Elevated Intracellular Calcium Levels Research Articles

Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes.

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Protective effect of caffeic acid against beta-amyloid-induced neurotoxicity by the inhibition of calcium influx and tau phosphorylation

Aims The progressive accumulation of beta-amyloid peptide (Aβ), in the form of senile plaques, has been recognized as one of the major causes of Alzheimer's disease (AD) pathology. Increased production of Aβ and the aggregation of Aβ to oligomers have been reported to trigger neurotoxicity, oxidative damage and inflammation. Furthermore, Aβ-induced tau hyperphosphorylation and neurotoxicity are downstream of Aβ. Therefore, we studied the possible neuroprotective effects of caffeic acid against Aβ-induced toxicity. Main methods Treatment of PC12 cells with 10 μM Aβ (25–35) for 24 h significantly decreased the cell viability; this was accompanied by an increase in intracellular calcium levels and tau phosphorylation with GSK-3β (glycogen synthase kinase-3β) activation (phosphorylation). Key findings However, pretreatment of the PC12 cells with 10 and 20 μg/ml of caffeic acid, for 1 h prior to Aβ, significantly reversed the Aβ-induced neurotoxicity by attenuating the elevation of intracellular calcium levels and tau phosphorylation. Significance Taken together, these results suggest that caffeic acid protected the PC12 cells against Aβ-induced toxicity. In addition, the neuroprotective mechanisms of caffeic acid against Aβ attenuated intracellular calcium influx and decreased tau phosphorylation by the reduction of GSK-3β activation.

Analysis of Protein Complex Hierarchy in Living Cells

The identification and analysis of protein complexes is usually achieved by performing in vitro experiments. We describe a translocation-based method for studying protein complexes in living cells. Annexin A4, a phospholipid- and calcium-binding protein, translocates from the cytoplasm and nucleoplasm to plasma and nuclear membranes in response to elevated intracellular calcium levels. By fusing a bait protein, for instance, a core component of a protein complex or a similarly relevant peptide, to the annexin, translocation of both bait and its interacting target proteins are readily monitored in living cells in response to a single treatment. Proteins of interest are fused to a variety of fluorescent proteins suitable for multiparameter imaging. Using this generally applicable approach, we were able to visualize the formation of protein complexes in their natural environment. Specifically, we detected the hierarchical assembly of four-component protein complexes in single cells.

2,3,7,8-TCDD neurotoxicity in neuroblastoma cells is caused by increased oxidative stress, intracellular calcium levels, and tau phosphorylation

One of the most notorious environmental toxicants, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), easily accumulates in the environment and most organisms, including humans, because of its high lipophilicity and resistance to degradation. TCDD exposure causes a variety of adverse health effects in humans including immunotoxicity, hepatotoxicity, neurotoxicity, and carcinogenesis. For the most part, studies regarding the adverse effects of TCDD on the central nervous system (CNS) have been limited to neurodevelopmental processes. In this study, we investigated the neurotoxicity of TCDD in neuronal cells using a neuroblastoma cell line (clone N2a) and explored the possible mechanisms of action. MTT and Comet assays were conducted to determine if TCDD is cytotoxic and if it causes DNA damage, respectively. The results of these assays revealed that treatment with 100, 300, 500 and 1000 nM TCDD decreased the viability of N2a cells and increased DNA damage in a dose-dependent manner compared to controls. Additionally, a malondialdehyde (MDA) assay was performed to determine if TCDD induces lipid peroxidation. The results of this assay revealed that 100, 300 and 500 nM TCDD induced lipid peroxidation in a dose-dependent manner. Finally, TCDD neurotoxicity (300 nM or higher) in N2a cells was accompanied by elevated intracellular calcium levels. These increased calcium levels increased the phosphorylation of tau via up-regulation of phospho-glycogen synthase kinase-3β (GSK-3β). Taken together, these results indicate that TCDD exposure induces neurotoxicity in N2a cells by increasing DNA damage, oxidative stress and intracellular calcium levels. The TCDD-mediated increase of tau phosphorylation in particular indicates an important role for tau hyperphosphorylation in TCDD-induced neurotoxicity.

