Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP₃) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP₃ levels by rapid photolysis of caged IP₃ has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP₃ uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.     

Cytosolic and nuclear calcium signaling in atrial myocytes: IP3-mediated calcium release and the role of mitochondria

In rabbit atrial myocytes Ca signaling has unique features due to the lack of transverse (t) tubules, the spatial arrangement of mitochondria and the contribution of inositol-1,4,5-trisphosphate (IP₃) receptor-induced Ca release (IICR). During excitation-contraction coupling action potential-induced elevation of cytosolic [Ca] originates in the cell periphery from Ca released from the junctional sarcoplasmic reticulum (j-SR) and then propagates by Ca-induced Ca release from non-junctional (nj-) SR toward the cell center. The subsarcolemmal region between j-SR and the first array of nj-SR Ca release sites is devoid of mitochondria which results in a rapid propagation of activation through this domain, whereas the subsequent propagation through the nj-SR network occurs at a velocity typical for a propagating Ca wave. Inhibition of mitochondrial Ca uptake with the Ca uniporter blocker Ru360 accelerates propagation and increases the amplitude of Ca transients (CaTs) originating from nj-SR. Elevation of cytosolic IP₃ levels by rapid photolysis of caged IP₃ has profound effects on the magnitude of subcellular CaTs with increased Ca release from nj-SR and enhanced CaTs in the nuclear compartment. IP₃ uncaging restricted to the nucleus elicites ‘mini’-Ca waves that remain confined to this compartment. Elementary IICR events (Ca puffs) preferentially originate in the nucleus in close physical association with membrane structures of the nuclear envelope and the nucleoplasmic reticulum. The data suggest that in atrial myocytes the nucleus is an autonomous Ca signaling domain where Ca dynamics are primarily governed by IICR.     

MiR-204 regulates type 1 IP3R to control vascular smooth muscle cell contractility and blood pressure

MiR-204 is expressed in vascular smooth muscle cells (VSMC). However, its role in VSMC contraction is not known. We determined if miR-204 controls VSMC contractility and blood pressure through regulation of sarcoplasmic reticulum (SR) calcium (Ca²⁺) release. Systolic blood pressure (SBP) and vasoreactivity to VSMC contractile agonists (phenylephrine (PE), thromboxane analogue (U46619), endothelin-1 (ET-1), angiotensin-II (Ang II) and norepinephrine (NE) were compared in aortas and mesenteric resistance arteries (MRA) from miR-204^−/− mice and wildtype mice (WT). There was no difference in basal systolic blood pressure (SBP) between the two genotypes; however, hypertensive response to Ang II was significantly greater in miR-204^−/− mice compared to WT mice. Aortas and MRA of miR-204^−/− mice had heightened contractility to all VSMC agonists. In silico algorithms predicted the type 1 Inositol 1, 4, 5-trisphosphate receptor (IP₃R1) as a target of miR-204. Aortas and MRA of miR-204^−/− mice had higher expression of IP₃R1 compared to WT mice. Difference in agonist-induced vasoconstriction between miR-204^−/− and WT mice was abolished with pharmacologic inhibition of IP₃R1. Furthermore, Ang II-induced aortic IP₃R1 was greater in miR-204^−/− mice compared to WT mice. In addition, difference in aortic vasoconstriction to VSMC agonists between miR-204^−/− and WT mice persisted after Ang II infusion. Inhibition of miR-204 in VSMC in vitro increased IP₃R1, and boosted SR Ca²⁺ release in response to PE, while overexpression of miR-204 downregulated IP₃R1. Finally, a sequence-specific nucleotide blocker that targets the miR-204-IP₃R1 interaction rescued miR-204-induced downregulation of IP₃R1. We conclude that miR-204 controls VSMC contractility and blood pressure through IP₃R1-dependent regulation of SR calcium release.     

