Endogenous Calcium Research Articles

Over-accumulation of putrescine induced by cyclohexylamine interferes with chromium accumulation and partially restores pollen tube growth in Actinidia deliciosa

Both trivalent and hexavalent chromium, i.e., Cr(III) and Cr(VI), respectively, were previously demonstrated to affect invitro germination and ultrastructure of kiwifruit (Actinidia deliciosa) pollen. In the present work, the response to chromium in germinating pollen was evaluated in terms of changes in the polyamine profile. Slight, though significant, increases in free spermidine and spermine occurred after exposure to Cr(III), while the levels remained almost unchanged after Cr(VI) treatment. The spermidine synthase inhibitor cyclohexylamine (CHA) caused a dramatic increase in free putrescine in both chromium-treated and untreated samples, while spermidine content was not affected. Interestingly, CHA positively affected the performance of chromium-treated pollen by partially, though significantly, restoring pollen tube growth. The major growth recovery was registered with 1mM CHA in the presence of Cr(VI), concomitant with a considerable reduction in uptake of the metal. Conversely, endogenous calcium levels were more heavily affected in Cr(III)-treated pollen. The effect of CHA on production of reactive oxygen species also varied depending on the chromium species. The response of pollen to the CHA-induced putrescine excess was compared with that exerted by an exogenous supply of the same diamine. Results show that in Cr(III)-treated pollen, putrescine over-accumulation induced by CHA exerted similar effects as exogenous putrescine, while this was not true in the Cr(VI) treatment. It appears that the diamine was able to improve pollen tolerance to metal stress through different mechanisms, mostly depending upon the chromium species, namely via reduced metal uptake or by substituting for calcium.

The PI3K/Akt and ERK1/2 signaling pathways mediate the erythropoietin-modulated calcium influx in kainic acid-induced epilepsy

Erythropoietin (EPO) suppresses epileptogenesis and limits the neuronal damage associated with recurrent seizures, but the neurocellular mechanism is unclear. Dysregulation of intracellular calcium homeostasis is a key pathogenic event leading to the progression of epileptic activity, suggesting that EPO may suppress seizures by stabilizing intracellular calcium. In this study, we examined the effects of EPO on voltage-gated Ca(2+) influx in cultured rat hippocampal neurons and population spike (PS) amplitude in kainic acid (KA)-induced rats and the mechanisms responsible. KA injection markedly increased EPO and EPO receptor expression and the amplitude of PS in the hippocampal CA3 region, evoked by perforant pathway stimulation. Intracerebroventricular injection of exogenous rat recombinant EPO reversed KA-induced PS amplitude in the hippocampal CA3 region. Similarly, rat recombinant EPO pretreatment attenuates the increased voltage-gated calcium current's (I(Ca)) amplitude and density induced by KA in cultured hippocampal neurons. In contrast, transient transfection of rat EPO small interfering RNS (siRNA) further enhanced I(Ca) amplitude and density in the presence of KA, whereas a scrambled control siRNA had no effect. Further, EPO activates the PI3K and ERK1/2 pathways in cultured hippocampal neurons, and the PI3K/Akt inhibitor LY294002 and ERK1/2 inhibitor U0126 both blocked, at least in part, the suppressive effect of exogenous EPO on KA-induced calcium currents. This study indicates that both endogenous and exogenous EPO decrease KA-sensitive calcium influx and concomitant hyperexcitability in hippocampal neurons. The results also demonstrate that the PI3K/Akt and ERK1/2 signaling pathways mediate the EPO-modulated calcium influx in KA-induced epilepsy.

The Kelch‐Like 1 Actin Binding Protein Modulates Neuronal Voltage Gated Calcium Channels

Kelch‐like 1 (KLHL1) is a neuronal actin‐binding protein first identified in humans as part of the genetic locus of the neurological movement disorder Spinocerebellar Ataxia type 8. KLHL1 is expressed in the cytosol, axons and dendrites. KLHL1 KO causes loss of post‐synaptic structures, loss of motor coordination and gait abnormalities. KLHL1 is an ion channel modulator and upregulates HVA and LVA calcium currents in vitro by increased recycling endosome activity and by alteration of the kinetics of τdeactivation. We analyzed the endogenous HVA and LVA calcium currents in neurons from WT and KLHL1 KO mice, and compared these data with the acute down‐regulation of KLHL1 using a knock‐down shRNA (KD) strategy. KLHL1 KD caused decreased HVA and LVA calcium current expression. In contrast, the KO had no differences in total LVA current density, yet changes in current kinetics suggest there is a down‐regulation of α1H and compensatory expression of α1I. HVA current density was also decreased in KO (40%) at 4–6 DIV but not at 7–10 DIV, suggesting this deficit was also compensated for after 7 DIV. Our data confirm that KLHL1 modulates LVA and HVA channel function in vivo; the compensatory modulation of currents in the KO may contribute to the mild motor phenotype seen in these mice.This material is based upon work supported by the National Science Foundation under Grant No. 1022075

Inhibition of endothelial cell Ca²⁺ entry and transient receptor potential channels by Sigma-1 receptor ligands.

