Internal Ca2 Research Articles

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.

To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For β-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. Antimalarial activity of 4a and 4b was IC50 = 1 and 4 μm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 μm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of β-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).

Force enhancement after stretch in mammalian muscle fiber: no evidence of cross-bridge involvement.

Stretching of activated skeletal muscles induces a force increase above the isometric level persisting after stretch, known as residual force enhancement (RFE). RFE has been extensively studied; nevertheless, its mechanism remains debated. Unlike previous RFE studies, here the excess of force after stretch, termed static tension (ST), was investigated with fast stretches (amplitude: 3-4% sarcomere length; duration: 0.6 ms) applied at low tension during the tetanus rise in fiber bundles from flexor digitorum brevis (FDB) mouse muscle at 30°C. ST was measured at sarcomere length between 2.6 and 4.4 μm in normal and N-benzyl-p-toluene sulphonamide (BTS)-added (10 μM) Tyrode solution. The results showed that ST has the same characteristics and it is equivalent to RFE. ST increased with sarcomere length, reached a peak at 3.5 μm, and decreased to zero at ∼4.5 μm. At 4 μm, where active force was zero, ST was still 50% of maximum. BTS reduced force by ∼75% but had almost no effect on ST. Following stimulation, ST developed earlier than force, with a time course similar to internal Ca(2+) concentration: it was present 1 ms after the stimulus, at zero active force, and peaked at ∼3-ms delay. At 2.7 μm, activation increased the passive sarcomere stiffness by a factor of ∼7 compared with the relaxed state All our data indicate that ST, or RFE, is independent of the cross-bridge presence and it is due to the Ca(2+)-induced stiffening of a sarcomeric structure identifiable with titin.

The role of NFATc2 in chronic autoimmune neuroinflammation

Dimethyl fumarate (DMF), a fumaric acid ester with potential immunomodulatory and neuroprotective effect, was recently approved as treatment for relapsing–remitting multiple sclerosis (MS). DMF ameliorates the clinical course of experimental autoimmune encephalomyelitis (EAE), the murine model of MS, where it exerts a neuroprotective action, reducing demyelination and axonal loss. We hypothesized that these effects are mediated, at least in part, through its action on microglia. We used a microglial cell line (N9) activated with lipopolysaccharide (LPS) to analyze the effect of monomethyl fumarate (MMF), a bioactive metabolite of DMF, in vitro. We show that MMF reverts the molecular phenotype of LPS-activatedmicroglia from pro(M1-like) to anti(M2-like) inflammatory. Thus, MMF significantly reduced production and expression of neurotoxic molecules, TNF-alpha, IL1-beta and iNOS, by activated microglia, while significantly increasing the production of receptors typically expressed by alternatively activated microglia, NURR1, CD200R and CX3CR1. We validated MMF effect at functional level, with the observation that MMF increases microglial internal calcium concentration and phagocytosis, itself associated with an increased expression of TREM2, which facilitates debris clearance in the absence of inflammation. Previous studies suggest thatMMF effects are related to activation of the Nrf2-anti-oxidant pathway; however such a possibility may not account for inhibitory effects on pro-inflammatory cytokines in microglia. In line with the observation that MMF upregulates NURR1, a molecule involved in inhibition of NFkB, we postulated that MMF exerts its anti-inflammatory effect through inhibiting NFkB. MMF is an agonist of HCA2 and activation of this receptor leads to an increase in intracellular Ca2+, which we observed upon exposure of activated microglia to MMF. We therefore addressed the possibility that MMF could signal through binding to HCA2.We show that HCA2 is expressed onN9 and blocking HCA2 reverts the effect ofMMF on the expression of pro-inflammatory cytokines and of NURR1. We have postulated and validated a downstream pathway activated by internal Ca2+ increase induced through MMF binding to HCA2, which leads to inhibition of NFkB activation by de-acetylation via the AMPK/Sirt axis. We show in vivo and ex vivo that treatment of EAE-affected mice with DMF upregulates M2-phenotype markers in the brain, and confirm the neuroprotective effect of MMF.

