The effects of reserpine and haloperidol on tyrosine hydroxylase activity in the brains of aged rats

Many neurotransmitter systems appear to be altered with aging. The effects of aging on the regulation of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of catecholamines in the brain has been examined. The endogenous basal activity of tyrosine hydroxylase was lower in the hypothalamus of 24 month old Fisher 344 rats than in the hypothalamus of 3 month old or 6 month old animals. There was no difference in the basal activity of tyrosine hydroxylase in the locus ceruleus, frontal cortex, hippocampus, substantia nigra, or the striatum of rats of ages 3 months, 6 months and 24 months. Tyrosine hydroxylase activity was increased in the striatum of 3 month old (60%) and 6 month old (28%) rats after treatment with haloperidol or reserpine, whereas no change in enzyme activity followed administration of these drugs to 24 month old animals. In conclusion, increases in tyrosine hydroxylase activity in the brain that normally occur in the striatum of 3 month old rats after haloperidol or reserpine treatment are significantly decreased in 6 month old rats and not apparent in 24 month old rats.     

Long-lasting effects of chronic ethanol administration on the activity of antioxidant enzymes

The aim of this study was to investigate the long-lasting effects of prolonged administration of ethanol doses on oxidative processes of aged rats. We determined the activity of superox-ide dismutase (SOD), catalase (CAT), and gluta-thione peroxidase (GPx) in erythrocytes of rats 15, 20, and 24 months old treated with an average daily dose of 1.5 g/Kg of ethanol or saline administered intraperitoneally for 13 weeks and after a 2 month period of withdrawal from treatment. The activity of all three enzymes decreased significantly with aging in the controls, while no age-related changes were found among treated rats. These findings are important since they are the first to show a long-lasting toxic effect of low ethanol doses observed in association with the aging process.     

Biochemical changes in the gastric mucosa after injury in young and aged rats

Changes in gastric mucosal thymidine kinase (TK) activity (an indicator of proliferative activity) were examined in young (4 month) and aged (24 month) Fischer-344 male rats 6 h after intragastric administration of either 2 M NaCl (1 ml/130 g b.w.) or an equivalent volume of water (control). These changes were related to the expression of c-myc gene, tyrosine kinase (Tyr-K) activity and tyrosine-specific phosphorylation of proteins in the gastric mucosa. Basal gastric mucosal TK activity (data from the controls) in the aged rats was found to be 75% (P less than 0.001) above the young animals. This was accompanied by increased expression of c-myc gene and a 67% (P less than 0.001) enhancement in Tyr-K activity. Intragastric administration of 2 M NaCl resulted in gastric mucosal damage (as evidenced by lesions index) in both age groups. However, in aged rats, the lesions index was found to be about 75% higher than in their younger counterparts. In young rats, mucosal injury resulted in a 95% rise in TK activity, whereas in aged rats it was increased by only 38%, when compared with corresponding controls. This 2-fold rise in TK activity in young rats was also associated with increased expression of the c-myc gene. In young rats, administration of hypertonic saline caused a 90% (P less than 0.001) increment in Tyr-K activity and significantly stimulated tyrosine-specific phosphorylation of five mucosal proteins with an apparent molecular mass of 170, 120, 100, 55 and 43 kDa. On the other hand, administration of hypertonic saline to the aged rats caused only a small 16% (P less than 0.025) increase in Tyr-K activity, and produced no apparent change in either expression of c-myc gene or tyrosine-specific phosphorylation of any of the proteins in the gastric mucosa, when compared with the corresponding controls. We conclude that aging increases the susceptibility of the gastric mucosa to damaging agents and diminishes its regenerative capacity. We also suggest that Tyr-K may play a role in determining these events.     

Potentiated effect of systemic administration of oxytocin on hypertonic NaCl intake in food-deprived male rats

Subcutaneous administration of oxytocin (OT) increases water intake and sodium/urine excretion in food-deprived male rats. This study analyzes the effect of OT administration (at 0830 and 1430h) on the consumption of water and hypertonic NaCl (1.5%). In the first experiment, injections of OT increased the intake of hypertonic NaCl (but not of water) in food-deprived rats but not in ad lib-fed animals during the second 12 h (2030 to 0830) of the treatment day. The net concentration of the fluid consumed by OT/deprived animals was close to isotonic. In the second experiment, the initial effect of OT administration was an increase in urine volume and urinary sodium excretion and concentration by food-deprived animals during the first 12 h (0830 to 2030). These findings suggest that in food-deprived animals, systemic administration of OT induces NaCl intake as a consequence of previous urine loss and urinary sodium excretion.     

