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Background: Endothelial cell TRPV4 (transient receptor potential vanilloid 4) channels provide a control point that is pivotal in regulating blood vessel diameter by mediating the Ca 2+ -dependent release of endothelial-derived vasoactive factors. In hypertension, TRPV4-mediated control of vascular function is disrupted, but the underlying mechanisms and precise physiological consequences remain controversial., Methods: Here, using a comprehensive array of methodologies, endothelial TRPV4 channel function was examined in intact mesenteric resistance arteries from normotensive Wistar-Kyoto and spontaneously hypertensive rats., Results: Our results show there is a notable shift in vascular reactivity in hypertension characterized by enhanced endothelium-dependent vasodilation at low levels of TRPV4 channel activation. However, at higher levels of TRPV4 activity, this vasodilatory response is reversed, contributing to the aberrant vascular tone observed in hypertension. The change in response, from dilation to constriction, was accompanied by a shift in intracellular Ca 2+ signaling modalities arising from TRPV4 activity. Oscillatory TRPV4-evoked IP 3 (inositol triphosphate)-mediated Ca 2+ release, which underlies dilation, decreased, while the contraction inducing sustained Ca 2+ rise, arising from TRPV4-mediated Ca 2+ influx, increased. Our findings also reveal that while the sensitivity of endothelial cell TRPV4 to activation was unchanged, expression of the channel is upregulated and IP 3 receptors are downregulated in hypertension., Conclusions: These data highlight the intricate interplay between endothelial TRPV4 channel expression, intracellular Ca 2+ signaling dynamics, and vascular reactivity. Moreover, the data support a new unifying hypothesis for the vascular impairment that accompanies hypertension. Specifically, endothelial cell TRPV4 channels play a dual role in modulating blood vessel function in hypertension.

Background and Purpose: The single layer of cells lining all blood vessels, the endothelium, is a sophisticated signal co-ordination centre that controls a wide range of vascular functions including the regulation of blood pressure and blood flow. To co-ordinate activities, communication among cells is required for tissue level responses to emerge. While a significant form of communication occurs by the propagation of signals between cells, the mechanism of propagation in the intact endothelium is unresolved., Experimental Approach: Precision signal generation and targeted cellular manipulation was used in conjunction with high spatiotemporal mesoscale Ca 2+ imaging in the endothelium of intact blood vessels., Key Results: Multiple mechanisms maintain communication so that Ca 2+ wave propagation occurs irrespective of the status of connectivity among cells. Between adjoining cells, regenerative IP 3 -induced IP 3 production transmits Ca 2+ signals and explains the propagated vasodilation that underlies the increased blood flow accompanying tissue activity. The inositide is itself sufficient to evoke regenerative phospholipase C-dependent Ca 2+ waves across coupled cells. None of gap junctions, Ca 2+ diffusion or the release of extracellular messengers is required to support this type of intercellular Ca 2+ signalling. In contrast, when discontinuities exist between cells, ATP released as a diffusible extracellular messenger transmits Ca 2+ signals across the discontinuity and drives propagated vasodilation., Conclusion and Implications: These results show that signalling switches underlie endothelial cell-to-cell signal transmission and reveal how communication is maintained in the face of endothelial damage. The findings provide a new framework for understanding wave propagation and cell signalling in the endothelium., (© 2024 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Protease-activated receptor-1 & -2 (PAR1 and PAR2) are expressed widely in cardiovascular tissues including endothelial and smooth muscle cells. PAR1 and PAR2 may regulate blood pressure via changes in vascular contraction or relaxation mediated by endothelial Ca 2+ signaling, but the mechanisms are incompletely understood. By using single-cell Ca 2+ imaging across hundreds of endothelial cells in intact blood vessels, we explored PAR-mediated regulation of blood vessel function using PAR1 and PAR2 activators. We show that PAR2 activation evoked multicellular Ca 2+ waves that propagated across the endothelium. The PAR2-evoked Ca 2+ waves were temporally distinct from those generated by muscarinic receptor activation. PAR2 activated distinct clusters of endothelial cells, and these cells were different from those activated by muscarinic receptor stimulation. These results indicate that distinct cell clusters facilitate spatial segregation of endothelial signal processing. We also demonstrate that PAR2 is a phospholipase C-coupled receptor that evokes Ca 2+ release from the IP 3 -sensitive store in endothelial cells. A physiological consequence of this PAR2 signaling system is endothelium-dependent relaxation. Conversely, PAR1 activation did not trigger endothelial cell Ca 2+ signaling nor relax or contract mesenteric arteries. Neither did PAR1 activators alter the response to PAR2 or muscarinic receptor activation. Collectively, these results suggest that endothelial PAR2 but not PAR1 evokes mesenteric artery relaxation by evoking IP 3 -mediated Ca 2+ release from the internal store. Sensing mediated by PAR2 receptors is distributed to spatially separated clusters of endothelial cells., (© 2023 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Arteries and veins are lined by nonproliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis, rather than mitochondrial oxidative phosphorylation, to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells, and little is known about energy production in endothelial cells within the fully formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial respiration disrupts endothelial control of vascular tone. Basal, mechanically activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. Agonist-evoked calcium activity was also inhibited by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. The role for mitochondria in endothelial cell energy production is independent of species, sex, or vascular bed. These data show that a mitochondrial ATP supply is necessary for calcium-dependent, nitric oxide-mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

Every blood vessel is lined by a single layer of highly specialized, yet adaptable and multifunctional endothelial cells. These cells, the endothelium, control vascular contractility, hemostasis, and inflammation and regulate the exchange of oxygen, nutrients, and waste products between circulating blood and tissue. To control each function, the endothelium processes endlessly arriving requests from multiple sources using separate clusters of cells specialized to detect specific stimuli. A well-developed but poorly understood communication system operates between cells to integrate multiple lines of information and coordinate endothelial responses. Here, the nature of the communication network has been addressed using single-cell Ca2+ imaging across thousands of endothelial cells in intact blood vessels. Cell activities were cross-correlated and compared to a stochastic model to determine network connections. Highly correlated Ca2+ activities occurred in scattered cell clusters, and network communication links between them exhibited unexpectedly short path lengths. The number of connections between cells (degree distribution) followed a power-law relationship revealing a scale-free network topology. The path length and degree distribution revealed an endothelial network with a “small-world” configuration. The small-world configuration confers particularly dynamic endothelial properties including high signal-propagation speed, stability, and a high degree of synchronizability. Local activation of small clusters of cells revealed that the short path lengths and rapid signal transmission were achieved by shortcuts via connecting extensions to nonlocal cells. These findings reveal that the endothelial network design is effective for local and global efficiency in the interaction of the cells and rapid and robust communication between endothelial cells in order to efficiently control cardiovascular activity.

