Age-associated differences in beta adrenergic-stimulated lipolysis in adipocytes from Sprague-Dawley and Fischer 344 rats

We compared the ability of adipocytes isolated from the epididymal fat pads of Sprague-Dawley and Fischer 344 rats of different ages and weights to release glycerol in response to beta-adrenergic stimulation. Twelve-month-old Sprague-Dawley rats weighed approximately three times more than did 2-month-old rats of the same strain (761 +/- 61 g vs 223 +/- 8 g, p less than .001). Basal glycerol release was increased in the adipocytes of the 12-month-old rats (128 +/- 8 nmol/10(5) cells/L compared to the 2-month-old rats 51 +/- 3 nmol/10(5) cells/L). However, the ability of isoproterenol to stimulate glycerol release above basal was markedly decreased in the older and fatter Sprague-Dawley rats (178 +/- 15 nmol/10(5) cells/L vs 482 +/- 20 nmol/10(5) cells/L, p less than .001), and significant correlation coefficients between isoproterenol-stimulated glycerol release and both total body (r = .76, p less than .001) and fat pad (r = .83, p less than .001) weight were seen in Sprague-Dawley rats. Total body weights of 2-month (188 +/- 16 g), 12-month (393 +/- 15 g), and 27-month-old (402 +/- 36 g) Fischer 344 rats were less disparate. Isoproterenol-stimulated glycerol release was similar in the three groups of Fischer 344 rats, and there was no correlation between either total body or fat pad weight and lipolysis in these rats.     

Age-related changes in renal metabolism of acetaminophen in male Fischer 344 rats

Male Fischer 344 rats classified as young (2–4 months), middle age (12–15 months) and aged (22–25 months) were examined for changes in renal metabolism of acetaminophen. Renal microsomal cytochrome P-450 levels were 30% and 48% lower in middle age and aged animals, respectively, compared to young; however, no age-related changes were observed in NADPH-cytochrome c reductase activity. Renal mixed-function oxidase production of the reactive intermediate of acetaminophen was reduced 50% in aged rats. The deacetylation of acetaminophen to the nephrotoxic metabolite p-aminophenol by the renal 10,000 × g supernatant fraction was not changed by age. Thus, p-aminophenol becomes the proportionately greater nephrotoxic metabolite of acetaminophen produced by the aged rat kidney. Whole kidney reduced and oxidized glutathione content tended to be higher in aged rats, while glutathione S-transferase activity was 46% lower in aged animals. These results demonstrate that age alters several indices of renal metabolism of acetaminophen in male Fischer 344 rats.     

Age-related changes in the acoustic startle reflex in Fischer 344 and Long Evans rats

The behavioral consequences of age-related changes in the auditory system were studied in Fischer 344 (F344) rats as a model of fast aging and in Long Evans (LE) rats as a model of normal aging. Hearing thresholds, the strength of the acoustic startle responses (ASRs) to noise and tonal stimuli, and the efficiency of the prepulse inhibition (PPI) of ASR were assessed in young-adult, middle-aged, and aged rats of both strains. Compared with LE rats, F344 rats showed larger age-related hearing threshold shifts, and the amplitudes of their startle responses were mostly lower. Both rat strains demonstrated a significant decrease of startle reactivity during aging. For tonal stimuli, this decrease occurred at an earlier age in the F344 rats: middle-aged F344 animals expressed similar startle reactivity as aged F344 animals, whereas middle-aged LE animals had similar startle reactivity as young-adult LE animals. For noise stimuli, on the other hand, a similar progression of age-related ASR changes was found in both strains. No significant relationship between the hearing thresholds and the ASR amplitudes was found within any age group. Auditory PPI was less efficient in F344 rats than in LE rats. An age-related reduction of the PPI of ASR was observed in rats of both strains; however, a significant reduction of PPI occurred only in aged rats. The results indicate that the ASR may serve as an indicator of central presbycusis.     