Pathophysiology of Peripheral Nerve Injury During Regional Anesthesia

Despite attention to technical details in performance of regional anesthetics, damage to nerves continues to be a concern. Understanding of pathophysiological mechanisms may aid in decreasing the incidence and severity of such injuries. Studies from both clinical and basic science perspective are reviewed. Exposure of peripheral nerves to local anesthetics may result in axonal damage, particularly if the solution is injected intrafascicularly, if the concentration is high, and if duration of exposure is prolonged. Disruption of numerous cellular functions may contribute to neuronal damage by local anesthetics, but elevated intracellular calcium levels may play a central role. Needle penetration of a nerve results in minimal lasting damage unless this is combined with local anesthetic administration within the nerve fascicle. Direct compression by a pronged tourniquet application may damage axons particularly of large myelinated fibers. Ischemia may also contribute to neuronal injury in proportion to the duration of blood flow interruption. The relative importance of these pathogenic factors in cases of nerve injury after regional anesthesia is not resolved.

T.P.4.11 Effect of calpain and proteasome inhibition on calcium-dependent proteolysis and muscle histopathology in the mdx mouse

The progressive muscle weakness as observed in Duchenne muscular dystrophy (DMD) is a consequence of dystrophin deficiency. Loss of this functional protein leads to elevated levels of intracellular calcium, a key step in the cellular pathology of DMD. The cysteine protease calpain is activated in dystrophin-deficient muscle, and its inhibition is regarded as a potential therapeutic approach. In addition, previous work has shown that the ubiquitin/proteasome system also contributed to muscle protein breakdown in dystrophic muscle and, therefore, also qualifies as a potential target for therapeutic intervention in DMD.

Salidroside attenuates glutamate-induced apoptotic cell death in primary cultured hippocampal neurons of rats

Salidroside, a compound of natural origin, has displayed a broad spectrum of pharmacological properties. This study aimed to evaluate the inhibitory effects of salidroside on glutamate-induced cell death in a primary culture of rat hippocampal neurons as compared to brain-derived neurotrophic factor (BDNF), a usual positive control. MTT and LDH assays, together with Hoechst 33342 staining, terminal deoxynucleotidyl transferase dUTP-mediated nicked end labeling (TUNEL) assay and flow cytometric analysis using annexin-V and propidium (PI) label, indicated that salidroside pretreatment attenuated glutamate-induced apoptotic cell death in primary cultured hippocampal neurons, showing a dose-dependent pattern. Furthermore, caspase-3 activity assay and calcium measurements with Fluo 4-AM, respectively, revealed that salidroside pretreatment antagonized activation of caspase-3 and elevation of intracellular calcium level, both of which were induced by glutamate stimulation, thus adding to the understanding of how salidroside offered neuroprotection against glutamate excitotoxicity.

Cytoprotective roles of senescence marker protein 30 against intracellular calcium elevation and oxidative stress

Senescence marker protein 30 (SMP30) is identified as an important aging marker molecule and known to play multifunctional roles as an intracellular calcium regulatory protein in the signaling process. To elucidate the functional significance of SMP30, we established the stably transfected P19 cell line with SMP30 expression vector. Overexpression of SMP30 slightly suppressed the proliferation of P19 cells. However, SMP30 overexpression was cytoprotective against calcium-mediated stress such as calcium ionophore (A23187), and thapsigargin. We found that SMP30 overexpression reduced the elevated intracellular calcium levels induced by A23187, but not by thapsigargin. In addition, SMP30 transfected P19 cells were more protective to tert-butylhydroperoxide induced cytotoxicity, indicating the antioxidative properties of SMP30. Taken together, our results suggest that external calcium regulation and antioxidant properties are involved in the cytoprotective mechanism of SMP30.

