Exogenous NADPH Research Articles

Rapid assessment of induced cytochrome P4501 a protein and catalytic activity in fish hepatoma cells grown in multiwell plates: Response to TCDD, TCDF, and two planar PCBS

Induction of cytochrome P450 1A1 (CYP1A1) in cultured cells can be used to determine taxon-specific relative potencies of Ah receptor agonists. This report describes optimized methods for growth and treatment of PLHC-1 fish hepatoma cells in multiwell plates, in situ analysis of ethoxyresorufin O-deethylase (EROD) activity, and measurement of CYP1A protein by immunoblotting of cell lysates. EROD activity was undetectable (< 1 pmol min{sup {minus}1} mg{sup {minus}1}) in untreated or dimethyl sulfoxide-treated cells, but was highly induced (up to 150 pmol min{sup {minus}1} mg{sup {minus}1}) in cells exposed to Ah receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), or plant chlorobiphenyls (CB). Addition of exogenous NADPH was not required for measurement of EROD activity in PLHC-1 cells. As inducers of EROD activity, TCDD, TCDF, 3,3{prime},4,4{prime},5-pentachlorobiphenyl (CB-126), and 3,3{prime},4,4{prime}-tetrachlorobiphenyl (CB-77) differed both in potency and in apparent efficacy (maximal level of induced activity). In each case, EROD induction was biphasic, with stronger induction at lower concentrations and an attenuated response at higher concentrations. In contrast, the content of immunodetectable CYP1A protein increased monotonically with dose of CB, and the maximum level achieved was similar for all inducers. The discrepancy in results obtained for EROD activity versus CYP1A protein may result frommore » inhibition or inactivation of catalytic function at high concentrations of inducer. By reducing peak EROD values, this inhibition leads to lower apparent EC50 values and thus the overestimation of relative potencies or toxic equivalency factors (TEFs) for many inducers. These studies demonstrate the necessity of measuring both EROD activity and immunodetectable CYP1A protein for the accurate assessment of CYP1A induction and relative potencies in cultured cells.« less

Direct evidence for the presence of two external NAD(P)H dehydrogenases coupled to the electron transport chain in plant mitochondria

Exogenous NADPH oxidation by purified mitochondria from both potato tuber and Arum maculatum spadix was completely and irreversibly inhibited by sub-micromolar diphenyleneiodonium (DPI), while exogenous NADH oxidation was inhibited to only a small degree. Addition of DPI caused the collapse of the membrane potential generated by NADPH oxidation, while the potential generated by NADH was unaffected. We conclude that there are two distint enzymes on the outer surface of the inner membrane of plant mitochondria, one specific for NADH, the other relatively specific for NADPH, with both enzymes linked to the electron transport chain.

Identification by photoaffinity labelling of a pyridine nucleotide-dependent tri-iodothyronine-binding protein in the cytosol of cultured astroglial cells

High-affinity 3,3',5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3',5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3'5'-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).

Separation of intact and damaged hepatocytes in sucrose following non-enzymatic liver perfusion

This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP(+)) indicates a predominantly mitochondrial localization of TPP(+) in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP(+), and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll.

The histochemical G6PDH reaction but not the LDH reaction with neotetrazolium is suitable for the oxygen sensitivity test to detect cancer cells.

We used the oxygen sensitivity of the histochemical reaction to detect glucose-6-phosphate dehydrogenase (G6PDH) activity based on neotetrazolium (NT) reduction to discriminate cancer cells from normal cells. Formazan generation was strongly reduced in normal but not in malignant cells when the incubation was performed in oxygen instead of nitrogen. Competition for reductive equivalents between NT and oxygen via superoxide dismutase (SOD) has been suggested. Since SOD activity is usually decreased in cancer cells, NT reduction would not be hampered in these cells. We tested this hypothesis by demonstrating NAD-dependent lactate dehydrogenase (LDH) activity instead of NADP-dependent G6PDH activity in normal rat liver and colon, in human colon carcinoma, and in experimentally induced metastases of colon carcinoma in rat livers. Reactions for both enzymes were determined cytophotometrically in an atmosphere of pure oxygen or nitrogen. G6PDH acted as described previously, showing distinct activity in cancer cells but strongly reduced activity in normal cells after incubation in oxygen, but this was not the case with LDH because formazan was also generated in normal tissue in oxygen. It appeared that after 5 min of incubation at 37 degrees C the residual activity of G6PDH in an atmosphere of oxygen compared with nitrogen was 0% in normal liver tissue and 15% in normal colon epithelium, whereas in colon carcinoma and in colon carcinoma metastasis in liver it was 48% and 33%, respectively. The residual activity of LDH in oxygen was 30% in normal female rat liver, 75% in normal male rat liver, and 38% in normal colon epithelium, whereas the residual activity in colon carcinoma and metastases in liver was 54% and 24%, respectively. These experiments clearly indicate that the oxygen sensitivity phenomenon is not solely an effect of competition for reducing equivalents between NT and oxygen via SOD, because NADPH generated by G6PDH and NADH generated by LDH have a similar redox potential. Apparently the system is more complex. The role of specifically NADPH-converting cellular systems such as NADPH-cytochrome P450 reductase was excluded because incubations in the presence of exogenous NADPH as substrate for these systems revealed oxygen sensitivity. Involvement of NADPH-dependent lipid peroxidation in the oxygen sensitivity test is discussed.

