Lipid oxidation by heart mitochondria from young adult and senescent rats

1. State-3 (i.e. ADP-stimulated) rates of O(2) uptake with palmitoylcarnitine, palmitoyl-CoA plus carnitine, pyruvate plus malonate plus carnitine and octanoate as respiratory substrate were all diminished in heart mitochondria isolated from senescent (24-month-old) rats compared with mitochondria from young adults (6 months old). By contrast, State-3 rates of O(2) uptake with pyruvate plus malate or glutamate plus malate were the same for mitochondria from each age group. 2. Measurements of enzyme activities in disrupted mitochondria showed a decline with senescence in the activity of acyl-CoA synthetase (EC 6.2.1.2 and 6.2.1.3), carnitine acetyltransferase (EC 2.3.1.7) and 3-hydroxy-acyl-CoA dehydrogenase (EC 1.1.1.35), but no change in the activity of carnitine palmitoyltransferase (EC 2.3.1.21) or acyl-CoA dehydrogenase (EC 1.3.99.3). 3. Measurement of dl-[(3)H]carnitine (in)/acetyl-l-carnitine (out) exchange in intact mitochondria showed decreased rates when the animals used were senescent. However, this followed from a decreased intramitochondrial pool of exchangeable carnitine, such that calculated first-order rate constants for exchange were identical in mitochondria from the two age groups. 4. The decline in acyl-CoA synthetase activity is thought to be the reason for the diminished rate of O(2) uptake with octanoate in senescence. The decline in carnitine acetyltransferase activity is considered to be the cause of the diminished rate of O(2) uptake with acetylcarnitine or with pyruvate plus malonate plus carnitine as substrate. The mechanism of the diminished rate of O(2) uptake with palmitoylcarnitine in senescence is discussed.     

The composition and fluidity of adipocyte membranes prepared from young and adult rats

Membranes from adipocytes of adult and young rats have been compared. Phospholipid fatty acids from adult rats were more saturated than those from young rats. This difference was associated with a decreased fluidity in the membranes of the adult rats, which was inferred from measurements of fluorescence polarisation of the fluorescent probe, 1,6-diphenylhexa-1,3,5-triene.     

Fluorimetric comparison of protomitochondria and mitochondria

Protomitochondria (PRM) are intracellular mitochondrial germ organelles, precursors of mitochondria in specialized cells of animals. PRM have been isolated from rat liver by centrifugation and filtration through Millipore filters with pore diameters of 0.1 to 0.45 μm and characterized by fluorescence-based assay. PRM, having the volume many times smaller than that of light mitochondria (LM), did not differ much from the latter in protein, lipid and DNA composition, membrane charge, and some other parameters. At the same time, the activity of some enzymes of the respiratory chain (NADH dehydrogenase, succinate dehydrogenase) in PRM was even higher than that in mature mitochondria. The results obtained are important for understanding the processes of mitochondrial biogenesis in specialized cells of mammals.     

Regulation by calcium of the proliferation of heart cells from young adult rats

Summary Cells of primary and secondary cultures of rat heart ventricle were grown in a medium supplemented with homologous plasma to avoid the non-specific proliferogenic effects of foreign sera. Specific chelation of calcium from the medium arrested the cells' progression through their cycle at the G1 phase. A permanent or brief restoration of the extracellular calcium level (48 hr later) was followed, after a 5 to 6 hr delay, by a burst of DNA synthesis, and the resumption of mitotic activity. NRCC No. 14976.     

Glutamate-induced differential mitochondrial response in young and adult rats

Excitatory amino acid glutamate is involved in neurotransmission in the nervous system but it becomes a potent neurotoxin under variety of conditions. However, the molecular mechanism of excitotoxicity is not known completely. We have studied the influence of glutamate on intracellular calcium and mitochondrial functions in cortical slices from young and adult rats. The slices from both the age groups exhibited comparable intracellular calcium changes upon glutamate stimulation. Glutamate treatment caused a decrease in adenosine 5'-diphosphate/adenosine 5'-triphosphate (ADP/ATP) and an increase in nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide reduced form (NAD/NADH) ratio in both the age groups but the magnitude and the nature of temporal change was different. Glutamate-induced decrease in ATP/ADP and increase in NAD/NADH ratio was significantly higher in slices from the adult as compared to the young rats. The slices from young rats elicited slightly higher mitochondrial depolarization than adult rats. However, the formation of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) release were significantly higher in adult rats as compared to young rats. Glutamate-induced mitochondrial depolarization, ROS formation and LDH release were highly dependent on the presence of Ca(2+) in the extracellular medium. The treatment of slices with mitochondrial inhibitors rotenone and oligomycin inhibited ROS formation and LDH release substantially. Our results suggest that the glutamate-induced increase in intracellular calcium is not the only factor responsible for neuronal cell death but the mitochondrial functions could be crucial in excitotoxicity.     

