Arrhythmic Effects Evaluated on Caenorhabditis elegans: The Case of Polypyrrole Nanoparticles.

Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes' pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs' biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs.

SERCA inhibition improves lifespan and healthspan in a chemical model of Parkinson disease in Caenorhabditis elegans.

Introduction: The high prevalence of neurodegenerative diseases in our population and the lack of effective treatments encourage the search for new therapeutic targets for these pathologies. We have recently described that submaximal inhibition of the Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA), the main responsible for ER calcium storage, is able to increase lifespan in Caenorhabditis elegans worms by mechanisms involving mitochondrial metabolism and nutrient-sensitive pathways. Methods: We have studied here the effects of submaximal SERCA inhibition in a chemical model of Parkinson's disease (PD) induced in C. elegans worms by treatment with the mitochondrial complex I inhibitor rotenone. For specific SERCA inhibition, we treated worms with RNAi against sca-1, the sole orthologue of SERCA in C. elegans. Results and Discussion: Our results show that rotenone produces alterations in worms that include decreased lifespan, smaller size, reduced fertility, decreased motility, defecation and pumping rate, increased mitochondrial ROS production, reduced mitochondrial membrane potential and oxygen consumption rate, altered mitochondrial structure, and altered ethanol preference in behavioral studies. Most of these alterations were either fully or partially reversed in worms treated with sca-1 RNAi, suggesting that SERCA inhibition could be a novel pharmacological target in the prevention or treatment of neurodegeneration.

Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins.

The molecular mechanisms that mediate and regulate calcium (Ca2+) fluxes through the membranes of intracellular organelles play a key role in the generation and shaping of the local and global cytosolic Ca2+ signals triggering the process of regulated exocytosis in chromaffin cells. Beyond that role, intraorganellar Ca2+ homeostasis also regulates organelle-specific processes such as oxidative phosphorylation in mitochondria, maturation of secretory granules, or stress in the endoplasmic reticulum. In this chapter, we describe current methods to study mitochondrial, endoplasmic reticulum, and secretory vesicle calcium homeostasis in living chromaffin cells using engineered targeted aequorins.

Modeling Alzheimer's Disease in Caenorhabditis elegans.

Alzheimer's disease (AD) is the most frequent cause of dementia. After decades of research, we know the importance of the accumulation of protein aggregates such as ß-amyloid peptide and phosphorylated tau. We also know that mutations in certain proteins generate early-onset Alzheimer's disease (EOAD), and many other genes modulate the disease in its sporadic form. However, the precise molecular mechanisms underlying AD pathology are still unclear. Because of ethical limitations, we need to use animal models to investigate these processes. The nematode Caenorhabditis elegans has received considerable attention in the last 25 years, since the first AD models overexpressing Aß peptide were described. We review here the main results obtained using this model to study AD. We include works studying the basic molecular mechanisms of the disease, as well as those searching for new therapeutic targets. Although this model also has important limitations, the ability of this nematode to generate knock-out or overexpression models of any gene, single or combined, and to carry out toxicity, recovery or survival studies in short timeframes with many individuals and at low cost is difficult to overcome. We can predict that its use as a model for various diseases will certainly continue to increase.

The Mitochondrial Na+/Ca2+ Exchanger Inhibitor CGP37157 Preserves Muscle Structure and Function to Increase Lifespan and Healthspan in Caenorhabditis elegans.

We have reported recently that the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 extends lifespan in Caenorhabditis elegans by a mechanism involving mitochondria, the TOR pathway and the insulin/IGF1 pathway. Here we show that CGP37157 significantly improved the evolution with age of the sarcomeric regular structure, delaying development of sarcopenia in C. elegans body wall muscle and increasing the average and maximum speed of the worms. Similarly, CGP37157 favored the maintenance of a regular mitochondrial structure during aging. We have also investigated further the mechanism of the effect of CGP37157 by studying its effect in mutants of aak-1;aak-2/AMP-activated kinase, sir-2.1/sirtuin, rsks-1/S6 kinase and daf-16/FOXO. We found that this compound was still effective increasing lifespan in all these mutants, indicating that these pathways are not involved in the effect. We have then monitored pharynx cytosolic and mitochondrial Ca2+ signalling and our results suggest that CGP37157 is probably inhibiting not only the mitochondrial Na+/Ca2+ exchanger, but also Ca2+ entry through the plasma membrane. Finally, a transcriptomic study detected that CGP37157 induced changes in lipid metabolism enzymes and a four-fold increase in the expression of ncx-6, one of the C. elegans mitochondrial Na+/Ca2+ exchangers. In summary, CGP37157 increases both lifespan and healthspan by a mechanism involving changes in cytosolic and mitochondrial Ca2+ homeostasis. Thus, Ca2+ signalling could be a promising target to act on aging.

