Anti-CD3 activation of human CD4+ T cells increases expression of the intracellular beta-endorphin endopeptidase (IDE/gamma-EpGE).

In this study, increased expression of an endopeptidase hydrolyzing beta-endorphin (beta-Ep) to gamma-endorphin (gamma-Ep, beta-Ep1-17) was observed upon immobilized anti-CD3 stimulated activation of human peripheral blood CD4+ T cells (hCD4+ T cells). Although freshly isolated hCD4+ T cells are devoid of significant beta-Ep endopeptidase activity ( < 0.1 nmol h(-1) 10(6) cells (-1)), activation of these cells with immobilized anti-CD3 results in a time dependent appearance of beta-Ep endopeptidase activity which reaches a maximal value of 17.4+/-0.48 nmol h(-1) 10(6) cells(-1) after 48 h of culture. Significant up-regulation of both mRNA encoding IDE/gamma-EpGE and immunoreactive protein are observed in anti-CD3 stimulated hCD4+ T cells, indicating transcription and translation of IDE/gamma-EpGE may be elevated. No significant hydrolysis of exogenous beta-Ep is observed with intact hCD4+ T cells whether quiescent or activated or from preparations of hCD4+ T cell membranes. Therefore, this activity appears to be intracellular. Immunoreactive IDE/gamma-EpGE is detected inside activated hCD4+ T cells. Analysis of metabolites generated upon hydrolysis of beta-Ep with lysed activated hCD4+ T cell preparations identified the presence of: beta-Ep1-18, beta-Ep2-18, beta-Ep1-17, beta-Ep2-17, beta-Ep18-31, beta-Ep19-31, beta-Ep1-13, beta-Ep2-13, beta-Ep18-26, and beta-Ep20-31 as major metabolites and the majority of these are consistent with beta-Ep hydrolytic activity attributable to IDE/gamma-EpGE.

Increased expression of an endopeptidase (gamma-EGE/IDE) hydrolyzing beta-endorphin during differentiation and maturation of bone marrow macrophages.

The presence and regulated expression of peptidase activity is a powerful mechanism with the potential to terminate or alter receptor recognition, cell membrane signal transduction, and physiological responses of immune cells to exogenous opioid peptides. In this study, the expression of an endopeptidase that hydrolyzes beta-endorphin to gamma-endorphin and other peptide products was investigated during in vitro differentiation and maturation of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) -derived, bone marrow-derived macrophages. In freshly isolated intact isolated mouse bone marrow cells the rate of beta-endorphin hydrolysis is undetectable (<0.1 nmol beta-endorphin hydrolyzed/h/10[6] cells). However, total intracellular beta-endorphin hydrolytic activity was increased significantly to 20.0 +/- 1.7 nmol/h/10(6) cells in the mature mouse macrophages derived in vitro by culture with rGM-CSF. rGM-CSF-derived macrophages expressed significantly higher levels of both protein and mRNA for the major beta-endorphin endopeptidase, gamma-endorphin-generating enzyme/insulin-degrading enzyme (gamma-EGE/IDE). Moreover, this enzymatic activity appears to be responsible for cleavage of exogenous beta-endorphin by intact rGM-CSF-derived macrophages or peritoneal macrophages to generate gamma-endorphin and other peptide products.

Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3-(13)C3]propionate.

Simple equations that relate glucose and glutamate 13C-NMR multiplet areas to gluconeogenesis and pyruvate recycling during metabolism of [1,2,3-(13)C3]propionate are presented. In isolated rat livers, gluconeogenic flux was 1.2 times TCA cycle flux and about 40% of the oxaloacetate pool underwent recycling to pyruvate prior to formation of glucose. The 13C spectra of glucose collected from rats after gastric versus intravenous administration of [1,2,3-(13)C3]propionate indicated that pyruvate recycling was slightly higher in vivo (49%) while glucose production was unchanged. This indicates that a direct measure of gluconeogenesis and pyruvate recycling may be obtained from a single 13C-NMR spectrum of blood collected after oral administration of enriched propionate.

