Internal Stores Research Articles

Generation and Regulation of Spontaneous Contractions in theProstate.

Spontaneous myogenic contractions have been shown to be significantly upregulated in prostate tissue collected from men with Benign Prostatic Hyperplasia (BPH), an extremely common disorder of the ageing male. Although originally thought likely to be involved in 'housekeeping' functions, mixing prostatic secretions to prevent stagnation, these spontaneous myogenic contractions provide a novel opportunity to understand and treat BPH. This treatment potential differs from previous models, which focused exclusively on attenuating nerve-mediated neurogenic contractions. Previous studies in the rodent prostate have provided an insight into the mechanisms underlying the regulation of myogenic contractions. 'Prostatic Interstitial Cells' (PICs) within the prostate appear to generate pacemaker potentials, which arise from the summation of number of spontaneous transient depolarisations triggered by the spontaneous release of Ca2+ from internal stores and the opening of Ca2+-activated Cl- channels. Pacemaker potentials then conduct into neighbouring smooth muscle cells to generate spontaneous slow waves. These slow waves trigger the firing of 'spike-like' action potentials, Ca2+ entry and contraction, which are not attenuated by blockers of neurotransmission. However, these spontaneous prostatic contractions can be modulated by the autonomic nervous system. Here, we discuss the mechanisms underlying rodent and human prostate myogenic contractions and the actions of existing and novel pharmacotherapies for the treatment of BPH. Understanding the generation of human prostatic smooth muscle tone will confirm the mechanism of action of existing drugs, inform the identification and effectiveness of new pharmacotherapies, as well as predict patient outcomes.

Antispasmodic Effects of <i>Salvia officinalis</i> in Isolated Ileum of Rabbit

Salvia officinalis has been commonly used in Syria and other Middle East countries in popular medicine for many medical purposes. One of the strong claims about its properties is the spasmolytic effect. The aim of this research was to investigate the spasmolytic effect of ethanolic extract of Salvia officinalis in isolated rabbit ileum preparations. Male rabbits were sacrificed and portions of ileum were isolated and mounted in Tyrode’s solution. Afterwards, the experiments were performed according to different groups with different spasmogenic agents: the first group was only with spontaneous contraction, the second was KCl-induced contractions and the third was Carbachol-induced contractions. The examined extract was added cumulatively in all previous groups. Verapamil was used as a reference. The results showed profound relaxing effect of S. officinalis extract in mentioned groups, especially in the group of KCl-induced contraction. These findings suggest that the main mechanism of the spasmolytic action is related to the inhibition of calcium entry through cell membrane as well as via internal stores.

Disentangling the net: concomitant xylem and over-bark size measurements reveal the phloem-generated turgor signal behind daytime stem swelling in the mangrove Avicennia marina.

Daytime stem shrinking is a well recorded phenomenon: trees dip into their internal water stores to meet imbalances between water supply and demand. Uncertainty surrounds the mechanisms behind the unusual pattern of daytime stem swelling, presented by species like the mangrove Avicennia marina (Forssk.) Vierh., and which is thought to originate in the osmotic adjustment of storage tissues. We performed on-xylem radius change (XRC) and whole-stem radius change (SRC) measurements with point dendrometers to ascertain if the swelling of the stem is due to an increase in the thickness of the phloem and inner-bark (BRC). We measured leaf water potential (ψleaf), sap flow, leaf turgor and microclimate to understand the coupling between BRC and transpiration-driven changes in XRC. Our results present direct evidence of the type of mechanism responsible for daytime stem swelling. Inner-bark thickness increase concurrent with XRC decrease, sap-flow increase and leaf turgor loss, reveals a phloem-generated turgor signal behind daytime stem swelling of tree stems. On-xylem measurements were highly heterogeneous due to the variability in the three dimensional fish-net wood structure of the stem of A. marina. As daytime decreases in XRC were compensated by BRC and correlated with conditions of high water demand, we suggest a potential role of daytime stem swelling in the hydraulic safety of A. marina.

