Intracellular Ca Release Research Articles

Cathepsin K knockout alleviates aging-induced cardiac dysfunction.

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload-induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age-dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca2+ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura-2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24-month-old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross-sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca2+ release compared to young (6-month-old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged-cathepsin K knockout mice compared to their wild-type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age-induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis-inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin-induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age-related decline in cardiac function via suppressing caspase-dependent and caspase-independent apoptosis.

Vasodilatory Effect of Wogonin on the Rat Aorta and Its Mechanism Study.

Wogonin, a natural flavonoid, is one of the bioactive compounds of the medicinal herb Eucommia ulmoides OLIV. widely used in southeastern Asia for treating hypertension. However, the molecular mechanisms for the therapeutic benefits remain largely unclear. The present study investigated the vasodilatory effect of wogonin and its possible mechanisms. The flavonoid (0.1-100 µM) caused concentration-dependent relaxations in endothelium-intact aortic rings precontracted with norepinephrine (NE, 1 µM) or potassium chloride (KCl, 60 mM). Preincubation with wogonin (10, 100 µM) for 20 min significantly inhibited the contractile responses to NE (0.1, 1, 10 µM) or KCl (7.5, 15, 30, 60 mM). Relaxant responses to wogonin were not inhibited by N(G)-nitro-L-arginine methylester (100 µM) or endothelial denudation. In a Ca(2+)-free Krebs' solution, wogonin not only blocked Ca(2+) influx-dependent vasoconstriction by either NE (1 µM) or KCl (100 mM), but also inhibited NE (1 µM)-induced tonic contraction, which is dependent on intracellular Ca(2+) release. Wogonin also suppressed the elevation of [Ca(2+)]i induced by KCl (60 mM) after exhausting the calcium store in sarcoplasmic and endoplasmic reticula with thapsigargin (1 µM) or by ATP (100 µM) in primary vascular smooth muscle cells. These findings suggest that wogonin-induced responses are mainly due to the inhibition of both intracellular Ca(2+) release and extracellular Ca(2+) influx.

The Resveratrol-induced Relaxation of Cholecystokinin Octapeptide- or KCl-induced Tension in Male Guinea Pig Gallbladder Strips Is Mediated Through L-type Ca2+Channels.

Background/AimsResveratrol (3,5,4′-trihydroxystilbene) is a polyphenolic compound (stilbene) and a phytoalexin. The purpose of this study was to determine the mechanism which mediated the resveratrol-induced relaxation of cholecystokinin octapeptide- or KCl-induced tension in male guinea pig gallbladder strips.MethodsGallbladder strips were prepared and suspended in in vitro chambers filled with Krebs-Henseleit solution. The strips were attached to force displacement transducers, and the changes in tension were recorded on a polygraph. All reagents were added directly into the chambers.ResultsTo determine if intracellular Ca2+ release mediated the resveratrol-induced relaxation of cholecystokinin octapeptide-induced tension, 2-aminoethoxydiphenylborane (2-APB) was used. 2-APB significantly (P < 0.01) decreased the amount of RSVL-induced relaxation. To determine if protein kinase A (PKA) mediated the resveratrol-induced relaxation, PKA inhibitor 14-22 amide myristolated (PKA-IM) was used. PKA-IM had no effect on resveratrol-induced relaxation. Neither KT5823, NG-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor, nor fulvestrant had a significant effect on the amount of resveratrol-induced relaxation. Genistein, a protein tyrosine kinase inhibitor, significantly (P < 0.01) increased the RSVL-induced relaxation. To determine if protein kinase C mediated the RSVL-induced relaxation, the protein kinase C inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together, and a significant (P < 0.05) increase in resveratrol-induced relaxation was observed. The pretreatment of the strips with resveratrol significantly (P < 0.001) decreased the amount of KCl- and cholecystokinin octapep-tide-induced tension.ConclusionsResveratrol-induced relaxation is mediated by its effects on L-type Ca2+ channels and intracellular Ca2+ release.

