Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl? Secretion by Human Airway Epithelia

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro- or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl⁻ and fluid secretion in the cystic fibrosis transmembrane regulator (CFTR)-expressing airway epithelia. 3O-C12 does not stimulate Cl⁻ secretion in CF cells, suggesting that lactone activates the CFTR. 3O-C12 also appears to directly activate the inositol trisphosphate receptor and release Ca²⁺ from the endoplasmic reticulum (ER), lowering [Ca²⁺] in the ER and thereby activating the Ca²⁺-sensitive ER signaling protein STIM1. 3O-C12 increases cytosolic [Ca²⁺] and, strikingly, also cytosolic [cAMP], the known activator of CFTR. Activation of Cl⁻ current by 3O-C12 was inhibited by a cAMP antagonist and increased by a phosphodiesterase inhibitor. Finally, a Ca²⁺ buffer that lowers [Ca²⁺] in the ER similar to the effect of 3O-C12 also increased cAMP and I_Cl. The results suggest that 3O-C12 stimulates CFTR-dependent Cl⁻ and fluid secretion in airway epithelial cells by activating the inositol trisphosphate receptor, thus lowering [Ca²⁺] in the ER and activating STIM1 and store-operated cAMP production. In CF airways, where CFTR is absent, the adaptive ability to rapidly flush the bacteria away is compromised because the lactone cannot affect Cl⁻ and fluid secretion.     

The calcium transporter ANNEXIN1 mediates cold‐induced calcium signaling and freezing tolerance in plants

The transient elevation of cytosolic free calcium concentration ([Ca²⁺]_cyt) induced by cold stress is a well‐established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca²⁺‐permeable transporter ANNEXIN1 (AtANN1) mediates cold‐triggered Ca²⁺ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold‐induced [Ca²⁺]_cyt increase and upregulation of the cold‐responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca²⁺ transport activity, thereby potentiating Ca²⁺ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold‐induced [Ca²⁺]_cyt elevation in the ost1‐3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1‐AtANN1 to cold‐induced Ca²⁺ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.     

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells

Two active calcium (Ca²⁺) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca²⁺ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl⁻-stimulated, NO₃⁻-inhibited ATPase activity on sucrose density gradients. Ca²⁺ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N′-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The K_m for MgATP and Ca²⁺ were 0.1 mm and 21 micromolar, respectively. The predominant Ca²⁺ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca²⁺ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I₅₀ = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca²⁺ dependent kinetics were complex with an apparent K_m ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca²⁺/H⁺ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca²⁺ transport systems are proposed to restore and maintain cytoplasmic Ca²⁺ homeostasis under changing cellular and environmental conditions.     

Abnormal Calcium Handling and Exaggerated Cardiac Dysfunction in Mice with Defective Vitamin D Signaling

Aim

Altered vitamin D signaling is associated with cardiac dysfunction, but the pathogenic mechanism is not clearly understood. We examine the mechanism and the role of vitamin D signaling in the development of cardiac dysfunction.

Methods and Results

We analyzed 1α-hydroxylase (1α-OHase) knockout (1α-OHase^−/−) mice, which lack 1α-OH enzymes that convert the inactive form to hormonally active form of vitamin D. 1α-OHase^−/− mice showed modest cardiac hypertrophy at baseline. Induction of pressure overload by transverse aortic constriction (TAC) demonstrated exaggerated cardiac dysfunction in 1α-OHase^−/− mice compared to their WT littermates with a significant increase in fibrosis and expression of inflammatory cytokines. Analysis of calcium (Ca²⁺) transient demonstrated profound Ca²⁺ handling abnormalities in 1α-OHase^−/− mouse cardiomyocytes (CMs), and treatment with paricalcitol (PC), an activated vitamin D₃ analog, significantly attenuated defective Ca²⁺ handling in 1α-OHase^−/− CMs. We further delineated the effect of vitamin D deficiency condition to TAC by first correcting the vitamin D deficiency in 1α-OHase^−/− mice, followed then by either a daily maintenance dose of vitamin D or vehicle (to achieve vitamin D deficiency) at the time of sham or TAC. In mice treated with vitamin D, there was a significant attenuation of TAC-induced cardiac hypertrophy, interstitial fibrosis, inflammatory markers, Ca²⁺ handling abnormalities and cardiac function compared to the vehicle treated animals.