Curcumin protected PC12 cells against beta-amyloid-induced toxicity through the inhibition of oxidative damage and tau hyperphosphorylation

One of the pathological hallmarks of Alzheimer’s disease is the progressive accumulation of beta-amyloid (Aβ) in the form of senile plaques, and Aβ insult to neuronal cells has been identified as one of the major causes of the onset of the disease. Curcumin, the major and most active antioxidant of Curcuma longa, protects neuronal cells against Aβ-induced toxicity. Therefore, in this study, we investigated the neuroprotective mechanisms by which curcumin acts against Aβ (25–35)-induced toxicity in PC12 cells. Following the exposure of PC12 cells to 10 μM Aβ (25–35) for 24 h, significant increases in the level of antioxidant enzymes, and DNA damage were observed, and these increases were accompanied by a decrease in cell viability, and an increase in intracellular calcium levels and tau hyperphosphorylation. In addition, pretreatment of PC12 cells with 10 μg/ml curcumin for 1 h significantly reversed the effect of Aβ, by decreasing the oxidative stress, and DNA damage induced by Aβ, as well as attenuating the elevation of intracellular calcium levels and tau hyperphosphorylation induced by Aβ. Taken together, these data indicate that curucmin protected PC12 cells against Aβ-induced neurotoxicity through the inhibition of oxidative damage, intracellular calcium influx, and tau hyperphosphorylation.

Loss of the Calmodulin-Dependent Inhibition of the RyR1 Calcium Release Channel upon Oxidation of Methionines in Calmodulin

The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its effect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility determined by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle. The functional effect of CaM oxidation on the open probability of the RyR1 calcium release channel was assessed through measurements of [3H]ryanodine binding using a heavy sarcoplasmic reticulum preparation enriched in RyR1. There is a biphasic regulation of RyR1 by unoxidized CaM, in which calcium-activated CaM acts to enhance the calcium sensitivity of channel closure, while apo-CaM functions to enhance channel activity at resting calcium levels. We find that physiological levels of CaM oxidation preferentially weaken the CaM-dependent inhibition of the RyR1 calcium release channel observed at activating micromolar levels of calcium. In contrast, the oxidation of CaM resulted in minimal functional changes in the CaM-dependent activation of RyR1 at resting nanomolar calcium levels. Oxidation does not significantly affect the high-affinity binding of calcium-activated CaM to the CaM-binding sequence of RyR1; rather, methionine oxidation disrupts interdomain interactions between the opposing domains of CaM in complex with the CaM-binding sequence of RyR1 that normally function as part of a conformational switch associated with RyR1 inhibition. These results suggest that the oxidation of CaM can contribute to observed elevations in intracellular calcium levels in response to conditions of oxidative stress observed during biological aging. We suggest that the sensitivity of RyR1 channel activity to CaM oxidation may function as part of an adaptive cellular response that enhances the duration of calcium transients to promote enhanced contractility.

The Calcineurin/NFAT Signaling Pathway Is a Target for the Collagen Type I-Induced Calcium Response in Megakaryocytes and Mediates Expression of Megakaryocytic Genes.

Megakaryocytes, as precursor cells of platelets, comprise a cell population crucial for the maintenance of adult hematopoiesis. Since platelets are anuclear cellular fragments, the platelet transcriptome as well as the proteome are largely determined by the megakaryocyte. Calcium signaling in response to agonists such as collagen or thrombin has a central function in platelet activation and therefore likely represents an important signaling pathway in megakaryocytes as well. Indeed, megakaryocytes show considerable elevation of intracellular calcium levels in response to selected platelet agonists, but the downstream effector molecules of calcium signaling in these cells or the transcriptional responses induced are unknown. We here establish calcineurin and the NFAT (Nuclear Factor of Activated T cells) family of transcription factors as components of a calcium-induced signaling cascade in megakaryocytes. In resting megakaryocytes, NFAT is cytoplasmic and inactive, but can be activated by fibrillar collagen type 1, a physiological agonist of platelets and megakaryocytes known to induce a sustained increase in intracellular calcium levels in these cells. In contrast, treatment with SDF-1a, thrombopoietin, or VEGF remained without noticeable effect, presumably because of the short duration of the calcium transients induced by these agents. Collagen-induced NFAT activation in megakaryocytes requires dephosphorylation by calcineurin and is completely sensitive to the calcineurin inhibitor cyclosporin A. Activation of NFAT by collagen was paralleled by the induction of the expression of Down Syndrome Critical Region I (DSCR1) and Fas Ligand (FasL), two genes recently identified as NFAT targets in megakaryocytes. Collagen-induced expression of DSCR1 and FasL occurred in a calcineurin- and NFAT-dependent manner, as it was blocked by both cyclosporin A as well as the specific peptide inhibitor of NFAT, VIVIT. These experiments show that the calcineurin pathway is a target for selected physiological ligands capable of inducing sustained calcium mobilization in megakaryocytes and regulates megakaryocyte gene expression in a cyclosporin A- and NFAT-dependent manner.

