The role of calcium stores in long-term potentiation and synaptic tagging and capture in mouse hippocampus.

The roles of Ca2+-induced calcium release in synaptic plasticity and metaplasticity are poorly understood. The present study has addressed the role of intracellular Ca2+ stores in long-term potentiation (LTP) and a form of heterosynaptic metaplasticity known as synaptic tagging and capture (STC) at CA1 synapses in mouse hippocampal slices. The effects of two compounds, ryanodine and cyclopiazonic acid (CPA), were examined on LTP induced by three distinct induction protocols: weak (w), compressed (c) and spaced (s) theta-burst stimulation (TBS). These compounds did not significantly affect LTP induced by the wTBS (one episode of TBS; 25 stimuli) or cTBS (three such episodes with a 10 s inter-episode interval (IEI); 75 stimuli) but substantially inhibited LTP induced by a sTBS (10 min IEI; 75 stimuli). Ryanodine and CPA also prevented a small heterosynaptic potentiation that was observed with the sTBS protocol. Interestingly, these compounds also prevented STC when present during either the sTBS or the subsequent wTBS, applied to an independent input. All of these effects of ryanodine and CPA were similar to that of a calcium-permeable AMPA receptor blocker. In conclusion, Ca2+ stores provide one way in which signals are propagated between synaptic inputs and, by virtue of their role in STC, may be involved in associative long-term memories. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Comparison of the structure-function of five newly members of the calcin family.

Calcins are a group of scorpion toxin peptides specifically binding to ryanodine receptors (RyRs) with high affinity, and have the ability to activate and stabilize RyR in a long-lasting subconductance state. Five newly calcins synthesized compounds exhibit typical structural characteristics of a specific family through chemical synthesis and virtual analysis. As the calcins from the same species, Petersiicalcin1 and Petersiicalcin2, Jendekicalcin2 and Jendekicalcin3, have only one residue difference. Both Petersiicalcin1 and Petersiicalcin2 exhibited different affinities in stimulating [3H]ryanodine binding, but the residue mutation resulted in a 2.7 folds difference. Other calcins also exhibited a stimulatory effect on [3H]ryanodine binding to RyR1, however, their affinities were significantly lower than that of Petersiiicalcin1 and Petersiiicalcin2. The channel domain of RyR1 was found to be capable of binding with the basic residues of these calcins, which also exhibited interactions with the S6 helices on RyR1. Dynamic simulations were conducted for Petersiicalcin1 and Petersiicalcin2, which demonstrated their ability to form a highly stable conformation and resulting in an asymmetric tetramer structure of RyR1. The discovery of five newly calcins further enriches the diversity of the natural calcin family, which provides more native peptides for the structure-function analysis between calcin and RyRs.

Everolimus-induced hyperpermeability of endothelial cells causes lung injury.

The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca2+-dependent manner and can lead to lung injury through mTOR-independent mechanisms.

Ryanodine receptor inhibition with acute dantrolene treatment reduces arrhythmia susceptibility in human hearts.

Ryanodine receptor 2 (RyR2) hyperactivity is observed in structural heart diseases that are a result of ischemia or heart failure. It causes abnormal calcium handling and calcium leaks that cause metabolic, electrical, and mechanical dysfunction, which can trigger arrhythmias. Here, we tested the antiarrhythmic potential of dantrolene (RyR inhibitor) in human hearts. Human hearts not used in transplantation were obtained, and right ventricular outflow tract (RVOT) wedges and left ventricular (LV) slices were prepared. Pseudo-ECGs were recorded to determine premature ventricular contraction (PVC) incidences. Optical mapping was performed to determine arrhythmogenic substrates. After baseline optical recordings, tissues were treated with 1) isoproterenol (250 nM), 2) caffeine (200 mM), and 3) dantrolene (2 or 10 mM). Optical recordings were obtained after each treatment. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Isoproterenol shortened action potential duration (APD) in the RV, RVOT, and LV regions and shortened calcium transient duration (CaTD) in the LV. Caffeine further shortened APD in the RV, did not modulate APD in the RVOT, and prolonged APD in the LV. In addition, in the LV, CaTD prolongation was also observed. More importantly, adding dantrolene did not alter APD in the RV or RVOT regions but produced a trend toward APD prolongation and significant CaTD prolongation in the LV, restoring these parameters to baseline values. In conclusions, dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates in the human heart and could be a novel antiarrhythmic therapy in patients with structural heart disease.NEW & NOTEWORTHY Ryanodine receptor 2 hyperactivity is observed in structural heart diseases caused by ischemia or heart failure. It causes abnormal calcium leaks, which can trigger arrhythmias. To prevent arrhythmias, we applied dantrolene in human hearts ex vivo. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates and could be a novel antiarrhythmic therapy in patients with structural heart disease.