IP3R3 silencing induced actin cytoskeletal reorganization through ARHGAP18/RhoA/mDia1/FAK pathway in breast cancer cell lines

Cell morphology is altered in the migration process, and the underlying cytoskeleton remodeling is highly dependent of intracellular Ca²⁺ concentration. Many calcium channels are known to be involved in migration. Inositol 1,4,5-trisphosphate receptor (IP₃R) was demonstrated to be implicated in breast cancer cells migration, but its involvement in morphological changes during the migration process remains unclear. In the present work, we showed that IP₃R3 expression was correlated to cell morphology. IP₃R3 silencing induced rounding shape and decreased adhesion in invasive breast cancer cell lines. Moreover, IP₃R3 silencing decreased ARHGAP18 expression, RhoA activity, Cdc42 expression and ^Y861FAK phosphorylation. Interestingly, IP₃R3 was able to regulate profilin remodeling, without inducing any myosin II reorganization. IP₃R3 silencing revealed an oscillatory calcium signature, with a predominant oscillating profile occurring in early wound repair. To summarize, we demonstrated that IP₃R3 is able to modulate intracellular Ca²⁺ availability and to coordinate the remodeling of profilin cytoskeleton organization through the ARHGAP18/RhoA/mDia1/FAK pathway.     

Initiation and propagation of a neuronal intracellular calcium wave

The ability to image calcium movement within individual neurons inspires questions of functionality including whether calcium entry into the nucleus is related to genetic regulation for phenomena such as long term potentiation. Calcium waves have been initiated in hippocampal pyramidal cells with glutmatergic signals both in the presence and absence of back propagating action potentials (BPAPs). The dendritic sites of initiation of these calcium waves within about 100 μm of the soma are thought to be localized near oblique junctions. Stimulation of synapses on oblique dendrites leads to production of inositol 1,4,5-trisphosphate (IP₃) which diffuses to the apical dendrite igniting awaiting IP₃ receptors (IP₃Rs) and initiating and propagating catalytic calcium release from the endoplasmic reticulum. We construct a reduced mathematical system which accounts for calcium wave initiation and propagation due to elevated IP₃. Inhomogeneity in IP₃ distribution is responsible for calcium wave initiation versus subthreshold or spatially uniform suprathreshold activation. However, the likelihood that a calcium wave is initiated does not necessarily increase with more calcium entering from BPAPs. For low transient synaptic stimuli, timing between IP₃ generation and BPAPs is critical for calcium wave initiation. We also show that inhomogeneity in IP₃R density can account for calcium wave directionality. Simulating somatic muscarinic receptor production of IP₃, we can account for the critical difference between calcium wave entry into the soma and failure to do so.     

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic sarcoplasmic reticulum vesicles

Inositol 1,4,5-trisphosphate-induced calcium release from canine aortic smooth muscle sarcoplasmic reticulum vesicles was examined using the calcium indicator antipyrylazo III. Calcium release was initiated by addition of inositol 1,4,5-trisphosphate (IP₃) to aortic vesicles 7 min after initiation of ATP-supported calcium uptake. Half-maximal calcium release occurred at 1 μM IP₃, with maximal calcium release amounting to 25±2% of the intravesicular calcium (n=12, 9 preparations). Ruthenium red (10–20 μM), which has been reported to block IP₃-induced calcium release from skeletal muscle sarcoplasmic reticulum, did not inhibit aortic IP₃-induced calcium release. Elevation of Mg²⁺ concentration from 0.06 to 7.8 mM inhibited aortic IP₃-induced calcium release 75%, which contrasts with the Mg²⁺-insensitive IP₃-induced calcium release from platelet reticular membranes. The IP₃-dependence of aortic calcium release suggested that Mg²⁺ acted as a noncompetitive inhibitor. Thus, aortic sarcoplasmic reticulum vesicles contain an IP₃-sensitive calcium pathway which is inhibited by millimolar concentrations of Mg²⁺, but which is not inhibited by Ruthenium red and so differs from the previously described IP₃-sensitive calcium pathways in skeletal muscle and platelet reticular membranes.     

