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The purpose of this paper was to investigate the bone regeneration effect of autologous adipose tissue mesenchymal stem cells (ATMSC) in a small animal model. Twelve Wistar rats were given bilateral critical-size defects in the mandible. The defects were filled with coralline hydroxyapatite alone or combined with autologous undifferentiated ATMSC obtained from the dorsal fat pad. Studies were conducted at three and six weeks. Descriptive histology and histomorphometry revealed a significant (p < 0.05) increased bone regeneration values in the cell-treated defects at both three and six weeks. ATMSC promoted the formation of new bone in the central areas of the defects and in the scaffold micropores, both in a higher state of maturation. Autologous undifferentiated ATMSC enhanced bony healing of mandibular critical-size defects in rats when implanted with a coralline hydroxyapatite scaffold.

Excitability in astroglia is controlled by Ca2+ fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsP3R) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca2+-gated Ca2+ release channels is less explored in astroglia. Here we address this process by monitoring Ca2+ dynamics directly in the cytosol and the ER of astroglial cells. Cultured astrocytes exhibited spontaneous and high-K-evoked cytosolic Ca2+ transients, both of them reversibly abolished by external Ca2+ removal, addition of plasma membrane channel blockers or ER Ca2+ depletion with SERCA inhibitors. Resting astrocyte [Ca2+]ER averaged 400 μM and maximal stimulation with ATP provoked a complete and reversible ER discharge. Direct monitoring of Ca2+ in the lumen of ER showed that high-K induced a Ca2+ release from the ER, and its amplitude was proportional to the [K]. Furthermore, by combining the low affinity GAP3 indicator targeted to the ER with the high affinity cytosolic Rhod-2, we simultaneously imaged ER- and cytosolic-Ca2+ signals, in astrocytes in culture and in situ. Plasma membrane Ca2+ entry triggered a fast ER Ca2+ release coordinated with an increase in cytosolic Ca2+. Thus, we identify a Ca2+-induced Ca2+-release (CICR) mechanism that is likely to participate in spontaneous astroglial oscillations, providing a graded amplification of the cytosolic Ca2+ signal., This work was supported by grants from The Spanish MINECO (BFU2017-83066-P) and the Junta de Castilla y León (GR175). Macarena Rodriguez-Prados and Jonathan Rojo-Ruiz were supported by fellowships from the Spanish MINECO.

Producción Científica, Ocular stem cell transplantation derived from either autologous or allogeneic donor corneoscleral junction is a functional cell therapy to manage extensive and/or severe limbal stem cell deficiencies that lead to corneal epithelial failure. Mesenchymal stem cells have been properly tested in animal models of this ophthalmic pathology, but never in human eyes despite their potential advantages. We conducted a 6- to 12-month proof-of-concept, randomized, and double-masked pilot trial to test whether allogeneic bone marrow-derived mesenchymal stem cell transplantation (MSCT], n = 17) was as safe and as equally efficient as allogeneic cultivated limbal epithelial transplantation (CLET), (n = 11) to improve corneal epithelial damage due to limbal stem cell deficiency. Primary endpoints demanded combination of symptoms, signs, and the objective improvement of the epithelial phenotype in central cornea by in vivo confocal microscopy. This proof-of-concept trial showed that MSCT was as safe and efficacious as CLET. Global success at 6–12 months was 72.7%–77.8% for CLET cases and 76.5%–85.7% for MSCT cases (not significant differences). Central corneal epithelial phenotype improved in 71.4% and 66.7% of MSCT and CLET cases, respectively at 12 months (P = 1.000). There were no adverse events related to cell products. This trial suggests first evidence that MSCT facilitated improvement of a diseased corneal epithelium due to lack of its stem cells as efficiently as CLET. Consequently, not only CLET but also MSCT deserves more preclinical investigational resources before the favorable results of this proof-of-concept trial could be transformed into the larger numbers of the multicenter trials that would provide stronger evidence. (ClinicalTrials.gov number, NCT01562002.), Ministerio de Sanidad, Consumo y Bienestar Social (project SAS/2481/2009), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León (grant SAN 1178/200), Red de Terapia Celular TerCel (project RD12/0019/0036)

The adenohypophysis contains five secretory cell types (somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs), each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca2+ signals ([Ca2+]C), which can be generated either by Ca2+ entry through the plasma membrane and/or by Ca2+ release from the endoplasmic reticulum (ER). In addition, Ca2+ entry signals can eventually be amplified by ER release via calcium-induced calcium release (CICR). We have investigated the contribution of ER Ca2+ release to the action of physiological agonists in pituitary gland. Changes of [Ca2+] in the ER ([Ca2+]ER) were measured with the genetically encoded low-affinity Ca2+ sensor GAP3 targeted to the ER. We used a transgenic mouse strain that expressed erGAP3 driven by a ubiquitous promoter. Virtually all the pituitary cells were positive for the sensor. In order to mimick the physiological environment, intact pituitary glands or acute slices from the transgenic mouse were used to image [Ca2+]ER. [Ca2+]C was measured simultaneously with Rhod-2. Luteinizing hormone-releasing hormone (LHRH) or thyrotropin releasing hormone (TRH), two agonists known to elicit intracellular Ca2+ mobilization, provoked robust decreases of [Ca2+]ER and concomitant rises of [Ca2+]C. A smaller fraction of cells responded to thyrotropin releasing hormone (TRH). By contrast, depolarization with high K+ triggered a rise of [Ca2+]C without a decrease of [Ca2+]ER, indicating that the calcium-induced calcium-release (CICR) via ryanodine receptor amplification mechanism is not present in these cells. Our results show the potential of transgenic ER Ca2+ indicators as novel tools to explore intraorganellar Ca2+ dynamics in pituitary gland in situ.