Astrocytic CB1R enhances GLAST activity in a Ca2+ dependent manner

Event Abstract Back to Event Astrocytic CB1R enhances GLAST activity in a Ca2+ dependent manner Joana Gonçalves-Ribeiro1, 2, Tatiana P. Morais3, Oksana Savchak1, 2, Carlos Meneses4, Ana M. Sebastião1, 2 and Sandra H. Vaz1, 2* 1 Laboratório de Farmacologia Clínica e Terapêutica, Faculdade de Medicina da Universidade de Lisboa, Portugal 2 Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal 3 School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, United Kingdom 4 Instituto Superior de Engenharia de Lisboa, Portugal Astrocytes perform many functions at the synapse level, including glutamate uptake, which is mainly mediated by specific glutamate transporters, GLT-1 and GLAST transporter (Perego et al., 2000) that are responsible for preventing excitotoxicity and for modulating synaptic function. Astrocytes can communicate with surrounding astrocytes and neurons by changes in the intracellular Ca2+ (Perea and Araque, 2005), being this achieved through the expression of several types of receptors in their membranes such as the cannabinoid type one receptor (CB1R) whose activation leads to Ca2+ mobilization from internal cellular stores (Navarrete and Araque, 2008). Thus, the aim of this work was to study the role of astrocytic CB1R activation-mediated Ca2+ signaling upon GLAST in rat primary astrocytic cultures. Primary cortical astrocytic cultures were prepared from Sprague Dawley pups (0-2 days old). Variations in [Ca2+]i were detected by Ca2+ imaging technique using fura 2-acetoxymethyl ester (Fura2AM, 5 M). The transport was initiated by addition of [3H]glutamate and the specific transport mediated by GLAST was accessed in presence of UCPH-101. Ca2+ signalling and [3H]-glutamate uptake experiments were performed with 18-23 DIC astrocytes. It was observed that astrocytic CB1R activation with ACEA, a CB1R specific agonist, led to Ca2+ transients, whose amplitude and frequency was dose-dependent for the two tested ACEA concentrations (0.5 and 1.0 µM, n = 3-7 independent cultures). The occurrence of these transients were impaired in the presence of a PLC antagonist (U73122, 3 µM) and a intracellular Ca2+ stores depletory (CPA, 10 µM). Low extracellular Ca2+ concentration decreased both frequency and amplitude of the transients. Regarding glutamate uptake, astrocytic CB1R activation increased significantly the maximum velocity of transport (Vmax) of GLAST transporter without significant changes in affinity (Km). This enhancement was lost in the presence of U73122 and a Ca2+ chelant (BAPTA, 20 µM), but not with a PKC antagonist (GF 10109203X, 1 µM). In conclusion, in astrocytes, CB1R activation triggers Ca2+ transients by PLC activation, in a dose-dependent manner enhancing GLAST activity probably by increasing the number of functional transporters at the astrocytic membrane. Acknowledgements This project was funded by Fundação para a Ciência e a Tecnologia (PTDC/BTM-SAL/32147/2017). Keywords: Astrocytes, CB1R, Glutamate Uptake, Calcium, Cannabinoids Conference: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019. Presentation Type: Poster presentation Topic: Glia / Neuroinflammation Citation: Gonçalves-Ribeiro J, Morais TP, Savchak O, Meneses C, Sebastião AM and Vaz SH (2019). Astrocytic CB1R enhances GLAST activity in a Ca2+ dependent manner. Front. Cell. Neurosci. Conference Abstract: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019). doi: 10.3389/conf.fncel.2019.01.00039 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Dr. Sandra H Vaz, Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Portugal, svaz@medicina.ulisboa.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joana Gonçalves-Ribeiro Tatiana P Morais Oksana Savchak Carlos Meneses Ana M Sebastião Sandra H Vaz Google Joana Gonçalves-Ribeiro Tatiana P Morais Oksana Savchak Carlos Meneses Ana M Sebastião Sandra H Vaz Google Scholar Joana Gonçalves-Ribeiro Tatiana P Morais Oksana Savchak Carlos Meneses Ana M Sebastião Sandra H Vaz PubMed Joana Gonçalves-Ribeiro Tatiana P Morais Oksana Savchak Carlos Meneses Ana M Sebastião Sandra H Vaz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Latent Sex Differences in Molecular Signaling That Underlies Excitatory Synaptic Potentiation in the Hippocampus.

