Antenatal Glucocorticoid Administration Promotes Cardiac Structure and Energy Metabolism Maturation in Preterm Fetuses.

Although the rate of preterm birth has increased in recent decades, a number of preterm infants have escaped death due to improvements in perinatal and neonatal care. Antenatal glucocorticoid (GC) therapy has significantly contributed to progression in lung maturation; however, its potential effects on other organs remain controversial. Furthermore, the effects of antenatal GC therapy on the fetal heart show both pros and cons. Translational research in animal models indicates that constant fetal exposure to antenatal GC administration is sufficient for lung maturation. We have established a premature fetal rat model to investigate immature cardiopulmonary functions in the lungs and heart, including the effects of antenatal GC administration. In this review, we explain the mechanisms of antenatal GC actions on the heart in the fetus compared to those in the neonate. Antenatal GCs may contribute to premature heart maturation by accelerating cardiomyocyte proliferation, angiogenesis, energy production, and sarcoplasmic reticulum function. Additionally, this review specifically focuses on fetal heart growth with antenatal GC administration in experimental animal models. Moreover, knowledge regarding antenatal GC administration in experimental animal models can be coupled with that from developmental biology, with the potential for the generation of functional cells and tissues that could be used for regenerative medical purposes in the future.

Prenatal glucocorticoid administration enhances bilirubin metabolic capacity and increases Ugt1a and Abcc2 gene expression via glucocorticoid receptor and PXR in rat fetal liver.

AIM: Jaundice is especially common in premature infant born before 35 weeks. Because the premature infant liver is not fully developed at birth it may be incomplete the bilirubin metabolism. The purpose was to evaluate the metabolism and the excretion of bilirubin in the premature infant rat liver following prenatal glucocorticoid (GC) administration. METHODS: Dexamethasone (DEX) was administered subcutaneously to pregnant Wistar rats for two consecutive days on gestational days 17 and 19. The fetus were delivered by cesarean section in gestational days 19 and 21. The mRNA levels and protein levels of bilirubin-metabolic enzymes and transporters in the fetal liver tissues were analyzed using RT-PCR immunohistochemistry staining and ELISA, respectively. We evaluated that the effect of bilirubin-metabolic enzymes in the primary fetal rat hepatocytes treated with DEX after pretreated with glucocorticoid receptor (GR, Nr3c1) and Pxr (Nr1i2) siRNA. RESULTS: Ugt1a1 and Bsep (Abcb11) mRNA levels were significantly increased in the fetuses by prenatal GC administration. The mRNA levels of nuclear transcription factors Nr1i2, Car (Nr1i3), and Rxrα (Nr2b1) were also significantly increased in the fetuses by prenatal GC administration. In addition, DEX increased Nr1i2, Ugt1a1, and Abcc2 (Mrp2) mRNA levels in the primary fetal hepatocytes. The Nr3c1 or Nr1i2 siRNA-mediated knockdown suppressed the increases of Ugt1a1, and Abcc2 mRNA levels induced by DEX, indicating that DEX are mediated by GC receptor and PXR in primary fetal hepatocytes. CONCLUSIONS: These results suggest that prenatal GC administration increases bilirubin-metabolic ability, in the premature liver, which may prevent jaundice in neonates.

Prenatal glucocorticoid administration accelerates the maturation of fetal rat hepatocytes.

BACKGROUND: Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because their livers are immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats. METHODS AND RESULTS: Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, hepatocyte nuclear factor-4 alpha (HNF4α), hepatocyte growth factor (HGF), thymus cell antigen 1 (Thy-1), cyclin B, and Cyclin-dependent kinase 1 (CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production. The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration. CONCLUSIONS: The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in preterm fetuses.

Identification of interleukin-16 production on tumor aggravation in hepatocellular carcinoma by a proteomics approach.