Producción Científica, Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca2+ peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca2+ concentration ([Ca2+]SR) is at the origin of this loss of Ca2+ homeostasis. We engineered Drosophila melanogaster to express the Ca2+ indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca2+]SR in vivo. [Ca2+]SR fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca2+]SR was, Ministerio de Economía, Industria y Competitividad (grant BFU2017-83066-P), Junta de Castilla y León (project GR175)

Tissue engineering combining cross-linked serum scaffolds with bone-derived mesenchymal stem cells has displayed excellent results for repair of maxillofacial bone defects in animal models, but it had not been tested in humans yet. We present here a pilot clinical trial using autologous bone-derived mesenchymal stem cells (H-MSV) grown in a serum cross-linked scaffold (BioMax) for treatment of maxillary cysts in 9 patients. Cells obtained from alveolar bone were seeded in the BioMax scaffold prepared from autologous serum, expanded under GMP conditions, and subjected to osteogenic differentiation for 3–4 weeks before application. Evolution of the cystic cavity was followed by computerized tomography (CT) for 7 months. There was no inflammation or other adverse effects, and the CT density of the cyst interior increased significantly after the treatment. The ratio of the CT values after/before treatment was (mean ± SE) 2.52 ± 0.45; in contrast, the density of the contralateral control area of spongy alveolar bone without treatment did not change (ratio after/before, 0.99 ± 0.14). In conclusion, cell therapy with BioMax could be considered as an alternative therapy for maxillary bone defects and other losses of bone substance. Further research with allogeneic cells would be useful for reducing costs and improving logistics., Financial support from the Red de Terapia Cellular (RD16/0011/0003 and RD16/0011/0022) of the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, and the Centro en Red de Medicina Regenerativa de Castilla y León is gratefully acknowledged. The clinical trial was supported by grant EC10-255 from the Ministerio de Sanidad y Política Social (to LMR) and grant PI10/01566 from the Fondo de Investigaciones Sanitarias (FIS Intrasalud), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (to AS). EU co-financed these grants through the European Regional Development Fund. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

Excitability in astroglia is controlled by Ca

Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca

Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson’s disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases., Research from the authors’ laboratories is supported by the European Research Council-ERC (2012-StG-311736-PD-HUMMODEL), the European Union’s Horizon 2020 Program (MESOBRAIN project; grant agreement No 713140), the Spanish Ministry of Economy and Competitiveness-MINECO (SAF2015-69706-R; BFU2017-83066-P; and BFU2016-80870-P), Instituto de Salud Carlos III-ISCIII/FEDER (Red de Terapia Celular - TerCel RD16/0011/0003 and RD16/0011/0024; PIE12/00061), AGAUR (2017-SGR-899 and 2017-SGR-1061), and CERCA Program/Generalitat de Catalunya. C.C. is supported by the PD-HUMMODEL European Research Council (ERC)- Ideas PhD fellowship. G.C. is partially supported by pre-doctoral fellowship from Spanish Economy and Competitiveness-MINECO (BES-2014-069603).

Cytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10−7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3–1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR., This work was supported by grants from The Spanish MINECO [BFU2017-83066-P] and the Junta de Castilla y León [GR175]. J.R.-R., M.R.-P., and A.D.-L. were supported by fellowships from the Spanish MINECO.

Animal and human studies have suggested the potential of mesenchymal stromal cells (MSCs) to treat systemic lupus erythematosus (SLE). Here, we present the results of compassionate MSC treatments for three SLE patients to provide the proof of concept for a randomized and controlled clinical trial. Three patients of different ethnicities who suffer from chronic SLE, and who presented with class IV active proliferative nephritis confirmed by biopsy, were treated with allogeneic MSCs from healthy donors. Ninety million cells were infused intravenously into each patient during high and very high activity disease flare-ups and follow-up was continued for 9 months. Multi-organic affectation was quantified by the SLE disease activity index (SLEDAI), and indicators of lupus nephritis activity, such as proteinuria, as well as lymphocyte and monocyte antigens and anti-HLA antibodies were measured at 1, 3, 6, and 9 months after treatment. Proteinuria levels improved dramatically during the 1st month after treatment and the ameliorations were sustained throughout the follow-up period. SLEDAI scores revealed early, durable, and substantial remissions that were complete for two patients and partial for the third patient and that permitted medication doses to be reduced 50-90%. These favourable outcomes support completion of the randomized and controlled MSC trial for SLE.

[Background]: Osteoarthritis is themost prevalent joint disease and a common cause of joint pain, functional loss, and disability. Conventional treatments demonstrate only modest clinical benefits without lesion reversal. Autologous mesenchymal stromal cell (MSC) treatments have shown feasibility, safety, and strong indications for clinical efficacy. We performed a randomized, active control trial to assess the feasibility and safety of treating osteoarthritis with allogeneic MSCs, and we obtain information regarding the efficacy of this treatment. [Methods]: We randomized 30 patients with chronic knee pain unresponsive to conservative treatments and showing radiological evidence of osteoarthritis into 2 groups of 15 patients. The test group was treated with allogeneic bone marrow MSCs by intra-articular injection of 40 × 106 cells. The control group received intra-articular hyaluronic acid (60 mg, single dose). Clinical outcomes were followed for 1 year and included evaluations of pain, disability, and quality of life. Articular cartilage quality was assessed by quantitative magnetic resonance imaging T2 mapping. [Results]: Feasibility and safety were confirmed and indications of clinical efficacy were identified. The MSC-treated patients displayed significant improvement in algofunctional indices versus the active controls treated with hyaluronic acid. Quantification of cartilage quality by T2 relaxation measurements showed a significant decrease in poor cartilage areas, with cartilage quality improvements inMSC-treated patients. [Conclusions]: Allogeneic MSC therapy may be a valid alternative for the treatment of chronic knee osteoarthritis that is more logistically convenient than autologous MSC treatment. The intervention is simple, does not require surgery, provides pain relief, and significantly improves cartilage quality., Financial support was provided by the Program for Support of Independent Clinical Research of the Spanish Ministerio de Sanidad (EC11-309), Red de Terapia Celular (RD06/0010/0000 and RD12/0019/0036) of the Instituto de Salud Carlos III, Ministerio de Economia y Competitividad, and the Centro en Red de Medicina Regenerativa de Castilla y León.