A secreted peptidase involved in T cell beta-endorphin metabolism.

Beta-endorphin metabolism by CD4+ and CD8+ T cells, and the thymoma cell line, EL4, was investigated. In all three cell types, extracellular beta-endorphin was metabolized exclusively by a secreted, metal-dependent, thiol peptidase. The enzyme activity is expressed constitutively in EL4 cells and following activation of CD4+ and CD8+ T cells with anti-CD3 antibody. The enzyme is not one of the proteinases associated with cytolytic T cells and does not appear to be identical with any previously described beta-endorphin metabolizing enzyme. The enzyme cleaves beta-endorphin at approximately equal rates at either of two sites to yield beta-endorphin(1-17) (which is gamma-endorphin), beta-endorphin(1-18), beta-endorphin(18-31) and beta-endorphin(19-31). Evidence in the literature indicates that these N- and C-terminal peptides which contain, respectively, the opioid and non-opioid receptor binding domains of beta-endorphin, are biologically active. Thus, it is likely that this new T cell peptidase has important immunoregulatory activity.

In vivo effects of lipopolysaccharide on hepatic free-NAD(P)(+)-linked redox states and cytosolic phosphorylation potential in 48-hour-fasted rats.

This study was performed to determine the magnitude and time of onset of in vivo changes in hepatic bioenergetics in response to a sublethal dose of lipopolysaccharide (LPS), a bacterial endotoxin. Male rats (48-hour-fasted) were administered an intraperitoneal injection of LPS (5 mg/kg body weight) or vehicle alone, and the livers were freeze-clamped 5, 30, or 180 minutes or 24 hours later. Liver tissue was extracted with perchloric acid, and the metabolites necessary to calculate NAD(+)- and NADP(+)-linked redox states and the cytosolic phosphorylation potential were measured. There was no significant difference in hepatic cytosolic phosphorylation potential between LPS and control groups at any of the times investigated. This indicated that the ability of the liver to synthesize adenosine triphosphate (ATP) was not compromised under the conditions of the study. No changes in hepatic redox states were observed 5 or 30 minutes after LPS treatment. Three hours after LPS treatment, hepatic cytosolic and mitochondrial free-[NAD+]/[NADH] redox states and the cytosolic free-[NADP+]/[NADPH] redox state were more oxidized. By 24 hours, only NAD(+)-linked redox states were more oxidized than the time-matched controls. Hepatic urea content was elevated at both 3 and 24 hours, compatible with an increased rate of urea synthesis as a consequence of increased amino acid metabolism, whereas hepatic beta-hydroxybutyrate and total ketone bodies were decreased 24 hours after LPS treatment, indicating decreased hepatic ketogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of G alpha i2 mimics several aspects of LPS priming in a murine macrophage-like cell line.

Priming of macrophages with low concentrations of lipopolysaccharide (LPS) enhances the ability of substances that act through heterotrimeric G proteins to stimulate immune cell functions. Although LPS-induced alterations in the expression and functions of G proteins of the alpha i family have been reported in hematopoietic cells, their effects on subsequent steps in LPS priming of macrophages have not been defined. To study the role of G alpha i2 in priming of macrophages by LPS, we expressed a mutant, activated form of alpha i2 (alpha i2Q205L) in P388D1 cells, and compared its effects on PAF-dependent C alpha signalling and arachidonic acid release to those in cells treated with LPS. In control P388D1 cells, treatment with LPS (100 ng/ml) for 1 hr increased the amount of alpha i2 protein 2-fold. Both LPS treatment and expression of alpha i2Q205L increased the rate of PAF-induced C alpha influx across the cell membrane and arachidonic acid release, although neither altered release of C alpha from intracellular stores by PAF. Expression of alpha i2Q205L is sufficient to mimic the effects of LPS on the PAF-induced C alpha i signal and enhanced arachidonic acid release. Consequently, although increasing the expression of alpha i2 may not be the sole mechanism by which LPS enhances signalling by PAF, increased alpha i2 expression can account for the alterations in PAF-induced C alpha i regulation, and arachidonic acid release in LPS-primed P388D1 cells.

