Local Calcium Signals Research Articles

Coupled Activation of Mechanosensitive and Calcium-Dependent Potassium Channels in 3T3 and 3T3-SV40 Cells

Using the patch–clamp method, mechanosensitive regulation of ion channels was studied in cultivated 3T3 and 3T3-SV40 fibroblasts. The activity of mechanosensitive cation channels with a conductivity 25 pS in response to plasma-membrane stretching was observed in both cell lines. Despite obvious differences in the actin network in normal and transformed cells, the threshold values of the stimulus required for the channel activation were close and were approximately 55 mm Hg. The frequency of channels was significantly higher in transformed 3T3-SV40 fibroblasts than in their untransformed 3T3 analogs. Coupled activation of mechanosensitive calcium-permeable channels and potassium calcium-controlled channels was found in both cell lines. The analysis of flows through single channels allows to detect functional interaction of different channels: stretch-induced local calcium entry activates potassium channels that do not have their own mechanosensitivity. The results of a comparative study show that there is a fundamental similarity between the ion mechanisms of cellular mechanotransduction in normal and transformed fibroblasts. The quantitative differences, first of all, concern the level of functional activity of mechanosensitive channels that provide the development of the local calcium signal in the near-membrane cell region.

An open source tool for automatic spatiotemporal assessment of calcium transients and local 'signal-close-to-noise' activity in calcium imaging data.

Local and spontaneous calcium signals play important roles in neurons and neuronal networks. Spontaneous or cell-autonomous calcium signals may be difficult to assess because they appear in an unpredictable spatiotemporal pattern and in very small neuronal loci of axons or dendrites. We developed an open source bioinformatics tool for an unbiased assessment of calcium signals in x,y-t imaging series. The tool bases its algorithm on a continuous wavelet transform-guided peak detection to identify calcium signal candidates. The highly sensitive calcium event definition is based on identification of peaks in 1D data through analysis of a 2D wavelet transform surface. For spatial analysis, the tool uses a grid to separate the x,y-image field in independently analyzed grid windows. A document containing a graphical summary of the data is automatically created and displays the loci of activity for a wide range of signal intensities. Furthermore, the number of activity events is summed up to create an estimated total activity value, which can be used to compare different experimental situations, such as calcium activity before or after an experimental treatment. All traces and data of active loci become documented. The tool can also compute the signal variance in a sliding window to visualize activity-dependent signal fluctuations. We applied the calcium signal detector to monitor activity states of cultured mouse neurons. Our data show that both the total activity value and the variance area created by a sliding window can distinguish experimental manipulations of neuronal activity states. Notably, the tool is powerful enough to compute local calcium events and ‘signal-close-to-noise’ activity in small loci of distal neurites of neurons, which remain during pharmacological blockade of neuronal activity with inhibitors such as tetrodotoxin, to block action potential firing, or inhibitors of ionotropic glutamate receptors. The tool can also offer information about local homeostatic calcium activity events in neurites.

Extension of Rapid Buffering Approximation to Ca2+ Buffers with Two Binding Sites

Fundamental cell processes such as synaptic neurotransmitter release, endocrine hormone secretion, and myocyte contraction are controlled by highly localized calcium (Ca2+) signals resulting from brief openings of trans-membrane Ca2+ channels. On short temporal and spatial scales, the corresponding local Ca2+ nanodomains formed in the vicinity of a single or several open Ca2+ channels can be effectively approximated by quasi-stationary solutions. The rapid buffering approximation (RBA) is one of the most powerful of such approximations, and is based on the assumption of instantaneous equilibration of the bimolecular Ca2+ buffering reaction, combined with the conservation condition for the total Ca2+ and buffer molecule numbers. Previously, RBA has been generalized to an arbitrary arrangement of Ca2+ channels on a flat membrane, in the presence of any number of simple Ca2+ buffers with one-to-one Ca2+ binding stoichiometry. However, many biological buffers have multiple binding sites. For example, buffers and sensors phylogenetically related to calmodulin consist of two Ca2+-binding domains (lobes), with each domain binding two Ca2+ ions in a cooperative manner. Here we consider an extension of RBA to such buffers with two interdependent Ca2+ binding sites. We show that in the presence of such buffers, RBA solution is given by the solution to a cubic equation, analogous to the quadratic equation describing RBA in the case of a simple, one-to-one Ca2+ buffer. We examine in detail the dependence of RBA accuracy on buffering parameters, to reveal conditions under which RBA provides sufficient precision.

