Transient Rise In Intracellular Ca2 Research Articles

Modulating Ca2+ influx into adrenal chromaffin cells with short duration nanosecond electric pulses

Isolated bovine adrenal chromaffin cells exposed to single 2, 4 or 5 ns pulses undergo a rapid, transient rise in intracellular Ca2+ mediated by Ca2+ entry via voltage-gated Ca2+ channels (VGCC), mimicking the activation of these cells in vivo by acetylcholine. However, pulse durations 150 ns or longer elicit larger amplitude and longer-lived Ca2+ responses due to Ca2+ influx via both VGCC and a yet to be identified plasma membrane pathway(s). To further our understanding of the differential effects of ultrashort versus longer pulse durations on Ca2+ influx, chromaffin cells were loaded with Calcium Green-1 and exposed to single 3, 5, 11, 25 or 50 ns pulses applied at their respective Ca2+ activation threshold electric fields. Increasing pulse duration from 3 or 5 ns to only 11 ns was sufficient to elicit increased amplitude and longer-lived Ca2+ responses in the majority of cells, a trend that continued as pulse duration increased to 50 ns. The amplification of Ca2+ responses was not the result of Ca2+ release from intracellular stores and was accompanied by a decreased effectiveness of VGCC inhibitors to block the responses and a reduced reliance on extracellular Na+ and membrane depolarization to evoke the responses. Inhibitors of pannexin channels, P2X receptors or non-selective cation channels failed to attenuate 50 ns-elicited Ca2+ responses, ruling out these Ca2+-permeable channels as secondary Ca2+ entry pathways. Analytical calculations and numerical modeling suggest that the parameter that best determines the response of chromaffin cells to increasing pulse durations is the time the membrane charges to its peak voltage. These results highlight the pronounced sensitivity of a neuroendocrine cell to pulse durations differing by only tens of nanoseconds, which has important implications for the future development of nanosecond pulse technologies enabling electrostimulation applications for spatially focused and graded in vivo neuromodulation.

Physical memory of astrocytes

Traumatic brain injury (TBI) is a major risk factor for development of neurodegenerative disorders later in life. Short, repetitive, mechanical impacts can lead to pathology that appears days or months later. The cells have a physical “memory” of mechanical events. The origin of this memory is not known. To examine the properties of this memory, we used a microfluidic chip to apply programmed fluid shear pulses to adherent adult rat astrocytes. These caused a transient rise in intracellular Ca2+. In response to repeated stimuli, 6 to 24 hrs apart, the Ca2+ response increased. This effect lasted longer than 24 hrs. The Ca2+ responses were more sensitive to the number of repetitions than to the rest time between stimuli. We found that inhibiting the Ca2+ influx during conditioning stimulus did not eliminate the stress potentiation, suggesting that mechanical deformation during the primary injury is accountable for the later response. The mechanical mechanism that triggers this long term “memory” may act by plastic deformation of the cytoskeleton.

Palmitoylation Controls NMDA Receptor Function and Steroid Sensitivity.

NMDARs are ligand-gated ion channels that cause an influx of Na+ and Ca2+ into postsynaptic neurons. The resulting intracellular Ca2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+ (Ca2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μm PAhPim had virtually no effect on NMDAR responses; however, following the Ca2+ challenge, it inhibited the responses by 62%; similarly, the Ca2+ challenge induced a 3.7-fold decrease in the steroid IC50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.

2-ns Electrostimulation of Ca2+ Influx into Chromaffin Cells: Rapid Modulation by Field Reversal

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca2+ levels due to Ca2+ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca2+ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca2+ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.

Interdependent allosteric free fatty acid receptor 2 modulators synergistically induce functional selective activation and desensitization in neutrophils

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y2R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca2+). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca2+. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling.

