Dopamine beta-hydroxylase in rat serum and lymph: changes with age and effect of cold exposure.

The pH optimum for rat serum dopamine beta-hydroxylase (DBH) (3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating)(EC 1.14.17.1) was 4.0 in acetate buffer; other requirements were as reported by others. DBH activity in serum of 20-day-old fetuses is slightly higher than in that of their mothers. Levels of the enzyme in blood a few hours after birth are almost five times greater than in the adult, remain high during the suckling period, then drop rapidly during the 4th week after birth to about three times the adult level, which is then slowly reached over the next few weeks. These fluctuations in serum DBH activity coincide with the period of intense development and maturation of the sympathetic nervous system. There was not significant effect of cold exposure on blood DBH activity when newborn, suckling, weanling or adult warm- and cold-acclimated rats were exposed to cold. Similarly, exposure to cold that elicited two- to three-fold increases in O2 consumption failed to increase DBH activity in thoracic duct lymph. Therefore serum and lymph DBH activities are not sensitive indices of sympathetic secretory activity in the intact rat.     

Comparative analysis of indices of central dopaminergic functions in man

Various postulated indices of central dopaminergic activity - cerebrospinal fluid (CSF) dopamine (DA), dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), noradrenaline (NA), plasma NA, serum prolactin, serum dopamine-β-hydroxylase (DBH), and platelet monoamine oxidase (MAO) activity - were measured in 30 drug-free inpatients. The mean values and the ranges were similar to those described in the literature. Plasma NA showed significant positive correlation with age. Significant positive correlation was found between CSF DA and its metabolites DOPAC and HVA. Serum DBH activity showed a slight but significant inverse correlation with CSF DA and its two metabolites. CSF NA showed a significant positive correlation with CSF DOPAC, but only in females. Serum DBH activity had no significant correlation either with CSF or with plasma NA levels. These findings suggest that either CSF HVA or DOPAC and DA may be useful indicators of DA metabolism in humans. Serum DBH activity may be in relationship with the central dopaminergic functions.     

Regulation of succinyl-CoA:3-oxoacid CoA-transferase in developing rat brain: responsiveness associated with prenatal but not postnatal hyperketonemia

Activities of ketone body-metabolizing enzymes in rat brain rise 3- to 5-fold during the suckling period, then fall more than 50% after weaning. Our purpose was to determine the mechanism of the developmental changes in activity of 3-oxoacid CoA-transferase in rat brain and to study its regulation by dietary modification. Purified rat brain 3-oxoacid CoA-transferase was used to generate specific antibody. Immunotitrations of the enzyme from brains of 4-, 24-, and 90-day-old rats indicated that changes in 3-oxoacid CoA-transferase activity during development are due to changes in content of the enzyme protein. Pulse-labeling studies showed that changes in enzyme specific activity reflected changes in its relative rate of synthesis, which increased 2.5-fold between the nineteenth day of gestation and the third postnatal day, remained at this high level until the twelfth postnatal day, and declined thereafter, returning by Day 38 to the level observed in utero. The enzyme is apparently degraded very slowly during early postnatal life. Fetal hyperketonemia induced by feeding pregnant rats a high-fat diet was associated with an increase in the relative rate of synthesis of 3-oxoacid CoA-transferase in brains of 19-day-old fetuses and newborn rats and with an increase in the specific activity of the enzyme at birth. To examine the role of postnatal hyperketonemia in the development of the enzyme in brains of suckling rats, neonates received intragastric cannulas and were fed, for up to 13 days, a modified milk formula low in fat. Postnatal hyperketonemia was abolished but cerebral 3-oxoacid CoA-transferase specific activity on Days 10 and 17 was not significantly affected. Thus, the physiological hyperketonemia caused by the high fat content of rat milk is not required for the normal development of 3-oxoacid CoA-transferase in rat brain.     

