In JoVE (1)

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Articles by Roland Malli in JoVE

 JoVE Biology

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

1Institute of Molecular Biology and Biochemistry, Medical University of Graz

JoVE 55486

Other articles by Roland Malli on PubMed

Oxidized Phospholipids Stimulate Tissue Factor Expression in Human Endothelial Cells Via Activation of ERK/EGR-1 and Ca(++)/NFAT

Blood. Jan, 2002  |  Pubmed ID: 11756172

Activation of endothelial cells by lipid oxidation products is a key event in the initiation and progression of the atherosclerotic lesion. Minimally modified low-density lipoprotein (MM-LDL) induces the expression of certain inflammatory molecules such as tissue factor (TF) in endothelial cells. This study examined intracellular signaling pathways leading to TF up-regulation by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a biologically active component of MM-LDL. OxPAPC induced TF activity and protein expression in human umbilical vein endothelial cells (HUVECs). However, OxPAPC neither induced phosphorylation or degradation of I kappa B alpha nor DNA binding of nuclear factor-kappa B (NF-kappa B). Furthermore, OxPAPC-induced TF expression was not inhibited by overexpression of I kappa B alpha. These results strongly indicate that OxPAPC-induced TF expression is independent of the classical NF-kappa B pathway. However, OxPAPC stimulated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and expression of early growth response factor 1 (EGR-1). Inhibitors of mitogen-activated kinase/ERK (MEK) or protein kinase C (PKC) blocked elevation of both EGR-1 and TF. Furthermore, overexpression of NAB2, a corepressor of EGR-1, inhibited effects of OxPAPC. In addition, OxPAPC induced rapid and reversible elevation of free cytosolic Ca(++) levels and nuclear factor of activated T cells (NFAT)/DNA binding. Induction of TF expression by OxPAPC was partially inhibited by cyclosporin A, known to block calcineurin, a Ca(++)-dependent phosphatase upstream of NFAT. Treatment of OxPAPC with phospholipase A(2) destroyed its biologic activity and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine was identified as one biologically active component of OxPAPC that induces TF expression. Together, the results demonstrate that OxPAPC induces TF expression in HUVECs through activation of PKC/ERK/EGR-1 and Ca(++)/calcineurin/NFAT pathways rather than by NF-kappa B-mediated transcription. Thus, oxidized phospholipids may contribute to inflammation by activating pathways alternative to the classical NF-kappa B pathway.

Tissue-specific Expression of Human Lipoprotein Lipase in the Vascular System Affects Vascular Reactivity in Transgenic Mice

British Journal of Pharmacology. Jan, 2002  |  Pubmed ID: 11786490

1. The role of smooth muscle-derived lipoprotein lipase (LPL) that translocates to the endothelium surface on vascular dysfunction during atherogenesis is unclear. Thus, the role of vascular LPL on blood vessel reactivity was assessed in transgenic mice that specifically express human LPL in the circulatory system. 2. Aortic free fatty acids (FFAs) were increased by 69% in the transgenic mice expressing human LPL in aortic smooth muscle cells (L2LPL) compared with their non-transgenic littermates (L2). 3. Contractility to KCl was increased by 33% in aortae of L2LPL mice. Maximal contraction to phenylephrine (PE) was comparable in L2 and L2LPL animals, while the frequency of tonus oscillation to PE increased by 104% in L2LPL mice. 4. In L2LPL animals, *NO mediated relaxation to acetylcholine (ACh) and ATP was reduced by 47 and 32%, respectively. In contrast, endothelium-independent relaxation to sodium nitroprusside (SNP) was not different in both groups tested. 5. ATP-initiated Ca(2+) elevation that triggers *NO formation was increased by 41% in single aortic endothelial cells freshly isolated from L2LPL animals. 6. In aortae from L2LPL mice an increased *O(2)(-) release occurred that was normalized by removing the endothelium and by the NAD(P)H oxidase inhibitor DPI and the PKC inhibitor GF109203X. 7. The reduced ACh-induced relaxation in L2LPL animals was normalized in the presence of SOD, indicating that the reduced relaxation is due, at least in part, to enhanced *NO scavenging by *O(2)(-). 8. These data suggest that despite normal lipoprotein levels increased LPL-mediated FFAs loading initiates vascular dysfunction via PKC-mediated activation of endothelial NAD(P)H oxidase. Thus, vascular LPL activity might represent a primary risk factor for atherosclerosis independently from cholesterol/LDL levels.

Subplasmalemmal Endoplasmic Reticulum Controls K(Ca) Channel Activity Upon Stimulation with a Moderate Histamine Concentration in a Human Umbilical Vein Endothelial Cell Line

The Journal of Physiology. Apr, 2002  |  Pubmed ID: 11927670

This study was designed to elucidate the role of the subplasmalemmal endoplasmic reticulum (sER) in autacoid-induced stimulation of Ca(2+)-dependent K(+) channels in the umbilical vein endothelial cell-derived cell line EA.hy926. Cells were transfected with the Ca(2+) probe cameleon targeted to the ER for visualization of the ER network. A patch pipette was then placed close to or far (> 5 microm away) from the sER, single channel recordings (patch clamp technique) were monitored simultaneously with measurements of either ER Ca(2+) concentration (using the Ca(2+) probe Cam4-ER) or cytosolic free Ca(2+) concentration ([Ca(2+)](i); using fura-2) using a deconvolution imaging device. A voltage-dependent, large conductance Ca(2+)-dependent K(+) channel (BK(Ca); single channel conductance (gamma), 250 pS) was found. At membrane potentials of +40 and -40 mV, the EC(50) for Ca(2+) was 2.7 and 49.7 microM, respectively. In the vicinity of the sER, the BK(Ca) channel activity induced by 10 microM histamine was 32 times higher (open probability (P(o)) = 0.083 +/- 0.026) than in areas away from the sER (P(o) = 0.0026 +/- 0.002). However, at supramaximal histamine stimulation (100 microM), BK(Ca) channel activation was similar in patches in the vicinity of or away from the sER (P(o) = 0.18 +/- 0.09 and 0.25 +/- 0.07, respectively). In contrast to BK(Ca) channel activity, ER Ca(2+) depletion (Cam4-ER) and elevation of [Ca(2+)](i) in response to 10 and 100 microM histamine were not influenced by the pipette position. We conclude that in endothelial cells, the activation of BK(Ca) channels in response to moderate histamine concentration essentially depends on the proximity of the sER domains to the mouth of this K(+) channel. These findings further support our concept of the subplasmalemmal Ca(2+) control unit (SCCU) and add the local activation of Ca(2+)-activated K(+)-channels to the function of the SCCU.

Functional Analysis of Histamine Receptor Subtypes Involved in Endothelium-mediated Relaxation of the Human Uterine Artery

Clinical and Experimental Pharmacology & Physiology. Aug, 2002  |  Pubmed ID: 12100006

1. This work was designed to introduce human uterine arteries as a new model for cardiovascular research. Advantages of this model include considerable availability of tissue because of the appearance of uterus myomatosus in post-menopausal women who undergo surgery and the chance to work on dysfunctional and healthy vessels. 2. Histamine evoked relaxation of the uterine artery that was prevented by removal of the endothelium or by the presence of N(G)-nitro-L-arginine. 3. Receptor antagonists for histamine H(1) (mepyramine) and H(2) (ranitidine) receptors increased the EC(50) of histamine by 112- and 67-fold, respectively. 4. Remarkably, isolated uterine arteries could be stored in incubators for 5 days without any change in contractility to phenylephrine and endothelium-dependent relaxation to acetylcholine and histamine. 5. Endothelial cells could be isolated and cultured in high purity, as demonstrated by histochemical staining of factor VIII, low CD45-RO for macrophages and no smooth muscle alpha-actin. In addition, cultured human uterine artery endothelial cells could be used for single cell Ca(2+) measurements. 6. In agreement with our findings in the intact vessel, histamine-initiated elevation of the intracellular free Ca(2+) concentration was reduced in the presence of mepyramine and ranitidine by 59 and 55%, respectively. 7. These data indicate that, in the human uterine artery, H(1) and H(2) receptors are involved in histamine-induced endothelium-dependent relaxation that is mediated by nitric oxide. 8. In addition, this vessel can be stored for possible virus-mediated gene expression for 5 days without any loss of reagibility. 9. Finally, endothelial cells can be isolated and cultured from the human uterine artery and maintain their reactivity to histamine in culture.