Mitochondrial Ca

Producción Científica, Aequorins are excellent tools for measuring intra-organellar Ca2+ and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal. We also focus on bioluminescent Ca2+ imaging in nucleus or mitochondria of living cells. Finally, recent advances with a new chimeric GFP-aequorin protein (GAP), which can be used either as luminescent or fluorescent Ca2+ probe, are presented. GAP is robustly expressed in transgenic flies and mice, where it has proven to be a suitable Ca2+ indicator for monitoring physiological Ca2+ signaling ex vivo and in vivo., Ministerio de Economía, Industria y Competitividad (Project BFU2014-53469P), Instituto de Salud Carlos III (TerCel, RD16/0011/0003)

Producción Científica, GFP-Aequorin Protein (GAP) can be used to measure [Ca2 +] inside intracellular organelles, both by luminescence and by fluorescence. The low-affinity variant GAP3 is adequate for ratiometric imaging in the endoplasmic reticulum and Golgi apparatus, and it can be combined with conventional synthetic indicators for simultaneous measurements of cytosolic Ca2 +. GAP is bioorthogonal as it does not have mammalian homologues, and it is robust and functionally expressed in transgenic flies and mice, where it can be used for Ca2 + measurements ex vivo and in vivo to explore animal models of health and disease. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech., Ministerio de Economía, Industria y Competitividad (Project BFU2014-53469P), Instituto de Salud Carlos III (TerCel; RD16/0011/0003)

International audience; Mitochondrial Ca2+ homeostasis is crucial for regulating vital functions such as respiration or apoptosis. Targeted aequorins are excellent probes to measure subcellular Ca2+. Ca2+ concentration in mitochondria ([Ca2+]M) is low at rest (about 10-7 M) and can increase to the micromolar or even approach the millimolar range, upon cell activation. Here we describe a new quantitative luminescent protocol to directly measure mitochondrial Ca2+ uptake, optimized for high throughput. The sensitivity of the method allows detection of changes in either the capacity or the affinity of mitochondrial Ca2+ transport.

Producción Científica, The necessity for more effective therapies for chronic osteoarticular diseases has led to the development of treatments based on mesenchymal stem cells (MSCs), the natural precursors of musculoskeletal tissue. Treatments with autologous MSCs yielded excellent results, with nearly 70% improvement of pain and disability in osteoarthritis and degenerative disc disease. Using allogeneic MSCs is logistically more convenient and would widen the pool of eligible patients, but potential immune rejection should be considered. In this context, MSCs are purportedly immune evasive and better tolerated than other cell types. Methods We used samples collected during the performance of 2 randomized clinical trials using allogeneic bone marrow MSCs for treatment of osteoarthritis (NCT01586312) and degenerative disc disease (NCT01860417). Serum samples were used to determine anti-HLA antibodies, whereas either blood or MSC samples were used for HLA typing of recipients and donors, respectively. Algofunctional indexes were used as indicators of clinical evolution, and the correlation between the number of donor-host HLA mismatches and the efficacy of treatment was determined. Results Immune response was weak and transient, with reactivity decaying during the first year. Consistently, better donor-recipient HLA matching did not enhance efficacy. Conclusions This lack of reactivity is presumably due to the cooperation of 2 factors, (1) downregulation of the host immune responses by the transplanted MSCs and (2) effective insulation of these cells inside the articular cavity or the intervertebral disc, respectively. Interestingly, better HLA matching did not enhance efficacy. These observations have medical relevance as they support the clinical use of allogeneic cells, at least as a single-dose administration. Multiple-dose applications will require further research to exclude possible sensitization., Red de Terapia Celular of the Instituto de Salud Carlos III (RD16/0011/0003) and Centro en Red de Medicina Regenerativa de Castilla y León

The characterization and isolation of various stem cell populations, from embryonic to tissue-derived stem cells and induced pluripotent stem cells (iPSCs), have led to a rapid growth in the field of stem cell research and its potentially clinical application in the field of regenerative medicine and tissue repair. Stem cell therapy has recently progressed from the preclinical to the early clinical trial arena for a variety of diseases states, although further knowledge on action mechanisms, long-term safety issues, and standardization and characterization of the therapeutic cell products remains to be thoroughly elucidated. In this paper we summarize the current state of the art of basic and clinical research that were highlighted at the 2012 meeting of the Spanish Cell Therapy Network. This includes the current research involving in genomic and transcriptomic characterization of selected stem cell populations, studies of the role of resident and transplanted stem cells during tissue regeneration and their mechanism of action, improved new strategies of tissue engineering, transplantation of mesenchymal stem cells (MSCs) in different animal models of disease, disease correction by iPSCs, and preliminary results of cell therapy in human clinical trials.