Background and Purpose: Ca 2+ influx via TRPV4 channels triggers Ca 2+ release from the IP 3 -sensitive internal store to generate repetitive oscillations. Although mitochondria are acknowledged regulators of IP 3 -mediated Ca 2+ release, how TRPV4-mediated Ca 2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca 2+ -induced Ca 2+ release at IP 3 receptors to being independent of Ca 2+ influx and instead mediated by ATP release via pannexins., Experimental Approach: TRPV4-evoked Ca 2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520., Key Results: TRPV4 activation with GSK1016790A (GSK) generated repetitive Ca 2+ oscillations that required Ca 2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca 2+ waves in the absence of external Ca 2+ . The ATP synthase inhibitor oligomycin did not potentiate TRPV4-mediated Ca 2+ signals. GSK-evoked Ca 2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca 2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid., Conclusion and Implications: These results highlight a previously unknown role of mitochondria in shaping TRPV4-mediated Ca 2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP 3 -evoked Ca 2+ release., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Photouncaging delivers compounds with high spatial and temporal control to induce or inhibit biological processes but the released compounds may diffuse out. We here demonstrate that sulfonate anions can be photocaged so that a membrane impermeable compound can enter cells, be uncaged by photoirradiation and trapped within the cell.

Background and Purpose: Coordinated endothelial control of cardiovascular function is proposed to occur by endothelial cell communication via gap junctions and connexins. To study intercellular communication, the pharmacological agents carbenoxolone (CBX) and 18β-glycyrrhetinic acid (18βGA) are used widely as connexin inhibitors and gap junction blockers., Experimental Approach: We investigated the effects of CBX and 18βGA on intercellular Ca 2+ waves, evoked by inositol 1,4,5-trisphosphate (IP 3 ) in the endothelium of intact mesenteric resistance arteries., Key Results: Acetycholine-evoked IP 3 -mediated Ca 2+ release and propagated waves were inhibited by CBX (100 μM) and 18βGA (40 μM). Unexpectedly, the Ca 2+ signals were inhibited uniformly in all cells, suggesting that CBX and 18βGA reduced Ca 2+ release. Localised photolysis of caged IP 3 (cIP 3 ) was used to provide precise spatiotemporal control of site of cell activation. Local cIP 3 photolysis generated reproducible Ca 2+ increases and Ca 2+ waves that propagated across cells distant to the photolysis site. CBX and 18βGA each blocked Ca 2+ waves in a time-dependent manner by inhibiting the initiating IP 3 -evoked Ca 2+ release event rather than block of gap junctions. This effect was reversed on drug washout and was unaffected by small or intermediate K + -channel blockers. Furthermore, CBX and 18βGA each rapidly and reversibly collapsed the mitochondrial membrane potential., Conclusion and Implications: CBX and 18βGA inhibit IP 3 -mediated Ca 2+ release and depolarise the mitochondrial membrane potential. These results suggest that CBX and 18βGA may block cell-cell communication by acting at sites that are unrelated to gap junctions., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Obesity is a major risk factor for diabetes and cardiovascular diseases such as hypertension, heart failure, and stroke. Impaired endothelial function occurs in the earliest stages of obesity and underlies vascular alterations that give rise to cardiovascular disease. However, the mechanisms that link weight gain to endothelial dysfunction are ill-defined. Increasing evidence suggests that endothelial cells are not a population of uniform cells but are highly heterogeneous and are organized as a communicating multicellular network that controls vascular function., Purpose: To investigate the hypothesis that disrupted endothelial heterogeneity and network-level organization contribute to impaired vascular reactivity in obesity., Methods and Results: To study obesity-related vascular function without complications associated with diabetes, a state of prediabetic obesity was induced in rats. Small artery diameter recordings confirmed nitric-oxide mediated vasodilator responses were dependent on increases in endothelial calcium levels and were impaired in obese animals. Single-photon imaging revealed a linear relationship between blood vessel relaxation and population-wide calcium responses. Obesity did not alter the slope of this relationship, but impaired calcium responses in the endothelial cell network. The network comprised structural and functional components. The structural architecture, a hexagonal lattice network of connected cells, was unchanged in obesity. The functional network contained sub-populations of clustered specialized agonist-sensing cells from which signals were communicated through the network. In obesity there were fewer but larger clusters of sensory cells and communication path lengths between clusters increased. Communication between neighboring cells was unaltered in obesity. Altered network organization resulted in impaired, population-level calcium signaling and deficient endothelial control of vascular tone., Conclusions: The distribution of cells in the endothelial network is critical in determining overall vascular response. Altered cell heterogeneity and arrangement in obesity decreases endothelial function and provides a novel framework for understanding compromised endothelial function in cardiovascular disease., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Recent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood., Methods: We used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC., Results: We propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients., Conclusion: We identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

