Dihydropyridine (DHP) Ca2+ antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca2+ oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT3 receptor antagonist suppressed spontaneous Ca2+ activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT3 receptor agonist, restored it. GR113808, a selective antagonist for 5-HT4, and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT3 receptors, had little effect on ICC Ca2+ activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT3 receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT3 receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome.Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HT's actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca Special interstitial cells with abundant c-Kit receptors on their surface are distributed throughout the gastrointestinal tract. These cells are referred to as interstitial cells of Cajal (ICC) due to the histological features of the network Serotonin (5-hydroxytryptamine: 5-HT), well known for mood control in the brain, also plays a crucial role in cellular signalling in the gut. Indeed, enterochromaffin cells release the majority (>90%) of 5-HT in the human body in response to the pressure of intraluminal content and other noxious stimuli 2+ concentration ([Ca2+]i) in ICC cells underlie gut pacemaker activity. Namely, periodic activation of Ca2+-sensitive ion channels in the plasma membrane generates pacemaker potentials 2+]i oscillations in ICC, and that coordinated actions of intracellular Ca2+ release channels and transmembrane Ca2+ influx pathways underlie ICC [Ca2+]i oscillations It is thought that oscillations of the intracellular (cytosolic) Ca2+ imaging and potential mapping of ICC pacemaker activity using fluorescent Ca2+ probes and microelectrode array (MEA), respectively, and found that 5-HT enhances both Ca2+ and electric activities of ICC via 5-HT3 receptors, which are nonselective cation channels permeable to Ca2+. We also carried out RT-PCR and immunostaining to confirm the expression of 5-HT3 receptors in ICC. Our findings suggest that 5-HT modulation of ICC activity should also be considered for gut motility disorders, for example, irritable bowel syndrome with a prevalence of around 10% In the present study, we provide evidence that 5-HT regulates ICC pacemaker activity. We performed CavW/W mice (3–6 weeks after birth) were killed by cervical dislocation, after being anaesthetized with diethyl ether. Unless otherwise stated, BALB/c mice were used in all experiments.Animals used in the present study were treated ethically. All procedures were approved by the Institutional Animal Care and Use Committee. BALB/c (wild-type) and 2+ imaging 2+ activity in isolated ileal musculature segments containing the myenteric plexus , trypsin inhibitor , ATP , and bovine serum albumin  for 40 min at 37°C. The musculature preparation was then triturated with fire-blunted glass pipettes. The resultant cell clusters were plated onto a lab-made culture dish, and kept in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% foetal bovine serum (Sigma) and antibiotics  at 37°C for 2–3 days.The musculature along with the myenteric plexus were carefully dissected from the ileum, and incubated in CaF) were expressed as Ft/F0, where F0 is the basal fluorescence intensity. Ratio-images were constructed by dividing each Ca2+ image with a Ca2+ image acquired at a basal [Ca2+]i time after subtracting background fluorescence. The frequency of spontaneous [Ca2+]i oscillations in the presence of nifeipine (and TTX) did not differ from that of spontaneous oscillatory inward currents previously measured by the patch clamp technique 2+]i activity reflected pacemaker activity in ICC. This notion also agrees well with previous reports in which dihydropyridine (DHP) Ca2+ antagonists selectively suppress [Ca2+]i activity in smooth muscle by blocking L-type Ca2+ channels The cultured cell cluster preparations were incubated in ‘normal’ solution containing approximately 8 µM Fluo3-AM  and detergents  for 3–4 h at room temperature. A CCD camera system  was used to continuously monitor digital images of Fluo-3 emission light. The cell clusters were illuminated at 488 nm and emission light of 515–565 nm was detected. The temperature of the recording chamber was kept at 35°C using a micro-warm plate system . Digital images (0.963 µm/pixel) were normally collected at 300 ms intervals. Changes in fluorescence emission intensity . The active area of [Ca2+]i oscillations was nearly the same (102% of the control) at 1–3 µM, and increased to ∼145% at 10 µM and to ∼149% at 50 µM. We thus compared the effects of 5-HT and other 5-HT receptor agonists at 10 µM  connected to a 64-channel amplifier  was used to simultaneously record electrical field potentials of ∼1 mmIn field potential data processing, the digital resolution was reduced to 50 ms (20 Hz) by thinning out the recording points at 1∶1000, and an FFT-based digital band-pass filter (0.04–0.5 Hz) was additionally applied. The effects of 5-HT and 2-Me-5-HT on ICC pacemaker electrical activity were evaluated using a power spectrum (9.4–27.0 cpm). Two-dimensional field potential images were constructed by calculating the values at the desired location via spline interpolation (with 50 points between each electrode), using the MATLAB software package  A longer (50 min) enzymatic incubation and more complete trituration with glass pipettes were performed to obtain isolated cells. The digestive enzymes used were the same as described for cell cluster preparations. The resultant cell suspension was incubated in a ‘normal’ extracellular solution containing phycoerythrin-conjugated anti-mouse CD117 (c-Kit) antibody  in 1/100 v/v for 10 min, and then centrifuged and rinsed with ‘normal’ solution three times. About 5–10 isolated smooth muscle cells and c-Kit-immunopositive cells were separately collected into sterile tubes, using patch pipettes  with 10–20 µm tip diameter under a fluorescent microscope. The samples were kept at −80°C until use. The procedures for subsequent RT-PCR were the essentially the same as previously described 2B : 5′-GATCAACCCTGCCATGTACCA-3′ (+) and 5′-CGCCATCGTTTTCAGTGAGA-3′ (-); 5-HT3A : 5′-GACTCCTGAGGACTTCGACAATG-3′ (+) and 5′-ACTTCCCCACGTCCACAAACT-3′ (-); 5-HT3B : 5′-ACTCTTCTGGCACCATTAGAACC-3′ (+) and 5′-GAGGCTGCAGTTCTGGATATCA-3′ (-); 5-HT4 : 5′-CTTTCCTCTGGCTTGGCTATATCA-3′ (+) and 5′-GTCTTTTGTAGCGCTCATCATCAC-3′ (-); c-Kit : 5′-GAGCCTTCCTGTGACAGTTCAAAT-3′ (+) and 5′-TCTATTCTTGCGGATCTCCTCTTG-3′ (-); glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) : 5′-CATGGCCTTCCGTGTTCCT-3′ (+) and 5′-CCTGCTTCACCACCTTCTTGA-3′ (-); CD68 (a marker for mast cells) : 5′-CACCTGTCTCTCTCATTTCC-3′(+) and 5′-CTTAGAGAGAGCAGGTGAAG-3′ (-); Cma1 (a marker for macrophages) : 5′-CGGGAAGGTCTATAACAGTCCTCC-3′ (+) and 5′-CTGGTGAAGTGTTTGCAGGCT-3′(-). The pair of primers for 5-HT4 was also designed to cover 5-HT4A, 5-HT4B, 5-HT4E, and 5-HT4F.The following PCR primers were used: 5-HT3 antibody  and anti-mouse CD117 (c-Kit) antibody  in 100 mM PBS  overnight. The PBS contained 1% BSA in order to block non-specific reactions. This was followed by incubation with secondary antibodies, Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 555-conjugated anti-rat IgG  at a concentration of 15 µg/ml for 1 h. Double-stained small segments were mounted on a slide glass with an anti-fading agent (ProLong: Molecular Probes) and scanned using a confocal microscope . Controls were prepared by omitting the primary antibodies. The reactivity was negligible in network-forming cells in the myenteric plexus (i.e. ICC). The antibody used for staining the 5-HT3 receptor (sc-28958: Santa Cruz Biotechnology) is a rabbit polyclonal antibody raised against amino acids 341–478 mapping at the C-terminus of 5-HT3A of human origin, and is used in human, mouse and rat specimens.Small segments (10 mm×20 mm) of smooth muscle layers (including the myenteric plexus) isolated from the mouse ileum, were fixed with 4% paraformaldehyde (4°C) for 30 min, and permeabilized with 0.1% triton X-100 and 5% BSA  for 1 h. The tissue was double stained sequentially with anti-5-HT2+]i imaging and electrical recording was (in mM): NaCl, 125; KCl, 5.9; MgCl2 1.2; CaCl2 2.4; glucose 11; Tris-HEPES 11.8 (pH 7.4). Nifedipine, LY-278584, 2-methyl-5-HT , GR113808 and O-methyl-5-HT (hydrochloride) were purchased from Sigma. SK&F96365 was from Calbiochem . Stock solutions of nifedipine were prepared by dissolving the drug in ethanol, while other drugs were dissolved in dimethyl sulfoxide (DMSO). The working concentrations of the solvents were less than 1%. In preliminary experiments, we observed that applications of this concentration of either solvent alone had little effect on [Ca2+]i oscillation in cell cluster preparations. DMEM and other reagents for cell culture were purchased from Sigma.The composition of the ‘normal’ extracellular solution used in [Cat-tests. The probability (P)<0.05 was taken as a statistically significant difference.Numerical data are expressed as mean±SD. Differences between means were evaluated by paired 2+]i oscillations 2+]i activity in ICC, we used cell cluster preparations (100–200 µm in diameter) obtained from the mouse ileum. This preparation contains ICC, smooth muscle cells and enteric neurons, and is considered to be an integrated model system to investigate gut pacemaker activity. Dihydropyridine (DHP) Ca2+ antagonists can selectively suppress [Ca2+]i and contractile activities of smooth muscle cells by blocking L-type Ca2+ channels, while pacemaking [Ca2+]i oscillations in ICC persist 2+]i imaging in the presence of nifedipine.ICC generate pacemaker activity employing [Ca2+]i oscillations were observed in a small limited area (near (x)) [control: Panels (a–b) in 2+]i oscillations [Panels (c–d)]. The time course of changes in [Ca2+]i (Ft/F0) is plotted in 2+]i oscillations with an amplitude of more than 1.2 (Ft/F0), while such activity was negligible in ROI (y) and (z). After application of 5-HT, the area yielding spontaneous [Ca2+]i oscillations enlarged, covering all three regions (x–z) synchronously. The peak amplitude in ROI (y) reached 80–90% of that in (x). On average, the area yielding [Ca2+]i oscillations increased from 11.5±6.0 to 42.5±18.3% (n = 22), while the frequency was little affected   . Essenti2+]i oscillations. 