Nitric Oxide Inhibits Capacitative Ca2+ Entry by Suppression of Mitochondrial Ca2+ Handling

British Journal of Pharmacology. Nov, 2002  |  Pubmed ID: 12411413

1. Nitric oxide (NO) is a key modulator of cellular Ca(2+) signalling and a determinant of mitochondrial function. Here, we demonstrate that NO governs capacitative Ca(2+) entry (CCE) into HEK293 cells by impairment of mitochondrial Ca(2+) handling. 2. Authentic NO as well as the NO donors 1-[2-(carboxylato)pyrrolidin-1-yl]diazem-1-ium-1,2-diolate (ProliNO) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) suppressed CCE activated by thapsigargin (TG)-induced store depletion. Threshold concentrations for inhibition of CCE by ProliNO and DEANO were 0.3 and 1 micro M, respectively. 3. NO-induced inhibition of CCE was not mimicked by peroxynitrite (100 micro M), the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1, 100 micro M) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 1 mM). In addition, the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a] quinoxalin-1-one (ODQ, 30 micro M) failed to antagonize the inhibitory action of NO on CCE. 4. DEANO (1-10 micro M) suppressed mitochondrial respiration as evident from inhibition of cellular oxygen consumption. Experiments using fluorescent dyes to monitor mitochondrial membrane potential and mitochondrial Ca(2+) levels, respectively, indicated that DEANO (10 micro M) depolarized mitochondria and suppressed mitochondrial Ca(2+) sequestration. The inhibitory effect of DEANO on Ca(2+) uptake into mitochondria was confirmed by recording mitochondrial Ca(2+) during agonist stimulation in HEK293 cells expressing ratiometric-pericam in mitochondria. 5. DEANO (10 micro M) failed to inhibit Ba(2+) entry into TG-stimulated cells when extracellular Ca(2+) was buffered below 1 micro M, while clear inhibition of Ba(2+) entry into store depleted cells was observed when extracellular Ca(2+) levels were above 10 micro M. Moreover, buffering of intracellular Ca(2+) by use of N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)] bis [N-[25-[(acetyloxy) methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA/AM) eliminated inhibition of CCE by NO, indicating that the observed inhibitory effects are based on an intracellular, Ca(2+) dependent-regulatory process. 6. Our data demonstrate that NO effectively inhibits CCE cells by cGMP-independent suppression of mitochondrial function. We suggest disruption of local Ca(2+) handling by mitochondria as a key mechanism of NO induced suppression of CCE.

Novel High Energy Intermediate Analogues with Triazasterol-related Structures As Inhibitors of Ergosterol Biosynthesis. Part I: Synthesis and Antifungal Activity of N-alkyl-N'-(phenethyl- and Cyclohexenylethyl)guanidines and N2-substituted 2-imidazolinamines

Archiv Der Pharmazie. 2002  |  Pubmed ID: 12596218

A series of N-alkyl-N'-(phenethyl- and cyclohexenylethyl) guanidines and N(2)- and N(2), 4-substituted imidazolin-2-amine hydrochlorides with triazasterol-related structures was designed and synthesized as stable analogues to mimic high energy intermediates of ergosterol biosynthesis. The in vitro antifungal susceptibility tests with a standard panel of pathogenic fungi revealed moderate to strong antimycotic effects of the sixteen prepared compounds, in some cases comparable with the activity observed for itraconazole.

Mitochondria Efficiently Buffer Subplasmalemmal Ca2+ Elevation During Agonist Stimulation

The Journal of Biological Chemistry. Mar, 2003  |  Pubmed ID: 12529366

In endothelial cells, local Ca(2+) release from superficial endoplasmic reticulum (ER) activates BK(Ca) channels. The resulting hyperpolarization promotes capacitative Ca(2+) entry (CCE), which, unlike BK(Ca) channels, is inhibited by high Ca(2+). To understand how the coordinated activation of plasma membrane ion channels with opposite Ca(2+) sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca(2+) concentration ([Ca(2+)](pm)) was investigated. For organelle visualization, cells were transfected with DsRed and yellow cameleon targeted to mitochondria and ER. The patch pipette was placed far from any organelle (L1), close to ER (L3), or mitochondria (L2) and activity of BK(Ca) channels was used to estimate local [Ca(2+)](pm). Under standard patch conditions (130 mm K(+) in the bath), histamine increased [Ca(2+)](pm) at L1 and L3 to approximately 1.6 microm, whereas close to mitochondria [Ca(2+)](pm) remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca(2+)](pm) at L2 increased in response to histamine. Under standard patch conditions Ca(2+) entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mm K(+) in the bath), changes in [Ca(2+)](pm) to histamine could be monitored without cell depolarization and, thus, in conditions where Ca(2+) entry occurred. Here, histamine induced an initial transient Ca(2+) elevation to > or =3.5 microm followed by a long lasting plateau at approximately 1.2 microm in L1 and L3, whereas mitochondria kept neighboring [Ca(2+)](pm) low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca(2+) allowing simultaneous activation of BK(Ca) and CCE, despite their opposite Ca(2+) sensitivity.

Sustained Ca2+ Transfer Across Mitochondria is Essential for Mitochondrial Ca2+ Buffering, Sore-operated Ca2+ Entry, and Ca2+ Store Refilling

The Journal of Biological Chemistry. Nov, 2003  |  Pubmed ID: 12941956

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.

Anandamide Initiates Ca(2+) Signaling Via CB2 Receptor Linked to Phospholipase C in Calf Pulmonary Endothelial Cells

British Journal of Pharmacology. Dec, 2003  |  Pubmed ID: 14645143

The endocannabinoid anandamide has been reported to affect neuronal cells, immune cells and smooth muscle cells via either CB1 or CB2 receptors. In endothelial cells, the receptors involved in activating signal transduction are still unclear, despite the fact that anandamide is produced in this cell type. The present study was designed to explore in detail the effect of this endocannabinoid on Ca2+ signaling in single cells of a calf pulmonary endothelial cell line. Anandamide initiated a transient Ca2+ elevation that was prevented by the CB2 receptor antagonist SR144528, but not by the CB1 antagonist SR141716A. These data were confirmed by molecular identification of the bovine CB2 receptor in these endothelial cells by partial sequencing. The phospholipase C inhibitor 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5dione and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenylborate prevented Ca2+ signaling in response to anandamide. Using an improved cameleon probe targeted to the endoplasmic reticulum (ER), fura-2 and ratiometric-pericam, which is targeted to the mitochondria, anandamide was found to induce Ca2+ depletion of the ER accompanied by the activation of capacitative Ca2+ entry (CCE) and a transient elevation of mitochondrial Ca2+. These data demonstrate that anandamide stimulates the endothelial cells used in this study via CB2 receptor-mediated activation of phospholipase C, formation of inositol 1,4,5-trisphosphate, Ca2+ release from the ER and subsequent activation of CCE. Moreover, the cytosolic Ca2+ elevation was accompanied by a transient Ca2+ increase in the mitochondria. Thus, in addition to its actions on smooth muscle cells, anandamide also acts as a powerful stimulus for endothelial cells.

Heterozygous Missense Mutations in BSCL2 Are Associated with Distal Hereditary Motor Neuropathy and Silver Syndrome

Nature Genetics. Mar, 2004  |  Pubmed ID: 14981520

Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy (OMIM #182960) is a heterogeneous group of disorders characterized by an almost exclusive degeneration of motor nerve fibers, predominantly in the distal part of the limbs. Silver syndrome (OMIM #270685) is a rare form of hereditary spastic paraparesis mapped to chromosome 11q12-q14 (SPG17) in which spasticity of the legs is accompanied by amyotrophy of the hands and occasionally also the lower limbs. Silver syndrome and most forms of dHMN are autosomal dominantly inherited with incomplete penetrance and a broad variability in clinical expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed linkage to the locus SPG17, which was confirmed in 16 additional families with a phenotype characteristic of dHMN or Silver syndrome. After refining the critical region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy (BSCL2) and identified two heterozygous missense mutations resulting in the amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes the protein seipin, were previously shown to be associated with autosomal recessive Berardinelli-Seip congenital lipodystrophy (OMIM #269700). We show that seipin is an integral membrane protein of the endoplasmic reticulum (ER). The amino acid substitutions N88S and S90L affect glycosylation of seipin and result in aggregate formation leading to neurodegeneration.

Kisspeptin-10, a KiSS-1/metastin-derived Decapeptide, is a Physiological Invasion Inhibitor of Primary Human Trophoblasts

Journal of Cell Science. Mar, 2004  |  Pubmed ID: 15020672

Trophoblast invasion of the uterine extracellular matrix, a critical process of human implantation and essential for fetal development, is a striking example of controlled invasiveness. To identify molecules that regulate trophoblast invasion, mRNA signatures of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared using U95A GeneChip microarrays yielding 220 invasion/migration-related genes. In this 'invasion cluster', KiSS-1 and its G-protein-coupled receptor KiSS-1R were expressed at higher levels in first trimester trophoblasts than at term of gestation. Receptor and ligand mRNA and protein were localized to the trophoblast compartment. In contrast to KiSS-1, which is only expressed in the villous trophoblast, KiSS-1R was also found in the extravillous trophoblast, suggesting endocrine/paracrine activation mechanisms. The primary translation product of KiSS-1 is a 145 amino acid polypeptide (Kp-145), but shorter kisspeptins (Kp) with 10, 13, 14 or 54 amino acid residues may be produced. We identified Kp-10, a dekapeptide derived from the primary translation product, in conditioned medium of first trimester human trophoblast. Kp-10, but not other kisspeptins, increased intracellular Ca(2+) levels in isolated first trimester trophoblasts. Kp-10 inhibited trophoblast migration in an explant as well as transwell assay without affecting proliferation. Suppressed motility was paralleled with suppressed gelatinolytic activity of isolated trophoblasts. These results identified Kp-10 as a novel paracrine/endocrine regulator in fine-tuning trophoblast invasion generated by the trophoblast itself.

