The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium.Circulating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction. The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium. The endothelium is a complex sensory system that acts as an innate mechanotransducer and enables the vascular system to regulate cardiovascular function. The endothelium detects frictional shear stresses generated by the flow of blood Davies,  and tranet al. et al. et al. In addition to mechanical stimuli, the endothelium also responds to biochemical signalling molecules to affect a diverse range of vascular functions, such as vascular tone  transduction in the endothelium of intact arteries exposed to flow, and also to address whether local cholinergic mechanisms provide a mechanochemical transduction pathway responsible for promoting flow‐mediated dilatation. We show that endothelial organic cation transporters (OCTs) release ACh in response to mechanical activation by shear stress. The release of ACh requires mitochondrial generation of acetyl‐coenzyme A (acetyl‐CoA) and 2 or (ii) an overdose of pentobarbital sodium  as described.All animal care and experimental procedure were carried out with the approval of the University of Strathclyde Local Ethical Review Panel , under UK Home Office regulations. All experiments used either common carotid arteries or second‐order mesenteric arteries (as described) obtained from male Sprague–Dawley rats , killed by either (i) an overdose of COen face carotid artery preparations, using a modification of a procedure for visualization of endothelial Ca2+ signalling , 0.02% Pluronic F‐127 and 0.35% DMSO in physiological saline solution (PSS) for 60 min at room temperature. Following incubation, arteries were gently washed in PSS before the Sylgard blocks were placed face down on 0 grade thickness microscope coverslips fixed to the bottom of a custom bath chamber  and left for a further 60 min to allow intracellular de‐esterification of DAF‐FM. Stainless steel pins (diameter 200 μm) were used as spacers to ensure that the endothelium did not contact the coverslip, as well as to enable the endothelium to be exposed to fluid flow. With the Sylgard block in the chamber, laminar flow was provided by a syringe pump that was connected to the chamber via silicone tubing.Nitric oxide production was assessed in the endothelium of tw (dyne cm−2) was calculated  using the equation:Q is volumetric flow rate (cm3 s−1) and η is the fluid viscosity (0.0089 dyne cm−2 for water). The endothelium was imaged using an inverted epi‐fluorescence microscope . DAF‐FM was excited with 488 nm wide‐field epifluorescence illumination provided by a monochromator  and fluorescence emission was imaged at 10 Hz using a 40× objective lens , a 0.7× coupling lens and a back‐illuminated electron‐multiplying charge‐coupled device (EMCCD) camera  (1× binning). DAF‐FM fluorescence intensity measurements, averaged across the field‐of‐view, are expressed as baseline‐corrected fluorescence intensity (F/F0), where F is DAF‐FM fluorescence at time t and F0 is basal fluorescence intensity. Because nitric oxide does not dissociate from the dye, DAF‐FM F/F0 measurements represent the cumulative production of nitric oxide /dt] traces. d(F/F0)/dt was obtained by convolving F/F0 traces with the first derivative of a Gaussian kernel in the programming language, Python. Discrete derivate [d(F/F0)/dt] signals are analogous to the first derivative of F/F0, if F/F0 was continuous in the time domain.The dimensions of the flow chamber were 3 × 1.5 × 0.2 cm (length × width × height). The level of endothelial shear stress, 2+ signalling was monitored in the endothelium of en face carotid artery and second‐order mesenteric artery preparations. The endothelium of en face preparations were incubated with a loading solution containing the fluorescent Ca2+ indicator, Cal‐520 acetoxymethyl ester  (5 μm), 0.02% Pluronic F‐127 and 0.35% DMSO in PSS for 30 min at 37 °C. Cal‐520/AM was used throughout as the indicator is reported to offer the highest signal‐to‐noise ratio of the most commonly available Ca2+ dyes  as described above for nitric oxide production. In some flow experiments, a Nikon Ti‐S microscope with no coupling lens and an Andor iXON EMCCD (2X binning) camera were used. Following equilibration and confirmation that flow (1.5 ml min−1) elicited repeatable Ca2+ responses, flow‐mediated (1.5 ml min−1) endothelial Ca2+ responses were monitored before and after various treatments, as described in the text. Unless indicated otherwise, all treatments were tested after 20, 40 and 60 min of incubation. All summary data shown correspond to 20 min time point, unless otherwise stated. The results for all incubation times are summarized in Table Endothelial Ca2+ signalling was examined in response to local photolysis of caged IP3. The endothelium of en face arteries was first loaded with Cal‐520/AM (5 μm), as described above, and then incubated with a second loading solution containing a membrane permeant caged IP3, caged IP3 4,5‐dimethoxy‐2‐nitrobenzyl (10 μm), 0.02% Pluronic F‐127 and 0.35% DMSO in PSS for 30 min at 37 °C. Photolysis of caged IP3 was achieved using a frequency tripled neodymium: yttrium aluminium garnet  laser  attached directly to the TE2000U microscope system  was added to the MOPS perfusion solution (MOPS PSS) and the endothelium was incubated 10 min. Subsequently, TMRE (120 nm) was present in all perfusion solutions. Ca2+ and TMRE images were acquired sequentially using the TE2000U microscope system described above equipped with a 100× objective . The exposure on each channel was 50 ms, resulting in an acquisition rate of 10 Hz for each channel. Minimal photobleaching of TMRE was observed over the 5 min recording periods used.Ca2+ signalling was imaged in large fields (∼150 cells) of intact endothelium with high spatial resolution . To facilitate visual inspection of endothelial Ca2+ signals in all cells, image stacks were generated to show propagating Ca2+ wavefronts. These Ca2+ wavefront image stacks were generated by calculating the forward difference of changes in fluorescence intensity   of intensity of the sequential subtraction image stacks . Throughout the present study, STDev images are presented overlaid on average‐intensity projections to create colour composites showing Ca2+ activity .Ca fields ∼50 cells  fields ∼50 cells  fields ∼50 cells 2+ waves. The whole‐cell, or ‘global’ signals are similar to those seen when the endothelium is activated by exogenous ACh. We previously developed an automated analysis routine for analysing these signals. This procedure enables rapid extraction of Ca2+ signals and quantification of signalling metrics for every cell in the field‐of‐view and, furthermore, enables the response of individual cells to be matched throughout lengthy experimental protocols  and thus enables the quantification of whole‐cell Ca2+ signalling metrics. The action of the algorithm, which we now name WAVE (Whole‐cell AVErage) Ca2+ signal analysis, is: (1) all individual cells within the field‐of‐view are identified and, for each cell, a ROI that encompasses the majority of the cell area is created; (2) time‐dependent fluorescence intensities  are extracted from each ROI; (3) each Ca2+ signal is normalized to its corresponding baseline; (4) Ca2+ signalling metrics, such as peak amplitudes and oscillation frequency, are rapidly and objectively determined for all cells; and (5) Ca2+ signals and summary data are presented in pictorial form.As reported in the Results, fluid flow evoked whole‐cell propagating CaAa). Sharpened images were obtained by applying an unsharp mask filter. ROIs were then generated by applying a threshold to intensity levels. The resulting ROIs encompassed the majority of the area of each cell. These whole‐cell ROIs were verified and any erroneous ROIs were corrected manually. Our longitudinal experimental design required a comparison of Ca2+ responses before and after pharmacological intervention (at multiple time‐points) in the same cells. Because of the incubation/equilibration periods, these data were acquired across multiple recordings. Occasionally, the microscope field‐of‐view drifted slightly between acquisitions, and so ROI sets were aligned, across separate image acquisitions, using an automated alignment plug‐in in FIJI .To enable ROIs to be generated by intensity thresholding, we first created average‐intensity projections of each image stack Fig. Aa. Shar2+ signals were extracted from the fluorescence intensity (F) within each of the whole‐cell ROIs for each frame of the image recordings   by dividing each fluorescence intensity trace by the average value of a 50‐frame (5 s) baseline‐period (F0) at the start of each trace. F0 was determined for each cell. F/F0 traces were then smoothed using 11‐point (1.1 s), third‐order polynomial Savitzky–Golay filter.Time‐dependent whole‐cell Ca0) Fig. B–D, top2+ (F/F0) signal  were determined using an automated computer algorithm. First, for each whole‐cell Ca2+ signal, we calculated discrete (first) derivative Ca2+ signals [d(F/F0)/dt] , as described for nitric oxide measurements above. These derivate signals can be used to infer Ca2+ activity in the corresponding F/F0 signal  of a spike in a F/F0 Ca2+ signal, the derivative changes sign from positive to negative (or negative to positive). Thus, we used a ‘zero‐crossing detector’ to identify the times at which the sign of each derivate signal changed from positive to negative or negative to positive. The zero‐crossing detector identifies all times of ‘zero‐crossing’ . For each whole‐cell Ca2+ signal, these times were organized into sequential pairs. The magnitude of the critical point (peak or nadir) between each pair was then extracted. Any peak or nadir in the derivate trace with a magnitude less than three times the standard deviation of baseline noise (5 s of each derivate trace) was discarded; three times the standard deviation was considered to be the threshold. The sign of each critical point that was greater in magnitude than the threshold level of the derivate signal was used to determine whether the ‘zero‐crossing’ pair corresponded to a detectable rise or fall in the F/F0 signal. Thus, the zero‐crossing detector enabled the times of all peaks (above threshold) in the derivate signal to be extracted. These peaks correspond to the rising edge in the F/F0 signal. The time at which the peak occurred in the F/F0 signal was then extracted by measuring the maximum F/F0 value in a 5 s window following the identified rising edge. The occurrence of any detectable peak in each trace was used to determine whether the corresponding cell had responded. If cells responded, they were defined as ‘Active’. The times of occurrence of the first peak in each Ca2+ response were used to