Biomedical subjects

F C Kauffman

Publications and source records attributed to F C Kauffman.

At least 91 records · Page 5Linked to original sources

PubMed1984

Effects of soman on neuritic outgrowth and substrate utilization by explants of the rat superior cervical ganglion.

The influence of the organophosphate, soman (pinacolyl methylphosphonofluoridate), on neuritic growth and substrate utilization by mammalian autonomic neural tissue was studied using explants of the rat superior cervical ganglion as a model. Soman produced a dose-dependent decrease in neuritic outgrowth from explants of this tissue maintained in a serum-free medium. Acetylcholinesterase in the explant as well as in culture media also decreased. In contrast, the effects of soman on explant metabolism were modest. Total protein and DNA content of the tissue was not affected. Only marginal changes in substrate utilization were detected; glucose use was unaltered, lactate production increased 20% with the highest soman tested. Soman increased the content of phosphocreatine in ganglion explants. This increase occurred in the absence of changes in the oxidation-reduction state of NAD calculated from pyruvate/lactate ratios. The results indicated that soman inhibited neuritic outgrowth from explants of the rat superior cervical ganglion in the absence of major effects on substrate utilization by this tissue.

Acetylcholinesterase↗

PubMed1984

Stimulation of hepatic microsomal beta-glucuronidase by calcium.

Hydrolysis of 3-methylumbelliferyl glucuronide by liver microsomal beta-glucuronidase is enhanced about 2-fold by micromolar concentrations of Ca2+; half-maximal stimulation occurs with 0.35 microM Ca2+. Dissociation of the enzyme from microsomal membranes by various treatments increases basal beta-glucuronidase activity and markedly decreases the sensitivity of the enzyme to Ca2+. Under similar conditions, the soluble lysosomal form of the enzyme is insensitive to Ca2+. Ca2+ stimulation was unaltered by addition of calmodulin inhibitors or exogenous calmodulin. Thus, interaction of cytosolic Ca2+ with membrane bound beta-glucuronidase may modulate glucuronidation in intact hepatocytes via a novel, calmodulin-independent mechanism.

Animals↗

PubMed1984

Calcium-mediated inhibition of glucuronide production by epinephrine in the perfused rat liver.

Rates of production of p-nitrophenyl glucuronide by isolated perfused livers from fed or fasted phenobarbital-treated rats were estimated by monitoring the concentration of glucuronide in the effluent perfusate. Infusion of epinephrine decreased the steady state level of p-nitrophenyl glucuronide by about 39% (half-maximal inhibition at approximately 5 microM). This result was unexpected because epinephrine activated glycogenolysis and elevated hepatic UDP-glucuronic acid contents. The effect of epinephrine can be attributed to its interaction with alpha-adrenergic receptors, since the inhibition of glucuronide production by epinephrine was reversed by an alpha-antagonist (phentolamine) but not by a beta-antagonist, propranolol. Since alpha-adrenergic agonists increase the cytosolic free calcium concentration, we investigated the possibility that the decrease in glucuronide production elicited by epinephrine was mediated by calcium. Removal of calcium from the perfusion fluid diminished the inhibition of glucuronide production by epinephrine, while increasing extracellular calcium from 0 to 150 microM restored the inhibition in a dose-dependent manner. In the presence of extracellular calcium, glucuronide production was inhibited by the addition of the calcium ionophore A23187 or angiotensin II, a hormone which increases cytosolic calcium. Concentrations of ionized calcium comparable to physiological intracellular levels (0.1-2 microM) increased microsomal beta-glucuronidase activity by 50 to 100% but had no effect on microsomal glucuronosyl-transferases . These results indicate that activation of hepatic alpha-adrenergic receptors increases cytosolic calcium which stimulates microsomal beta-glucuronidase activity. This decreases net glucuronide formation by the liver. In support of this hypothesis, rates of glucuronide production were unaffected by epinephrine in perfused livers from beta-glucuronidase-deficient C3H/HeJ mice.

Animals↗

PubMed1984

Cholinesterase activity in the rat superior cervical ganglion: effect of denervation and axotomy.