Excitatory synapses can be potentiated by chemical neuromodulators, including 17β-estradiol (E2), or patterns of synaptic activation, as in long-term potentiation (LTP). Here, we investigated kinases and calcium sources required for acute E2-induced synaptic potentiation in the hippocampus of each sex and tested whether sex differences in kinase signaling extend to LTP. We recorded EPSCs from CA1 pyramidal cells in hippocampal slices from adult rats and used specific inhibitors of kinases and calcium sources. This revealed that, although E2 potentiates synapses to the same degree in each sex, cAMP-activated protein kinase (PKA) is required to initiate potentiation only in females. In contrast, mitogen-activated protein kinase, Src tyrosine kinase, and rho-associated kinase are required for initiation in both sexes; similarly, Ca2+/calmodulin-activated kinase II is required for expression/maintenance of E2-induced potentiation in both sexes. Calcium source experiments showed that L-type calcium channels and calcium release from internal stores are both required for E2-induced potentiation in females, whereas in males, either L-type calcium channel activation or calcium release from stores is sufficient to permit potentiation. To investigate the generalizability of a sex difference in the requirement for PKA in synaptic potentiation, we tested how PKA inhibition affects LTP. This showed that, although the magnitude of both high-frequency stimulation-induced and pairing-induced LTP is the same between sexes, PKA is required for LTP in females but not males. These results demonstrate latent sex differences in mechanisms of synaptic potentiation in which distinct molecular signaling converges to common functional endpoints in males and females.SIGNIFICANCE STATEMENT Chemical- and activity-dependent neuromodulation alters synaptic strength in both male and female brains, yet few studies have compared mechanisms of neuromodulation between the sexes. Here, we studied molecular signaling that underlies estrogen-induced and activity-dependent potentiation of excitatory synapses in the hippocampus. We found that, despite similar magnitude increases in synaptic strength in males and females, the roles of cAMP-regulated protein kinase, internal calcium stores, and L-type calcium channels differ between the sexes. Therefore, latent sex differences in which the same outcome is achieved through distinct underlying mechanisms in males and females include kinase and calcium signaling involved in synaptic potentiation, demonstrating that sex is an important factor in identification of molecular targets for therapeutic development based on mechanisms of neuromodulation.

Neomycin: An Effective Inhibitor of Jasmonate-Induced Reactions in Plants

Jasmonates are important phytohormones involved in both plant developmental processes as well as defense reactions. Many JA-mediated plant defense responses have been studied in model plants using mutants of the jasmonate signaling pathway. However, in plant species where JA-signaling mutants are not accessible, the availability of a tool targeting JA signaling is crucial to investigate jasmonate-dependent processes. Neomycin is a poly-cationic aminoglycoside antibiotic that blocks the release of Ca2+ from internal stores. We examined the inhibitory activities of neomycin on different jasmonate-inducible responses in eight different plant species: Intracellular calcium measurements in Nicotiana tabacum cell culture, Sporamin gene induction in Ipomoea batatas, PDF2.2 gene expression in Triticum aestivum, Nepenthesin protease activity measurement in Nepenthes alata, extrafloral nectar production in Phaseolus lunatus, nectary formation in Populus trichocarpa, terpene accumulation in Picea abies, and secondary metabolite generation in Nicotiana attenuata. We are able to show that neomycin, an easily manageable and commercially available compound, inhibits JA-mediated responses across the plant kingdom.

Calcium Pathways in Human Neutrophils-The Extended Effects of Thapsigargin and ML-9.

