Nitric oxide synthase in cat brain: cofactors--enzyme-substrate interaction.

Nitric oxide, derived from L-arginine by the enzyme nitric oxide synthase, is an activator of the soluble guanylate cyclase and a cellular messenger. This work demonstrates that, in cat brain, the neuronal constitutive nitric oxide synthase activity is a) NADPH/calcium dependent, b) independent upon exogenous calmodulin in crude brain supernatant, c) significantly enhanced by exogenous FAD and tetrahydrobiopterin (Vmax: 118 instead of 59.4 pmol of citrulline formed .mg of prot.-1 min-1, d) inhibited by calcium chelators and calmodulin antagonist, and e) present in several neuroanatomical structures. Moreover, the Km value for L-arginine was of 11 microM instead of 41 microM in the presence of FAD and tetrahydrobiopterin in the incubation mixture, thus demonstrating that these cofactors are able to stabilize the enzyme-substrate interactions.

Dietary calcium supplementation and dopamine-beta-hydroxylase in spontaneously hypertensive rats.

Spontaneously hypertensive 4-week-old male rats were fed, before and after the onset of hypertension, with either commercial chow (control) or commercial chow combined with different forms of milk proteins with or without calcium supplementation. After 40 weeks, rats were still hypertensive, and dopamine-beta-hydroxylase enzyme activity measured simultaneously in serum and adrenal was found to be higher than in the controls. The enzyme activity in rats fed diets with milk proteins was increased significantly in both serum and adrenal compared with the control, and such enhancement was significantly higher than that observed in animals fed the commercial diet supplemented with calcium (1.2%), suggesting that dietary calcium intake associated with dietary protein of high digestibility, such as casein, potentiates the endogenous mechanisms regulating the homeostasis of calcium more than calcium supplementation itself. Moreover, the selective and additive effect of diets supplemented with milk proteins and calcium on adrenal enzyme activity clearly suggests a relationship between cardiovascular diseases involving the genesis of hypertension and stress mechanisms through the hypothalamo-pituitary adreno-sympathetic axis.

Cortisol and catecholamines response to venisection by humans with different blood groups.

Hormonal response following a minor physiological stress induced by bleeding in a blood donor clinic was investigated in 134 humans with different blood groups. After venisection, serum cortisol concentration (mean +/- SD) was found highest in blood group A donors (455 +/- 217 nmol/L), followed by group B (364 +/- 206), AB (325 +/- 154) and O (297 +/- 110). In 14 subjects of blood group A compared to an equal number of blood group B subjects, mean plasma adrenaline level was higher in group A (0.23 +/- 0.09 nmol/L) than in group B (0.15 +/- 0.12) but the difference was not statistically significant. Blood group A individuals responded to a stressful situation with higher levels of cortisol, and possibly of adrenaline. These observations tend to support findings of previous studies demonstrating a high risk of diseases related to stress (coronary heart diseases and gastrointestinal diseases), in men with A blood group.

Effects of chlordiazepoxide on acquisition and performance of a go-no go successive discrimination task, and on brain biogenic amines in cats.

1. Normal and chlordiazepoxide (0.4 mg/kg/day, per os) treated cats were trained on a symmetric go-no go successive discrimination task with positive reinforcement. 2. The treatment impaired the acquisition, but not the performance once the task was learned. 3. Serotonin, 5-hydroxyindoleacetic acid and noradrenaline were assayed in 12 brain areas in trained cats, and in normal and treated cats which did not undergo the training. 4. The treatment produced localized changes in the serotonin: 5-hydroxyindoleacetic acid ratio and the noradrenaline level; these effects were modified by the training. 5. The drug induced an acquisition deficit rather than a general behavioral disinhibition, and produced neurochemical effects which were dependent upon the brain area, the treatment duration and the behavioral state.

Dietary protein and carbohydrate effects on blood parameters related to stress in cat.

Adult cats were adapted to hypoglucidic semi-purified diets containing casein or soya as the protein source to study the effects of a 2 hr immobilization period. Body weight of cats fed hypoglucidic diets was significantly decreased. The control casein group showed higher plasma dopamine-beta-hydroxylase activity but lower pyridoxal 5'-phosphate level than control soya group. Cats fed hypoglucidic casein diet, plasma glucose, insulin and pyridoxal 5'-phosphate levels were increased whereas cats fed hypoglucidic soya diet, pyridoxal 5'-phosphate levels were decreased and dopamine-beta-hydroxylase activity increased when data were compared to their respective control groups. A 2 hr immobilization period induced hyperglycemia in all groups whereas cats fed soya diets, plasma insulin level and dopamine-beta-hydroxylase activity were significantly increased and pyridoxal 5'-phosphate content significantly decreased. These results demonstrate that dietary casein and soya protein might be differentiated on a physiological basis and immobilization emphasized the biochemical disturbances observed between the groups thus suggesting a greater resistance to stress in casein groups than in soya groups.

