Catecholamine biosynthesis in vitro and in vivo in the chromaffin tissue of the Atlantic cod, Gadus morhua

1. The biosynthesis of [3H]catecholamines from [3H]L-tyrosine in the intact chromaffin tissue of cod posterior cardinal veins was studied in vitro and in vivo at 10 degrees C. 2. The tritiated products dopamine, noradrenaline and adrenaline were separated from the [3H]tyrosine by paper chromatography of tissue extracts and the radioactivity of 1 cm strips of the chromatogram was determined by liquid scintillation spectrophotometry. DOPA could never be demonstrated in the tissue extracts from any of the experiments performed. 3. The content of [3H]noradrenaline in pieces of the cardinal veins incubated in vitro was found to increase rapidly. The tissue content of dopamine and adrenaline remained at lower levels which were reached during the first few hours of the incubation. A similar pattern could be demonstrated in the chromaffin tissue in vivo after infusion of [3H]tyrosine, but the total content of the [3H]catecholamines was lower than in the in vitro experiments. 4. The results are consistent with the view that the methylation of noradrenaline is the rate-limiting step in the biosynthesis of adrenaline in cod chromaffin tissue.     

Changes in the catecholamine content of brain structures in rats subjected to immobilization stress

The changes in DOPA and catecholamine (adrenaline, noradrenaline, dopamine) levels were investigated in noradrenaline- and dopamine-synthesizing brain nuclei of Wistar rats after prolonged immobilization stress on catecholamine analyzer (BAS, USA) using HPLC technique. Distinct DOPA and catecholamine changes were observed in locus ceruleus + nucleus subceruleus (1. c + n. sc) and substantia nigra at any stage after immobilization (right after immobilization and 15 and 30 days later). The most prominent alterations in noradrenaline content were detected in 1. c + n. sc. 30 days after immobilization NA level in these nuclei was 1.5 times higher, as compared to the control one. It is suggested that the increasing noradrenaline level in 1. c + n. sc. plays a defensive role in survival of rats after immobilization stress.     

The cholinergic reaction of the amnion in the chick embryo

Studies have been made of the spontaneous contractions of the amnion and acetylcholine sensitivity of amniotic membrane in 8--14-day chick embryos. In 12--14-day embryos, the spontaneous rhythmic contractions were rather rare as compared to those in 8--9-day ones, their frequency being also lower. On the basis of kinetic analysis, it was concluded that both the dissociation constant (K) and the value of Pmax do not exhibit significant changes for tonotropic reaction from the 8th to the 14th day and for chronotropic reaction--from the 8th to the 10th day of incubation. After the 10th day of incubation, dose-effect chronotropic reaction not expressed. The spontaneous activity of the amnion and acetylcholine sensitivity of the amniotic membrane depend on the temperature being maximal at 38 degrees C. Possible regulatory mechanisms of contractile activity in chick amnion are discussed.     

Enkephalins and the function of the sympathetic-adrenal system in acute myocardial ischemia

It has been shown in experiments on albino rats that acute myocardial ischemia (AMI) produces a noticeable increase in excretion of adrenaline, noradrenaline, DOPA and dopamine with urine. Intraperitoneal injection of leu-enkephalin analogs (D-Ala2-Leu5-Arg6 and D-Ala2-D-Leu5-D-Arg6) to rats with AMI was accompanied by a noticeable prevention of activation of the sympathoadrenal system.     

