Nescient helix-loop-helix 1 (Nhlh1) is a novel activating transcription factor 5 (ATF5) target gene in olfactory and vomeronasal sensory neurons in mice.

Activating transcription factor 5 (ATF5) is a transcription factor that belongs to the cAMP-response element-binding protein/ATF family and is essential for the differentiation and survival of sensory neurons in mouse olfactory organs. However, transcriptional target genes for ATF5 have yet to be identified. In the present study, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed to verify ATF5 target genes in the main olfactory epithelium and vomeronasal organ in the postnatal pups. ChIP-qPCR was conducted using hemagglutinin (HA)-tagged ATF5 knock-in olfactory organs. The results obtained demonstrated that ATF5-HA fusion proteins bound to the CCAAT/enhancer-binding protein-ATF response element (CARE) site in the enhancer region of nescient helix-loop-helix 1 (Nhlh1), a transcription factor expressed in differentiating olfactory and vomeronasal sensory neurons. Nhlh1 mRNA expression was downregulated in ATF5-deficient (ATF5-/-) olfactory organs. The LIM/homeobox protein transcription factor Lhx2 co-localized with ATF5 in the nuclei of olfactory and vomeronasal sensory neurons and bound to the homeodomain site proximal to the CARE site in the Nhlh1 gene. The CARE region of the Nhlh1 gene was enriched by the active enhancer marker, acetyl-histone H3 (Lys27). The present study identified Nhlh1 as a novel target gene for ATF5 in murine olfactory organs. ATF5 may upregulate Nhlh1 expression in concert with Lhx2, thereby promoting the differentiation of olfactory and vomeronasal sensory neurons.

Changes in physical activity level during hospitalization in patients with stroke and those with fracture: a prospective longitudinal study.

[Purpose] To examine changes in physical activity levels between admission and discharge in patients hospitalized after stroke and fracture. [Participants and Methods] Patients with stroke (n=36) or fracture (n=41) wore an accelerometer during the daytime for three days after admission and before discharge. Physical activity was divided into sedentary behavior (SB), light-intensity (LIPA), and moderate-to-vigorous (MVPA), and then compared between hospital admission and discharge using the Wilcoxon signed-rank test. The characteristics of patients with or without changes in SB during hospitalization were compared using the Mann-Whitney U test. [Results] The median LIPA time in patients after stroke and fracture increased from 107.5 and 106.7 minutes on admission to 122.0 and 127.3 minutes at discharge, and the median MVPA time increased from 2.7 and 0.7 minutes on admission to 4.2 and 2.7 minutes at discharge, respectively. In particular, LIPA in non-therapy time increased for patients both after stroke and fracture. No differences in characteristics were observed between with or without changes in SB regardless of differences in diagnoses. [Conclusion] These findings indicate that while physical activity levels increased during hospitalization, they remained below World Health Organization recommendations for MVPA, and patient characteristics alone may not account for increased activity levels.

Activating transcription factor 5 (ATF5) controls intestinal tuft and goblet cell expansion upon succinate-induced type 2 immune responses in mice.

Intestinal tuft cells, a chemosensory cell type in mucosal epithelia that secrete interleukin (IL)-25, play a pivotal role in type 2 immune responses triggered by parasitic infections. Tuft cell-derived IL-25 activates type 2 innate lymphoid cells (ILC2) to secrete IL-13, which, in turn, acts on intestinal stem or transient amplifying cells to expand tuft cells themselves and mucus-secreting goblet cells. However, the molecular mechanisms of tuft cell differentiation under type 2 immune responses remain unclear. The present study investigated the effects of the deletion of activating transcription factor 5 (ATF5) on the type 2 immune response triggered by succinate (a metabolite of parasites) in mice. ATF5 mRNAs were expressed in the small intestine, and the loss of the ATF5 gene did not affect the gross morphology of the tissue or the basal differentiation of epithelial cell subtypes. Succinate induced marked increases in tuft and goblet cell numbers in the ATF5-deficient ileum. Tuft cells in the ATF5-deficient ileum are assumed to be a subtype of intestinal tuft cells (Tuft-2 cells) marked by the transcription factor Spib. Exogenous IL-25 induced similar increases in tuft and goblet cell numbers in wild-type and ATF5-deficient ilea. IL-13 at a submaximal dose enhanced tuft cell differentiation more in ATF5-deficient than in wild-type intestinal organoids. These results indicate that the loss of ATF5 enhanced the tuft cell-ILC2 type 2 immune response circuit by promoting tuft cell differentiation in the small intestine, suggesting its novel regulatory role in immune responses against parasitic infections.