NADPH Oxidase-derived Reactive Oxygen Intermediates Regulate Tyrosine Phosphorylation in Human Neutrophils

Activation of the NADPH-oxidase in neutrophils results in the production of reactive oxygen intermediates (ROI) including superoxide (O2-) and hydrogen peroxide (H2O2) which have traditionally been viewed as functioning in host defense. Recent attention has been directed at a physiologic role for ROI as intracellular signaling molecules. Phosphorylation of proteins on tyrosine residues has been implicated in the control of cell activation, and the tyrosine kinase ltk in lymphocytes has been shown to be regulated by oxidants. As neutrophils produce prodigious quantities of ROI, we hypothesized that these compounds function in a physiological manner in the regulation of tyrosine phosphorylation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immuno-blotting with antiphosphotyrosine antibodies were used to assess tyrosine phosphorylation in human neutrophils. Exposure of neutrophils to diamide, a thiol-oxidizing agent, resulted in marked tyrosine phosphorylation of several proteins suggesting the regulation of this pathway by oxidant-sensitive enzymes. The following observations suggested that ROI produced by the NADPH-oxidase regulate tyrosine phosphorylation in a physiological manner: (1) direct activation of the oxidase by stimulation of G-proteins in electropermeabilized neutrophils by GTPγS or fluoroaluminate (AlF4-) resulted in marked tyrosine phosphorylation of bands of Mr = 30, 42, 60, and 90 in a time and concentration-dependent manner. Importantly, the latter effect was observed only when the cells were supplied with exogenous NADPH; (2) in the presence of NADPH but when the function of the oxidase was inhibited by diphenyleneiodonium, GTPγS-induced tyrosine phosphorylation was minimal; and (3) neutrophils from a patient with X-linked (X91°) chronic granulomatous disease (which are unable to produce ROI) did not demonstrate enhancement of GTPγS-induced tyrosine phosphorylation in the presence of NADPH. Using an in vitro gel renaturation assay with myelin basic protein as the substrate, we observed stimulation of kinase activity of a 42-kd band in cells treated with diamide suggesting that MAP kinase activity could be modulated by oxidants.

Inhibition of Biotransformation of Xenobiotic p-Nitroanisole after Cryopreservation of Isolated Rat Hepatocytes

Cryopreservation of isolated rat hepatocytes with dimethyl sulfoxide (Me2SO) using a rapid two-step freezing protocol allows significant preservation of cell recovery and xenobiotic p-nitroanisole (p-NA) metabolism. A decrease in the rate of O-demethylation of p -NA after cryopreservation correlates with a change in cellular viability. A freeze-thaw procedure in the absence of a cryoprotectant results in a sharp inhibition and subsequent washing causes a complete loss of the ability of cells to metabolize the xenobiotic. In the presence of exogenous NADPH the rate of p-NA biotransformation is restored. The content of cytochrome P-450 does not change during a hepatocyte freeze-thaw procedure in the presence or absence of Me2SO. Separation of cells after cryopreservation using a Percoll solution permits the isolation of a suspension, the viability of which does not differ from that of the freshly isolated control, but which metabolizes p-NA at a slower rate. During incubation, viability of cryopreserved cells was reduced. The data indicate that inhibition of monooxygenative activity after hepatocyte cryopreservation results from the loss of endogenous reduced pyridine nucleotides. The loss of pyridine nucleotides results from damage of plasma membrane during both cryopreservation and subsequent incubation in the presence of xenobiotic.