The motor activity of the chick embryo amnion in the late stages of development. The role of serotonin and noradrenaline]

Experiments with 13- to 19-day-old chicken embryos maintained at 37 degrees C were conducted using direct registration of embryonic movements and amnion pulsation. It is shown that in the non-innervated and lacking blood vessels non-striated-muscled amnion, motoric activity could be observed nearly up to the end of the embryonic development, not only during 5 to 14 days old interval as it was supposed earlier. In accordance with our previous results indicating important role of biogenic amines (serotonin and noradrenaline) in the regulation of motoric activity of the chicken embryo amnion at earlier and middle ages, this study provides some evidences of the humoral regulatory mechanism even at later embryonic stages. After being injected into amniotic fluid of older embryos, the serotonin stimulates and noradrenaline inhibits amnion motoric activity (the both are taken at final concentration of 10(-7) to 10(-6) M). Serotonin's receptors blocator, the cyprogeptadin, suppress while beta-adrenoreceptors' blocator, the propranolol, activates intact amnion motorics ( both are taken at final concentration of 10(-7) to 10(-5) M).     

Catecholamines in sow graafian follicles at proestrus and at diestrus

Endogenous dopamine, noradrenaline, and adrenaline were detected in the sow graafian follicular wall and in the follicular fluid. Noradrenaline represented the highest level and adrenaline the lowest. Dopamine and noradrenaline concentrations found in the follicular fluid were lower at early proestrus than at mid-diestrus, whereas adrenaline levels in the fluid did not differ at either stage of the estrous cycle. The sow follicular wall contained less dopamine, noradrenaline and adrenaline at early proestrus than at mid-diestrus. Concomitantly, a decrement of [3H]-dopamine and [3H]-noradrenaline uptake, and of dopamine-beta-hydroxylase activity was detected at early proestrus compared to levels detected at mid-diestrus. The findings in sow graafian follicles show the existence of relationships between hormonal status, dopamine, noradrenaline and adrenaline endogenous levels and uptake, and dopamine-beta-hydroxylase activity. Possible links between estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels during the pig estrous cycle and ovarian catecholamines are discussed, as is a plausible involvement of these neurotransmitters in the contractile activity of the theca layer and the processes of follicular rupture and ovulation.     

Changes in the balance of biogenic monoamines and their metabolites in the organs of rats with oxygen-induced epilepsy

The content of some biogenic monoamines and their metabolites in rat brain and heart in different periods of oxygen epilepsia was studied using high performance liquid chromatography with electrochemical detection. It was shown that already at the 5th minute of exposure to oxygen adrenaline, DOPA and some noradrenaline metabolites disappeared in the brain and noradrenaline level reduced. At this period in rat heart the reduction of catecholamine content was the most distinct and serotonin level was unchanged. At the beginning of convulsive period the modifications of biogenic amines content were nonparallel in brain regions: in the heart the reduction of catecholamine level went on, especially in right ventricle. In the terminal phase of oxygen epilepsia brain biogenic amines increased, however, not up to normal meaning, heart catecholamines at this period were at the same level as at the beginning of the convulsive period.     

Comparison of the specific binding of cortisol in human amnion, amniotic fluid, and plasma

The purpose of this study was to compare the specific cortisol-binding protein found associated with human amnion with specific cortisol binding in human amniotic fluid and plasma. The electrophoretic mobility on polyacrylamide gels of the specific cortisol binding in amnion, amniotic fluid, and maternal plasma was identical. The influence of pH on cortisol binding activity was similar in all tissues and the cortisol binding was immunoprecipitable by a polyclonal antibody raised against human corticosteroid-binding globulin. The interaction of the cortisol binding protein with concanavalin A was studied in preterm amniotic fluid, term amniotic fluid, term amnion, and plasma from pregnant women at term and women under oral contraceptive treatment. Binding to concanavalin A was similar in term amnion and term amniotic fluid but was less than that found with both preterm amniotic fluid and term plasma. These results indicate that the cortisol binding protein associated with human amnion has similar characteristics to plasma corticosteroid-binding globulin, but that its state of glycosylation appears to be more like that of the cortisol binding protein in term amniotic fluid rather than in plasma.     

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.     

