Identification of Tissue miRNA Signatures for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), a neoplasm of the gastrointestinal tract, is the most common pancreatic malignancy (90%) and the fourth highest cause of cancer mortality worldwide. Surgery intervention is currently the only strategy able to offer an advantage in terms of overall survival, but prognosis remains poor even for operated patients. Therefore, the development of robust biomarkers for early diagnosis and prognostic stratification in clinical practice is urgently needed. In this work, we investigated deregulated microRNAs (miRNAs) in tissues from PDAC patients with high (G3) or low (G2) histological grade and with (N+) or without (N-) lymph node metastases. miRNA expression profiling was performed by a comprehensive PCR array and subsequent validation by RT-qPCR. The results showed a significant increase in miR-1-3p, miR-31-5p, and miR-205-5p expression in G3 compared to G2 patients (** p < 0.01; *** p < 0.001; *** p < 0.001). miR-518d-3p upregulation and miR-215-5p downregulation were observed in N+ compared to N- patients. A statistical analysis performed using OncomiR program showed the significant involvement (p < 0.05) of two miRNAs (miR-31 and miR-205) in the histological grade of PDAC patients. Also, an expression analysis in PDAC patients showed that miR-31 and miR-205 had the highest expression at grade 3 compared with normal and other tumor grades. Overall, survival plots confirmed that the overexpression of miR-31 and miR-205 was significantly correlated with decreased survival in TCGA PDAC clinical samples. A KEGG pathway analysis showed that all three miRNAs are involved in the regulation of multiple pathways, including the Hippo signaling, adherens junction and microRNAs in cancer, along with several target genes. Based on in silico analysis and experimental validation, our study suggests the potential role of miR-1-3p, miR-31-5p, and miR-205-5p as useful clinical biomarkers and putative therapeutic targets in PDAC, which should be further investigated to determine the specific molecular processes affected by their aberrant expression.

Prospective Evaluation of Radiotherapy-Induced Immunologic and Genetic Effects in Colorectal Cancer Oligo-Metastatic Patients with Lung-Limited Disease: The PRELUDE-1 Study.

BACKGROUND: in recent years, the management of advanced colorectal cancer (CRC) has been greatly improved with integrated strategies including stereotactic radiation therapy (SRT). The administration of SRT has been demonstrated, particularly in oligo-metastatic (om) CRC, to be a safe and effective option. Interestingly, it has been demonstrated that SRT can induce regression of tumors in non-irradiated regions ("abscopal effect") through stimulation of anti-tumor immune effects ("radiation-induced immunity"). We have recently shown that lung-limited omCRC is characterized by regression of tumor clones bearing specific key driver gene mutations. AIMS: to assess the genetic evolution on tumor cancer cells induced by SRT in lung-limited omCRC. Secondary objectives included descriptions of the abscopal effect, responses' duration, toxicity, and progression-free survival. A translational research will be performed to evaluate tumor genetic evolution (through liquid biopsies and Next Generation Sequencing), HLA class I repertoire, peripheral immune cells, and cytokine dynamics. METHODS: PRELUDE-1 is a prospective translational study. SRT will be administered only to the largest nodule (with a maximum diameter ≤ 25 mm) in omCRC with two or three radiologically evident lesions. The sample size is based on the innovative hypothesis that radiation-induced immunity could induce regression of tumor clones bearing KRAS oncogene mutations. According to the binomial test, considering the frequency of KRAS mutations and assuming a probability of mutant KRAS→wild type KRAS of p0 = 0.0077, with α = 0.05 and 1-ß = 0.60, the final sample size is 25 patients.

Polydatin Induces Differentiation and Radiation Sensitivity in Human Osteosarcoma Cells and Parallel Secretion through Lipid Metabolite Secretion.