Mechanism of the lifespan extension induced by submaximal SERCA inhibition in C. elegans.

We have reported recently that submaximal inhibition of the Sarco Endoplasmic Reticulum Ca2+ ATPase (SERCA) produces an increase in the lifespan of C. elegans worms. We have explored here the mechanism of this increased survival by studying the effect of SERCA inhibition in several mutants of signaling pathways related to longevity. Our data show that the mechanism of the effect is unrelated with the insulin signaling pathway or the sirtuin activity, because SERCA inhibitors increased lifespan similarly in mutants of these pathways. However, the effect required functional mitochondria and both the AMP kinase and TOR pathways, as the SERCA inhibitors were ineffective in the corresponding mutants. The same effects were obtained after reducing SERCA expression with submaximal RNAi treatment. The SERCA inhibitors did not induce ER-stress at the concentrations used, and their effect was not modified by inactivation of the OP50 bacterial food. Altogether, our data suggest that the effect may be due to a reduced ER-mitochondria Ca2+ transfer acting via AMPK activation and mTOR inhibition to promote survival.

Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms.

Mitochondrial [Ca2+] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca2+ overload. This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.

The Role of Ca2+ Signaling in Aging and Neurodegeneration: Insights from Caenorhabditis elegans Models.

Ca2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the "calcium hypothesis" of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (ß-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration.

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release from the endoplasmic reticulum by AMP-activated kinase modulators.

The 5' AMP-activated protein kinase (AMPK) is a nutrient-sensitive kinase that plays a key role in the control of cellular energy metabolism. We have explored here the relationship between AMPK and Ca2+ signaling by looking at the effect of an AMPK activator (A769662) and an AMPK inhibitor (dorsomorphin) on histamine-induced Ca2+-release from the endoplasmic reticulum (ER) in HeLa cells. Our data show that incubation with A769662 (EC50 = 29 µM) inhibited histamine-induced Ca2+-release from the ER in intact cells, as well as inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release in permeabilized cells. On the contrary, dorsomorphin (EC50 = 0.4 µM) activated both histamine and IP3-induced Ca2+-release and reversed the effect of A769662. These results suggest a direct effect of AMPK regulation on IP3 receptor (IP3R) function. A phosphoproteomic study did not reveal changes in IP3R phosphorylation, but showed significant changes in phosphorylation of proteins placed upstream in the IP3R interactome and in several proteins related with Ca2+ metabolism, which could be candidates to mediate the effects observed. In conclusion, our data suggest that AMPK negatively regulates IP3R. This effect constitutes a novel and very important link between Ca2+ signaling and the AMPK pathway.

Inhibition of Sarco-Endoplasmic Reticulum Ca2+ ATPase Extends the Lifespan in C. elegans Worms.

The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range and is therefore the main controller of the ER [Ca2+] level ([Ca2+]ER), which has a key role in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient-sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone (2,5-BHQ) as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone (2,6-BHQ) as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging.

The Neuroprotector Benzothiazepine CGP37157 Extends Lifespan in C. elegans Worms.

The benzothiazepine CGP37157 has shown neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. Although its mechanism of neuroprotection is unclear, it is probably related with some of its effects on Ca2+ homeostasis. CGP37157 is a well-known inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX). However, it is not very specific and also blocks several other Ca2+ channels and transporters, including voltage-gated Ca2+ channels, plasma membrane Na+/Ca2+ exchanger and the Ca2+ homeostasis modulator 1 channel (CALHM1). In the present work, we have studied if CGP37157 could also induce changes in life expectancy. We now report that CGP37157 extends C. elegans lifespan by 10%-15% with a bell-shaped concentration-response, with high concentrations producing no effect. The effect was even larger (25% increase in life expectancy) in worms fed with heat-inactivated bacteria. The worm CGP37157 concentration producing maximum effect was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and was close to the IC50 for inhibition of the Na+/Ca2+ exchanger. CGP37157 also extended the lifespan in eat-2 mutants (a model for caloric restriction), suggesting that caloric restriction is not involved in the mechanism of lifespan extension. Actually, CGP37157 produced no effect in mutants of the TOR pathway (daf15/unc24) or the insulin/insulin-like growth factor-1 (IGF-1) pathway (daf-2), indicating that the effect involves these pathways. Moreover, CGP37157 was also ineffective in nuo-6 mutants, which have a defect in the mitochondrial respiratory chain complex I. Since it has been described that neuroprotection by this compound in cell cultures is abolished by mitochondrial inhibitors, this suggests that life extension in C. elegans and neuroprotection in cell cultures may share a similar mechanism involving mitochondria.