In neutrophils, intracellular Ca2+ levels are regulated by several transporters and pathways, namely SERCA [sarco(endo)plasmic reticulum Ca2+-ATPase], SOCE (store-operated calcium entry), and ROCE (receptor-operated calcium entry). However, the exact mechanisms involved in the communication among these transporters are still unclear. In the present study, thapsigargin, an irreversible inhibitor of SERCA, and ML-9, a broadly used SOCE inhibitor, were applied in human neutrophils to better understand their effects on Ca2+ pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca2+ flux were evaluated in freshly isolated human neutrophils, using a microplate reader for monitoring fluorimetric kinetic readings. The obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca2+ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca2+ levels due to its influx by SOCE. Moreover, it was obvious that ML-9 enhanced the thapsigargin-induced emptying of the internal stores. Indeed, ML-9 does not have this effect by itself, which indicates that, in neutrophils, thapsigargin does not act only on the influx by SOCE, but also by other Ca2+ pathways, that, in the future, should be further explored.

Spontaneous oscillation of the ciliary beat frequency regulated by release of Ca2+ from intracellular stores in mouse nasal epithelia

Ciliary beating frequency (CBF) was investigated in ciliated nasal epithelial cells (cMNECs) isolated from mice using video microscopy equipped with a high-speed camera. In cMNECs, a spontaneous CBF oscillation was observed. The CBF oscillation was abolished by BAPTA-AM but not by Ca2+-free solution. The addition of thapsigargin, which depletes Ca2+ from internal stores, also abolished CBF oscillation. Moreover, the intracellular Ca2+ concentration [Ca2+]i, spontaneously oscillated even with the Ca2+-free solution. Moreover, 2APB (an inhibitor of the IP3 receptor) abolished CBF oscillation in cMNECs. Overall, these findings suggest that the CBF oscillation in cMNECs is triggered by the release of Ca2+ from the IP3-sensitive internal stores. Moreover, IBMX, an inhibitor of phosphodiesterase, did not affect CBF oscillation in cMNECs, although it slightly increased CBF. These results suggest that CBF oscillations were induced by [Ca2+]i oscillation controlled via the release of Ca2+ from IP3-sensitive stores, rather than via cAMP accumulation. CBF oscillation possibly plays a crucial role in maintaining an efficient mucociliary clearance in the nasal epithelia.

Ade2 Functions in the Drosophila Fat Body To Promote Sleep.

Metabolic state is a potent modulator of sleep and circadian behavior, and animals acutely modulate their sleep in accordance with internal energy stores and food availability. Across phyla, hormones secreted from adipose tissue act in the brain to control neural physiology and behavior to modulate sleep and metabolic state. Growing evidence suggests the fat body is a critical regulator of complex behaviors, but little is known about the genes that function within the fat body to regulate sleep. To identify molecular factors functioning in non-neuronal tissues to regulate sleep, we performed an RNAi screen selectively knocking down genes in the fat body. We found that knockdown of Phosphoribosylformylglycinamidine synthase/Pfas (Ade2), a highly conserved gene involved the biosynthesis of purines, sleep regulation and energy stores. Flies heterozygous for multiple Ade2 mutations are also short sleepers and this effect is partially rescued by restoring Ade2 to the Drosophila fat body. Targeted knockdown of Ade2 in the fat body does not alter arousal threshold or the homeostatic response to sleep deprivation, suggesting a specific role in modulating baseline sleep duration. Together, these findings suggest Ade2 functions within the fat body to promote both sleep and energy storage, providing a functional link between these processes.