Endothelial Mitochondria Regulate Local Calcium Signalling in Mesenteric Arteries From Mice

Local calcium (Ca2+) signals are crucial for endothelial functions, including regulation of underlying smooth muscle cell contractility. Ca2+ pulsar is an endothelial IP3R‐dependent Ca2+ signal restricted to myoendothelial projections (MEP). Targets for this spatially‐discrete Ca2+ signal such as KCa3.1 channels, NOS3 and CaMKII have been previously described. However, determinants for Ca2+ pulsar kinetics and stochasticity remains poorly understood. Mitochondria are an important regulator of intracellular Ca2+ dynamics in several cell types such as pancreatic acinar cells. It has thus been hypothesized that mitochondria are an important modulator of Ca2+ pulsar. Mitochondrial influence on Ca2+ pulsar was explored on cut‐opened mesenteric resistance arteries using a high‐speed confocal microscope. Inhibition of mitochondrial uniporter‐dependent Ca2+ uptake (Ru360) increased the surface covered by a Ca2+ pulsar (+30%). Conversely, stimulation of mitochondrial Ca2+ uptake (kaempferol) reduced Ca2+ pulsar's spreading (−20%), suggesting that mitochondria could limit Ca2+ pulsars propagation. Interestingly, inhibition of mitochondrial Ca2+ efflux (CsA for mPTP; CGP37157 for NCLX) had little if any effect on spatial dispersion of a Ca2+ pulsar. However, de novo Ca2+ pulsar sites were observed following exposure to CsA/CGP. Mitochondria might thus control subcellular Ca2+ levels within MEP to modulate IP3R activity. Electron microscopy showed clustering of mitochondrial distribution near the MEP. These results suggest that mitochondria might play a significant role in regulating local endothelial Ca2+ signalling and then, modulate vascular tone.Support or Funding InformationFRQS, CFI, SQHA and CIHR

Cellular defects by deletion of ANO10 are due to deregulated local calcium signaling

TMEM16K (ANO10) belongs to a family of ion channels and phospholipid scramblases. Mutations in ANO10 cause neurological and immunological defects, and abrogated ion transport. Here we show that Ano10 knockout in epithelial cells leads to defective ion transport, attenuated volume regulation and deranged Ca2+ signaling. Intestinal epithelial cells from Ano10 null mice are reduced in size and demonstrate an almost abolished spontaneous and TNFα-induced apoptosis. Similar defects were found in mouse peritoneal Ano10 null macrophages and in human THP1 macrophages with reduced ANO10 expression. A cell cycle dependent colocalization of Ano10 with acetylated tubulin, centrioles, and a submembranous tubulin containing compartment was observed in Fisher rat thyroid cells. Axs, the Drosophila ortholog of ANO10 is known for its role in mitotic spindle formation and association with the endoplasmic reticulum and Ca2+ signaling. We therefore propose that mutations in ANO10 cause cellular defects and genetic disorders through deranged local Ca2+ signaling.

Mitochondria Modulates Calcium Pulsars Kinetics in Native Endothelial Cells from Mouse Mesenteric Arteries

Endothelial control of vascular tone is modulated by local calcium (Ca2+) signaling events like Ca2+ pulsars, an IP3R‐dependant Ca2+ release restricted to myoendothelial projections (MEP). Aside from the recent report of Ca2+ pulsars being modulated by CaMKII, mechanisms regulating endothelial spontaneous Ca2+ release remain unidentified. Mitochondria are important modulators of intracellular Ca2+ dynamics in various cell types and, we hypothesized mitochondria may control endothelial Ca2+ pulsars. High‐speed confocal microscopy was used to investigate mitochondria's influence on endothelial Ca2+ pulsar in cut‐open mouse mesenteric arteries (en face configuration). Preventing mitochondrial Ca2+ uptake by uncoupling with FCCP uncovered a new Ca2+ pulsar site population that had a lower frequency (≈+30%) but larger diffusional spread (+57%) than controls. Inhibition of mitochondrial Ca2+ uptake via the uniporter using Ru360 increased the spread of Ca2+ pulsars (≈+30%), though kinetics were not significantly altered. Significantly, inhibition of mitochondrial Ca2+ export (CGP37157, CsA) increased the number of active Ca2+ pulsar sites, each with a decreased bursting frequency. In summary, these results suggest that mitochondrial uptake and release of Ca2+ acts as a critical local modulator of Ca2+ pulsar kinetics and may therefore be crucial to endothelial control of vascular tone.Support or Funding InformationFRQS, HSFC, CFI, SQHA and CIHR

Editorial: Determinants of Synaptic Information Transfer: From Ca(2+) Binding Proteins to Ca(2+) Signaling Domains.