Purified IgGs from Type 2 Diabetes with Atrial Fibrillation Induce Intracellular Calcium Release in Cardiomyocytes through IP3 Pathway

Higher risk of developing life threatening cardiac arrhythmias, such as atrial fibrillation (AF) has been found in type 2 diabetes mellitus (T2DM), but the mechanism underlying the association is largely unknown. Our previous studies showed that circulating IgGs from certain advanced T2DM patients displayed anti-endothelial effects and often induced intracellular Ca release in endothelial cells. The present study is to test the hypothesis that circulating IgGs from T2DM patients with AF perturb intracellular Ca homeostasis in atrial cardiomyocytes which in turn trigger AF. Protein A-purified IgGs were obtained from a cohort of T2DM subjects with AF and subjects without AF or other cardiovascular complications. The data show that IgG (1 µg/mL) from T2DM AF patients caused acute intracellular Ca release in cultured mouse atrial cardiomyocytes HL-1 cells compared to control groups (p < 0.001). The IgG-mediated Ca release was insensitive to verapamil (20 µM), mibefradil (25 µM) or BTP-2 (5 µM), indicating that the Ca release is not through voltage-gated Ca channels or store-operated Ca entry. On the other hand, application of Xestospongin C (10 µM) or 2-APB (100 µM), two membrane-permeable IP3 receptor antagonists, or knockdown of IP3 receptor, significantly decreased Ca release stimulated by the IgGs from T2DM AF patients (p < 0.01). Taken together, our data suggest that circulating IgGs alter intracellular Ca homeostasis in cardiomyocytes through IP3 pathway in T2DM AF patients. Our studies may provide the mechanistic understanding on a causal role of circulating IgGs in T2DM patients with AF and other symptomatic cardiac conduction diseases. The IgGs may serve as a biomarker for identifying a subset of T2DM patients with increased risk for development of AF.

The mechanisms of calcium cycling and action potential dynamics in cardiac alternans.

Alternans is a risk factor for cardiac arrhythmia, including atrial fibrillation. At the cellular level alternans manifests as beat-to-beat alternations in contraction, action potential duration (APD), and magnitude of the Ca(2+) transient (CaT). Electromechanical and CaT alternans are highly correlated, however, it has remained controversial whether the primary cause of alternans is a disturbance of cellular Ca(2+) signaling or electrical membrane properties. To determine whether a primary failure of intracellular Ca(2+) regulation or disturbances in membrane potential and AP regulation are responsible for the occurrence of alternans in atrial myocytes. Pacing-induced APD and CaT alternans were studied in single rabbit atrial and ventricular myocytes using combined [Ca(2+)]i and electrophysiological measurements. In current-clamp experiments, APD and CaT alternans strongly correlated in time and magnitude. CaT alternans was observed without alternation in L-type Ca(2+) current, however, elimination of intracellular Ca(2+) release abolished APD alternans, indicating that [Ca(2+)]i dynamics have a profound effect on the occurrence of CaT alternans. Trains of 2 distinctive voltage commands in form of APs recorded during large and small alternans CaTs were applied to voltage-clamped cells. CaT alternans was observed with and without alternation in the voltage command shape. During alternans AP-clamp large CaTs coincided with both long and short AP waveforms, indicating that CaT alternans develop irrespective of AP dynamics. The primary mechanism underlying alternans in atrial cells, similarly to ventricular cells, resides in a disturbance of Ca(2+) signaling, whereas APD alternans are a secondary consequence, mediated by Ca(2+)-dependent AP modulation.

Dantrolene rescues aberrant N-terminus intersubunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors.

