Sialorphin Potentiates Effects of [Met5]Enkephalin without Toxicity by Action other than Peptidase Inhibition.

This dose-response study investigated the effects of sialorphin on [Met5]enkephalin (ME)-induced inhibition of contractions in mouse vas deferens and antinociception in male rats. Differences were compared among combinations of three chemical peptidase inhibitors: amastatin, captopril, and phosphoramidon. The ratio of potencies of ME in mouse vas deferens pretreated with both sialorphin (100 µM) and a mixture of the three peptidase inhibitors (1 µM each) was higher than that with the mixture of peptidase inhibitors alone at any dose. Intrathecal administration of sialorphin (100-400 nmol) significantly and dose dependently increased ME (3 nmol)-induced antinociception with the mixture of three peptidase inhibitors (10 nmol each). The degree of antinociception with a combination of any two of the peptidase inhibitors (10 nmol each) in the absence of sialorphin was less than that in the presence of sialorphin (200 nmol). Pretreatment with both sialorphin (200 nmol) and the mixture of three peptidase inhibitors (10 nmol each) produced an approximately 100-fold augmentation in ME (10 nmol)-induced antinociception, but without signs of toxicity such as motor dysfunction in rats. Radioligand receptor binding assay revealed that sialorphin did not affect either binding affinity or maximal binding capacity of [d-Ala2,N-MePhe4,Gly-ol5]enkephalin. These results indicate that sialorphin potentiates the effects of ME without toxicity by a mechanism other than peptidase inhibition and with no effect on its affinity to µ-opioid receptors. SIGNIFICANCE STATEMENT: Sialorphin is regarded as an endogenous peptidase inhibitor that interacts with enkephalin-degrading enzymes. The results of these in vitro and in vivo studies confirm that sialorphin potentiates the effects of [Met5]enkephalin without toxicity by an action other than peptidase inhibition. This suggests that sialorphin offers the advantage of reducing or negating the side effects of opioid drugs and endogenous opioid peptides.

Cognitive Function Related to the Sirh11/Zcchc16 Gene Acquired from an LTR Retrotransposon in Eutherians.

Gene targeting of mouse Sushi-ichi-related retrotransposon homologue 11/Zinc finger CCHC domain-containing 16 (Sirh11/Zcchc16) causes abnormal behaviors related to cognition, including attention, impulsivity and working memory. Sirh11/Zcchc16 encodes a CCHC type of zinc-finger protein that exhibits high homology to an LTR retrotransposon Gag protein. Upon microdialysis analysis of the prefrontal cortex region, the recovery rate of noradrenaline (NA) was reduced compared with dopamine (DA) after perfusion of high potassium-containing artificial cerebrospinal fluid in knockout (KO) mice. These data indicate that Sirh11/Zcchc16 is involved in cognitive function in the brain, possibly via the noradrenergic system, in the contemporary mouse developmental systems. Interestingly, it is highly conserved in three out of the four major groups of the eutherians, euarchontoglires, laurasiatheria and afrotheria, but is heavily mutated in xenarthran species such as the sloth and armadillo, suggesting that it has contributed to brain evolution in the three major eutherian lineages, including humans and mice. Sirh11/Zcchc16 is the first SIRH gene to be involved in brain function, instead of just the placenta, as seen in the case of Peg10, Peg11/Rtl1 and Sirh7/Ldoc1.

The effects of 2,3-dimercapto-1-propanesulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA) on the nephrotoxicity in the mouse during repeated cisplatin (CDDP) treatments.

