Effects of L-arginine and L-NAME on chronic partial bladder outlet obstruction in rabbit

Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). NOS can be inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and stimulated by supplementing the diet with L-arginine. The aim of this study was to investigate the influence of NOS activity on the response of rabbits to chronic partial bladder outlet obstruction (PBOO). Surgical PBOOs (2 and 8 wk) were performed on male New Zealand White rabbits. Before obstruction, one-third of the animals were premedicated for 7 days with L-NAME and another third with L-arginine. The results are summarized as follows. First, bladder weight after 8-wk PBOO was significantly lower in animals treated with L-arginine compared with both untreated and rabbits treated with L-NAME. Second, contractile function decreased progressively with PBOO duration. However, after 8 wk of PBOO, the L-arginine group had significantly greater contractile function compared with the no-treatment group, and the L-NAME group had significantly lower contractile function compared with the no-treatment group. Third, at 8 wk following PBOO, the level of protein oxidation and nitration was lowest for the L-arginine group and highest in the L-NAME group. These studies clearly demonstrated that increasing blood flow by stimulating NOS significantly protected the bladder from PBOO dysfunctions, whereas inhibiting blood flow by L-NAME enhanced the dysfunctions mediated by PBOO.     

Effect of outlet obstruction on pyruvate metabolism of the rabbit urinary bladder

Bladder function is dependent upon cellular metabolism of substrates and the adequate generation of high-energy phosphate compounds. Partial outlet obstruction induces a marked decrease in bladder function which is associated with a significant decrease in the oxidative metabolism of glucose.The current investigation was designed to determine whether the time course of the decrease in mitochondrial oxidation in the hypertrophied urinary bladder is similar to the time course of the contractile dysfunction observed. In these studies we determined: 1) the rate of ¹⁴C-pyruvate metabolism to ¹⁴CO₂ in control and obstructed tissue (1, 3, 5 and 7 days), and 2) the mitochondrial enzymatic activities of malate dehydrogenase and citrate synthase.The results can be summarized as follows: 1) The rate of pyruvate metabolism decreases by over 50% within one day following partial outlet obstruction, and remains at this level for the seven day period of study. 2) Kinetic analysis demonstrates that the change in enzymatic activity is related to a decrease in Vmax; the K_d for pyruvate is similar for control and after all time periods of obstruction. 3) The enzymatic activity of malate dehydrogenase and citrate synthase is reduced by over 50% within one day following partial obstruction, and remains at this level throughout the 7 day study period. These metabolic results correlate in time and duration with the decreased ability of the bladder to empty following partial outlet obstruction.     

Dietary supplementation of old mice with flavonoid dihydroquercetin causes recovery of the mitochondrial enzyme activities in skeletal muscles

The effect of a potent antioxidant, flavonoid dihydroquercetin on the activity of three mitochondrial enzymes in mouse skeletal muscles has been investigated. An ability of this substance to restore the activity of mitochondrial enzymes in old animals was demonstrated. The activities of citrate synthase, NADHcoenzymeQ1-oxidoreductase (complex 1) and cytochromc-oxidase (complex 4) were assessed using spectro-photometric analysis in a quadriceps muscle homogenate. It was shown that the citrate synthase activity decreased moderately and the activities of complexes 1 and 4 in skeletal muscles dropped significantly in old mice. Supplementation of drink water with dihydroquercetin for a few weeks led to an increase of citrate synthase and complex 1 activity (P < 0.1) in muscles of old animals. Activity of complex 4 returned to the level found in the tissue of young mice. Maximal activity of citrate synthase and complex 1 was found in muscles of young mice. Sensitivity of NADH-coenzymeQ1-oxidoreductase to a specific inhibitor rotenone differed in all three groups of mice. Young and old mice exhibited about 95% and 84% of the total sensitivity, respectively, while in old mice receiving dihydroquercetin the sensitivity of complex 1 to the inhibitor increased up to 98%. The biochemical alterations entailed an increase in animals’ mobility as well as an improvement of fur and skin condition. Fatty acid composition of homogenate in muscle tissue of all three groups was also investigated. A reliable decline of the amount of linoleic acid and an increase in stearic and docosanoic acid contents as well as an increase of total amount of fatty acids in muscles of old mice were found. Statistically significant changes in fatty acid composition in muscles of old mice in the control group and in old mice receiving antioxidant were not observed.     