Osteosarcoma is a bone cancer characterized by the production of osteoid tissue and immature bone from mesenchymal cells. Osteosarcoma mainly affects long bones (femur is most frequently site) and occur in children and young adults with greater incidence. Here, we investigated the role accomplished by polydatin, a natural antioxidative compound, in promoting osteogenic differentiation alone or after radiation therapy on osteosarcoma cells. In vitro, polydatin significantly induced cell cycle arrest in S-phase and enhanced bone alkaline phosphatase activity. Moreover, the differentiation process was paralleled by the activation of Wnt-ß-catenin pathway. In combination with radiotherapy, the pretreatment with polydatin promoted a radiosensitizing effect on osteosarcoma cancer cells as demonstrated by the upregulation of osteogenic markers and reduced clonogenic survival of tumor cells. Additionally, we analyzed, by mass spectrometry, the secretion of sphingolipid, ceramides, and their metabolites in osteosarcoma cells treated with polydatin. Overall, our results demonstrate that polydatin, through the secretion of sphingolipids and ceramide, induced osteogenic differentiation, alone and in the presence of ionizing therapy. Future investigations are needed to validate the use of polydatin in clinical practice as a potentiating agent of radiotherapy-induced anticancer effects.

Pomolic acid reduces contractility and modulates excitation-contraction coupling in rat cardiomyocytes.

Pomolic acid (PA) isolated from Licania pittieri has hypotensive effects in rats, inhibits human platelet aggregation and elicits endothelium-dependent relaxation in rat aortic rings. The present study was designed to investigate the effects of PA on cardiomyocytes. Trabeculae and enzymatically isolated cardiomyocytes from rats were used to evaluate the concentration-dependent effects of PA on cardiac muscle tension and excitation-contraction coupling (ECC) by recording Ca2+ transients reported with Fluo-3 and Fura-2, as well as L-type Ca2+ currents (LTCC). PA reduced the contractile force in rat cardiac trabeculae with an EC50 = 14.3 ±â€¯2.4 µM. PA also reduced the amplitude of Ca2+ transients in a concentration-dependent manner, with an EC50 = 10.5 ±â€¯1.3 µM, without reducing sarcoplasmic reticulum (SR) Ca2+ loading. PA decreased the half width of the Ca2+ transient by 31.7 ±â€¯3.3% and increased the decay time and decay time constant (τ) by 7.6 ±â€¯2.7% and 75.6 ±â€¯3.7%, respectively, which was associated with increased phospholamban (PLN) phosphorylation. PA also reversibly reduced the macroscopic LTCC in the cardiomyocyte membrane, but did not demonstrate any effects on skeletal muscle ECC. In conclusion, PA reduces LTCC, Ca2+ transients and cardiomyocyte force, which along with its vasorelaxant effects explain its hypotensive properties. Increased PLN phosphorylation protected the SR from Ca2+ depletion. Considering the effects of PA on platelet aggregation and the cardiovascular system, we propose it as a new potential, multitarget cardiovascular agent with a demonstrated safety profile.

Cholesterol removal from adult skeletal muscle impairs excitation-contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins.

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction (E-C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-ß-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca(2+) transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na(+)/K(+)-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47(phox) and gp91(phox)), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E-C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.

Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation-contraction coupling supramolecular complex.

The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.

Tetanic Ca2+ transient differences between slow- and fast-twitch mouse skeletal muscle fibres: a comprehensive experimental approach.

One hundred and eighty six enzymatically dissociated murine muscle fibres were loaded with Mag-Fluo-4 AM, and adhered to laminin, to evaluate the effect of modulating cytosolic Ca(2+) buffers and sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA), mitochondria, and Na(+)/Ca(2+) exchanger (NCX) on the differential tetanic Ca(2+) transient kinetics found in different fibre types. Tetanic Ca(2+) transients were classified as morphology type I (MT-I) or type II (MT-II) according to their shape. The first peak of the MT-I (n = 25) and MT-II (n = 23) tetanic Ca(2+) transients had an amplitude (∆F/F) of 0.41 ± 0.03 and 0.83 ± 0.05 and a rise time (ms) of 1.35 and 0.98, respectively. MT-I signals had a time constant of decay (τ1, ms) of 75.9 ± 4.2 while MT-II transients showed a double exponential decay with time constants of decay (τ1 and τ2, ms) of 18.3 ± 1.4 and 742.2 ± 130.3. Sarcoendoplasmic reticulum Ca(2+) ATPase inhibition demonstrated that the decay phase of the tetanic transients mostly rely on SERCA function. Adding Ca(2+) chelators in the AM form to MT-I fibres changed the morphology of the initial five peaks to a MT-II one, modifying the decay phase of the signal in a dose-dependent manner. Mitochondria and NCX function have a minor role in explaining differences in tetanic Ca(2+) transients among fibre types but still help in removing Ca(2+) from the cytosol in both MT-I and MT-II fibres. Cytoplasmic Ca(2+) buffering capacity and SERCA function explain most of the different kinetics found in tetanic Ca(2+) transients from different fibre types, but mitochondria and NCX have a measurable role in shaping tetanic Ca(2+) responses in both slow and fast-twitch muscle fibre types. We provided experimental evidence on the mechanisms that help understand the kinetics of tetanic Ca(2+) transients themselves and explain kinetic differences found among fibre types.

