Francisco E. Martín-Cano

Melatonin induces neural SOD2 expression independent of the NF-kappaB pathway and improves the mitochondrial population and function in old mice

Abstract:  Aging is commonly defined as a physiological phenomenon associated with morphological and functional deleterious changes in which oxidative stress has a fundamental impact; therefore, readjusting the oxidative balance should have beneficial effects. In our study, we tested the antioxidant melatonin in old mouse brains and showed positive effects at the cellular and mitochondrial levels. Melatonin attenuated β‐amyloid protein expression and α‐synuclein deposits in the brain compared to aged group. Furthermore, oxidative stress was increased by aging and induced the nuclear translocation of nuclear factor‐kappa B (NF‐κB), which was suppressed by melatonin treatment. The antioxidant mitochondrial expression, superoxide dismutase 2 (SOD2), was increased in both control and melatonin‐treated old mice, despite the different activation states of the NF‐κB pathway. The NF‐κB pathway was activated in the old mice, which may be explained by this group’s response to the increased oxidative insult; this insult was inhibited in melatonin‐treated animals, showing this group an increase in active mitochondria population that was not observed in old group. We also report that melatonin is capable of restoring the mitochondrial potential of age‐damaged neurons. In conclusion, melatonin’s beneficial effects on brain aging are linked to the increase in mitochondrial membrane potential and SOD2 expression, which probably reduces the mitochondrial contribution to the oxidative stress imbalance.

Melatonin, and to a lesser extent growth hormone, restores colonic smooth muscle physiology in old rats

Abstract:  There is increasing evidence that aging is associated with oxidative damage, inflammation, and apoptosis in different cell types. However, there is limited information regarding aging mechanisms in colon smooth muscle. Old male Wistar rats (22 months) were treated for 10 wks with melatonin or growth hormone (GH). Animals were sacrificed at 24 months of age by decapitation. The colon was dissected and the smooth muscle homogenized. H2O2 and malonyl dialdehyde (MDA) content and catalase and glutathione peroxidase (GPX) activities were determined using colorimetric kits. Expression of nuclear factor kappa B (NF‐κB), cyclooxygenase 2 (COX‐2), caspase‐3, and caspase‐9 were determined by Western blot. Aging of colon smooth muscle correlated with an increase in H2O2 and MDA levels when compared with young animals in both proximal and distal segments; these changes were associated with a decrease in the catalase activity in the distal colon. Oxidative stress correlated with an increase in COX‐2 and NF‐κB expression, which were accompanied by an enhanced expression of the pro‐apoptotic enzyme caspase‐3 and its upstream enzyme, caspase‐9. Melatonin treatment normalized the oxidative, inflammatory, and apoptotic patterns, whereas GH replacement, although effective in reducing oxidative stress in distal colon, did not reverse the age‐related inflammation or apoptosis. These results suggest that melatonin should be the treatment of choice to most effectively recover physiological functions in aged colonic smooth muscle.

Age‐related changes in mitochondrial function of mouse colonic smooth muscle: beneficial effects of melatonin

Aging is a multifactorial process that involves biochemical, structural, and functional changes in mitochondria. The ability of melatonin to palliate the alterations induced by aging is based on its chronobiologic, antioxidant, and mitochondrial effects. There is little information about the effects of melatonin on the in situ mitochondrial network of aging cells and its physiological implications. We have studied the ability of melatonin to prevent the functional alterations of in situ mitochondria of smooth muscle cells and its impact on contractility. Mitochondrial membrane potential was recorded in isolated colonic smooth muscle cells from young mice (3 month old), aged mice (22–24‐month old), and aged mice treated with melatonin (starting at 14‐month age). Aging induced a partial mitochondrial depolarization in resting conditions and reduced the depolarizing response to cellular stimulation. Use of oligomycin indicated that aging enhanced the resting activity of the mitochondrial ATP synthase, whereas in young cells, the enzyme operated mainly in reverse mode. Melatonin treatment prevented all these changes. Aging reduced both spontaneous and stimulated contraction of colonic strips and shifted the metabolic dependence of contraction from mitochondria to glycolysis, as indicated the use of mitochondrial and glycolysis inhibitors. These functional alterations were also palliated by melatonin treatment. Aging effects were not related to a decrease in Ca2+ store mobilization, because this was enhanced in aged cells and restored by melatonin. In conclusion, melatonin prevents the age induced in situ mitochondrial potential alterations in smooth muscle cells and the associated changes in contractility and metabolism.