Module 3: Ion Channels

Ion channels have two main signalling functions: either they can generate second messengers or they can function as effectors by responding to such messengers. Their role in signal generation is mainly centred on the Ca2+ signalling pathway, which has a large number of Ca2+ entry channels and internal Ca2+ release channels, both of which contribute to the generation of Ca2+ signals. Ion channels are also important effectors in that they mediate the action of different intracellular signalling pathways. There are a large number of K+ channels and many of these function in different aspects of cell signalling. The voltage-dependent K+ (KV) channels regulate membrane potential and excitability. The inward rectifier K+ (Kir) channel family has a number of important groups of channels such as the G protein-gated inward rectifier K+ (GIRK) channels and the ATP-sensitive K+ (KATP) channels. The two-pore domain K+ (K2P) channels are responsible for the large background K+ current. Some of the actions of Ca2+ are carried out by Ca2+-sensitive K+ channels and Ca2+-sensitive Cl− channels. The latter are members of a large group of chloride channels and transporters with multiple functions. There is a large family of ATP-binding cassette (ABC) transporters some of which have a signalling role in that they extrude signalling components from the cell. One of the ABC transporters is the cystic fibrosis transmembrane conductance regulator (CFTR) that conducts anions (Cl− and HCO3−) and contributes to the osmotic gradient for the parallel flow of water in various transporting epithelia. Many of these epithelia also express aquaporins, which are water channels that increase the flux of water during fluid secretion or absorption. The cation channel of sperm (CatSper) is sensitive to intracellular pH and has a vital role to play in maintaining sperm hyperactivity prior to fertilization. Finally, there are hyperpolarizing-activated cyclic nucleotide-gated (HCN) channels that play an important role in various pacemaker mechanisms both in the heart and nervous system. Hemichannels provide membrane channels responsible for releasing ions and messengers from cells. On the other hand, gap junctions provide an intercellular pathway to transfer ions and messengers between cells. Mechanosensitive channels that open in response to membrane deformation have multiple functions especially in sensory systems such as touch and hair cell mechanoelectrical transduction. A large number of genetic diseases have been traced to mutations in various channels. For example, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel is the cause of cystic fibrosis. Many of the other channelopathies have been linked to changes in the channels that gate either the entry or the release of Ca2+.

Physiological Epidermal Growth Factor Concentrations Activate High Affinity Receptors to Elicit Calcium Oscillations

Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

Altered platelet calsequestrin abundance, Na+/Ca2 + exchange and Ca2 + signaling responses with the progression of diabetes mellitus

IntroductionDownregulation of calsequestrin (CSQ), a major Ca2+ storage protein, may contribute significantly to the hyperactivity of internal Ca2+ ([Ca2+]i) in diabetic platelets. Here, we investigated changes in CSQ-1 abundance, Ca2+ signaling and aggregation responses to stimulation with the progression of diabetes, especially the mechanism(s) underlying the exaggerated Ca2+ influx in diabetic platelets. Materials and methodsType 1 diabetes was induced by streptozotocin in rats. Platelet [Ca2+]i and aggregation responses upon ADP stimulation were assessed by fluorescence spectrophotometry and aggregometry, respectively. CSQ-1 expression was evaluated using western blotting. ResultsDuring the 12-week course of diabetes, the abundance of CSQ-1, basal [Ca2+]i and ADP-induced Ca2+ release were progressively altered in diabetic platelets, while the elevated Ca2+ influx and platelet aggregation were not correlated with diabetes development. 2-Aminoethoxydiphenyl borate, the store-operated Ca2+ channel blocker, almost completely abolished ADP-induced Ca2+ influx in normal and diabetic platelets, whereas nifedipine, an inhibitor of the nicotinic acid adenine dinucleotide phosphate receptor, showed no effect. Additionally, inhibition of Na+/Ca2+ exchange induced much slower Ca2+ extrusion and more Ca2+ influx in normal platelets than in diabetic platelets. Furthermore, under the condition of Ca2+-ATPase inhibition, ionomycin caused greater Ca2+ mobilization and Ca2+ influx in diabetic platelets than in normal platelets. ConclusionsThese data demonstrate that platelet hyperactivity in diabetes is caused by several integrated factors. Besides the downregulation of CSQ-1 that mainly disrupts basal Ca2+ homeostasis, insufficient Na+/Ca2+ exchange also contributes, at least in part, to the hyperactive Ca2+ response to stimulation in diabetic platelets.

Cardiac alternans and intracellular calcium cycling.