Redox modulation of calcium entry and release of intracellular calcium by thimerosal in GH4C1 pituitary cells

In the present work we have investigated the actions of the oxidizing sulfhydryl reagent thimerosal on different mechanisms which regulate intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in GH₄C₁ pituitary cells. In intact Fura-2 loaded cells, low concentrations of thimerosal potentiated the spike phase of the TRH-induced (thyrotropin-releasing hormone) rise in [Ca²⁺]_i, whereas high thimerosal concentrations inhibited it. The effect of thimerosal on the plateau phase was always inhibitory.The effect of thimerosal on the IP₃-induced calcium release (IICR) was studied in permeabilized cells using the Ca²⁺ indicator Fluo-3. A low concentration of thimerosal (10 μM) stimulated IICR: the Ca²⁺ release induced by 300 nM inositol-1,4,5-trisphosphate (IP₃) was enhanced in cells treated with thimerosal for 1 or 6 min (67 ± 11 nM and 34 ± 5 nM, respectively) as compared to control cells (17 ± 2 nM). On the other hand, a high concentration of thimerosal (100 μ inhibited IICR: when IP₃ (10 μM) was added after a 5 min preincubation with thimerosal, the IP₃-induced rise in [Ca²⁺]_i (46 ± 14 nM) was 57% smaller as compared with that seen in control cells (106 ± 10 nM).The effect of thimerosal on the voltage-operated Ca ²⁺ channels (VOCCs) was studied by depolarizing intact Fura-2 loaded cells by addition of 20 mM K⁺ to the cuvette. The depolarization-evoked increase in [Ca²⁺]_i was inhibited in a dose-dependent manner by thimerosal. Direct evidence for an inhibitory effect of thimerosal on VOCCs was obtained by using the whole-cell configuration of the patch-clamp technique: thimerosal (100 μM) potently inhibited the Ba²⁺ currents through VOCCs.In addition, our results indicated that thimerosal inhibited the caffeine-induced increase in [Ca²⁺]_i, and activated a capacitative Ca²⁺ entry pathway. The actions of thimerosal were apparently due to its oxidizing activity because the effects were mostly reversed by the thiol-reducing agent dithiothreitol (DTT).We conclude that, in GH₄C₁ pituitary cells, the mobilization of intracellular calcium and the different Ca²⁺ entry pathways are sensitive to redox modulation.     

Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through σ1R/IP<Subscript>3</Subscript>R1 Complex

Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs). Aim of this work was to investigate possible changes in IP₃Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP₃Rs (IP₃R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP₃Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP₃R1s elevated cytosolic calcium and generated ER stress. IP₃R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.     

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.Key words: autophagy, calcium, Fyn, IP₃ receptor, dexamethasone     

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.     

The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP3 receptor channels

Ca²⁺ signalling plays an important role in various physiological processes in vertebrates. In mammals, the highly conserved anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein is an important modulator of the inositol 1,4,5-trisphosphate receptor (IP₃R), i.e. the main intracellular Ca²⁺ - release channel located at the endoplasmic reticulum (ER). The Bcl-2 Homology (BH) 4 domain of Bcl-2 (BH4-Bcl-2) is a critical determinant for inhibiting IP₃Rs, by directly targeting a region in the modulatory domain of the receptor (domain 3). In this paper, we aimed to track the evolutionary history of IP₃R regulation by the BH4 domain of Bcl-2 orthologues from different classes of vertebrates, including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. The high degree of conservation of the BH4 sequences correlated with the ability of all tested peptides to bind to the domain 3 of mouse IP₃R1 in GST-pull downs and their overall ability to inhibit IP₃-induced Ca²⁺ release (IICR) in permeabilized cells. Nevertheless, the BH4 domains differed in their potency to suppress IICR. The peptide derived from X. laevis was the least potent inhibitor. We identified a critical residue in BH4-Bcl-2 from H. sapiens, Thr7, which is replaced by Gly7 in X. laevis. Compared to the wild type X. laevis BH4-Bcl-2, a “humanized” version of the peptide (BH4-Bcl-2 Gly7Thr), displayed increased IP₃R-inhibitory properties. Despite the differences in the inhibitory efficiency, our data indicate that the BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as a binding partner and inhibitor of IP₃R channels.     