5-HT (10 µM) caused the activity towards a continuous oscillation pattern, with a significant enlargement of the oscillation active area (n = 6) (An interesting response was observed in preparations showing intermittent [Ca (n = 6) .2+]i imaging was carried out in ileal cell cluster preparations from vW/W mice, which have few pacemaking interstitial cells in this part of the small intestine   . This re2+]i activity. In these experiments, we used preparations showing synchronous [Ca2+]i oscillations (like the preparation shown in 3) significantly decreased the amplitude of the oscillations, and the intervals between them often became irregular  .Next, we assessed which receptors are responsible for 5-HT-mediated pacemaker [Ca3 receptors. Application of O-Me-5-HT (10 µM) caused no recovery of spontaneous [Ca2+]i activity in the presence of LY-278584 (10 µM) is a known non-selective 5-HT receptor agonist, but lacking affinity for 5-HTM)n = 4, . Essenti3 antagonists, application of 10 µM GR113808, a selective antagonist for 5-HT4, had little effect on spontaneous [Ca2+]i oscillations in ICC .To confirm expression of 5-HT receptors in ileal ICC, we performed RT-PCR. After enzymatic dispersion, c-Kit immunopositive interstitial cells  and smooth muscle cells were individually collected. A transcript for 5-HTle cells . We alsole cells . In addi3 antibodies (3 and c-Kit (orange cells in left panel) exist near the myenteric plexus, which contains neurons with only 5-HT3 immunoreactivity (green). In addition, these network-forming cells have large nuclei (a single cell is shown expanded in the right panels). The network-like structure and large nuclei are known features of ICC Immunohistochemistry was also performed. Double-labelled immunostaining with anti-c-Kit and anti-5-HTtibodies  revealed2 area were simultaneously monitored through a multi-channel amplifier and recording system  with a polar distance of 150 µm, and field potentials of a ∼1 mmstem See . To suppA potential map was constructed by using the simultaneous recordings at 64 channels and spline interpolation . In this3 receptor agonist (Pw9.4-27.0cpm) was used to evaluate the ICC electrical activity, based on a comparison between wild-type and vW/W mice Pw9.4–27.0cpm (from 64 channels) ranged from 1.0 to 19.0×10−3 µV2 (n = 24). Application of 5-HT increased Pw9.4–27.0cpm to 168±45% , and 2-Me-5-HT increased it to 153±35% .The effect of 2-Me-5-HT (10 µM), a 5-HT agonist , on ICC ICC act as gut pacemaker cells. Moreover, fairly recent studies suggest that these cells also coordinate peristaltic movements through their network-forming structure 3 receptors are responsible for the 5-HT-mediated enhancement of ICC Ca2+ activity. Lines of studies have suggested that the primary pacemaking activity is the spontaneous [Ca2+]i oscillations in ICC 2+]i oscillations in ICC periodically activate plasmalemmal Ca2+-activated ion channels: Ca2+-activated Cl− channels 2+-activated non-selective cation channels 2+ release from the intracellular Ca2+ stores, presumably endoplasmic reticulum (ER), appears to be the major Ca2+ source of [Ca2+]i oscillations in ICC, while Ca2+ influx from the extracellular space is required to maintain this [Ca2+]i activity (periodic Ca2+ release) 2+ pathways are not yet understood. Since 5-HT3 receptors are ligand-gated non-selective cation channels, permeable to Ca2+2+ influx pathway to enhance pacemaker activity in ICC  are known to form the 5-HT3 receptor complex, and changes in the composition alter Ca2+-permeability of this channel 3A-E receptor subtypes and how 5-HT3 receptors are coupled to Ca2+ release channels in intracellular Ca2+ stores to generate pacemaker [Ca2+]i activity. Polymorphism of these 5-HT3 receptor subunit genes seems likely to affect gut motility by modulating ICC as well as neuronal activities 2 and 5-HT4 receptor genes 2B receptor antagonists reduced proliferation of cultured ICC, and that the small intestine of mice lacking 5-HT2B receptors contains less ICC in the myenteric and deep muscular plexuses, although intestinal transit is not significantly slowed 4 receptor antagonists impair the regeneration of enteric neurons after surgical operation and their development in gut-like organs derived from mouse embryonic stem cells, with indistinguishable changes in the ICC network Five subunits (i.e. 5-HT2+ antagonists), in addition to network-forming processes in ICC, the smooth muscle bundle conducts a part of electric current generated by a group of ICC, and amplifies pacemaker activity in adjacent ICC, because it is thought that ICC possess a mechanism to transform depolarisation in the plasma membrane into activation of [Ca2+]i oscillations for pacemaking The scenario for 5-HT augmentation of ICC activity is possibly modified by the roles of adjacent cells in the actual gut. In the present study, we applied nifedipine and TTX to clearly demonstrate the effect of 5-HT on ICC; however, smooth muscle cells and enteric neurones suppressed by these inhibitors may also be involved, because coordinated actions of these cells produce gut motility 3 receptors implies pharmacological interventions on gut motility disorders. For example, irritable bowel syndrome, classified into two types, i.e. diarrhoea- and constipation-dominant IBS (d-IBS and c-IBS), is known to involve 5-HT-related mechanisms along with infectious and inflammatory changes. Excess 5-HT due to impairment of reuptake transport is ascribed to some populations of d-IBS 3 receptors. It is speculated that stimulation of 5-HT3 receptors in enteric neurons and ICC synergically facilitates gut contractility and afferent neural activity toward the brain. Thereby, 5-HT3 receptors in the gut may contribute to the gut-brain axis. As seen in murine ileal ICC, we have also observed that 5-HT3 receptor agonists potentiate, while antagonists suppress both Ca2+ and electric pacemaker activities in the murine stomach in preliminary experiments. Although extensive studies are required in model animals and humans, 5-HT is likely to enhance ICC pacemaker activity throughout the gastrointestinal tract.The present finding on augmentation of ICC activity via 5-HT2+]i and electric activities have already been demonstrated. In the light of regulatory mechanisms of ICC and ICC-like cells, investigating functional disorders related to a wide range of peripheral spontaneous rhythmicity, e.g. irritable bladder, is merited.Similar regulatory mechanisms may underlie other peripheral spontaneous activities. Evidence is being accumulated that ICC-like interstitial cells ubiquitously exist in many organs and tissues that are effectors of the autonomic nervous system, such as the ureter, urinary bladder, urethra, uterus, lymph ducts, veins, etc, suggesting their possible contribution to spontaneous activity 3 receptors. Since 5-HT3 receptors are Ca2+-permeable nonselective cation channels, this effect on ICC activity is presumably through enhancement of Ca2+ influx from the extracellular space, through itself and simultaneous activation of voltage-gated Ca2+-permeable channels. ICC appear to be a promising target in functional motility disorders in the gut.In summary, the present study has shown 5-HT augmentation of ICC pacemaker activity via 5-HTFigure S12+]i oscillations in an ileal musculature preparation. Ileal musculature segments (∼5 mm×20 mm) containing the myenteric plexus, the same preparation used in 8×8 MEA measurements, were loaded with Fluo-3AM. Fluo-3 emission light images were continuously monitored the same as cell cluster experiments. The extracellular solution contained 1 µM nifedipine and 250 nM TTX. A) A control fluorescent image. B and C) Series of ratio images of a [Ca2+]i oscillation cycle in control and in the presence of 5-HT (10 µM), respectively. Each image was acquired at 300 ms intervals. Ft/F0 ratio of ∼1 in cell cluster preparations (An example of 5-HT-augmentation of ICC pacemaker [Caarations  to displ(TIF)Click here for additional data file.Figure S22+]i oscillations after application of 5-HT. A) Ca2+ images acquired from a cell cluster preparation in control (a–c) and 5 min after 5-HT (10 µM) application . This preparation showed intermittent [Ca2+]i oscillations in control condition. B) Time course of pacemaker [Ca2+]i activity recorded in the square (x) indicated in A: control (left) and 5 min after application of 10 µM 5-HT (right). Dotted lines correspond to the times when images (a–e) were acquired. C–E) Bar graphs showing changes in the peak amplitude (C), frequency (D), and active area (E) of spontaneous pacemaker [Ca2+]i activity .Regular occurrence of pacemaker i oscillations presumably (1) by facilitating Ca2+ influx via 5-HT3 receptors, and (2) simultaneous activation of voltage-gated Ca2+-permeable insensitive to DHP Ca2+ antagonists (DHP(-)VGCC). In ICC, [Ca2+]i oscillations periodically activate Ca2+-activated ion channels in the plasmamembrane, i.e. Ca2+-activated Cl− channels (ClCa) and/or Ca2+-activated nonselective cation channels (NSCCCa), and thereby generate electric pacemaker activity. In smooth muscle (SM) cells, conducted pacemaker activity via gap junction (GJ) channels activates DHP-sensitive voltage-gated Ca2+ channels (DHP(+)VGCC), i.e. L-type Ca2+ channels, causing periodic contraction. In the present study, to differentiate ICC activity, all experiments were carried out in the presence of a DHP Ca2+ antagonist, nifedipine.Possible underlying mechanisms for 5-HT-enhancement of gut pacemaker activity and contractility. It is thought that intracellular Ca(TIF)Click here for additional data file.Video S12+]i oscillations measured in a musculature preparation in normal condition, corresponding to [Ca(MPG)Click here for additional data file.Video S22+]i oscillations measured in a musculature preparation in the presence of 5-HT (10 µM), corresponding to [Ca(MPG)Click here for additional data file.Video S32.Field potential oscillations acquired under a control condition, corresponding to (MPG)Click here for additional data file.Video S4Field potential oscillations acquired 5 min after application of 10 µM 5-HT corresponding to (MPG)Click here for additional data file.