Background and Purpose: FK506 and rapamycin are modulators of FK-binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side-effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP 3 receptors (IP 3 R) and ryanodine receptors (RyR) to alter Ca 2+ signalling in endothelial cells., Experimental Approach: We investigated the effects of FK506 and rapamycin on Ca 2+ release via IP 3 R and RyR in hundreds of endothelial cells, using the indicator Cal-520, in intact mesenteric arteries from male Sprague-Dawley rats. IP 3 Rs were activated by acetylcholine or localised photo-uncaging of IP 3 , and RyR by caffeine., Key Results: While FKBPs were present, FKBP modulation with rapamycin did not alter IP 3 -evoked Ca 2+ release. Conversely, FK506, which modulates FKBP and blocks calcineurin, increased IP 3 -evoked Ca 2+ release. Inhibition of calcineurin (okadiac acid or cypermethrin) also increased IP 3 -evoked Ca 2+ release and blocked FK506 effects. When calcineurin was inhibited, FK506 reduced IP 3 -evoked Ca 2+ release. These findings suggest that IP 3 -evoked Ca 2+ release is not modulated by FKBP, but by FK506-mediated calcineurin inhibition. The RyR modulators caffeine and ryanodine failed to alter Ca 2+ signalling suggesting that RyR is not functional in native endothelium., Conclusion and Implications: The hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at the documented cellular targets of Ca 2+ release and altered FKBP binding to IP 3 and RyR., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Hydrogen peroxide (H 2 O 2 ) is a mitochondrial-derived reactive oxygen species (ROS) that regulates vascular signalling transduction, vasocontraction and vasodilation. Although the physiological role of ROS in endothelial cells is acknowledged, the mechanisms underlying H 2 O 2 regulation of signalling in native, fully-differentiated endothelial cells is unresolved. In the present study, the effects of H 2 O 2 on Ca 2+ signalling were investigated in the endothelium of intact rat mesenteric arteries. Spontaneous local Ca 2+ signals and acetylcholine evoked Ca 2+ increases were inhibited by H 2 O 2 . H 2 O 2 inhibition of acetylcholine-evoked Ca 2+ signals was reversed by catalase. H 2 O 2 exerts its inhibition on the IP 3 receptor as Ca 2+ release evoked by photolysis of caged IP 3 was supressed by H 2 O 2 . H 2 O 2 suppression of IP 3 -evoked Ca 2+ signalling may be mediated by mitochondria. H 2 O 2 depolarized mitochondria membrane potential. Acetylcholine-evoked Ca 2+ release was inhibited by depolarisation of the mitochondrial membrane potential by the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or complex 1 inhibitor, rotenone. We propose that the suppression of IP 3 -evoked Ca 2+ release by H 2 O 2 arises from the decrease in mitochondrial membrane potential. These results suggest that mitochondria may protect themselves against Ca 2+ overload during IP 3 -linked Ca 2+ signals by a H 2 O 2 mediated negative feedback depolarization of the organelle and inhibition of IP 3 -evoked Ca 2+ release., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Endothelial cells line all blood vessels and are critical regulators of vascular tone. In hypertension, disruption of endothelial function alters the release of endothelial-derived vasoactive factors and results in increased vascular tone. Although the release of endothelial-derived vasodilators occurs in a Ca 2+ -dependent manner, little is known on how Ca 2+ signaling is altered in hypertension. A key element to endothelial control of vascular tone is Ca 2+ signals at specialized regions (myoendothelial projections) that connect endothelial cells and smooth muscle cells. This work describes disruption in the operation of this key Ca 2+ signaling pathway in hypertension. We show that vascular reactivity to phenylephrine is increased in hypertensive (spontaneously hypertensive rat) when compared with normotensive (Wistar Kyoto) rats. Basal endothelial Ca 2+ activity limits vascular contraction, but that Ca 2+ -dependent control is impaired in hypertension. When changes in endothelial Ca 2+ levels are buffered, vascular contraction to phenylephrine increased, resulting in similar responses in normotension and hypertension. Local endothelial IP 3 (inositol trisphosphate)-mediated Ca 2+ signals are smaller in amplitude, shorter in duration, occur less frequently, and arise from fewer sites in hypertension. Spatial control of endothelial Ca 2+ signaling is also disrupted in hypertension: local Ca 2+ signals occur further from myoendothelial projections in hypertension. The results demonstrate that the organization of local Ca 2+ signaling circuits occurring at myoendothelial projections is disrupted in hypertension, giving rise to increased contractile responses.

Rapid imaging of multiple focal planes without sample movement may be achieved through remote refocusing, where imaging is carried out in a plane conjugate to the sample plane. The technique is ideally suited to studying the endothelial and smooth muscle cell layers of blood vessels. These are intrinsically linked through rapid communication and must be separately imaged at a sufficiently high frame rate in order to understand this biologically crucial interaction. We have designed and implemented an epifluoresence-based remote refocussing imaging system that can image each layer at up to 20fps using different dyes and excitation light for each layer, without the requirement for optically sectioning microscopy. A novel triggering system is used to activate the appropriate laser and image acquisition at each plane of interest. Using this method, we are able to achieve axial plane separations down to 15  μ m, with a mean lateral stability of ≤ 0.32  μ m displacement using a 60x, 1.4NA imaging objective and a 60x, 0.7NA reimaging objective. The system allows us to image and quantify endothelial cell activity and smooth muscle cell activity at a high framerate with excellent lateral and good axial resolution without requiring complex beam scanning confocal microscopes, delivering a cost effective solution for imaging two planes rapidly. We have successfully imaged and analysed Ca 2 + activity of the endothelial cell layer independently of the smooth muscle layer for several minutes., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.)

