Calcio y Función Celular

The eighth ECS workshop on “Calcium signaling in aging and neurodegenerative diseases”

Tue, 01 Jan 2019 00:00:00 GMT

We are very proud to present in this special issue, not only a full meeting report, but also a nice slate of up-to-date reviews and research papers reflecting the groundbreaking work performed by the participants of the eighth ECS workshop on “Calcium signaling in aging and neurodegenerative diseases”.

Effects of cellular interactions on calcium dynamics in prolactin-secreting cells

Thu, 01 Jan 1998 00:00:00 GMT

Signals derived from other pituitary cells can have a dramatic effect on PRL gene expression and secretion by mammotropes. However, the intracellular mechanisms by which these effects are manifested on the target cell remain unexplored. Inasmuch as calcium is a key modulator of both gene expression and hormone export in mammotropes, we evaluated the effects of cell to cell contact vs. specific cellular interactions on calcium dynamics within these cells. This was accomplished by digital-imaging fluorescence microscopy of fura-2 in pituitary cells that were isolated in culture (singles) or adjoining one other cell (doublets). After calcium imaging, we then subjected cells to immunocytochemistry for PRL. Doublets were further categorized into mammotropes attached to another mammotrope (M-M) or to a nonmammotrope (M-nonM). We then calculated and compared Mean[ Ca2+]i values as well as Oscillation Indices (which reflect the oscillatory behavior of cells) in singles and doublets and found that they were not different (P> 0.05). However, the phenotype of the adjoining cell had a profound influence on both of these calcium parameters, such that the presence of one mammotrope could consistently decrease (P < 0.05) the Mean [Ca2+]i value (39.17 ± 3.83 vs. 56.24 ± 5.56 in M-nonM) and Oscillation Index (10.19 ± 1.76 vs. 21.21 ± 3.73 in M-nonM) of its neighboring counterpart. A more detailed analysis of oscillatory patterns in these cells revealed that nonoscillators were more abundant in M-M (23%) than in M-nonM (12%) doublets. Taken together, our results indicate that PRL-secreting cells convey a signal that dampens the oscillatory behavior of neighboring mammotropes. Thus, it appears that it is the phenotype rather than the physical presence of a neighbor that controls intercellular regulation of calcium dynamics among mammotropes.

Dynamics of stimulus-expression coupling as revealed by monitoring of prolactin promoter-driven reporter activity in individual, living mammotropes

Fri, 01 Jan 1999 00:00:00 GMT

Single-cell paradigms have greatly expanded our knowledge about stimulus-secretion coupling, but the understanding of stimulus-gene expression coupling has lagged behind for lack of a dynamic model sufficiently sensitive to provide single-cell resolution. In the present study, we made continuous indirect measurements within individual, living cells of expression dynamics both before and after treatment with a gene-activating secretagogue. This was accomplished by transfecting (via microinjection) individual, primary mammotropes with a PRL promoter-driven luciferase reporter plasmid, and then quantifying the rate of photonic emissions (reflective of endogenous gene activity). We found that individual cells exhibit spontaneous, random, short-term fluctuations of basal reporter activity and are extremely heterogeneous in terms of responses to a stimulatory agent (TRH). In addition, we found that responses are affected by several factors including the secretory status of the pituitary donor, the manner in which the stimulus is presented, and by the initial level of reporter activity. Moreover, the responsiveness of an individual cell can fluctuate dramatically over time. These results invite speculation that a given cell can “sense” its gene activation state and regulate its response accordingly to satisfy requirements for the corresponding secretory product.

RhoA–ROCK and p38MAPK-MSK1 mediate vitamin D effects on gene expression, phenotype, and Wnt pathway in colon cancer cells

Tue, 01 Jan 2008 00:00:00 GMT

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation and promotes differentiation of colon cancer cells through the activation of vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Additionally, 1,25(OH)2D3 has several nongenomic effects of uncertain relevance. We show that 1,25(OH)2D3 induces a transcription-independent Ca2+ influx and activation of RhoA–Rho-associated coiled kinase (ROCK). This requires VDR and is followed by activation of the p38 mitogen-activated protein kinase (p38MAPK) and mitogen- and stress-activated kinase 1 (MSK1). As shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, RhoA–ROCK, and p38MAPK-MSK1 activation is necessary for the induction of CDH1/E-cadherin, CYP24, and other genes and of an adhesive phenotype by 1,25(OH)2D3. RhoA–ROCK and MSK1 are also required for the inhibition of Wnt–β-catenin pathway and cell proliferation. Thus, the action of 1,25(OH)2D3 on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA–ROCK and p38MAPK-MSK1.

Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion

Wed, 01 Jan 1997 00:00:00 GMT

The classic view for hypothalamic regulation of anterior pituitary (AP) hormone secretion holds that release of each AP hormone is controlled specifically by a corresponding hypothalamic-releasing hormone (HRH). In this scenario, binding of a given HRH (thyrotropin-, growth hormone-, corticotropin-, and luteinizing hormone-releasing hormones) to specific receptors in its target cell increases the concentration of cytosolic Ca2+ ([Ca2+]i), thereby selectively stimulating the release of the appropriate hormone. However, “paradoxical” responses of AP cells to the four well-established HRHs have been observed repeatedly with both in vivo and in vitro systems, raising the possibility of functional overlap between the different AP cell types. To explore this possibility, we evaluated the effects of HRHs on [Ca2+]i in single AP cells identified immunocytochemically by the hormone they stored. We found that each of the five major AP cell types contained discrete subpopulations that were able to respond to several HRHs. The relative abundance of these multi-responsive cells was 59% for lactotropes, 33% for thyrotropes, and in the range of 47–55% for gonadotropes, corticotropes, and somatotropes. Analysis of prolactin release from single living cells revealed that each of the four HRHs tested were able to induce hormone release from a discrete lactotrope subpopulation, the size of which corresponded closely to that in which [Ca2+]i changes were induced by the same secretagogues. When viewed as a whole, our diverse functional measurements of multi-responsiveness suggest that hypothalamic control of pituitary function is more complicated than previously envisioned. Moreover, they provide a cellular basis for the so-called “paradoxical” behavior of pituitary cells to hypothalamic hypophysiotropic agents.

The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons

Sat, 01 Jan 2000 00:00:00 GMT

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1–43 fluorescence to monitor changes of secretion in individual GT1–7 cells and then combined this approach with simultaneous measurement of intracellular free calcium ([Ca2+]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K+ (75 mM) or N-methyl-D-aspartate (NMDA, 100μ M) predictably evoked dynamic increases of both FM1–43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that[ Ca2+]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells.

Mitochondrial [Ca2+] oscillations driven by local high [Ca2+] domains generated by spontaneous electric activity

Mon, 01 Jan 2001 00:00:00 GMT

Mitochondria take up calcium during cell activation thus shaping Ca2+ signaling and exocytosis. In turn, Ca2+ uptake by mitochondria increases respiration and ATP synthesis. Targeted aequorins are excellent Ca2+ probes for subcellular analysis, but single-cell imaging has proven difficult. Here we combine virus-based expression of targeted aequorins with photon-counting imaging to resolve dynamics of the cytosolic, mitochondrial, and nuclear Ca2+ signals at the single-cell level in anterior pituitary cells. These cells exhibit spontaneous electric activity and cytosolic Ca2+oscillations that are responsible for basal secretion of pituitary hormones and are modulated by hypophysiotrophic factors. Aequorin reported spontaneous [Ca2+] oscillations in all the three compartments, bulk cytosol, nucleus, and mitochondria. Interestingly, a fraction of mitochondria underwent much larger [Ca2+] oscillations, which were driven by local high [Ca2+] domains generated by the spontaneous electric activity. These oscillations were large enough to stimulate respiration, providing the basis for local tune-up of mitochondrial function by the Ca2+ signal.

Calcium dynamics and resting transcriptional activity regulates prolactin gene expression

Tue, 01 Jan 2002 00:00:00 GMT

Research on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca2+ concentration ([Ca2+]i) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca2+ release, followed by sustained Ca2+ influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca2+]i increase. In addition, transcriptional responses were related to the rate of Ca2+ entry but not Ca2+ release. Finally, cells lacking transcriptional responses (but showing [Ca2+]i rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca2+ induced by TRH (but not the Ca2+ release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.

Multifunctional cells in human pituitary adenomas: Implications for paradoxical secretion and tumorigenesis

Thu, 01 Jan 2004 00:00:00 GMT

Pituitary adenomas are very common in humans. They are of monoclonal origin, very heterogeneous, and produce frequently paradoxical secretion. The normal anterior pituitary (AP) contains some unorthodox multifunctional cells able to store more than one AP hormone (polyhormonal) and/or to express multiple hypothalamic-releasing hormone receptors (multiresponsive). Multifunctional AP cells seem to be involved in plasticity processes such as transdifferentiation or paradoxical secretion. Here, we have characterized the single-cell phenotypes of 15 human pituitary tumors, including prolactinomas, nonfunctioning adenomas, and adenomas from multiple endocrine neoplasia type I (MEN-I) and pituitary Cushing’s disease patients. Individual tumor cells were typed according to expression of AP hormones and hypothalamic-releasing hormone receptors by combination of calcium imaging and multiple sequential immunocytochemistry in the same cells. We found a large heterogeneity among the different tumors. In eight of the 15 tumors studied, more than 80% of the cells presented a multifunctional phenotype. This may explain the occurrence of paradoxical secretion. In addition, our results suggest that human pituitary adenomas might derive from multifunctional cells. This is consistent with the existence of a link between pituitary plasticity and tumorigenesis.