generate latency profiles of cellular Ca2+ responses. These latency profiles are presented as histograms with time t = 0 corresponding to the time of the first detected peak . The times of occurrence of each peak in the Ca2+ data were also used to generate rastergrams of Ca2+ activity . The rastergram plots show peaks in the Ca2+ signals as dots at the time of their occurrence. These plots provide a convenient display of the oscillatory activity of many cells and permit visual identification of cells that respond to various stimuli. The times were also used to extract conventional measurements from the corresponding F/F0 data as described below.The time of occurrence and amplitude of peaks in each whole‐cell Canal Fig. B were dity Fig. E. The rThe endothelial preparations used in the present study exhibited minimal photobleaching, or response ‘run‐down’, and remained in focus for extended periods of time (>10 min) permitting repeated imaging of the same large field of intact endothelia over several hours. Routine recordings typically consisted of imaging periods of ∼2 min performed every 20 min of the duration of each experiment. A typical experimental protocol consisted of two control recordings . These two control recordings were followed by another recording of 5 min in duration, during which an initial control response was obtained and a pharmacological intervention was performed . This permitted us to record the effects drugs during their addition. After treatment, we then recorded another three responses .2+ data in a convenient pictorial form, F/F0 signals are shown with respect to the time at which flow was initiated  . These F/F0 signals are displayed ‘locked’ to the time of stimulus and termed ‘stimulus‐locked’. However, there was a considerable spread in the time at which each cell responded to stimuli. Therefore, we automatically aligned the F/F0 traces based on the time at which the first peak occurred in each and every cell . Thus, these aligned F/F0 signals are ‘locked’ to the first response of each cell  and are termed ‘response‐locked’ . In cases where no peak was identified in a given trace, those Ca2+ signals were instead extracted with respect to the average time at which all other cells responded. Alignment of the signals in this way enabled Ca2+ signalling metrics to be extracted with ease. Measurements extracted from the aligned signals included the the amplitude of the first peak (peak ΔF/F0), the average signal level during the first 60 s following the first peak (average ΔF/F0) and the inverse of the number of peaks occurring within this 60 s period (i.e. oscillation frequency). Because all signals were aligned so that the initial peak of each coincided in time , peak amplitudes (peak ΔF/F0) were calculated by measuring the values of each response‐locked signal at this time. In signals where no peak was identified, peak ΔF/F0 was taken to be zero. Thus, our measure of peak Ca2+ response, as shown in summary data  throughout the present study, accounts for the Ca2+ response integrated across all cells within the field‐of‐view. Time‐average signal levels were calculated by taking the average of the 60 s period of all signals following this time. Furthermore, the frequency (events cell–1 min−1) of each active whole‐cell Ca2+ signal was calculated by taking inverse of the number of peaks occurring within this same 60 s period. Because of possible errors arising from signals falling below baseline values, area under the curve measurements were not calculated.To present the Ca2+ traces from all cells within a field‐of‐view are presented throughout the present study as composite plots where the colour of each trace  represents the amplitude of the initial peak in the response to flow. A full heat map range was used to colour the traces . In scatter plots showing the  peak Ca2+ responses or time‐averaged Ca2+ responses, datapoints are colour‐coded according to the plotting density of individual points and pairing of responses are indicated by the connecting lines. Linescan kymograph images were generated using the ‘reslice’ function in FIJI. These images were generated by measuring fluorescence across a specified single (or multipoint) line placed on raw image stacks, and their evolution over time is displayed as a pseudocoloured representation. 3‐D surface plots were generated using custom macros employing the ‘3D_Surface_Plot’ function in FIJI. In some images, ‘full‐field’ average responses are shown. Full‐field average responses are derived from a single ROI encompassing the entire field‐of‐view.Representative whole‐cell Caace Fig. F and G 2+ signal analysis algorithm, together with sample experimental data, is currently under preparation. This will be freely available via the University of Strathclyde data repository, ‘KnowledgeBase’ . In the meantime, readers are encouraged to contact the authors if they would like to share the current tools described above.A release of the source code for our WAVE Ca2+ activity for extended periods of time. We recorded basal endothelial Ca2+ activity in carotid and mesenteric endothelial preparations for periods of 60 s. The occurrence of spontaneous Ca2+ activity in these recordings was first confirmed by visual inspection and by placing subcellular regions of interest within the boundaries of cells exhibiting spontaneous activity. Rigorous manual analysis of these data was impractical as a result of the large number of cells visualized. As such, we performed an analysis of this data using the WAVE Ca2+ signal analysis algorithm described above. However, as described in the Results, WAVE does not assess the spatial profile of extracted Ca2+ signals and may miss very small‐amplitude subcellular events, and so may not be well‐suited to the analysis of highly localized spontaneous Ca2+ signals. Instead, we used an alternative algorithm, developed by an independent group specifically for the automated detection and analysis of localized Ca2+ signals in camera‐based imaging data  and lack of smooth muscle tone/rhymicity enabled recordings of endothelial CaF/FAVG) of the increase in fluorescence (ΔF) of that pixel relative to its mean fluorescence (FAVG) throughout the recording. The standard deviation of the resulting image stack was then normalized to 1 by dividing each frame in the stack by the standard deviation of all frames, and the resultant stack further processed by applying a Guassian blur (two pixel radius). These image stacks were then converted into binary form by applying a threshold. The resulting binary representation is thus a matrix where a pixel value of 1 (or 0) indicates the presence (or absence) of a Ca2+ event above threshold. All image pre‐processing was performed in FLIKA, an interactive image processing suite written in Python (http://flika-org.github.io/).Raw image stacks from recordings of spontaneous activity were first processed by dividing each frame in the stack by the mean of all frames and subtracting a value of 1 from every pixel. The resulting fluorescence of each pixel thus represents a ratio  co‐ordinates that encompass each Ca2+ event, identified in binary ΔF/FAVG image stacks. Unique Ca2+ events are first identified using a clustering algorithm based on the premise that a pixel corresponding to a cluster centre may be recognized as a local density maxima with a relatively large distance from points with higher densities  by a user‐defined number of pixels in the xy planes, and a mean spatial image of each event is created by averaging each pixel intensity within the time window. These images are then normalized to the highest pixel value, and a 2‐D elliptical Gaussian function is fitted to this mean spatial image. The Gaussian fitting function reports the x and y centroid positions, x and y standard deviations, and angle of the long axis of the resulting elliptical function. Ca2+ event traces are then extracted from the ΔF/FAVG image stack by averaging pixel values within square ROIs of user‐defined width centered around each of the centroids calculated by the Gaussian fitting process for a user‐defined number of frames preceding and following event. Once these event traces are extracted, the maximum amplitude of the event is calculated automatically. Because multiple events may arise from the same site, detected events that occur within a user‐defined distance from each other are grouped together and considered to arise from a single site. The centroid location of each of these sites is calculated by taking the mean (unweighted) of a square that encompasses the centroids of all the grouped events. Ca2+ traces for each site are then extracted by averaging fluorescence intensity within a square ROI centered on each group centroid. In the present study, the settings used were: ROI width = 5 pixels (∼2.9 um); number of frames following/preceding event = 25, xy padding = 40 pixels (∼23 um), group radius = 15 pixels (∼8.5 um) that occurred within a 20 pixel (∼11.5 um) radius were grouped and considered to be arising from the same site. The results are presented as peak event amplitude (ΔF/FAVG), spatial spread (μm2), frequency of Ca2+ events per site  and the fraction of cells exhibiting sites of spontaneous Ca2+ events. The spatial spread of each event was determined by calculating the elliptical area under the fitted 2‐D Gaussian.Spontaneous Ca2+ events and flow‐evoked Ca2+ waves were examined in the same artery. In these experiments, basal activity was recorded after an equilibration period (no flow) of 20 min. Flow‐evoked Ca2+ activity was then recorded in the same field of endothelial cells. Single images showing regions of spontaneous activity were created by generating STDev images of the corresponding sequential subtraction image stack of the basal recording. Similarly, single images indicating the sites of origin of flow‐evoked Ca2+ waves were created by generating STDev images of a 2 s period (20 frames) of the sequential subtraction image stack immediately following the response to flow. STDev images were converted to binary form using Huang's method for thresholding in FIJI, and the extent of co‐localization was assessed by calculating the amount of area that overlapped for each set of binary STDev images.In some experiments, the sites of spontaneous basal Ca−1 and raising the height of the waste reservoir, until a pressure of 120 mmHg was reached. Any buckle resulting from reapplication of pressure was then removed by straightening the vessel and arteries were equilibrated at 37 °C for 30 min. The carotid artery exhibits minimal spontaneous tone  and then assessed for endothelial viability by extraluminal application of ACh (100 μm). Note that ACh is less effective applied extralumenally than when applied directly to the endothelium of a large artery, such as the rat carotid artery. Indeed, in a