However, this knowledge is based on dispersed outcomes that do not set up a consensus model of the Ca2+-dependent control of autophagy and endocytosis. Here, we will provide a critical synopsis of insights from the last decade on the involvement of Ca2+-sensor proteins in the activation of autophagy and in fusion events of endocytic vesicles, autophagosomes and lysosomes.Autophagy and endocytosis are two evolutionarily conserved catabolic processes that comprise vesicle trafficking events for the clearance of the sequestered intracellular and extracellular cargo. Both start differently but end in the same compartment, the lysosome. Mounting evidences from the last years have established the involvement of proteins sensitive to intracellular Ca Lysosomes are ubiquitous organelles that degrade material sequestered by two main dynamic processes: autophagy and endocytosis. Both processes comprise a complex traffic of vesicles that finally ends with the clearance of their contents by the lysosomal acid hydrolases.1): microautophagy, chaperone-mediated autophagy and macroautophagy. Microautophagy involves the internalization of cytosolic components by various modifications of the lysosomal membrane [ATGs), most of them essential for autophagosome formation and whose mammalian homologues are well identified [Autophagy is an important pathway responsible for the turnover of intracellular macromolecules and even whole organelles . At leasmembrane . It has membrane . Finallymembrane . Severalmembrane , 6, plasmembrane , Golgi cmembrane  and endomembrane , appear membrane , but themembrane -13. Oncemembrane . Analysientified . Many reentified , 16), an1). One of the best-characterized forms of endocytosis is receptor-mediated endocytosis, responsible for the selective internalization of specific ligands recognized by their receptors at the cell surface [Endocytosis is the process whereby extracellular and plasma membrane materials are internalized and transported to lysosomes by vesicles . During  surface . Phagocy surface . Differe surface , 19. 2+ is a second messenger that is involved in the regulation of several physiological cell functions, such as gene transcription, metabolism, secretion and apoptosis, and perturbations in its homeostasis have been implicated in various pathological processes, such as disorders of the nervous system, cardiac and vascular pathologies and diabetes mellitus [2+ with signalling and trafficking steps related with autophagy and endocytosis, but several details still remain unknown. Here we will review, consecutively, the role of Ca2+ in the regulation of: i) autophagy, ii) endocytosis, and iii) their final convergence into lysosomes for the degradation of the material taken up by these two processes. Camellitus , 21. Ins11.12+ signaling activates autophagy was provided in a study performed in MCF-7, NIH3T3 and HeLa cells, where increasing cytosolic Ca2+ levels with pharmacological agents such as ionomycin induced autophagy in a Beclin 1- and ATG7-dependent manner [2A). Autophagy was activated by a signaling pathway, involving Ca2+/calmodulin-dependent kinase kinase-beta (CAMKK-β) and AMP-activated protein kinase (AMPK), which inhibits the serine-threonine kinase mammalian target of rapamycin (mTOR). This inhibition of mTOR occurs via the GTPase activating protein Tuberous Sclerosis Complex (TSC1/2) and its substrate, the Ras-family GTP binding protein Rheb that directly regulates the activity of mTOR [2+. Under these conditions, the CAMKK-β-AMPK signalling pathway becomes activated and inhibits mTOR, leading to the autophagic degradation of amyloid-β [Direct evidence that cytosolic Cat manner   and bypasses AMPK [2+ in the induction of autophagy was derived from transfection experiments with calcium-phosphate precipitates in which it was observed that these precipitates activate autophagy in a Beclin 1- and ATG5-dependent way [However, it has been reported that Cases AMPK . Furtherdent way .2+ on autophagy . Thus, using Ca2+ channel antagonists, such as verapamil, which inhibit a family of Ca2+-activated cysteine proteases, the calpains, autophagy was activated by a pathway independent of mTOR [2+ channel agonists inhibit autophagy via the cleavage of ATG5 by calpains, which in turn decreases the formation of the ATG12-ATG5 conjugate that is indispensable for the formation of autophagosomes [However, other results are in conflict with those described above, since they support an inhibitory effect of cytosolic Ca of mTOR , whereasagosomes .2+ activate or inactivate autophagy is still a matter of discussion. Of note, studies supporting inactivation of autophagy by cytosolic Ca2+ are based on the modulation of voltage-dependent Ca2+ channels  that exist only in excitable cells [2+ has been reported in non-excitable cells [2+ is mainly provided from the extracellular space by voltage-activated channels, whereas in non-excitable cells it is mainly released from intracellular stores via second messengers ) [2+-sensor proteins in both groups of cells activate distinct signalling routes that lead to opposite autophagic responses. Therefore, whether rises in the cytosolic Cale cells , 29, whele cells , 26, 27.1.22+ storage within cell compartments in autophagy stimulation [2+ for the autophagic activity has been confirmed by several experimental evidences. The ER lumen constitutes both the main intracellular Ca2+ store and the major site in the secretory pathway for the proper folding of proteins, which is carried out by a group of chaperones, most of them Ca2+-dependent [2+ homeostasis inside the ER cause stress that compromises the functionality of this organelle and of the cell. Earlier studies demonstrated a role of Camulation . Since tependent -33. Ther1.2.12+ efflux from the ER and autophagy came from the observation of an induction of autophagy by thapsigargin [2+ transport into the ER through Ca2+-ATPase pumps, rendering this store depleted of Ca2+ and, subsequently, provokes ER stress [2+ rather than ER stress is important for the induction of autophagy by thapsigargin, since its effect is abolished by the potent cell permeant Ca2+ chelator BAPTA-AM ethane-N,N,N’,N’-tetraacetic acid (acetoxy methyl ester)) [2+ from the ER induce autophagy without altering the unfolded protein response [2+ to the activation of autophagy independently of ER stress. The first direct evidence of a possible connection between Casigargin . This coR stress , 35. Sev ester)) , 36. In  ester)) ), while  ester))  and otheresponse . All the2+-dependent activation of autophagy by thapsigargin has been reported to occur in simple eukaryotes such as Dictyostelium [Dictyostelium ATG1 is shown to be required [2+-dependent autophagy activation has been described to occur either via CAMKK-b-AMPK-mTOR signalling [2+-dependent phosphorylation of PKCθ that recruits this PKC isoform to the autophagic vesicles [The Caostelium , as wellostelium , 38, 41.required , whereasgnalling  that actgnalling  and our gnalling , 41 or a2+-dependent activation of calpains [2+ and hence of the Ca2+ supplied to the cytosol in this process. However, examples of non-excitable cells where autophagy is inhibited [2+, since, for example, the use of BAPTA-AM in some of these studies does not rule out the involvement of Ca2+ present in other organelles. In fact, thapsigargin treatments at high concentrations and/or during prolonged times inhibit for example Ca2+-ATPase pumps at the Golgi complex [2+ released by thapsigargin from the ER activates or inhibits autophagy in non-excitable cells is still under debate.However, other studies have shown the opposite effect of thapsigargin , 43, 44,calpains . It seemnhibited , 30 or anhibited , 41, 45  complex . Therefo2+-ATPase pumps that control Ca2+ entry to the ER lumen, Ca2+ homeostasis in this organelle is also affected by Ca2+ release through the IP3 receptor (IP3R), an aspect that we discuss below.Apart from Ca1.2.22+ from the ER is mainly regulated by interaction of the second messenger IP3 with IP3R, resulting in the formation of a Ca2+ release channel at the ER [3 is generated through the cleavage of phosphatidylinositol 4, 5-bisphosphate (PIP2) by phospholipase C (PLC), which can be activated by inositol recycled from inositol monophosphate by dephosphorylation [3 in the regulation of autophagy  [2+ release through IP3R prevents autophagy, since inhibitors of this receptor, such as xestospongin B or dexamethasone, or the knockdown/knockout of all three IP3R isoforms induce autophagy [2+ released to the cytosol through IP3R appears to be only relevant under nutrient rich conditions, because in this situation, but not under starvation [3R isoforms decreases mTOR activity and results in an increase of basal autophagy [Efflux of Cat the ER . IP3 is rylation . InhibitFig. 3B) , 50. In utophagy . This nearvation , the knoutophagy . 3R forming a complex. Although Bcl-2 is not necessary for the in vitro binding of Beclin 1 to IP3R, it is indispensable for the complex formation in a cellular context and under full nutrient conditions [3R/Bcl-2 [3R under starvation conditions [3R by its knockdown or by xestospongin B disrupts the complex and leads to autophagy activation [3R probably acts as a scaffold to recruit proteins of the autophagic machinery under nutrient rich conditions.Moreover, this channel has been associated with two autophagy-related proteins, Bcl-2 and Beclin 1, which interact with IPnditions . However3R/Bcl-2 , 55 and 3R/Bcl-2 . Of notenditions -60. Alsotivation , 55. Thu3R function as a Ca2+ channel, it also seems to be dependent on the nutritional state of the cell, at least for the autophagy inducer Beclin 1. Under full nutrient conditions this protein does not affect Ca2+ release through IP3R [2+ from the ER by IP3R in response to IP3 [3R, reduces Ca2+ release through IP3R by a still unknown mechanism [As for the role of these autophagy-related proteins in IPugh IP3R , wherease to IP3 . Moreoveechanism -64. 2+ discharge from the ER through IP3R on autophagy appears to depend on two factors: the nutritional state of the cell and the scaffold properties of this channel to recruit autophagy-related proteins. Under full nutrient conditions, IP3R sequesters proteins essential for autophagy activation that do not affect Ca2+ release through this channel, whereas under starvation conditions these proteins are liberated and this increases both autophagy and Ca2+ release.In conclusion, the impact of Ca2+ efflux from the ER via IP3R, produce a similar effect  on autophagy via extracellular signalling-regulated kinase (ERK1/2) [3A). However, these chemicals are not necessarily specific for IP3R. For example, 2-aminoethoxydiphenyl borate is not a selective inhibitor of IP3R, because it also alters the activity of store-operated Ca2+ channels and Endoplasmic Reticulum Ca2+-ATPase (SERCA) pumps at the plasma membrane [2+ derived from the ER through IP3R.  