Functional and signaling characterization of the neutrophil FPR2 selective agonist Act-389949

Despite the steadily increased numbers of formyl peptide receptor (FPR) ligands identified over the years, few have been characterized in studies using animal disease models and even less have entered clinical trials in human subjects. A small-molecule compound, Act-389949, was however recently tested in a phase I clinical trial and found to be safe and well tolerated in healthy human subjects. The desired anti-inflammatory property of Act-389949 was proposed to be mediated through FPR2, one of the FPRs expressed in neutrophils, but no basic characterization was included in the study. To gain more insights into FPR2 recognition of this first-in-class compound for future utility of the agonist, we have in this study determined the receptor preference and down-stream signaling characteristics induced by Act-389949 in human blood neutrophils isolated from healthy donors. Our data demonstrate that Act-389949 is an agonist for FPR2 that triggers functional/signaling repertoires comparable to what has been earlier described for other FPR2 agonists, including neutrophil chemotaxis, granule mobilization and activation of the NADPH-oxidase. In fact, Act-389949 was found to be as potent as the prototype FPR2 peptide agonist WKYMVM and had the advantage of being resistant to oxidation by MPO-H2O2-halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gαq-independent transient rise in intracellular Ca2+ and recruitment of β-arrestin. In summary, our data show that Act-389949 serves as an excellent tool-compound for further dissection of FPR2-regulated activities in vitro and in vivo. Potent and stable FPR ligands such as Act-389949 may therefore be used to develop the next generation of FPR signaling regulating anti-inflammatory therapeutics.

Participation of membrane calcium channels in erythropoietin-induced endothelial cell migration

Calcium (Ca2+) plays an important role in angiogenesis, as it activates the cell migration machinery. Different proangiogenic factors have been demonstrated to induce transient Ca2+ increases in endothelial cells. This has raised interest in the contribution of Ca2+ channels to cell migration, and in a possible use of channel-blocking compounds in angiogenesis-related pathologies. We have investigated the ability of erythropoietin (Epo), a cytokine recently involved in angiogenesis, to induce Ca2+ influx through different types of membrane channels in EA.hy926 endothelial cells. The voltage-dependent Ca2+ channel antagonists amlodipine and diltiazem inhibited an Epo-triggered transient rise in intracellular Ca2+, similarly to a specific inhibitor (Pyr3) and a blocking antibody against the transient potential calcium channel 3 (TRPC3). Unlike diltiazem, amlodipine and the TRPC3 inhibitors prevented the stimulating action of Epo in cell migration and in vitro angiogenesis assays. Amlodipine was also able to inhibit an increase in endothelial cell migration induced by Epo in an inflammatory environment generated with TNF-α. These results support the participation of Ca2+ entry through voltage-dependent and transient potential channels in Epo-driven endothelial cell migration, highlighting the antiangiogenic activity of amlodipine.

Transthyretin—A Key Gene Involved in Regulating Learning and Memory in Brain, and Providing Neuroprotection in Alzheimer Disease via Neuronal Synthesis of Transthyretin Protein

Transthyretin (TTR), a carrier protein present in the liver and choroid plexus of the brain, has been shown to be responsible for binding thyroid hormone thyroxin (T4) and retinol in plasma and cerebrospinal fluid (CSF). TTR aids in sequestering of beta-amyloid peptides Aβ deposition, and protects the brain from trauma, ischemic stroke and Alzheimer disease (AD). Accordingly, hippocampal gene expression of TTR plays a significant role in learning and memory as well as in simulation of spatial memory tasks. TTR via interacting with transcription factor CREB regulates this process and decreased expression leads to memory deficits. By different signaling pathways, like MAPK, AKT, and ERK via Src, TTR provides tropical support through megalin receptor by promoting neurite outgrowth and protecting the neurons from traumatic brain injury. TTR is also responsible for the transient rise in intracellular Ca2+ via NMDA receptor, playing a dominant role under excitotoxic conditions. In this review, we tried to shed light on how TTR is involved in maintaining normal cognitive processes, its role in learning and memory, under memory deficit conditions; by which mechanisms it promotes neurite outgrowth; and how it protects the brain from Alzheimer disease (AD).