Background and PurposeThe Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels.Experimental ApproachIntracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies.Key ResultsIn endothelial cells, 10–100 μM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3.Conclusions and ImplicationsThe data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.

Statistical inference for ratiometric imaging of excitable cells: a self‐organizing state‐space model

AbstractConcentrations of free intracellular calcium ions ([Ca2+]i) in excitable cells are often measured using indicator dyes, such as fura‐2. Of note, however, these indicator dyes are divalent metal ion chelators that affect intrinsic changes in [Ca2+]i. To examine whether indicator dyes alter calcium signaling, we estimated [Ca2+]i using a novel statistical inference method that examines fluorescence signals and the calcium current through the cell membrane. We first constructed a model of transient [Ca2+]i, which was then translated into a state‐space model with such state variables as [Ca2+]i, endogenous calcium buffer, and calcium indicators. Then, a self‐organizing state‐space model was defined by augmenting a state vector with unknown parameters from the original state‐space model. In the model, some unknown parameters were estimated with the original state vector. Next, we used a recursive Bayesian estimation to obtain a set of state vectors and the unknown parameters associated with a set of observation vectors. To calculate the recursive Bayesian estimation, we used a sequential Monte Carlo method, which is referred to as a particle filter method. To verify the effectiveness of the proposed method, we carried out experiments with a set of test data from a model with known parameters. The results show that our proposed method properly estimated the temporal profiles of [Ca2+]i, the indicator dye concentration, and certain model parameters in a noisy environment. © 2012 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.

TRPC1 protein forms only one type of native store-operated channels in HEK293 cells

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.

Elucidation of molecular mechanism involved in neuroprotective effect ofCoenzymeQ10 in alcohol‐induced neuropathic pain

The aim of the present investigation was to evaluate the effect of Coenzyme Q10 and its combination with vitamin E in alcohol-induced chronic neuropathic pain. Male Wistar rats were orally treated with alcohol (10 g/kg, 35% v/v, b.i.d.) for 10 weeks. Coenzyme Q10 (25, 50, and 100 mg/kg) and vitamin E (100 mg/kg) were coadministered orally for 1 h after ethanol administration for 10 weeks. Various nerve functions, biochemical, and molecular parameters were assessed. Chronic administration of ethanol for 10 weeks resulted significant development of neuropathic pain. Treatment with Coenzyme Q10 (50 and 100 mg/kg) for 10 weeks showed significant and dose dependently increased in level of nociceptive threshold, endogenous antioxidant, and Na,K-ATPase enzyme. Coenzyme Q10 (50 and 100 mg/kg) significantly restored the levels of motor nerve conduction velocity and sensory nerve conduction velocity. It also showed significant decrease in levels of endogenous calcium, oxidative-nitrosative stress, TNF-α, IL-1β, and IL-4 level. Alteration in protein expression of polymerase gamma (pol γ) was significantly restored the Coenzyme Q10 treatment. The important finding of the study is that, Coenzyme Q10 (100 mg/kg) and α-tocopherol (100 mg/kg) combination-treated rats showed more significant prevention of behavioral, biochemical, and molecular neurotoxic effect of alcohol administration than Coenzyme Q10 or α-tocopherol alone treated group. It is evident from the finding of present investigation that plethora of mechanism including inhibition of oxido-nitrosative stress, release of pro-inflammatory cytokine, modulation of endogenous biomarker, and protection of pol γ protein expression simultaneously orchestrate to exhibits neuroprotective effect of Coenzyme Q10, vitamin E and their combination.