Cardiac alternans refers to a condition in which there is a periodic beat-to-beat oscillation in electrical activity and the strength of cardiac muscle contraction at a constant heart rate. Clinically, cardiac alternans occurs in settings that are typical for cardiac arrhythmias and has been causally linked to these conditions. At the cellular level, alternans is defined as beat-to-beat alternations in contraction amplitude (mechanical alternans), action potential duration (APD; electrical or APD alternans) and Ca(2+) transient amplitude (Ca(2+) alternans). The cause of alternans is multifactorial; however, alternans always originate from disturbances of the bidirectional coupling between membrane voltage (Vm ) and intracellular calcium ([Ca(2+) ]i ). Bidirectional coupling refers to the fact that, in cardiac cells, Vm depolarization and the generation of action potentials cause the elevation of [Ca(2+) ]i that is required for contraction (a process referred to as excitation-contraction coupling); conversely, changes of [Ca(2+) ]i control Vm because important membrane currents are Ca(2+) dependent. Evidence is mounting that alternans is ultimately caused by disturbances of cellular Ca(2+) signalling. Herein we review how two key factors of cardiac cellular Ca(2+) cycling, namely the release of Ca(2+) from internal stores and the capability of clearing the cytosol from Ca(2+) after each beat, determine the conditions under which alternans occurs. The contributions from key Ca(2+) -handling proteins (i.e. surface membrane channels, ion pumps and transporters and internal Ca(2+) release channels) are discussed.

Cav1.3 channels control D2-autoreceptor responses via NCS-1 in substantia nigra dopamine neurons.

Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca(2+) channels both contribute to Parkinson's disease pathology. L-type Ca(2+) channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca(2+) channel activity, internal Ca(2+), and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca(2+) channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca(2+) channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca(2+) channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.

Insulin Dosage Optimization Based on Model Predictive Control with the Model Parameters Estimation

Diabetes is a disease characterized by the inability of the pancreas to regulate blood glucose concentration. The development of the closed-loop control method for the treatment of type 1 diabetes is a highly desired endeavor for patients, physicians and scientists. Focusing on the need for maintaining normal glycaemia, the paper proposes an integrated closed-loop control method, which is the model predictive control (MPC) method combined with model parameters estimation. Using the static glucose model to be the internal model can’t achieve the good performance in different objects because of the individual differences. By updating the internal controller model with current measurement information using the parameters estimation, mismatch between the actual controlled object and the internal controller model is significantly reduced. Therefore, the predictions of future glucose values using the updated model are more accurate than those of the static input–output model. Finally, the integrated control method was tested on the T1DM simulator which was accepted by the FDA. Four objects are selected randomly in T1DM simulator. The control results showed it can modulate the glucose concentration to 85~120 mg/dl without severe hypoglycemia in the effective time. In conclusion, it is promising for the control of glucose concentration in subjects with type 1 diabetes.

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Patch-clamp measurement of ICRAC and ORAI channel activity.

Depletion of internal Ca(2+) stores activates store-operated Ca(2+) channels. The most prominent members of this class of channels are Ca(2+) release-activated Ca(2+) (CRAC) channels, which are present in a variety of cell types including immune cells. CRAC channels are composed of ORAI proteins, which are activated by endoplasmic reticulum-bound STIM proteins on Ca(2+) store depletion. The underlying Ca(2+) current is called ICRAC, which is required for many cellular functions including T-cell activation, mast cell activation, Ca(2+)-dependent gene expression, and refilling of internal Ca(2+) stores. To analyze ICRAC or the Ca(2+) current through heterologously expressed ORAI channels, whole-cell patch clamp is the technique of choice. It allows the direct analysis of ion currents through CRAC/ORAI channels. The patch-clamp technique has been used to determine selectivity, permeability, rectification, inactivation, and several other biophysical and pharmacological properties of the channels, and is the most direct and reliable technique to analyze ICRAC.

Glutamate involvement in calcium-dependent migration of astrocytoma cells.

