Regulation Of Intracellular Ca2 Research Articles

Overexpression of Calreticulin in Pre-eclamptic Placentas: Effect on Apoptosis, Cell Invasion and Severity of Pre-eclampsia

Endoplasmic reticulum (ER) stress has recently been identified as an important process involved in the pathology of pre-eclampsia (PE). Calreticulin (CRT) is an important ER resident protein which participates in the regulation of intracellular Ca(2+) homeostasis, cell adhesion, and cell apoptosis. In order to clarify the role of this protein in normal human pregnancy and in PE, this study has examined the expression of CRT in pre-eclamptic placenta compared with control placenta. The expression of CRT mRNA and protein was elevated in the pre-eclamptic placentas in comparison with control placentas. Furthermore, the expression level was related to the severity of symptoms experienced by PE patients. Therefore, this study aimed to identify the biological characteristics of the CRT gene in trophoblast cells. A CRT-expressing vector was transfected into the JEG-3 human choriocarcinoma cell line. Investigations showed that both proliferation and invasion were inhibited and apoptosis was promoted by CRT expression in JEG-3 cells. These data suggest that augmentation of CRT in the placenta may induce cell apoptosis and impair the invasion of extravillous trophoblast cells, thus leading to shallow placentation in PE.

The effect of MII α-conotoxin and its N-terminal derivatives on Ca2+- and Na+-signals induced by nicotine in SH-SY5Y neuroblastoma cells

Nicotinic acetylcholine receptors (nAChRs) are implicated in the regulation ofintracellular Ca2+-dependent processes in cells both in normal and pathological states, alpha-Conotoxins isolated from Conus snails venom are a valuable tool for the study of pharmacological properties and functional role of nAChRs. In the present study the alpha-conotoxin MII analogue with the additional tyrosine attached to the N terminus (Y0-MII) was prepared. Also we synthesized analogs with the N-terminal glycine residue labeled with the Bolton- Hunter reagent (BH-MII) or fluorestsein isothiocyanate (FITC-MII). Fluorescence microscopy studies of the neuroblastoma SH-SY5Y cells loaded with Ca2+ indicator Fura-2 or with Ca2+ and Na+ indicators Fluo-4 and SBFI were performed to examine effect of MII modification on its ability to inhibit nicotin-induced increases in intracellular free Ca2+ and Na+ concentrations ([Ca2+] and [Na+]i respectively). Monitoring of individual cell [Ca2+]i and [Na+]i signals revealed different kinetics of [Ca2+]i and [Na+]i rise and decay in responses to brief nicotine (Nic) applications (10-30 microM, 3-5 min), which indicates to different mechanisms of Ca2+ and Na+ homeostasis control in SH-SY5Y cells. MII inhibited in concentration-dependent manner the both [Ca2+]i and [Na+]i increase induced by Nic. Additional tyrosine in the Y0-MII or, especially, more sizeable label in FITC-MII significantly reduced the inhibitory effect of MII. Whereas the efficiency of the Ca2+ response inhibition by BH-MII was found to be close to the efficiency of its inhibition by natural alpha-conotoxin MII, radioiodinated derivatives BH-MII can be used in radioligand assay.

L-SERINE DECREASES BASAL CA2+ IN RAT AORTIC VASCULAR SMOOTH MUSCLE CELLS

L-serine decreases mean arterial pressure (MAP) in both normotensive and hypertensive rats. However, the molecular mechanism is not clear. Therefore, we examined the effect of L-serine on intracellular calcium (Ca2+) in cultured aortic vascular smooth muscle cells (VSMC) isolated from normotensive and hypertensive rat models. Ca2+ fluorescence intensity was studied by using confocal imaging. L-serine evoked a concentration dependent (5-30 μM) decrease in basal Ca2+ fluorescence intensity in normotensive rat models. This decrease in basal Ca2+ fluorescence intensity level was also profound in hypertensive rat models despite the fact that basal Ca2+ fluorescence intensity was higher in these models compared to the normotensive rats. We also observed L-serine (20-30 μM) decreases nuclear Ca2+ fluorescence intensity in both the rat models. In presence of a neutral amino acid transporter inhibitor, 2-amino-2-norbornanecarboxylic acid (BCH), L-serine did not have any effect on basal Ca2+ fluorescence intensity in VSMC of either rat models, suggesting the inhibition of amino acid transporter blocked the entry of L-serine in to the cell to have an effect. Hence this data indicates the possible role of L-serine in regulation of intracellular Ca2+ in these rat models.