Hyperglycemic Conditions Affect Shape and Ca2+ Homeostasis of Mitochondria in Endothelial Cells

Journal of Cardiovascular Pharmacology. Oct, 2004  |  Pubmed ID: 15454850

In this study the contribution of alternating architecture and Ca2+ handling of mitochondria to cytosolic Ca2+ homeostasis was elucidated under normoglycemic and hyperglycemic (HGC) conditions in the human endothelial cell line EA.hy926. Exposure of endothelial cells to hyperglycemic medium elevated basal cytosolic free Ca2+ concentration ([Ca2+]cyto), the histamine-initiated cytosolic Ca2+ signaling, and the mitochondrial Ca2+ content after cell stimulation. The latter was possibly due to the prolonged mitochondrial Ca2+ elevation in response to agonists found in HGC-pretreated cells. Moreover, under HGC mitochondrial free radical production was increased and mitochondrial shape changed from a mainly tubular, highly interconnected network toward multiple, isolated singular structures. Such changes could not be correlated with HGC-induced alterations of cytosolic Ca2+ signaling that became normalized with antimycin A, an inhibitor of the respiratory chain. These data suggest that although mitochondrial structure changes considerably during HGC, alterations in cytosolic Ca2+ signaling are more likely due to the enhanced energy status/metabolism of the mitochondria. On the other hand, in normoglycemic cells of unforced fragmentation of mitochondria yielded elevated basal [Ca2+]cyto, while the global Ca2+ signaling in response to histamine remained unchanged. Thus, mitochondrial architecture (ie, tubular versus fragmented structure) per se does not have a detectable impact on agonist-initiated global cytosolic Ca2+ signaling, while this organelle represents an early target in hyperglycemia leading to alterations in cytosolic Ca2+ signaling.

Twenty Years of Calcium Imaging: Cell Physiology to Dye For

Molecular Interventions. Apr, 2005  |  Pubmed ID: 15821159

The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca(2+) in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca(2+) signaling is accomplished. In excitable cells, the coordination of changing Ca(2+) concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca(2+) signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes.

A New Type of Non-Ca2+-buffering Apo(a)-based Fluorescent Indicator for Intraluminal Ca2+ in the Endoplasmic Reticulum

The Journal of Biological Chemistry. Feb, 2006  |  Pubmed ID: 16368693

Genetically encoded Ca2+ indicators are outstanding tools for the assessment of intracellular/organelle Ca2+ dynamics. Basically, most indicators contain the Ca2+-binding site of a (mutated) cytosolic protein that interacts with its natural (mutated) interaction partner upon binding of Ca2+. Consequently, a change in the structure of the sensor occurs that, in turn, alters the fluorescent properties of the sensor. Herein, we present a new type of genetically encoded Ca2+ indicator for the endoplasmic reticulum (ER) (apoK1-er (W. F. Graier, K. Osibow, R. Malli, and G. M. Kostner, patent application number 05450006.1 at the European patent office)) that is based on a single kringle domain from apolipoprotein(a), which is flanked by yellow and cyan fluorescent protein at the 3'- and 5'-ends, respectively. Notably, apoK1-er does not interact with Ca2+ itself but serves as a substrate for calreticulin, the main constitutive Ca2+-binding protein in the ER. ApoK1-er assembles with calreticulin and the protein disulfide isomerase ERp57 and undergoes a conformational shift in a Ca2+-dependent manner that allows fluorescence resonance energy transfer between the two fluorophores. This construct primarily offers three major advantages compared with the already existing probes: (i) it resolves perfectly the physiological range of the free Ca2+ concentration in the ER, (ii) expression of apoK1-er does not affect the Ca2+ buffering capacity of the ER, and (iii) apoK1-er is not inactivated by binding of constitutive interaction partners that prevent Ca2+-dependent conformational changes. These unique characteristics of apoK1-er make this sensor particularly attractive for studies on ER Ca2+ signaling and dynamics in which alteration of Ca2+ fluctuations by expression of any additional Ca2+ buffer essentially has to be avoided.

Mitochondria Maintain Maturation and Secretion of Lipoprotein Lipase in the Endoplasmic Reticulum

The Biochemical Journal. May, 2006  |  Pubmed ID: 16466345

Considering the physiological Ca2+ dynamics within the ER (endoplasmic reticulum), it remains unclear how efficient protein folding is maintained in living cells. Thus, utilizing the strictly folding-dependent activity and secretion of LPL (lipoprotein lipase), we evaluated the impact of ER Ca2+ content and mitochondrial contribution to Ca2+-dependent protein folding. Exhaustive ER Ca2+ depletion by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases caused strong, but reversible, reduction of cell-associated and released activity of constitutive and adenovirus-encoded human LPL in CHO-K1 (Chinese-hamster ovary K1) and endothelial cells respectively, which was not due to decline of mRNA or intracellular protein levels. In contrast, stimulation with the IP3 (inositol 1,4,5-trisphosphate)-generating agonist histamine only moderately and transiently affected LPL maturation in endothelial cells that paralleled a basically preserved ER Ca2+ content. However, in the absence of extracellular Ca2+ or upon prevention of transmitochondrial Ca2+ flux, LPL maturation discontinued upon histamine stimulation. Collectively, these data indicate that Ca2+-dependent protein folding in the ER is predominantly controlled by intraluminal Ca2+ and is largely maintained during physiological cell stimulation owing to efficient ER Ca2+ refilling. Since Ca2+ entry and mitochondrial Ca2+ homoeostasis are crucial for continuous Ca2+-dependent protein maturation in the ER, their pathological alterations may result in dysfunctional protein folding.

Mg2+ Deprivation Elicits Rapid Ca2+ Uptake and Activates Ca2+/calcineurin Signaling in Saccharomyces Cerevisiae

Eukaryotic Cell. Apr, 2007  |  Pubmed ID: 17337637

To learn about the cellular processes involved in Mg(2+) homeostasis and the mechanisms allowing cells to cope with low Mg(2+) availability, we performed RNA expression-profiling experiments and followed changes in gene activity upon Mg(2+) depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg(2+) depletion are also induced by high Ca(2+) and/or alkalinization. Among the genes significantly up-regulated by Mg(2+) starvation, Ca(2+) stress, and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p (calcineurin-responsive zinc finger protein) signaling pathway. Similarly to Ca(2+) stress, Mg(2+) starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg(2+) starvation depends on extracellular Ca(2+). Using fluorescence resonance energy transfer microscopy, we demonstrate that removal of Mg(2+) results in an immediate increase in free cytoplasmic Ca(2+). This effect is dependent on external Ca(2+). The results presented indicate that Mg(2+) depletion in yeast cells leads to enhanced cellular Ca(2+) concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg(2+) depletion stress.

Uncoupling Proteins 2 and 3 Are Fundamental for Mitochondrial Ca2+ Uniport

Nature Cell Biology. Apr, 2007  |  Pubmed ID: 17351641

Mitochondrial Ca(2+) uptake is crucial for the regulation of the rate of oxidative phosphorylation, the modulation of spatio-temporal cytosolic Ca(2+) signals and apoptosis. Although the phenomenon of mitochondrial Ca(2+) sequestration, its characteristics and physiological consequences have been convincingly reported, the actual protein(s) involved in this process are unknown. Here, we show that the uncoupling proteins 2 and 3 (UCP2 and UCP3) are essential for mitochondrial Ca(2+) uptake. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that UCP2 and UCP3 are elementary for mitochondrial Ca(2+) sequestration in response to cell stimulation under physiological conditions - observations supported by isolated liver mitochondria of Ucp2(-/-) mice lacking ruthenium red-sensitive Ca(2+) uptake. Our results reveal a novel molecular function for UCP2 and UCP3, and may provide the molecular mechanism for their reported effects. Moreover, the identification of proteins fundemental for mitochondrial Ca(2+) uptake expands our knowledge of the physiological role for mitochondrial Ca(2+) sequestration.