BackgroundAstrocytoma are known to have altered glutamate machinery that results in the release of large amounts of glutamate into the extracellular space but the precise role of glutamate in favoring cancer processes has not yet been fully established. Several studies suggested that glutamate might provoke active killing of neurons thereby producing space for cancer cells to proliferate and migrate. Previously, we observed that calcium promotes disassembly of integrin-containing focal adhesions in astrocytoma, thus providing a link between calcium signaling and cell migration. The aim of this study was to determine how calcium signaling and glutamate transmission cooperate to promote enhanced astrocytoma migration.MethodsThe wound-healing model was used to assay migration of human U87MG astrocytoma cells and allowed to monitor calcium signaling during the migration process. The effect of glutamate on calcium signaling was evaluated together with the amount of glutamate released by astrocytoma during cell migration.ResultsWe observed that glutamate stimulates motility in serum-starved cells, whereas in the presence of serum, inhibitors of glutamate receptors reduce migration. Migration speed was also reduced in presence of an intracellular calcium chelator. During migration, cells displayed spontaneous Ca2+ transients. L-THA, an inhibitor of glutamate re-uptake increased the frequency of Ca2+ oscillations in oscillating cells and induced Ca2+ oscillations in quiescent cells. The frequency of migration-associated Ca2+ oscillations was reduced by prior incubation with glutamate receptor antagonists or with an anti-β1 integrin antibody. Application of glutamate induced increases in internal free Ca2+ concentration ([Ca2+]i). Finally we found that compounds known to increase [Ca2+]i in astrocytomas such as thapsigagin, ionomycin or the metabotropic glutamate receptor agonist t-ACPD, are able to induce glutamate release.ConclusionOur data demonstrate that glutamate increases migration speed in astrocytoma cells via enhancement of migration-associated Ca2+ oscillations that in turn induce glutamate secretion via an autocrine mechanism. Thus, glutamate receptors are further validated as potential targets for astrocytoma cancer therapy.

STIM1 controls neuronal Ca²⁺ signaling, mGluR1-dependent synaptic transmission, and cerebellar motor behavior.

In central mammalian neurons, activation of metabotropic glutamate receptor type1 (mGluR1) evokes a complex synaptic response consisting of IP3 receptor-dependent Ca(2+) release from internal Ca(2+) stores and a slow depolarizing potential involving TRPC3 channels. It is largely unclear how mGluR1 is linked to its downstream effectors. Here, we explored the role of stromal interaction molecule 1 (STIM1) in regulating neuronal Ca(2+) signaling and mGluR1-dependent synaptic transmission. By analyzing mouse cerebellar Purkinje neurons, we demonstrate that STIM1 is an essential regulator of the Ca(2+) level in neuronal endoplasmic reticulum Ca(2+) stores. Both mGluR1-dependent synaptic potentials and IP3 receptor-dependent Ca(2+) signals are strongly attenuated in the absence of STIM1. Furthermore, the Purkinje neuron-specific deletion of Stim1 causes impairments in cerebellar motor behavior. Together, our results demonstrate that in the mammalian nervous system STIM1 is a key regulator of intracellular Ca(2+) signaling, metabotropic glutamate receptor-dependent synaptic transmission, and motor coordination.

Regulation of Store‐Operated Ca2+ Entry in the Vascular Endothelium by the G Protein‐coupled Estrogen Receptor 1 (LB570)

The novel G protein‐coupled estrogen receptor 1 (GPER/GPR30) has been found to participate in numerous cardiovascular functions. Store‐operated Ca2+ entry (SOCE) is an essential mechanism that is required for many endothelial cell functions. We found that activation of GPER using the GPER specific agonist G1 causes a dose‐dependent inhibition of SOCE in primary vascular endothelial cells. Heterologous of GPER in HEK 293 cells is associated with substantial decreases in the total Ca2+ signal triggered by thapsigargin, and a 40% decrease in the rate of SOCE. Knockdown of GPER in endothelial cells using antisense directed against GPER increases SOCE by approximately 50% compared to cells transfected scrambled oligo. Interestingly, GPER specific antagonist G15 triggers total Ca2+ signals via SOCE by releasing the same internal Ca2+ stores as does thapsigargin. In primary endothelial cells, GPER forms a complex with the stromal interaction molecule 1 (Stim1), an essential molecular switcher of SOCE. In addition, Stim1‐ECFP colocalizes with GPER‐tetracysteine when heterologously expressed in HEK 293 cells. Heterologous expression of GPER‐DsRed2 fusion substantially decreases total Ca2+ signal stimulated by thapsigargin. Interestingly, SOCE measured in cells stably expressing fusions between EYFP and individual sub‐membrane domains (SMDs) 1 ‐ 4 of GPER does not differ from internal controls. These data indicate that GPER may be an important regulatory input of store‐operated Ca2+ entry via its interaction with Stim1. Since the EYFP‐SMD fusions are all cytoplasmically expressed, the data may also suggest that proper membrane docking (PM or ER) of full‐length GPER is required for it to exert its effect on SOCE.Grant Funding Source: National Institutes of Health