Pharynx mitochondrial [Ca2+] dynamics in live C. elegans worms during aging.

Progressive decline in mitochondrial function is generally considered one of the hallmarks of aging. We have expressed a Ca2+ sensor in the mitochondrial matrix of C. elegans pharynx cells and we have measured for the first time mitochondrial [Ca2+] ([Ca2+]M) dynamics in the pharynx of live C. elegans worms during aging. Our results show that worms stimulated with serotonin display a pharynx [Ca2+]M oscillatory kinetics that includes both high frequency oscillations (up to about 1Hz) and very prolonged "square-wave" [Ca2+]M increases, indicative of energy depletion of the pharynx cells. Mitochondrial [Ca2+] is therefore able to follow "beat-to-beat" the fast oscillations of cytosolic [Ca2+]. The fast [Ca2+]M oscillations kept steady frequency values during the whole worm life, from 2 to 12 days old, but the height and width of the peaks was progressively reduced. [Ca2+]M oscillations were also present with similar kinetics in respiratory chain complex I nuo-6 mutant worms, although with smaller height and frequency than in the controls, and larger width. In summary, Ca2+ fluxes in and out of the mitochondria are relatively well preserved during the C. elegans life, but there is a clear progressive decrease in their magnitude during aging. Moreover, mitochondrial Ca2+ fluxes were smaller in nuo-6 mutants with respect to the controls at every age and decreased similarly during aging.

Long-term monitoring of Ca2+ dynamics in C. elegans pharynx: an in vivo energy balance sensor.

Ca2+ is a key signal transducer for muscle contraction. Continuous in vivo monitoring of intracellular Ca2+-dynamics in C. elegans pharynx muscle revealed surprisingly complex Ca2+ patterns. Despite the age-dependent decline of pharynx pumping, we observed unaltered fast Ca2+ oscillations both in young and old worms. In addition, sporadic prolonged Ca2+ increases lasting many seconds or minutes were often observed in between periods of fast Ca2+ oscillations. We attribute them to the inhibition of ATP-dependent Ca2+-pumps upon energy depletion. Accordingly, food deprivation largely augmented the frequency of prolonged [Ca2+] increases. However, paradoxically, prolonged [Ca2+] increases were more frequently observed in young worms than in older ones, and less frequently observed in energy-deficient mitochondrial respiratory chain nuo-6 mutants than in wild-type controls. We hypothesize that young animals are more susceptible to energy depletion due to their faster energy consumption rate, while nuo-6 mutants may keep better the energy balance by slowing energy consumption. Our data therefore suggest that the metabolic state of the pharynx during feeding stimulation depends mainly on the delicate balance between the instant rates of energy production and consumption. Thus, in vivo monitoring of muscle Ca2+ dynamics can be used as a novel tool to study cellular energy availability.

Functional roles of MICU1 and MICU2 in mitochondrial Ca(2+) uptake.

MICU1 and MICU2 are the main regulators of the mitochondrial Ca(2+)-uniporter (MCU), but their precise functional role is still under debate. We show here that MICU2 behaves as a pure inhibitor of MCU at low cytosolic [Ca(2+)] ([Ca(2+)]c), though its effects decrease as [Ca(2+)]c is increased and disappear above 7 µM. Regarding MICU1, studying its effects is more difficult because knockdown of MICU1 leads also to loss of MICU2. However, while knockdown of MICU2 induces only a persistent increase in mitochondrial Ca(2+) uptake, knockdown of MICU1 also induces a peculiar use-dependent transient activation of MCU that cannot be attributed to the parallel loss of MICU2. Therefore, MICU1 is endowed with a specific inhibitory effect on MCU at low [Ca(2+)]c, separate and kinetically different from that of MICU2. On the other hand, we and others have shown previously that MICU1 activates MCU at [Ca(2+)]c above 2.5 µM. Thus, MICU1 has a double role in MCU regulation, inhibitory at low [Ca(2+)]c and activatory at high [Ca(2+)]c.