Gintonin: A Novel Ginseng-Derived Ligand that Targets G Protein- Coupled Lysophosphatidic Acid Receptors

Ginseng, the root of Panax ginseng C.A. Meyer, is one of the oldest herbal medicines and induces a variety of physiological and pharmacological effects. Ginseng contains saponins called ginsenosides, which are considered as the biologically active ingredients in ginseng. Increasing evidences indicate that ginsenosides are non-selective in their actions and high concentrations are required for cellular effects. Crude ginseng total saponin (cGTS) fraction is about 50% ginsenoside in composition, but the remainder is not defined clearly. Biochemically, cGTS fraction triggers differing patterns of ginsenoside activity; for example, the cGTS fraction itself functions as an agent that activates a G protein-coupled receptor because it induces endogenous calcium (Ca2+)-activating chloride (Cl-) channel activations in Xenopus oocytes via the same signaling pathways used by Gαq/11 protein-coupled receptors. Recent reports have revealed that the cGTS fraction contains novel glycolipoproteins, designated the gintonin. It was discovered that gintonin, but not ginsenosides, interacts with unidentified membrane proteins to generate [Ca2+]i transient in mammalian cells and to activate endogenous Ca2+-activating Cl- channels in Xenopus oocytes. Further studies have shown that gintonin is a novel lysophosphatidic acids (LPAs)-ginseng protein complex and can selectively activate LPA receptors with an affinity greater than that of LPA. This review aims to characterize gintonin as an LPA receptor ligand, explain the reasons why gintonin remained unidentified for a long time, list the advantages of LPA-ginseng protein complexes over free LPAs, compare the activities of gintonin and ginsenoside, and describe the pharmacological applications of gintonin as a drug that targets LPA receptors.

A Review of a Family of Ultra-Rapid-Acting Insulins: Formulation Development

This review summarizes the clinical development of a family of ultra-rapid-acting recombinant human insulin formulations. These formulations use ethylenediaminetetraacetic acid (EDTA) to chelate zinc and thereby destabilize insulin hexamers. In addition, insulin monomer surface charges are chemically masked with citrate to prevent reaggregation. The first phase 1 trials were performed using BIOD-090, an acidic 25 unit U/ml insulin formulation, which contained disodium-EDTA (NaEDTA). When compared with regular human insulin (RHI) and/or insulin lispro in multiple phase 1 studies, BIOD-090 consistently showed more rapid absorption and/or onset of action. A standard meal challenge study also demonstrated improved postprandial glucose profiles associated with BIOD-090. However, increased patient exposure in larger phase 3 trials showed that this formulation was associated with an increased incidence of local injection site reactions, most commonly pain. A next generation formulation, BIOD-100, contained the same excipients as a standard insulin concentration of 100 U/ml. BIOD-100 maintained an ultra-rapid action profile and was associated with modest but significantly improved toleration when compared with BIOD-090. In order to further improve toleration, the hypothesis that NaEDTA contributed to discomfort by chelating endogenous calcium was tested by either substituting calcium-EDTA for NaEDTA or by adding calcium chloride to the NaEDTA formulation. These calcium formulations essentially eliminated the excess discomfort associated with BIOD-090 but were associated with less optimal pharmacokinetic profiles in humans. Recent efforts have succeeded in developing ultra-rapid-acting human insulin formulations with acceptable injection site toleration by optimizing concentrations of calcium (BIOD-125) and with the use of magnesium sulfate to mitigate discomfort (BIOD-123). Similar formulation technology has also been shown to accelerate absorption of insulin analogs in animal models.

Effect of Calcium Sprays on Mechanical Strength and Cell Wall Fractions of Herbaceous Peony (Paeonia Lactiflora Pall.) Inflorescence Stems

Calcium is an essential element and imparts significant structural rigidity to the plant cell walls, which provide the main mechanical support to the entire plant. In order to increase the mechanical strength of the inflorescence stems of herbaceous peony, the stems are treated with calcium chloride. The results shows that preharvest sprays with 4% (w/v) calcium chloride three times after bud emergence are the best at strengthening “Da Fugui” peonies’ stems. Calcium sprays increased the concentrations of endogenous calcium, total pectin content as well as cell wall fractions in herbaceous peonies stems, and significantly increased the contents of them in the top segment. Correlation analysis showed that the breaking force of the top segment of peonies’ stems was positively correlated with the ratio of water insoluble pectin to water soluble pectin (R = 0.673) as well as lignin contents (R = 0.926) after calcium applications.

