Ionomycin-induced Ca2 Research Articles

Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

The primary cilium is an antenna-like organelle protruding from the cell surface that can detect physical and chemical stimuli in the extracellular space to activate specific signaling pathways and downstream gene expressions. Calcium ion (Ca2+ ) signaling regulates a wide spectrum of cellular processes, including fertilization, proliferation, differentiation, muscle contraction, migration, and death. This study investigated the effects of the regulation of cytosolic Ca2+ levels on ciliogenesis using chemical, genetic, and optogenetic approaches. We found that ionomycin-induced Ca2+ influx inhibited ciliogenesis and Ca2+ chelator BATPA-AM-induced Ca2+ depletion promoted ciliogenesis. In addition, store-operated Ca2+ entry and the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) negatively regulated ciliogenesis. Moreover, an optogenetic platform was used to create different Ca2+ oscillation patterns by manipulating lighting parameters, including density, frequency, exposure time, and duration. Light-activated Ca2+ -translocating channelrhodopsin (CatCh) is activated by 470-nm blue light to induce Ca2+ influx. Our results show that high-frequency Ca2+ oscillations decrease ciliogenesis. Furthermore, the inhibition of cilia formation induced by Ca2+ may occur via the activation of Aurora kinase A. Cilia not only induce Ca2+ signaling but also regulate cilia formation by Ca2+ signaling.

Storing stallion sperm in SpermSafe™ at 17°C may improve fertility by reducing mPTP formation

Stallions with sperm that do not tolerate cooling have limited commercial potential. Cooling increases sperm intracellular calcium (Ca2+i; White, Reproduction, Fertility and Development. 1993;5:639-58), which in human spermatozoa triggers formation of the mitochondrial permeability transition pore (mPTP), leading to apoptosis (Treulen et al., Human Reproduction. 2015;30:767-76). However, the mechanism of sperm mPTP formation is yet to be described in the horse. As such, this study investigated whether mPTP formation occurs in stallion spermatozoa following Ca2+i increase and whether storing spermatozoa at higher temperatures can minimize Ca2+i increase and mPTP formation, thereby improving fertility. This would be investigated by alternatively storing sperm at 17°C in SpermSafe™ (BREED Diagnostics), a medium designed for liquid-storage at higher temperatures. Experiment 1 utilised three ejaculates from each of three pony stallions (n=9) to investigate the relationship between Ca2+i and mPTP formation. Semen was collected using an artificial vagina (AV) and extended 2:1 (extender:semen) using EquiPlus (Minitube). Spermatozoa were isolated from seminal plasma and extender using single-layer colloidal centrifugation before being resuspended to 20 × 106/ml in Biggers, Whitten, and Whittingham medium. Spermatozoa were pre-loaded with calcein-AM (fluorescent mPTP probe) and exposed to various doses of ionomycin to induce the Ca2+i increase. Data were checked for normaldistribution (Shapiro-Wilk test), with oneway ANOVA (treatment effect) and Dunnets test (comparing treatments and the control) used to identify significant differences, and ‘stallion’ used as a blocking term. The mPTP formed (loss of calcein fluorescence) following ionomycin-induced Ca2+i increase at 5 nM (100±16.4 AFU vs. 44.3±8.3 AFU for control and 5 nM ionomycin, respectively; P≤0.001), indicating that stallion spermatozoa do undergo mPTP formation in response to elevated Ca2+i. Experiment 2 utilised a fertile (83% per-cycle conception rate [PCCR] with fresh semen; n=6), 5-year-old Warmblood stallion with a history of poor fertility using cooled semen. Semen was collected and extended as described above and was washed via cushioned centrifugation. Sperm pellets were resuspended to 50 × 106/ml in either EquiPlus (cooled in a standard commercial shipper) or in SpermSafe (stored at 17°C in an EquOcyte shipper) for 24 h. Due to the logistical constraints of commercial practice, split ejaculates could not be utilized, so each sample was a separate ejaculate. Fixed-time AI was performed 24 h following semen collection and ovulation induction. Pregnancy rates of 0% (0/6) and 67% (8/12) were achieved following AI with cooled vs. SpermSafe-stored spermatozoa, respectively (Chi Square; P≤0.01). In conclusion, these data suggest that storing stallion sperm at 17°C in SpermSafe before AI may improve fertility in stallions with poor cooling tolerance, potentially due to reduced mPTP formation.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism

N-Methyl-d-aspartate (NMDA) receptors are Ca2+-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca2+-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca2+, we observed robust CDI of whole-cell GluN2A currents (0.42 ± 0.05) but no CDI in GluN2B currents (0.08 ± 0.07). In contrast, when Ca2+ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 ± 0.03 and 0.67 ± 0.02, respectively). To examine how the source of Ca2+ affects CDI, we recorded one-channel Na+ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca2+ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (Po). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high Po GluN2B receptors (GluN1A652Y/GluN2B). Together, these results show that Ca2+ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel Po. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca2+ load during high Po activation.

Protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial Ca2+ stress by melatonin's cascade metabolites C3-OHM and AFMK in RBA1 astrocytes.

Cyclic 3-hydroxymelatonin (C3-OHM) and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) are two major cascade metabolites of melatonin. We previously showed melatonin provides multiple levels of mitochondria-targeted protection beyond as a mitochondrial antioxidant during ionomycin-induced mitochondrial Ca2+ (mCa2+ ) stress in RBA1 astrocytes. Using noninvasive laser scanning fluorescence coupled time-lapse digital imaging microscopy, this study investigated whether C3-OHM and AFMK also provide mitochondrial levels of protection during ionomycin-induced mCa2+ stress in RBA1 astrocytes. Interestingly, precise temporal and spatial dynamic live mitochondrial images revealed that C3-OHM and AFMK prevented specifically mCa2+ -mediated mitochondrial reactive oxygen species (mROS) formation and hence mROS-mediated depolarization of mitochondrial membrane potential (△Ψm ) and permanent lethal opening of the MPT (p-MPT). The antioxidative effects of AFMK, however, were less potent than that of C3-OHM. Whether C3-OHM and AFMK targeted directly the MPT was investigated under a condition of "oxidation free-Ca2+ stress" using a classic antioxidant vitamin E to remove mCa2+ -mediated mROS stress and the potential antioxidative effects of C3-OHM and AFMK. Intriguingly, two compounds still effectively postponed "oxidation free-Ca2+ stress"-mediated depolarization of △Ψm and p-MPT. Measurements using a MPT pore-specific indicator Calcein further identified that C3-OHM and AFMK, rather than inhibiting, stabilized the MPT in its transient protective opening mode (t-MPT), a critical mechanism to reduce overloaded mROS and mCa2+ . These multiple layers of mitochondrial protection provided by C3-OHM and AFMK thus crucially allow melatonin to extend its metabolic cascades of mitochondrial protection during mROS- and mCa2+ -mediated MPT-associated apoptotic stresses and may provide therapeutic benefits against astrocyte-mediated neurodegeneration in the CNS.

Aging-related increase in store-operated Ca2+ influx in human ventricular fibroblasts.