Mitochondria and Ca(2+) Signaling: Old Guests, New Functions

Pflugers Archiv : European Journal of Physiology. Dec, 2007  |  Pubmed ID: 17611770

Mitochondria are ancient endosymbiotic guests that joined the cells in the evolution of complex life. While the unique ability of mitochondria to produce adenosine triphosphate (ATP) and their contribution to cellular nutrition metabolism received condign attention, our understanding of the organelle's contribution to Ca(2+) homeostasis was restricted to serve as passive Ca(2+) sinks that accumulate Ca(2+) along the organelle's negative membrane potential. This paradigm has changed radically. Nowadays, mitochondria are known to respond to environmental Ca(2+) and to contribute actively to the regulation of spatial and temporal patterns of intracellular Ca(2+) signaling. Accordingly, mitochondria contribute to many signal transduction pathways and are actively involved in the maintenance of capacitative Ca(2+) entry, the accomplishment of Ca(2+) refilling of the endoplasmic reticulum and Ca(2+)-dependent protein folding. Mitochondrial Ca(2+) homeostasis is complex and regulated by numerous, so far, genetically unidentified Ca(2+) channels, pumps and exchangers that concertedly accomplish the organelle's Ca(2+) demand. Notably, mitochondrial Ca(2+) homeostasis and functions are crucially influenced by the organelle's structural organization and motility that, in turn, is controlled by matrix/cytosolic Ca(2+). This review intends to provide a condensed overview on the molecular mechanisms of mitochondrial Ca(2+) homeostasis (uptake, buffering and storage, extrusion), its modulation by other ions, kinases and small molecules, and its contribution to cellular processes as fundamental basis for the organelle's contribution to signaling pathways. Hence, emphasis is given to the structure-to-function and mobility-to-function relationship of the mitochondria and, thereby, bridging our most recent knowledge on mitochondria with the best-established mitochondrial function: metabolism and ATP production.

Mitochondrial Ca2+, the Secret Behind the Function of Uncoupling Proteins 2 and 3?

Cell Calcium. Jul, 2008  |  Pubmed ID: 18282596

The underlying molecular action of the novel uncoupling proteins 2 and 3 (UCP2 and UCP3) is still under debate. The proteins have been implicated in many cell functions, including the regulation of insulin secretion and regulation of reactive oxygen species (ROS) generation. These effects have mainly been explained by suggesting that the proteins establish a proton leak through the inner mitochondrial membrane (IMM). However, accumulating data question this mechanism and suggest that UCP2 and UCP3 may play other roles, including carrying free fatty acids from the matrix towards the intermembrane space, or contributing to the mitochondrial Ca(2+) uniport. Accordingly, in this review we reflect on these actions of UCP2/UCP3 and discuss alternative explanations for the molecular mechanisms by which UCP2/UCP3 might contribute to aspects of cell function. Based on the potential role of UCP2/UCP3 in regulating mitochondrial Ca(2+) uptake, we propose a scheme whereby these proteins integrate Ca(2+)-dependent signal transduction and energy metabolism in order to meet the energy demand of the cell for its continuous response, adaptation, and stimulation to environmental input.

The C-terminal Region of Human Adipose Triglyceride Lipase Affects Enzyme Activity and Lipid Droplet Binding

The Journal of Biological Chemistry. Jun, 2008  |  Pubmed ID: 18445597

Adipose triglyceride lipase (ATGL) catalyzes the first step in the hydrolysis of triacylglycerol (TG) generating diacylglycerol and free fatty acids. The enzyme requires the activator protein CGI-58 (or ABHD5) for full enzymatic activity. Defective ATGL function causes a recessively inherited disorder named neutral lipid storage disease that is characterized by systemic TG accumulation and myopathy. In this study, we investigated the functional defects associated with mutations in the ATGL gene that cause neutral lipid storage disease. We show that these mutations lead to the expression of either inactive enzymes localizing to lipid droplets (LDs) or enzymatically active lipases with defective LD binding. Additionally, our studies assign important regulatory functions to the C-terminal part of ATGL. Truncated mutant ATGL variants lacking approximately 220 amino acids of the C-terminal protein region do not localize to LDs. Interestingly, however, these mutants exhibit substantially increased TG hydrolase activity in vitro (up to 20-fold) compared with the wild-type enzyme, indicating that the C-terminal region suppresses enzyme activity. Protein-protein interaction studies revealed an increased binding of truncated ATGL to CGI-58, suggesting that the C-terminal part interferes with CGI-58 interaction and enzyme activation. Compared with the human enzyme, the C-terminal region of mouse ATGL is much less effective in suppressing enzyme activity, implicating species-dependent differences in enzyme regulation. Together, our results demonstrate that the C-terminal region of ATGL is essential for proper localization of the enzyme and suppresses enzyme activity.

Integrin Clustering Enables Anandamide-induced Ca2+ Signaling in Endothelial Cells Via GPR55 by Protection Against CB1-receptor-triggered Repression

Journal of Cell Science. May, 2008  |  Pubmed ID: 18445684

Although the endocannabinoid anandamide is frequently described to act predominantly in the cardiovascular system, the molecular mechanisms of its signaling remained unclear. In human endothelial cells, two receptors for anandamide were found, which were characterized as cannabinoid 1 receptor (CB1R; CNR1) and G-protein-coupled receptor 55 (GPR55). Both receptors trigger distinct signaling pathways. It crucially depends on the activation status of integrins which signaling cascade becomes promoted upon anandamide stimulation. Under conditions of inactive integrins, anandamide initiates CB1R-derived signaling, including Gi-protein-mediated activation of spleen tyrosine kinase (Syk), resulting in NFkappaB translocation. Furthermore, Syk inhibits phosphoinositide 3-kinase (PI3K) that represents a key protein in the transduction of GPR55-originated signaling. However, once integrins are clustered, CB1R splits from integrins and, thus, Syk cannot further inhibit GPR55-triggered signaling resulting in intracellular Ca2+ mobilization from the endoplasmic reticulum (ER) via a PI3K-Bmx-phospholipase C (PLC) pathway and activation of nuclear factor of activated T-cells. Altogether, these data demonstrate that the physiological effects of anandamide on endothelial cells depend on the status of integrin clustering.

Cytosolic Ca2+ Prevents the Subplasmalemmal Clustering of STIM1: an Intrinsic Mechanism to Avoid Ca2+ Overload

Journal of Cell Science. Oct, 2008  |  Pubmed ID: 18765567

The stromal interacting molecule (STIM1) is pivotal for store-operated Ca(2+) entry (SOC). STIM1 proteins sense the Ca(2+) concentration within the lumen of the endoplasmic reticulum (ER) via an EF-hand domain. Dissociation of Ca(2+) from this domain allows fast oligomerization of STIM1 and the formation of spatially discrete clusters close to the plasma membrane. By lifetime-imaging of STIM1 interaction, the rearrangement of STIM1, ER Ca(2+) concentration ([Ca(2+)](ER)) and cytosolic Ca(2+) signals ([Ca(2+)](cyto)) we show that [Ca(2+)](cyto) affects the subcellular distribution of STIM1 oligomers and prevents subplasmalemmal STIM clustering, even if the ER is depleted. These data indicate that [Ca(2+)](cyto), independently of the ER Ca(2+) filling state, crucially tunes the formation and disassembly of subplasmalemmal STIM1 clusters, and, thus, protects cells against Ca(2+) overload resulting from excessive SOC activity.

Mitochondrial Protein Phosphorylation: Instigator or Target of Lipotoxicity?

Trends in Endocrinology and Metabolism: TEM. May, 2009  |  Pubmed ID: 19356948

Lipotoxicity occurs as a consequence of chronic exposure of non-adipose tissue and cells to elevated concentrations of fatty acids, triglycerides and/or cholesterol. The contribution of mitochondria to lipotoxic cell dysfunction, damage and death is associated with elevated production of reactive oxygen species and initiation of apoptosis. Although there is a broad consensus on the involvement of these phenomena with lipotoxicity, the molecular mechanisms that initiate, mediate and trigger mitochondrial dysfunction in response to substrate overload remain unclear. Here, we focus on protein phosphorylation as an important phenomenon in lipotoxicity that harms mitochondria-related signal transduction and integration in cellular metabolism. Moreover, the degradation of mitochondria by mitophagy is discussed as an important landmark that leads to cellular apoptosis in lipotoxicity.

Lysophosphatidic Acid Receptor Activation Affects the C13NJ Microglia Cell Line Proteome Leading to Alterations in Glycolysis, Motility, and Cytoskeletal Architecture

Proteomics. Jan, 2010  |  Pubmed ID: 19899077

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

Mitochondrial Ca2+ Channels: Great Unknowns with Important Functions

FEBS Letters. May, 2010  |  Pubmed ID: 20074570

Mitochondria process local and global Ca(2+) signals. Thereby the spatiotemporal patterns of mitochondrial Ca(2+) signals determine whether the metabolism of these organelles is adjusted or cell death is executed. Mitochondrial Ca(2+) channels of the inner mitochondrial membrane (IMM) actually implement mitochondrial uptake from cytosolic Ca(2+) rises. Despite great efforts in the past, the identity of mitochondrial Ca(2+) channels is still elusive. Numerous studies aimed to characterize mitochondrial Ca(2+) uniport channels and provided a detailed profile of these great unknowns with important functions. This mini-review revisits previous research on the mechanisms of mitochondrial Ca(2+) uptake and aligns them with most recent findings.

Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

Molecular Biology of the Cell. Mar, 2010  |  Pubmed ID: 20089833

The significance and extent of Ca(2+) regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca(2+) with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca(2+) in the early secretory pathway. Specific depletion of luminal Ca(2+) in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca(2+) in COPII vesicle homotypic fusion was demonstrated in vitro using Ca(2+) chelators. The EF-hand-containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca(2+)-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca(2+) on homotypic fusion. Immunoisolation established that Ca(2+) chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca(2+), acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca(2+)-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.