Microsomal cytochrome P450 1A1 dependent monooxygenase activity in guinea pig heart: induction, inhibition, and increased activity by addition of exogenous NADPH–cytochrome P450 reductase

Characterization of cytochrome P450 1A1 dependent monooxygenases in guinea pig heart revealed low rates of 7-ethoxyresorufin O-deethylation, which are markedly increased (20-fold) by treatment with beta-naphthoflavone, a polycyclic aromatic hydrocarbon. Both 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation were found to be approximately 4-fold higher in microsomes prepared from the ventricle than the atrium of beta-naphthoflavone-induced guinea pigs. The low rates of 7-ethoxyresorufin O-deethylation in cardiac microsomes were due, at least in part, to a deficiency of the flavoprotein NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase dramatically increased 7-ethoxyresorufin O-deethylation in cardiac microsomes of guinea pigs, before and after treatment with beta-naphthoflavone. N-Benzyl-1-aminobenzotriazole, a suicide substrate of cytochrome P450 1A1 in guinea pig, was able to inhibit almost all of the 7-ethoxyresorufin O-deethylase and 7-methoxyresorufin O-demethylase activities in polycyclic aromatic hydrocarbon induced guinea pig heart (88 and 71%, respectively), suggesting that cytochrome P450 1A1 coupled to NADPH--cytochrome P450 reductase in these microsomes inactivates itself by a suicidal mechanism. Addition of alpha-naphthoflavone, an inhibitor of cytochrome P450 1A isozymes, to cardiac microsomes from beta-naphthoflavone-induced guinea pigs resulted in greater than 95% inhibition of 7-ethoxyresorufin O-deethylase activity. The biological significance of these low levels of cytochrome P450 1A1 monooxygenase activity in guinea pig heart and their induction by polycyclic aromatic hydrocarbons are not currently understood.

Biosynthesis of 12(S)‐hydroxyeicosatetraenoic acid by bovine corneal epithelium

Microsomes of bovine corneal epithelium metabolized [14C]arachidonic acid extensively to [14C]12-hydroxyeicosatetraenoic acid. The biosynthesis was not stimulated by exogenous NADPH and it could be inhibited by over 90% with two lipoxygenase inhibitors, esculetin and eicosatetraynoic acid. Chiral phase high performance liquid chromatography showed that 12-HETE consisted of the 12S enantiomer. The results indicate biosynthesis by 12S-lipoxygenase of bovine corneal epithelium rather than by cytochrome P450.

Deficient glucose phosphorylation as a possible common denominator and its relation to abnormal leucocyte function, in glycogen storage disease 1b patients

Patients with glycogen storage disease (GSD) 1b suffer from recurrent bacterial infections related to neutropenia and impairment of neutrophil functions. One of these functions is the oxidative burst activity which is initiated by NADPH oxidase and depends on the availability of glucose. This activity was markedly reduced in the patient's intact neutrophils when either N-formyl-methionyl-leucyl-phenylalanine (fMLP), or phorbol myristate acetate were used as stimulants. In disrupted GSD 1b polymorphonuclear leucocytes (PMNs), in the presence of exogenous NADPH, this activity was within the normal range. Degranulation, which is calcium dependent but glucose independent, was not significantly different in neutrophils from the patients as compared to controls. Resting cytosolic calcium concentration was indistinguishable from controls. Activation with 10(-7) M fMLP, in the presence or absence of glucose, triggered a prompt and rapid elevation of cytosolic calcium both in the control and the patients' cells. We have previously shown that hexose monophosphate (HMP) shunt activity and glycolytic rate were found to be lower by 70% in intact PMN cells of the patients compared with controls. These activities were normal in disrupted neutrophils. The uptake of the non-metabolized glucose analogues 2-deoxyglucose (2-DOG) and 3-O-Methylglucose (3-OMG) into PMN of GSD 1b patients was studied. 2-DOG is phosphorylated within the cells, thus its uptake rate reflects hexose transport at low concentrations, as long as phosphorylation is not rate limiting. Under those conditions (5 microM 2-DOG) transport was found to be similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Transplantation of transgenic photoreceptors to RD mouse retina