Background and Purpose: The TRPV4 ion channels are Ca 2+ permeable, non-selective cation channels that mediate large, but highly localized, Ca 2+ signals in the endothelium. The mechanisms that permit highly localized Ca 2+ changes to evoke cell-wide activity are incompletely understood. Here, we tested the hypothesis that TRPV4-mediated Ca 2+ influx activates Ca 2+ release from internal Ca 2+ stores to generate widespread effects., Experimental Approach: Ca 2+ signals in large numbers (~100) of endothelial cells in intact arteries were imaged and analysed separately., Key Results: Responses to the TRPV4 channel agonist GSK1016790A were heterogeneous across the endothelium. In activated cells, Ca 2+ responses comprised localized Ca 2+ changes leading to slow, persistent, global increases in Ca 2+ followed by large propagating Ca 2+ waves that moved within and between cells. To examine the mechanisms underlying each component, we developed methods to separate slow persistent Ca 2+ rise from the propagating Ca 2+ waves in each cell. TRPV4-mediated Ca 2+ entry was required for the slow persistent global rise and propagating Ca 2+ signals. The propagating waves were inhibited by depleting internal Ca 2+ stores, inhibiting PLC or blocking IP 3 receptors. Ca 2+ release from stores was tightly controlled by TRPV4-mediated Ca 2+ influx and ceased when influx was terminated. Furthermore, Ca 2+ release from internal stores was essential for TRPV4-mediated control of vascular tone., Conclusions and Implications: Ca 2+ influx via TRPV4 channels is amplified by Ca 2+ -induced Ca 2+ release acting at IP 3 receptors to generate propagating Ca 2+ waves and provide a large-scale endothelial communication system. TRPV4-mediated control of vascular tone requires Ca 2+ release from the internal store., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The endothelium is the single layer of cells lining all blood vessels, and it is a remarkable cardiovascular control centre. Each endothelial cell has only a small number (on average six) of interconnected neighbours. Yet this arrangement produces a large repertoire of behaviours, capable of controlling numerous cardiovascular functions in a flexible and dynamic way. The endothelium regulates the delivery of nutrients and removal of waste by regulating blood flow and vascular permeability. The endothelium regulates blood clotting, responses to infection and inflammation, the formation of new blood vessels, and remodelling of the blood vessel wall. To carry out these roles, the endothelium autonomously interprets a complex environment crammed with signals from hormones, neurotransmitters, pericytes, smooth muscle cells, various blood cells, viral or bacterial infection and proinflammatory cytokines. It is generally assumed that the endothelium responds to these instructions with coordinated responses in a homogeneous population of endothelial cells. Here, we highlight evidence that shows that neighbouring endothelial cells are highly heterogeneous and display different sensitivities to various activators. Cells with various sensitivities process different extracellular signals into distinct streams of information in parallel, like a vast switchboard. Communication occurs among cells and new 'emergent' signals are generated that are non-linear composites of the inputs. Emergent signals cannot be predicted or deduced from the properties of individual cells. Heterogeneity and emergent behaviour bestow capabilities on the endothelial collective that far exceed those of individual cells. The implications of heterogeneity and emergent behaviour for understanding vascular disease and drug discovery are discussed., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pressure myography, one of the most commonly used techniques in vascular research, measures the diameter of isolated, pressurized arteries to assess the functional activity of smooth muscle and endothelial cells. Despite the widespread adoption of this technique for assessing vascular function, there are only a small number of commercial systems and these are expensive. Here, we introduce a complete, open source pressure myograph system and analysis software, VasoTracker, that can be set-up for approximately 10% of the cost of commercial alternatives. We report on the development of VasoTracker and demonstrate its ability to assess various components of vascular reactivity. A unique feature of the VasoTracker platform is the publicly accessible website (http://www.vasotracker.com/) that documents how to assemble and use this affordable, adaptable, and expandable pressure myograph.

Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca 2+ signaling or modify reactive oxygen species (ROS). To control Ca 2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca 2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca 2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP 3 )-mediated Ca 2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca 2+ signals occurred even when mitochondria were far from Ca 2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca 2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP 3 -mediated Ca 2+ release events. ROS buffering did not significantly alter local Ca 2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP 3 -evoked local Ca 2+ release in intact endothelium., (© 2019 Wilson et al.)

Blood flow, blood clotting, angiogenesis, vascular permeability, and vascular remodeling are each controlled by a large number of variable, noisy, and interacting chemical inputs to the vascular endothelium. The endothelium processes the entirety of the chemical composition to which the cardiovascular system is exposed, carrying out sophisticated computations that determine physiological output. Processing this enormous quantity of information is a major challenge facing the endothelium. We analyzed the responses of hundreds of endothelial cells to carbachol (CCh) and adenosine triphosphate (ATP) and found that the endothelium segregates the responses to these two distinct components of the chemical environment into separate streams of complementary information that are processed in parallel. Sensitivities to CCh and ATP mapped to different clusters of cells, and each agonist generated distinct signal patterns. The distinct signals were features of agonist activation rather than properties of the cells themselves. When there was more than one stimulus present, the cells communicated and combined inputs to generate new distinct signals that were nonlinear combinations of the inputs. Our results demonstrate that the endothelium is a structured, collaborative sensory network that simplifies the complex environment using separate cell clusters that are sensitive to distinct aspects of the overall biochemical environment and interactively compute signals from diverse but interrelated chemical inputs. These features enable the endothelium to selectively process separate signals and perform multiple computations in an environment that is noisy and variable., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Ciliopathies are a group of genetically heterogeneous disorders, characterized by defects in cilia genesis or maintenance. Mutations in the RPGR gene and its interacting partners, RPGRIP1 and RPGRIP1L , cause ciliopathies, but the function of their proteins remains unclear. Here we show that knockdown (KD) of RPGR, RPGRIP1 or RPGRIP1L in hTERT-RPE1 cells results in abnormal actin cytoskeleton organization. The actin cytoskeleton rearrangement is regulated by the small GTPase RhoA via the planar cell polarity (PCP) pathway. RhoA activity was upregulated in the absence of RPGR, RPGRIP1 or RPGRIP1L proteins. In RPGR, RPGRIP1 or RPGRIP1L KD cells, we observed increased levels of DVl2 and DVl3 proteins, the core components of the PCP pathway, due to impaired proteasomal activity. RPGR, RPGRIP1 or RPGRIP1L KD cells treated with thapsigargin (TG), an inhibitor of sarcoendoplasmic reticulum Ca 2+ - ATPases, showed impaired store-operated Ca 2+ entry (SOCE), which is mediated by STIM1 and Orai1 proteins. STIM1 was not localized to the ER-PM junction upon ER store depletion in RPGR, RPGRIP1 or RPGRIP1L KD cells. Our results demonstrate that the RPGR protein complex is required for regulating proteasomal activity and for modulating SOCE, which may contribute to the ciliopathy phenotype., Competing Interests: CONFLICTS OF INTEREST No conflicts of interest were declared.