Role of motility in embryonic development I: Embryo movements and amnion contractions in the chick and the influence of illumination

This study provides a quantitative analysis of the active movements of the chick embryo and of the contractions of the amnion over the entire developmental period of 21 days. Four types of embryo movements are distinguished. The motor activity of the embryo shows two characteristic peaks, with maximum contraction frequencies on the 12th and on the 16th day. In contrast, the amnion activity is higher at earlier stages and decreases as the body activity increases. The amnion activity is largely independent of the body activity. Illumination has a strong influence on embryo movements. It is shown that increases of light intensity affect the patterns of activity of both the embryo and the amnion. While the effect of light on the embryo can be interpreted as being transmitted via the optic system, the mechanism of the amniotic response is unclear. The results suggest that the amnion itself may be sensitive to light. J. Exp. Zool. (Mol. Dev. Evol.) 291:186-194, 2001.     

Simultaneous radioenzymatic determination of plasma and tissue adrenaline,noradrenaline and dopamine within the femtomole range

A radiometric-enzymatic assay for measuring simultaneously femtomole quantities of adrenaline, noradrenaline and dopamine has been developed. The three catecholamines are first converted to their O-methylated analogues by catechol-O-methyltransferase in the presence of S-adenosyl-methionine-³H and thereafter extracted following addition of sodium tetraphenylborate. This extraction, together with an improved quick chromatographic separation and the oxidation of the adrenaline and noradrenaline derivatives to vanillin, yields an extremely high sensitivity and specificity of the method.The present assay allows the determination of adrenaline, noradrenaline and dopamine in tissue samples with a protein content of 100 μg or less and in plasma volumes of 20 – 100 μl. The amine content of 40 – 50 samples can be determined in two days by one person.Due to the high sensitivity achieved, this method promises to be a valid alternative to the gas chromatography-mass spectrometry technique.     

Regulation of Substances in Allantoic and Amniotic Fluid of the Chicken Embryo

Traditionally, the avian allantois has been considered a respiratory organ and a dumping ground for metabolic wastes. We tested the hypothesis that the allantoic fluid is also a depot for free amino acids and related compounds. To gain further insight in the specific role of the allantoic fluid, we included plasma and the amniotic fluid in this study. The work was carried out in 13- and 14-day-old chicken embryos. Using an HPLC-fluorometric technique, 40 of the 41 amino acids and related compounds investigated were detected. The amniotic fluid contained 32 compounds, while plasma and allantoic fluid contained 38 and 39 compounds, respectively. The glucose concentration was determined with a hexokinase technique. It was highest in plasma and lowest in the amniotic fluid. We identified three barriers that hyper- and hyporegulate a number of compounds: (1) a blood/allantois barrier, (2) a blood/amnion barrier, and (3) an allantois/amnion barrier. Compared with plasma and allantoic fluid, the amniotic fluid is a mostly hyporegulated environment.     

Pharmacokinetic analysis of alpha- and beta-adrenoreceptors in the chick embryo amnion

Following a stimulation with acetylcholine, the beta-adrenergic agonists adrenaline (A), noradrenaline (NA), isoproterenol (Iso) and salbutamol (Sal) induced a concentration-dependent decrease in the tone and (or) rate of amnion contraction with EC50 ISO < NA < A < Sal. Metaprolol, a specific beta 1-antagonist, induced a rightward shift in the dose-response curves of Iso, NA and A, whereas beta-antagonist butoxamine was ineffective. pA2 values for beta-antagonists were propranolol 8.3, metoprolol 7.0, butoxamine 5.6. EC50 values of alpha-adrenergic agonists form a sequence: clonidine < NA < methoxamine < phenylephrine. Specific alpha-antagonists yohimbine and idazoxan were found to antagonise competitively the effects of NA. The data obtained characterize the adrenergic receptors mediating stimulation of amniotic contractile activity as alpha 2-adrenergic receptors. Inhibition of contractile receptors in amnion is mainly mediated by beta 1-adrenergic receptor activation.     