Previously, we reported that specific lower dose of sodium 2,3-dimercapto-1-propanesulfonic acid (DMPS) which is an antidote to heavy metal intoxication, inversely enhanced cisplatin (CDDP)-induced antitumor activity to S-180 cell-bearing mouse. This activity was only weak with meso-2,3-dimercaptosuccinic acid (DMSA), however. This study investigated the effects of lower doses of DMPS or DMSA on the nephrotoxicity and kinetics of CDDP. Kidney and blood isolated from female mice which received CDDP with or without DMPS or DMSA once daily for 4 days were provided for measuring levels of blood urea nitrogen (BUN) and transporter proteins (OCT2: organic cation transporter; MATE1: multidrug and toxin extrusion) mRNA, and CDDP-originated platinum, and TUNEL staining of renal tubular cells. DMPS or DMSA reduced effectively CDDP-induced BUN, and caused a moderate reduction of platinum in kidney. Additionally, both dimercapto-compounds restored the CDDP-reduced mRNA levels of transporter proteins (OCT2 and MATE1), and apparently suppressed the CDDP-induced apoptosis. These results suggest that DMPS, as well as DMSA, at approximate 17-fold dose (µmol/kg) of CDDP, has an enough potential to reverse the CDDP nephrotoxicity, and concomitant use of DMPS considering both dose and timing for administration is potentially useful for preventing nephrotoxicity and enhancing antitumor activity during CDDP chemotherapy.

Effect of three peptidase inhibitors on antinociceptive potential and toxicity with intracerebroventricular administration of dynorphin A (1-17) or (1-13) in the rat.

PURPOSE: The N- and C-terminal regions of dynorphin (Dyn) A (1-17) activate opioid and N-methyl-D-aspartate receptors, respectively. Earlier studies demonstrated that Dyn-converting enzyme cleaved Dyn A (1-17) mainly at the Arg(6)-Arg(7) bond, resulting in the production of N- and C-terminal region peptide fragments, and that this enzyme was not inhibited by a mixture of the three peptidase inhibitors (PIs) amastatin (A), captopril (C), and phosphoramidon (P). The purpose of the present study was to evaluate antinociceptive potential and toxicity with intracerebroventricular administration of Dyn A (1-17) or (1-13) under pretreatment with a mixture of A, C, and P and/or Dyn-converting enzyme inhibitor (p-hydroxymercuribenzoate). METHODS: Peptide fragments from Dyn A (1-17) following incubation with membrane preparation under pretreatment with a mixture of the three PIs was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Infusion of drugs and peptides into the third ventricle in rats was performed via indwelling cannulae. Induction of antinociception and toxicity by Dyn A (1-17), Dyn A (1-13), Dyn A (1-6), or Dyn A (7-17) were determined by the tail-flick test and induction of barrel rotation, respectively. The effects of the PIs on antinociception and toxicity were evaluated by a dose-response study and a comparison of differences among various combinations of Dyn A (1-17) or Dyn A (1-13) and the three PIs and p-hydroxymercuribenzoate. RESULTS: MALDI-TOF-MS analysis identified Dyn A (1-6) and Dyn A (1-10) fragments as products following incubation of Dyn A (1-17) with membrane preparation of rat midbrain under pretreatment with a mixture of the three PIs. Pretreatment with a mixture of the three PIs produced an approximately 30-fold augmentation in antinociception induced by low-dose intracerebroventricular administration of Dyn A (1-17) or (1-13) in a µ-, δ- and κ-opioid receptor antagonist-reversible manner, but without signs of toxicity such as barrel rotation in the rat. Dyn A (1-17)-induced antinociception and toxicity was greater than that of Dyn A (1-6), Dyn A (1-13), or Dyn A (7-17) at the same dose. Dyn A (1-17)-induced antinociception and toxicity under pretreatment with various combinations of the three PIs and p-hydroxymercuribenzoate was greater than that with a mixture of the three PIs alone. CONCLUSION: These findings suggest that administration of a mixture of the three PIs increases Dyn A (1-17)- or (1-13)-induced antinociception under physiological conditions without toxicity.

Antinociceptive effect of intracerebroventricular administration of D-serine on formalin-induced pain.