Age-related differences in the incorporation of 3H-arginine into vasopressin in Fischer 344 rats

We studied the effect of aging on the incorporation of 3H-arginine into vasopressin (VP) molecule in the hypothalamus (HT) and the rate of its transport into the neurohypophysis (NH) of male Fischer 344 rats of different ages: 2-3 months (young, n = 28), 11-13 months (adult, n = 30) and 30 months (old, n = 27). 3H-arginine was injected in a single dose through a permanent cannula into the lateral ventricle of an awake animal. The rats were decapitated 1, 6, and 24 h after the injection. VP was extracted from individual HTs and NHs (without pars intermedia), purified in a chromatography column and determined by RIA. Hypothalamus: One hour after the injection the 3H-activity was highest in the young, lower in the adult and lowest in the old rats, whereas the specific activity (cpm/pg VP) was similar in all age groups, reflecting endogenous VP content to be highest in the young, lower in the adult and lowest in the old rats. In the young rats both activities decreased 6 and 24 h after the injection. The adult rats also exhibited declining activities, whereas in the old rats the activities remained unchanged from 1 to 24 h after the injection. Neurohypophysis: One hour after the injection of radioactive label 3H-activity and specific activity were significantly lower in the NH of the old than in those of young and adult rats. The activities increased 6 h after the injection in all age groups with the lowest values in the old rats. After 6 h, both activities in the old rats reached only the 1-h values of the young and adult rats suggesting a delay of at least 6 h in the axonal transport of newly synthetized VP in aged rats. Twenty-four hours after the injection both activities increased in all age groups exhibiting no difference among the age groups, indicating a decreased release of the newly synthetized VP from the NH of aged rats. These data demonstrate an age-related decrease in the rate of incorporation of 3H-arginine into VP, in the rate of its axonal transport and in the release of newly synthetized VP from the NH of Fischer 344 rats.     

Neurohypophyseal aging: differential changes in oxytocin and vasopressin release, studied in Fischer 344 and Sprague-Dawley rats

We had previously shown that the hypothalamo-neurohypophyseal vasopressin secreting system is suppressed in aged rats. In the present study, using aged (26 months) male Fischer 344 (F344) rats, we showed that in contrast to vasopressin, oxytocin plasma concentration and hypothalamic content were unaltered in comparison with young (2-3 months) rats; however, based on data from our past and current studies, the neurohypophyseal concentrations of both hormones were found to be decreased in aged rats. We also compared the effect of aging on the oxytocin and vasopressin in secretory functions. Superfusion technique was employed to examine oxytocin and vasopressin release from isolated neural lobes of young (2-3 months) and old (26 months) male F344 and young (2-3 months), middle-aged (12 months) and old (30 months) Sprague-Dawley (SD) rats. Aging affected basal release of oxytocin and vasopressin in a differential manner. Expressed per gland, basal release of oxytocin increased in aged rats of both strains; whereas vasopressin release decreased in SD, and did not change in F344, old rats. The vasopressin responses to electrical stimulation, 56 mM K+ and initial traumatic release were decreased in aged rats; whereas oxytocin responses were either unaltered or decreased much less. All age-related changes were more pronounced in SD than in F344 rats. Thus, while the aging process is associated with a significant impairment in the vasopressin secretory function, the oxytocin secretory function is much less affected by that process. Significant strain differences were observed in the effects of aging on oxytocin and vasopressin release.     

Age-related changes in brain neurotransmission

In experiments on 2-, 10- and 22-month-old rats it was found that the Bmax values of dopamine (DA2), serotonin (5-HT1) and enkephalin (Enk) receptors as well as the 5-HT level in the three brain regions (cortex, striatum and hypothalamus) decreased with age; the DA level in the brain cortex and striatum and the noradrenaline (NA) content in the brain cortex decreased, while the NA level in the striatum and the 5-hydroxyindolacetic acid (5-HIAA) level in the brain cortex and the striatum as well as the MAO-T and MAO-A activities in the three brain structures increased. It is suggested that these and other changes observed in brain neurotransmission are an important element in the neurochemical bases of the age-related changes in behavior.     

