Iloprost for the attenuation of ischaemia/reperfusion injury in a distant organ

The objective of this study was to investigate antioxidant and cytoprotective properties of iloprost in a distant organ after ischaemia reperfusion injury. Male Wistar rats were divided into two groups. After application of anesthaesia both hindlimbs were occluded. A 2-h reperfusion procedure was carried out after 60 min of ischemia. Study group (STU) rats (n=10) received 10 microg kg(-1) iloprost in 1 ml of saline from the tail vein 10 min before reperfusion. Control (CON) group rats (n=10) received an equal amount of saline. The rats were sacrificed by injection of a high dose of thiopentone sodium. Blood and tissue samples (right kidneys) were taken for analysis. Differences in malondialdehyde (MDA), myeloperoxidase (MPO), Na+-K+ ATPase and total antioxidant capacity (TAC) between the groups were analysed. MPO, MDA and TAC levels in the sera of CON and STU groups were 1.60+/-0.26 U l(-1), 11.42+/-5.23 nmol ml(-1), 8.30 x 10(-2)+/- 3.93 x 10(-2) nmol ml(-1) h(-1) and 1.07+/-0.11 U l(-1), 7.60+/-1.81 nmol ml(-1) and 0.15+/-3.23 x 10(-2) nmol ml(-1) h(-1) (p=0.0001, p=0.043 and p=0.0001 respectively). MPO, ATPase and MDA levels in kidneys for CON and STU groups were 1.24+/-0.58 U g(-1), 85.70+/-52.05 nmol mg(-1), 17.90+/-7.40 nmol ml(-1) and 0.78+/-0.31 U g(-1), 195.90+/-56.13 nmol mg(-1) and 10.10+/-0.99 nmol ml(-1) (p=0.046, p=0.0001 and p=0.009 respectively). When given prior to reperfusion, the positive effect of iloprost in the attenuation of distant organ reperfusion injury has been demonstrated.     

Metabolic differences between attached and free-living marine bacteria: inadequacy of liquid cultures for describing in situ bacterial activity

Marinobacter sp. strain CAB was cultivated with or without porous glass beads as solid support. Two substrates were used: the hydrophilic sodium lactate and a hydrophobic C(18)-isoprenoid ketone (6,10,14-trimethylpentadecan-2-one (TMP)). The substrate adsorption onto the beads was measured. Bacterial adhesion was determined by a direct count technique and amounted to 70% of total cells. In the immobilised cell cultures (ICC), generation times were 1.5 and 1.8 times shorter than in the planktonic cultures (FCC) with sodium lactate and with TMP, respectively. In ICC, the growth yields were lower (15.3(FCC) x 10(9) and 0.8(ICC) x 10(9) bacteria mg(-1) of sodium lactate; 50(FCC) x 10(9) and 35(ICC) x 10(9) bacteria mg(-1) of TMP). The mineralisation of substrates was estimated after mass spectrometric determination of the CO2 production rates of both free and immobilised cell cultures. The results indicated a higher specific CO2 production rate in the ICC with sodium lactate (3.1(FCC)+/-0.2 and 3.5(ICC)+/-0.3 nmol CO2 mg(-1) protein min(-1)) but not in the ICC with TMP (1.9(FCC)+/-0.7 and 0.5(ICC)+/-0.3 nmol CO2 mg(-1) protein min(-1)). The affinities for the two substrates were lower in the presence of the solid support (K(m,ICC)=18.2+/-0.2 microM and 37.1+/-2.0 microM, for sodium lactate and TMP, respectively) than without support (K(m,FCC)=8.5+/-1.5 microM and 8.4+/-1.2 microM, for sodium lactate and TMP, respectively). Moreover, the presence of a solid support showed a lower inhibition by the TMP (K(i,FCC)=3.8+/-1.0 microM and K(i,ICC)=12.2+/-2.5 microM) which may explain why the immobilised cell cultures degraded hydrophobic TMP more efficiently than the planktonic cultures.     

Magnesium uptake by intestinal brush-border membranes of spontaneously hypertensive rats.