Cholinesterase activity and its various forms in the superior cervical ganglion of the rat were studied after denervation, axotomy and double section. The response of this activity to various inhibitors was also studied. The specific activity of cholinesterase activity was 323 +/- 25 nmol/mg protein/min in homogenates of normal ganglia and the total enzyme activity was 78 +/- 5 nmol/ganglion/min. Axotomy produced approximately a 40% decrease in total activity in ganglia at 3 days compared to contralateral ganglia. Activity in the contralateral ganglia was also decreased by about 30% compared to normal ganglia. Decentralization alone for 3 days did not affect the specific or total cholinesterase activity. However, decentralization followed by axotomy 3 days later resulted in a greater loss in activity compared to axotomy and a significant change in the forms of cholinesterase. After chromatography on Sepharose 6B-100 columns, 3 forms (a, c, d) of cholinesterase activity could be separated from homogenates of single ganglia. The 3 forms had estimated molecular weights of 1,934,000, 129,000 and 59,000 daltons, respectively. A fourth form (b) presented as a shoulder and had an estimated molecular weight of 404,000 daltons. Decentralization or axotomy decreased forms a and d. A reduction in the proportion of form c was also observed in the axotomized ganglion. Decentralization followed by axomoty resulted in large decreases in forms a, b and c and complete loss of form d. Assay of the separated forms in the presence of the specific inhibitor of acetylcholinesterase, BW284C-51, indicated that the majority of the enzyme recovered after chromatography is acetylcholinesterase.

Acetylcholinesterase↗

PubMed1984

p-Nitroanisole O-demethylation in perfused hamster liver. High rates of pentose cycle-independent mixed-function oxidation.

Rates of p-nitroanisole O-demethylation in perfused livers from Syrian golden hamsters were three to four times greater than comparable rates measured in preparations from Sprague-Dawley rats. Hamsters also had greater microsomal p-nitroanisole O-demethylase activity and cytochrome P-450 contents than rats. In general, phenobarbital caused similar increases in these properties in both species. Fasting of hamsters for 24 hr increased p-nitroanisole O-demethylase activity in microsomes but did not affect rates in perfused livers. Rates were also unaffected in the perfused liver by pretreatment with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt. Hamster livers had low activities of pentose cycle enzymes but high activities of malic enzyme and isocitrate dehydrogenase compared to rats. In hamster livers, maximal rates of p-nitroanisole O-demethylation were not maintained but declined steadily over 40 min with prolonged p-nitroanisole infusion. The decreased rates of mixed-function oxidation in the non-recirculating perfusion system could not be explained by diminished tissue viability or degradation of cytochrome P-450 but were likely due to a decline in the formation of reduced cofactor. Hepatic concentrations of alpha-ketoglutarate and malate increased during p-nitroanisole infusion. Furthermore, rates of p-nitroanisole O-demethylation were inhibited by ethanol and aminooxyacetate, agents which inhibit the generation and/or movement of mitochondrial reducing equivalents into the cytosol. The infusion of pyruvate stimulated p-nitroanisole O-demethylation in perfused livers from fasted hamsters. This effect was maximal with 0.1 mM pyruvate, did not require gluconeogenesis, and was insensitive to 6-aminonicotinamide treatment. Thus, stimulation of p-nitroanisole metabolism by pyruvate in hamster livers is likely related to the mitochondrial oxidation of pyruvate, rather than to increased NADPH generation via the pentose phosphate cycle. These data indicate that mitochondrial sources of NADPH supply reducing equivalents for mixed-function oxidation in hamster liver.

Aminooxyacetic Acid↗

PubMed1984

Inhibition of mixed-function oxidation in perfused rat liver by fluoroacetate treatment.

The effect of fluoroacetate, an inhibitor of the citric acid cycle, on the mixed-function oxidation of p-nitroanisole in isolated perfused livers from fed rats was studied. The citric acid cycle was inhibited by injection of 5 mg/kg sodium fluoroacetate into rats 3 hr prior to liver perfusion experiments. Inhibition of the citric acid cycle was marked by accumulation of citrate (5-fold) and decreases in rates of glycolysis and glycogenolysis by 50-90%. Fluoroacetate treatment inhibited mixed function oxidation in the perfused liver by about 50% without affecting p-nitroanisole O-demethylation by isolated microsomes. Fluorocitrate, at concentrations up to 50 microM, did not inhibit microsomal p-nitroanisole O-demethylation in vitro. These data support the hypothesis that mixed-function oxidation in intact hepatocytes is dependent upon reducing equivalents generated via the citric acid cycle.