Effects of long-term feeding of the polyphenols resveratrol and kaempferol in obese mice.

The effect of the intake of antioxidant polyphenols such as resveratrol and others on survival and different parameters of life quality has been a matter of debate in the last years. We have studied here the effects of the polyphenols resveratrol and kaempferol added to the diet in a murine model undergoing long-term hypercaloric diet. Using 50 mice for each condition, we have monitored weight, survival, biochemical parameters such as blood glucose, insulin, cholesterol, triglycerides and aspartate aminotransferase, neuromuscular coordination measured with the rotarod test and morphological aspect of stained sections of liver and heart histological samples. Our data show that mice fed since they are 3-months-old with hypercaloric diet supplemented with any of these polyphenols reduced their weight by about 5-7% with respect to the controls fed only with hypercaloric diet. We also observed that mice fed with any of the polyphenols had reduced levels of glucose, insulin and cholesterol, and better marks in the rotarod test, but only after 1 year of treatment, that is, during senescence. No effect was observed in the rest of the parameters studied. Furthermore, although treatment with hypercaloric diets induced large changes in the pattern of gene expression in liver, we found no significant changes in gene expression induced by the presence of any of the polyphenols. Thus, our data indicate that addition of resveratrol or kaempferol to mice food produces an initial decrease in weight in mice subjected to hypercaloric diet, but beneficial effects in other parameters such as blood glucose, insulin and cholesterol, and neuromuscular coordination, only appear after prolonged treatments.

Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells.

MICU1 (Ca2+ uptake protein 1, mitochondrial) is an important regulator of the MCU (Ca2+ uniporter protein, mitochondrial) that has been shown recently to act as a gatekeeper of the MCU at low [Ca2+]c (cytosolic [Ca2+]). In the present study we have investigated in detail the dynamics of MCU activity after shRNA-knockdown of MICU1 and we have found several new interesting properties. In MICU1-knockdown cells, the rate of mitochondrial Ca2+ uptake was largely increased at a low [Ca2+]c (<2 µM), but it was decreased at a high [Ca2+]c (>4 µM). In the 2-4 µM range a mixed behaviour was observed, where mitochondrial Ca2+ uptake started earlier in the MICU1-silenced cells, but at a lower rate than in the controls. The sensitivity of Ca2+ uptake to Ruthenium Red and Ru360 was similar at both high and low [Ca2+]c, indicating that the same Ca2+ pathway was operating in both cases. The increased Ca2+-uptake rate observed at a [Ca2+]c below 2 µM was transient and became inhibited during Ca2+ entry. Development of this inhibition was slow, requiring 5 min for completion, and was hardly reversible. Therefore MICU1 acts both as a MCU gatekeeper at low [Ca2+]c and as a cofactor necessary to reach the maximum Ca2+-uptake rate at high [Ca2+]c. Moreover, in the absence of MICU1, the MCU becomes sensitive to a slow-developing inhibition that requires prolonged increases in [Ca2+]c in the low micromolar range.

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin.

We use here a new very low-Ca(2+)-affinity targeted aequorin to measure the [Ca(2+)] in the endoplasmic reticulum ([Ca(2+)]ER). The new aequorin chimera has the right Ca(2+)-affinity to make long-lasting measurements of [Ca(2+)]ER in the millimolar range. Moreover, previous Ca(2+)-depletion of the ER is no longer required. The steady-state [Ca(2+)]ER obtained is 1-2 mM, higher than previously reported. In addition, we find evidence that there is significant heterogeneity in [Ca(2+)]ER among different regions of the ER. About half of the ER had a [Ca(2+)]ER of 1 mM or below, and the rest had [Ca(2+)]ER values above 1mM and in some parts even above 2 mM. About 5% of the ER was also found to have high [Ca(2+)]ER levels but to be thapsigargin-insensitive and inositol trisphosphate insensitive. The rate of refilling with Ca(2+) of the ER was almost linearly dependent on the extracellular [Ca(2+)] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca(2+)] microdomains connecting the store operated Ca(2+) channels with the ER Ca(2+) pumps may be important during refilling.

