Release Of Intracellular Calcium Stores Research Articles

Organelle calcium-derived voltage oscillations in pacemaker neurons drive the motor program for food-seeking behavior in Aplysia.

The expression of motivated behaviors depends on both external and internally arising neural stimuli, yet the intrinsic releasing mechanisms for such variably occurring behaviors remain elusive. In isolated nervous system preparations of Aplysia, we have found that irregularly expressed cycles of motor output underlying food-seeking behavior arise from regular membrane potential oscillations of varying magnitude in an identified pair of interneurons (B63) in the bilateral buccal ganglia. This rhythmic signal, which is specific to the B63 cells, is generated by organelle-derived intracellular calcium fluxes that activate voltage-independent plasma membrane channels. The resulting voltage oscillation spreads throughout a subset of gap junction-coupled buccal network neurons and by triggering plateau potential-mediated bursts in B63, can initiate motor output driving food-seeking action. Thus, an atypical neuronal pacemaker mechanism, based on rhythmic intracellular calcium store release and intercellular propagation, can act as an autonomous intrinsic releaser for the occurrence of a motivated behavior.

The chemical cue tetrabromopyrrole induces rapid cellular stress and mortality in phytoplankton

Eukaryotic phytoplankton contribute to the flow of elements through marine food webs, biogeochemical cycles, and Earth’s climate. Therefore, how phytoplankton die is a critical determinate of the flow and fate of nutrients. While heterotroph grazing and viral infection contribute to phytoplankton mortality, recent evidence suggests that bacteria-derived cues also control phytoplankton lysis. Here, we report exposure to nanomolar concentrations of 2,3,4,5-tetrabromopyrrole (TBP), a brominated chemical cue synthesized by marine γ-proteobacteria, resulted in mortality of seven phylogenetically-diverse phytoplankton species. A comparison of nine compounds of marine-origin containing a range of cyclic moieties and halogenation indicated that both a single pyrrole ring and increased bromination were most lethal to the coccolithophore, Emiliania huxleyi. TBP also rapidly induced the production of reactive oxygen species and the release of intracellular calcium stores, both of which can trigger the activation of cellular death pathways. Mining of the Ocean Gene Atlas indicated that TBP biosynthetic machinery is globally distributed throughout the water column in coastal areas. These findings suggest that bacterial cues play multiple functions in regulating phytoplankton communities by inducing biochemical changes associated with cellular death. Chemically-induced lysis by bacterial infochemicals is yet another variable that must be considered when modeling oceanic nutrient dynamics.

Temporal reprogramming of calcium signalling via crosstalk of gonadotrophin receptors that associate as functionally asymmetric heteromers

Signal crosstalk between distinct G protein-coupled receptors (GPCRs) is one mechanism that underlies pleiotropic signalling. Such crosstalk is also pertinent for GPCRs activated by gonadotrophic hormones; follicle-stimulating hormone (FSH) and luteinising hormone (LH), with specific relevance to female reproduction. Here, we demonstrate that gonadotrophin receptor crosstalk alters LH-induced Gαq/11-calcium profiles. LH-induced calcium signals in both heterologous and primary human granulosa cells were prolonged by FSHR coexpression via influx of extracellular calcium in a receptor specific manner. LHR/FSHR crosstalk involves Gαq/11 activation as a Gαq/11 inhibitor abolished calcium responses. Interestingly, the enhanced LH-mediated calcium signalling induced by FSHR co-expression was dependent on intracellular calcium store release and involved Gβγ. Biophysical analysis of receptor and Gαq interactions indicated that ligand-dependent association between LHR and Gαq was rearranged in the presence of FSHR, enabling FSHR to closely associate with Gαq following LHR activation. This suggests that crosstalk may occur via close associations as heteromers. Super-resolution imaging revealed that LHR and FSHR formed constitutive heteromers at the plasma membrane. Intriguingly, the ratio of LHR:FSHR in heterotetramers was specifically altered following LH treatment. We propose that functionally significant FSHR/LHR crosstalk reprograms LH-mediated calcium signalling at the interface of receptor-G protein via formation of asymmetric complexes.

