Anaerobic citrate metabolism and its regulation in enterobacteria

Several species of enterobacteria are able to utilize citrate as carbon and energy source. Under oxic conditions in the presence of a functional tricarboxylic acid cycle, growth on this compound solely depends on an appropriate transport system. During anaerobiosis, when 2-oxoglutarate dehydrogenase is repressed, some species such as Klebsiella pneumoniae and Salmonella typhimurium, but not Escherichia coli, are capable of growth on citrate by a Na+-dependent pathway forming acetate, formate, and CO2 as products. During the last decade, several novel features associated with this type of fermentation have been discovered in K. pneumoniae. The biotin protein oxaloacetate decarboxylase, one of the key enzymes of the pathway besides citrate lyase, is a Na+ pump. Recently it has been shown that the proton required for the decarboxylation of carboxybiotin is taken up from the side to which Na+ ions are pumped, and a membrane-embedded aspartate residue that is probably involved both in Na+ and in H+ transport was identified. The Na+ gradient established by oxaloacetate decarboxylase drives citrate uptake via CitS, a homodimeric carrier protein with a simultaneous-type reaction mechanism, and NADH formation by reversed electron transfer involving formate dehydrogenase, quinone, and a Na+-dependent NADH:quinone oxidoreductase. All enzymes specifically required for citrate fermentation are induced under anoxic conditions in the presence of citrate and Na+ ions. The corresponding genes form a cluster on the chromosome and are organized as two divergently transcribed operons. Their co-ordinate expression is dependent on a two-component system consisting of the sensor kinase CitA and the response regulator CitB. The citAB genes are part of the cluster and are positively autoregulated. In addition to CitA/CitB, the cAMP receptor protein (Crp) is involved in the regulation of the citrate fermentation enzymes, subjecting them to catabolite repression.     

Dopaminergic activity and the discriminative stimulus effects of mu opioids in pigeons: importance of training dose and attenuation by the D3 agonist (+/-)-7-OH-DPAT

Chlorophyll fluorescence measurements were performed on osmotically lysed potato chloroplasts in order to characterize the reactions involved in the dark reduction of photosynthetic inter-system chain electron carriers. Addition of NADH or NADPH to lysed chloroplasts increased the chlorophyll fluorescence level measured in the presence of a non-actinic light until reaching Fmax, thus indicating an increase in the redox state of the plastoquinone (PQ) pool. The fluorescence increase was more pronounced when the experiment was carried out under anaerobic conditions and was about 50% higher when NADH rather than NADPH was used as an electron donor. The NAD(P)H-PQ oxidoreductase reaction was inhibited by diphenylene iodonium, N-ethylmaleimide and dicoumarol, but insensitive to rotenone, antimycin A and piericidin A. By comparing the substrate specificity and the inhibitor sensitivity of this reaction to the properties of spinach ferredoxin-NADP+-reductase (FNR), we infer that FNR is not involved in the NAD(P)H-PQ oxidoreductase activity and conclude to the participation of rotenone-insensitive NAD(P)H-PQ oxidoreductase. By measuring light-dependent oxygen uptake in the presence of DCMU, methyl viologen and NADH or NADPH as an electron donors, the electron flow rate through the NAD(P)H-PQ oxidoreductase is estimated to about 160 nmol O2 min-1 mg-1 chlorophyll. The nature of this enzyme is discussed in relation to the existence of a thylakoidal NADH dehydrogenase complex encoded by plastidial ndh genes. Copyright 1998 Elsevier Science B.V.     

Mitochondrial malic enzymes. An association between NAD(P)+-dependent malic enzyme and cell renewal in Sprague-Dawley rat tissues

The activities of the mitochondrial NAD(P)+- and NADP+-dependent malic enzymes were measured in 11 tissues of the male Sprague-Dawley rat. The NAD(P)+-dependent malic enzyme was present in small intestinal mucosa, spleen, thymus, lung, and testis. Each of these tissues contain cells that are undergoing active rates of renewal. The NADP+-dependent malic enzyme was not confined to tissues undergoing cell renewal, and was present in mitochondria from brain, skeletal, and heart muscle, kidney, and lung and testis. Both enzymes were absent or at a low activity in normal and regenerating liver. The results support, and extent to nonneoplastic tissues, our proposal that cells which show active and sustained rates of renewal contain the NAD(P)+-dependent malic enzyme and have a unique intramitochondrial pathway for malate oxidation (Sauer, L.A., Dauchy, R.T., Nagel, W.O., and Morris, H.P. (1980) J. Biol. Chem. 255, 3844-3848).     

