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Biomedical subjects

F C Kauffman

Publications and source records attributed to F C Kauffman.

At least 109 records · Page 6Linked to original sources

Muscarinic receptor binding and oxidative enzyme activities in the adult rat superior cervical ganglion: effects of 6-hydroxydopamine and nerve growth factor.

Administration of 6-hydroxydopamine to adult rats results in changes in the superior cervical ganglion similar to those noted after axotomy; namely, a decrease in muscarinic receptor binding and increases in activities of the oxidative enzymes of the pentose phosphate pathway. These changes were either prevented or attenuated markedly by the systemic administration of nerve growth factor. Administration of nerve growth factor alone did not significantly increase N-methylscopolamine binding in the ganglion or reduce the activities of the oxidative enzymes. Explants of the ganglion maintained in serum-free medium over a period of 3 days demonstrated increases in oxidative enzyme activity and a decrease in N-methylscopolamine binding. Addition of 20 nM nerve growth factor to the culture medium prevented the decline in N-methylscopolamine binding in ganglion explants. The increases in oxidative enzyme activities were unaltered. Addition of high amounts of nerve growth factor, 200 nM, resulted in a significant increase in tyrosine hydroxylase activity but no further increase in N-methylscopolamine binding in ganglion explants. Glucocorticoids added to the culture medium did not affect the muscarinic binding or enzyme activities. Thus, decreases in muscarinic binding activity which occur in the superior cervical ganglion after axotomy or 6-hydroxydopamine treatment may be explained by a loss of nerve growth factor supplied to the ganglion. Increases in the oxidative enzymes of the pentose phosphate pathway that occur in the ganglion after axonal injury appear to involve additional factors.

Animals↗

Regulation of NADPH-dependent mixed-function oxidation in perfused livers. Comparative studies with sorbitol and ethanol.

Sorbitol and ethanol were shown to have opposite effects on p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Sorbitol (2 mM) stimulated drug metabolism by 50% while ethanol (20 mM) caused 80% inhibition. Both sorbitol and ethanol infusion decreased the NAD+/NADH ratio and increased fluorescence of pyridine nucleotides monitored from the liver surface; however, the NADP+/NADPH ratio was decreased by sorbitol but tended to be 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, but was not affected by aminooxyacetate, a transaminase inhibitor. These results indicate that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. Ethanol and sorbitol caused changes in intracellular concentrations of NADPH in livers from fasted rats which correlated directly with changes in hepatic levels of citrate and aspartate. Furthermore, aspartate infusion reduced the inhibition of p-nitroanisole O-demethylation by ethanol. This inhibition was also reversed partially by sorbitol in livers from 6-aminonicotinamide-treated rats. It is concluded that ethanol inhibits mixed-function oxidation primarily by decreasing the concentrations of citric acid cycle intermediates which leads to depletion of cytosolic NADPH.

Animals↗

Benzo(a)pyrene phenol production by perfused rat liver and its inhibition by ethanol.

A rapid and inexpensive method has been developed to estimate rates of benzo(a)pyrene phenol production by perfused rat liver. This method is based on the measurement of benzo(a)pyrene phenols utilizing a simple fluorometric procedure. Within 2 to 3 min after infusion of benzo(a)pyrene bound to serum albumin, phenols are excreted into the perfusate, primarily as glucuronide and sulfate conjugates. Maximal rates of phenol release were 8 to 10 nmol/g/hr in livers from control rats and 40 to 42 nmol/g/hr in livers from 3-methylcholanthrene-treated rats. Fasting of 3-methylcholanthrene-treated rats for 24 hr prior to perfusion experiments did not affect either the rate of phenol production or the extent of their conjugation. Ethanol (20 mM) inhibited rates of phenol formation by 50% in livers from fasted, 3-methylcholanthrene-treated rats but had no effect on benzo(a)pyrene hydroxylase activity in isolated hepatic microsomes. These data indicate that ethanol inhibits phenol formation from benzo(a)pyrene in intact liver, probably by diminishing the supply of the cofactor reduced nicotinamide adenine dinucleotide phosphate.

