Activated Rho/Rho kinase and modified calcium sensitivity in cryopreserved human saphenous veins

Background

We have shown previously that cryopreservation of human internal mammary arteries activates protein kinase C and enhances intracellular Ca²⁺ [Ca²⁺]_i. We now present evidence that in human saphenous veins (HSV) cryoinjury is associated with activation of the Rho/Rho kinase signaling pathways and enhanced [Ca²⁺]_i.

Methods

HSV were investigated in vitro either unfrozen within 12 h after removal or after storage at −196 °C in a cryomedium containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose as cryoprotectant additives.

Results

Cryostorage diminished responses to receptor-mediated contractile agonists such as noradrenaline, 5-HT and endothelin-1 by up to 30% whereas responses to KCl were attenuated by about 50%. Concentration-response curves for CaCl₂ on unfrozen and cryopreserved HSV revealed similar inhibitory activities of both blocking 1,4-dihydropyridine derivatives nifedipine and the (−)-(R) enantiomer of SDZ 202-791 whereas the Ca²⁺ channel activating (+)-(S) enantiomer of SDZ 202-791 was 10 times less effective at enhancing contractions to CaCl₂ when tested after cryostorage. These functional effects were reflected by changes in [Ca²⁺]_i as demonstrated by fluorescence of Fluo-3AM loaded veins. The diminished activity of (+)-(S) SDZ 202-791 in cryopreserved HSV was reversed partially when the potassium channel opener pinacidil (1 μM) was present during the freezing/thawing process. Blockade of Rho kinase by HA-1077 proved to be significantly more effective at attenuating contractile responses to both endothelin-1 and KCl after cryostorage.

Conclusions

Data suggested that cryopreservation modified [Ca²⁺]_i of venous smooth muscle cells (1) through depolarization-induced changes in Ca²⁺ influx and (2) through activation of Rho kinase signaling pathways.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells

Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca²⁺ action potentials due to the activation of voltage-dependent Ca²⁺ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca²⁺ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca²⁺ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca²⁺ from the endolysosomal system, resulting in localized Ca²⁺ signals. We show here that NAADP-mediated Ca²⁺ release from endolysosomal Ca²⁺ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca²⁺ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca²⁺ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca²⁺ release from acidic Ca²⁺ storage organelles in stimulus-secretion coupling in β cells.     

Changes in iron-regulatory gene expression occur in human cell culture models of Parkinson's disease

Background

Neuronal iron accumulation is thought to be relevant to the pathogenesis of Parkinson’s disease (PD), although the mechanism remains elusive. We hypothesized that neuronal iron uptake may be stimulated by functional mitochondrial iron deficiency.

Objective

To determine firstly whether the mitochondrial toxin, 1-methyl-4-phenylpyridinium iodide (MPP⁺), results in upregulation of iron-import proteins and transporters of iron into the mitochondria, and secondly whether similar changes in expression are induced by toxins with different mechanisms of action.

Methods

We used quantitative PCR and Western blotting to investigate expression of the iron importers, divalent metal transporter, transferrin receptor 1 and 2 (TfR1 and TfR2) and mitoferrin-2 and the iron exporter ferroportin in differentiated SH-SY5Y cells exposed to three different toxins relevant to PD, MPP⁺, paraquat (a free radical generator) and lactacystin (an inhibitor of the ubiquitin-proteasome system (UPS)).

Results

MPP⁺ resulted in increased mRNA and protein levels of genes involved in cellular iron import and transport into the mitochondria. Similar changes occurred following exposure to paraquat, another inducer of oxidative stress. Lactacystin also resulted in increased TfR1 mRNA levels, although the other changes were not found.

Conclusion

Our results support the hypothesis of a functional mitochondrial iron deficit driving neuronal iron uptake but also suggest that differences exist in neuronal iron handling induced by different toxins.     

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP₃Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP₃Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca²⁺ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP₃Rs at different cytosolic free Ca²⁺ concentrations ([Ca²⁺]_c) indicates that the association between these proteins is finely regulated by cytosolic Ca²⁺ via association of CaM and displacement of the IP₃Rs at high [Ca²⁺]_c. Thrombin-stimulated association of TRPC6 with CaM or the IP₃Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca²⁺-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP₃Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca²⁺ entry in the presence of low or high, respectively, extracellular Ca²⁺ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca²⁺ entry and aggregation through its interaction with CaM and IP₃Rs.     