Effect of castration on male rabbit lower urinary tract tissue enzymes

Objectives The influence of testosterone on the prostate and corpus cavernosum have been studied extensively. However, the influence of testosterone on the lower urinary tract (bladder and urethra) have not been investigated to any great extent. The aim of this study was to determine whether androgen deprivation alters lower urinary tract metabolism. Methods A total of 16 rabbits were divided into four groups of four rabbits each. Each rabbit in groups 1–3 underwent surgical bilateral castration for duration of 1, 2 , and 4 weeks, and group 4 underwent sham operations. Sections of bladder body and base wall and mucosa, urethra and corpora were isolated, frozen, and stored under liquid nitrogen. The activities of citrate synthase-thapsigargin sensitive Ca²⁺ ATPase (Sarco/Endoplasmic Reticulum Ca²⁺ ATPase [SERCA]), and choline acetyl-transferase were examined as markers for mitochondrial function, sarcoplasmic reticular calcium storage and release, and cholinergic nerve function, respectively. Results The activity of SR function indicator, Ca²⁺ ATPase was significantly higher in the control corpora than in the control bladder or urethra. Castration resulted in decreased activity in the mitochondria specific enzyme, citrate synthase, the activity of which was greatest in the urethra and lowest in the corpora. Cholinergic nerve density indicator, choline acetyl-transferase activity was greatest in the bladder body and lowest in the urethra. Conclusions Our data indicate that (1) significant differences exist in the activities of all three enzymes in the various organs associated with the lower urinary tract; and (2) that castration results in significant alterations in the activities of all three enzymes in the bladder body, base, urethra, and corpora.     

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)     

Dietary supplementation of old rats with hydrogenated peanut oil restores activities of mitochondrial respiratory complexes in skeletal muscles

The effect of dietary supplementation of old rats (26–33 months) with hydrogenated peanut oil on the activity of mitochondrial enzymes in skeletal muscles has been studied. The activities of NADH-coenzyme Q1 oxidoreductase, cytochrome c oxidase, and citrate synthase were determined spectrophotometrically in muscle homogenates. The activities of respiratory complexes I and IV were shown to significantly decrease with the age compared to the activity of the same enzymes in young animals, while the activity of citrate synthase was virtually unchanged. The fatty acid composition of muscle homogenates of old rats differed from that of young animals by a reduced content of myristic, oleic, linoleic, and α-linolenic acids and enhanced content of dihomo-γ-linolenic, arachidonic, and docosahexaenoic acids. Per oral supple-mentation of the old rats with hydrogenated peanut oil completely restored the activity of complex IV and increased the activity of complex I to 80% of the value observed in muscles of young animals, reducing the content of stearic, dihomo-γ-linolenic, arachidonic, eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids relative to that in the groups of old and young rats. The content of oleic and linoleic acids increased relatively to that in the group of the old rats, as well as young animals. The possible mechanisms of the restoration of the activity of the respiratory enzymes under the administration of hydrogenated peanut oil are discussed.     

Increased corpus cavernosum smooth muscle tone associated with partial bladder outlet obstruction is mediated via Rho-kinase

Numerous studies have now demonstrated that lower urinary tract symptoms (LUTS) are associated with erectile dysfunction (ED) in men independent of age or general health. Because one-third of men over the age of 50 will develop LUTS and a recent study showed ED in 62% of patients presenting for LUTS, the importance of determining the mechanistic link between these two pathologies is clear. Using a rabbit model of partial bladder outlet obstruction (PBOO), a primary cause of LUTS, we have identified an increased basal corpus cavernosum smooth muscle (CCSM) tone associated with an elevated level of smooth muscle myosin (SMM) phosphorylation in PBOO compared with sham-operated control rabbits (sham). Results from in vitro kinase and phosphatase assays using purified smooth muscle myosin showed increased kinase and decreased phosphatase activities in cellular extracts from corpora cavernosa isolated from PBOO compared with sham rabbits. Increased Rho-kinase expression in the CCSM of PBOO rabbits was suggested by the observations that Rho-kinase inhibitors attenuated the increased kinase activity and were less effective in relaxing CCSM strips from PBOO vs. sham rabbits. This hypothesis was then confirmed by RT-PCR and Western blotting, which demonstrated increased expression of both isoforms of Rho-kinase (ROKalpha and ROKbeta). Increased SMM basal phosphorylation (necessary for SM contraction) in the CCSM of PBOO rabbits, mediated via an increase in Rho-kinase expression/activity, would be expected to make the CCSM more difficult to relax (necessary for erection), which suggests that the RhoA/Rho-kinase pathway as being involved in the mechanism for LUTS-associated ED.     