The ryanodine receptor (RyR2) is an intracellular Ca(2+) release channel essential for cardiac excitation-contraction coupling. Abnormal RyR2 channel function results in the generation of arrhythmias and sudden cardiac death. The present study was undertaken to investigate the mechanistic basis of RyR2 dysfunction in inherited arrhythmogenic cardiac disease. We present several lines of complementary evidence, indicating that the arrhythmia-associated L433P mutation disrupts RyR2 N-terminus self-association. A combination of yeast two-hybrid, co-immunoprecipitation, and chemical cross-linking assays collectively demonstrate that a RyR2 N-terminal fragment carrying the L433P mutation displays substantially reduced self-interaction compared with wild type. Moreover, sucrose density gradient centrifugation reveals that the L433P mutation impairs tetramerization of the full-length channel. [(3)H]Ryanodine-binding assays demonstrate that disrupted N-terminal intersubunit interactions within RyR2(L433P) confer an altered sensitivity to Ca(2+) activation. Calcium imaging of RyR2(L433P)-expressing cells reveals substantially prolonged Ca(2+) transients and reduced Ca(2+) store content indicating defective channel closure. Importantly, dantrolene treatment reverses the L433P mutation-induced impairment and restores channel function. The N-terminus domain constitutes an important structural determinant for the functional oligomerization of RyR2. Our findings are consistent with defective N-terminus self-association as a molecular mechanism underlying RyR2 channel deregulation in inherited arrhythmogenic cardiac disease. Significantly, the therapeutic action of dantrolene may occur via the restoration of normal RyR2 N-terminal intersubunit interactions.

Neurotensinergic Excitation of Dentate Gyrus Granule Cells via Gαq-Coupled Inhibition of TASK-3 Channels.

Neurotensin (NT) is a 13-amino acid peptide and serves as a neuromodulator in the brain. Whereas NT has been implicated in learning and memory, the underlying cellular and molecular mechanisms are ill-defined. Because the dentate gyrus receives profound innervation of fibers containing NT and expresses high density of NT receptors, we examined the effects of NT on the excitability of dentate gyrus granule cells (GCs). Our results showed that NT concentration dependently increased action potential (AP) firing frequency of the GCs by the activation of NTS1 receptors resulting in the depolarization of the GCs. NT-induced enhancement of AP firing frequency was not caused indirectly by releasing glutamate, GABA, acetylcholine, or dopamine, but due to the inhibition of TASK-3 K(+) channels. NT-mediated excitation of the GCs was G protein dependent, but independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Immunoprecipitation experiment demonstrates that the activation of NTS1 receptors induced the association of Gαq/11 and TASK-3 channels suggesting a direct coupling of Gαq/11 to TASK-3 channels. Endogenously released NT facilitated the excitability of the GCs contributing to the induction of long-term potentiation at the perforant path-GC synapses. Our results provide a cellular mechanism that helps to explain the roles of NT in learning and memory.

Investigating the molecular mechanisms through which FTY720‐P causes persistent S1P1 receptor internalization

The molecular mechanism underlying the clinical efficacy of FTY720-P is thought to involve persistent internalization and enhanced degradation of the S1P1 receptor subtype (S1P1R). We have investigated whether receptor binding kinetics and β-arrestin recruitment could play a role in the persistent internalization of the S1P1R by FTY720-P. [(3) H]-FTY720-P and [(33) P]-S1P were used to label CHO-S1P1/3Rs for binding studies. Ligand efficacy was assessed through [(35) S]-GTPγS binding and β-arrestin recruitment. Metabolic stability was evaluated using a bioassay measuring intracellular Ca(2+) release. CHO-S1P1/3R numbers were determined, following FTY720-P treatment using flow cytometry. The kinetic off-rate of [(3) H]-FTY720-P from the S1P1R was sixfold slower than from the S1P3R, and comparable to [(33) P]-S1P dissociation from S1P1/3Rs. S1P and FTY720-P stimulated [(35) S]-GTPγS incorporation to similar degrees, but FTY720-P was over 30-fold less potent at S1P3Rs. FTY720-P stimulated a higher level of β-arrestin recruitment at S1P1Rs, 132% of the total recruited by S1P. In contrast, FTY720-P was a weak partial agonist at S1P3R, stimulating just 29% of the total β-arrestin recruited by S1P. Internalization experiments confirmed that cell surface expression of the S1P1R but not the S1P3R was reduced following a pulse exposure to FTY720-P, which is metabolically stable unlike S1P. FTY720-P and S1P activation of the S1P1R results in receptor internalization as a consequence of an efficient recruitment of β-arrestin. The combination of slow off-rate, efficacious β-arrestin recruitment and metabolic stability all contribute to FTY720-P's ability to promote prolonged S1P1R internalization and may be critical factors in its efficacy in the clinic.