Other drugs that increase (Cadmium) or inhibit (2-aminoethoxydiphenyl borate) Ca(ERK1/2)  , two enzymes of the Krebs cycle (isocitrate dehydrogenase and ketoglutarate dehydrogenase), and the F1F0 ATPase. Activation of PDH occurs by its dephosphorylation produced by the Ca2+-dependent stimulation of the PDH phosphatase (PDP). Although some cells, such as hepatocytes, express a PDP isoform whose activity is Ca2+-independent [2+-dependent activation of PDH by PDP seems to be a key step in many cells to supply them with NADH and ATP [2+-dependent activity, when a moderate extent of Ca2+ (in the low micromolar range) is delivered to mitochondria, ATP increases, AMPK is inhibited and this restrains autophagy by an mTOR-independent signalling pathway [4A). IPnnection . Thus, aependent , the Ca2 and ATP -72. Thus pathway   into mitochondria occurs as a consequence of its depolarization. This provokes the disruption of the integrity of the mitochondrial outer membrane and a rise in mitochondrial permeability [4B). On the contrary, under situations that may induce cell death, such as oxidative stress, a massive entry of Caeability , 74. In eability , 76 (see2+ overload and autophagy are provided by some proteins. The proapoptotic proteins Bcl-2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) and BNIP3-like, also known as NIX, participate in mitophagy induction in various cell types, including tumors, and localize on the outer mitochondrial membrane [2+ transfer from the ER to mitochondria in cardiac cells under stress conditions that may induce cell death [2+ overload and to generalize these observations to other cell types. Indirect links between mitochondrial Camembrane . As NIX ll death , it is p2+ overload inside this organelle activates the cytosolic Ca2+-dependent phosphatase calcineurin [4B) by dephosphorylation and inhibition of IP3R, constituting in this way a negative feedback to control Ca2+ release and to preserve mitochondrial homeostasis [2+-mediated activation of calcineurin . Moreover, permeabilization of mitochondrial membranes under Cacineurin , which feostasis , 80. Sineostasis , a nucleeostasis , it is p2+ regulates autophagy in two opposite ways. Moderate Ca2+ levels provided from the ER within mitochondria produce ATP that represses autophagy via inhibition of AMPK. On the other hand, when cells run into stress conditions, an excessive mitochondrial Ca2+ upload occurs that activates mitophagy by mechanisms involving pro-apoptotic proteins and probably calcineurin. In summary, mitochondrial Ca2+ stores in non-excitable cells, it seems that this cation and its sensor proteins in the cytosol induce autophagy when cells encounter conditions that require this process. Ca2+ release from the ER and mitochondria to the cytosol activates autophagy under stress conditions, whereas in healthy state, the storage of this cation inside these two organelles maintains low levels of autophagy. Thus, Ca2+ seems to participate in the adaptation of the autophagic level of the cells to their physiological state. As for excitable cells, although less attention has been paid to the Ca2+ impact on their autophagy, cytosolic Ca2+ seems to have the opposite effect on autophagy, probably because, as pointed above, the characteristics of the Ca2+-sensor proteins implicated in autophagy in these cells are different from the corresponding proteins in non-excitable cells.Taken together the different Ca22+ appears to be relevant in fusion/fission events [5) [2+ from endolysosomal compartments is thought to trigger fusion/fission events near Ca2+ release sites [2+ chelators BAPTA and ethylene glycol tetraacetic (EGTA). In membrane fusion assays, BAPTA but not EGTA inhibits the fusion of late endosomes with early endosomes [2+ in less time (0.3 μs) than EGTA (1.2 ms) [2+ diffusion rate in the cytosol is 20 nm/ms [2+ release source is situated at 20 nm or less from the site where fusion occurs, a reasonable distance to consider the lumen of vesicles committed to a fusion event as the source of this Ca2+. In fact, the depletion of luminal Ca2+ from these vesicles has the same effect on their fusion than BAPTA [During endocytosis, Can events , 84. Theents [5) -88. In sents [5) . After dse sites , 91. Thindosomes , lysosomndosomes  or yeast(1.2 ms) , and sin20 nm/ms , this sean BAPTA , 96.2+-sensors transduce these Ca2+ signalling into a fusion response. The best studied sensor is calmodulin, which has been shown to be crucial in homotypic [2+ binding to calmodulin leads to interactions between this protein and specific targets, such as calmodulin-dependent kinase II (CAMKII) [2+/calmodulin may not only play the role of recruiting fusion effectors to early endosomes, but it can also recycle tethering molecules such as EEA1. It is believed that specific endolysosomal Caomotypic , 97 and omotypic  fusions.(CAMKII)  and the (CAMKII) , to prom(CAMKII) . Thus, Clinked gene-2 (ALG-2), has been also proposed as a Ca2+-sensor for later fusion events in the endolysosomal system through its Ca2+-dependent interaction with the transient receptor potential cation channel, mucolipin subfamily 1 (TRPML1) [2+ and other ions from the lysosomal lumen to the cytosol [2+ from the lumen of vesicles is essential for their fusion, this channel may provide Ca2+ from endolysosomes for their fusion with endosomes and autophagic vacuoles [2+ in these processes that we will discuss in the following section.Apoptosis-(TRPML1) , a putat cytosol -102. Sinvacuoles , 103-1063+ transport growth defect 1) in their regulation [2+ is a key player [2+ derived from lysosomes in the fusion of these organelles with autophagosomes and endosomes. Fusion of autophagosomes and endosomes with lysosomes to deliver their respective cargo constitutes a late step in autophagy and endocytosis. Both groups of fusions share certain features, such as the involvement of RAB7 and the AAA ATPase SKD  and two-pore channels (TPCs). TRPM2, whose expression is restricted to specific cells, like pancreatic β cells, is mainly expressed at the plasma membrane, but it has been also localized on lysosomes, where it has been proposed to regulate luminal Ca2+ release [2+ from acidic compartments [Two other candidates to function as lysosomal/endosomal Ca release . TPC1 an release -123. Botartments , 125.2+ from endolysosomal compartments, knowledge on their specific role in autophagy and endocytosis remains rudimentary. In this regard, it has been suggested that Ca2+ release through NAADP-sensitive channels contributes, at least, to fusions between lysosomes and endosomes, since these channels are localized on these organelles [Although NAADP-regulated TRPM/TPCs channels can release Caganelles -123. 2+ is also regulated by pH. In fact, disruption of lysosomal pH by lysosomotropic agents, like bafilomycin A1, chloroquine diphosphate or nigericin, prevents Ca2+ storage in the lysosomal lumen and arrests the fusion of lysosomes with autophagosomes [2+ in the lysosomal lumen, a requirement to induce fusions between lysosomes and endosomes or autophagosomes upon Ca2+ release from the lumen of these vesicles. In accordance with this concept, an in vitro study with isolated autophagosomes and lysosomes revealed that fusion between both organelles requires a minimum of 250 μM of calcium chloride [Lysosomal Caagosomes . Therefochloride .3.22+ that is relevant in autophagy and endocytosis consists on its ability to promote the fusion of vesicles by inducing local segregations of specific lipids such as phosphatidic acid [in vivo and in vitro data support that these lipid domains are stabilized by proteins that bind to the membranes [2+ release. Subsequently, Ca2+-binding proteins (that we will discuss below: see Table 1) are activated, probably organizing a scaffold upon the membranes that initiates the fusion processes. Finally, after dissipation of the Ca2+ gradient, these proteins remain activated until fusion is accomplished [Another physiological feature of Cadic acid , 129. Seembranes -132. Themplished . 2+-sensitive protein associated to early endosomes. When bound to a still undefined SNARE protein on membranes of early endosomes, HRS prevents homotypic membrane fusions, thus negatively regulating the fusogenic function of SNAREs [2+ release from the endosomal lumen dissociates HRS from the SNARE complex and abolishes this effect, enabling in this way endocytic fusion [A peculiar protein from the SNARE complex is Hepatocyte responsive serum phosphoprotein (HRS), a Caf SNAREs . Ca2+ rec fusion . 2+-binding protein, annexin A5, has been shown to be recruited to lysosomal membranes in a Ca2+ dependent way, to induce autophagosome fusion with lysosomes and to inhibit endocytosis [2+ release from the autophagic/ endolysosomal vesicles may control the role of these proteins in autophagy and endocytosis. On the other hand, this protein has been also shown to partially colocalize with autophagosomes and to promote their maturation . Somewhaocytosis , 137. Th2+-sensor of SNAREs. The first evidences of this role were obtained in yeast, where calmodulin was identified within a protein complex involved in homotypic vacuole fusion [Calmodulin has been also proposed to be a Cae fusion , 138. Ine fusion , 140. In vitro studies showed the requirement of annexin A1 in fusions between early endosomes in a Ca2+-dependent manner [in vivo analysis attributed to annexins A2, A5 and A6 the abilities to mediate the fusions of early endosomes [Moreover, some members of the annexin family are associated with fusion events in the endolysosomal system. t manner , whereasndosomes , autophandosomes , and latndosomes . 2+-dependent effectors of fusions between autophagosomes, endosomes and lysosomes belong to a wide range of subgroups such as SNAREs, EF-hand proteins and annexins, with some common characteristics, including the requirement of Ca2+ binding. However, the molecular mechanisms by which they control these events are still poorly understood.Overall, Ca2+ controls endocytosis and autophagy. Its effect on autophagy occurs both at the level of the signalling pathways that initiate it or, later, when autophagosomes fuse with endolysosomal compartments.Growing evidences support that Ca2+ on autophagy depends on the cell type, since excitable and non-excitable cells exhibit opposite autophagic responses  to this cation. Although less attention has been paid to excitable cells, Ca2+ rise within them restrains autophagy and this effect is mainly due to the activation of calpains that cleave proteins essential for autophagy. To decide whether other Ca2+-sensor proteins, specific or not for these cells, are also involved in this effect requires further work that would help to better understand the autophagic behavior of these cells.The effect of Ca2+ on autophagy depends on the nutritional state of the cells and, probably, on the Ca2+ levels within the cytosol. Under full nutrient conditions, Ca2+ levels in the cytosol are low and maintain a basal autophagy. Starvation and stress conditions induce a rise of cytosolic Ca2+ originated, respectively, from the ER and mitochondria overloaded with Ca2+. Subsequently, these conditions trigger autophagy via various pathways that depend on Ca2+-sensor proteins . Thus, in non-excitable cells, Ca2+ seems to play a protective role by adapting autophagic activity to extracellular conditions. Therefore, manipulation of intracellular Ca2+ levels in situations of defective autophagy may be useful to recuperate cellular homeostasis.In non-excitable cells, the effect of Ca2+ derived from their lumen and, subsequently, Ca2+-sensor proteins transduce this Ca2+ signalling into fusion events. Concerning endocytosis, the traffic of endocytic vesicles is controlled by Ca2+ originated from lysosomes/late endosomes and on proteins that are subsequently activated by this cation. However, the involvement of Ca2+ and its effects on sensor proteins in these final autophagic and endocytic stages remain poorly understood. Although various members of these proteins have been identified, further investigations are needed to identify new Ca2+ effectors and their role in the regulation of the different steps of autophagy and endocytosis.Finally, the convergence of the autophagic and endocytic vesicles to lysosomes shares several features that depend on Ca


