Underactive bladder in aging rats is associated with a reduced number of serotonin‐expressing cells in the urethra and is improved by serotonin application to the urethra

The aim of this study was to determine whether aging‐related detrusor underactivity (DU) involves a decrease in 5‐hydroxytryptamine (5‐HT‐positive)‐expressing urethral cells and whether 5‐HT stimulation of urethral sensory fibers improves detrusor function. Cystometries were performed in young (6 months) and aged (18–24 months) female Wistar rats. Aged rats with voiding contractions (VC) that were 2SD below the mean of those in young rats were considered to have DU. Bladder voiding efficiency (BVE) was calculated during saline or 5‐HT solution cystometries. Rats were perfusion‐fixed with a fixative solution (paraformaldehyde, PFA 4%) through the circulatory system and the urethra sectioned to count the number of 5‐HT‐immunoreactive (IR) cells. Isovolumetric cystometry was performed while irrigating the urethra with saline or 5μM‐HT solution. Two‐tailed unpaired t tests were used to determine the significance of differences. In aged DU rats, the mean (±SD) VC frequency was 0.24 ± 0.07 per minute, with an amplitude of 15 ± 3 cm H₂O. The mean (±SD) number of 5‐HT‐IR cells in the urethra of aged DU and young rats was 90 ± 11 and 182 ± 25, respectively (P < 0.01). 5‐HT improved the mean (±SD) BVE of aged DU rats from 49 ± 3% to 78 ± 2% (P < 0.001). In isovolumetric cystometries, detrusor pressure during irrigation of the urethra with saline was 18 ± 1 cm H₂O, compared with 39 ± 2 cm H₂O during irrigation with 5‐HT solution (P < 0.05). In rats, DU associated with aging is accompanied by a decrease in the number of 5‐HT‐positive cells. The results suggest that decreased 5‐HT availability decreases urethral sensory fiber excitation, leading to a decrease the number of effective VC.     

Concentration of plasma calcium in response to human calcitonin in female rats during aging.

The effects of aging and estradiol on the concentration of plasma calcium in response to human calcitonin (hCT) were investigated in rats. Old (20 months) and young (2 months) female rats were catheterized via the right jugular vein and the left femoral vein before an infusion of 1 nM hCT (0.1 ml/min) into the left femoral vein. Young ovariectomized (Ovx) rats were injected subcutaneously with estradiol benzoate (EB, 25 micrograms/kg) or oil once daily for 3 days before hCT challenge. Blood samples were collected from the jugular catheter before and after the hCT infusion. The basal level of plasma calcium was not affected by age. The infusion of 1 nM hCT decreased the plasma calcium concentration by 18% in old and by 29% in young female rats. The post-infusion levels of plasma calcium were comparatively higher in old rats than in young rats (p less than .05 at 30 min and p less than .02 at 90 min). In young rats, the responses of plasma calcium to hCT infusion were reduced after ovariectomy. Short-term estrogen injection, however, did not restore the sensitivity of young Ovx rats to the CT infusion. These findings suggest that the plasma calcium level in response to calcitonin is decreased in rats during aging. The reduction of the hypocalcemic effect of calcitonin in old female rats at least was estradiol-independent.     

A pyridoindole antioxidant SMe1EC2 regulates contractility,relaxation ability,cation channel activity,and protein-carbonyl modifications in the aorta of young and old rats with or without diabetes mellitus