Shiga toxin (Stx)1B and Stx2B induce von Willebrand factor secretion from human umbilical vein endothelial cells through different signaling pathways

AbstractDiarrhea-associated hemolytic uremic syndrome (D+HUS) is caused by the ingestion of Escherichia coli that produce Shiga toxin (Stx), which is composed of a cytotoxic A subunit and pentameric B subunits that bind globotriaosylceramide on susceptible cells. Stx occurs in 2 types, Stx1 and Stx2. B subunits of either type stimulate von Willebrand factor (VWF) secretion from human umbilical vein endothelial cells (HUVECs), and Stx2B can cause thrombotic microangiopathy in Adamts13−/− mice. We have now determined that Stx1B and Stx2B activate different signaling pathways in HUVECs. VWF secretion induced by Stx1B is associated with a transient rise in intracellular Ca2+ level that is blocked by chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester, removal of extracellular Ca2+, the phospholipase C inhibitor U73122, the protein kinase inhibitor staurosporine, or small interfering RNA knockdown of protein kinase Cα. In contrast, Stx2B-induced VWF secretion is associated with activation of protein kinase A (PKA) and is blocked by the PKA inhibitor H89 or small interfering RNA knockdown of PKA. Stx2B does not increase cAMP levels and may activate PKA by a cAMP-independent mechanism. The activation of distinct signaling pathways may be relevant to understanding why E coli that express Stx2 are more likely to cause D+HUS than are E coli expressing only Stx1.

The FPR2-induced rise in cytosolic calcium in human neutrophils relies on an emptying of intracellular calcium stores and is inhibited by a gelsolin-derived PIP2-binding peptide

BackgroundThe molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific G-protein-coupled cell surface receptors (GPCRs). Human neutrophils express two pattern recognition GPCRs, FPR1 and FPR2, which belong to the family of formyl peptide receptors. The high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. The aims of this study were to determine whether FPR2 triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived PIP2-binding peptide, PBP10, selectively inhibits FPR2-mediated transient rise in intracellular Ca2+.ResultsThe transient rise in intracellular Ca2+ induced by agonists for FPR1 or FPR2 in human neutrophils occurred also in the presence of a chelator of Ca2+ (EGTA). PBP10 inhibited not only FPR2-induced oxidase activity, but also the transient rise in intracellular Ca2+.ConclusionsCa2+ signaling mediated via FPR2 follows the same route as FPR1, which involves initial emptying of the intracellular stores. PBP10 inhibits selectively the signals generated by FPR2, both with respect to NADPH-oxidase activity and the transient rise in intracellular Ca2+ induced by agonist exposure.

B Subunits of Shiga Toxin 2 Induce VWF Secretion by a cAMP-Independent Protein Kinase A Signaling Pathway.