Functional validation of epitope-tagged ATF5 knock-in mice generated by improved genome editing of oviductal nucleic acid delivery (i-GONAD).

Activating transcription factor 5 (ATF5) is a stress-responsive transcription factor that belongs to the cAMP response element-binding protein (CREB)/ATF family, and is essential for the differentiation and survival of sensory neurons in murine olfactory organs. However, the study of associated proteins and target genes for ATF5 has been hampered due to the limited availability of immunoprecipitation-grade ATF5 antibodies. To overcome this issue, we generated hemagglutinin (HA)-tag knock-in mice for ATF5 using CRISPR/Cas9-mediated genome editing with one-step electroporation in oviducts (i-GONAD). ATF5-HA fusion proteins were detected in the nuclei of immature and some mature olfactory and vomeronasal sensory neurons in the main olfactory epithelium and vomeronasal organ, respectively, as endogenous ATF5 proteins were expressed, and some ATF5-HA proteins were found to be phosphorylated. Chromatin immunoprecipitation (ChIP) experiments revealed that ATF5-HA bound to the CCAAT/enhancer-binding protein (C/EBP)-ATF response element site in the promotor region of receptor transporting protein 1 (Rtp1), a chaperone gene responsible for proper olfactory receptor expression. These knock-in mice may be used to examine the expression, localization, and protein-protein/-DNA interactions of endogenous ATF5 and, ultimately, the function of ATF5 in vivo.

Co-expression of C/EBPγ and ATF5 in mouse vomeronasal sensory neurons during early postnatal development.

The differentiation of sensory neurons involves gene expression changes induced by specific transcription factors. Vomeronasal sensory neurons (VSNs) in the mouse vomeronasal organ (VNO) consist of two major subpopulations of neurons expressing vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo, which differentiate from a common neural progenitor. We previously demonstrated that the differentiation and survival of VSNs were inhibited in ATF5 transcription factor-deficient mice (Nakano et al. Cell Tissue Res 363:621-633, 2016). These defects were more prominent in V2r/Gαo-type than in V1r/Gαi2-type VSNs; however, the molecular mechanisms responsible for the differentiation of V2r/Gαo-type VSNs by ATF5 remain unclear. To identify a cofactor involved in ATF5-regulated VSN differentiation, we investigated the expression and function of CCAAT/enhancer-binding protein gamma (C/EBPγ, Cebpg), which is a major C/EBP family member expressed in the mouse VNO and dimerizes with ATF5. The results obtained showed that C/EBPγ mRNAs and proteins were broadly expressed in the postmitotic VSNs of the neonatal VNO, and their expression decreased by the second postnatal week. The C/EBPγ protein was expressed in the nuclei of approximately 70% of VSNs in the neonatal VNO, and 20% of the total VSNs co-expressed C/EBPγ and ATF5 proteins. We examined the trans-acting effects of C/EBPγ and ATF5 on V2r transcription and found that the co-expression of C/EBPγ and ATF5, but not C/EBPγ or ATF5 alone, increased Vmn2r66 promoter reporter activity via the C/EBP:ATF response element (CARE) in Neuro2a cells. These results suggest the role of C/EBPγ on ATF5-regulated VSN differentiation in early postnatal development.

Characterization of chylomicron in preterm infants.

BACKGROUND: The aim of this study was to investigate cholesterol and triglyceride levels in the chylomicron fraction of preterm infants at birth and during the early postnatal period. METHODS: The subjects consisted of 133 infants (81 boys and 52 girls): 74 were term infants born at 37-41 weeks of gestation and 59 were preterm infants born at 29-36 weeks of gestation. Cholesterol and triglyceride in the chylomicron fraction were measured using high-performance liquid chromatography. RESULTS: Compared with term infants, preterm infants had higher cholesterol and lower triglyceride in the chylomicron fraction, both in cord blood and at 1 month after birth. Thus, the chylomicron triglyceride/cholesterol ratio was significantly lower in preterm infants than in term infants in cord blood and at 1 month of age. On single regression analysis the chylomicron triglyceride/cholesterol ratio correlated positively with gestational age at birth (r = 0.331, P = 0.0003) and at 1 month (r = 0.221, P = 0.0119). CONCLUSIONS: Preterm infants have a less-lipidated chylomicron composition at birth and at 1 month of age. Some prenatal factors may persist to influence chylomicron lipidation during the early postnatal period.