This sensor is based on the high-affinity Mg2+ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg2+ affinity (Kd = 148 µM) with a 50% increase in emission ratio upon Mg2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca2+, this affinity is sufficiently attenuated (Kd of 10 µM) to make the sensor insensitive to known Ca2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg2+ sensors could prove very useful in understanding intracellular Mg2+ homeostasis and signaling.Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg In addition to its omnipresent role as an essential enzymatic cofactor, Mg2+, the intracellular regulation of Mg2+ homeostasis and the putative role of Mg2+ in intracellular signal transduction are not well understood. In part this is because of a lack of convenient molecular tools to image the intracellular Mg2+ concentration in single living cells in real time 2+ levels in different muscle cells, revealing concentrations between 0.7 and 0.9 mM 2+ is the measurement of the ratio of Mg2+-bound and Mg2+-free ATP using 31P NMR 31P NMR measures Mg2+ indirectly and averaged over a large collection of cells 2+2+ and often bind Ca2+ with low micromolar affinity 2+ is higher than for Ca2+ , rendering these dyes completely insensitive to physiological changes in cytosolic Ca2+ concentration 2+ dyes, the KMG dyes are intensiometric, making Mg2+ quantification challenging and sensitive to changes in sensor concentration. A few ratiometric Mg2+ fluorescent dyes (e.g. Mag-Fura and Mag-Indo) exist, yet these have the disadvantage that they require potentially cytotoxic UV excitation Despite the abundance and importance of Mg2+2++Genetically encoded fluorescent sensor proteins provide an attractive alternative to small-molecule fluorescent sensors, because they do not require cell-invasive procedures, their concentration can be tightly controlled and they can be targeted to different locations in the cell 2+. One of the specific challenges in this case is metal binding specificity. Mg2+ and Ca2+ show similar coordination chemistry and often bind to the same metal binding proteins, with Ca2+ typically showing stronger binding. Here we report the first genetically encoded fluorescent sensor (MagFRET-1) for Mg2+ by taking advantage of the particular metal binding properties of the N-terminal part of the HsCen3 protein, which binds both Ca2+ and Mg2+ with high affinity 2+ binding to MagFRET-1 induces folding from a molten-globule state that results in an increase in FRET. Mutagenesis of metal binding site residues allowed further tuning of the metal binding properties, yielding MagFRET variants with Kd values for Mg2+ binding ranging between 0.15 and 15 mM. While also responsive to Ca2+in vitro, we show that the Ca2+ affinities of the MagFRET sensors are sufficiently attenuated that they are not responsive to normal Ca2+ fluctuations in situ.Surprisingly, no genetically encoded sensors have thus far been reported for MgNheI and NcoI yielded an insert fragment that was compatible with a pET28a acceptor vector encoding for His6-Cerulean-(GGS)18-Citrine SpeI (creating an NheI-compatible cohesive overhang) and NcoI. A ligation was carried out at equimolar vector-to-insert ratio using T4 DNA ligase  at 16°C for 1 hour following the manufacturer's instructions, resulting in pET28a-MagFRET-1 . The SpeI restriction site in the pET28a-His6-Cerulean-(GGS)18-Citrine acceptor vector was located 8 residues upstream of the Cerulean C-terminus, such that in the final MagFRET construct, the native flexible C-terminus of Cerulean was deleted, resulting in tighter allosteric coupling between changes in HsCen3 conformation and changes in the fluorescent domains' interchromophore distance. The mammalian expression vector for MagFRET-1 was obtained by digesting pET28a-MagFRET-1 using restriction enzymes AgeI and NotI. Ligation into a peCALWY-1 vector Table S1. To obtain the mammalian expression vector encoding for a nuclear-targeted MagFRET-1 (MagFRET-1-NLS), a pUC57 vector containing a synthetic gene encoding for the final part of Citrine together with three PKKKRKV repeats was digested using restriction enzymes HindIII and NotI, followed by ligation into a pCMV-MagFRET-1 plasmid that was treated with the same restriction enzymes . The correct open reading frame of each sensor was confirmed by Sanger dideoxy sequencing .DNA encoding the N-terminal fragment of HsCen3 (residues 23 to 98 E. coli BL21(DE3) cells were used for protein expression. A single colony was used to inoculate 5 mL LB medium  supplemented with 30 µg/mL kanamycin which was grown overnight at 225 rpm at 37°C. Overnight cultures were diluted in 500 mL LB medium containing kanamycin (30 µg/mL) and grown until an optical density of 0.6–0.8 was reached at 600 nm wavelength. Protein expression was induced by the addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside . Bacteria were cultured overnight at 225 rpm at 25°C and harvested by centrifugation at 10,000 g for 10 minutes at 4°C. The cell pellets were lysed using Bugbuster reagent (Novagen) according to the manufacturer's instructions. The resulting soluble protein fraction was used for further purification. The expressed MagFRET proteins contain an N-terminal hexahistidine-tag. Ni2+-NTA resin  was used for affinity chromatography following the manufacturer's instructions. After elution of the protein using 0.5 M imidazole, the protein was dialyzed overnight against 100 volumes of 20 mM Tris-HCl (pH 8.4), 150 mM NaCl and 2.5 mM CaCl2 using a 12–14 kDa Molecular Weight Cut-Off (MWCO) dialysis membrane (Spectropore) at 4°C. The hexahistidine-tag was subsequently removed by the addition of 0.3 U thrombin protease (Novagen) per mg protein at a 0.2 mg/mL protein concentration and incubated for 24 hours at 4°C. His-tags and uncleaved proteins were removed using Ni2+ affinity chromatography. The flow-through was further purified using size exclusion chromatography (SEC) on a Sephacryl S-200 High resolution column . Fractions containing pure proteins were pooled, concentrated, frozen in liquid nitrogen and stored in aliquots at −80°C. The purity and correct molecular weight of the obtained proteins was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis  analysis. The protein concentration was determined using the absorption at 515 nm (ND-1000 Nanodrop) and a molar extinction coefficient of 77,000 M−1cm−1 for Citrine 2 and CaCl2 (both from Sigma) were added at increasing concentrations from a concentrated stock solution in water. To determine the MagFRET-1 dissociation constant (dK) for Mg2+, the emission ratio (R) as a function of MgCl2 concentration ([2+Mg]) was fit to SR is the starting emission ratio in absence of Mg2+ and ΔR the difference in emission ratio between the Mg2+-free and Mg2+-saturated form of MagFRET-1. To determine the MagFRET dK1 and dK2 values associated with the first and second Ca2+-binding events respectively, the emission ratio (R) as a function of CaCl2 concentration ([Ca2+]) was fit to a double binding event using SR is the starting emission ratio in absence of Ca2+, ΔR1 is the difference between the emission ratio in the Ca2+-free state and the state in which a single Ca2+ ion has bound to the first metal binding loop of HsCen3, ΔR2 the difference between the latter state and the state in which a second Ca2+ ion has bound to EF-hand II. For the metal specificity measurements, either BaCl2, NiSO4, CuSO4, ZnCl2 or FeCl3 was added from 1000× concentration stock solutions to a final concentration of 10 µM, followed by addition of 1 mM MgCl2. For titrations  testing the effect of ionic strength on the sensor, a low salt buffer was used, consisting of 20 mM Hepes (pH 7.1), 10 mM NaCl and 10% (v/v) glycerol. To test for pH sensitivity of MagFRET-1, MgCl2 and CaCl2 titrations were carried out in buffers where Hepes was replaced with either MES (2-(N-morpholino)ethanesulfonic acid) or Tris (tris(hydroxymethyl)aminomethane) in the standard measurement buffer. The MES-containing buffer was prepared at pH 6 while the Tris-containing buffer was prepared at pH 8.Unless otherwise mentioned, magnesium and calcium titrations were performed in 150 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.1), 100 mM NaCl, 10% (v/v) glycerol. Fluorescence emission spectra were recorded between 450 and 600 nm at a 0.2 µM protein concentration on a Varian Cary Eclipse fluorometer with an excitation wavelength of 420 nm. MgCl−1 penicillin and 100 µg mL−1 streptomycin at 37°C and 5% CO2. Cells were plated on poly-L-lysine (Sigma) treated glass coverslips and transfected with 1.5 µg of plasmid DNA and 5 µg polyethyleneimine (PEI). Cells were imaged for transient expression 2 days after transfection. In addition to the pCMV-MagFRET-1 and pCMV-MagFRET-1-NLS constructs described above, cells were also transfected with peZinCh-NB HEK293 cells were grown in Dulbecco's modified Eagle medium  containing 10% (vol/vol) fetal bovine serum , 3 mM glucose, 2 mM glutamine, 100 units mLTo demonstrate correct localization and subcellular targeting of MagFRET-1, confocal microscopy (Leica TCS SP5 X) was used to image the sensor with high spatial resolution. Samples were excited using a 405 nm laser and emission was detected using a hybrid APD/PMT detector . Spectral emission windows were set to 460–490 nm for the Cerulean channel and 510–550 nm for the Citrine channel, using an acousto optical beam splitter . Widefield fluorescence microscopy for FRET measurements was performed on an Axio observer D.1 (Zeiss) equipped with an Axiocam MRm monochrome digital camera (Zeiss) using Axiovision 4.7 software. Samples were excited using a HXP 120 Mercury lamp (Zeiss) and Cerulean and Citrine emission was recorded sequentially using filter set 47  and 48  (Zeiss) in a motorized filter turret. Emission of Oregon Green-BAPTA was recorded using filterset 38 HE  (Zeiss). Images were acquired using an apochromat 40× objective, with an exposure time of 200–300 ms.2+ or EDTA, cells were permeabilized by a 6 minute incubation of HEK293 cells in 400 µL intracellular buffer (IB) containing 10 µg/ml digitonin (Sigma). IB comprised 20 mM Hepes (pH 7.05), 140 mM KCl, 10 mM KH2PO4, 100 µM ATP, 2 mM Na+ succinate and 5.5 mM glucose. After 6 minutes, recordings were started and buffers containing increasing concentrations of EDTA or MgCl2 were added as stated in the main text. When adding MgCl2, KCl concentrations were reduced accordingly to maintain the Cl− concentration at 140 mM. Imaging frequency was 0.1 Hz.Prior to addition of Mg2PO4, 1.5 mM CaCl2, 25 mM NaHCO3 and 3 mM glucose. Where indicated, PAR-1 agonist peptide  or ATP (Sigma) were added to a final concentration of 50 µM. Control experiments were performed using non-transfected HEK293 cells that were loaded with 10 µM Oregon Green-BAPTA-AM  in phosphate buffered saline (PBS) with 0.01% (w/v) Pluronic F-127 (Life Technologies) for 30 minutes. At the end of each experiment in which Oregon Green-BAPTA-AM was used, 20 µM of calcium ionophore A23187 (Sigma) was added. Imaging frequency was 0.2 Hz.Intracellular calcium specificity measurements were performed on HEK293 cells transfected as described above. Modified Krebs-bicarbonate buffer was used, consisting of 10 mM Hepes (pH 7.4), 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2+ requires the availability of a metal binding domain that undergoes a large conformational change and displays a relatively high affinity for Mg2+ compared to Ca2+. Cox and coworkers previously reported that a truncated version of HsCen3 containing the first two of its four native EF-hand metal binding sites, undergoes a dramatic change in conformation upon metal binding from a molten globular (MG) state to a compact, natively-folded state 2+ orders of magnitude more strongly than Mg2+, HsCen3's first EF hand is a high-affinity mixed Mg2+/Ca2+ binding site, with a reported Kd for Mg2+ of 10–28 µM and a Kd for Ca2+ of 1.5–8 µM. The second metal binding site was reported to bind only Ca2+, but with a much weaker affinity (Kd = 140 µM). HsCen3 is one of the four isoforms of human Centrin, a family of proteins that is involved in centriole duplication. We based our design on the structure of HsCen2, which shows high homology to HsCen3 and is the only isoform for which an X-ray structure has been determined  and fuse it to the fluorescent proteins Cerulean and Citrine  and purified using Ni2+-affinity and size exclusion chromatography. A relatively high ratio of Citrine to Cerulean emission of 3.6 was observed in the absence of Mg2+, indicating that the molten globule state of the metal binding domain is relatively compact bringing the fluorescent domains close together . When the Mg2+ titration was repeated in a low ionic strength buffer, the emission ratio of the apo form decreased to 2.2, whereas the emission ratio in the Mg2+-bound state was the same  and the Mg2+ affinity remained mostly unaffected (Kd = 231±10 µM). This ionic strength dependence most likely reflects the influence of ionic strength on the compactness of the molten globule structure of HsCen3. Although the effect is less pronounced at physiologically relevant salt concentrations, it does mean that large changes in ionic strength should be avoided when applying MagFRET-1 in situ.To allow characterization of MagFRET-1 st EF hand  resulted in a reduction of both the Mg2+ and Ca2+ affinity to the millimolar regime, indicating that these residues are indeed directly involved in high affinity metal binding  did not alter the affinity for Mg2+ or Ca2+ , rendering this variant effectively Ca2+ insensitive. In an effort to abolish Ca2+ binding to the weakly Ca2+-binding EF hand II, we replaced aspartic acid 1 (MagFRET-7) and glycine 6 (MagFRET-8) at that site by positively charged lysine residues. Surprisingly, upon titration of Ca2+, both sensor variants still displayed the same biphasic response as seen with MagFRET-1 . In addition, transfection of cells with a construct containing three repeats of the nuclear localization sequence PKKKRKV Kd of MagFRET-1 for Ca2+ determined in vitro is an order of magnitude higher than the maximum Ca2+ concentration that is typically observed in the cytosol during signaling, it was still important to verify that the MagFRET-1 sensor does not respond to stimuli that are known to transiently induce increases in cytosolic Ca2+ concentrations. Ca2+ signaling in HEK293 cells was activated via addition of 50 µM of the protease activated receptor-1 (PAR-1) agonist peptide 2+ dye Oregon Green-BAPTA did show a transient increase in fluorescence upon addition of PAR-1 agonist peptide, confirming that the expected increase in intracellular Ca2+ concentration was induced under these conditions , while cells loaded with Oregon Green-BAPTA showed a clear response to the same treatment .To assess the sensor properties of MagFRET-1 2+ fluctuations, we next characterized MagFRET-1's ability to report on changes in cytosolic [Mg2+]free. Surprisingly, attempts to perturb the free concentration of Mg2+ in the cytosol of intact HEK293 cells using previously reported procedures did not induce a significant ratiometric response in HEK293 cells transfected with MagFRET-1 (not shown). These protocols included incubation of the cells in 50 mM MgCl22+ competitor Li+2+/Na+ exchanger 2+ or EDTA concentrations . To verify that the sensor could also monitor a decrease in cytosolic Mg2+ levels, the metal chelator EDTA was added to permeabilized cells expressing MagFRET-1. Upon addition of EDTA, cells expressing MagFRET-1 showed a decrease in emission ratio that is consistent with a decrease in cytosolic Mg2+ levels . The difference in metal binding affinity might be explained by the fact that the central α-helix that connects the 2nd and 3rd EF hands in HsCen3 was reduced to half its length in MagFRET-1, possibly further destabilizing the N-terminal domain, resulting in a net decrease in Mg2+ affinity. Fortunately, in this case the attenuation yielded a sensor that is sensitive to physiologically relevant Mg2+ concentrations, and insensitive to normal cytosolic Ca2+ concentrations. The limited number of metal binding domain mutations that were explored in this study revealed that mutations in the first EF hand typically result in strongly attenuated metal binding affinities. A more subtle, 4–6 fold attenuation of metal binding affinity was obtained after introduction of positively charged amino acids in the 2nd EF hand. These effects could be due to direct allosteric coupling between the two EF hands in the metal-bound state, but alternatively could also result from further stabilization of the molten globule state. An important goal is to develop sensor variants that are less sensitive to Ca2+ yet retain affinity for Mg2+, as this would allow targeting to organelles known to have much higher resting levels of Ca2+. The similar coordination chemistries of Ca2+ and Mg2+ make rational design of such variants challenging, although mutations in an EF-hand-like protein have been reported that decreased the Ca2+ affinity 100-fold while simultaneously doubling the Mg2+ affinity 2+ sensor GECO The MgIn situ characterization of MagFRET-1 in HEK293 cells revealed that the sensor is readily expressed in the cytosol and can be targeted to the nucleus. Two ligands that are known to induce Ca2+ signaling in cells, PAR-1 agonist peptide and ATP, did not affect the emission ratio of the MagFRET-1 sensor in HEK 293 cells. This result was expected based on the affinity of MagFRET-1 that was determined in vitro (Kd = 10 µM) and previous reports that show that bulk cytosolic Ca2+ concentrations typically reach a maximum of 1 µM during signaling 2+ dyes with similar affinity to Ca2+ as MagFRET-1 have been reported to respond to Ca2+. This may be explained by the fact that MagFRET-1 is exclusively localized in the cytosol, whereas synthetic dyes sometimes partially mislocalize to Ca2+-rich organelles such as the ER or even leak into the external buffer 2+ concentration. A possible explanation is that the free concentration of Mg2+ in the cytosol is tightly buffered and controlled and not easily changed by external stimuli. The free concentration of Mg2+ in the cytosol is at least 1000-fold higher than that of Ca2+ and 106-fold higher than that of Zn2+. For this reason and because Mg2+ is essential to such a wide variety of biological processes, it would not be surprising that manipulation of intracellular free Mg2+ is much more difficult than that of other metals. Nonetheless, it is conceivable that while the overall free Mg2+ concentration in cells is relatively constant, substantial and physiologically relevant fluctuations in Mg2+ concentration could still occur locally, e.g. at the plasma membrane near Mg2+-specific ion channels. Although MagFRET-1 responded to changes in Mg2+ concentration in the order of seconds both in vitro and in cells, protein-based sensors often display slower kinetics than small molecule sensors, so that MagFRET-1 might fail to respond to extremely fast Mg2+ transients, should they occur. In addition, overall Mg2+ levels could change over longer periods of time, e.g. as a function of the cell cycle. These changes may be more reliably monitored using lifetime imaging, which might also be the preferred method to allow quantification of intracellular Mg2+ concentrations. Although we confirmed that MagFRET-1 is responsive to changes in Mg2+ concentration in permeabilized cells, we cannot completely rule out that for some unknown reason MagFRET-1 is less responsive in intact cells. The potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging therefore remain to be further established.Figure S1Nucleotide sequence of bacterial expression vector pET28a-MagFRET-1 ORF. The DNA sequence is shown in lowercase, with the single letter amino acid code shown beneath each codon in uppercase. The His-tag is highlighted in bright green, the thrombin cleavage site in pink, Cerulean in turquoise, HsCen3 in red and Citrine in yellow. The two EF-hand motifs are underlined in white.(PDF)Click here for additional data file.Figure S2Nucleotide sequence of mammalian expression vector pCMV-MagFRET-1 ORF. The DNA sequence is shown in lowercase, with the single letter amino acid code shown beneath each codon in uppercase. Cerulean is highlighted in turquoise, HsCen3 in red and Citrine in yellow. The two EF-hand motifs are underlined in white.(PDF)Click here for additional data file.Figure S3Nucleotide sequence of mammalian expression vector pCMV-MagFRET-1-NLS ORF. The DNA sequence is shown in lowercase, with the single letter amino acid code shown beneath each codon in uppercase. Cerulean is highlighted in turquoise, HsCen3 in red, Citrine in yellow and the three PKKKRKV repeats in grey. The two EF-hand motifs are underlined in white.(PDF)Click here for additional data file.Figure S4Effect of pH on MagFRET-1. To check for pH sensitivity, the MagFRET-1 emission ratio was followed as a function of Mg2+  and Ca2+  concentration, at pH 6  and pH 8 . Fitting of the data revealed a MagFRET-1 Kd for Mg2+ of 230±35 µM at pH 6 and 99±18 µM at pH 8. The sensor's Kd for Ca2+ (first binding event) at pH = 6 was found to be 5.6±1.7 µM, while at pH 8 it was 5.9±1.9 µM. Buffers used were 150 mM MES (pH 6), 100 mM NaCl and 10% glycerol for pH 6 and 150 mM Tris (pH 8), 100 mM NaCl and 10% glycerol for pH 8.(TIF)Click here for additional data file.Figure S5Effect of ionic strength on MagFRET-1. (A) Emission ratio of MagFRET-1 at increasing concentrations of ammonium acetate or NaCl in a buffer with low ionic strength. (B) Emission ratio of MagFRET-1 as a function of Mg2+ concentration in a buffer with low ionic strength. The low ionic strength buffer used in  was 20 mM Hepes (pH 7.1), 10 mM NaCl, 10% (v/v) glycerol. Fitting of the data using a single binding event revealed a Kd for Mg2+ of 231±10 µM.(TIF)Click here for additional data file.Figure S6Western blot analysis of MagFRET-1 expressing HEK293 cells. A molecular weight marker  was loaded in the left-hand lane. Lane 1 displays the lysate of HEK293 cells transfected with a vector encoding for MagFRET-1 under control of a CMV promoter. The blotting membrane was incubated with mouse anti-GFP , followed by HRP-functionalized goat anti-mouse antibody (Dako). The calculated molecular weight for MagFRET-1 is 62 kDa.(TIF)Click here for additional data file.Figure S72+ induced by ATP.Investigation of MagFRET-1 response to elevated cytosolic Ca (A) Emission ratio over time of intact HEK293 cells expressing MagFRET-1 measured by widefield fluorescence microscopy. At t = 104 s, 50 µM ATP was added to activate Ca2+ signaling. (B) To confirm Ca2+ signaling took place in stimulated cells, the fluorescence intensity of intact HEK293 cells loaded with Ca2+-dye Oregon Green–BAPTA was followed. At t = 104 s, 50 µM ATP was added to activate Ca2+ signaling, and at t = 226 s, 20 µM of the Ca2+ ionophore A23187 was added. In A and B, each trace represents the response of an individual cell, with ratio (A) or intensity (B) normalized to the value at t = 0 s.(TIF)Click here for additional data file.Method S1Western blotting.(PDF)Click here for additional data file.Table S1Primers used for mutagenesis of HsCen3.(PDF)Click here for additional data file.


Meanwhile, heme oxygenase-1 (HO-1) expression increased in the AVE0991 + Ang II group (10−5 mol/L or 10−6 mol/L). However, the beneficial effects of AVE0991 were completely abolished when the VSMC were pretreated with A-779 (10−6 mol/L). Furthermore, treatment with the HO-1 inhibitor ZnPPIX attenuated the inhibitory effect of AVE0991 on Ang II-induced p38MAPK phosphorylation. These results suggest that AVE0991 attenuates Ang II-induced VSMC proliferation in a dose-dependent fashion and that this effect is associated with the Mas/HO-1/p38 MAPK signaling pathway.The nonpeptide AVE0991 is an agonist of the angiotensin-(1–7) (Ang-(1–7)) Mas receptor and is expected to be a putative new drug for treatment of cardiovascular disease. However, the mechanisms involved in the antiproliferative effects of AVE0991 are not fully understood. We saw that the compound attenuated proliferation in an angiotensin II-induced rat vascular smooth muscle cells (VSMC) proliferation model. Moreover, treatment with AVE0991 (10 Mas-deficient mice Thymidine (20 Ci/mmol) was obtained from the Atomic Energy Institute of China . Fetal bovine serum (FBS) was purchased from the Sijiqing Company . AVE0991 was kindly provided by Dr. Juergen Puenter of Aventis Pharma  and male Sprague-Dawley rats (10 to 12 weeks old) were obtained from the Experimental Animal Facility of Sun Yat-sen University, China.The peptides Ang II and the Ang-(1–7) Mas receptor antagonist A779 were obtained from Bachem . The μg/mL penicillin, and 100 U/mL streptomycin (Invitrogen). The cell preparations were cultured at 37°C in a humidified atmosphere with 5% CO2. The culture purity was assessed by immunostaining with a monoclonal antibody against smooth muscle α-actin, followed by an anti-mouse fluorescein-conjugated goat IgG antibody. Using these methods, the purity of VSMCs reached more than 98%. Cells between passages 4 and 7 were used for all of the experiments thymidine incorporation efficiency of VSMCs was inhibited by AVE0991 in a dose-dependent manner  (−5 mol/L and 10−7 mol/L), the [3H]thymidine incorporation efficiency decreased significantly when compared to the Ang II group , although the efficiency was still higher than the control group. Nevertheless, when the cells were treated with Ang II + AVE0991 + A-779 (10−6 mol/L), this AVE0991-mediated effect which inhibited [3H]thymidine incorporation efficiency of VSMCs was abolished. However, when the VSMCs were treated with 10−5 mol/L AVE0991 or 10−6 mol/L A-779, both reagents affected the [3H]thymidine incorporation efficiency of the VSMCs. Compared to the control group, the [3H]thymidine incorporation efficiency was decreased when the cells were treated with AVE0991 alone . Conversely, the efficiency increased when the cells were treated with A-779 alone .Secondly, treatment with 10 < 0.01) . When th−6 mol/L) compared to the control group (−6 mol/L) + AVE0991-H (10−5 mol/L) group . However, neither AVE0991 nor A-779 alone had a significant effect on ROS expression.We sought to determine whether AVE0991 inhibition of Ang II-stimulation has any effect on ROS production afterwards. The ROS level was significantly increased in the Ang II group (10ol group . AVE09913H]thymidine incorporation efficiency of the VSMCs that were pretreated with ZnPPIX increased significantly when compared to the Ang II + AVE0991 (10−5 mol/L) group , although the efficiency was lower than the Ang II group   .