Conclusions

Our results provide insight into the mechanism of cardiac dysfunction, which is associated with severely defective Ca²⁺ handling and defective vitamin D signaling in 1α-OHase^−/− mice.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca²⁺ entry (SOCE) with Orai1 as principal Ca²⁺ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca²⁺ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1^−/− and Orai1^−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca²⁺ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2^−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca²⁺ signals of Stim1^−/− and Orai1^−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1^−/− and Orai1^−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, a blocker of (receptor-operated) Ca²⁺ entry, inhibited Ca²⁺ and procoagulant responses even in Stim1^−/− and Orai1^−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca²⁺ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca²⁺ entry and PS exposure, only one relying on STIM1-Orai1 interaction.     

Transient Receptor Potential Channel 1 Deficiency Impairs Host Defense and Proinflammatory Responses to Bacterial Infection by Regulating Protein Kinase Cα Signaling

Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca²⁺ homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1^−/− mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca²⁺ entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca²⁺ entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca²⁺ entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit.     

Ca2+ Overload and Sarcoplasmic Reticulum Instability in tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K⁺, Cl⁻, and H⁺ channels may facilitate charge neutralization during Ca²⁺ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca²⁺ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a^−/− skeletal muscle showed Ca²⁺ overload inside the SR with frequent formation of Ca²⁺ deposits compared with the wild type muscle. This elevated SR Ca²⁺ pool in the tric-a^−/− muscle could be released by caffeine, whereas the elemental Ca²⁺ release events, e.g. osmotic stress-induced Ca²⁺ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a^−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca^{2+ ATPase} function could lead to aggravation of the stress-induced alternans in the tric-a^−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca²⁺ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca²⁺ movement across the SR membrane. The observed alternan behavior with the tric-a^−/− muscle may reflect a skeletal muscle version of store overload-induced Ca²⁺ release that has been reported in the cardiac muscle under stress conditions.     

The crystal structure of TRPM2 MHR1/2 domain reveals a conserved Zn2+‐binding domain essential for structural integrity and channel activity

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺‐permeable, nonselective cation channel involved in diverse physiological processes such as immune response, apoptosis, and body temperature sensing. TRPM2 is activated by ADP‐ribose (ADPR) and 2′‐deoxy‐ADPR in a Ca²⁺‐dependent manner. While two distinct binding sites exist for ADPR that exert different functions dependent on the species, the involvement of either binding site regarding the superagonistic effect of 2′‐deoxy‐ADPR is not clear yet. Here, we report the crystal structure of the MHR1/2 domain of TRPM2 from zebrafish (Danio rerio), and show that both ligands bind to this domain and activate the channel. We identified a so far unrecognized Zn²⁺‐binding domain that was not resolved in previous cryo‐EM structures and that is conserved in most TRPM channels. In combination with patch clamp experiments we comprehensively characterize the effect of the Zn²⁺‐binding domain on TRPM2 activation. Our results provide insight into a conserved motif essential for structural integrity and channel activity.     

Characterization of the Effect of the Mitochondrial Protein Hint2 on?Intracellular Ca2+ dynamics

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca²⁺ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca²⁺ dynamics in this cell type. We found that in hepatocytes isolated from Hint2^−/− mice, the frequency of Ca²⁺ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca²⁺ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2^−/− mice; we found that Hint2 accelerates Ca²⁺ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca²⁺ in suspensions of mitochondria. This prediction was then confirmed experimentally.     

Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

The second messenger NAADP triggers Ca²⁺ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2^−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca²⁺ responses as assessed by single-cell Ca²⁺ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P₂ were only partially dependent on TPCs. In Tpcn1/2^−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca²⁺-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca²⁺ release. High-affinity [³²P]NAADP binding still occurs in Tpcn1/2^−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca²⁺-permeable channels indispensable for NAADP signalling.     

Corticotropin-releasing Factor Receptor 2 Mediates Sex-Specific Cellular Stress Responses

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2–deficient (Crhr2^−/− ) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF₂) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2^−/− mice. Crhr₂^−/− mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2^−/− male mice during pancreatitis. WT and Crhr2^−/− female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2^−/− male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2^−/− mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2^−/−mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.     