Photoreceptors of nocturnal geckos are transmuted cones that acquired rod morphological and physiological properties but retained cone-type phototransduction proteins. We have used microspectrophotometry and microfluorometry of solitary isolated green-sensitive photoreceptors of Tokay gecko to study the initial stages of the visual cycle within these cells. These stages are the photolysis of the visual pigment, the reduction of all-trans retinal to all-trans retinol, and the clearance of all-trans retinol from the outer segment (OS) into the interphotoreceptor space. We show that the rates of decay of metaproducts (all-trans retinal release) and retinal-to-retinol reduction are intermediate between those of typical rods and cones. Clearance of retinol from the OS proceeds at a rate that is typical of rods and is greatly accelerated by exposure to interphotoreceptor retinoid-binding protein, IRBP. The rate of retinal release from metaproducts is independent of the position within the OS, while its conversion to retinol is strongly spatially non-uniform, being the fastest at the OS base and slowest at the tip. This spatial gradient of retinol production is abolished by dialysis of saponin-permeabilized OSs with exogenous NADPH or substrates for its production by the hexose monophosphate pathway (NADP + glucose-6-phosphate or 6-phosphogluconate, glucose-6-phosphate alone). Following dialysis by these agents, retinol production is accelerated by several-fold compared to the fastest rates observed in intact cells in standard Ringer solution. We propose that the speed of retinol production is set by the availability of NADPH which in turn depends on ATP supply within the outer segment. We also suggest that principal source of this ATP is from mitochondria located within the ellipsoid region of the inner segment.

Regulation of 5-Aminolevulinic Acid Synthesis in Developing Chloroplasts

5-Aminolevulinic acid synthesis in isolated, intact, developing chloroplasts from greening cucumber (Cucumis sativus) cotyledons was inhibited by broken chloroplast fragments. It was shown that the inhibitory constituent was associated with the thylakoid membrane system. The inhibitor was resistant to boiling, was not a form of ribonuclease, and did not inhibit Mg-chelatase, indicating that massive organelle destruction was not involved. The inhibitor was also found in etioplast and mature chloroplasts; and it was found in barley as well as cucumber. 5-Aminolevulinate synthesis in the dark with exogenous ATP and NADPH, or in the light without added cofactors, were inhibited approximately equally. In the dark, 5-aminolevulinate synthesis and protochlorophyllide synthesis from glutamate were inhibited to about equal extent. The inhibition was decreased when the membranes were washed with aqueous acetone prior to incubation. The inhibition by the unknown factor was compared to the inhibition by gabaculine, 4-amino-5-hexynoic acid, protoheme, and glutathione. The unknown inhibitor appeared to have a number of similarities with protoheme.

Lipid peroxidation in the liver of carcinogen-resistant rats

Recently, we developed a new strain of rats that exhibit marked resistance to the hepatotoxic and carcinogenic actions of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) and some other carcinogens. In this work, we compared lipid peroxidation in the liver of these carcinogen-resistant (R) rats and the parental Donryu strain rats that are sensitive (S) to hazardous actions of these carcinogens. The liver microsomal fractions of the R group contained less amounts of polyunsaturated fatty acids. Microsomal lipid peroxidation in the presence of exogenous NADPH was much lower in R rats than in S rats. Liver microsomes of R rats were much less active than those of S rats also in producing 4-hydroxynonenal, carbonyl compounds and conjugated diene. The hepatic contents of ascorbic acid, glutathione, α-tocopherol and coenzyme Q in the R rats were similar to those in S rats. The activities of the free radical scavenger enzymes, Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), in the two groups were also similar. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are both thought to function in disposal of these cytotoxic aldehydes. The liver microsomal and mitochondrial ALDH activities of the two groups were similar. The ADH activity of the liver cytosolic fraction of R rats was nearly twice that of S rats, as measured with 4-hydroxynonenal as substrate. The higher ADH activity may explain the decreased lipid peroxidation in R rats at least partly, if this enzyme is involved in lipid peroxidation.

Propargite Metabolism in Bulb Mite (Acari: Acaridae)

When 50-mg samples of bulb mites, Rhizoglyphus echinopus (Fumouze and Robin), were placed for 1 h in glass vials coated with 0.53 μg of radiocarbon-labeled propargite, about 12% (1.27 μg/g) of the acaricide was transferred to the mites. Mites treated with propargite were kept in holding vials and the extent of metabolism was evaluated after 6, 12, 24, and 48 h. Internal levels of radiocarbon ranged from 14.8 to 17.4% of the dose and were divided about equally between the mite extract and mite residue fractions. Levels of propargite in the internal extract decreased from 5.9 at 6 h to 2.5 at 48 h when expressed as percentage of total recovered radiocarbon and from 61.5 at 6 h to 29.8 at 48 h when expressed as relative percentage of radiocarbon in the internal extract. Thus, propargite was degraded slowly by the mites. The metabolites 4- tert -butylphenol and 2-4- tert (-butylphenoxy) cyclohexanol (glycol ether) were present at low levels (<0.5%). Propargite wasdegraded in vitro by bulb mite homogenates with the 12,000 x g supernatant being about four times more active than the corresponding pellet. Exogenous NADPH and glutathione greatly enhanced propargite degradation by the supernatant fraction. However, not all of the degradation in the presence of glutathione was enzymatic; about 15% of the propargite was converted to 4- tert -butylphenol when the acaricide was incubated with this cofactor in buffer in the absence of the mite supernatant. In vitro propargite degradation products consisted mainly of unidentified polar metabolites, but low levels of 4- tert -butylphenol and glycol ether also were present.