We studied the effects of treatment with SMe1EC, a hexahydropyridoindole antioxidant, on vascular reactivity, endothelial function, and oxidonitrosative stress level of thoracic aorta in young and old rats with or without diabetes mellitus. The rats were grouped as young control (YC 3 months old), old control (OC 15 months old), young diabetic (YD), old diabetic (OD), young control treated (YCT), old control treated (OCT), young diabetic treated (YDT), and old diabetic treated (ODT). Diabetes was induced by streptozotocin injection and subsequently SMe1EC2 (10 mg/kg/day, p.o.) was administered to YCT, OCT, YDT, and ODT rats for 5 months. In young and old rats, diabetes resulted in hypertension, weight loss, hyperglycemia, and hypertriglyceridemia, which were partially prevented by SMe1EC2. SMe1EC2 also inhibited the diabetes-induced increase in aorta levels of AGEs (advanced glycosylation end-protein adducts), 4-HNE (4-hydroxy-nonenal-histidine), 3-NT (3-nitrotyrosine), and RAGEs (receptors for AGEs). The contractions of the aorta rings to phenylephrine (Phe) and KCL did not significantly change, but acetylcholine (ACh) and salbutamol relaxations were reduced in OC compared to YC rats. Diabetes induction increased Phe contractions in YC and OC rats, KCL contractions in YC rats, and did not cause further inhibition in already inhibited ACh and salbutamol relaxations in OC rats. We have achieved the lowest levels of ACh relaxation in YD rats compared to other groups. SMe1EC2 did not change the response of aorta to ACh, salbutamol and Phe in YC rats, and ameliorated ACh relaxations in OC and YD but not in OD rats. In YDT and ODT rats, increased Phe and KCL contractions, high blood pressure, and impaired salbutamol relaxations were amended by SMe1EC2. Phe contractions observed in YD and OD rats as well as KCl contractions observed in OC rats were the lowest levels when the rats were treated with SMe1EC2. When the bath solution was shifted to cyclopiazonic acid (CYP) or CYP plus Ca²⁺-free medium, the contraction induced by a single dose of Phe (3?×?10^?6 M) was more inhibited in YD and OD than in YC but not in OC rats. In SMe1EC2-treated rats, neither the presence of CFM nor CFM plus CYP exhibited a significant change in response of aorta to a single dose of Phe. These findings suggest that α1-adrenergic receptor signaling is activated in both age groups of diabetic rats, diabetes activates K⁺-depolarization and calcium mobilization via Ca_V especially in the aorta of young rats, and sensitizes the aorta of old rats to the regulating effect of SMe1EC2. ACh relaxations were inhibited in YC rats, increased in OC rats and unchanged in YD and OD rats when aortic rings pretreated with TEA, an inhibitor of calcium-activated K⁺ channels (K_Ca), or 4-aminopyridine (4-AP), an inhibitor of voltage-sensitive K⁺ channels (K_V). ACh relaxations were inhibited in YCT, OCT, and YDT rats in the presence of 4-AP or TEA. In ODT rats, 4-AP did not change ACh relaxation but TEA inhibited. These findings suggest that the contribution of K_v and K_Ca to ACh relaxation is likely upregulated by SMe1EC2 when the relaxations were inhibited by aging or diabetes. We conclude that SMe1EC2 might be a promising agent for aging and diabetes related vascular disorders.     

Aging differentially modifies agonist-evoked mouse detrusor contraction and calcium signals

Although aging-induced changes in urinary bladder neurotransmission have been studied in some detail, information regarding alterations in detrusor muscle is scanty and addresses only partial aspects of the myogenic response of detrusor. Rodent bladder aging shows several features similar to those reported in humans. The aim of this study was to characterize in aged mouse the alterations of detrusor muscle contraction and the putative underlying changes in Ca²⁺ signals. We studied in vitro the myogenic contraction induced by agonists in detrusor strips from adult (3 months old) or aged (23–25 months old) mice. In addition, we determined the agonist-induced [Ca²⁺]_i signals by epifluorescence microscopy in fura-2 loaded isolated detrusor cells. Aging impaired the contractile response of bladder strips to cholinergic stimulation with bethanechol and to chemical depolarization with KCl-containing solutions. On the contrary, the response to purinergic stimulation (ATP) was enhanced. Aging also diminished the transient Ca²⁺ signal evoked by bethanechol and the Ca²⁺ influx induced by KCl in bladder strips. Treatments aimed to release calcium from intracellular stores (caffeine and a low level of ionomycin in Ca²⁺-free medium) showed that aging reduces the size of agonist-releasable stores. Similar to contraction, the mobilization of Ca²⁺ by ATP was increased in aged cells. Therefore, the differential effects of aging on detrusor contraction are associated to alterations of [Ca²⁺]_i signals: the cholinergic inhibition is due to inhibition of voltage-operated Ca²⁺ influx and reduction of the size of intracellular Ca²⁺ stores, while the age-induced ATP response is accompanied by an enhanced Ca²⁺ mobilization.     

