Crabtree effect induced by fructose in hepatocytes isolated from developing rats.

The inhibition of respiration caused by fructose is investigated in hepatocytes isolated from fetal and developing rats. The respiration rate of the hepatocytes increases in the early days after birth. Adult values for oxygen uptake are reached by hepatocytes isolated from 14-day-old rats. After weaning, the Crabtree effect induced by fructose appears simultaneously with changes in energy metabolism.     

EFFECTS OF ALTERED OSMOLALITY ON RESPIRATION AND MORPHOLOGY OF MITOCHONDRIA FROM THE DEVELOPING BRAIN

Abstract— The effects of altered osmolality on respiration and fine structure were studied in isolated cerebral mitochondria from mature rats (60-100 days of age) and from rat pups in the first month of postnatal life (5, 10, 20 and 30 days). In the mature cerebral mitochondria, ADP-dependent respiration was inhibited in media of decreased osmolality. There was a transient inhibition of ADP-dependent respiration and a sustained increase in ADP-independent respiration in media of increased osmolality. In contrast, cerebral mitochondria from 5-day-old rats showed both inhibition of ADP-dependent respiration and increased ADP-independent respiration in hypo-osmolal media. In these mitochondria, inhibition of ADP-dependent respiration was stable and ADP-independent respiration was unchanged in media of increased osmolality. The transition to the mature respiratory response occurring with altered osmolality took place between 10 and 30 days of age. During this same age period, cerebral mitochondria showed an increasing resistance to matrix condensation in media of normal and increased osmolality.     

Microgradients of Microbial Oxygen Consumption in a Barley Rhizosphere Model System

A microelectrode technique was used to map the radial distribution of oxygen concentrations and oxygen consumption rates around single roots of 7-day-old barley seedlings. The seedlings were grown in gel-stabilized medium containing a nutrient solution, a soil extract, and an inert polymer. Oxygen consumption by microbial respiration in the rhizosphere (<5 mm from the root) and in bulk medium (>30 mm from the root) was determined by using Fick's laws of diffusion and an analytical approach with curve fitting to measured microprofiles of oxygen concentration. A marked increase of microbial respiration was observed in the inner 0- to 3-mm-thick, concentric zone around the root (rhizosphere). The volume-specific oxygen consumption rate (specific activity) was thus 30 to 60 times higher in the innermost 0 to 0.01 mm (rhizoplane) than in the bulk medium. The oxygen consumption rate in the root tissue was in turn 10 to 30 times higher than that in the rhizoplane. Both microbial respiration and oxygen uptake by the root varied between different roots. This was probably due to a between-root variation of the exudation rate for easily degradable carbon compounds supporting the microbial oxygen consumption.     

Chronic hypoxia modulates diaphragm function in the developing rat.

We studied the effect of chronic hypoxia on contractile properties and neuromuscular transmission in the developing rat diaphragm. We hypothesized that chronic hypoxia delays maturation of neuromuscular transmission. Phrenic nerve hemidiaphragm preparations were harvested from 3- to 26-day-old rats and littermates raised in 9.5% oxygen. Specific force, contraction time, and one-half relaxation time were measured. Each diaphragm was stimulated directly or via its nerve with 1-s trains at 10-100 Hz. Contraction time and one-half relaxation time decreased with advancing age in both groups, with a greater rate of decrease in hypoxic diaphragms. Specific force was lower for hypoxic diaphragms compared with controls. Diaphragms from the 3- to 10-day-old control and hypoxic groups generated less force in response to stimulation at frequencies >40 Hz but did so to a greater degree with nerve stimulation. Nerve stimulation of diaphragms from 11- to 18-day-old hypoxic rats showed a greater decrease in force with increasing frequency compared with age-matched controls. Diaphragms from 19- to 26-day-old rats showed no difference between the hypoxic and control groups. We conclude that chronic hypoxia leads to diaphragms that generate lower specific force as well as to a delayed maturation of mechanisms involved in neuromuscular transmission.     

The contribution of ATP turnover by the Na+/K+-ATPase to the rate of respiration of hepatocytes. Effects of thyroid status and fatty acids

In less than 1 min ouabain maximally inhibits oxygen consumption due to gramicidin-induced ATP turnover by the Na+/K+-ATPase in hepatocytes. Ouabain rapidly inhibits respiration on palmitate or glucose by only 6-10% indicating that the Na+/K+-ATPase plays a minor role in cell ATP turnover. 29% of the extra oxygen consumption of hepatocytes isolated from hyperthyroid rats was inhibited by ouabain showing that the Na+/K+-ATPase is responsible for some but not the majority of the stimulation of respiration induced by thyroid hormone.     