Animals↗

Growth of adult superior cervical ganglion explants in serum-free media.

Neurite outgrowth from explants of superior cervical ganglion from adult rats can be achieved in a serum-free medium. Extensive neurite outgrowth occurred from ganglion explants maintained in Eagle's minimum essential medium supplemented with either 10% (V/V) fetal calf serum or 1% (W/V) bovine serum albumin and nerve growth factor. After one week in culture, the ATP content of explants maintained in the serum-free medium was slightly higher than that noted in explants cultured in the presence of fetal calf serum and amounts of phosphocreatine were significantly lower. Despite these differences in high energy phosphate content, the abundance and morphology of neuritic outgrowth were essentially the same from explants cultured in the two types of media. Comparable activities of a number of NADP+-dependent dehydrogenases were noted in explants maintained in the two types of media. Increases in the activities of the oxidative enzymes of the pentose pathway, which occur in axotomized ganglia in vivo, were observed in the cultured ganglion explants. NADP+-dependent isocitrate dehydrogenase activity remained constant in ganglion explants in vitro, and measurements of this activity were employed in a new method to quantitate neurite outgrowth. The activity of isocitrate dehydrogenase in lyophilized neurite processes that had grown out onto a Millipore filter substrate correlated well with visual estimates of neuritic outgrowth. Substitution of delipidated for normal bovine serum albumin in the growth medium resulted in a significant decrease in neuritic outgrowth from ganglion explants from both adult and weanling rats. Addition of fatty acids to media containing delipidated bovine serum albumin enhanced neuritic outgrowth in explants of weanling rats. Thus, lipophilic substances bound to bovine serum albumin including fatty acids appear necessary for optimal growth of neurites from explants of the rat superior cervical ganglion.

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Inhibition of calcium-dependent ATPase from sarcoplasmic reticulum by a new class of indolizidine alkaloids, pumiliotoxins A, B, and 251D.

Pumiliotoxins (PTX) A, B, and 251D, members of a new class of indolizidine alkaloids isolated from the skin of poison frogs of the family Dendrobatidae, inhibit Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from frog and rat hind-limb muscles. PTX-B and PTX-A appear to be relatively specific inhibitors of Ca2+-ATPase; PTX-A is much less potent than PTX-B. PTX-251D is a potent inhibitor of Ca2+-ATPase, and was also found to inhibit Na+, K+, and Mg2+-ATPases in rat brain synaptosomes. Caffeine and verapamil, two drugs known to affect calcium translocation, are very weak inhibitors of the Ca2+-ATPase. The Ki values for inhibition of the Ca2+-ATPase of rat and frog sarcoplasmic reticulum by PTX-B were comparable and ranged between 22 and 36 microM. Inhibition of calcium-dependent ATPase in sarcoplasmic reticulum by pumiliotoxin-B is noncompetitive with calcium and is not readily reversible. Based on structure-activity profiles, it is concluded that inhibition of Ca2+-ATPase by the indolizidine alkaloids is responsible for the alkaloid-elicited prolongation of twitch in intact muscle.

Alkaloids↗

Role of reducing equivalents from fatty acid oxidation in mixed-function oxidation: studies with 2-bromooctanoate in the perfused rat liver.