Evidence for an extended duration of GABA‐mediated excitation in the developing male versus female hippocampus

Gamma-aminobutyric acid (GABA) is as an excitatory neurotransmitter during brain development. Activation of GABA(A) receptors in neonatal rat hippocampus results in chloride efflux and membrane depolarization sufficient to open voltage sensitive calcium channels. As development progresses, there is a decline in the magnitude of calcium influx subsequent to GABA(A) receptor activation and the number of cells that respond to GABA with excitation. By the second postnatal week in the rat, GABA action in the hippocampus is predominantly inhibitory. The functional consequences and endogenous regulation of developmental GABA-mediated excitation remains under-explored. Hippocampal neurons in the newborn male and female rat respond to GABA(A) receptor activation with increased intracellular calcium and are susceptible to GABA-mediated damage -- both being indicative of the excitatory nature of GABA. In the present study we observed that by postnatal day 7, only males are susceptible to GABA(A) agonist-induced damage and respond to GABA(A) agonist administration with elevated levels of intracellular calcium in cultured hippocampal neurons. By postnatal day 14, GABA(A) agonist administration was without effect on intracellular calcium in both males and females. The age-related sex difference in the impact of GABA(A) receptor activation correlates with a sex difference in chloride co-transporter expression. Males have elevated protein levels of pNKCC1 on PN0 and PN7, with no sex difference by PN14. In contrast, females displayed elevated levels of KCC2 on PN7. This converging evidence infers that sex affects the duration of GABA(A) receptor-mediated excitation during normal hippocampal development, and provides a mechanism by which the effect is mediated.

Exogenous nitric oxide triggers classic ischemic preconditioning by preventing intracellular Ca 2+ overload in cardiomyocytes

The involvement of nitric oxide (NO) in the late phase of ischemic preconditioning is well established. However, the role of NO as a trigger or mediator of “classic preconditioning” remains to be determined. The present study was designed to investigate the effects of NO on calcium homeostasis in cultured newborn rat cardiomyocytes in normoxia and hypoxia. We found that treatment with the NO donor, sodium nitroprusside (SNP) induced a sustained elevation of intracellular calcium level ([Ca 2+] i) followed by a decrease to control levels. Elevation of extracellular calcium, which generally occurs during ischemia, caused an immediate increase in [Ca 2+] i and arrhythmia in cultures of newborn cardiomyocytes. Treatment with SNP decreased [Ca 2+] i to control levels and re-established synchronized beating of cardiomyocytes. A decrease in extracellular [Na +], which inhibits the Na +/Ca 2+ exchanger, did not prevent [Ca 2+] i reduction by SNP. In contrast, application of thapsigargin, an inhibitor of sarcoplasmic reticulum Ca 2+-ATPase (SERCA2a), increased [Ca 2+] i, and in its presence, SNP did not reduce [Ca 2+] i, indicating that Ca 2+ reduction is achieved via activation of SERCA2a. The results obtained suggest that activation of SERCA2a by SNP increases Ca 2+ uptake into the sarcoplasmic reticulum (SR) and prevents cytosolic Ca 2+ overload, which might explain the protective effect of SNP from hypoxic damage.