Contribution of the OBSCN Nonsynonymous Variants to Aspirin Exacerbated Respiratory Disease Susceptibility in Korean Population

Airway remodeling and exacerbated airway narrowing in asthma have been attributed to the regulation of intracellular Ca(2+) by sarcoplasmic reticulum (SR) of the airway smooth muscle cells. The protein encoded by obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF (OBSCN) is a crucial factor in determining the SR architecture in Obscn(-/-) mice. This study genotyped a total of 55 common single-nucleotide polymorphisms (SNPs) in 592 Korean asthmatics including 163 aspirin exacerbated respiratory disease (AERD) cases and 429 aspirin-tolerant asthma (ATA) controls. Eight SNPs, including two nonsynonymous polymorphisms rs1188722C>T (Leu2116Phe) and rs1188729G>C (Cys4642Ser), and one haplotype BL2_ht1 showed statistically significant associations with AERD development (p=0.003-0.03). Two variants, rs1188722C>T (Leu2116Phe) and rs369252C>A, also revealed nominal association with FEV1 decline by aspirin provocation in asthmatics (p=0.03-0.04). Intriguingly, rs1188722C>T (Leu2116Phe) is a highly conserved amino acid residue among species, suggesting its functional relevance to AERD. In addition, the A allele of rs369252C>A, which was more prevalent in AERD than in ATA, was predicted as a potential branch point (BP) site for alternative splicing (BP score=4.29). Although further functional evaluation is required, our findings suggest that OBSCN polymorphisms, in particular, highly conserved nonsynonymous Leu2116Phe variant, might contribute to aspirin hypersensitivity in asthmatics.

Cyclic ADP ribose is a novel regulator of intracellular Ca2+ oscillations in human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca2+ signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca2+ (Ca2+i) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca2+i oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca2+i oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca2+i oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca2+ oscillation via activation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca2+ messenger for mobilizing the internal Ca2+ stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied.

Molecular and electrophysiological evidence for the expression of BK channels in oligodendroglial precursor cells

Changes in intracellular Ca(2+) play a key role in regulating gene expression and developmental changes in oligodendroglial precursor cells (OPCs). However, the mechanisms by which Ca(2+) influx in OPCs is controlled remains incompletely understood. Although there are several mechanisms that modulate Ca(2+) influx, in many systems the large-conductance, voltage- and Ca(2+) -activated K(+) channel (BK channel) plays an important role in regulating both membrane excitability and intracellular Ca(2+) levels. To date, the role of the BK channel in the regulation of intracellular Ca(2+) in oligodendroglial lineage cells is unknown. Here we investigated whether cells of the oligodendroglial lineage express BK channels and what potential role they play in regulation of Ca(2+) influx in these cells. In oligodendrocytes derived from differentiated adult neural precursor cells (NPCs, obtained from C57bl6 mice) we observed outward currents that were sensitive to the BK channel blocker iberiotoxin (IbTx). Further confirmation of the expression of the BK channel was obtained utilizing other blockers of the BK channel and by confocal immunofluoresence labelling of the BK channel on oligodendroglia. Using Fura-2AM to monitor intracellular Ca(2+) , it was observed that inhibition of the BK channel during glutamate-induced depolarization led to an additive increase in intracellular Ca(2+) levels. Electrophysiological difference currents demonstrated that the expression levels of the BK channel decrease with developmental age. This latter finding was further corroborated via RT-PCR and Western blot analysis. We conclude that the BK channel is involved in regulating Ca(2+) influx in OPCs, and may potentially play a role during differentiation of oligodendroglial lineage cells.

The specific Na +/Ca 2+ exchange inhibitor SEA0400 prevents nitric oxide-induced cytotoxicity in SH-SY5Y cells