The excitation-contraction coupling mechanism in skeletal muscle.

First coined by Alexander Sandow in 1952, the term excitation-contraction coupling (ECC) describes the rapid communication between electrical events occurring in the plasma membrane of skeletal muscle fibres and Ca2+ release from the SR, which leads to contraction. The sequence of events in twitch skeletal muscle involves: (1) initiation and propagation of an action potential along the plasma membrane, (2) spread of the potential throughout the transverse tubule system (T-tubule system), (3) dihydropyridine receptors (DHPR)-mediated detection of changes in membrane potential, (4) allosteric interaction between DHPR and sarcoplasmic reticulum (SR) ryanodine receptors (RyR), (5) release of Ca2+ from the SR and transient increase of Ca2+ concentration in the myoplasm, (6) activation of the myoplasmic Ca2+ buffering system and the contractile apparatus, followed by (7) Ca2+ disappearance from the myoplasm mediated mainly by its reuptake by the SR through the SR Ca2+ adenosine triphosphatase (SERCA), and under several conditions movement to the mitochondria and extrusion by the Na+/Ca2+ exchanger (NCX). In this text, we review the basics of ECC in skeletal muscle and the techniques used to study it. Moreover, we highlight some recent advances and point out gaps in knowledge on particular issues related to ECC such as (1) DHPR-RyR molecular interaction, (2) differences regarding fibre types, (3) its alteration during muscle fatigue, (4) the role of mitochondria and store-operated Ca2+ entry in the general ECC sequence, (5) contractile potentiators, and (6) Ca2+ sparks.

Quantifying SOCE fluorescence measurements in mammalian muscle fibres. The effects of ryanodine and osmotic shocks.

We have quantified Ca(2+) entry through store operated calcium channels in mice muscle fibres, measuring the rates of change of myoplasmic [Ca(2+)], d[Ca(2+)](myo)/dt, and of Ca(2+) removal, d[Ca(2+)](Removal)/dt, turning store operated calcium entry (SOCE) ON, and OFF, by switching on or off external Ca(2+). In depleted fibres, poisoned with 10 µM cyclopiazonic acid SOCE influx was about 3 µM/s. Ryanodine (50 µM) caused a robust, nifedipine (50 µM) independent, increase in SOCE activation to 8.6 µM/s. Decreasing medium osmolarity from 300 to 220 mOsm/L, decreased SOCE to 0.9 µM/s, while increasing osmolarity from 220 to 400 mOsm/L potentiated SOCE to 43.6 µM/s. Ryanodine inhibited the effects of hypotonicity. Experiments using 2-aminoethoxydiphenyl borate, nifedipine, or Mn(2+) quenching, strongly suggest that the increased [Ca(2+)](myo) by ryanodine or hypertonic shock is mediated by potentiated SOCE activation. The Ca(2+) response decay, quantified by d[Ca(2+)](Removal)/dt, indicates a robust residual Ca(2+) removal mechanism in sarco-endoplasmic reticulum calcium ATPase poisoned fibres. SOCE high sensitivity to osmotic shocks, or to ryanodine receptor (RyR) binding, suggests its high dependency on the structural relationship between its molecular constituents, Orai1 and stromal interaction molecule and the sarcoplasmic reticulum and plasma membranes, in the triadic junctional region, where RyRs, are conspicuously present. This study demonstrates that SOCE machinery is highly sensitive to structural changes caused by binding of an agonist to its receptor or by imposed osmotical volume changes.