Flavanone Glycoside Biosynthesis in Citrus

Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP-glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP-glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP-glucose:flavanone-7-O-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species.

Enzymatic formation of nitrogen oxides from L-arginine in bovine brain cytosol

In dialyzed bovine brain cytosol, the enzymatic formation of nitrogen oxides was directly determined. The basal formation of nitrite and nitrate was concentration-dependently enhanced by L-arginine (EC 50 about 3·10 −5 M). Both the basal and L-arginine induced formations were inhibited by N G-monomethyl-L-arginine (EC 50 about 2·10 −4 M). In the presence of L-arginine, a concomitant formation of citrulline was detected. L-Arginine methyl ester also served as a substrate, but neither D-arginine, D-arginine methyl ester nor N α-benzoyl-L-arginine ethyl ester did so. The formation of nitrite and nitrate was time-dependent, increased linearly with the protein concentration of the cytosol and was not observed when the cytosolic proteins were heat-denaturated. Exogenous NADPH (or NADP +) concentration-dependently enhanced the formation of nitrite and nitrate, whereas NADH, NAD +, FAD, Ca 2+, Mg 2+ and calmodulin were ineffective. These results indicate that bovine brain contains a cytosolic enzyme which uses NADPH or NADP + as cofactors to form nitrogen oxides from both an endogenous non-dialyzable substrate and from L-arginine.

Receptor-mediated activation of electropermeabilized neutrophils. Evidence for a Ca2+- and protein kinase C-independent signaling pathway.

Electrically permeabilized neutrophils were used to study the mechanism of activation of the respiratory burst by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (fMLP). Permeabilization was assessed by flow cytometry, radioisotope trapping, and by the requirement for exogenous NADPH for oxygen consumption. A respiratory burst could be elicited by fMLP, phorbol ester, or diacylglycerol in permeabilized cells suspended in EGTA-buffered medium with 100 nM free Ca2+. The fMLP response persisted even in cells depleted of intracellular Ca2+ stores by pretreatment with ionomycin. Therefore, a change in cytosolic free Ca2+ ([Ca2+]i) is not required for receptor-mediated stimulation of the respiratory burst. The responses induced by phorbol ester and diacylglycerol were largely inhibited by H7, a protein kinase C antagonist. In contrast, the stimulation of oxygen consumption by fMLP was unaffected by H7. These results suggest that a third signaling pathway, distinct from changes in [Ca2+]i and activation of protein kinase C, is involved in the response of neutrophils to chemoattractants.

The adrenal gland of stranded whales ( Kogia breviceps and Mesoplodon europaeus): In vitro modulation of mitochondrial steroid enzyme activities

The effects of adrenocorticotropic hormone (ACTH), cyclic AMP (cAMP), NADPH, Krebs cycle intermediates (KCI), and metyrapone on the two key mitochondrial reactions in the biosynthesis of glucocorticoids—11β-hydroxylation and cholesterol cleavage—were studied in preparations from the adrenal glands of stranded whales ( Kogia breviceps and Mesoplodon europaeus) and some terrestrial mammals. ACTH (30 p M) and cAMP (1.0 m M) enhanced the 11β-hydroxylation of [11- 3H]deoxycorticosterone ([ 3H]DOC) in monolayer cultures of whale adrenal cells during a 4-hr incubation period. Mitochondria from whale and beef adrenals responded in a similar dose-related fashion to NADPH generated by the addition of increasing amounts of NADP (0–0.6 m M) to the in vitro system; at each level of NADPH, 11β-hydroxylation of [ 14C]DOC was several-fold greater than the cleavage of [ 14C]cholesterol. Metyrapone interfered in a dose-related manner with both the 11β-hydroxylation of [ 14C]DOC and the cleavage of [ 14C]cholesterol by mitochondria from whale and beef adrenals; inhibition of 11β-hydroxylation exceeded 60% at 0.1 m M metyrapone and was virtually complete at 1.0 m M in both species, while inhibition of [ 14C]cholesterol cleavage averaged 25% at 0.1 m M metyrapone and 50% at 1.0 m M. The effect of exogenous NADPH in supporting the 11β-hydroxylation of [ 14C]DOC could be maintained in beef and rat adrenal mitochondria to the extent of 70–100% by substitution with any of the KCI. This phenomenon was not found in similar whale studies where the KCI were all ineffective. This finding represents the major biochemical difference between adrenal steroidogenesis in whales and in terrestrial mammals, but it is not clear whether this distinction represents a physiologic adaptation to the aquatic environment or an effect of the unknown pathologic process associated with the stranding of these marine mammals.