Rapid changes in anterior pituitary cell phenotypes in male and female mice after acute cold stress

Tue, 01 Jan 2008 00:00:00 GMT

The anterior pituitary (AP) is made of five different cell types. The relative abundance and phenotype of AP cells may change in different physiological situations as an expression of pituitary plasticity. Here, we analyze in detail the phenotype of mouse corticotropes and the effects of acute cold stress on AP cell populations. The hormone content and the expression of hypothalamic-releasing hormone (HRH) receptors in all the five AP cell types were studied in the male and female mice at rest and after a 30-min cold stress. Expression of HRH receptors was evidenced by imaging the single-cell cytosolic Ca2+ responses in fura-2-loaded cells. Hormone contents were studied by multiple, simultaneous immunofluorescence of all the five hormones. Corticotropes displayed a striking sexual dimorphism, even in the resting condition. Male corticotropes showed the orthodox phenotype. They were monohormonal, storing only ACTH, and monoreceptorial, responding only to CRH. In contrast, female corticotropes were made of about equal parts of orthodox cells and multifunctional cells, which co-stored additional AP hormones and expressed additional HRH receptors. Cold stress did not modify the number of ACTH containing cells, but, according to immunostaining, it increased the relative abundance of other AP cell types at the expense of the pool of cells storing no hormones. Cold stress also modified the response to CRH and other HRHs. Most of these phenotypical changes presented a strong sexual dimorphism. These results indicate that pituitary plasticity is even larger than previously thought.

Mitochondrial Ca2+ overload underlies Aβ oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs

Tue, 01 Jan 2008 00:00:00 GMT

Dysregulation of intracellular Ca2+ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca2+ in neurons and promote mitochondrial Ca2+ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca2+ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca2+ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca2+ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca2+ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.

Nonsteroidal anti-inflammatory drugs inhibit vascular smooth muscle cell proliferation by enabling the Ca2+-dependent inactivation of calcium release-activated calcium/Orai channels normally prevented by mitocondria

Sat, 01 Jan 2011 00:00:00 GMT

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca2+ homeostasis and Ca2+-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca2+ entry (SOCE) and voltage-operated Ca2+ entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca2+ released-activated Ca2+ (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca2+-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca2+ uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca2+ uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca2+ uptake, suggesting that they favor the Ca2+-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca2+ buffering conditions but not in strong Ca2+ buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca2+-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca2+.

Irvalec inserts into the plasma membrane causing rapid loss of integrity and necrotic cell death in tumor cells

Sat, 01 Jan 2011 00:00:00 GMT

Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca2+ influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn2+. Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity.

Regulation of mitochondrial permeability transition pore by PINK1

Sun, 01 Jan 2012 00:00:00 GMT

Background: Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson’s disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains. Results: In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1−/− cells recapitulate the respiratory defect in isolated mitochondria from PINK1−/− mouse brains, suggesting that these PINK1−/− cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1−/− cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1−/− cells, and this genotypic difference between PINK1−/− and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1−/− cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1−/− and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1−/− compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1. Conclusions: Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1−/− cells.

Pharmacological characterization of the mechanisms involved in delayed calcium deregulation in SH-SY5Y cells challenged with methadone

Sun, 01 Jan 2012 00:00:00 GMT

Previously, we have shown that SH-SY5Y cells exposed to high concentrations of methadone died due to a necrotic-like cell death mechanism related to delayed calcium deregulation (DCD). In this study, we show that, in terms of their Ca2+ responses to 0.5 mM methadone, SH-SY5Y cells can be pooled into four different groups. In a broad pharmacological survey, the relevance of different Ca2+-related mechanisms on methadone-induced DCD was investigated including extracellular calcium, L-type Ca2+ channels, -opioid receptor, mitochondrial inner membrane potential, mitochondrial ATP synthesis, mitochondrial Ca2+/2Na+-exchanger, reactive oxygen species, and mitochondrial permeability transition. Only those compounds targeting mitochondria such as oligomycin, FCCP, CGP 37157, and cyclosporine A were able to amend methadone-induced Ca2+ dyshomeostasis suggesting that methadone induces DCD by modulating the ability of mitochondria to handle Ca2+. Consistently, mitochondria became dramatically shorter and rounder in the presence of methadone. Furthermore, analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial Ca2+ uptake in a respiratory substrate-dependent way. We conclude that methadone causes failure of intracellular Ca2+ homeostasis, and this effect is associated with morphological and functional changes of mitochondria. Likely, this mechanism contributes to degenerative side effects associated with methadone treatment.

A reciprocal shift in transient receptor potential channel 1 (TRPC1) and stromal interaction molecule 2 (STIM2) contributes to Ca2+remodeling and cancer hallmarks in colorectal carcinoma cells

Wed, 01 Jan 2014 00:00:00 GMT

We have investigated the molecular basis of intracellular Ca2+ handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca2+ stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca2+ levels, agonist-induced Ca2+ increases, store-operated Ca2+ entry (SOCE), and store-operated currents (ISOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca2+ stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca2+ release-activated Ca2+ currents (ICRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced ICRAC plus a nonselective ISOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca2+ store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca2+ store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca2+ remodeling and tumor features in colon cancer.