Aging impairs Ca2+ sensitization pathways in gallbladder smooth muscle

Calcium sensitization is an important physiological process in agonist-induced contraction of smooth muscle. In brief, calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca²⁺]_i by mean of inhibition of myosin light chain phosphatase. Aging has negative impacts on gallbladder contractile response due to partial impairment in calcium signaling and alterations in the contractile machinery. However, information regarding aging-induced alterations in calcium sensitization is scanty. We hypothesized that the calcium sensitization system is negatively affected by age. To investigate this, gallbladders were collected from adult (4 months old) and aged (22–24 months old) guinea pigs. To evaluate the contribution of calcium sensitization pathways we assayed the effect of the specific inhibitors Y-27632 and GF109203X on the “in vitro” isometric gallbladder contractions induced by agonist challenges. In addition, expression and phosphorylation (as activation index) of proteins participating in the calcium sensitization pathways were quantified by Western blotting. Aging reduced bethanechol- and cholecystokinin-evoked contractions, an effect associated with a reduction in MLC20 phosphorylation and in the effects of both Y-27632 and GF109203X. In addition, there was a drop in ROCK I, ROCK II, MYPT-1 and PKC expression and in the activation/phosphorylation of MYPT-1, PKC and CPI-17 in response to agonists. Interestingly, melatonin treatment for 4 weeks restored gallbladder contractile responses due to re-establishment of calcium sensitization pathways. These results demonstrate that age-related gallbladder hypocontractility is associated to alterations of calcium sensitization pathways and that melatonin treatment exerts beneficial effects in the recovery of gallbladder contractility.     

Genetic diversity contributes to abnormalities in pain behaviors between young and old rats

Aging has profound yet unpredictable effects on pain perception and incidence of anxiety disorders. However, the mechanisms underlying age-related pathologies are confounded by contradictory observations in rodent models. Therefore, the goal of our study was to test the hypothesis that genetic variability contributes to age-related pain behaviors and susceptibility to anxiety. To address this hypothesis, we examined pain and anxiety-like behavior in young or old Brown Norway (BN), Fisher 344, and BN/F344 (F1), three rat strains used in studies to evaluate the effect of aging. Mechanosensitive thresholds were assessed using the Von Frey assay, and visceral pain sensitivity was measured via the visceromotor response to colorectal distension. Anxiety-like behavior and exploration was quantified in the elevated plus maze. In the BN strain, old rats exhibited increased mechanosensitive thresholds compared to young rats; however, age did not affect visceral sensitivity in this strain. In F344-BN rats, the number of abdominal contractions induced by the highest colonic distension pressure was significantly lower in old rats. However, following colonic sensitization, a difference was no longer apparent. In the F-344 strain, visceral hypersensitivity following afferent sensitization was evident in young rats at all distension pressures but was not observed in older animals at 20 mmHg. Aging significantly reduced maze exploration across all strains. Our data demonstrate that age- and strain-related alterations exist in pain behavior and highlight the effects of aging on exploratory behavior. These findings suggest that strain differences contribute to the controversial data on the effects of aging on pain perception.     

Aging increases neuronal nitric oxide release and superoxide anion generation in mesenteric arteries from spontaneously hypertensive rats

We hypothesized that neuronal NO release as well as its bioavailability and vasomotor response could be affected when aging and hypertension are present simultaneously. The neuronal nitric oxide (NO) release, its metabolism and vasomotor response induced by electrical field stimulation was analyzed in isolated segments of endothelium-denuded mesenteric arteries from young and old spontaneously hypertensive rats (SHR). The nitric oxide synthase (NOS) inhibitor N(G)-nitro-arginine-methyl ester (L-NAME) and NOS inhibitor 7-nitroindazole both strengthened electrical field stimulation-elicited contractions more in arteries from young than aged SHR rats. Superoxide dismutase (SOD) potentiated the L-NAME effect in segments from old rats, whereas catalase decreased the contractions induced by electrical field stimulation and noradrenaline but did not modify the L-NAME effect. In noradrenaline-precontracted segments, sodium nitroprusside induced a similar relaxation in arteries from both experimental groups. This relaxation was increased by SOD in old SHR. 8Br cGMP induced greater relaxation in segments from old than from young SHR. Electrical field stimulation induced a tritium release in arteries preincubated with [(3)H]-noradrenaline, that was similar in both young and old SHR rats. Electrical field stimulation induced NO(2)(-) formation, which was greater in segments from old than young SHR rats. Basal cGMP levels and those stimulated with sodium nitroprusside were similar in segments from both groups. Superoxide anion production was greater from old than young SHR rats. Peroxynitrite production induced by electrical field stimulation was only detected in segments from old SHR. The results obtained in mesenteric arteries from old SHR showed increased neuronal NO release and superoxide anion production with respect to those observed in arteries from young SHR rats. This induced decreased NO bioavailability through peroxynitrite formation. The final effect is to decrease the involvement of neuronal NO in electrical field stimulation-induced vasomotor response in arteries from old SHR rats.     