−5 mol/L). The ROS level was significantly higher in the Ang II + AVE0991 (10−5 mol/L) + ZnPPIX group compared to the Ang II + AVE0991 (10−5 mol/L) group . However, this AVE0991-mediated effect was also abolished by the Ang-(1–7) Mas receptor antagonist A-779  group compared to the control group  (−5 mol/L) compared to the AVE0991 low-concentration treatment group . However, when the cells were treated with Ang II + AVE0991 and 10−6 mol/L of A-779, there was no change in the level of p38 phosphorylation compared to the Ang II + AVE0991 group.The phosphorylation of p38 was significantly higher in the Ang II (10 < 0.01) . AVE0991−6 mol/L) and control groups . This increase in HO-1 protein expression was more pronounced in the AVE0991-H treatment group (10−5 mol/L) compared to the AVE0991-L treatment group (10−7 mol/L) . However, when the cells were treated with Ang II + AVE0991 (10−5 mol/L) + A-779 (10−6 mol/L), there was no change in HO-1 expression compared to the Ang II + AVE0991 (10−5 mol/L) group. Neither AVE0991 nor A-779 treatment alone has a significant effect on HO-1 protein expression.HO-1 protein expression was not significantly different between the Ang II (10l groups . However−6 mol/L) group was significantly higher than that in the control group. AVE0991 treatment (10−5 mol/L) significantly inhibited Ang II-mediated p38 phosphorylation , although p38 phosphorylation was higher than that in the control group . However, when the cells were pretreated with the HO-1 inhibitor ZnPPIX (10−5 mol/L), the p38 phosphorylation level in the Ang II + AVE0991 + ZnPPIX group was significantly higher than that in the Ang II + AVE0991 group , although the phosphorylation level was lower than that in the Ang II group.We next sought to determine whether AVE0991 modulates p38 phosphorylation via HO-1 expression. As The major finding of this study is that AVE0991, a nonpeptide analog of Ang (1–7), attenuates Ang II-induced VSMCs proliferation by inducing heme oxygenase-1 expression and by downregulating p-38 MAPK phosphorylation. In addition, the present study shows that AVE0991 suppresses the Ang II-stimulated ROS production in VSMCs. Furthermore, experiments with the HO-1 inhibitor ZnPPIX indicate that AVE0991 decreases p-38 MAPK phosphorylation via induction of HO-1 protein expression in Ang II-induced VSMCs proliferation. In addition, treatment with AVE0991 attenuated proliferation of the VSMCs in a dose-dependent manner. However, all of the beneficial effects of AVE0991 were completely blocked by pretreatment with the Ang-(1–7) receptor antagonist A-779.3H]leucine incorporation and [3H]-Thymidine incorporation -Thymidine incorporation experiments -Thymidine incorporation) in vitro compared to the control group, which agrees with the previously published findings. Treatment with AVE0991 + Ang II significantly suppressed DNA synthesis. Moreover, when the concentration of AVE0991 was increased from 10−8 mol/L to 10−5 mol/L, AVE0991 inhibited the Ang II-mediated increase in DNA synthesis of the VSMCs in a dose-dependent manner. When the cells were pretreated with the Ang-(1–7) Mas receptor antagonist A-779, the beneficial effect of AVE0991 was completely abolished, which also suggested that the AVE0991 is a nonpeptide Mas receptor agonist. In addition, treatment with AVE0991 alone significantly suppressed VSMCs DNA synthesis. Conversely, A-779 alone also elicited a significant increase in DNA synthesis. However, differently to the results that were seen with DNA synthesis, neither treatment with AVE0991 alone nor treatment with A779 alone can alter ROS production and p38 phosphorylation. Future studies are needed to confirm these results.The present study indicates that Ang II promotes VSMCs DNA synthesis H oxidase enzyme system and promotes the generation of ROS, such as the superoxide anion and hydrogen peroxide which stimulate smooth muscle cell proliferation , 11, 25.Recently, induction of heme oxygenase- (HO-) 1 expression in vivo has been reported to suppress NADPH oxidase-derived oxidative stress. Additionally, HO-1 overexpression suppressed the Ang II-induced hypertrophic response in cardiomyocytes via decreasing the ROS production stress . Ang II-In summary, our results suggest that the ACE2-Ang-(1–7)-Mas pathway may play a more important antiproliferative role than our current understanding of endogenous Ang-(1–7) in RAS. Moreover, Ang II treatment upregulates p38 phosphorylation and ROS production, which contribute to VSMCs proliferation. Treatment with AVE0991 attenuates Ang II-induced VSMCs proliferation in a dose-dependent manner, which may be associated with regulation of Mas/HO-1/p38 signaling pathway.


Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca2+response. RPE cells from Atrap−/− mice showed smaller AngII-evoked Ca2+peak (by 22%) and loss of sustained Ca2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca2+transients in the RPE by releasing Ca2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca2+elevation.Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca The classical view of the Renin-Angiotensin System (RAS) as a systemic regulator of blood pressure has been extended, and a substantial number of studies have highlighted the importance of local RAS in a variety of extra-renal tissues, including adrenal glands Angiotensin II (AngII) mediates its biological effects through the activation of AT1R and angiotensin II type 2 (AT2R) receptors. It is, however, through AT1R activation that AngII elicits most of its well known effects, including vasoconstriction, electrolyte homeostasis, fibrosis, inflammation and proliferation. In the eye, AT1R activation has been implicated in the pathogenesis of many ocular disorders such as diabetic retinopathy In addition, proteins that directly interact with AT1R have received considerable attention because of their crucial roles in the first steps in signal transduction. Among them, the angiotensin receptor-associated protein (Atrap) is the best described The RPE plays an important role in maintaining retinal function by transporting nutrients, isomerizing all-trans to 11-cis retinal, and phagocytosing shed photoreceptor outer segments 2+from the endoplasmic reticulum (ER) 2+entry during AngII stimulation in juxtaglomerular cells is well established 2+channels 2+by AngII. However, the identity of molecules responsible of such Ca2+influx remains controversial. In the RPE, different Ca2+channel types are functionally expressed potentially enabling the AngII-mediated Ca2+entry such as CaV1.3, Orai,  TRPC1/4 and TRPV2 2+responses.However, how the RPE would accomplish AT1R-dependent signaling transduction is unknown. It has been proposed that activation of AT1R by AngII triggers a variety of complex intracellular signaling events, such as activation of phospholipase-D In contrast to the pathology role of the RAS in the retina, which is well characterized, its physiological function is less understood Atrap−/−) adult mice (C57BL/6x129SvEv) from a local colony were used in the study. . Atrap−/− and Atrap+/+littermates were used from heterozygous breeding pairs. All experimental procedures were performed following the guidelines approved by the Institutional Animal Care and Use Committee at University of Regensburg and the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in vision research. All animal experiments were formally approved by the German authorities  under the number 54-2532.1-06/10.Atrap knockout  RPE, retina, kidney and lung was carried out by isolation of total RNA either from cultured cells or from the corresponding tissues using the NucleoSpin RNAII kit . Reverse transcription was assessed using the RevertAid M-MuLV reverse transcriptase . After killing the mice, one eye from each subject was dissected and fixed in 4% (wt/vol) paraformaldehyde for 2 h at room temperature. Immunolabeling was performed on 3-µm paraffin sections, dewaxed, and treated in citrate buffer  pH 6.0. Sections were incubated for 5 min in phosphate-buffered saline (PBS) containing Tween 0.2% pH 7.2 (PBS-Tween). Sections were permeabilized with blocking/permeabilization solution  for 20 min. After three washing steps with PBS, eye sections were then labeled overnight at 4°C with anti-angiotensin II type 1 receptor (AT1R) antibody  diluted 1∶100 or with anti-Atrap antibody Western Blot analysis was performed as previously described in detail 2+/Mg2+free (Buffer I) for 20 min at 37°C. Eyes were transferred to buffer II  for 25 min at 37°C. The retinae were carefully removed from the RPE in buffer I and the eye cups were transferred into the activated enzyme solution . After incubation for 23 min at 37°C, RPE cells were isolated and collected in culture medium (Hams/F-12/DMEM (PAA)): 10% fetal calf serum (PAA), 1% penicillin/streptomycin (PAA), 0.5 µl/ml insulin transferring selenium A (GIBCO), 1% nonessential amino acids (PAA) and 5 mM Hepes solution (PAA)] and maintained at 37°C and 5% CO2. Within 7 to10 days, cells from wild-type and Atrap−/− mice were all densely pigmented, showed uniform cubical morphology and were ready to use.Culture of mouse primary RPE cells (passage 0) was performed as described previously by Culture of porcine primary RPE cells (passage 0) was performed as described previously by 2PO4, 1.6 K2PO4, 5 glucose, 1 MgCl2, and 1.3 Ca2+-gluconate pH 7.4 (310–320 mOsm). AngII, U73122∶1-amino]hexyl]-1H-pyrrole-2,5-dione, U73343∶1-amino]hexyl]-2,5-pyrrolidinedione, xestospongin C and losartan, were purchased from Sigma. SKF96365∶1-[β-(3-(4-Methoxyphenyl) propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride, 1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole and cannabidiol were purchased from Tocris Bioscience.During the recording, unless otherwise specified, cells were bathed in Ringer’s solution composed of the following (in mM): 145 NaCl, 0.4 KH2+imaging or protein isolation.A mixture of three different small interfering RNAs (siRNA) were designed in duplexes of 25 nucleotides and synthesized by Invitrogen . All three sense strands of the siRNA used to silence the porcine TRPV2 gene contained Alexa488 fluorescent dye. The three sense oligonucleotides are: 5′-Alexa 488-CACUGAGCUGGUGACCCUGAUGUAU-3′; 5′-Alexa 488-AGGUUCCUCAUCAACUUCCUGUGUU-3; 5′-Alexa 488-ACCUCCUUCGUGGACAGCUACUUUG-3′. A scrambled sequence siRNA double-stranded oligomer not homologous to any known gene (fluorescent Alexa 488-coupled oligonucleotide) served as a control. Transfection of primary porcine RPE cells was carried out 1 week after seeding using a lipophilic transfection reagent  in Opti-MEM. After 24–48 h, cells were used for patch clamping, Ca2+were performed as we previously described 2+imaging system and continuously superfused with Ringeŕs solution. For Ca2+measurements, fluorescence was elicited at 340 nm and 380 nm excitation wavelengths, emission was filtered with a 510 nm filter and detected by a cooled charged-coupled device camera . Data were collected and analyzed with MetaFlour software . We identified transfected cells by the expression of the fluorescent Alexa 488-coupled oligonucleotide and the given image was combined with the corresponding fura-2 AM image using ImageJ 1.44p software  for data analysis. Cells were stimulated during 80 seconds by bath perfusion of 100 nM AngII or with different blockers. The ratio of F340/F380 was converted to approximate [Ca2+]i as described by Measurements of intracellular free Cat tests was applied to determine whether densitometry measurements of western blot following a given treatment (mock or RNAi treatment) were significant. Data presented in bar graph shows mean ± SEM, p<0.05 was accepted as significant. n = number of cells from 4–9 (for poricine) or 9–16 (for mouse), independent experiments = cell cultures). The n-number were handled as individual cells because there was no indication that AngII-evoked Ca2+-raises spread among the cells due to gap-junction coupling. Only individual cells in a mono layer have responded to the AngII applications.Statistical significance for all calcium experiments was tested using ANOVA with Tukey’s multiple comparison post test. Statistical analysis using paired Student’s 2+(2+concentration were performed at the resting (before AngII application), peak (during AngII) and 60 s after the Ca2+-peak (delayed recovery phase) for all the experiments. In all control experiments, as they will be shown later on (non-transfected and transfected porcine or non-transfected mouse RPE cells) at 60 s after the Ca2+-peak, the intracellular calcium differed to the resting Ca2+by 23–50 nM (p>0.05). In order to have an internal control for each cell in experiments using different blockers, we used an experimental paradigm consisting of a double application of AngII in sequence. Control experiments were performed to show that repeated AngII stimulation for 80 seconds leads to comparable Ca2+transients. Application of AngII at 100 nM to pRPE cells with 7 minutes of wash out between applications (until [Ca2+]i returned back to the resting level) led to transient rise in [Ca2+]i concentration (In a recent study we showed that cultured porcine RPE (pRPE) represent a reliable model to study AngII signaling in the RPE 2+ which lantration . Importantration .2+release from the endoplasmic reticulum (ER), and the subsequent sustained Ca2+entry from the extracellular compartment or a combination of both 2+]i has returned back to the resting level, then AngII was applied a second time in the presence of a blocker for these two pathways. Perfusion of the phospholipase C (PLC) blocker U73122 (10 µM) completely inhibited the AngII-dependent calcium increase observed in pRPE  by RT-PCR in isolated mouse (Atrap+/+and Atrap−/−) RPE cells (Atrap+/+) RPE cells, we detected the expression of the two AT1 receptor paralogs (AT1R 1A and AT1R 1B) (Atrap+/+) and Atrap-deficient (Atrap−/−) mice  PE cells  and AtraPE cells  in isolaAT1R 1B)  and alsoAT1R 1B) . MoreoveAT1R 1B) . AT1R ex/−) mice  while At+/+mice . Notably+/+mice  insets i smaller . Remarkape cells .