9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

It is well established that intracellular calcium ([Ca²⁺]_i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca²⁺ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC_Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC_Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca²⁺ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca²⁺ oscillations followed by an overall increase in [Ca²⁺]_i. The latter occurs also in HL-1 cells in Ca²⁺-free solution and after depletion of sarcoplasmic reticulum Ca²⁺ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca²⁺ oscillations followed by a compensatory increase in [Ca²⁺]_i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. 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.     

Structural and stoichiometric determinants of Ca release-activated Ca (CRAC) channel Ca-dependent inactivation

Depletion of intracellular Ca^2 + stores in mammalian cells results in Ca^2 + entry across the plasma membrane mediated primarily by Ca^2 + release-activated Ca^2 + (CRAC) channels. Ca^2 + influx through these channels is required for the maintenance of homeostasis and Ca^2 + signaling in most cell types. One of the main features of native CRAC channels is fast Ca^2 +-dependent inactivation (FCDI), where Ca^2 + entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca^2 + entry. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca^2 + influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states.     

Up-modulation of phorbol dibutyrate receptors by carbachol and arachidonic acid in rat prostatic epithelial cells

Phorbol dibutyrate (PDBu) binding to rat prostatic epithelial cells has been measured as an indirect determination of protein kinase C in this cell system. Analysis of [³H]PDBu binding using competitive displacement demonstrated a single class of PDBu receptors with a Kd=141 nM and a binding capacity of 4.8 pmol PDBu bound/mg cell protein. Raising cytosolic Ca²⁺ levels by redistribution of intracellular Ca²⁺ after cell treatment with carbachol or arachidonic acid (which also affects the bulk biophysical properties of the cell membrane) resulted in up-regulation of the available number of PDBu receptors. These results appear to be a consequence of PKC translocation from the cytosolic compartment to the plasma membrane after a cytosolic Ca²⁺ increase, confirming previous results in other cell systems.     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

Mammary gland involution is associated with rapid down regulation of major mammary Ca-ATPases

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca²⁺-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca²⁺-ATPases and mammary calcium transport is unknown. We found that 24 h after stopping milk production, PMCA2 and secretory pathway Ca²⁺-ATPases 1 and 2 (SPCA1 and 2) expression decreased 80-95%. PMCA4 and Sarco/Endoplasmic Reticulum Ca²⁺-ATPase 2 (SERCA2) expression increased with the loss of PMCA2, SPCA1, and SPCA2 but did not increase until 72-96 h of involution. The rapid loss of these Ca²⁺-ATPases occurs at a time of high mammary tissue calcium. These results suggest that the abrupt loss of Ca²⁺-ATPases, required by the mammary gland to regulate the large amount of calcium associated with milk production, could lead to accumulation of cell calcium, mitochondria Ca²⁺ overload, calcium mediated cell death and thus play a part in early signaling of mammary involution.     

The C-terminus of human Cav2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines

Ca_v2.3 containing voltage-activated Ca^2 + channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca^2 + which can both, facilitate and inhibit the influx of Ca^2 + ions through Ca_v2.3. The facilitated Ca^2 + influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca^2 + mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca_v2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca_v2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Altered Ca signaling in cancer cells: Proto-oncogenes and tumor suppressors targeting IP3 receptors

Proto-oncogenes and tumor suppressors critically control cell-fate decisions like cell survival, adaptation and death. These processes are regulated by Ca^2 + signals arising from the endoplasmic reticulum, which at distinct sites is in close proximity to the mitochondria. These organelles are linked by different mechanisms, including Ca^2 +-transport mechanisms involving the inositol 1,4,5-trisphosphate receptor (IP₃R) and the voltage-dependent anion channel (VDAC). The amount of Ca^2 + transfer from the endoplasmic reticulum to mitochondria determines the susceptibility of cells to apoptotic stimuli. Suppressing the transfer of Ca^2 + from the endoplasmic reticulum to the mitochondria increases the apoptotic resistance of cells and may decrease the cellular responsiveness to apoptotic signaling in response to cellular damage or alterations. This can result in the survival, growth and proliferation of cells with oncogenic features. Clearly, proper maintenance of endoplasmic reticulum Ca^2 + homeostasis and dynamics including its links with the mitochondrial network is essential to detect and eliminate altered cells with oncogenic features through the apoptotic pathway. Proto-oncogenes and tumor suppressors exploit the central role of Ca^2 + signaling by targeting the IP₃R. There are an increasing number of reports showing that activation of proto-oncogenes or inactivation of tumor suppressors directly affects IP₃R function and endoplasmic reticulum Ca^2 + homeostasis, thereby decreasing mitochondrial Ca^2 + uptake and mitochondrial outer membrane permeabilization. In this review, we provide an overview of the current knowledge on the proto-oncogenes and tumor suppressors identified as IP₃R-regulatory proteins and how they affect endoplasmic reticulum Ca^2 + homeostasis and dynamics.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes

Oxidative stress remodels Ca²⁺ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca²⁺ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca²⁺ current (I_Ca,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H₂O₂ induced an increase in I_Ca,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H₂O₂-induced LTF of I_Ca,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca²⁺ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H₂O₂ via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.     