Animals↗

PubMed1984

Mitochondrial-microsomal interactions in vitro: use of microsomal 7-ethoxycoumarin O-deethylase to study succinate-stimulated malate efflux from mitochondria.

An assay system was developed utilizing microsomal 7-ethoxycoumarin O-deethylase to monitor the formation and efflux of malate from mitochondria in vitro. Malate was coupled by means of malic enzyme, NADP+, and the microsomal mixed-function oxidase system to the production of highly fluorescent 7-hydroxycoumarin. Under conditions of the assay, steady-state concentrations of NADPH could be maintained, and half-maximal rates of microsomal 7-ethoxycoumarin O-deethylase were observed with about 0.2 microM NADPH and 250 microM malate. Addition of succinate to mixtures of intact mitochondria and microsomes enhanced rates of 7-hydroxycoumarin formation by facilitating the generation and transport of malate from mitochondria (half-maximal stimulation was observed with 0.4 mM succinate). Stimulation of 7-hydroxycoumarin formation by succinate, under conditions where concentrations of cofactors and substrate were adjusted to resemble those in the intact cell, provides further evidence that reducing equivalents for mixed-function oxidation may arise via malate efflux from mitochondria.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1984

Reducing equivalents for mixed function oxidation in periportal and pericentral regions of the liver lobule in perfused livers from normal and phenobarbital-treated rats.

The supply of NADPH for cytochrome P-450-dependent mixed function oxidation from the pentose cycle and mitochondria in periportal and pericentral regions of the liver lobule was evaluated in perfused rat liver. Rates of 7-ethoxycoumarin O-deethylation in livers from fed, normal rats monitored with micro-light guides placed on periportal and pericentral regions were 1.2 mumol/g/hr in both regions of the liver lobule. In livers from fed, phenobarbital-treated rats, rates were 3.6 and 7.0 mumol/g/hr in periportal and pericentral regions, respectively. Following treatment of rats with 6-aminonicotinamide, an inhibitor of the pentose cycle, rates of 7-hydroxycoumarin production were approximately 0.9 mumol/g/hr in both regions of the lobule in livers from normal rats and 2.1 and 3.4 mumol/g/hr in periportal and pericentral regions, respectively, in livers from phenobarbital-treated rats. Based on the difference in rates of 7-hydroxycoumarin production in the presence and absence of 6-aminonicotinamide, we conclude that the pentose cycle supplies NADPH for 7-ethoxycoumarin metabolism at rates around 0.3 mumol/g/hr in both regions of the liver lobule in livers from normal rats and 1.5 and 3.6 mumol/g/hr in periportal and pericentral regions, respectively, in livers from phenobarbital-treated rats. Potassium cyanide, an inhibitor of mitochondrial oxidation, reduced rates of 7-ethoxycoumarin O-deethylation to approximately 0.6 mumol/g/hr in both regions of the liver lobule in livers from fed, normal rats and to around 0.2 mumol/g/hr after fasting or treatment with 6-aminonicotinamide. In livers from fasted, phenobarbital-treated rats, 7-hydroxycoumarin was produced at rates of 0.3 and 0.7 mumol/g/hr in periportal and pericentral regions, respectively, in the presence of KCN. Decreases in rates of 7-hydroxycoumarin production during KCN infusion indicate that the mitochondria supply about 0.7 mumol of NADPH/g/hr for 7-ethoxycoumarin metabolism in both regions in livers from normal rats and 1.3 and 2.7 mumol/g/hr in periportal and pericentral regions in livers from phenobarbital-treated rats. The sum of KCN and 6-aminonicotinamide-sensitive rates of 7-ethoxycoumarin metabolism closely approximated rates measured in the absence of the inhibitors. These data indicate that mitochondria supply 50 to 70% of the reducing equivalents for mixed function oxidation of 7-ethoxycoumarin in both regions of the liver lobule in livers from fed rats.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1984

Glucuronidation of 7-hydroxycoumarin in periportal and pericentral regions of the liver lobule.