Cross-talk between organelles and plasma membrane Ca(2+) channels is essential for modulation of the cytosolic Ca(2+) ([Ca(2+)]C) signals, but such modulation may differ among cells. In chromaffin cells Ca(2+) entry through voltage-operated channels induces calcium release from the endoplasmic reticulum (ER) that amplifies the signal. [Ca(2+)]C microdomains as high as 20-50 μm are sensed by subplasmalemmal mitochondria, which accumulate large amounts of Ca(2+) through the mitochondrial Ca(2+) uniporter (MCU). Mitochondria confine the high-Ca(2+) microdomains (HCMDs) to beneath the plasma membrane, where exocytosis of secretory vesicles happens. Cell core [Ca(2+)]C is much smaller (1-2 μm). By acting as a Ca(2+) sink, mitochondria stabilise the HCMD in space and time. In non-excitable HEK293 cells, activation of store-operated Ca(2+) entry, triggered by ER Ca(2+) emptying, also generated subplasmalemmal HCMDs, but, in this case, most of the Ca(2+) was taken up by the ER rather than by mitochondria. The smaller size of the [Ca(2+)]C peak in this case (about 2 μm) may contribute to this outcome, as the sarco-endoplasmic reticulum Ca(2+) ATPase has much higher Ca(2+) affinity than MCU. It is also possible that the relative positioning of organelles, channels and effectors, as well as cytoskeleton and accessory proteins plays an important role.

Aequorins are excellent tools for measuring intra-organellar Ca

GFP-Aequorin Protein (GAP) can be used to measure [Ca

et al., [Background]: Degenerative disc disease often causes severe low-back pain, a public health problem with huge economic and life quality impact. Chronic cases often require surgery, which may lead to biomechanical problems and accelerated degeneration of the adjacent segments. Autologous mesenchymal stromal cells (MSC) treatments have shown feasibility, safety and strong indications of clinical efficacy. We present here a randomized, controlled trial using allogeneic MSC, which are logistically more convenient than autologous cells. [Methods]: We randomized 24 patients with chronic back pain diagnosed with lumbar disk degeneration and unresponsive to conservative treatments into 2 groups. The test group received allogeneic bone marrow MSCs by intradiscal injection of 25 × 10 cells per segment under local anesthesia. The control group received a sham infiltration of paravertebral musculature with the anesthetic. Clinical outcomes were followed up for 1 year and included evaluation of pain, disability, and quality of life. Disc quality was followed up by magnetic resonance imaging. [Results]: Feasibility and safety were confirmed and indications of clinical efficacy were identified. MSC-treated patients displayed a quick and significant improvement in algofunctional indices versus the controls. This improvement seemed restricted to a group of responders that included 40% of the cohort. Degeneration, quantified by Pfirrmann grading, improved in the MSC-treated patients and worsened in the controls. [Conclusions]: Allogeneic MSC therapy may be a valid alternative for the treatment of degenerative disc disease that is more logistically convenient than the autologous MSC treatment. The intervention is simple, does not require surgery, provides pain relief, and significantly improves disc quality., Financial support from the Red de Terapia Celular (RD12/0019/0036, RD12/0019/0001 and RD16/0011/0003), Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, and the Centro en Red de Medicina Regenerativa de Castilla y León is gratefully acknowledged.

The concept of stimulus–secretion coupling was born from experiments performed in chromaffincells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca2+) entry and this generates a transient elevation of the cytosolic Ca2+ concentration ([Ca2+]c) that triggers the exocytotic release of catecholamines. The control of the [Ca2+]csignal is complex and depends on various classes of plasmalemmal calcium channels, cytosoliccalcium buffers, the uptake and release of Ca2+ from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca2+ extrusion mechanisms, such as the plasma membrane Ca2+-stimulated ATPase, and the Na+/Ca2+ exchanger. Computation of the rates of Ca2+ fluxes between the different cell compartments support the proposal that the chromaffincell has developed functional calcium tetrads formed by calcium channels, cytosoliccalcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca2+ transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytoticresponses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca2+]c transients occurring at different cell compartments, and generated by redistribution and release of Ca2+ by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo–endocytotic activities with energy production and protein synthesis., The work of the author’s laboratories has been supported by the following institutions. To AGG: (1) SAF2010-21795, Ministerio de Ciencia e Innovación (MCINN); (2) RETICS RD06/0009, Instituto de Salud Carlos III (MICINN); (3) S-SAL-0275-2006, Comunidad Autónoma de Madrid; (4) NDG07/9 y NDG09/8, Agencia Laín Entralgo, Comunidad Autónoma de Madrid, Spain. (5) Fundación Teófilo Hernando. To JGS: grants from the EU-ERA-Net programme, the Spanish Ministerio de Ciencia e Innovación (MICINN; SAF2008- 03175-E and BFU2010-17379), the Instituto de Salud Carlos III (RD06/0010/0000) and the Junta de Castilla y León (gr175).

[Background]: Degenerative disc disease may cause severe low-back pain, a large public health problem with significant economic and life quality impact. Chronic cases often require surgery, which may lead to biomechanical problems and accelerated degeneration of the adjacent segments. Cell-based therapies may circumvent these problems and have exhibited encouraging results in vitro and in animal studies. We designed a pilot study to assess feasibility and safety and to obtain early indications on efficacy of treatment with mesenchymal stem cells (MSC) in humans. [Methods]: Ten patients with chronic back pain diagnosed with lumbar disc degeneration with intact annulus fibrosus were treated with autologous expanded bone marrow MSC injected into the nucleus pulposus area. Clinical evolution was followed for 1 year and included evaluation of back pain, disability, and quality of life. Magnetic resonance imaging measurements of disc height and fluid content were also performed. [Results]: Feasibility and safety were confirmed and strong indications of clinical efficacy identified. Patients exhibited rapid improvement of pain and disability (85% of maximum in 3 months) that approached 71% of optimal efficacy. This outcome compares favorably with the results of other procedures such as spinal fusion or total disc replacement. Although disc height was not recovered, water content was significantly elevated at 12 months. [Conclusions]: MSC therapy may be a valid alternative treatment for chronic back pain caused by degenerative disc disease. Advantages over current gold standards include simpler and more conservative intervention without surgery, preservation of normal biomechanics, and same or better pain relief. © 2011 by Lippincott Williams & Wilkins.