BACKGROUND: Cytokines play an important role in the immune response, angiogenesis, cell growth, and differentiation in hepatocellular carcinoma (HCC). OBJECTIVE: We performed a comprehensive study to identify tumor-related cytokines and pathways involved in HCC pathogenesis. METHODS: Cytokine production was evaluated in human HCC tissues and adjacent non-tumor tissues using an antibody-based protein array technique. We compared cytokine expression in HCC tissues with that of hepatic hemangioma (HH), liver metastasis of colorectal cancer, and noncancerous liver tissues from transplantation donors. The protein levels and localization of the candidate cytokines were analyzed by western blotting and immunohistochemistry. RESULTS: Increased expression of interleukin (IL)-1 receptor antagonist, macrophage migration inhibitory factor, and IL-16 was observed in HCC and paired adjacent non-tumor tissues compared with noncancerous livers. In addition, there were increased IL-16 levels in HCC tissues compared with HH. IL-16 treatment significantly increased cell proliferation in vitro. The expression of extracellular signal-regulated kinase (ERK)1/2 and cyclin D1 was markedly increased in cells from two HCC cell lines, Huh7 and HepG2, in a dose- and time-dependent manner. Phosphorylated to total ERK1/2 ratio was increased in Huh7 cells following IL-16 50 ng/ml, but not HepG2 cells. ERK phosphorylation have occurred earlier than protein accumulation at 48 h. Pretreatment with the ERK inhibitor, FR18024, or an anti-IL-16 antibody reduced the increase in IL-16 production in HCC cells. CONCLUSIONS: These results suggest that cell proliferation induced by IL-16 is mediated through the ERK pathway, thus, we identified a new factor associated with HCC tumor growth.

Cinacalcet corrects biased allosteric modulation of CaSR by AHH autoantibody.

Biased agonism is a paradigm that may explain the selective activation of a signaling pathway via a GPCR that activates multiple signals. The autoantibody-induced inactivation of the calcium-sensing receptor (CaSR) causes acquired hypocalciuric hypercalcemia (AHH). Here, we describe an instructive case of AHH in which severe hypercalcemia was accompanied by an increased CaSR antibody titer. These autoantibodies operated as biased allosteric modulators of CaSR by targeting its Venus flytrap domain near the Ca2+-binding site. A positive allosteric modulator of CaSR, cinacalcet, which targets its transmembrane domain, overcame this autoantibody effect and successfully corrected the hypercalcemia in this patient. Hence, this is the first study to our knowledge that identifies the interaction site of a disease-causing GPCR autoantibody working as its biased allosteric modulator and demonstrates that cinacalcet can correct the AHH autoantibody effects both in vitro and in our AHH patient. Our observations provide potentially new insights into how biased agonism works and how to design a biased allosteric modulator of a GPCR. Our observations also indicate that the diagnosis of AHH is important because the severity of hypercalcemia may become fatal if the autoantibody titer increases. Calcimimetics may serve as good treatment options for some patients with severe AHH.

Vasoactive intestinal peptide increases apoptosis of hepatocellular carcinoma by inhibiting the cAMP/Bcl-xL pathway.

Vasoactive intestinal peptide (VIP) is a modulator of inflammatory responses. VIP receptors are expressed in several tumor types, such as colorectal carcinoma. The study described herein was conducted to confirm the presence of VIP and its receptors (VPAC1 and VPAC2) in surgically resected hepatocellular carcinoma (HCC) tissues and in the HCC cell line Huh7. The mechanism responsible for apoptosis of HCC cells was then examined because VIP treatment (10-10  M) significantly suppressed proliferation of Huh7 cells. In examining apoptosis-related proteins, we found caspase-3 to be significantly increased and Bcl-xL and cyclic AMP (cAMP) response element-binding protein (CREB) to be significantly decreased in Huh7 cells cultured with VIP. Furthermore, the CREB level and phosphorylation were reduced. These effects were reversed by the addition of VIP receptor antagonist or cAMP antagonist Rp-cAMPS. Pretreatment with cAMP analogue blocked the increased apoptosis, suggesting that VIP induces apoptosis via a PKA-independent signaling mechanism. Our data indicate that VIP prevents the progression of HCC by apoptosis through the cAMP/Bcl-xL pathway.