Rates of glucuronidation were measured at high substrate concentrations in specific zones of the liver lobule using micro-light guides placed on periportal and pericentral regions on the surface of livers from phenobarbital-treated rats. Livers were perfused with sulfate-free buffer under normoxic conditions, and fluorescence of free 7-hydroxycoumarin was monitored in the tissue. The formation of nonfluorescent 7-hydroxycoumarin glucuronide was then inhibited completely by perfusion with N2-saturated perfusate containing 20 mM ethanol. Under these conditions, fluorescence recorded from the surface of the liver was directly proportional to the concentration of substrate infused. The difference in 7-hydroxycoumarin fluorescence between N2 plus ethanol and normoxic perfusion was due to glucuronidation. Maximal rates of glucuronidation in periportal and pericentral regions of the liver lobule calculated with this new method were 9.6 and 35 mumoles/g/hr, respectively. Glucuronidation was half-maximal with 25-50 microM 7-hydroxycoumarin in both regions. Glucuronosyltransferase activity assayed in microdissected, freeze-dried tissue samples in vitro was 3-fold greater in pericentral areas than in periportal areas. This activity was half-maximal with 0.2 mM UDP-glucuronic acid and 54 microM 7-hydroxycoumarin in both regions of the liver lobule. Thus, the maximal capacity of the glucuronidation system determined in vitro is about 3-fold greater in pericentral than in periportal regions of the liver lobule, a difference which correlates well with measured rates of glucuronidation of 7-hydroxycoumarin in the two zones of the lobule in the intact, perfused liver.

Animals↗

PubMed1984

Trypan blue uptake as a new method to investigate hepatotoxicity in periportal and pericentral regions of the liver lobule: studies with allyl alcohol in the perfused liver.

A new method based on trypan blue uptake was developed to study zonal hepatotoxicity in specific regions of the liver lobule in perfused livers. Allyl alcohol (350 microM), a hepatotoxin that causes necrosis of periportal regions, was infused into livers from phenobarbital-treated rats for 60 min. The subsequent infusion of trypan blue (0.2 mM) stained nuclei in periportal but not midzonal or pericentral regions of the liver lobule. With this new method of dye uptake, comparison of the temporal relationship between metabolic changes and the onset of cellular damage was possible. During the first 10 min of allyl alcohol infusion, allyl alcohol was taken up at rates of 64 mumol/g/h concomitant with an increase in oxygen uptake of 18 mumol/g/h. Allyl alcohol infusion also increased bile flow from 69 to 167 microliters/min and release of oxidized glutathione from 127 to 217 nmol/g/h, respectively. However, during the next 20-min phase of exposure to allyl alcohol, oxygen uptake was inhibited by 60% and allyl alcohol uptake declined by 80%. Bile flow and release of oxidized glutathione also declined continuously between 10 and 30 min and by 30 min stopped completely. Concomitant with the inhibition of oxygen uptake, rates of glycogenolysis and glycolysis increased by 130 and 85%, respectively. Within minutes after maximal inhibition of oxygen uptake, between 30 and 40 min after initiation of allyl alcohol infusion, trypan blue was taken up by hepatocytes in periportal zones of the liver lobule. Subsequently, lactate dehydrogenase began to appear in the effluent perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Propanol↗

PubMed1984

Characterization of mutagenic glucuronide formation from benzo(a)pyrene in the nonrecirculating perfused rat liver.