Low dose of bradykinin selectively increases intracellular calcium in glioma cells

To investigate the underlying basis for the selective modulation of the permeability of the blood–brain barrier by small doses of bradykinin, we first established cell lines of rat brain microvascular endothelial cells (BMECs) and astrocytes by primary cultures from neonatal rats. BMECs, astrocytes and C6 glioma cells were treated with different concentrations of BK (range from 10 – 8 M to 10 – 4 M), and changes of intracellular calcium levels were measured by fluorescence spectrophotometry. Expression levels of B2 receptors were analyzed by Western blot analysis. We found that a low dose (10 – 6 M) of BK could trigger an elevation of intracellular calcium level in C6 glioma cells, whereas astrocytes responded to a higher concentration of BK (10 – 5 M), and the BMECs remained unresponsive to BK. Western blot results showed that C6 glioma cells expressed the highest level of B2 receptors compared with primary astrocytes or BMECs. B2 receptors are highly expressed in glioma cells and a low dose of BK selectively increases the intracellular calcium level in tumor cells which, in turn, could contribute to the selective increase in the permeability of the blood–tumor barrier by small doses of BK.

T-lymphocyte navigation and migration: beyond the PI3K paradigm

The co-ordinated and directional trafficking of T-lymphocytes in lymphoid and peripheral tissues is an important process in lymphoid development, immunosurveillance and immune responses. Members of the chemokine superfamily play a key role in providing navigational cues for T-cells and chemokine receptors couple with a wide range of biochemical signals including phosphoinositide lipid metabolism, elevation of intracellular calcium levels, activation of a wide array of protein kinases as well as small GTPases. One of the most robust biochemical signals elicited by chemokines in T-lymphocytes is the activation of several members of the PI3K (phosphoinositide 3-kinase) family. In many cell systems, PI3Ks are known to contribute to several aspects of the migratory machinery, although their role in T-cell migration has been unclear and will be considered in the present paper.

Aging is associated with elevated intracellular calcium levels and altered calcium homeostatic mechanisms in hippocampal neurons

Aging is associated with increased vulnerability to neurodegenerative conditions such as Parkinson's and Alzheimer's disease and greater neuronal deficits after stroke and epilepsy. Emerging studies have implicated increased levels of intracellular calcium ([Ca 2+] i) for the neuronal loss associated with aging related disorders. Recent evidence demonstrates increased expression of voltage gated Ca 2+ channel proteins and associated Ca 2+ currents with aging. However, a direct comparison of [Ca 2+] i levels and Ca 2+ homeostatic mechanisms in hippocampal neurons acutely isolated from young and mid-age adult animals has not been performed. In this study, Fura-2 was used to determine [Ca 2+] i levels in CA1 hippocampal neurons acutely isolated from young (4–5 months) and mid-age (12–16 months) Sprague–Dawley rats. Our data provide the first direct demonstration that mid-age neurons in comparison to young neurons manifest significant elevations in basal [Ca 2+] i levels. Upon glutamate stimulation and a subsequent [Ca 2+] i load, mid-age neurons took longer to remove the excess [Ca 2+] i in comparison to young neurons, providing direct evidence that altered Ca 2+ homeostasis may be present in animals at significantly younger ages than those that are commonly considered aged (≥24 months). These alterations in Ca 2+ dynamics may render aging neurons more vulnerable to neuronal death following stroke, seizures or head trauma. Elucidating the functionality of Ca 2+ homeostatic mechanisms may offer an understanding of the increased neuronal loss that occurs with aging, and allow for the development of novel therapeutic agents targeted towards decreasing [Ca 2+] i levels thereby restoring the systems that maintain normal Ca 2+ homeostasis in aged neurons.