Exposure of immature rat heart to antenatal glucocorticoid results in cardiac proliferation.

BACKGROUND: ATP synthesis and cardiac contraction-related protein production are accelerated in the immature fetal heart by antenatal glucocorticoids (GC). This study investigated the structural maturity of the myocardium and underlying signal pathway associated with cardiac growth in fetal rats that received antenatal GC. METHODS AND RESULTS: Dexamethasone (DEX) was given to pregnant rats for 2 days from day 17 or day 19 of gestation, and the hearts of 19 and 21 day fetuses and 1-day-old neonates were analyzed. Although irregular myofibril orientation was observed morphologically in 19 day fetal hearts, the myofibril components were organized in fetuses after DEX. The cross-sectional area of the myocardium and Ki-67-positive cells were significantly increased in fetal DEX groups, suggesting that cardiac enlargement resulted from myocyte proliferation. Glycogen synthase kinase-3ß (GSK-3ß) protein was significantly decreased in fetal DEX groups. ß-Catenin and vascular endothelial growth factor protein were also significantly increased. Furthermore, increased cardiomyocyte proliferation appeared to be mediated by GC receptors after culture with DEX in vitro. CONCLUSIONS: Antenatal DEX induces structural maturity accompanying cardiomyocyte proliferation in the premature fetal rat heart, and GSK-3ß and ß-catenin are thought to contribute to cardiac growth.

Dysregulation of miRNA in chronic hepatitis B is associated with hepatocellular carcinoma risk after nucleos(t)ide analogue treatment.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). Nucleos(t)ide analogue (NA) therapy effectively reduces the incidence of HCC, but it does not completely prevent the disease. Here, we show that dysregulation of microRNAs (miRNAs) is involved in post-NA HCC development. We divided chronic hepatitis B (CHB) patients who received NA therapy into two groups: 1) those who did not develop HCC during the follow-up period after NA therapy (no-HCC group) and 2) those who did (HCC group). miRNA expression profiles were significantly altered in CHB tissues as compared to normal liver, and the HCC group showed greater alteration than the no-HCC group. NA treatment restored the miRNA expression profiles to near-normal in the no-HCC group, but it was less effective in the HCC group. A number of miRNAs implicated in HCC, including miR-101, miR-140, miR-152, miR-199a-3p, and let-7g, were downregulated in CHB. Moreover, we identified CDK7 and TACC2 as novel target genes of miR-199a-3p. Our results suggest that altered miRNA expression in CHB contributes to HCC development, and that improvement of miRNA expression after NA treatment is associated with reduced HCC risk.

Isolation of Adipose-Derived Stem/Stromal Cells from Cryopreserved Fat Tissue and Transplantation into Rats with Spinal Cord Injury.

Adipose tissue contains multipotent cells known as adipose-derived stem/stromal cells (ASCs), which have therapeutic potential for various diseases. Although the demand for adipose tissue for research use remains high, no adipose tissue bank exists. In this study, we attempted to isolate ASCs from cryopreserved adipose tissue with the aim of developing a banking system. ASCs were isolated from fresh and cryopreserved adipose tissue of rats and compared for proliferation (doubling time), differentiation capability (adipocytes), and cytokine (hepatocyte growth factor and vascular endothelial growth factor) secretion. Finally, ASCs (2.5 × 106) were intravenously infused into rats with spinal cord injury, after which hindlimb motor function was evaluated. Isolation and culture of ASCs from cryopreserved adipose tissue were possible, and their characteristics were not significantly different from those of fresh tissue. Transplantation of ASCs derived from cryopreserved tissue significantly promoted restoration of hindlimb movement function in injured model rats. These results indicate that cryopreservation of adipose tissue may be an option for clinical application.

Evaluation of CYP2D6 Protein Expression and Activity in the Small Intestine to Determine Its Metabolic Capability in the Japanese Population.