Deteriorated gene expression of selected calcium transporters in streptozotocin-induced diabetic hearts of Wistar rats

Abstract Aim: The aim is to identify the possible changes in the expression of genes, that regulate calcium homeostasis in cardiomyocytes in diabetes mellitus. Methods: Male Wistar rats were randomized into two experimental protocols: short-term 5-days streptozotocin-induced diabetes protocol with 20 weeks old animals at the end of the protocol (total N = 20) and long-term 4-weeks protocol with 18 weeks of age at the end of the protocol (total N = 38). 50 mg/kg of streptozotocin (STZ) was administered in both protocols by a single intraperitoneal injection in 0,1M citrate buffer (pH = 4.5). Control group (CON) received only vehiculum. Gene expressions in samples of left heart ventricle were measured by RT-qPCR method. Results: The expression of SERCA2a in short-term protocol was decreased. In long-term protocol, decreased SERCA2a, TRPC4 and TRPC6 mRNA levels were observed (*p < 0.05). SERCA2a and TRPC4 mRNA levels exhibited statistical monotonic correlation in STZ-treated group in long-term protocol. Conclusions: In diabetes mellitus, the calcium homeostasis in cardiomyocytes is altered and there could be a relation between alteration of internal sarcoplasmatic stores and store-operated calcium entry.

Astrocytes integrate and drive action potential firing in inhibitory subnetworks

Many brain functions depend on the ability of neural networks to temporally integrate transient inputs to produce sustained discharges. This can occur through cell-autonomous mechanisms in individual neurons and through reverberating activity in recurrently connected neural networks. We report a third mechanism involving temporal integration of neural activity by a network of astrocytes. Previously, we showed that some types of interneurons can generate long-lasting trains of action potentials (barrage firing) following repeated depolarizing stimuli. Here we show that calcium signaling in an astrocytic network correlates with barrage firing; that active depolarization of astrocyte networks by chemical or optogenetic stimulation enhances; and that chelating internal calcium, inhibiting release from internal stores, or inhibiting GABA transporters or metabotropic glutamate receptors inhibits barrage firing. Thus, networks of astrocytes influence the spatiotemporal dynamics of neural networks by directly integrating neural activity and driving barrages of action potentials in some populations of inhibitory interneurons.

Calcium stores regulate excitability in cultured rat hippocampal neurons.

Extracellular calcium ions support synaptic activity but also reduce excitability of central neurons. In the present study, the effect of calcium on excitability was explored in cultured hippocampal neurons. CaCl2 injected by pressure in the vicinity of a neuron that is bathed only in MgCl2 as the main divalent cation caused a depolarizing shift in action potential threshold and a reduction in excitability. This effect was not seen if the intracellular milieu consisted of Cs+ instead of K-gluconate as the main cation or when it contained ruthenium red, which blocks release of calcium from stores. The suppression of excitability by calcium was mimicked by caffeine, and calcium store antagonists cyclopiazonic acid or thapsigargin blocked this action. Neurons taken from synaptopodin-knockout mice show significantly reduced efficacy of calcium modulation of action potential threshold. Likewise, in Orai1 knockdown cells, calcium is less effective in modulating excitability of neurons. Activation of small-conductance K (SK) channels increased action potential threshold akin to that produced by calcium ions, whereas blockade of SK channels but not big K channels reduced the threshold for action potential discharge. These results indicate that calcium released from stores may suppress excitability of central neurons. NEW & NOTEWORTHY Extracellular calcium reduces excitability of cultured hippocampal neurons. This effect is mediated by calcium-gated potassium currents, possibly small-conductance K channels. Release of calcium from internal stores mimics the effect of extracellular calcium. It is proposed that calcium stores modulate excitability of central neurons.

Characterization of potential TRPP2 regulating proteins in early Xenopus embryos.

Transient receptor potential cation channel-2 (TRPP2) is a nonspecific Ca2+ -dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+ ]i) from internal stores of endoplasmic reticulum, and calcium-dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left-right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+ ]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase-D1, and disheveled-2 has been confirmed by immunoblotting analysis of TRPP2-coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase-D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells.

Flagellar hyperactivation of bull and boar spermatozoa.