et al., [Background]: Osteoarthritis is the most prevalent joint disease and a frequent cause of joint pain, functional loss, and disability. Osteoarthritis often becomes chronic, and conventional treatments have demonstrated only modest clinical benefits without lesion reversal. Cell-based therapies have shown encouraging results in both animal studies and a few human case reports. We designed a pilot study to assess the feasibility and safety of osteoarthritis treatment with mesenchymal stromal cells (MSCs) in humans and to obtain early efficacy information for this treatment. [Methods]: Twelve patients with chronic knee pain unresponsive to conservative treatments and radiologic evidence of osteoarthritis were treated with autologous expanded bone marrow MSCs by intra-articular injection (40×10 cells). Clinical outcomes were followed for 1 year and included evaluations of pain, disability, and quality of life. Articular cartilage quality was assessed by quantitative magnetic resonance imaging T2 mapping. [Results]: Feasibility and safety were confirmed, and strong indications of clinical efficacy were identified. Patients exhibited rapid and progressive improvement of algofunctional indices that approached 65% to 78% by 1 year. This outcome compares favorably with the results of conventional treatments. Additionally, quantification of cartilage quality by T2 relaxation measurements demonstrated a highly significant decrease of poor cartilage areas (on average, 27%), with improvement of cartilage quality in 11 of the 12 patients. [Conclusions]: MSC therapy may be a valid alternative treatment for chronic knee osteoarthritis. The intervention is simple, does not require hospitalization or surgery, provides pain relief, and significantly improves cartilage quality.

7 páginas, 5 figuras.-- El pdf es la versión post-print del artículo., STIM1 and Orai1 are the main players in capacitative calcium entry (CCE). STIM1 senses [Ca2+] inside the endoplasmic reticulum (ER) and, when it decreases, opens Orai1, a store-operated calcium channel (SOC) in the plasma membrane that promotes Ca2+ entry and increases cytosolic Ca2+. The final destination of the entering Ca2+ is the ER, which refills very efficiently (capacitatively) with it. We propose here that SERCA is the third element of CCE, to which is tightly coupled to favour rapid Ca2+ pumping from the high Ca2+ microdomains, generated at the SOC's mouth, to the ER. We find that, on depletion of the intracellular Ca2+ stores, SERCA co-localizes with STIM1 at puncta. Adequate coupling of CCE and ER Ca2+ pumping requires correct proportions of STIM1, Orai1 and SERCA. Overexpression of Orai1 decreased modestly Ca2+ entry, but produced a dramatic fall of Ca2+ uptake into ER, which was rescued by STIM1 co-expression or by increasing external Ca2+. In permeabilized cells, Ca2+ uptake into the ER was indistinguishable in the Orai1-expressing and in the control cells. We propose that excess Orai1 uncouples SERCA from Ca2+ entry in the intact cell by disturbing the fine topology of Ca2+ pumping complexes within the ER-plasma membrane junctions., This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICINN, BFI2007-60157 and SAF2009- 03175-E) and the Instituto de Salud Carlos III. IMM was supported by a fellowship from the Spanish MICINN.

Cytosolic Ca2+ signals are followed by mitochondrial Ca2+ uptake, which, in turn, modifies several biological processes. Mg2+ is known to inhibit Ca2+ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca2+ signals associated with parallel changes in cytosolic [Mg2+]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca2+ influx on cytosolic Mg2+ concentration ([Mg2+]c) was negligible. Uncaged Ca2+ displaced Mg2+ from cytosolic binding sites, but for an equivalent Ca2+ signal, the change in [Mg2+] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca2+ and Mg2+ from internal stores in permeabilized cells. The increase of [Mg2+] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca2+ uptake in permeabilized cells without affecting mitochondrial Ca2+ efflux. Therefore, the Mg2+ signal generated by Ca2+ mobilizing agonists may attenuate mitochondrial Ca2+ uptake.

et al., Simultaneous control of different functions by calcium signals is possible because of subcellular compartmentalization. Targeted chemiluminescent Ca 2+ probes, such as aequorins (AEQs) are optimal for detecting signals originating in different subcellular domains, but imaging is difficult because of low photon yield causing poor spatiotemporal resolution. To overcome this problem, we have co-expressed two spectrally distinct AEQs in different subcellular locations within the same cells. Seven chimeric proteins containing either green- or red-emitting AEQs, with different targeting sequences and Ca2+ affinities, have been designed and tested. We show here evidence for physical and functional independence of the different probes. Cytosolic Ca2+ signals were mirrored in the nucleus, but amplified inside mitochondria and endoplasmic reticulum, and had different time courses in the various locations. Our results demonstrate that these novel tools permit simultaneous and independent monitoring of [Ca2+] in different subcellular domains of the same cell. © 2007 Springer-Verlag., We thank the Spanish Ministerio de Educación y Ciencia (MEC; BFU2004-02765/BFI, and BFU2005-02078) and Junta de Castilla y León (VA-088/A06) for financial support. IMMand BD held predoctoral fellowships from MEC and Junta de Comunidades de Castilla-La Mancha, respectively.

Financial support from Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Red de Terapia Celular, from Fundación de Investigación Médica de la Mutua Madrileña and from Ministerio de Educación y Ciencia, Grant BFU2004-0265, is gratefully acknowledged.