Changes of dopamine-beta-hydroxylase activity during ontogenesis in healthy subjects and in an experimental model (rats)

In children and adolescents (250 healthy subjects) serum dopamine-beta-hydroxylase (DBH) activity (23.9+/-5.2 to 57.1+/-17.5 micromol/min/ml) increases with the age between 3-10 years, later it decreases approximately by the age of 10-14 years. At the age of 21 to 60 years DBH level is stable. Our study described decreasing DBH activity in adolescents at the age of 10-14 years in the studied sample of healthy persons. Experimental animals (200 Wistar rats, 5-120 days old) show the same trend of enzymatic activity, similarly as in humans. DBH activity in rats is between 0.85+/-0.1 to 2.8+/-0.05 micromol/min/ml. This activity is highest in 5-day-old rats; it decreases till the age of 14 days and increases mainly in 14- to 35-day-old animals. Decrease of DBH activity in rats between 35 to 40 days is significant and corresponds to the reduction of DBH activity in adolescent humans (10-14 years). Adult rats (aged 90-120 days) show a stable DBH activity. DBH activity intermediately decreases in 10- to 14-year-old children. This decrease corresponds to the intermediate developmental changes of electrophysiological parameters (decreasing EEG activity in healthy adolescents occurs in 10-14 years old children). Puberty is coupled with intermediate decreasing of DBH activity in man and also in experimental animals in the period of prominent psychological and physiological changes.     

Effect of weaning on the activities of glycogen synthase and phosphorylase in rat liver

The effects of weaning on the level of glycogen and the activities of glycogen synthase and phosphorylase were determined in rat liver. Glycogen levels in rat liver increased at the start of the weaning period and reached a plateau on postnatal day 20. The active form of glycogen synthase increased until postnatal day 19 and then declined. Total glycogen synthase (active + inactive) remained high during the suckling period and declined to a new low level during the weaning period. The activity ratio (active/total) increased from day 16 to days 18-22 and then decreased to the same level as found during the suckling period. At the onset of weaning the active form of phosphorylase decreased, whereas total phosphorylase initially increased and then decreased after postnatal day 20. Both forms of phosphorylase increased again at the end of the weaning period. The activity ratio decreased at the start of weaning and remained low throughout the rest of the weaning period. The effects of premature weaning were similar to those observed in normally weaned animals, but the changes occurred sooner and were more pronounced.     

Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats

We asked whether cyclooxygenase (COX) activity controls the renin-angiotensin system in the postnatal period. During kidney development, renin peaked at postnatal days 0-1 at the mRNA, tissue protein [renal renin concentration (RRC)], and plasma renin concentration (PRC) levels and was widely expressed along preglomerular vessels. PRC and renin mRNA expression was elevated until weaning in the 4th postnatal week compared with adult rats. Renocortical COX-2 was restricted to Tamm-Horsfall protein-positive cells in the thick ascending limb of Henle's loop, and cortical COX-2 mRNA and protein expression were elevated along with PRC in the 2nd and 3rd postnatal weeks. In contrast, cortical COX-1 expression was constant, but medullary COX-1 expression increased eightfold from the 1st to 4th postnatal week. A COX-2-selective blocker, parecoxib, and a nonselective blocker, indomethacin, given in a period with COX-2 induction from postnatal day 6 to day 12, markedly decreased PRC, but not renin mRNA or RRC. Inhibition of angiotensin AT(1) receptors by candesartan from postnatal day 1 to day 5 increased COX-2 mRNA (2.5-fold), protein, and distribution, renin mRNA (7-fold) and PRC (20- to 70-fold), but had no influence on COX-1 mRNA. Thus, due to very low levels of expression, COX-2 is unlikely to be responsible for the birth peak of renin, but COX-2 activity supports renin secretion later in the suckling period. ANG II negatively feeds back on renocortical COX-2 expression in the 1st postnatal days with high activity of the renin system. We suggest that suckling in the rat is correlated to an enhanced, COX-2-mediated, secretory activity of renin-producing juxtaglomerular cells.     