DNA damage,p53 gene expression and p53 protein level in the rat brain aging

The aging induces free radicals leading to DNA damage (8‐oxo‐2′‐deoxyguanosine, 8‐oxo2dG). DNA injury causes increased expression of p53 gene and p53 protein. Levels of 8‐oxo2dG (HPLC), p53 mRNA (PCR) and p53 protein (Western blot) were estimated in gray matter (GM), white matter (WM), cerebellum (C) and medulla oblongata (MO) of control, 12‐ and 24‐month‐old rats. The level of 8‐oxo2dG increased with age in C (P < 0.05 in 12‐month‐old and P < 0.01 in 24‐month‐old rats) and MO. In 12‐month‐old animals the level of 8‐oxo2dG in GM and WM was higher than in controls. In 12‐month‐old animals p53 gene expression decreased while amounts of p53 protein increased, depending on the oxidative DNA damage. In 24‐month‐old rats, expression of p53 increased in all structures (P ≤ 0.05) while p53 protein showed decreased levels in most of structures of central nervous system (WM, C, MO). Aging leads to increased 8‐oxo2dG and augmented p53 gene expression, accompanied by a lowered expression of p53 protein.     

Combined effect of vanadium(V) and chromium(III) on lipid peroxidation in liver and kidney of rats

Since chromium(III) was demonstrated to have antioxidative action, we have decided to study the effect of this element on V-induced LPO in liver and kidney of rats. Outbred 2-month-old, albino male Wistar rats received daily, for a period of 12 weeks: group I (control), deionized water to drink; group II, sodium metavanadate (SMV) solution at a concentration of 0.100mgV/mL; group III, chromium chloride (CC) solution at a concentration of 0.004mgCr/mL and group IV, SMV-CC solution at a concentration of 0.100mgV and 0.004mgCr/mL. The particular experimental groups took up with drinking water about 8.6mgV/kg b.w./24h (group II), 0.4mgCr/kg b.w./24h (group III), 9mgV and 0.36mgCr/kg b.w./24h (group IV). The V- or Cr-treated groups had higher concentrations of these two elements in liver and kidney compared to the controls. The administration of vanadium alone caused a significant decrease in fluid intake and in body weight gain compared to the controls. In liver supernatants obtained from all tested rats a statistically significant increase in MDA concentration was demonstrated in spontaneous LPO in comparison with the control rats. Moreover, in rats intoxicated with vanadium alone a statistically significant increase in liver MDA level was observed in the presence of 100microM NaVO(3). Instead, in supernatants of liver received from rats treated with chromium alone, a statistically significant increase in MDA concentration in comparison with the controls was found in the presence of 400microM NaVO(3). In kidney supernatants obtained from rats treated with chromium alone, a statistically significant increase in lipid peroxidation was shown in the presence of 30microM FeSO(4) and 400microM NaVO(3). These results show that the tested doses of vanadium(V) and chromium(III) ingested by rats with their drinking water caused significant alterations in internal organs, especially in liver. Under the conditions of our experiment, Cr(III) did not demonstrate antioxidant action, it rather had an oxidant effect.     

Effects of aging and cyclosporin treatment on the hepatobiliary efflux of glutathione

The aim of this study was to investigate the effects of cyclosporin (CyA) treatment on biliary glutathione efflux in rats of different ages (1, 2, 4, and 24 months). CyA treatment reduced the liver content of total glutathione in 1-, 2- and 24 month old rats (-30%, -43% and -30%, respectively). By contrast, oxidized glutathione (GSSG) concentration in liver tended to increase, although non significantly, in the rats aged 4 and 24 month (+36% and +28%, respectively). The oxidized-to-reduced glutathione ratio was significantly increased in 2-, 4- and 24 month old animals (+23%, +36% and >100%, respectively). Regarding biliary glutathione, our data indicate that efflux rates of total glutathione in control (untreated) rats increased to a maximum at 4 months, and decreased (-56%) in 24 month old rats, although values were still higher than those from young animals. CyA treatment significantly reduced biliary glutathione secretion except in 24 month old rats (-98%, -66% and -32%, at 1, 2 and 4 month, respectively). In addition, following inhibition of the intrabiliary catabolism of the tripeptide by acivicin, glutathione efflux rates into bile were significantly reduced by the drug only in 1- and 2 month old rats (-29% and -55%, respectively) and even tended to increase, although non significantly, in oldest animals. Our data indicate that inhibition of biliary glutathione efflux by CyA was greater in younger rats and support the view that increased intrabiliary catabolism of the tripeptide and inhibition of its canalicular transport could contribute to the decline in biliary glutathione secretion induced by the drug.     