CYP2D6 plays an important role in the metabolism of many drugs such as opioids and antidepressants. Polymorphisms of the CYP2D6 gene are widely observed in the Japanese population, and can affect the first-pass metabolism of orally administered drugs. Several CYP enzymes have been identified in the small intestine of Caucasians, but intestinal CYP enzymes have not been reported in the Japanese population, except for CYP3A4 and CYP2C19. In this study, we evaluated the CYP2D6 metabolic capacity by measurement of CYP2D6 mRNA and protein levels and activity in the small intestine of Japanese individuals. Normal jejunal tissues were obtained from 31 patients who had undergone pancreaticoduodenectomy, and the CYP2D6*10 variant was identified in these tissues. CYP2D6 mRNA and CYP2D6 protein levels were analyzed using real-time RT-PCR and Western blotting, respectively. Bufuralol 1'-hydroxylation, a marker of CYP2D6 activity, was analyzed using HPLC. Frequencies of the CYP2D6*1/*1, *1/*10, and *10/*10 genotypes in the jejunal tissue were 29.0% (n=9), 35.5% (n=11), and 35.5% (n=11), respectively. CYP2D6 protein and activity levels did not differ significantly between the genotypes. A positive correlation was found between CYP2D6 protein and activity levels. Furthermore, CYP2D6 protein levels and activity in the small intestine were significantly lower than those in the liver. These findings suggest that the metabolic capacity of CYP2D6 in the small intestine of the Japanese population has a relatively small effect on drug metabolism.

Intravenous infusion of adipose-derived stem/stromal cells improves functional recovery of rats with spinal cord injury.

BACKGROUND AIMS: Adipose tissue has therapeutic potential for spinal cord injury (SCI) because it contains multipotent cells known as adipose-derived stem/stromal cells (ASCs). In this study, we attempted intravenous ASC transplantation in rats with SCI to examine the effect on functional recovery. METHODS: ASCs (2.5 × 106) were intravenously infused into SCI rats, after which hindlimb motor function was evaluated. Distribution of transplanted ASCs was investigated and growth factor/cytokine levels were determined. RESULTS: Intravenous transplantation of ASCs promoted the functional recovery in SCI rats and reduced the area of spinal cord cavitation. A distribution study revealed that ASCs gradually accumulated at the site of injury, but long-term survival of these cells was not achieved. Levels of growth factors increased only slightly in the spinal cord after ASC transplantation. Unexpectedly, cytokine-induced neutrophil chemoattractant (CINC)-1 showed a transient but substantial increase in the spinal cord tissue and blood of the ASC group. CINC-1 was secreted by ASCs in vitro, and the sponge implantation assay showed that CINC-1 and ASCs induced angiogenesis. CINC-1 promoted functional recovery in SCI rats, which was similar to the ASCs. Expression of glial cell line-derived neurotrophic factor was greater in the ASC group than in the CINC-1 group, although both promoted extracellular signal-regulated kinase (ERK)1/2 phosphorylation; Akt phosphorylation was enhanced in the spinal cord after ASC transplantation. CONCLUSIONS: Our findings indicated that intravenously transplanted ASCs gradually accumulated in the injured spinal cord, where cytokines such as CINC-1 activated ERK1/2 and Akt, leading to functional recovery.

The Rho kinase inhibitor fasudil is involved in p53-mediated apoptosis in human hepatocellular carcinoma cells.