Biomineralizations: insights and prospects from crustaceans

For growing, crustaceans have to molt cyclically because of the presence of a rigid exoskeleton. Most of the crustaceans harden their cuticle not only by sclerotization, like all the arthropods, but also by calcification. All the physiology of crustaceans, including the calcification process, is then linked to molting cycles. This means for these animals to find regularly a source of calcium ions quickly available just after ecdysis. The sources of calcium used are diverse, ranging from the environment where the animals live to endogenous calcium deposits cyclically elaborated by some of them. As a result, crustaceans are submitted to an important and energetically demanding calcium turnover throughout their life. The mineralization process occurs by precipitation of calcium carbonate within an organic matrix network of chitin-proteins fibers. Both crystalline and stabilized amorphous polymorphs of calcium carbonate are found in crustacean biominerals. Furthermore, Crustacea is the only phylum of animals able to elaborate and resorb periodically calcified structures. Notably for these two previous reasons, crustaceans are more and more extensively studied and considered as models of choice in the biomineralization research area.

Designed Red Fluorescent Calcium Sensor with Ultrafast Kinetic Properties

Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium store, as well as membrane calcium channels and vesicles/endosomes. To probe calcium responses in such high calcium environments simultaneously with the calcium transients in intracellular plasma, there is a pressing need to develop a calcium sensor featured with 1) fast kinetic properties, 2) a different spectral window from other calcium indicators, and 3) pH-independent fluorescence change. In this study, we first report our development of fast red calcium sensors using our protein design instead of using endogenous calcium binding proteins or natural calcium binding motifs. Calcium binding results in fluorescence signal increase with a Kd in the submilimolar level. The red calcium sensors with fluorescence emission in the near Infrared region allow us to monitor calcium signaling together with GFP-based calcium indicators. The pH stability of the red sensors was dramatically enhanced, with pKa below 5, compared to the GFP-derived calcium sensors. High resolution NMR has been applied to probe the mechanism of calcium-induced conformational change of designed calcium binding proteins. Our results show the developed calcium sensors are able to monitor calcium dynamics at several cellular environments responding to perturbations of extracellular calcium signaling.

Calcium Accumulation Only during Rapid Growth in Female Rats

AbstractCalcium retention varies with developmental state, which may be partially under the control of insulin-like growth factor 1 (IGF-1). IGF-1 levels can be manipulated through dietary and therapeutic interventions. We investigated the relationship between IGF-1 endogenous production and calcium utilization and bone accretion during growth as well as the effects of IGF-1 treatment on calcium utilization during rapid and slowed growth in intact female Sprague-Dawley rats. In 33 rats killed at 11 time points (n= 3 each) from age 4 to 24 wk, femoral and vertebral bone mass were paralleled by plasma IGF-1 up to 9 wk. Fractional calcium absorption was maximal at 9 wk, reduced by one-half at 12 wk, and there was no further change at 20 wk. From this study, we selected 2 stages of growth, rapid and slow, for a subsequent intervention study. A 4-wk intervention was initiated at 6 or 8 wk when rats (n= 15/group) received either continuous rhIGF-1/IGF binding protein 3 (IGFBP3) infusion (0.3 mg/d) or vehicle (control) by osmotic mini-pumps. In rapidly growing IGF-1/IGFBP3-treated rats compared to controls, but not in slowly growing treated compared to control rats, IGF-1 treatment increased (P< 0.05) calcium absorption (35 vs. 21%), bone calcium balance (0.55 vs. 0.3 mmol/d), and femoral calcium content (31 vs. 24% of dry weight). Exogenous IGF-1/IGFBP3 treatment increased calcium accretion during rapid growth, but rats past rapid growth were no longer as sensitive to this dose of IGF-1/IGFBP3. Thus, interventions designed to improve bone mass through increased IGF-1 will have the greatest impact during rapid growth.

Prismatic shell repairs by hemoctyes in the extrapallial fluid of the Pacific Oyster, Crassostrea gigas

To understand the role of hemocytes in the shell repair process, a hole was drilled in the right valves of the Pacific oyster, Crassostrea gigas, and the repair process was observed. Histological observations suggested that the exterior surface of the shell was repaired by aggregated hemocytes. The nuclei of the hemocytes were cleary stained in the regenerated shell while appearing fragmented after calcification at the <TEX>$7^{th}$</TEX> day. Globular calcium granules were genegenerated from the hemocytic monolyer after 6 days of incubation which were morphologically and chemically identical with those from prismatic shell. Our finding suggested that the repaired prismatic shell was composed by aggregated hemocytes and that their endogenous calcium component might support the nucleation of calcium biomineralization during shell repair.

Expression changes of parvalbumin and microtubule-associated protein 2 induced by chronic constriction injury in rat dorsal root ganglia.