Mixed-function oxidation of p-nitroanisole in isolated perfused livers from fasted rats was studied in the presence and absence of 2-bromooctanoate, an inhibitor of the beta-oxidation of acyl CoA compounds. These experiments were designed to test the hypothesis that reducing equivalents from fatty acid oxidation may play a role in drug metabolism in the fasted state. Complete inhibition of ketogenesis, as indicated by beta-hydroxybutyrate and acetoacetate production, was achieved with the addition of 0.6 mM 2-bromooctanoate to the perfusate. This concentration of 2-bromooctanoate had no effect on p-nitroanisole O-demethylation measured in isolated microsomes but diminished maximal rates of p-nitroanisole O-demethylation by 78% in perfused livers of fasted, phenobarbital-treated rats. No inhibition of p-nitroanisole O-demethylation was seen in perfused livers from fed rats. Intracellular concentrations of ATP, NADH, glutamate, alpha-ketoglutarate and isocitrate decreased significantly in the presence of 2-bromooctanoate; oxygen uptake also decreased by about 50%. These data indicate that by inhibiting endogenous acyl CoA oxidation, 2-bromooctanoate diminishes beta-oxidation of fatty acids, thereby decreasing the supply of NADH for the generation of ATP. This decreases the synthesis of NADPH, an obligatory cofactor for drug metabolism. 2-Bromooctanoate also decreased the concentration of intermediates which act through substrate shuttle mechanisms to move reducing equivalents from the mitochondrial into the extramitochondrial space. It is concluded that the decrease in mixed-function oxidation observed when beta-oxidation of fatty acids is inhibited with 2-bromooctanoate is due to diminished availability of mitochondrial reducing equivalents for the generation of cytosolic NADPH. 24CA-23080

Adenine Nucleotides↗

Regulation of p-nitroanisole O-demethylation in perfused rat liver. Adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase.

Perfusion of rat livers with 10 mM-fructose or pretreatment of the rat with 6-aminonicotinamide (70 mg/kg) 6 h before perfusion decreased intracellular ATP concentrations and increased the rate of p-nitroanisole O-demethylation. This increase was accompanied by a decrease in the free [NADP+]/[NADPH] ratio calculated from concentrations of substrates assumed to be in near-equilibrium with isocitrate dehydrogenase. After pretreatment with 6-aminonicotinamide the [NADP+]/[NADPH] ratio also declined. Reduction of NADP+ during mixed-function oxidation may be explained by inhibition of of one or more NADPH-generating enzymes. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and "malic" enzyme, partially purified from livers of phenobarbital-treated rats, were inhibited by ATP and ADP. Inhibitor constants of ATP for the four dehydrogenases varied considerably, ranging from 9 micrometer for "malic" enzyme to 1.85 mM for glucose 6-phosphate dehydrogenase. NADPH-cytochrome c reductase was also inhibited by ATP (Ki 2.8 mM) and by ADP (Ki 0.9 mM), but not by AMP. Concentrations of ATP and ADP that inhibited glucose 6-phosphate dehydrogenase and the reductase were comparable with concentrations in the intact liver. Thus agents that lower intracellular ATP may accelerate rates of mixed-function oxidation by a concerted mechanism involving deinhibition of NADPH-cytochrome c reductase and one or more NADPH-generating enzymes.

6-Aminonicotinamide↗

Retrograde alteration of 6-phosphogluconate dehydrogenase in axotomized superior cervical ganglia of the rat.

Mechanisms underlying increased activity of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase [decarboxylating] EC 1.1.1.44) in axotomized rat superior cervical ganglia were explored using a highly sensitive micro-immunochemical assay employing antibodies raised in rabbits against the purified enzyme. 6-Phosphogluconate dehydrogenase was purified from rat brain more than 1700-fold by salt fractionation, anion exchange, and immunoaffinity chromatography. The purified enzyme consisted of identical subunits having molecular weights of about 48,800 which could aggregate to catalytically active isomers of various sizes; however, only one form of the enzyme was detected in freshly prepared homogenates of rat neural tissue. Physical and immunological properties of the enzyme from rat brain were similar to those from superior cervical ganglia and liver. Augmented 6-phosphogluconate dehydrogenase activity noted in superior cervical ganglia 2 days after transection of major postganglionic nerve trunks was accompanied by a parallel increase in immunoreactive protein. Michaelis constants of the enzyme were the same in control and axotomized ganglia, and the presence of activators and inhibitors was not detected. It is concluded that increases in 6-phosphogluconate dehydrogenase subsequent to axotomy can be accounted for entirely by an increase in the steady state concentration of this protein.

Animals↗