Myricetin Inhibits the Release of Glutamate in Rat Cerebrocortical Nerve Terminals

The excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders. The purpose of the present study was to investigate the effect and possible mechanism of myricetin, a naturally occurring flavonoid with a neuroprotective profile, on endogenous glutamate release in the nerve terminals (synaptosomes) of the rat cerebral cortex. The release of glutamate was evoked by the K(+) channel blocker 4-aminopyridine (4-AP) and measured by one-line enzyme-coupled fluorometric assay. We also used a membrane potential-sensitive dye to assay the synaptosomal plasma membrane potential, and a Ca(2+) indicator Fura-2 to monitor cytosolic Ca(2+) concentrations ([Ca(2+)]C). Results show that myricetin inhibited 4-AP-evoked glutamate release, and this effect was prevented by chelating extracellular Ca(2+) ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate had no effect on myricetin action. Myricetin did not alter the synaptosomal membrane potential, but decreased 4-AP-induced increases in the cytosolic free Ca(2+) concentration. Furthermore, the myricetin effect on 4-AP-evoked glutamate release was prevented by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release. These results suggest that myricetin inhibits glutamate release from cerebrocortical synaptosomes by attenuating voltage-dependent Ca(2+) entry. This implies that the inhibition of glutamate release is an important pharmacological activity of myricetin that may play a critical role in the apparent clinical efficacy of this compound.

Analysis of tubular membrane networks in cardiac myocytes from atria and ventricles.

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

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).

Ryanodine receptor 2 contributes to hemorrhagic shock-induced bi-phasic vascular reactivity in rats.

Ryanodine receptor 2 (RyR2) is a critical component of intracellular Ca(2+) signaling in vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the role of RyR2 in abnormal vascular reactivity after hemorrhagic shock in rats. SD rats were hemorrhaged and maintained mean arterial pressure (MAP) at 40 mmHg for 30 min or 2 h, and then superior mesenteric arteries (SMA) rings were prepared to measure the vascular reactivity. In other experiments, SMA rings of normal rats and rat VSMCs were exposed to a hypoxic medium for 10 min or 3 h. SMA rings of normal rats and VSMCs were transfected with siRNA against RyR2. Intracellular Ca(2+) release in VSMCs was assessed using Fura-2/AM. The vascular reactivity of the SMA rings from hemorrhagic rats was significantly increased in the early stage (30 min), but decreased in the late stage (2 h) of hemorrhagic shock. Similar results were observed in the SMA rings exposed to hypoxia for 10 min or 3 h. The enhanced vascular reactivity of the SMA rings exposed to hypoxia for 10 min was partly attenuated by transfection with RyR2 siRNA, whereas the blunted vascular reactivity of the SMA rings exposed to hypoxia for 3 h was partly restored by transfection with RyR2 siRNA. Treatment with the RyR agonist caffeine (1 mmol/L) significantly increased Ca(2+) release in VSMCs exposed to hypoxia for 10 min or 3 h, which was partially antagonized by transfection with RyR2 siRNA. RyR2-mediated Ca(2+) release contributes to the development of bi-phasic vascular reactivity induced by hemorrhagic shock or hypoxia.

Serotonin 5-HT3 receptor-mediated vomiting occurs via the activation of Ca2+/CaMKII-dependent ERK1/2 signaling in the least shrew (Cryptotis parva).

Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca2+) and upon stimulation allows increased Ca2+ influx. We examined the contribution of Ca2+/calmodulin-dependent protein kinase IIα (Ca2+/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca2+ levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca2+ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca2+ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca2+ mobilization via extracellular Ca2+ influx through 5-HT3Rs/L-type Ca2+ channels, and intracellular Ca2+ release via RyRs on ER, initiate Ca2+-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis.

Invited: Nanowire Field-Effect Transistor-Based Biosensors as a Tool for Life Science

Silicon nanowire field-effect transistors (SiNW-FETs) have been used as a highly sensitive biosensor for label-free, real-time, and target selective detections in biomedical diagnosis and cellular-recording investigation. For practical uses, a reusable SiNW-FET was made possible with a reversible surface functionalization method, allowing for calibratable and quantitative analysis (Figure 1). In recent studies, we have applied SiNW-FETs for probing neurotransmitter release from living neurons, screening protein-protein interactions (Figure 2), detecting influenza virus, and monitoring extracellular K+ flux from stimulated chromaffin cells. In the study of protein-protein interactions, we integrated SiNW-FET with mass spectrometry (MS) for not only fast screening (by NW-FET) but also spectrally identifying (by MS) the unknown interacting proteins that were captured on the probe protein-modified SiNW-FET. To improve the sensitivity of SiNW-FETs, we designed a modification method to immobilize probe molecules only on the SiNW channel without contaminating the surrounding substrate, thus substantially enhancing the detection sensitivity. Recently, we developed an ultrasensitive biosensor for selective dopamine detection by an aptamer-modified multiple-parallel-connected (MPC) SiNW-FET with a detection limit of ~10 pM (Figure 3). This MPC SiNW-FET was applied to monitor the dopamine release under hypoxic stimulation from living PC12 cells. We resolved the controversial pathways that the increase in intracellular Ca2+ to trigger dopamine secretion is dominated by an extracellular Ca2+ influx, rather than the release of intracellular Ca2+ stores.In conclusion, SiNW-FET biosensors have been successfully applied in biological and cellular studies, including the label-free and real-time recordings of neurotransmitter release, protein-protein interaction, virus detection, dopamine release from stimulated excitable cells, and clinical diagnosis. Finally, this novel bio-nano-electronic FET device, which is capable of integrating with living cell systems, provides a promising tool for biological analysis and cellular investigation and adds a new item to the biosensor toolbox for the future studies of cell biology and clinical disease diagnosis.This work is partially supported by the Ministry of Science and Technology of Taiwan under NSC 101-2113-M-002-016-MY2 and NSC 102-2627-M-002-001.Figure 1. Taking advantage of the reversible association-dissociation between glutathione (GSH) and glutathione S-transferase (GST), we built a reusable SiNW-FET biosensor for fast screening protein-protein interactions. The strategy used in the study includes the association of GST-tagged calmodulin (represented by CaM-GST) with a GSH-modified SiNW-FET (referred to as GSH/SiNW-FET) to form CaM/SiNW-FET, the subsequent detection of interacting proteins with CaM, and the removal of bound proteins via the dissociation of GSH-GST with concentrated GSH (³1 mM) washing solution. (PNAS, 107, 1047 (2010))Figure 2. (a) An illustration for the detection of membrane fractions containing N-type voltage-gated Ca2+ channels (VGCCs) utilizing a CaM/SiNW-FET. (b–c) Real-time electrical detections of the binding of N-type VGCCs to the CaM/SiNW-FET in 0.1× PS (phosphate solution, consisting of 0.76 mM Na2HPO4 and 0.24 mM NaH2PO4 at pH 7.4) supplemented with (b) 100 mM Ca2+; (c) 500 mM EDTA. (d) Two control tests were conducted separately. (top graph) Electrical detection of the membrane fraction without the a1b subunit by CaM/SiNW-FET in 0.1× PS supplemented with 100 mM Ca2+. (bottom graph) Electrical detection of N-type VGCCs by GST/SiNW-FET (without CaM) in 0.1× PS supplemented with 100 mM Ca2+. (PNAS, 107, 1047 (2010))Figure 3. (a) Schematics illustrate (top graph) the experimental setup of a DNA-aptamer/MPC SiNW-FET for detecting dopamine exocytosis from living PC12 cells under hypoxic stimulation and (bottom graph) the procedure for immobilization of the DNA-aptamer on the surface of SiNW. (b) An illustration about how the hypoxic condition triggers the dopamine (DA) exocytosis by the escalation of intracellular Ca2+ concentration via the hypothetical pathway of either extracellular Ca2+ influx (solid line) or intracellular Ca2+ store release (dashed line). (c) Real-time recordings for the dopamine exocytosis from living PC12 cells under different hypoxic stimulations: (upper trace) 35% [O2] reduced buffer, (middle trace) 70% [O2] reduced buffer, and (bottom trace) 70% [O2] reduced buffer containing 1 mM CdI2. (JACS, 135, 16034 (2013))