Abstract 3046Poster Board II-1022Shiga toxin (Stx) is composed of an A subunit that inactivates ribosomes and 5 B subunits that bind globotriaosylceramice (Gb3) on cell surfaces to facilitate internalization and retrograde transport of the toxin to the cytosol. Stx-producing E. coli cause hemolytic uremic syndrome (HUS) by damaging renal microvascular endothelium. In animal experiments, Stx causes fatal thrombotic microangiopathy in ADAMTS13-deficient mice by inducing the secretion of von Willebrand factor (VWF). Using cultured human umbilical vein endothelial cells (HUVECs), we have shown that the B subunits of either Stx1 or Stx2 are sufficient to stimulate VWF secretion, independent of the catalytically-active A subunit. HUVECs were perfused in a parallel plate flow chamber with Stx preparations and fluorescently-labeled anti-VWF, and secreted VWF strings were visualized in real time by immunofluorescence microscopy. Either Stx1B or Stx2B rapidly induced the secretion of long strings of VWF that attached to the cell surface and bound platelets with high affinity. VWF string formation was maximal within 5 min and was blocked by soluble analogs of Gb3 or anti-Stx B subunit antibodies. Stx1B subunits activated phospholipase C (PLC), increased intracellular Ca2+, and activated protein kinase C (PKC). Stx2B is homologous to Stx1B, but their amino acid sequences differ by 34% and Stx2B activates distinct signaling pathways. For example, pretreatment of HUVECs with a chelator of intracellular Ca2+ (0.1 mM BAPTA-AM, 30 min), Ca2+-free medium (pH 7.2 PBS, 30 min), a phospholipase C (PLC) inhibitor (5 μM U73122, 15 min) or a protein kinase C inhibitor (5 μM 19-31, 1 h) blocked VWF secretion in response to Stx1B, but not Stx2B. The role of intracellular Ca2+ was also assessed using a fluorescent BD Calcium Assay. Stx1B (5 nM), histamine (0.1 mM) or thrombin (1 U/ml) induced a similar transient rise in intracellular Ca2+ level that peaked by 30 sec and declined to baseline over 5 min, whereas Stx2B did not affect the intracellular Ca2+ level. Conversely, pretreatment with a protein kinase A inhibitor (5 μM H89, 30 min) had no effect on Stx1B-induced VWF string formation, but decreased Stx2B-induced VWF string formation more than 90%. Neither Stx1B nor Stx2B increased intracellular cAMP in cell extracts within 5 min. To more directly detect cAMP production, HUVECs were transfected with a CFP-Epac-YFP construct that exhibits decreased FRET upon binding cAMP, which is detected by confocal microscopy. Forskolin caused the expected increase in CFP/YPF ratio, indicating a rise in cAMP, whereas Stx1B and Stx2B did not. Despite the absence of a change in cAMP, Stx2B caused an increase in PKA activity comparable to that induced by forskolin, when assayed in cell extracts with a synthetic peptide substrate (Assay Designs) kit. In addition, treatment of HUVECs with Stx2B increased the phosphorylation of the endogenous PKA substrate, 14-3-3ζ. These results indicate that Stx2B subunits stimulate VWF secretion through a previously unsuspected signaling pathway that involves binding to cell surface Gb3 and cAMP-independent activation of PKA. Therefore, Stx2 may contribute to the pathogenesis of HUS by at least two mechanisms that affect microvascular endothelium: Stx2A subunit-dependent inhibition of protein synthesis, and Stx2B subunit-dependent induction of VWF secretion. DisclosuresSadler:Baxter BioSciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ablynx: Consultancy.

Aromatic D-amino acids act as chemoattractant factors for human leukocytes through a G protein-coupled receptor, GPR109B

GPR109B (HM74) is a putative G protein-coupled receptor (GPCR) whose cognate ligands have yet to be characterized. GPR109B shows a high degree of sequence similarity to GPR109A, another GPCR that was identified as a high-affinity nicotinic acid (niacin) receptor. However, the affinity of nicotinic acid to GPR109B is very low. In this study, we found that certain aromatic D-amino acids, including D-phenylalanine, D-tryptophan, and the metabolite of the latter, D-kynurenine, decreased the activity of adenylate cyclase in cells transfected with GPR109B cDNA through activation of pertussis toxin (PTX)-sensitive G proteins. These D-amino acids also elicited a transient rise of intracellular Ca(2+) level in cells expressing GPR109B in a PTX-sensitive manner. In contrast, these D-amino acids did not show any effects on cells expressing GPR109A. We found that the GPR109B mRNA is abundantly expressed in human neutrophils. D-phenylalanine and D-tryptophan induced a transient increase of intracellular Ca(2+) level and a reduction of cAMP levels in human neutrophils. Furthermore, knockdown of GPR109B by RNA interference inhibited the D-amino acids-induced decrease of cellular cAMP levels in human neutrophils. These D-amino acids induced chemotactic activity of freshly prepared human neutrophils. We also found that D-phenylalanine and D-tryptophan induced chemotactic responses in Jurkat cells transfected with the GPR109B cDNA but not in mock-transfected Jurkat cells. These results suggest that these aromatic D-amino acids elicit a chemotactic response in human neutrophils via activation of GPR109B.