PURPOSE: In a previous study using the tail-flick test, we found that intracerebroventricular administration of D-serine, an endogenous co-agonist at the glycine sites of N-methyl-D-aspartate (NMDA) receptors, elicited an antinociceptive effect on thermal nociception. The purpose of the present study was to evaluate the effect of intracerebroventricular administration of D-serine on nociception induced by tissue damage or inflammation using the formalin test. METHODS: Infusion of drugs into the third ventricle in rat was performed via indwelling cannulae. Drugs were infused at a volume of 10 µl over 2 min, and the infusion cannula was left in place for 2 min before removal. The formalin test was performed 10 min after drug administration. RESULTS: Intracerebroventricular administration of D-serine significantly and dose-dependently decreased the number of flinches in both the early and late phases in the formalin test. This antinociceptive effect was antagonized by intracerebroventricular administration of L-701,324, a selective antagonist at the glycine sites of NMDA receptors. CONCLUSION: The present data suggest that activation of NMDA receptors via glycine sites at the supraspinal level induces an antinociceptive effect on both acute and tonic pain.

Potentiation of [Met5]enkephalin-induced antinociception by mixture of three peptidase inhibitors in rat.

PURPOSE: Previous in vitro studies have shown that degradation of opioid peptides during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), namely, amastatin, captopril, and phosphoramidon. In the present in vivo study, we evaluate the effects of intrathecal administration of these PIs on antinociception by [Met(5)]enkephalin (ME) or PIs themselves. METHODS: Drugs were administered into the thoracolumbar level of the spinal cord in the intrathecal space in rat. Induction of antinociception was measured by the tail immersion assay, with 55 °C as the nociceptive stimulus. Effects of PIs on antinociception were evaluated by dose-response study (ME, 1-20 nmol; PIs, 1-20 nmol each), by comparison of differences among two combinations of PIs (amastatin and captopril; captopril and phosphoramidon; amastatin and phosphoramidon) and three PIs (amastatin, captopril, and phosphoramidon), and by using opioid receptor selective antagonists. RESULTS: Intrathecal administration of ME with these three PIs or PIs alone significantly and dose dependently increased antinociception in a µ- and δ-opioid receptor antagonist-reversible manner; moreover, the degree of antinociception with a combination of any two of these was less than that with all three, indicating that any residual single peptidase could inactivate significant amounts of ME. CONCLUSION: The present data, together with those of earlier studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play an important role in inactivation of opioid peptides at the spinal level.

mRNA expression and localization of LPS-induced ß-defensin isoforms in rat salivary glands.

ß-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of ß-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat ß-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. ß-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. ß-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce ß-defensins.

d-Serine Increases Release of Acetylcholine in Rat Submandibular Glands.

d-serine has been observed in submandibular gland tissue in rats, but its functions remain to be clarified. Oral administration of d-serine, but not l-serine, increased its concentrations in the submandibular gland and pilocarpine-induced salivary secretion. In vivo microdialysis was used to collect the d- and l-enantiomers of amino acids from local interstitial fluid in the rat submandibular gland. The proportion of the d-form of serine in interstitial fluid was higher than that in plasma or saliva. Perfusion of the rat submandibular gland with d-serine and l-glutamic acid via the submandibular gland artery resulted in a significant increase in salivary secretion after stimulation of muscarinic receptors with carbachol. In vivo microdialysis applied to the submandibular glands of rats showed that infusion of d-serine along with l-glutamate through the microdialysis probe significantly elevated acetylcholine levels in local interstitial fluids in the submandibular glands of anesthetized rats as compared to that with l-glutamate alone in an N-methyl-d-aspartate receptor glycine site antagonist-sensitive manner. These results indicate that d-serine augments salivary secretion by increasing acetylcholine release in the salivary glands.

Terahertz Spectroscopy and Ab-Initio Vibrational Analysis of Two Crystalline Forms of 5,5-Diethylbarbituric Acid.

