Ca2+ sparks which originated from subsarcolemmal junctional sarcoplasmic reticulum (j-SR) release sites in atrial myocytes were anisotropic and elongated in the longitudinal direction of the cell. Ca2+ sparks in atrial cells originating from non-junctional SR and in ventricular myocytes were symmetrical. Ca2+ spark recording in line scan mode at 40,000 lines/s uncovered step-like increases of [Ca2+]i. 2-D imaging of Ca2+ transients revealed an asynchronous activation of release sites and allowed the sequential recording of Ca2+ entry through surface membrane Ca2+ channels and subsequent activation of Ca2+-induced Ca2+ release. With a latency of 2.5 ms after application of an electrical stimulus, Ca2+ entry could be detected that was followed by SR Ca2+ release after an additional 3 ms delay. Maximum Ca2+ release was observed 4 ms after the beginning of release. The timing of Ca2+ entry and release was confirmed by simultaneous [Ca2+]i and membrane current measurements using the whole cell voltage-clamp technique. In atrial cells activation of discrete individual release sites of the j-SR led to spatially restricted Ca2+ release events that fused into a peripheral ring of elevated [Ca2+]i that subsequently propagated in a wave-like fashion towards the center of the cell. In ventricular myocytes asynchronous Ca2+ release signals from discrete sites with no preferential subcellular location preceded the whole-cell Ca2+ transient. In summary, ultra-fast confocal imaging allows investigation of Ca2+ signals with a time resolution similar to patch clamp technique, however in a less invasive fashion.The spatio-temporal properties of Ca Calcium that enters the cell activates sarcoplasmic reticulum (SR) Ca2+ release channels  through a process known as calcium-induced calcium release  refers to the process that links cell membrane depolarization to Case CICR; . RyRs anits CRU; . Togethe2+ release. As a consequence of these ultrastructural arrangements AP-induced Ca2+ release is spatially inhomogeneous  it is severely limited by the fact that a cellular event such as a Ca2+ spark, a Ca2+ wave or the whole-cell Ca2+ transient occurs in a 3-dimensional space, however its optical recording by line scan imaging is reduced to one spatial dimension. Therefore, line scan data notoriously suffer from out-of-focus problems  and how reliably complex 3-dimensional spatial properties can be captured with this approach. Recently, novel technical imaging tools, consisting of a fast 2-D (or x-y) confocal scanning mechanism (LSM 5 LIVE slit scanner) in conjunction with the ability to move the plane of focus vertically in rapid and reproducible manner, have enabled us to achieve x-y-z-t (or 4-D) imaging of sparks with a reasonable time resolution und to gain novel insight into the properties of cardiac Ca2+ sparks 2+ sparks and make the observation that sparks had a modal amplitude distribution. Here, we extended fast confocal imaging to the study of AP-induced Ca2+ transients in atrial and ventricular myocytes with the goal to gain new insight into Ca2+ entry, local Ca2+ release and cellular spread of activation during ECC, and to explore Ca2+ sparks at high temporal resolution to reveal the characteristics of the underlying Ca2+ release and gating properties of the channels involved.The fact that such critical questions have remained inconclusive is, at least in part, linked to methodological and technical limitations hampering the recording of locally restricted and rapidly changing subcellular Ca−1, I.P.). After thoracotomy, hearts were excised, mounted on a Langendorff apparatus, and retrogradely perfused via the aorta with oxygenated collagenase (cats) or Liberase Blendzyme TH (rabbits) containing solution (37°C). Myocytes were used for experimentation within 1–6 hours after isolation.Single myocytes were isolated from cat 2 2; MgCl2 1; D-glucose 10; HEPES 10; pH 7.4 adjusted with NaOH). All experiments were performed at room temperature (22–25°C). APs were elicited by electrical field stimulation by applying 1 ms voltage pulses of suprathreshold amplitude through a pair of platinum electrodes.Chemicals were obtained from Sigma-Aldrich  unless otherwise noted. During experiments myocytes were superfused continuously with normal Tyrode solution  at room temperature. 15–20 min were allowed for de-esterification of the dye. Fluo-4 was excited with the 488 nm line of an argon ion laser and emitted fluorescence was measured at wavelengths >515 nm. Confocal imaging at high temporal resolution was performed with a slit scanning confocal microscope  equipped with a 63×, 1.20 n.a. water-immersion objective . High-speed data acquisition was achieved by bi-directional scanning. 2-dimensional (2-D) imaging  yielded images of 512×53 or 512×31 pixels (pixel distance of 0.3 µm) recorded at 1038 and 1719 Hz, respectively. The x-dimension is defined as the longitudinal axis of the cell. The y-dimension refers to the transverse cell axis and the z-dimension defines the axial  cell dimension. The point-spread function of the system had full-width at half maximum (FWHM) values of 0.52, 0.46, and 1.25 µm, respectively, in the x, y, and z directions 2+]i are presented as background-subtracted normalized fluorescence (ΔF/F0) where F is the fluorescence intensity, F0 is resting fluorescence recorded under steady-state conditions at the beginning of an experiment, and ΔF = F–F0. Average data are presented as mean ± SEM.Changes of [Ca2+]i and membrane Ca2+ current measurements were performed in rabbit ventricular myocytes using the voltage-clamp technique in the whole-cell ruptured patch configuration, using an Axopatch 200A amplifier, the Axon Digidata 1440A interface and pCLAMP 10.2 software . The patch pipette solution contained (in mM): 120 L-aspartate; 120 CsOH; 20 TEA-Cl; 20 HEPES; 1 L-gluthathione (reduced); 1 DM-nitrophen ; 4 ATP-Na, 0.25 CaCl2 and 0.1 Fluo-4 pentapotassium salt (Molecular Probes); pH 7.2 adjusted with CsOH). Currents were recorded at 100 kHz and low-pass filtered at 5 kHz.Experiments for simultaneous [Ca2+ transients during normal ECC in atrial myocytes. AP-dependent Ca2+ transients were elicited by electrical field stimulation (0.7 Hz). As shown previously cat atrial myocytes are devoid of a t-tubular network 2+ release channels are present throughout the cell with the exception of the nuclear region 2+]i images of the early phase of an electrically evoked Ca2+ transient in an atrial cell, recorded at 1719 Hz from a central axial plan, i.e. at equal distance from the top and bottom cell borders in the axial (z) dimension. Ca2+ release appeared spatially inhomogeneous and initiated at discrete sites in the periphery where the j-SR is located. Ca2+ release was also found to be asynchronous among peripheral j-SR release sites and required 3.0±0.1 ms  for all detectable peripheral release sites to be activated , and also centripetally (y-dimension). 