Long-term effect of low-dose combined steroid contraceptives on body iron status.

The present study was aimed to evaluate iron metabolism in active and healthy adult women having taken oral contraceptives (OC) long-term. Mean dietary iron intake in age-matched control and experimental groups was adequate. Serum ferritin used as a marker for body iron stores was marginal in both groups underlying a high prevalence of deficient-iron reserves among subjects. This parameter was not correlated to the iron content of the diet. The serum iron concentration was significantly higher in OC users than control subjects (p less than 0.001). Biochemical results commanded a discussion on the pertinence of evaluating the total dietary iron intake and on the sensitivity of biochemical methods used to assess the iron status.

Chlordiazepoxide, go-nogo successive discrimination and brain biogenic amines in cats.

Chlordiazepoxide (CDP; 0.4 mg/kg/day, per os) was administered to cats during either the acquisition (CDP 21-22 days) of a go-nogo successive discrimination task (SD) or the performance (CDP 10 days) of the previously learned SD task. Endogenous levels of serotonin, 5-hydroxyindoleacetic acid, noradrenaline and dopamine were assayed in 12 brain areas, in trained as well as in untrained cats. This study has shown that (1) CDP strongly impaired the acquisition but not performance of the SD task, revealing a dissociation of the effects of CDP on these two stages of training; (2) the CDP administration, as well as the SD training, produced regional changes in brain levels of biogenic amines, suggesting the involvement of particular monoaminergic neurons in the behavioral effects of CDP and in operant behavior; and (3) in particular brain areas, interactions were observed between the effects of the SD training and those of the CDP administration on monoamines, indicating that the behavioral state may interfere with the neurochemical effects of CDP.

Monoamine roles in retention and reversal of delayed response in cats.

Two experiments were performed to study the effects of manipulation of central monoamines upon behavior in a delayed response (DR) situation. In the first study, serotonin (5-HT) levels were increased by administration of 5-hydroxy-L-tryptophan (5-HTP) and RO4-4602, a decarboxylase inhibitor, to cats that had overlearned the DR. This intervention had no significant effect upon performance in the 0 sec delay condition, but significantly increase error and non-response scores during delay trials; the effect is specific to an "information holding" demand upon the animal and according to the neurochemical analysis appears to be due to a central effect of 5-HT. In a second experiment, dopamine levels were raised by L-DOPA administration during a reversal of the original DR situation, and the effect of L-DOPA on the evolution of response strategies was observed. All animals developed a position habit that proved impossible to correct but L-DOPA animals developed a significant position habit more quickly than controls, thus suggesting a possible relationship between the neostriatal dopamine accumulation and behavioral plasticity.

Effects of vitamin B12 supplementation on choline acetyltransferase activity in cat brain.

Vitamin B12 was daily supplemented to a semi-purified casein diet and given per os during 17 consecutive days to cats in a dosage of 25 micrograms/kg every morning at the meal time. The specific choline acetyltransferase activity, measured in several structures in cat brain, was significantly increased in the hypothalamus, piriform lobe, hippocampus, pons and pons raphe nuclei and significantly decreased in the medulla raphe nuclei, respectively. These results demonstrate for the first time that vitamin B12 involved as coenzyme in biochemical reactions related to the liberation of methyl synthesis synthesis through the bioavailability of choline, the enzyme substrate of choline acetyltransferase. These results might contribute to a better understanding of some neurochemical mechanisms related to Alzheimer-type dementia.

The nutritional status of subjects with anorexia nervosa.

This study characterized the dietary pattern of 25 subjects with anorexia nervosa from a diet history. The patients mean age was 18 +/- 5 years and mean weight loss from preillness weight was 26%. Diets of anorectics were found to be significantly lower in total energy, reflecting a disturbance in nutrient intake when data were compared to a normal population. The percentage of energy derived from macronutrients was higher for protein and lower for concentrated carbohydrates, but total carbohydrates and lipids were found similar to the ones observed in normal subjects. Dietary intake of calcium, iron, thiamin, riboflavin, niacin and ascorbic acid of anorectics were significantly lower than in normal subjects. Such a particular pattern was paralleled by high serum cortisol levels and circadian cortisol cycle, low morning glycemia, high carotenemia, hypokaliemia and hyperalbuminemia. Results are discussed on the basis of the nutritional adequacy of the anorexia nervosa dietary pattern and its relationship with some aspects of the symptomatology of the disease.