The effect of age on protein composition of rat cerebral microvessels

The effect of age on protein composition of cerebral microvessels was investigated by examining the content of glycosylation endproducts in cerebral microvessels isolated from young (3–6 month old), intermediate age (18 month) and aged (24–26 month old) Fischer 344 male rats and by quantitating various protein spots identified with two dimensional (2D) electrophoresis. The results indicate that aging in rats is not associated with significant increase in glycosylation of microvessel proteins. Of the 26 proteins in cerebral microvessels identified on the 2-D gel, ten showed significant age-related changes (p<0.0004) and in two of these the changes were significant as early as 18-months of age. A large acidic protein with a molecular weight of 144,000 and isoelectric point (pI) of 5.4 (Spot #1) was found only in aged rats. The results indicate that aging is associated with significant quantitative changes in protein composition of cerebral microvessels. It is possible that Spot #1 may be a novel biochemical marker of aging blood-brain barrier.     

Plasma corticosterone level of rats after bilateral lesion of the globus pallidus and elimination of the sex-dependence of the body weight loss following pallidal lesion by bilateral adrenalectomy

Plasma corticosterone (CST) level and body weight were studied in male and female rats after bilateral pallidal lesion (GPL). The effect of high dose of glycocorticoids on body weight of food and water deprived, non-lesioned rats was also studied since high CST levels due to the stress caused by the aphagia and adipsia could be anticipated. Weight changes in lesioned-adrenalectomized animals were in investigated as well. A weight loss, higher in males than females, developed after the pallidal lesion and the CST level was higher in lesioned females than in lesioned males. This sex-difference was present in non-lesioned, food and water deprived animals as well. The weight loss was higher in lesioned males than in food and water deprived male/female controls. Adrenalectomy eliminated weight loss differences between the sexes after lesion, by reducing the weight loss in males. CST administration reduced body weight loss in food and water deprived males but not in familes, and cortisol facilitated it in both sexes.     

Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system

The cardiac β-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (?) ³H-DHA in membranes prepared from cardiac ventricles was 21.1 ± 2.78 (SD) fmoles/mg protein in 3–4 month old rats (young rats) and 31.2 ± 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, K_D was 4.3 ± 1.8 nM and 6.7 ± 1.7 nM for young and old rats, respectively. Various compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3–4 month). (?)Isoproterenol (10^?5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 μM), fluoride (10 mM), and forskolin (100 μM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (?)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3–4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein (s) with β-adrenergic receptor and/or catalytic adenylate cyclase.     

Cadmium toxicity and liver mitochondria. I. Different effects of cadmium administered in vivo to adult, young, and ethionine-fed rats

The changes in liver mitochondrial respiratory activities and cytochrome concentrations were investigated when cadmium chloride was administered orally to adult, young, and ethionine-fed rats. Following a seven-day administration of 30 ppm cadmium in drinking water, adult rats showed no change, while young rats and ethionine-fed rats exhibited a marked increase in mitochondrial respiration with concomitant decrease of respiratory control index and P/O ratio. The concentrations of cytochromes aa3, b, and c + c1 in liver mitochondria were unchanged in adult rats, but increased significantly in ethionine-fed rats. In young rats receiving cadmium the liver mitochondrial protein increased with a slight change in the cytochrome concentration in mitochondria. It was further found that in adult rats a higher concentration (300 ppm) of cadmium in drinking water was toxic to the liver mitochondrial functions. Thus, the effect of oral administration of cadmium on the liver mitochondrial function depends on the condition of the animals.     

Protein synthesis in the livers of aging mice studied by electron microscopic radioautography.

The application of 3H-leucine results in labeling of the liver cells of mice in which protein is synthesized at various ages of the animals. Quantitative changes of protein synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains in the hepatocytes were mainly located over the rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, cytoplasmic matrix, and a few over the nuclei. The number of silver grains in the cytoplasm and nuclei of the hepatocytes gradually increased after birth, reached the maximum at 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the hepatocyte cytoplasm was more than that in nuclei at various ages. The number of silver grains in the rough surfaced endoplasmic reticulum and mitochondria gradually increased from embryo to 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the Golgi apparatus showed almost no change from fetal stage to 6 months after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the cytoplasmic matrix gradually increased from fetal stage to 2 months after birth, then decreased with aging until the 24th month. These changes reflect the quantity of protein synthesized in each cell organelle at various ages of animals.     