Activation of ryanodine-sensitive calcium store drives pseudo-allergic dermatitis via Mas-related G protein-coupled receptor X2 in mast cells.

Mast cell (MC) activation is implicated in the pathogenesis of multiple immunodysregulatory skin disorders. Activation of an IgE-independent pseudo-allergic route has been recently found to be mainly mediated via Mas-Related G protein-coupled receptor X2 (MRGPRX2). Ryanodine receptor (RYR) regulates intracellular calcium liberation. Calcium mobilization is critical in the regulation of MC functional programs. However, the role of RYR in MRGPRX2-mediated pseudo-allergic skin reaction has not been fully addressed. To study the role of RYR in vivo, we established a murine skin pseudo-allergic reaction model. RYR inhibitor attenuated MRGPRX2 ligand substance P (SP)-induced vascular permeability and neutrophil recruitment. Then, we confirmed the role of RYR in an MC line (LAD2 cells) and primary human skin-derived MCs. In LAD2 cells, RYR inhibitor pretreatment dampened MC degranulation (detected by ß-hexosaminidase retlease), calcium mobilization, IL-13, TNF-α, CCL-1, CCL-2 mRNA, and protein expression activated by MRGPRX2 ligands, namely, compound 48/80 (c48/80) and SP. Moreover, the inhibition effect of c48/80 by RYR inhibitor was verified in skin MCs. After the confirmation of RYR2 and RYR3 expression, the isoforms were silenced by siRNA-mediated knockdown. MRGPRX2-induced LAD2 cell exocytosis and cytokine generation were substantially inhibited by RYR3 knockdown, while RYR2 had less contribution. Collectively, our finding suggests that RYR activation contributes to MRGPRX2-triggered pseudo-allergic dermatitis, and provides a potential approach for MRGPRX2-mediated disorders.

To the Mechanism of the Antiarrhythmic Action of Compound ALM-802: the Role of Ryanodine Receptors.

The effect of the compound N1-(2,3,4-trimethoxy)-N2-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) on the amplitude of the Ca2+ response in the cell was studied in in vitro experiments. The concentration of intracellular calcium was assessed using a Fura-2 two-wave probe. The experiments were performed on a culture of isolated rat hippocampal neurons. The effect of compound ALM-802 on the activity of ryanodine receptors (RyR2) was studied on an isolated strip of rat myocardium. The compound ALM-802 (69.8 µM) in hippocampal neurons causes a significant decrease in the amplitude of the Ca2+ response induced by addition of KCl to the medium. Experiments performed on an isolated myocardial strip showed that compound ALM-802 (10-5 M) almost completely blocked the positive inotropic reaction of the strip to the RyR2 agonist caffeine (5×10-5 M). The data obtained indicate that the decrease in the concentration of Ca2+ ions in the cell caused by ALM-802 is due to its ability to block RyR2 located on the membrane of the sarcoplasmic reticulum, which can be associated with the antiarrhythmic activity of the compound.

Bacterial lipopolysaccharide hyperpolarizes the membrane potential and is antagonized by the K2p channel blocker doxapram.

Exposure of Drosophila skeletal muscle to bacterial lipopolysaccharides (LPS) rapidly and transiently hyperpolarizes membrane potential. However, the mechanism responsible for hyperpolarization remains unclear. The resting membrane potential of the cells is maintained through multiple mechanisms. This study investigated the possibility of LPS activating calcium-activated potassium channels (KCa) and/or K2p channels. 2-Aminoethyl diphenylborinate (2-APB), blocks uptake of Ca2+ into the endoplasmic reticulum (ER); thus, limiting release from ryanodine-sensitive internal stores to reduce the function of KCa channels. Exposure to 2-APB produces waves of hyperpolarization even during desensitization of the response to LPS and in the presence of doxapram. This finding in this study suggests that doxapram blocked the acid-sensitive K2p tandem-pore channel subtype known in mammals. Doxapram blocked LPS-induced hyperpolarization and depolarized the muscles as well as induced motor neurons to produce evoked excitatory junction potentials (EJPs). This was induced by depolarizing motor neurons, similar to the increase in extracellular K+ concentration. The hyperpolarizing effect of LPS was not blocked by decreased extracellular Ca2+or the presence of Cd2+. LPS appears to transiently activate doxapram sensitive K2p channels independently of KCa channels in hyperpolarizing the muscle. Septicemia induced by gram-negative bacteria results in an increase in inflammatory cytokines, primarily induced by bacterial LPS. Currently, blockers of LPS receptors in mammals are unknown; further research on doxapram and other K2p channels is warranted. (220 words).