Magnesium uptake by intestinal brush-border membranes (BBM) was studied in duodenal and jejunal vesicles of the spontaneously hypertensive rat (SHR) and normotensive control, the Wistar-Kyoto (WKY) rat. In the duodenum, no statistical difference was evidenced between the two types of rats. By contrast, initial rates of magnesium uptake in jejunal vesicles were lower in SHR (5.4 +/- 2.1 nmol/mg protein x 10 sec) in comparison to WKY rats (11.0 +/- 2.5 nmol/mg protein x 10 sec) at a magnesium concentration of 1 mM (P less than 0.01). In jejunal BBM, kinetic analysis of magnesium uptake showed three components in WKY rats, with one being diffusional. In SHR, only two components were seen, with the diffusional one being absent. The two saturable components showed Vmax of 6.5 +/- 1.3 and 26.2 +/- 6.0 nmol/mg protein x 10 sec and apparent Km of 0.22 +/- 0.12 mM and 1.9 +/- 0.4 mM in WKY rats, and Vmax of 10.9 +/- 3.5 and 14.8 +/- 5.9 nmol/mg protein x 10 sec and apparent Km of 0.43 +/- 0.23 mM and 1.3 +/- 0.2 mM in SHR. Only the component with the lowest apparent affinity appeared statistically different in SHR as compared with WKY rats for both Vmax and apparent Km (P less than 0.05). Time course evolution of magnesium uptake in jejunal BBM indicated, by extrapolation at zero time, that 2.5 and 5.1 nmol magnesium/mg protein in SHR and WKY rats, respectively, would be in the bound state. The study of the influence of medium osmolarity on 60-min magnesium uptakes was also indicative of a smaller binding compartment in jejunal BBM of SHR (3.70 and 8.26 nmol/mg protein in SHR and WKY rats, respectively); at the four osmolarities assayed, the 60-min uptakes were significantly lower in SHR as compared with WKY rats (P less than 0.01). From 60-min glucose uptakes, a smaller volume of jejunal BBM vesicles was determined for SHR as compared with WKY rats (0.34 +/- 0.06 and 0.63 +/- 0.17 microliter/mg of protein in SHR and WKY rats respectively, P less than 0.05), this volume being significantly augmented by the presence of 1 mM MgCl2 (0.48 +/- 0.05 and 1.27 +/- 0.02 microliter/mg of protein in SHR and WKY rats respectively, P less than 0.01). These results suggest that magnesium uptake and binding by jejunal BBM are altered in SHR in comparison to WKY rats, implying a possible role of the small intestine in the abnormalities of magnesium metabolism in genetic hypertension.     

Simultaneous determination of N-acetylaspartic acid, N-acetylglutamic acid, and N-acetylaspartylglutamic acid in whole brain of 3-mercaptopropionic acid-treated rats using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

The measurement of N-acetylaspartic acid (NAA), N-acetylglutamic acid (NAG), and N-acetylaspartylglutamic acid (NAAG) in the whole brain of 3-mercaptopropionic acid (3-MPA)-treated rats has been developed using liquid chromatography-mass spectrometry with an atmospheric pressure ionization interface system. The recoveries of these compounds were 90.85 +/- 3.43% for NAA, 91.62 +/- 5.47% for NAG, and 92.29 +/- 4.44% for NAAG. The detection limits for NAA, NAG, and NAAG were 12, 15, and 20 microg/ml, respectively. After administration of 3-MPA, the concentrations of NAA, NAG, and NAAG in the whole brain over 10 min increased 177.25, 134.23, and 127.70%, respectively. These concentrations then decreased over the next 60 min. The simultaneous determination of NAA, NAG, and NAAG using this method was found to be very useful for studies of metabolism of NAA, NAG, and NAAG in biological samples.     

Preservation of myocardial function after adenoviral gene transfer in isolated myocardium

Adenoviral gene transfer to the heart represents a promising model for structure-function analyses. Rabbit hearts were subjected to an ex vivo perfusion protocol that achieves gene transfer in >90% of cardiac myocytes. Contractile function of isolated myocardial preparations of these hearts was then observed for 2 days in a recently developed trabecula culture system. In sham-infected hearts, the initial developed force (F(init)) (15.6 +/- 3.7 mN/mm(2); n = 12) did not change significantly after 48 h (17.0 +/- 1.9 mN/mm(2); P = 0.46). In adenovirus-infected preparations, F(init) (14.3 +/- 1. 8 mN/mm(2); n = 21) did not significantly differ from the control (P = 0.75) and was unchanged after 48 h (15.3 +/- 2.5 mN/mm(2); P = 0. 93). After 2 days of continuous contractions, we observed homogenous and high-level expression of the reporter genes LacZ coding for beta-galactosidase and Luc coding for firefly luciferase. Luciferase activity increased more than 2,500-fold from background levels of 8. 7 x 10(3 )+/- 5.0 x 10(3) relative light units (RLU)/mg protein (from hearts transfected with promotorless adenovirus with luciferase transgene construct AdNULLLuc, n = 5) to 23.4 x 10(6)+/- 11.1 x 10(6)RLU/mg protein (from hearts tranfected with adenovirus with Rous sarcoma virus promotor and luciferase transgene construct AdRSVLuc, n = 5) in infected myocardial preparations (P < 0.005). Our results demonstrate a new ex vivo approach to achieve homogenous and high-level expression of recombinant adenoviral genes in contracting myocardium without adverse functional effects.     