Thermogenic capacity of the brown adipose tissue of developing rats; effects of rearing temperature

A chronological study was performed to investigate the postnatal development of the thermogenic capacity of the brown adipose tissue (BAT) comparing rats born and reared at 16 degrees C (cold) or 28 degrees C (control). Mitochondrial mass, cytochrome-c-oxidase activity (index of oxidative capacity) and GDP binding to mitochondria (uncoupling test) were investigated in rats from 1 to 33 days of age. Specific cytochrome-c-oxidase activity was the same in both groups during the first week, then increased in the cold group and decreased in controls; from the 9th day it was always twice as high in the former as in the latter. Specific binding of GDP to mitochondrial proteins remained almost constant in control rats during the first week contrasting with a rapid increase in that for cold rats. Afterwards it decreased in both groups until weaning but remained five times as high in cold rats as in control rats. As growth of BAT is faster and mitochondrial content greater in cold reared rats, the capacity of the tissue for thermogenesis appeared to be greatly temperature dependent soon after birth and during the entire suckling period. However the mechanisms of this stimulation remain to be elucidated.     

Adipose-tissue-specific increase in glyceraldehyde-3-phosphate dehydrogenase activity and mRNA amounts in suckling pre-obese Zucker rats. Effect of weaning.

The regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was studied during the onset of obesity in the genetically obese (fa/fa) rat by determination of GAPDH activity and hybridizable mRNA amounts in adipose tissue and liver from suckling and weanling rats. GADPH activity remained low throughout the suckling period, and a burst of activity occurred after weaning in both lean and obese pups. As early as 7 days of age, adipose tissue from pre-obese rats displayed a significant increase in enzyme activity, whereas no difference could be detected in the liver. In both suckling (16 days of age) and weanling (30 days of age) obese rats a proportionate increase in GAPDH activity and mRNA amounts was observed in adipose tissue, but not in liver. It is concluded that the obese genotype influences GAPDH gene expression at a pretranslational level and in a tissue-specific manner. This phenomenon could partly contribute to the hyperactive fat accretion in the obese rat, since glycolysis is the major metabolic pathway for lipogenic substrates in adipose tissue.     

CHARACTERIZATION OF THE BASIS FOR DIFFERENCES IN SERUM DBH ACTIVITY IN IMMATURE AND ADULT RATS BY USE OF HOMOLOGOUS ANTIBODY

Abstract– Rat serum dopamine-β-hydroxylase (DBH) activity decreased 5-7-fold between 15 and 60 days of age. Immunoprecipitation performed with homologous antibody (guinea-pig anti-rat adrenal DBH) showed that during this time period the quantity of antibody necessary to precipitate 50% of the enzymatic activity (AD₅₀) decreased 5-fold from 0.25 to 0.05 μl/ml. The biochemical properties of rat serum DBH at 15 and 60 days of age were compared to test the hypothesis that there might be different biochemical forms of the enzyme in the blood of immature and adult rats. Thermal stability, apparent K_m for tyramine, electrophoretic mobility, pH optima and elution profile on gel filtratioh chromatography were all found to be similar for rat serum DBH at both ages. On the basis of homospecific activity and multiple similarities in biochemical characteristics, it appears that differences in serum activity at the two ages reflect differences in the steady-state levels of enzyme. To determine the turnover of serum DBH in the two age groups, the recovery of enzyme activity was monitored after acute clearance of the circulating pool of DBH by treatment with the homologous antiserum. Immunotitration of DBH activity in vivo indicated that the total pool of serum enzyme was 4-fold greater in the mature rat than in 4-day-olds. After treatment of adult rats with 2μl of homologous antiserum, serum DBH activity was reduced by 85% with a half-life of recovery of 3.0 ± 0.6 days; the estimated fractional rate of degradation was 0.23 ± 0.06 day^?1 and the rate of entrance was 2.3 ± 0.2 units/ml/day. After treatment of 4-day-old rats with 1 μl of homologous antiserum, serum DBH activity was reduced by 95% with a half-life of recovery of 3.3 ± 0.5 days: the estimated average fractional rate of degradation was 0.22 ± 0.06 day^?1 and the average rate of entrance was 10.7 ± 1.6 units/ml/day. Thus, the several-fold difference in steady-state levels of serum DBH in rat pups as compared to adult rats appears to be due to greatly increased rates of entrance of the enzyme in the immature rats.     