The role of liver glutathione in the acute toxicity of retrorsine to rats.

(1) Two procedures have been used to change the glutathione concentration in the livers of male rats. The glutathione level is increased to about double that of the controls, 0.5 h after the administration of cysteine (200 mg/kg, i.p.) and to about 25% that of controls, 1 h after the administration of 2-chloroethanol (30 mg/kg, i.p.). (2) The acute LD50 of retrorsine to rats (42 mg/kg) is increased by pretreatment with cysteine to 83 mg/kg and decreased by pre-treatment with chloroethanol to 23 mg/kg. In all three groups, deaths are accompanied by haemorrhagic centrilobular necrosis of the liver. (3) 2 h after the administration of retrorsine to rats (60 mg/kg), the levels of pyrrolic metabolites in the livers of animals pre-dosed with cysteine or chloroethanol are respectively about 60% and 200% those of rats given no pre-treatment. (4) Neither in the normal nor in the pre-treated rats dose retrorsine (60 mg/kg) cause a detectable fall in liver glutathione concentration 0.5-4 h after dosing. By 24 h, the glutathione concentration in the livers of the retrorsine-dosed rats is higher than those of the corresponding controls. There was no significant change in the liver weights of the treated rats relative to the controls. (5) Treatment of rats with retrorsine (60 mg/kg) causes a fall in the liver concentrations of cytochrome P-450, 24 h after dosing. This loss of cytochrome P-450 is increased in rats pre-treated with chloroethanol. The concentrations of cytochrome b5 in the same animals are not significantly reduced.     

Endurance exercise training causes adrenal medullary hypertrophy in young and old Fischer 344 rats.

The purpose of the present investigation was to determine the effects of endurance exercise training on adrenal medullary volume and epinephrine content in young (5 month) and old (23 month) female Fischer 344 rats. Animals from each group underwent 10 weeks of treadmill running (60 minutes per day, 5 days per week). 72 hours following the last training session animals were killed and the adrenal glands removed for subsequent analysis. Plantaris muscle citrate synthase activity increased with training in both young and old animals (39.8% young; 36.4% old). Trained animals had larger adrenal medullary volumes (48% increase in young, and 18% in old) than untrained controls. Trained animals also had higher total adrenal medullary epinephrine content (36% increase in young, and 24% in old). There were no differences in adrenal medullary epinephrine or norepinephrine concentration (micrograms/microliters medulla). It was concluded that the training-induced increase in adrenal epinephrine content is due to an increase in the size of the medulla, and not to a greater medullary epinephrine concentration. Furthermore, similar responses to training occur in both old and young animals.     

Effects of water-deprivation on aldosterone production in Brattleboro male rats congenitally lacking vasopressin

Effects of water-deprivation on several metabolic parameters and on plasma aldosterone concentration have been investigated in male Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI) and in male Long-Evans rats (LE) as controls. Two separate experiments were performed over a period of 72 hours: 1) to determine the global effect of water-deprivation, water deprived rats were compared with hydrated animals, 2) to elucidate the specific effect of dehydration alone, water-deprived rats were compared with similar food-restricted, but water-supplied DI and LE rats. In hydrated animals, plasma aldosterone concentration was close to 50% less in DI rats than in LE rats. After 72 hours, plasma aldosterone values increased mainly because of dehydration and this increase was greater in DI rats than in LE rats. At the same time, plasma aldosterone concentration remained lower in DI rats compared to LE rats. The changes in plasma aldosterone concentration after dehydration and possible reasons for the impairment of aldosterone production in DI rats are discussed.     