Suppressive effect of coenzyme Q10 on phospholipase A2 activation in cardiac cells after prolonged swimming.

Phospholipase A2 (PLA2) activity is elevated in cardiac microsomal fractions and phospholipids (PL) are much reduced in both the cardiac mitochondria and microsomal fractions from rats subjected to prolonged swimming. Preadministration of coenzyme Q10 (CoQ10 i.v. 30 mg/kg) significantly suppressed these changes. Two groups of 8-week-old male Wistar rats were trained to swim, receiving 30 min of training for 4 days. On the fifth day they were given an intravenous injection of either 30 mg/kg CoQ10 in saline or 1 ml saline. Thirty minutes later they began to swim for 3 hours carrying a weight representing 3% of body weight. On completion of the swim they were sacrified by instantaneous decapitation, and cardiac mitochondria were isolated. Mitochondria were also prepared from saline injected, unexercised control rats. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) concentrations were measured with HPLC and PLA2 activity was assayed fluorometrically. The mitochondrial concentrations (means +/- SEM, n = 6) of PE and PC were respectively 126 +/- 22 and 140 +/- 22 nmol/mg protein in the exercise-CoQ10 group against 66 +/- 4 and 50 +/- 10 nmol/mg protein in the exercise-saline group. The specific PLA2 activities (expressed as nmol degraded dipyrene phosphorylethanolamine substrate/hr/mg protein) in the microsomes was 0.20 +/- 0.02 in the exercise-CoQ10 group against 0.30 +/- 0.02 in the exercise-saline group. These results suggest CoQ10 has a protective effect against an excessive reduction in mitochondrial membrane phospholipids during prolonged exercise.     

N-Acetyl-Aspartyl-Glutamate: Regional Levels in Rat Brain and the Effects of Brain Lesions as Determined by a New HPLC Method

Abstract: An isocratic HPLC method to measure endogenous N -acetyl-aspartyl-glutamate (NAAG) and N -acetyl-aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG-50 resin, the eluate was subject to HPLC anion-exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10-fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsi-lateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain. Combined with the fact that NAAG interacts with a subpopulation of glutamate receptors, these results suggest that NAAG may serve as an excitatory neurotransmitter.     

Competitive Inhibition of N-Acetylated-α-Linked Acidic Dipeptidase Activity by N-Acetyl-L-Aspartyl-β-Linked L-Glutamate

The endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) fulfills several criteria required to be accepted as a neurotransmitter. NAAG inactivation may proceed through enzymatic hydrolysis into N-acetyl-L-aspartate and glutamate by an N-acetylated-alpha-linked acidic dipeptidase (NAALADase). Therefore, some properties of NAALADase activity were investigated using crude membranes from the rat forebrain. Kinetic parameters of the hydrolysis of [Glu-3H]NAAG were determined first (Km = 0.40 +/- 0.05 microM; Vmax = 155 +/- 20 pmol/min/mg of protein). The enzymatic activity, i.e., NAALADase, was inhibited noncompetitively by the glutamatergic agonist quisqualate (Ki = 1.9 +/- 0.3 microM), and competitively by N-acetyl-L-aspartyl-beta-linked L-glutamate (beta-NAAG; Ki = 0.70 +/- 0.05 microM). To determine whether glutamate-containing dipeptides, such as NAAG, beta-NAAG, N-acetyl-L-aspartyl-D-glutamate, L-aspartyl-L-glutamate, L-alanyl-L-glutamate, L-glutamyl-L-glutamate, and L-glutamyl-gamma-linked L-glutamate, were substrates of NAALADase, rat brain membranes were immobilized on a C-8 column. Thus, endogenous trapped glutamate was washed away and formation of unlabelled glutamate could be estimated using an o-phthaldialdehyde/reverse-phase HPLC detection procedure. beta-NAAG was shown to be a nonhydrolyzable competitive inhibitor of NAALADase. L-Aspartyl-L-glutamate was hydrolyzed faster than NAAG, suggesting that the acetylated moiety is not essential for NAALADase specificity. Rat brain membranes also contained nonspecific peptidase activities (insensitive to both quisqualate and beta-NAAG), which, in the case of L-alanyl-L-glutamate, for instance, accounted for all observed hydrolysis.     

