Calcium-dependent facilitation and graded deactivation of store-operated calcium entry in fetal skeletal muscle

Activation of store-operated Ca²⁺ entry (SOCE) into the cytoplasm requires retrograde signaling from the intracellular Ca²⁺ release machinery, a process that involves an intimate interaction between protein components on the intracellular and cell surface membranes. The cellular machinery that governs the Ca²⁺ movement in muscle cells is developmentally regulated, reflecting maturation of the junctional membrane structure as well as coordinated expression of related Ca²⁺ signaling molecules. Here we demonstrate the existence of SOCE in freshly isolated skeletal muscle cells obtained from embryonic days 15 and 16 of the mouse embryo, a critical stage of muscle development. SOCE in the fetal muscle deactivates incrementally with the uptake of Ca²⁺ into the sarcoplasmic reticulum (SR). A novel Ca²⁺-dependent facilitation of SOCE is observed in cells transiently exposed to high cytosolic Ca²⁺. Our data suggest that cytosolic Ca²⁺ can facilitate SOCE whereas SR luminal Ca²⁺ can deactivate SOCE in the fetal skeletal muscle. This cooperative mechanism of SOCE regulation by Ca²⁺ ions not only enables tight control of SOCE by the SR membrane, but also provides an efficient mechanism of extracellular Ca²⁺ entry in response to physiological demand. Such Ca²⁺ signaling mechanism would likely contribute to contraction and development of the fetal skeletal muscle.     

Decrease in Ca2+-Activated K+ Conductance in Differentiated C6-Glioma Cells

Ca2+-activated K+ channels were studied in C6-glioma cells in an attempt to correlate changes in expression with cell proliferation and differentiation. In this study, we treated C6-glioma cells with thapsigargin for 48 h. Cell proliferation was markedly inhibited, and cell morphology changed from round to a spindle differentiated shape. Furthermore, intracellular calcium concentration was initially increased during acute treatment with thapsigargin. The internal [Ca2+]i pool was eventually depleted after a 48-h thapsigargin treatment. We have characterized Ca2+-activated K+ currents in less differentiated C6 cells. After differentiation of C6 cells induced by thapsigargin, Ca2+-activated K+ currents were selectively suppressed. These data lend further support to the notion that the expression of Ca2+-activated K+ channels is intimately associated with the proliferation of C6-glioma cells, and the suppression of Ca2+-activated K+ channels coincides with the inhibition of proliferation and subsequent induction of cell differentiation.     

Role of Intracellular Calcium Stores on the Effect of Metabotropic Glutamate Receptors on Phosphorylation of Glial Fibrillary Acidic Protein in Hippocampal Slices from Immature Rats

Phosphorylation of glial fibrillary acidic protein (GFAP) in slices from immature rats is stimulated by glutamate via a group II metabotropic glutamate receptor (mGluR II) and by absence of external Ca2+ in reactions that are not additive (Wofchuk and Rodnight, Neurochem. Int. 24:517-523, 1994). These observations suggested that glutamate, via an mGluR, inhibits Ca(2+)-entry through L-type Ca2+ channels and down-regulates a Ca(2+)-dependent dephosphorylation event coupled to GFAP. Because ryanodine receptors are present on internal Ca2+ stores and are associated with L-type Ca(2+)-channels, we investigated the possibility that the glutamatergic modulation of GFAP phosphorylation involves internal Ca2+ stores regulated by ryanodine receptors and whether the Ca2+ originating from these stores acts in a similar manner to external Ca2+. The results showed that the ryanodine receptor-agonists, caffeine and ryanodine and thapsigargin, all of which in appropriate doses increase cytoplasmic Ca2+, reversed the stimulation of GFAP phosphorylation given by 1S,3R-ACPD, an mGluR II agonist.     

Adipokinetic hormone-induced influx of extracellular calcium into insect fat body cells is mediated through depletion of intracellular calcium stores

Adipokinetic hormone I (AKH I) needs extracellular Ca²⁺ for its activating action on glycogen phosphorylase in locust fat body in vitro. TMB-8 reduces this AKH effect significantly, indicating that for a major part, hormone action also requires the mobilization of Ca²⁺ from intracellular stores. Using ⁴⁵Ca²⁺, AKH was shown to stimulate both the influx and the efflux of Ca²⁺. Thapsigargin also enhances the influx of extracellular Ca²⁺ into the fat body cells, indicating that the stimulating effect of AKH on Ca²⁺ influx may be mediated through depletion of intracellular Ca²⁺ stores as well. AKH is known to enhance cAMP levels in locust fat body. We show that elevation of cAMP with forskolin or theophylline leads to activation of glycogen phosphorylase, both in the presence and in the absence of extracellular Ca²⁺. The present data are discussed in an attempt to elucidate further the mechanism underlying transduction of the hormonal signal in locust fat body.     