Editorial: Determinants of Synaptic Information Transfer: From Ca(2+) Binding Proteins to Ca(2+) Signaling Domains.

Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo

Aging is associated with vascular dysfunction that impairs tissue perfusion, physical activity, and the quality of life. Calcium signaling in endothelial cells (ECs) is integral to vasomotor control, exemplified by localized Ca(2+) signals within EC projections through holes in the internal elastic lamina (IEL). Within these microdomains, endothelium-derived hyperpolarization is integral to smooth muscle cell (SMC) relaxation via coupling through myoendothelial gap junctions. However, the effects of aging on local EC Ca(2+) signals (and thereby signaling between ECs and SMCs) remain unclear, and these events have not been investigated in vivo. Furthermore, it is unknown whether aging affects either the number or the size of IEL holes. In the present study, we tested the hypothesis that local EC Ca(2+) signaling is impaired with advanced age along with a reduction in IEL holes. In anesthetized mice expressing a Ca(2+)-sensitive fluorescent protein (GCaMP2) selectively in ECs, our findings illustrate that for mesenteric arteries controlling splanchnic blood flow the frequency of spontaneous local Ca(2+) signals in ECs was reduced by ∼85% in old (24-26 mo) vs. young (3-6 mo) animals. At the same time, the number (and total area) of holes per square millimeter of IEL was reduced by ∼40%. We suggest that diminished signaling between ECs and SMCs contributes to dysfunction of resistance arteries with advanced age.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/aging-impairs-endothelial-ca2-signaling/.

Dyadic Remodeling as a Cause of Impaired E-C Coupling in Early Stages of Myocardial Injury

In failing myocytes, impaired E-C coupling is often associated with loss of t-tubules and appearance of orphaned calcium release units. Impairment of communication between Ca-channels of sarcolemma and of sarcoplasmic reticulum (SR) has been observed in non-failing stages of cardiac hypertrophy. We therefore studied the relationship between Ca-signaling and ultrastructure of dyadic junctions in early stages of myocardial injury. Myocardial injury (IMI) was induced by a single dose of isoproterenol (150 mg/kg, i.p.) in adult male rats. On day 15, isolated myocytes were studied using laser scanning fluorescence confocal microscopy: Ca-spikes were activated by whole-cell patch-clamp and t-tubules were visualized by di-8-ANEPPS. Ultrastructure of dyadic junctions was evaluated from electron-microscopic images. Our results show that remodeling of IMI myocytes transpired as increased proportion of longitudinal elements of tubular system as well as increased proportion of dyads with defective contact between the membranes of sarcolemma and SR; however, there was no actual loss of t-tubules and ultrastructure of t-tubules in dyads of IMI myocytes was similar to control (CTR). In parallel, Ca-spike activation was slower and less synchronized in IMI. In both CTR and IMI, distribution of latencies of Ca-spikes and the time profile of Ca-release flux measured from whole cells consisted of two components. The fraction of the slow component of calcium release in IMI was close to the fraction of dyads with defective contact between the membranes of sarcolemma and SR. We conclude that overload of cardiac myocytes of the working part of injured myocardium leads to damage of tubulo-reticular junctions of dyadic microdomains, which results in impairment of local calcium signaling. Support: APVV-0721-10, VEGA 2/047/14 and VEGA 2/0095/15

Postnatal Development of Calcium Signaling in Rat Cardiomyocytes

During development, cardiac myocytes undergo rapid growth and structural remodeling. With increased cell size and increased requirements for calcium for contraction, excitation-contraction coupling becomes increasingly dependent on calcium-induced calcium release (CICR). Extending into the interior of the cell, t-tubules form a network securing signal transduction into the whole volume of the cell.We studied the development of L-type calcium currents and of t-tubules in ventricular myocytes of neonatal and young rats (ages 2-18 and 28 days). Calcium currents were recorded in isolated ventricular myocytes (80 ms depolarization from −50 to 0 mV) using patch-clamp. Plasma membrane-specific fluorescent probe FM 4-64 was used to observe formation of t-tubules in myocytes using isolated cells and intact myocardial tissue by laser-scanning confocal microscopy.Formation of t-tubules started around day 9 (D9) as small membrane invaginations. Invaginations formed a loose network with predominating longitudinal elements around D14, and after D17 developed into a semi-regular tubular network. Cell size, area and membrane capacitance was increasing throughout the observed age interval and around D28 reached values typical for cells from adult rat hearts. During early development, calcium current density increased, saturating around D11. The rate of Ca-current decay increased with age, reaching values typical for adult myocytes after D10.These data suggest that functional local calcium signaling is present already at D11, when the t-tubules only start to form. This is supported especially by the dynamics of calcium current decay, which at D11 becomes as fast as in adult cardiac myocytes.Support: VEGA 2/0095/15 and VEGA 2/0147/14.