Linking muscarinic receptor activation to UPS-mediated object memory destabilization: Implications for long-term memory modification and storage

Consolidated memories can become destabilized during reactivation, resulting in a transient state of instability, a process that has been hypothesized to underlie long-term memory updating. Consistent with this notion, relatively remote memories, which are resistant to standard destabilization procedures, are reliably destabilized when novel information (i.e., the opportunity for memory updating) is present during reactivation. We have also shown that cholinergic muscarinic receptor (mAChR) activation can similarly destabilize consolidated object memories. Synaptic protein degradation via the ubiquitin proteasome system (UPS) has previously been linked to destabilization of fear and object-location memories. Given the role of calcium in regulating proteasome activity, we hypothesized that activation of cholinergic receptors, specifically M1 mAChRs, stimulates the UPS via inositol triphosphate receptor (IP3R)-mediated release of intracellular calcium stores to facilitate object memory destabilization. We present converging evidence for this hypothesis, which we tested using a modified spontaneous object recognition task for rats and microinfusions into perirhinal cortex (PRh), a brain region strongly implicated in object memory. We extend our previous findings by demonstrating that M1 mAChRs are necessary for novelty-induced object memory destabilization. We also show that proteasome inhibition or IP3R antagonism in PRh prevents object memory destabilization induced by novelty or M1 mAChR stimulation. These results establish an intracellular pathway linking M1 receptors, IP3Rs, and UPS activity to object memory destabilization and suggest a previously unacknowledged role for cholinergic signaling in long-term memory modification and storage.

231 Super resolution imaging unveiling the dyadic ultrastructure in atrial and ventricular cardiomyocytes

Calcium-induced calcium release drives contraction in cardiomyocytes. Located on the sarcoplasmic reticulum, ryanodine receptors (RyR) are responsible for the release of intracellular calcium stores. It has recently become apparent that...

Aristoyunnolin H attenuates extracellular matrix secretion in cardiac fibroblasts by inhibiting calcium influx.

Aristoyunnolin H is a novel aristophyllene sesquiterpenoid isolated from the traditional Chinese medicine Aristolochia yunnanensis Franch. The present research was designed to explore the anti-fibrotic effects of aristoyunnolin H in adult rat cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II). Western blot analysis data showed that aristoyunnolin H reduced the upregulation of fibronectin (FN), connective tissue growth factor and collagen I(Col I) production induced by Ang II in CFs. By studying the dynamic intracellular changes of Ca2+, we further found that while aristoyunnolin H relieved the calcium influx, it has no effect on intracellular calcium store release. Meanwhile, aristoyunnolin H also inhibited the Ang II-stimulated phosphorylation of Ca2+/calmodulin-dependent protein kinase II. In conclusion, aristoyunnolin H may attenuate extracellular matrix secretion in vitro by inhibiting Ang II-induced calcium signaling.

Effects of Fatty Acids on Intracellular [Ca2+], Mitochondrial Uncoupling and Apoptosis in Rat Pachytene Spermatocytes and Round Spermatids.

The aim of this work was to explore the ability of free arachidonic acid, palmitic acid and the unsaturated fatty acids oleic acid and docosahexaenoic acid to modify calcium homeostasis and mitochondrial function in rat pachytene spermatocytes and round spermatids. In contrast to palmitic acid, unsaturated fatty acids produced significant increases in intracellular calcium concentrations ([Ca2+]i) in both cell types. Increases were fatty acid specific, dose-dependent and different for each cell type. The arachidonic acid effects on [Ca2+]i were higher in spermatids than in spermatocytes and persisted when residual extracellular Ca2+ was chelated by EGTA, indicating that the increase in [Ca2+]i originated from release of intracellular calcium stores. At the concentrations required for these increases, unsaturated fatty acids produced no significant changes in the plasma membrane potential of or non-specific permeability in spermatogenic cells. For the case of arachidonic acid, the [Ca2+]i increases were not caused by its metabolic conversion to eicosanoids or anandamide; thus we attribute this effect to the fatty acid itself. As estimated with fluorescent probes, unsaturated fatty acids did not affect the intracellular pH but were able to induce a progressive decrease in the mitochondrial membrane potential. The association of this decrease with reduced reactive oxygen species (ROS) production strongly suggests that unsaturated fatty acids induced mitochondrial uncoupling. This effect was stronger in spermatids than in spermatocytes. As a late event, arachidonic acid induced caspase 3 activation in a dose-dependent manner both in the absence and presence of external Ca2+. The concurrent but differential effects of unsaturated fatty acids on [Ca2+]i and mitochondrial functions are additional manifestations of the metabolic changes that germ cells undergo during their differentiation.