Changes in the cytosolic Ca2+ concentration ([Ca2+]c) are essential for triggering neurotransmitter release from presynaptic nerve terminals. Calcium-induced Ca2+ release (CICR) from the endoplasmic reticulum (ER) may amplify the [Ca2+]c signals and facilitate neurotransmitter release in sympathetic neurons. In adrenal chromaffin cells, functional triads are formed by voltage-operated Ca2+ channels (VOCCs), CICR sites and mitochondria. In fact, mitochondria take up most of the Ca2+ load entering the cells and are essential for shaping [Ca2+]c signals and exocytosis. Here we have investigated the existence of such functional triads in sympathetic neurons. The mitochondrial Ca2+ concentration ([Ca2+]m) in soma and neurites of individual mouse superior cervical ganglion (SCG) neurons was monitored by bioluminescence imaging of targeted aequorins. In soma, Ca2+ entry through VOCCs evoked rapid, near millimolar [Ca2+]m increases in a subpopulation of mitochondria containing about 40% of the aequorin. Caffeine evoked a similar [Ca2+]m increase in a mitochondrial pool containing about 30% of the aequorin and overlapping with the VOCC-sensitive pool. These observations suggest the existence of functional triads similar to the ones described in chromaffin cells. In neurites, mitochondria were able to buffer [Ca2+]c increases resulting from activation of VOCCs but not those mediated by caffeine-induced Ca2+ release from the ER. The weaker Ca2+ buffering by mitochondria in neurites could contribute to facilitate Ca2+-induced exocytosis at the presynaptic sites.

We have recently described a new class of genetically encoded Ca(2+) indicators composed of two jellyfish proteins, a variant of green fluorescent protein (GFP) and the calcium binding protein apoaequorin, named GAP (Rodriguez-García et al., 2014). GAP is a unique dual-mode Ca(2+) indicator, able to function either as a fluorescent or a luminescent probe, depending on whether the photoprotein aequorin is in its apo-state or reconstituted with its cofactor coelenterazine. We describe here a novel application of GAP as a low affinity bioluminescent indicator, suitable for measurements of [Ca(2+)] in ER or in Golgi apparatus. We used the low affinity variant, GAP1, which carries mutations in two EF-hands of aequorin, reconstituted with coelenterazine n. In comparison to previous bioluminescent aequorin fusions, the decay rate of GAP1 was decreased 8 fold and the affinity for Ca(2+) was lowered one order of magnitude. This improvement allows long-term measurements in high Ca(2+) environments avoiding fast aequorin consumption. GAP1 was targeted to the ER of various cell types, where it monitored resting Ca(2+) concentrations in the range from 400 to 600 μM. ER could be emptied of calcium by stimulation with ATP, carbachol or histamine in intact cells, and by challenge with inositol tris-phosphate in permeabilized cells. GAP1 was also targeted to the Golgi apparatus where it was able to precisely monitor long-term calcium dynamics. GAP1 provides a novel and robust indicator applicable to bioluminescent high-throughput quantitative assays.

Producción Científica, We have recently described a new class of genetically encoded Ca2+ indicators composed of two jellyfish proteins, a variant of green fluorescent protein (GFP) and the calcium binding protein apoaequorin, named GAP (Rodriguez-García et al., 2014). GAP is a unique dual-mode Ca2+ indicator, able to function either as a fluorescent or a luminescent probe, depending on whether the photoprotein aequorin is in its apo-state or reconstituted with its cofactor coelenterazine. We describe here a novel application of GAP as a low affinity bioluminescent indicator, suitable for measurements of [Ca2+] in ER or in Golgi apparatus. We used the low affinity variant, GAP1, which carries mutations in two EF-hands of aequorin, reconstituted with coelenterazine n. In comparison to previous bioluminescent aequorin fusions, the decay rate of GAP1 was decreased 8 fold and the affinity for Ca2+ was lowered one order of magnitude. This improvement allows long-term measurements in high Ca2+ environments avoiding fast aequorin consumption. GAP1 was targeted to the ER of various cell types, where it monitored resting Ca2+ concentrations in the range from 400 to 600 μM. ER could be emptied of calcium by stimulation with ATP, carbachol or histamine in intact cells, and by challenge with inositol tris-phosphate in permeabilized cells. GAP1 was also targeted to the Golgi apparatus where it was able to precisely monitor long-term calcium dynamics. GAP1 provides a novel and robust indicator applicable to bioluminescent high-throughput quantitative assays., Ministerio de Economía, Industria y Competitividad (Project BFU2014-534698P), Instituto de Salud Carlos III (RD06/0010/0000 and RD12/0019/0036)

High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. In addition, the Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis/cis-to-medial-Go (cGo) or the trans-Go (tGo). Ca2+ homoeostasis in these compartments and in the endoplasmic reticulum (ER) has been studied and compared. Moreover, the relative size of each subcompartment was estimated from aequorin consumption. We found that the cGo accumulates Ca2+ to high concentrations (150-300 μM) through the sarco plasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). The tGo, in turn, is divided into two subcompartments: tGo1 and tGo2. The subcompartment tGo1 contains 20% of the aequorin and has a high internal [Ca2+]; Ca2+ is accumulated in this subcompartment via the secretory pathway Ca2+-ATPase 1 (SPCA-1) at a very high affinity (K50=30 nM). The subcompartment tGo2 contains 80% of aequorin, has a lower [Ca2+] and no SPCA-1 activity; Ca2+ uptake happens through SERCA and is slower than in tGo1. The two tGo subcompartments, tGo1 and tGo2, are diffusionally isolated. Inositol trisphosphate mobilizes Ca2+ from the cGo and tGo2, but not from tGo1, whereas caffeine releases Ca2+ from all the Golgi regions, and nicotinic acid dinucleotide phosphate and cADP ribose from none., This work was supported by a grant from the Spanish Ministerio de Economía y Competitividad (MEC) [grant numbers BFU2010–17379] and the Instituto de Salud Carlos III [grant numbers RD06/0010/0000 and RD12/0019/0036]. F.J.A. was supported by an FPI fellowship from MEC.