The Na +/Ca 2+ exchanger (NCX) plays a role in the regulation of intracellular Ca 2+ levels, and nitric oxide (NO) is involved in many pathological conditions including neurodegenerative disorders. We have previously found that sodium nitroprusside (SNP), an NO donor, causes apoptotic-like cell death in cultured glial cells via NCX-mediated pathways and the mechanism for NO-induced cytotoxicity is cell type-dependent. The present study examined using the specific NCX inhibitor 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) whether NCX is involved in NO-induced injury in cultured neuronal cells. The treatment of neuroblastoma SH-SY5Y cells with SNP resulted in apoptosis and the cytotoxicity was blocked by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126 and the p38 MAP kinase (MAPK) inhibitor SB203580, but not by the c-Jun N-terminal kinase (JNK) inhibitor SP60012. SNP increased Ca 2+ influx and intracellular Ca 2+ levels. In addition, SNP increased ERK and p38 MAPK phosphorylation, and production of reactive oxygen species (ROS) in an extracellular Ca 2+-dependent manner. These effects of SNP were prevented by SEA0400. SNP-induced cytotoxicity was not affected by inhibitors of the Ca 2+, Na + and store-operated/capacitative channels. Moreover, SNP-induced increase in intracellular Ca 2+ levels, ROS production and decrease in cell viability were blocked by a cGMP-dependent protein kinase (PKG) inhibitor. These results suggest that Ca 2+ influx via the reverse of NCX is involved in the cascade of NO-induced neuronal apoptosis and NO activates the NCX through guanylate cyclase/PKG pathway.

Hadp1, a newly identified pleckstrin homology domain protein, is required for cardiac contractility in zebrafish

SUMMARYThe vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca2+ handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca2+ handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4)P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca2+ handling in the heart through its interaction with phosphatidylinositols.

Na+–Ca2+Exchanger (NCX3) Knock-Out Mice Display an Impairment in Hippocampal Long-Term Potentiation and Spatial Learning and Memory

Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca(2+) homeostasis, including Ca(2+) transporters. One of the major players in the regulation of intracellular Ca(2+) ([Ca(2+)](i)) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca(2+) clearance in the synaptic sites of hippocampal neurons. Because NCX3, one of the three brain isoforms of the NCX family, is highly expressed in the hippocampal subfields involved in LTP, we hypothesized that it might represent a potential candidate for LTP modulation. To test this hypothesis, we first examined the effect of ncx3 gene ablation on NCX currents (I(NCX)) and Ca(2+) homeostasis in hippocampal neurons. ncx3(-/-) neurons displayed a reduced I(NCX), a higher basal level of [Ca(2+)](i), and a significantly delayed clearance of [Ca(2+)](i) following depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed that ncx3(-/-) mice had an impaired basal synaptic transmission. Moreover, hippocampal slices from ncx3(-/-) mice exhibited a worsening in LTP compared with congenic ncx3(+/+). Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus of ncx3(-/-) mice both Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared with ncx3(+/+). Interestingly, ncx3(-/-) mice displayed a reduced spatial learning and memory performance, as revealed by the novel object recognition, Barnes maze, and context-dependent fear conditioning assays. Collectively, our findings demonstrate that the deletion of the ncx3 gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.

Differential Regulation and Recovery of Intracellular Ca2+ in Cerebral and Small Mesenteric Arterial Smooth Muscle Cells of Simulated Microgravity Rat

BackgroundThe differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca2+ determined by the alterations in the functions of plasma membrane CaL channels and ryanodine-sensitive Ca2+ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.Methodology/Principal FindingsSprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of CaL channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca2+ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of CaL channels and ryanodine-sensitive Ca2+ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.ConclusionsThe differential regulation of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca2+, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.

Recovery of rhythmic activity in a central pattern generator: analysis of the role of neuromodulator and activity-dependent mechanisms

The pyloric network of decapods crustaceans can undergo dramatic rhythmic activity changes. Under normal conditions the network generates low frequency rhythmic activity that depends obligatorily on the presence of neuromodulatory input from the central nervous system. When this input is removed (decentralization) the rhythmic activity ceases. In the continued absence of this input, periodic activity resumes after a few hours in the form of episodic bursting across the entire network that later turns into stable rhythmic activity that is nearly indistinguishable from control (recovery). It has been proposed that an activity-dependent modification of ionic conductance levels in the pyloric pacemaker neuron drives the process of recovery of activity. Previous modeling attempts have captured some aspects of the temporal changes observed experimentally, but key features could not be reproduced. Here we examined a model in which slow activity-dependent regulation of ionic conductances and slower neuromodulator-dependent regulation of intracellular Ca(2+) concentration reproduce all the temporal features of this recovery. Key aspects of these two regulatory mechanisms are their independence and their different kinetics. We also examined the role of variability (noise) in the activity-dependent regulation pathway and observe that it can help to reduce unrealistic constraints that were otherwise required on the neuromodulator-dependent pathway. We conclude that small variations in intracellular Ca(2+) concentration, a Ca(2+) uptake regulation mechanism that is directly targeted by neuromodulator-activated signaling pathways, and variability in the Ca(2+) concentration sensing signaling pathway can account for the observed changes in neuronal activity. Our conclusions are all amenable to experimental analysis.