2+]i at an individual j-SR release site and a peripheral subsarcolemmal (SS) non-release site at a lateral distance of ∼1 µm, revealing a time delay that was characteristic for subsarcolemmal Ca2+ diffusion. Traces representing the SS j-SR region were recorded within <1 µm from the edge of the cell. Ca2+ release from peripheral j-SR sites eventually gave rise to a rather homogeneous elevation of [Ca2+]i in the cell periphery, obscuring individual release sites. On average this homogeneous ‘ring’ of increased [Ca2+]i was complete 10.1±1.5 ms after application of the electrical stimulus. As shown in 2+]i started to rise with a delay at a central (CT) subcellular location distant from the cell membrane. At this location, which corresponds to the nj-SR, [Ca2+]i rose much more slowly and peaked at a level that was below peak [Ca2+]i in the cell periphery. As previously established 2+ release from release units of the nj-SR in atrial cells occurs by propagating CICR or a ‘diffuse-fire-diffuse’ mechanism where Ca2+ released from a peripheral release site diffuses over a short distance (1–2 µm) to a neighboring more centrally located release site where it activates CICR. Through this sequence of events activation of Ca2+ release in atrial cells becomes spatially inhomogeneous and propagates from the periphery to the center of the myocyte in a Ca2+ wave-like fashion.In a first set of experiments we investigated the spatio-temporal organization of Cactivated . This is2+ transient is the result of spatial and temporal summation of elementary Ca2+ release events from individual CRU, termed Ca2+ sparks 2+ transient in atrial cells we investigated spontaneous Ca2+ sparks from peripheral j-SR CRUs. 2+ spark. Images were recorded at a sampling rate of 1038 Hz. Sparks in the cell periphery arising from atrial j-SR CRUs are highly anisotropic with a preferential spread in the longitudinal direction. FWHM in longitudinal (x-dimension) direction was 2.50±0.07 µm , whereas the average FWHM in transverse direction was 1.51±0.05 µm. Below the panel of 2-D images, two line scan images (x-t- and y-t-images) are shown that were constructed from the stack of 2-D images. For the x-t image a single line of pixels in the x-dimension was extracted at a fixed value of y from each consecutive 2-D image and the lines were stacked from left to right along the time axis. Analogously, a single line of pixels in the y-dimension was extracted at a fixed value for x. While the x-t-image shows a symmetrical spread of [Ca2+]i from the site of initiation, the y-t-image clearly shows an anisotropic spread. The degree of anisotropy was quantified as eccentricity (e). Eccentricity was defined as the ratio of FWHM of Ca2+ sparks measured along two perpendicular axis within the focal plane and by convention the larger value was divided by the smaller measurement, or e = FWHMwide/FWHMnarrow. For peripheral j-SR Ca2+ sparks in atrial myocytes the average eccentricity was e = 1.71±0.05, i.e. peripheral sparks were approximately 70% wider along the longitudinal cell axis with all sparks analyzed extending more in the longitudinal dimension.It is generally agreed that the AP-induced whole-cell CaIn contrast, sparks recorded from central (nj-SR) regions revealed a different geometry . Sparks 2+ when imaged in a single confocal plane cutting across such a coupling. As discussed above, during ECC Ca2+ release propagates by CICR from the cell periphery to the center of the cell. We therefore explored the spatio-temporal properties of peripheral Ca2+ sparks for patterns that indicate transverse or centripetal spread of Ca2+. For this purpose we determined frame-by-frame the x-y-coordinates of maximal fluorescence which identifies a point source of Ca2+ release. As shown in 2+ out of the diadic cleft of the peripheral couplings. In contrast, in transverse (y) direction the location of maximal fluorescence shifted by ∼0.5 µm from the cell membrane towards cell center over the time course of 20 ms, thus facilitating the spread of excitation in centripetal direction. This spatial pattern cannot be explained by simple Ca2+ diffusion away from a fixed point source of Ca2+ and may suggest a preferential direction of Ca2+ release propagation possibly within a peripheral CRU.The structural arrangements of surface cell and SR membranes in the peripheral couplings of the j-SR can explain the preferential spread of Ca2+ channels. Through this arrangement membrane depolarization by an AP allows the synchronous delivery of a triggering amount of Ca2+ into the diadic cleft (formed by t-tubular and SR membranes) to induce CICR. Because the t-tubular system guarantees synchronicity for CICR activation, the onset of Ca2+ release appears spatially and temporally rather homogeneous, although upon closer examination inhomogeneities have been observed Ventricular myocytes are equipped with an extensive t-tubular membrane system that is organized in a sarcomeric pattern and functionally allows conduction of the AP into deeper regions of the myocyte. Transverse tubules provide the structural basis that CRUs are all in close contact with the cell membrane that hosts voltage-gated Ca2+ transient from a ventricular myocytes. The images were recorded from a central focal plane equidistant from the bottom and the top of the cell at a sampling rate of 1719 Hz. Several characteristic features of a ventricular Ca2+ transient become evident: (1) localized Ca2+ release signals from individual CRUs appeared randomly throughout the cell with no obvious preferential subcellular location; (2) recruitment of CRUs is not entirely synchronized. As shown in 2+]i from peripheral and central CRUs that were recruited at the same time, were essentially indistinguishable , ventricular j-SR and atrial nj-SR (CT). The time course of the rapid rising phase of the ventricular and the atrial SS signals were virtually identical, whereas the rise of [Ca2+]i at the atrial CT site was significantly slower. Thus, in respect to the dynamics of local Ca2+ release ventricular and atrial j-SR sites behaved remarkably similar, whereas release from atrial nj-SR CRUs revealed a distinctly different spatio-temporal pattern.In atrial myocytes, the arrangement of surface membrane and j-SR membranes of the peripheral couplings have a certain resemblance with ventricular myocytes. The common features are that the peripheral couplings form diadic clefts were both membranes associate closely and voltage-gated Ca2+ sparks result from the concerted opening of a finite number of RyR Ca2+ release channels organized in clusters in the SR membrane. It is, however still a matter of debate how many individual release channels contribute to a spark signal, and the reported numbers vary widely, ranging from small numbers of less than a dozen to over a hundred such channels (see Introduction and Discussion).It is generally agreed that Ca2+ sparks typically employs scanning speeds in the 0.5–1 kHz range. Such time resolution can impose severe limitations for the fidelity of recording of fast components of a Ca2+ spark such as its rising phase, and may hamper the interpretation of such signals in terms of the underlying channel gating and number of channels involved. Taking advantage of the high temporal resolution capabilities of the LSM 5 LIVE, we recorded Ca2+ sparks in line scan (x-t) mode with an acquisition rate of 40 kHz (25 µs/line) as shown in 0) obtained from the center of the spark. The spark shown had a rise time (time-to-peak) of ∼6 ms. Detailed analysis of the rising phase of the Ca2+ spark revealed step-like increases of [Ca2+]i /dt; 0 signal represents an approximation of the underlying Ca2+ fluxes 0)/dt revealed several discrete peaks identifying maxima of Ca2+ release flux. The timing of these peaks  was used to identify discrete steps in the [Ca2+]i signal marked by solid black lines in 0 signal could be identified with this approach. The improved temporal resolution of Ca2+ spark recording may open new avenues to investigate RyR gating within a CRU in-situ by a non-invasive approach as will be discussed below.Conventional confocal line scan imaging of Ca2+ entry through voltage-gated Ca2+ channels that is followed by CICR from the SR. Thus, the global Ca2+ transient is the result of Ca2+ flux across the cell membrane and a much larger Ca2+ flux through RyR SR Ca2+ release channels. In the next set of experiments we set out to identify these two flux components with high speed Ca2+ imaging. 2+ transient (ΔF/F0) induced by an AP in a ventricular cell and averaged over a region of interest of ∼550 µm2 (200×31 pixels) to obtain a low noise signal. Marker ‘1’ identifies the time when the electrical stimulus was applied to trigger an AP. 2+]i signal (d(ΔF/F0)/dt) representing the underlying Ca2+ flux. The first derivative signal revealed three distinct phases. The first phase, defined by the time of application of the electrical stimulus (marker ‘1’) and the begin of the rise of the d(ΔF/F0)/dt signal (marker ‘2’) was defined as the latency period between electrical stimulus and activation of Ca2+ flux, presumably resulting from Ca2+ entry. The latency period was 2.5±0.3 ms (n = 9 ventricular myocytes). During the following phase (between markers ‘2’ and ‘3’) d(ΔF/F0)/dt increased steadily at a slow rate. At t = 5.5±0.2 ms (marker ‘3’) the flux rate rather abruptly increased and peaked (marker ‘4’) on average at d(ΔF/F0)/dt = 748±71 s−1, 9.9±0.7 ms after application of the electrical stimulus. 2+ fluxes in control ventricular cells and myocytes with a disabled SR. The SR was disabled by complete depletion with caffeine (10 mM) and prevention of refilling by treatment with the SERCA blocker thapsigargin (1 µM). Elimination of SR Ca2+ release abolished the large increase of Ca2+ flux normally observed after marker ‘3’, however the latency period  and the subsequent slow rise of the d(ΔF/F0)/dt signal were preserved. The d(ΔF/F0)/dt signal peaked at 50±5 s−1, 7.7±0.7 ms after the electrical stimulus. In conclusion, these observations are consistent with the hypothesis that phase ‘1–2’ (latency) marks the time interval that is required for the spread of depolarization and activation of surface membrane ion channels. Phase ‘2–3’ represents Ca2+ entry flux, and was identical in control myocytes and cells with disabled SR function. Phase ‘3–4’ is identified as Ca2+ release flux from the SR, since it was completely abolished when the SR was eliminated.During ECC AP depolarization enables Ca2+ fluxes were further analyzed separately for SS (j-SR) and CT (nj-SR) regions. 2+]i (ΔF/F0) induced by electrical field stimulation applied at marker ‘1’, revealing the delayed and slower rise of [Ca2+]i in the CT region. 0 representing the underlying Ca2+ fluxes. In the SS region, similar to ventricular cells, a latency period , was followed by a slow increase (starting at marker ‘2’) that changed to a fast increase of d(ΔF/F0)/dt at 6.4±0.3 ms after application of the electrical stimulus (marker ‘3’) and peaked at 1183±180 s−1 after 11.8±0.6 ms. In contrast the CT d(ΔF/F0)/dt signal did not reveal an obvious slow rising phase and did not start to rise until 2.2±0.3 ms after the first change of the SS signal (marker ‘2’) was observed. Maximal flux in the CT region was 3.5±0.3 times less than in the periphery and peaked 5.3±1.7 ms later. 