Shiga Toxin 1(Stx1) Stimulates Endothelial Cell VWF Secretion through a Ca2+/PKC Signaling Pathway That Does Not Require the Active Toxin Stx1A Subunit

Shiga toxins (Stx) consist of 5 B (binding) subunits that interact with cell surface globotriaosylceramide (Gb3), and a single A subunit that is retrotranslocated into the cytoplasm where it enzymatically inactivates ribosomal RNA. E. coli O157:H7 can express several variants of Shiga toxin (Stx) that cause hemolytic uremic syndrome (HUS) by damaging renal microvascular endothelium. The Stx A subunit is required for cytopathic effects on endothelium. In addition, Stx causes fatal thrombotic microangiopathy in ADAMTS13-deficient mice, but not in wild-type mice, and this effect requires the presence of von Willebrand factor (VWF). When added to cultured human endothelial cells under conditions of laminar flow, Stx rapidly induces the acute secretion of long strings of VWF that attach to the cell surface and bind platelets with high affinity, which suggests that VWF-induced platelet aggregation might contribute to the pathogenesis of HUS. Whether VWF secretion depends on ribotoxic stress is unknown, and the mechanism of Stx-induced VWF secretion has not been characterized. To address these questions, we investigated VWF secretion by human umbilical vein endothelial cells (HUVECs) treated with Stx1 holotoxin (AB5) or binding subunits (B5). Recombinant Stx1 B5 was expressed in E. coli and purified to homogeneity. The pentameric composition and purity of B5 preparations were demonstrated by gel filtration chromatography and Western blotting. Endotoxin was removed from Stx AB5 and B5 preparations by affinity chromatography. HUVECs were perfused in a parallel plate flow chamber with fluorescently labeled anti-VWF and Stx preparations, and secreted VWF strings were visualized in real time by immunofluorescence microscopy. Unexpectedly, we found that Stx1 B5 and Stx1 AB5 were equally potent in stimulating the secretion of VWF strings. String formation was maximal after 5 min and was blocked by soluble analogs of Gb3 or anti-Stx1 B subunit antibodies. Pretreatment of HUVECs with a chelator of intracellular Ca2+ (0.1 mM BAPTA-AM, 30 min), a phospholipase C (PLC) inhibitor (5 μM U73122, 15 min), or a protein kinase C inhibitor (50 nM staurosporine, 30 min) decreased the secretion of VWF strings by 95%, 82%, or 90%, respectively. Treatment with a protein kinase A inhibitor (5 μM H89, 30 min) did not affect VWF string formation. To more directly assess Stx1-induced PLC activation, HUVECs were transfected with a plasmid expressing a PLC-delta PH domain-GFP construct, which binds membrane-associated phosphatidylinositol 4,5-bisphosphate (PIP2). When HUVECs were observed by confocal microscopy, both Stx1 AB5 and Stx1 B5 caused a rapid (<60 s) redistribution of fluorescent signal from plasma membrane to cytosol, indicating the acute activation of PLC and hydrolysis of PIP2. In addition, Stx1 AB5 or B5 induced a transient rise in intracellular Ca2+ level that peaked by 30 sec and declined to baseline over 5 min. Stx1-induced Ca2+ transients were comparable to those induced by 0.1 mM histamine or 1 U/ml thrombin. Stx-induced Ca2+ responses were inhibited by BAPTA-AM or U73122, but not by staurosporine or H89. Treatment with Stx1 AB5 or B5 had no effect on intracellular levels of cAMP. These results indicate that Stx1 B subunits stimulate VWF secretion through a previously unsuspected signaling pathway that involves binding to cell surface Gb3, PIP2 hydrolysis by PLC, increased intracellular Ca2+, and activation of PKC. Therefore, Stx1 secreted by enterohemorrhagic E. coli may contribute to the pathogenesis of HUS through at least two mechanisms that affect microvascular endothelium: cell death caused by A subunit-induced ribotoxic stress, and VWF secretion caused by an independent B subunit-induced cell signaling pathway.