The dual face of connexin-based astroglial Ca communication: A key player in brain physiology and a prime target in pathology

For decades, studies have been focusing on the neuronal abnormalities that accompany neurodegenerative disorders. Yet, glial cells are emerging as important players in numerous neurological diseases. Astrocytes, the main type of glia in the central nervous system , form extensive networks that physically and functionally connect neuronal synapses with cerebral blood vessels. Normal brain functioning strictly depends on highly specialized cellular cross-talk between these different partners to which Ca^2 +, as a signaling ion, largely contributes. Altered intracellular Ca^2 + levels are associated with neurodegenerative disorders and play a crucial role in the glial responses to injury. Intracellular Ca^2 + increases in single astrocytes can be propagated toward neighboring cells as intercellular Ca^2 + waves, thereby recruiting a larger group of cells. Intercellular Ca²⁺ wave propagation depends on two, parallel, connexin (Cx) channel-based mechanisms: i) the diffusion of inositol 1,4,5-trisphosphate through gap junction channels that directly connect the cytoplasm of neighboring cells, and ii) the release of paracrine messengers such as glutamate and ATP through hemichannels (‘half of a gap junction channel’). This review gives an overview of the current knowledge on Cx-mediated Ca^2 + communication among astrocytes as well as between astrocytes and other brain cell types in physiology and pathology, with a focus on the processes of neurodegeneration and reactive gliosis. Research on Cx-mediated astroglial Ca^2 + communication may ultimately shed light on the development of targeted therapies for neurodegenerative disorders in which astrocytes participate. 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.     

Crosstalk between insulin receptor and G protein-coupled receptor signaling systems leads to Ca oscillations in pancreatic cancer PANC-1 cells

We examined crosstalk between the insulin receptor and G protein-coupled receptor (GPCR) signaling pathways in individual human pancreatic cancer PANC-1 cells. Treatment of cells with insulin (10 ng/ml) for 5 min markedly enhanced the proportion of cells that display an increase in intracellular [Ca²⁺] induced by picomolar concentrations of the GPCR agonist neurotensin. Interestingly, insulin increased the proportion of a subpopulation of cells that exhibit intracellular [Ca²⁺] oscillations in response to neurotensin at concentrations as low as 50-200 pM. Insulin enhanced GPCR-induced Ca²⁺ signaling in a time- and dose-dependent manner; a marked potentiation was obtained after an exposure to a concentration of 10 ng/ml for 5 min. Treatment with the mTORC1 inhibitor rapamycin abrogated the increase in GPCR-induced [Ca²⁺]_i oscillations produced by insulin. Our results identify a novel aspect in the crosstalk between insulin receptor and GPCR signaling systems in pancreatic cancer cells, namely that insulin increases the number of [Ca²⁺]_i oscillating cells induced by physiological concentrations of GPCR agonists through an mTORC1-dependent pathway.     

Annexin A6—Linking Ca signaling with cholesterol transport

Annexin A6 (AnxA6) belongs to a conserved family of Ca²⁺-dependent membrane-binding proteins. Like other annexins, the function of AnxA6 is linked to its ability to bind phospholipids in cellular membranes in a dynamic and reversible fashion, in particular during the regulation of endocytic and exocytic pathways. High amounts of AnxA6 sequester cholesterol in late endosomes, thereby lowering the levels of cholesterol in the Golgi and the plasma membrane. These AnxA6-dependent redistributions of cellular cholesterol pools give rise to reduced cytoplasmic phospholipase A2 (cPLA₂) activity, retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. In addition to regulating cholesterol and caveolin distribution, AnxA6 acts as a scaffold/targeting protein for several signaling proteins, the best characterized being the Ca²⁺-dependent membrane targeting of p120GAP to downregulate Ras activity. AnxA6 also stimulates the Ca²⁺-inducible involvement of PKC in the regulation of HRas and possibly EGFR signal transduction pathways. The ability of AnxA6 to recruit regulators of the EGFR/Ras pathway is likely potentiated by AnxA6-induced actin remodeling. Accordingly, AnxA6 may function as an organizer of membrane domains (i) to modulate intracellular cholesterol homeostasis, (ii) to create a scaffold for the formation of multifactorial signaling complexes, and (iii) to regulate transient membrane-actin interactions during endocytic and exocytic transport. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Role of the beta1 subunit in large-conductance Ca(2+)-activated K(+) channel gating energetics. Mechanisms of enhanced Ca(2+) sensitivity

