1 mRNA expression and attenuating morphine‐induced analgesia in CXBK mice. These recent findings suggest that the MOR‐1 3′UTR is involved in mRNA expression and in the difference in response to morphine. This possible genetic mechanism may provide a good starting point for designing effective pain treatments with opiates.

Involvement of annexin A8 in the properties of pancreatic cancer.

Although Annexin A8 (ANXA8), a member of a superfamily of calcium and phospholipid binding proteins, is physiologically expressed in a tissue‐specific manner, recent microarray studies reported that ANXA8 was also ectopically expressed in pancreatic cancers. We investigated the molecular mechanism of expression of ANXA8 in cancer cells and its functional role in pancreatic cancer cells. ANXA8 was diversely expressed in human cancer cell lines. Expression was enhanced by treatment with 5‐aza‐dC and butyrate, and correlated with methylation status at CpG in the promoter‐exon 1 region. Inhibition of ANXA8 using siRNA in BxPC‐3 cells which express ANXA8 at a high level elevated caspase‐3 and ‐7 activities. In in vitro invasion assay, inhibition of ANXA8 using siRNA in BxPC‐3 reduced the numbers of migrating cells, and down‐regulated HIF‐1α mRNA transcription. Overexpression of ANXA8 increased the number of viable cells and BrdU incorporation in PANC‐1 cells, which express ANXA8 at a low level. Expression of ANXA8 was induced under conditions of nutrient deprivation, and overexpression of ANXA8 showed resistance against serum starvation in PANC‐1 cells. In a promoter assay, co‐transfection with the expression vector of ANXA8 and the vector of a reporter gene containing the promoter of HIF‐1α enhanced HIF‐1α promoter activity. In contrast, this effect of ANXA8 was inhibited by administration of BAPTA‐AM, an intracellular Ca2+ chelator. These results suggest that ectopic ANXA8 expression in cancer cells might involve an epigenetic mechanism. ANXA8 might play an important role in calcium fluctuation‐mediated HIF‐1α transcriptional activation and cell viability.

Exclusive expression of VMAT2 in noradrenergic neurons increases viability of homozygous VMAT2 knockout mice

The vesicular monoamine transporter 2 (VMAT2) translocates monoamine neurotransmitters from the neuronal cytoplasm into synaptic vesicles. Since VMAT2-/- mice die within a few days of birth, it is difficult to analyze the detailed VMAT2 functions using these mice. In this study, we generated human VMAT2 transgenic mice that expressed VMAT2 in noradrenergic neurons with the aim to rescue the lethality of VMAT2 deletion. The expression of human VMAT2 in noradrenergic neurons extended the life of VMAT2-/- mice for up to three weeks, and these mice showed severe growth deficiency compared with VMAT2+/+ mice. These results may indicate that VMAT2 expressed in noradrenergic neurons has crucial roles in survival during the first several weeks after birth, and VMAT2 functions in other monoaminergic systems could be required for further extended survival. Although VMAT2 rescue in noradrenergic neurons did not eliminate the increased morbidity and lethality associated with VMAT2 deletion, the extension of the lifespan in VMAT2 transgenic mice will enable behavioral, pharmacological and pathophysiological studies of VMAT2 function.

Induction of activation-induced cytidine deaminase by a not-directly mutagenic carcinogen: a novel potential molecular mechanism

AbstractObjective The molecular mechanisms underlying the carcinogenic activity of not-directly mutagenic (Ames mutagenicity test-negative) carcinogens are not fully understood. Given recent findings that ectopic expression of activation-induced cytidine deaminase (AID) in somatic cells plays a critical role in carcinogenesis, we investigated whether several of the established not-directly mutagenic carcinogens induce AID expression.Methods We prepared cells with stable expression of luciferase reporter gene containing the promoter of AID. We then used this system to examine the AID promoter activity of the non-genotoxic carcinogen: butyl benzyl phthalate, bisphenol A, di (2-ethylhexyl) phthalate, cadmium chloride (Cd), and butylated hydroxyanisole.ResultsResults showed that Cd increased the promoter activity of AID and actually induced AID gene expression.ConclusionA not-directly mutagenic carcinogen, cadmium, has the potential to induce the AID gene, suggesting that this might represent a novel molecular mechanism of carcinogenesis of cadmium.