Parvalbumin (PV), as a mobile endogenous calcium buffer, plays an important role in affecting temporospatial characteristics of calcium transients and in modulating calcium homeostasis. PV is expressed in neurons in the dorsal root ganglion (DRG) and spinal dorsal horn and may be involved in synaptic transmission through regulating cytoplasm calcium concentrations. But the exact role of PV in peripheral sensory neurons remains unknown. Microtubule-associated protein 2 (MAP-2), belonging to structural microtubule-associated protein family, is especially vulnerable to acute central nervous system (CNS) injury, and there will be rapid loss of MAP-2 at the injury site. The present study investigated the changes of PV expressing neurons and the MAP-2 neurons in the DRG after an operation for chronic constriction injury to the unilateral sciatic nerve (CCI-SN), in order to demonstrate the possible roles of PV and MAP-2 in transmission and modulation of peripheral nociceptive information. Seventy-two adult male Sprague-Dawley (SD) rats, weighing 180 - 220 g, were randomly divided into two groups (36 rats in each group), the sham operation group and chronic constriction injury (CCI) group. Six rats in each group were randomly selected to receive mechanical and thermal sensitivity tests at one day before operation and 1, 3, 5, 7, and 14 days after surgery. After pain behavioral test, ipsilateral lumbar fifth DRGs were removed and double immunofluorescence staining was performed to assess the expression changes of PV and of MAP2 expressing neurons in the L5 DRG before or after surgery. The animals with CCI-SN showed obvious mechanical allodynia and thermal hyperalgesia (P < 0.05). Both the thermal and mechanical hyperalgesia decreased to their lowest degree at 7 days after surgery compared to the baseline before surgery (P < 0.01). In normal rats before surgery, a large number of neurons were MAP-2 single labeled cells, and just a small number of PV-expressed neurons were found. PV-positive neurons, PV-positive nerve fibers and PV-negative neurons, formed a direct or close contact for cross-talk. We used immunocytochemical staining to quantify the time course of changes to PV and MAP-2 expressing neurons in tissue, and found that the number of PV expressing neurons began to slightly decrease at 3 days after surgery, and had a significant reduction at CCI day 5, day 7 (P < 0.05). But MAP-2 neurons significantly decreased on just the 3rd day after CCI (P < 0.05). No changes in PV and MAP-2 expression were almost found in sham operated rats. The number of PV positive neurons, was positively correlated with the hyperalgesia threshold. A sharp decline in MAP-2 neurons may be the early response to surgical injury, and PV positive neurons were much more effective at affecting the changes of pain behaviors, indicating that the down-regulation of PV protein could participate in, at least in part, the modulation of nociceptive transmission.

Effects of Calretinin on Ca2+ Signals in Cerebellar Granule Cells: Implications of Cooperative Ca2+ Binding

Calretinin is thought to be the main endogenous calcium buffer in cerebellar granule cells (GrCs). However, little is known about the impact of cooperative Ca(2+) binding to calretinin on highly localized and more global (regional) Ca(2+) signals in these cells. Using numerical simulations, we show that an essential property of calretinin is a delayed equilibration with Ca(2+). Therefore, the amount of Ca(2+), which calretinin can accumulate with respect to equilibrium levels, depends on stimulus conditions. Based on our simulations of buffered Ca(2+) diffusion near a single Ca(2+) channel or a large cluster of Ca(2+) channels and previous experimental findings that 150 μM 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid (BAPTA) and endogenous calretinin have similar effects on GrC excitability, we estimated the concentration of mobile calretinin in GrCs in the range of 0.7-1.2 mM. Our results suggest that this estimate can provide a starting point for further analysis. We find that calretinin prominently reduces the action potential associated increase in cytosolic free Ca(2+) concentration ([Ca(2+)]( i )) even at a distance of 30 nm from a single Ca(2+) channel. In spite of a buildup of residual Ca(2+), it maintains almost constant maximal [Ca(2+)]( i ) levels during repetitive channel openings with a frequency less than 80 Hz. This occurs because of accelerated Ca(2+) binding as calretinin binds more Ca(2+). Unlike the buffering of high Ca(2+) levels within Ca(2+) nano/microdomains sensed by large conductance Ca(2+)-activated K(+) channels, the buffering of regional Ca(2+) signals by calretinin can never be mimicked by certain concentration of BAPTA under all different experimental conditions.

