Cristina Camello-Almaraz

Ca2+ accumulation into acidic organelles mediated by Ca2+- and vacuolar H+-ATPases in human platelets

Most physiological agonists increase cytosolic free [Ca2+]c (cytosolic free Ca2+ concentration) to regulate a variety of cellular processes. How different stimuli evoke distinct spatiotemporal Ca2+ responses remains unclear, and the presence of separate intracellular Ca2+ stores might be of great functional relevance. Ca2+ accumulation into intracellular compartments mainly depends on the activity of Ca2+- and H+-ATPases. Platelets present two separate Ca2+ stores differentiated by the distinct sensitivity to thapsigargin and TBHQ [2,5-di-(t-butyl)-1,4-hydroquinone]. Although one store has long been identified as the dense tubular system, the nature of the TBHQ-sensitive store remains uncertain. Treatment of platelets with GPN (glycylphenylalanine-2-naphthylamide) impaired Ca2+ release by TBHQ and reduced that evoked by thrombin. In contrast, GPN did not modify Ca2+ mobilization stimulated by ADP or AVP ([arginine]vasopressin). Treatment with nigericin, a proton carrier, and bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, to dissipate the proton gradient into acidic organelles induces a transient increase in [Ca2+]c that was abolished by previous treatment with the SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) 3 inhibitor TBHQ. Depleted acidic stores after nigericin or bafilomycin A1 were refilled by SERCA 3. Thrombin, but not ADP or AVP, reduces the rise in [Ca2+]c evoked by nigericin and bafilomycin A1. Our results indicate that the TBHQ-sensitive store in human platelets is an acidic organelle whose Ca2+ accumulation is regulated by both Ca2+- and vacuolar H+-ATPases.

Role of mitochondria in Ca2+ oscillations and shape of Ca2+ signals in pancreatic acinar cells

Abstract We studied the role of mitochondria in Ca2+ signals in fura-2 loaded exocrine pancreatic acinar cells. Mitochondrial depolarization in response to carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone or rotenone (assessed by confocal microscopy using rhodamine-123) induced a partial but statistically significant reduction in the decay of Ca2+ signals under different experimental conditions. Spreading of Ca2+ waves evoked by the pancreatic secretagogue cholecystokinin cholecystokinin octapeptide was accelerated by mitochondrial inhibitors, whereas the cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to physiological levels of this hormone were suppressed by rotenone and carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone. Oligomycin, an inhibitor of mitochondrial ATP synthase, did no affect either propagation of calcium waves nor [Ca2+]i oscillations. Individual mitochondria of rhod-2 loaded acinar cells showed heterogeneous matrix Ca2+ concentration increases in response to oscillatory and maximal levels of cholecystokinin octapeptide. On the other hand, using Ba2+ for unequivocal study of capacitative calcium entry we found that mitochondrial inhibitors did not affect this process. Our results show that although the role of mitochondria as a Ca2+ clearing system in exocrine cells is quantitatively secondary, they play an essential role in the spatial propagation of Ca2+ waves and in the development of [Ca2+]i oscillations.

Age-related alterations in Ca2+ signals and mitochondrial membrane potential in exocrine cells are prevented by melatonin

Abstract:  Information regarding age‐induced Ca2+ signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca2+ signals and mitochondrial potential, a parameter involved in both Ca2+ signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca2+ homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22–24 months old) mice by collagenase dispersion. Ca2+ signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca2+ oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca2+ homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca2+ responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca2+ signals and mitochondrial potential of aged exocrine cells.

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 Treatment Reverts Age-Related Changes in Guinea Pig Gallbladder Neuromuscular Transmission and Contractility

The incidence of gallbladder illness increases with age, but the altered mechanisms leading to gallbladder dysfunction are poorly understood. Here we determine the age-related alterations in gallbladder contractility and the impact of melatonin treatment. Isometric tension changes in response to electrical field stimulation and to agonists were recorded from guinea pig gallbladder muscle strips. [Ca2+]i was determined by epifluorescence microscopy in fura-2 loaded isolated gallbladder smooth muscle cells, and F-actin content was quantified by confocal microscopy. Aging reduced neurogenic contractions, which was associated with the impairment of nitrergic innervation and with increased responsiveness of capsaicin-sensitive relaxant nerves, possibly involving calcitonin gene-related peptide. Melatonin treatment for 4 weeks restored neurogenic responses to normal values, with an associated recovery of nitrergic function and the disappearance of the capsaicin-sensitive component. Aging also reduced the contractile responses to cholecystokinin and Ca2+ influx. The impaired contractility only correlated with diminished Ca2+ mobilization in response to activation of Ca2+ influx. Melatonin improved contractility and increased smooth muscle F-actin content without changing Ca2+ homeostasis. In conclusion, aging impairs gallbladder function as the result of changes in the inhibitory neuromodulation of smooth muscle contractility and the reduction in the myogenic response to contractile agonists. Impaired contractility seems to be related to decreased Ca2+ influx and damage of contractile proteins. Melatonin significantly ameliorated these age-related changes.

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.

Pharmacological Approaches to Improve Ageing

Aging is generally considered as a progressive and irreversible set of structural and functional changes, due both to the genetic background of the individual and the oxidative damage and modifications of intracellular signaling mechanisms. Although the anatomical and physiological alterations associated to aging (e.g. sarcopenia, cognitive and sensorial decline, functional loss in cardiovascular system...) are not a disease, they reduce the functional reserve of the organism, ultimately leading to pathological alterations and death.

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.