Uropathogenic Escherichia coli alters muscle contractions in rat urinary bladder via a nitric oxide synthase-related signaling pathway

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a common cause of urinary-tract infection. The mechanisms by which bacteria cause the symptoms of cystitis remain unclear. METHODS: The contractions of isolated rat detrusor strips evoked by electrical field stimulations (EFS) or by exogenous agonists and immunoblotting for the detection of protein expression in the bladder were measured in the short (1 h) and long (24 h) term after the intravesical instillation of J96 (O4:K6) strain or UPEC isolated from patients with acute E. coli pyelonephritis or E. coli lipopolysaccharide (LPS). RESULTS: One hour after the instillation of UPEC, the level of endothelial nitric oxide synthase (eNOS) and the contractile response, but not protein kinase C (PKC) and extracellular signal-regulated kinase (ERK)-1/2 activation, were higher. Twenty-four hours after UPEC treatment, detrusor contractions were decreased, and inducible (i) NOS protein expression and ERK1/2 phosphorylation, but not PKC activation, were observed. Both aminoguanidine and PD98059 treatment markedly reversed the decrease of EFS- and acetylcholine-evoked detrusor contractions induced by UPEC. The instillation of LPS triggered PKC activation but not ERK1/2 phosphorylation. CONCLUSIONS: Short-term intravesical instillation of UPEC enhances detrusor contractions through an eNOS-related pathway, but iNOS-regulated ERK1/2 signaling may be involved in long-term UPEC treatment-induced responses. There are different mechanisms involved in the responses induced by UPEC and LPS.     

Impaired autophagic function in rat islets with aging

Type 2 diabetes is characterized by a deficit in β-cell function and mass, and its incidence increases with age. Autophagy is a highly regulated intracellular process for degrading cytoplasmic components, particularly protein aggregates and damaged organelles. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. Autophagy may be necessary to maintain structure, mass, and function of pancreatic β-cells. In this study, we investigated the effects of age on β-cell function and autophagy in pancreatic islets of 4-month-old (young), 14-month-old (adult), and 24-month-old (old) male Wistar rats. We found that islet β-cell function decreased gradually with age. Protein expression of the autophagy markers LC3/Atg8 and Atg7 exhibited a marked decline in aged islets. The expression of Lamp-2, a good indicator of autophagic degradation rate, was significantly reduced in the islets of old rats, suggesting that autophagic degradation is decreased in the islets of aged rats. However, protein expression of beclin-1/Atg6, which plays an important role in the induction and formation of the pre-autophagosome structure by associating with a multimeric complex of autophagy regulatory proteins (Atg14, Vps34/class 3 PI3 kinase, and Vps15), was most prominent in the islets of adult rats, and was higher in 24-month-old islets than in 4-month-old islets. The levels of p62/SQSTM1 and polyubiquitin aggregates, representing the functions of autophagy and proteasomal degradation, were increased in aging islets. 8-Hydroxydeoxyguanosine, a marker of mitochondrial and nuclear DNA oxidative damage, exhibited strong immunostaining in old islets. Analysis by electron microscopy demonstrated swelling and disintegration of cristae in the mitochondria of aged islets. These results suggest that β-cell and autophagic function in islets decline simultaneously with increasing age in Wistar rats, and that impaired autophagy in the islets of older rats may cause accumulation of misfolded and aggregated proteins and reduce the removal of abnormal mitochondria in β-cells, leading to reduced β-cell function. Dysfunctional autophagy in islets during the aging process may be an important mechanism leading to the development of type 2 diabetes.     