Differential effect of pentobarbital on chick neurons and astrocytes grown in culture

Primary cultures of neurons and astrocytes prepared from brains of 8-day-old and 15-day-old chick embryos. respectively, were grown for periods between 3 and 23 days. Cellular oxygen consumption was measured at various times in the presence of either pyruvate or succinate as substrate. Neuronal oxygen consumption was significantly higher than glial respiration, irrespective of the substrate employed. Dose-response curves for the effect of pentobarbital on respiratory activity of each cell type were constructed with the two substrates. In the presence of succinate neuronal respiration was more sensitive to pentobarbital than that of glial cells with a shift in the dose-effect curve by at least one order of magnitude. In the presence of pyruvate, glial cell respiration was inhibited at pentobarbital concentrations more than ten times lower than those effective in neurons. It is concluded that the differential sensitivity to pentobarbital between neurons and glia is due to differences in their respective energy metabolism.     

The effect of CdCl2 on the respiration of rat small intestine mucosa

Process of oxygen consumption of rat small intestine have already been studied at the level of whole intestinal wall or mucosa only by means of manometric or polarographic methods. The influence of cadmium on mucosal respiration of three sections of rat small intestine was determined because of its toxicological importance. Oxygen consumption was measured polarographically with Clark electrode at 38 degrees C in Krebs Ringer phosphate medium with 0.011 mol/l glucose. Control as well as cadmium affected respiration values were measured one after the other on the same mucosa. High cadmium concentration (applied as CdCl2) that is 2.2 and 1.4 . 10(-2) mol/l significantly inhibited the respiration in duodenum, jejunum and ileum. Observed inhibition of 80 and 50% on the average approximately for these two concentrations was related (similarly as following effects) to 100% value of control respiration. Concentrations 7.8 and 4.5. 10(-3) mol/l caused mostly lower inhibition of oxygen consumption (with the exception of significant 53% inhibition of jejunal respiration for 7.8. 10(-3) mol/l CdCl2). Low concentrations 10(-4) - 10(-6) mol/l CdCl2 had either no effect or stimulated oxygen uptake in intestinal mucosa. In older rats (weight 300-350 g) the respiration of duodenal and jejunal mucosa was inhibited with cadmium chloride concentration of 10(-6) mol/l.     

Acceleration of p-aminohippurate excretion in immature rats by dexamethasone treatment

In 5-, 10- and 15-day-old rats repeated administration of dexamethasone caused a dose-dependent increase of p-aminohippurate (PAH) excretion. Remarkably, dexamethasone has no influence on PAH excretion in adult rats. The effect of treatment in young rats cannot be explained by an increase in the glomerular filtration rate. In 10-day-old rats GFR shows a tendency to increase. In renal cortical slices from 5-day-old rats PAH transport is increased following dexamethasone treatment, whereas in 10- and 15-day-old rats an increase of kidney mass seems to be responsible for acceleration of renal excretion of PAH after administration of dexamethasone. In 5-day-old rats only the protein content is statistically significantly increased after dexamethasone treatment.     

Proteolytic and peptidase activities of the jejunum and ileum of the rat during postnatal development

1. Proteolytic (substrate nitrocasein), tripeptidase (substrate glycylglycylgly-cine) and aminopeptidase (substrate leucyl-beta-naphthylamide) activities were studied in homogenates of jejunal and ileal mucosa of 7-, 10-, 14-, 21-, 35- and 60-day-old rats. 2. Proteolytic activity was practically the same in jejunum of 7-, 10-, 14- and 21-day-old rats, but after day 21 a significant increase was observed. The activity of the ileum changed very little during postnatal development and was always higher than that of the jejunum. 3. Tripeptidase activity was low in the jejunum of 7- and 14-day-old rats, an increase was observed between day 14 and 21, but later no substantial changes were found. There were no changes in the ileum. The activity in the jejunum of 7- and 14-day-old rats was lower than in the ileum, but later the jejunum was more active than the ileum. 4. Aminopeptidase activity had a similar developmental pattern to tripeptidase activity. A low activity was found in the jejunum of 7- and 14-day-old rats, the maximum was in 21-day-old rats and then a decrease was observed, though values for 60-day-old rats were still higher than for 7- and 10-day-old rats. The activity in the ileum was practically the same in all age groups studied except in 14- and 21-day-old rats, where a transient peak was observed.     