A screening campaign in sea urchin egg homogenate as a platform for discovering modulators of NAADP-dependent Ca2+ signaling in human cells

The Ca²⁺ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. Targeting NAADP-evoked Ca²⁺ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. To aid discovery of novel scaffolds that modulate NAADP-evoked Ca²⁺ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. Known pharmacological inhibitors of NAADP-evoked Ca²⁺ release (but not cADPR- or IP₃-evoked Ca²⁺ release) in this invertebrate system strongly correlated with inhibition of MERS-pseudovirus infectivity in a human cell line. A primary screen of 1534 compounds yielded eighteen ‘hits’ exhibiting >80% inhibition of NAADP-evoked Ca²⁺ release. A validation pipeline for these candidates yielded seven drugs that inhibited NAADP-evoked Ca²⁺ release without depleting acidic Ca²⁺ stores in a human cell line. These candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of NAADP-evoked Ca²⁺ signals correlated well with observed inhibition of infectivity of a Middle East Respiratory syndrome coronavirus (MERS-CoV) pseudovirus. These experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of NAADP, cADPR and IP₃-dependent Ca²⁺ signaling with potential therapeutic value.     

Organelle-specific Subunit Interactions of the Vertebrate Two-pore Channel Family

The organellar targeting of two-pore channels (TPCs) and their capacity to associate as homo- and heterodimers may be critical to endolysosomal signaling. A more detailed understanding of the functional association of vertebrate TPC1–3 is therefore necessary. We report here that when stably expressed in HEK293 cells, human (h) TPC1 and chicken (c) TPC3 were specifically targeted to different subpopulations of endosomes, hTPC2 was specifically targeted to lysosomes, and rabbit (r) TPC3 was specifically targeted to both endosomes and lysosomes. Intracellular dialysis of NAADP evoked a Ca²⁺ transient in HEK293 cells that stably overexpressed hTPC1, hTPC2, and rTPC3, but not in cells that stably expressed cTPC3. The Ca²⁺ transients induced in cells that overexpressed endosome-targeted hTPC1 were abolished upon depletion of acidic Ca²⁺ stores by bafilomycin A₁, but remained unaffected following depletion of endoplasmic reticulum stores by thapsigargin. In contrast, Ca²⁺ transients induced via lysosome-targeted hTPC2 and endolysosome-targeted rTPC3 were abolished by bafilomycin A₁ and markedly attenuated by thapsigargin. NAADP induced marked Ca²⁺ transients in HEK293 cells that stably coexpressed hTPC2 with hTPC1 or cTPC3, but failed to evoke any such response in cells that coexpressed interacting hTPC2 and rTPC3 subunits. We therefore conclude that 1) all three TPC subtypes may support Ca²⁺ signaling from their designate acidic stores, and 2) lysosome-targeted (but not endosome-targeted) TPCs support coupling to the endoplasmic reticulum.     

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells

Background

It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca²⁺ mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca²⁺ release from Ca²⁺ stores in adrenal chromaffin cells.

Methods

We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca²⁺ in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2 h.

Results

Exposure to the magnetic field significantly reduced the increase in [Ca²⁺]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca²⁺ release by the exposure was unaffected.

Conclusions and general significance

These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca²⁺ release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca²⁺]i.     

Acidic Ca(2+) stores come to the fore

Changes in the concentration of cytosolic Ca²⁺ form the basis of a ubiquitous signal transduction pathway. Accumulating evidence implicates acidic organelles in the control of Ca²⁺ dynamics in organisms across phyla. In this special issue, we discuss Ca²⁺ signalling by these “acidic Ca²⁺ stores” which include acidocalcisomes, vacuoles, the endo-lysosomal system, lysosome-related organelles, secretory vesicles and the Golgi complex. Ca²⁺ release from these morphologically very different organelles is mediated by members of the TRP channel superfamily and two-pore channels. Inositol trisphosphate and ryanodine receptors which are traditionally viewed as endoplasmic reticulum Ca²⁺ release channels can also mobilize acidic Ca²⁺ stores. Ca²⁺ uptake into acidic Ca²⁺ stores is driven by Ca²⁺ ATPases and Ca²⁺/H⁺ exchangers. In animal cells, the Ca²⁺-mobilizing messenger NAADP plays a central role in mediating Ca²⁺ signals from acidic Ca²⁺ stores through activation of two-pore channels. These signals are important for several physiological processes including muscle contraction and differentiation. Dysfunctional acidic Ca²⁺ stores have been implicated in diseases such as acute pancreatitis and lysosomal storage disorders. Acidic Ca²⁺ stores are therefore emerging as essential components of the Ca²⁺ signalling network and merit extensive further study.     