Postmortem Degradation of N-Acetyl Aspartate and N-Acetyl Aspartylglutamate: An HPLC Analysis of Different Rat CNS Regions

N-acetyl aspartate (NAA), a putative marker of neuronal injury, can be measured non-invasively in patients by magnetic resonance spectroscopy (MRS). Interpretation of in vivo MRS data, however, requires neuropathological correlates to NAA alterations using autopsy or biopsy material. Since detailed hydrolysis data is lacking, NAA and the related dipeptide N-acetyl aspartylglutamate (NAAG) were quantified by high performance liquid chromatography (HPLC) in different rat CNS regions over 24 h postmortem. Both molecules decreased rapidly 1-4 h postmortem, and subsequently slower with time. The average reduction at 24 h was 46% and 38% for NAA and NAAG respectively. The NAA reduction was proportionally smaller in cortical areas (34-37%) compared to more caudal regions (54-58%). An exception was the optic nerve, a pure white matter tract, where NAA and NAAG hydrolysis was slower. The NAA/NAAG ratio remained relatively constant, but exhibited marked regional differences. The data show a significant postmortem degradation of NAA and NAAG that needs to be considered when these compounds are studied ex-vivo.     

Quantitative determination of Ca2+-dependent Mg2+-ATPase from sarcoplasmic reticulum in muscle biopsies.

The possibility of quantifying the total concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum was investigated by measurement of the Ca2+-dependent steady-state phosphorylation from [gamma-32P]ATP and the Ca2+-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity in crude muscle homogenates. The Ca2+-dependent phosphorylation at 0 degree C (mean +/- S.E.) was 40.0 +/- 2.5 (n = 6) and 6.2 +/- 0.7 (n = 4) nmol/g wet wt. in rat extensor digitorum longus (EDL) and soleus muscle, respectively (P less than 0.001). The Ca2+-dependent 3-O-MFPase activity at 37 degrees C was 1424 +/- 238 (n = 6) and 335 +/- 56 (n = 4) nmol/min per g wet wt. in rat EDL and soleus muscle, respectively (P less than 0.01). The molecular activity calculated from these measurements amounted to 35 +/- 5 min-1 (n = 6) and 55 +/- 10 min-1 (n = 4) for EDL and soleus muscle respectively. These values were not different from the molecular activity calculated for purified Ca2+-ATPase (36 min-1). The Ca2+-dependent 32P incorporation in soleus muscle decreased in the order mice greater than rats greater than guinea pigs. In EDL muscles from hypothyroid rats at a 30% reduction of the Ca2+-dependent phosphorylation was observed. The Ca2+-dependent phosphorylation in vastus lateralis muscle from three human subjects amounted to 4.5 +/- 0.8 nmol/g wet wt. It is concluded that measurement of the Ca2+-dependent phosphorylation allows rapid and reproducible quantification of the concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum. Since only 20-60 mg of tissue is required for the measurements, the method can also be used for biopsies obtained in clinical studies.     

Opposite changes in imidazoline I2 receptors and alpha2-adrenoceptors density in rat frontal cortex after induced gliosis

Opposite age-dependent changes in alpha2-adrenoceptor and imidazoline I2 receptor (I2-IRs) density have been related to brain gliosis development with aging. To check this hypothesis we applied in rats a model of reactive gliosis induced by heat. The specific binding of [3H]idazoxan (0.5-20 nM) in the presence of (-)adrenaline (5 x 10(-6) M) to membranes from rat brain cortex showed that the density of I(2)-IRs was significantly higher in membranes of injured cortex (Bmax=60+/-6 fmol/mg protein; n=9) than in control (Bmax=38+/-3 fmol/mg protein; n=9; p=0.0053). Conversely, the density of alpha2-adrenoceptors, measured by [3H]clonidine (0.25-16 nM), in the injured cortex (Bmax=75+/-4 fmol/mg protein; n=9) was significantly lower than in sham membranes (Bmax=103+/-7 fmol/mg protein; n=9; p=0.0035). No significant differences in receptor's affinity were observed between both groups. These results support the hypothesis that gliosis induces opposite changes in alpha2-adrenoceptor and I2-IR density.     