Regulation of intracellular Ca2+ by reactive oxygen species in osteoblasts treated with antimycin A

This study evaluated the effects of antimycin A (AMA), an inhibitor of electron transport in mitochondria, on the release of intracellular calcium ion ([Ca(2+) ](i) ), ROS and bone resorbing factors in osteoblastic MC3T3-E1 cells. Pretreatment of osteoblasts with trolox, a ROS scavenger, and cyclosporin A, a potent inhibitor of calcium release from mitochondria, prevented the AMA-induced increases in [Ca(2+) ](i) . However, [Ca(2+) ](i) increase by AMA was unaffected by dantrolene, which blocks the ryanodine receptor channel of the endoplasmic reticulum. BAPTA/AM (an intracellular Ca(2+) chelator), dantrolene and cyclosporine A did not reverse the effect of AMA on ROS release. We also investigated whether intracellular calcium release inhibitor and antioxidant protect against AMA-induced bone resorbing cytokine release. Trolox prevented the release of receptor activator of nuclear factor-κB ligand (RANKL), IL-6, and TNF-α induced by AMA. Moreover, the increased IL-6 and TNF-α release by AMA was markedly reduced by BAPTA/AM and cyclosporin A. However, BAPTA/AM did not reverse the effect of AMA on osteoprotegerin and RANKL. Taken together, these results demonstrate that mitochondrial ROS generation and Ca(2+) influx by AMA is required for osteoblast death and bone resorbing cytokine release.

The Influence of Age on Bupivacaine Cardiotoxicity

The susceptibility of children and newborns to cardiotoxicity from racemic bupivacaine, RS(±)-bupivacaine, is controversial. Some studies indicate that newborns can sustain higher bupivacaine plasma levels than adults, without severe toxicity. In this study, we compared the influence of age on cardiotoxicity from RS(±)-bupivacaine and S(-)-bupivacaine in rats. The effects of these local anesthetics (LAs) on the regulation of intracellular Ca(2+) concentrations in cardiac fibers were also investigated. The lethal dose was determined in ventilated male Wistar rats at 2, 4, 8, and 16 weeks of age by monitoring when cardiac electrical activity stopped after infusion of RS(±)-bupivacaine and S(-)-bupivacaine (4 mg · kg(-1) · min(-1)). The effects on cardiac muscle contraction were investigated by in vitro measurement of papillary muscle twitches in the presence and absence of RS(±)-bupivacaine or S(-)-bupivacaine. Skinned ventricular fibers were used to investigate the intracellular effects on Ca(2+) regulation induced by both LAs. The lethal dose for RS(±)-bupivacaine and S(-)-bupivacaine in 2-week-old animals (46.0 ± 5.2 and 91.3 ± 4.9 mg · kg(-1), respectively) was higher than in 16-week-old animals (22.7 ± 1.3 and 22.0 ± 2.7 mg · kg(-1), respectively). Papillary muscle twitches were reduced in a dose-dependent manner, with significant difference between young and adult hearts. In adults, the muscle twitches were reduced to 8.6% ± 0.8% of control by RS(±)-bupivacaine, and to 18.1% ± 2.7% of control by S(-)-bupivacaine (100 μM). S(-)-bupivacaine had a positive inotropic effect at <10 μM, but only in 2-week-old animals. In chemically skinned ventricular fibers, RS(±)-bupivacaine and S(-)-bupivacaine induced similar increases in Ca(2+) release from the sarcoplasmic reticulum (SR) preactivated with caffeine (1 mM), and this effect was greater in younger rats than adults. In 16-week-old rats, caffeine-induced tension was 53.9% ± 1.7% of the maximal fiber response with RS(±)-bupivacaine, and 54.1% ± 3.2% with S(-)-bupivacaine. The caffeine response in 2-week-old rats was 81.1% ± 3.7% of the maximal response with RS(±)-bupivacaine, and 78.1% ± 4.5% with S(-)-bupivacaine. The Ca(2+) sensitivity of contractile proteins was equally increased at both ages tested, with RS(±)-bupivacaine or S(-)-bupivacaine. Ca(2+) uptake from the SR was not altered by the LA or by age. Differences in the mechanisms for regulating intracellular SR Ca(2+) may contribute to the decreased susceptibility of young animals to cardiodepression induced by RS(±)-bupivacaine and S(-)-bupivacaine.

Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (α1H) T-type Ca2+ Channels

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.

Intracellular calcium level is upregulated by interleukin-32 in auditory cells

Interleukin (IL)-32 has been associated with inflammation, apoptosis, and chemokine induction. The intracellular Ca 2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. Here, we document for the first time that IL-32 highly increased intracellular calcium level and IL-1β expression in an auditory cell line, HEI-OC1 cells. Treatment with 1, 2-bis (2-aminophenoxy) ethane-N, N, N’, N’-tetraacetic acid acetoxymethyl ester, a chelator of intracellular calcium, inhibited IL-32-induced IL-1 β production and caspase-1 activation. Thus, IL-32 may contribute to modulation of the inflammatory reaction through the regulation of intracellular Ca 2+ in the inner ear.

Regulation of store-operated channels by endoplasmic reticulum chaperons

Calreticulin is a conserved Ca2+ binding chaperone protein that is localized to the lumen of the endoplasmic reticulum (ER). The protein is implicated in many cellular functions such as the regulation of intracellular Ca2+ homeostasis, the regulation of gene expression, the folding of the newly synthesized proteins, cell adhesion, cancer and auto-immunity. The role of calreticulin in Ca2+ homeostasis regulation through Ca2+ storage and signaling might be the key to explaining the involvement of the protein in many biological functions inside and outside the ER. In this study, we examined whether calreticulin is responsible for regulating store-operated channels (SOC) on the plasma membrane by assessing changes in Ca2+ levels and SOC activity in mouse embryonic fibroblasts that are deficient in calreticulin. Wild type and calreticulin deficient fibroblasts were loaded with fura2-AM and the release of Ca2+ from the ER stores was induced using Thapsigargin or Ionomycin, and Ca2+ levels were measured using InC lm2 computer program. Calreticulin-defiecint fibroblasts showed a marked decrease in Ca2+ influx through SOC compared to wild type. To investigate the mechanism that underlines this decrease in SOC activity we performed western blots using antibodies for ORAI (Calcium release-activated calcium channel protein) and STIM (stromal interacting molecule). Our data showed a significant decrease in the expression of ORAI in the calreticulin-deficient fibroblasts compared to wild type while STIM expression in calreticulin-defiecint fibroblasts did not significantly differ from wild type. We concluded from our data that calreticulin controls SOC activity and this seems to be through regulating the expression of ORAI on the plasma membrane.

Echinacea-induced cytosolic Ca2+ elevation in HEK293

BackgroundWith a traditional medical use for treatment of various ailments, herbal preparations of Echinacea are now popularly used to improve immune responses. One likely mode of action is that alkamides from Echinacea bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca2+. Here, we show that unidentified compounds from Echinacea purpurea induce cytosolic Ca2+ elevation in non-immune-related cells, which lack CB2 receptors and that the Ca2+ elevation is not influenced by alkamides.MethodsA non-immune human cell line, HEK293, was chosen to evaluate E. purpurea root extracts and constituents as potential regulators of intracellular Ca2+ levels. Changes in cytosolic Ca2+ levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor, were tested to determine the mechanism of this Ca2+ signaling pathway. E. purpurea root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity.ResultsA rapid transient increase in cytosolic Ca2+ levels occurs when E. purpurea extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP3 receptor dependent. Echinacea-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca2+ elevation is sustained after the Echinacea-induced Ca2+ release from intracellular Ca2+ stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from E. purpurea root extracts, six induce cytosolic Ca2+ increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of Echinacea do not explain the observed Ca2+ response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity.ConclusionsOur data indicate that as yet unidentified constituents from Echinacea stimulate an IP3 receptor and phospholipase C mediation of cytosolic Ca2+ levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.