2+ fluxes in control and myocytes with disabled SR (TG+Caff). In the SS region d(ΔF/F0)/dt started to increase 2.1±0.1 ms  after the electrical stimulus was applied and flux reached a maximum (164±30 s−1) after 6.5±0.5 ms, approximately at the time when in control cells SR Ca2+ release flux started. Ca2+ release flux in the CT region was 4.0±0.7 times smaller compared to the SS region.2+ imaging enabled us to identify Ca2+ fluxes associated with Ca2+ entry and Ca2+ release from the SR. Next, we attempted to validate the optically measured Ca2+ entry and release signals (2+]i and membrane Ca2+ currents (ICa) recorded with the patch clamp technique. Spatially averaged [Ca2+]i and ICa measurements were obtained from single ventricular myocytes (2+]i (ΔF/F0), Ca2+ flux (d(ΔF/F0)/dt), ICa and the command voltage. The Ca2+ flux signal showed a slow steady increase shortly after the begin of the depolarization pulse (marker ‘a’). The slow increase of Ca2+ flux abruptly increased at marker ‘b’, giving way to a rapid rise of the Ca2+ flux rate. The distinct change in flux rate (marker ‘b’) occurred 5.7 ms after the begin of the depolarization pulse and correlated well with the begin of SR Ca2+ release determined by optical methods  in atrial and ventricular myocytes with 2-D and line scan confocal imaging at high recording speeds. The key findings of our investigation were as follows:In this study, we investigated the behavior and interactions of CRUs during electrically evoked Ca2+ imaging allowed to resolve a distinct sequency of events during a Ca2+ transient, consisting of a latency period, Ca2+ entry and SR Ca2+ release, by an optophysiological approach 2+]i during the rising phase of Ca2+ sparks that may provide novel insights into RyR gating properties within individual CRUs.First, the recruitment of individual CRUs by AP depolarization was spatially inhomogeneous and temporally asynchronous. Spatial inhomogeneity was particularly pronounced in atrial myocytes that lack a t-tubular membrane system. Second, high-speed confocal Ca2+ release in atrial myocytes was asynchronous at two different levels. As has been described before 2+ release originating at the peripheral couplings of the j-SR preceded Ca2+ release from the nj-SR in central regions of the cell, and peak CT [Ca2+]i lagged behind by tens of milliseconds during AP-triggered Ca2+ transients  and 6.4 ms  after the begin of the pulse, represents Ca2+ entry across the surface membrane which was confirmed with simultaneous patch clamp and [Ca2+]i measurements  do not have the characteristics of a typical couplon. Specifically, the RyRs are not facing into a narrow diadic cleft where upon activation of LCCs [Ca2+]i rapidly increases to concentrations that are several orders magnitude higher than resting [Ca2+]i. With the inherent low Ca2+-sensitivity of the RyR 2+]i can rise sufficiently high in the vicinity of RyRs of the nj-SR to lead to channel opening and Ca2+ release. Furthermore, it has even been suggested that Ca2+ sequestering mitochondria in the subsarcolemmal space of atrial myocytes constitute a Ca2+ barrier 2+ flux (∼11 pA) and large numbers of channels involved (20–30 RyRs) with an enhanced propensity to open and a diminished influence of negative feedback mechanisms as well as an extended spatial spread , to whole-cluster activation with possibly contribution from several hundred channels 2+ currents through single cardiac RyR channels reconstituted in lipid bilayer and recorded under physiological ionic conditions gave unitary current values of 0.3–0.7 pA 2+ signals of known magnitude arising from LCCs 2+ sparks, it could be demonstrated that Ca2+ sparks have a quantal substructure and that Ca2+ spark release flux is made up of quanta of 1.2 pA. Furthermore, the number of quanta in a spark varies dynamically from one to ≤8 0)/dt signal shown in 2+ sparks involve release from 20–30 RyRs 0)/dt signal in 2+ release since quantal release consisting of a small number of quanta (even 1 or 2 quanta only) has been demonstrated The question how many RyRs are found in an individual CRUs has remained rather elusive. Earlier reports suggested the number to be in the range of 50–250 RyRs 2+ release found in subsarcolemmal atrial CRUs , strong Ca2+-dependent RyR inactivation or the recently proposed ‘pernicious attrition’ Ca2+ release termination mechanism 2+ binding to the RyR could be responsible for such a strong inhibitory mechanism While entirely speculative at this point, the progressive activation of RyR sub-clusters within a CRU may also explain the centripetal propagation of Caial CRUs . Further2+ dynamics and intricacies of single SR Ca2+ release units. With this technique we showed that dynamics of Ca2+ entry and CICR can reliably be distinguished at the level of individual CRUs in intact cells, that Ca2+ sparks of atrial myocytes have inherent properties that facilitate centripetal propagation of activation, and that the rising phase of Ca2+ sparks reveals step-like kinetics which bears potentially valuable information about intra-CRU channel gating. These findings have important ramifications for the understanding of the structural and functional organization of CRUs, CICR and ECC.In summary, we demonstrate here that high-speed confocal imaging with sampling rates in the 1–40 kHz range in conjunction with high quantum yield fluorescent indicators allows to study Ca


Drosophila erecta, a close relative of Drosophila melanogaster and specialist on screw pine fruits (Pandanus spp.). In comparison with three sympatric sibling species, D. erecta shows specific alterations in its olfactory system towards detection and processing of a characteristic Pandanus volatile . We show that D. erecta is more sensitive towards this substance, and that the increased sensitivity derives from a numerical increase of one olfactory sensory neuron (OSN) class. We also show that axons from these OSNs form a complex of enlarged glomeruli in the antennal lobe, the first olfactory brain centre, of D. erecta. Finally, we show that 3M2BA induces oviposition in D. erecta, but not in D. melanogaster. The presumed adaptations observed here follow to a remarkable degree those found in Drosophila sechellia, a specialist upon noni fruit, and suggest a general principle for how specialization affects the sense of smell.Finding appropriate feeding and breeding sites is crucial for all insects. To fulfil this vital task, many insects rely on their sense of smell. Alterations in the habitat—or in lifestyle—should accordingly also be reflected in the olfactory system. Solid functional evidence for direct adaptations in the olfactory system is however scarce. We have, therefore, examined the sense of smell of  An animal exposed to a novel environment or niche will presumably alter its olfactory system over evolutionary time to encompass the composition of new chemical volatiles. Insects are well suited for this line of study, because they possess a rich repertoire of odour-guided behaviours (such as mating and breeding), and their nervous system is accessible as well as numerically reduced compared with the nervous system of vertebrates .Drosophila sechellia —a close relative of the laboratory work-horse D. melanogaster—is a well-known model system for studying questions relating to adaptive host specialization, particularly regarding the olfactory system [D. sechellia–Morinda system has, however, a number of shortcomings as a model of host plant-driven sensory specialization. Ample evidence suggests that D. sechellia has a very small effective population size [Morinda association  and specializes on ripe fruits of Pandanus spp. Parkinson  were trapped in banana baits between two Pandanus fruiting events.anaceae; ,15; figutinguish . From thl Africa , D. erecsyncarps –20. The n Africa . Pandanu& Tsacas  found a D. erecta, its sibling D. sechellia relies exclusively on fruits of the noni tree  for feeding and breeding [D. melanogaster and Drosophila simulans. Pandanus syncarps are also visited by other drosophilids, including the sympatric sibling species D. yakuba (F. capensis (Moraceae), Theobroma cacao  and Landolphia hirsuta (Apocynaceae) [D. yakuba (like most of the other visiting drosophilids) uses Pandanus fruits only for feeding and not for breeding [Drosophila orena, which is only known from a single collection event on Mount Lefo, Cameroon.In contrast to breeding ,21. Fres. yakuba , a generynaceae) . Althougbreeding . By contD. erecta is reflected in its olfactory system. We demonstrate that the D. erecta–Pandanus association has resulted in alterations in the olfactory system, similar to those found in D. sechellia. Specifically, we show that D. erecta, in contrast to its non-specialized sympatric relatives D. orena, D. yakuba and D. melanogaster, is more sensitive towards the Pandanus volatile 3-methyl-2-butenyl acetate . The enhanced sensitivity is accomplished by increasing a specific class of olfactory sensory neurons (OSNs); intriguingly, this is the same class of OSNs affected in D. sechellia. Moreover, we show that the numerical increase in the periphery is accompanied by the formation of enlarged glomeruli in the antennal lobes (ALs), the first olfactory neuropil in the fly brain. Furthermore, 3M2BA triggers oviposition in D. erecta.Here, we ask whether the specialized lifestyle of 2.(a)Drosophila Species Stock Centre (https://stockcenter.ucsd.edu): D. erecta (14021–0224.01), D. orena (14021–0245.01) and D. yakuba (14021–0261.01); except for D. melanogaster (wild-type Berlin). For functional imaging, transgenic fly lines of D. melanogaster used were Orco-Gal4 [GCaMP3.0 flies were kept for 36 h at 29°C before experiments to enhance UAS-reporter gene expression. All flies were reared on standard cornmeal medium at 25°C, except for D. orena (18°C), 12 L : 12 D photoperiod and 70 per cent relative humidity. All experiments were carried out with mated female flies, 4–10 days post-eclosion, except for oviposition experiments.Wild-type flies were obtained from the rco-Gal4  and UAS- 14021–02.01, D. o(b)Pandanus sp. fruits were wrapped in polyester bags , and volatiles were trapped with Super Q adsorbent filters . Sample collection was done for 6 h (1.0 l min−1) using a vacuum pump . Adsorbed volatiles were desorbed by eluting filters with 300 µl dichloromethane . Samples were stored at −20°C.Ripe (c)Pandanus fruit volatiles were analysed by gas chromatography-mass spectrometry (GC-MS) . The GC was equipped with a non-polar HP5 column  with helium as a carrier gas (1.1 ml min−1 flow rate). One microlitre of sample was injected splitless at 265°C. Temperature program was 40°C for 3 min, rising to 280°C at 5°C min−1, held for 10 min. Compounds were identified by their mass spectra in a National Institute of Standards and Technology library search, and were confirmed by comparing the Kovats index with the indices of synthetic compounds.(d)Pandanus fruit volatiles. Flies were mounted following standard procedures [Pandanus extract or synthetic compound , was injected splitless into an Agilent GC 6890 equipped with a non-polar column (for details see above). A 1 : 1 effluent splitter allowed for the simultaneous flame ionization detection (FID) and the EAD of the separated compounds. Helium was the carrier gas; injector and detector temperatures were 250°C and 300°C, respectively. Column temperature was held at 40°C for 1 min, rising to 300°C at 20°C min−1, held for 10 min. The GC-separated components were introduced into a continuous, filtered and humidified air stream flowing over the antennae (1 l min−1). The EAD and FID signals were simultaneously recorded and analysed .Gas chromatography coupled with electroantennographic detection (GC-EAD) and electroantennography (EAG)  were useocedures . For GC-iji [iji (as ‘text image’) and colour-coded (‘smart’). Headspace compounds were determined as biologically active when eliciting reproducible responses in the fly antennae. EAD responses were manually quantified with the Syntech software [Determination of GC-EAD active compounds was simplified by converting the antennal responses into false-colour-coded heat maps using Fiji . Therefosoftware , and norPrincipal component analysis (PCA) was then applied (variance–covariance matrix), and the antennal responses were displayed within a three-dimensional space. With one-way analysis of similarity (ANOSIM), we tested by which degree the antennal responses of the four species differed from each other . The similarity percentage (SIMPER) method  indicate−1 to 10−5) to record dose–response curves. Prior to each experiment, 10 µl of diluted odours was freshly loaded onto a small piece of filter paper , and placed inside a glass Pasteur pipette. The antennae were held in a continuous, filtered and humidified air stream produced by a stimulus controller , whose flow was 1 l min−1. Odour stimuli were applied for 0.5 s into the constant air stream. Antennal signals were recorded