previous study of endothelial Ca2+ imaging, we found the EC50 for ACh to be 1000‐fold lower in en face carotid artery preparations, in which ACh had free access to the endothelium, than for pressurized carotid artery preparations, in which ACh had to traverse the vascular wall  was assessed in the pre‐constricted arteries. In all experiments, arteries were imaged using a 2.5× objective and a charge‐coupled‐device camera, and pressure was monitored using two pressure transducers contained within the pressure myograph. Data were streamed to a computer, and arterial diameter was measured using online video dimension analyser software .All pre‐constricted arteries exhibited >50% dilatation to ACh and were thus considered viable. After confirmation of viability, flow‐mediated dilatation (2 ml mininh172) were obtained from Tocris . Cal‐520/AM and TTX were obtained from Abcam . Pluronic F‐127 was obtained from Invitrogen . All other drugs and chemicals were obtained from Sigma . The PSS consisted of (mm): 145 NaCL, 4.7 KCl, 2.0 MOPS, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, 1.17 MgCl2, 2.0 CaCl2, adjusted to pH 7.4 with NaOH. High‐K+ PSS contained 70 mm KCl, which replaced NaCl on an equimolar basis. All solutions were freshly prepared each day and pyruvate (S8636) was replaced on a weekly basis.SKF‐96365, U73122, vesamicol, corticosterone and cystic fibrosis transmembrane regulator inhibitor 172 . The mean ± SEM is reported for the n biological replicates. In some cases, the total number of cells from which averaged measurements were made is reported as N technical replicates. Apart from experiments performed in High‐K+ PSS, the Ca2+ responses of the same individual cells were paired. In those experiments using High‐K+ PSS, arteries contracted significantly and, although there was some overlap in the cells imaged, pairing individual cells was not possible. Unless indicated otherwise, all values were normalized to control responses. Responses were analysed statistically using one‐way ANOVA with Dunnet's post hoc test, as appropriate. All statistical analyses were performed using Prism, version 6.0 . P < 0.01 was considered statistically significant.Summary data are presented graphically, as averaged, paired responses from each of m phenylephrine) dilated to exogenous ACh in a concentration‐dependent manner  and responded to the induction of flow  with a rapid dilatation that persisted until flow was stopped . The maximal relaxation to flow was 32 ± 6% (n = 5). Flow‐mediated dilatation of rat carotid arteries is dependent on an intact endothelium and is attenuated by blockade of nitric oxide synthase using l‐NAME  loaded with the fluorescent indicator, DAF‐FM. Figure D (black line) shows a representative trace of DAF‐FM fluorescence intensity from an experiment in which the endothelium was stimulated by fluid flow (1.5 ml min−1). Because nitric oxide irreversibly binds to DAF‐FM, measured fluorescence intensities represent the total accumulation of nitric oxide. To show the time‐dependent changes in nitric oxide production, we calculated derivate DAF‐FM fluorescence signals  illustrates that the onset of flow induces a rapid increase in nitric oxide generation, whereas steady flow maintains sustained nitric oxide production.To examine the physiological relevance of endothelial mechanotransduction, ACh‐ and flow‐mediated dilatations were studied in pressurized rat carotid arteries mounted in a pressure/flow myograph. Pre‐constricted arteries . Flow  caused a rapid rise in [Ca2+]i in all cells across the field‐of‐view (∼150 cells per experiment) . As measured from whole‐cell ROIs, the rise in [Ca2+]i of individual cells was heterogeneous and oscillatory  and arose from the wave‐like propagation of whole‐cell Ca2+ waves within individual cells and throughout clusters of endothelial cells. The frequency of whole‐cell Ca2+ waves ranged from 0.017 to 0.267 Hz . The frequency, averaged across individual experiments, was 0.106 ± 0.020 Hz (n = 5). Because of the apparent multicellular nature of these waves, the endothelial Ca2+ response was spatiotemporally complex . However, when the responses of each cell were matched to itself across three separate fluid flow activations , the Ca2+ responses measured each time were approximately reproducible (n = 5) .The generation of nitric oxide is a Ca2+ signalling was observed in the endothelium of second‐order mesenteric arteries . The magnitude of flow‐induced cellular Ca2+ responses in individual endothelial cells was dependent on flow rate , ceased rapidly upon termination of flow  and increased in a stepwise manner in response to stepwise increases in flow . Despite the persistence of complex endothelial Ca2+ signalling during ongoing flow, endothelial Ca2+ activity was augmented when exogenous ACh was added to the perfusion solution (100 nm) . Individual cellular Ca2+ levels after exogenous ACh treatment were ranked in a similar order to those after flow activation . The converse was also true  .A similar profile of multicellular Caate Fig. A–C, cealow Fig. D and E low Fig. E. Despi2+ imaging recordings revealed that flow‐evoked Ca2+ waves originated in discrete subcellular locations, in multiple separate cells across the field‐of‐view  was first confirmed by manually placing subcellular ROIs within the boundaries of cells exhibiting spontaneous activity . Spontaneous Ca2+ events were often apparent in traces obtained from whole‐cell ROIs  and were observed as restricted waves of Ca2+ activity that mostly remained confined within subcellular regions. However, with ∼150 endothelial cells in focus throughout each field‐of‐view, manual and systematic analysis of multiple ROIs for each cell was impractical. Instead, we analysed spontaneous Ca2+ events by WAVE Ca2+ signal analysis. Using this analysis technique, spontaneously occurring transients were automatically detected in whole‐cell Ca2+ traces . Spontaneous events were detected infrequently in carotid artery endothelia  but frequently in mesenteric artery preparations . In those cells displaying spontaneous activity, the frequency of detected events ranged from 0.017 to 0.050 Hz  in carotid arteries and from 0.017 to 0.083 Hz  in mesenteric arteries. The mean frequency of detected Ca2+ events in active cells was 0.027 ± 0.004 Hz in carotid arteries and 0.026 ± 0.002 Hz in mesenteric arteries.Close inspection of Ca2+ signal analysis is capable of assessing the observed spontaneous Ca2+ signalling. However, recent studies of endothelial Ca2+ suggest that whole‐cell measurements may not detect low amplitude subcellular Ca2+ events  for automated analysis of localized Ca2+ events  than in mesenteric arteries . Each of these event sites identified by FLIKA corresponded to a unique endothelial cell. Thus, using this analysis method, on average 2.16 ± 1.38% of endothelial cells displayed spontaneous activity in carotid arteries , whereas 28.5 ± 5.3% displayed spontaneous activity in mesenteric arteries, N = 963 cells, n = 6) .These results suggest that WAVE Ca2+ events in active cells was 0.012 ± 0.004 Hz in carotid arteries 0.025 ± 0.003 Hz in mesenteric arteries.The frequency of events at each site ranged from 0.017 to 0.1 Hz  in carotid arteries and from 0.017 to 0.117 Hz  in mesenteric arteries. The mean frequency of detected CaF/F0, the mean event duration was 4.05 ± 0.29 s and the mean spatial spread was 145.23 ± 34.87 μm2. In mesenteric artery endothelium (437 events), the mean event amplitude was 0.33 ± 0.01 F/F0, the mean event duration was 2.59 ± 0.08 s and the mean spatial spread was 123.07 ± 9.44 μm2. Analysis of the distribution of Ca2+ events in mesenteric endothelium revealed approximately log‐normal distributions of event amplitudes, durations and spatial spreads . This analysis shows that the spontaneous Ca2+ signals have a non‐quantal, continuous distributions. These results suggest that, under the experimental conditions of the present study, the endothelium displays a continuum of spontaneous subcellular Ca2+ waves. By contrast to the propagation of Ca2+ waves throughout the whole cell upon activation by flow , 3‐D surface plots  and line scan kymographs . 3‐D surface plots and kymograph analyses also demonstrate that events arising from a single location may differ in both magnitude and spatial spread .A pooled analysis of all 33 events identified in carotid artery endothelium revealed that the mean event amplitude was 0.17 ± 0.02 ads Fig. G–I. Thi2+ signals, we compared the spatial distribution of spontaneously occurring events with the spatial distribution of the initial Ca2+ response evoked by flow in mesenteric arteries. Some sites of spontaneous activity did give rise to flow‐evoked Ca2+ waves  of the area in which spontaneous endothelial activity occurred overlapped with flow‐evoked Ca2+ wave origination sites, whereas 13.95 ± 2.2% (n = 5) of the area in which flow‐evoked Ca2+ waves originated overlapped with sites of spontaneous activity. These results suggest that some regions of spontaneous activity may give rise to flow activation.To investigate the extent of co‐localization between spontaneous and flow‐evoked Caves Fig. . Howeverves Fig. . In meseet al., et al. et al. et al. et al. 2+ responses in the absence of extracellular ATP. Barbiturates have previously been reported to influence endothelial function  . In these same arteries, the majority of cells responded to exogenous ACh  . Moreover, flow‐mediated endothelial Ca2+ signalling in the endothelium of carotid arteries obtained from rats killed by CO2 was abolished by the addition of pentobarbital to the perfusion solution . By contrast, another brand of pentobarbital sodium, Euthatal, did not inhibit flow‐evoked endothelial Ca2+ signalling when used for animal dispatch . These experiments suggest that some formulations of pentobarbital sodium abolish flow‐mediated Ca2+ signalling but not the response to exogenous ACh, perhaps because of the different percipients used.Some previous studies of flow‐mediated dilatation of intact arteries required the presence of ATP in the perfusion solution to obtain consistent responses (Liu  7) Fig. A and B.2+: (1) release of Ca2+ from the intracellular store (i.e. the endoplasmic reticulum) and (2) influx of Ca2+ across the plasma membrane. To examine the contributions of Ca2+ entry and Ca2+ release to flow‐induced endothelial Ca2+ signalling, we first examined responses in the absence of Ca2+ in the perfusion solution  and then exchanged the PSS for Ca2+‐free PSS that contained EGTA . Flow‐evoked endothelial Ca2+ signalling persisted in Ca2+‐free PSS (n = 3) . Indeed, as shown by whole field averages, the sustained phase of the flow‐evoked response was lost upon removal of Ca2+ from the bath and [Ca2+]i levels fell to below baseline levels . There was also a gradual reduction in the