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: Characterization and putative role

Background

The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors.

Scope of review

In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used.

Major conclusions

Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca²⁺.

General significance

Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.     

Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells

Background and aims

Steroid hormones target K⁺ channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3.

Results

Whole cell patch-clamp studies revealed E2 (10 nM) rapidly activates a whole cell K⁺ conductance, which is abolished by clotrimazole (30 μM), an inhibitor of the intermediate conductance calcium activated K⁺ channel (IK_Ca). The estrogen receptor (ER) antagonist ICI 182, 780 had no effect on this E2 activated K⁺ conductance, suggesting an estrogen receptor independent mechanism of activation. Confocal microscopy studies revealed under basal conditions that the IK_Ca channel is located within the cell cytoplasm and in the presence of E2, rapidly translocates to both the apical and basolateral membrane. In the presence of E2, tyrosine phosphorylation of calmodulin, which is known to regulate trafficking of the IK_Ca channel, is increased, and treatment of cells with the calmodulin inhibitor trifluoperazine (TFP) prevents the E2-induced translocation.

Conclusions

Estradiol rapidly regulates a K⁺ conductance through the IK_Ca channel in an estrogen receptor independent manner. E2 stimulates the translocation of IK_Ca to the cell membrane in a calmodulin dependent manner, representing a novel paradigm of estrogen action in sweat gland epithelial cells.     

The contractile vacuole in Ca2+-regulation in Dictyostelium: its essential function for cAMP-induced Ca2+-influx

Background  

cAMP-induced Ca²⁺-influx in Dictyostelium is controlled by at least two non-mitochondrial Ca²⁺-stores: acidic stores and the endoplasmic reticulum (ER). The acidic stores may comprise the contractile vacuole network (CV), the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca²⁺-store was investigated.     

Departure gate of acidic Ca2+ confirmed

More potent, but less known than IP₃ that liberates Ca²⁺ from the ER, NAADP releases Ca²⁺ from acidic stores. The notion that TPC channels mediate this Ca²⁺ release was questioned recently by studies suggesting that TPCs are rather PI(3,5)P₂‐activated Na⁺ channels. Ruas et al (2015) now partially reconcile these views by showing that TPCs significantly conduct both cations and confirm their activation by both NAADP and PI(3,5)P₂. They attribute the failure of others to observe TPC‐dependent NAADP‐induced Ca²⁺ release in vivo to inadequate mouse models that retain partial TPC function.     

Epithelial transport and barrier function in occludin-deficient mice

Background and Aims

This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model.

Methods

Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (R^e, epithelial resistance). Conductance scanning differentiated transcellular (G^c) and tight junctional conductance (G^tj). The pH-stat technique quantified gastric acid secretion.

Results

In occludin+/+ mice, R^e was 23±5 Ω cm² in jejunum, 66±5 Ω cm² in distal colon and 33±6 Ω cm² in gastric corpus and was not altered in heterozygotic occludin+/− or homozygotic occludin−/− mice. Additionally, [³H]mannitol fluxes were unaltered. In the control colon, G^c and G^tj were 7.6±1.0 and 0.3±0.1 mS/cm² and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin−/− mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control R^t was 5.8±0.8 kΩ cm² in urinary bladder and 33±6 Ω cm² in stomach and not altered in occludin−/− mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin−/− mice.

Conclusion

Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.     