[[omega-(Heterocyclylamino)alkoxy]benzyl]-2,4-thiazolidinediones as potent antihyperglycemic agents.

A series of [(ureidoethoxy)benzyl]-2,4-thiazolidinediones and [[(heterocyclylamino)alkoxy]-benzyl]-2,4-thiazolidinediones was synthesized from the corresponding aldehydes. Compounds from the urea series, exemplified by 16, showed antihyperglycemic potency comparable with known agents of the type such as pioglitazone and troglitazone (CS-045). The benzoxazole 49, a cyclic analogue of 16, was a very potent enhancer of insulin sensitivity, and by modification of the aromatic heterocycle, an aminopyridine, 37, was identified as a lead compound from SAR studies. Evaluation of antihyperglycemic activity together with effects on blood hemoglobin content, to determine the therapeutic index, was performed in 8-day repeat administration studies in genetically obese C57 Bl/6 ob/ob mice. From these studies, BRL 49653 (37) has been selected, on the basis of antihyperglycemic potency combined with enhanced selectivity against reductions in blood hemoglobin content, for further evaluation.

Methionine enkephalin is hydrolyzed by aminopeptidase N on CD4+ and CD8+ spleen T cells.

Exogenous methionine enkephalin incubated with CD4+ or CD8+ T cells purified from murine spleen is metabolized primarily, if not exclusively, by aminopeptidase N (aminopeptidase M, EC 3.4.11.2), a membrane-anchored ectopeptidase. The enzyme activity is identified by its substrate specificity, sensitivity to inhibition by amastatin, and immunoreactivity with antibody to rat kidney aminopeptidase N. Activation of CD4+ T cells results in a small increase per cell in aminopeptidase N activity.

Orientation-conserved transfer of symmetric Krebs cycle intermediates in mammalian tissue.

Metabolism of [2-13C]-, [3-13C]-, and [1,2,3-13C]propionate in perfused rat livers and [2-13C]-acetate in perfused rat hearts has been examined in tissue extracts by 13C NMR. Label from [2-13C]-propionate was preferentially incorporated into the C2 carbon of lactate, alanine, and aspartate in liver tissue while label from [3-13C]propionate appeared preferentially in the C3 carbon of those same molecules. These data suggest that 13C may not be completely randomized in the symmetric citric acid cycle intermediates succinate and fumarate as is normally assumed but that some fraction of those intermediates may be transferred between enzymes in this span of the cycle with conservation of spatial orientation, consistent with recent results obtained in yeast [Sumegi et al. (1990) Biochemistry 29, 9106-9110]. This was confirmed by performing similar experiments with [1,2,3-13C]propionate. Time-dependent asymmetry was also observed between the intensities of the glutamate C2 and C3 resonances and between the aspartate C2 and C3 resonances in 13C NMR spectra of intact hearts and heart extracts during early perfusion with [2-13C]-acetate. A model is presented which predicts that isotopic asymmetry is observed only during the first 2-3 turns of the cycle pools when isotope enters the cycle via acetyl-CoA even if all symmetric cycle intermediates retain a unique molecular orientation on each pass through the citric acid cycle.

Methionine enkephalin metabolism by murine macrophage ectopeptidase(s).

Ectopeptidases which hydrolyze opioid and other neuropeptides have been identified in brain, kidney and intestine. In this study, identification of the enzymes metabolizing the opioid peptide methionine enkephalin (YGGFM) in murine macrophages was undertaken. Incubation of methionine enkephalin with intact murine peritoneal macrophages results in five products identified as Y, F, FM, GFM and GGFM by amino acid analysis and peptide microsequencing after fractionation by HPLC. The spectrum of metabolites results from at least two distinct aminopeptidase activities. The enzyme hydrolyzing YGGFM to GGFM is identified as the membrane-anchored aminopeptidase N (ApN; EC 3.4.11.2) based on its substrate specificity and inhibitor profile. A distinct bestatin and amastatin sensitive aminopeptidase catalyzes hydrolysis of GGFM to GFM. The macrophage ApN protein has a larger mass and is antigenically distinct from murine kidney ApN, which is suggested to result from glycosylation differences rather than expression of a distinct protein. The ApN catalytic activity and mRNA levels are increased in thioglycollate-elicited as compared to resident peritoneal macrophages. RT-PCR analysis identified a 0.7 kb fragment of the ApN coding sequence which was identical in mouse kidney and thioglycollate-elicited peritoneal macrophages and which has 89% identity with the corresponding rat kidney ApN cDNA sequence.