Senescence-related fibrosis contributes to cardiac dysfunction. Profibrotic processes are Ca2+ dependent. The effect of aging on the Ca2+ mobilization processes of human ventricular fibroblasts (hVFs) is unclear. Therefore, we tested whether aging altered intracellular Ca2+ release and store-operated Ca2+ entry (SOCE). Disease-free hVFs from 2- to 63-yr-old trauma victims were assessed for cytosolic Ca2+ dynamics with fluo 3/confocal imaging. Angiotensin II or thapsigargin was used to release endoplasmic reticulum Ca2+ in Ca2+-free solution; CaCl2 (2 mM) was then added to assess SOCE, which was normalized to ionomycin-induced maximal Ca2+. The angiotensin II experiments were repeated after phosphoenolpyruvate pretreatment to determine the role of energy status. The expression of genes encoding SOCE-related ion channel subunits was assessed by quantitative PCR, and protein expression was assessed by immunoblot analysis. Age groups of <50 and ≥50 yr were compared using unpaired t-test or regression analysis. Ca2+ release by angiotensin II or thapsigargin was not different between the groups, but SOCE was significantly elevated in the ≥50-yr group. Regression analysis showed an age-dependent phosphoenolpyruvate-sensitive increase in SOCE of hVFs. Aging did not alter the mRNA expression of SOCE-related genes. The profibrotic phenotype of hVFs was evident by sprouty1 downregulation with age. Thus, an age-associated increase in angiotensin II- and thapsigargin-induced SOCE occurs in hVFs, independent of receptor mechanisms or alterations of mRNA expression level of SOCE-related ion channel subunits but related to the cellular bioenergetics status. Elucidation of mechanisms underlying enhanced hVF SOCE with aging may refine SOCE targets to limit aging-related progression of Ca2+-dependent cardiac fibrosis. NEW & NOTEWORTHY Human ventricular fibroblasts exhibit an age-related increase in store-operated Ca2+ influx induced by angiotensin II, an endogenous vasoactive hormone, or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, independent of receptor mechanisms or genes encoding store-operated Ca2+ entry-related ion channel subunits. Selective inhibition of this augmented store-operated Ca2+ entry could therapeutically limit aging-related cardiac fibrosis.

Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G1/S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca2+i chelation or CaM inhibition. Ionomycin-induced Ca2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.

Aspirin induces cell death by directly modulating mitochondrial voltage-dependent anion channel (VDAC)

Aspirin induces apoptotic cell death in various cancer cell lines. Here we showed that silencing of VDAC1 protected HeLa cells from aspirin-induced cell death. Compared to the wild type cells, VDAC1 knocked down cells showed lesser change of mitochondrial membrane potential (Δψm), upon aspirin treatment. Aspirin augmented ATP and ionomycin-induced mitochondrial Ca2+ uptake which was abolished in VDAC1 knocked down cells. Aspirin dissociated bound hexokinase II (HK-II) from mitochondria. Further, aspirin promoted the closure of recombinant human VDAC1, reconstituted in planar lipid bilayer. Taken together, these results imply that VDAC1 serves as a novel target for aspirin. Modulation of VDAC1 is possibly associated with the cell death and anticancer effects of aspirin.

Autumnalamide targeted proteins of the immunophilin family

Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ionomycin Ca2+ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca2+ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca2+ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca2+ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The KD obtained for Cyp D- autumnalamide was 1.51±1.399. Moreover, the KD for Cyp A- autumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08±1.23 and 6.85±1.1μM respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca2+ channels.

Desensitization-resistant and -sensitive GPCR-mediated inhibition of GABA release occurs by Ca2+-dependent and -independent mechanisms at a hypothalamic synapse.

Whereas the activation of Gαi/o-coupled receptors commonly results in postsynaptic responses that show acute desensitization, the presynaptic inhibition of transmitter release caused by many Gαi/o-coupled receptors is maintained during agonist exposure. However, an exception has been noted where GABAB receptor (GABABR)-mediated inhibition of inhibitory postsynaptic currents (IPSCs) recorded in mouse proopiomelanocortin (POMC) neurons exhibit acute desensitization in ∼25% of experiments. To determine whether differential effector coupling confers sensitivity to desensitization, voltage-clamp recordings were made from POMC neurons to compare the mechanism by which μ-opioid receptors (MORs) and GABABRs inhibit transmitter release. Neither MOR- nor GABABR-mediated inhibition of release relied on the activation of presynaptic K(+) channels. Both receptors maintained the ability to inhibit release in the absence of external Ca(2+) or in the presence of ionomycin-induced Ca(2+) influx, indicating that inhibition of release can occur through a Ca(2+)-independent mechanism. Replacing Ca(2+) with Sr(2+) to disrupt G-protein-mediated inhibition of release occurring directly at the release machinery did not alter MOR- or GABAB -mediated inhibition of IPSCs, suggesting that reductions in evoked release can occur through the inhibition of Ca(2+) channels. Additionally, both receptors inhibited evoked IPSCs in the presence of selective blockers of N- or P/Q-type Ca(2+) channels. Altogether, the results show that MORs and GABABRs can inhibit transmitter release through the inhibition of calcium influx and by direct actions at the release machinery. Furthermore, since both the desensitizing and nondesensitizing presynaptic receptors are similarly coupled, differential effector coupling is unlikely responsible for differential desensitization of the inhibition of release.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