Faecal corticosterone and immunoglobulin A in young adult rats

Quantitative analyses of relevant molecules in faeces may have potential as future non-invasive measures of stress. This study examined levels of faecal corticosterone and immunoglubulin A (IgA) in young adult rats and how these levels varied according to age, gender and time of day. Faecal samples were collected from 40 young adult rats (7 weeks old, n = 20 and 10 weeks old, n = 20) of both sexes from two time windows: day and night. The concentrations of corticosterone and IgA were measured by ELISAs following organic solvent extraction and aqueous extraction, respectively, of the molecules from faecal pellets. The production of faeces per time unit was higher in males than in females, and linear correlations were found between the faecal concentrations of corticosterone and IgA and total amounts of the respective molecules excreted in faeces per kg body weight per hour. In all further analyses the levels of the two molecules were calculated as amounts secreted per kg of body weight per hour. There was no gender difference between females and males in the production of corticosterone and IgA, but 7-week-old animals excreted significantly higher amounts of both molecules than did 10-week-old rats. The levels of IgA excreted by female rats were higher in the evening than in the morning, and male rats excreted higher concentrations of corticosterone in the morning than in the evening.     

Changes in nonhistone chromatin proteins from liver of young and adult chickens following D-galactosamine administration

The chromatin proteins soluble in 0.35 M NaCl altered during chicken liver development. Administration of D-galactosamine causes variations in the gel patterns of chromatin proteins from chicken liver. The amount of nonhistone protein with a mol. wt of 150,000 decreased both during liver growth and galactosamine application. The majority of age-specific chromatin proteins were, however, unaffected by galactosamine.     

Mitochondrial respiration and triiodothyronine concentration in liver from postpubertal and adult rats.

The purpose of this study was to investigate the decline in rat liver mitochondria respiration found in adult rats compared to younger ones, and to find a link between this respiratory impairment and a tissue hypothyroidism state. To this end, hepatic concentration and serum levels of triiodothyronine were measured in postpubertal rats (60 days old) and adult rats (180 days old). In addition, in these rats we measured oxidative phosphorylation in homogenate together with coupled and uncoupled respiration in isolated mitochondria using succinate or durohydroquinone as substrate. We found that mitochondria from adult rats consumed less oxygen compared to younger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4 condition, mitochondria from adult rats consumed less oxygen than mitochondria from young rats. Finally, we found a decrease in liver triiodothyronine concentration in adult rats. In conclusion, the results of this study show that hepatic mitochondria in adult rats have a decreased ATP synthesis capacity and proton permeability, both consistent with the tissue hypothyroidism found in the liver of adult rats.     

Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats

The transport of phosphate by primary cultures of renal cells from young (5-6 weeks) and adult (10-12 months) rats was studied. Renal tubule cells isolated from young and adult groups exhibited typical epithelial morphology and similar growth rates. The Na-dependent phosphate uptake was saturable with a Km of 5-7 microM over a substrate range of 1-500 microM. A decrease in Na-dependent phosphate uptake in adult cells (30%) was found compared to that of young cells. The Na-independent component of phosphate uptake did not vary with age. In addition, the inhibition of phosphate uptake by a variety of compounds (ouabain, gramicidin, 2,4-dinitrophenol, KCN, and arsenate) were similar in both age groups. Kinetic analysis showed that a significant reduction in Vmax (4.4 +/- 0.4 vs. 3.1 +/- 0.2 nmol Pi/mg protein/10 min in young and adult cells, respectively), but not Km, resulted in this decreased uptake of phosphate in adult groups. There was no difference in the efflux of phosphate from both age groups. When cells were preincubated in a phosphate-free medium for 24 hours, the uptake of phosphate was increased to 46% and 24% of their corresponding controls in young and adult cells, respectively. The decreased phosphate uptake and limited adaptation to a phosphate-free medium by the adult renal cells may account for the hypophosphatemia and phosphaturia seen in adult and old animals in vivo.     