Target value of oxygen saturation during the first 10 min after birth.

BACKGROUND: During neonatal resuscitation, careful oxygenation is needed. Pulse oximetry is recommended to evaluate the need for oxygenation, but it is not clear whether peripheral perfusion is adequate for the evaluation of arterial oxygen saturation (SpO2 ). Additionally, there has been no study on the changes in SpO2 immediately after birth in Japan, despite the indispensable need for definitive oxygenation criteria. METHODS: A prospective observational study was performed in neonates at gestational age 35-41 weeks. An SpO2 measurement probe was attached to the neonates immediately after birth at the right palm or wrist, and the perfusion index (PI), pulse rate, and SpO2 were measured until 10 min after birth. RESULTS: Sixty neonates were examined. Stable PI was obtained soon after birth, preceding SpO2 measurement. The median PI (%) was constant at approximately 1.3, and the median SpO2 at 2-10 min was 70%, 81%, 82%, 87%, 89%, 92%, 92%, 94%, and 95%, respectively. The current target value for SpO2 in the Neonatal Cardiopulmonary Resuscitation (NCPR) guideline in Japan is approximately the 25th percentile. CONCLUSION: PI is stable and sufficient in the early postnatal period, meaning that peripheral perfusion is adequate for the measurement of SpO2 . The current target SpO2 used in the NCPR guidelines is at approximately the 25th percentile and is thought to be sufficient for meeting oxygenation criteria.

Activating transcription factor 5 (ATF5) is essential for the maturation and survival of mouse basal vomeronasal sensory neurons.

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of transcription factors, which is highly expressed in olfactory chemosensory tissues, the main olfactory epithelium and vomeronasal epithelium (VNE) in mice. The vomeronasal sensory neurons in the VNE detect pheromones in order to regulate social behaviors such as mating and aggression; however, the physiological role of ATF5 in the vomeronasal sensory system remains unknown. In this study, we found that the differentiation of mature vomeronasal sensory neurons, assessed by olfactory marker protein expression, was inhibited in ATF5-deficient VNE. In addition, many apoptotic vomeronasal sensory neurons were evident in ATF5-deficient VNE. The vomeronasal sensory neurons consist of two major types of neuron expressing either vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo. We demonstrated that the differentiation, survival and axonal projection of V2r/Gαo-type rather than V1r/Gαi2-type vomeronasal sensory neurons were severely inhibited in ATF5-deficient VNE. These results suggest that ATF5 is one of the transcription factors crucial for the vomeronasal sensory formation.

Two Alkaloids from Bulbs of Lycoris sanguinea MAXIM. Suppress PEPCK Expression by Inhibiting the Phosphorylation of CREB.

In the fasting state, gluconeogenesis is upregulated by glucagon. Glucagon stimulates cyclic adenosine monophosphate production, which induces the expression of key enzymes for gluconeogenesis, such as cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), which are involved in gluconeogenesis through the protein kinase A/cAMP response element-binding protein (CREB) pathway. Using a luciferase reporter gene assay, a methanol extract of the bulbs of Lycoris sanguinea MAXIM. var. kiushiana Makino was found to suppress cAMP-enhanced PEPCK-C promoter activity. In addition, two alkaloids, lycoricidine and lycoricidinol, in the extract were identified as active constituents. In forskolin-stimulated human hepatoma cells, these alkaloids suppressed the expression of a reporter gene under the control of cAMP response element and also prevented increases in the endogenous levels of phosphorylated CREB and PEPCK mRNA expression. These results suggest that lycoricidine and lycoricidinol suppress PEPCK-C expression by inhibiting the phosphorylation of CREB and may thus have the potential to prevent excessive gluconeogenesis in type 2 diabetes. Copyright © 2016 John Wiley & Sons, Ltd.

N-terminal hydrophobic amino acids of activating transcription factor 5 (ATF5) protein confer interleukin 1ß (IL-1ß)-induced stabilization.