Targeting microparticle biogenesis: a novel approach to the circumvention of cancer multidrug resistance.

Microparticles (MPs) are released from most eukaryotic cells after the vesiculation of the plasma membrane and serve as vectors of long and short-range signaling. MPs derived from multidrug resistant (MDR) cancer cells carry molecular components of the donor cell such as nucleic acids and proteins, and can alter the activity of drug-sensitive recipient cells through the transfer of their cargo. Given the substantial role of MPs in the acquisition and dissemination of MDR, we propose that the inhibition of MP release provides a novel therapeutic approach. This study characterises the effect of a panel of molecules known to act on MP-biosynthetic pathways. We demonstrate a differential effect by these molecules on MP inhibition that appear dependent on the release of intracellular calcium stores following activation with the calcium ionophore A23187. Calpain inhibitor, PD-150606; a selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK), Y-27632; and the vitamin B5 derivative pantethine, inhibited MP release only upon prior activation with A23187. Calpain inhibitor II showed significant inhibition in the absence of cell activation, whereas the vitamin B5 derivatives cystamine dihydrochloride and cysteamine hydrochloride showed no effect on MP inhibition under either condition. In contrast the classical pharmacological inhibitor of MDR, the calcium channel blocker Verapamil, showed an increase in MP formation on resting cells. These results suggest a potential role for calcium in the mechanism of action for PD-150606, Y-27632 and pantethine. These molecules, together with calpain inhibitor II have shown promise as modulators of MP release and warrant consideration as potential candidates for the development of an alternative therapeutic strategy for the prevention of MP-mediated MDR in cancer.

Calcium waves occur as Drosophila oocytes activate

Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.

Lysophosphatidic acid receptor 5 inhibits B cell antigen receptor signaling and antibody response.

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.

The buzz on caffeine in invertebrates: effects on behavior and molecular mechanisms

A number of recent studies from as diverse fields as plant-pollinator interactions, analyses of caffeine as an environmental pollutant, and the ability of caffeine to provide protection against neurodegenerative diseases have generated interest in understanding the actions of caffeine in invertebrates. This review summarizes what is currently known about the effects of caffeine on behavior and its molecular mechanisms in invertebrates. Caffeine appears to have similar effects on locomotion and sleep in both invertebrates and mammals. Furthermore, as in mammals, caffeine appears to have complex effects on learning and memory. However, the underlying mechanisms for these effects may differ between invertebrates and vertebrates. While caffeine's ability to cause release of intracellular calcium stores via ryanodine receptors and its actions as a phosphodiesterase inhibitor have been clearly established in invertebrates, its ability to interact with invertebrate adenosine receptors remains an important open question. Initial studies in insects and mollusks suggest an interaction between caffeine and the dopamine signaling pathway; more work needs to be done to understand the mechanisms by which caffeine influences signaling via biogenic amines. As of yet, little is known about whether other actions of caffeine in vertebrates, such as its effects on GABAA and glycine receptors, are conserved. Furthermore, the pharmacokinetics of caffeine remains to be elucidated. Overall behavioral responses to caffeine appear to be conserved amongst organisms; however, we are just beginning to understand the mechanisms underlying its effects across animal phyla.

Rab8 Modulates Metabotropic Glutamate Receptor Subtype 1 Intracellular Trafficking and Signaling in a Protein Kinase C-Dependent Manner

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.

Membrane-initiated estradiol actions mediate structural plasticity and reproduction.