24-Hour variation in gene expression of redox pathway enzymes in rat hypothalamus: effect of melatonin treatment

Abstract

The 24-h changes in medial basal hypothalamic (MBH) gene expression of redox pathway enzymes nitric oxide synthase (NOS)-1 and NOS-2, heme oxygenase (HO)-1 and HO-2, Cu/Zn- and Mn-superoxide dismutases (SOD) and catalase were examined in adult male Wistar rats kept under an alternating regimen of light/dark. Half of the animals received melatonin (～60 μg/day) in the drinking water. After 1 month, rats were killed at six different time intervals, throughout a 24-h cycle. MBH mRNA levels were measured by real-time PCR analysis. In controls, gene expression of NOS-2 and HO-2 peaked at the early light phase while that of HO-1 showed a maximum at the middle of the dark phase. None of MBH mRNAs encoding NOS-1, Cu/Zn-SOD, Mn-SOD and catalase exhibited significant 24-h variations in control rats. Melatonin administration decreased significantly mRNAs for NOS-1, NOS-2, HO-1 and HO-2 as well as changed their 24-h profile. Melatonin augmented gene expression of the antioxidant enzymes Cu/Zn-SOD, Mn-SOD or catalase at certain time intervals only. The results are compatible with the view that the principal indirect (i.e. gene expression of redox pathway enzymes) effect of melatonin on redox pathway in the hypothalamus is mainly exerted via down-regulation of pro-oxidant enzyme mRNAs.     

Deficits in the Activation and Phosphorylation of Hippocampal Tyrosine Hydroxylase in the Aged Fischer 344 Rat Following Intraventricular Administration of 6-Hydroxydopamine

Abstract: Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 µg of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45–55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 m M 6-methyl-5,6,7,8-tetrahydropterin) and an ∼35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 m M 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pl isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.     

The relationship of gestational age to vitamin A induced postnatal dysfunction

In an effort to determine the relationship between time of administration and consequent behavioral effects on progeny, a uniform subteratogenic dose of vitamin A (80,000 I.U./KG) was administered to gravid Sprague-Dawley rats during one of five periods of gestation (days 5-7, 8-10, 11-13, 14-16 and 17-19). Offspring were examined for changes in rate of weight gain, locomotor activity and maze learning ability (T-maze with return to nest as reward and multiple T water maze escape). Vitamin A 8-10 animals were hyperactive, vitamin A 11-13 animals acquired T-maze slower than controls and both vitamin A 8-10 and 11-13 acquired water maze slower than controls. Vitamin A 11-13 animals were significnatly lighter than controls and all other vitamin A groups.     

Age and food restriction alter the porphyrin concentration and mRNA levels for 5-aminolevulinate synthase in rat Harderian gland.

The effects of age and food restriction on the porphyrin concentration in Harderian glands were studied in male Fisher 344 rats. Harderian gland porphyrin concentrations increased with age; this was statistically significant in 20 month old animals compared with 3 month old animals. Food restriction (by 40%) prevented the age-associated rise in porphyrins; thus, in 20 month old food restricted rats had porphyrin concentrations similar to those found in young animals. In a second experiment, we correlated the age-associated rise in Harderian gland porphyrin concentrations with an increase in mRNA levels for 5-aminolevulinate synthase (ALV-S). Both the porphyrin concentration and ALV-S mRNA rose at 12 and 18 months of age, but decreased by 24 months of age. It is concluded that, a) porphyrin biosynthesis in the Harderian glands increases up to 20 months of age but decreases in rats that are 24 months old, and b) food restriction prevents the porphyrin rise associated with age in the Harderian gland of male Fisher 344 rats.     

Identification of common and differential mechanisms of glomerulus and tubule senescence in 24‐month‐old rats by quantitative LC–MS/MS

Kidney aging together with related renal disease had become a major clinical problem. Understanding the mechanisms of aging was important for suspending senescence and decreasing the incidence of aging‐related diseases. In the present work, 24‐month‐old F344 rats were used as aging rats and 3‐month‐old rats were used as young controls. Senescence‐associated‐β‐galactosidase staining results showed that the degree of senescence in renal tubules was more severe than that in glomeruli. We performed quantitative LC–MS to assess the differential protein expression profiles of senescent glomeruli and tubules. Bioinformatics analysis showed that aging, response to oxidative stress, nucleotide metabolism, amine acid metabolism, and inflammatory response were common mechanisms of glomerulus and tubule senescence. Differentially expressed proteins network mediated Golgi vesicle transport, actin filament based process, and regulation of cell death were associated with tubule senescence. More importantly, we found that the changes of four and a half LIM protein 2 (FHL2) were opposite in senescent glomeruli and tubules, and FHL2 could regulate p16 by suppressing T‐box 3, which was involved in regulation of senescence in glomeruli and tubules. In conclusion, we assessed the mechanisms of senescence in aging glomeruli and tubules, and the results yielded new insight into kidney senescence.