Activating transcription factor 5 (ATF5) is a stress-response transcription factor that responds to amino acid limitation and exposure to cadmium chloride (CdCl2) and sodium arsenite (NaAsO2). The N-terminal amino acids contribute to the destabilization of the ATF5 protein in steady-state conditions and serve as a stabilization domain in the stress response after CdCl2 or NaAsO2 exposure. In this study, we show that interleukin 1ß (IL-1ß), a proinflammatory cytokine, increases the expression of ATF5 protein in HepG2 hepatoma cells in part by stabilizing the ATF5 protein. The N-terminal domain rich in hydrophobic amino acids that is predicted to form a hydrophobic network was responsible for destabilization in steady-state conditions and served as an IL-1ß response domain. Furthermore, IL-1ß increased the translational efficiency of ATF5 mRNA via the 5' UTRα and phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). ATF5 knockdown in HepG2 cells up-regulated the IL-1ß-induced expression of the serum amyloid A 1 (SAA1) and SAA2 genes. Our results show that the N-terminal hydrophobic amino acids play an important role in the regulation of ATF5 protein expression in IL-1ß-mediated immune response and that ATF5 is a negative regulator for IL-1ß-induced expression of SAA1 and SAA2 in HepG2 cells.

Early postnatal alteration of body composition in preterm and small-for-gestational-age infants: implications of catch-up fat.

The concept of the developmental origins of health and disease is based on studies by Barker et al. They proposed a hypothesis that undernutrition in utero permanently changes the body's structure, function, and metabolism in ways that lead to atherosclerosis and insulin resistance in later life. In addition, profound effects on the extent of body fatness and insulin sensitivity are demonstrated, if there is a "mismatch" between prenatal and postnatal environments. In previous studies, undernutrition in utero has been evaluated simply by birth weight itself or birth weight for gestational age, and the degree of mismatch has been estimated by postnatal rapid weight gain. Recently, we investigated subcutaneous fat accumulation in small-for-gestational-age infants and found that a rapid catch-up in skinfold thickness developed prior to the body weight catch-up. Furthermore, insulin-like growth factor-I and lipoprotein lipase mass concentrations also demonstrate rapid increase during the neonatal period with fat accumulation. Investigating the precise mechanisms of developmental origins of health and disease including mediating metabolic and hormonal factors may provide a new approach to prevent atherosclerosis and insulin resistance. Better management of undernutrition during gestation and neonatal growth during the early postnatal period is an important theme for future health.

Activating transcription factor 5 is required for mouse olfactory bulb development via interneuron.

Activating transcription factor 5 (ATF5) is a stress response transcription factor of the cAMP-responsive element-binding/ATF family. Earlier, we reported that ATF5 expression is up-regulated in response to stress, such as amino acid limitation or arsenite exposure. Although ATF5 is widely expressed in the brain and the olfactory epithelium, the role of ATF5 is not fully understood. Here, the olfactory bulbs (OBs) of ATF5-deficient mice are smaller than those of wild-type mice. Histological analysis reveals the disturbed laminar structure of the OB, showing the thinner olfactory nerve layer, and a reduced number of interneurons. This is mainly due to the reduced number of bromodeoxyuridine-positive proliferating cells in the subventricular zone, where the interneuron progenitors are formed and migrate to the OBs. Moreover, the olfaction-related aggressive behavior of ATF5-deficient mice is reduced compared to wild-type mice. Our data suggest that ATF5 plays a crucial role in mouse OB development via interneuron.

Residual blood volume in the umbilical cord of extremely premature infants.

BACKGROUND: The aim of this study was to investigate residual blood volume in the umbilical cord of extremely premature infants. METHODS: Twenty extremely premature infants were held at or below the placenta while the umbilical cord was clamped and cut at approximately 2-3 cm from the umbilicus within 30 s after birth. The umbilical cord was then clamped near the placenta to obtain a length of approximately 30 cm and cut. The residual blood volume in the segment of cord was drained and measured in milliliters. RESULTS: Mean birthweight was 846 ± 172 g (range, 587-1180 g). The average length of the clamped segment of umbilical cord was 29.8 ± 1.5 cm (range, 27-32 cm). Total residual blood volume and residual blood volume per cm were 15.5 ± 6.7 mL (range, 6-25 mL) and 0.5 ± 0.2 mL/cm (range, 0.2-0.8 mL/cm), respectively. The residual cord blood volume per kilogram of infant weight per 30 cm was 17.7 ± 5.5 mL/kg/30 cm (range, 8.9-29.0 mL/kg/30 cm). CONCLUSION: Infants could receive approximately 18 mL/kg of whole blood by one-time milking of 30 cm umbilical cord. With an average hematocrit of 40%, this volume is equivalent to approximately 13 mL of packed red blood cells (hematocrit 55%).