A method for obtaining 13C isotopomer populations in 13C-enriched glucose.

13C NMR analysis of 13C-enriched glucose containing multiple isotopomers is hampered by chemical shift similarities of several carbon resonances and by the presence of two anomeric forms. A convenient and quantitative method of enzymatically oxidizing glucose to gluconate in tissue and perfusate extracts is presented. The six carbon resonances of the resulting 13C-enriched gluconate are fully resolved at high pH, thereby allowing a determination of the fractional population of each 13C isotopomer by 13C NMR. The utility of this method is demonstrated using the effluent from an isolated perfused liver containing 13C-enriched glucose produced by hepatic metabolism of sodium [1,2,3-13C3]propionate via the citric acid cycle and gluconeogenesis. An analysis of the gluconate C2 and C5 resonances in this sample showed that pentose phosphate activity was insignificant during this perfusion protocol. As demonstrated, this method provides a means of fully describing 13C isotopomer populations in enriched glucose samples where isotope may be derived from multiple metabolic pathways, thus expanding the scope of experimental design and enrichment strategies.

Endotoxin stimulation of liver parenchymal cell phosphofructokinase activity requires nonparenchymal cells.

The rate of carbohydrate flux through phosphofructokinase (measured as the rate of [3-3H]glucose detritiation) was increased fourfold in rat liver parenchymal cells incubated with conditioned medium from lipopolysaccharide-stimulated adherent liver non-parenchymal cells. The rate was not affected in parenchymal cells incubated either with lipopolysaccharide directly or with conditioned medium from non-stimulated non-parenchymal cells. The stimulation of carbohydrate flux through phosphofructokinase by conditioned medium was not duplicated by peptide cytokines known to be released by lipopolysaccharide-activated liver non-parenchymal cells (interleukin-1, interleukin-6, tumor necrosis factor-alpha, and transforming growth factor-beta) or platelet activating factor. Furthermore, formation of the active conditioned medium was not prevented by inclusion of cycloheximide or dexamethasone to inhibit cytokine synthesis, or indomethacin or BW755c to inhibit arachidonic acid metabolism, during lipopolysaccharide-stimulation of the non-parenchymal cells. The results indicate that intercellular communication between lipopolysaccharide-stimulated liver non-parenchymal cells and parenchymal cells by soluble mediators is responsible for the stimulation of liver phosphofructokinase activity during endotoxin-induced shock. Studies to isolate and identify the factor(s) in the conditioned medium are currently in progress.

Pertussis toxin and H-7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide-primed macrophages. Eicosanoid release from lipopolysaccharide-primed macrophages.

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1-2 micrograms/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 microM) or with phorbol 12-myristate 13-acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12-myristate 13-acetate. The presence of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12-myristate 13-acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis-toxin-sensitive, guanine-nucleotide-binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide-activated macrophages in response to A23187 and phorbol 12-myristate 13-acetate.

Endotoxin increases the liver fructose 2,6-bisphosphate concentration in fasted rats.

Following endotoxin administration to fasted rats, the liver fructose 2,6-bisphosphate level is significantly increased within 1 hr, is elevated 2.3-fold by 3 hrs, and remains elevated 2 to 3-fold for at least 24 hrs. This increase in the potent allosteric activator of phosphofructokinase occurs when there is no change in the liver Glc 6-P, glycogen or cAMP concentrations, or in the activities of phosphoenolpyruvate carboxykinase or pyruvate kinase. The increase in fructose 2,6-bisphosphate concentration accounts for the increased phosphofructokinase activity previously observed in hepatocytes isolated 18 hours following endotoxin administration to rats (1). By stimulating the phosphofructokinase/Fru 1,6-bisphosphate cycle in the direction of glycolysis, fructose 2,6-bisphosphate is likely the factor responsible for decreased gluconeogenesis in endotoxemia.