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.     

Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase

Murine macrophage nitric oxide synthase (NOS) was expressed in E. coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B). Isolation of active enzyme required the coexpression of calmodulin. Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages. H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers. H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates. No product formation from arginine was observed under either condition. The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn). Nitrite and nitrate were also formed. Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction. The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH. NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate. At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM. HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM.     

Transport of tricarballylate by intestinal brush-border membrane vesicles from steers

Tricarballylic acid is a non-metabolizable rumen bacterial fermentation product of the naturally occurring tricarboxylic acid trans-aconitic acid. The aim of the present study was to investigate intestinal absorption of tricarballylate using brush-border membrane vesicles (BBMVs) isolated from the proximal jejunum of steers by a Ca2+ precipitation method with subsequent differential centrifugation. Transport of tricarballylate was investigated indirectly (influence of tricarballylate on the uptake of 14C-labelled citrate) as well as directly (uptake of 3H-labelled tricarballylate). Citrate as well as tricarballylate uptake (at a concentration of 0.05 mmol l-1) was strongly stimulated by an inwardly directed initial Na+ gradient. Furthermore, transport of both tricarboxylates under Na+ gradient conditions was clearly enhanced by lowering the extravesicular pH from 7.8 to 5.6. The imposition of an inwardly directed H+ gradient (pH(out)/pH(in) = 5.6/7.8) further enhanced the intravesicular accumulation of citrate as well as of tricarballylate compared with pH(out)/pH(in) = 5.6/5.6. Unequivocal evidence for a common transport site for tricarballylate and citrate was obtained from 'cis-inhibition' and 'trans-stimulation' of Na(+)-dependent citrate uptake by tricarballylate. In further experiments the influence of different substances on the uptake of 3H-labelled tricarballylate was evaluated. Unlabelled tricarballylate, citrate, succinate as well as trans- and cis-aconitate significantly inhibited the accumulation of 3H-labelled tricarballylate by BBMVs. Tricarballylate uptake as a function of the tricarballylate concentration revealed a Na(+)-dependent saturable component (apparent kinetic parameters: maximal transport capacity (Vmax) = 119 pmol (mg protein)-1 (3s)-1; affinity constant (Km) = 0.097 mmol l-1) and a Na(+)-independent diffusional component (diffusion constant: 169 nl (mg protein)-1 (3s)-1). It is concluded that tricarballylate and citrate are transported across the intestinal brush-border membrane by a common, Na(+)-dependent transport mechanism. The stimulatory influence of a low extravesicular pH most probably indicates that the protonated forms of tricarboxylates are better transported than the trivalent species.     

The low-affinity dihydropyridine receptor and Na+/Ca2+ exchanger are associated in adrenal medullary mitochondria

The effect of Ca2+ channel-acting drugs on bovine adrenal mitochondria Ca2+ movements was investigated. Mitochondrial Ca2+ uptake is performed by an energy-driven Ca2+ uniporter with a Km of 20.9 +/- 3.2 microM and Vmax of 148.1 +/- 7.2 nmol 45Ca2+ min-1 mg-1. Ca2+ release is performed through an Na+/Ca2+ antiporter with a Km for Na+ of 4.2 +/- 0.5 mM, a Vmax of 7.5 +/- 0.4 nmol 45Ca2+ min-1 mg-1, and a Hill coefficient of 1.4 +/- 0.2 Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger was inhibited by several dihydropyridines (nitrendipine, felodipine, nimodipine, (+)isradipine) and by the benzothiazepine diltiazem with similar potencies. In contrast, neither CGP 28392, Bay-K-8644, amlodipine, nor verapamil had any effect on Ca2+ efflux. Nitrendipine at 20 microM modified neither the Km nor the Hill coefficient for Na+, whereas the Vmax was reduced to 2.9 nmol 45Ca2+ min-1 mg-1, thus demonstrating noncompetitive modulation of the Na+/Ca2+ exchanger. None of the Ca2+ channel-acting drugs assayed at 100 microM affected Ca2+ influx through the uniporter. Ca2+ channel blockers inhibited the Na+/Ca2+ antiporter and displaced the specific binding of [3H]nitrendipine to intact mitochondria with Ki values similar to the IC50s obtained for the inhibition of the Ca2+ efflux. Ca2+ channel-acting drugs that did not inhibit the Na+/Ca2+ exchanger (amlodipine, CGP 28392, Bay-K-9644, and verapamil, at concentrations of 100 microM or higher) had no effect on [3H]nitrendipine binding. These results suggest that the adrenomedullary mitochondrial dihydropyridine receptor is associated with the Na+/Ca2+ exchanger.     