The endothelium is the single layer of cells that lines the entire cardiovascular system and regulates vascular tone and blood-tissue exchange, recruits blood cells, modulates blood clotting, and determines the formation of new blood vessels. To control each function, the endothelium uses a remarkable sensory capability to continuously monitor vanishingly small changes in the concentrations of many simultaneously arriving extracellular activators that each provides cues to the physiological state. Here we suggest that the extraordinary sensory capabilities of the endothelium do not come from single cells but from the combined activity of a large number of endothelial cells. Each cell has a limited, but distinctive, sensory capacity and shares information with neighbours so that sensing is distributed among cells. Communication of information among connected cells provides system-level sensing substantially greater than the capabilities of any single cell and, as a collective, the endothelium solves sensory problems too complex for any single cell., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Key Points: 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., Abstract: 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., (© 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: Smooth muscle cell (SMC) phenotypic conversion from a contractile to a migratory phenotype is proposed to underlie cardiovascular disease but its contribution to vascular remodelling and even its existence have recently been questioned. Tracking the fate of individual SMCs is difficult as no specific markers of migratory SMCs exist. This study used a novel, prolonged time-lapse imaging approach to continuously track the behaviour of unambiguously identified, fully differentiated SMCs. In response to serum, highly-elongated, contractile SMCs initially rounded up, before spreading and migrating and these migratory cells displayed clear phagocytic activity. This study provides a direct demonstration of the transition of fully contractile SMCs to a non-contractile, migratory phenotype with phagocytic capacity that may act as a macrophage-like cell., Abstract: Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully differentiated, contractile SMCs reprogramme into a 'synthetic' migratory phenotype, so-called phenotypic modulation, whilst plaque macrophages are thought to derive from blood-borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long-term, high-resolution, time-lapse microscopy to track the fate of unambiguously identified, fully-differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly elongated, contractile SMCs initially rounded up, for 1-3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell-cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage-like behaviour was confirmed by their internalisation of 1 μm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low-density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling., (© 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: Age is proposed to be associated with altered structure and function of mitochondria; however, in fully-differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel-by-pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during 'flickers' of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly-elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly-motile. Mitochondria that are motile are located more peripherally in the cell than non-motile mitochondria., Abstract: Mitochondrial function, motility and architecture are each central to cell function. Age-associated mitochondrial dysfunction may contribute to vascular disease. However, mitochondrial changes in ageing remain ill-defined because of the challenges of imaging in native cells. We determined the structure of mitochondria in live native cells, demarcating boundaries of individual organelles by inducing stochastic 'flickers' of membrane potential, recorded as fluctuations in potentiometric fluorophore intensity (flicker-assisted localization microscopy; FaLM). In freshly-isolated myocytes from rat cerebral resistance arteries, FaLM showed a range of mitochondrial X-Y areas in both young adult (3 months; 0.05-6.58 μm(2) ) and aged rats (18 months; 0.05-13.4 μm(2) ). In cells from young animals, most mitochondria were small (mode area 0.051 μm(2) ) compared to aged animals (0.710 μm(2) ). Cells from older animals contained a subpopulation of highly-elongated mitochondria (5.3% were >2 μm long, 4.2% had a length:width ratio >3) that was rare in younger animals (0.15% of mitochondria >2 μm long, 0.4% had length:width ratio >3). The extent of mitochondrial motility also varied. 1/811 mitochondria observed moved slightly (∼0.5 μm) in myocytes from older animals, whereas, in the younger animals, directed and Brownian-like motility occurred regularly (215 of 1135 mitochondria moved within 10 min, up to distance of 12 μm). Mitochondria positioned closer to the cell periphery showed a greater tendency to move. In conclusion, cerebral vascular myocytes from young rats contained small, motile mitochondria. In aged rats, mitochondria were larger, immobile and could be highly-elongated. These age-associated alterations in mitochondrial behaviour may contribute to alterations in cell signalling, energy supply or the onset of proliferation., (© 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium., (© The Author(s).)

Aging is the summation of many subtle changes which result in altered cardiovascular function. Impaired endothelial function underlies several of these changes and precipitates plaque development in larger arteries. The endothelium transduces chemical and mechanical signals into changes in the cytoplasmic calcium concentration to control vascular function. However, studying endothelial calcium signaling in larger arteries in a physiological configuration is challenging because of the requirement to focus through the artery wall. Here, pressure- and agonist-sensitive endothelial calcium signaling was studied in pressurized carotid arteries from young (3-month-old) and aged (18-month-old) rats by imaging from within the artery using gradient index fluorescence microendoscopy. Endothelial sensitivity to acetylcholine increased with age. The number of cells exhibiting oscillatory calcium signals and the frequency of oscillations were unchanged with age. However, the latency of calcium responses was significantly increased with age. Acetylcholine-evoked endothelial calcium signals were suppressed by increased intraluminal pressure. However, pressure-dependent inhibition of calcium signaling was substantially reduced with age. While each of these changes will increase endothelial calcium signaling with increasing age, decreases in endothelial pressure sensitivity may manifest as a loss of functionality and responsiveness in aging., (© 2017 The Author(s) Published by S. Karger AG, Basel.)

Changes in high localised concentrations of Ca 2+ ions are fundamental to cell signalling. The synthesis of a dual excitation, ratiometric calcium ion sensor with a K d of 90 μM, is described. It is tagged with an azido group for bioconjugation, and absorbs in the blue/green and emits in the red region of the visible spectrum with a large Stokes shift. The binding modulating nitro group is introduced to the BAPTA core prior to construction of a benzofuran-2-yl carboxaldehyde by an allylation-oxidation-cyclisation sequence, which is followed by condensation with an azido-tagged thiohydantoin. The thiohydantoin unit has to be protected with an acetoxymethyl (AM) caging group to allow CuAAC click reaction and incorporation of the KDEL peptide endoplasmic reticulum (ER) retention sequence.

Key Points: Increased pressure suppresses endothelial control of vascular tone but it remains uncertain (1) how pressure is sensed by the endothelium and (2) how the vascular response is inhibited. This study used a novel imaging method to study large numbers of endothelial cells in arteries that were in a physiological configuration and held at normal blood pressures. Increased pressure suppressed endothelial IP3 -mediated Ca(2+) signals. Pressure modulated endothelial cell shape. The changes in cell shape may alter endothelial Ca(2+) signals by modulating the diffusive environment for Ca(2+) near IP3 receptors. Endothelial pressure-dependent mechanosensing may occur without a requirement for a conventional molecular mechanoreceptor., Abstract: The endothelium is an interconnected network upon which haemodynamic mechanical forces act to control vascular tone and remodelling in disease. Ca(2+) signalling is central to the endothelium's mechanotransduction and networked activity. However, challenges in imaging Ca(2+) in large numbers of endothelial cells under conditions that preserve the intact physical configuration of pressurized arteries have limited progress in understanding how pressure-dependent mechanical forces alter networked Ca(2+) signalling. We developed a miniature wide-field, gradient-index (GRIN) optical probe designed to fit inside an intact pressurized artery that permitted Ca(2+) signals to be imaged with subcellular resolution in a large number (∼200) of naturally connected endothelial cells at various pressures. Chemical (acetylcholine) activation triggered spatiotemporally complex, propagating inositol trisphosphate (IP3 )-mediated Ca(2+) waves that originated in clusters of cells and progressed from there across the endothelium. Mechanical stimulation of the artery, by increased intraluminal pressure, flattened the endothelial cells and suppressed IP3 -mediated Ca(2+) signals in all activated cells. By computationally modelling Ca(2+) release, endothelial shape changes were shown to alter the geometry of the Ca(2+) diffusive environment near IP3 receptor microdomains to limit IP3 -mediated Ca(2+) signals as pressure increased. Changes in cell shape produce a geometric microdomain regulation of IP3 -mediated Ca(2+) signalling to explain macroscopic pressure-dependent, endothelial mechanosensing without the need for a conventional mechanoreceptor. The suppression of IP3 -mediated Ca(2+) signalling may explain the decrease in endothelial activity as pressure increases. GRIN imaging provides a convenient method that gives access to hundreds of endothelial cells in intact arteries in physiological configuration., (© 2015 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic "flickers" of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension.