Passive transfer of Sjogren's syndrome IgG produces the pathophysiology of overactive bladder

OBJECTIVE: The presence, in patients with primary and secondary Sjogren's syndrome (SS), of autoantibodies that acutely inhibit M(3) muscarinic receptor (M3R)-mediated bladder contractions is difficult to reconcile with the fact that symptoms of detrusor overactivity and other features of cholinergic hyperresponsiveness occur in this disease. This study was undertaken to examine the in vivo effects of these autoantibodies on bladder function by examining bladder responsiveness and compliance following passive transfer of patient IgG to mice. METHODS: Contractile responses of isolated bladder strips both to the muscarinic agonist carbachol and to electrically evoked acetylcholine release were measured 48 hours after injection of mice with patient or control IgG. A whole bladder assay with intact neuronal pathways was developed to assess bladder wall compliance on filling cystometry. Expression of M3R in bladders from IgG-injected mice was assessed by immunohistochemistry. RESULTS: Passive transfer of SS IgG with inhibitory anti-M3R activity produced a paradoxical increase in contractile responses of detrusor strips to cholinergic stimulation. Cystometry of whole bladders revealed a corresponding decrease in bladder wall compliance and phasic detrusor contractions upon filling, replicating the urodynamic features of an overactive bladder. The features of cholinergic hyperresponsiveness were associated with increased postsynaptic M3R expression and were reproduced by injecting mice with a rabbit antibody against the second extracellular loop of M3R. CONCLUSION: These findings are consistent with the notion that there is initial inhibition of parasympathetic neurotransmission by antagonistic autoantibodies to M3R, which produces a compensatory increase in M3R expression in vivo. The enhanced cholinergic responses during bladder distention result in detrusor overactivity. We conclude that the overactive bladder associated with SS is an autoantibody-mediated disorder of the autonomic nervous system, which may be part of a wider spectrum of cholinergic hyperresponsiveness.     

Aged spontaneously hypertensive rats exhibit a selective loss of EDHF-mediated relaxation in the renal artery

Endothelium-dependent relaxation is frequently attenuated in hypertension. We hypothesized that the contribution of the endothelium-derived hyperpolarizing factor (EDHF) to the acetylcholine (ACh)-induced, endothelium-dependent relaxation is attenuated with aging in the renal artery of spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) rats. ACh-induced, NO-mediated relaxation was identical in young (8-week-old) WKY and SHR, whereas EDHF-mediated relaxations (assessed in the presence of Nomega-nitro-l-arginine and diclofenac) were much more pronounced in SHR than WKY. KCl-induced relaxations were more pronounced in vessels from young WKY rats than from young SHR. The cytochrome P450 inhibitor sulfaphenazole significantly inhibited EDHF-mediated relaxation in vessels from young SHR but not WKY. Vessels from old (22 months) SHR exhibited a slightly reduced NO-mediated relaxation but a complete loss of EDHF-mediated responses. In contrast, aging did not affect EDHF-mediated responses in WKY. Moreover, ACh-induced hyperpolarization and resting membrane potential were decreased in old SHR but not in WKY. KCl-induced relaxation increased with age in WKY, whereas no response to KCl was recorded in arteries from aged SHR. In vessels from old WKY but not old SHR, mRNA expression of the Na-K-ATPase subunit alpha2 was increased by 2-fold compared with young animals. These data indicate that the increase in EDHF responses in renal arteries from aged WKY can be attributed to the release of K+ ions from the endothelium, whereas increased EDHF responses in renal arteries from young SHR can be attributed to a sulfaphenazole-sensitive cytochrome P450-dependent EDHF.     

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.     

Carotid body function in aged rats: responses to hypoxia,ischemia, dopamine,and adenosine

The carotid body (CB) is the main arterial chemoreceptor with a low threshold to hypoxia. CB activity is augmented by A₂-adenosine receptors stimulation and attenuated by D₂-dopamine receptors. The effect of aging on ventilatory responses mediated by the CB to hypoxia, ischemia, and to adenosine and dopamine administration is almost unknown. This study aims to investigate the ventilatory response to ischemia and to adenosine, dopamine, and their antagonists in old rats, as well as the effect of hypoxia on adenosine 3′,5′-cyclic monophosphate (cAMP) accumulation in the aged CB. In vivo experiments were performed on young and aged rats anesthetized with pentobarbitone and breathing spontaneously. CB ischemia was induced by bilateral common carotid occlusions. cAMP content was measured in CB incubated with different oxygen concentrations. Hyperoxia caused a decrease in cAMP in the CB at all ages, but no differences were found between normoxia and hypoxia or between young and old animals. The endogenous dopaminergic inhibitory tonus is slightly reduced. However, both the ventilation decrease caused by exogenous dopamine and the increase mediated by A_2A-adenosine receptors are not impaired in aged animals. The bradycardia induced by adenosine is attenuated in old rats. The CB’s peripheral control of ventilation is preserved during aging. Concerns have also arisen regarding the clinical usage of adenosine to revert supraventricular tachycardia and the use of dopamine in critical care situations involving elderly people.     