BackgroundIn mammals, flagellar hyperactivation is indispensable to sperm fertilization with oocytes in vivo, although there are species differences in regulatory mechanisms for this event. In this study, I reviewed researches regarding hyperactivation of bull and boar spermatozoa, in comparison with those of spermatozoa from other species.MethodsRecent publications regarding sperm hyperactivation were collected and summarized.Results (Main findings)In bull and boar spermatozoa, there are two types of hyperactivation “full‐type hyperactivation and nonfull‐type hyperactivation” which are equivalent to anti‐hock hyperactivation and pro‐hock hyperactivation of mouse spermatozoa, respectively, on the basis of the flagellar parts exhibiting asymmetrical beating. Full‐type hyperactivation is initiated in response to a rapid increase of cytoplasmic Ca2+ in the connecting/middle and principal pieces by the mobilization of this divalent ion from extracellular space and internal store through cation channels. Regulatory molecules for the increase of cytoplasmic Ca2+ in the connecting/middle pieces are probably different from those in the principal pieces.ConclusionI have proposed a hypothesis on the regulation of full‐type hyperactivation by the distinct signaling cascades leading to the increase in cytoplasmic Ca2+ between the connecting/middle and principal pieces of bull and boar spermatozoa.

Inhibition of endocytosis suppresses the nitric oxide-dependent release of Cl- in retinal amacrine cells.

Our lab has previously shown that nitric oxide (NO) can alter the synaptic response properties of amacrine cells by releasing Cl- from internal acidic compartments. This alteration in the Cl- gradient brings about a positive shift in the reversal potential of the GABA-gated current, which can convert inhibitory synapses into excitatory synapses. Recently, we have shown that the cystic fibrosis transmembrane regulator (CFTR) Cl- channel is involved in the Cl- release. Here, we test the hypothesis that (acidic) synaptic vesicles are a source of NO-releasable Cl- in chick retinal amacrine cells. If SVs are a source of Cl-, then depleting synaptic vesicles should decrease the nitric oxide-dependent shift in the reversal potential of the GABA-gated current. The efficacy of four inhibitors of dynamin (dynasore, Dyngo 4a, Dynole 34–2, and MiTMAB) were evaluated. In order to deplete synaptic vesicles, voltage-steps were used to activate V-gated Ca2+ channels and stimulate the synaptic vesicle cycle either under control conditions or after treatment with the dynamin inhibitors. Voltage-ramps were used to measure the NO-dependent shift in the reversal potential of the GABA-gated currents under both conditions. Our results reveal that activating the synaptic vesicle cycle in the presence of dynasore or Dyngo 4a blocked the NO-dependent shift in EGABA. However, we also discovered that some dynamin inhibitors reduced Ca2+ signaling and L-type Ca2+ currents. Conversely, dynasore also increased neurotransmitter release at autaptic sites. To further resolve the mechanism underlying the inhibition of the NO-dependent shift in the reversal potential for the GABA-gated currents, we also tested the effects of the clathrin assembly inhibitor Pitstop 2 and found that this compound also inhibited the shift. These data provide evidence that dynamin inhibitors have multiple effects on amacrine cell synaptic transmission. These data also suggest that inhibition of endocytosis disrupts the ability of NO to elicit Cl- release from internal stores which may in part be due to depletion of synaptic vesicles.

PLC and IP3-evoked Ca2+ release initiate the fast block to polyspermy in Xenopus laevis eggs.

The prevention of polyspermy is essential for the successful progression of normal embryonic development in most sexually reproducing species. In external fertilizers, the process of fertilization induces a depolarization of the egg's membrane within seconds, which inhibits supernumerary sperm from entering an already-fertilized egg. This fast block requires an increase of intracellular Ca2+ in the African clawed frog, Xenopus laevis, which in turn activates an efflux of Cl- that depolarizes the cell. Here we seek to identify the source of this intracellular Ca2+ Using electrophysiology, pharmacology, bioinformatics, and developmental biology, we explore the requirement for both Ca2+ entry into the egg from the extracellular milieu and Ca2+ release from an internal store, to mediate fertilization-induced depolarization. We report that although eggs express Ca2+-permeant ion channels, blockade of these channels does not alter the fast block. In contrast, insemination of eggs in the presence of Xestospongin C-a potent inhibitor of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from the endoplasmic reticulum (ER)-completely inhibits fertilization-evoked depolarization and increases the incidence of polyspermy. Inhibition of the IP3-generating enzyme phospholipase C (PLC) with U73122 similarly prevents fertilization-induced depolarization and increases polyspermy. Together, these results demonstrate that fast polyspermy block after fertilization in X. laevis eggs is mediated by activation of PLC, which increases IP3 and evokes Ca2+ release from the ER. This ER-derived Ca2+ then activates a Cl- channel to induce the fast polyspermy block. The PLC-induced cascade of events represents one of the earliest known signaling pathways initiated by fertilization.