Probabilistic encoding of stimulus strength in astrocyte global calcium signals.

Astrocyte calcium signals can range in size from subcellular microdomains to waves that spread through the whole cell (and into connected cells). The differential roles of such local or global calcium signaling are under intense investigation, but the mechanisms by which local signals evolve into global signals in astrocytes are not well understood, nor are the computational rules by which physiological stimuli are transduced into a global signal. To investigate these questions, we transiently applied receptor agonists linked to calcium signaling to primary cultures of cerebellar astrocytes. Astrocytes repetitively tested with the same stimulus responded with global signals intermittently, indicating that each stimulus had a defined probability for triggering a response. The response probability varied between agonists, increased with agonist concentration, and could be positively and negatively modulated by crosstalk with other signaling pathways. To better understand the processes determining the evolution of a global signal, we recorded subcellular calcium "puffs" throughout the whole cell during stimulation. The key requirement for puffs to trigger a global calcium wave following receptor activation appeared to be the synchronous release of calcium from three or more sites, rather than an increasing calcium load accumulating in the cytosol due to increased puff size, amplitude, or frequency. These results suggest that the concentration of transient stimuli will be encoded into a probability of generating a global calcium response, determined by the likelihood of synchronous release from multiple subcellular sites.

Biochemical and molecular characterization of the calcineurin in Echinococcus granulosus larval stages

Calcineurin (CaN) is a Ca2+-calmodulin activated serine–threonine protein phosphatase that couples the local or global calcium signals, thus controlling important cellular functions in physiological and developmental processes. The aim of this study was to characterize CaN in Echinococcus granulosus (Eg-CaN), a human cestode parasite of clinical importance, both functionally and molecularly. We found that the catalytic subunit isoforms have predicted sequences of 613 and 557 amino acids and are substantially similar to those of the human counterpart, except for the C-terminal end. We also found that the regulatory subunit consists of 169 amino acids which are 87% identical to the human ortholog. We cloned a cDNA encoding for one of the two catalytic subunit isoforms of CaN (Eg-can-A1) as well as the only copy of the Eg-can-B gene, both constitutively transcribed in all Echinococcus larval stages and responsible for generating a functionally active heterodimer. Eg-CaN native enzyme has phosphatase activity, which is enhanced by Ca2+/Ni2+ and reduced by cyclosporine A and Ca2+ chelators. Participation of Eg-CaN in exocytosis was demonstrated using the FM4-64 probe and Eg-CaN-A was immunolocalized in the cytoplasm of tegumental cells, suckers and excretory bladder of protoscoleces. We also showed that the Eg-can-B transcripts were down-regulated in response to low Ca2+ intracellular level, in agreement with decreased enzyme activity. Confocal microscopy revealed a striking pattern of Eg-CaN-A in discrete fluorescent spots in the protoscolex posterior bladder and vesicularized protoscoleces beginning the vesicular differentiation. In contrast, Eg-CaN-A was undetectable during the pre-microcyst closing stage while a high DDX-like RNA helicase expression was evidenced. Finally, we identified and analyzed the expression of CaN-related endogenous regulators.

Variations in local calcium signaling in adjacent cardiac myocytes of the intact mouse heart detected with two-dimensional confocal microscopy.