Excretion of mutagenic metabolites of benzo(a)pyrene into bile from livers of corn oil- or 3-methylcholanthrene-treated Sprague-Dawley rats perfused with a nonrecirculating perfusion system was quantitated. Mutagenic benzo(a)pyrene metabolites were detected using Salmonella typhimurium (strain TA 98) grown in the presence of limiting amounts of histidine. Microsomes were not included in the bacterial assay since metabolic activation was carried out by the perfused liver. Mutagenic activity was detected only if beta-glucuronidase was added to the assay mixture or if bile was treated with acid to hydrolyze glucuronides prior to assay. When livers were perfused with 20 microM benzo(a)pyrene, stable, mutagenic glucuronides were exported from corn oil-treated livers at maximal rates of 149 +/- 24 (S.E.) revertants/g/hr and at rates of 225 +/- 22 revertants/g/hr in livers from 3-methylcholanthrene-treated rats. Chromatography of bile by high-performance liquid chromatography demonstrated that two peak areas contained phenolic glucuronides which were hydrolyzed by beta-glucuronidase. These two peaks, one which cochromatographed with authentic 3-benzo(a)pyrenyl-beta-D-glucuronide, accounted for all of the mutagenic activity in bile from livers perfused with benzo(a)pyrene. A good correlation (r = 0.86) between rates of mutagen production and rates of formation of phenolic glucuronides was observed under a variety of experimental conditions. The mutagenic activity observed with pure 3-benzo(a)pyrenyl-beta-D-glucuronide exposed to beta-glucuronidase was 4 revertants/nmol. When the rate of mutagen production was divided by the rate of production of 3-benzo(a)pyrenyl-beta-D-glucuronide by the perfused liver, a value of 4 revertants/nmol was also obtained. Therefore, it is concluded that mutagens exported in bile from livers perfused with benzo(a)pyrene can be accounted for predominantly by hydrolysis products of phenolic glucuronides.

Animals↗

PubMed1984

Rates of allyl alcohol metabolism in periportal and pericentral regions of the liver lobule.

Rates of allyl alcohol metabolism in periportal and pericentral regions of the liver lobule were measured to determine whether the zonal toxicity due to allyl alcohol results from its selective metabolism in periportal regions. Infusion of allyl alcohol into perfused livers from fed, phenobarbital-treated rats caused an increase in NADH fluorescence (366 leads to 450 nm) measured with a large-tipped (2-mm) light guide placed on the surface of the liver. A linear increase in NADH fluorescence was observed when 25-150 microM allyl alcohol was infused; however, when allyl alcohol exceeded 200 microM, oxygen uptake by the liver was inhibited 30-40%, and a large increase in NADH fluorescence occurred. Miniature oxygen electrodes were then placed on periportal and pericentral regions of the liver lobule and local rates of oxygen uptake were determined [Matsumura and Thurman, Am. J. Physiol. 244:G656-G659 (1983)]. Allyl alcohol (350 microM) or acrolein (200 microM) inhibited oxygen uptake only in periportal regions. Micro-light guides were placed on periportal and pericentral regions of livers perfused in either the anterograde or retrograde direction. The maximal increase in NADH fluorescence due to allyl alcohol infusion (100 microM) was greater in pericentral than in periportal regions. 4-Methylpyrazole (80 microM), an inhibitor of alcohol dehydrogenase, prevented the fluorescence increase due to allyl alcohol in both regions, indicating that the changes were due entirely to NADH generated from alcohol dehydrogenase-dependent allyl alcohol metabolism. Using the correlation (r = 0.91) between rates of allyl alcohol uptake and the increase in NADH fluorescence established for the whole organ, local rates of allyl alcohol metabolism were 23 and 31 mumoles/g/hr in periportal and pericentral regions, respectively. These results indicate that metabolism of allyl alcohol occurs at slightly greater rates in pericentral than in periportal regions of the liver lobule. Thirty minutes after the i.p. injection of a necrogenic dose of allyl alcohol in vivo, the concentrations of allyl alcohol in the portal vein and vena cava were 1210 and 530 microM, respectively. Thus, both periportal and pericentral regions of the liver lobule were exposed to concentrations of allyl alcohol (e.g., greater than 200 microM) which were metabolized in the perfused liver. Since allyl alcohol is metabolized in both regions of the liver lobule, the hypothesis that the zone-specific hepatotoxicity results from its exclusive metabolism to acrolein in periportal regions seems unlikely.

1-Propanol↗

PubMed1983

Stimulation of mixed-function oxidation of 7-ethoxycoumarin in periportal and pericentral regions of the perfused rat liver by xylitol.