Norepinephrine content in cardiovascular tissues from the aged Fischer 344 rat.

The major aim of the present study was to examine cardiovascular tissues and to determine if norepinephrine (NE) stores declined in aged (22-30 months old) male Fischer 344 (F344) rats. NE content was measured by HPLC in the whole heart, ventricle, atria, caudal artery, renal arteries, portal vein and kidney. The release of [3H]NE from the caudal artery of adult and aged rats was assessed as a functional index of sympathetic innervation. Renal alpha 2-adrenergic binding sites were also determined using 2.0 nM [3H]rauwolscine. In general, total NE content per organ was not altered in aged rats. The exception was the ventricle of aged rats which exhibited a 25% decrease in NE content. In contrast, NE content per gram of tissue weight was significantly decreased in the whole heart, atria, ventricle, kidney and caudal artery of aged rats when compared to that of adults (6-8 months old). Neither basal nor potassium-stimulated NE release was altered in the caudal artery of aged rats. Presynaptic regulation of NE release by taurine and alpha 2-adrenoceptors was also not affected by age. Renal alpha 2-adrenergic binding sites were decreased 23% in aged rats. Tissue growth that occurs as a function of aging does not appear to receive a concomitant increase in sympathetic growth as indexed by NE content. The findings of the present study would lead to the conclusion that with the exception of the ventricle, sympathetic nerve terminals of the aged male F344 rat are relatively intact.     

Decreased aldosterone receptor affinity in aortic cells from the Fischer 344 rat

The Fischer 344 rat strain represents a uniform population that is immune to salt induced hypertension and resistant to mineralocorticoid hypertension. We have compared aldosterone binding in aortic cells cultured from salt-resistant Fischer 344 rats to that from salt-sensitive Wistar-Kyoto controls for aldosterone binding. Aortic smooth muscle cells of both strains contain two classes of aldosterone binding sites: corticoid receptor I with high affinity and low capacity and corticoid receptor II with low affinity and high capacity. The corticoid receptor I of Fischer 344 rats has a significantly (P less than 0.001) lower affinity than that of age and sex-matched Wistar-Kyoto controls, but the binding capacity was the same. There was no difference between the strains in the affinity or binding capacity of corticoid receptor II. These results indicate that mineralocorticoid binding may be important in susceptibility and resistance to hypertension and support the contention that mineralocorticoids regulate blood pressure in part by direct action on vascular smooth muscle cells.     

The in vitro kinetics of senescence of Fischer 344 rat embryo fibroblasts

The kinetics of replicative senescence have been determined in three independent cultures of fibroblasts derived from Fischer rat embryos (FREF). In each case the growth fraction was measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and by metabolic labeling using bromodeoxyuridine (BrdU). FREF cultures entered senescence at an average of 20.4 population doublings. The growth fraction declined smoothly as measured by both kinetic techniques. The average rate of decline of the growth fraction observed using BrdU was −0.79 ± 0.12% population doubling⁻¹ which was closely comparable to the loss of PCNA reactivity (−0.80 ± 0.11% population doubling⁻¹). We conclude that senescence in FREF cultures occurs as the result of a progressive loss of the culture growth fraction rather than a sudden abrupt collapse.     