Lack of Evidence for Presenilins as Endoplasmic Reticulum Ca2+ Leak Channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.

Role of Mitochondrial Na-Ca Exchange on BCR-Mediated Ca2+ Signalling in B Lymphocytes

To clarify the role of mitochondrial Na-Ca exchange (NCXm) on antigen-receptor (BCR)-mediated Ca2+ signaling of B lymphocytes, we first carried out mathematical modeling of BCR-mediated Ca2+ signaling. Our computer simulation suggested that NCXm modulates both endoplasmic reticulum (ER) Ca2+ content and BCR-mediated cytoplasmic Ca2+ (Ca2+i) rise. The model predictions were validated by following experiments with DT40 B lymphocytes whose NCXm gene (NCLX) was knocked-out (NCLX+/-). In NCLX+/- cells, expression of NCLX protein was markedly reduced and cytoplasmic Na+-dependent Ca2+m efflux were significantly attenuated. ER Ca2+ content, which was estimated as ionomycin-induced Ca2+i increase in the nominally Ca2+-free solution, was reduced to about 21% of control cells (WT). ER Ca2+ uptake via Ca2+ pump (SERCA) was decreased by about 42% in NCLX+/- cells, while it was comparable to WT when mitochondria functions were suppressed. No significant difference was observed in content of SERCA mRNA. In consequence, BCR stimulation by anti-IgM antibody hardly induced Ca2+i rise in NCLX+/- cells. Inhibition of NCXm by CGP-37157 induced similar results in WT. Essentially the same results were obtained by silencing NCLX in A 20 B lymphocytes with small interfering RNA. Taken together, we concluded that NCXm supports ER Ca2+ filling and pivotal in BCR-mediated Ca2+ signaling in B lymphocytes.

Minocycline Suppresses Activation of Nuclear Factor of Activated T Cells 1 (NFAT1) in Human CD4+ T Cells

Minocycline is a tetracycline family antibiotic that has anti-inflammatory and immunomodulatory properties. These properties have shown promise in the treatment of conditions such as rheumatoid arthritis, Huntington disease, and multiple sclerosis. As lymphocyte activation is involved in the pathogenesis of many of these diseases, T cells are postulated to be a primary target in minocycline therapy. Previous studies have demonstrated attenuation of CD4(+) T cell activation by minocycline, but a specific mechanism has not been elucidated. In this study, we investigated the effect of minocycline on the activity of three key transcription factors regulating CD4(+) T cell activation: NF-κB, AP-1 (activator protein 1), and NFAT (nuclear factor of activated T) cells. Our data demonstrate that minocycline selectively impairs NFAT-mediated transcriptional activation, a result of increased phosphorylation and reduced nuclear translocation of the isoform NFAT1. Minocycline increased the activity of the NFAT kinase GSK3 and decreased intracellular Ca(2+) flux, both of which facilitate NFAT1 phosphorylation. These findings provide a novel mechanism for minocycline induced suppression of CD4(+) T cell activation and may better inform the application of minocycline as an immunomodulatory agent.