Regulation of pollen tube growth by reversible protein phosphorylation

The tip-growth of pollen tube is regulated by diverse signaling and metabolic processes, including Rop GTPase signaling pathway, phosphatidylinositol signaling pathway, Ca(2+) signaling, actin dynamics, vesicular trafficking, and cell wall re-modulation. These processes are regulated by reversible protein phosphorylation events: (1) The activities of Rop regulatory proteins (GEF, GDI, and GAP) that regulate Rop GTPase are variable under different protein phosphorylation states. In addition, various protein kinases function as the downstream effectors of Rop GTPase, and participate in the downstream pathways of Rop signaling. (2) Reversible protein phosphorylation can activate/inactivate the plasma membrane (PM) Ca(2+) channels and/or Ca(2+) pump, as well as control the release of intracellular Ca(2+), thereby regulating the formation of Ca(2+) gradient in pollen tube tip. In addition, protein kinases function as Ca(2+) sensors and phosphorylate the target proteins involved in the downstream regulatory pathways of Ca(2+) signaling. (3) The dynamics of actin polymerization and depolymerization are regulated by reversible phosphorylation of actin binding proteins (e.g., ADF and profilin). (4) Reversible protein phosphorylation regulates the activities of endo/exocytosis-related proteins and PM phospholipid metabolism that are involved in the membrane trafficking. (5) Reversible protein phosphorylation participates in the pollen tube cell wall re-modulation through regulating the function and subcellular distribution of cytoplasmic serine/threonine protein kinase and sucrose synthase. (6) The activities of transcription regulatory protein and eukaryotic translation initiation factor are regulated by protein phosphorylation, modulating the RNA transcription and protein synthesis. In this review, we present an overview of the functions of protein reversible phosphorylation in the aforementioned processes during the pollen tube tip-growth.

Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50% in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

Cypermethrin induces astrocyte damage: Role of aberrant Ca2+, ROS, JNK, P38, matrix metalloproteinase 2 and migration related reelin protein

Cypermethrin is a synthetic type II pyrethroid, derived from a natural pyrethrin of the chrysanthemum plant. Cypermethrin-mediated neurotoxicity is well studied; however, relatively less is known of its effect on astrocyte development and migration. Astrocytes are the major components of blood brain barrier (BBB), and astrocyte damage along with BBB dysfunction impair the tight junction (TJ) proteins resulting in altered cell migration and neurodegeneration. Here, we studied the mechanism of cypermethin mediated rat astrocyte damage and BBB disruption, and determined any change in expression of proteins associated with cell migration. Through MTT assay we found that cypermethrin reduced viability of cultured rat astrocytes. Immunolabelling with astrocyte marker, glial fibrillary acidic protein, revealed alteration in astrocyte morphology. The astrocytes demonstrated an enhanced release of intracellular Ca++ and ROS, and up-regulation in p-JNK and p-P38 levels in a time-dependent manner. Cypermethrin disrupted the BBB (in vivo) in developing rats and attenuated the expression of the extracellular matrix molecule (ECM) and claudin-5 in cultured astrocytes. We further observed an augmentation in the levels of matrix metalloproteinase 2 (MMP2), known to modulate cellular migration and disrupt the developmental ECM and BBB. We observed an increase in the levels of reelin, involved in cell migration, in cultured rat astrocytes. The reelin receptor, α3β1integrin, and a mammalian cytosolic protein Disabled1 (Dab1) were also up-regulated. Overall, our study demonstrates that cypermethrin induces astrocyte injury via modulation in Ca++, ROS, JNK and P38 pathways, which may alter MMP expression and reelin dependent astrocyte migration during brain development.