Glucose-stimulated Cdc42 Signaling Is Essential for the Second Phase of Insulin Secretion

The small Rho family GTPases Cdc42 and Rac1 have each been shown to function in insulin exocytosis and are presumed to function in actin remodeling and insulin granule mobilization. However, whether either GTPase is required for the mobilization phase of insulin release (second phase) and are linked in a common signaling pathway has remained unknown. Here we demonstrate that small interfering RNA-mediated depletion of Cdc42 from isolated islets results in the selective loss of second phase insulin release. Consistent with a role in this nutrient-dependent phase, Cdc42 activation was detected exclusively in response to D-glucose and was unresponsive to KCl or non-metabolizable glucose analogs in MIN6 beta-cells. Cdc42 activation occurred early in secretion (3 min), whereas Rac1 activation required approximately 15-20 min, suggesting Cdc42 as proximal and Rac1 as distal regulators of second-phase secretion. Importantly, Rac1 activation and function was linked in a common pathway downstream of Cdc42; Cdc42 depletion ablated glucose-induced Rac1 activation, and expression of constitutively active Rac1 in Cdc42-depleted cells functionally restored glucose-stimulated insulin secretion. Occurring at a time midway between Cdc42 and Rac1 activations was the phosphorylation of p21-activated-kinase 1 (Pak1), and this phosphorylation event required Cdc42. Moreover, small interfering RNA-mediated Pak1 depletion abolished Rac1 activation and glucose-stimulated insulin release, suggesting that Pak1 may mediate the link between Cdc42 and Rac1 in this pathway. Taken together, these data substantiate the existence of a novel signaling pathway in the islet beta-cell whereby Cdc42 functions as a key proximal transmitter of the glucose signal early in stimulus-secretion coupling to support the later stage of insulin release.

Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells

Oxidative stress plays an important role in Alzheimer's disease and other neurodegenerative disorders. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L, shows potent antioxidant property. In this paper, the neuroprotective effects of salidroside on hydrogen peroxide (H2O2)-induced apoptosis in SH-SY5Y cells were investigated. Pretreatment with salidroside markedly attenuated H2O2-induced cell viability loss and apoptotic cell death in a dose-dependent manner. The mechanisms by which salidroside protected neuron cells from oxidative stress included the induction of several antioxidant enzymes, thioredoxin, heme oxygenase-1, and peroxiredoxin-I; the downregulation of pro-apoptotic gene Bax and the upregulation of anti-apoptotic genes Bcl-2 and Bcl-XL. Furthermore, salidroside dose-dependently restored H2O2-induced loss of mitochondrial membrane potential as well as the elevation of intracellular calcium level. These results suggest that salidroside has protective effects against oxidative stress-induced cell apoptosis, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases implicated with oxidative stress.

Mitochondrial DNA-dependent effects of valproate on mitochondrial calcium levels in transmitochondrial cybrids

Calcium plays important roles in various cellular processes. Using transmitochondrial hybrid cells (cybrids) carrying fluorescent calcium indicators, we previously found two mitochondrial DNA (mtDNA) polymorphism sites, 8701 and 10398, that alter intracellular calcium signalling and mitochondrial pH. The 10398A polymorphism is reportedly associated with bipolar disorder, Parkinson's disease, Alzheimer's disease, and cancer, whereas 10398G is associated with longevity. In bipolar disorder, elevation of intracellular calcium levels in the platelets and lymphocytes is a well-replicated finding. Thus, we examined whether two mood stabilizers, lithium and valproate, affect the intracellular calcium signalling in cybrids with these mtDNA polymorphisms. After cybrids with 8701A/10398A and 8701G/10398G (three cell lines for each) derived from healthy controls were pretreated with lithium (0.75 mm or 1.5 mm) or valproate (0.6 mm or 1.2 mm) for 7 d, they were stimulated by 10 mum histamine. Valproate decreased mitochondrial calcium levels, compared with untreated cybrids, only in cybrids with 8701A/10398A. Moreover, valproate decreased cytosolic calcium levels at plateau after stimulation in cybrids with 8701A/10398A. These finding suggest that valproate may stabilize intracellular calcium only in cells with high mitochondrial calcium levels.

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin.

We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 microM Ca(2+), suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 microM in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens.