Calcium-induced Release Research Articles

Rectification of muscle and nerve deficits in paralyzed ryanodine receptor type 1 mutant embryos

Locomotion and respiration require motor axon connectivity and activation of the neuromuscular junction (NMJ). Through a forward genetic screen for muscle weakness, we recently reported an allele of ryanodine receptor type 1 (Ryr1AG). Here we reveal a role for functional RyR1 during acetylcholine receptor (AChR) cluster formation and embryonic synaptic transmission. Ryr1AG homozygous embryos are non-motile. Motor axons extend past AChR clusters and enlarged AChR clusters are found under fasciculated nerves. Using physiological and pharmacological methods, we show that contractility can be resumed through the masking of a potassium leak, and evoked vesicular release can be resumed via bypassing the defect in RyR1 induced calcium release. Moreover, we show the involvement of ryanodine receptors in presynaptic release at the NMJ. This data provides evidence of a role for RyR1 on both the pre- and postsynaptic sides of the NMJ.

Conformational and biological properties of Bauhinia bauhinioides kallikrein inhibitor fragments with bradykinin-like activities.

Proteinase inhibitors extracted form medicinal plants are an interesting source of new drugs. Modifications in the structure of some of this kind of macromolecules could also lead to compounds of interesting biological properties. In this work, we synthesized and tested one fragment containing the reactive site of the Bauhinia bauhinioides kallikrein inhibitor (BbKI), denoted BbKI51-62 , and a related analog (P2 ) in which a proline residue was inserted in order to mimic the N-terminal region of the bradykinin molecule. The related retro-inverso counterparts Ri-BbKI51-62 and Ri-P2 were also included. The ability of these peptides to induce contraction of stomach fundus isolated from mouse was evaluated as well as their capability to induce calcium release from a cell culture of smooth muscle from guinea pig ileum. The conformational properties of the peptides were evaluated by circular dichroism and their resistance to enzymatic degradation by exposure to human blood plasma. Our results show that neither the parent BbKI51-62 nor its Ri-BbKI51-62 analog exhibit bradykinin-like activity, although the retro-inverso isomer was resistant to blood plasma degradation. On the other hand, the peptides P2 and Ri-P2 presented contractile activities on gastric smooth muscle from stomach fundus possibly by acting via B-2 receptor. Both compounds also induce calcium release from guinea pig ileum muscle cells in a manner similar to bradykinin. Moreover, both compounds also inhibited porcine pancreatic kallikrein. However, conformational analysis did not reveal any direct correlation between structure and biological effects.

Turning off of GluN2B subunits and turning on of CICR in hippocampal LTD induction after developmental GluN2 subunit switch.

NMDA receptors (NMDARs) are essential for the induction of synaptic plasticity that mediates activity-dependent refinement of neural circuits during development. GluN2B subunits of NMDARs are abundant at synapses in the immature hippocampus and begin to be replaced by GluN2A subunits with the help of casein kinase 2 activity in the second postnatal week, the critical period for the GluN2 subunit switch (Sanz-Clemente et al. (2000) Neuron 67:984-996). However, the physiological role of GluN2B subunits in the hippocampus during this critical period has not been elucidated. Here, we report that GluN2B subunits mediate the induction of long-term depression (LTD) in the CA1 region of the hippocampus only until this period. Ifenprodil and Ro25-6981, selective inhibitors of NMDARs containing GluN2B subunits, blocked LTD in postnatal Day 11-14 (P11-14) rat hippocampal slices but not in P18-22 hippocampus. Just a few days after P14, synaptic NMDAR currents became narrower than those at P11-14, and calcium influx through NMDARs must be reduced. We found that calcium-induced calcium release (CICR) through ryanodine receptors starts to support the induction of NMDAR-dependent LTD at P18-22. Intracellular application of thapsigargin and ryanodine, inhibitors of Ca2+ -ATP pumps on internal stores and ryanodine receptors, respectively, did not at all affect LTD in the hippocampus at P11-14 but completely blocked LTD in the P18-22 hippocampus. Therefore, calcium influx through NMDAR with GluN2B subunits is sufficient to induce LTD at P11-14, after which CICR compensates for the decrease in calcium influx during LTD induction.

Discovery of novel N-aryl piperazine CXCR4 antagonists.

