Although not well understood this allows normally contractile VSMCs to become proliferative following vessel injury, a process essential for repair but which also contributes to vascular remodelling, atherogenesis and restenosis. Here we show that the Ca2+/HCO3−-sensitive enzyme, soluble adenylyl cyclase (sAC), links Ca2+ influx in human coronary artery smooth muscle cells (hCASMCs) to 3′,5′-cyclic adenosine monophosphate (cAMP) generation and phosphorylation of the transcription factor Ca2+/cAMP response element binding protein (CREB). Store-operated Ca2+ entry (SOCE) into hCASMCs expressing the FRET-based cAMP biosensor H187 induced a rise in cAMP that mirrored cytosolic [Ca2+]. SOCE also activated the cAMP effector, protein kinase A (PKA), as determined by the PKA reporter, AKAR4-NES, and induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and CREB. Transmembrane adenylyl cyclase inhibition had no effect on the SOCE-induced rise in cAMP, while sAC inhibition abolished SOCE-generated cAMP and significantly reduced SOCE-induced VASP and CREB phosphorylation. This suggests that SOCE in hCASMCs activates sAC which in turn activates the cAMP/PKA/CREB axis. sAC, which is insensitive to G-protein modulation but responsive to Ca2+, pH and ATP, may therefore act as an overlooked regulatory node in vascular Ca2+-transcription coupling.Ca Quiescent VSMCs change to a synthetically active and proliferative phenotype in response to stimuli such as mechanical stress, growth factors or inflammatory mediators2. This plasticity is important for vessel repair at times of vascular injury, but is also a central feature of vascular disease. Proliferation of VSMCs in the vessel wall contributes to atherogenesis, vascular remodelling in pulmonary hypertension, and to restenosis, a narrowing of the vessel lumen following balloon angioplasty and bypass vein grafting5. The altered pattern of gene expression that drives changes in VSMC phenotype correlates with different patterns of Ca2+ signaling7, although the molecular basis of Ca2+-regulated gene expression in VSMCs is not well understood.In healthy blood vessels, the majority of vascular smooth muscle cells (VSMCs) are quiescent, contractile and proliferate slowly2+-induced gene expression can be mediated in VSMCs by the transcription factor Ca2+/cyclic AMP response element binding protein (CREB)6. CREB binds as a dimer to a conserved cyclic AMP response element (CRE) in the promoter of pro-survival genes and activates transcription in response to a variety of diverse extracellular signals8. Multiple Ca2+-regulated signaling cascades converge to phosphorylate CREB at a critical Ser133 residue, which induces recruitment of coactivators and the formation of an active transcriptional complex9. Ser133 is a substrate for a number of kinases that are either directly or indirectly activated by Ca2+, including protein kinase A (PKA), protein kinase C, members of the calcium/calmodulin-dependent kinase (CaMK) family and ribosomal S6 kinase (RSK) −26. Intriguingly, Ca2+ signals from different sources trigger different patterns of CRE-regulated VSM gene expression10. Voltage-dependent Ca2+ influx through L-type Ca2+ channels induces CREB phosphorylation in both cultured VSMCs and in intact mouse cerebral arteries12 and correlates with an increase in transcription of proliferation-associated CRE-containing genes such as c-fos and early growth response-1, egr-112. In agreement with this, cerebral arteries from hypertensive rats are depolarized compared to normotensive animals and have elevated cytosolic Ca2+ and increased CREB phosphorylation/c-fos expression, which can be reversed by L-type Ca2+ channel inhibition13. Ca2+ influx in response to depletion of the sarcoplasmic reticulum (SR) Ca2+ stores  also activates CREB in both cultured VSMCs and intact arteries. Here, CREB activation leads to the transcription of a set of CRE-regulated genes that overlaps, but is distinct from, the genes expressed following L-type Ca2+ channel activation14. Several transient receptor potential (TRP) channels as well as the Ca2+ release-activated channel protein 1 (Orai1) and the SR Ca2+ sensor stromal interaction molecule 1 (STIM1) are expressed in VSM and may mediate SOCE16. Orai1 and STIM1 are both upregulated in response to vascular injury17 and are also required for the activation of CREB involved in VSMC proliferation18. In line with this, STIM1 knockdown decreases SOCE-induced CREB activation in human coronary artery SMCs and suppresses cell growth19. STIM2 is also upregulated in pulmonary artery hypertension, which raises basal Ca2+ levels and enhances pulmonary artery VSMC proliferation and vascular remodelling at least partially through activation CREB20. Indeed, inhibition of Orai-mediated Ca2+ entry and the resultant suppression of Ca2+-transcription coupling has been suggested to underlie the anti-proliferative effects of stent-coating drug sirolimus (rapamycin) in human arteries21.Ca25, and anti-proliferative outcomes27, and this complex behaviour is also reflected in vivo. In rat models, phospho-CREB is elevated in neointimal VSMCs that appear after balloon injury of the carotid artery28, and increased phospho-CREB levels correlate with high proliferation rates in cerebral artery following several forms of vessel injury linked with susceptibility to stroke29. In contrast, analysis of arteries taken from bovine systemic and pulmonary circulation show a reverse correlation between CREB phosphorylation and proliferation, with phospho-CREB high in quiescent, contractile smooth muscle cells from the healthy vessel’s medial layer30. Here, chronic exposure of animals to lower than normal oxygen levels leads to increased VSMC proliferation in pulmonary vessels and decreased levels of both CREB and phospho-CREB30. This highlights our incomplete understanding of how different environmental cues are integrated by VSMCs to activate gene transcription.In cultured VSMCs CREB activation is associated with both pro-2+ entering the cytosol binds to calmodulin (CaM) and that the Ca2+/CaM complex then activates Ca2+-sensitive transmembrane adenylyl cyclases (tmACs), leading to generation of cyclic AMP and the activation of PKA8. An alternative source of cyclic AMP comes from soluble adenylyl cyclase (sAC), a novel enzyme that lacks the two hydrophobic domains that embed tmACs in the membrane and which thus distributes about the cell in regions remote from the plasma membrane31. Here, we demonstrate that SOCE activates sAC to generate cyclic AMP and induce phosphorylation of CREB at Ser133 in human coronary artery smooth muscle cells. sAC is insensitive to G-protein modulation and forskolin activation, but directly activated by Ca2+ (independently of CaM) and HCO3− and responds to physiologically relevant ATP concentrations32. Since CO2 is in equilibrium with HCO3− due to the activity of carbonic anhydrases, sAC functions as a CO2/HCO3−, and thus indirect, pH sensor. This ability to sense and integrate fluctuations in intracellular signals suggests sAC may be an important and overlooked regulatory node in the VSMC Ca2+-transcription pathway.The current model for PKA-mediated phosphorylation of CREB in VSMCs is that Ca2+ entry (SOCE), hCASMC internal Ca2+ stores were depleted by bathing fura-2-AM-loaded cells in nominally Ca2+-free solution for 10 minutes before addition of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, thapsigargin  to the same solution. Ca2+ leaks passively from the stores through permanently open leak channels and is subsequently removed from the cytosol either by extrusion across the plasma membrane or reuptake into the stores by SERCA. Inhibition of SERCA thus blocks reuptake and causes a transient rise in intracellular Ca2+ as leak Ca2+ briefly builds up in the cytosol before being moved out of the cell by plasma membrane Ca2+ ATPases  to the extracellular solution induces a rapid rise in cytosolic Ca2+ due to SOCE  and one soluble isoform , which are differentially regulated by Ca2+ 36. Of the tmACS, AC5 and AC6 are inhibited by sub-micromolar concentrations of Ca2+ while AC1 and AC8, and to a lesser extent AC3, are stimulated by Ca2+ via CaM binding36. Consistent with other findings39, reverse transcription-PCR, using cDNA derived from hCASMCs as a template and primers designed to detect AC isoforms 1–10, confirmed the presence of transcripts for AC3, 4, 6, 7, 9 and 10  is a well-characterised cell-permeable pharmacological tool for distinguishing between tmACs and sAC. In whole-cell lysates DDA inhibits tmACs with an IC50 of ~10 μM and sAC with an IC50 of over 500 μΜ41. Preincubation of hCASMCs in DDA (100 μM) significantly reduced the transient increase in cyclic AMP seen upon addition of thapsigargin, but had no effect on SOCE-generated cyclic AMP .The Ca 10 Fig. . Since AAMP Fig. . In paraAMP Fig. . Pre-incOCE Fig. . Aside fa2+ Fig. .Figure 22+ reintroduction protocol  significantly reduced SOCE-induced VASP phosphorylation  or 4CE (20 μM) significantly reduced SOCE-induced phosphorylation of VASP Fig. . For thecol Figs , 2 in thion Fig. . The eff133 phospho-site  to the extracellular solution induces significant phosphorylation of CREB at Ser 133. Pre-incubation with the catechol estrogens 2CE and 4CE 40. Acute inhibition of sAC abolished the intracellular increases in cyclic AMP seen during thapsigargin-induced store depletion and subsequent SOCE, suggesting that activation of sAC is a prerequisite for cyclic AMP generation via both processes. To our knowledge, SOCE has not previously been reported to activate sAC, although Ca2+ influx through voltage-gated Ca2+ channels activates sAC in insulinoma cells43.Our data suggest that Ca2+ influx through store-operated channels activates CREB and induces transcription of a distinct set of genes in VSMCs10, but the pool of cyclic AMP responsible for PKA-mediated CREB activation was believed to be generated exclusively by tmACs. More recent work shows that CREB can mediate both pro- and anti-mitogenic responses in VSMCs dependent upon different modes of activation44. Activation of CREB by cyclic AMP generated by either forskolin or agonists at G protein-coupled receptors (both of which exclusively activate tmACs) is independent of Ser133 phosphorylation but dependent on nuclear translocation of CREB Regulated Transcription Coactivators (CRTCs)45. This form of CREB activation is anti-proliferative. In contrast, stimulation of VSMCs by mitogens significantly increases CREB activity via Ser133 phosphorylation but not CRTC activation. CREB activated by this mechanism induces pro-mitogenic responses since CREB silencing under these circumstances inhibits basal and growth factor-induced proliferation44. Our studies, which were conducted in the presence of serum and show Ser133 phosphorylation, suggest that sAC-generated cyclic AMP may contribute to this latter pro-proliferative pathway.By using biosensors to monitor real-time changes in the activity of the major cyclic AMP effector, PKA, our data also show that SOCE/sAC-generated cyclic AMP activates PKA. This induces phosphorylation of a number of PKA substrates within the cell, including VASP and the transcription factor, CREB. Previous studies have shown that Ca46, the only previously reported role for vascular sAC involves activation by oxidative stress to promote the intrinsic apoptotic pathway47. Similar roles in apoptotic signaling have been described in coronary endothelial cells48 and cardiomyocytes49. In cardiomyocytes there is evidence of roles for sAC outside of death pathways in the development of cardiac hypertrophy50, and in matching energy production to nutrient levels in mitochondria51. Here, CO2/HCO3− generated by the citric acid cycle activates sAC localized in the matrix