Mitochondrial free [Ca(2+)] dynamics measured with a novel low-Ca(2+) affinity aequorin probe.

Mitochondria have a very large capacity to accumulate Ca(2+) during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca(2+)](M) (mitochondrial [Ca(2+)]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca(2+)](M) during prolonged stimulation has been previously precluded by the high Ca(2+) affinity of the probes available. We have now developed a mitochondrially targeted double-mutated form of the photoprotein aequorin which is able to measure [Ca(2+)] in the millimolar range for long periods of time without problems derived from aequorin consumption. We show in the present study that addition of Ca(2+) to permeabilized HeLa cells triggers an increase in [Ca(2+)](M) up to an steady state of approximately 2-3 mM in the absence of phosphate and 0.5-1 mM in the presence of phosphate, suggesting buffering or precipitation of calcium phosphate when the free [Ca(2+)] reaches 0.5-1 mM. Mitochondrial pH acidification partially re-dissolved these complexes. These millimolar [Ca(2+)](M) levels were stable for long periods of time provided the mitochondrial membrane potential was not collapsed. Silencing of the mitochondrial Ca(2+) uniporter largely reduced the rate of [Ca(2+)](M) increase, but the final steady-state [Ca(2+)](M) reached was similar. In intact cells, the new probe allows monitoring of agonist-induced increases of [Ca(2+)](M) without problems derived from aequorin consumption.

Dynamics of mitochondrial [Ca(2+)] measured with the low-Ca(2+)-affinity dye rhod-5N.

Available methods to measure mitochondrial [Ca(2+)] ([Ca(2+)](M)) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca(2+)](M) values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca(2+)-affinity dye rhod-5N provides [Ca(2+)](M) values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca(2+)-affinity of the fluorescent dyes used. We find rhod-5N has an apparent in situ intramitochondrial Kd around 0.5mM. Addition of Ca(2+) buffers containing between 4.5 and 10µM [Ca(2+)] to permeabilized cells loaded with rhod-5N induced increases in calibrated [Ca(2+)](M) up to the 100µM-1mM range, which were dependent on mitochondrial membrane potential. Ca(2+) release from mitochondria was largely dependent on [Na(+)]. We have then used rhod-5N loaded cells to investigate the [Ca(2+)](M) response to agonist stimulation at the single-cell and subcellular level. The [Ca(2+)](M) peaks induced by histamine varied by nearly 10-fold among different cells, with a mean about 25µM. In the presence of the Ca(2+) uniporter stimulator kaempferol, the [Ca(2+)](M) peaks induced by histamine were also highly variable, and the mean [Ca(2+)](M) peak was 3-fold higher. Simultaneous measurement of cytosolic and mitochondrial [Ca(2+)] peaks showed little correlation among the heights of the peaks in both compartments. Studying the [Ca(2+)](M) peaks at the subcellular level, we found significant heterogeneities among regions in the same cell. In particular, the [Ca(2+)](M) increase in mitochondrial regions close to the nucleus was more than double that of mitochondrial regions far from the nucleus.

Ca2+ dynamics in the secretory vesicles of neurosecretory PC12 and INS1 cells.

We have investigated the dynamics of the free [Ca(2+)] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca(2+)-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca(2+)] ([Ca(2+)](SG)] was around 20-40 µM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca(2+) pumps with thapsigargin largely blocked Ca(2+) uptake by the granules in Ca(2+)-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type Ca(2+) pump inhibitor benzohydroquinone induced a rapid release of Ca(2+) from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca(2+) pumps is essential to maintain the steady-state [Ca(2+)](SG). Both inositol 1,4,5-trisphosphate (InsP(3)) and caffeine produced a rapid Ca(2+) release from the granules, suggesting the presence of InsP(3) and ryanodine receptors in the granules. The response to high-K(+) depolarization was different in both cell types, a decrease in [Ca(2+)](SG) in PC12 cells and an increase in [Ca(2+)](SG) in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type. Finally, increasing the glucose concentration triggered a decrease in [Ca(2+)](SG) in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca(2+) through SERCA-type Ca(2+) pumps and can release it through InsP(3) and ryanodine receptors, supporting the hypothesis that secretory granule Ca(2+) may be released during cell stimulation and contribute to secretion.