with EagProV2 (Syntech). Control stimuli consisted of filter paper with mineral oil, which was applied before each odours set. Response traces were baseline corrected with the mean of the first second and normalized to the control. Statistics  were done with the software InStat v. 3.06 .For EAG experiments, synthetic compounds were diluted in mineral oil  in decadic steps (10(e)−5, 10−3, 10−1) in mineral oil .To specify which glomerulus is activated by 3M2BA, we performed functional calcium imaging experiments as previously described . Pure od(f)D. erecta and D. melanogaster females. Pure odorants were diluted (10−2–10−8) in mineral oil . Single sensillum recording (SSR) measurements were performed as outlined in [To test the activation of the Or22a receptor by different 3M2BA concentrations, dose–response experiments were performed on the large basiconic sensilla in lined in . Respons(g)Drosophila ringer solution as described by Wu & Luo [Anesthetized flies were dissected in Wu & Luo . The braWhole-brain mounts were studied with a Zeiss LSM 510 Meta confocal laser scanning microscope . Scans were performed for every 0.5 µm stack with a 40 × 10 water immersions objective . Structures labelled with Alexa Fluor 633 were excited with a HeNe laser at 633 nm.D. melanogaster and D. erecta females were analysed and compared. For each species, a template AL was chosen based on staining quality and shape. The terminology of the glomeruli is based on Couto [D. melanogaster 63638.14 ± 3992.36 s.e.; D. erecta 68111.61 ± 3847.56 s.e.).Three-dimensional reconstructions and volumetric measurements of glomeruli were made with the segmentation software AMIRA v. 5.4.1 . The ALs of three on Couto . Per spe(h)D. erecta females were performed as previously described [g).To trail the convergence of the OSNs of the ab3 sensilla, anterograde-neurobiotin backfills  of escribed . Treated(i)D. melanogaster and the specialist D. erecta to 3M2BA. Tests were generally performed as previously described [By using a two-choice assay, we tested the oviposition site preference (OSP) of the generalist escribed  with somPrior to the analyses numbers of eggs were log-transformed.To test for the effect of odour and spatial information on the OSP, linear mixed effects models were performed using the computing environment R v. 2.15.2 . Spatial3.(a)D. erecta to its host, fresh fruits of the screw pine tree Pandanus. We, therefore, followed a comparative approach using electrophysiology in four sympatric melanogaster sibling species . First, we sampled volatiles from fresh Pandanus sp. fruits. The collected headspace was then examined for antennal activity in linked GC-EAD experiments [D. melanogaster, D. yakuba, D. orena and D. erecta as natural odorant detectors, we found a total of 19 volatiles eliciting reproducible antennal responses among the Pandanus-emitted volatiles . We identified the biologically active compounds via GC-MS, through library comparison and co-injection of synthetic standards. We succeeded in identifying all but three minor peaks .The purpose of this study was to unravel insect–host-derived adaptations within the olfactory system of eriments . PerformPandanus bouquet evoked species-specific response spectra on the fly antenna. However, only few and typically fruit-related compounds [figure 2a and electronic supplementary material, figure S1). We next asked whether the antennal response spectra towards the Pandanus bouquet varied between the four different species. First, we examined if the antennal response spectra simply mirrored the phylogenetic relationship between these species. To address this question, we performed a PCA based on the quantitative EAD responses to the 19 active peaks in the Pandanus headspace, and plotted the results within three-dimensional space (figure 2b). The D. erecta measurements formed a distinct cluster, which did not overlap with D. orena, its closest relative. Instead, D. erecta grouped closer with D. yakuba, a species also attracted to Pandanus syncarps [D. orena clustered closer to the generalist D. melanogaster. The PCA, hence indicates that the generalist D. yakuba and the specialist D. erecta show similarities in their antennal response spectra, perhaps owing to the common food source [figure 2a). We thus conclude that the recorded olfactory responses of the different fly species probably reflect lifestyle rather than phylogenetic relationship.Notably, the ompounds  induced syncarps . On the d source . The dis(b)Pandanus syncarps [Pandanus fruit examined here, it evoked strong antennal responses in the GC-EAD experiments (a). Accordingly, we next examined the sensitivity of the four species towards this novel Drosophila odour ligand in more detail. In dose–response experiments, we recorded electroantennogram (EAG) activity from the four species, using synthetic 3M2BA as stimulus. The specialist D. erecta was considerably more sensitive than its relatives towards this Pandanus volatile (c). Our findings thus parallel earlier studies from other insects demonstrating increased sensitivity of specialist species towards host-specific volatiles [D. erecta to 3M2BA represents a host-specific adaptation towards use of Pandanus syncarps. Next, we wondered which olfactory receptors (ORs) are activated by this ligand and where the respective OSNs target in the brain.Whereas, isoamyl acetate and phenethyl alcohol are common natural fruit ligands , 3M2BA asyncarps . Althougeriments a. Accordvolatile c. Our fiolatiles ,39. In s(c)D. erecta are still not available, we performed these experiments with D. melanogaster females expressing the Ca2+ sensitive reporter GCaMP3.0 [Orco promoter to get an indication of which glomeruli and, consequently, which ORs were activated by 3M2BA. Stimulation with diagnostic odours and comparison with the map of the fly AL [a–c). 