Different influences of two types of diets commonly used for rats on a series of parameters related to the metabolism of central serotonin and noradrenaline.

A commercial chow and a semipurified diet fed for 14 days to Sprague-Dawley male rats kept under standardized conditions of temperature, humidity, and light had different effects on a series of parameters related to the metabolism of central serotonin and noradrenaline. Rats fed the commercial chow had (1) a lower serum level of the six neutral amino acids (valine, isoleucine leucine, tyrosine, phenylalanine, and methionine) known to compete with tryptophan for its entry into the brain, (2) a higher ratio of tryptophan to the sum of the six neutral amino acids, (3) a lower ratio of tyrosine to the other five neutral amino acids, (4) a lower ratio of serotonin to 5-hydroxyindoleacetic acid in hypothalamus, (5) a higher tryptophan hydroxylase activity in raphe nuclei, and (6) a higher content of noradrenaline in hypothalamus. It is suggested that chow fed rats had a more active central serotonin metabolism in hypothalamus than rats fed the semipurified diet.

Biochemical properties and kinetic parameters of dihydroxyphenylalanine--5-hydroxytryptophan decarboxylase in brain, liver, and adrenals of cat.

Biochemical properties and kinetic parameters of nonpurified dihydroxyphenylalanine-5-hydroxytryptophan decarboxylase extracted from brain and two peripheral organs, liver and adrenals, were studied in the cat. This study shows that decarboxylase activity in brain is lower than in peripheral organs and that 5-hydroxytryptophan can be decarboxylated without exogenous addition of pyridoxal-5'-phosphate (PLP). However, the addition of PLP substantially increases the enzyme activity. Excess of coenzyme (greater than 60 muM) induces inhibition in adrenals and liver but not in the central nervous system (CNS). The observed inhibition might be related to the presence of a tetrahydroisoquinoline derivative formed in the medium. Differentiation between mechanisms of action of decarboxylase in the CNS and peripheral organs is suggested.

Difference in monoamine oxidase activity measured by either liquid ion exchange or ion exchange resin chromatography in rat and cat brain.

Cat and rat brain monoamine oxidase (MAO) activity was measured with a radioisotopic procedure and two extraction methods. Results indicated an underestimation of MAO activity when liquid ion exchange chromatography (LIEC) was used instead of an ion exchange chromatographic method (IEC) to separate the different products of the deaminated tyramine, phenylethylamine, or serotonin. MAO produced aldehydic products which may be found in the incubation medium and may be extracted with the substrate in the chloroform phase by the LIEC method. In cat brain, the resulting underestimation of the MAO activity was prevented by the addition of nicotinamide adenine dinucleotide (10(-3) M) in the incubation medium or by allowing a 2-h period between the end of incubation and the LIEC extraction procedure. In the rat brain, the same result was obtained by the addition of an equimolar mixture of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate in reduced form (NAD-NADPH, 10(-3) M). Using the IEC method, the NAD decreased only the deamination of tyramine and serotonin in rat brain. This study suggests that the use of an IEC method to evaluate MAO activity is more accurate for the estimation of the enzymatic activity.

Comparative effects of carbohydrate restriction vs starvation on biochemical parameters related to neurotransmitters in rat.

Adult rats were submitted to a 4-day starvation period or maintained on a 50% carbohydrate-restricted diet for 8 consecutive days to obtain a body weight loss of 20-30%. Serum dopamine-beta-hydroxylase (DBH) activity and amino acids content were measured as well as brain tryptophan and tyrosine levels. Moreover, brain serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), noradrenaline (NA), and dopamine (DA) contents were assayed in five brain areas. In 4-day starved and 8-day carbohydrate-restricted rats, the serum tyrosine and total tryptophan contents as well as tyrosine to the sum of six neutral amino acids ratios were lowered. Moreover, in these groups, free tryptophan to the sum of six neutral amino acids ratio remained normal and serum DBH activity increased. In the brain, to a decreased tyrosine content observed in 4-day starved and 8-day carbohydrate-restricted rats corresponded a high DA to NA ratio in the hypothalamus, thalamus, and raphe nuclei, thus suggesting a low DA utilization whereas a low DA to NA ratio was found in the neostriatum. On the other hand, brain tryptophan content was decreased in 4-day starved rats and increased in 8-day carbohydrate-restricted rats. In the former group, a high 5-HT to 5-HIAA ratio characteristic of a low 5-HT utilization was found in the hypothalamus and neostriatum whereas in the latter group a significant decrease in this ratio was only observed in the thalamus. These results suggest that the biochemical response to starvation vs carbohydrate restriction can be differentiated on neurochemical and neuroanatomical bases.