to produce cyclic AMP, which mediates a PKA-dependent increase in the activity of the electron transport chain and oxidative phosphorylation52. A source of cyclic AMP that is distinct from the G-protein-responsive plasma membrane-localized tmACs is likely to be an important general feature in signaling. Cyclic AMP has a limited diffusion distance largely due to the action of phosphodiesterases that restrict its spread throughout the cell to ensure the fidelity of signal transfer to intended targets53. sAC is able to locate to several intracellular compartments, including the cytosol, the nucleus54, and the mitochondria55. Indeed, in skin cells, sAC localizes to the nucleus where it complexes with CREB56. Zippin et al. demonstrated the existence of a functional signaling complex consisting of sAC, the PKA holoenzyme and CREB in the nucleus of human cell lines and in a subpopulation of rat liver cells54. This complex ensures rapid CREB activation in response to changes in HCO3− and was distinct from the slower hormone or neurotransmitter CREB activation via tmACs.Compared to the relatively well-studied tmACs, our knowledge of the role of sAC in the vasculature is limited. Although widely expressed throughout the cardiovascular system2+ and HCO3−-sensitive sAC, which generates cyclic AMP to activate the PKA/CREB axis. Being located away from the plasma membrane may allow sAC access to pools activators and effectors that are distinct from the membrane-confined tmAC microdomains. This coupled with its ability to respond rapidly to changes in the intracellular environment suggests it may act as a regulatory point in VSMC Ca2+-transcription coupling. Although the functional consequences of sAC-induced CREB phosphorylation are undetermined, recent evidence suggests it may link to pro-mitogenic responses in VSMCs44. A single gene encodes sAC, which is widely expressed in mammalian tissues31. Alternative splicing generates multiple sAC isoforms57, suggesting that VSMC sAC may warrant investigation as a potentially novel drug target for proliferative vascular disease. It will also be important to determine if specific subsets of phosphodiesterases control the dispersal of sAC-generated cyclic AMP in VSMCs as these ‘druggable’ targets may offer an additional route to manipulate the signaling pathway.In conclusion, our data suggest that in hCASMCs SOCE activates the CaH-benzimidazol-2-ylthio)propanoic acid 2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide (KH7) and forskolin were purchased from Tocris Bioscience . Fura-2-AM and Fluo-4 AM were purchased from ThermoFisher Scientific . All other chemicals, including 2-hydroxyestradiol (2-CE), 4-hydroxyestradiol (4-CE), and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma Aldrich .2′-5′dideoxyadenosine (DDA) and thapsigarin were purchased from Merck Millipore . (6)-2-(1−1), epidermal growth factor (0.5 ng mL−1) and insulin . Cells between passages 6–12 were cultured in a humidified incubator at 37 °C and 5% CO2 atmosphere, with culture media changed every 2–3 days. Cells were routinely sub-cultured at 70–80% confluency.Human coronary artery smooth muscle cells  were cultured in smooth muscle cell growth medium 2 containing 5% (v/v) foetal calf serum supplemented with basic fibroblast growth factor (2 ng mLAdenoviruses encoding H187 [AD(RGD)-CMV-H187] and AKAR4 [AD(RGD)-CMV-AKAR4-NES] were cloned, packaged, amplified and titrated by Vector Biosystems Inc . For FRET-based imaging studies, viral particles of either AD(RGD)-CMV-H187 or AD(RGD)-CMV-AKAR4-NES in a solution containing Dulbecco’s Modified Eagle Medium , 2% (w/v) bovine serum albumin  and 2.5% (v/v) glycerol, were added directly to hCASMC suspensions at 10 MOI (multiplicity of infection). Cells were then seeded onto glass-bottomed culture dishes  for imaging 24 or 48 hours later.58. This sensor consists of the cAMP effector Epac1 fused to a cyan fluorescent protein (CFP) donor (mTurquoise2) and an optimized yellow fluorescent protein (YFP) acceptor . Binding of cAMP causes a conformational change resulting in a loss of energy transfer between the two fluorophores that can be detected as an increase in the CFP/YFP fluorescence ratio. To prevent construct enzymatic activity affecting downstream effectors, Epac has been rendered catalytically dead (CD) and a membrane-targeting domain is deleted (ΔDEPmutant) to allow measurement of cAMP in the bulk cytosol. Real-time PKA catalytic activity was measured in hCASMCs using the FRET-based PKA reporter A-Kinase Activity Reporter-Nuclear Export Signal, AKAR4-NES59 . AKAR4 contains a PKA substrate and a phospho-amino acid binding domain (PAABD) between the FRET pair Cerulean and Venus. The PAABD binds to the PKA substrate when it is phosphorylated by active PKA, causing a decrease in distance between the fluorophores and an increase in FRET. Prior to each experiment, H187 and AKAR4-expressing hCASMCs were rinsed twice in a Na+-HEPES-based solution containing (mM): 140 NaCl, 4.7 KCl, 1.13 MgCl2, 10 HEPES, and 10 glucose ; adjusted to pH 7.4 (referred to as HEPES solution hereafter). This HEPES solution was also supplemented with 1% (v/v) foetal calf serum (0.4 ng mL−1), epidermal growth factor (0.1 ng mL−1), insulin (1 μg mL−1) and 1.8 mM CaCl2 unless stated otherwise. All test solutions, including controls, were made using this HEPES solution.Real-time cyclic AMP changes were measured in live hCASMCs transduced with adenoviruses encoding the biosensor H187 -tdcp173Venus; plasmid used for adenoviral construction a gift from Kees Jalink, Univ of Amsterdam, Netherlands)Cells were imaged using an Olympus IX71 based inverted imaging system . Excitation of fluorescence was provided by a 150 W xenon high stability lamp and a polychrome V monochromator wavelength control . Emission light was collected using band or high pass filters and sent to an iXon DV885 cooled EM CCD camera . Camera binning was set to 2 both horizontally and vertically which provided 512 × 512 pixel points per image. TillVision software  was used to control the parameters of image acquisition and selection of regions of interest, and allowed for the measurement of fluorescence while the experiment was in progress. H187 and AKAR4-expressing hCASMCs were both excited at 405 nm, close to the excitation maximum of the donor, CFP. Emissions of both CFP and YFP fluorescence were measured simultaneously using an Optical Insights Dual-View Filter cube with D480 + 30 m and D535 + 40 m Filter Sets,  which only allows emission wavelengths of 480 nm and 535 nm to reach the camera. During each experiment non-fluorescent cells or regions containing no cells were used as background fluorescence from which the reading was subtracted.2+ dye Fura-2 AM dissolved in HEPES solution for 1 hour at room temperature in the dark. Fura-2 AM-loaded cells were then rinsed twice with HEPES solution, before being placed into the perfusion chamber for live cell imaging using the Olympus IX71 based inverted imaging system (above). The cell was illuminated alternately with 340 and 380 nm light (bandpass 5 nm) and the emission signal was detected at 510 nm (40 nm bandpass). Temporal change in photon counts was measured, backgrounds were subtracted, and the complete ratio was obtained at 50 Hz. As in FRET experiments, cells that were not fluorescent or regions containing no cells were used as background from which the reading was subtracted. For indicated experiments, cells were incubated with 5 µM Fluo-4 AM in glass-bottomed dishes , washed as described above and imaged using a Zeiss LSM 710 confocal microscope. Cells were excited with 488 nm light and the emission signal was detected within a range of 493–622 nm.hCASMCs were incubated in 5 µM of the ratiometric Ca2 atmosphere until 70–80% confluence. Cells were then incubated in vehicle control (0.1% DMSO) or incubated in zero extracellular Ca2+ for 5 mins before being treated with thapsigargin  for 5 min in zero extracellular Ca2+ followed by introduction of 1.8 mM extracellular CaCl2 for a further 5 min. As a positive control cells were exposed to saturating concentrations of forskolin (20 μM) and IBMX (500 μM) only. In some experiments, cells were incubated in the tmAC inhibitor DDA (100 μM) or the sAC inhibitors KH7 or 2CE or 4CE (20 μM) for at least 15 minutes before TG treatment. Inhibitors were then present throughout the experiment. The solution used for all treatments was the same supplemented HEPES solution used for fluorescent imaging. Following each treatment, cells were then lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 1% (v/v) protease inhibitor cocktail and 1x PhosSTOP phosphatase inhibitor cocktail (both Sigma-Aldrich). The resultant lysates were rotated for 15 minutes at 4 °C and then centrifuged at 18,220 × g for 15 minutes at 4 °C. Resultant supernatants were mixed 1:3 (v/v) with 4x sodium dodecyl sulfate-polyacrylamide (SDS) sample buffer, before heating to 98 °C for 10 minutes. Protein concentrations were quantified using a PierceTM BCA Protein Assay Kit (Thermofisher Scientific). Samples were kept at −20 °C until use. Proteins within the lysates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 10–15% polyacrylamide gels and transferred electrophoretically onto nitrocellulose membranes . Immunoblotting was performed as previously described60.hCASMCs were seeded onto 6-well plates and allowed to grow at 37 °C and 5% COα actin  was from Sigma-Aldrich. Anti-phospho-CREB  and anti-GAPDH (1: 2000) were purchased from Abcam . The secondary antibodies used were: anti-mouse IgG (H + L) horseradish peroxidase (HRP) conjugated polyclonal antibody and anti-rabbit IgG (H + L) HRP conjugated polyclonal antibody , both used at a dilution of 1:5000.For immunoblotting the primary antibodies used were against: total CREB, vasodilator-stimulated phosphoprotein (VASP) and phospho-PKA substrates (RRXS*/T*); all purchased from Cell Signaling Technologies  and used at a dilution of 1:1000. Anti-smooth muscle 61.Total RNA extraction and reverse transcription was carried out as previously described61. Primers used to amplify specific adenylyl cyclase isoforms were previously published62. Products were analysed by running on a 3% agarose gel containing Midori Green  for ~1 hour at 80 V. Bands were excised under ultraviolet light and products purified using a QIAquick Gel Extraction Kit (Qiagen) according to manufacturer’s instructions. All products were verified by sequencing .Touchdown PCR was carried out as previously described2+/cyclic AMP measurements, n numbers indicate number of cells from at least 3 experimental repeats conducted on separate days. For immunoblots n numbers indicate number of experimental repeats.Statistical tests stated throughout. Results are expressed as the mean ± standard error of mean. For real-time Ca


Human mesenchymal stem cells (hMSC) are becoming increasingly popular in tissue engineering. They are the most frequently used stem cell source for clinical applications due to their high potential to differentiate into several lineages. Cartilage is known for its low capacity for self-maintenance and currently there are no efficient methods to improve cartilage repair. Chondrogenic differentiation of hMSC isolated from different tissues is widely employed due to a high clinical demand for the improvement of cartilage regeneration. Calcium channels that are regulated by physical stimuli seem to play a pivotal role in chondrogenic differentiation of MSCs. These channels increase intracellular calcium concentration, which leads to the initiation of the relevant cellular processes that are required for differentiation. This review will focus on the impact of different physical stimuli, including electrical, electromagnetic/magnetic and mechanical on various calcium channels and calcium signaling mechanisms during chondrogenic differentiation of hMSC. The poor regenerative capacity of human articular cartilage is a major challenge for researchers and clinicians that are working in the area of cartilage repair and it has gained particular attention during the past decades. The degradation of cartilage tissue, caused by trauma or chronic and progressive degenerative joint disease, e.g., osteoarthritis (OA) or rheumatoid arthritis (RA), has become a global problem for which no efficient therapy is available . The ult2+) play a crucial role in regulating cell functions [2+ was shown to improve cell differentiation potential [Differentiation of MSCs includes multiple stages that generate specific proteins according to each lineage. A number of biological/chemical factors can regulate the differentiation process. For instance, steroid hormones and growth factors, such as tumor growth factors-β (TGF-βs), Activin A, and bone morphogenetic proteins (BMP)-2,-4,-7 are crucial to induce chondrogenesis and synthesize collagen type 2, aggrecan and other extracellular matrix (ECM) proteins [unctions , whereasotential .2+ can enter cells through different calcium channels, primarily voltage operated calcium channels (VOCCs), transient receptor potential (TRP) channels, and purinergic receptors have been shown to be regulated by physical stimuli [Although Ca stimuli ,6,7,8. S stimuli . Members stimuli . Therefo2+ levels control a number of crucial processes in cells, including exocytosis, apoptosis, motility, gene transcription, and differentiation [2+ can diffuse into mitochondrias or nucleus, where interplays in several counteracting processes, which are then divided into Ca2+ “on” and “off” processes, depending whether they increase or decrease intracellular Ca2+. When the cells are in their “resting” state, Ca2+ concentration balance lies in favor to “off” processes, decreasing Ca2+ up to 100 nmol/L [2+ can be increased up to 1 µmol/L. Intracellular Ca2+ binds to different proteins that take part in Ca2+ signaling pathways [2+ metabolic pathway induction, but also in silencing Ca2+ signaling, 4, 5 bisphosphate (PIP2), C2 domain, calcineurin, and others [2+ sources for generating intracellular signals, one of which is Ca2+ entry through the plasma membrane and the other is the release of intracellular Ca2+ stores. Intracellularly Ca2+ is stored in the endoplasmic reticulum, where two main channels, namely, inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR), may release Ca2+ if its concentration is getting low [2+ transport across the plasma membrane occurs via two distinct pathways—VOCCs, and agonist-dependent Ca2+ entry pathways, which include a number of different Ca2+ channels.Intracellular Cantiation . Intrace0 nmol/L . Howeverpathways . One of d others . Most ofting low . Ca2+ tr2+, even though Na+ concentration in an extracellular space is 70-fold higher [2+ homeostasis, L-type channels are considered to be the major HVA channels. They are the main route for calcium influx in cardiac, skeletal, and smooth muscle cells [2+ signaling [VOCCs comprise a group of membrane proteins that are predominantly found in “excitable” cells . These channels are well-known for their involvement in electrical current generation, however, they are also abundantly expressed in a range of “non-excitable” cells, including osteoblasts and chondrocytes . VOCCs ad higher . VOCCs ad higher . The LVAd higher , see Figd higher . Due to le cells . All HVAignaling   and categorized into seven subfamilies: TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, and TRPV. As TRP channels are prevalent in almost all mammalian cells and are involved in regulation of Ca2+ concentration and signaling is significant in both excitable and non-excitable cells [TRP channels are ion channels that are induced by various physical and chemical stimuli, ranging from PIP2, Cal inputs . When acle cells . Some TRle cells . Other cle cells . Blockinle cells .2+ influx into the cell in response to interactions with ATP molecules. P2X4 in particular was shown to regulate Ca2+ entry into the cytosol in chicken mesenchymal cells, which was linked to cartilage formation [2+ concentration that is needed for hMSC differentiation [Purinergic receptors are activated by purines or their nucleotides. Their function is mostly associated with purinergic signaling pathways that are involved in neurological processes , howeverormation  and contntiation . The P2Xntiation .2+ signaling in MSCs has gained increasing interest during the past decades. In 2002, Kawano and colleagues were the first to describe Ca2+ signaling in MSCs, suggesting that Ca2+ plays an important role in controlling many cellular processes, including cell growth, fertilization, transformation, secretion, smooth muscle contraction, sensory perception, and neuronal signaling [2+ oscillations in MSCs and the transporters that may be responsible for those oscillations, as reviewed elsewhere [2+ oscillations in undifferentiated hMSCs in the absence of any agonist stimulations have been observed, suggesting that Ca2+ oscillations are predominantly referred to Ca2+ release from intracellular stores, but not to the Ca2+ entry through plasma membrane [2+ channels in osteogenesis [2 [2+ entry through VOCCs might modulate the differentiation capacity of different cell types.The role of Caignaling . Particulsewhere . Howevermembrane . During ogenesis . Howeverogenesis . Furthernesis [2 , which s2+, inhibiting voltage-gated calcium channels, and depleting intracellular calcium stores suppressed the beneficial effect of hydrostatic pressure on chondrogenesis, indicating that Ca2+ mobility might play an important role in the mechanotransduction of mechanical pressure [2+ is known to be involved in wide range of Ca2+ sensitive signaling pathways, including various protein kinase systems, which may enable distinct gene expression profiles via differential activation of key transcription factors , giving rise to chondrogenic differentiation [2+ for their activation [2+/calmodulin signaling pathways on explants from bovine articular cartilage has demonstrated the importance of intracellular Ca2+ in aggrecan mRNR synthesis [2+/calmodulin-dependent signaling processes in bovine articular chondrocytes [The crucial role of intracellular ion homeostasis in chondrocyte function has been extensively demonstrated . Mature pressure ,34. Durintiation . Among ativation . Concepttivation . A studyynthesis . The tradrocytes .2+ influx for oscillations [Enhancement of chondrogenic differentiation-associated gene expression by intracellular calcium has been demonstrated in hMSCs cultured on 3D scaffolds . Increasllations ,32, as illations ,39. Invollations . It was llations . NeverthElectrical stimulation (ES) is an effective inductor that guides the development and regeneration of many tissues. It can lead to the secretion of various growth factors by different cells, including TGF-β . ES prom2+ [2+, leading to activated calmodulin and an increase in TGF-β mRNA expression [2+ through VOCC . This permeabilized state may persist up to several minutes, followed by the self-recovery of the cells. During electropermeabilization, the organization of the cell plasma membrane is affected and transmembrane ion transport is induced, resulting in a burst of intracellular Ca2+ . Severalpression ,36. Furtugh VOCC .2+ oscillations in MSC by modulating VOCCs, their implication in chondrogenesis has been suggested [SOX9 genes and the partial inhibition of the expression of type I and X collagens [Chondrogenic differentiation of MSCs has been shown to be efficiently induced using exclusively ES, even in the absence of growth factors . ES enhauggested ,45,46. Aollagens .The increase in intracellular calcium concentration under the application of magnetic field (MF) was reported more than 20 years ago when the effect of an imposed 60 Hz magnetic field on T lymphocytes was investigated . Althoug2+, these predictions are still under investigation  of VOCC gained even more scientific attention as compared to ES, as EMF induced efficient enhancement of osteogenesis and chondrogenesis of MSCs with no negative effects documented so far. Out of twenty three different studies that demonstrated how EMF exposures activate VOCCs, as reviewed in 2013, the majority implicated L-type VOCCs . The pose growth . Further channel .2+ transient [VOCCs are the key signal transducers of electrical excitability, converting the electrical signal of the action potential in the cell surface membrane to an intracellular Caransient . In 2015ransient . Noteworransient . TRP charansient . Howeverransient .2+ channels, and, of course their potential in developing novel therapies based on cell chondrogenic differentiation capability, first of all the parameters, such as frequency, intensity, and time of exposure have to be optimized for either living system where the experimental and environmental conditions are clearly analyzed, thereby allowing for the optimization of a treatment that may have beneficial regenerative effects.Therefore, in order to understand the mechanism of action of ES, EMF, or magnetic field on either cell type and mechanotransductive Ca2+ levels [2+ responses of chondrocytes in the loaded cartilage [2+ channels, resulting in further Ca2+ influx, see 2+ influx in response to mechanical stimuli in mice models [2+ entry and regulating downstream molecular signals to enhance chondrogenesis remain to be clarified.Mechanical forces are crucial factors in the maintenance of cartilage in vivo . Chondro+ levels . Severalartilage . Engineeartilage . The useartilage . Moreoveartilage . Inhibite models . Taken tEffective stimulation of chondrogenic differentiation in human mesenchymal stem cells is highly desirable and a clinically relevant process due to the poor regenerative capabilities of cartilage. Current techniques that are proposed to enhance chondrogenic differentiation in vitro often include the use of a range of growth factors—TGF-β, Activin A, and BMP-2. Additional application of different physical stimuli—mechanical, electrical, electromagnetic, or magnetic field might further lead to the enhanced calcium concentration and ultimately to the enhanced activation of chondrogenic gene expression and the formation of extracellular matrix in cartilage. Different studies propose electrical stimulation as the most potential stimuli to activate VOCCs, while others claim that mechanical stimuli is necessary when it comes to cartilage tissue formation in vitro. Magnetic field is still under research, though several studies suggest it as a potential mean in the chondrogenic differentiation capacity of MSCs. Although none of these studies clarify the interplay between the channels in chondrogenic differentiation, in most of the cases, it was associated with the increased calcium influx. The parameters of the applied physical stimulation, including strength, duration, intervals, etc., seem to have the essential impact on their efficacy in stimulation of the ECM production . In conc


FLT3 mutations are one of the most common findings in acute myeloid leukemia (AML). FLT3 inhibitors have been in active clinical development. Midostaurin as the first-in-class FLT3 inhibitor has been approved for treatment of patients with FLT3-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101. New generation FLT3 inhibitors and combination therapies may overcome resistance to first-generation agents. This mutation causes resistance to conventional chemotherapy. Although patients with AML can be cured with hematopoietic stem cell transplantation (HSCT), most of these patients are at high risk for relapse. Thus, the overall cure rate of AML is only 30–40% [Acute myeloid leukemia (AML) remains a highly resistant disease to conventional chemotherapy, with a median survival of only 4 months for relapsed and/or refractory disease . Moleculy 30–40% .FLT3/ITD gene is found in approximately 30% of patients with AML with normal cytogenetics. FLT3/ITD belongs to the type III family of receptor tyrosine kinases . The FLTMultiple FLT3 inhibitors are in clinical trials for treating patients with FLT3/ITD-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101.50 of 69.3 ng/ml [Sorafenib is an oral multikinase inhibitor of RAF-1, VEGF, c-KIT, PDGFR, ERK, and FLT3. Currently, sorafenib is approved for treating hepatocellular carcinoma and renal cell carcinoma. Sorafenib also has a potent anti-leukemic effect on FLT3-mutated AML. It completely inhibits FLT3/ITD activity with an IC.3 ng/ml .+ cell invasion in the epidermis, high proportion of CD8+ lymphocytes in the bone marrow, and high expression levels of COL4A3, TLR9, FGF1, and IL-12 genes have been observed in patients treated with sorafenib [There are several mechanisms that explain the efficacy of sorafenib for treating AML. First, sorafenib promotes secretion of IL-15 by FLT3/ITD-mutated leukemic cells and improves the survival time of patients with FLT3/ITD-positive AML . Secondlorafenib . Sorafenorafenib .2/d for 7 days and orally administrated sorafenib 400 mg continuously. Response rate (RR) was 46%, complete remission with incomplete count recovery (CRi) 27%, complete remission (CR) 16%, and partial remission (PR) 3%. Recently, Mahdi et al. used the same doses of azacytidine and sorafenib to successfully treat a pregnant patient with FLT3/ITD-mutated AML [Sorafenib combined with conventional chemotherapy is being explored for AML therapy. Sorafenib and decitabine have been used in preclinical and clinical trials to treat FLT3/ITD-mutated AML in vitro and in vivo . Both drated AML . After 1ated AML . Sorafenated AML . The resSorafenib has shown encouraging results in HSCT for patients with FLT3/ITD-positive AML. In a retrospective analysis, 17 patients with FLT3/ITD-positive AML received sorafenib in combination with allo-HSCT . Among tSunitinib (SU11248) is a small-molecule FLT3 inhibitor with selectivity for PDGFR, VEGFR1, VEGFR2, KIT, and FLT3 . It has The mechanism of sunitinib’s effect against AML is similar to that of sorafenib . One stuIn the past few years, more clinical trials of sunitinib with chemotherapy have been conducted. In a phase I/II clinical trial, sunitinib and intensive chemotherapy were chosen for 22 patients with FLT3/ITD-mutated AML aged over 60 years . Thirtee50 against FLT3 phosphorylation. Interestingly, lestaurtinib is cytotoxic to human AML cell lines expressing both mutant and wild-type FLT3, and it prolongs the survival of FLT3/ITD leukemia in a mouse model [Lestaurtinib (CEP-701) is an orally bioavailable indolocarbazole alkaloid compound derived from the bacterial fermentation product K-252a. It has activities against tropomyosin receptor kinases, neurotrophin receptors, FLT3, and JAK2 –37. Diffse model .In a phase II trial, lestaurtinib was used as a monotherapy in untreated older patients with AML . LestaurTandutinib  is a novel quinazoline-based inhibitor of the type III receptor tyrosine kinases, FLT3, PDGFR, and KIT. Tandutinib, at a concentration that does not affect normal colony formation, was shown to inhibit blast growth in patients with FLT3/ITD-positive AML . TandutiThe clinical effect of tandutinib in patients with AML was examined in a phase I trial. Tandutinib showed anti-leukemic activity and decreased the number of blasts in the peripheral blood as well as in the bone marrow in 40 patients with AML or high-risk MDS . In addiMidostaurin  is a small-molecule tyrosine kinase inhibitor (TKI) and was approved by the US FDA in 2017 for the treatment of FLT3-mutated AML . It has The clinical activity of midostaurin has been investigated in multiple clinical trials. In a phase I trial, midostaurin was administered with bortezomib alone or in combination with mitoxantrone, etoposide, and cytarabine to patients with refractory or relapsed AML . The oveQuizartinib (AC220) is a selective and highly potent second-generation class III receptor TKI . Quizart2/day.The optimum dosages and efficacy of quizartinib alone and in combination with chemotherapy in patients with AML were investigated. A phase I open-label, sequential group dose-escalation trial was the first to evaluate the safety and tolerability of quizartinib in combination with chemotherapy in 19 patients newly diagnosed with AML . Out of 2/day 3 times a day in another single-center phase II study in 10 patients with relapsed/refractory AML who progressed after HSCT  is a novel dual FLT3/AXL inhibitor. Gilteritinib significantly reduced the colony-forming capacity of FLT3/ITD-positive leukemia cells . GilteriCabozantinib is an oral inhibitor of multiple receptor tyrosine kinases VEGFR-1, VEGFR-2, VEGFR-3, Kit, MET, AXL, KIT, FLT3, and RET , 73. It /ITD inhibitor. SEL24-B489 suppresses the growth of AML cell lines. Unlike selective FLT3/ITD or PIM inhibitors, SEL24-B489 exhibits significantly broader on-target activity in AML cell lines, primary AML blasts, and FLT3-TKD-mutated cells [Sel24-B489 is a novel dual pan-PIM and FLT3ed cells ./ITD-positive as well as FLT3/WT- cell lines. It displayed potent anti-leukemic activity toward bone marrow blasts from patients with AML, including those with little or only minor responses to agents like AC220 or PKC412 [G-749 is a novel FLT3 inhibitor against FLT3-ITD, D835Y, ITD/F691 L, and ITD/N676D. G-749 was shown to have sustained inhibition of FLT3 phosphorylation and downstream effectors in FLT3r PKC412 . G-749 w/ITD-mutated AML and overcome drug resistance [AMG 925 is a highly bioavailable dual kinase inhibitor of cyclin-dependent kinase 4 (CDK4) and FLT3 and active against many FLT3 mutants reported to date . AMG 925sistance .TTT-3002 is a novel FLT3 inhibitor with the most potent activity against a broad spectrum of FLT3-activating point mutations, including D835 and F691 L gatekeeper mutations. Compared with several other TKIs currently in clinical trials, TTT-3002 is only moderately protein-bound. TTT-3002 maintains its effect on cells isolated from patients with relapsed AML that are resistant to sorafenib and AC220. Tumor burden in an FLT3 TKI-resistant transplant mouse model was significantly reduced by oral administration of TTT-3002 . TTT-300FF-10101 is a novel selective and irreversible FLT3 inhibitor with activities against FLT3/ITD, MOLM-13, MOLM-14, MV4-11, D835, Y842, and F691. FF-10101 covalently binds to the cysteine residue at 695 of FLT3 kinase and was shown to have high selectivity and inhibitory activity against FLT3 kinases. It significantly suppresses the growth of 32D cells with FLT3/ITD/D835Y-or FLT3/ITD/F691 L-expressing cells and primary AML cells with FLT3-ITD or FLT3-D835 mutations both in vitro and in vivo . These eMany studies have shown that FLT3 inhibitors have favorable clinical activities for AML patients with FLT3/ITD, but response duration remains short because of the rapid development of resistance. Resistance to FLT3 inhibitors was attributed to the emergence of new mutations. The secondary FLT3 tyrosine kinase domain (TKD) mutation was one of the new mutations in the patients who showed resistance to FLT3 inhibitors , 84. TheCombining FLT3 inhibitors with other agents is the major drive in the clinical trials to overcome the resistance to current FLT3 TKIs. Dayal successfully used a collaborative FLT3 inhibitor, HSD 1169, to act against FLT3/ITD and sorafenib-resistant cell lines . In a reFLT3 inhibitors have shown promising efficacies in aggressive AML. However, the duration of clinical response is short because of the rapid development of resistance. Novel next-generation FLT3 inhibitors are in active development to concur the resistance. Combining FLT3 inhibitors with other targeted agents are additional areas of investigation to minimize resistance to current FLT3 inhibitors.


Clinical studies have shown that empagliflozin (Jardiance™) has cardiovascular benefits, however the mechanisms have not been fully elucidated. This study aimed to investigate whether empagliflozin modulates cardiac electrical activity as well as Ca2+/Na+ homeostasis in DM cardiomyopathy. Electrocardiography, echocardiography, whole-cell patch-clamp, confocal microscopic examinations, and Western blot, were performed in the ventricular myocytes of control and streptozotocin-induced DM rats, with or without empagliflozin (10 mg/kg for 4 weeks). The results showed that the control and empagliflozin-treated DM rats had smaller left ventricular end-diastolic diameters and shorter QT intervals than the DM rats. In addition, the prolonged action potential duration in the DM rats was attenuated in the empagliflozin-treated DM rats. Moreover, the DM rats had smaller sarcoplasmic reticular Ca2+ contents, intracellular Ca2+ transients, L-type Ca2+, reverse mode Na+-Ca2+exchanger currents, lower protein expressions of sarcoplasmic reticulum ATPase, ryanodine receptor 2 (RyR2), but higher protein expressions of phosphorylated RyR2 at serine 2808 than the control and empagliflozin-treated DM rats. The incidence and frequency of Ca2+ sparks, cytosolic and mitochondrial reactive oxygen species, and late Na+ current and Na+/hydrogen-exchanger currents were greater in the DM rats than in the control and empagliflozin-treated DM rats. Empagliflozin significantly changed Ca2+ regulation, late Na+ and Na+/hydrogen-exchanger currents and electrophysiological characteristics in DM cardiomyopathy, which may contribute to its cardioprotective benefits in DM patients.Diabetes mellitus (DM) has significant effects on cardiac calcium (Ca Cardiovascular (CV) complications are major causes of mortality in patients with both type 1 and type 2 diabetes mellitus (DM) ,4. Sodiu2+) and sodium (Na+) in the heart is important to appropriately regulate heart rhythm, myocardial signal transduction, energy production and respiration of the myocardium i transients  than the control  and empagliflozin-treated DM  ventricular myocytes by 35% and 41.2%, respectively. However, the control and empagliflozin-treated DM ventricular myocytes had similar [Ca2+]i transients  and empagliflozin-treated DM  ventricular myocytes, whereas the control and empagliflozin-treated DM ventricular myocytes had similar decay times of [Ca2+]i transients. We measured the sarcoplasmic reticular (SR) Ca2+ content and found that the DM ventricular myocytes had significantly smaller Ca2+ stores  as measured by integrating the Na+-Ca2+exchanger (NCX) current after caffeine than the control  and empagliflozin-treated DM  ventricular myocytes  compared to the control  and empagliflozin-treated DM  ventricular myocytes. In addition, the DM ventricular myocytes had a longer duration and larger width of Ca2+ sparks  than the control  and empagliflozin-treated DM  ventricular myocytes. The peak amplitude of Ca2+ sparks was similar in the different groups.As shown in Ca-L in the DM ventricular myocytes was smaller than those in the control and empagliflozin-treated DM ventricular myocytes  was greater than those in the control  and empagliflozin-treated DM  ventricular myocytes  had a larger Na+/H+ exchanger current than the control  and empagliflozin-treated DM  ventricular myocytes.The DM ventricular myocytes  had higher levels of cytosolic reactive oxygen species (ROS) than the control  and empagliflozin-treated DM  ventricular myocytes, by 28.8% and 29.8%, respectively. The control and empagliflozin-treated DM ventricular myocytes had similar cytosolic ROS levels. In addition, the level of mitochondrial ROS in the DM ventricular myocytes  was greater than those in the control  and empagliflozin-treated DM  ventricular myocytes by 44.3% and 10.5%, respectively (+ ([Na+]i) level in the DM ventricular myocytes  was greater than those in the control  and empagliflozin-treated DM  ventricular myocytes by 13.2% and 7.8%, respectively , and APD50  in DM ventricular myocytes. In addition, empagliflozin (1 μM) reduced INa-Late from 0.66 ± 0.07 pA/pF to 0.28 ± 0.04 pA/pF  and cytosolic ROS levels from 108 ± 4 F/F0 to 81 ± 4 F/F0  in DM ventricular myocytes.In order to study whether empagliflozin may directly affect cardiac disorder in DM rats, we investigated the acute effects of empagliflozin (1 μM) on isolated DM rat cardiomyocytes. As shown in 2+ regulatory proteins in control, DM and empagliflozin-treated DM rat ventricles using Western blotting (p < 0.005) and RyR2  in the DM group (N = 7) were downregulated, compared with those in control (N = 7) and empagliflozin-treated DM groups .We compared the expressions of Cablotting . The pro2+]i signaling i in late-onset type-2 DM rat myocytes which displayed a cardiac phenotype that closely resembled human DM cardiomyopathy [a-lateN and Na+/H+-exchanger activity may contribute to the elevation of [Na+]i of DM myocytes, leading to Ca2+ overload, with an increased risk of arrhythmias and oxidative stress [2+]i transient amplitude, prolonged transient decay, and reduced Ca2+ store i and aids in the binding of Ca2+ to myofilaments and initiating cardiac contractions [2+]i transients in our DM hearts may have been caused by a decrease in SR Ca2+ content, resulting in impaired excitation-contraction coupling efficiency and myocardial dysfunction. The reduction in SR Ca2+ stores might have been exacerbated in DM hearts due to a decrease in SERCA2a protein. We also found a prolonged [Ca2+]i transient decay associated with the depletion of SR Ca2+ contents in the DM hearts. The increase in the [Ca2+]i transient decay may have been due to derangement in either the SERCA pump resulting in a decrease in the removal rate of cytoplasmic Ca2+ [2+]i transient of DM ventricular myocytes may also have been affected by the prolonged APD, altered [Ca2+]i handling, or a combination of both. We also found that decreases in [Ca2+]i transients and Ca2+ stores with prolonged [Ca2+]i decay in the DM hearts were attenuated in the DM rats treated with empagliflozin. These effects may have been caused by enhanced myocardial SERCA2a function as demonstrated in a previous study by Hammoudi et al. [2+]i after [Ca2+]i release in the DM rats treated with empagliflozin. In addition, the Ca2+ regulatory effects of empagliflozin in DM hearts may have contributed to the improved cardiac function measured by echocardiography. The increase in SERCA2a protein in the DM rats treated with empagliflozin attenuated the negative impact of DM on Ca2+ dysregulation. These findings suggest that empagliflozin has potential to be a therapeutic strategy for cardiac electrical dysfunction in DM.Caractions . The decmic Ca2+ ,27, and i et al.  resultin2+ entry through molecular mediators that permit the Ca2+ influx required for excitation-contraction coupling i transients and SR Ca2+ contents. In this study, the reverse mode (Ca2+ influx) of NCX was depressed in the DM ventricular myocytes, indicating that a reduction in Ca2+ influx may have contributed to the decrease in [Ca2+]i. We also found that empagliflozin treatment restored the diminished NCX current.DM has been shown to modify Cacoupling . Several rodents ,33,34. Ile force . This ma2+ sparks are brief releases of Ca2+ that are considered to be essential events in the excitation-contraction of cardiomyocyten addition, the frequency and incidence of Ca2+ sparks in the DM rats were also reversed after empagliflozin treatment. Thus, empagliflozin may have a beneficial effect in preventing increased Ca2+ leakage in the SR that may cause Ca2+ deficiency leading to myocardial dysfunction in DM cardiomyopathy. The decrease in RyR phosphorylation in DM hearts receiving empagliflozin may increase the amplitude of [Ca2+]i transients and improve myocardial contractility.CaCa-L and Na+-H+ exchanger currents in DM ventricular myocytes. We demonstrated that chronic treatment with empagliflozin lowered intracellular Na+ levels, and that INa-late and Na+-H+ exchange currents were ameliorated after empagliflozin treatment. This may at least in part be related to the effect of empagliflozin on cardiac ion homeostasis, and may contribute to the cardiac benefits in DM patients. Baartscheer et al. i was recorded in single myocytes. Cells were loaded with a fluorescent Ca2+ (10 μM) fluo-3/AM for 30 min at room temperature. Excess extracellular dye was removed by changing the bath solution and allowing the intracellular hydrolysis of fluo-3/AM after 30 min. Fluo-3 fluorescence was excited with a 488-nm line of an argon ion laser. The emission was recorded at >515 nm. For line scan imaging (8-bit), cells were repetitively scanned at 2-ms intervals. Fluorescence imaging was performed using a laser scanning confocal microscopic examination  and an inverted microscope . The fluorescent signals were corrected for variation in dye concentration by normalizing the fluorescence (F) against baseline fluorescence (F0), to obtain reliable information about transient intracellular Ca2+ ([Ca2+]i) changes from the baseline values (F/F0) and to exclude variations in the fluorescence intensity by different volumes of injected dye. The [Ca2+]i transient, and decayed portion of the [Ca2+]i transient were measured during a 1-Hz field-stimulation with 10-ms twice-threshold strength square-wave pulses. The [Ca2+]i was determined by the monoexponential least-squares fit. After achieving steady-state Ca2+ transients with repeated pulses from -40 to 0 mV (1 Hz for 5 s), the SR Ca2+ content was estimated by integrating the NCX current following application of 20 mM of caffeine within 0.5 s while resting with the membrane potential clamped to −40 mV to cause SR Ca2+ release [2+ content (expressed as millimoles of cytosol) was determined using this equation: SR Ca2+ content = [(1 + 0.12) (Ccaff/F × 1000)]/(Cm × 8.31 × 8.44), where Cm is the membrane capacitance; F is Faraday’s number; and the cell surface to volume ratio is 6.44 pF/pL [Freshly isolated rat ventricular myocytes were enzymatically dissociated using collagenase  and protease  after euthanasia as described previously . [Ca2+]i44 pF/pL ,50.2+ sparks were detected using the line scan mode along a line parallel to the longitudinal axis of single freshly isolated rat ventricular myocytes, while avoiding the nuclei. Each line was composed of 512 pixels. Ca2+ sparks were detected as an increase in the signal mass (more than 1.3 F/F0) of a 5-µm section through the center of a Ca2+ spark, with no detectable increase in the adjacent 5-µm section. The Ca2+ spark frequency was determined for each cell and normalized to the scanned cell length. Images were analyzed using both ImageJ and custom-made routines based on IDL .Ca2 1, MgATP 5, HEPES 10, EGTA 10, NaGTP 0.1, and Na2 phosphocreatine 5, titrated to a pH of 7.2 with CsOH for ICa-L; NaCl 20, CsCl 110, MgCl2 0.4, CaCl2 1.75, tetraethylammonium 20, 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) 5, glucose 5, MgATP 5, and HEPES 10, titrated to pH of 7.25 for NCX currents; 10 NaCl, 130 CsCl, 5 EGTA, 5 HEPES, 5 glucose, and 5 ATP-Mg for the INa-Late; and KCl 20, K aspartate 110, MgCl2 1, MgATP 5, HEPES 10, EGTA 0.5, LiGTP 0.1, and Na2 phosphocreatine 5, titrated to a pH of 7.2 with KOH for the APs. Voltage command pulses were generated with a 12-bit digital-to-analog converter controlled using pCLAMP software (Axon Instruments). Recordings were low pass-filtered at half the sampling frequency. The APA was determined by measuring the difference between the RMP and the peak of AP depolarization. The APD at repolarization extents of 90%, 50%, and 20% of the amplitude were measured, and respectively designated as APD90, APD50, and APD20.A whole-cell patch-clamp using an Axopatch 1D amplifier  was performed in the freshly isolated ventricular myocytes of rats at 35 ± 1 °C. Borosilicate glass electrodes  were used with tip resistances of 3~5 MΩ. Before the formation of the membrane-pipette seal, the tip potentials were zeroed in Tyrode’s solution. Junction potentials between the bath and pipette solution (9 mV) were corrected for AP recordings. APs driven at 1 Hz were recorded in the current-clamp mode, and ionic currents were measured in the voltage-clamp mode. A small hyperpolarizing step from a holding potential of −50 mV to a test potential of −55 mV for 80 ms was delivered at the beginning of each experiment. The area under the capacitative currents curve was divided by the applied voltage step to obtain the total cell capacitance . In mostCa-L was measured as an inward current during depolarization from a holding potential of −50 mV to test potentials ranging from −40 to +60 mV in 10-mV steps for 300 ms at a frequency of 0.1 Hz. NaCl and KCl in the external solution were respectively replaced by tetraethylammonium chloride and CsCl. The NCX current was elicited by depolarizing pulses between −100 and +100 mV from a holding potential of −40 mV for 300 ms at a frequency of 0.1 Hz. Amplitudes of NCX currents were measured as 10-mM nickel-sensitive currents. The external solution consisted of (in mM) NaCl 140, CaCl2 2, MgCl2 1, HEPES 5, and glucose 10, with the pH adjusted to 7.4 and 10 μM strophanthidin (to block the Na+/K+ pump), 10 μM nitrendipine (a dihydropyridine antagonist), and 100 μM niflumic acid (to block Ca2+-activated Cl− currents).INa-Late was recorded with an external solution containing (in mM): NaCl 130, CsCl 5, MgCl2 1, CaCl2 1, HEPES 10, and glucose 10 at a pH of 7.4 with NaOH by a step/ramp protocol . The tetrodotoxin (30 μM)-sensitive portion of the current traces, obtained when the voltage was ramped back to −100 mV, was measured as the INa-Late current [I current .+/H+ exchanger current, a whole-cell camp with patch electrodes of 1.5–3.0 MΩ resistance were filled with solution consisting of (in mM) 20 KCl, 130 K-Aspartate, 1 MgCl2 (6 H2O), 10 HEPES/KOH (pH 7.3), and 0.005 EGTA. The external solution consisted of (in mM) 150 NaCl, 5.4 KCl, 3.6 CaCl2, 1.2 MgCl2 (6 H2O), 20 glucose, and 5 HEPES/NaOH (pH 7.4). All groups of freshly isolated rat ventricular myocytes were stimulated with 40 ms depolarizing pulses from −45 to 0 mV at 3 Hz [To measure Na at 3 Hz .+ concentration of freshly isolated ventricular myocytes in the control, DM, and empagliflozin-treated DM rats. Experiments were carried out using a laser scanning confocal microscope  and an inverted microscope (Axiovert 100) with a 63x1.25 numerical aperture oil immersion objective, as described previously [2 1.8, MgCl2 0.5, and HEPES 10, with appropriate fluorescent dye of 10 μM CellROX green, 2 μM MitoSOX Red, or 5 μmol/L Asante NaTRIUM Green-2 AM. CellROX green, MitoSOX Red, and Asante NaTRIUM Green-2 AM were excited at 488 nm, and fluorescence signals were acquired at wavelengths of >505 nm in the XY mode of the confocal system. In this experiment, ventricular myocytes were paced at 1 Hz. Fluorescent images were analyzed using Image-Pro plus 6.0 and Sigma plot 12 software, as described previously [The cross-sectional area in the isolated single ventricular myocytes were imaged using a confocal laser scan microscope  and images were processed by ImageJ measurement tools . We usedeviously . FreshlyEqual amounts of proteins were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis as described previously . Blots wTargeted bands were normalized to cardiac glyceraldehyde 3-phosphate dehydrogenase  to confirm equal protein loading.t-test or one-way analysis of variance (ANOVA) with Duncan’s method, using Systat software SigmaPlot version 12  for multiple comparisons as appropriate. A p value of <0.05 was considered to be statistically significant. The sample size was calculated according to the ejection fraction with an estimated standard deviation of 3. The required sample size was 7 under a type I error of 5% with a statistical power of 80%.All quantitative data are expressed as the mean ± standard error of the mean (SEM). Statistical significance between different groups was determined using an unpaired