A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

High-resolution analysis of the cytosolic Ca2+ events in ß cell collectives in situ.

The release of peptide hormones is predominantly regulated by a transient increase in cytosolic Ca2+ concentration ([Ca2+]c). To trigger exocytosis, Ca2+ ions enter the cytosol from intracellular Ca2+ stores or from the extracellular space. The molecular events of late stages of exocytosis, and their dependence on [Ca2+]c, were extensively described in isolated single cells from various endocrine glands. Notably, less work has been done on endocrine cells in situ to address the heterogeneity of [Ca2+]c events contributing to a collective functional response of a gland. For this, ß cell collectives in a pancreatic islet are particularly well suited as they are the smallest, experimentally manageable functional unit, where [Ca2+]c dynamics can be simultaneously assessed on both cellular and collective level. Here, we measured [Ca2+]c transients across all relevant timescales, from a subsecond to a minute time range, using high-resolution imaging with a low-affinity Ca2+ sensor. We quantified the recordings with a novel computational framework for automatic image segmentation and [Ca2+]c event identification. Our results demonstrate that under physiological conditions the duration of [Ca2+]c events is variable, and segregated into three reproducible modes, subsecond, second, and tens of seconds time range, and are a result of a progressive temporal summation of the shortest events. Using pharmacological tools we show that activation of intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in ß cell collectives, and that a subset of [Ca2+]c events could be triggered even in the absence of Ca2+ influx across the plasma membrane. In aggregate, our experimental and analytical platform was able to readily address the involvement of intracellular Ca2+ receptors in shaping the heterogeneity of [Ca2+]c responses in collectives of endocrine cells in situ.NEW & NOTEWORTHY Physiological glucose or ryanodine stimulation of ß cell collectives generates a large number of [Ca2+]c events, which can be rapidly assessed with our newly developed automatic image segmentation and [Ca2+]c event identification pipeline. The event durations segregate into three reproducible modes produced by a progressive temporal summation. Using pharmacological tools, we show that activation of ryanodine intracellular Ca2+ receptors is both sufficient and necessary for glucose-dependent [Ca2+]c oscillations in ß cell collectives.

Intestinal enteroendocrine cells rely on ryanodine and IP3 calcium store receptors for mechanotransduction.

Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5-HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na+ , K+ , Ca2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP-1) resulted in a rapid increase in cytoplasmic Ca2+ and a slower decrease in ER stores Ca2+ , suggesting the involvement of intracellular Ca2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca2+ store receptors, a similar expression of IP3 receptors, but a >30-fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca2+ responses and 5-HT release. In tissue, pressure-induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca2+ stores to amplify mechanically induced Ca2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca2+ signalling, 5-HT release and epithelial secretion. KEY POINTS: A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca2+ ) with a longer duration than the stimulus, suggesting intracellular Ca2+ signal amplification. In this study, we profiled the expression of intracellular Ca2+ store receptors and found an enriched expression of the intracellular Ca2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5-HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.

The ryanodine receptor-calstabin interaction stabilizer S107 protects hippocampal neurons from GABAergic synaptic alterations induced by Abeta42 oligomers.