Dyssynchronous local Ca release within individual cardiac myocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. In a new approach we quantify variation in local Ca transients between adjacent myocytes in the whole heart. Langendorff-perfused mouse hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize cell membranes. A spinning disc confocal microscope with a fast camera allowed us to record Ca signals within an area of 465 μm by 315 μm with an acquisition speed of 55 fps. Images from multiple transients recorded at steady state were registered to their time point in the cardiac cycle to restore averaged local Ca transients with a higher temporal resolution. Local Ca transients within and between adjacent myocytes were compared with regard to amplitude, time to peak and decay at steady state stimulation (250 ms cycle length). Image registration from multiple sequential Ca transients allowed reconstruction of high temporal resolution (2.4 ± 1.3 ms) local CaT in 2D image sets (N = 4 hearts, n = 8 regions). During steady state stimulation, spatial Ca gradients were homogeneous within cells in both directions and independent of distance between measured points. Variation in CaT amplitudes was similar across the short and the long side of neighboring cells. Variations in TAU and TTP were similar in both directions. Isoproterenol enhanced the CaT but not the overall pattern of spatial heterogeneities. Here we detected and analyzed local Ca signals in intact mouse hearts with high temporal and spatial resolution, taking into account 2D arrangement of the cells. We observed significant differences in the variation of CaT amplitude along the long and short axis of cardiac myocytes. Variations of Ca signals between neighboring cells may contribute to the substrate of cardiac remodeling.

GnRH evokes localized subplasmalemmal calcium signaling in gonadotropes.

The binding of GnRH to its receptor initiates signaling cascades in gonadotropes, which result in enhanced LH and FSH biosynthesis and secretion. This process is necessary for follicular maturation and ovulation. Calcium influx activates MAPKs, which lead to increased transcription of LH and FSH genes. Previous research suggests that two MAPK signaling pathways, ERK and jun-N-terminal kinase, are activated by either calcium influx through L-type calcium channels or by global calcium signals originating from intracellular stores, respectively. Here we continued this investigation to further elucidate molecular mechanisms transducing GnRH receptor stimulation to ERK activation. Although it is known that GnRH activation of ERK requires calcium influx through L-type calcium channels, direct evidence supporting an underlying local calcium signaling mechanism was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence microscopy to visualize discrete sites of calcium influx (calcium sparklets) in gonadotrope-derived αT3-1 cells in real time. GnRH increased localized calcium influx and promoted ERK activation. The L-type calcium channel agonist FPL 64176 enhanced calcium sparklets and ERK activation in a manner indistinguishable from GnRH. Conversely, the L-type calcium channel antagonist nicardipine inhibited not only localized calcium sparklets but also ERK activation in response to GnRH. GnRH-dependent stimulation of L-type calcium channels was found to require protein kinase C and a dynamic actin cytoskeleton. Taken together, we provide the first direct evidence for localized L-type calcium channel signaling in αT3-1 cells and demonstrate the utility of our approach for investigating signaling mechanisms and cellular organization in gonadotropes.

Abstract 20662: Variations in Local Calcium Signaling in Adjacent Cardiomyocytes of the Intact Mouse Heart Detected With Two-Dimensional Confocal Microscopy

Coordinated Ca release in the heart ensures efficient myocardial contraction and electrical stability. Dyssynchronous Ca release within individual cardiomyocytes has been linked to cellular contractile dysfunction. Differences in Ca kinetics in adjacent cells may also provide a substrate for inefficient contraction and arrhythmias. We present an approach to quantify local Ca signals related to the geometry of adjacent cardiomyocytes within the intact heart using 2D confocal microscopy. We observe significant differences in Ca kinetics between adjacent cardiomyocytes. Methods: Ca transients (CaT, Fluo-8 AM) and cell membranes (Di-4 ANEPPS) were visualized in an epicardial area of 465 μm by 315 μm (12 to 20 cells) in Langendorff-perfused mouse hearts. Images (55/second) from a series of CaT at steady state stimulation (250 ms cycle length) were registered to their relative timepoint in the cardiac cycle to restore local CaT with a temporal resolution of 2 ms. Variation of local CaT within a cardiomyocyte (INTRA) and between adjacent cardiomyocytes (INTER) were compared with regard to amplitude, time to peak (TTP) and time constant of decay (TAU). Results: Temporal registration of images from successive CaT yielded local signals with good signal/noise ratio (N= 4 hearts, n= 8 regions). Along the long axis but not along the short axis of cardiomyocyte orientation the variation of CaT amplitude was significantly larger between that within cells (INTER 51.49 ±23.57% vs. INTRA <0.01 ±0.001% of global Ca amplitude). Variation in TTP Ca was comparable in all directions, but TAU also tended to vary more along the long axis (INTER 68.50 ±27.03% vs. INTRA 37.74 ±13.17%). Isoproterenol (10 μM) significantly increased the differences in TAU between adjacent cardiomyocytes along the short axis but not along the long axis. Summary: We present an approach to detect and analyze local Ca signals in Langendorff-perfused intact mouse hearts with high temporal and spatial resolution, taking into account 2D orientation of the cells. We observed significant differences in the synchrony of Ca signals along the long and short axis of adjacent cardioymyocytes. Alterations in synchrony of Ca signals in neighboring cells may contribute to the substrate in cardiac remodeling.