Evidence that quantal Ca2+ release in HSG cells is not due to ‘all-or-none’ release from discrete Ca2+ stores with differing sensitivities to IP3

We demonstrate that the application of the muscarinic agonist carbachol to the human salivary epithelial cell line HSG elicits the now well-known phenomenon of quantal Ca²⁺-release; namely, that the application of a submaximal concentration of agonist results in the release of only a portion of the agonist-sensitive intracellular Ca²⁺ pool. One explanation that has been proposed to account for this effect is that there are multiple intracellular Ca²⁺ stores, each with a different agonist sensitivity, which release Ca²⁺ in an all-or-none fashion. We test this hypothesis in intact HSG cells with an experimental protocol designed to preferentially load less-agonist-sensitive stores with ⁴⁵Ca²⁺ and more-agonist-sensitive stores with ⁴⁵Ca²⁺. However, contrary to the expectations of the above explanation, these cells do not preferentially release ⁴⁵Ca²⁺ in response to low concentrations of agonist. Thus our data suggest that quantal Ca²⁺ release must arise from some other property of the stores or their Ca²⁺ release channels.     

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities

Purpose

Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs).

Methods

Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25 mmol/l for 24 h. l-Glucose was used at a concentration of 25 mmol/l as a control.

Results

Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB⁺FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment.

Conclusion

These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.     

TPC2 mediates new mechanisms of platelet dense granule membrane dynamics through regulation of Ca2+ release

Platelet dense granules (PDGs) are acidic calcium stores essential for normal hemostasis. They develop from late endosomal compartments upon receiving PDG-specific proteins through vesicular trafficking, but their maturation process is not well understood. Here we show that two-pore channel 2 (TPC2) is a component of the PDG membrane that regulates PDG luminal pH and the pool of releasable Ca²⁺. Using a genetically encoded Ca²⁺ biosensor and a pore mutant TPC2, we establish the function of TPC2 in Ca²⁺ release from PDGs and the formation of perigranular Ca²⁺ nanodomains. For the first time, Ca²⁺ spikes around PDGs—or any organelle of the endolysosome family—are visualized in real time and revealed to precisely mark organelle “kiss-and-run” events. Further, the presence of membranous tubules transiently connecting PDGs is revealed and shown to be dramatically enhanced by TPC2 in a mechanism that requires ion flux through TPC2. “Kiss-and-run” events and tubule connections mediate transfer of membrane proteins and luminal content between PDGs. The results show that PDGs use previously unknown mechanisms of membrane dynamics and content exchange that are regulated by TPC2.     

Serum miR-146a and miR-223 as potential new biomarkers for sepsis

Objective

Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified.

Methods

Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay.

Results

Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively.

Conclusion

Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.)     

Structural insights into the calcium-dependent interaction between calbindin-D28K and caspase-3

The regulation of apoptosis involves a complicated cascade requiring numerous protein interactions including the pro-apoptotic executioner protein caspase-3 and the anti-apoptotic calcium-binding protein calbindin-D28K. Using isothermal titration calorimetry, we show that calbindin-D28K binds caspase-3 in a Ca²⁺-dependent fashion. Molecular docking and conformational sampling studies of the Ca²⁺-loaded capase-3/calbindin-D28K interaction were performed in order to isolate potentially crucial intermolecular contacts. Residues in the active site loops of caspase-3 and EF-hands 1 and 2 of calbindin-D28K were shown to be critical to the interaction. Based on these studies, a model is proposed to help understand how calbindin-D28K may deactivate caspase-3 upon binding.

Structured summary of protein interactions

Calbindin-D28K and Caspase-3bind by isothermal titration calorimetry(View interaction)     

Sleep deprivation impairs calcium signaling in mouse splenocytes and leads to a decreased immune response

Background

Sleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca^2 +) plays a critical signaling role. We performed live cell measurements of cytosolic Ca^2 + mobilization to understand the changes in Ca^2 + signaling that occur in splenic immune cells after various periods of sleep deprivation (SD).

Methods

Adult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72 h) and Ca^2 + intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca^2 + mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca^2 + channel gene expression was evaluated

Results

Splenocytes showed a progressive loss of intracellular Ca^2 + maintenance from endoplasmic reticulum (ER) stores. Transient Ca^2 + buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca^2 + channels.

Conclusions and general significance

These novel data suggest that SD impairs Ca^2 + signaling, most likely as a result of ER stress, leading to an insufficient Ca^2 + supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function.