Noradrenaline release from rat sympathetic neurones triggered by activation of B2 bradykinin receptors

1. The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [3H]-noradrenaline and the outflow of radioactivity was determined under continuous superfusion. 2. Bradykinin (100 nmol l[-1] applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 micromol l[-1]) and nicotine (10 micromol l[-1]) caused elevations in 3H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm[-1], 1.0 Hz). 3. When bradykinin was applied for periods of 2 min, the evoked 3H overflow was half-maximal at 12 nmol l(-1) and reached a maximum of 2.3% of cellular radioactivity. The preferential B1 receptor agonist des-Arg9-bradykinin failed to alter 3H outflow. The B2 receptor antagonists, [D-Phe7]-bradykinin (1 micromol l[-1]) and Hoe 140 (10 nmol l[-1]), per se did not alter 3H outflow, but shifted the concentration-response curve for bradykinin-evoked 3H overflow to the right by a factor of 7.9 and 4.3, respectively. 4. Bradykinin-induced overflow was abolished in the absence of extracellular Ca2+ and in the presence of either 1 micromol l(-1) tetrodotoxin or 300 micromol l(-1) Cd2+, as was electrically-induced overflow. Activation of alpha2-adrenoceptors by 1 micromol l(-1) UK 14,304 reduced both bradykinin- and electrically-triggered overflow. The Ca2+-ATPase inhibitor thapsigargin (0.3 micromol l[-1]) failed to alter either type of stimulated overflow. Caffeine (10 mmol l[-1]) enhanced bradykinin-induced overflow, but reduced overflow triggered by electrical field stimulation. 5. Inclusion of Ba2+ (0.1 to 1 mmol l[-1]) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin-triggered overflow by almost 400%. Application of 1 mmol l(-1) Ba2+ for periods of 2 min triggered 3H overflow, and this overflow was abolished by 1 micromol l(-1) tetrodotoxin and enhanced by 10 mmol l(-1) caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l[-1]) in the superfusion buffer caused similar increases of bradykinin- and electrically evoked 3H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter 3H outflow. 6. Treatment of cultures with 100 ng ml(-1) pertussis toxin caused a significant increase in bradykinin-, but not in electrically-, evoked tritium overflow. Treatment with 100 ng ml(-1) cholera toxin reduced both types of stimulated 3H overflow. 7. These data reveal bradykinin as a potent stimulant of action potential-mediated and Ca2+-dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B2-receptors, presumably linked to pertussis- and cholera toxin-insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K+ (KM) channels, like caffeine and Ba2+, indicate that the secretagogue action of bradykinin probably involves inhibition of these K+ channels.     

Clinical spectrum of nonmenstrual toxic shock syndrome (TSS): comparison with menstrual TSS by multivariate discriminant analyses

