The regulation of glutamine and ketone-body metabolism in the small intestine of the long-term (40-day) streptozotocin-diabetic rat.

The small intestine is the major site of glutamine utilization in the mammalian body. During prolonged (40-day) streptozotocin-diabetes in the rat there is a marked increase in both the size and the phosphate-activated glutaminase activity of the small intestine. Despite this increased capacity, intestinal glutamine utilization ceases in diabetic rats. Mean arterial glutamine concentration fell by more than 50% in diabetic rats, suggesting that substrate availability is responsible for the decrease in intestinal glutamine use. When arterial glutamine concentrations in diabetic rats were elevated by infusion of glutamine solutions, glutamine uptake across the portal-drained viscera was observed. The effect of other respiratory fuels on intestinal glutamine metabolism was examined. Infusions of ketone bodies did not affect glutamine use by the portal-drained viscera of non-diabetic rats. Prolonged diabetes had no effect on the activity of 3-oxoacid CoA-transferase in the small intestine or on the rate of ketone-body utilization in isolated enterocytes. Glutamine (2 mM) utilization was decreased in enterocytes isolated from diabetic rats as compared with those from control animals. However, glutaminase activity in homogenates of enterocytes was unchanged by diabetes. In enterocytes isolated from diabetic rats the addition of ketone bodies or octanoate decreased glutamine use. It is proposed that during prolonged diabetes ketone bodies, and possibly fatty acids, replace glutamine as the major respiratory fuel of the small intestine.     

Studies on the interactions of human pancreatic elastase 2 with human alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin.

Several intermediates in the reaction of 2-methylglutamate with glutamate decarboxylase from Escherichia coli were detected by stopped-flow spectrophotometry and by rapid-scanning spectrophotometry after conventional mixing. Structures were assigned to intermediates on the basis of kinetic and spectral evidence. In the early stages of the reaction an intermediate with the properties expected of a geminal diamine accumulated significantly. Changes consistent with the conversion of this species into the external aldimine were also observed. The course of product formation was determined and linked with spectral changes taking place in the bound coenzyme. The effect of the minor decarboxylation-dependent transamination that accompanies the major reaction was analysed.     

Phorbol ester inhibits phosphatidylserine synthesis in human promyelocytic leukaemia HL60 cells. Possible involvement of free radicals and correlation with phosphorylation of nuclear protein 1b.

Treatment of human promyelocytic leukaemia HL60 cells in conditioned medium with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 4 h resulted in 25-30% inhibition of labelling of phosphatidylserine (PS) with [U-14C]serine. PS labelling was 40% lower, and no inhibitory TPA effect was observed when the experiments were performed in fresh medium. Cycloheximide or puromycin also inhibited PS labelling by 38-44%; their inhibitory effects were non-additive with that of TPA and occurred only in conditioned medium. Catalase (CAT) and superoxide dismutase (SOD), both free-radical scavengers, and H7, a protein kinase C inhibitor, reversed to various extents the inhibitory effect of TPA on PS synthesis. On the other hand, chlorobenzoic acid, a free-radical-generating agent, also inhibited PS synthesis by 22% after 4 h treatment when conditioned medium was used. When ethanolamine was added to cells in conditioned medium to quench PS formation through the exchange of free serine with the ethanolamine moiety of phosphatidylethanolamine (PE), PS labelling was decreased by 33% and the inhibitory TPA effect was significantly decreased. On the other hand, ethanolamine had marginal quenching effect on PS labelling when added to cells in fresh medium. TPA increased the phosphorylation of various proteins in the cells, including protein lb (Mr 80,000; pI 5.5) shown to be localized mainly in the nuclear fraction. Chlorobenzoic acid selectively stimulated the phosphorylation of protein lb, whereas CAT and SOD specifically attenuated the TPA-stimulated phosphorylation of this protein. All these agents affected phosphorylation of protein lb only if conditioned medium was used. The findings suggested that net synthesis of PS through the base-exchange mechanism was stimulated in HL60 cells by cell products present in the conditioned medium. TPA inhibited this stimulated PS synthesis by a mechanism which appeared to involve active oxygen species and protein synthesis and might be related to the phosphorylation of protein lb.     

Chemical modification of human alpha 1-proteinase inhibitor by tetranitromethane. Structure-function relationship.

Nitration of tyrosine residues of alpha 1-proteinase inhibitor (alpha 1-PI) by tetranitromethane yielded a product that maintained its inhibitory activity against trypsin but lost most of its inhibitory activity against elastase. Chemical analysis of the product showed that four out of the six tyrosine residues in alpha 1-PI had been nitrated to various degrees: Tyr-38 and Tyr-297 were not nitrated, whereas Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were nitrated to extents in the range 40-80%. We interpreted these data to mean that modification of these tyrosine residues decreased the association constant between alpha 1-PI and the proteinases and that the decrease differs from one proteinase to the other. When either alpha 1-PI-trypsin or alpha 1-PI-elastase complex was nitrated, nitration took place only to a very slight extent at these latter four tyrosine residues. On the other hand, Tyr-38 and Tyr-297 underwent nitration to about 20%. We concluded that Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were located on the surface of alpha 1-PI that interacts with either trypsin or elastase in the formation of complexes, and were therefore protected from nitration.     

Stimulation of phospholipid hydrolysis and arachidonic acid mobilization in human uterine decidua cells by phorbol ester.