Modeling the intracellular organization of calcium signaling

Calcium (Ca²⁺) is a key signaling ion that plays a fundamental role in many cellular processes in most types of tissues and organisms. The versatility of this signaling pathway is remarkable. Depending on the cell type and the stimulus, intracellular Ca²⁺ increases can last over different periods, as short spikes or more sustained signals. From a spatial point of view, they can be localized or invade the whole cell. Such a richness of behaviors is possible thanks to numerous exchange processes with the external medium or internal Ca²⁺ pools, mainly the endoplasmic or sarcoplasmic reticulum and mitochondria. These fluxes are also highly regulated. In order to get an accurate description of the spatiotemporal organization of Ca²⁺ signaling, it is useful to resort to modeling. Thus, each flux can be described by an appropriate kinetic expression. Ca²⁺ dynamics in a given cell type can then be simulated by a modular approach, consisting of the assembly of computational descriptions of the appropriate fluxes and regulations. Modeling can also be used to get insight into the mechanisms of decoding of the Ca²⁺ signals responsible for cellular responses. Cells can use frequency or amplitude coding, as well as take profit of Ca²⁺ oscillations to increase their sensitivity to small average Ca²⁺ increases.

Mechanisms of Intracellular Calcium Homeostasis in Developing and Mature Bovine Corpora Lutea1

Although calcium (Ca(2+)) is accepted as an intracellular mediator of prostaglandin F2 alpha (PGF2alpha) actions on luteal cells, studies defining mechanisms of Ca(2+) homeostasis in bovine corpora lutea (CL) are lacking. The increase in intracellular Ca(2+) concentration ([Ca(2+)]i) induced by PGF2alpha in steroidogenic cells from mature CL is greater than in those isolated from developing CL. Our hypothesis is that differences in signal transduction associated with developing and mature CL contribute to the increased efficacy of PGF2alpha to induce a Ca(2+) signal capable of inducing regression in mature CL. To test this hypothesis, major genes participating in Ca(2+) homeostasis in the bovine CL were identified, and expression of mRNA, protein, or activity, in the case of phospholipase Cbeta (PLCbeta), in developing and mature bovine CL was compared. In addition, we examined the contribution of external and internal Ca(2+) to the PGF2alpha stimulated rise in [Ca(2+)]i in LLCs isolated from developing and mature bovine CL. Three differences were identified in mechanisms of calcium homeostasis between developing and mature CL, which could account for the lesser increase in [Ca(2+)]i in response to PGF2alpha in developing than in mature CL. First, there were lower concentrations of inositol 1,4,5-trisphosphate (IP3) after similar PGF2alpha challenge, indicating reduced phospholipase C beta (PLCbeta) activity, in developing than mature CL. Second, there was an increased expression of sorcin (SRI) in developing than in mature CL. This cytoplasmic Ca(2+) binding protein modulates the endoplasmic reticulum (ER) Ca(2+) release channel, ryanodine receptor (RyR), to be in the closed configuration. Third, there was greater expression of ATP2A2 or SERCA, which causes calcium reuptake into the ER, in developing than in mature CL. Developmental differences in expression detected in whole CL were confirmed by Western blots using protein samples from steroidogenic cells isolated from developing and mature CL. Localization of these genes in steroidogenic luteal cells was confirmed by immunohistochemistry. Therefore, it is concluded that the cellular mechanisms that allow PGF2alpha to induce a calcium signal of greater magnitude in mature than in developing CL involve 1) greater PLCbeta activity with enhanced generation of IP3, 2) an enhanced Ca(2+) release from the ER via unrestrained RYR2 due to a decrease in SRI expression, and 3) a reduction in calcium reuptake to the ER due to lower expression of ATP2A2. Accordingly, the increase in [Ca(2+)]i induced by PGF2alpha in mature large steroidogenic cells had less dependency from extracellular calcium than in those isolated from immature CL.