Influence of age, sex and hypoxia on plasma dopamine-beta-hydroxylase activity in the rat

Using rats (Wistar strain) of our own breed, we studied dopamine-beta-hydroxylase (E.C. 1.14.17.1) (DBH) activity in the plasma of animals of different ages (in correlation to sex) under normal conditions and after exposure to altitude hypoxia (corresponding to 7000 or 9000 m and lasting 20 min). The enzyme was determined by the method of Kato et al. (1974). We found that the given plasma enzyme activity was significantly higher in females than in males, throughout the whole life-span. In addition, we found that minimum activity was reached on about the 14th and 21st day of postnatal life and again on the 40th day, while maximum activity was recorded at the ages of 5, 30 and 35 days and in adult rats. In adult animals (males and females), exposure to altitude hypoxia was followed by a statistically significant increase in plasma DBH activity, which was much more pronounced in females than in males. In males, 240 min after terminating hypoxia plasma DBH activity had returned to normal, but in females it was still significantly raised; after 48 h, plasma DBH activity in females was identical to the activity before exposure to hypoxia. In rats aged 5 and 35 days, hypoxia evoked a fluctuating response. A decrease in activity immediately after terminating hypoxia was followed at 60 min by a return to normal, but at 240 min there was again a significant decrease. In 21-day-old rats, hypoxia did not induce any significant change in plasma DBH activity (the initial activity level in this group was very low).     

Influence of chronic treatment with iopanoic acid on thyroxine metabolism in the newborn rat

Parameters of the peripheral metabolism of thyroxine (T4) were studied in the early postnatal period. Iopanoic acid (IOP) was administered to newborn rats that were either euthyroid or rendered hypothyroid in utero by propylthiouracil (PTU) or methimazole (MMI) administration to the mothers during gestation and injected with thyroxine on postnatal days 6 and 7. In euthyroid newborn rats given IOP from postnatal day 6, the plasma T4 level increased (+50%) while the plasma 3,3',5'-triiodothyronine (T3) level slightly decreased (-18%). Peripheral deiodination of T4 was also reduced (about -50%) as estimated by thyroid 125I uptake after injection of 125I (3'-5')L-T4. In the newborn rats rendered hypothyroid in utero and given T4 on postnatal days 6 and 7, IOP treatment started on day 4 decreased the constant rate of elimination (-50%), the distribution volume (-43%) and the metabolic clearance (-74%) of plasma T4. The results were the same in PTU- and MMI-treated newborn rats. The differences between newborn and adult animals under IOP treatment are discussed.     

Development of insulin resistance and hyperphagia in Zucker fatty rats

The onset of hyperphagia in the Zucker fatty (fa/fa) rat occurs on a single day in postnatal development and could be driven by an increase in insulin sensitivity. To test this hypothesis, we performed insulin tolerance tests at several points in development. In rapidly growing juvenile rats, fatty rats are as insulin sensitive as lean rats at 4 wk of age but become increasingly insulin resistant as they became obese. During the suckling to weaning transition, fatty rats are insulin resistant at 2 wk of age, when they are exclusively suckling; they are also insulin resistant at 3 wk of age, when they are suckling and consuming solid food, but not hyperphagic. By 4 wk of age, when fatty rats are hyperphagic, they are as insulin sensitive as their lean littermates. These data indicate that fatty rats experience two phases of insulin resistance, punctuated by a brief period of insulin sensitivity that follows the onset of hyperphagia. To determine whether the increase in insulin sensitivity could be driving the onset of hyperphagia, insulin tolerance tests were performed from 21 to 27 days of age. Obese and lean rats became increasingly insulin resistant from 21 to 23 days of age and then became as insulin sensitive as lean rats by 25 days of age. These data show that increased insulin resistance precedes the onset of hyperphagia and increased insulin sensitivity follows the onset of hyperphagia. This pattern suggests that developmental perturbations in insulin signaling are likely to be involved in the onset of hyperphagia.     

Changes in the lipoprotein lipase (clearing-factor lipase) activity of white adipose tissue during development of the rat.

