Ana Maria Kelmer-Bracht

Effects of four monoterpenes on germination, primary root growth, and mitochondrial respiration of maize

The effects of four monoterpenes—camphor, eucaliptol, limonene, and α-pinene—on the oxidative metabolism of mitochondria isolated from maize root (Zea mays), on maize seed germination, and on primary root growth were investigated. The effects of individual monoterpenes on respiration were variable. α-Pinene concentrations of 0.05–1.0 mM stimulated respiration with a mixture of substrates composed of NADH, L-malate, succinate, and L-glutamate, and in the absence of exogenously added ADP (basal respiration). However, at concentrations higher than 1.0 mM, α-pinene inhibited respiration both in the absence (basal respiration) and presence of ADP (coupled respiration). Limonene at 0.1 mM or above stimulated basal respiration and inhibited in parallel the coupled respiration. Similar effects were promoted by eucaliptol, but at a higher concentration range (1.0 mM or above). Camphor was less active. At 10 mM concentration, it caused stimulation of basal respiration but did not affect coupled respiration. In the concentration range 0.1–10.0 mM, limonene was inactive on seed germination and primary root growth. Camphor and eucaliptol did not inhibit germination but reduced fresh and/or dry weight of roots at 5.0 mM and above. α-Pinene inhibited both seed germination and fresh weight of primary roots at 10.0 mM concentration. The results indicate that intact seeds and primary roots are less sensitive than isolated mitochondria. The relatively more lipophilic monoterpenes α-pinene and limonene had less activity than the more water-soluble oxygenated monoterpenes camphor and eucaliptol in inhibiting seed germination and/or primary root growth, despite the fact that they had a higher activity on the oxidative metabolism of isolated mitochondria. The findings suggest that the solubility of monoterpenes may be the major factor implicated in these differences.

Gluconeogenesis in the liver of arthritic rats.

The gluconeogenic response in the liver from rats with chronic arthritis to various substrates and the effects of glucagon were investigated. The experimental technique used was the isolated liver perfusion. Hepatic gluconeogenesis in arthritic rats was generally lower than in normal rats. The difference between normal and arthritic rats depended on the gluconeogenic substrate. In the absence of glucagon the following sequence of decreasing differences was found: alanine (−71·8 per cent)∽glutamine (−71·7 per cent)>pyruvate (−60 per cent)>lactate+pyruvate (−44·9 per cent)>xylitol (n.s.=non‐significant)∽glycerol (n.s.). For most substrates glucagon increased hepatic gluconeogenesis in both normal and arthritic rats. The difference between normal and arthritic rats, however, tended to diminish, as revealed by the data of the following sequence: alanine (−48·9 per cent)∽pyruvate (−47·6 per cent)>glutamine (−33·8 per cent)>glycerol (n.s.)∽lactate+pyruvate (n.s.)∽xylitol (n.s.). The causes for the reduced hepatic gluconeogenesis in arthritic rats are probably related to: (a) lower activities of key enzymes catalyzing most probably steps preceding phosphoenolpyruvate (e.g. phosphoenolpyruvate carboxykinase, pyruvate carboxylase, etc.); (b) a reduced availability of reducing equivalents in the cytosol; (c) specific differences in the situations induced by hormones or by the individual substrates. Since glycaemia is almost normal in chronically arthritic rats, it seems that lower gluconeogenesis is actually adapted to the specific needs of these animals. Copyright

The urea cycle in the liver of arthritic rats.

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (−32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.

Effects of rutin and quercetin on mitochondrial metabolism and on ATP levels in germinating tissues of Glycine max

The effects of quercetin and its glycoside rutin, flavonoid compounds widely distributed in higher plants, on mitochondria isolated from soybean hypocotyls and on ATP levels of soybean embryonic axes were, investigated. Quercetin at 0.5 mM, and lower, inhibited respiration with a mixture of substrates composed of NADH, L-malate, succinate and L-glutamate, both in the absence or in the presence of exogenously added ADP. At concentrations higher than 0.5 mM, the effects of quercetin on basal respiration were shifted toward stimulation. Respiration of uncoupled mitochondria was inhibited by quercetin, irrespective of the nature of the substrates. Swelling driven by phosphate uptake was also inhibited by quercetin. Rutin had no effect on isolated mitochondria. The ATP levels of embryonic axes decreased in the presence of either quercetin or rutin. These results indicate that quercetin impairs the respiratory activity of soybean hypocotyl mitochondria by at least three mechanisms: (a) it inhibits substrate oxidation, probably by a direct action on the electron transfer chain; (b) it inhibits phosphate uptake; and (c) it possibly acts as an uncoupler of oxidative phosphorylation at concentrations higher than 0.5 mM. It was concluded that the action of quercetin on energy metabolism in mitochondria might be the cause of the reduction of the ATP levels in embryonic axes.

Glycogen levels and glycogen catabolism in livers from arthritic rats

Hepatic glycogen catabolism and glycogen levels in rats with chronic arthritis were investigated. At 9:00 a.m., the hepatic glycogen contents of ad libitum fed arthritic and normal rats were 225.5 ± 17.7 and 332.1 ± 28.6 μmol glucosyl units × (g liver)–1, respectively. Food intake of arthritic and normal rats was equal to 100.1 ± 6.7 and 105.0 ± 3.1 mg × (g body w)–1 × (per 24 h)–1, respectively. In isolated perfused livers from normal and arthritic rats the rates of glucose, lactate and pyruvate release were the same when substrate- and hormone-free perfusion was performed. During an infusion period of 20 min glucagon caused an increment in glucose release of 35.3 ± 4.7 μmol × (g liver)–1 in livers from arthritic rats; in the normal condition the corresponding increment was 69.6 ± 5.7 μmol × (g liver)–1. Lactate and pyruvate productions (indicators of glycolysis) were diminished by glucagon in livers from normal rats; in the arthritic condition an initial stimulation was found, followed by a slow decay, which did not result in significant inhibition at the end of the glucagon infusion period (20 min). The actions of cAMP and dibutyryl-cAMP were similar to those of glucagon. It was concluded that livers from arthritic rats show an impaired capacity of releasing glucose under the stimulus of glucagon. This can be partly due to the lower glycogen levels and partly to a smaller capacity of inhibiting glycolysis. Reduction in glycogen levels was not associated with reduction in food intake or failure in the energetic state of the hepatic cells. These changes in glycogen metabolism may be related to reduced gluconeogenic capacity of the livers and/or to production of inflammatory mediators observed in the arthritis disease.

Effect of Stryphnodendron adstringens (barbatimao) on energy metabolism in the rat liver

The action of a barbatimão extract on hepatic energy metabolism was investigated using isolated mitochondria and the perfused rat liver. In mitochondria the barbatimão extract inhibited respiration in the presence of ADP and succinate. Stimulation occurred, however, after ADP phosphorylation (state IV respiration). The ADP/O and respiratory control ratios were reduced. The activities of succinate-oxidase, NADH-oxidase and the oxidation of ascorbate were inhibited. The ATPase of intact mitochondria was stimulated, but the ATPases of uncoupled and disrupted mitochondria were inhibited. In perfused livers the extract caused stimulation of oxygen consumption, inhibition of gluconeogenesis and stimulation of glycolysis. Glucose release due to glycogenolysis was stimulated shortly after the introduction of the extract, but inhibition gradually developed as the infusion was continued. Apparently the barbatimão extract impairs the hepatic energy metabolism by three mechanisms: (1) uncoupling of oxidative phosphorylation, (2) inhibition of mitochondrial electron transport, and (3) inhibition of ATP-synthase.

Transport, distribution space and intracellular concentration of the anti- inflammatory drug niflumic acid in the perfused rat liver

Transport and distribution space of niflumic acid in the perfused rat liver were investigated employing the multiple-indicator dilution technique with constant infusion of the drug (step input). Niflumic acid permeated the cell membrane in both directions at very high rates and its distribution in the cellular space was flow-limited; at least at 37 degrees, the rates of influx and efflux could not be measured. Dissociation of the niflumic acid-albumin complex also occurred at very high rates. The apparent space of distribution of niflumic acid in the liver depended on the concentration of the drug and varied between 4.37 (1 mM) and 43.5 (10 microM) times the water space; even with 90% extracellular binding to albumin, the apparent space of distribution of niflumic acid was 5.1 times greater than the water space. The high apparent spaces of distribution reflected the high intracellular concentrations. The ratio of intracellular bound plus free concentration to the extracellular bound plus free concentration (Ci/Ce) varied between 6.62 (1 mM portal niflumic acid) and 71.0 (10 microM portal niflumic acid). Metabolic transformation depended on the concentration of the free form. Intracellular binding is probably the major reason for the high concentration of the drug in the hepatic tissue.

Kinetic properties of the glucose 6-phosphatase of the liver from arthritic rats.

According to previous reports, adjuvant-induced arthritic rats present reduced activities of the hepatic glucose 6-phosphatase. A kinetic study was done in order to characterize this phenomenon. Microsomes were isolated from livers of arthritic and control rats (Holtzman strain) and the glucose 6-phosphatase was measured at various temperatures (13-37 degrees C) and glucose 6-phosphate concentrations. Irrespective of the temperature, the enzyme from arthritic rats presented a reduction of both V(max) and K(M). Detergent treatment of liver microsomes from control rats increased the activity, but no increase was found when microsomes from arthritic rats were treated in the same way. The mannose 6-phosphatase activity of detergent-treated microsomes from arthritic rats was only 25% of the activity found with detergent-treated microsomes from control rats. Without detergent treatment, the mannose 6-phosphatase activities of both control and arthritic rats were minimal. The activation energy, derived from V(max), was not changed by arthritis. In vivo arthritic rats presented higher hepatic glucose 6-phosphate concentrations, a phenomenon that is consistent with a reduced activity of glucose 6-phosphatase. It was concluded that in arthritic rats, the hydrolase is probably reduced, without a similar change in the translocase activity.

Effects of the nonsteroidal anti-inflammatory drug piroxicam on rat liver mitochondria

1. The effects of piroxicam, a nonsteroidal anti-inflammatory drug, on rat liver mitochondria were investigated in order to obtain direct evidence about a possible uncoupling effect, as suggested by a previous work with the perfused rat liver. 2. Piroxicam increased respiration in the absence of exogenous ADP and decreased respiration in the presence of exogenous ADP, the ADP/O ratios and the respiratory control ratios. 3. The ATPase activity of intact mitochondria was increased by piroxicam. With 2,4-dinitrophenol uncoupled mitochondria, inhibition was observed. The ATPase activity of freeze-thawing disrupted mitochondria was insensitive to piroxicam. 4. Swelling driven by the oxidation of several substrates and safranine uptake induced by succinate oxidation were inhibited. 5. The results of this work represent a direct evidence that piroxicam acts as an uncoupler, thus, decreasing mitochondrial ATP generation.

The action of extracellular NAD+ on gluconeogenesis in the perfused rat liver

In the rat liver NAD+ infusion produces increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption. The aim of the present work was to investigate the possible action of this agent on gluconeogenesis using lactate as a gluconeogenic precursor. Hemoglobin-free rat liver perfusion in antegrade and retrograde modes was used with enzymatic determination of glucose production and polarographic assay of oxygen uptake. NAD+ infusion into the portal vein (antegrade perfusion) produced a concentration-dependent (25–100 μM) transient inhibition of oxygen uptake and gluconeogenesis. For both parameters inhibition was followed by stimulation. NAD+ infusion into the hepatic vein (retrograde perfusion) produced only transient stimulations. During Ca2+-free perfusion the action of NAD+ was restricted to small transient stimulations. Inhibitors of eicosanoid synthesis with different specificities (indo-methacin, nordihydroguaiaretic acid, bromophenacyl bromide) either inhibited or changed the action of NAD+. The action of NAD+ on gluconeogenesis is probably mediated by eicosanoids synthesized in non-parenchymal cells. As in the fed state, in the fasted condition extracellular NAD+ is also able to exert two opposite effects, inhibition and stimulation. Since inhibition did not manifest significantly in retrograde perfusion it is likely that the generating signal is located in pre-sinusoidal regions.