Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes.

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca(2+) signals and replenishment of Ca(2+) stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP(3)R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca(2+) entry between 1-10 microm. 2-APB activated authentic SOCs because the entry was totally selective for Ca(2+) (no detectable entry of Ba(2+) or Sr(2+) ions), and highly sensitive to La(3+) ions (IC(50) 30-100 nm). To assess the role of InsP(3)Rs in this response, we used the DT40 triple InsP(3)R-knockout (ko) cell line, DT40InsP(3)R-ko, in which the absence of full-length InsP(3)Rs or InsP(3)R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP(3)R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP(3)R-ko, cells indicating InsP(3)Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca(2+) entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca(2+) release from stores in intact DT40wt cells but not in DT40InsP(3)R-ko cells, indicating an InsP(3)R-mediated effect. In contrast, 2-APB blocked InsP(3)Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP(3)Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP(3)R activation but did not alter InsP(3)R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP(3)Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca(2+) entry.     

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.     

ITPRs/inositol 1,4,5-trisphosphate receptors in autophagy: From enemy to ally

ITPRs (inositol 1,4,5-trisphosphate receptors), the main endoplasmic reticulum (ER) Ca²⁺-release channels, were originally proposed as suppressors of autophagy. Yet, new evidence has accumulated over recent years supporting a crucial, stimulatory role for ITPRs in driving the autophagic flux. Here, we provide an integrated view on how ITPR-mediated Ca²⁺ signaling can have a dual impact on autophagy, depending on the characteristics of the spatio-temporal Ca²⁺ signals, including the existence of ER-mitochondrial and ER-lysosomal Ca²⁺ signaling microdomains.     

Endogenously bound calmodulin is essential for the function of the inositol 1,4,5-trisphosphate receptor

Calmodulin (CaM) is a ubiquitous Ca2+ sensor protein that plays an important role in regulating a large number of Ca2+ channels, including the inositol 1,4,5-trisphosphate receptor (IP3R). Despite many efforts, the exact mechanism by which CaM regulates the IP3R still remains elusive. Here we show, using unidirectional 45Ca2+ flux experiments on permeabilized L15 fibroblasts and COS-1 cells, that endogenously bound CaM is essential for the proper activation of the IP3R. Removing endogenously bound CaM by titration with a high affinity (pM) CaM-binding peptide derived from smooth muscle myosin light-chain kinase (MLCK peptide) strongly inhibited IP3-induced Ca2+ release. This inhibition was concentration- and time-dependent. Removing endogenously bound CaM affected the maximum release capacity but not its sensitivity to IP3. A mutant peptide with a strongly reduced affinity for CaM did not affect inhibited IP3-induced Ca2+ release. Furthermore, the inhibition by the MLCK peptide was fully reversible. Re-adding exogenous CaM, but not CaM1234, reactivated the IP3R. These data suggest that, by using a specific CaM-binding peptide, we removed endogenously bound CaM from a high affinity CaM-binding site on the IP3R, and this resulted in a complete loss of the IP3R activity. Our data support a new model whereby CaM is constitutively associated with the IP3R and functions as an essential subunit for proper functioning of the IP3R.     

The suppressor domain of inositol 1,4,5-trisphosphate receptor plays an essential role in the protection against apoptosis

The N-terminal 1-225 amino acids (aa) of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) function as a suppressor/coupling domain. In this study we used IP(3)R-deficient B-lymphocytes to investigate the effects of modifications in this domain on IP(3) binding and Ca(2+)-release activity. Although the N-terminal 1-225 aa of IP(3)R3 had the same role as in IP(3)R1, the suppression of IP(3) binding for IP(3)R1 was lost when the suppressor/coupling domains were exchanged between the two isoforms. Resulting chimeric receptors showed a higher sensitivity to IP(3)-induced activation (IICR). Deletion of 11 aa in IP(3)R1 ([Delta76-86]-IP(3)R1) or replacing aa 76-86 of the IP(3)R1 in the suppressor/coupling domain by 13 aa of IP(3)R3 ([75-87 T3]-IP(3)R1) also resulted in increased IP(3) binding and sensitivity of IICR. These residues constitute the only part of the suppressor/coupling domain that is strikingly different between the two isoforms. Expression of [Delta76-86]-IP(3)R1 and of [75-87 T3]-IP(3)R1 increased the propensity of cells to undergo staurosporine-induced apoptosis, but had no effect on the Ca(2+) content in the endoplasmic reticulum. In the cell model used, our observations suggest that the sensitivity of the Ca(2+)-release activity of IP(3)R1 to IP(3) influences the sensitivity of the cells to apoptotic stimuli and that the suppressor/coupling domain may have an anti-apoptotic function by attenuating the sensitivity of IICR.     