A novel series of CXCR4 antagonists with substituted piperazines as benzimidazole replacements is described. These compounds showed micromolar to nanomolar potency in CXCR4-mediated functional and HIV assays, namely inhibition of X4 HIV-1(IIIB) virus in MAGI-CCR5/CXCR4 cells and inhibition of SDF-1 induced calcium release in Chem-1 cells. Preliminary SAR investigations led to the identification of a series of N-aryl piperazines as the most potent compounds. Results show SAR that indicates type and position of the aromatic ring, as well as type of linker and stereochemistry are significant for activity. Profiling of several lead compounds showed that one (49b) reduced susceptibility towards CYP450 and hERG, and the best overall profile when considering both SDF-1 and HIV potencies (6-20 nM).

Calcium signaling, excitability, and synaptic plasticity defects in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) and aging result in impaired ability to store memories, but the cellular mechanisms responsible for these defects are poorly understood. Presenilin 1 (PS1) mutations are responsible for many early-onset familial AD (FAD) cases. The phenomenon of hippocampal long-term potentiation (LTP) is widely used in studies of memory formation and storage. Recent data revealed long-term LTP maintenance (L-LTP) is impaired in PS1-M146V knock-in (KI) FAD mice. To understand the basis for this phenomenon, in the present study we analyzed structural synaptic plasticity in hippocampal cultures from wild type (WT) and KI mice. We discovered that exposure to picrotoxin induces formation of mushroom spines in both WT and KI cultures, but the maintenance of mushroom spines is impaired in KI neurons. This maintenance defect can be explained by an abnormal firing pattern during the consolidation phase of structural plasticity in KI neurons. Reduced frequency of neuronal firing in KI neurons is caused by enhanced calcium-induced calcium release (CICR), enhanced activity of calcium-activated potassium channels, and increased afterhyperpolarization. As a result, "consolidation" pattern of neuronal activity converted to "depotentiation" pattern of neuronal activity in KI neurons. Consistent with this model, we demonstrated that pharmacological inhibitors of CICR (dantrolene), of calcium-activated potassium channels (apamin), and of calcium-dependent phosphatase calcineurin (FK506) are able to rescue structural plasticity defects in KI neurons. Furthermore, we demonstrate that incubation with dantrolene or apamin also rescued L-LTP defects in KI hippocampal slices, suggesting a role for a similar mechanism. This proposed mechanism may be responsible for memory defects in AD but also for age-related memory decline.

Unifying principles of calcium wave propagation — Insights from a three-dimensional model for atrial myocytes

Atrial myocytes in a number of species lack transverse tubules. As a consequence the intracellular calcium signals occurring during each heartbeat exhibit complex spatio-temporal dynamics. These calcium patterns arise from saltatory calcium waves that propagate via successive rounds of diffusion and calcium-induced calcium release. The many parameters that impinge on calcium-induced calcium release and calcium signal propagation make it difficult to know a priori whether calcium waves will successfully travel, or be extinguished. In this study, we describe in detail a mathematical model of calcium signalling that allows the effect of such parameters to be independently assessed. A key aspect of the model is to follow the triggering and evolution of calcium signals within a realistic three-dimensional cellular volume of an atrial myocyte, but with low computational costs. This is achieved by solving the linear transport equation for calcium analytically between calcium release events and by expressing the onset of calcium liberation as a threshold process. The model makes non-intuitive predictions about calcium signal propagation. For example, our modelling illustrates that the boundary of a cell produces a wave-guiding effect that enables calcium ions to propagate further and for longer, and can subtly alter the pattern of calcium wave movement. The high spatial resolution of the modelling framework allows the study of any arrangement of calcium release sites. We demonstrate that even small variations in randomly positioned release sites cause highly heterogeneous cellular responses. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Calcium-Induced Calcium Release during Action Potential Firing in Developing Inner Hair Cells

In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signaling mechanisms involved in early developmental processes.

Calcium, a multitasking signaling actor in airway smooth muscle cells, as a target of novel strategies to limit airway disease?

Asthma, Chronic Obstructive Pulmonary Disease (COPD),… words that have entered our everyday vocabulary, but more especially diseases whose prevalence is growing steadily and which faithfully illustrate the continuous increase in respiratory diseases. The quantified estimations, suggesting that asthma affects 300,000 persons worldwide and that COPD will become the third cause of death in 2020, give a very clear vision of the future of these diseases. Thus, the investigation of the cellular mechanisms of the functioning of the respiratory system has led to the identification of signaling pathways in which modifications could be responsible for the appearance of pathophysiological situations. The ubiquitous nature of the role of calcium as a signaling actor in smooth muscle cells (SMC) associated with the fact that the SMC are major constituents of the airways, are two basic findings that clearly justify the importance of this book edited by Yong-Xiao Wang. Thus, the union of the 25 articles by almost 50 contributors that make up the 460 pages of this book clearly illustrates the variety and importance of the role of calcium in airway SMC (ASMC). However, more than a simple illustration, this book is a form of “state of the art” of the role of calcium in the physiology of ASMC and then, in its potential involvement in various pathophysiological conditions. However, it is first necessary to recognize the importance of Yong-Xiao Wang, who has gathered contributors recognized as experts in the field. The numerous works published by Yong-Xiao Wang on this subject therefore confer him the legitimacy to be the editor of such a book based on a simple observation: the number of scientific research is steadily increasing without a “comprehensive book compiling and detailing the (our) state-of-the-art advances” (preface p. v) is available. One reason for this increased interest in the role of calcium in ASCM directly comes from the involvement of alterations in regulating the calcium movements in pathophysiological situations. In this context, although the findings presented by Anderson in 1983 in one of the 1st review available on this subject seem very distant, he was clearly establishing the basis for all work presented here: “It cannot be excluded that changes in one or more of these mechanisms, induced by mediators, hormones, or other agents may be a contribution factor to airway hyperreactivity” (Anderson, 1983). Therefore, in the context of recent data collection on this theme, a book review on “calcium signaling in airway smooth muscle cells” should probably aim to try to offer an overview of this subject. As mention above, since this book is clearly a state of the art in the field, it seems to me that a figure summarizing the physiological pathways involving calcium in SMC could be the main asset that a short “book review” can provide (Figure ​(Figure1).1). It will enable readers to get an overview of the calcium-dependent pathways that are targets to consider in pathophysiological situations. Figure 1 Calcium-dependent pathways in airways smooth muscle cells. This figure summarizes the main effects of intracellular calcium dynamics (Ca2+-Sparks/Oscillations) from calcium entry into cell and local Calcium-Induced Calcium-Release (CICR) resulting from ... Figure ​Figure11 shows the intracellular mechanisms involving the calcium in the operation of the ASCM. The main roles presented here show the involvement of calcium in the control of the contraction and relaxation of ASCM but also its role in the control of cell proliferation. Although the main calcium-dependent pathways in ASMC are recalled in Figure ​Figure1,1, they can be modulated by sex hormones or maturation as well-described by Y.S. Prakash et al. in two chapters (pp. 322–332; 333–357). Moreover, since the respiratory pathologies are unanimously recognized as associated with inflammatory situations, the three chapters describing the influence of inflammatory mediators (H. Matsumoto pp. 359–379; Y. Amrani pp. 423–439; Y. Su pp. 441–457) on Ca-dependent pathways in ASMC perfectly complement this book. Finally this synthesis can be well-completed by the two chapters presenting an interesting approach based on mathematical simulation of calcium oscillation in the SMC. Therefore, the mathematical models of calcium dynamics proposed by Sneyd et al. (pp. 341–357) have to be related with the observations of E. Roux (pp. 147–175) on the kinetics of the mechanisms involved in calcium homeodynamics. In conclusion by drawing an updated synthesis of advanced knowledge of calcium-dependent intracellular pathways in the ASCM, this authoritative book can claim to be an important tool to the use not only for researchers but also for teachers and students.

Serca Located in the Junctional SR Shapes Calcium Release in Cardiac Myocytes

Calcium-induced calcium release is in principle an all-or-nothing mechanism and self-propagation of Ca2+ waves is a pathological manifestation of this positive feedback loop. However, since local release of Ca2+ only elicits elementary Ca2+ release events (Ca2+ sparks) that do not develop into Ca2+ waves, Ca2+ release can be graded. This local control is possible because of the spatial arrangement of L-type Ca2+ channels and clusters of ryanodine receptors at the sites of Ca2+ release (dyads) (Stern 1992). We propose that the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) also contributes to local control by limiting diffusion of Ca2+. Six days following conditional SERCA knockout, Ca2+ sparks exhibited broadened geometry and slowed kinetics (increase in spark width, time to peak, and duration by 33%, 33%, and 51%, respectively). At this time point, SERCA protein levels are reduced by 53% and SR Ca2+ content is decreased by 25% (Stokke et al. 2010). To determine the precise localization of SERCA we employed cryo immuno-gold electron microscopy on sections from the mouse papillary muscle. Normal cardiac myocytes showed preferential SERCA expression near the Z-lines (nearly twofold higher labeling density at the Z-line than the A-band) with SERCA molecules clearly located in the junctional SR in close proximity to the dyads. The conditional SERCA knockout resulted in preferential loss of labeling at these locations. From resin-embedded cross-sections of papillary muscles, we observed more abundant SR in the I-band compared to the A-band (by factor of 1.7 and 1.6 for control and KO respectively). These results suggest that SERCA contributes to local control of Ca2+ release by limiting diffusion of Ca2+ from the dyad. Thus, the processes of Ca2+ release and re-uptake are closely linked by a population of SERCA molecules in the junctional SR.

Contribution of synaptic release mechanisms to the building of sensory maps

Numerous neurotransmitters have been implicated in neurodevelopmental processes. In addition, developing neurons show an abundance of vesicles in the growth cones, and express proteins of the SNARE complex early on. This has led to propose a role for vesicular fusion machinery in axonal growth and synapse formation. However, as the molecular machinery of vesicular fusion started to unveil, and knockouts for the major proteins of this complex were generated, it came as a surprise that none of these proteins was essential for the construction of brain architecture, although they were crucial for vital functions of the organism, leading to early mortality of exocytosis mutants. Because of this early death, conditional ablation of these genes in well-defined neuronal populations was necessary to study their role at later stages of neural circuit development, when activity-dependent mechanisms are best defined. Early studies showed that mutants of Munc18-1, a gene essential for both constitutive and calcium triggered release, were required for target dependent cell survival but not for axon growth or early refinement of topographic targeting, at least in the retinotectal system. Conditional knockout of the Rim1 and Rim2 genes allowed to interrogate more specifically the role of calcium-triggered release. Rims (rab interacting molecules) play a key role in the assembly of calcium channels and their coupling to the SNARE complex alters calcium-triggered release with little effect on constitutive release. When Rim1/Rim2 genes were ablated in the thalamus, layer IV neurons failed to organize into barrel structures, and to form the characteristic asymmetric distribution of their dendrites. More surprisingly, thalamocortical axons still organized in precise topographic maps and formed well differentiated synapses despite considerable reduction of calcium-induced synaptic release. However, this reduction in release probability altered axon targeting in the visual system where axons from both eyes compete for the same target. Thus, genetic tools targeting the exocytosis machinery are allowing to dissect more precisely the contribution of synaptic and non-synaptic mechanisms to activity-dependent circuit wiring.

MG56, a Member of the MBOAT Family of Proteins, Regulates Intracellular Calcium Signaling in Striated Muscle

Calcium-induced-calcium-release (CICR) from the sarcoplasmic reticulum (SR) plays an integral role in excitation-contraction coupling - the driving mechanism behind synchronous cardiac muscle contractions. Alterations in CICR are commonly found in individuals suffering from cardiac arrhythmias, and closely associated with ventricular fibrillation, tachycardia, and sudden cardiac death. The ryanodine receptor 2 (RyR2) is a key mediator of CICR, functioning as a calcium channel along the SR. Elucidating the role of proteins that modulate RyR2-mediated CICR represents a major interest in cardiovascular research. We recently discovered a novel SR-resident membrane protein named mitsugumin 56 (MG56), which belongs to the membrane-bound o-acyltransferase (MBOAT) family of proteins. Knockout of MG56 produced a postnatal lethal phenotype. We have observed elevated Ca spark activity in MG56 null muscle fibers when compared with the wild type littermates. Using HEK293 cells with inducible RYR2 expression, we found that over-expression of MG56 led to suppression of RyR2-mediated store-overload induced calcium release (SOICR) from the endoplasmic reticulum (ER). The findings suggest the possibility that MG56 functions as a stabilizer of RyR channel function. Since MBOAT family proteins contain acyltransferase activity, we hypothesize that the putative acyltransferase function for MG56 may be involved in the Ca regulatory function. In preliminary studies, we found that pretreatment of muscle fibers with 2-bromohexanoic acid, an inhibitor of palmitoyl acyltransferases, led to suppression of Ca spark activity. The effect of 2- bromohexanoic acid was also observed in HEK293 cells expressing RyR2, where SOICR activity was reduced upon treatment. Future studies are required to dissect the functional interaction between MG56 and RyR2, and to delineate the potential role for MG56-mediated lipid modification of RyR2 in the overall Ca signaling in cardiac muscle.