The oligomeric form of the peptide amyloid beta 42 (Abeta42) contributes to the development of synaptic abnormalities and cognitive impairments associated with Alzheimer's disease (AD). To date, there is a gap in knowledge regarding how Abeta42 alters the elementary parameters of GABAergic synaptic function. Here we found that Abeta42 increased the frequency and amplitude of miniature GABAergic currents as well as the amplitude of evoked inhibitory postsynaptic currents. When we focused on paired pulse depression (PPD) to establish whether GABA release probability was affected by Abeta42, we did not observe any significant change. On the other hand, a more detailed investigation of the presynaptic effects induced by Abeta42 by means of multiple probability fluctuation analysis and cumulative amplitude analysis showed an increase in both the size of the readily releasable pool responsible for synchronous release and the number of release sites. We further explored whether ryanodine receptors (RyRs) contributed to exacerbating these changes by stabilizing the interaction between RyRs and the accessory protein calstabin. We observed that the RyR-calstabin interaction stabilizer S107 restored the synaptic parameters to values comparable to those measured in control conditions. In conclusion, our results clarify the mechanisms of potentiation of GABAergic synapses induced by Abeta42. We further suggest that RyRs are involved in the control of synaptic activity during the early stage of AD onset and that their stabilization could represent a new therapeutical approach for AD treatment. KEY POINTS: Accumulation of the peptide amyloid beta 42 (Abeta42) is a key characteristic of Alzheimer's disease (AD) and causes synaptic dysfunctions. To date, the effects of Abeta42 accumulation on GABAergic synapses are poorly understood. The findings reported here suggest that, similarly to what is observed on glutamatergic synapses, Abeta42 modifies GABAergic synapses by targeting ryanodine receptors and causing calcium dysregulation. The GABAergic impairments can be restored by the ryanodine receptor-calstabin interaction stabilizer S107. Based on this research, RyRs stabilization may represent a novel pharmaceutical strategy for preventing or delaying AD.

Statin activation of skeletal ryanodine receptors (RyR1) is a class effect but separable from HMG-CoA reductase inhibition.

BACKGROUND AND PURPOSE: Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolaemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. Using a range of structurally diverse statin analogues, we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property. EXPERIMENTAL APPROACH: Compounds were screened for RyR1 activity utilising [3 H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers. KEY RESULTS: All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca2+ . We synthesised the one essential structural moiety that all statins must possess for HMG-CoA reductase inhibition, the R-3,5-dihydroxypentanoic acid unit, and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four that lacked the ability to modulate RyR1. CONCLUSION AND IMPLICATIONS: That cerivastatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin regulation of RyR1 and HMG-CoA reductase are separable effects will allow the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds we have identified, allowing development of effective cardioprotective statins with improved patient tolerance.

How does flecainide impact RyR2 channel function?

Flecainide, a cardiac class 1C blocker of the surface membrane sodium channel (NaV1.5), has also been reported to reduce cardiac ryanodine receptor (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ release. It has been introduced as a clinical antiarrhythmic agent for catecholaminergic polymorphic ventricular tachycardia (CPVT), a condition most commonly associated with gain-of-function RyR2 mutations. Current debate concerns both cellular mechanisms of its antiarrhythmic action and molecular mechanisms of its RyR2 actions. At the cellular level, it targets NaV1.5, RyR2, Na+/Ca2+ exchange (NCX), and additional proteins involved in excitation-contraction (EC) coupling and potentially contribute to the CPVT phenotype. This Viewpoint primarily addresses the various direct molecular actions of flecainide on isolated RyR2 channels in artificial lipid bilayers. Such studies demonstrate different, multifarious, flecainide binding sites on RyR2, with voltage-dependent binding in the channel pore or voltage-independent binding at distant peripheral sites. In contrast to its single NaV1.5 pore binding site, flecainide may bind to at least four separate inhibitory sites on RyR2 and one activation site. None of these binding sites have been specifically located in the linear RyR2 sequence or high-resolution structure. Furthermore, it is not clear which of the inhibitory sites contribute to flecainide's reduction of spontaneous Ca2+ release in cellular studies. A confounding observation is that flecainide binding to voltage-dependent inhibition sites reduces cation fluxes in a direction opposite to physiological Ca2+ flow from SR lumen to cytosol. This may suggest that, rather than directly blocking Ca2+ efflux, flecainide can reduce Ca2+ efflux by blocking counter currents through the pore which otherwise limit SR membrane potential change during systolic Ca2+ efflux. In summary, the antiarrhythmic effects of flecainide in CPVT seem to involve multiple components of EC coupling and multiple actions on RyR2. Their clarification may identify novel specific drug targets and facilitate flecainide's clinical utilization in CPVT.