Over the past few years, it has become clear that an important mechanism by which large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca²⁺ 10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca²⁺. With this study, we address the question: which aspects of BK_Ca gating are altered by β1 to bring about these effects: Ca²⁺ binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BK_Ca α subunit in Xenopus oocytes, and then to compare β1''s steady state effects over a wide range of Ca²⁺ concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes in the aspects of gating listed above. The results of our analysis suggest that much of β1''s steady state effects can be accounted for by a reduction in the intrinsic energy the channel must overcome to open and a decrease in its voltage sensitivity, with little change in the affinity of the channel for Ca²⁺ when it is either open or closed. Interestingly, however, the small changes in Ca²⁺ binding affinity suggested by our analysis (K_c 7.4 μM → 9.6 μM; K_o = 0.80 μM → 0.65 μM) do appear to be functionally important. We also show that β1 affects the mSlo conductance–voltage relation in the essential absence of Ca²⁺, shifting it +20 mV and reducing its apparent gating charge 38%, and we develop methods for distinguishing between alterations in Ca²⁺ binding and other aspects of BK_Ca channel gating that may be of general use.     

Modulation of Ca2+ mobilization by protein kinase C in the submandibular duct cell line A253

The expression of protein kinase C (PKC) isoforms and the modulation of Ca²⁺ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP₃ formation, Ca²⁺ release and Ca²⁺ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP₃ formation but inhibited the Ca²⁺ release and the Ca²⁺ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca²⁺ release induced by TG, but reduced the influx of Ca²⁺ seen in the presence of this Ca²⁺-ATPase inhibitor. These results suggest that PKC modulates elements of the IP₃/Ca²⁺ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca²⁺ channels to IP₃, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca²⁺ ATPase, and (3) modulating Ca²⁺ entry pathways.     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

ORAI-mediated calcium influx in T cell proliferation, apoptosis and tolerance

Ca²⁺ homeostasis controls a diversity of cellular processes including proliferation and apoptosis. A very important aspect of Ca²⁺ signaling is how different Ca²⁺ signals are translated into specific cell functions. In T cells, Ca²⁺ signals are induced following the recognition of antigen by the T cell receptor and depend mainly on Ca²⁺ influx through store-operated CRAC channels, which are mediated by ORAI proteins following their activation by STIM proteins. The complete absence of Ca²⁺ influx caused by mutations in Stim1 and Orai1 leads to severe immunodeficiency. Here we summarize how Ca²⁺ signals are tuned to regulate important T cell functions as proliferation, apoptosis and tolerance, the latter one being a special state of immune cells in which they can no longer respond properly to an otherwise activating stimulus. Perturbations of Ca²⁺ signaling may be linked to immune suppressive diseases and autoimmune diseases.     

TGF-β-activated kinase 1 mediates mechanical stress-induced IL-6 expression in osteoblasts

Mechanical stress plays a key role in bone remodeling. Previous studies showed that loading of mechanical stretch induces a rapid Ca²⁺ influx and subsequent activation of stress-activated protein kinase pathways in osteoblasts. However, the activation mechanism and its significance in bone remodeling have not been fully elucidated. Here we show that TAK1 MAPKKK was activated by cyclic stretch loading of MC3T3-E1 cells. Knockdown of TAK1 attenuated the stretch-induced activation of JNK, p38, and NF-κB. Extracellular (EGTA) or intracellular (BAPTA/AM) Ca²⁺ chelator prevented the stretch-induced activation of TAK1. Activation of TAK1 and its associated downstream signaling pathways were also suppressed by CaMKII inhibitors (KN-93 and KN-62). Furthermore, TAK1-mediated downstream pathways cooperatively induced the expression of IL-6 mRNA in the stretched MC3T3-E1 cells. We also confirmed that TAK1 mediates cyclic stretch-induced IL-6 protein synthesis in the cells using immunoblotting and ELISA. Finally, stretch loading of murine primary osteoblasts induced the expression of IL-6 mRNA via TAK1. Collectively, these data suggest that stretch-dependent Ca²⁺ influx activates TAK1 via CaMKII, leading to the enhanced expression of IL-6 through JNK, p38, and NF-κB pathways in osteoblasts.