PURPOSE: Rho kinase is an important factor in tumor progression. We demonstrated that Rho kinase-associated coil-containing protein kinase (ROCK) is expressed in hepatic tissues in hepatocellular carcinoma (HCC) and confirmed its roles in cell survival in HCC cells using the ROCK inhibitor, fasudil. METHODS: ROCK protein levels were estimated in hepatic tissues with HCC compared with healthy liver tissues or hepatic hemangioma tissues using immunohistochemistry. Furthermore, HepG2 and Huh7 cells were cultured with ROCK inhibitor, fasudil for 24 h in vitro. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, and apoptotic cells were detected by cell death ELISA. The expression apoptosis-related proteins were analyzed using Western blotting. RESULTS: Fasudil significantly decreased cell proliferation and induced apoptosis mediated by increases in p53, Bax, caspase-9, and caspase-3 in HepG2 and Huh7 cells. The induction of apoptosis was inhibited in HCC cells precultured with p53 decoy oligodeoxynucleotide. CONCLUSION: These results suggest that ROCK inhibits the p53-mediated apoptosis pathway in HCC. Fasudil may thus be a beneficial approach to HCC therapy.

Comparison of the effects of omeprazole and rabeprazole on ticlopidine metabolism in vitro.

The thienopyridine derivative ticlopidine (TCL) is an inhibitor of adenosine diphosphate-induced platelet aggregation. Combination therapy with a thienopyridine derivative and aspirin is standard after coronary stenting, although more hemorrhagic complications occur with the combination therapy than with aspirin alone. A proton pump inhibitor (PPI) is required for prevention or treatment of upper gastrointestinal bleeding in such cases. We examined the effects of PPIs [omeprazole (OPZ) and rabeprazole (RPZ)] on TCL metabolism using pooled human liver microsomes prepared from various human liver blocks and 12 individual human liver microsomes. We calculated the K(i) values of each PPI for TCL metabolic activity and compared the inhibitory effect of each PPI on TCL metabolism. The K(i) values of OPZ and RPZ were 1.4 and 12.7 µM, respectively. The inhibitory effect of OPZ (78.6 ± 0.05%) was significantly greater than that of RPZ (24.2 ± 0.05%) (P < 0.001). Interestingly, a negative correlation existed between the inhibitory effect of OPZ and CYP2C19 activity (r = -0.909, P < 0.001). These results suggest that the inhibitory effect of OPZ is more potent than that of RPZ in vitro. In conclusion, RPZ appears preferable when administering TCL, aspirin, and a PPI in combination.

Orange juice and its component, hesperidin, decrease the expression of multidrug resistance-associated protein 2 in rat small intestine and liver.

We investigated the effects of orange juice (OJ) or hesperidin, a component of OJ, on the pharmacokinetics of pravastatin (PRV) and the expression of both protein and mRNA of multidrug resistance-associated protein 2 (Mrp2) in the rat small intestine and liver. Eight-week-old male Sprague-Dawley rats were used in this study. OJ or a 0.079% hesperidin suspension was administered orally for 2 days. Tap water was given as a control. A single dose of PRV at 100 mg/kg p.o. was administered after 2 days of OJ, hesperidin, or tap water ingestion. The AUC, C(max), and t(1/2) values of PRV were significantly increased in OJ group. Mrp2 protein and mRNA levels in the small intestine and liver, respectively, were significantly decreased after the ingestion of OJ. The same results were obtained with hesperidin. These results suggest that the changes in PRV pharmacokinetic parameters and the decrease in Mrp2 expression caused by OJ are due to hesperidin in the juice.

Comparative study of culture conditions for maintaining CYP3A4 and ATP-binding cassette transporters activity in primary cultured human hepatocytes.

The aim of this study was to determine suitable culture conditions for maintaining the activity of cytochrome p450 (CYP) 3A4 and drug transporters in primary cultured human hepatocytes. Human hepatocytes were isolated using the two-step collagenase perfusion technique and were cultured with four different media, serum-free William's E medium (serum-free WEM), WEM containing fetal calf serum (FCS-WEM), WEM with human serum (HS-WEM), and Lanford's medium. The albumin levels were maintained for 7 days in hepatocytes. Although CYP3A4 mRNA levels gradually decreased from 3 days, CYP3A4 and hepatocyte nuclear factor-4α alpha protein levels and activities were maintained for 7 days in hepatocytes cultured with serum-free WEM and Lanford's but not in those with FCS-WEM and HS-WEM. Furthermore, CYP3A4 protein levels were significantly increased by the addition of rifampicin and dexamethasone to the culture media, indicating that the induction potential was maintained. The protein levels of P-glycoprotein, multi-drug-resistance-2, and breast cancer-resistance protein were maintained for 7 days in all media. Serum-free WEM and Lanford's also maintained protein levels of CYP2C19, CYP2D6, and organic anion transporter polypeptide in the hepatocytes. Serum-free WEM and Lanford's may be appropriate culture media for maintaining CYP3A4 and drug transporter protein levels in primary cultured hepatocytes.