A dynamic model of calcium signaling in mast cells and LTC4 release induced by mechanical stimuli

Mast cells (MCs) play an important role in the immune system. It is known that mechanical stimuli can induce intracellular Ca2+ signal and release a variety of mediators, including leukotriene C4 (LTC4), leading to other cellular and physiological changes. In this paper, we present a mathematical model to explore signalling pathways in MCs, by including cellular mechanisms for intracellular $$\hbox{Ca}^{2+}$$ increase and LTC4 release in response to mechanical stimuli, thapsigargin (TG, SERCA pump inhibitor), and LTC4 stimuli. We show that (i) mechanical stimuli activate mechano-sensitive ion channels and induce inward ion fluxes and Ca2+ entry which increases intracellular Ca2+ concentration and releases LTC4; (ii) TG inhibits SERCA pumps, empties the internal Ca2+ stores, which activates Ca2+ release-activated Ca2+ channels and results in sustained intracellular Ca2+ increase; and (iii) LTC4 activates receptors on MCs surface and increases intracellular Ca2+ concentration. Our results are consistent with experimental observations, and furthermore, they also reveal that mechanical stimuli can increase intracellular Ca2+ even when LTC4 release is blocked, which suggests a feed forward loop involved in LTC4 production. This study may facilitate our understanding of the mechanotransduction process in MCs and provide a useful modeling tool for quantitatively analyzing immune mechanisms involving MCs.

DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca2+ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca2+ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca2+-activated Cl- channels (CaCCs) and increased intracellular calcium concentrations ([Ca2+]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca2+]i in mouse salivary gland cells and human corneal epithelial cells. [Ca2+]i increase of DA-6034 was dependent on the Ca2+ entry from extracellular and Ca2+ release from internal Ca2+ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca2+ stores. These results suggest that DA-6034 induces Ca2+ signaling via extracellular Ca2+ entry and RyRs-sensitive Ca2+ release from internal Ca2+ stores in epithelial cells.

Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death.

ABSTRACTThe model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca2+ ([Ca2+]c) dynamics and a distinct Ca2+ signature that involves Ca2+ influx from the external medium and internal Ca2+ stores. We investigated the molecular basis of this Ca2+ response by using [Ca2+]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca2+ signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca2+ entry does not occur through the hitherto described high- and low-affinity Ca2+ uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca2+ release from internal stores following IP3 formation combines with the extracellular Ca2+ influx.

Charged Residues on the Intracellular Intersubunit Assembly Interface Contribute to Calcium-Sensitivity of BK Channels

The large-conductance calcium-activated K+ (BK) channel contains a large C-terminal intracellular Ca2+-sensing region formed by two RCK domains. The RCK2 domain harbors a Ca2+-binding site of clustered negatively charged residues, known as the Ca2+-bowl, while the RCK1 domain is believed to contain another Ca2+-binding site (RCK1-site). Recent X-ray crystallographic structures of the Ca2+-free and -bound intracellular regions revealed an interesting location of the Ca2+-bowl near the tertrameric intersubunit assembly interface. We investigated the role of the intersubunit assembly interface on Ca2+-gated BK channel activation through mutational analyses of charged residues Glu955, Glu956, Arg786, and Arg790 which may electrostatically stabilize RCK2 intersubunit interactions by formation of intersubunit salt-bridges. We observed that mutations E955Q, E956Q, and E955Q/E956Q caused ∼50%, ∼30%, and ∼70% loss, respectively, of Ca2+-sensitivity as determined at 0, 7.3, and 90.4 μM internal free Ca2+. Combined analyses with Ca2+-bowl or RCK1-site null mutations indicated that E955Q mutation eliminated most Ca2+-sensitivity from the Ca2+-bowl and ∼50% sensitivity from the RCK1-site. The charge neutralization mutation R786A/R790A on the interacting subunits also led to ∼50% loss of Ca2+-sensitivity, suggesting involvement of intersubunit salt-bridge(s) between residues E955 and R786 or R790. Furthermore, intersubunit charge reversal mutations induced recovery of Ca2+-sensitivity. In contrast to the drastic effects of these charged residue mutations, which are located distant (≥20A) to the bound Ca2+, mutation of the Asn449 residue, which may coordinate the RCK2 Ca2+ from a different subunit, displayed little effect on Ca2+-sensitivity. We conclude that the intersubunit interfacial electrostatic interactions play an important role in Ca2+-dependent BK channel gating. We propose that such interactions are unlikely to be involved in direct Ca2+-binding but may provide intersubunit supporting points to mediate Ca2+-gated RCK gating ring expansion or conformational coupling to channel activation.