1. The present experiments were undertaken in order to characterize further the apparently irreversible inhibition of the contraction of depolarized rat aorta caused by lacidipine, a 1,4-dihydropyridine calcium antagonist. 2. We studied the effect of lacidipine on contraction evoked by 100 mM KCl solution in rat aorta, treated by N omega-nitro-L-arginine (0.1 mM), an inhibitor of nitric oxide (NO) synthesis. We compared the effect of prolonged depolarization on lacidipine and (+)-isradipine inhibition and the reversal of this inhibition after washout in the absence of dihydropyridines. Assuming that the onset of lacidipine-evoked inhibition was a pseudo-first order association kinetics, we estimated the dissociation rate constant (k-1 = 0.031 min-1), the association rate constant (k1 = 2.70 x 10(8) M-1 min-1) and the dissociation constant (KD = k-1/k1 = 115 pM) which was close to the IC50 value in steady-state conditions (160 pM). 3. The inhibitory effects of lacidipine and (+)-isradipine on rat aorta contraction were reversibly enhanced after preincubation with the drug in a 40 mM KCl-solution. Washout with drug-free 40 mM K(+)-depolarizing solution reversed inhibition in the (+)-isradipine-treated preparations, but not in the lacidipine-treated ones. 4. Radioligand binding studies were performed with [3H]-lacidipine and [3H]-isradipine in microsomes from rat aorta and rat ileum. Both ligands bound to a homogeneous population of binding sites (for[3H]-lacidipine: KD = 23 +/- 2.6 pM, Bmax = 380 +/- 21 fmol mg-1 protein in membranes from aorta; KD =23 +/- 3.1 pM, Bmax = 790 +/- 60 fmol mg-1 protein in membranes from ileum; for [3H]-isradipine:KD = 140 +/- 46 pM, Bmax = 350 +/- 64 fmol mg-1 protein in membrane from aorta; KD = 68 +/- 14 pM,Bmax = 760 +/- 75 fmol mg-1 protein in membranes from ileum). After isotopic dilution, [3H]-lacidipine and [3H]-isradipine dissociated according to a monoexponential kinetics. In membranes from ileum, the calculated dissociation rate constants (kappa_ 1) were 0.0257 min-1 and 0.0595 min-1, for [3H]-lacidipine and[3H]-isradipine, respectively.5. The non specific binding of [3H]-lacidipine and [3H]-isradipine, was measured in intact rat aorta preparations incubated under the conditions of the functional experiments, in the presence of nifedipine(1 microM). After incubation with [3H]-lacidipine 77.6 +/- 1.9 pM for 2 h the concentration of drug in the tissue was 15.15 +/- 1.18 fmol mg-1 w.wt. and still amounted to 7.24 +/- 0.61 fmol mg-1 w.wt. after 3.5 h washout in drug-free solution. After incubation with [3H]-isradipine 47.2 +/- 0.4 pM for 2 h it was 2.26 +/-0.07 fmol mg-1 w.wt. and was undetectable after 3.5 h washout in a drug-free solution.6. It is concluded that lacidipine interacts reversibly with dihydropyridine binding sites and that the apparent irreversible inhibition of contraction in depolarized preparations could be related to a nonspecific binding in a tissue compartment different from the plasma membrane.     

Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart

BACKGROUND: Increased expression of Ca2+-sensitive protein kinase C (PKC) isoforms may be important markers of heart failure. Our aim was to determine the relative expression of PKC-beta1, -beta2, and -alpha in failed and nonfailed myocardium. METHODS AND RESULTS: Explanted hearts of patients in whom dilated cardiomyopathy or ischemic cardiomyopathy was diagnosed were examined for PKC isoform content by Western blot, immunohistochemistry, enzymatic activity, and in situ hybridization and compared with nonfailed left ventricle. Quantitative immunoblotting revealed significant increases of >40% in PKC-beta1 (P<0.05) and -beta2 (P<0.04) membrane expression in failed hearts compared with nonfailed; PKC-alpha expression was significantly elevated by 70% in membrane fractions (P<0.03). PKC-epsilon expression was not significantly changed. In failed left ventricle, PKC-beta1 and -beta2 immunostaining was intense throughout myocytes, compared with slight, scattered staining in nonfailed myocytes. PKC-alpha immunostaining was also more evident in cardiomyocytes from failed hearts with staining primarily localized to intercalated disks. In situ hybridization revealed increased PKC-beta1 and -beta2 mRNA expression in cardiomyocytes of failed heart tissue. PKC activity was significantly increased in membrane fractions from failed hearts compared with nonfailed (1021+/-189 versus 261+/-89 pmol. mg-1. min-1, P<0.01). LY333531, a selective PKC-beta inhibitor, significantly decreased PKC activity in membrane fractions from failed hearts by 209 pmol. min-1. mg-1 (versus 42.5 pmol. min-1. mg-1 in nonfailed, P<0.04), indicating a greater contribution of PKC-beta to total PKC activity in failed hearts. CONCLUSIONS: In failed human heart, PKC-beta1 and -beta2 expression and contribution to total PKC activity are significantly increased. This may signal a role for Ca2+-sensitive PKC isoforms in cardiac mechanisms involved in heart failure.     