Hormonal studies of young lean and obese Zucker rats

Plasma concentrations of insulin, corticosterone, T3, T4 and glucose were measured at 6 hour intervals throughout 24 hours in undisturbed, 34-day-old lean (Fa/?) and genetically obese (fa/fa) Zucker rats. fa/fa rats had higher plasma concentrations of insulin at all sampling times and higher plasma concentrations of corticosterone at 0300 and 0900 hours. Neither T3 nor T4 levels differed between phenotypes at any sampling time. Fasting for 24 hours at 34 days abolished the hyperinsulinaemia of fa/fa rats and raised the plasma corticosterone concentrations of both phenotypes. Before weaning there were no phenotypic differences in the plasma insulin or corticosterone concentrations measured at two sampling times in undisturbed rats. Following an intra-gastric glucose load however, fa/fa rats became hyper-insulinaemic compared with similarly treated Fa/? animals. Pancreatic insulin contents were higher in fa/fa rats at 34 days of age, but not before weaning. Somatostatin contents of the pancreas, hypothalamus and cerebral cortex did not differ between phenotypes at either 18 or 34 days of age. In conclusion, the elevated plasma concentrations of insulin and corticosterone in young fa/fa rats may contribute to their greater lipid deposition and lower protein deposition.     

Effect of adrenaline and triamcinolone on cytochrome oxidase activity in the brain and liver of young and adult rats.

The effect of adrenaline (0.15 i.p./kg b.w.) and of the synthetic glucocorticoid triamcinolone (40 mg i.p./kg b.w.) on cytochrome oxidase activity, the terminal enzyme of the cytochrome system, was studied in homogenates of the cerebral cortex, subcortical formations (including the basal ganglia, the thalamus and the hypothalamus), the medulla oblongata and the liver of 5-day-old and adult rats. Activity in the above mentioned homogenates was measured polarographically 15 and 30 min after administering adrenaline or 48 h after administering triamcinolone. Fifteen minutes after its injection, adrenaline caused a statistically significant drop in cytochrome oxidase activity in the cerebral cortex, subcortical formations and liver of 5-day-old rats. The decrease still persisted 30 min after administration of the hormone, but was intensified only in the liver. In adult rats, on the other hand, a significant increase in activity was observed in the cerebral cortex and liver after adrenaline. Triamcinolone had no effect on cytochrome oxidase activity in any of the given parts of the brain in either young or adult rats. It significantly stimulated cytochrome oxidase activity in the liver of 5-day-old rats, but severely inhibited it in the liver of adult rats.     

Submandibular gland secretory function in young adult and aged rats

An evaluation of submandibular gland secretory function was performed in young adult and aged male and female rats. No significant alterations in submandibular salivary flow rate or the concentrations of total protein, Na+,K+ and neutral sugar in the secreted saliva, were observed between different aged animals. Significant sex differences in salivary flow rate and total protein content were found.     

Hypertension after bilateral kidney irradiation in young and adult rats

The mechanism of a rise in blood pressure after kidney irradiation is unclear but most likely of renal origin. We have investigated the role of the renin-angiotensin system and dietary salt restriction in the development of systolic hypertension after bilateral kidney irradiation in young and adult rats. Three to 12 months after a single X-ray dose of 7.5 or 12.5 Gy to both kidneys of young and adult rats, the systolic blood pressure (SBP) and plasma renin concentration (PRC) were measured regularly. A single X-ray dose of 12.5 Gy caused a moderate rise in SBP and a slight reduction in PRC in both young and adult rats. A dose of 7.5 Gy did not significantly alter the SBP or PRC during the follow-up period of 1 year. In a second experiment, the kidneys of young rats received an X-ray dose of 20 Gy. Subsequently, rats were kept on a standard diet (110 mmol sodium/kg) or a sodium-poor diet (10 mmol sodium/kg). On both diets, SBP started to rise rapidly 3 months after kidney irradiation. Sodium balance studies carried out at that time revealed an increased sodium retention in the irradiated rats compared to controls on the same diet. In rats on a low sodium intake, there was neither a delay nor an alleviation in the development of hypertension. Compared to controls, the PRC tended to be lower in irradiated rats up to 4 months after irradiation. Subsequently, malignant hypertension developed in all 20 Gy rats, resulting in pressure natriuresis, stimulating the renin-angiotensin system. Our findings indicated that hypertension after bilateral kidney irradiation was not primarily the result of an activation of the renin-angiotensin system. Although there were some indications that sodium retention played a role, dietary sodium restriction did not influence the development of hypertension.     

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.