Metaplastic control of the endocannabinoid system at inhibitory synapses in hippocampus

The modifiability of neuronal response plasticity is called "metaplasticity." In suppressing synaptic inhibition and facilitating induction of long-term excitatory synaptic plasticity, endocannabinoids (eCBs) act as agents of metaplasticity. We now report the discovery of a calcium-dependent mechanism that regulates eCB mobilization by metabotropic glutamate receptor (mGluR) activation. The switch-like mechanism primes cells to release eCBs and requires a transient rise in intracellular Ca2+ concentration ([Ca2+]i) but not concurrent activation of mGluRs. Conversely, short-term, [Ca2+]i-dependent eCB release can be persistently enhanced by mGluR activation. Hence, eCBs are also objects of metaplasticity, subject to higher levels of physiological control.

A Conserved Docking Site Modulates Substrate Affinity for Calcineurin, Signaling Output, and In Vivo Function

Calcineurin, the conserved Ca(2+)/calmodulin-regulated protein phosphatase, mediates diverse aspects of Ca(2+)-dependent signaling. We show that substrates bind calcineurin with varying strengths and examine the impact of this affinity on signaling. We altered the calcineurin-docking site, or PxIxIT motif, in Crz1, the calcineurin-regulated transcription factor in S. cerevisiae, to decrease (Crz1(PVIAVN)) or increase (Crz1(PVIVIT)) its affinity for calcineurin. As a result, the Ca(2+)-dependent dephosphorylation and activation of Crz1(PVIAVN) are decreased, whereas Crz1(PVIVIT) is constitutively dephosphorylated and hyperactive. Surprisingly, the physiological consequences of altering calcineurin-Crz1 affinity depend on the growth conditions. Crz1(PVIVIT) improves yeast growth under several environmental stress conditions but causes a growth defect during alkaline stress, most likely by titrating calcineurin away from other substrates or regulators. Thus, calcineurin-substrate affinity determines the Ca(2+) concentration dependence and output of signaling in vivo as well as the balance between different branches of calcineurin signaling in an overall biological response.

Mitochondrial Ca2+ transport in frog early distal tubule

A global and transient rise of intracellular Ca2+ (Ca2+i) is central to the operation of pump-leak coupling in the frog early distal tubule (EDT). The endoplasmic reticulum (ER) is the site of this Ca2+ release and reuptake; however, it is likely that other intracellular pools, such as mitochondria, also contribute to cellular Ca2+ homeostasis. The present study was performed to seek evidence of mitochondrial Ca2+ transport in the frog EDT. Experiments were performed on isolated and permeabilized EDT segments from the frog kidney loaded with the low-affinity, Ca2+-sensitive fluorescent indicator, mag-fura-2. Ca2+ uptake in the absence of SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) activity (inhibition by 2,5-di-t-butyl hydroquinone, TBQ) was evident at a bath [Ca2+] of 1 microm, but not at 200 nm, in the presence of ATP. This uptake was sensitive to the protonophore FCCP and the ATP-synthase inhibitor oligomycin. Ca2+ uptake was also stimulated by respiratory substrates; this uptake was enhanced by oligomycin and reversed by the application of FCCP. These findings provide the first evidence of mitochondrial Ca2+ transport in renal tubules, which appears to occur via a low-affinity pathway and which will act as a physiological Ca2+ buffer, protecting the cell from large increases in Ca2+i.

Nongenomic stimulation of vacuolar H+-ATPases in intercalated renal tubule cells by aldosterone.