3M2BA activated three glomeruli, DM2, DM5 and DM6, of which DM2 showed the strongest response (a). In D. melanogaster, DM2 receives input from ab3A type neurons that express the olfactory receptor Or22a [D. melanogaster [melanogaster species group, including D. erecta. The signals measured from the DM5 and DM6 glomeruli probably stem from light scattering from the strongly activated neighbouring DM2 glomerulus.To identify OR(s) activated by 3M2BA, we next performed functional imaging of the AL. Briefly, the AL consists of subunits, the so-called glomeruli, which are formed by afferents of the OSNs; these in turn are housed in the sensilla that cover the insect antenna. OSNs expressing the same receptor converge onto one specific glomerulus . BecauseGCaMP3.0  from thee fly AL ,41 enablresponse a. In D. or Or22a . This nenogaster  as well D. erecta underlie the responsiveness towards 3M2BA, we next performed SSR from the three large basiconic sensilla in this species. Indeed, these OSNs respond to 3M2BA in D. erecta (d). In addition, we also noted responses from the ab2A neuron towards 3M2BA, albeit only at high concentrations. Dose–response experiments performed from OSNs in ab2 and ab3 sensilla also demonstrate the high sensitivity of ab3A neurons to 3M2BA (e). We note that in the functional imaging experiments, no significant increased activity was observed from the DM4. This discrepancy is probably owing to the low sensitivity of the ab2A OSNs towards this substance, making it difficult to observe the signal with conventional functional imaging. In line with the notion that the signals from the DM5 stem from light scattering from DM2, we also recorded no increased spike firing in response to stimulation with 3M2BA from the ab2B OSNs, whose axons target DM5.To verify that ab3A OSNs in . erecta d. In addto 3M2BA e. We notD. erecta towards Pandanus thus appears to involve the same pathway as affected in D. sechellia. In D. erecta, the ab3A/DM2 pathway mediates information regarding the Pandanus volatile 3M2BA, whereas in D. sechellia the homologous pathway handles the major noni volatile methyl hexanoate. In D. sechellia, the increased sensitivity to the noni volatile is also accompanied by morphological alterations within the AL. Hence, we wondered if we would also find similar modifications in D. erecta.Interestingly, the specialization of (d)D. erecta, and compared these with the well-established AL structure of D. melanogaster (a). The AL of D. erecta revealed a complex of four enlarged glomeruli at the medial lateral site. Comparing these with the AL atlas of D. melanogaster [D. erecta, compared with D. melanogaster ).To identify potential changes in the morphology of the olfactory system, we next reconstructed ALs of nogaster a. The ALnogaster ,41,43, wD. erecta AL, the most enlarged glomeruli (DM2 and VM5d), presumably both receive input from OSNs housed in ab3 type sensilla. Indeed, using data from a previous study [D. erecta compared with the other species (c). The increased volume of both DM4 and DM5, which receive input from OSNs housed in ab2 type sensilla can also be explained by a proportional increase of these sensilla in D. erecta. The larger spiking neuron in this sensillum type, which targets the DM4 glomerulus, also responded to 3M2BA, albeit only at high concentrations.The main factor contributing to the increase in glomerular volume is probably enhanced antennal input, i.e. a higher number of a certain sensilla . In the us study , we esti species c. The inD. erecta receive input from ab3 type sensilla, we next labelled OSNs via anterograde-neurobiotin backfills, allowing us to connect physiological response with the neuronal correlates in the AL [D. erecta target the enlarged glomeruli (b).To further verify that the enlarged glomeruli in lomeruli b.Pandanus in D. erecta parallel to a striking degree those found for the noni-specialist D. sechellia, even involving the same subpopulation of OSNs and receptor genes. However, some distinctions have to be emphasized here. First, in D. erecta, unlike D. sechellia, increased sensitivity is not based on the complete replacement of another type of sensilla (c). Second, while ab3A neurons in D. erecta are increased in numbers, the homologous neurons in D. sechellia also display a change in sensitivity (e).In short, adaptations towards use of sensilla c. Secondsitivity e.(e)D. erecta females, we next examined the behavioural relevance of 3M2BA. Egg laying in Drosophila is a behaviour that is under strong selection pressure [D. erecta and generalist D. melanogaster within a two-choice oviposition experiment by offering them either a control or 3M2BA (d). There are indications that flies preferentially lay eggs along vertical structures [Having unveiled the olfactory detection of host-derived signals in pressure . We, theor 3M2BA d. There ructures ,46. We, D. erecta and D. melanogaster clearly preferred the vertical medium surface over the vertical gap around the odour cup, and over the horizontal medium surface . However, 3M2BA only triggered oviposition in D. erecta, but not in D. melanogaster . More strikingly, this effect was strongly enhanced when combined with spatial information . In combination with 3M2BA, D. erecta laid significantly more eggs inside the vertical gap around the odour cup than along the vertical medium surface .Considering the effect of spatial information, both Pandanus in D. erecta is unlikely. Most probably, it is a combination of Pandanus volatiles that guides gravid females to the fruits and induces egg-laying. However, an enhanced sensitivity towards a host odour with limited availability outside of the host, such as 3M2BA, would be evolutionary beneficial.Preferring vertical edges for egg-laying might be advantageous for flies in general, because it may provide protection from predators and desiccation. It is conceivable that both odour and spatial information are needed to indicate reliable egg-laying places. The potential role of 3M2BA as the sole stimulus triggering oviposition preference for 4.D. erecta, and provide evidence for adaptational changes in the insect towards its host and oviposition site, fruits of Pandanus. We not only found D. erecta being more sensitive towards host volatiles than its non-specialist siblings, we also demonstrate that the antennal response spectrum displays the life history rather than the phylogenetic relationship of the four flies investigated in this study. The higher sensitivity in D. erecta towards host volatiles is accompanied by changes in the AL morphology, specifically, a complex of enlarged glomeruli. We further uncovered the