Over the years, our ideas about estrogen signaling have greatly expanded. In addition to estradiol having direct nuclear actions that mediate transcription and translation, more recent experiments have demonstrated membrane-initiated signaling. Both direct nuclear and estradiol membrane signaling can be mediated by the classical estrogen receptors, ERα and ERβ, which are two of the numerous putative membrane estrogen receptors. Thus far, however, only ERα has been shown to play a prominent role in regulating female reproduction and sexual behavior. Because ERα is a ligand-gated transcription factor and not a typical membrane receptor, trafficking to the cell membrane requires post-translational modifications. Two necessary modifications are palmitoylation and association with caveolins, a family of scaffolding proteins. In addition to their role in trafficking, caveolin proteins also serve to determine ERα interactions with metabotropic glutamate receptors (mGluRs). It is through these complexes that ERα, which cannot by itself activate G proteins, is able to initiate intracellular signaling. Various combinations of ERα-mGluR interactions have been demonstrated throughout the nervous system from hippocampus to striatum to hypothalamus to dorsal root ganglion (DRG) in both neurons and astrocytes. These combinations of ER and mGluR allow estradiol to have both facilitative and inhibitory actions in neurons. In hypothalamic astrocytes, the estradiol-mediated release of intracellular calcium stores regulating neurosteroid synthesis requires ERα-mGluR1a interaction. In terms of estradiol regulation of female sexual receptivity, activation of ERα-mGluR1a signaling complex leads to the release of neurotransmitters and alteration of neuronal morphology. This review will examine estradiol membrane signaling (EMS) activating a limbic-hypothalamic lordosis regulating circuit, which involves ERα trafficking, internalization, and modifications of neuronal morphology in a circuit that underlies female sexual receptivity.

Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes

Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca 2+] i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca 2+ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca 2+] i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca 2+ entry.

Possible mechanism of perfluorooctane sulfonate and perfluorooctanoate on the release of calcium ion from calcium stores in primary cultures of rat hippocampal neurons

Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are anthropogenic compounds manufactured since the 1950s and are distributed worldwide. Now, the pollutants are being challenged by entering into the brain and the toxic effect on the central nervous system due to calcium disorder, mainly through channels on cell membrane. However, little is known about the role of calcium store in PFOS- and PFOA-evoked abnormal calcium increase. In the present study, PFOA and PFOS were measured in primary cultures of rat hippocampal neurons by LC/MS/MS analysis. Flow cytometry was used to examine altered calcium patterns in neurons labeled with fluo-3/AM and to disclose the mechanism by which PFOS and PFOA induced calcium increase in cultured neurons. The results indicate that both PFOS and PFOA can accumulate in cultured neurons and elevate calcium concentrations via release of intracellular calcium stores. Furthermore, inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) were found to take part in PFOS or PFOA inducing calcium release from calcium stores. IP3Rs seem to serve a predominant role in PFOS-induced calcium release. Calcium release from intracellular stores may partially account for the perturbation of calcium homeostasis caused by PFOS or PFOA.

Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells

Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.

A New Class of Human Mast Cell and Peripheral Blood Basophil Stabilizers that Differentially Control Allergic Mediator Release

Treatments for allergic disease block the effects of mediators released from activated mast cells and blood basophils. A panel of fullerene derivatives was synthesized and tested for their ability to preempt the release of allergic mediators in vitro and in vivo. The fullerene C(70)-tetraglycolic acid significantly inhibited degranulation and cytokine production from mast cells and basophils, while C(70)-tetrainositol blocked only cytokine production in mast cells and degranulation and cytokine production in basophils. The early phase of FcepsilonRI inhibition was dependent on the blunted release of intracellular calcium stores, elevations in reactive oxygen species, and several signaling molecules. Gene microarray studies further showed the two fullerene derivatives inhibited late phase responses in very different ways. C(70)-tetraglycolic acid was able to block mast cell-driven anaphylaxis in vivo, while C(70)-tetrainositol did not. No toxicity was observed with either compound. These findings demonstrate the biological effects of fullerenes critically depends on the moieties added to the carbon cage and suggest they act on different FcepsilonRI-specific molecules in mast cells and basophils. These next generation fullerene derivatives represent a new class of compounds that interfere with FcepsilonRI signaling pathways to stabilize mast cells and basophils. Thus, fullerene-based therapies may be a new approach for treating allergic diseases.