Purification and characterization of a feruloyl esterase from the fungus Penicillium expansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and rho-coumaric acid from methyl esters of the acids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->3)-O-beta- D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and O-[5-O-((E)-rho-coumaroyl)-alpha-L-arabinofuranosyl]- (1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (PAXX). The esterase was purified 360-fold in successive steps involving ultrafiltration and column chromatography by gel filtration, anion exchange and hydrophobic interaction. These chromatographic methods separated the phenolic acid esterase from alpha-L-arabinofuranosidase, pectate and pectin lyase, polygalacturonase, xylanase and beta-D-xylosidase activities. The phenolic acid esterase had an apparent mass of 65 kDa under non-denaturing conditions and a mass of 57.5 kDa under denaturing conditions. Optimal pH and temperature were 5.6 and 37 degrees C, respectively and the metal ions Cu2+ and Fe3+ at concentrations of 5 mmol 1-1 inhibited feruloyl esterase activity by 95% and 44%, respectively, at the optimum pH and temperature. The apparent Km and Vmax of the purified feruloyl esterase for methyl ferulate at pH 5.6 and 37 degrees C were 2.6 mmol 1-1 and 27.1 mumol min-1 mg-1. The corresponding constants of rho-coumaroyl esterase for methyl coumarate were 2.9 mmol 1-1 and 18.6 mumol min-1 mg-1.     

Intensity, duration, and frequency of physical activity and coronary risk factors

Relationships between coronary risk factors and intensity, duration, and frequency of leisure activity were studied in 5943 men and 6039 women, ages 25-69. Age, smoking, socioeconomics, season, body mass index (BMI), urbanization, occupational activity, and liquid, alcohol, and saturated/total fat intake were adjusted using multivariate regressions. Among men each 100 kcal.kg-1.wk-1 spent on vigorous activities (7.5-9.0 MET) was associated with: significant (P < 0.01) average differences of -0.36 mmol.L-1 total cholesterol, +0.17 mmol.L-1 HDL cholesterol (P < 0.001), +0.05 HDL/total cholesterol (P < 0.001), -0.33 mmol.L-1 triglycerides, -3 mm Hg diastolic blood pressure, -10 beats.min-1 heart rate (P < 0.001), +30 L.min-1 peak flow, and -1.1 kg.m-2 BMI. Among women it was associated with: -7 mm Hg systolic blood pressure, -6 beats.min-1 heart rate (P < 0.001), +50 L.min-1 peak flow (P < 0.001), and -1.4 kg.m-2 BMI (P < 0.05). Moderate activity (either 3.0-4.5 MET or 5.0-7.0 MET) was significantly (P < 0.05) associated with HDL cholesterol, BMI, and, for men, heart rate; for women, it was associated with HDL/total cholesterol, triglycerides, diastolic blood pressure, and peak flow. With duration and intensity constant, increasing frequency by one time per wk was significantly (P < 0.05) associated with -0.014 mmol.L-1 total cholesterol, +0.001 HDL/total cholesterol, -0.36 beats.min-1 heart rate, -0.093 kg.m-2 BMI among men, and +0.009 mmol.L-1 HDL cholesterol, +0.001 HDL/total cholesterol, -0.014 mmol.L-1 triglycerides, -0.31 beats.min-1 heart rate, and -0.098 kg.m-2 BMI among women. Serum lipids and BMI showed stronger associations with frequency than with intensity or duration.     