Oxidation of acetoacetate and palmitylcarnitine by brain and liver mitochondria from suckling and adult rats

Experiments in which we investigated the possible oxidative utilization of lipoid substrates by brain and liver mitochondria were carried out with rats aged 5 and 90 days, kept under completely standardized conditions. Brain mitochondria were isolated on a Ficoll gradient after Clark and Nicklas (1970). Respiratory activity (or the respiratory control index-R.C.) was determined in the manner described in an earlier paper (Dobesová and Mourek 1980). Na succinate or Na malate was used as the testing substrate; palmityl carnitine, acetyl carnitine and acetoacetate were used as lipoid substrates. Oxygen consumption was measured with a Clark's oxygen electrode and respiration was expressed in nAt oxygen per min per mg protein, which was measured by the method of Lowry et al. (1951). When using succinate or malate, in agreement with our previous results we did not find any development changes in the respiratory activity of the brain mitochondria. The oxidation of acetoacetate by the brain mitochondria of 5-day-old rats was about five times greater, and of acetyl carnitine over two times greater, compared with the CNS mitochondria of adult rats. The oxidative utilization of lipoid substrates by the liver mitochondria of 5-day-old rats was significantly greater than their utilization by CNS mitochondria (in the case of palmityl carnitine three times greater, for example) and was always significantly greater than in the liver mitochondria of adult rats. We demonstrated that mitochondria isolated from the brain of 5-day-old rats are equipped with an enzymatic apparatus which allows them to utilize lipoid substrates on a significantly greater scale than in adulthood.     

Stimulation of the respiratory and phosphorylating activities in rat brain mitochondria by idebenone (CV-2619), a new agent improving cerebral metabolism

The effects of 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (CV-2619) on the respiration and non-respiratory oxygen consumption induced by ascorbate and Fe2+ in rat brain mitochondria were studied. When CV-2619 (100 and 300 mg/kg) was orally administered to rats for 3 days, it increased the state 3 respiration stimulated by ADP, slightly decreased the state 4 respiration after the consumption of ADP and resulted in a significant increase of the respiratory control index (RCI) by 14-19% for glutamate oxidation (p less than 0.01) and 10-17% for succinate oxidation (p less than 0.05), respectively. The RCI increasing effect of CV-2619 was dose dependent, but the compound had no effect on the ADP/O ratio. CV-2619 significantly suppressed by about 10% the non-respiratory oxygen consumption (p less than 0.02), which closely associated with non-enzymatic reactions such as lipid peroxidation, membrane lysis and swelling of mitochondria. Thus, CV-2619 may contribute to stimulate the net ATP formation by the well-coupling of electron and energy transfer, and by the reduction of non-respiratory oxygen consumption in cerebral metabolism.     

Macromolecular synthesis in mitochondria isolated from different regions of developing rat brain

DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-old rats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.     

Exercise and ovarian steroid hormones: their effects on mitochondrial respiration

The effect of exercise on mitochondria respiration was studied in gastrocnemius muscle of ovariectomized rats, pseudopregnant rats, and estrous rats. The estrous cycles were followed by vaginal smears. Rats were made pseudopregnant (PSP) by 45 s cervical stimulation with a glass rod on the day of estrous. The treadmill protocol (21 m/min, 10 grade uphill) induced a significant decrease in state 3 oxygen consumption (oxidative phosphorylation) in estrous (0.26 +/- 0.02 vs. 0.49 +/- 0.05 microatoms O min(-1) mg protein(-1)) and ovariectomized rats (0.18 +/- 0.03 vs. 0.40 +/- 0.03 microatoms O min(-1) mg protein(-1)). In contrast, pseudopregnant and progesterone-treated ovariectomized rats did not decrease state 3 nor state 4 respiratory rates. These results show that the effect of exercise on mitochondria respiration does vary according to the hormonal status.     

Oxidative activity in mitochondria isolated from rat liver at different stages of development

The purpose of this study was to evaluate the oxidative capacities in hepatic mitochondria isolated from prepubertal, young adult and adult rats (40, 90 and 180 days of age, respectively). In these rats, mitochondrial respiratory rates using FAD- and NAD-linked substrates as well as mitochondrial protein mass were measured. The results show that only the oxidative capacity of FAD-linked pathways significantly declined in mitochondria from 180-day-old rats compared with those from younger animals. When we consider FAD-linked respiration expressed per g liver, no significant difference was found among rats of different ages because of an increased mitochondrial protein mass found in 180-day-old rats. However, when FAD-linked and lipid-dependent respiratory rates were expressed per 100 g body weight, significant decreases occurred in 180-day-old rats. Therefore, the decrease in liver weight expressed per 100 g body weight rather than an impaired hepatic cellular activity may be the cause of body energy deficit in 180-day-old rats. Copyright © 1998 John Wiley & Sons, Ltd.     