Functional changes of the coronary microvasculature with aging regarding glucose tolerance,energy metabolism,and oxidative stress

This study was aimed at characterizing the functional progression of the endothelial (ECs) and smooth muscle cells (SMCs) of the coronary microvasculature between youth and old age, as well as at determining the mechanisms of the observed changes on the basis of the glucose tolerance, mitochondrial energy metabolism, and oxidative stress. Male rats were divided into four age groups (3, 6, 11, and 17 months for the young (Y), young adult (YA), middle-aged (MA), and old (O) animals). The cardiac mechanical function, endothelial-dependent dilatation (EDD) and endothelial-independent dilatation (EID) of the coronary microvasculature were determined in a Langendorff preparation. The mitochondrial respiration and H₂O₂ production were evaluated and completed by ex vivo measurements of oxidative stress. EDD progressively decreased from youth to old age. The relaxation properties of the SMCs, although high in the Y rats, decreased drastically between youth and young adulthood and stabilized thereafter, paralleling the reduction of mitochondrial oxidative phosphorylation. The ECs dilatation activity, low at youth, was stimulated in YA animals and returned to their initial level at middle age. That parameter followed faithfully the progression of the amount of active cardiac endothelial nitric oxide synthase and whole body glucose intolerance. In conclusion, the progressive decrease in EDD occurring with aging is due to different functional behaviors of the ECs and SMCs, which appear to be associated with the systemic glucose intolerance and cardiac energy metabolism.     

Effects of aging on respiration, ATP levels and calcium transport in rat liver mitochondria. Response to theophylline

The concept that aging results in an impairment of mitochondrial biochemistry has been tested on organelles isolated from the liver of 3-4-month-old and 24-month-old rats of the Wistar strain. Our data suggest that aging results in significant decreases in succinate-supported respiration, ATP levels and calcium uptake. When theophylline was added to the incubation mixture, both respiration and calcium uptake were depressed in approximately the same proportion in the mitochondria from old rats, although the mitochondrial ATP of young animals was significantly decreased by this substance.     

Melatonin restores impaired contractility in aged guinea pig urinary bladder

Abstract:  Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin-treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca²⁺]_i) was estimated by epifluorescence microscopy of fura-2-loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca²⁺]_i in response to the stimuli, suggesting a reduced Ca²⁺sensitivity. Indeed, the agonist-induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca²⁺ sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and with the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca²⁺ handling and Ca²⁺ sensitizations pathways. Moreover, the indoleamine restored age-induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging-related urinary bladder contractile impairment.     

Estrogen replacement reduces age-associated remodeling in rat mesenteric arteries

Estrogen replacement therapy significantly decreases the incidence of cardiovascular disease in postmenopausal women. In aging, there is an increase in vascular stiffness along with a decrease in matrix metalloproteinase (MMP) activity. Our hypothesis was that estrogen replacement would increase MMPs and therefore reduce the vascular stiffness that is associated with aging. Female Sprague-Dawley rats were implanted with a placebo or 17ss-estradiol-containing pellet (0.5 mg/pellet, 60-day release) at 10 months of age (n=6, each). Six young rats (3 months old) were also studied. After a 2-month exposure to the pellet, mesenteric arteries were studied on a pressurized arteriograph system. Distensibility and wall thickness were measured in response to stepwise increases in intraluminal pressure in Ca(2+)-free physiological saline solution buffer with papaverine (10(-4) mol/L). In response to increasing pressure, aged placebo rats exhibited a significant decrease in distensibility compared with young rats (P<0.05) that was accompanied by an increase in wall thickness (P<0.05). Conversely, estrogen replacement increased distensibility and decreased wall thickness in aged rats (old estrogen-replaced versus old placebo, P<0.05). Zymography data indicated that MMP-2 activity decreased in aging but was increased by estrogen replacement. In summary, estrogen replacement in aging female rats reduces age-associated vascular remodeling.     