Platelet-derived Growth Factor-mediated Induction of the Synaptic Plasticity Gene Arc/Arg3.1

Platelet-derived growth factor (PDGF) is a pleiotropic protein with critical roles in both developmental as well as pathogenic processes. In the central nervous system specifically, PDGF is critical for neuronal proliferation and differentiation and has also been implicated as a neuroprotective agent. Whether PDGF also plays a role in synaptic plasticity, however, remains poorly understood. In the present study we demonstrated that in the rat hippocampal neurons PDGF regulated the expression of Arc/Arg3.1 gene that has been implicated in both synapse plasticity and long term potentiation. Relevance of these findings was further confirmed in vivo by injecting mice with intracerebral inoculations of PDGF, which resulted in a rapid induction of Arc in the hippocampus of the injected mice. PDGF induced long term potentiation in rat hippocampal slices, which was abolished by PDGF receptor-tyrosine kinase inhibitor STI-571. We also present evidence that PDGF-mediated induction of Arc/Arg3.1 involved activation of the MAPK/ERK (MEK) pathway. Additionally, induction of Arc/Arg3.1 also involved the upstream release of intracellular calcium stores, an effect that could be blocked by thapsigargin but not by EGTA. Pharmacological approach using inhibitors specific for either MAPK/ERK phosphorylation or calcium release demonstrated that the two pathways converged downstream at a common point involving activation of the immediate early gene Egr-1. Chromatin immunoprecipitation assays demonstrated the binding of Egr-1, but not Egr-3, to the Arc promoter. These findings for the first time, thus, suggest an additional role of PDGF, that of induction of Arc.

Regulation of bone turnover by calcium‐regulated calcium channels

Calcium plays multiple roles in osteoclast formation, survival, and activity. Intracellular calcium is determined both by the release of intracellular stores and the influx of extracellular calcium through a variety of calcium channels. Osteoclasts express several classes of calcium channels, including ryanodine receptors (RyRs), inositol-1,4,5-trisphosphate receptors (IP(3)Rs), and calcium release-activated calcium channels (CRACs), which respond to depletion of intracellular stores. IP(3)R2 is expressed in osteoclast precursors and activated by cytokines that stimulate osteoclast differentiation. In mature osteoclasts, the IP(3)R1 isoform is highly expressed and is implicated in nitric oxide-cGMP-stimulated processes. RyR calcium channels may contribute to the release of intracellular calcium stores, while RyR2 in the plasma membrane may act to limit osteoclast activity based on extracellular calcium concentration. Orai, through regulation by endoplasmic reticular store-sensing proteins, including Stim-1, may also mediate calcium influx and act as a signal amplifier for calcium release by other calcium channels. Together, these receptors allow intracellular Ca(2+) signals to modulate bone turnover and, through calcium-sensing functions, allow coupling of osteoclast activity to extracellular conditions and integrating additional cytokine and nitric oxide signals via transient intracellular calcium signals.

Non‐canonical Wnt Signaling in Embryonic Liver Development

In addition to activating canonical beta‐catenin mediated signaling events, Wnt proteins also initiate beta‐catenin‐independent, non‐canonical signaling pathways: the planar cell polarity and Wnt/Ca2+ pathways. The planar cell polarity pathway promotes asymmetric cytoskeletal organization to establish cellular as well as tissue polarity in developing organs. Activation of the Wnt/Ca2+ pathway triggers release of intracellular calcium stores which activate signaling molecules and transcription factors and is critical for axis establishment and cell migration in embryos. These pathways play roles in the development of multiple tissues, but their contribution to liver development remains unexplored. We have investigated the expression by qPCR of the non‐canonical Frizzleds (Fz3, 4, 6) as well as their Wnt ligands (Wnts 4, 5a, 6, 7a and 11) at embryonic stages E12.5, E14.5, E16.5, E17.5, E18.5 and found a marked increase in Wnt5a expression in late liver development, peaking at E17.5. Consistent with activation of the Wnt/Ca2+ pathway, we have also seen an increase in Wnt5a protein as well as active CamKII by western blot, though we do not observe NFAT localization in nuclei coinciding with Wnt 5a. In summary, non‐canonical Wnt signaling may be playing an important role in late embryonic liver development.