Mitochondrial Ca²⁺ uptake contributes important feedback controls to limit the time course of Ca²⁺ signals. Mitochondria regulate cytosolic [Ca²⁺] over an exceptional breath of concentrations (~200 nM to >10 μM) to provide a wide dynamic range in the control of Ca²⁺ signals. Ca²⁺ uptake is achieved by passing the ion down the electrochemical gradient, across the inner mitochondria membrane, which itself arises from the export of protons. The proton export process is efficient and on average there are less than three protons free within the mitochondrial matrix. To study mitochondrial function, the most common approaches are to alter the proton gradient and to measure the electrochemical gradient. However, drugs which alter the mitochondrial proton gradient may have substantial off target effects that necessitate careful consideration when interpreting their effect on Ca²⁺ signals. Measurement of the mitochondrial electrochemical gradient is most often performed using membrane potential sensitive fluorophores. However, the signals arising from these fluorophores have a complex relationship with the electrochemical gradient and are altered by changes in plasma membrane potential. Care is again needed in interpreting results. This review provides a brief description of some of the methods commonly used to alter and measure mitochondrial contribution to Ca²⁺ signaling in native smooth muscle., (© 2013 John Wiley & Sons Ltd.)

Increases in bulk average cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) are derived from the combined activities of many Ca(2+) channels. Near (<100 nm) the mouth of each of these channels the local [Ca(2+)](c) rises and falls more quickly and reaches much greater values than occurs in the bulk cytoplasm. Even during apparently uniform, steady-state [Ca(2+)] increases large local inhomogeneities exist near channels. These local increases modulate processes that are sensitive to rapid and large changes in [Ca(2+)] but they cannot easily be visualized with conventional imaging approaches. The [Ca(2+)] changes near channels can be examined using total internal reflection fluorescence microscopy (TIRF) to excite fluorophores that lie within 100 nm of the plasma membrane. TIRF is particularly powerful when combined with electrophysiology so that ion channel activity can be related simultaneously to the local subplasma membrane and bulk average [Ca(2+)](c). Together these techniques provide a better understanding of the local modulation and control of Ca(2+) signals.

The diversity of mitochondrial arrangements, which arise from the organelle being static or moving, or fusing and dividing in a dynamically reshaping network, is only beginning to be appreciated. While significant progress has been made in understanding the proteins that reorganise mitochondria, the physiological significance of the various arrangements is poorly understood. The lack of understanding may occur partly because mitochondrial morphology is studied most often in cultured cells. The simple anatomy of cultured cells presents an attractive model for visualizing mitochondrial behaviour but contrasts with the complexity of native cells in which elaborate mitochondrial movements and morphologies may not occur. Mitochondrial changes may take place in native cells (in response to stress and proliferation), but over a slow time-course and the cellular function contributed is unclear. To determine the role mitochondrial arrangements play in cell function, a crucial first step is characterisation of the interactions among mitochondrial components. Three aspects of mitochondrial behaviour are described in this review: (1) morphology, (2) motion and (3) rapid shape changes. The proposed physiological roles to which various mitochondrial arrangements contribute and difficulties in interpreting some of the physiological conclusions are also outlined., (© 2013 S. Karger AG, Basel.)

Objective: Mitochondria are widely described as being highly dynamic and adaptable organelles, and their movement is thought to be vital for cell function. Yet, in various native cells, including those of heart and smooth muscle, mitochondria are stationary and rigidly structured. The significance of the differences in mitochondrial behavior to the physiological function of cells is unclear and was studied in single myocytes and intact resistance-sized cerebral arteries. We hypothesized that mitochondrial dynamics is controlled by the proliferative status of the cells., Methods and Results: High-speed fluorescence imaging of mitochondria in live vascular smooth muscle cells shows that the organelle undergoes significant reorganization as cells become proliferative. In nonproliferative cells, mitochondria are individual (≈ 2 μm by 0.5 μm), stationary, randomly dispersed, fixed structures. However, on entering the proliferative state, mitochondria take on a more diverse architecture and become small spheres, short rod-shaped structures, long filamentous entities, and networks. When cells proliferate, mitochondria also continuously move and change shape. In the intact pressurized resistance artery, mitochondria are largely immobile structures, except in a small number of cells in which motility occurred. When proliferation of smooth muscle was encouraged in the intact resistance artery, in organ culture, the majority of mitochondria became motile and the majority of smooth muscle cells contained moving mitochondria. Significantly, restriction of mitochondrial motility using the fission blocker mitochondrial division inhibitor prevented vascular smooth muscle proliferation in both single cells and the intact resistance artery., Conclusions: These results show that mitochondria are adaptable and exist in intact tissue as both stationary and highly dynamic entities. This mitochondrial plasticity is an essential mechanism for the development of smooth muscle proliferation and therefore presents a novel therapeutic target against vascular disease.

Increases in cytosolic Ca(2+) concentration ([Ca(2+)](c)) mediated by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3), hereafter InsP(3)] regulate activities that include division, contraction and cell death. InsP(3)-evoked Ca(2+) release often begins at a single site, then regeneratively propagates through the cell as a Ca(2+) wave. The Ca(2+) wave consistently begins at the same site on successive activations. Here, we address the mechanisms that determine the Ca(2+) wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP(3) receptors (InsP(3)R) to InsP(3) nor regional clustering of muscarinic receptors (mAChR3) or InsP(3)R1 explained the selection of an initiation site. However, examination of the overlap of mAChR3 and InsP(3)R1 localisation, by centre of mass analysis, revealed that there was a small percentage (∼10%) of sites that showed colocalisation. Indeed, the extent of colocalisation was greatest at the Ca(2+) wave initiation site. The initiation site might arise from a selective delivery of InsP(3) from mAChR3 activity to particular InsP(3)Rs to generate faster local [Ca(2+)](c) increases at sites of colocalisation. In support of this hypothesis, a localised subthreshold 'priming' InsP(3) concentration applied rapidly, but at regions distant from the initiation site, shifted the wave to the site of the priming. Conversely, when the Ca(2+) rise at the initiation site was rapidly and selectively attenuated, the Ca(2+) wave again shifted and initiated at a new site. These results indicate that Ca(2+) waves initiate where there is a structural and functional coupling of mAChR3 and InsP(3)R1, which generates junctions in which InsP(3) acts as a highly localised signal by being rapidly and selectively delivered to InsP(3)R1.