Postnatal development of uterine response to estradiol-17β in the rat

The gradual acquisition of uterine responsiveness to a single injection of estradiol-17β was studied in rats aged 5–30 days with respect to wet weight and the content of DNA, RNA, and protein. No significant response in any of these parameters was found in 5- or 10-day-old rats. In 15- and 20-day-old rats all the parameters, except DNA content, were elevated by the hormone treatment; only in 30-day-old rats was there a significant increase in DNA content.     

Anti-apoptotic factor humanin is expressed in the testis and prevents cell-death in leydig cells during the first wave of spermatogenesis

Humanin (HN) is a 24 amino acids peptide with potent neuro-survival properties that protects against damage associated with Alzheimer's disease. In the present report, we have demonstrated by immunohistochemical analysis and Western blotting the pattern of expression of rat humanin (HNr) in the testis of 10- to 60-day-old rats. The Leydig cells of 10- and 40- day-old rats expressed this peptide at high levels; and in the testis of 60-day-old rats the expression of HNr expanded to include Leydig, endothelial, peritubular and germ cells. As monitored by Western blotting, HNr was released into the medium of cultures of Leydig cells isolated from 10-, 40-, and 60-days-old rats. HNr stimulated the incorporation of [(3)H]TdR into DNA of Leydig cells from 10-days-old rats, in a manner that indicated promotion of cell survival rather than an increase in the rate of cell multiplication. This peptide also enhanced steroidogenesis by cultured Leydig cells from 10- to 40-day-old rats both alone and synergistically with IGF-I. The expression of HNr in cultured Leydig cells increased in response to GH and IGF-I. In summary, we demonstrated here that HNr was expressed at all stages of maturation in the rat testis. This peptide promoted the survival of Leydig cells in culture and interacted with IGF-I to stimulate DNA synthesis and steroidogenesis. We propose that HNr is a novel testicular anti-apoptotic factor.     

Cellular energetics and the oxygen dependence of respiration in cardiac myocytes isolated from adult rat

The oxygen dependence of mitochondrial respiration was investigated using suspensions of mitochondria and quiescent ventricular myocytes isolated from adult rat hearts. A new optical method was used to determine oxygen concentration in the suspending media. The P50 for respiration for coupled mitochondria at a high [ATP]/[ADP].[Pi] ratio and oxidizing glutamate/malate was 0.45 +/- 0.03 microM but was increased to 0.57 +/- 0.02 microM by the addition of succinate to the substrate mixture. This value was decreased to less than 0.06 +/- 0.01 microM when the ATP/ADP.Pi ratio was decreased with the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The P50 value in resting myocytes was 2.23 +/- 0.13 microM at a Vmax of 13.22 +/- 1.38 nmol of O2/g, dry weight/min. During resting conditions, the creatine phosphate/creatine and ATPfree/ADPfree ratios were high in these cells, 6.81 +/- 1.11 and 1131 +/- 185, respectively. Addition of 1 mM Ca2+ to the suspending media increased the P50 by 50% whereas respiration rose by only 10%. Respiratory rate was increased up to about 10-fold by uncoupling the cells, but the P50 increased by less than 3-fold. When these uncoupled cells were inhibited with Amytal to lower the rate of oxygen consumption to that of resting cells, the P50 fell to 1.25 +/- 0.14 microM. Diffusion models indicate that in resting myocytes, the oxygen concentration difference from sarcolemma to cell core was approximately 1.84 microM with an additional difference of about 0.27 microM attributed to the unstirred layer of media surrounding each cell. The intracellular oxygen diffusivity coefficient in myocytes was calculated to be 0.30 x 10(-5) cm2/s. The results show that the oxygen dependence of respiration is modulated by the cellular metabolic state. At near maximal levels of respiration or on recovery from hypoxic episodes, oxygen diffusion may become an important determinant of the oxygen dependence of myocardial respiration.     

Age-dependent modulation by galanin of growth hormone release from rat pituitary cells in culture.