However, the underlying mechanism for this Ca2+-dependent, internalisation of CFTR is unknown. To gain a better understanding of the effect of Ca2+ on CFTR, we performed whole cell current recordings to study the temporal effect of raising cytosolic Ca2+ on CFTR function. We show that an increase in cytosolic Ca2+ induced a time-dependent reduction in whole cell CFTR conductance, which was paralleled by a loss of cell surface CFTR expression, as measured by confocal and widefield fluorescence microscopy. The decrease in CFTR conductance and cell surface expression were both dynamin-dependent. Single channel reconstitution studies showed that raising cytosolic Ca2+ per se had no direct effect on CFTR. In fact, the loss of CFTR plasma membrane activity correlated with activation of calcineurin, a Ca2+-dependent phosphatase, suggesting that dephosphorylation of CFTR was linked to the loss of surface expression. In support of this, the calcineurin inhibitor, cyclosporin A, prevented the Ca2+-induced decrease in cell surface CFTR. These results provide a hitherto unrecognised role for cytosolic Ca2+ in modulating the residency of CFTR at the plasma membrane through a dynamin- and calcineurin-dependent mechanism.The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated, apical anion channel that regulates ion and fluid transport in many epithelia including the airways. We have previously shown that cigarette smoke (CS) exposure to airway epithelia causes a reduction in plasma membrane CFTR expression which correlated with a decrease in airway surface hydration. The effect of CS on CFTR was dependent on an increase in cytosolic CaThe online version of this article (10.1007/s00018-018-2989-3) contains supplementary material, which is available to authorized users. CFTR is a cAMP- and ATP-regulated anion channel whose function is critical for transepithelial anion and fluid secretion in multiple organs, including the conducting airways, intestines, reproductive tracts, and kidneys , 2. The CFTR itself is composed of two membrane spanning domains, each of which contains six transmembrane regions and two nucleotide binding domains (NBDs) which bind ATP. Unique to CFTR is the regulatory ‘R’ domain. The functional properties of CFTR have been classically characterised using electrophysiology; CFTR has a single channel conductance of 8–10 pS and a linear current–voltage relationship , 7. CFTR2+. This Ca2+ emanated from intracellular stores  and did not involve Ca2+ influx across the plasma membrane, consistent with other studies showing that CS and its constituents cause increases in cytosolic Ca2+ [2+ prevented CFTR internalisation [2+ to the loss of CFTR function. Within airway epithelia, Ca2+ regulates processes including Cl− secretion through Ca2+-activated Cl− channels, mucus secretion, and ciliary beat frequency [2+ through mechanisms other than via exposure to CS could modulate the residency of CFTR at the plasma membrane has not been previously investigated. To investigate this possibility, we have measured the temporal effect of raising cytosolic Ca2+ by a range of Ca2+ agonists on CFTR activity using real-time measurements of single channel and whole cell CFTR current recordings, as well the temporal effect of raising Ca2+ on cell surface expression as determined by confocal and widefield fluorescence microscopy.We have previously demonstrated that acute exposure of fully differentiated primary airway epithelial cultures to CS caused a time-dependent loss of CFTR from the plasma membrane, which correlated with a reduction in airway surface liquid (ASL) volume (i.e. dehydration) , 22. Furlic Ca2+ . Importarequency –28. Howel-glutamine, 1% non-essential amino acids, 10% foetal bovine serum (FBS), 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. HEK293T cells used for imaging experiments were cultured in DMEM supplemented with 10% FBS, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. CF bronchial epithelial (CFBE41o−) cells stably expressing the mCherry-Flag-WT-CFTR construct were cultured in DMEM supplemented with 10% FBS, 10 µg ml−1 blasticidin and 2 µg ml−1 puromycin. This construct contains a reporter under the control of a Tet-ON promoter [2 atmosphere.Human embryonic kidney (HEK) 293T cells for patch clamp experiments were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM promoter . Cells w−1 and added to cells. Following 6 h incubation at 37 °C, cells were left to incubate overnight in Opti-MEM with 10% FBS after which cells were transferred back to culture media. Cells were studied 48–72 h post transfection at ~ 50% confluency. For imaging experiments, cells were transfected with 1 µg wild-type CFTR with a GFP tag on the N terminus of the channel, CFTR containing an external HA-tag [HEK293T cells were transiently transfected with the bicistronic pIRES2-EGFP-CFTR vector to co-express wild-type CFTR and enhanced green fluorescent protein (GFP) for patch clamp experiments . Brieflyl HA-tag , 22 or a2, 0.2 EGTA, 10 HEPES and 1 Na2ATP, set to pH 7.2 with CsOH. Pipette solution osmolarity was typically 260 mOsm. Pipette solution contained low EGTA to prevent a buffering of increases in cytosolic Ca2+ [2, 1 MgCl2, 10 HEPES, 10 glucose and 20 mannitol, set to pH 7.4 with HCl. For nominally Ca2+-free bath solution, CaCl2 was omitted and 2 mM MgCl2 was added to maintain the Cl− gradient. Bath solution osmolarity was typically 310 mOsm. Experiments were carried out using an Axopatch 200B (Molecular Devices Inc) patch clamp amplifier and data were captured using pClamp10 software. The reference electrode was a silver/silver chloride wire connected to a 150-mM NaCl agar bridge. Cells were held at 0 mV and voltage steps were applied between ± 100 mV in 20 mV increments. Each voltage step lasted 250 ms and there was a 1-s interval between subsequent steps. Data were filtered at 1 kHz and sampled at 2 kHz with a four-pole Bessel filter. The average current obtained between a 100-ms period starting 80 ms into the voltage step was used to construct current–voltage (I–V) plots. Liquid junction potentials were corrected for and applied to membrane potentials. The Axopatch 200B amplifier was used to compensate for series resistance; 70% compensation was used for all whole cell recordings. The input capacitance of cells was measured before each experiment and compensated for. Slope conductance was calculated by fitting a linear regression to each I–V plot. Single cell slope conductance was divided by cell capacitance (pF) to normalise data to cell size and is expressed as nS/pF.Whole cell recordings were carried out on single cells and all experiments were conducted at room temperature as previously described , 33. Piplic Ca2+ , 35. The2+-free conditions  and a Ca2+-containing solution  on the cytosolic face of CFTR. An all points histogram by multipeak Gaussian was fitted to the data and single channel conductance was calculated using the distance between peaks on the all points histogram. Channel open probability was calculated using the ratio of the area under the peak when the channel was open compared to the total area.Single channel CFTR activity was measured using purified CFTR expressed in planar lipid bilayers as previously described , 37. ChaHEK293T cells were imaged using a Leica TCS SP8 confocal laser scanning microscope. Images were captured using a 63 × 1.3 NA oil immersion lens, with a bidirectional scan frequency of 700 Hz and a pinhole of 1 airy unit. GFP was excited with the 488 nm line of an argon laser. Images were captured using the Leica Application Suite: Advanced Fluorescence (LAS AF) software.Images were analysed offline using ImageJ by manually selecting 6 regions of equal size from the plasma membrane and 6 regions from the intracellular space. Brightfield images were used to select regions of interest from the plasma membrane where fluorescence had been lost due to internalisation of CFTR. Any membranes connecting two adjacent cells were excluded from analysis. The average intensity of the six regions was then determined for each cell. The average values of all the cells from either vehicle-treated and air-exposed cells were collected and were taken as one; all other treatments were normalised as previously described .− mCherry-Flag-WT-CFTR cells were seeded onto 384-well plates (2.5 × 103 cells/well) using a Multidrop Combi peristaltic dispenser (Thermo Scientific). The next day, CFTR expression was induced with antibiotic-free medium supplemented with 1 µg ml−1 doxycycline. 24 h after induction of CFTR expression, cells were treated with DMSO, thapsigargin or ionomycin for up 2 h. Thereafter, extracellular Flag-tags were immunostained in non-permeabilised cells as previously described [CFBE41oescribed . Cell imescribed .−1 tetramethylrhodamine conjugated to 10 kDa dextran. Before imaging, the cultures were transferred to Ringer’s solution and 100 µl perfluorocarbon  was added mucosally to prevent evaporation of the ASL. Compounds added apically in these experiments were suspended as a dry powder in PFC and sonicated for 10 min.HBECs were washed with PBS for 1 h to remove excess mucus before all experiments. Cells were then loaded with 1 mg mlASL was imaged using a XZ scan on a confocal laser scanning microscope (Leica SP5) with a 63× glycerol immersion lens and the 561 nm laser. Images were taken at 12 predefined points on the transwell. Images were analysed offline using ImageJ software.2, cultures were fixed in 4% PFA and permeabilised with 1% Triton-X. DAPI was added for 30 min for nuclear staining and cells were washed three more times with PBS and then imaged using a Leica SP8 confocal microscope with a 63 × 1.3 NA oil immersion lens. Fluorophores were excited with 405, 488 and 647 nm laser lines and images were collected using sequential scanning to avoid fluorescent bleed through. To determine the percentage co-localisation between CFTR and calnexin, images were overlaid, and the mean Pearson’s correlation coefficient, as an indicator of colocalisation, was quantified using the LAS-AF software (Leica). The following calculation was automatically used by the LAS AF software to determine the percentage colocalization between colocalized areas and background within the ROI, with single cells being chosen as an ROI. Percent colocalisation = colocalisation area/area foreground, where area foreground = image area/area background.For surface labeling of CFTR, HEK293T cells were transfected with HA-CFTR using Lipofectamine 2000 and plated on 35 mm glass bottom cell culture dishes. 