Age-related alterations in prolactin binding sites in the female rat

Aging is associated with various neuroendocrine alterations, including in the rat a hypersecretion of PRL with maintained ovulations (repetitive pseudo-pregnancy) and a reduced activity of the hypothalamic dopaminergic neurons with loss of the neuron responsiveness to PRL, suggestive of age-related alterations in PRL receptors. In this study we have investigated PRL binding sites in the hypothalamus as well as in the mammary glands, the ovaries and the liver of young and old nulliparous female rats. The old rats (26-28 months) displayed spontaneous repetitive pseudopregnancies and they were compared with young (4-6 months) pseudopregnant rats; the binding studies were performed by saturation analysis using 125I-oPRL as ligand and particulate membrane preparations. In the hypothalamus, a negligible binding of PRL was observed in all fragments studied, mediobasal hypothalamus, median eminence, in both young and old rats and no characterization of the binding sites could be achieved. In the mammary glands, the number of PRL binding sites was appreciable in spite of the nulliparity of the rats, but it was smaller in the old than in the young rats (9.0 +/- 1.4 vs 14.9 +/- 1.2 fmol/mg protein; mean +/- SEM; p less than 0.02). In the ovaries, the density of PRL binding sites was similar in the old and young rats (112.6 +/- 9.7 vs 115.0 +/- 8.9 fmol/mg protein), illustrative of a maintained luteotropic effect of PRL with age in the rat. In contrast, in the liver a greater number of binding sites was found in the old than in the young rats (261.9 +/- 36.6 vs 63.6 +/- 5.8 fmol/mg protein; p less than 0.001), supportive of the ability of PRL to induce its own receptors in that tissue. The affinity constant of PRL binding was not altered with age in the tissues studied. These results are illustrative of tissue-specific modifications in the number of PRL binding sites with age and they are suggestive of a sustained biological activity of PRL in the old rats.     

Bioenergetics in the aging Fischer 344 rat: effects of exercise and food restriction

The capacity for energy production was evaluated in male, Fischer 344 rats as they advanced from adulthood through senescence. At 10 months of age, the animals were divided into three groups: sedentary, fed ad libitum (S); exercised by treadmill running, fed ad libitum (E); and sedentary, caloric restricted by alternate day feeding (R). Activities of selected enzymes, ADP-stimulated respiration and levels of cytochromes, were determined in homogenates of liver and gastrocnemius muscle prepared from young controls (10-month old S) and 18-, 24-, and 30-month old animals. In liver, age-linked decrements were found in the activities of 3-hydroxyacyl-CoA dehydrogenase (S, E, and R) and citrate synthase (S), and in cytochrome c content (S and E), whereas substrate-catalysed oxidations were unaffected. In the gastrocnemius muscle (S, E, and R), respiration, activities of enzymes of the Krebs cycle and glycolysis, and cytochrome content were decreased after the age of 18 months. Oxidative capacity was increased in muscle through exercise (about 40%) and in liver by food restriction (about 20%). Body and soleus muscle mass declined similarly in all groups (about 14% from 30 to 18 months of age), whereas the loss of weight in the gastrocnemius muscle was much greater (34%). The data indicate that energy metabolism in the senescent animal is competent to meet its needs and age-related declines in energy metabolism are secondary to the aging process.     

Changes in basic fibroblast growth factor coincident with estradiol-induced hyperplasia of the anterior pituitaries of Fischer 344 and Sprague-Dawley rats

Adult female Fischer 344 (F344) and Sprague-Dawley (SD) rats were treated with estradiol via Silastic implants for 10 and 20 days. This treatment period in F344 rats is sufficient to produce dramatic hyperplasia of anterior pituitary lactotropes, activation of folliculo-stellate cells (FS) as phagocytes, and reorganization of the blood supply, i.e. hemorrhagic lakes and arteriogenesis from vessels in the adjacent meninges. Estradiol-treated SD rats do not demonstrate a comparable response. We now report intense focal concentrations of cells immunopositive for basic fibroblast growth factor (FGF) in estradiol-treated F344 rats predominantly near the posterolateral edge of the anterior pituitary, a zone rich in gonadotropes and lactotropes. Immunostaining for FGF, by both light and electron microscopy, revealed that the immunopositive cells were gonadotropes, and that the immunoprecipitate was cytosolic and was most abundant in the cytosol facing the capillaries. Immunostaining for extracellular matrix-associated FGF also revealed foci of positivity at the postero-lateral edge. Estradiol-treated SD rats did not reveal comparable localization for FGF. Morphological analysis and immunolocalization of S-100 protein, a marker for FS cells, revealed that the periphery of the anterior pituitary of estradiol-treated F344 rats included numerous disrupted gonadotropes and, furthermore, was largely devoid of FS cells. This zone was more intact in control F344 rats, but lacked FS cells. The peripheral parenchyma of control and estradiol-treated SD rats was intact compared to that of F344 rats and consistently included FS cells. These results suggest that disruptions of gonadotropes at the pituitary periphery may release FGF, which could then stimulate angiogenesis from blood vessels within the adjacent meninges. The resultant systemic blood supply would stimulate lactotrope hypertrophy and hyperplasia. Since FS cells are known phagocytes within the anterior pituitary, their absence from the periphery of F344 rats may intensify or prolong the effect of the peripherally released FGF.     