The Contribution of UCP2 and UCP3 to Mitochondrial Ca(2+) Uptake is Differentially Determined by the Source of Supplied Ca(2+)

Cell Calcium. May, 2010  |  Pubmed ID: 20403634

The transmission of Ca(2+) signals to mitochondria is an important phenomenon in cell signaling. We have recently reported that the novel uncoupling proteins UCP2 and UCP3 (UCP2/3) are fundamental for mitochondrial Ca(2+) uniport (MCU). In the present study we investigate the contribution of UCP2/3 to mitochondrial accumulation of Ca(2+) either exclusively released from the ER or entering the cell via the store-operated Ca(2+) entry (SOCE) pathway. Using siRNA we demonstrate that constitutively expressed UCP2/3 are essentially involved in mitochondrial sequestration of intracellularly released Ca(2+) but not of that entering the cells via SOCE. However, overexpression of UCP2/3 yielded elevated mitochondrial Ca(2+) uptake from both sources, though it was more pronounced in case of entering Ca(2+), indicating that the expression levels of UCP2/3 are crucial for the capacity of mitochondria to sequester entering Ca(2+). Our data point to distinct UCP2/3-dependent and UCP2/3-independent modes of mitochondrial Ca(2+) sequestration, which may meet the various demands necessary for an adequate organelle Ca(2+) loading from different Ca(2+) sources in intact cells.

Mitochondrial Ca2+ Uptake and Not Mitochondrial Motility is Required for STIM1-Orai1-dependent Store-operated Ca2+ Entry

Journal of Cell Science. Aug, 2010  |  Pubmed ID: 20587595

Store-operated Ca(2+) entry (SOCE) is established by formation of subplasmalemmal clusters of the endoplasmic reticulum (ER) protein, stromal interacting molecule 1 (STIM1) upon ER Ca(2+) depletion. Thereby, STIM1 couples to plasma membrane channels such as Orai1. Thus, a close proximity of ER domains to the plasma membrane is a prerequisite for SOCE activation, challenging the concept of local Ca(2+) buffering by mitochondria as being essential for SOCE. This study assesses the impact of mitochondrial Ca(2+) handling and motility on STIM1-Orai1-dependent SOCE. High-resolution microscopy showed only 10% of subplasmalemmal STIM1 clusters to be colocalized with mitochondria. Impairments of mitochondrial Ca(2+) handling by inhibition of mitochondrial Na(+)-Ca(2+) exchanger (NCX(mito)) or depolarization only partially suppressed Ca(2+) entry in cells overexpressing STIM1-Orai1. However, SOCE was completely abolished when both NCX(mito) was inhibited and the inner mitochondrial membrane was depolarized, in STIM1- and Orai1-overexpressing cells. Immobilization of mitochondria by expression of mAKAP-RFP-CAAX, a construct that physically links mitochondria to the plasma membrane, affected the Ca(2+) handling of the organelles but not the activity of SOCE. Our observations indicate that mitochondrial Ca(2+) uptake, including reversal of NCX(mito), is fundamental for STIM1-Orai1-dependent SOCE, whereas the proximity of mitochondria to STIM1-Orai1 SOCE units and their motility is not required.

Acyl Chain-dependent Effect of Lysophosphatidylcholine on Endothelial Prostacyclin Production

Journal of Lipid Research. Oct, 2010  |  Pubmed ID: 20610733

Previously we identified palmitoyl-lysophosphatidylcholine (16:0 LPC), linoleoyl-LPC (18:2 LPC), arachidonoyl-LPC (20:4 LPC), and oleoyl-LPC (18:1 LPC) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein. In the present study, the impact of those LPC on prostacyclin (PGI(2)) production was examined in vitro in primary human aortic endothelial cells (HAEC) and in vivo in mice. Although 18:2 LPC was inactive, 16:0, 18:1, and 20:4 LPC induced PGI(2) production in HAEC by 1.4-, 3-, and 8.3-fold, respectively. LPC-elicited 6-keto PGF1α formation depended on both cyclooxygenase (COX)-1 and COX-2 and on the activity of cytosolic phospholipase type IVA (cPLA2). The LPC-induced, cPLA2-dependent (14)C-arachidonic acid (AA) release was increased 4.5-fold with 16:0, 2-fold with 18:1, and 2.7-fold with 20:4 LPC, respectively, and related to the ability of LPC to increase cytosolic Ca(2+) concentration. In vivo, LPC increased 6-keto PGF(1α) concentration in mouse plasma with a similar order of potency as found in HAEC. Our results indicate that the tested LPC species are capable of eliciting production of PGI(2), whereby the efficacy and the relative contribution of underlying mechanisms are strongly related to acyl-chain length and degree of saturation.

GPR55-dependent and -independent Ion Signalling in Response to Lysophosphatidylinositol in Endothelial Cells

British Journal of Pharmacology. Sep, 2010  |  Pubmed ID: 20735417

The glycerol-based lysophospholipid lysophosphatidylinositol (LPI) is an endogenous agonist of the G-protein-coupled receptor 55 (GPR55) exhibiting cannabinoid receptor-like properties in endothelial cells. To estimate the contribution of GPR55 to the physiological effects of LPI, the GPR55-dependent and -independent electrical responses in this cell type were investigated.

Uncoupling Protein 3 Adjusts Mitochondrial Ca(2+) Uptake to High and Low Ca(2+) Signals

Cell Calcium. Nov, 2010  |  Pubmed ID: 21047682

Uncoupling proteins 2 and 3 (UCP2/3) are essential for mitochondrial Ca(2+) uptake but both proteins exhibit distinct activities in regard to the source and mode of Ca(2+) mobilization. In the present work, structural determinants of their contribution to mitochondrial Ca(2+) uptake were explored. Previous findings indicate the importance of the intermembrane loop 2 (IML2) for the contribution of UCP2/3. Thus, the IML2 of UCP2/3 was substituted by that of UCP1. These chimeras had no activity in mitochondrial uptake of intracellularly released Ca(2+), while they mimicked the wild-type proteins by potentiating mitochondrial sequestration of entering Ca(2+). Alignment of the IML2 sequences revealed that UCP1, UCP2 and UCP3 share a basic amino acid in positions 163, 164 and 167, while only UCP2 and UCP3 contain a second basic residue in positions 168 and 171, respectively. Accordingly, mutants of UCP3 in positions 167 and 171/172 were made. In permeabilized cells, these mutants exhibited distinct Ca(2+) sensitivities in regard to mitochondrial Ca(2+) sequestration. In intact cells, these mutants established different activities in mitochondrial uptake of either intracellularly released (UCP3(R171,E172)) or entering (UCP3(R167)) Ca(2+). Our data demonstrate that distinct sites in the IML2 of UCP3 effect mitochondrial uptake of high and low Ca(2+) signals.

Triacylglycerol Accumulation Activates the Mitochondrial Apoptosis Pathway in Macrophages

The Journal of Biological Chemistry. Mar, 2011  |  Pubmed ID: 21196579

Programmed cell death of lipid-laden macrophages is a prominent feature of atherosclerotic lesions and mostly ascribed to accumulation of excess intracellular cholesterol. The present in vitro study investigated whether intracellular triacylglycerol (TG) accumulation could activate a similar apoptotic response in macrophages. To address this question, we utilized peritoneal macrophages isolated from mice lacking adipose triglyceride lipase (ATGL), the major enzyme responsible for TG hydrolysis in multiple tissues. In Atgl(-/-) macrophages, we observed elevated levels of cytosolic Ca(2+) and reactive oxygen species, stimulated cytochrome c release, and nuclear localization of apoptosis-inducing factor. Fragmented mitochondria prior to cell death were indicative of the mitochondrial apoptosis pathway being triggered as a consequence of defective lipolysis. Other typical markers of apoptosis, such as externalization of phosphatidylserine in the plasma membrane, caspase 3 and poly(ADP-ribose) polymerase cleavage, were increased in Atgl(-/-) macrophages. An artificial increase of cellular TG levels by incubating wild-type macrophages with very low density lipoprotein closely mimicked the apoptotic phenotype observed in Atgl(-/-) macrophages. Results obtained during the present study define a novel pathway linking intracellular TG accumulation to mitochondrial dysfunction and programmed cell death in macrophages.

Sequential Synthesis and Methylation of Phosphatidylethanolamine Promote Lipid Droplet Biosynthesis and Stability in Tissue Culture and in Vivo

The Journal of Biological Chemistry. May, 2011  |  Pubmed ID: 21454708

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt(-/-) mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt(-/-) animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.

The GPR55 Agonist Lysophosphatidylinositol Directly Activates Intermediate-conductance Ca2+ -activated K+ Channels

Pflugers Archiv : European Journal of Physiology. Aug, 2011  |  Pubmed ID: 21603896

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca(2+) and voltage-gated potassium (BK(Ca)) channels. Here, we explored the effect of LPI on intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channels using excised inside-out patches from endothelial cells. The functional expression of IK(Ca) was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IK(Ca) channels with a slope conductance of 39 pS in symmetric K(+) gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3-10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IK(Ca) single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IK(Ca) single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.