amplitude and then loss of these initial responses with successive stimulation in Ca2+‐free PSS  the reintroduction of extracellular Ca2+ resulted in an immediate increase in [Ca2+]i, indicating the activation of Ca2+ entry mechanisms and (2) subsequent flow‐evoked responses in the presence of Ca2+ were comparable to control levels  canonical (TRPC) channel antagonist, SKF 96365 (50 μm), abolished the ongoing sustained Ca2+entry when applied to the perfusion solution (not shown) but did not abolish subsequent initial flow‐evoked Ca2+ signals . These results suggest that both Ca2+ release from the internal store(s) and store‐operated/receptor‐operated Ca2+ ‐entry through TRPC channels contribute to flow‐induced endothelial Ca2+ signals, and that Ca2+ entry through TRPC channels is required to refill the internal store(s).In endothelial cells, there are two main mechanisms responsible for an increase in cytosolic Caion Fig. . In thes2+:  release 2+:  release 2+:  release et al. et al. et al. et al. et al. et al. et al. 2+ signals described, we studied the effects of the the broad‐spectrum TRPV channel antagonist, ruthenium red (RuR) at a concentration (5 μm) five times greater than that required to block both 4 ‐phorbol‐12,13‐didecanoate‐induced TRPV4 currents in intact rat carotid endothelial cells in situ and vasodilatation of pressurized arteries . To confirm that RuR does indeed inhibit endothelial TRPV4 channels, we examined endothelial responses stimulated by the selective TRPV4 agonist, GSK1016790A, in the absence and presence of RuR. It is reported that the endothelium is particularly sensitive and exhibits a particularly steep concentration response to GSK1016790A (in the nanomolar range). Concentrations of 30 nm are reported to stimulate local, subcellular Ca2+ events  induce Ca2+ overload in the endothelium  activated 95.8 ± 3.4% of endothelial cells  (n = 3). Initially, GSK1016790A (30 nm) caused local Ca2+ events . However, prolonged activation with 30 nm GSK1016790A caused global Ca2+ increases and large‐scale propagating Ca2+ waves between cells , as seen by in other studies using 100 nm GSK1016790A  by pre‐incubation with RuR (5 μm for 20 min). These results suggest that TRPV4 channels contribute little to the flow‐induced endothelial Ca2+ signalling described in the present study.TRP vanilloid 4 (TRPV4) channels are reported to contribute to flow‐induced vasodilatation  inhibitor, cyclopiazonic acid (CPA) (10 μm), to prevent Ca2+ sequestration by the endoplasmic reticulum. The introduction of CPA during sustained flow resulted in an increase in basal [Ca2+] . Following incubation with CPA , basal [Ca2+] remained at a steady, elevated level, presumably as a result of activation of store‐operated Ca2+‐entry. However, subsequent re‐initiation of flow still triggered an increase in endothelial [Ca2+]i . All cells did respond with a slow and sustained increase in [Ca2+]  (n = 3), indicative of Ca2+ entry. These data, along with the persistence of transient flow‐mediated Ca2+ signalling in Ca2+‐free PSS, suggest that flow‐induced Ca2+ signals result from the interplay of two separate components: a fast transient increase in [Ca2+]i as a result of the release of Ca2+ from the endoplasmic reticulum, as well as a slower sustained increase in [Ca2+]i as a result of Ca2+ entry from the extracellular space.To confirm involvement of Ca2+] Fig. A. Follo+]i Fig. . This inid (CPA) 0 μm, to 2+] Fig. D (n = 32+ release from the intracellular store(s) may occur via IP3 receptors (IP3Rs) or ryanodine (Ry) receptors (RyRs) or both  (100 μm) and the selective RyR modulator, ryanodine (Ry) (30 μm). In addition to being a potent inhibitor of IP3‐mediated Ca2+ release, 2‐APB is also reported to inhibit Ca2+ influx in a number of cell types . However, unlike in Ca2+‐free PSS , suggesting that 2‐APB inhibits Ca2+ entry channels, as well as Ca2+ release via the IP3R, in the endothelium of intact arteries. To confirm that 2‐APB inhibits endothelial IP3Rs, we examined the effects of 2‐APB on Ca2+ signals evoked by flash‐releasing a photo‐activatable form (caged) of IP3 in specific endothelial cells. Localized, subcellular, photo‐release of caged IP3 generated rapid, repeatable Ca2+ transients in the targeted endothelial cells . However, after 2‐APB (100 μm), photo‐release of caged IP3 evoked no Ca2+ response  . 2‐APB also did not prevent Ca2+ increases stimulated by the selective activator of TRPV4 channels, GSK1016790A . Thus, 2‐APB reliably blocks IP3Rs in native endothelium. We did not use xestospongin because, in our experience in smooth muscle cells, xestospongin C does not block Ca2+ increases evoked by selective activation of IP3Rs using caged IP3 . Other studies have also found that neither xestospongin C, nor xestospongin D blocked any subtype of IP3R , suggesting that RyRs contribute little to the observed flow‐evoked responses.In endothelial cells, Caels Fig. A. Howev (2‐APB) 0 μm and  (2‐APB) 00 μm andlls Fig. E–G. Hownse Fig. E–G (n = (2‐APB) 00 μm and3, we inhibited phospholipase C (PLC)‐dependent IP3 production using the PLC inhibitor, U73122 (5 μm). U73122 completely prevented both flow‐mediated Ca2+ release and entry  in carotid artery endothelia. However, the peak amplitude of Ca2+ responses evoked by local photolysis of caged IP3 (thus bypassing PLC) in the presence of U73122 was 102 ± 13% of those obtained prior to the introduction of U73122  . Taken together, the data presented thus far suggest that flow‐mediated endothelial Ca2+ signalling arises from IP3‐mediated Ca2+ release from the endoplasmic reticulum and PLC‐dependent (store‐ or receptor‐operated) Ca2+ entry.To further examine the contribution of IPtry Fig. D–F in c122 Fig. G–I  with exogenous ACh was similar of that arising from flow . To test whether muscarinic receptor activation contributes to endothelial Ca2+ signalling evoked by fluid flow, we examined Ca2+ responses in the presence of low concentrations of the muscarinic receptor inhibitor, atropine (100 nm). Atropine caused an immediate fall in endothelial [Ca2+]i to below baseline levels  and prevented subsequent applications of flow stimulating endothelial Ca2+ release or entry in carotid  (n = 3). In mesenteric endothelia  (n = 3), atropine also inhibited large‐scale flow‐mediated Ca2+ responses, although the occurrence of more localized flow‐induced Ca2+ signals persisted. Additionally, atropine failed to inhibit Ca2+ increases as a result of photo‐release of caged IP3  , suggesting that the block of the flow response in carotid and mesenteric arteries by atropine is a result of the inhibition of muscarinic receptor‐mediated Ca2+ release and muscarinic‐receptor mediated store/receptor operated Ca2+ entry.The inhibition of flow‐evoked endothelial Caels Fig. A and prIP3 Fig. G–I , and the cholinesterase inhibitor, neostigmine (10 μm), on flow‐evoked endothelial Ca2+ signalling. AChE abolished flow‐mediated endothelial Ca2+ signalling in the endothelium of both carotid  (n = 3) and mesenteric  (n = 3) arteries. Unexpectedly, when neostigmine was present, we no longer observed robust endothelial Ca2+ signalling upon commencement of flow . However, closer inspection of the data revealed significant basal activity in carotid endothelia (in the absence of flow) after (but not before) neostigmine had been introduced and incubated under no flow conditions for 20 min  (n = 3). Hence, the endothelium retains endogenous cholinesterase activity. These data, along with the inhibition by AChE, demonstrate that flow‐mediated endothelial Ca2+ signalling arises from the release of ACh endogenous to the arterial wall.Because a large variety of Gtid Fig. A and B ric Fig. C        (n = 3). Taken together, these results suggest that flow does not activate ACh release from nerves, but rather evokes a non‐vesicular release of ACh from the endothelium.We next aimed to investigate whether or not flow‐evoked endothelial Caase Fig. . We founμm) Fig. A and E  Zimmer,  and the  m) Fig. B and E et al. 2+ signalling, we pharmacologically dissipated the mitochondrial membrane potential (ΔΨm) using the proton uncoupler, carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) (5 μm). The ATP‐synthase blocker, oligomycin (6 μm), was also included to prevent ATP consumption by mitochondria as a result of reversal of ATP‐synthase. In these experiments, we simultaneously monitored endothelial [Ca2+]i and ΔΨm by dual‐loading the endothelium with the Ca2+ indicator, Cal‐520/AM, and the mitochondria indicator, TMRE.ACh is produced when the acetyl group from the coenzyme, acetyl‐CoA, is transferred to choline. Acetyl‐CoA is produced in the mitochondrial matrix, and mitochondrial uncoupling can reduce the supply of cytosolic acetyl‐CoA . The introduction of a CCCP/oligomycin caused a rapid loss of punctate TMRE staining and an increase in fluorescence throughout the cytoplasm of individual cells , indicative of mitochondrial membrane depolarization and movement of TMRE from mitochondria to the cytoplasm. Significantly, collapsing the mitochondrial membrane potential abolished flow‐evoked endothelial Ca2+ signalling  (n = 3). Thus, flow‐evoked ACh production is dependent on polarized endothelial mitochondria.As shown in Fig. lls Fig. A and B,ing Fig. C–E . These results suggest that a sufficient supply of pyruvate is required to enable endothelial production of ACh and that endothelial glycolysis, which may produce sufficient pyruvate, is not necessary for ACh production when exogenous substrates are present.In mammalian cells, acetyl‐CoA is produced from pyruvate by the pyruvate dehydrogenase complex. Thus far, the experiments described were performed using a standard MOPS PSS, which contained 2 moth Fig. . Removalill Fig.  (n = 3).et al. et al. m; n = 3)  and decynium 22   significantly reduced and abolished flow‐mediated endothelial Ca2+ signalling, respectively. Efflux of ACh by OCTs is electrogenic . Depolarization with high potassium PSS, which does not block endogenous ACh‐mediated endothelial Ca2+ signalling  (n = 3). Depolarizing solutions may also induce Ca2+ independent ACh release  Fig. A and de 3) Fig. B signifing Fig. C   (n = 5), or the chloride channel inhibitors, DIDS (10 μm)  (n = 3) or CFTR inhibitor 172 (CFTRinh172 (20 μm)    Fig. D (n = 5μm) Fig. E    , suggesting that regenerative ATP release does not contribute to the flow‐evoked endothelial Ca2+ signals described in the present study.The results obtained show that flow activates a muscarinic receptor/PLC/IPcid Fig.   of the muscarinic receptor blocker, atropine, abolished flow‐induced Ca2+ activity; AChE, which would degrade any endogenously released ACh, abolished flow‐induced Ca2+ activity; and neostigmine, which would prevent the breakdown of locally released ACh, enhanced Ca2+ activity. The endothelium expresses VAChT, ChAT and AChE  used in the present study. It may be that the mechanisms reported in the present study are relevant to the physiological diastolic flow rates.The diversity of mechanisms reported to explain flow‐induced endothelial responses perhaps reflects different pathways by which shear stress may be transduced by the endothelium and a redundancy required to maintain various short‐ and long‐term vascular responses. Alternatively, the nature of the flow‐mediated Ca2+ signalling in cultured endothelial cells, presumably as a result of a rapid decline in muscarinic receptor expression in culture  . These were dependent on the artery studied and the mechanisms of activation. Under non‐stimulated  conditions, local Ca2+ waves were observed in both carotid and mesenteric endothelia. Spontaneous endothelial activity was observed far less frequently in carotid arteries than in mesenteric arteries. By contrast, activation of the endothelium with fluid flow resulted in whole‐cell propagating Ca2+ waves that began in small subcellular regions. Given the sensitivity of the flow response to inhibitors, ACh and IP3R are probably the major contributors to the initiation of the flow‐evoked wave.In the present study, two endothelial Caet al. et al. et al. et al. et al. et al. et al. et al. 2+ signalling, whereas it was not observed third‐order mesenteric arteries ex vivo . However, it is unclear whether the lack of a description of spontaneous events in some of these studies is a result of their absence, an inability to resolve such events because of insufficient temporal resolution, or because investigative focus was on stimulated Ca2+ signalling.Spontaneous events of various sizes have previously been reported in the endothelium of pressurized rat carotid  that result from the opening of a single IP3R. The second level includes slightly larger (> 50 nm) and longer lasting (<360 ms) Ca2+ release events (‘Ca2+ puffs’) that arise from the concerted opening of several IP3Rs within a cluster. Ultimately, these elementary Ca2+ signals events may co‐ordinate to generate global Ca2+ release events, Ca2+ waves, throughout the cell. In cultured endothelial cells, the smallest events, Ca2+ blips or small Ca2+ puffs, are reported to have a mean amplitude of 23 nm and a spread of 1–3 μm and these may precede the occurrence of Ca2+ waves , although they occur infrequently under control (store intact) conditions, with one event occurring only every ∼10 min (∼0.0016 Hz).In intact endothelium, two additional elementary Ca2+ events had a very wide spread of amplitudes and durations, and occurred with a range of frequencies (0.05–0.017 Hz). These events ranged from subcellular, local Ca2+ waves to whole‐cell Ca2+ waves. Very brief Ca2+ events (i.e. puffs and pulsars) were not observed, presumably as a result of insufficient temporal resolution. Other studies  to Ca2+ waves that propagated though part (i.e. abortive waves)  or the entirety of the cell (i.e. whole‐cell waves). Each of these events  may vary in amplitude, frequency and spatial spread. Indeed, as reported in the present study and elsewhere  may be a consequence of the Ca2+ imaging method. For the smallest of local events , the upstroke and amplitude is directly related to both the open time of the release channel and the number of channels that are gated together. By contrast to electrophysiological single channel recordings, a single channel opening for a duration twice as long as a previous channel opening will generate a local Ca2+ signal with an amplitude double that of the previous opening . Thus, given the exponential distribution of open times of single channels  and larger events (whole‐cell waves) as observed in the present study, the amplitude and spread of the signal also depend on the number of channels/clusters recruited. In our experiments, imaged at 10 Hz, we did not limit the progression of a Ca2+ signal from one IP3R cluster to the next and spontaneous Ca2+ events were observed as either abortive or whole‐cell waves. Thus, the distribution of durations, amplitudes, and spread of these spontaneous Ca2+ waves presumably arises from variations in: (1) the duration of channel opening times; (2) the number of channels gated; and (3) the extent of recruitment of neighbouring IP3R clusters. In addition to these biophysical variables, the methods used to measure cytoplasmic Ca2+ , the vascular bed under study and the experimental conditions  probably all determine whether spontaneous events occur and whether they may be resolved.In the present study, the continuum of spontaneous endothelial Cas Hille, , the amp2+]i. Using this technique, we were able to image fields of endothelium containing ∼150 cells at 10 Hz and resolve a range of spontaneously occurring subcellular Ca2+ waves, and stimulated (flow) whole‐cell propagating waves. This approach is consistent with other endothelial Ca2+ imaging studies utilizing confocal microscopy  are imaged for relatively short periods of time (e.g. 30 s). However, it is impractical when large numbers of cells (>100) are imaged for long times (e.g. 2 min) where the number of events occurring may run into thousands. The method used in the present study  is a refinement of a previously developed largely‐automated analysis for large‐scale endothelial Ca2+ imaging data . The method is objective, removes user bias and permits large quantities of data to be processed in a few minutes. However, because it assigns whole‐cell regions of interest (rather than subcellular regions), the analysis may not resolve particularly low amplitude, subcellular Ca2+ events. The limitation is not in the visualization of a low‐amplitude event but, instead, is a result of the signal being averaged out because of a lack of a signal elsewhere within the cell  than in the present study (10 Hz), combined with advanced cross‐correlation techniques  to multicellular Ca2+ waves, to be determined.Endothelial cells form a network of interconnected cells. The network structure and existence of intercellular gap junctions may facilitate the transmission of activity between cells by permitting the passage of IPin vivo physiological consequences of shear stress induced release of ACh from the endothelium. However, it is tempting to speculate on the physiological consequences of flow‐induced ACh release. In all vascular beds, increasing tissue activity results in increased regional blood flow. For example, during exercise, blood flow to skeletal muscle increases in proportion to the metabolic demand of the tissue  and the British Heart Foundation (PG/11/70/29086), whose support is gratefully acknowledged.Disclaimer: Supporting information has been peer‐reviewed but not copyedited.Movie S1. Flow‐mediated Ca2+ signals in the endothelium of a carotid artery. Ca2+ signalling was examined in a cut‐opened (en face) preparation of an artery in which the endothelium was loaded with the Ca2+ indicator, Cal‐520/AM. The movie shows a raw 5 min long Ca2+ imaging recording  and baseline‐corrected Ca2+ signals (F/F0) for a single cell  and for the whole field‐of‐view (red). The timescale of the Ca2+ imaging recording is indicated at the top right of the movie panel. Flow (1.5 ml min−1) was initiated at approximately t = 15 s. Scale bar = 50 μm.Click here for additional data file.Movie S2. Flow‐mediated Ca2+ signals in the endothelium of a second‐order mesenteric artery. Ca2+ signalling was examined in a cut‐opened (en face) preparation of an artery in which the endothelium was loaded with the Ca2+ indicator, Cal‐520/AM. The timescale of the Ca2+ imaging recording is indicated at the top right of the movie panel. Flow (1.5 ml min−1) was initiated at approximately t = 15 s. Scale bar = 50 μm.Click here for additional data file.Movie S3. Exogenous ACh augments flow‐mediated Ca2+ signalling. Ca2+ signalling was examined in a cut‐opened (en face) preparation of an carotid artery in which the endothelium was loaded with the Ca2+ indicator, Cal‐520/AM. The timescale of the Ca2+ imaging recording is indicated at the top right of the movie panel. Flow (1.5 ml min−1) was initiated at approximately t = 15 s and ACh (100 nm) was added at approximately t = 15 s. The movie corresponds to data shown in Fig. F. Scale bar = 50 μm.Click here for additional data file.Movie S4. Flow‐mediated Ca2+ signals originate in distinct subcellular locations. The movie shows active Ca2+ wavefronts  overlaid on a single Ca2+ image . The movie begins at the onset of flow, and is first played before being rewound and played at double speed. The movie corresponds to data shown in Fig. Click here for additional data file.Movies S5. Spontaneous Ca2+ signals in the endothelium of a mesenteric artery. Ca2+ signalling was examined in a cut‐opened (en face) preparation of an artery in which the endothelium was loaded with the Ca2+ indicator, Cal‐520/AM. The movie shows a raw 60 s long Ca2+ imaging recording (left) and a binary image highlighting locations of spontaneous activity. In the binary image, pixels corresponding to a single event arising from a single location are all displayed with the same colour. Scale bar = 50 μm.Click here for additional data file.Movie S6. Activation of TRPV4 channels induces local Ca2+ signals followed by global Ca2+ increases in en face carotid artery preparations. The movie begins just prior to the onset of activation (t = 20 s) by the specific TRPV4 agonist, GSK1016790A (30 nm). Scale bar = 50 μm.Click here for additional data file.Movie S7. Prolonged activation of TRPV4 channels induces large‐scale propagating Ca2+ waves in en face carotid artery preparations. The movie shows active Ca2+ wavefronts  overlaid on a single Ca2+ image . Scale bar = 50 μm.Click here for additional data file.Movie S8. Flow‐mediated Ca2+ signalling in carotid artery endothelia is abolished by atropine. The movie shows three sequential experimental recordings obtained from a single en face carotid artery preparation. The first recording shows a control response to flow (flow was initiated at t = 15 s). The second recording shows a control response during which atropine (100 nm) was added to the perfusion solution (flow was initiated at t = 20 s and atropine was introduced at t = 115 s). The third recording shows a response to flow in the presence of atropine . Scale bar = 50 μm.Click here for additional data file.Movie S9. Flow‐mediated Ca2+ signalling in mesenteric artery endothelia is abolished by atropine. The movie shows two sequential experimental recordings obtained from a single en face carotid artery preparation. The first recording shows a control response to flow (flow was initiated at t = 17 s). The second recording shows a response to flow in the presence of atropine . Large‐scale, flow‐mediated Ca2+ signalling is absent following atropine treatment. Scale bar = 50 μm.Click here for additional data file.