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC_17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC_17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC_17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders. bladder remodeling; bladder dysfunction; SM-A; LC_17a; benign prostatic hyperplasia     

Creatine kinase activity in normal and hypertrophied rabbit urinary bladder tissue (following partial outlet obstruction)

The urinary bladder depends on intracellular ATP to support a number of essential intracellular processes including contraction. The concentration of ATP is maintained by mitochondrial oxidative phosphorylation, cytosolic glycolysis and the cytosolic activity of creatine kinase, the enzyme that catalysis the rapid transfer of a phosphate from creatine phosphate (CP) to ADP resulting in the formation of ATP.Prior studies in this lab and others have demonstrated that mitochondrial respiration is significantly lower in hypertrophied bladder tissue (induced by partial outlet obstruction of the white New Zealand Rabbit). In addition to decreased mitochondrial respiration, there are significant increases in glycolysis and lactic acid formation in the hypertrophied tissue.In view of the increased glycolysis and decreased mitochondrial function in the hypertrophied tissue, and the importance in creatine kinase in maintaining cytosolic levels of ATP, the current study was designed to determine if outlet obstruction induces any changes in the activity of creatine kinase.The following is a summary of the results: 1) The bladder mass increased from 2.2 ± 0.2 gm to 11.5 ±1.6 gm at 7 days following outlet obstruction. 2) The intracellular concentrations of both ATP and CP were significantly reduced in the bladder tissue following 7 days of obstruction. 3) The percent of protein (per tissue mass) was significantly lower in the obstructed bladders, although the percent of soluble protein was similar. 4) Creatine kinase activity of control bladders showed linear kinetics with a V_max = 1120 nmoles/mg protein/4 min and K_m = 147 µM CP. 2) The creatine kinase activity of obstructed bladders also displayed linear kinetics with a V_max = 1125 nmoles/mg protein/4 min tissue, and K_m = 276 µM CP.These studies demonstrate that whereas both control and obstructed bladders have virtually identical maximum creatine kinase activities, the K_m for the obstructed tissue is significantly higher than the K_m for the control tissue. This may indicate that under cellular conditions (at sub-maximum substrate concentrations), the creatine kinase activity of the obstructed bladders may be significantly lower than the activity of the control bladders. In addition, the reduced tissue concentrations of ATP and CP would certainly be consistent with the reduced functional response to bethanechol and field stimulation.     

Evaluation of muscle oxidative potential by 31P-MRS during incremental exercise in old and young humans

The purpose of this study was to compare muscle oxidative capacity between moderately active young and old humans by measuring intracellular threshold (IT) during exercise with ³¹P-magnetic resonance spectroscopy (³¹P-MRS). Changes in phosphocreatine, inorganic phosphate, and intracellular pH were measured by ³¹P-MRS during a progressive unilateral ankle plantar flexion exercise protocol in groups of moderately active old (n=12, mean age 66.7 years) and young (n=13, mean age 26.2 years) individuals. From muscle biopsy samples of the lateral gastrocnemius, citrate synthase (CS) activity was determined in six subjects from each group, and fibre type composition was determined in nine old and ten young subjects. The old group had a lower IT for pH, as a percentage of peak work rate (P<0.05), despite a similar CS activity compared to the young. IT was significantly correlated with CS activity (R=0.59; P<0.05), but not with fibre type composition. It was concluded that metabolic responses to exercise are affected by ageing, as indicated by a lower IT in old compared to young individuals. Accepted: 7 May 1998     

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.     

Effect of bilateral in vivo ischemia/reperfusion on the activities of superoxide dismutase and catalase: Response to a standardized grape suspension