Individual metabolic capacity evaluation of cytochrome P450 2C19 by protein and activity in the small intestinal mucosa of Japanese pancreatoduodenectomy patients.

Some P450 enzymes are expressed not only in the liver but also in the small intestine, and these enzymes play an important role in first-pass drug metabolism in the small intestine. Cytochrome P450 (CYP)2C19 has been confirmed to exist in the small intestine of white people, but not yet in Japanese. We investigated the mRNA level, protein level, and activity of CYP2C19 in the small intestine in a Japanese population. Samples were obtained from the healthy portions of resected small intestines from 18 patients who had undergone pancreatoduodenectomy. The microsomes were extracted from the epithelium of the small intestinal tissues. CYP2C19 mRNA and protein levels were analyzed using real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CYP2C19 activity in the microsomes was evaluated based on the 5-hydroxylation of lansoprazole using HPLC. CYP2C19 mRNA and protein levels and activities in the small intestine showed interindividual differences. CYP2C19 mRNA levels were not correlated with protein levels or its activity. On the other hand, there was significant correlation between CYP2C19 protein levels and its activity. Further, CYP2C19 protein levels and activities in the small intestine were approximately equal to those in liver. These results suggest the metabolic capacity of CYP2C19 in Japanese small intestine may play as important a role as the liver in drug metabolism. Analyses of the protein level or protein activity of CYP2C19 rather than its mRNA level should be required for predicting the individual metabolic capacity of CYP2C19 in the small intestine.

Antenatal glucocorticoid therapy accelerates ATP production with creatine kinase increase in the growth-enhanced fetal rat heart.

BACKGROUND: Previous study has demonstrated the increase of several cardiac function-related proteins, including creatine kinase (CK) as an important enzyme in the process of ATP synthesis in the fetal heart of rats administered glucocorticoid (GC) antenatally. In the present study the effect of antenatal GC administration on the CK expression in fetal and neonatal hearts was demonstrated. METHODS AND RESULTS: Dexamethasone was administered to pregnant rats on days 19 and 20 of gestation. The mRNA levels of the CK isoforms, CK-M and Mi-CK, in 21-day-old fetal and 1-day-old neonatal hearts were significantly increased after antenatal GC administration. CK protein levels were also increased in both cultured cardiomyocytes and the mitochondria of the hearts. Uptake of 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-benzimidazolocarbocyanine iodide by mitochondria was significantly increased. An increased ATP level accompanied the CK increase in the neonatal hearts. Furthermore, in vitro these effects were mediated though the GC receptor of cardiomyocytes. Peroxisome proliferator-activated receptor gamma as the upstream transcription factor of CK was significantly increased in fetal hearts. CONCLUSIONS: These results suggest that antenatal GC administration accelerates ATP synthesis through increased CK and may contribute to maturation of the premature heart so that it is ready for preterm delivery. (Circ J 2010; 74: 171 - 180).

Potential of lansoprazole as a novel probe for cytochrome P450 3A activity by measuring lansoprazole sulfone in human liver microsomes.