Impact of calcium signaling during infection of Neisseria meningitidis to human brain microvascular endothelial cells.

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca2+) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca2+ from the sarcoplasmic reticulum (SR). The Ca2+ leak out of the SR is an important process for cellular Ca2+ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca2+ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca2+ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca2+ sparks and robust Ca2+ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca2+ spark and nonspark-based SR Ca2+ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca2+-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca2+ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca2+ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca2+ release properties due to variations in inter-RyR coupling via local subspace Ca2+ concentration ([Ca2+]ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.

Cardioprotection by ranolazine in perfused rat heart.

: We used the isolated working rat model to evaluate the effect of therapeutic concentrations (5-10 μM) of ranolazine on contractile performance, oxygen consumption, irreversible ischemic injury, and sarcoplasmic reticulum (SR) function. Ischemic injury was induced by 30 minutes of global ischemia followed by 120 minutes of Langendorff reperfusion and evaluated on the basis of triphenyltetrazolium chloride staining. SR function was determined on the basis of [H]-ryanodine binding, the kinetics of calcium-induced calcium release, measured by quick filtration technique, and oxalate-supported calcium uptake. In working hearts, ranolazine significantly reduced oxygen consumption (P = 0.031), in the absence of significant changes in contractile performance, and decreased irreversible ischemic injury (P = 0.011), if administered either before ischemia-reperfusion (25.4% ± 4.7% vs. 42.7% ± 6.0%) or only at the time of reperfusion (20.2% ± 5.2% vs. 43.7% ± 9.9%). In SR experiments, treatment with ranolazine determined a significant reduction in [H]-ryanodine binding (P = 0.029), because of decreased binding site density (369 ± 9 vs. 405 ± 12 fmol/mg), and in the kinetics of SR calcium release (P = 0.011), whose rate constant was decreased, whereas active calcium uptake was not affected. Ranolazine effectiveness at reperfusion and its ability to module SR calcium release suggest that this drug might be particularly useful to induce cardioprotection during coronary revascularization interventions, although the relevance of the effects on calcium homeostasis remains to be determined.

Structure of a mammalian ryanodine receptor.

Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here, we report the closed-state structure of the 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+.

Abstract 18342: Inducible Cardiomyocyte-Specific Deletion of Stromal Interaction Molecules Results in Cardiomyopathy in Mice

Background: Stromal interaction molecules (STIM1 and STIM2) are sarco/endoplasmic calcium sensors ubiquitously expressed in mammalian cells. Upon Ca2+ store discharge STIM proteins oligomerize in proximity of the plasma membrane to activate the Ca2+ selective channels (Orai) and trigger Ca2+ entry. While this mechanism is widely accepted to be the main source of calcium in non-excitable cells, in cardiomyocytes the calcium-induced calcium-release is the major controller of myocardial function, and the presence of STIM in these cells appears somewhat puzzling. Here we investigated the role of STIM in cardiomyocytes and its role in regulating cardiac contractility and intracellular Ca2+ dynamics. Methods: We have generated an inducible cardiac-specific STIM1 knockout (STIM KO) mouse model to elucidate the role of STIM in the myocardium. Echocardiography was used to evaluate structure and function of the left ventricle. Heart rate was monitored continuously in conscious mice by telemetry. Cell dimensions, shortening, and relaxation were determined by videomicroscopy and calcium transients by fura 2. Results: Echocardiographic analyses revealed development of dilated cardiomyopathy with a significant reduction of left ventricular fractional shortening (39.91±4.05% vs. 22.41±7.92%, p<0.01). Histological and morphological analyses confirmed a dilated cardiomyopathy characterized by enlarged ventricular chambers. Electrocardiography showed higher heart rate in the STIM1 KO mice with no differences in P-wave duration and QRS interval while Q-T interval was reduced in the STIM1 KO mice as compared to the WT mice. Sarcomere length and cell shortening measurements in freshly isolated cardiomyocytes from STIM1 KO mice confirmed the reduced contractility associated with a reduction of time to 50% relaxation (283±13ms vs. 258±8ms, p<0.05) . Intracellular calcium transient analysis in STIM1 KO cardiomyocytes showed a higher peak amplitude and a time to 50% decay of calcium significantly accelerated. Conclusions: Our data demonstrate that STIM proteins play an important role in the maintaining normal cardiac function in the adult heart and reveal that STIM plays an important role during cardiac repolarization.