What Is Required for Neuronal Calcium Waves? A Numerical Parameter Study

Neuronal calcium signals propagating by simple diffusion and reaction with mobile and stationary buffers are limited to cellular microdomains. The distance intracellular calcium signals can travel may be significantly increased by means of calcium-induced calcium release from internal calcium stores, notably the endoplasmic reticulum. The organelle, which can be thought of as a cell-within-a-cell, is able to sequester large amounts of cytosolic calcium ions via SERCA pumps and selectively release them into the cytosol through ryanodine receptor channels leading to the formation of calcium waves. In this study, we set out to investigate the basic properties of such dendritic calcium waves and how they depend on the three parameters dendrite radius, ER radius and ryanodine receptor density in the endoplasmic membrane. We demonstrate that there are stable and abortive regimes for calcium waves, depending on the above morphological and physiological parameters. In stable regimes, calcium waves can travel across long dendritic distances, similar to electrical action potentials. We further observe that abortive regimes exist, which could be relevant for spike-timing dependent plasticity, as travel distances and wave velocities vary with changing intracellular architecture. For some of these regimes, analytic functions could be derived that fit the simulation data. In parameter spaces, that are non-trivially influenced by the three-dimensional calcium concentration profile, we were not able to derive such a functional description, demonstrating the mathematical requirement to model and simulate biochemical signaling in three-dimensional space.

CB1 receptor activation induces intracellular Ca2+ mobilization and 2-arachidonoylglycerol release in rodent spinal cord astrocytes

Accumulating evidence supports the role of astrocytes in endocannabinoid mediated modulation of neural activity. It has been reported that some astrocytes express the cannabinoid type 1 receptor (CB1-R), the activation of which is leading to Ca2+ mobilization from internal stores and a consecutive release of glutamate. It has also been documented that astrocytes have the potential to produce the endocannabinoid 2-arachidonoylglycerol, one of the best known CB1-R agonist. However, no relationship between CB1-R activation and 2-arachidonoylglycerol production has ever been demonstrated. Here we show that rat spinal astrocytes co-express CB1-Rs and the 2-arachidonoylglycerol synthesizing enzyme, diacylglycerol lipase-alpha in close vicinity to each other. We also demonstrate that activation of CB1-Rs induces a substantial elevation of intracellular Ca2+ concentration in astrocytes. Finally, we provide evidence that the evoked Ca2+ transients lead to the production of 2-arachidonoylglycerol in cultured astrocytes. The results provide evidence for a novel cannabinoid induced endocannabinoid release mechanism in astrocytes which broadens the bidirectional signaling repertoire between astrocytes and neurons.

Voltage-gated Ca2+ channel activity modulates smooth muscle cell calcium waves in hamster cremaster arterioles.