Age-related alterations of active pumping mechanisms in rat thoracic duct

OBJECTIVE: To evaluate the age-related changes in active pumping in thoracic duct (TD) from 24-month-old Fisher-344 rats comparing with TD pumping in 9-month rats. METHODS: Lymphatic diameters, contraction amplitude and frequency, ejection fraction, and fractional pump flow were determined in isolated TD preparations. Western blot analyses were performed to evaluate relative levels of eNOS and iNOS in 9- and 24-month-old TD. RESULTS: Stretch-dependent regulation was altered in aged TD especially at higher levels of pressure: the negative inotropy, negative chronotropy and diminished minute pumping (2- to 3-fold decrease) were observed. Physiological NO/imposed-flow-dependent inhibition was completely abolished in aged TD, yet NO-synthase blockade by L-NAME (10(-4) M) increased pumping in a flow-independent manner. Western blot analyses indicated that the relative levels of eNOS were decreased approximately 7-fold in the 24-month-old TD when compared with 9-month-old TD; whereas iNOS levels were increased approximately 10-fold in 24-month-old TD. CONCLUSIONS: These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function.     

Age-related alterations in adenylyl cyclase system of rat hearts.

Cardiac membranes from 26-, 52- and 104-week-old Wistar rats were used to investigate the age-related alterations in the beta-adrenergic receptor-adenylyl cyclase system. The densities and affinities of beta-adrenoceptors did not change with aging. There were no significant changes in the total amount of stimulatory G-protein (Gs), and in Gs activity measured in a reconstitution assay using human platelet membranes. The major isoform of Gs alpha, however, changed from a 45,000 to 52,000 dalton peptide with aging. The total amount of pertussis toxin substrates (Gi2 and Go) decreased significantly with aging. This finding was supported by the fact that pertussis toxin-induced potentiation of adenylyl cyclase activity was markedly reduced in the aged group. The activity of catalytic protein assessed by forskolin-stimulated adenylyl cyclase activity was decreased at 104 weeks. On the other hand, GTP analogue-stimulated adenylyl cyclase activity was significantly potentiated in the same group. These results suggest that the decreased sensitivity to catecholamines observed in aged hearts is mainly due to a dysfunction of catalytic protein, and that decreased Gi activity partially compensates for this catalytic dysfunction.     

Age-related electrophysiological changes in cerebellar noradrenergic receptors

Noradrenergic transmission in the central nervous system declines early in the aging process. This decline can be demonstrated as subsensitivity to the depressant effects of norepinephrine on cerebellar Purkinje neurons of aged rats. In young rats alpha₁ alpha₂ and beta adrenergic receptors are present and functional in the cerebellar cortex. The purpose of this study was to determine which of these receptor subtypes alter their response with age. Inhibition of the spontaneous activity of Purkinje neurons by selective noradrenergic agonlsts was compared in young (3-month-old) and aged (18-and 26–28-month-old) Fischer 344 rats. These agonists were applied to Purkinje neurons by pressure microejection from multi-barreled micropipettes and the change in neuronal action potential discharge rate was recorded. Purkinje cells of both groups of aged rats were significantly less sensitive to locally-applied isoproterenol, a beta-adrenerglc agonist, than Purkinje cells of young rats. Subsensitivity to the alpha, agonist phenylephrine and the alpha₂ agonist clonidine was not observed in the aged rats. These results suggest an age-related functional decline in an adenylate cyclase-linked beta receptor system with no concomitant functional change in receptor systems linked to other second messengers. Recipient of the Walter R. Nicolai Student Award for 1988.     