Rates of O-deethylation of 7-ethoxycoumarin by perfused livers from fasted, phenobarbital-treated rats were 3.7 mumol X g-1 X h-1. Approximately 50% of the product was conjugated. When rates of 7-ethoxycoumarin O-deethylation were varied by infusing different concentrations of substrate, a good correlation (r = 0.91) was found between rates of O-deethylation of 7-ethoxycoumarin and fluorescence of 7-hydroxycoumarin detected from the liver surface. Micro-light guides (tip diameter 170 microns) placed on periportal and pericentral regions on the liver surface were used to monitor the conversion of nonfluorescent 7-ethoxycoumarin to fluorescent 7-hydroxycoumarin. The O-deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin increased fluorescence 64% and 28% in pericentral and periportal regions of the liver lobule, respectively. Rates of 7-ethoxycoumarin O-deethylation estimated from these increases in fluorescence were 5.2 mumol X g-1 X h-1 in pericentral and 2.2 mumol X g-1 X h-1 in periportal regions of the liver. During mixed-function oxidation of 7-ethoxycoumarin, the oxidation:reduction state of NADP(H) was similar in both regions of the liver lobule. Xylitol (2 mM) decreased the NADP+/NADPH ratio and stimulated rates of drug metabolism in both regions of the liver lobule. This indicates that conditions exist where the supply of NADPH is an important rate-determining factor for 7-ethoxycoumarin metabolism in both periportal and pericentral regions of the liver lobule.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1983

Genetic regulation of NADPH supply in perfused mouse liver. Role of the Ah locus during induction by 3-methylcholanthrene.

Rates of p-nitroanisole O-demethylation in perfused livers from control DBA/2J and C57BL/6J, as well as from 3-methylcholanthrene-treated DBA/2J mice, reached maximal values of 15-17 mumol/g/h after 10 min of p-nitroanisole (0.2 mM) infusion. During the subsequent 30 min of p-nitroanisole infusion these rates declined steadily to 50% of the maximal rate. Infusion of 20 mM glucose completely reversed this decline in rate. Phenobarbital treatment of C57BL/6J and DBA/2J mice increased rates of p-nitroanisole O-demethylation about 2.5-fold to 44.4 +/- 3.4 and 35.1 +/- 3.4 mumol/g/h, respectively, which also declined during subsequent perfusion. In contrast, the kinetics of p-nitroanisole O-demethylation in perfused livers from 3-methylcholanthrene- and beta-naphthoflavone-treated C57BL/6J were distinctly different. These livers maintained high maximal rates of p-nitroanisole O-demethylation (41.1 +/- 4.7 mumol/g/h) throughout the 40 min of p-nitroanisole infusion. 3-Methylcholanthrene-treated (C57BL/6J)(DBA/2J)F1 perfused livers also maintained high rates of monooxygenation throughout the perfusion. All livers from the 3-methylcholanthrene-treated, Ah locus-responsive F1 X DBA/2J backcross group maintained high rates of p-nitroanisole O-demethylation throughout perfusion; however, 3-methylcholanthrene-treated, Ah locus-nonresponsive perfused livers resembled livers from untreated mice. The NADP+/NADPH ratio in livers from 3-methylcholanthrene-treated C57BL/6J but not DBA/2J mice was significantly higher than controls in the absence of p-nitroanisole. Addition of p-nitroanisole increased this ratio in both groups; however, higher values were observed in livers from C57BL/6J than DBA/2J mice. These data provide genetic evidence that the ability of mouse liver to sustain high rates of monooxygenation following 3-methylcholanthrene treatment is an autosomal dominant trait corresponding with the Ah locus and most likely is due to enhanced NADPH turnover.

Animals↗

PubMed1983

Inhibition of p-nitroanisole O-demethylation in perfused rat liver by potassium cyanide.

The effect of potassium cyanide on p-nitroanisole O-demethylation in perfused rat livers has been examined. Cyanide (2 mM), an inhibitor of cytochrome oxidase, diminished p-nitroanisole O-demethylation by 50-75% in perfused livers from normal and phenobarbital-treated rats, but had much less effect on hepatic microsomal p-nitroanisole O-demethylation. The inhibition was also observed in livers where the activity of the pentose phosphate shunt was abolished by pretreatment with 6-aminonicotinamide. Cyanide infusion decreased hepatic ATP/ADP ratios and cellular concentrations of glutamate, alpha-ketoglutarate, and isocitrate, but caused an increase in the NADP+/NADPH ratio. Rates of NADPH generation via the pentose phosphate shunt were unchanged by cyanide, and hepatic concentrations of glucose 6-phosphate were markedly increased by cyanide. Thus, inhibition of p-nitroanisole metabolism could not be explained solely by a direct interaction of cyanide with mixed-function oxidases or diminished NADPH generation via the pentose cycle. These data indicate that cyanide inhibits mixed-function oxidation in intact cells by diminishing the generation of NADPH from sources other than the pentose cycle. Further, these data are consistent with the hypothesis that some NADPH for mixed-function oxidation arises from cyanide-sensitive mitochondrial sources.