Sodium-calcium exchanger modulates the L-glutamate Ca(i) (2+) signalling in type-1 cerebellar astrocytes.

We have previously demonstrated that rat type-1 cerebellar astrocytes express a very active Na(+)/Ca(2+) exchanger which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of Ca (i) (2+) induced by physiological agonist. In this chapter, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signalling in rat cerebellar astrocytes. Laser-scanning confocal microscopy experiments using immunofluorescence labelling of Na(+)/Ca(2+) exchanger and RyRs demonstrated that they are highly co-localized. The most important finding presented in this chapter is that L-glutamate activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing a Na(+) entry through the electrogenic Na(+)-glutamate co-transporter and not through the ionophoric L-glutamate receptors as confirmed by pharmacological experiments with specific blockers of ionophoric L-glutamate receptors, electrogenic glutamate transporters and the Na/Ca exchange.

Kinetic changes in tetanic Ca²âº transients in enzymatically dissociated muscle fibres under repetitive stimulation.

We used enzymatically dissociated flexor digitorum brevis (FDB) and soleus fibres loaded with the fast Ca(2+) dye Magfluo-4 AM, and adhered to Laminin, to test whether repetitive stimulation induces progressive changes in the kinetics of Ca(2+) release and reuptake in a fibre-type-dependent fashion. We applied a protocol of tetani of 350 ms, 100 Hz, every 4 s to reach a mean amplitude reduction of 25% of the first peak. Morphology type I (MT-I) and morphology type II (MT-II) fibres underwent a total of 96 and 52.8 tetani (P < 0.01 between groups), respectively. The MT-II fibres (n = 18) showed significant reductions of the amplitude (19%), an increase in rise time (8.5%) and a further reduction of the amplitude/rise time ratio (25.5%) of the first peak of the tetanic transient after 40 tetani, while MT-I fibres (n = 5) did not show any of these changes. However, both fibre types showed significant reductions in the maximum rate of rise of the first peak after 40 tetani. Two subpopulations among the MT-II fibres could be distinguished according to Ca(2+) reuptake changes. Fast-fatigable MT-II fibres (fMT-II) showed an increase of 32.2% in the half-width value of the first peak, while for fatigue-resistant MT-II fibres (rMT-II), the increase amounted to 6.9%, both after 40 tetani. Significant and non-significant increases of 36.4% and 11.9% in the first time constant of decay (t(1)) values were seen after 40 tetani in fMT-II and rMT-II fibres, respectively. MT-I fibres did not show kinetic changes in any of the Ca(2+) reuptake variables. All changes were reversed after an average recovery of 7.5 and 15.4 min for MT-I and MT-II fibres, respectively. Further experiments ruled out the possibility that the differences in the kinetic changes of the first peak of the Ca(2+) transients between fibres MT-I and MT-II could be related to the inactivation of Ca(2+) release mechanism. In conclusion, we established a model of enzymatically dissociated fibres, loaded with Magfluo-4 and adhered to Laminin, to study muscle fatigue and demonstrated fibre-type-dependent, fatigue-induced kinetic changes in both Ca(2+) release and reuptake.

Myosin heavy chain isoform composition and Ca(2+) transients in fibres from enzymatically dissociated murine soleus and extensor digitorum longus muscles.