48 h later, cultures were incubated for 1 h at 4 °C, with a blocking solution (1% BSA/1% NGS) and another 1 h at 4 °C with mouse anti-HA antibody (ab) conjugated to Alexa-488  or rabbit anti-calnexin ab  in blocking solution. Cultures were washed three times at 4 °C in ice-cold PBS to remove excess ab, placed in HEK293T media warmed up to 37 °C and exposed to vehicle or thapsigargin. After 1 h incubation at 37 °C with 5% COFor on-cell Western blot, HEK293T cells were transiently transfected with a HA-CFTR and/or the dynamin K44A plasmid using Lipofectamine 2000 as per the manufacturers’ instructions (ThermoFisher Scientific). After overnight incubation, transfected cells were plated in 384-well plates  and studied 24 h later. Cultures were treated with thapsigargin or vehicle diluted in HEK293T media at 0, 15, 30 and 60 min. The media was then replaced with standard Ringer’s solution (50 μl/well). To halt the experiment, cultures were placed in ice-cold media at 4 °C, followed by a 1-h incubation at 4 °C, with a blocking solution (10% FBS and 1% BSA). This solution was replaced with antibody solution  and incubated for 1 h at 4 °C. Cultures were then fixed using 4% PFA and rinsed 3× with cold PBS. Fluorescence (488 nm) was measured using a Cytation5 cell imaging multi-mode reader (Biotek). Cultures were then stained using DAPI nuclear dye to give an indicator of cell number. Background fluorescence was determined by exposing non-transfected/antibody-exposed HEK293T cells to HA-488 antibody and each well was background-subtracted. Images were analysed offline using ImageJ software; regions of interest were selected using the threshold function and data were then background subtracted against non-transfected wells.Whole cigarette smoke was generated manually using 3R4F reference cigarettes from the University of Kentucky . Cells were exposed to 13 × 35 ml puffs of 2 s duration every 30 s, which was the equivalent of one research cigarette. The particulate fraction of smoke is highly autofluorescent and was removed by placing a Cambridge filter pad in the output line. Before exposure, cells were rinsed in pyruvate Ringer’s solution and left to incubate in Ringer’s for 5 min at 37 °C. Following incubation, the excess Ringer’s solution was tipped off, and cells were then exposed to smoke or room air in dedicated chambers for each. Following smoke exposure, cells were placed back into culture media and incubated for the times indicated before fixation.2+; all experiments were carried out at room temperature. Cells were subsequently placed in chambers with entry and exit ports for CS or air and manually exposed to either one filtered reference cigarette or the equivalent of room air as described above. Single cells were selected as regions of interest and images were acquired every 30 s with HCImageLive software (Hamamatsu). Experiments were carried out using an inverted microscope  and were viewed under an oil immersion lens . The microscope was equipped with a 200-W metal halide lamp and shutter that contained 340 nm and 380 nm bandpass filters; emission at 510 nm was measured using an Orca CCD camera (Hamamatsu). The emission ratio of the 340 nm and 380 nm wavelengths was taken to be a read out of cytosolic Ca2+. Counts were corrected for background fluorescence. Changes in cytosolic Ca2+ are expressed as maximum changes in the 340/380 ratio induced by each agent from the resting ratio. Area under the curve (AUC) was also chosen to indicate changes in Ca2+ as this gives an estimation of the magnitude of any changes as well as any subsequent recovery.HEK293T were loaded with Fura-2 acetoxymethylester  to measure changes in cytosolic CaCalcineurin phosphatase activity was assayed using a colourimetric test from Enzo life sciences kit as per manufacturer’s instructions. HEK293T cells were seeded onto 60 mm tissue culture dishes and treated 24 h later. Total phosphatase activity was measured in 3 µg of sample.t test for all other experiments, as appropriate. A p value < 0.05 was considered statistically significant.Data are presented as mean ± SEM. Analyses were carried out on Graphpad Prism v5. Significance was tested using Friedman test with Dunn’s post-test for patch clamp experiments, Kruskal–Wallis with Dunn’s post-test or a one sample 2+ on CFTR channel activity, we first studied the effect of altering cytosolic Ca2+ on whole cell CFTR Cl− currents in CFTR-transfected HEK293T cells. To activate CFTR cells were exposed to forskolin, which caused a time-dependent increase in whole cell conductance  [To investigate the effect of raising cytosolic Ca 10 µM) , caused 0 µM , ca2+-dependent inhibition of CFTR [2+ ATPase (SERCA) pump inhibitor, thapsigargin, which causes release of Ca2+ from the ER  [2+ buffer, EGTA (0.2 mM), which allows Ca2+ to increase upon agonist stimulation [inh-172 sensitive conductance remained  . Note thmulation , 35. Fig2+ on CFTR trafficking, we used confocal microscopy to assess changes in the cellular location of CFTR. These experiments revealed CFTR was lost from the plasma membrane after exposure to thapsigargin with a half-life of 11.9 min and a τ of 17.2. Intracellular fluorescence increased consistent with an increase in cytosolic Ca2+ inducing the internalisation of CFTR. However, when cells were exposed to vehicle, there was little change in either plasma membrane or intracellular fluorescence . Importantly, we found the absence of extracellular Ca2+ did not have any effect on the thapsigargin-induced reduction in CFTR-mediated conductance . Thus, these data suggested that like CS exposure, Ca2+ release from cytosolic stores was sufficient to cause a reduction in CFTR at the plasma membrane . As with earlier experiments, CFTR was first activated by forskolin and then cells exposed to the ionophore, ionomycin (1 µM) for 10 min, with CFTR currents monitored for a further 10 min after the wash out of ionomycin. In comparison to the thapsigargin experiments, exposure to ionomycin caused an increased rate of loss of CFTR-mediated conductance, with only 22.9 ± 7.2% of the forskolin-activated conductance remaining after 20 min exposure  [inh-172 sensitive conductance remaining at the end of the exposure period . To further validate our findings in HEK293T cells that increases in Ca2+-induced internalisation of CFTR, we measured changes in surface labelled CFTR, using a HA-tag linked to the extracellular domain of CFTR, following exposure to thapsigargin or CS. Our data showed that increases in Ca2+ caused a loss of surface labelled CFTR from the plasma membrane. Furthermore, this internalised CFTR co-localised with the endoplasmic reticulum marker, calnexin, suggesting CFTR was being relocated to this organelle  . Overalliod Fig. c, d. Of ane Fig. e–g. In tlle Fig. a–c. To p2+ could be replicated in epithelial cells, using the CFBE41o− model cell line stably expressing a double tagged CFTR construct that allows assessment of both surface and total CFTR levels in a single cell [We next tested whether the observed effect of increases in cytosolic Cagle cell . Similar2+ reduced CFTR function at the plasma membrane, we measured ASL height in primary human bronchial epithelial cells, as a measure of CFTR-dependent Cl− secretion [− efflux via CFTR, and inhibition of ENaC by S18 [As a further proof that increases in cytosolic Caecretion . When cuC by S18 . However2+ acted via the same mechanism, HEK293T cells were pre-treated with the dynamin GTPase inhibitor, dynasore (80 µM) for 30–60 min before exposure to thapsigargin. Pre-treatment with dynasore significantly blunted the thapsigargin-induced decrease in CFTR-mediated conductance, consistent with thapsigargin inducing a dynamin-dependent internalisation of CFTR  as a negative control to inhibit total calcineurin activity, and human recombinant calcineurin (40 U) as a positive control. In total, these controls inhibited or caused a large response in calcineurin activity, respectively, when compared to total cellular calcineurin activity  found that exposure of HT-29 cells to agents that increased the intracellular concentration of divalent cations, including Ca2+, caused a reduction in CFTR mRNA and protein in a time- and dose-dependent manner [2+ alone are able to reduce CFTR activity.Tobacco smoke has been shown to cause internalisation of CFTR through a smoke-induced increase in cytosolic Ca2+ . Howevert manner , validat2+-free conditions suggested that extracellular Ca2+ was not necessary for the loss of CFTR-mediated conductance, and, therefore, store-operated Ca2+ entry was not required. Similarly, Rasmussen and colleagues found that if cells were exposed to CS in the absence of extracellular Ca2+, the increase in cytosolic Ca2+ was similar to that seen when Ca2+ was present [2+, derived from internal stores, was sufficient to cause CFTR internalisation.Experiments testing the effect of thapsigargin on CFTR activity under nominally Ca present . These d2+ had no effect on either the conductance or open probability of purified CFTR studied in lipid bilayers. These findings are consistent to those reported by Clunes et al. who carried out similar experiments in bilayers that had been exposed to CS [2+, causes a reduction in CFTR function via an effect on the number of channels present on the membrane and not the gating of the channel. However, Bozoky and colleagues found that an increase in Ca2+ in the presence of forskolin caused a reduction in CFTR activity [2+ in excised patches versus purified CFTR in an artificial bilayer is not known. One possibility is that in the excised patch experiments, there was a Ca2+-dependent phosphatase present which, if activated, could decrease CFTR activity via dephosphorylation. This protein would be lost during purification for the bilayer experiments reported here.Single channel experiments showed that an increase in cytosolic Caed to CS . The datactivity , when me2+ from ER stores [2 receptor causes activation of the Gq pathway and the activation of PKC via this pathway results in the phosphorylation of CFTR which potentiates the effect of PKA [2B receptor [2B receptor has been linked to cAMP accumulation via Gs [2+ induced by ATP caused a loss of CFTR channels at the cell surface, but the recovery was due to one of the effects listed above. Most cells tested in our experiments showed some decrease in conductance, either as an initial decrease followed by a plateau or a gradual decline in conductance. This suggests that the increase in cytosolic Ca2+ caused by ATP may have overwhelmed the ability of the various mechanisms listed above to prevent a loss in conductance.To test the effect of a physiological agonist on CFTR-mediated conductance, cells were exposed to ATP. Usually released in response to the mechanical stresses seen during breathing, ATP causes the release of CaR stores . A propot of PKA . Furtherreceptor . Likewisn via Gs , 47. The2+. However, ATP likely caused physiological increases in Ca2+. In our study, the dosage used may account for the proportion of cells which showed a reduction in CFTR activity. However, it has been suggested that low levels of increases in cytosolic Ca2+ can stimulate Ca2+-dependent adenylyl cyclase and PKA phosphorylation of CFTR, suggesting that physiological increases in cytosolic Ca2+ are likely to cause an increase in CFTR activity [2+ activated tyrosine kinases, such as Pyk2, can activate CFTR through tyrosine phosphorylation of the channel and activation of the Pyk2/Src complex [Thapsigargin and ionomycin typically cause supraphysiological increases in cytosolic Caactivity . Furtheractivity . Indeed, complex .2+-induced loss of CFTR from the plasma membrane and subsequent internalisation could be replicated in either a bronchial epithelial cell line (CFBE41o−) or in fully differentiated primary human bronchial cells, where CFTR signalling is highly compartmentalised. Furthermore, the loss of CFTR from primary cultures was associated with a failure of these bronchial epithelial cells to respond to physiological agonist, as measured by a change in ASL height. As an abnormal ASL height has been linked to an impairment of mucociliary clearance and an increased prevalence of bacterial infections [2+ could result in detrimental effects on the airway microenvironment via changes in CFTR expression at the plasma membrane.Our data showed that the Cafections , our dat2+-induced decrease in conductance, suggesting