Propofol Causes Sustained Ca2+ Elevation in Endothelial Cells by Stimulating Ryanodine Receptor and Suppressing Plasmalemmal Ca2+ Pump.

ABSTRACT: Propofol, a general anesthetic administered intravenously, may cause pain at the injection site. The pain is in part due to irritation of vascular endothelial cells. We here investigated the effects of propofol on Ca2+ transport and pain mediator release in human umbilical vein endothelial cells (EA.hy926). Propofol mobilized Ca2+ from cyclopiazonic acid (CPA)-dischargeable pool but did not cause Ca2+ release from the lysosomal Ca2+ stores. Propofol-elicited Ca2+ release was suppressed by 100 µM ryanodine, suggesting the participation of ryanodine receptor channels. Propofol did not affect ATP-triggered Ca2+ release but abolished the Ca2+ influx triggered by ATP; in addition, propofol also suppressed store-operated Ca2+ entry elicited by CPA. Ca2+ clearance during CPA-induced Ca2+ discharge was unaffected by a low Na+ (50 mM) extracellular solution, but strongly suppressed by 5 mM La3+ (an inhibitor of plasmalemmal Ca2+ pump), suggesting Ca2+ extrusion was predominantly through the plasmalemmal Ca2+ pump. Propofol mimicked the effect of La3+ in suppressing Ca2+ clearance. Propofol also stimulated release of pain mediators, namely, reactive oxygen species and bradykinin. Our data suggest propofol elicited Ca2+ release and repressed Ca2+ clearance, causing a sustained cytosolic [Ca2+]i elevation. The latter may cause reactive oxygen species and bradykinin release, resulting in pain.

Mechanisms underlying spontaneous phasic contractions and sympathetic control of smooth muscle in the rat caudal epididymis.

Here we investigate mechanisms underlying spontaneous phasic contractions (SPCs) and sympathetic control of contractility in the rat epididymis, a long tubular duct involved in transportation and maturation of sperm. Longitudinal contractions of short segments (~ 1.5 mm) of rat proximal and distal caudal epididymal duct were measured + / - nerve stimulation. The extent of sympathetic innervation of these duct regions was determined by immunohistochemistry. Proximal caudal duct segments (150-300 µm dia.) exhibited SPCs, while distal segments (350-500 µm) were quiescent in ~ 80% of preparations. SPC amplitude and frequency were reduced by the L-type voltage-dependent Ca2+ channel (LVDCC) blocker nifedipine (1 µM), with the T-type voltage-dependent Ca2+ channel (TVDCC) blocker ML218 (1 µM) specifically decreasing SPC frequency. SPCs were inhibited upon blockade of the SR/ER Ca2+-ATPase (CPA 10 µM). SPCs were also inhibited by caffeine (1 µM), 2-APB (100 µM), niflumic acid (100 µM), or by lowering extracellular [Cl-] from 134.4 to 12.4 mM but not by ryanodine (25 µM) or tetracaine (100 µM). Electrical field stimulation (EFS) at 2 Hz for 60 s caused a sustained α1-adrenoceptor-sensitive contraction in distal segments and enhanced and/or induced α2-adrenoceptor-sensitive oscillatory phasic contractions in proximal and distal segments, the latter mimicked by application of the α2-adrenoceptor agonist clonidine. We hypothesise that SPCs in the proximal cauda are triggered by pacemaker mechanisms involving rhythmic IP3 receptor-operated SR/ER store Ca2+ release and resultant activation of CaCC with TVDCCs and possibly LVDCCs subserving in this process. Sympathetic nerve-released noradrenaline induces α2-adrenoceptor-mediated phasic contractions in the proximal and distal cauda. These findings provide new pharmacological targets for male infertility and contraception.

Functional Microstructure of CaV-Mediated Calcium Signaling in the Axon Initial Segment.

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (CaV). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of CaVs. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: CaV3 generates hotspot regions of calcium influx coupled to calcium stores, whereas CaV2 channels underlie diffuse calcium influx between hotspots. Therefore, different CaV classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes.

Efavirenz, atazanavir, and ritonavir disrupt sarcoplasmic reticulum Ca2+ homeostasis in skeletal muscles.