Adenosine 5'-triphosphate (ATP) mediates a variety of biological functions following nerve-evoked release, via activation of either G-protein-coupled P2Y- or ligand-gated P2X receptors. In smooth muscle, ATP, acting via P2Y receptors (P2YR), may act as an inhibitory neurotransmitter. The underlying mechanism(s) remain unclear, but have been proposed to involve the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] by phospholipase C (PLC), to evoke Ca(2+) release from the internal store and stimulation of Ca(2+)-activated potassium (K(Ca)) channels to cause membrane hyperpolarization. This mechanism requires Ca(2+) release from the store. However, in the present study, ATP evoked transient Ca(2+) increases in only ∼10% of voltage-clamped single smooth muscle cells. These results do not support activation of K(Ca) as the major mechanism underlying inhibition of smooth muscle activity. Interestingly, ATP inhibited Ins(1,4,5)P(3)-evoked Ca(2+) release in cells that did not show a Ca(2+) rise in response to purinergic activation. The reduction in Ins(1,4,5)P(3)-evoked Ca(2+) release was not mimicked by adenosine and therefore, cannot be explained by hydrolysis of ATP to adenosine. The reduction in Ins(1,4,5)P(3)-evoked Ca(2+) release was, however, also observed with its primary metabolite, ADP, and blocked by the P2Y(1)R antagonist, MRS2179, and the G protein inhibitor, GDPβS, but not by PLC inhibition. The present study demonstrates a novel inhibitory effect of P2Y(1)R activation on Ins(1,4,5)P(3)-evoked Ca(2+) release, such that purinergic stimulation acts to prevent Ins(1,4,5)P(3)-mediated increases in excitability in smooth muscle and promote relaxation.

Ca(2+) may selectively activate various processes in part by the cell's ability to localize changes in the concentration of the ion to specific subcellular sites. Interestingly, these Ca(2+) signals begin most often at the plasma membrane space so that understanding subplasma membrane signals is central to an appreciation of local signaling. Several experimental procedures have been developed to study Ca(2+) signals near the plasma membrane, but probably the most prevalent involve the use of fluorescent Ca(2+) indicators and fall into two general approaches. In the first, the Ca(2+) indicators themselves are specifically targeted to the subplasma membrane space to measure Ca(2+) only there. Alternatively, the indicators are allowed to be dispersed throughout the cytoplasm, but the fluorescence emanating from the Ca(2+) signals at the subplasma membrane space is selectively measured using high resolution imaging procedures. Although the targeted indicators offer an immediate appeal because of selectivity and ease of use, their limited dynamic range and slow response to changes in Ca(2+) are a shortcoming. Use of targeted indicators is also largely restricted to cultured cells. High resolution imaging applied with rapidly responding small molecule Ca(2+) indicators can be used in all cells and offers significant improvements in dynamic range and speed of response of the indicator. The approach is technically difficult, however, and realistic calibration of signals is not possible. In this review, a brief overview of local subplasma membrane Ca(2+) signals and methods for their measurement is provided., (Copyright © 2012 Wiley Periodicals, Inc.)

The cytosolic Ca²⁺ concentration ([Ca²⁺]c) controls virtually every activity of smooth muscle, including contraction, migration, transcription, division and apoptosis. These processes may be activated by large (>10 μM) amplitude [Ca²⁺]c increases, which occur in small restricted regions of the cell or by smaller (<1 μM) amplitude changes throughout the bulk cytoplasm. Mitochondria contribute to the regulation of these signals by taking up Ca²⁺. However, mitochondria's reported low affinity for Ca²⁺ is thought to require the organelle to be positioned close to ion channels and within a microdomain of high [Ca²⁺]. In cultured smooth muscle, mitochondria are highly dynamic structures but in native smooth muscle mitochondria are immobile, apparently strategically positioned organelles that regulate the upstroke and amplitude of IP₃-evoked Ca²⁺ signals and IP₃ receptor (IP₃R) cluster activity. These observations suggest mitochondria are positioned within the high [Ca²⁺] microdomain arising from an IP₃R cluster to exert significant local control of channel activity. On the other hand, neither the upstroke nor amplitude of voltage-dependent Ca²⁺ entry is modulated by mitochondria; rather, it is the declining phase of the transient that is regulated by the organelle. Control of the declining phase of the transient requires a high mitochondrial affinity for Ca²⁺ to enable uptake to occur over the normal physiological Ca²⁺ range (<1 μM). Thus, in smooth muscle, mitochondria regulate Ca²⁺ signals exerting effects over a large range of [Ca²⁺] (∼200 nM to at least tens of micromolar) to provide a wide dynamic range in the control of Ca²⁺ signals.

Mitochondria may function as multiple separate organelles or as a single electrically coupled continuum to modulate changes in [Ca2+]c (cytoplasmic Ca2+ concentration) in various cell types. Mitochondria may also be tethered to the internal Ca2+ store or plasma membrane in particular parts of cells to facilitate the organelles modulation of local and global [Ca2+]c increases. Differences in the organization and positioning contributes significantly to the at times apparently contradictory reports on the way mitochondria modulate [Ca2+]c signals. In the present paper, we review the organization of mitochondria and the organelles role in Ca2+ signalling.