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Paracetamol (acetaminophen) decreases hydrogen sulfide tissue concentration in brain but increases it in the heart, liver and kidney in mice

The biological action ofN-acetyl-p-aminophenol - paracetamol (acetaminophen) has been demonstrated to involve different mechanisms and is still not clear. Hydrogen sulfide (H2S) has been shown to play an important role in many physiological and pathological processes including nociception. The interaction between acetaminophen and endogenous H2S is unknown. Twenty four female CBA strain mice were administered intraperitoneal injections of N-acetyl-p-aminophenol solution: paracetemol in doses of 30 mg/kg b.w. per day (group D1, n = 8) or 100 mg/kg b.w. per day (group D2, n = 8).. The control group (n = 8) received physiological saline in portions of the same volume--0.2 ml. The measurements of tissue H2S concentration were performed with the Siegel spectrophotometric modified method. In the brain, the H2S tissue level decreased, but more significantly in the lower drug dose group. Conversely, there was a significant rise in the H2S tissue concentration in D1 and D2 groups in heart and kidney with the increase more pronounced in the group with the lower paracetamol dose. In the liver only the higher acetaminophen dose elicited a change in H2S concentration, increasing after administration of acetaminophen at 100 mg/kg. Our study demonstrates that paracetamol induces H2S tissue concentration changes in different mouse organs.     

A dual role for Ca(2+) in autophagy regulation

Autophagy is a cellular process responsible for delivery of proteins or organelles to lysosomes. It participates not only in maintaining cellular homeostasis, but also in promoting survival during cellular stress situations. It is now well established that intracellular Ca²⁺ is one of the regulators of autophagy. However, this control of autophagy by intracellular Ca²⁺ signaling is the subject of two opposite views. On the one hand, the available evidence indicates that intracellular Ca²⁺ signals, and mainly inositol 1,4,5-trisphosphate receptors (IP₃Rs), suppress autophagy. On the other hand, elevated cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyt) were also shown to promote the autophagic process. Here, we will provide a critical overview of the literature and discuss both hypotheses. Moreover, we will suggest a model explaining how changes in intracellular Ca²⁺ signaling can lead to opposite outcomes, depending on the cellular state.     

The IP3 receptor-mitochondria connection in apoptosis and autophagy

The amount of Ca²⁺ taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca²⁺ ions mainly originate from the inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores of the endoplasmic reticulum (ER). The uptake of these Ca²⁺ ions in the mitochondria depends on the functional properties and the subcellular localization of the IP₃ receptor (IP₃R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca²⁺ ions from the ER to the mitochondria, structural interactions between IP₃Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

IFN-γ promoter gene polymorphism in psoriasis vulgaris

This study was performed to investigate the association between interferon (IFN)-γ single nucleotide polymorphism (SNP) and susceptibility for psoriasis vulgaris. DNA from 78 patients with psoriasis vulgaris (54 patients with type I psoriasis, 24 with type II psoriasis) and 74 healthy volunteers was investigated. IFN-γ promoter gene SNP in position 874 was evaluated by polymerase chain reaction with sequence-specific primers (PCR-SSP) and the results were compared between a group of psoriatic patients, divided into early onset of psoriasis (type I) and late onset of psoriasis (type II) subgroups, and healthy control subjects. A significant difference in the genotype frequencies between psoriasis patients and healthy controls was found (p <0.02) and no significant differences were observed analyzing subsets of psoriatic patients (gender, type of disease) also in carriage and allele frequencies. The results suggest that IFN-γ polymorphism is associated with susceptibility to psoriasis vulgaris.