[Objective]: To evaluate outcomes of cultivated limbal epithelial transplantation (CLET) for management of ocular surface failure due to limbal stem cell deficiency (LSCD). [Design]: Prospective, noncomparative, interventional case series and extensive comparison with recent similar studies. [Participants]: Twenty eyes with LSCD underwent CLET (11 autologous; 9 allogeneic) and were followed up for 3 years. Etiologies were divided into 3 prognostic categories: Group 1, chemical injuries (7 eyes); Group 2, immune-based inflammation (4 eyes); and Group 3, noninflammatory diseases (9 eyes). [Intervention]: Autologous and allogeneic limbal epithelial cells were cultivated on amniotic membranes and transplanted. Evaluations were based on clinical parameters, survival analysis, and in vivo confocal microscopy (IVCM). European Union Tissues/Cells Directive and good manufacturing procedures were followed. [Main Outcome Measures]: Improved clinical parameters, absence of epithelial defects, and improved central corneal epithelial phenotype. [Results]: Success rate was 80% at 1-2 years and 75% at 3 years. Autografts and allografts had similar survival. Success rate was significantly lower in prognostic Group 1 (42.9%) than in Groups 2-3 (100% each). All clinical parameters improved substantially. By IVCM, 80% of cases improved in epithelial status. [Conclusions]: CLET improved corneal epithelium quality, with subsequent improvement in symptoms, quality of life, and vision. These results confirm that CLET is a valid therapy for ocular surface failure., This study received a financial support from the Advanced Therapies Program (SAS/2481/2009), Ministry of Health, Spain; Regional Center for Regenerative Medicine and Cell Therapy, SAN 1178/200, Castilla y León, Spain. Beatriz E. Ramírez held a predoctoral scholarship from The Carolina Foundation, Ministry of Foreign Affairs, Spain.

Endomembranes modify the progression of the cytosolic Ca2+ wave and contribute to generate Ca2+ microdomains, both in the cytosol and inside the own organella. The concentration of Ca2+ in the cytosol ([Ca2+]C), the mitochondria ([Ca2+]M) and the nucleus ([Ca2+]N) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca2+ from the high [Ca2+]C microdomains generated during cell activation near Ca2+ channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca2+ signal to the bulk cytosol. This shaping of [Ca2+]C signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca2+]M signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca2+]C signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca2+]N signals could be generated by direct release of stored Ca2+ into the nucleoplasm. Ca2+ release could even be restricted to subnuclear domains. Putative Ca2+ stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca2+ into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain. © 2006 Elsevier Ltd. All rights reserved., This work was funded by grants from the Ministerio de Educación y Ciencia (BFU2004-02765/BFI and BFU2005-05464) and from Junta de Castilla y León (VA016A05). Pablo Chamero holded a predoctoral fellowship from the Basque government.

Simple ex vivo or in vitro models are most useful for testing putative cell therapy protocols, as they allow quick and controlled screening of variants and possible improvements. We discuss here three different models: coculture of precursors of human bone marrow cells (BMCs) with mouse heart slices bearing a cryogenic lesion; coculture of human BMCs and rat cardiomyocytes separated by a porous membrane that allows passage of soluble substances but prevents migration of nuclear material; and injection of human BMCs in developing chick heart bearing burn lesions. Our results indicate that the damaged areas express specific genes such as MPC1 and SDF1, and that some human BMCs migrate and graft near the lesion, where they can originate cells with a cardiac phenotype that produce human cardiac proteins. The frequency of this transformation is, however, very low. Understanding the factors that determine and regulate nuclear reprogramming and transdifferentiation would be crucial to appraising the contribution of these phenomena to cardiac regeneration and, eventually, to modulating them with therapeutic intent.

Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx pathway involved in control of multiple cellular and physiological processes including cell proliferation. Recent evidence has shown that SOCE depends critically on mitochondrial sinking of entering Ca2+ to avoid Ca2+-dependent inactivation. Thus, a role of mitochondria in control of cell proliferation could be anticipated. We show here that activation of SOCE induces cytosolic high [Ca2+] domains that are large enough to be sensed and avidly taken up by a pool of nearby mitochondria. Prevention of mitochondrial clearance of the entering Ca2+ inhibited both SOCE and cell proliferation in several cell types including Jurkat and human colon cancer cells. In addition, we find that therapeutic concentrations of salicylate, the major metabolite of aspirin, depolarize partially mitochondria and inhibit mitochondrial Ca2+ uptake, as revealed by mitochondrial Ca2+ measurements with targeted aequorins. This salicylate-induced inhibition of mitochondrial Ca2+ sinking prevented SOCE and impaired cell growth of Jurkat and human colon cancer cells. Finally, direct blockade of SOCE by the pyrazole derivative BTP-2 was sufficient to arrest cell growth. Taken together, our results reveal that cell proliferation depends critically on mitochondrial Ca2+ uptake and suggest that inhibition of tumour cell proliferation by salicylate may be due to interference with mitochondrial Ca2+ uptake, which is essential for sustaining SOCE. This novel mechanism may contribute to explaining the reported anti-proliferative and anti-tumoral actions of aspirin and dietary salicylates.