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity., (© 2011 American Chemical Society)

Smooth muscle responds to IP(3)-generating agonists by producing Ca(2+) waves. Here, the mechanism of wave progression has been investigated in voltage-clamped single smooth muscle cells using localized photolysis of caged IP(3) and the caged Ca(2+) buffer diazo-2. Waves, evoked by the IP(3)-generating agonist carbachol (CCh), initiated as a uniform rise in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) over a single though substantial length (approximately 30 microm) of the cell. During regenerative propagation, the wave-front was about 1/3 the length (approximately 9 microm) of the initiation site. The wave-front progressed at a relatively constant velocity although amplitude varied through the cell; differences in sensitivity to IP(3) may explain the amplitude changes. Ca(2+) was required for IP(3)-mediated wave progression to occur. Increasing the Ca(2+) buffer capacity in a small (2 microm) region immediately in front of a CCh-evoked Ca(2+) wave halted progression at the site. However, the wave front does not progress by Ca(2+)-dependent positive feedback alone. In support, colliding [Ca(2+)](c) increases from locally released IP(3) did not annihilate but approximately doubled in amplitude. This result suggests that local IP(3)-evoked [Ca(2+)](c) increases diffused passively. Failure of local increases in IP(3) to evoke waves appears to arise from the restricted nature of the IP(3) increase. When IP(3) was elevated throughout the cell, a localized increase in Ca(2+) now propagated as a wave. Together, these results suggest that waves initiate over a surprisingly large length of the cell and that both IP(3) and Ca(2+) are required for active propagation of the wave front to occur.

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP(3))-mediated increases in cytosolic Ca2+ concentration ([Ca2+](c)). Local Ca2+ release from an InsP(3) receptor (InsP(3)R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP(3)R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP(3)-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP(3)R. Mitochondria may regulate InsP(3)-mediated Ca2+ signals by operating between or within InsP(3)R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP(3)-mediated Ca2+ release by operating within (local) or between (global) InsP(3)R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP(3) in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5-4.0 F/F(0), durations of approximately 112 ms at half-maximum amplitude, and were abolished by the InsP(3)R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized DeltaPsi(M) to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by approximately 66 or approximately 60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by approximately 62%. The "fast" Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acted like mitochondria to prolong InsP(3)-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP(3)R communication at the intra-cluster level and that mitochondria regulate both local and global InsP(3)-mediated Ca2+ signals.

n vascular smooth muscle cells, Ca2+ release via IP(3) receptors (IP(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca2+ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca2+ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca2+ store is a single continuous interconnected network or multiple separate Ca2+ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca2+ available throughout the store was examined using localized photolysis of caged-IP3 and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP(3)R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP(3)R and RyR was regulated by the Ca2+ concentration within the SR (luminal [Ca2+]). As the luminal [Ca2+] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP(3)R. The SR Ca2+ store is a single luminally continuous entity which contains both IP(3)R and RyR and within which Ca2+ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP3R and RyR by luminal [Ca2+] explains the appearance of multiple stores in some functional studies.

Mitochondria modulate cellular Ca2+ signals by accumulating the ion via a uniporter and releasing it via Na+- or H+-exchange. In smooth muscle, inhibition of mitochondrial Ca2+ uptake inhibits Ca2+ release from the sarcoplasmic reticulum (SR) via inositol-1,4,5-trisphosphate-sensitive receptors (IP(3)R). At least two mechanisms may explain this effect. First, localised uptake of Ca2+ by mitochondria may prevent negative feedback by cytosolic Ca2+ on IP(3)R activity, or secondly localised provision of Ca2+ by mitochondrial efflux may maintain IP(3)R function or SR Ca2+ content. To distinguish between these possibilities the role of mitochondrial Ca2+ efflux on IP(3)R function was examined. IP(3) was liberated in freshly isolated single colonic smooth muscle cells and mitochondrial Na+-Ca2+ exchanger inhibited with CGP-37157 (10microM). Mitochondria accumulated Ca2+ during IP(3)-evoked [Ca2+](c) rises and released the ion back to the cytosol (within approximately 15s) when mitochondrial Ca2+ efflux was active. When mitochondrial Ca2+ efflux was inhibited by CGP-37157, an extensive and sustained loading of mitochondria with Ca2+ occurred after IP(3)-evoked Ca2+ release. IP(3)-evoked [Ca2+](c) rises were initially unaffected, then only slowly inhibited by CGP-37157. IP(3)R activity was required for inhibition to occur; incubation with CGP-37157 for the same duration without IP(3) release did not inhibit IP(3)R. CGP-37157 directly inhibited voltage-gated Ca2+ channel activity, however SR Ca2+ content was unaltered by the drug. Thus, the gradual decline of IP(3)R function that followed mitochondrial Na+-Ca2+ exchanger inhibition resulted from a gradual overload of mitochondria with Ca2+, leading to a reduced capacity for Ca2+ uptake. Localised uptake of Ca2+ by mitochondria, rather than mitochondrial Ca2+ efflux, appears critical for maintaining IP(3)R activity.

The study of mitochondria and mitochondrial Ca2+ signalling in localised regions is hampered by the lack of tools that can uncouple the mitochondrial membrane potential (DeltaPsi(m)) in a spatially predefined manner. Although there are a number of existing mitochondrial uncouplers, these compounds are necessarily membrane permeant and therefore exert their actions in a spatially unselective manner. Herein, we report the synthesis of the first caged (photolabile protected) mitochondrial uncouplers, based on the tyrphostin AG10. We have analysed the laser photolysis of these compounds, using (1)H NMR and HPLC, and demonstrate that the major product of caged AG10 photolysis is AG10. It is shown that photolysis within single smooth muscle cells causes a collapse of DeltaPsi(m) consistent with photorelease of AG10. Furthermore, the effect of the photoreleased AG10 is localised to a subcellular region proximal to the site of photolysis, demonstrating for the first time spatially predefined mitochondrial uncoupling.

In smooth muscle, the gating of dihydropyridine-sensitive Ca(2+) channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca(2+) influx and determining the bulk average cytoplasmic Ca(2+) concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca(2+) signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (-70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca(2+) current (I(Ca)) and evoked a rise in [Ca(2+)] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca(2+)] increase ([Ca(2+)](c)) was approximately uniform, whereas that of the subplasma membrane space ([Ca(2+)](PM)) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca(2+) channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca(2+) channels were not normally constitutively active. The repetitive localized [Ca(2+)](PM) rises ("persistent Ca(2+) sparklets") that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca(2+) channels regulate the bulk average [Ca(2+)](c). A dihydropyridine blocker of Ca(2+) channels, nimodipine, which blocked I(Ca) and accompanying [Ca(2+)](c) rise, reduced neither the resting bulk average [Ca(2+)](c) (at -70 mV) nor the rise in [Ca(2+)](c), which accompanied an increased electrochemical driving force on the ion by hyperpolarization (-130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca(2+) channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca(2+)](c) regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca(2+)].