Functional Characterization and High-Throughput Screening of Positive Allosteric Modulators of α7 Nicotinic Acetylcholine Receptors in IMR-32 Neuroblastoma Cells

α7 nicotinic acetylcholine receptors (nAChRs) are characterized by relatively low ACh sensitivity, rapid activation, and fast desensitization kinetics. ACh/agonist evoked currents at the α7 nAChR are transient, and, typically, calcium flux responses are difficult to detect using conventional fluorometric assay techniques. One approach to study interactions of agonists with the α7 nAChR is by utilizing positive allosteric modulators (PAMs). In this study, we demonstrate that inclusion of type II PAMs such as PNU-120596, but not type I, can enable detection of endogenous α7 nAChR-mediated calcium responses in human neuroblastoma (IMR-32) cells. Using this approach, we characterized the pharmacological profile of nicotine, epibatidine, choline, and other nAChR agonists such as PNU-282987, SSR-180711, GTS-21, OH-GTS21, tropisetron, NS6784, and A-582941. The rank order potency of agonists well correlated with α7 nAChR binding affinities measured in brain membranes. Inhibition of calcium response by methyllycaconitine in the presence of increasing concentrations of PNU-282987 or PNU-120596 revealed that the IC(50) value of methyllycaconitine was sensitive to varying concentrations of the agonist, but not that of the PAM. This format demonstrated the feasibility of this approach for high-throughput screening to identify small molecule, PAMs, which were further confirmed in electrophysiological assays of human α7 nAChR expressed in oocytes.

EGF-like peptide-enhanced cell motility in Dictyostelium functions independently of the cAMP-mediated pathway and requires active Ca2+/calmodulin signaling

Current knowledge suggests that cell movement in the eukaryotic slime mold Dictyostelium discoideum is mediated by different signaling pathways involving a number of redundant components. Our previous research has identified a specific motility-enhancing function for epidermal growth factor-like (EGFL) repeats in Dictyostelium, specifically for the EGFL repeats of cyrA, a matricellular, calmodulin (CaM)-binding protein in Dictyostelium. Using mutants of cAMP signaling (carA−, carC−, gpaB−, gpbA−), the endogenous calcium (Ca2+) release inhibitor TMB-8, the CaM antagonist W-7, and a radial motility bioassay, we show that DdEGFL1, a synthetic peptide whose sequence is obtained from the first EGFL repeat of cyrA, functions independently of the cAMP-mediated signaling pathways to enhance cell motility through a mechanism involving Ca2+ signaling, CaM, and RasG. We show that DdEGFL1 increases the amounts of polymeric myosin II heavy chain and actin in the cytoskeleton by 24.1±10.7% and 25.9±2.1% respectively and demonstrate a link between Ca2+/CaM signaling and cytoskeletal dynamics. Finally, our findings suggest that carA and carC mediate a brake mechanism during chemotaxis since DdEGFL1 enhanced the movement of carA−/carC− cells by 844±136% compared to only 106±6% for parental DH1 cells. Based on our data, this signaling pathway also appears to involve the G-protein β subunit, RasC, RasGEFA, and protein kinase B. Together, our research provides insight into the functionality of EGFL repeats in Dictyostelium and the signaling pathways regulating cell movement in this model organism. It also identifies several mechanistic components of DdEGFL1-enhanced cell movement, which may ultimately provide a model system for understanding EGFL repeat function in higher organisms.

Regulator of G Protein Signaling 3 Modulates Wnt5b Calcium Dynamics and Somite Patterning

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Gα subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.

CJun integrates calcium activity and tlx3 expression to regulate neurotransmitter specification

Neuronal differentiation is accomplished through cascades of intrinsic genetic factors initiated in neuronal progenitors by external gradients of morphogens. Activity was thought to be important only late in development, but recent evidence indicates that activity also regulates early neuronal differentiation. Activity in post-mitotic neurons prior to synapse formation can regulate phenotypic specification, including neurotransmitter choice, but the mechanisms are not clear. Here we identify a mechanism that links endogenous calcium spike activity with an intrinsic genetic pathway to specify neurotransmitter choice in neurons in the dorsal embryonic spinal cord of Xenopus tropicalis. Early activity modulates transcription of the GABAergic/glutamatergic selection gene tlx3 and requires a variant cAMP response element (CRE) in its promoter. The cJun transcription factor binds to this CRE site, modulates transcription, and regulates neurotransmitter phenotype through its transactivation domain. Calcium signals through cJun N-terminal phosphorylation, thus integrating activity-dependent and intrinsic neurotransmitter specification. This mechanism provides a basis for early activity to regulate genetic pathways at critical decision points, switching the phenotype of developing neurons.