Functional expression of GFP-VR1 Ca2+ channel. Single-cell [Ca2+]c image kinetic analysis of short pulse-treated cells expressing GFP-VR1. Transiently transfected JHM12 cells were loaded with fura 4F and subjected to digital imaging fluorescence microscopy, and [Ca2+]c images were obtained as indicated under “Experimental Procedures.” The first color frame (GFP) corresponds to the green fluorescence image, and subsequent ratiometric images for [Ca2+]c were recorded every 5 s. Representative frames corresponding to the different [Ca2+]c peaks were taken at the indicated time points . [Ca2+]c profiles were averaged for the two cell populations (GFP-VR1+, n = 45; GFP-VR1−, n = 62) and represented at the bottom of the Fig. 2 In Jambrina et al.. Cells were superfused during the indicated periods with the different solutions containing the VR1 agonist capsaicin (0.1 μm, CAPS) and the HM1R agonist carbachol (50 μm, CBCh), in the presence or the absence (Ca2+0) of calcium in the external medium. The capsaicin-sensitive cells corresponded to cells expressing GFP-VR1, as assessed by superimposition of fluorescence images excited at 490 nm (to visualize GFP) and the ratiometric Ca2+ images (fura-4F, excited at 340 and 380 nm; see upper panels in Fig. 2 A of Jambrina et al.). Color code included in Fig. 2; Blue represents 0 calcium whereas red is high calcium., We address the specific role of cytoplasmic Ca2+ overload as a cell death trigger by expressing a receptor-operated specific Ca2+ channel, vanilloid receptor sub-type 1 (VR1), in Jurkat cells. Ca2+ uptake through the VR1 channel, but not capacitative Ca2+ influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca2+] rises, exposure of phosphatidylserine, and cell death. Ca2+ influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca2+induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca2+ influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis., This work was supported by grants from Ministerio de Ciencia y Tecnología/European Union and Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (FEDER, 1FD97-1725-C02-01 and C02-02) and Comisión Interministerial de Ciencia y Tecnología, Spain, Grant SAF2000-0118-C03-02).

In the last years, intracellular organella have emerged as key components in the generation and transduction of Ca(2+) signals in adrenal chromaffin cells. Therefore, accurate measurements of Ca(2+) inside cytoplasmic organella are essential for a comprehensive analysis of the Ca(2+) redistribution that follows cell stimulation. We have engineered the Ca(2+)-sensitive photoprotein aequorin to monitor selectively Ca(2+) within the endoplasmic reticulum and the mitochondria. The targeted aequorins were delivered to the appropriate organelles of bovine chromaffin cells by using a herpes simplex virus-based amplicon vector, which permits efficient gene transfer and high levels of expression in infected cells. We have investigated the relationship between the caffeine and InsP(3)-sensitive Ca(2+) pools and the presence of the Ca(2+)-induced Ca(2+) release (CICR) mechanism in chromaffin cells. We find that ER Ca(2+) pools responding to caffeine and to InsP(3) mostly overlap and that CICR can be induced by Ca(2+) entry elicited by high K(+) depolarization. Moreover, the activation of Ca(2+) channels, either the voltage-gated Ca(2+) channels on the plasma membrane or the channels on the endoplasmic reticulum (ER), generates subplasmalemmal high [Ca(2+)](c) domains that induce Ca(2+) uptake by mitochondria. Interestingly, only a subpopulation of mitochondria, the one contained in the pool located close to the plasma membrane and the ryanodine receptors, take up Ca(2+) efficiently, and the [Ca(2+)](M) reaches values of 300-500 micro M.

Treatment of GH3 pituitary cells with p-chloromercurybenzenesulfonate (PCMBS) increased the cytosolic Ca2+ concentration ([Ca2+]i). This effect was reversed by dithiothreitol and blocked by L-type Ca2+ channel antagonists or Na+ removal. PCMBS increased membrane conductance and depolarized the plasma membrane. Apart from minor effects on K+ and Ca2+ channels, PCMBS increased (6 times at −80 mV) an inward Na+ current whose properties were similar to those of a background Na+conductance (BNC) described previously, necessary for generation of spontaneous electrical activity. In rat lactotropes and somatotropes in primary culture, PCMBS also produced a Na+-dependent [Ca2+]i increase, whereas little or no effect was observed in thyrotropes, corticotropes, and gonadotropes. The Na+ conductance elicited by PCMBS in somatotropes seemed to be the same as that stimulated by the hypothalamic growth hormone (GH)-releasing hormone, which regulates membrane excitability and GH secretion. The BNC studied here could play a physiological role, regulating excitability and spontaneous activity, and explains satisfactorily the [Ca2+]i-increasing actions of the mercurials reported previously in several excitable tissues.

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca(2+) microdomains described previously.

Genetically encoded calcium indicators allow monitoring subcellular Ca2+ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners.We describe here a novel family of fluorescent Ca2+ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg2+, tunable Ca2+ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca2+ indicators for organelles and becomes a valuable tool for in vivo Ca2+ imaging applications., This work was supported by grants from the European Research Area Net (ERA-Net) program, the Spanish Ministerio de Economía y Competitividad (SAF2008-03175-E, BFU2010-17379), and the Instituto de Salud Carlos III (RD06/0010/0000).

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca 2+ signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy. In contrast, ER was sparse in the cortex and more abundant in deep cytoplasmic regions. The mitochondrial distribution might be due to organellar transport, which experiences increasing restrictions in the cell cortex. Further study of organelle distribution in relation to the position of SNARE microdomains and the granule fusion sites revealed that a third of the cortical mitochondria colocalized with exocytotic sites and another third located at a distance closer than two vesicle diameters. ER structures were also present in the vicinity of secretory sites but at a lower density. Therefore, mitochondria and ER have a spatial distribution that suggests a specialized role in modulation of exocytosis that fits with the role of cytosolic Ca 2+ microdomains described previously.