JNK1/2 regulates ER–mitochondrial Ca2+ cross-talk during IL-1β–mediated cell death in RINm5F and human primary β-cells

Elevated interleukin-1β (IL-1β) induces apoptosis in pancreatic β-cells through endoplasmic reticulum (ER) stress induction and subsequent c-jun-N-terminal kinase 1/2 (JNK1/2) activation. In earlier work we showed that JNK1/2 activation is initiated before ER stress and apoptotic induction in response to IL-1β. However, the detailed regulatory mechanisms are not completely understood. Because the ER is the organelle responsible for Ca²⁺ handling and storage, here we examine the effects of IL-1β on cellular Ca²⁺ movement and mitochondrial dysfunction and evaluate the role of JNK1/2. Our results show that in RINm5F cells and human primary β-cells, IL-1β alters mitochondrial membrane potential, mitochondrial permeability transition pore opening, ATP content, and reactive oxygen species production and these alterations are preceded by ER Ca²⁺ release via IP₃R channels and mitochondrial Ca²⁺ uptake. All these events are prevented by JNK1/2 small interfering RNA (siRNA), indicating the mediating role of JNK1/2 in IL-1β–induced cellular alteration. This is accompanied by IL-1β–induced apoptosis, which is prevented by JNK1/2 siRNA and the IP₃R inhibitor xestospongin C. This suggests a regulatory role of JNK1/2 in modulating the ER-mitochondrial-Ca²⁺ axis by IL-1β in apoptotic cell death.     

Novel small molecule inhibition of IKK/NF‐κB activation reduces markers of senescence and improves healthspan in mouse models of aging

Constitutive NF‐κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF‐κB family members are enriched in centenarians. We recently identified a novel small molecule (SR12343) that inhibits IKK/NF‐κB activation by disrupting the association between IKKβ and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence‐associated beta‐galactosidase (SA‐β‐gal) activity in oxidative stress‐induced senescent mouse embryonic fibroblasts as well as in etoposide‐induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1 ^{−/ ∆} and Zmpste24 ^−/− mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2‐year‐old WT mice. Taken together, these results demonstrate that IKK/NF‐κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age‐related diseases.     

CD36 Protein Influences Myocardial Ca2+ Homeostasis and Phospholipid Metabolism: CONDUCTION ANOMALIES IN CD36-DEFICIENT MICE DURING FASTING*

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca²⁺ flux and activation of Ca²⁺-dependent phospholipases A₂ that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca²⁺ handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18–22 h) CD36^−/− and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36^−/− mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36^−/− mice documented prolongation of Ca²⁺ transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca²⁺. Hearts of CD36^−/− mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36^−/− hearts. Abnormal Ca²⁺ homeostasis in the CD36^−/− myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A₂ activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca²⁺ homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca²⁺ may trigger apoptosis in β-cells. Hence, the control of intracellular Ca²⁺ may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca²⁺-ATPase (PMCA) overexpression on Ca²⁺-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca²⁺] using a combination of aequorins with different Ca²⁺ affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. Overexpression of PMCA decreased [Ca²⁺] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. PMCA overexpression depletes intracellular [Ca²⁺] stores and, despite a decrease in mitochondrial [Ca²⁺], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca²⁺ homeostasis that could decrease β-cell apoptosis in diabetes.     

Na+ Dysregulation Coupled with Ca2+ Entry through NCX1 Promotes Muscular Dystrophy in Mice

Unregulated Ca²⁺ entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na⁺-Ca²⁺ exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd^−/−), Dysf^−/−, and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd^−/− mice. Measured increases in baseline Na⁺ and Ca²⁺ in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca²⁺ influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca²⁺ levels. Indeed, Atp1a2^+/− (encoding Na⁺-K⁺ ATPase α2) mice, which have reduced Na⁺ clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na⁺-K⁺ ATPase inhibitor digoxin. Treatment of Sgcd^−/− mice with ranolazine, a broadly acting Na⁺ channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology.     

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.