Dehydroisoandrosterone prevention of autoimmune disease in NZB/W F1 mice: lack of an effect on associated immunological abnormalities.

The effect of dietary dehydroisoandrosterone (DHA) on several immunological abnormalities associated with the development of systemic lupus erythematosus in New Zealand Black/New Zealand White F1 (NZB/W) female mice was examined. Despite the extraordinary benefits in prolonged survival and decreased synthesis of antibodies to double-stranded DNA obtained by adding DHA (0.4% w/v) to the diet fed to these mice (Lucas et al. (1985) J. Clin. Invest. 75, 2091-2093), remarkably small changes in the chemistry and function of the immune system were detected. DHA prevented the increases in spleen mass and in peritoneal cell number which occur with age in NZB/W female mice, but did not prevent the development of hypergammaglobulinemia. DHA did not affect peritoneal macrophage functions as measured by the phagocytosis of opsonized and non-opsonized sheep erythrocytes, or the zymosan-stimulated release of PGE2, 6-ketoPGF1 alpha, TXB2 and LTC4. In spleen, DHA delayed the loss of T-cell mitogenic responses until 5.5 months of age, but did not alter the spleen lymphocyte population.

Wheelchair obstacle course performance in right cerebral vascular accident victims.

The initial experiment of this paper investigated the role of hemispatial neglect in wheelchair-related accidents of right-hemisphere stroke victims. Twelve subjects with and 12 subjects without neglect of left space drove their wheelchairs through an obstacle course. Two types of obstacle course errors were evaluated: direct hits and sideswipes. The neglecting group made significantly more direct hits but there were no significant differences between groups in sideswipe errors. In Experiment 2, the nonneglecting group's data were compared with a left-hemisphere stroke group without neglect and three motor control groups to investigate if their errors resulted from motor deficits. The right-hemisphere stroke group made significantly more left-sided errors and errors in contralateral space than any other group. In Experiment 3, 13 subjects with neglect were taught to scan to the left which resulted in significant decreases in direct hits but not in sideswipes on the obstacle course. These results suggest that obstacle course performance is sensitive to more than hemispatial neglect.

Liver composition and lipid metabolism in NZB/W F1 female mice fed dehydroisoandrosterone.

The beneficial effects obtained with dehydroisoandrosterone (DHA) feeding in the treatment of murine systemic lupus erythematosus are similar to those obtained with caloric restriction or with dietary manipulation of essential fatty acid availability. In this study, the fatty acid composition of selected tissues was examined in NZB/W F1 mice fed a diet containing 0.4% DHA. The effect of the DHA diet on liver composition and the activity of key hepatic enzymes involved in fatty acid synthesis and glucose metabolism was also investigated. The content of the essential fatty acid, arachidonate, was decreased in plasma cholesteryl esters and liver and kidney phospholipids in mice fed the DHA diet, yet no significant decrease in arachidonate content was observed in plasma phospholipid. The most striking change in both plasma and liver phospholipid was an increase in palmitic acid and a decrease in stearic acid, which could result from a decreased ability for fatty acid elongation. The liver mass was dramatically increased in the mice fed DHA, primarily from parenchymal cell hypertrophy, and contained little lipid. Significant changes in the activities of malic enzyme, glucose-6-phosphate dehydrogenase and pyruvate kinase, similar to those changes which occur with fasting, were observed during the initial adaptation to the DHA diet. The pyruvate kinase activity remained low, suggesting a decrease in liver glycolysis. These results are consistent with the concept that diets containing DHA result in an altered metabolism with a decreased dependence on carbohydrate metabolism and an increased metabolism of lipids.