Enhanced superoxide anion generation but reduced leukotriene B4 productivity in thioglycollate-elicited peritoneal macrophages

Lipoxygenase metabolism of arachidonic acid was compared between peritoneal macrophages from untreated rats and those from rats on day 7 after intraperitoneal injection of thioglycollate broth (TG). Resident macrophages (M phi) from untreated rats produced mainly LTB4 (303 +/- 25 pmol/5 x 10(6) cells) and 5-HETE (431 +/- 56 pmol/5 x 10(6) cells) when stimulated with 5 micrograms/ml calcium ionophore A23187 for 20 min at 37 degrees C. On the other hand, TG-elicited M phi generated less amounts of lipoxygenase metabolites (157 +/- 10 pmol LTB4 and 319 +/- 19 pmol 5-HETE/5 x 10(6) cells) with the same stimulus. Then, leukotriene productivity was examined by using subcellular fractions of each M phi lysate and an unstable epoxide intermediate, leukotriene A4. LTA4 hydrolase activity was mainly contained in soluble fractions from the both groups of M phi. The cytosol fraction from the resident M phi exhibited the following specific and total activity; 2.2 +/- 0.1 nmol LTB4/mg protein/5 min and 12.2 +/- 0.5 nmol LTB4/5 min per 10(8) cells. On the contrary, the cytosol fraction from the TG-elicited M phi showed 1.9 +/- 0.1 nmol LTB4/mg protein/5 min and 9.6 +/- 0.3 nmol LTB4/5 min per 10(8) cells. The resident M phi, however, generated 0.14 +/- 0.04 nmol O2-/min/4 x 10(5) cells whereas the TG-elicited M phi did 0.49 +/- 0.13 nmol O2-/min/4 x 10(5) cells when stimulated with wheat germ lectin. These results suggest that the TG-elicited macrophages show enhanced superoxide production but generate less lipoxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperglycemia in diabetic rats reduces the glutathione content in the aortic tissue

The glutathione redox cycle plays a major role in scavenging hydrogen peroxide (H2O2) under physiological conditions. Recently, we demonstrated that a high glucose concentration in the culture medium reduced the level of H2O2 scavenging activity of human vascular smooth muscle cells (hVSMCs). We also showed that a high glucose concentration reduced the intracellular glutathione (GSH) content and the rate of uptake of cystine, which itself is a rate-limiting factor that maintains the GSH level (FEBS Lett.421: 19-22,1998). In the present study, we investigated whether the hyperglycemic condition in diabetic rats impairs the glutathione content in the aortic tissue in vivo. Wistar rats were divided into the following three groups: streptozotocin-induced diabetic rats (STZ-D, n=7), insulin-treated STZ-D rats (I-STZ-D, n=8), and non-diabetic controls (C, n=7). Fourteen days after streptozotocin injection, the aortic tissue was extracted and the GSH content in the aortic tissue was measured. Furthermore, the relationship between the GSH content in the aortic tissue and blood glucose level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 30 weeks, which developed diabetes spontaneously, was investigated. The GSH content in the aortic tissue of the STZ-D group (0.99+/-0.14 nmol/mg protein) was significantly lower than that of the control group (1.68+/-0.15 nmol/mg protein). Insulin treatment to the diabetic rats restored the GSH content in the aortic tissue (I-STZ-D group; 1.45+/-0.11 nmol/mg protein). Among the 22 Wistar rats, the GSH content in the aortic tissue was negatively correlated with the blood glucose level (r=-0.69, p<0.01, n=22). Among the OLETF rats, a similar negative correlation between the GSH content in the aortic tissue and blood glucose level was seen (r=-0.64, p<0.05, n=10). We demonstrated in vivo that the hyperglycemic condition in STZ-induced diabetic Wistar rats and OLETF rats reduced the GSH content in aortic tissue. This suggested reduced glutathione redox cycle function of aorta.     

Caco-2 cells in culture synthesize and degrade dopamine and 5-hydroxytryptamine: a comparison with rat jejunal epithelial cells

To explore the usefulness of Caco-2 cells in the study of intestinal dopaminergic and 5-hydroxytryptaminergic physiology, we have undertaken the study of aromatic L-amino acid decarboxylase (AADC), catechol-O-methyltransferase (COMT) and type A and B monoamine oxidase (MAO-A and MAO-B) activities in these cells using specific substrates. The activity of these enzymes was also evaluated in isolated rat jejunal epithelial cells. The results showed that Vmax values (in nmol mg protein(-1) h(-1)) for AADC, using L-DOPA as the substrate, in rat jejunal epithelial cells (127.3+/-11.4) were found to be 6-fold higher than in Caco-2 cells (22.5+/-2.6). However, Km values in Caco-2 cells (1.24+/-0.37 mM) were similar to those observed in rat jejunal epithelial cells (1.30+/-0.29 mM). Similar results were obtained when AADC activity was evaluated using L-5HTP as substrate; in rat jejunal epithelial cells Vmax values (in nmol mg prot(-1) h(-1)) were found to be 5-fold that in Caco-2 cells (16.3+/-1.0 and 3.0+/-0.2, respectively), and Km values in Caco-2 cells (0.23+/-0.08 mM) were again similar to those observed in rat intestinal epithelial cells (0.09+/-0.03 mM). Caco-2 cells were not able to O-methylate dopamine, in contrast to rat jejunal epithelial cells (Vmax = 8.6+/-0.4 nmol mg protein(-1)(h-1); Km = 516+/-57 microM). Vmax values (in nmol mg protein(-1)(h-1)) for type A and B MAO in Caco-2 cells (19.0+/-0.6 and 5.4+/-0.6, respectively) were found to be significantly lower (P<0.05) than those in rat jejunal epithelial cells (46.9+/-3.1 and 9.6+/-1.2, respectively); however, no significant differences in the Km values were observed between Caco-2 and rat jejunal epithelial cells for both type A and B MAO. In conclusion, Caco-2 cells in culture are endowed with the synthetic and metabolic machinery needed to form and degrade DA and 5-HT, though, no COMT activity could be detected in these cells.     

Impaired hepatic fatty acid oxidation in rats with short-term cholestasis: characterization and mechanism

Rats with long-term cholestasis have reduced ketosis during starvation. Because it is unclear whether this is also the case in short-term cholestasis, we investigated hepatic fatty acid metabolism in rats with bile duct ligation for 5 days (BDL5, n = 11) or 10 days (BDL10, n = 11) and compared the findings with those made with pair-fed control rats (CON5 and CON10, n = 11). The plasma beta-hydroxybutyrate concentration was reduced in BDL rats (0.54 +/- 0.10 vs. 0.83 +/- 0.30 mM at 5 days and 0.59 +/- 0.24 vs. 0.88 +/- 0.09 mM at 10 days in BDL and control rats, respectively). In isolated liver mitochondria, state 3 oxidation rates for various substrates were not different between BDL and control rats. Production of ketone bodies from [(14)C]palmitate was reduced by 40% in mitochondria from BDL rats at both time points, whereas production of (14)CO(2) was maintained. These findings indicated intact function of the respiratory chain, Krebs cycle, and beta-oxidation and suggested impaired ketogenesis (HMG-CoA pathway). Accordingly, the formation of acetoacetate from acetyl-CoA by disrupted mitochondria was reduced in BDL rats at 5 days (2.1 +/- 1.0 vs. 4.8 +/- 1.9 nmol/min per mg protein) and at 10 days (1.7 +/- 1.0 vs. 6.2 +/- 1.9 nmol/min per mg protein). The principal defect could be localized at the rate-limiting enzyme of the HMG-CoA pathway, HMG-CoA synthase, which revealed decreased activity, and reduced hepatic mRNA and protein levels. We conclude that short-term cholestasis in rats leads to impaired hepatic fatty acid metabolism due to impaired ketogenesis. Ketogenesis is impaired because of decreased mRNA levels of HMG-CoA synthase, leading to reduced hepatic protein levels and to decreased activity of this key enzyme of ketogenesis. - Lang, C., M. Sch?fer, D. Serra, F. G. Hegardt, L. Kr?henbühl, and S. Kr?henbühl. Impaired hepatic fatty acid oxidation in rats with short-term cholestasis: characterization and mechanism. J. Lipid Res. 2001. 42: 22;-30.     

Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Intestinal brush border calcium uptake in spontaneously hypertensive rats and their genetically matched WKY rats

The current studies were designed to characterize calcium transport by intestinal brush border membrane in the spontaneously hypertensive rat (SHR) and normotensive control, the Wistar-Kyoto (WKY) rat. The biochemical and functional purity of the intestinal brush border membranes in SHR and WKY rats was validated by marker enzymes and the ability to transiently transport D-glucose in the presence of Na+ gradient. Calcium transport into duodenal and jejunal vesicles represented a minor binding component and transmembrane movement as evident by initial rate studies, A23187 studies, and lanthanum displacement experiments. Initial rate and time course of calcium uptake was lower in SHR compared with WKY rats. Kinetic analysis of calcium uptake by the jejunum (total uptake minus binding component) showed a Vmax of 6.98 +/- 0.2 and 1.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.76 +/- 0.04 and 0.87 +/- 0.1 mM for WKY rats and SHR, respectively. Similar kinetic analysis of calcium uptake by the duodenal segments showed a Vmax of 10.3 +/- 0.8 and 2.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.01). Km values were 0.7 +/- 0.2 and 0.3 +/- 0.06 mM (P greater than 0.05). Vmax of calcium uptake in the 2-week-old rats (prehypertensive period) was 6.0 +/- 0.3 and 3.53 +/- 0.3 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.60 +/- 0.07 and 0.5 +/- 0.01 mM, respectively. These results suggest that calcium binding and uptake by duodenal and jejunal intestinal brush border membranes of SHR is significantly decreased compared with WKY rats. The decrease in transmembrane calcium uptake is secondary to decrease in Vmax and is present before the appearance of hypertension, implying a genetically determined defect in calcium uptake in intestinal brush border membranes of the SHR.     

Kinetic studies on arachidonate 5-lipoxygenase from rat basophilic leukemia cells

Arachidonate 5-lipoxygenase of a 10,000 x g supernatant from RBL-1 cell homogenate was studied by a continuous assay measuring enzyme-catalysed oxygen consumption. Parallel HPLC and TLC analysis of arachidonic acid metabolites revealed that the oxygen consumption measured is solely due to 5-lipoxygenation of arachidonic acid. Oxygen consumption by this lipoxygenase was strictly dependent upon Ca2+, ATP and 5-HPETE. Removal of any of these three cofactors caused a complete inhibition of enzyme activity. Addition of the missing cofactor instantly restored the 5-lipoxygenase-dependent consumption of oxygen which remained linear for 10-20 s. Later on the velocity of the reaction decreased and after 2-3 min the enzyme became inactivated. Kinetic data were obtained from the initial velocity of the reaction using constant and saturating concentrations of CaCl2 and ATP. From Lineweaver-Burk plots substrate inhibition is evident for arachidonic acid concentrations greater than 45-50 microM. Km(app) for arachidonic acid is 182 +/- 16 microM (mean +/- SD, n = 5) and Vmax(app) is 425 +/- 140 nmol O2/(min x mg protein) (mean +/- SD, n = 5).     

N-Acetylsuccinimidylglutamate, a Cyclic Imide Form of the Peptide N-Acetylaspartylglutamate, Is Present in Low Micromolar Concentrations in Murine and Bovine CNS

Abstract: N -Acetylsuccinimidylglutamate [(asu)NAAG], a cyclic form of the peptide N -acetylaspartylglutamate (NAAG) in which the aspartyl residue is linked to glutamate via the α- and β-carboxylates, was identified and quantified by HPLC in the murine and bovine CNS. In the rat, the highest concentrations of (asu)NAAG were detected in the spinal cord (1.83 ± 0.15 pmol/mg of wet tissue weight) and brainstem (1.16 ± 0.08 pmol/mg wet weight), whereas the levels were below the limit of detection in cerebellum, hippocampus, and cerebral cortex. (Asu)NAAG was also detected in significant amounts in the superior colliculus and lateral genicutale nucleus (1.17 ± 0.05 and 0.82 ± 0.13 pmol/mg wet weight, respectively). Although the tissue content of (asu)NAAG was about three orders of magnitude lower than that of NAAG, levels of both peptides were positively correlated among different CNS regions ( r = 0.74, p < 0.003). In the rat spinal cord, (asu)NAAG levels progressively increased from week 2 to month 12 after birth. In bovine spinal cord, the contents of (asu)NAAG and NAAG were comparable in gray and white matter as well as in the dorsal and ventral horns. These results suggest that NAAG and (asu)-NAAG are closely related metabolically and raise the question of the physiological significance of such a cyclic peptide.