Simulation of the Metabolism of Absorbed Energy-Yielding Nutrients in Young Sheep: Efficiency of Utilization of Acetate

A previously described mathematical model that simulates the metabolism of absorbed nutrients was used to examine factors influencing the efficiency of utilization of acetate in a sheep weighing 25 kg. A significant component of the model was a function representing substrate cycles that degraded ATP when its concentration reached twice initial levels. Acetate utilization was simulated when up to 2 g mol/d were added to four diets (representing forages and concentrates) that supplied varying proportions of absorbed volatile fatty acids, glucose and protein. The basal diets provided either 4.375 (near energy balance) or 8.75 MJ/d absorbed energy. The predicted net efficiency of utilization (kf) of the energy in added acetate was high (0.58–0.70) for all combinations of absorbed nutrients at the low level of energy absorption, whereas at the higher level, it ranged from 0.16 (forage) to 0.49 (high protein concentrate). Low kf values were always associated with a considerable flux of ATP through the degradation pathway. The increase in ATP concentration that caused this pathway to operate was linked to an inhibition in the utilization of acetyl-CoA for fatty acid synthesis and an increase in its oxidation. The simulated addition of exogenous NADPH or its precursors (particularly glucose and propionate) to these diets repartitioned acetyl-CoA flux towards fatty acid synthesis instead of oxidation, decreased the flux of ATP through the degradation pathway and increased the kf of added acetate. Although a negative relationship was predicted between kf of added acetate and NADPH production for diets with increasing protein content, kf still depended on ATP flux through the degradation pathway. Addition of glucose to the high protein diets decreased this flux by decreasing acetyl-CoA oxidation and increasing fatty acid synthesis. The predictions suggest that the efficiency of acetate utilization in ruminants may be influenced by NADPH availability when a considerable amount of absorbed energy is derived from acetate. Increased absorption of glucose or propionate enhanced NADPH production, but the major effect on acetate utilization of increasing dietary protein was not through NADPH production.

The regulation of exogenous NAD(P)H oxidation in spinach (Spinacia oleracea) leaf mitochondria by pH and cations.

Essentially chlorophyll-free mitochondria were isolated from green leaves of spinach (Spinacia oleracea L. cv. Viking II). Uncoupled oxidation of exogenous NADPH (1 mM) to oxygen had an optimum at pH 6.0, and activity was relatively low at pH 7.0, even in the presence of 1 mM-CaCl2. There was a proportional increase in the apparent Km for NADPH with decreasing H+ concentrations, suggesting that NADPH protonated on the 2'-phosphate group was the true substrate. Exogenous NADH was oxidized by oxygen with an optimum at pH 6.9. Under low-cation conditions, EGTA or EDTA (both 1 mM) had no effect on the Vmax. of NADH oxidation, although the removal of bivalent cations from the membrane surface by the chelators could be observed by use of 9-aminoacridine fluorescence. In contrast, under high-cation conditions, chelators lowered the Vmax. by about 50%, probably due to a better approach of the negatively charged chelators to the negative membrane surface than under low-cation conditions. In a low-cation medium, the Vmax. of NADH oxidation was increased by about 50% by the addition of cations. This was caused by a lowering of the size of the negative surface potential through charge screening. In contrast with other cations, La3+ inhibited NADH oxidation, possibly through binding to lipids essential for NADH oxidation. The apparent Km for NADH varied 6-fold in response to changes in the size of the surface potential, suggesting that the approach of the negatively charged NADH to the active site is hampered by the negative surface potential. The results demonstrate that the spinach leaf cell can regulate the mitochondrial NAD(P)H oxidation through several mechanisms: the pH; the cation concentration in general; and the concentration of Ca2+ in particular. The results also emphasize the importance of electrostatic considerations when investigating the kinetic behaviour of membrane-bound enzymes.