Terahertz (THz) absorption spectra of the two crystalline forms of 5,5-diethylbarbituric acid (barbital) were measured with THz time-domain spectroscopy (THz-TDS). The spectra exhibited the dissimilarity between the two forms. Identification of the polymorphs and quantitative analysis of the two forms are possible by THz-TDS. Further, we performed ab-initio calculations of the vibrational modes considering the crystal structures of the two forms, and the results were in good agreement with the experimental data. We discuss the difference between the THz absorption spectra of the two forms.

Free d-Amino Acids in Salivary Gland in Rat.

Free d-amino acids, which are enantiomers of l-amino acids, are found in mammals, including humans, and play an important role in a range of physiological functions in the central nervous system and peripheral tissues. Several d-amino acids have been observed in saliva, but their origin and the enzymes involved in their metabolism and catabolism remain to be clarified. In the present study, large amounts of d-aspartic acid and small amounts of d-serine and d-alanine were detected in all three major salivary glands in rat. No other d-enantiomers were detected. Protein expression of d-amino acid oxidase and d-aspartate oxidase, the enzymes responsible for the oxidative deamination of neutral and dicarboxylic d-amino acids, respectively, were detected in all three types of salivary gland. Furthermore, protein expression of the d-serine metabolic enzyme, serine racemase, in parotid glands amounted to approximately 40% of that observed in the cerebral cortex. The N-methyl-d-aspartic acid subunit proteins NR1 and NR2D were detected in all three major salivary glands. The results of the present study suggest that d-amino acids play a physiological role in a range of endocrine and exocrine function in salivary glands.

Diazepam enhances production of diazepam-binding inhibitor (DBI), a negative saliva secretion regulator, localized in rat salivary gland.

Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase-activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

Diazepam Enhances Production of Diazepam-Binding Inhibitor (DBI), a Negative Saliva Secretion Regulator, Localized in Rat Salivary Gland.

Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase-activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

Proteomic analysis of lipopolysaccharide-treated submandibular gland in rat.

We investigated changes in the protein profile of submandibular gland (SMG) with inflammation induced by exposure to lipopolysaccharide (LPS) with the aim of identifying potential molecular markers of injured gland. Lipopolysaccharide (2.5µg) was directly administered into rat SMG unilaterally by retrograde ductal injection. At 12hr after treatment, the gland was excised and the proteins identified by two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Many proteins in the LPS-treated gland showed a marked change compared to those in the contralateral gland. Of particular note were increases in ubiquitin, a highly-conserved small regulatory protein and in calgranulin B, which has an immunological function in inflammation. Proteins related to apoptosis and stress also showed change in the inflamed gland. The results of this study suggest that the ubiquitin system of protein modification is involved in LPS-induced inflammation in salivary gland, and that a number of specific proteins might be applicable as molecular markers in the monitoring of inflamed or injured gland.

In Vivo Monitoring of Acetylcholine Release from Nerve Endings in Salivary Gland.

A microdialysis technique was used to monitor acetylcholine levels in the local interstitial fluid in rat submandibular glands, with the aim of determining parasympathetic nerve activity in vivo. The dialysis probe housed a 10 × 0.22 mm semipermeable membrane (molecular weight cutoffs: 50,000 Da). When the probe was perfused at 2 µL/min in vitro, the mean relative recovery of acetylcholine was 41.7% ± 2.5%. The dialysis probes were implanted in the submandibular glands of anesthetized rats and perfusion with Ringer's solution, at 2 µL/min, was performed. Acetylcholine concentrations in the dialysate were measured by high-performance liquid chromatography and electrochemical detection. The results revealed the following: (1) that mixing Eserine with Ringer's solution allowed acetylcholine in the salivary glands to be quantified; (2) that acetylcholine concentrations in the dialysate were highly variable and unstable over the first 120 min after probe implantation, but reached a nearly stable level (4.8 ± 2.7 nM) thereafter in the presence of 100 µM of Eserine; and (3) that electrical stimulation of the chorda tympani nerve, or perfusion with high potassium Ringer's solution, significantly increased acetylcholine concentrations in the dialysate. These results indicate that the present microdialysis technique offers a powerful tool for detecting changes in parasympathetic activity within the salivary glands.