Animals↗

PubMed1983

Increased lipid content in the rat axotomized superior cervical ganglion.

Transection of postganglionic nerves of the rat superior cervical ganglion results in significant increases in the concentration of lipid as well as the total content of lipid in the ganglion. The purpose of this work was to characterize the effect of axotomy on the major classes of lipid in the ganglion and to examine possible relationships among increases in lipid content and changes in steady-state concentrations of metabolites utilized in lipid biosynthesis. Major classes of lipids in axotomized and contralateral intact ganglia were compared using the Folch extraction and partitioning scheme. Analyses of lipids were carried out 7 days after axotomy because total lipid content was increased maximally at that time. The increase in lipid content in axotomized ganglia was related primarily to an increase in phospholipids. Amounts of gangliosides, cerebrosides, cholesterol, and proteolipid protein did not change significantly after axotomy. The elevation of phospholipid content was accompanied by increases in steady-state concentrations of alpha-glycerophosphate and decreases in Coenzyme A in the ganglion; however, concentrations of acetyl CoA and long-chain acyl CoA compounds did not change significantly in axotomized ganglia. Increases in alpha-glycerophosphate do not appear to arise from enhanced utilization of glucose because alpha-glycerophosphate dehydrogenase activity was decreased in axotomized ganglia and the incorporation of [U-14C] glucose into lipids in explants of the axotomized ganglia in vitro was not elevated. In contrast, incorporation of [U-14C] glycerol into lipids of explants from axotomized ganglia was increased in vitro. The results suggest that utilization of extracellular glycerol for phospholipid biosynthesis may be enhanced in the rat's axotomized superior cervical ganglion.

Animals↗

PubMed1983

Effects of ethanol and sorbitol on mixed-function oxidation in perfused rat livers.

Ethanol (20 mM) caused 50-90% inhibition of rates of mixed-function oxidation of p-nitroanisole, 7-ethoxycoumarin and benzo(a)pyrene in perfused rat livers; however, the microsomal metabolism of these substrates was unaltered by low concentrations of ethanol. The metabolism of ethanol was required for this inhibition in the perfused liver. In contrast to ethanol, sorbitol stimulated rates of p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Both sorbitol and ethanol infusion decreased the hepatic NAD+/NADH ratio; however, the NADP+/NADPH ratio was decreased by sorbitol but increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt. These data indicated that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. The changes in intracellular concentrations of NADPH produced by ethanol and sorbitol correlated directly with changes in hepatic content of citrate and aspartate. These data suggest that inhibition of the citric acid cycle by ethanol decreases the movement of mitochondrial reducing equivalents into the cytosol via substrate shuttle mechanisms.

Acetaldehyde↗

PubMed1983

Metabolic heterogeneity in the perfused rat liver.

New methods have been developed to monitor metabolic events non-invasively within periportal and pericentral regions of perfused rat livers. These techniques utilize two-fiber micro-light guides and miniature oxygen electrodes positioned on identified lobular regions of the perfused liver based on differential pigmentation of periportal and pericentral areas. Two-fiber micro-light guides detect the fluorescence of native and introduced fluors and are used to monitor redox changes of endogenous pyridine nucleotides and the generation of fluorescent products (e.g., 7-hydroxycoumarin) from exogenous substrates. Changes in fluorescence detected with two-fiber micro-light guides are correlated with changes measured with large, multi-fiber light guides and with whole organ rates of metabolism. Subsequently, local rates are estimated. With these techniques, we show that (a) rates of ethanol and acetaldehyde metabolism are similar in periportal and pericentral regions of the liver lobule; (b) mixed-function oxidation predominantes in pericentral regions in livers from phenobarbital-treated rats; (c) rates of sulfation of 7-hydroxycoumarin are greater in periportal than in pericentral hepatocytes; and (d) oxygen uptake is approximately 3-fold greater in periportal than in pericentral areas of the liver lobule.

Acetaldehyde↗