In Vivo Calcium Recordings and Channelrhodopsin-2 Activation through an Optical Fiber

We describe here an approach for the fluorometric monitoring of population activity in neurons in live mice combined with the activation of optogenetic actuators in vivo. In this protocol, a thin multimode fiber, which is used for both delivering excitation light and collecting emitted fluorescence signals, is inserted into the skull of a mouse. When combined with multicell bolus loading of Ca(2+) indicators, this optical fiber and its associated fluorescence detection system can be used for the in vivo recording of brain Ca(2+) signals from a local cluster of coactive neurons. The fiber can also be used for the optogenetic stimulation of light-activated ion channels, such as channelrhodopsin-2, allowing the monitoring of local calcium signals evoked by optogenetic stimulation.

Excitability in a stochastic differential equation model for calcium puffs

Calcium dynamics are essential to a multitude of cellular processes. For many cell types, localized discharges of calcium through small clusters of intracellular channels are building blocks for all spatially extended calcium signals. Because of the large noise amplitude, the validity of noise-approximating model equations for this system has been questioned. Here we revisit the master equations for local calcium release, examine the multiple scales of calcium concentrations in the cluster domain, and derive adapted stochastic differential equations. We show by comparison of discrete and continuous trajectories that the Langevin equations can be made consistent with the master equations even for very small channel numbers. In its deterministic limit, the model reveals that excitability, a dynamical phenomenon observed in many natural systems, is at the core of calcium puffs. The model also predicts a bifurcation from transient to sustained release which may link local and global calcium signals in cells.

An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging

Local Ca2+ transients such as puffs and sparks form the building blocks of cellular Ca2+ signaling in numerous cell types. They have traditionally been studied by linescan confocal microscopy, but advances in TIRF microscopy together with improved electron-multiplied CCD (EMCCD) cameras now enable rapid (>500framess−1) imaging of subcellular Ca2+ signals with high spatial resolution in two dimensions. This approach yields vastly more information (ca. 1Gbmin−1) than linescan imaging, rendering visual identification and analysis of local events imaged both laborious and subject to user bias. Here we describe a routine to rapidly automate identification and analysis of local Ca2+ events. This features an intuitive graphical user-interfaces and runs under Matlab and the open-source Python software. The underlying algorithm features spatial and temporal noise filtering to reliably detect even small events in the presence of noisy and fluctuating baselines; localizes sites of Ca2+ release with sub-pixel resolution; facilitates user review and editing of data; and outputs time-sequences of fluorescence ratio signals for identified event sites along with Excel-compatible tables listing amplitudes and kinetics of events.

Bitter taste receptor (TAS2R) agonists inhibit IgE-dependent mast cell activation

Bitter taste receptor (TAS2R) agonists inhibit IgE-dependent mast cell activation

Major role of ryanodine type 2 receptors in global and local intracellular calcium release in arterial smooth muscle (1067.7)

Ryanodine receptors (RyR) are calcium‐sensitive ion channels in the sarcoplasmic reticulum (SR) membrane, and are important effectors in arterial smooth muscle excitation‐contraction (EC) coupling. While three different RyR isoforms are expressed in arterial smooth muscle cells (SMCs), the relative contribution of each RyR isoform to vascular function remains unresolved. We generated tamoxifen‐inducible SMC specific knock‐out mice of RyR2 to test the hypothesis of RyR2 contributing in arterial EC coupling, vasoregulation, and systemic blood pressure. Myography and videomicroscopy of isolated arteries, calcium imaging and patch clamp current recordings on isolated SMCs, and radiotelemetric blood pressure measurements were used. Targeted deletion of the gene for RyR2 resulted in a loss in functional coupling of calcium sparks to associated BK potassium channel activation, and increased arterial tone and systemic blood pressure. Aortas, femoral, cerebral and mesenteric arteries from RyR2‐deficient mice showed no contraction in response to RyR activation by 10 mmol/L caffeine. Our data demonstrate that RyR2 are key elements in Cav1.2 channel‐induced EC coupling in large and small arteries. Furthermore, RyR2, by activating BK channels, are important molecular SR calcium release components in translating local calcium signals (Ca2+‐sparks) to vasoregulation and arterial blood pressure.Grant Funding Source: Supported by the Deutsche Forschungsgemeinschaft (DFG)