+/+Atrap), RPE cells from −/−Atrap mouse showed two main features in the AngII-evoked Ca2+signals: a reduced initial peak and sustained component remarkably smaller. Classical Ca2+signals elicited from G protein-coupled receptors comprises an initial peak that results from the release of Ca2+from the cytosolic stores, while the subsequently occurring phase results from the influx of extracellular Ca2+into the cell. In the next step we aimed to identify the molecular entity contributing to the delayed recovery phase which, most likely, results from activation of Ca2+-conducting ion channels. Human RPE cells express functional IGF-1 receptor-activated TRPV2 channels 2+rises in the RPE cells. With RT-PCR we found that porcine RPE cells also express TRPV2 channels (2+response to cannabidiol (a TRPV channel activator) and SKF96365 (a TRPV channel inhibitor). This was done in two separated set of experiments. In one cannabidiol was applied alone and in the second set cannabidiol was applied in the presence of SKF96365. Application of 15 µM cannabidiol, a broad range TRPV channel opener 2+from a resting concentration of 93.6±11.9 nM to a peak of 648.1±61.8 nM (2+rise by 48.61% (from a resting concentration of 81.6±7.7 nM to a peak of 369.1±42.9 nM ) when compared to control . In summary, this pharmacologic profile suggests that porcine RPE cells express functional SKF96365-sensitive TRPV2 channels likely contributing to the delay in recovery phase of AngII-induced Ca2+rises.When compared to wild type  in TRPV2-RNAi cells occurring 60 seconds after the peak AngII-response (arrows) was significantly reduced when compared with non-transfected or mock transfected cells (2+levels (delayed recovery phase), which were much higher than those observed for TRPV2-RNAi treated cells at 60 seconds  reduced endogenous TRPV2 channel protein levels to about 55% (green bar) of those in mock transfected cells (gray bar), according to densitometry analysis  of westee groups . Howevered cells , green.  seconds . Togethe2+responses in the RPE involves both calcium mobilization from intracellular stores and extracellular Ca2+entry, most likely through TRPV2 channel activation. Furthermore, a new role of Atrap for AngII-dependent Ca2+-signalling was found in the RPE.The present study was performed to evaluate the calcium signaling pathway triggered by AngII in the RPE. The most remarkable result presented here is that AngII-dependent Ca2+-imaging experiments. As we have reported previously cultured pRPE cells showed biphasic increases in intracellular free Ca2+upon stimulation by AngII. Since in the presence of the specific AT1R blocker losartan AngII failed to evoke Ca2+transients, the AngII-stimulated effect was due to specific activation of AT1R receptors and not AT2 receptors, which is in accordance with previously published data Milenkovic et al. (2010) reported recently that systemic infusion of AngII into mice caused a significant decrease in renin expression in the kidney and a reduction of the renin mRNA levels in both RPE cells and neuronal retina, whereas systemic application of ACE inhibitor increased the renin expression in the RPE by 20-fold 2+from the ER through the activation of PLC and concomitant generation of IP3. Incubation with the PLC blocker U73122 completely abolished the AngII Ca2+response and inhibition of calcium release by using the IP3R blocker xestospongine C also prevented the calcium rise evoked by AngII. In agreement with our finding, Fellner and Arendshorst (2005), using calcium imaging techniques on isolated afferent arterioles reported that the peak response to AngII was attenuated by inhibiting the IP3 receptors with 8- octyl 3,4,5-trimethoxybenzoate (TMB). These IP3R blockers also inhibited AngII-mediated AT1R induced calcium release from the ER through PLC/IP3 pathways in preglomerular renal vascular smooth muscle cells 2+mobilization in vivo, since TMB decreased AngII-dependent renal vasoconstriction 2+]i in a biphasic manner, with both an initial peak and plateau phase. Thus in the RPE as in other AngII sensitive cells the AngII-dependent rise of [Ca2+]i depends on release of Ca2+from cytosolic stores.Our study shows that AngII stimulation of AT1R receptor leads to release of Ca−/−Atrap mice demonstrated AT1R expression as it was shown by immunohistochemistry on retina sections. In addition to that cultured RPE cells from −/−Atrap mice show AngII-evoked Ca2+responses which prove that the functional expression of AT1R is maintained in the cell culture. Thus, we can use mouse RPE cells to study AngII-mediated Ca2+transients in the absence of Atrap. Also in the mouse RPE, AngII led to a biphasic increase in intracellular Ca2+, which consists of an initial peak followed by sustained phases. We found that −/−Atrap RPE cells showed a smaller Ca2+peak and a smaller intracellular Ca2+during the recovery phase (measured at 60 s), which implies that Atrap plays a role in mediating the different events of AngII-mediated Ca2+signaling. This observation points to a new function of Atrap. Atrap had been considered to be a negative regulator of the surface expression of AT1R −/−Atrap retina sections. The smaller Ca2+signals would indicate more likely a role as a positive regulator of AT1R-mediated signaling cascade. Due to the limited material of the RPE cells in mice it was not possible to measure the AT1R surface expression in wild-type and Atrap knock-out mice. Because the absence of Atrap reduces the Ca2+peak, Atrap might directly influence the mechanism of Ca2+signaling, perhaps by depleting cytosolic Ca2+stores. However, without knowing other proteins with which Atrap might interact it is uncertain what the nature of this influence might be and in which parts of the signaling pathway Atrap might be involved in.Since Atrap is a protein bound to the AT1R, it is possible that Atrap is involved in the ignition of the cascade of AT1R signaling in the RPE. We found that mouse RPE cells in situ and in vitro and porcine RPE in vitro express Atrap. Both AT1R and Atrap were found at the basolateral membrane of the RPE in the wild-type. This implies a functional association of the proteins. However, a proportion of Atrap was also found at the apical membrane of the RPE which implies possible functions of Atrap other than AngII-signaling. 2+entry pathways following stimulation by AngII that generate a plateau phase, both in RPE cells and in other cell types. In renal vascular smooth muscle cells, removal of extracellular Ca2+attenuated the AngII-mediated peak response to about 50% of the control value, with the remaining proportion likely stemming from intracellular stores 2+transients likely results from a Ca2+influx into the cell through activation of Ca2+conducting ion channels. It has been long known that many TRP channels can be stimulated subsequent to PLC activation by G protein-coupled receptors, by accumulation of DAG in the membrane after PLC activation, by tyrosine kinase receptors, or by intracellular Ca2+directly, this last feature a property of the majority of these ion channels i and Go-protein blocker) in CHO and HEK cells suggested that lysophosphatydilcholine-(LPC) dependent TRPV2 activation is mediated by G-protein pathways. In the human RPE, functional expression of TRPV2 channels has also been shown 2+in porcine RPE cells. Although cannabidiol is known to activate TRPV1,2,3 and 4, it is more selective to TRPV2 2+signaling. SKF96365 is known to block TRPC channels, T-type Ca2+channels and TRPV2 channels 2+channels only TRPV2 is known to be activated by cannabidiol. Thus, this experiment clearly demonstrates the functional presence of TRPV2 channels in porcine RPE cells. However, the cannabidiol response was not fully blocked by SKF96365. This can be explained by the fact that the RPE also express the TRPV 1, 3 and 4 channels which are also known to be activated by cannabidiol. It is likely that the SKF96365-insensitive part of the cannabidiol response is due the activation of TRPV1, 3 or 4 channels.In general, less well understood are the mechanisms involved in the activation of Ca2+rises. Indeed, SKF96365 prevented the AngII-evoked Ca2+response in pRPE which strongly reinforces the concept of a role for Ca2+entry from the extracellular compartments and implies a contribution of TRPV2 channels. However, nothing has been previously reported about TRPV2 channel activation by AngII. Because both mRNA and protein TRPV2 were shown to be expressed in pRPE cells, we investigated the implications of TRPV2 activation on the AngII-mediated Ca2+signaling in RPE cells by means of RNAi techniques. Our calcium imaging data clearly show that knockdown of endogenous TRPV2 channels with TRPV2-RNAi strongly reduced the delay in the recovery phase AngII-mediated Ca2+response (later 60 seconds after the peak burst) and showed the same magnitude as in SKF96365-treated cells. Thus, the remaining Ca2+elicited after AngII stimulation in RNAi treated cells very likely comes from Ca2+release from the stores. This observation strongly indicates that TRPV2 contributes to the AngII-induced Ca2+entry in the RPE.Based on the pharmacologic analysis discussed above SKF96365 was used to gain insight to whether TRPV2 channels might contribute to the delayed recovery phase of AngII-evoked CaThe AngII signaling at the RPE is of importance to understand inflammatory and/or neovascular diseases of the retina. In recent years the effects of AngII signaling in diseases such as diabetic retinopathy or age-related macular degeneration has gained increasing attention 2+by activation of PLC and subsequent release of Ca2+from intracellular Ca2+stores via activation of IP3-receptors. This leads to an initial peak of the Ca2+response followed by a Ca2+influx into the cell through TRPV2 channels, which yields a sustained Ca2+increase. The generation of the initial peak and the sustained response requires the activation of Atrap. In particular, the sustained Ca2+response requires the presence of Atrap, as suggested by the fact that it was almost abolished in RPE cells from Atrap knock-out mice. This also appears to be the first physiological demonstration of a role of TRPV2 in the intraocular RAS. Together, our results constitute a significant and novel addition to our understanding regarding how the systemic RAS interacts with the RAS in the eye.In summary the following picture of AT1R signaling in RPE cells emerges from our data. Activation of AT1R leads to an increase in intracellular free CaFigure S12+response.Effect of U73343, the inactive analof of U73122, on AngII-evoked CaA: Application of AngII (100 nM) for 80 seconds (bars) caused transient Ca2+response in pRPE cells. Bath application of 100 nM AngII (bars) produced a Ca2+response that was not abolished by co-application of 10 µM U73343 (gray shadow), the inactive analog of the phospholipase C (PLC) blocker U73122. B: summary of data from experiments shown in A. Bars in 2+responses before (open bars) during the peak (black bars) and at 60 s after the maximum AngII-elicited calcium response (gray bars).  p<0.05, ** p<0.0001; repeated measures ANOVA. n = number of cells from 9 independent experiments.(TIF)Click here for additional data file.