Developmental changes in Ca2+ homeostasis and contractility in gallbladder smooth muscle

Relatively little is known about the contribution of Ca(2+)-dependent and -independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We therefore studied Ca(2+) homeostasis and Ca(2+) sensitization mechanisms in 10-day-old and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, intracellular Ca(2+) concentration was estimated by epifluorescence microscopy of fura-2-loaded isolated cells, and protein expression and phosphorylation were assessed by Western blot analysis. The neonatal gallbladder contracted significantly less to CCK than adult tissue, but this correlated with an increased Ca(2+) mobilization, suggesting immaturity of Ca(2+) sensitization mechanisms. The enhanced Ca(2+) release in the newborn gallbladder was the result of the increase in the size of the releasable Ca(2+) pool. Moreover, in neonatal smooth muscle cells, neither the plasma membrane Ca(2+) pump nor the Na(+)/Ca(2+) exchanger collaborate in the extrusion of Ca(2+). In contrast, in these cells, there is an increase in phospholamban phosphorylation, which could drive to an overactivity of the sarco(endo)plasmic reticulum Ca(2+)-ATPase pump. The reduced Ca(2+) sensitivity in neonatal tissues was demonstrated by the lack of effect to Y-27362, an inhibitor of Rho kinase (ROCK), and GF-109203X, an inhibitor of PKC, on agonist-induced contraction. In addition, the neonatal gallbladder showed lower levels of RhoA, ROCK, PKC, and two effectors [C-kinase-dependent inhibitor of 17 kDa (CPI-17) and myosin phosphatase targetting 1 (MYPT1)] as well as an absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation.

PINK1 deficiency enhances autophagy and mitophagy induction

Parkinsons disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control.

Characterization of the motor inhibitory role of colonic mucosa under chemical stimulation in mice

The main roles of the colonic mucosa are the absorption of water and electrolytes and the barrier function that preserves the integrity of the colonic wall. The mediators and mechanisms to accomplish these functions are under continuous investigation, but little attention has been paid to a possible control of colonic motility by the mucosa that would fine tune the relationship between absorption and motility. The purpose of this study was to establish the role of the mucosa in the control of induced colonic contractility. Young ICR-CD1 mice (3-5 mo old) were studied. Isometric tension transducers were used to record contractility in full-thickness (FT) and mucosa-free (MF) strips from proximal colon. Proximal FT strips showed lower KCl- and bethanechol-induced responses than MF strips. The difference was not due to mechanical artefacts since the contractile response of FT strips to electrical field stimulation was around 50% lower than in MF. The inhibitory effects of the mucosa on FT strips were mimicked by immersion of separate strips of mucosa in the organ bath but not by addition of mucosal extract, suggesting gaseous molecules as mediators of this effect. Incubation of MF strips with synthase inhibitors of nitric oxide, carbon monoxide, and hydrogen sulfide abolished the inhibition caused by addition of the mucosal strip, indicating that mucosal gasotransmitters are the mediators of these effects. This suggests that the control of colonic motility exerted by the mucosa could fine tune the balance between transit and absorption.

Propagation of Intracellular Ca2+ Signals in Aged Exocrine Cells

There is little information on the effects of aging in the propagation of calcium signals and its underlying mechanisms. We studied the effects of aging on propagation of Ca(2+) signals in pancreatic acinar cells. Fura-2 loaded cells isolated from young (3-4 months old) and aged (24 months old) mouse responded to acetylcholine (ACh) and cholecystokinin (CCK) with a polarized Ca(2+) response initiated at the secretory pole before spreading to the basal one. Aging slowed down the propagation of the response to ACh but enhanced the velocity of the CCK response. This pattern can be explained by the age-induced depolarization of mitochondria, because it can be reproduced in young cells by mitochondrial inhibitors. Aging also increased the role of acidic stores in the CCK signal, as judged by the folimycin-induced suppression of the polarization in aged but not in young cells. The involvement of ryanodine receptors in the ACh response was also enhanced, as indicated by the loss of polarization after the treatment with 8Br-cyclic ADP ribose. Therefore, we conclude that aging modifies differentially the propagation of ACh and CCK-evoked Ca(2+) signals through mitochondrial depolarization and changes in the role of the acidic Ca(2+) stores and ryanodine receptors in the initiation of the signals.