Cytochrome P450 (CYP) 3A enzymes are responsible for the metabolism of many drugs. It is useful to know CYP3A activity in individual patients undergoing drug therapy so as to predict the efficacies or adverse events. Lansoprazole is metabolized to Lansoprazole sulfone (LS) by CYP3A, while to 5-hydroxylansoprasole by CYP2C19. The aim of this study was to evaluate whether lansoprazole can be used to assess CYP 3A activity in human liver. Lansoprazole sulfoxidation activity in 14 human liver microsomes was determined as the ratio of lansoprazole/LS, measuring these parameters by high-performance liquid chromatography. Testosterone 6beta-hydroxylation (T6beta-OH) activity, a known marker for CYP3A activity was also measured together with lansoprazole sulfoxidation activity. Lansoprazole sulfoxidation activity was also analyzed in microsomes preincubat-ed with anti-CYP2C19 antibody. Interindividual variation was observed in lansoprazole sulfoxidation activity and T6beta-OH activities of those microsomes, respectively. Lansoprazole sulfoxidation activity was significantly correlated with T6beta-OH activity and CYP3A protein level. Lansoprazole sulfoxidation activity in microsomes with anti-CYP2C19 antibody was closely correlated with T6beta-OH activity. In contrast, lansoprazole 5-hydroxylation activity was correlated with the CYP2C19 activity. These results suggest that metabolism of lansoprazole to LS by CYP3A occurs independently of metabolism by CYP2C19. LS can be used as a new marker of CYP3A activity.

Antenatal glucocorticoid therapy increase cardiac alpha-enolase levels in fetus and neonate rats.

AIMS: Antenatal glucocorticoid therapy has been shown to prevent acute diseases including infant respiratory distress syndrome and reduce mortality, although little is known about the effects on cardiac function-related proteins in the fetus or neonate. We investigated whether cardiac function-related proteins were altered in cardiac tissues of fetuses and neonates born to pregnant rats treated by glucocorticoid. MAIN METHODS: Dexamethasone (DEX) was administered to pregnant rats for 2 days on day 17 and 18 or day 19 and 20 of gestation to simulate antenatal DEX therapy, and cardiac tissues of 19- and 21-day fetuses and 1-, 3-, and 5-day neonates were analyzed using a proteomic technique with liquid chromatography-mass spectrometry/mass spectrometry. KEY FINDINGS: The identified five proteins; alpha-enolase, creatine kinase-M type, beta-tubulin, troponin T, and ATP synthase beta-chain, were significantly increased in fetal cardiac tissues with DEX administration. We observed that significant increase of alpha-enolase in the 19-day fetuses by DEX using Western blotting and immunohistochemistry. ATP and cAMP levels were also increased in the fetal heart tissue. In addition, pyruvate levels were significantly increased in the fetus groups by DEX. SIGNIFICANCE: These results suggest that increased alpha-enolase may contribute to acceleration of glycolysis in the preterm heart.

Effects of antenatal dexamethasone on antioxidant enzymes and nitric oxide synthase in the rat lung.

We investigated the effects of prenatal dexamethasone (DEX) administration on antioxidant enzymes (AOEs) and nitric oxide synthase (NOS) in fetal and neonatal rat lungs. DEX (1 mg/kg, s.c., for 2 days) or vehicle alone was administered to pregnant rats, and the lungs of fetuses on days 19 and 21 of gestation and of 1- and 3-day-old neonates were examined. We measured protein levels of the AOEs manganese superoxide dismutase and copper-zinc superoxide dismutase (Mn SOD and Cu-Zn SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and inducible and endothelial nitric oxide synthase (i-NOS and e-NOS). Mn SOD, GSH-Px, and e-NOS expression gradually increased with increasing gestational and postnatal age in the lungs of the control groups. Cu-Zn SOD, CAT, and i-NOS expression did not change with increasing gestational and postnatal age in the lungs of the control groups. DEX administration had significant effects on i-NOS and e-NOS protein and mRNA expression. The increased Mn SOD, GSH-Px, and e-NOS expressions during the perinatal period suggests that antenatal developmental changes in AOEs in the lungs of premature fetuses could be reduced by reactive oxygen species-mediated injury at birth. Furthermore, antenatal glucocorticoid treatment may accelerate the development of lungs via the two types of NOS.