Spontaneous Ca^2＋ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca^2＋ pools

INTRODUCTION In vascular smooth muscle, as in other types of muscle,an increase in intracellular Ca2 is the immediate triggerfor contraction, which ultimately determines vascular toneand peripheral resistance. In the past 12 years, investiga-tors have …     

Inhibition of the Ecto-ATPdiphosphohydrolase of Schistosoma mansoni by Thapsigargin

ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable ofhydrolyzing nucleoside di- and triphosphates. Although a number ofpossible physiological roles have been proposed for ATPDases, detailedstudies on structure-function relationships have generally been hamperedby the lack of specific inhibitors of these enzymes. We have previouslycharacterized a Ca²⁺-activated ATPDase on the external surface ofthe tegument of Schistosoma mansoni, the etiologic agent of humanschistosomiasis. In the present work, we have examined the effectsof thapsigargin, a sesquiterpene lactone known as a high affinityinhibitor of sarco-endoplasmic reticulum calcium transport (SERCA)ATPase, on ATPDase activity. Whereas other lactones tested had littleor no inhibitory action, thapsigargin inhibited ATP hydrolysis by theATPDase (K _i20 M). Interestingly, hydrolysis of ADP was notinhibited by thapsigargin. The lack of inhibition of ATPase activityby orthovanadate, a specific inhibitor of P-type ATPases, and theinhibition of the Mg²⁺-stimulated ATP hydrolysis by thapsigarginruled out the possibility that the observed inhibition of the ATPDaseby thapsigargin could be due to the presence of contaminating SERCAATPases in our preparation. Kinetic analysis indicated that a singleactive site in the ATPDase is responsible for hydrolysis of both ATPand ADP. Thapsigargin caused changes in both V _max and K _m for ATP,indicating a mixed type of inhibition. Inhibition by thapsigarginwas little or not affected by changes in free Ca²⁺ or Mg²⁺concentrations. These results suggest that interaction of thapsigarginwith the S. mansoni ATPDase prevents binding of ATP or its hydrolysisat the active site, while ADP can still undergo catalysis.     

Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands

The effect of ATP on mitochondrial membrane depolarization in rat submandibular glands was investigated. Exposure of the cell suspension to high concentrations of ATP induced a sustained depolarization of mitochondrial membrane. This effect was blocked in the presence of magnesium and reproduced by low concentrations of 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), suggesting the implication of the P2X(7) purinergic receptor. This point was confirmed by comparison of the response to ATP by wild-type and P2X(7) knock-out (P2X(7)R(-/-)) mice. Mitochondria took up calcium after ATP stimulation but the depolarization of the mitochondrial membrane by ATP was not affected by the removal of calcium from the extracellular medium. It was nearly fully suppressed in the absence of sodium and partially blocked by the mitochondrial Na/Ca exchanger inhibitor 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Both ATP and monensin increased the uptake of extracellular sodium (as shown by the depolarization of the plasma membrane) but the sodium ionophore did not affect the mitochondrial membrane potential. It is concluded that the activation of P2X(7) receptors depolarizes the mitochondrial membrane. The uptake of extracellular sodium is necessary but not sufficient to induce this response.     

Mitochondria adjust Ca signaling regime to a pattern of stimulation in salivary acinar cells

The salivary acinar cells have unique Ca²⁺ signaling machinery that ensures an extensive secretion. The agonist-induced secretion is governed by Ca²⁺ signals originated from the endoplasmic reticulum (ER) followed by a store-operated Ca²⁺ entry (SOCE). During tasting and chewing food a frequency of parasympathetic stimulation increases up to ten fold, entailing cells to adapt its Ca²⁺ machinery to promote ER refilling and ensure sustained SOCE by yet unknown mechanism. By employing a combination of fluorescent Ca²⁺ imaging in the cytoplasm and inside cellular organelles (ER and mitochondria) we described the role of mitochondria in adjustment of Ca²⁺ signaling regime and ER refilling according to a pattern of agonist stimulation. Under the sustained stimulation, SOCE is increased proportionally to the degree of ER depletion. Cell adapts its Ca²⁺ handling system directing more Ca²⁺ into mitochondria via microdomains of high [Ca²⁺] providing positive feedback on SOCE while intra-mitochondrial tunneling provides adequate ER refilling. In the absence of an agonist, the bulk of ER refilling occurs through Ca²⁺-ATPase-mediated Ca²⁺ uptake within subplasmalemmal space. In conclusion, mitochondria play a key role in the maintenance of sustained SOCE and adequate ER refilling by regulating Ca²⁺ fluxes within the cell that may represent an intrinsic adaptation mechanism to ensure a long-lasting secretion.     

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.     

The role of MAPK signalling pathways in the response to endoplasmic reticulum stress

Perturbations in endoplasmic reticulum (ER) homeostasis, including depletion of Ca^2 + or altered redox status, induce ER stress due to protein accumulation, misfolding and oxidation. This activates the unfolded protein response (UPR) to re-establish the balance between ER protein folding capacity and protein load, resulting in cell survival or, following chronic ER stress, promotes cell death. The mechanisms for the transition between adaptation to ER stress and ER stress-induced cell death are still being understood. However, the identification of numerous points of cross-talk between the UPR and mitogen-activated protein kinase (MAPK) signalling pathways may contribute to our understanding of the consequences of ER stress. Indeed, the MAPK signalling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses. In this article, we review UPR signalling and the activation of MAPK signalling pathways in response to ER stress. In addition, we highlight components of the UPR that are modulated in response to MAPK signalling and the consequences of this cross-talk. We also describe several diseases, including cancer, type II diabetes and retinal degeneration, where activation of the UPR and MAPK signalling contribute to disease progression and highlight potential avenues for therapeutic intervention. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.