that the Ca2+-dependent decrease in whole cell conductance was due to a dynamin-dependent endocytosis of CFTR. These data were supported by expression of a dominant negative dynamin mutant, which blunted the thapsigargin-induced CFTR internalisation. Dynamin is the primary protein involved in scission of clathrin-coated pits from the cell membrane and dynasore inhibits the GTPase activity of dynamin by preventing the hydrolysis of GTP [2+, resembles that of the normal trafficking process [2+ increases have been linked to regulating the balance between endocytosis and exocytosis. In synaptosomes, Marks and McMahon showed that an increase in Ca2+ was able to cause endocytosis at concentrations lower than that needed for exocytosis [2+, and binding of Ca2+ to calmodulin, has been shown to be linked to the initiation of endocytosis [The dynamin GTPase inhibitor, dynasore , prevents of GTP . Previou process . Interesocytosis . Furtherocytosis .2+ [2+ and loss of CFTR.As we have previously reported, we found that acute exposure to the volatile phase of CS caused internalisation of CFTR , 22. Won2+ . Indeed,2+ . Thus, o2+-induced internalisation of CFTR; however, it did prevent CS-induced loss of CFTR. Whilst no other studies have investigated the effect of an increase in cytosolic Ca2+ on CFTR internalisation, others have found that forskolin prevented endocytosis of CFTR in pancreatic and colonic cells [2+, phosphorylation via PKA is not able to protect CFTR. Arguably, CS exposure would cause numerous effects within a cell. It is possible that besides the increase in cytosolic Ca2+, another consequence of CS exposure is responsible for this discrepancy between the effect of CS and an increase in cytosolic Ca2+; for instance, CS contains constituents which could activate cell surface or intracellular signalling pathways that are not directly linked to changes in cytosolic calcium [Our study revealed that forskolin pre-treatment had no effect on Caic cells , 59. The calcium , but whi calcium , 62 furt2+ caused activation of the Ca2+-dependent phosphatase, calcineurin, which is known to regulate CFTR activity. Fischer et al. [2+ may cause the movement of calcineurin to bring it into close proximity of CFTR to enable dephosphorylation of the channel. In this regard, Lai et al. [2+-dependent interaction between calcineurin and dynamin 1. Further, an increase in cytosolic Ca2+ was linked to the dephosphorylation of dynamin, resulting in the translocation of the calcineurin–dynamin complex to a cluster of proteins involved in clathrin-mediated endocytosis [2+-induced internalisation of CFTR. Our data suggested that inhibition of calcineurin may be beneficial in cells exposed to CS, as cyclosporin A and analogues such as tacrolimus are currently in clinical use as immunosuppressants [We showed that an increase in Car et al.  found thr et al. . Thus, ai et al.  found thocytosis . Given tressants . Howeverressants .2+ are able to modulate the cell surface expression of CFTR at the plasma membrane, through a dynamin- and calcineurin-dependent mechanism. These findings help provide a better mechanistic understanding of how changes in cytosolic Ca2+ negatively impact CFTR function and help explain the action of CS on CFTR function. Given that CS is a major cause of lung disease and that loss of CFTR expression induced by CS is Ca2+-dependent, our data may help in the development of new strategies to minimise the deleterious effect of CS on CFTR function. Together, we hypothesise that these strategies would prevent dehydration of the ASL and consequently could be beneficial to lung health by offsetting the CS-induced loss of CFTR.In conclusion, we have shown that agonists that promote a rise in cytosolic CaFig. S1 Spatiotemporal changes in cytosolic Ca2+induced by agonists used in this study Cultures were exposed to forskolin  followed by a Ca2+ agonist in the presence of forskolin. The agonist was subsequently washed out of the perfusate and cells were exposed to CFTRinh-172 . Mean changes in cytosolic Ca2+, as indicted by changes in Fura-2 AM ratio, induced by thapsigargin  in a (a) bath solution containing 1 mM Ca2+ or (b) nominally Ca2+-free solution. Changes in cytosolic Ca2+ induced by (c) ionomycin (1 µM) and (d) ATP (100 µM). Correlation between changes in cytosolic Ca2+ and inhibition of CFTR-mediated conductance determined by the average rate of inhibition of CFTR-mediated conductance and (e) maximum change in Fura-2 ratio or (f) area under the curve (AUC) induced by Ca2+ agonists. Data are mean ± SEM (n = 3 independent experiments). (JPEG 1548 kb)Fig. S2 K44A Dynamin prevents a thapsigargin-induced CFTR internalisation. Changes in plasma membrane localised HA-CFTR following exposure to thapsigargin (TG) for times indicated with, or without, co-transfection with the dominant negative dynamin mutant. K44A. Data are mean ± SEM (n = 34-44 cells from 3 independent experiments). * = p < 0.05 compared to t = 0 † = p < 0.05 compared to thapsigargin at 60 min. (JPEG 1082 kb)Below is the link to the electronic supplementary material.


Optical harmonic generation, e.g., second- (SHG) and third-harmonic generation (THG), provides intrinsic contrasts for three-dimensional intravital microscopy. Contrary to two-photon excited fluorescence (TPEF), however, they have found relatively specialized applications, such as imaging collagenous and non-specific tissues, respectively. Here we review recent advances that broaden the capacity of SHG and THG for imaging the central nervous system in particular. The fundamental contrast mechanisms are reviewed as they encode novel information including molecular origin, spectroscopy, functional probes, and image analysis, which lay foundations for promising future applications in neuroscience. Even when such a system exists, questions still linger whether it recapitulates in vivo phenomena properly so that we can accept the data for inductive reasoning. Visualizing native tissue is therefore crucial for unraveling the complex dynamics of health and disease. Light microscopy is the method of choice for intravital studies of small animals. Combining non-invasiveness and ~1 μm resolution, it is irreplaceable for studying cellular dynamics. The field of view of ~1 mm2 and the imaging depth up to ~1 mm achievable via non-linear optical excitation are ideal for visualizing model embryos and whole organisms.Our knowledge is inherently limited for a biological process lacking an experimental replica, e.g., Historically, second-harmonic generation (SHG) was the first non-linear optical process to be observed experimentally  from single dipole moments, i.e., harmophores.ijk, which is a sum of the microscopic hyperpolarizabilities βijk of an ensemble of harmophores within the coherence volume of the excitation beam. It is well-known that an organic compound with a large asymmetric charge transfer (the “push-pull chromophore”) has a substantial hyperpolarizability  becomesThe SHG intensity is proportional to the density of harmophores squared in contrast to TPEF which is proportional to the density of chromophores. Consequently, even from molecules with HRS weaker than TPEF by ~4 to 10 orders of magnitude , only two of them are independent and non-vanishing - χzxx and χzzz. Then the ratio is related to the average polar angle, φ, of harmophores relative to the symmetry axis.For the simplest form of harmophore with As a result, the average orientation of harmophores can be obtained regardless of the details of the hyperpolarizability. Experimentally, γ can be determined by measuring the SHG intensity as the linear polarization of the excitation is rotated at an angle θ relative to the symmetry axis.The measured ratio can then be compared with the predicted value from the Protein Data Bank (PDB). This method is referred to as SHG polarization anisotropy or polarization-resolved SHG (pSHG).SHG polarization anisotropy has been demonstrated for collagen . Straight GTP-bound tubulins are readily incorporated into microtubule lattices whereas the mechanical strain produced by curved GDP-bound tubulins can lead to a catastrophic breakdown   than in structural proteins (millimolar) the hyperpolarizability of individual harmophores becomes crucial for measureable SHG signals. Organic compounds have been employed as exogenous agents for SHG contrast, e.g., styryl dyes  SHG effect is negligible, with the relative magnitude of the second- to third-order susceptibilities in the order of the atomic field strength (χ(2)/χ(3)~109 to 1011 V/m) . SHG voltage-sensors have a number of advantages over fluorescent probes: Since the voltage-sensitive SHG is mainly an electrooptic effect occurring without significant molecular rearrangements  Boyd, . However3)( SHG component independently from the χ2) is still low compared to fluorephores and many line-scans must be averaged to obtain a reasonable SNR. The susceptibility can be enhanced by means of near-resonance excitation at the expense of low background and the safety of the illumination. Consequently, reducing photodamage is key to the success of the voltage-sensing SHG technique . With the peptide bonds identified as SHG harmophores, it seems plausible to engineer genetically-encoded SHG probes. Synthetic peptides have been designed as SHG probes for studying various dynamics such as membrane penetration  of the three-photon excitation, h  .optical edge detection. Below are a few selected applications following this insight.Notably, gradient magnitude is a well-known operator of edge detection in image processing . Optical metrology of the myelin sheath is not without shortcomings. While sufficiently accurate to distinguish the subtle difference between wild-type (WT) and haploinsufficient NRG1 knockout (NRG1 +/–) mice, the precision of THG morphometry is limited when the spacing between the adaxonal and abaxonal membranes is much smaller than the wavelength, leading to a sampling bias  and schizophrenia, the morphology of individual myelinated fibers in living animals cannot be determined by current clinical modalities, e.g., magnetic resonance imaging (MRI). Furthermore, the standard procedure for measuring the ratio of axon to fiber diameter (the g-ratio), which is a principal biometric for the conduction speed, is prohibitively laborious and time-consuming (~days), involving fixation, imbedding, sectioning, and electron microscopy (EM). Various methods of optical metrology have been developed to address this issue (Kwon et al., ing bias . The g-ring bias  (Figure ing bias . Howevering bias  as long THG has been adopted to visualize many cell types and tissues (Aptel et al., The development of exogenous THG agents has been exploratory to date. Contrary to SHG, to design molecules with large susceptibility has not been a major avenue of development because the refractive index mismatch within the focal volume, thus the geometrical factors, is far more essential for THG imaging. Instead, a successful approach pertinent to intravital studies has been to enhance the THG contrast by way of a near-resonance excitation of organic aggregates, such as porphyrin. Since an initial demonstration of THG enhancement by the absorption band of hemoglobin (Clay et al., We have introduced recent tricks of optical harmonic generation which allownovel information to be extracted about the CNS of living animals. Innovations in deep-tissue microscopy have motivated ingenious intravital preparations for observing the intra- and intercellular dynamics beyond 2D cultured cells, thus opening new fields of investigation. Similar synergy is expected to follow the empowered intravital SHG and THG microscopy, enabling novel discoveries in neuroscience.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.