The role of TASK1 in aldosterone production and its expression in normal adrenal and aldosterone‐producing adenomas

Aldosterone production in the adrenal glomerulosa is mainly regulated by angiotensin II and K+. Adrenal glomerulosa cells are uniquely sensitive to extracellular K+. Genetic deletion of subunits of K+-selective leak-channels (KCNK), TASK1 and/or TASK3, in mice generates animals with hyperaldosteronism and histological changes in the adrenal cortex. Herein, we studied the expression of TASK1 in human adrenocortical cells, as well as its role in aldosterone production in H295R cells. TASK1 expression was investigated by comparative microarray analysis of aldosterone-producing adenomas (APA) and normal adrenals (NAs). The effects of TASK1 knockdown by siRNA transfection were investigated in H295R cells. Fluo-4 fluorescent measurements of intracellular Ca2+ and pharmacological inhibition of Ca2+ -dependent calmodulin kinases (CaMK) were performed to better define the effects of TASK1 on Ca2+ signalling pathways. Microarray analysis of APA and NA showed similar expression of TASK1 between these two groups. However, in APA, NA and H295R cells the expression of TASK1 was predominant when compared with other KCNK family members. Knockdown of TASK1 (with siRNA) induced the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2), and also stimulated pregnenolone and aldosterone production. Cells transfected with siTASK1 had increased intracellular Ca2+, leading to activation of CaMK and increased expression of CYP11B2. Our study reveals the predominant expression of TASK1 over other KCNK family genes in the human adrenal cortex. Herein, we also described the role of TASK1 in the regulation of human aldosterone production through regulation of intracellular Ca2+ and CaMK signalling pathways.

The expression of estrogen receptors in rat genioglossus muscle-derived satellite cells and its relationship to intracellular Ca 2+ mobilization

Genioglossus (GG) is the most important pharyngeal dilator muscle in maintaining upper airway (UA) patency in human; therefore, its dysfunction plays an important role in pathogenesis of sleep-related breathing disorder. Recently, the expression of estrogen receptors (ERs) on mRNA and protein level has been evidenced in GG muscle; however, the cellular localization of two subtypes of ER in GG myoblasts remains unclear. The present study was designed to clarify the expression and cellular distribution of ERs in rat GG muscle-derived satellite cells (MDSCs) and further probe the effect of ERs expression on regulation of intracellular Ca 2+. The immunocytochemistry revealed positive staining for both ERα and ERβ in nuclei and cytoplasm of GG MDSCs. Noticeably, positive signals for ERα and ERβ were comparable in cytoplasm, whereas the positive staining of ERα in nuclear was obviously strong than that of ERβ. More intriguingly, by using Fluo 4-AM as a fluorescent Ca 2+ indicator and 17β-estradiol (E2) as a stimulant, we observed that the level of intracellular Ca 2+ was not affected by E2 application, which implied that Ca 2+ signaling may not be involved in ER-mediated estrogenic effects on GG MDSCs. Taken together, the present study clearly indicates the differential cellular localization of ERs in rat GG MDSCs; moreover, ER-mediated estrogenic effect in rat GG MDSCs bears no relationship to intracellular Ca 2+ mobilization. In addition, the GG MDSCs express both ERα and ERβ and therefore, provide a suitable and convenient in vitro cell model for investigating the molecular mechanisms of estrogenic effects on rat GG muscle.

The calmodulin inhibitor N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalene sulphonamide directly blocks human ether à‐go‐go‐related gene potassium channels stably expressed in human embryonic kidney 293 cells

N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) is a well-known calmodulin inhibitor used to study calmodulin regulation of intracellular Ca(2+) signalling-related process. Here, we have determined whether W-7 would inhibit human ether gene (hERG or K(v) 11.1) potassium channels, hK(v) 1.5 channels or hK(IR) 2.1 channels expressed in human embryonic kidney (HEK) 293 cells. The hERG channel current, hK(v) 1.5 channel current or hK(IR) 2.1 channel current was recorded with a whole-cell patch clamp technique. It was found that the calmodulin inhibitor W-7 blocked hERG, hK(v) 1.5 and hK(IR) 2.1 channels. W-7 decreased the hERG current (I(hERG) ) in a concentration-dependent manner (IC(50) : 3.5 µM), and the inhibition was more significant at depolarization potentials between +10 and +60 mV. The hERG mutations in the S6 region Y652A and F656V, and in the pore helix S631A, had the IC(50) s of 5.5, 9.8 and 25.4 µM respectively. In addition, the compound inhibited hK(v) 1.5 and hK(IR) 2.1 channels with IC(50) s of 6.5 and 13.4 µM respectively. These results indicate that the calmodulin inhibitor W-7 exerts a direct channel-blocking effect on hERG, hK(v) 1.5 and hK(IR) 2.1 channels stably expressed in HEK 293 cells. Caution should be taken in the interpretation of calmodulin regulation of ion channels with W-7.