Cremaster muscle arteriolar smooth muscle cells (SMCs) display inositol 1,4,5-trisphosphate receptor-dependent Ca2+ waves that contribute to global myoplasmic Ca2+ concentration and myogenic tone. However, the contribution made by voltage-gated Ca2+ channels (VGCCs) to arteriolar SMC Ca2+ waves is unknown. We tested the hypothesis that VGCC activity modulates SMC Ca2+ waves in pressurized (80 cmH2O/59 mmHg, 34°C) hamster cremaster muscle arterioles loaded with Fluo-4 and imaged by confocal microscopy. Removal of extracellular Ca2+ dilated arterioles (32 ± 3 to 45 ± 3 μm, n = 15, P < 0.05) and inhibited the occurrence, amplitude, and frequency of Ca2+ waves ( n = 15, P < 0.05), indicating dependence of Ca2+ waves on Ca2+ influx. Blockade of VGCCs with nifedipine (1 μM) or diltiazem (10 μM) or deactivation of VGCCs by hyperpolarization of smooth muscle with the K+ channel agonist cromakalim (10 μM) produced similar inhibition of Ca2+ waves ( P < 0.05). Conversely, depolarization of SMCs with the K+ channel blocker tetraethylammonium (1 mM) constricted arterioles from 26 ± 3 to 14 ± 2 μm ( n = 11, P < 0.05) and increased wave occurrence (9 ± 3 to 16 ± 3 waves/SMC), amplitude (1.6 ± 0.07 to 1.9 ± 0.1), and frequency (0.5 ± 0.1 to 0.9 ± 0.2 Hz, n = 10, P < 0.05), effects that were blocked by nifedipine (1 μM, P < 0.05). Similarly, the VGCC agonist Bay K8644 (5 nM) constricted arterioles from 14 ± 1 to 8 ± 1 μm and increased wave occurrence (3 ± 1 to 10 ± 1 waves/SMC) and frequency (0.2 ± 0.1 to 0.6 ± 0.1 Hz, n = 6, P < 0.05), effects that were unaltered by ryanodine (50 μM, n = 6, P > 0.05). These data support the hypothesis that Ca2+ waves in arteriolar SMCs depend, in part, on the activity of VGCCs. NEW & NOTEWORTHY Arterioles that control blood flow to and within skeletal muscle depend on Ca2+ influx through voltage-gated Ca2+ channels and release of Ca2+ from internal stores through inositol 1,4,5-trisphosphate receptors in the form of Ca2+ waves to maintain pressure-induced smooth muscle tone.

Mechanistic Role of IP3R Calcium Release Channel in Pancreatic Beta-Cell Function

Background: According to the classic paradigm, insulin secretion is triggered by the influx of extracellular Ca2+ via voltage-dependent channels, leading to the fusion of insulin granules. Instead, the mechanisms involved in Ca2+ mobilization from internal stores are less defined. The main intracellular Ca2+ release channels are inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), whereas Ca2+ is returned to the ER primarily by the activity of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump. We recently demonstrated the importance of RyR in type 2 diabetes mellitus (T2DM), showing that it is essential in glucose-stimulated insulin secretion (GSIS). Conversely, the exact role of IP3R in GSIS remains not fully understood. Methods and Results: Three isoforms of IP3R have been identified in mammalian cells. Channel opening is stimulated by the binding of second messenger IP3 and by changes in Ca2+ concentrations. Studies in rodent and human samples indicate that β cells express all IP3R isoforms. We demonstrated that the expression of all isoforms is significantly increased in human islets from T2DM cadaveric donors T2DM compared with nondiabetic individuals. These results were also confirmed in diabetic db/db mice and in mice fed high-fat diet. Moreover, pancreatic β cells from T2DM patients exhibited dysmorphic and dysfunctional mitochondria, with markedly altered Ca2+ uptake. Similar features were found in clonal β cells chronically exposed to high glucose. In vitro, overexpression of IP3Rs was associated with impaired GSIS, whereas IP3R silencing improved β cell function, mitochondrial Ca2+ uptake and function, ER stress, and insulin release in response to different secretagogues. Conclusions: Taken together, our data indicate that IP3Rs are upregulated in human islets from T2DM donors, leading to mitochondrial dysfunction and pancreatic β cell failure, identifying in these intracellular Ca2+ release channels a novel therapeutic target to treat T2DM. Disclosure J. Shu: None. J. Gambardella: None. D. Sorriento: None. G. Santulli: None.