Japanese neonate with congenital chloride diarrhea caused by SLC26A3 mutation.

Congenital chloride diarrhea (CCD) beginning in utero is a rare autosomal recessive inherited disorder characterized by impairment of Cl(-) /HCO3 (-) exchange in an otherwise normal distal ileum and colon. Life-long secretory diarrhea is caused by mutations in solute carrier family 26, member 3, (SLC26A3), which disrupt epithelial Cl(-) /HCO3 (-) transport in the ileum and colon. Although 55 mutations in SLC26A3 have been identified throughout the world, few Japanese cases have been confirmed on genetic analysis. We report the successful treatment of a Japanese neonate with CCD caused by SLC26A3 mutation.

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.

Effects of a liquid diet on the temporomandibular joint of growing rats.

OBJECTIVE: The aim of the present study was to clarify the effects of a liquid diet on the temporomandibular joint (TMJ) in growing rats. MATERIALS AND METHODS: Twenty-four male Wistar rats were weaned at 21 days and divided into control and experimental groups (12 in each group). Control rats were fed a solid diet and experimental rats were fed a liquid diet from 1 to 8 weeks. After injection with 5-bromo-2'-deoxyuridine (BrdU), the animals were perfused and the heads were removed. Serial coronal sections of the TMJ were stained with hematoxylin and eosin, or BrdU immunohistochemistry was done (12 rats in each group). Three dimensions and the thicknesses of the cartilage layers of the TMJ were measured, and cell proliferation in the TMJ was examined. RESULTS: After 4 weeks, the height and width of the mandibular fossa and the width and length of the mandibular condyle were smaller in the experimental groups than in the control groups. The cartilage layer in these areas was also thinner at 4 weeks. The BrdU levels in the intermediate zone of the mandibular fossa (at 4 weeks) and the mandibular condyle (at 1 and 4 weeks) were lower in the experimental groups than in the controls. CONCLUSION: These findings suggest that the growth of the mandibular fossa and mandibular condyle of rats was inhibited by the low proliferative activity of intermediate zone cells induced by liquid feeding.

Isolation and total syntheses of cytotoxic cryptolactones A1, A2, B1, and B2: α,ß-unsaturated δ-lactones from a Cryptomyzus sp. aphid.

The cryptolactones A1, A2, B1, and B2, which are α,ß-unsaturated δ-lactones, were isolated from a Cryptomyzus sp. aphid. The structures were established by 1-D and 2-D NMR spectra and CI-HRMS. Their absolute configurations were determined with the Kusumi-Mosher method, combined with asymmetric total syntheses. The syntheses were accomplished with the Mukaiyama aldol reaction and olefin metathesis, which utilized the second-generation Grubbs catalyst for the key steps. These compounds exhibited cytotoxic activity against human promyelocytic leukemia HL-60 cells with IC50 values of 0.97-5.3 µM.

A proposed core curriculum for dental English education in Japan.

BACKGROUND: Globalization of the professions has become a necessity among schools and universities across the world. It has affected the medical and dental professions in terms of curriculum design and student and patient needs. In Japan, where medicine and dentistry are taught mainly in the Japanese language, profession-based courses in English, known as Medical English and Dental English, have been integrated into the existing curriculum among its 83 medical and 29 dental schools. Unfortunately, there is neither a core curriculum nor a model syllabus for these courses. METHODS: This report is based on a survey, two discussion forums, a workshop, and finally, the drafting of a proposed core curriculum for dental English approved by consensus of the participants from each university. RESULTS: The core curriculum covers the theoretical aspects, including dental English terms and oral pathologies; and practical aspects, including blended learning and dentist-patient communication. It is divided into modules and is recommended to be offered for at least two semesters. CONCLUSIONS: The core curriculum is expected to guide curriculum developers in schools where dental English courses are yet to be offered or are still in their early development. It may also serve as a model curriculum to medical and dental schools in countries in Asia, Europe, Africa, and Central and South America, where English is not the medium of instruction.

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.