Intracallosal neuronal nitric oxide synthase neurons colocalize with neurokinin 1 substance P receptor in the rat.

The corpus callosum (cc) contains nitric oxide (NO)-producing neurons. Because NO is a potent vasodilator, these neurons could translate neuronal signals into vascular responses that can be detected by functional brain imaging. Substance P (SP), one of the most widely expressed peptides in the CNS, also produces vasomotor responses by inducing calcium release from intracellular stores through its preferred neurokinin 1 (NK1) receptor, thus inducing NO production via activation of neuronal NO synthase (nNOS). Single- and double-labeling experiments were performed to establish whether NK1-immunopositive neurons (NK1IP -n) are found in the rat cc and the extent of NK1 colocalization with nNOS. NK1IP -n were seen to constitute a large neuronal population in the cc and had a distribution similar to that of nNOSIP neurons (nNOSIP -n). NK1IP -n were numerous in the lateral cc and gradually decreased in the more medial portions, where they were few or absent. Intracallosal NK1IP -n and their dendritic trees were intensely labeled, allowing classification into four morphological types: bipolar, round, polygonal, and pyramidal. Confocal microscopic examination demonstrated that nearly all NK1IP -n contained nNOS (96.43%) and that 84.59% of nNOSIP -n co-expressed NK1. These data suggest that the majority of intracallosal neurons can release NO as a result of the action of SP. A small proportion of nNOSIP -n does not contain NK1 and is not activated by SP; these neurons may release NO via alternative mechanisms. The possible mechanisms by which intracallosal neurons release NO are also reviewed.

Cellular and molecular changes to cortical neurons following low intensity repetitive magnetic stimulation at different frequencies.

Repetitive transcranial magnetic stimulation is increasingly used as a treatment for neurological dysfunction. Therapeutic effects have been reported for low intensity rTMS (LI-rTMS) although these remain poorly understood. Our study describes for the first time a systematic comparison of the cellular and molecular changes in neurons invitro induced by low intensity magnetic stimulation at different frequencies. We applied 5 different low intensity repetitive magnetic stimulation (LI-rMS) protocols to neuron-enriched primary cortical cultures for 4 days and assessed survival, and morphological and biochemical change. We show pattern-specific effects of LI-rMS: simple frequency pulse trains (10Hz and 100Hz) impaired cell survival, while more complex stimulation patterns (theta-burst and a biomimetic frequency) did not. Moreover, only 1 Hz stimulation modified neuronal morphology, inhibiting neurite outgrowth. To understand mechanisms underlying these differential effects, we measured intracellular calcium concentration during LI-rMS and subsequent changes in gene expression. All LI-rMS frequencies increased intracellular calcium, but rather than influx from the extracellular milieu typical of depolarization, all frequencies induced calcium release from neuronal intracellular stores. Furthermore, we observed pattern-specific changes in expression of genes related to apoptosis and neurite outgrowth, consistent with our morphological data on cell survival and neurite branching. Thus, in addition to the known effects on cortical excitability and synaptic plasticity, our data demonstrate that LI-rMS can change the survival and structural complexity of neurons. These findings provide a cellular and molecular framework for understanding what low intensity magnetic stimulation may contribute to human rTMS outcomes.