Caffeine Ingestion by Rats Increases Noradrenaline Turnover and Results in Self-Biting

The effects of caffeine on the activity of central and peripheral catecholaminergic structures have been studied in rats ingesting high doses of caffeine. The activities of the enzymes tyrosine hydroxylase and dopamine-beta-hydroxylase were measured as well as 3,4-dihydroxyphenylethylamine (dopamine), adrenaline, and noradrenaline concentrations, in brain (striatum and hypothalamus), heart, and adrenal gland. At the peripheral level, we observed a significant increase in the dopamine and adrenaline plus noradrenaline content in the heart, but an increase in dopamine content only was found in the adrenal gland. Dopamine-beta-hydroxylase activity in serum was increased, but the only significant enzymic change in brain was an increase in the dopamine-beta-hydroxylase activity of the hypothalamus. However, an increase in catecholamine content was observed in both structures of the brain. These data suggest that the mechanisms involved in caffeine-induced self-biting in rats are not limited to the dopaminergic system, because we have also observed an increase in noradrenaline turnover.     

Cholinesterase in the amnion of chick embryos

Cholinesterase (ChE) activity in amnion was studied in developing chick embryos and the enzyme's substrate-inhibitory characteristics were established. The enzyme activity increased until the 8th day of incubation and then gradually decreased; on day 12-15 the activity is 40% only of the maximal one. On the basis of substrate-inhibitory analysis the enzyme was referred to propionyl-cholinesterases. Relations between age changes in ChE activity and morphological structure of smooth-muscle amnion tissue, its differentiation during development and functional activity of amnion are discussed.     

Proteome analysis of human amnion and amniotic fluid by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Proteome analysis by 2-DE and PMF by MALDI-TOF MS was performed on human amnion and amniotic fluid at term. Ninety-two soluble and nineteen membrane proteins were identified from amnion. Thirty-five proteins were identified from amniotic fluid. Calgranulin A and B were found in all patients infected with Ureaplasma urealyticum, but not in any of the patients without infection, indicating that they are potential markers of intrauterine infection. Identity of calgranulin A and B was confirmed by MALDI-TOF/TOF MS. This study represents the first extensive analysis of the human amnion and amniotic fluid proteome at term and demonstrates that 2-DE and MALDI-TOF MS is a useful tool for identifying clinically significant biomarkers of problematic pregnancies.     

Developmental changes in the glycosylation and binding properties of human fibronectins. Characterization of the glycan structures and ligand binding of human fibronectins from adult plasma, cord blood and amniotic fluid

Fibronectins from human adult plasma, fetal plasma and from amniotic fluid obtained during early and late gestation were compared with respect to (i) their reactivity with lectins, (ii) their binding to the physiological ligands gelatin and heparin, and (iii) the role of the carbohydrate residues in the binding to these two ligands. The two fibronectin isoforms displayed distinct developmental differences in both glycosylation and binding properties: (i) Proportions of tri/tetraantennary complex glycans compared to the fraction of biantennary structures, as inferred from the reactivity with concanavalin A, were highest in amniotic fluid fibronectin from late pregnancy, lower in amniotic fluid fibronectin from early gestation, and even lower in fetal and adult plasma fibronectins. Likewise, fucose (alpha 1-6) linked to the innermost N-acetylglucosamine of the chitobiosyl core, defined by reactivity with Lens culinaris agglutinin (LCA), was present primarily in amniotic fluid fibronectin, and decreased in content during gestation from the 2nd. to the 3rd. trimenon. Both fetal and adult plasma fibronectins were only weakly reactive with LCA, indicating a low content of (alpha 1-6) linked fucose residues. After prior treatment with sialidase, both plasma and amniotic fluid fibronectins strongly reacted with erythrocyte phytohaemagglutinin (E-PHA), indicating that both fibronectin isoforms contain bisecting (beta 1-4) N-acetylglucosamine residues. Amniotic fluid fibronectins showed much greater reactivity than adult and fetal plasma fibronectins with wheat germ agglutinin; binding of this lectin to amnion fluid fibronectins was not decreased by desialylation indicating the presence of poly(N-acetyllactosamine) units. Whereas amniotic fluid fibronectins were strongly reactive with peanut agglutinin, neither adult nor fetal plasma fibronectins did bind to this lectin unless after prior desialylation. Hence, both fibronectin isoforms contain O-glycan residues that are fully sialylated in fetal and adult plasma fibronectins, but only partly sialylated in amniotic fluid fibronectins. According to these differences, glycosylation of plasma and amniotic fluid fibronectins is under developmental regulation. (ii) Amniotic fluid fibronectins had a significantly lower binding activity for both heparin and gelatin than plasma fibronectins. Moreover, amnion fibronectin from late gestation displayed a significantly lower binding to these two ligands than amnion fibronectin from early gestation. Fetal plasma fibronectins had a lower binding activity for gelatin than adult plasma fibronectin. (iii) Treatment of fibronectins with sialidase, fucosidase and removal of N-glycans with endoglycosidases H and F did not affect binding to gelatin and heparin, indicating that the interaction of plasma and amnion fibronectin with these two ligands is not influenced by their oligosaccharide moieties.     

Effect of a fall of blood pressure on the release of catecholamines in the hypothalamus

The posterior hypothalamus of anaesthetized cats was superfused through a push-pull cannula and the release of endogenous catecholamines was determined in the superfusate which was continuously collected in 15 min periods. Fall in blood pressure elicited by nitroprusside or bleeding led to an increased rate of release of noradrenaline, adrenaline and dopamine in the hypothalamus. Transection of the brain causal to hypothalamus greatly reduced the rate of resting release of the catecholamines and abolished the enhancing effects of bleeding and nitroprusside. Determination of the catecholamines in samples which were collected in 90 s periods suggested a different pattern of release of the three catecholamines. Further shortening of the collection period (10 s) showed that the fall in blood pressure immediately increased the release of dopamine, while the rates of release of noradrenaline and adrenaline were increased gradually. Hypotension did not influence the rates of release of the catecholamines in the anterior hypothalamus. It is concluded that dopamine, adrenaline and noradrenaline systems of the hypothalamus are involved in the regulation of the arterial blood pressure. The different patterns of release might indicate that dopamine exerts a different function from those of noradrenaline and adrenaline in the normalization of the blood pressure after acute hypotension.     

Changes in plasma free and sulfoconjugated catecholamines before and after acute physical exercise: experimental and clinical studies.

To elucidate whether sulfoconjugated catecholamines in plasma, especially dopamine, serve as a source of free catecholamines, we examined the change in afterload on the deconjugating activity of catecholamines in isolated Langendorff perfused rat hearts. Dopamine-sulfate was administered under ordinary or high-work-load conditions. Free dopamine in the effluent was increased by the high-work-load of the hearts, whereas conjugated dopamine showed an apparent decrease. These results indicate the possibility that deconjugation of sulfoconjugated catecholamines is accelerated by a high-work-load. To obtain further evidence in humans, we also examined the changes in the plasma levels of free and sulfoconjugated catecholamines in healthy volunteers before and after marathon running. Free dopamine increased 1.99-fold from the baseline value after exercise, whereas conjugated dopamine decreased by 12%. Similarly, the plasma levels of free noradrenaline and adrenaline increased after exercise to 2.45- and 1.51-fold their respective baseline values, while conjugated noradrenaline and adrenaline both decreased. These clinical results, as well as those of the experimental studies, suggest that the increase in plasma free catecholamines after exercise is due not only to increased release from the sympathoadrenal system but also to accelerated conversion from sulfoconjugated catecholamines in the plasma.     

Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices

The uptake of [3H]adrenaline and [3H]noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high-affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose-dependent manner, but the selective dopamine and 5-hydroxytryptamine uptake inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]-noradrenaline and by unlabelled noradrenaline with [3H]-adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline-containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high-affinity sites on noradrenergic nerve terminals.