These interventions were able to delineate the role of membrane versus intracellular signalling, previously identified as main factors in smooth muscle contraction and the generation of vessel tone. Each SMC was modelled by a system of nonlinear differential equations that account for intracellular ionic signalling, and in particular Ca2+ dynamics. Cytosolic Ca2+ concentrations formed the catalytic input to a cross-bridge kinetics model. Contractile output from these cellular components forms the input to the finite-element model of the arterial rings under isometric conditions that reproduces the experimental conditions. The model does not account for the role of the endothelium, as the nitric oxide production was suppressed by the action of L-NAME, and also due to the absence of shear stress on the arterial ring, as the experimental set-up did not involve flow. Simulations generated by the integrated model closely matched experimental observations qualitatively, as well as quantitatively within a range of physiological parametric values. The model also illustrated how increased intercellular coupling led to smooth muscle coordination and the genesis of vascular tone.Arterial wall dynamics arise from the synergy of passive mechano-elastic properties of the vascular tissue and the active contractile behaviour of smooth muscle cells (SMCs) that form the media layer of vessels. We have developed a computational framework that incorporates both these components to account for vascular responses to mechanical and pharmacological stimuli. To validate the proposed framework and demonstrate its potential for testing hypotheses on the pathogenesis of vascular disease, we have employed a number of pharmacological probes that modulate the arterial wall contractile machinery by selectively inhibiting a range of intracellular signalling pathways. Experimental probes used on ring segments from the rabbit central ear artery are: phenylephrine, a selective  Under physiological conditions the intracellular Ca2+ concentration exhibits modest variations, however, when operative conditions deviate far from normal, such as in the case of injury or under pharmacological interventions, significant fluctuations in intracellular Ca2+ concentrations may occur that are reflected in the pronounced dynamical variation in vascular tone [2+ dynamics of the smooth muscle has not been incorporated in a systematic way. This is particularly important as, according to classification by Fischer [2+ dynamics, has been proposed in [2+ catalyses cross-bridge (CB) kinetics, and the continuous where the contractile units (CUs), and therefore the arterial tissue, exhibit deformation and stress. Evaluation of the CB kinetics at cellular level relies on a modified version of the Hai & Murphy model [2+ dynamics scenarios, induced by drug interventions able to specifically modulate the SMCs contractile machinery.The arterial wall can be viewed as a control system able to regulate blood flow in order to satisfy local tissue oxygenation and nutrition requirements. This function is performed, in part, by spontaneous fluctuations in vascular tone and diameter, known as vasomotion, and is facilitated by the contractile apparatus located within the smooth muscle layer of the arterial wall . Arterielar tone –5. The clar tone –9. The dlar tone –14. Baselar tone –20. In s Fischer , the smoposed in ,23. Thisposed in . In thisposed in ,23, to ihy model ,26. The hy model ,19,20. Fhy model ,27 and ihy model . The per2.2+ dynamics is described by a system of three coupled nonlinear ordinary differential equations which express cytosolic Ca2+ concentration (χ), Ca2+ concentration within the sarcoplasmic reticulum (SR) (ζ) and the membrane potential (η). Physiological regulation of these variables is due to the coordinated effect of multiple ionic currents that have been incorporated in the formulation of the model and are described in detail in the following section. Cytosolic Ca2+ is the primary catalyst for the phosphorylation of CB formation that occurs within each SMC, and which facilitates the contractile response of the cellular cytoskeleton. At the level of the CU, four distinct states can represent the CB kinetics: dephosphorylated myosin (nM), phosphorylated myosin (nMp), attached actin–myosin filaments (nAM) and attached phosphorylated actin–myosin filaments (nAMp). The model assumes a spatial uniformity of CB within the cytosol. The state describing the fractions of attached actin–myosin filaments (represented as nAM+nAMp), along with variable λ that describes the physical deformation of the contractile elements (the stretch ratio in the tissue structure model), serve as inputs to the CU model, where the active free energy function is computed. The calculations performed in the CU model depend on the internal variable u and pressure p as variables), where the total (passive and active) tissue stress and deformation is evaluated. This accounts for the arterial wall structural response. Several stages of this physiological process may be probed through targeted pharmacological interventions (e.g. specific ion channel blockers) to reproduce and investigate the origin of vascular disease states. All the model parameters are listed in tables The multiscale modelling methodology that accounts for the contractile dynamics of the arterial wall is presented in 2.1.2.1.1.2+ concentrations are outlined in 2+ dynamics are due to the interplay of a membrane and an intracellular oscillator [2+ oscillator is Ca2+ induced-Ca2+ release (CICR) via the ryanodine receptor. Once released into the cytosol, Ca2+ is re-sequestered by the stores through the sarcoplasmic/endoplasmic reticulum ATPase (SERCA), thus completing the cycle. By contrast, the membrane oscillator is centred on cellular polarization and voltage-operated Ca2+ channels (VOCCs). Membrane potential is mainly influenced by the balance between K+ and Cl− gradients, and by Na+-Ca2+ exchange (NCX), depending on whether the exchanger operates in forward or reverse mode. The current model also includes the store operated Ca2+ channel (SOCC), which links directly store content with cytosolic Ca2+ influx [The fundamental processes involved in the regulation of cytosolic Cacillator . The mec+ influx . This ad2.1.1.1.ΦA, ΦS, ΦV and ΦN, which represent the Ca2+ permeable non-selective cation channels (NSCC), SOCC, voltage-operated cation channels (VOCC) and reverse mode Na+-Ca2+ exchange (NCX), respectively. In this study, the first current is assumed to be constant, while the others depends on χ and η via the following equations:AS, ζS, ECa, zCa1, RCa, ENCX, xNCX and zNCX are cellular model parameters. The SR acts as an inner store, uptaking cytosolic Ca2+ by means of the SERCA pump (ΦB(χ)) and releasing it into the cytosol through ryanodine-sensitive SR-Ca2+ release channel ). Store leakage ) is also accounted for. These fluxes are described through the following expressions:BSR, nSR, xSR, CRy, pRy, mRy, xRy, yRy and LSR are cellular model constants. The Ca2+ extrusion from the cytosol by ATPase pump ) is modelled as follows:DEX, kEX, zEX and REX are cellular model parameters. The chloride ) and potassium ) ion membrane fluxes are modelled as follows:ECl, xCl, zCl, EK, zK, βK and RK are cellular model constants.The extracellular influx is the sum of the concentration currents 2.1.1.2.2+ ion (JCa) and voltage (JV) currents. Assuming that the cellular size (ratio between the volume and surface) is uniform along the grid, we can define the net fluxes ith cell as follows:In a cellular cluster, each cell is able to communicate with its neighbours by exchanging Ca2.1.1.3.χ, ζ and η evolve in time according to the following system of nonlinear ordinary differential equations:γ is a scaling factor relating the net movement of ion fluxes to the membrane potential.The system variables 2.1.2.nAM), (ii) attached and phosphorylated (nAMp), (iii) detached and dephosphorylated (nA) and (iv) detached and phosphorylated (nAp). The temporal evolution of the four-state kinetics shown in τ1, τ2, τ3, τ4 and τ5 are the kinetic rates. Among these, τ1, depends on the intracellular Ca2+ concentration (χ) via [τ0 and χ0 are the material constants. As nM + nMp + nAMp + nAM=1, one of the states (nM in this work) can be assumed dependent on the other three states, leading to a three independent variables system.The kinetics is described through four different states representing the fraction of CB that are: (i) attached and dephosphorylated ( (χ) via :2.19whe2.2.2.2.1.et al. [nAMp, nAM) are considered elastic with the same mechanical stiffness. As mentioned previously, λ, the current stretch of the CU, can also be defined as the ratio between the current and the reference CU length. The average elastic elongation of the attached CB , which can also be seen as the (averaged) first Piola–Kirchhoff stress over the CU:μa behaves like an active shear modulus and where 2.2.2.Ψ) is split into volumetric (Ψvol) and isochoric components; the latter is then decomposed into active . Both λ), that can also be related to the fourth invariant asa0 is the direction of the media unstressed fibre. The active component depends directly on the CB attached , i.e.μp, cp1 and cp2 are material constants. More details regarding the solid mechanics formulation are provided in electronic supplementary material, S1.From the structural point of view, the medial tissue is considered as a hyper-elastic fibre-reinforced material ,27, with2.3.2.3.1.2+ dynamics and CB kinetics) may be considered as internal variables in the finite-element framework. The CU variables  are evaluated at the Gauss integration points of the finite-element, in order to take into account the spatial diversity of deformation over the element. For the structural problem, staggered finite-elements are used in which the displacement field is interpolated linearly while the pressure and dilation coefficient are constant over each element. The nonlinear problem is solved via a classical Newton–Raphson procedure.Each level of the framework is discretized by a spatial grid. The cellular network grid reflects the morphology of the tissue, so that each element represents a ‘real’ cell. For the continuous level, a finite-element discretization of the domain was carried out. By assigning each SMC to each finite-element the connectivity of the cellular grid and the mesh coincide. The variables computed at the cellular level (i.e. Ca2.3.2.2+ dynamics is solved by an explicit and adaptive scheme (Runge–Kutta Merson), whereas the time-dependent equations for both the CB kinetics and CU mechanics are solved by the forward Euler method (as in [λ) of the previous time step is used. By comparison, the tissue mechanics problem is solved in an implicit manner. The methodology proposed is valid for either quasi-static or dynamic problems, depending on whether the case considered is under isometric or non-isometric conditions. The same time step was employed for all subsystems. The solution procedure is presented step by step in The models/subsystems constituting the framework are solved in a block segregated fashion, as depicted in d (as in ). Note t3.The pharmacological studies employed in the validation of the modelling methodology along with the model set-up developed to reproduce the experimental settings are reported below.3.1.2; 5% CO2) Holman's buffer  at 37.0°C. Prior to any pharmacological interventions, the rings were maintained at a resting tension of 1 mN over a 60 min equilibration period, with frequent readjustments in baseline tension to correct for stress relaxation. The average inner and outer diameters of the annular segments were approximately 0.7 and 0.8 mm, respectively (a). Following the loading phase, the arterial rings obtained the deformed configuration shown in x-direction. Preparations were incubated for 30 min with both the endothelial nitric oxide synthase inhibitor N(G)-Nitro-l-arginine methyl ester  and the cyclooxygenase inhibitor indomethacin (10 μM) to inhibit prostanoid formation. Rings were then constricted with phenylephrine  and, once constrictor responses had reached a stable plateau, cumulative concentration–response curves to 10 and 30 μM CPA, and 10 and 30 μM ryanodine were obtained.Isolated rabbit ears were obtained as described previously , the cenectively a. Follow3.2.x-plane, while no translations along y and in the longitudinal direction (z) are expected. The system can thus be reduced to one-eighth of the ring. A finite-element mesh consisting of 5750 linear hexahedral elements is used in the calculations. The global force F applied to the hooks is evaluated by integrating the traction contribution of the reduced arterial system over the global contact surface. For the cellular cluster, each cell was associated with one element. At subcellular level, we associate a CU to each Gauss integration point. To account for cellular variability  the term corresponding to the influx via Ca2+ permeable non-selective cation channels (ΦA) is randomized with a normal distribution . Randomization of ΦA creates a population of cells exhibiting variable oscillatory frequency. This allows us to study the effect of increasing strengths of intercellular coupling as a factor of smooth muscle synchronization and generation of vascular tone. For all simulations, the initial values for χ, ζ and η are set equal to 0.1 μM, 0.2 μM and −0.02 V, respectively. The reference set of kinetic rates  necessary for solving the CB dynamics are taken from Parthimos et al. [From a structural point of view, each ring is assumed to deform symmetrically with respect to the s et al. .Table 44.4.1.