Vasopressin and oxytocin both stimulated inositol phosphate accumulation in isolated uterine decidua cells. Pretreatment of cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) prevented this agonist-induced phosphoinositide hydrolysis. TPA (0.1 microM) alone had no effect on basal inositol phosphate accumulation, but stimulated phosphoinositide deacylation, as indicated by a 2-fold increase in lysophosphatidylinositol and glycerophosphoinositol. TPA also stimulated a dose-related release of arachidonic acid from decidua-cell phospholipid [phosphatidylcholine (PC) much greater than phosphatidylinositol (PI) greater than phosphatidylethanolamine]. The phorbol ester 4 beta-phorbol 12,13-diacetate (PDA) at 0.1 microM had no effect on arachidonic acid mobilization. The TPA-stimulated increase in arachidonic acid release was apparent by 2 1/2 min (116% of control), maximal after 20 min (283% of control), and remained around this value (306% of control) after 120 min incubation. TPA also stimulated significant increases in 1,2-diacylglycerol and monoacylglycerol production at 20 and 120 min. Although the temporal increases in arachidonic acid and monoacylglycerol accumulation in the presence of TPA continued up to 120 min, that of 1,2-diacylglycerol declined after 20 min. In decidua cells prelabelled with [3H]choline, TPA also stimulated a significant decrease in radiolabelled PC after 20 min, which was accompanied by an increased release of water-soluble metabolites into the medium. Most of the radioactivity in the extracellular pool was associated with choline, whereas the main cellular water-soluble metabolite was phosphorylcholine. TPA stimulated extracellular choline accumulation to 183% and 351% of basal release after 5 and 20 min respectively and cellular phosphorylcholine production to 136% of basal values after 20 min. These results are consistent with a model in which protein kinase C activation by TPA leads to arachidonic acid mobilization from decidua-cell phospholipid by a mechanism involving phospholipase A-mediated PI hydrolysis and phospholipase C-mediated PC hydrolysis, coupled with further hydrolysis of the 1,2-diacylglycerol product.     

The leukotriene B4 paradox: neutrophils can, but will not, respond to ligand-receptor interactions by forming leukotriene B4 or its omega-metabolites.

Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.     

The effect of concanavalin A on the rat electro-olfactogram. Differential inhibition of odorant response.

When the rat olfactory mucosa is treated with concanavalin A, it subsequently shows diminished sensitivity towards 60% of the 112 odorants tested (as judged by the amplitude of the electro-olfactogram response). Odorants containing four to six carbon atoms tend to show the largest (absolute) diminutions, suggesting a receptor for this kind of odorant, although the structural specificity is weak. The receptor seems to be of particular importance in the detection of thiols, carboxylic acids and hydrocarbons of the above size, since these compounds loose the highest proportion of their original signal. The concanavalin A appears to be binding to the glycan of one or more cell-surface proteins. The binding may be at, or close to, at least one odorant receptor.     

C-terminal bombesin sequence requirements for binding and effects on protein synthesis in Swiss 3T3 cells.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.     

Metabolism of inositol 1,4,5-trisphosphate in guinea-pig hepatocytes.

Metabolism of inositol 1,4,5-trisphosphate was investigated in permeabilized guinea-pig hepatocytes. The conversion of [3H]inositol 1,4,5-trisphosphate to a more polar 3H-labelled compound occurred rapidly and was detected as early as 5 s. This material co-eluted from h.p.l.c. with inositol 1,3,4,5 tetrakis[32P]phosphate and is presumably an inositol tetrakisphosphate. A significant increase in the 3H-labelled material co-eluting from h.p.l.c. with inositol 1,3,4-trisphosphate occurred only after a definite lag period. Incubation of permeabilized hepatocytes with inositol 1,3,4,5-tetrakis[32P]phosphate resulted in the formation of 32P-labelled material that co-eluted with inositol 1,3,4-trisphosphate; no inositol 1,4,5-tris[32P]phosphate was produced, suggesting the action of a 5-phosphomonoesterase. The half-time of hydrolysis of inositol 1,3,4,5-tetrakis[32P]phosphate of approx. 1 min was increased to 3 min by 2,3-bisphosphoglyceric acid. Similarly, the rate of production of material tentatively designed as inositol 1,3,4-tris[32P]phosphate from the tetrakisphosphate was reduced by 10 mM-2,3-bisphosphoglyceric acid. In the absence of ATP there was no conversion of [3H]inositol 1,4,5-trisphosphate to [3H]inositol tetrakisphosphate or to [3H]inositol 1,3,4-trisphosphate, which suggests that the 1,3,4 isomer does not result from isomerization of inositol 1,4,5-trisphosphate. The results of this study suggest that the origin of the 1,3,4 isomer of inositol trisphosphate in isolated hepatocytes is inositol 1,3,4,5-tetrakisphosphate and that inositol 1,4,5-trisphosphate is rapidly converted to this tetrakisphosphate. The ability of 2,3-bisphosphoglyceric acid, an inhibitor of 5-phosphomonoesterase of red blood cell membrane, to inhibit the breakdown of the tetrakisphosphate suggests that the enzyme which removes the 5-phosphate from inositol 1,4,5-trisphosphate may also act to convert the tetrakisphosphate to inositol 1,3,4-trisphosphate. It is not known if the role of inositol 1,4,5-trisphosphate kinase is to inactivate inositol 1,4,5-trisphosphate or whether the tetrakisphosphate product may have a messenger function in the cell.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.