While muscle fatigue, pain and weakness are common co-morbidities in HIV-1 infected people, their underlying cause remain poorly defined. To this end, we evaluated whether the common antiretroviral drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) could be a contributing factor by pertubating sarcoplasmic reticulum (SR) Ca2+ cycling. In live-cell imaging, EFV (6.0 µM), ATV (6.0 µM), and RTV (3.0 µM) elicited Ca2+ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca2+ release channel blocker ryanodine (50 µM), slowed the rate and amplitude of Ca2+ release from and reuptake of Ca2+ into the SR. EFV, ATV and RTV (1 nM - 20 µM) potentiated and then displaced [3H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca2+ release channel (RyR1). These drugs at concentrations 0.25-31.2 µM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤5 µM) potentiated and >5µM inhibited the ability of sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA1) to hydrolyze ATP and transport Ca2+. RTV (2.5-31.5 µM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca2+ transport. EFV (0.25-31.5 µM) had no measurable effect on SERCA1's ability to hydrolyze ATP and transport Ca2+. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca2+ homeostasis.

Extra Virgin Olive Oil Phenols Vasodilate Rat MesentericResistance Artery via Phospholipase C (PLC)-CalciumMicrodomains-Potassium Channels (BKCa) Signals.

Recent evidence suggests that the reason Extra Virgin Olive Oil (EVOO) lowers blood pressure and reduces the risk of developing hypertension is partly due to minor components of EVOO, such as phenols. However, little is still known about the mechanism(s) through which EVOO phenols mediate anti-hypertensive effects. The aim of the present study was to investigate the mechanisms of action of EVOO phenols on mesenteric resistance arteries. A pressure myograph was used to test the effect of EVOO phenols on isolated mesenteric arteries in the presence of specific inhibitors of: 1) BKca channels (Paxillin, 10-5 M); 2) L-type calcium channels (Verapamil, 10-5 M); 3) Ryanodine receptor, RyR (Ryanodine, 10-5 M); 4) inositol 1,4,5-triphosphate receptor, IP3R, (2-Aminoethyl diphenylborinate, 2-APB, 3 × 10-3 M); 5) phospholipase C, PLC, (U73122, 10-5 M), and 6) GPCR-Gαi signaling, (Pertussis Toxin, 10-5 M). EVOO phenols induced vasodilation of mesenteric arteries in a dose-dependent manner, and this effect was reduced by pre-incubation with Paxillin, Verapamil, Ryanodine, 2-APB, U73122, and Pertussis Toxin. Our data suggest that EVOO phenol-mediated vasodilation requires activation of BKca channels potentially through a local increase of subcellular calcium microdomains, a pivotal mechanism on the base of artery vasodilation. These findings provide novel mechanistic insights for understanding the vasodilatory properties of EVOO phenols on resistance arteries.

Nitric Oxide/Cyclic GMP-Dependent Calcium Signalling Mediates IL-6- and TNF-α-Induced Expression of Glial Fibrillary Acid Protein.

Astrocyte activation is characterized by hypertrophy with increased glial fibrillary acidic protein (GFAP), whose expression may involve pro-inflammatory cytokines. In this study, the effects of pro-inflammatory IL-6 and TNF-α and anti-inflammatory cytokines IL-4 and IL-10 on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling, intracellular calcium concentration ([Ca2+]i) and GFAP expression were investigated. In human glioblastoma astrocytoma U-373 MG cells, IL-6 and TNF-α, but not IL-4 or IL-10, increased iNOS, cGMP, [Ca2+]i and GFAP expression. The inhibitors of iNOS (1400 W), soluble guanylyl cyclase (ODQ) and IP3 receptors (ryanodine and 2-APB) reversed the increase in cGMP or [Ca2+]i, respectively, and prevented GFAP expression. In rat striatal slices, IL-6 and TNF-α, at variance with IL-4 and IL-10, promoted a concentration-dependent increase in Ca2+ efflux, an effect prevented by 1400 W, ODQ and RY/2APB. These data were confirmed by in vivo studies, where IL-6, TNF-α or the NO donor DETA/NO injected in the striatum of anaesthetised rats increased cGMP levels and increased GFAP expression. The present findings point to NO/cGMP-dependent calcium signalling as part of the mechanism mediating IL-6- and TNF-α-induced GFAP expression. As this process plays a fundamental role in driving neurotoxicity, targeting NO/cGMP-dependent calcium signalling may constitute a new approach for therapeutic interventions in neurological disorders.

Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels.

In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics.NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.