αC, αV and the global Ca2+ dynamics. These simulations are carried out for an unloaded ring configuration. The variables associated with the Ca2+ dynamics are monitored for three different cells: cell1, cell2 and cell3 (b). In χ for  equal to 0.0, 0.1, 1.0 s−1 is shown. The plot shows clearly that coupling does not affect significantly the amplitude of χ signal. The cellular coupling tends to synchronize the χ beating pattern along the cluster.The proposed analysis is carried out for varying levels of cellular coupling in order to establish the dependency between the diffusion coefficients nd cell3 b. In figχ for two coupling levels  = 0.1 s−1, strongly coupled:  = 1.0 s−1) at two different time instants  are shown in The spatial distribution of 4.2.2+ homeostasis which is reflected in the contractile state of the smooth muscle. The actions of these pharmacological probes were simulated by the gradual variations of the associated model parameters, reported in t = 0 s (nM = 0.5 and nMp = 0.5), while the initial αC,αV) set equal to 1.0 s−1. The simulated drug interventions were performed only after the Ca2+ and CB variables reached stationary conditions. For each plotted result the drug intervention occurred at t = 0 s unless otherwise stated.A range of pharmacological interventions associated with the activation/inhibition of specific cellular mechanisms was employed for the validation of the model. To mimic the diffusion of pharmacological agents, parametric changes were applied in a graded fashion to all the cells constituting the network. The interventions selected  are associated with the modulation of the cellular CanAMp+nAM [2+ dynamics at the cellular level, see electronic supplementary material, S2.As the ring is clamped at the hooks, the resultant sum between internal and external forces at the nodes in contact with the hooks must be null. In this study, we are mainly interested in the global force developed at the hooks, which can be seen as the sum of nodal contributions along the contact surface. In addition to this, we compute also the force developed locally at cell1, which is assumed to be proportional to the sum nAMp+nAM . For a m4.2.1.α1-adrenergic receptor, associated with vasoconstriction. The action of phenylephrine at cellular level was modelled by increasing the cytosolic Ca2+ influx via NSCCs. A more complete picture of the action of phenylephrine involves an initial Ca2+ release from the SR, via inositol 1,4,5-trisphosphate-sensitive Ca2+ release channels (IP3R channels), followed by sustained Ca2+ influx into the cytosol through NSCCs. To simulate the action of phenylephrine, the coefficient ΦA of each individual cell was increased by a factor of 7. Therefore, the mean value of the ΦA distribution increased from 0.6 to 4.2 μM s−1. Two different drug dilution times (Δtdil = 50 and 100 s) were used, as shown in 2+ uptake, χ in cell1 starts to oscillate periodically. This pattern is reflected also in the store Ca2+ concentration variations (tdil involves longer transient before reaching stationary conditions.Phenylephrine is a selective agonist of riations . As expetdil) and different cellular coupling . By comparison to the simulated results, it appears that the drug was able to activate the muscle tissue at a dilution time Δtdil ∼ 50 s. We also observe that the final magnitude of the simulated and measured force is very similar.The forces generated by this intervention, normalized with respect to the force developed at the beginning of the intervention, are presented in 4.2.2.2+ store depletion. The effect of CPA, in terms of modulating the intracellular Ca2+ oscillator, is highly concentration-dependent as shown previously [BSR from 400 to 350 μM in 2000 s . In both the experiment and simulations CPA caused a small reduction in the oscillatory amplitude, while a regular waveform was maintained throughout the simulated intervention (iii)). This effect of weak cell–cell coupling is greatly enhanced in the case of force development, as seen in figure 11b. This is due to the fact that the force contributions of individual cells remain largely uncoordinated. Global force development is restored under strong intercellular coupling conditions (figure 11b(iii)).Cyclopiazonic acid (CPA) is an inhibitor of the SERCA pump, preventing refilling of the store and is thus associated with Caeviously . CPA wasrvention . A compa4.2.3.2+ release from the SR in a concentration-related fashion. The action of ryanodine was simulated according to [CRy at t = 100 s from 1250 μM s−1 down to 312.5 μM s−1. The temporal evolution of variables χ and ζ within the reference cell1 is shown in αC, αV) = 0.1 s−1 and  = 1.0 s−1). Gradual attenuation of Ca2+ release via the ryanodine receptor channels is associated with a decrease in frequency.Ryanodine was administrated at the arterial sample at concentrations of 10 and 30 μM. As discussed previously, the action of ryanodine can be simulated in different ways depending on the concentration ,23. Thisrding to , by linea,b. In this case, coefficient θ was equal to 0.5. The pattern of the forces follows Ca2+ concentrations, with the same gradual decreasing magnitude. To simulate this aspect of the experimental traces, we needed to employ the term in equation . A consistent vascular tone is restored when strong intercellular coupling is maintained.The forces obtained from the model are compared against the experimental values in figure 13equation  associat5.l-NAME to eliminate the inhibitory effect of endothelium-derived nitric oxide on the smooth muscle contractile apparatus [2+ and ionic uptake levels, which have been shown both theoretically and experimentally to greatly affect the oscillatory response of the arterial wall [2+ dynamics and the development of force at the tissue level. Indeed, the cellular events occur at the same timescale as the global tissue contraction. This finding is correlated to the increased cytosolic Ca2+ concentrations associated with the administration of phenylephrine. This observation highlights the central role of cytosolic Ca2+ influx in the generation of vascular tone and the onset of oscillatory activity observed in both experiments and simulations. Indeed, we have previously shown that the levels of Ca2+ influx can modulate other ionic signalling pathways in a specific, clinically relevant manner [2+ release through the IP3R channel pathway. We have previously employed different approaches to account for this effect. In [2+ (IICR) release pathways and assumed that the IICR results in a constant Ca2+ release current, which is essentially lumped in the overall Ca2+ uptake by the cell. Both effects are therefore accounted for by the same constant ΦA. We have shown that such an assumption still allowed the mathematical model of vasomotion to accurately reproduce an extensive range of pharmacological interventions [2+ influx to determine the natural frequency of the cell's contractile apparatus was the main reason that parameter ΦA was selected for randomization across the cellular population. By comparison, cellular dynamics are considerably less sensitive to variations of other system parameters, associated with alternative ionic fluxes [2+ dynamics under CPA was reproduced by the simulations. A noteworthy aspect of the function of CPA is its ability to transform arterial vasomotion in a controlled manner that follows hallmark transition routes out of chaotic behaviour [figure 11a,b although detailed investigation of nonlinear oscillatory transitions was not an aim of this work. Ryanodine receptor dysregulation is implicated in a range of neuromuscular disorders and arrhythmogenesis in cardiovascular diseases [We have developed a multi-component mathematical modelling framework that accounts for the structural response of the arterial wall under the active contraction of the media smooth muscle layer. The methodology was validated against a set of pharmacological interventions that modulate the contractile apparatus at the cellular level. The multiscale modelling approach combines dynamics and mechanics occurring at different levels, each requiring a specific solution strategy. With regard to the mechano-elastic component, the study was performed under isometric conditions, and thus the inertial force is neglected, allowing us to deal with a simplified system in which the evaluation of a number of dynamic parameters (such as density) was not necessary. Moreover, the ability to choose a finite-element discretization that conforms to the cellular grid, eliminated the need for spatial and temporal interpolation between the two subsystems. As a consequence, it was possible to adopt the same time step for all elements of the model. This is not a limitation, as different integration steps can be selected for each model level if required by the specific problem. This strategy can be implemented in conjunction with interpolation techniques that allow information transmission between the various contributing systems. Although necessary in many cases, this approach can result in loss of accuracy. All experiments in this study were performed following administration of pparatus . The invpparatus . Severalial wall ,23,30. Oial wall ,23. By st manner . As notefect. In , we haveventions . Furtherventions . The potc fluxes . Blockagehaviour ,38. Eviddiseases ,5. Considiseases ,40. Such


Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca2+-dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca2+-dependent phosphatase, calcineurin. Intracellular [Ca2+] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Cardiac arrhythmias are associated with raised intracellular [Ca2+]i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca2+]i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca2+-independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca2+]i. PP2A had no role. Conduction velocity was reduced by raised [Ca2+]i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca2+] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.Raised [CaThe online version of this article (doi:10.1007/s00424-016-1885-7) contains supplementary material, which is available to authorized users. Gj) [Gj including an increase of the intracellular Ca2+ concentration ([Ca2+]i) or altered Cx43 phosphorylation [2+] and GJ uncoupling i, Cn action, Cx43 phosphorylation state, Gj and AP conduction have not been characterised in myocardium. We hypothesised that with acute elevation of [Ca2+]i Cn, in synergy with PKC, controls Cx43 phosphorylation to decrease Gj and slow AP conduction, with possible intermediate roles for PP1 and PP2A. Intracellular [Ca2+] was elevated in isolated ventricular myocardial preparations by reducing the extracellular [Na+] and by increasing stimulation rate. Calcineurin activation by Ca2+ is sufficiently rapid and sensitive that both interventions are sufficient to activate this protein phosphatase i,:(i) superfusion with low-Na Tyrode’s solution to elevate [Ca2+]i via the Na+/Ca2+ exchanger; ii) an increase of electrical stimulation frequency from 1 up to 5 Hz. The effects of Cn, PKC or PP1 inhibitors on GJ conductance (Gj) and Cx43 phosphorylation status were measured in control conditions and during raised [Ca2+]i.Two interventions were used to increase , with or without Cn or PP1/PP2A inhibitors, for 20–30 min before new readings were taken. Tyrode’s solution was then reapplied for final control measurements.ent 0.02–00 kHz tolsewhere . After c2. Whole tissue protein lysate (30 μg) from each sample was prepared and then resolved by 12 % polyacrylamide SDS-PAGE and transferred to polyvinylidene difluoride membranes . Membranes were blocked with an Odyssey blocking buffer , probed with primary antibody (1:1000 dilution), then washed and incubated with secondary antibodies . Membranes were then stripped with a stripping buffer, washed and probed with another primary antibody followed by a secondary antibody. Resolved protein bands were imaged using an Odyssey infrared imaging system (UK) and then quantified with the Image-J software  in arbitrary units. The quantified band densities of pS368-Cx43 and pS365-Cx43 were normalised to corresponding total Cx43 bands. Similarly, the band densities of phosphorylated PP1 inhibitor-1 at threonine 35 (pThr35-I1) were normalised to total inhibitor-1. Total protein bands were normalised to corresponding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) band density (used as a loading control). Faint GAPDH bands were apparent in figures illustrating stripped membranes. Each sample is shown in triplicate in the relevant figures.Western blot analysis was performed, as previously described with slight modification . Hearts d, from the stimulating electrodes. Conduction velocity (CV) was calculated from the difference in latency (∆t) recorded by two separate microelectrode impalements, distance ∆d apart, as the ratio ∆d/∆t. At least five separate measurement pairs were made per preparation. To elicit APs in low-Na solution, stimulus duration (0.5–1.0 ms) was increased. In all preparations, CV was measured with a stimulus voltage 1.5 times the threshold value. Values of the maximum rate of depolarisation during the AP upstroke (dV/dtmax) and the time constant of the AP subthreshold region  were al2+]i with low-Na solution was measured in trabeculae with Ca2+-selective microelectrodes, filled with the Ca2+-ionophore ETH 1001  and in conjunction with 3 M-KCl-filled microelectrodes to record separately the membrane potential. Methods of manufacture, recording and calibration have been reported previously [2+]i with pacing were measured in freshly dispersed ventricular myocytes prepared by collagenase enzymatic dispersion using a Langendorff technique. Ventricular myocytes were loaded with Fura-2 (5 μM), superfused at 36 °C with Tyrode’s solution in a chamber on the stage of an inverted microscope. Cells were illuminated from a xenon-arc lamp that produces a continuous and uniform spectrum across the visible region. Excitation of the fluorochrome alternately at 340 and 380 nm was provided by interposing spectral band-pass filters (340 ± 5 and 380 ± 5 nm) within the light path, mounted in a wheel spinning at 32 Hz. Fluorescent light was recorded between 410 and 510 nm with a photomultiplier tube and output sample-and-hold amplifiers coordinated to the frequency of the spinning wheel. The ratio of emission intensity when illuminated at the two frequencies (R340/380) was used as an index of the intracellular [Ca2+] i was raised.The data thus far do not distinguish between a direct or indirect effect of Cn on pS365-Cx43 dephosphorylation. Caated PPI . This pon = 3, Fig. n = 3, Fig. Total I1 protein, normalised to GAPDH, was similar in control and low-Na solutions and also in the presence of CysA or CAIP  at a low concentration (5 nM) inhibits PP1 activity  and thus2+-independent protein phosphatase, that itself may influence the I1 pathway, in a way similar to that of Cn i is raised. It has been shown previously when using thin trabeculae, as used for Gj measurement, and bipolar stimulation at one end that there is one-dimensional (1-D) AP conduction along the longitudinal axis i was raised by increasing the stimulation rate. Increasing the rate from 1 to 2 or 5 Hz also decreased CV  that should mainly inhibit PP1 but with potentially a smaller effect on PP2A . PP2A alThe identity of the protein phosphatase(s) targeting pS365 site under pathological conditions is unclear, although some studies have proposed roles for PP1 and PP2A. Moreover, their relative importance varies with different cardiac pathologies and animal species; for example, PP1 mediates Cx43 dephosphorylation in ischaemic rat heart, but PP2A does not . Alterna2+]i is raised, there is an interplay between Cn and PP1 to influence gap junction electrical properties and AP conduction velocity; no role for PP2A is suggested. Moreover, this study has provided new evidence for an interplay between Cn-dependent dephosphorylation of Cx43 at S365 and phosphorylation at S368 by PKC. A consequence of this is that in normal and abnormal conditions AP conduction velocity is regulated through control of gap junction conductance.Because increased Cn expression and activity occur in most cardiac pathologies, it is plausible it contributes to the final effects of PP1 and/or PP2A, as both Cn and PP2A share similar substrates, such as I1, which once dephosphorylated at Thr35 activates PP1. Moreover, increased Cx43-dephosphorylation was observed in mouse cardiomyocytes overexpressing Cn . This stGj. Here, it was shown that with raised [Ca2+]i, slowed conduction and reduced Gj were mediated by the Ca2+-CaM dependent phosphatase calcineurin. Use of the cardiac glycoside ouabain or imposition of hypoxia to presumably raise [Ca2+]i has been shown to reduce CV as well as decrease total intracellular conductance, Gi i: a low-Na solution and rapid pacing, where CV and Gj could be independently measured; the former intervention was more convenient to raise [Ca2+]i in the oil-gap chamber.Reduced CV is a crucial determinant of re-entrant arrhythmias and occurs with rapid pacing , 24. Witnce, Gi , 45. The+ current and increased dependence of inward Ca2+ current, so it would be expected that CysA, through an action on Gj, should only partially restore CV. However, these observations are consistent with the independent demonstration that CysA, or the more specific CAIP, reversed the decrease of Gj when [Ca2+]i was raised. Thus, these data are consistent with the hypothesis that when [Ca2+]i is raised, Cn-dependent pathways reduce CV through a decrease of Gj. It has been previously shown that rapid pacing of myocardium between 4 and 6 Hz to significantly raise [Ca2+]i activates calcineurin [The Cn inhibitor, CysA, entirely reversed the slowed CV with rapid pacing and was partially effective in the low-Na solution. A slowed CV in low-Na solution would in part be due to reduced availability of Nacineurin , 43.Gj, was associated with increased dV/dtmax as local circuit current is concentrated nearer the propagating action potential (AP) wavefront. CysA reversed the increase of dV/dtmax as Gj was in turn normalised. In low-Na solution, dV/dtmax was decreased, due to reduced Na+ current during the AP upstroke, but was further reduced by CysA as CV itself partially recovered. This is also consistent with CysA increasing Gj under this condition.The actions of CysA on CV during rapid pacing and in low-Na solution are consistent with the biophysical basis of conduction , 8. RapiGj when CV when is altered in low-Na and rapid pacing conditions, in the presence and absence of CysA and under the above experimental conditions, may be made from 1-D cable theory , which could lead to mitochondrial swelling and eventual cell death. However, we suggest that this is not a significant effect as all interventions using low-Na solutions had reversible effects on electrophysiological function, suggesting no damaging effects to myocytes. Although CysA blocks the mPTP, the involvement of this mechanism may not impact significantly on the results presented here.Measurements of AP conduction velocity and gap junction conductance, is study . The incESM 1(DOCX 169 kb)


On the one hand, excessive mitochondrial Ca2+ leads to Ca2+-induced mitochondrial outer membrane permeabilization and thus apoptosis. On the other hand, mitochondria need Ca2+ in order to efficiently fuel the tricarboxylic acid cycle and maintain adequate mitochondrial bioenergetics. For obtaining this Ca2+, the mitochondria are largely dependent on close contact sites with the endoplasmic reticulum (ER), the so-called mitochondria-associated ER membranes. There, the inositol 1,4,5-trisphosphate receptors are responsible for the Ca2+ release from the ER. It comes as no surprise that this Ca2+ release from the ER and the subsequent Ca2+ uptake at the mitochondria are finely regulated. Cancer cells often modulate ER-Ca2+ transfer to the mitochondria in order to promote cell survival and to inhibit cell death. Important regulators of these Ca2+ signals and the onset of cancer are the B-cell lymphoma 2 (Bcl-2) family of proteins. An increasing number of reports highlight the ability of these Bcl-2-protein family members to finely regulate Ca2+ transfer from ER to mitochondria both in healthy cells and in cancer. In this review, we focus on recent insights into the dynamic regulation of ER–mitochondrial Ca2+ fluxes by Bcl-2-family members and how this impacts cell survival, cell death and mitochondrial energy production.Mitochondria are important regulators of cell death and cell survival. Mitochondrial Ca The mechanism involves binding of the Bcl-2 homology (BH) 3 domains of the pro-apoptotic proteins to the hydrophobic cleft formed by the BH1, BH2 and BH3 domains of the anti-apoptotic members, thereby inhibiting cell death (via a site that is distinct from Bax’s BH3 domain (The B-cell lymphoma 2 (Bcl-2)-protein family, consisting of both anti- and pro-apoptotic members, is critically involved in regulating cell death and survival –36. Dysrll death . A recenll death –42, is a3 domain . Moreove3 domain .2+ signaling at both organelles, and several Bcl-2-family members, including Bcl-2 and Bcl-XL, are present in the MAMs  also inhibits excessive pro-apoptotic IP3R-mediated Ca2+ release  2b activity. The effects were independent of the transcriptional roles of p53. Recruitment of p53 at the ER augmented the Ca2+ filling state of the ER stores, increasing the susceptibility to apoptotic stimuli and the likelihood for mitochondrial Ca2+ overload. Cells deficient in p53 did not display this effect and were resistant to chemotherapy. This resistance could be overcome by SERCA and/or MCU overexpression.Many chemotherapeutics trigger intracellular Ca effects . Moreoveeostasis , 77, 78.2+ transfer from ER to mitochondria has recently been proposed as an anti-cancer strategy  inhibited both Bcl-2 and Bcl-Xsurvival , 79. Morsurvival . Whether release . Howeverr [Ca2+] .3R/Bcl-XL complexes, ABT-737 has also been proposed to modulate the sensitivity of cancer cells to chemotherapy via a mechanism that involves remodeling of ER–mitochondrial contact sites , a stabilized TAT-linked peptide containing the 20 amino acids that represent the Bcl-2 interaction motif of IP3Rs, triggers intracellular Ca2+ overload and apoptotic cell death in a variety of cancer cell models, including chronic lymphocytic leukemia (2+ and by inhibiting IP3R activity (3R/Bcl-2 complex formation and raised cytosolic Ca2+ levels, although further work is needed to determine the contribution of Ca2+ signaling to BDA-366-induced cell death in cancer cells.Over the years, it has become clear that Bcl-2 inhibition reatment , 86–88. leukemia , diffuseleukemia , multiplleukemia , and smaleukemia . The celactivity , 89. Veractivity , 93. TheMcl-1 gene amplifications are frequently found in many types of cancer . Very re2+ transfer from ER to mitochondria is important for maintaining proper energy production and balance between cell survival and cell death. The anti-apoptotic Bcl-2-family members regulate these Ca2+ transfers at the level of the ER as well as of the mitochondria by directly targeting Ca2+-transport systems located at the ER and mitochondria. Moreover, the molecular determinants underlying the complex formation between the Bcl-2 proteins and these systems are emerging as a hot topic, which allows the development of strategies and tools to interfere with the Bcl-2 protein-mediated control of Ca2+-signaling. These mechanisms also appear to be exploited by cancer cells to promote survival and mitochondrial bioenergetics, to contribute to cell-death resistance and control metastasis. Thus, targeting the Ca2+-modulating abilities of Bcl-2 proteins may offer novel anti-cancer strategies. In addition to this, Ca2+ signaling might contribute to the cell-death properties of recently developed Bcl-2 inhibitors, including BH3-mimetics and BH4-domain antagonists.CaTV and GB drafted the manuscript. All authors critically read, amended, and/or corrected the manuscript. TV made the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.