The lipoprotein lipase (clearing-factor lipase) activity of the white adipose tissue from rats aged between 1 and 145 days was determined. Five adipose-tissue sites (epididymal, uterine, subcutaneous, perirenal and intramuscular) together with serum concentrations of triacylglycerol, cholesterol and glucose were studied. The pattern of enzyme-activity change was remarkably similar in all the sites studied, although the growth of the tissues proceeded non-uniformly. After a peak of activity early in suckling, lipoprotein lipase activity fell to low values by 20 days of age. At weaning (21 days) the activity increased sharply and within 5 days high values were regained. The serum triacylglycerol and cholesterol concentrations were low at birth and reached peaks of concentration coincidentally with the minima of white-adipose-tissue lipoprotein lipase activities, seen late in suckling. The changes in enzyme activity were related to other metabolic changes in adipose tissue and with the known changes in plasma insulin concentrations occurring during development.     

Effect of neonatal hypoxia on the development of hepatic lipase in the rat

Increases in plasma lipids occur during hypoxia in suckling but not in weaned rats and may result from altered hepatic enzyme activity. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Hypoxia led to an increase in hepatic lipid content in the suckling rat only. Hepatic lipase was decreased to approximately 45% of control in 7-day-old rats exposed to hypoxia but not in hypoxic 35-day-old rats. Hypoxic suckling rats also had a 50% reduction in lactate dehydrogenase activity, whereas transaminase activity and CYP1A and CYP3A protein content were not different between hypoxic and normoxic groups. Additional rats were studied 7 and 14 days after recovery from hypoxic exposure from birth to 7 days of age; hepatic lipase activity had recovered to 85% by 7 days and to 100% by 14 days in the rats previously exposed to hypoxia. Administration of dexamethasone to neonatal rats to simulate the hyperglucocorticoid state found in hypoxic 7-day-old rats led to a moderate decrease ( approximately 75% of control) in hepatic lipases. Developmentally, in the normoxic state, hepatic lipases increased rapidly after birth and reached levels more than twofold that of the newborn by 7 days of age. Hypoxia delays the maturation of hepatic lipases. We suggest that the decrease in hepatic lipase activity contributes to hyperlipemia in the hypoxic newborn rats.     

Dopamine-beta-hydroxylase inhibition acutely stimulates rats hypothalamic noradrenaline and dopamine neuronal activity as assessed from metabolic ratios and circulating glucose and ACTH responses

Because central noradrenaline neuronal activity is tonically inhibited by noradrenaline (NA) itself via an action at prejunctional alpha 2-adrenoceptors, it was hypothesised that the blockade of central NA synthesis following acute dopamine-beta -hydroxylase (DBH) inhibition might primarily deplete prejunctional NA levels and result in an increase in central NA neuronal activity through reduced NA autoinhibition. This hypothesis was tested in the rat following the acute administration of the DBH inhibitors diethyldithiocarbamate (DDC) and cysteamine (CSH). Computerised gas chromatography/mass spectrometry was used to precisely measure the hypothalamic levels of NA and dopamine (DA) together with those of their primary neuronal metabolites dihydroxyphenylethyleneglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC), respectively. Both DDC (at 4 h) and CSH (at 30 min.) caused approximately a 50% reduction of hypothalamic NA concentrations. However this was associated with marked and highly significant increases in hypothalamic DHPG levels (by 50-100%) and in the hypothalamic ratio DHPG/NA. Also, when measured after CSH, the hypothalamic levels of the DHPG metabolite 3-methoxy-4-hydroxyphenylethyleneglycol were highly significantly increased. Consistent with increased DA neuronal activity, both DBH inhibitors raised DA and DOPAC levels and also the ratio DOPAC/DA in the hypothalami of treated rats and markedly suppressed serum prolactin levels (all p less than 0.01). The rise in hypothalamic concentrations of DHPG indicates that an increase in hypothalamic NA neuronal activity occurs following DBH inhibition. Significant elevations of blood glucose, corticosterone and ACTH were also observed after DBH inhibition. As we have previously demonstrated that increased central NA activity is associated with elevations of blood glucose, corticosterone and ACTH, these data provide further evidence for a functional increase in central NA activity caused by acute DBH inhibition. It is proposed that the increase in hypothalamic NA activity after DBH inhibition results from a primary depletion of the prejunctional alpha 2-active autoregulatory pool of NA.     

Somatomedin production in the neonatal rat.