Renal collecting ducts play a critical role in acid-base homeostasis by establishing steep transepithelial pH gradients necessary for the almost complete reabsorption of bicarbonate and the effective secretion of ammonium into the urine. The mechanisms of urine acidification in collecting ducts involve active, electrogenic hydrogen (H+) secretion and, less importantly, potassium (K+)-H+ exchange. Deranged renal acidification and the inability to lower urine pH are hallmarks of distal tubular acidosis and often result from inborn errors of metabolism involving vacuolar H+-ATPase subunits in the collecting ducts. Three factors regulate H+-ATPase activity in intercalated cells of collecting ducts: the acid-base status, angiotensin II, and aldosterone. Most effects of aldosterone involve activation of the mineralocorticoid receptor and genomic changes in transcription and protein synthesis. Here we demonstrate a nongenomic pathway of vacuolar H+-ATPase activation in intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). In vitro exposure of isolated outer medullary collecting ducts to aldosterone (10 nM) for times as short as 15 min increases vacuolar H+-ATPase activity approximately 2- to 3-fold. Neither inhibition of mineralocorticoid receptors nor of transcription and protein synthesis prevented aldosterone-induced stimulation of H+-ATPase. Incubation with colchicine, however, abolished the stimulatory effect of aldosterone, suggesting a role of the microtubular network for H+-ATPase stimulation. Immunohistochemistry in kidneys from aldosterone-injected mice showed increased apical H+-ATPase staining in OMCD-intercalated cells. The stimulatory effect of aldosterone was associated with a transient rise in intracellular Ca2+ and required intact PKC. Thus, rapid nongenomic modulation of vacuolar H+-ATPase activity in OMCD-intercalated cells by aldosterone may play an additional role in hormonal control of systemic acid-base homeostasis.

Functional evidence for P2X receptors in isolated human vagus nerve.

Functional evidence for P2X receptors in isolated human vagus nerve.

Ca2+ signaling mediated by IP3-dependent Ca2+ releasing and store-operated Ca2+ channels in rat odontoblasts.

In the phospholipase-C (PLC) signaling system, Ca2+ is mobilized from intracellular Ca2+ stores by an action of inositol 1,4,5-trisphosphate (IP3). The depletion of IP3-sensitive Ca2+ stores activates a store-operated Ca2+ entry (SOCE). However, no direct evidence has been obtained about these signaling pathways in odontoblasts. In this study, we investigate the characteristics of the SOCE and IP3-mediated Ca2+ mobilizations in rat odontoblasts using fura-2 microfluorometry and a nystatin-perforated patch-clamp technique. In the absence of extracellular Ca2+ ([Ca2+]o), thapsigargin (TG) evoked a transient rise in intracellular Ca2+ concentration ([Ca2+]i). After TG treatment to deplete the store, the subsequent application of Ca2+ resulted in a rapid rise in [Ca2+]i caused by SOCE. In the absence of TG treatment, no SOCE was evoked. The Ca2+ influx was dependent on [Ca2+]o (KD = 1.29 mM) and was blocked by an IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate (2-APB), as well as La3+ in a concentration-dependent manner (IC50 = 26 microM). In TG-treated cells, an elevation of [Ca2+]o from 0 to 2.5 mM elicited an inwardly rectifying current at hyperpolarizing potentials with a positive reversal potential. The currents were selective for Ca2+ over the other divalent cations (Ca2+ > Ba2+ > Sr2+ >> Mn2+). In the absence of [Ca2+]o, carbachol, bradykinin, and 2-methylthioadenosine 5'triphosphate activated Ca2+ release from the store; these were inhibited by 2-APB. These results indicate that odontoblasts possessed Ca2+ signaling pathways through the activation of store-operated Ca2+ channels by the depletion of intracellular Ca2+ stores and through the IP3-induced Ca2+ release activated by PLC-coupled receptors.

Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons.

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.