Electrically elicited Ca(2+) transients reported with the fast Ca(2+) dye MagFluo-4 AM and myosin heavy chain (MHC) electrophoretic patterns were obtained in intact, enzymatically dissociated fibres from adult mice extensor digitorum longus (EDL) and soleus muscles. Thirty nine fibres (23 from soleus and 16 from EDL) were analysed by both fluorescence microscopy and electrophoresis. These fibres were grouped as follows: group 1 included 13 type I and 4 type IC fibres; group 2 included 2 type IIC, 3 IIA and 1 I/IIA/IIX fibres; group 3 included 4 type IIX and 1 type IIX/IIB fibres; group 4 included 2 type IIB/IIX and 9 type IIB fibres. Ca(2+) transients obtained in groups 1, 2, 3 and 4 had the following kinetic parameters (mean +/- s.e.m.): amplitude (F/F): 0.61 +/- 0.05, 0.53 +/- 0.08, 0.61 +/- 0.06 and 0.61 +/- 0.03; rise time (ms): 1.64 +/- 0.05, 1.35 +/- 0.05, 1.18 +/- 0.06 and 1.14 +/- 0.04; half-amplitude width (ms): 19.12 +/- 1.85, 11.86 +/- 3.03, 4.62 +/- 0.31 and 4.23 +/- 0.37; and time constants of decay (tau(1) and tau(2), ms): 3.33 +/- 0.13 and 52.48 +/- 3.93, 2.69 +/- 0.22 and 41.06 +/- 9.13, 1.74 +/- 0.06 and 12.88 +/- 1.93, and 1.56 +/- 0.11 and 9.45 +/- 1.03, respectively. The statistical differences between the four groups and the analysis of the distribution of the parameters of Ca(2+) release and clearance show that there is a continuum from slow to fast, that parallels the MHC continuum from pure type I to pure IIB. However, type IIA fibres behave more like IIX and IIB fibres regarding Ca(2+) release but closer to type I fibres regarding Ca(2+) clearance. In conclusion, we show for the first time the diversity of Ca(2+) transients for the whole continuum of fibre types and correlate this functional diversity with the structural and biochemical diversity of the skeletal muscle fibres.

Different fibre populations distinguished by their calcium transient characteristics in enzymatically dissociated murine flexor digitorum brevis and soleus muscles.

Enzymatically dissociated flexor digitorum brevis (FDB) and soleus fibres from mouse were used to compare the kinetics of electrically elicited Ca2+ transients of slow and fast skeletal muscle fibres, using the fast Ca2+ dye MagFluo4-AM, at 20-22 degrees C. For FDB two Ca2+ transient morphologies, types I (MT-I, 11 fibres, 19%) and II (MT-II, 47 fibres, 81%), were found, the kinetic parameters (amplitude, rise time, half width, decay time, and time constants of decay) being statistically different. For soleus (n = 20) only MT-I was found, with characteristics similar to MT-I from FDB. Correlations with histochemically determined mATPase, reduced nicotinamide adenine dinucleotide diaphorase and alpha-glycerophosphate dehydrogenase activities, as well as immunostaining and myosin heavy chain electrophoretic analysis of both muscles suggest that signals classified as MT-I may correspond to slow type I and fast IIA fibres while those classified as MT-II may correspond to fast IIX/D fibres. The results point to the importance of Ca2+ signaling for characterization of muscle fibres, but also to its possible role in determining fibre function.

Factors affecting SOCE activation in mammalian skeletal muscle fibers.

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca(2+) entry, SOCE. Sarcoplasmic reticulum (SR) Ca(2+) stores were depleted by repetitive exposures to high K(+) or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca(2+). SR Ca(2+) store replenishment was substantially reduced using 5 microM cyclopiazonic acid (CPA). Readmission of external Ca(2+) (5 mM) increased basal [Ca(2+)](i) under two modalities. In mode 1 [Ca(2+)](i) initially increased at a rate of 0.8 +/- 0.1 nM/s and later at a rate of 12.3 +/- 2.6 nM/s, reaching a final value of 477.8 +/- 36.8 nM in 215.7 +/- 25.9 s. In mode 2, [Ca(2+)](i) increased at a rate of 0.8 +/- 0.1 nM/s to a value of 204.9 +/- 20.6 nM in 185.4 +/- 21.1 s. FCCP, 2 microM, reduced this Ca(2+) entry. In nine FCCP-poisoned fibers, the initial rate of Ca(2+) increase was 0.34 +/- 0.1 nM/s (mean +/- SEM), reaching a plateau of 149.2 +/- 14.1 nM in 217 +/- 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca(2+), we show that during depletion [Ca(2+)](mito) initially increases and later diminishes. Finally, we show that the increase in basal [Ca(2+)](i), associated with SOCE activation, diminishes upon external Na(+) withdrawal. Na(+) entry through the SOCE pathway and activation of the reversal of Na(+)/Ca(2+) exchanger could explain this SOCE modulation by Na(+).

