Orai1α, but not Orai1ß, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells.

The identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1ß, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1ß differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1ß are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α-TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

Monochloramine effects on gallbladder contractility.

Digestive inflammatory processes induce motility alterations associated with an increase in reactive oxygen species production, including monochloramine (NH2 Cl). The aim of the study was to characterize the effects of the naturally occurring oxidant monochloramine in the guinea pig gallbladder. We used standard in vitro contractility technique to record guinea pig gallbladder strips contractions. NH2 Cl caused a concentration-dependent contraction which was reduced by inhibition of extracellular Ca2+ influx and tyrosine kinase pathways. The PKC antagonist GF109203X also reduced the response but not after previous tyrosine kinase inhibition, suggesting that PKC is activated by tyrosine kinase activity. The NH2 Cl contractile effect was also reduced by inhibitors of mitogen-activated protein kinase (MAPK), nitric oxide synthase, phospholipase A2 and cyclooxygenase. In addition, NH2 Cl impaired the responses to CCK, tissue depolarization and electrical field stimulation. In conclusion, we present new evidence that monochloramine impairs not only the gallbladder response to CCK but also to membrane depolarization and nervous plexus stimulation, and that tyrosine kinase, PKC, MAPK and NO pathways are involved in the contractile direct effect of monochloramine.

Age-Induced Differential Changes in the Central and Colonic Human Circadian Oscillators.

Aging modifies not only multiple cellular and homeostatic systems, but also biological rhythms. The circadian system is driven by a central hypothalamic oscillator which entrains peripheral oscillators, in both cases underlain by circadian genes. Our aim was to characterize the effect of aging in the circadian expression of clock genes in the human colon. Ambulatory recordings of the circadian rhythms of skin wrist temperature, motor activity and the integrated variable TAP (temperature, activity and position) were dampened by aging, especially beyond 74 years of age. On the contrary, quantitative analysis of genes expression in the muscle layer of colonic explants during 24 h revealed that the circadian expression of Bmal1, Per1 and Clock genes, was larger beyond that age. In vitro experiments showed that aging induced a parallel increase in the myogenic contractility of the circular colonic muscle. This effect was not accompanied by enhancement of Ca2+ signals. In conclusion, we describe here for the first time the presence of a molecular oscillator in the human colon. Aging has a differential effect on the systemic circadian rhythms, that are impaired by aging, and the colonic oscillator, that is strengthened in parallel with the myogenic contractility.

EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction.

BACKGROUND/AIMS: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. METHODS: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. RESULTS: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca2+ store depletion and their subsequent modulation via slow Ca2+-dependent inactivation. CONCLUSION: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction.

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.

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.

NFATc3 regulates trypsinogen activation, neutrophil recruitment, and tissue damage in acute pancreatitis in mice.

BACKGROUND & AIMS: The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice. METHODS: We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes. RESULTS: Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine-induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells. CONCLUSIONS: NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.

Secretion of Interleukin 6 in Human Skeletal Muscle Cultures Depends on Ca2+ Signalling.

The systemic effects of physical activity are mediated by the release of IL-6 and other myokines from contracting muscle. Although the release of IL-6 from muscle has been extensively studied, the information on the cellular mechanisms is fragmentary and scarce, especially regarding the role of Ca2+ signals. The aim of this study was to characterize the role of the main components of Ca2+ signals in human skeletal muscle cells during IL-6 secretion stimulated by the Ca2+ mobilizing agonist ATP. Primary cultures were prepared from surgical samples, fluorescence microscopy was used to evaluate the Ca2+ signals and the stimulated release of IL-6 into the medium was determined using ELISA. Intracellular calcium chelator Bapta, low extracellular calcium and the Ca2+ channels blocker La3+ reduced the ATP-stimulated, but not the basal secretion. Secretion was inhibited by blockers of L-type (nifedipine, verapamil), T-type (NNC55-0396) and Orai1 (Synta66) Ca2+ channels and by silencing Orai1 expression. The same effect was achieved with inhibitors of ryanodine receptors (ryanodine, dantrolene) and IP3 receptors (xestospongin C, 2-APB, caffeine). Inhibitors of calmodulin (calmidazolium) and calcineurin (FK506) also decreased secretion. IL-6 transcription in response to ATP was not affected by Bapta or by the T channel blocker. Our results prove that ATP-stimulated IL-6 secretion is mediated at the post-transcriptional level by Ca2+ signals, including the mobilization of calcium stores, the activation of store-operated Ca2+ entry, and the subsequent activation of voltage-operated Ca2+ channels and calmodulin/calcineurin pathways.

Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1).

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3' untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand-receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation.

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

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. H(2)O(2) 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 H(2)O(2) 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.

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

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 ß-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.

Tempol protects the gallbladder against ischemia/reperfusion.

Impairment in gallbladder emptying, increase in residual volume, and reduced smooth muscle contractility are hallmarks of acute acalculous cholecystitis and seem to be related to ischemia/reperfusion (I/R). This study was designed to determine the effects of tempol, a general antioxidant, on I/R-induced changes in gallbladder contractile capacity, the mechanisms involved in the contractile process, and the level of inflammatory mediators. Experimental gallbladder I/R was induced in male guinea pigs by common bile duct ligation for 2 days, then a deligation of the duct was performed and after 2 days the animals were sacrificed. A group of animals was treated with tempol, administered in the drinking water at 1 mmol/l for 10 days prior the bile duct ligation and until animal sacrifice. Isometric tension recordings showed that KCl and cholecystokinin-induced contractions were impaired by I/R, which correlated with decreased F-actin content and detrimental effects on Ca(2+) influx. In addition, I/R depolarized mitochondrial membrane potential, as indicated by the reduction of the heterogeneity of the rhodamine123 fluorescence signal, and increased the expression of NF-kappaB, COX-2, and iNOS. Tempol treatment improved contractility via normalization of Ca(2+) handling and improvement of F-actin content. Moreover, the antioxidant ameliorated mitochondrial polarity and normalized the expression levels of the inflammatory mediators. These results show that antioxidant treatment protects the gallbladder from I/R, indicating the potential therapeutic benefits of tempol in I/R injury.

Pancreatic calcium signaling: role in health and disease.

In order to control cell functions, extracellular agents, such as hormones or neurotransmitters among others, generate a diversity of calcium (Ca(2+)) signals in target cells. Here, we review the components involved in Ca(2+) handling and effectors, both members of the known calcium signaling pathways. In the pancreas, Ca(2+) signal appears as local increases, global elevations or Ca(2+) oscillations. Ca(2+) plays a key role in the pancreatic cells, regulating secretion in exocrine cells, a widely used model for studying the coupling between Ca(2+) signaling and secretion, and the release of insulin, glucagon and somatostatin in the exocrine pancreas. Interestingly, Ca(2+) deregulations have been related to pancreatitis and aging of the pancreas, and treatment with melatonin has shown beneficial effects suggesting that melatonin could be an adequate therapeutic approach.

Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells.

Orai1 plays a major role in store-operated Ca2+ entry (SOCE) in triple-negative breast cancer (TNBC) cells. This channel is inactivated via different mechanisms, including protein kinase C (PKC) and protein kinase A (PKA)-dependent phosphorylation at Ser-27 and Ser-30 or Ser-34, respectively, which shapes the Ca2+ responses to agonists. The Ca2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca2+- and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Here, we show that the breast cancer cell lines MCF7 and MDA-MB-231 exhibit enhanced expression of Orai1 and AC8 as compared to the non-tumoral breast epithelial MCF10A cell line. In these cells, AC8 interacts with the Orai1α variant in a manner that is not regulated by Orai1 phosphorylation. AC8 knockdown in MDA-MB-231 cells, using two different small interfering RNAs (siRNAs), attenuates thapsigargin (TG)-induced Ca2+ entry and also Ca2+ influx mediated by co-expression of Orai1 and the Orai1-activating small fragment (OASF) of STIM1 (stromal interaction molecule-1). Conversely, AC8 overexpression enhances SOCE, as well as Ca2+ entry, in cells co-expressing Orai1 and OASF. In MDA-MB-231 cells, we found that AC8 overexpression reduces the Orai1 phosphoserine content, thus suggesting that AC8 interferes with Orai1 serine phosphorylation, which takes place at residues located in the AC8-binding site. Consistent with this, the subset of Orai1 associated with AC8 in naïve MDA-MB-231 cells is not phosphorylated in serine residues in contrast to the AC8-independent Orai1 subset. AC8 expression knockdown attenuates migration of MCF7 and MDA-MB-231 cells, while this maneuver has no effect in the MCF10A cell line, which is likely attributed to the low expression of AC8 in these cells. We found that AC8 is required for FAK (focal adhesion kinase) phosphorylation in MDA-MB-231 cells, which might explain its role in cell migration. Finally, we found that AC8 is required for TNBC cell proliferation. These findings indicate that overexpression of AC8 in breast cancer MDA-MB-231 cells impairs the phosphorylation-dependent Orai1 inactivation, a mechanism that might support the enhanced ability of these cells to migrate.