Abstract 3237: Characterization of CM-572 and CM-769: Novel irreversible modulators of sigma-2 receptor function

Abstract Sigma-2 receptors are highly upregulated in a wide variety of cancer cells compared to normal cells, and increase by at least ten-fold in rapidly proliferating cancer cells compared to quiescent. Activation of the sigma-2 receptor induces apoptosis. Thus, there has been increasing interest in sigma-2 receptors as targets for cancer therapeutic and diagnostic agents and a need for small molecule tools to study function. We previously reported analogs of the sigma-2 antagonist, SN-79, that incorporate an isothiocyanate (ITC) group in order to impart irreversible binding to the receptor (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 54: #2242, 2013). Here we further characterize CM-572 (sigma-2 Ki = 320nM; ITC in heterocyclic ring) and CM-769 (sigma-2 Ki = 271nM; ITC in fluorophenyl ring) for their effects in SK-N-SH neuroblastoma, MCF-7 breast, and PANC-1 pancreatic cancer cell lines. Previous data showed that CM-572 potently and selectively acylated sigma-2 receptors over sigma-1, while CM-769 was less potent at irreversible binding, presumably due to non-optimal placement of the ITC group. CM-572 induced dose-dependent cell death in SK-N-SH neuroblastoma cells with ED50 about 5uM. CM-572 was also cytotoxic in MCF-7 breast and PANC-1 pancreatic cancer cells, however with reduced potency and efficacy (40% and 50% maximal cell death, respectively, at 30uM). The cytotoxic effect of CM-572 in SK-N-SH cells was retained after a 60min pretreatment of cells, washout of compound, and continued incubation for 24h, indicating irreversible agonist activity. Interestingly, CM-769 produced an apparent dose-dependent proliferative effect on SK-N-SH neuroblastoma cells. This effect was also retained after 60min pretreatment and washout, again indicating an irreversible affect. A subtoxic dose of CM-572 (300nM) was able to partially attenuate CM-769-induced cell proliferation, indicating that these two compounds act at the same receptor site. Cytosolic BID cleavage is a hallmark of sigma-2 receptor induced apoptosis in SK-N-SH cells. CM-572 (30uM) induced rapid BID cleavage beginning at 30min, with near completion at 6h. By contrast, CM-769 (30uM) did not induce BID cleavage, but rather appeared to increase cytosolic levels of full-length BID relative to control, consistent with the observed proliferative or protective effect. Together, the data indicate that CM-572 is an irreversible partial agonist at sigma-2 receptors. This is consistent with previously demonstrated effects of CM-572 on CB-64D (an agonist)-induced calcium release and cytotoxicity, where low-dose CM-572 attenuated CB-64D effects, and high-dose CM-572 mimicked CB-64D. CM-769 is an irreversible inverse agonist at the sigma-2 receptor, a novel effect not shown by any sigma-2 ligand to date. These two compounds represent novel tools for further study of sigma-2 receptor function. Citation Format: Hilary E. Nicholson, Pei Ling Chia, Anthony Comeau, Christophe Mesangeau, Christopher McCurdy, Wayne D. Bowen. Characterization of CM-572 and CM-769: Novel irreversible modulators of sigma-2 receptor function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3237. doi:10.1158/1538-7445.AM2014-3237

Histamine H1 receptor cell membrane chromatography online high‐performance liquid chromatography with mass spectrometry method reveals houttuyfonate as an activator of the histamine H1 receptor

Allergy is an abnormal reaction of the body to an allergen. Histamine is responsible for many of the acute symptoms of allergic diseases. Many of the allergic and inflammatory actions of histamine are mediated by the histamine H1 receptor. In the present study, we established a two-dimensional histamine H1 receptor/cell membrane chromatography with online high-performance liquid chromatography and mass spectrometry method for screening potential histamine-activating components in a traditional Chinese medicine injection. The specification of the method was validated by screening, separating, and identifying a mixed standard solution of diphenhydramine hydrochloride, gefitinib, tamsulosin, and nitrendipine. The Yujin injection, an example of traditional Chinese medicine injection, was screened and potential allergic components acting on the histamine H1 receptor were identified. A Ca(2+) flux assay showed that houttuyfonate and Yujin injection induced calcium release in a dose-dependent manner. This suggests that houttuyfonate is an activator of the histamine H1 receptor. The mechanism of houttuyfonate activation involves phosphorylation of the inositol-1,4,5-trisphosphate receptor. In conclusion, this two-dimensional method can rapidly detect and enrich target components isolated from the Yujin injection. This indicates that individuals with an overexpression of the histamine H1 receptor should be aware of possible allergic reactions when receiving the Yujin injection.