The activity of the Na+/Ca2+ exchanger largely modulates the Ca2+i signal induced by hypo-osmotic stress in rat cerebellar astrocytes. The effect of osmolarity on exchange activity.

We recently demonstrated that rat cerebellar Type-1 astrocytes express a very active Na(+)/Ca(2+) exchanger highly colocalized with ryanodine receptors (RyRs), which in turn play a key role in glutamate-induced Ca(2+) signaling through a calcium-induced calcium release (CICR) mechanism. In this work we have explored whether the Na(+)/Ca(2+) exchanger has any role in the Ca(2+)(i) signal induced by hypo-osmotic stress in these cells, using microspectrofluorometric measurements with Fura-2, pharmacological tools, and confocal microscopy image analysis. We present evidence for the first time that the increase in [Ca(2+)](i) in rat cerebellar Type-1 astrocytes, resulting from moderate hypotonic shock, is mediated by Ca(2+) release from ryanodine-operated Ca(2+)(i) stores, and that the magnitude of the intracellular Ca(2+) signal induced by hypotonicity in the short term (up to 240 s) is small and controlled by the activity of the Na(+)/Ca(2+) exchanger operating in its extrusion mode. With longer times in the hypotonic medium, intracellular Ca(2+) store depletion leads to Ca(2+) entry through store-operated Ca(2+) channels. We found it interesting that the activity of the Na(+)/Ca(2+) exchanger measured during this reverse mode operation (Ca(2+) entry in exchange for internal Na(+)) was found to be greatly increased in hypotonic solutions and decreased in hypertonic ones. The buffering of the [Ca(2+)](i) rise induced by hypo-osmotic stress may prevent excessive increases in [Ca(2+)](i), which otherwise might impair the normal function of this glial cell.

Effect of mitochondria poisoning by FCCP on Ca2+ signaling in mouse skeletal muscle fibers.

We have studied the effects of mitochondria poisoning by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) on Ca(2+) signaling in enzymatically dissociated mouse flexor digitorum brevis (FDB) muscle fibers. We used Fura-2AM to measure resting [Ca(2+)](i) and MagFluo-4AM to measure Ca(2+) transients. Exposure to FCCP (2 microM, 2 min) caused a continuous increase in [Ca(2+)](i) at a rate of 0.60 nM/s and a drastic reduction of electrically elicited Ca(2+) transients without much effect on their decay phase. Half of the maximal effect occurred at [Ca(2+)](i) = 220 nM. This effect was partially reversible after long recuperation and was not diminished by Tiron, a reactive oxygen species (ROS) scavenger. FCCP had no effects on fiber excitability as shown by the generation of action potentials. 4CmC, an agonist of ryanodine receptors, induced a massive Ca(2+) release. FCCP diminished the rate but not the amount of Ca(2+) released, indicating that depletion of Ca(2+) stores did not cause the decrease in Ca(2+) transient amplitude. Ca(2+) transient amplitude could also be diminished, but to a lesser degree, by increases in [Ca(2+)](i) induced by repetitive stimulation of fibers treated with ciclopiazonic acid. This suggests an important role for Ca(2+) in the FCCP effect on transient amplitude.

The use of CalciumOrange-5N as a specific marker of mitochondrial Ca2+ in mouse skeletal muscle fibers.

We report the use of the fluorescent dye CalciumOrange-5N (CaOr-5N) as a specific mitochondria Ca(2+) marker in enzymatically dissociated mouse FBD muscle fibers. Using laser scanning confocal microscopy and the dyes Mitotracker Green (MTG), di-8-ANEPPS and endoplasmic reticulum tracker green (ERTG), we determined the relative position of mitochondria, transverse tubules and sarcoplasmic reticulum in the sarcomere. Comparison with electron micrographies showed that mitochondria are mostly present at both sides of Z lines and near the triads located at the A-I band border. CaOr-5N fluorescence was mainly distributed in mitochondria, highly co-localised with MTG and basically excluded from the A band space. ERTG localised mostly between the two t-tubules present in each sarcomere. We studied the effect of the protonophore FCCP using CaOr-5N to measure mitochondrial Ca(2+) and JC-1 dye to measure mitochondria inner membrane potential (DeltaPsi(m)). After FCCP treatment, the CaOr-5N fluorescence diminished by about 33% in 80 s, while JC-1 fluorescence diminished by 36% in 200 s. Our results show the loss of Ca(2+) from mitochondria when DeltaPsi(m) is depolarised and demonstrate the usefulness of CaOr-5N to mark mitochondrial [Ca(2+)](m).

Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Ca(i)2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes.

We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing Na(+) entry through the electrogenic Na(+)-Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.

Calcium transients in developing mouse skeletal muscle fibres.

Ca(2)(+) transients elicited by action potentials were measured using MagFluo-4, at 20-22 degrees C, in intact muscle fibres enzymatically dissociated from mice of different ages (7, 10, 15 and 42 days). The rise time of the transient (time from 10 to 90% of the peak) was 2.4 and 1.1 ms in fibres of 7- and 42-day-old mice, respectively. The decay of the transient was described by a double exponential function, with time constants of 1.8 and 16.4 ms in adult, and of 4.6 and 105 ms in 7-day-old animals. The fractional recovery of the transient peak amplitude after 10 ms, F(2(10))/F(1), determined using twin pulses, was 0.53 for adult fibres and ranged between 0.03 and 0.60 in fibres of 7-day-old animals This large variance may indicate differences in the extent of inactivation of Ca(2)(+) release, possibly related to the difference in ryanodine receptor composition between young and old fibres. At the 7 and 10 day stages, fibres responded to Ca(2)(+)-free solutions with a larger decrease in the transient peak amplitude (25% versus 11% in adult fibres), possibly indicating a contribution of Ca(2)(+) influx to the Ca(2)(+) transient in younger animals. Cyclopiazonic acid (1 mum), an inhibitor of the sarcoplasmic reticulum (SR) Ca(2)(+)-ATPase, abolished the Ca(2)(+) transient decay in fibres of 7- and 10-day-old animals and significantly reduced its rate in older animals. Analysis of the transients with a Ca(2)(+) removal model showed that the results are consistent with a larger relative contribution of the SR Ca(2)(+) pump and a lower expression of myoplasmic Ca(2)(+) buffers in fibres of young versus old animals.

Inactivation of Ca2+ transients in amphibian and mammalian muscle fibres.

MagFluo-4 fluorescence (Ca2+) transients associated with action potentials were measured in intact muscle fibres, manually dissected from toads ( Leptodactylus insularis ) or enzymatically dissociated from mice. In toads, the decay phase of the Ca2+ transients is described by a single exponential with a time constant ( tau ) of about 7 ms. In mice, a double exponential function with tau 's of 1.5 and 15.5 ms, respectively gives a better fit. In both species the amplitude of Ca2+ transients diminished during repetitive stimulation: in amphibian muscle fibres, the decrease was about 20% with 1 Hz stimulation and 55% at 10 Hz. In mammalian fibres, repetitive stimulation causes a less conspicuous decrease of the transient amplitude: 10% at 1 Hz and 15% at 10 Hz. During tetanic stimulation at 100 Hz the transient amplitude decays to 20% in toad fibres and 40% in mouse fibres. This decrease could be associated with the phenomenon of inactivation of Ca2+ release, described by other authors. Recovery from inactivation, studied by a double stimuli protocol also indicates that in toad fibres the ability to release Ca2+ is abolished to a greater extent than in mouse fibres. In fact the ratio between the amplitudes of the second and first transient, when they are separated by a 10 ms interval, is 0.29 for toad and 0.58 for mouse fibres. In toad fibres, recovery from inactivation, to about 80 % of the initial value, occurs with a tau of 32 ms at 22 degrees C; while in mouse fibres recovery from inactivation is almost complete and occurs with a tau of 36 ms under the same conditions. The results indicate that Ca2+ release in enzymatically dissociated mammalian muscle fibres inactivates to a smaller extent than in intact amphibian muscle fibres.