Apoptosis induced clustering of IP(3)R1 in nuclei of non-differentiated PC12 cells

Inositol 1,4,5‐trisphosphate (IP₃) receptors are emerging as key sites for regulation by pro‐ and anti‐apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP₃ receptors in non‐differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP₃R's calcium release—2‐aminoethoxydiphenyl borate (2‐APB) and xestospongin—completely prevented Bax and caspase‐3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP₃R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP₃R1 protein, but it also causes formation of IP₃R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP₃R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP₃R1 in PC12 cells. The amount of IP₃ induced calcium release was higher in control than in AIK‐treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP₃ receptors translocation to nuclei that can boost the calcium transport through IP₃ receptors. J. Cell. Physiol. 226: 3147–3155, 2011. © 2011 Wiley Periodicals, Inc.     

Endoplasmic reticulum remodeling tunes IP₃-dependent Ca²+ release sensitivity

The activation of vertebrate development at fertilization relies on IP₃-dependent Ca²⁺ release, a pathway that is sensitized during oocyte maturation. This sensitization has been shown to correlate with the remodeling of the endoplasmic reticulum into large ER patches, however the mechanisms involved are not clear. Here we show that IP₃ receptors within ER patches have a higher sensitivity to IP₃ than those in the neighboring reticular ER. The lateral diffusion rate of IP₃ receptors in both ER domains is similar, and ER patches dynamically fuse with reticular ER, arguing that IP₃ receptors exchange freely between the two ER compartments. These results suggest that increasing the density of IP₃ receptors through ER remodeling is sufficient to sensitize IP₃-dependent Ca²⁺ release. Mathematical modeling supports this concept of ‘geometric sensitization’ of IP₃ receptors as a population, and argues that it depends on enhanced Ca²⁺-dependent cooperativity at sub-threshold IP₃ concentrations. This represents a novel mechanism of tuning the sensitivity of IP₃ receptors through ER remodeling during meiosis.     

Hydrogen sulphide inhibits cardiomyocyte hypertrophy by up-regulating miR-133a

Hydrogen sulphide (H₂S) has been shown to play a crucial role in cardiovascular physiology and disease. However, there is no information about the possible role of H₂S in cardiomyocyte hypertrophy (CH). Our results showed that pretreatment with NaHS, an H₂S donor, significantly reduced [³H]-leucine incorporation, cell surface area, mRNA expression of brain natriuretic peptide (BNP), intracellular reactive oxygen species (ROS), miR-21 and increased atrial natriuretic peptide (ANP) and miR-133a expression in hypertrophic cardiomyocytes. Anti-miR133a inhibitor transfection partly reduced the anti-hypertrophic effect of NaHS. In conclusion, H₂S is a direct inhibitor of CH; it acts by increasing miR-133a and inhibiting the increase in intracellular ROS.     

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.     

Probes for manipulating and monitoring IP3

Inositol 1,4,5-trisphosphate (IP₃) is an important second messenger produced via G-protein-coupled receptor- or receptor tyrosine kinase-mediated pathways. IP₃ levels induce Ca²⁺ release from the endoplasmic reticulum (ER) via IP₃ receptor (IP₃R) located in the ER membrane. The resultant spatiotemporal pattern of Ca²⁺ signals regulates diverse cellular functions, including fertilization, gene expression, synaptic plasticity, and cell death. Therefore, monitoring and manipulating IP₃ levels is important to elucidate not only the functions of IP₃-mediated pathways but also the encoding mechanism of IP₃R as a converter of intracellular signals from IP₃ to Ca²⁺.     

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP₃Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP₃R1 and IP₃R2, and barely to IP₃R3, and essentially all cellular Bok is IP₃R bound in cells that express substantial amounts of IP₃Rs. Binding to IP₃Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP₃Rs (amino acids 1895–1903 of IP₃R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP₃R binding, the following was observed: (i) Bok does not appear to control the ability of IP₃Rs to release ER calcium stores, (ii) Bok regulates IP₃R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP₃R degradation, and (iv) Bok protects IP₃Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP₃Rs and that the most significant consequence of this binding appears to be protection of IP₃Rs from proteolysis. Thus, Bok may govern IP₃R cleavage and activity during apoptosis.