Enhancement by food restriction of liver protein synthesis in the aging Fischer 344 rat

Age-related changes in liver protein synthesis of ad libitum fed and food-restricted SPF Fischer 344 rats have been measured. In ad libitum fed animals, valine incorporation increased from 3 to 6 mo of age, decreased to the 3-mo level at 12 mo, remained relatively constant through 18 mo, and then declined further at 24 mo of age. By 24 mo valine incorporation was 30% of the 6-mo value. Food restriction had no effect at 3 mo of age (6 wk of restriction) but at 6 mo the rate of valine incorporation was 35% greater than the control, an increment that was maintained throughout the life of the animal. Therefore, although food restriction does not prevent the age-related decline in protein synthesis, it does maintain the rate of liver valine incorporation at levels greater than those observed in the liver of the ad libitum fed animal.     

Rate of aging and dietary restriction: sensory and motor function in the Fischer 344 rat

Sensory and motor task performance was assessed at 3 to 4 month intervals in chronically underfed and ad libitum-fed control rats from maturity into senescence. Diet-restricted rats weighed less than controls and lived significantly longer. Diminished body mass improved the underfed rats' abilities to hang suspended from a wire, to maintain balance on a narrow beam, and to descend from a wire mesh pole in a coordinated fashion. Underfed rats, however, lost these abilities at the same rate as did control rats. Undernutrition did not affect the startle response to acoustic stimulation, nor did it influence auditory or visual lead stimulus inhibition of the startle response. Both groups of animals showed progressive, age-related losses of sensory-motor function which proceeded at the same rate in each group. Life-prolonging undernutrition did not appear to retard aging of these simple, reflexive behaviors.     

Age-related and sex-related alterations in beta-adrenergic receptors in different regions of rat brain

The binding of (-)[3H]dihydroalprenolol, an antagonist of norepinephrine, to beta-adrenergic receptors in different regions of the brain of male and female rats of various ages was measured. The binding to the synaptosomal fraction of corpus striatum, hypothalamus, cerebral cortex, cerebellum and the brainstems shows a significant decrease in the binding in old rats of both sexes. Only in the female corpus striatal region, the binding in the adult and the old is the same. In the case of females, the highest binding is seen in the young. In the male, an increase in binding occurs up to adulthood, after which it declines, suggesting a definite sex-related difference in the beta-adrenergic receptor.     

Age- and dose-dependent naproxen disposition in Fischer 344 rats

The effects of age and dose on the pharmacokinetics of naproxen were evaluated in young and senescent male Fischer 344 rats after 2.5 and 25 mg/kg doses. Pharmacokinetic parameters based on free naproxen concentrations demonstrated a significant decrease in free clearance and free steady-state volume of distribution in the aged rats. In vitro enzyme kinetic studies also demonstrated an age-related decline in the metabolic activity and affinity of the metabolic enzymes for naproxen in the aged rats. Plasma protein binding studies revealed a larger free fraction of drug in the plasma of senescent rats. Total clearance and steady-state volume of distribution were indistinguishable between young and old rats owing to the higher free fraction in aged rats. Dose had a significant effect with free clearance and free volume of distribution decreasing as dose increased. The binding of naproxen to plasma proteins was dependent on drug concentration. Unlike the parameters based on free naproxen concentrations, total plasma clearance and volume of distribution increased with increasing dose, due to the nonlinear protein binding.