Melatonin and amfenac modulate calcium entry, apoptosis, and oxidative stress in ARPE-19 cell culture exposed to blue light irradiation (405 nm)

Under conditions of oxidative stress, cell apoptosis is triggered through the mitochondrial intrinsic pathway. Increased levels of reactive oxygen species (ROS) are linked to excess cell loss and mediate the initiation of apoptosis in a diverse range of cell types. The aims of this study were to assess intracellular Ca(2+) release, ROS production, and caspase-3, and -9 activation in ARPE-19 cells during the blue light-mediated cell death, and to examine a potential protective effect of melatonin and amfenac, in the apoptotic cascade. ARPE-19 cells were cultured in their medium. First, MTT tests were performed to determine the protective effects of amfenac and melatonin. Cells were then exposed to blue light irradiation in an incubator. Intracellular Ca(2+) release experiments, mitochondrial membrane depolarization, apoptosis assay, glutathione (GSH), glutathione peroxidase (GSH-Px), and ROS experiments were done according to the method stated in the Materials and methods section. Cell death was clearly associated with increased levels of ROS production, as measured by 2',7'-dichlorofluorescein fluorescence, and associated increase in Ca(2+) levels, as measured by Fura-2-AM. Blue light-induced cell death was associated with an increased level of caspase-3 and 9, suggesting mediation via the apoptotic pathway. Cell death was also associated with mitochondrial depolarization. Melatonin was shown to delay these three steps. Melatonin, amfenac, and their combination protect ARPE-19 cells against blue light-triggered ROS accumulation and caspase-3 and -9 activation. The antiapoptotic effect of melatonin and amfenac at doses inhibiting caspase synthesis modified Ca(2+) release and prevented excessive ROS production, suggesting a new therapeutic approach to age-related macular degeneration.

BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 μM) and 6-OH-BDE-47 (0.2 μM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment.

Inhibition of ANKRD1 sensitizes human ovarian cancer cells to endoplasmic reticulum stress-induced apoptosis

High expression of Ankyrin Repeat Domain 1 (ANKRD1) in ovarian carcinoma is associated with poor survival, and in ovarian cancer cell lines is associated with platinum resistance. Importantly, decreasing ANKRD1 expression using siRNA increases cisplatin sensitivity. In this study, we investigated possible mechanisms underlying the association of ANKRD1 with cisplatin response. We first demonstrated that cisplatin-induced apoptosis in ovarian cancer cell lines was associated with endoplasmic reticulum (ER) stress, evidenced by induction of Glucose-Regulated Protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and increased intracellular Ca(2+) release. The level of sensitivity to cisplatin-induced apoptosis was associated with ANKRD1 protein levels and poly (ADP-ribose) polymerase (PARP) cleavage. COLO 316 ovarian cancer cells, which express high ANKRD1 levels, were relatively resistant to cisplatin, and ER stress-induced apoptosis, whereas OAW42 and PEO14 cells, which express lower ANKRD1 levels, are more sensitive to ER stress-induced apoptosis. Furthermore, we show that overexpression of ANKRD1 attenuated cisplatin-induced cytotoxicity, and conversely siRNA knockdown of ANKRD1 sensitized ovarian cancer cells to cisplatin and ER stress-induced apoptosis associated with induction of GADD153, and downregulation of BCL2 and BCL-XL. Taken together, these results suggest that ANKRD1 has a significant role in the regulation of apoptosis in human ovarian cancer cells, and is a potential molecular target to enhance sensitivity of ovarian cancer to chemotherapy.