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

Information regarding age-induced Ca(2+) signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca(2+) signals and mitochondrial potential, a parameter involved in both Ca(2+) signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca(2+) 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. Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca(2+) signals and mitochondrial potential of aged exocrine cells.

Protective effect of melatonin on Ca2+ homeostasis and contractility in acute cholecystitis.

Impaired Ca2+ homeostasis and smooth muscle contractility co-exist in acute cholecystitis (AC) leading to gallbladder dysfunction. There is no pharmacological treatment for this pathological condition. Our aim was to evaluate the effects of melatonin treatment on Ca2+ signaling pathways and contractility altered by cholecystitis. [Ca2+]i was determined by epifluorescence microscopy in fura-2 loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. Malondialdehyde (MDA) and reduced glutathione (GSH) contents were determined by spectrophotometry and cycloxygenase-2 (COX-2) expression was quantified by western blot. Melatonin was tested in two experimental groups, one of which underwent common bile duct ligation for 2 days and another that was later de-ligated for 2 days. Inflammation-induced impairment of Ca2+ responses to cholecystokinin and caffeine were recovered by melatonin treatment (30 mg/kg). This treatment also ameliorated the detrimental effects of AC on Ca2+ influx through both L-type and capacitative Ca2+ channels, and it was effective in preserving the pharmacological phenotype of these channels. Despite its effects on Ca2+ homeostasis, melatonin did not improve contractility. After de-ligation, Ca2+ influx and contractility were still impaired, but both were recovered by melatonin. These effects of melatonin were associated to a reduction of MDA levels, an increase in GSH content and a decrease in COX-2 expression. These findings indicate that melatonin restores Ca2+ homeostasis during AC and resolves inflammation. In addition, this indoleamine helps in the subsequent recovery of functionality.

Melatonin restores impaired contractility in aged guinea pig urinary bladder.

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 ([Ca(2+)](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 [Ca(2+)](i) in response to the stimuli, suggesting a reduced Ca(2+)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 Ca(2+) 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 Ca(2+) handling and Ca(2+) 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.

Ca2+ extrusion in aged smooth muscle cells.

We investigated the effects of aging in Ca(2+) extrusion mechanisms in smooth muscle bladder cells from 4 and 20-24-month-old guinea pigs using fluorescence microscopy and fura-2. Cells were challenged with a pulse of KCl immediately before perfusion with a Ca(2+) free solution containing no inhibitors (control, untreated cells) or inhibitors of plasma membrane Ca(2+) pump (PMCA, 1mM La(3+)), Na(+)/Ca(2+) exchanger (NCX, 1 microM SEA0400) or the sarcoendoplasmic Ca(2+) pump (SERCA, 1 microM thapsigargin). Treatment of young adult cells with the inhibitors allowed estimating a relative contribution of 55% for NCX, 27% for PMCA and 31% for SERCA. Combination of two inhibitors at the same time showed the presence of interaction between extrusion mechanisms. In aged cells the [Ca(2+)](i) extrusion was impaired due to decrease of PMCA activity, as revealed by the loss of effect of La(3+), and to inhibitory interactions between NCX and SERCA activities, indicated by acceleration of decay in response to their respective inhibitors. In conclusion, in smooth muscle cells aging decreases the overall Ca(2+) extrusion activity and modifies the interactions between the activities of the main Ca(2+) removing mechanisms.

Disruption of the filamentous actin cytoskeleton is necessary for the activation of capacitative calcium entry in naive smooth muscle cells.

It has been proposed that cytoskeleton plays a key positive role in the activation of capacitative calcium entry (CCE), which supported the secretion-like hypothesis for the mechanisms underlying this process. However, its role on CCE in native smooth muscle is unknown. Here we demonstrate that CCE in isolated gallbladder myocytes was enhanced by cytochalasin D or latrunculin A treatments (agents that cause actin disassembly) whereas it was reduced by jasplakinolide treatment (which causes actin polymerization), suggesting that actin cytoskeleton acts as a barrier in CCE. In addition, we show for the first time that depletion of intracellular Ca2+ stores by thapsigargin and cholecystokinin in BAPTA-loaded cells induced a decrease in F-actin content that was consistent with a link between CCE and actin reorganization. In conclusion, these data suggest an active participation of actin reorganization in the implementation of CCE and support a conformational coupling model for this process in naive smooth muscle cells.

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.