The hormonal stimulus to rat fetal and neonatal somatic and skeletal growth has been investigated by a study of ciruclating somatomedin (SM), growth hormone (GH) and insulin levels in rats from 21 days in utero to 40 days of post natal life. Somatomedin activity could not be detected in the serum of fetal rats in which circulating GH and insulin levels were high. In early post natal life GH and insulin levels remained high but gradually declined reaching normal adult levels at 19 days and 40 days respectively. Somatomedin activity was measurable only at low levels until 11 days after birth and thereafter remained steady throughout the time period studied. These studies suggest that somatomedin alone is not responsible for the rapid growth of the rat in early neonatal life and it is suggested that insulin may also be active as a growth factor in this period.     

Prenatal exposure of the fetal rat to excessive L-thyroxine or 3,5-dimethyl-3'-isopropyl-thyronine produces persistent changes in the thyroid control system

Studies were conducted to determine if brief exposure, in utero, to high levels of T4 or to the synthetic thyromimetic agent 3,5-dimethyl-3'-isopropyl-L-thyronine (DIMIT) can produce permanent disruption of the thyroid control system in a manner analogous to the changes in the "set point" reported to occur due to neonatal T4 exposure in the "neo-T4 syndrome". If such a change were to occur, it could explain the persistent thyroid disturbances seen in the progeny of hypothyroid mother rats. These latter progeny are exposed in utero to both low and high serum T4 levels. Maternal T4 treatment produced a 4-fold elevation in fetal serum T4 accompanied by a large decrease in serum TSH levels. The brief treatment in utero with high doses of T4 or of DIMIT resulted in higher neonatal mortality and the T4-treatment produce subsequent growth stunting. These treatments resulted in suppression of the fetal/neonatal thyroid which was very apparent at 5 days of age. At 30 days post-partum, the thyroid control system of the progeny of the T4 and DIMIT-treated animals was still abnormal with low serum T4 levels accompanied with normal serum TSH and T3 levels. At 60 days of age, serum T4 levels remained low in the progeny of the T4-treated animals and the TSH response to TRH was subnormal in both the progeny of the T4-treated and the DIMIT-treated animals. However, serum and pituitary TSH and serum T3 were normal. The thyroid control system of the rat is sensitive to prenatal exposure to hyperthyroxinemia as it is to postnatal exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of acetaminophen on enzymatic and non-enzymatic antioxidant factors and plasma total antioxidant capacity in developing and adult rats

Recently we reported that ferric reducing ability of plasma (FRAP) assay, as an index of total antioxidant activity, increases in growing rats in response to high dose of vitamin K. In this study, it was found that acetaminophen (APAP) can cause elevation in FRAP in suckling and adult rats. This study was initiated to assess the contribution of individual antioxidant factors on elevation in FRAP. A surge in FRAP, 1 h after high dose APAP (250 or 450 mg/kg BW) administration was recorded in both young as well as adults. Whereas, low dose drug (25 mg/kg) failed to alter FRAP in both the age groups. Time-course studies show that drug-dependent elevation in FRAP begin rapidly, reaching a maximum at 1 h (> 500%). Increased FRAP was associated with a marked increase (∼14-fold) in plasma bilirubin, 6 h after drug administration at 450 mg/kg only in suckling rats. Similarly, APAP-related increase in superoxide dismutase activity in erythrocytes was limited to young rats of both the age groups. Other factors measured during this period viz., plasma uric acid, bilirubin and total protein together with catalase activity of erythrocytes remained unchanged in treated rats. Under these circumstances, APAP-related depletion in liver glutathione was almost similar in both the age groups. During a 12 h study, the concentration of lipid peroxidation products, in liver of treated groups remained within the levels of respective controls. The endpoint hepatotoxic effects of APAP was almost similar in both the age groups, suggesting that like adults, immature rats can cope with toxic effects of APAP owing to their drug-dependent induction in certain antioxidant factors.     

Plasma level of amino-acids in the developing young rat after intra-uterine growth retardation

The plasma amino-acids of young rats with intra-uterine growth retardation were analysed quantitatively from birth to weaning. A decrease in levels of the essential amino-acids appeared immediately after birth and remained low for the next two days in the plasma of the experimental rats. On the contrary, the alanine level was higher in these same animals as compared to controls and remained elevated throughout the entire suckling period.