Purpose: Ischemia/reperfusion (I/R) is a major etiological factor in the bladder dysfunctions observed in men with lower tract obstruction, women with postmenopausal incontinence and with aging. A standardized grape suspension protects the rabbit urinary bladder from both the contractile dysfunctions and the morphologic changes mediated by I/R. Using a model of in vivo bilateral ischemia/reperfusion, the current study investigated the effect of this grape suspension on the endogenous antioxidant defense systems. Materials and methods: 24 NZW rabbits were separated into 6 groups of 4. Groups 1–3 were treated by gavage with aqueous grape suspensions; groups 4–6 received sugar-water vehicle. Groups 3 and 6 were controls. Groups 1 and 4 were subjected to bilateral ischemia for 2 h (I). Groups 2 and 5 underwent bilateral ischemia for 2 h and reperfusion for 1 week (I/R). For all rabbit bladders, the muscle and mucosa were separated by blunt dissection and analyzed separately. The effects of the various treatments on bladder antioxidant systems of cytoplasmic superoxide dismutase (Cu-Zn superoxide dismutase; SOD), and catalase (CAT) were evaluated. Results: The standardized grape suspension up-regulated both SOD and CAT activity of bladder muscle and mucosa in control animals. There were few differences in the grape suspension treated animals after ischemia, and in general the activities decreased following I/R. Conclusions: Increases of SOD and CAT activity in control animals as a result of grape suspension suggest a greater antioxidant capacity. This increase in the antioxidant defense system may explain the increased protection of grape suspension in the face of ischemia and I/R. However, the activities of both enzyme systems decreased in the smooth muscle subjected to I/R showing that reperfusion damages these systems probably via oxidation damage to the enzymes themselves.     

The effect of partial outlet obstruction on calpain and phospholipase-2 activities: analyzed by severity and duration

In an attempt to better understand the two pathways that lead to bladder decompensation following partial obstruction in rabbits one of which is caused by calcium-activated enzymes and the other by oxidative stress, calpain and phospholipase A₂ (PLA₂) biochemical assays were conducted to see how bladder decompensation is mediated by these two calcium-activated enzymes. Partial outlet obstructions of varying durations (4, 8, and 12 weeks plus controls) were performed on 32 New Zealand white rabbits. The rabbits were also grouped by severity: control, mild, intermediate, and severe. The activities of Calpain and PLA₂ on the muscle tissue of the bladders were analyzed. A stronger correlation was seen between activities and severities as opposed to between activities and durations for both PLA₂ and calpain. The activity for PLA₂ increased dramatically from control to mild and then stayed constant for both intermediate and severe obstructions. Calpain activity increased steadily from control to mild to intermediate to severe. Based on the increase in levels of the calcium-dependent enzymes, it was clearly shown that calcium levels increased in all stages of bladder decompensation most notably with the mild obstructions. Based on previous studies in which nitrotyrosine and dinitrophenol levels did not increase in mildly obstructed rabbits, the calcium overload pathway may predominate in mild decompensation because cells in mildly obstructed bladders are better able to cope with oxidative stress than increased calcium levels.     

Blebbistain, a myosin II inhibitor, as a novel strategy to regulate detrusor contractility in a rat model of partial bladder outlet obstruction

Partial bladder outlet obstruction (PBOO), a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB) and is associated with detrusor overactivity (DO). PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB) is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM) and reports suggest varied BLEB efficacy for different SM myosin (SMM) isoforms and/or SMM vs nonmuscle myosin (NMM). We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ～50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC) alternatively spliced isoform SM-A (associated with tonic-type SM) increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b) also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb) was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20%) only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable evidence that BLEB may be a novel type of potential therapeutic agent for regulation of myogenic and nerve-evoked DO in OAB.     

Cytoskeletal and Cytocontractile Protein Composition of Smooth Muscle Cells in Developing and Obstructed Rabbit Bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, α-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM α-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

Nodulating ability of Rhizobium tropici is conditioned by a plasmid-encoded citrate synthase

Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxalo-acetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino-sarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.     

Effects of physical exercise and aging on ascorbic acid and superoxide anion levels in peritoneal macrophages from mice and guinea pigs

The relationship of physical activity and aging, two processes with a high production of oxygen-free radicals to the ascorbate and superoxide anion (O ₂ ^- ) contents of peritoneal macrophages was studied in two animal species: guinea-pig (in which ascorbic acid is a vitamin) and mouse (in which ascorbic acid is not a vitamin). The effects of exhaustive exercise were examined in young and old animals. The results show that macrophages from old animals have a lower ascorbate content than those from young ones, whereas with exercise the ascorbate content increased in both old and young animals. This increase was higher in young than in old animals, and more evident in mice than in guinea-pigs. Aging also resulted in an increase in the O ₂ ^- levels of macrophages. With exercise these levels decreased in young mice but increased in young guinea-pigs. In old animals the exhaustive exercise did not change the O ₂ ^- levels. The results suggest in general a lack of correlation between the intracellular ascorbate and O ₂ ^- levels in relation to both physical exercise and aging.Abbreviations PBS phosphate buffered saline - NBT nitroblue tetrazolium - PEC peritoneal exudate cells - PMN polymorphonuclear