Analysis of the source of aggressiveness in gamecocks.

Although the fighting behaviour in gamecocks has evolved because of artificial selection, it is unknown whether the selection for aggressiveness affects neurotransmitter levels in the avian central nervous system. We sought to identify the source and origin of this trait. We collected the brain samples from 6 female Shamo gamecocks and 5 Shaver Brown chickens (control; bred for egg production). The midbrain levels of norepinephrine (NE) were significantly higher in Shamo gamecocks (P = 0.0087) than in the controls. Moreover, alleles encoding adrenergic receptors differed between the breeds in terms of response to NE. Gene mutations specific to Shamo and potentially associated with fighting behaviour were in sites T440N of ADRα1D; V296I of ADRα2A; and T44I, Q232R, and T277M of ADRß2. The evolutionary analysis indicated that the ADRß2 (T44I and Q232R) mutations were heritable in all Galliformes, whereas the T440N mutation of ADRα1D and V296I mutations of ADRα2A were unique to Shamo and originated by artificial selection. A high NE level may confer a selective advantage by enabling gamecocks to be aggressive and pain tolerant. Therefore, the strong fighting behaviour of Shamo has resulted from a combination of naturally inherited and mutant genes derived by artificial selection.

Inhibitory effect of low-dose pentazocine on the development of antinociceptive tolerance to morphine.

PURPOSE: The development of antinociceptive tolerance to morphine is one of the major problems in its clinical use. Therefore, exploring effective measures to prevent morphine tolerance is of great clinical relevance. We evaluated whether pentazocine could prevent morphine tolerance in mice. METHODS: Five groups of male ICR mice received repeated subcutaneous (s.c.) injections of morphine at a high dose (10 mg x kg(-1)) or saline, concomitantly with s.c. injections of pentazocine at low, subanalgesic doses (0.1, 0.3, or 1.0 mg x kg(-1)) or saline, respectively, once daily for 14 days. On day 15, mice received co-injections of morphine and pentazocine 120 min after pretreatment with nor-binaltorphimine (5 mg x kg(-1)), a selective kappa-opioid receptor antagonist. The tail pressure threshold was measured before and 60 min after the daily drug co-injections. RESULTS: Repeated s.c. co-injections of morphine and saline resulted in a progressive decrease in morphine-induced antinociception, due to the development of morphine tolerance. Co-injections of pentazocine (0.1, 0.3, and 1.0 mg x kg(-1)) with morphine potentiated the morphine-induced antinociception dose-dependently by preventing the development of morphine tolerance. Nor-binaltorphimine completely inhibited the chronic antinociception maintained by co-injections of morphine and pentazocine. CONCLUSION: When chronically co-administered with morphine, pentazocine at low, subanalgesic doses dose-dependently potentiated morphine-induced antinociception in morphine-tolerant mice, through its kappa-opioid-receptor-mediated tolerance-preventing activity. Because pentazocine is the only agonist-antagonist analgesic that has an effective oral formulation suitable for chronic administration, the results of the present study warrant clinical trials of pentazocine to assess its tolerance-preventing activity in patients with cancer pain.

Stress Characterization of the Interface Between Thermal Oxide and the 4H-SiC Epitaxial Layer Using Near-Field Optical Raman Microscopy.

Stresses induced in the silicon carbide (SiC) epitaxial layer near the interface between thermal silicon oxide and 4H-SiC epitaxial substrate were measured using a near-field optical Raman microscope equipped with a hollow pyramid probe (aperture size: approximately 250 nm). The E2 phonon was observed to undergo a 0.17 cm-1 redshift owing to reduction in oxide-layer thickness from 300 nm to 0 nm; this result was compared against that obtained using a standard Raman microprobe sans the pyramidal probe. The result indicates that the epitaxial layer near the SiO2-4H-SiC interface was maintained under a constant tensile stress of the order of 50 MPa. This agrees well with the result obtained using the finite element method (FEM). Based on results obtained using the said Raman microprobe and Fourier transform infrared (FT-IR) measurements, use of an inhomogeneity formation model at the SiO2-4H-SiC interface has been proposed in this study.