Leucine Zipper EF Hand-containing Transmembrane Protein 1 (Letm1) and Uncoupling Proteins 2 and 3 (UCP2/3) Contribute to Two Distinct Mitochondrial Ca2+ Uptake Pathways

The Journal of Biological Chemistry. Aug, 2011  |  Pubmed ID: 21613221

Cytosolic Ca(2+) signals are transferred into mitochondria over a huge concentration range. In our recent work we described uncoupling proteins 2 and 3 (UCP2/3) to be fundamental for mitochondrial uptake of high Ca(2+) domains in mitochondria-ER junctions. On the other hand, the leucine zipper EF hand-containing transmembrane protein 1 (Letm1) was identified as a mitochondrial Ca(2+)/H(+) antiporter that achieved mitochondrial Ca(2+) sequestration at small Ca(2+) increases. Thus, the contributions of Letm1 and UCP2/3 to mitochondrial Ca(2+) uptake were compared in endothelial cells. Knock-down of Letm1 did not affect the UCP2/3-dependent mitochondrial uptake of intracellularly released Ca(2+) but strongly diminished the transfer of entering Ca(2+) into mitochondria, subsequently, resulting in a reduction of store-operated Ca(2+) entry (SOCE). Knock-down of Letm1 and UCP2/3 did neither impact on cellular ATP levels nor the membrane potential. The enhanced mitochondrial Ca(2+) signals in cells overexpressing UCP2/3 rescued SOCE upon Letm1 knock-down. In digitonin-permeabilized cells, Letm1 exclusively contributed to mitochondrial Ca(2+) uptake at low Ca(2+) conditions. Neither the Letm1- nor the UCP2/3-dependent mitochondrial Ca(2+) uptake was affected by a knock-down of mRNA levels of mitochondrial calcium uptake 1 (MICU1), a protein that triggers mitochondrial Ca(2+) uptake in HeLa cells. Our data indicate that Letm1 and UCP2/3 independently contribute to two distinct, mitochondrial Ca(2+) uptake pathways in intact endothelial cells.

Studying Mitochondrial Ca(2+) Uptake - a Revisit

Molecular and Cellular Endocrinology. Apr, 2012  |  Pubmed ID: 22100614

Mitochondrial Ca(2+) sequestration is a well-known process that is involved in various physiological and pathological mechanisms. Using isolated suspended mitochondria one unique mitochondrial Ca(2+) uniporter was considered to account ubiquitously for the transfer of Ca(2+) into these organelles. However, by applying alternative techniques for measuring mitochondrial Ca(2+) uptake evidences for molecularly distinct mitochondrial Ca(2+) carriers accumulated recently. Herein we compared different methodical approaches of studying mitochondrial Ca(2+) uptake. Patch clamp technique on mitoplasts from endothelial and HeLa cells revealed the existence of three and two mitoplast Ca(2+) currents (I(CaMito)), respectively. According to their conductance, these channels were named small (s-), intermediate (i-), large (l-) and extra-large (xl-) mitoplast Ca(2+) currents (MCC). i-MCC was found in mitoplasts of both cell types whereas s-MCC and l-MCC or xl-MCC were/was exclusively found in mitoplasts from endothelial cells or HeLa cells. The comparison of mitochondrial Ca(2+) signals, measured either indirectly by sensing extra-mitochondrial Ca(2+) or directly by recording changes of the matrix Ca(2+), showed different Ca(2+) sensitivities of the distinct mitochondrial Ca(2+) uptake routes. Subpopulations of mitochondria with different Ca(2+) uptake capacities in intact endothelial cells could be identified using Rhod-2/AM. In contrast, cells expressing mitochondrial targeted pericam or cameleon (4mtD3cpv) showed homogeneous mitochondrial Ca(2+) signals in response to cell stimulation. The comparison of different experimental approaches and protocols using isolated organelles, permeabilized and intact cells, pointed to cell-type specific and versatile pathways for mitochondrial Ca(2+) uptake. Moreover, this work highlights the necessity of the utilization of multiple technical approaches to study the complexity of mitochondrial Ca(2+) homeostasis.

Endothelial Mitochondria--less Respiration, More Integration

Pflugers Archiv : European Journal of Physiology. Jul, 2012  |  Pubmed ID: 22382745

Lining the inner surface of the circulatory system, the vascular endothelium accomplishes a vast variety of specialized functions. Even slight alterations of these functions are implicated in the development of certain cardiovascular diseases that represent major causes of morbidity and mortality in developed countries. Endothelial mitochondria are essential to the functional integrity of the endothelial cell as they integrate a wide range of cellular processes including Ca²⁺ handling, redox signaling and apoptosis, all of which are closely interrelated. Growing evidence supports the notion that impairment of mitochondrial signaling in the endothelium is an early event and a causative factor in the development of diseases such as atherosclerosis or diabetic complications. In this review, we want to outline the significance of mitochondria in both physiology and pathology of the vascular endothelium.

Docosahexaenoic Acid-induced Unfolded Protein Response, Cell Cycle Arrest, and Apoptosis in Vascular Smooth Muscle Cells Are Triggered by Ca²⁺-dependent Induction of Oxidative Stress

Free Radical Biology & Medicine. May, 2012  |  Pubmed ID: 22391221

Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca(2+) release and entry of extracellular Ca(2+). Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca(2+) uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs.

Mutation in NSUN2, Which Encodes an RNA Methyltransferase, Causes Autosomal-recessive Intellectual Disability

American Journal of Human Genetics. May, 2012  |  Pubmed ID: 22541562

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.

Inhibition of Autophagy Rescues Palmitic Acid-induced Necroptosis of Endothelial Cells

The Journal of Biological Chemistry. Jun, 2012  |  Pubmed ID: 22556413

Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.

The Vascular Barrier-protecting Hawthorn Extract WS® 1442 Raises Endothelial Calcium Levels by Inhibition of SERCA and Activation of the IP3 Pathway

Journal of Molecular and Cellular Cardiology. Oct, 2012  |  Pubmed ID: 22814436

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442.

Acyl Chain-dependent Effect of Lysophosphatidylcholine on Cyclooxygenase (COX)-2 Expression in Endothelial Cells

Atherosclerosis. Oct, 2012  |  Pubmed ID: 22901457

Previously we identified palmitoyl-, oleoyl- linoleoyl-, and arachidonoyl-lysophosph-atidylcholine (LPC 16:0, 18:1, 18:2 and 20:4) as the most prominent LPC species generated by endothelial lipase (EL). In the present study, we examined the capacity of those LPC to modulate expression of cyclooxygenase (COX)-2 in vascular endothelial cells.

Mitochondrial Ca2+ Uptake 1 (MICU1) and Mitochondrial Ca2+ Uniporter (MCU) Contribute to Metabolism-secretion Coupling in Clonal Pancreatic β-cells

The Journal of Biological Chemistry. Oct, 2012  |  Pubmed ID: 22904319

In pancreatic β-cells, uptake of Ca(2+) into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca(2+) sequestration in clonal INS-1 832/13 pancreatic β-cells: the mitochondrial Ca(2+) uptake 1 (MICU1), mitochondrial Ca(2+) uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca(2+) sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca(2+) uptake upon both intracellular Ca(2+) release and Ca(2+) entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca(2+) uptake in response to either intracellular Ca(2+) release or Ca(2+) entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca(2+) uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca(2+) oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca(2+) uptake in pancreatic β-cells and their involvement in the positive feedback required for sustained insulin secretion.

Spatiotemporal Correlations Between Cytosolic and Mitochondrial Ca(2+) Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

PloS One. 2012  |  Pubmed ID: 23029314

The transfer of Ca(2+) from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca(2+) ([Ca(2+)](cyto)) can be excellently quantified with the ratiometric Ca(2+) probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca(2+) sensors, the cameleons, are efficiently used to specifically measure Ca(2+) within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca(2+)](cyto) and mitochondrial Ca(2+) ([Ca(2+)](mito)) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca(2+) probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca(2+)](cyto) and [Ca(2+)](mito) within same individual cells. Our data indicate that depending on the kinetics of [Ca(2+)](cyto) rises there is a significant lag between onset of [Ca(2+)](cyto) and [Ca(2+)](mito) signals, pointing to a certain threshold of [Ca(2+)](cyto) necessary to activate mitochondrial Ca(2+) uptake. The temporal correlation between [Ca(2+)](mito) and [Ca(2+)](cyto) as well as the efficiency of the transfer of Ca(2+) from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca(2+) extrusion and a desensitization of mitochondrial Ca(2+) uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca(2+) oscillations of pancreatic beta-cells in response to D-glucose.