Oxidative stress and mitochondrial function in skeletal muscle: Effects of aging and exercise training

The rate of oxidative phosphorylation was investigated in isolated mitochondria from hindlimb muscles of young (4.5 mo) and old (26.5 mo) male Fischer 344 rats with or without endurance training. Further, the susceptibility of the muscle mitochondria to exogenous reactive oxygen species was examined. State 3 and 4 respiration, as well as the respiratory control index (RCI), were significantly lower in muscle mitochondria from aged vs. young rats (P<0.05), using either the site 1 substrates malate-pyruvate (M-P) and 2-oxoglutarate (2-OG), or the site 2 substrate succinate. In both young and old rats, training increased state 4 respiration with M-P, but had no effect on state 3 respiration, resulting in a reduction of RCI. Training also increased state 4 respiration with 2-OG and decreased RCI in young rats. When muscle mitochondria were exposed to superoxide radicals (O₂ ^·−) and hydrogen peroxide (H₂O₂) generated by xanthine oxidase and hypoxanthine, or H₂O₂ alone in vitro, state 3 respiration and RCI in both age groups were severely hampered, but those from the old rats were inhibited to a less extent than the young rats. In contrast, state 4 respiration was impaired by O₂ ^·− and/or H₂O₂ to a greater extent in the old rats. Muscle mitochondria from trained young rats showed a greater resistance to the O₂ ^{· −} and/or H₂O₂-induced state 3 and RCI inhibition than those from untrained young rats. Muscle from aged rats had significantly higher total activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and glutathione reductase than that from young rats, however, training increased SOD and GPX activities in young but not old rats. The results of this study suggest that mitochondrial capacity for oxidative phosphorylation is compromised in aging skeletal muscle. Further, the increased mitochondrial resistance to reactive oxygen species demonstrated in aged and young trained muscles may be attributed to enhanced antioxidant enzyme activities.     

Urethral Sensations are Related to the Development of Detrusor Overactivity

Urgency is the core symptom of the overactive bladder symptom complex, but the underlying mechanisms are not fully understood. Clinical findings have led to the assumption that bladder outlet obstruction (BOO) caused by benign prostatic enlargement (BPE) induces storage symptoms and detrusor overactivity. Presumably, BOO by BPE accounts for urgency; however, urgency is not always caused by BOO. Sensory nerves in the wall of the urethra fire in response to urethral fluid flow, and this activity initiates bladder contractions in the quiescent bladder and augments ongoing contractions in the active bladder. In humans, prostatic urethral anesthesia results in significant increases in bladder capacity among BPH patients without neurological diseases, therefore sensory stimuli from an anatomically altered prostatic urethra has the possibility to induce urgency and detrusor overactivity. Studies in animals demonstrate the basis for an excitatory urethra to bladder reflex. Urethral stimulation by prostaglandin E₂ induces an excitatory effect on micturition reflex by activation of C‐fiber afferent nerves. α_1A‐adrenoceptor blocker has an inhibitory effect on the micturition reflex, suggesting excitatory urethra to bladder reflex is mediated by α_1A‐adrenoceptor. Even if there is no obstruction, increase in urethral sensory due to BPE may induce the development of the detrusor overactivity.     

Expression and function of vascular endothelial growth inhibitor in aged porcine bladder detrusor muscle cells

Aging of the bladder detrusor muscle plays an important role in lower urinary tract symptoms in elderly people. Our previous work demonstrated that elderly patients have increased levels of vascular endothelial growth inhibitor (VEGI) in bladder tissue. Therefore, we hypothesized that VEGI may play a role in aging of the bladder detrusor muscle cells. This study aims to develop and characterize primary cultures of aged porcine bladder detrusor muscle cells in order to explore the expression and function of VEGI. Bladder samples from female pigs were divided into two groups: the aged group (Model) and the young group (Control). We confirmed β-galactosidase expression, a marker for senescence, in aged muscle cells (identified by α-smooth muscle actin (α-SMA) staining), but not in the young group. mRNA levels of VEGI-251 and death receptor 3 (DR3) were up-regulated (P < 0.05) and total cell protein levels of VEGI-251, DR3 and nuclear factor-kappa B [NF-κB (p65)], membrane protein levels of DR3, and nuclear protein levels of NF-κB (p65) were significantly higher (P < 0.01) in the Model cells compared to Control cells. In conclusion, we have established a method to culture aged detrusor muscle cells derived from porcine bladder. Higher levels of VEGI-251, DR3 and NF-κB (p65) were observed in the aged cells. VEGI-251 may function by increasing DR3 on cellular membranes and promoting the transfer of NF-κB into the nucleus. This suggests that VEGI may be a target for reversing the aging process of bladder detrusor muscle cells.