The effect of galanin (Gal), a 29-amino acid peptide widely distributed in mammalian CNS, was investigated in cultured pituitary cells of rats of different ages. Gal (0.1-10/microM) stimulated GH release in a concentration-dependent manner in 5-and 10-day-old rat pituitaries (EC50: 0.87 and 1.73/microM, respectively) but was ineffective (0.01-1/microM) or even inhibitory (10/microM) in 40-day-old male rat pituitaries. Gal (0.1-10/microM) was ineffective to alter stimulation of GH release induced by GHRH (10 nM) in 5-day-and 40-day-old rat pituitary cells, but Gal (1/microM) slightly inhibited (24%) it in 10-day-old rat pituitaries. Gal (1 and 10/microM) also inhibited GH secretion (45 and 58%, respectively) from 40-day-old pituitary cells when a lower GHRH dose (0.1 nM) was used for stimulation. The results of this study indicate that Gal has the ability to either stimulate or inhibit GH release from dispersed pituitary cells and that its effects are closely related to the age of the rats.     

Effects of dieldrin on hepatic carbohydrate metabolism in the suckling and adult rat

Hepatic carbohydrate metabolism was studied in adult and suckling rats given age-specific LD50 doses of dieldrin po. These doses in 5-, 10-, and 60-day-old Wistar rats were 38, 28, and 63 mg/kg, respectively. Plasma glucose and free fatty acids (FFA), and hepatic glycogen, phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-diphosphatase (FDP), and glucose-6-phosphatase (G6P) were measured 1 and 3 h after administration of the insecticide. Plasma glucose concentrations were elevated (17%) in some 5-day-old rats after 1 h and in all adults after 1 and 3 h (45 and 30%, respectively). Plasma FFA concentrations were decreased (9%) in the 5-day-old rat 1 h after dieldrin. Hepatic glycogen content was reduced in both 5- and 10-day-old pups at 1 hour (22 and 17%, respectively). Hepatic FDP activity was elevated in the 5-day-old rat at 1 h (17%) and was decreased (10%) in the 10-day-old rat at 3 h. Hepatic PEPCK activity was increased in adult animals by 30% 1 h after dieldrin. Furthermore, PEPCK activity was increased at 3 h in rats of all ages (76%, 5-day-old pup; 115%, 10-day-old pup; 56%, 60-day-old adult). Hepatic G6P activity was unaltered by dieldrin. Thus only the activity of hepatic PEPCK is consistently elevated by dieldrin exposure. However, this enhanced PEPCK activity is associated with dieldrin-induced hyperglycemia only in the adult rat.     

Pharmacological and biochemical characterization of cholecystokinin/gastrin receptors in developing rat pancreas. Age-related expression of distinct receptor glycoforms.

Cholecystokinin/gastrin receptors in the pancreas of newborn (3-day-old) rats are of type A, as in control mature rats, revealed by pharmacological analysis of specific 125I-Bolton-Hunter-reagent-labelled [Thr34,Ahx37]cholecystokinin(31-39) (Ahx, aminohexanoic acid) binding. Also, by 1 day post-partum, pancreatic cholecystokinin receptors were shown to be coupled to guanine-nucleotide-binding regulatory (G) proteins. Scatchard analysis of 125I-Bolton-Hunter-reagent-labelled [Thr34,Ahx37]cholecystokinin(31-39) binding to pancreatic membranes from rats at different times after birth showed a slight increase in the binding capacity of cholecystokinin receptors between days 3 and 14 and a sixfold increase in 21-day-old rats, with no change in receptor affinity during development. SDS/PAGE analysis of pancreatic membranes affinity labelled with the photoactivable ligand 125I-[2-(p-azidosalicylamido)-1,3'-dithiopropionate]-labelled [Thr34,Ahx37]cholecystokinin-(31-39) identified cholecystokinin receptors of 100-135 kDa in 3-day-old rats, 96-130 kDa in 7-day-old rats, 90-125 kDa in 10-day-old rats and 85-100 kDa in 14-day-old and 21-day-old rats, as found in control adult rats. Endo-beta-N-acetylglucosaminidase F treatment yielded a core protein of 42 kDa in all developmental stages. These findings are consistent with an age-related postnatal expression of distinct glycoforms of pancreatic cholecystokinin receptors. Furthermore, it was observed that the period 2-3 weeks after birth, characterized by stabilization of the mass of the cholecystokinin receptor, precedes the dramatic increase in the receptor number.