The Endocannabinoid N-arachidonoyl Glycine (NAGly) Inhibits Store-operated Ca2+ Entry by Preventing STIM1-Orai1 Interaction

Journal of Cell Science. Feb, 2013  |  Pubmed ID: 23239024

The endocannabiniod anandamide (AEA) and its derivate N-arachidonoyl glycine (NAGly) have a broad spectrum of physiological effects, which are induced by both binding to receptors and receptor-independent modulations of ion channels and transporters. The impact of AEA and NAGly on store-operated Ca(2+) entry (SOCE), a ubiquitous Ca(2+) entry pathway regulating many cellular functions, is unknown. Here we show that NAGly, but not AEA reversibly hinders SOCE in a time- and concentration-dependent manner. The inhibitory effect of NAGly on SOCE was found in the human endothelial cell line EA.hy926, the rat pancreatic β-cell line INS-1 832/13, and the rat basophilic leukemia cell line RBL-2H3. NAGly diminished SOCE independently from the mode of Ca(2+) depletion of the endoplasmic reticulum, whereas it had no effect on Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Enhanced Ca(2+) entry was effectively hampered by NAGly in cells overexpressing the key molecular constituents of SOCE, stromal interacting molecule 1 (STIM1) and the pore-forming subunit of SOCE channels, Orai1. Fluorescence microscopy revealed that NAGly did not affect STIM1 oligomerization, STIM1 clustering, or the colocalization of STIM1 with Orai1, which were induced by Ca(2+) depletion of the endoplasmic reticulum. In contrast, independently from its slow depolarizing effect on mitochondria, NAGly instantly and strongly diminished the interaction of STIM1 with Orai1, indicating that NAGly inhibits SOCE primarily by uncoupling STIM1 from Orai1. In summary, our findings revealed the STIM1-Orai1-mediated SOCE machinery as a molecular target of NAGly, which might have many implications in cell physiology.

Characterization of Distinct Single-channel Properties of Ca²⁺ Inward Currents in Mitochondria

Pflugers Archiv : European Journal of Physiology. Jul, 2013  |  Pubmed ID: 23397170

Previous studies have demonstrated several molecularly distinct players involved in mitochondrial Ca(2+) uptake. In the present study, electrophysiological recordings on mitoplasts that were isolated from HeLa cells were performed in order to biophysically and pharmacologically characterize Ca(2+) currents across the inner mitochondrial membrane. In mitoplast-attached configuration with 105 mM Ca(2+) as a charge carrier, three distinct channel conductances of 11, 23, and 80 pS were observed. All types of mitochondrial currents were voltage-dependent and essentially depended on the presence of Ca(2+) in the pipette. The 23 pS channel exhibited burst kinetics. Though all channels were sensitive to ruthenium red, their sensitivity was different. The 11 and 23 pS channels exhibited a lower sensitivity to ruthenium red than the 80 pS channel. The activities of all channels persisted in the presence of cylosporin A, CGP 37187, various K(+)-channel inhibitors, and Cl(-) channel blockers disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate and niflumic acid. Collectively, our data identified multiple conductances of Ca(2+) currents in mitoplasts isolated from HeLa cells, thus challenging the dogma of only one unique mitochondrial Ca(2+) uniporter.

N-Arachidonoyl Glycine Suppresses Na⁺/Ca²⁺ Exchanger-mediated Ca²⁺ Entry into Endothelial Cells and Activates BK(Ca) Channels Independently of GPCRs

British Journal of Pharmacology. Jun, 2013  |  Pubmed ID: 23517055

N-Arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly's action on unstimulated and agonist-stimulated endothelial cells.

Molecularly Distinct Routes of Mitochondrial Ca2+ Uptake Are Activated Depending on the Activity of the Sarco/endoplasmic Reticulum Ca2+ ATPase (SERCA)

The Journal of Biological Chemistry. May, 2013  |  Pubmed ID: 23592775

The transfer of Ca(2+) across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca(2+) uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca(2+) fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca(2+) uptake pathway or establish distinct routes for mitochondrial Ca(2+) sequestration. In this study, we show that a modulation of Ca(2+) release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca(2+) signals and consequently switches mitochondrial Ca(2+) uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca(2+) sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca(2+) across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca(2+) uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca(2+) rises.

Mitochondrial Ca(2+) Uniporter (MCU)-dependent and MCU-independent Ca(2+) Channels Coexist in the Inner Mitochondrial Membrane

Pflugers Archiv : European Journal of Physiology. Jul, 2014  |  Pubmed ID: 24162235

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca(2+) uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca(2+) channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca(2+) and Na(+) currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca(2+) current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca(2+) current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca(2+) channels.

ATP Increases Within the Lumen of the Endoplasmic Reticulum Upon Intracellular Ca2+ Release

Molecular Biology of the Cell. Feb, 2014  |  Pubmed ID: 24307679

Multiple functions of the endoplasmic reticulum (ER) essentially depend on ATP within this organelle. However, little is known about ER ATP dynamics and the regulation of ER ATP import. Here we describe real-time recordings of ER ATP fluxes in single cells using an ER-targeted, genetically encoded ATP sensor. In vitro experiments prove that the ATP sensor is both Ca(2+) and redox insensitive, which makes it possible to monitor Ca(2+)-coupled ER ATP dynamics specifically. The approach uncovers a cell type-specific regulation of ER ATP homeostasis in different cell types. Moreover, we show that intracellular Ca(2+) release is coupled to an increase of ATP within the ER. The Ca(2+)-coupled ER ATP increase is independent of the mode of Ca(2+) mobilization and controlled by the rate of ATP biosynthesis. Furthermore, the energy stress sensor, AMP-activated protein kinase, is essential for the ATP increase that occurs in response to Ca(2+) depletion of the organelle. Our data highlight a novel Ca(2+)-controlled process that supplies the ER with additional energy upon cell stimulation.

Adaptations of Energy Metabolism Associated with Increased Levels of Mitochondrial Cholesterol in Niemann-Pick Type C1-deficient Cells

The Journal of Biological Chemistry. Jun, 2014  |  Pubmed ID: 24790103

Niemann-Pick type C1 (NPC1) is a late endosomal transmembrane protein, which, together with NPC2 in the endosome lumen, mediates the transport of endosomal cholesterol to the plasma membrane and endoplasmic reticulum. Loss of function of NPC1 or NPC2 leads to cholesterol accumulation in late endosomes and causes neuronal dysfunction and neurodegeneration. Recent studies indicate that cholesterol also accumulates in mitochondria of NPC1-deficient cells and brain tissue and that NPC1 deficiency leads to alterations in mitochondrial function and energy metabolism. Here, we have investigated the effects of increased mitochondrial cholesterol levels on energy metabolism, using RNA interference to deplete Chinese hamster ovary cells of NPC1 alone or in combination with MLN64, which mediates endosomal cholesterol transport to mitochondria. Mitochondrial cholesterol levels were also altered by depletion of NPC2 in combination with the expression of NPC2 mutants. We found that the depletion of NPC1 increased lactate secretion, decreased glutamine-dependent mitochondrial respiration, and decreased ATP transport across mitochondrial membranes. These metabolic alterations did not occur when transport of endosomal cholesterol to mitochondria was blocked. In addition, the elevated mitochondrial cholesterol levels in NPC1-depleted cells and in NPC2-depleted cells expressing mutant NPC2 that allows endosomal cholesterol trafficking to mitochondria were associated with increased expression of the antioxidant response factor Nrf2. Antioxidant treatment not only prevented the increase in Nrf2 mRNA levels but also prevented the increased lactate secretion in NPC1-depleted cells. These results suggest that mitochondrial cholesterol accumulation can increase oxidative stress and in turn cause increased glycolysis to lactate and other metabolic alterations.

IP3-mediated STIM1 Oligomerization Requires Intact Mitochondrial Ca2+ Uptake

Journal of Cell Science. Jul, 2014  |  Pubmed ID: 24806964

Mitochondria contribute to cell signaling by controlling store-operated Ca(2+) entry (SOCE). SOCE is activated by Ca(2+) release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma-membrane junctions and opens plasma membrane Ca(2+) channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca(2+) buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca(2+) uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca(2+) buffering or ER Ca(2+) depletion by sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca(2+) uptake. However, MCU-dependent mitochondrial sequestration of Ca(2+) entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca(2+) uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca(2+) micro-domains.

Characterization of Rat Serum Amyloid A4 (SAA4): a Novel Member of the SAA Superfamily

Biochemical and Biophysical Research Communications. Aug, 2014  |  Pubmed ID: 25044109

The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold during inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5'- and 3'RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/β and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.

TRPV1 Mediates Cellular Uptake of Anandamide and Thus Promotes Endothelial Cell Proliferation and Network-formation

Biology Open. Nov, 2014  |  Pubmed ID: 25395667

Anandamide (N-arachidonyl ethanolamide, AEA) is an endogenous cannabinoid that is involved in various pathological conditions, including cardiovascular diseases and tumor-angiogenesis. Herein, we tested the involvement of classical cannabinoid receptors (CBRs) and the Ca(2+)-channel transient receptor potential vanilloid 1 (TRPV1) on cellular AEA uptake and its effect on endothelial cell proliferation and network-formation. Uptake of the fluorescence-labeled anandamide (SKM4-45-1) was monitored in human endothelial colony-forming cells (ECFCs) and a human endothelial-vein cell line (EA.hy926). Involvement of the receptors during AEA translocation was determined by selective pharmacological inhibition (AM251, SR144528, CID16020046, SB366791) and molecular interference by TRPV1-selective siRNA-mediated knock-down and TRPV1 overexpression. We show that exclusively TRPV1 contributes essentially to AEA transport into endothelial cells in a Ca(2+)-independent manner. This TRPV1 function is a prerequisite for AEA-induced endothelial cell proliferation and network-formation. Our findings point to a so far unknown moonlighting function of TRPV1 as Ca(2+)-independent contributor/regulator of AEA uptake. We propose TRPV1 as representing a promising target for development of pharmacological therapies against AEA-triggered endothelial cell functions, including their stimulatory effect on tumor-angiogenesis.