Mice lacking d-amino acid oxidase activity exhibit marked reduction of methamphetamine-induced stereotypy.

The behavioral effects induced by methamphetamine (5.0 mg/kg) were compared in the mutant mice lacking d-amino acid oxidase activity and normal mice. The mutant mice exhibited marked decline in the methamphetamine-induced stereotypy compared to the normal mice, whereas the mutant mice displayed a drastic augmentation in the locomotor activity evoked by methamphetamine compared to the normal mice. Because the d-serine levels in the brain of the mutant mice are significantly higher than those in the normal mice, the enhanced d-serine in the brain of the mutant mice could antagonize the methamphetamine-induced stereotypy via the N-methyl-d-aspartate receptors.

Midazolam attenuates the antinociception induced by d-serine or morphine at the supraspinal level in rats.

Our recent study has shown that the intracerebroventricular administration of d-serine, an endogenous and selective agonist for the glycine site of the N-methyl-d-aspartate receptor, alone or in combination with morphine, leads to the potentiation of antinociception on the tail-flick response. Although there is a variety of information concerning the effects of benzodiazepines on opioid-induced antinociception, little is known about the effect of benzodiazepines on the N-methyl-d-aspartate receptor agonist-induced antinociception. To clarify the analgesic interactions among the benzodiazepine/GABA(A), N-methyl-d-aspartate and opioid receptors at the supraspinal level, we investigated the effects of intracerebroventricular administration of midazolam, a benzodiazepine receptor agonist, on the antinociception evoked by the intracerebroventricular application of d-serine or morphine. The intracerebroventricular administration of midazolam alone produced hyperalgesia on the tail-flick response in a benzodiazepine receptor antagonist, flumazenil-reversible manner. The antinociception induced by the intracerebroventricular application of d-serine or morphine was attenuated by the intracerebroventricular administration of midazolam. In addition, this inhibitory effect of midazolam on the antinociception of d-serine or morphine was antagonized by the intracerebroventricular administration of flumazenil. Together with the facts that d-serine and midazolam act as selective agonists for the glycine site of the N-methyl-d-aspartate receptor and benzodiazepine/GABA(A) receptor, respectively, these observations suggest a functional interaction between the NMDA and benzodiazepine/GABA(A) receptors in the regulation of antinociception at the supraspinal level.

Effects of MK-801 on the expression of serine racemase and d-amino acid oxidase mRNAs and on the D-serine levels in rat brain.

We have investigated the acute effects of the increasing doses of non-competitive N-methyl-d-aspartate receptor antagonist MK-801 (0.2-1.6 mg/kg) on the expression of serine racemase and d-amino acid oxidase (DAO) mRNAs in several brain areas of rats. We have also evaluated the effects of the chronic administration of MK-801 (0.4 mg/kg) on the gene expression of serine racemase and DAO and on the d-serine concentrations. A dose-dependent augmentation of the expression of serine racemase mRNA was seen in most brain areas at both 1 and 4 h after the administration. In contrast, a drastic decline in the expression of DAO mRNA was observed in most brain areas 1 h after the MK-801 administration, whereas a dose-dependent elevation in the expression of DAO mRNA was observed in most brain areas 4 h after the administration. The chronic MK-801 administration produced a significant increase in the expression of serine racemase mRNA in almost all brain areas, whereas no significant changes were found in the level of DAO mRNA in most brain areas. In addition, the chronic administration caused a slight but significant elevation in the concentrations of d-serine in the cortex and striatum. These present findings indicate that increasing the serine racemase mRNA and no changes in the DAO mRNA after the chronic administration could contribute to the elevation of the d-serine level in the forebrain, and that serine racemase and DAO could play an important role in the regulation of N-methyl-d-aspartate receptors via the d-serine metabolism.