Effect of the PEG component of certolizumab pegol on calcium flux in cellular systems

Background and objectivesCertolizumab pegol (the only polyethylene glycolylated (PEG) Fab' anti-tumour necrosis factor (CZP)) has a very low incidence of injection site pain (ISP) in clinical studies. This could be...

Sphingosine 1-Phosphate Has Dual Functions in the Regulation of Endothelial Cell Permeability and Ca2+Metabolism

Ca2+ signaling plays an important role in endothelial cell (EC) functions including the regulation of barrier integrity. Recently, the endogenous lipid derivative, sphingosine-1-phosphate (S1P), has emerged as an important modulator of EC barrier function. We investigated the role of endogenously generated S1P in Ca2+ metabolism and barrier function in human umbilical endothelial cells (HUVECs) stimulated by thrombin, histamine, or other agonists. Barrier function was assessed by dextran diffusion through HUVEC monolayers, and Ca2+ transients were measured using a fluoroprobe. Thrombin or histamine increased Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through store-operated channels (SOCs) that was accompanied by increased EC permeability. Inhibition of S1P synthesis by a specific sphingosine kinase inhibitor (SKI) decreased thrombin or histamine-induced increased permeability and decreased Ca2+ entry via SOC in a concentration-dependent fashion. SKI had minuscule effects on thrombin or histamine-induced Ca2+ release from ER. SKI also inhibited thapsigargin or ionomycin-induced Ca2+ entry via SOC without affecting Ca2+ release from the ER. In contrast to the effects of endogenously generated S1P, when S1P was administered externally, it initiated Ca2+ release from ER similar to thrombin and histamine while decreasing EC permeability. These observations indicate that after agonist-induced conditions, endogenously generated S1P functions as a positive modulator of Ca2+ entry via SOC and a mediator of increased cell permeability. In contrast, extracellular exposure to S1P has different signaling mechanisms and effects. Thus, the potential dual roles of endogenous and exogenous S1P on EC function need to be considered in pharmacological studies targeting sphingosine metabolism.

Lysophosphatidic acid triggers calcium entry through a non-store-operated pathway in human neutrophils

Lysophosphatidic acid (LPA) is a bioactive lipid, which is structurally similar to sphingosine 1-phosphate (S1P) and which can mobilize Ca2+ in multiple cell types. We recently showed that S1P induces Ca2+ entry directly through store-operated Ca2+ entry (SOCE) channels in human polymorphonuclear neutrophils (PMN). We therefore examined the mechanisms by which LPA induces intracellular Ca2+ mobilization in PMN. External application of low micromolar LPA caused dose-dependent Ca2+ influx without releasing Ca2+ stores, whereas G-protein-coupled (GPC) LPA receptors respond to nanomolar LPA. Additive Ca2+ influx by LPA compared with 100 nM ionomycin-induced Ca2+ influx suggests that LPA-induced Ca2+ influx does not pass through SOCE channels. Ca2+ influx was resistant to inhibition of Gi/o by pertussis toxin, of phospholipase C by U73122, and of G12/13/Rho by Y27632, all demonstrating GPC receptor independence. This Ca2+ influx was inhibited by Gd3+, La3+, Zn2+, or MRS1845 but not by Ni2+ or the sphingosine kinase inhibitor dimethylsphingosine. In addition, we found that LPA has no effect on neutrophil chemotaxis; however, it has stimulatory effects on neutrophil respiratory burst in a dose-response manner. These findings suggest that LPA-induced Ca2+ influx in PMN occurs through a mechanism other than SOCE channels, independent of Ca2+ store-depletion and S1P synthesis, and that the characteristics of LPA-induced Ca2+ influx are similar to those of S1P-induced influx in terms of sensitivity to inorganic inhibitors. Unlike S1P, LPA has stimulatory effects on neutrophil respiratory burst.

Tyrosyl‐phosphorylated proteins are involved in regulation of meiosis in the rat egg

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg. Mol. Reprod. Dev. 49:176–185, 1998. © 1998 Wiley-Liss, Inc.