Oleoyl-lysophosphatidylcholine Limits Endothelial Nitric Oxide Bioavailability by Induction of Reactive Oxygen Species

PloS One. 2014  |  Pubmed ID: 25419657

Previously we reported modulation of endothelial prostacyclin and interleukin-8 production, cyclooxygenase-2 expression and vasorelaxation by oleoyl- lysophosphatidylcholine (LPC 18:1). In the present study, we examined the impact of this LPC on nitric oxide (NO) bioavailability in vascular endothelial EA.hy926 cells. Basal NO formation in these cells was decreased by LPC 18:1. This was accompanied with a partial disruption of the active endothelial nitric oxide synthase (eNOS)- dimer, leading to eNOS uncoupling and increased formation of reactive oxygen species (ROS). The LPC 18:1-induced ROS formation was attenuated by the superoxide scavenger Tiron, as well as by the pharmacological inhibitors of eNOS, NADPH oxidases, flavin-containing enzymes and superoxide dismutase (SOD). Intracellular ROS-formation was most prominent in mitochondria, less pronounced in cytosol and undetectable in endoplasmic reticulum. Importantly, Tiron completely prevented the LPC 18:1-induced decrease in NO bioavailability in EA.hy926 cells. The importance of the discovered findings for more in vivo like situations was analyzed by organ bath experiments in mouse aortic rings. LPC 18:1 attenuated the acetylcholine-induced, endothelium dependent vasorelaxation and massively decreased NO bioavailability. We conclude that LPC 18:1 induces eNOS uncoupling and unspecific superoxide production. This results in NO scavenging by ROS, a limited endothelial NO bioavailability and impaired vascular function.

Assessment of Mitochondrial Ca²⁺ Uptake

Methods in Molecular Biology (Clifton, N.J.). 2015  |  Pubmed ID: 25631032

Mitochondrial Ca(2+) uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca(2+) signals and elaborating the mechanisms that accomplish mitochondrial Ca(2+) uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca(2+) concentration ([Ca(2+)]mito) in a wide range of applications.

Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum

Sensors (Basel, Switzerland). Jun, 2015  |  Pubmed ID: 26053751

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

UCP2 Modulates Single-channel Properties of a MCU-dependent Ca(2+) Inward Current in Mitochondria

Pflugers Archiv : European Journal of Physiology. Dec, 2015  |  Pubmed ID: 26275882

The mitochondrial Ca(2+) uniporter is a highly Ca(2+)-selective protein complex that consists of the pore-forming mitochondrial Ca(2+) uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca(2+) uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca(2+) uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca(2+) uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca(2+) currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca(2+) currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca(2+) currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38% decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca(2+) uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca(2+) inward current.

Rearrangement of MICU1 Multimers for Activation of MCU is Solely Controlled by Cytosolic Ca(2.)

Scientific Reports. Oct, 2015  |  Pubmed ID: 26489515

Mitochondrial Ca(2+) uptake is a vital process that controls distinct cell and organelle functions. Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca(2+) uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca(2+) channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca(2+) signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca(2+) rearranges MICU1 multimers with an EC50 of 4.4 μM, resulting in activation of mitochondrial Ca(2+) uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca(2+) rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca(2+) concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells.

Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (3rd Edition)

Autophagy. Jan, 2016  |  Pubmed ID: 26799652

Development of Novel FP-based Probes for Live-cell Imaging of Nitric Oxide Dynamics

Nature Communications. Feb, 2016  |  Pubmed ID: 26842907

Nitric oxide () is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca(2+) sensor allowed us to visualize and Ca(2+) signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

Filling a GAP-An Optimized Probe for ER Ca(2+) Imaging In Vivo

Cell Chemical Biology. Jun, 2016  |  Pubmed ID: 27341431

In this issue of Cell Chemical Biology, Navas-Navarro et al. (2016) demonstrate that fusion of engineered derivatives of the long-known jellyfish proteins green fluorescent protein (GFP) and aequorin yield optimized genetically encoded fluorescent probes for detecting Ca(2+) signals within the endoplasmic reticulum (ER) of living animals.

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria

Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 2016  |  Pubmed ID: 27606689

Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol.

PRMT1-mediated Methylation of MICU1 Determines the UCP2/3 Dependency of Mitochondrial Ca(2+) Uptake in Immortalized Cells

Nature Communications. Sep, 2016  |  Pubmed ID: 27642082

Recent studies revealed that mitochondrial Ca(2+) channels, which control energy flow, cell signalling and death, are macromolecular complexes that basically consist of the pore-forming mitochondrial Ca(2+) uniporter (MCU) protein, the essential MCU regulator (EMRE), and the mitochondrial Ca(2+) uptake 1 (MICU1). MICU1 is a regulatory subunit that shields mitochondria from Ca(2+) overload. Before the identification of these core elements, the novel uncoupling proteins 2 and 3 (UCP2/3) have been shown to be fundamental for mitochondrial Ca(2+) uptake. Here we clarify the molecular mechanism that determines the UCP2/3 dependency of mitochondrial Ca(2+) uptake. Our data demonstrate that mitochondrial Ca(2+) uptake is controlled by protein arginine methyl transferase 1 (PRMT1) that asymmetrically methylates MICU1, resulting in decreased Ca(2+) sensitivity. UCP2/3 normalize Ca(2+) sensitivity of methylated MICU1 and, thus, re-establish mitochondrial Ca(2+) uptake activity. These data provide novel insights in the complex regulation of the mitochondrial Ca(2+) uniporter by PRMT1 and UCP2/3.

Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin

The Journal of Biological Chemistry. Nov, 2016  |  Pubmed ID: 27679490

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN), resulting in activation of soluble guanylate cyclase (sGC) and cGMP-mediated vasodilation. We have previously shown that a minor reaction of ALDH2-catalyzed GTN bioconversion, accounting for about 5% of the main clearance-based turnover yielding inorganic nitrite, results in direct NO formation and concluded that this minor pathway could provide the link between vascular GTN metabolism and activation of sGC. However, lack of detectable NO at therapeutically relevant GTN concentrations (≤1 μm) in vascular tissue called into question the biological significance of NO formation by purified ALDH2. We addressed this issue and used a novel, highly sensitive genetically encoded fluorescent NO probe (geNOp) to visualize intracellular NO formation at low GTN concentrations (≤1 μm) in cultured vascular smooth muscle cells (VSMC) expressing an ALDH2 mutant that reduces GTN to NO but lacks clearance-based GTN denitration activity. NO formation was compared with GTN-induced activation of sGC. The addition of 1 μm GTN to VSMC expressing either wild-type or C301S/C303S ALDH2 resulted in pronounced intracellular NO elevation, with maximal concentrations of 7 and 17 nm, respectively. Formation of GTN-derived NO correlated well with activation of purified sGC in VSMC lysates and cGMP accumulation in intact porcine aortic endothelial cells infected with wild-type or mutant ALDH2. Formation of NO and cGMP accumulation were inhibited by ALDH inhibitors chloral hydrate and daidzin. The present study demonstrates that ALDH2-catalyzed NO formation is necessary and sufficient for GTN bioactivation in VSMC.

Intact Mitochondrial Ca(2+) Uniport is Essential for Agonist-induced Activation of Endothelial Nitric Oxide Synthase (eNOS)

Free Radical Biology & Medicine. Jan, 2017  |  Pubmed ID: 27923677

Mitochondrial Ca(2+) uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO(•)) production. However, it is not entirely clear if the organelles support or counteract NO(•) biosynthesis by taking up Ca(2+). The objective of this study was to verify whether or not mitochondrial Ca(2+) uptake influences Ca(2+)-triggered NO(•) generation by endothelial NO(•) synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO(•) probes, the geNOps, and Ca(2+) sensors to monitor single cell NO(•) and Ca(2+) dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP3)-generating agonist. Mitochondrial Ca(2+) uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca(2+) uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca(2+) uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca(2+)-triggered NO(•) increase independently of global cytosolic Ca(2+) signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca(2+) sequestration and Ca(2+)-induced NO(•) signals. The positive correlation between mitochondrial Ca(2+) elevation and NO(•) production was independent of eNOS phosphorylation at serine(1177). Our findings emphasize that manipulating mitochondrial Ca(2+) uptake may represent a novel strategy to control eNOS-mediated NO(•) production.

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