Pulmonary emphysema decreases hamster skeletal muscle oxidative enzyme capacity

Skeletal muscle oxidative enzyme capacity is impaired in patients suffering from emphysema and chronic obstructive pulmonary disease. This effect may result as a consequence of the physiological derangements because of the emphysema condition or, alternatively, as a consequence of the reduced physical activity level in these patients. To explore this issue, citrate synthase (CS) activity was measured in selected hindlimb muscles and the diaphragm of Syrian Golden hamsters 6 mo after intratracheal instillation of either saline (Con, n = 7) or elastase [emphysema (Emp); 25 units/100 g body weight, n = 8]. Activity level was monitored, and no difference between groups was found. Excised lung volume increased with emphysema (Con, 1.5 +/- 0.3 g; Emp, 3.0 +/- 0.3 g, P < 0.002). Emphysema significantly reduced CS activity in the gastrocnemius (Con, 45.1 +/- 2.0; Emp, 39.2 +/- 0.8 micromol . min-1 . g wet wt-1, P < 0.05) and vastus lateralis (Con, 48.5 +/- 1.5; Emp, 44.9 +/- 0.8 micromol . min-1 . g wet wt-1, P < 0.05) but not in the plantaris (Con, 47.4 +/- 3.9; Emp, 48.0 +/- 2.1 micromol . min-1 . g wet wt-1, P < 0.05) muscle. In contrast, CS activity increased in the costal (Con, 61.1 +/- 1.8; Emp, 65.1 +/- 1.5 micromol . min-1 . g wet wt-1, P < 0.05) and crural (Con, 58.5 +/- 2.0; Emp, 65.7 +/- 2.2 micromol . min-1 . g wet wt-1, P < 0.05) regions of the diaphragm. These data indicate that emphysema per se can induce decrements in the oxidative capacity of certain nonventilatory skeletal muscles that may contribute to exercise limitations in the emphysematous patient.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

Determination of human hepatic cytochrome P4501A2 activity in vitro use of tacrine as an isoenzyme-specific probe

Oxidative metabolism of the cognition activator tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is thought to be catalyzed by cytochrome P4501A2 (CYP1A2). In this study, the use of tacrine as a specific substrate to measure CYP1A2 activity in vitro was investigated. Tacrine metabolism was assessed in 16 human liver microsomal samples. Initially, the percentage conversion of tacrine to stable metabolites (i.e. 1-, 2-, 4-, and 7-hydroxytacrine) at a single time point was correlated with levels of CYP1A2 apoprotein. Apoprotein was detected by immunoquantification using a monospecific CYP1A2 antipeptide antibody. Significant correlations were seen between CYP1A2 content and the degree of 1-hydroxylation (r = 0.81, p < 0.001), 7-hydroxylation (r = 0.70, p < 0.001), and metabolism to all stable products (r = 0.82, p < 0.001). The major metabolite formed in all livers was 1-hydroxytacrine. The conversion of tacrine to this metabolite was examined in more detail. The rate of formation varied from 19.2 pmol min-1 mg-1 to 101.0 pmol min-1 mg-1. There was a significant correlation (r = 0.84, p < 0.001) between the rate of formation and CYP1A2 levels. Tacrine metabolism was also compared with the rate of formation of 3-methylxanthine, from theophylline, a reaction previously shown to be catalyzed by CYP1A2. Significant correlations were found between 3-methylxanthine formation and all quantified tacrine metabolites. The rate of 3-methylxanthine generation also correlated with CYP1A2 apoprotein levels. It is concluded, therefore, that tacrine is a valuable probe for the determination of human hepatic CYP1A2 activity in vitro.     

Inhibitory mechanisms of naloxone on human platelets

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.     

Ester synthesis by NAD(+)-dependent dehydrogenation of hemiacetal: production of methyl formate by cells of methylotrophic yeasts

A water-soluble ester, methyl formate, was detected as a metabolite in the culture medium of methylotrophic yeasts. Methyl formate synthase, which catalyses NAD(+)-dependent dehydrogenation of the hemiacetal adduct of methanol and formaldehyde, catalyses the ester synthesis. The enzyme activity was induced on a methanol medium and was increased further by the addition of formaldehyde. In the reaction system using intact cells of Pichia methanolica AKU 4262, 135 mM (8.1 g/liter) methyl formate was produced from 2 M methanol. This is a new biological process for ester synthesis that couples spontaneous formation of hemiacetal and alcohol dehydrogenase.     

In situ characterization of nonmitochondrial Ca2+ stores in individual pancreatic beta-cells

Free Ca2+ was measured in intracellular stores of individual mouse pancreatic beta-cells using dual-wavelength microfluorometry and the low-affinity Ca2+ indicator furaptra. Controlled permeabilization of the plasma membrane with 4 micromol/l digitonin revealed that 22% of the furaptra was trapped in intracellular nonnuclear compartments. When 3 mmol/l ATP and 200 nmol/l Ca2+ were simultaneously present, this cation rapidly accumulated in the organelle pool, reaching an average concentration of 200-500 micromol/l. Whereas agents affecting the mitochondrial function (5 mmol/l succinate, 2 micromol/l ruthenium red, or 10 micromol/l antimycin A + 2 microg/ml oligomycin) had little effects, the Ca2+-ATPase inhibitor thapsigargin released 92% of the Ca2+ mobilizable with the ionophore Br-A23187. Digital imaging revealed regional differences in the organelle Ca2+. The regions with the highest Ca2+ concentration were particularly responsive to inositol 1,4,5-trisphosphate (IP3). IP3 mobilized Ca2+ in a dose-dependent way with half-maximal and maximal effects at about 1 and 5 micromol/l, respectively. High concentrations of IP3 released about half of the thapsigargin-sensitive Ca2+, but there were no responses to agents known to activate ryanodine receptors, such as 10 mmol/l caffeine, 0.1-1 micromol/l ryanodine, or 1-5 micromol/l cyclic ADP ribose. The results reinforce the concept that mobilization of intracellular Ca2+ in the pancreatic beta-cell is mediated by IP3 receptors rather than ryanodine receptors.     

Integrating cytosolic calcium signals into mitochondrial metabolic responses

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.     

Ca2+-independent phospholipase A2 contributes to the insulinotropic action of cholecystokinin-8 in rat islets: dissociation from the mechanism of carbachol

Insulin secretion induced by cholecystokinin-8 (CCK-8) was recently suggested to involve phospholipase A2 (PLA2) activation. In this study, we examined whether CCK-8 stimulates the Ca2+-independent form of PLA2 in isolated rat islets, in comparison with stimulation by the PLA2-activating cholinergic agonist carbachol. We found that CCK-8 (100 nmol/l; 5.6 mmol/l glucose) induces lysophosphatidylcholine accumulation from [3H]palmitate-prelabeled islets (170 +/- 39%; P = 0.003) as well as arachidonic acid (AA) efflux from [3H]AA-prelabeled islets (190 +/- 13%; P < 0.001), and that p-amylcinnamoylantranilic acid (ACA) (50 micromol/l)-mediated PLA2 inhibition reduces CCK-8-induced AA efflux (52 +/- 11%; P = 0.001) and insulin secretion (67 +/- 16%; P < 0.001). Neither the Ca2+ channel antagonist verapamil (100 micromol/l) nor the Ca2+ATPase inhibitor thapsigargin (1 micromol/l) affected CCK-8-induced AA efflux and insulin secretion. Furthermore, despite removal of extracellular Ca2+, CCK-8 still increased AA efflux (48 +/- 14%; P = 0.006) and insulin secretion (105 +/- 46%; P = 0.025). In contrast, carbachol (100 micromol/l)-stimulated AA efflux was reduced by verapamil by 36 +/- 6% (P < 0.001) and abolished by removal of extracellular Ca2+. Overnight protein kinase C (PKC) downregulation by 12-O-tetradecanoyl phorbol-13-acetate (TPA) (500 nmol/l) reduced CCK-8-induced AA efflux (45 +/- 12%; P = 0.003) and insulin secretion (40 +/- 16%; P = 0.020). No additive action regarding either AA formation or insulin secretion was seen by combining TPA overnight and ACA, which implies the involvement of an additional PLA2- and PKC-independent signaling mechanism. The results show that CCK-8, in contrast to carbachol, activates Ca2+-independent PLA2 in islets and that the PLA2-activating capacity of CCK-8 is partly PKC dependent. Hence, Ca2+-independent PLA2 seems important for the insulinotropic effect of CCK-8, but not for that of carbachol.