glomerular activation pattern and the receptor activation of 3M2BA, a novel Drosophila fruit ligand, which seems to play an important role in OSP of D. erecta.Finding an appropriate breeding and feeding site is a crucial aspect in an insect's life, because the success of that choice directly influences the offspring's nutritional intake and consequential fitness. In this regard, the sense of smell is critically important for many insects. Here, we studied the olfactory system of the specialist D. erecta appear to have occurred at two levels: first, at the periphery, with an increased number of ab3 sensilla; and second (as direct consequence), a shift towards a complex of enlarged glomeruli in the AL. One of these glomeruli, the DM2, is involved in processing the Pandanus key compound 3M2BA. Our results thus not only support previous findings in the noni fruit specialist D. sechellia, but also provide evidence for a general pattern of olfactory adaptations in insect–host associations.In conclusion, olfactory adaptations in


We probed effects and underlying mechanisms of levosimendan on apoptosis and NCX activity in cultured human cardiomyocyte progenitor cells (CPC)-derived cardiomyocytes undergoing anoxia-reoxygenation (A/R), simulating I/R in vivo. Administration of levosimendan decreased apoptosis of CPC-derived cardiomyocytes induced by A/R. The increase in reverse-mode NCX activity after A/R was curtailed by levosimendan, and NCX1 was translocated away from the cell membrane. Concomitantly, endoplasmic reticulum (ER) stress response induced by A/R was attenuated in CPC-derived cardiomycytes treated with NCX-targeted siRNA or levosimendan, with no synergistic effect between treatments. Results indicated levosimendan inhibited reverse-mode NCX activity to protect CPC-derived cardiomyocytes from A/R-induced ER stress and cell death.Levosimendan, a known calcium sensitizer with positive inotropic and vasodilating properties, might also be cardioprotective during ischemia-reperfusion (I/R) insult. Its effects on calcium homeostasis and apoptosis in I/R injury remain unclear. Na Mechanisms mediating reperfusion injury include altered metabolism and Ca2+ concentration ([Ca2+]i) in cells subject to I/R plays a pivotal role in subsequent cellular damage: e.g., hypercontracture as a direct result of excessive [Ca2+]i. Along with other physiological derangements, excessive [Ca2+]i causes arrhythmia, myocardial stunning, and cell death by necrosis or apoptosis. In cardiomyocytes, excitation-contraction coupling is mediated by cyclic changes of [Ca2+]i, which in turn is regulated by several Ca2+ channels. The Na+/Ca2+ exchanger (NCX) on plasma membrane, 10–15 times more efficient than Ca2+-ATPase, is the major channel for Ca2+ extrusion during diastole normally + or Ca2+ gradients across plasma membrane, NCX functions in either forward mode (extracellular Na+ in exchange for intracellular Ca2+) as Ca2+ efflux mechanism in physiological conditions, or reverse mode (extracellular Ca2+ in exchange for intracellular Na+). NCX working in reverse mode is responsible for elevated [Ca2+]i that occurs during ischemia and early reperfusion +/K+ pump, main Na+ extrusion pathway, comes to a halt, leading to accumulation of Na+ in cytosol. Higher intracellular Na+ concentration in turn reverses direction of action of plasma membrane NCX, causing Ca2+ influx. In early reperfusion, as extracellular H+ is washed away by re-established blood flow, Na+ influx through Na+/H+ exchanger aggravates Na+ and again through the action of reverse mode NCX, Ca2+ overload.Cardinal feature of higher cytosolic Ca2+-sensitizing inotrope + channels.in vitro, plus involvement of NCX activity and ERS-induced apoptosis pathway in the mechanism. Among isoforms of NCX, ubiquitous NCX1 is highly expressed in the heart Ischemia induces accumulation of misfolded proteins in endoplasmic reticulum (ER) as well as endoplasmic reticulum stress (ERS) response This research was approved by China Medical University Hospital Institutional Review Board, written informed consent obtained from each patient’s guardian or caretaker. Human cardiomyocyte progenitor cells were isolated from right atrial auricle specimens of individuals undergoing cardiac surgery, which has been a frequently adopted method for isolation of human cardiac progenitor cells 2 and 5% CO2 for 2 or 14 hours in culture medium deprived of glucose and serum, with glucose-free DMEM (Gibco-BRC) supplemented with 100 U/ml penicillin and 100 ug/ml streptomycin. This anoxic period was followed by reoxygenation by transferring cells to a normoxic incubator in 95% air/5% CO2 for 2 hours in normal culture medium. Levosimendan 2 uM was added at the beginning of reoxygenation for the experimental group, experimental procedures illustrated in To simulate I/R, CPC-derived cardiomycytes were incubated in a hypoxic chamber (Biosphenix) filled with 95% NThe method to generate antibodies against NCX1 was described in our published report 4 cells/dish), incubated with 10 uM fura-2 acetoxymethyl ester (AM) at 37°C for 2 h, then loaded with Na+ by immersion in Ca2+-free Na+ loading buffer  for 10 min at 37°C. Isolated CPC-derived cardiomyocytes were selected to measure reverse-mode NCX1 activity by abruptly replacing Ca2+-free buffer with Na+-free buffer . Fura-2 fluorescence ratio obtained by 340 nm and 380 nm excitation (F340/F380) was monitored by inverted microscope  using a 40x oil immersion objective . Amplitude of [Ca2+]i rise upon replacement of buffers was then calculated as a measurement of reverse-mode NCX activity Three pieces of 12 mm cover-glass were put in a 3.5 cm dish. Human CPC-derived CMs were cultured on gelatin-coated 12 mm cover-glass  were analyzed by FACS (FL-3 channel). Ratio of apoptotic cells was detected by DiOC6 fluorescence, results obtained for both experimental and control groups.Apoptotic cells were quantified by propidium iodide (PI) and DiOC6 staining with FACS analysis Human CPC-derived CMs grown on cover slips were rinsed three times with PBS and fixed with 3.7% formaldehyde in PBS for 20 min at room temperature. Samples stained with appropriate primary antibodies were incubated with FITC-conjugated goat anti-mouse antiserum at 4°C overnight, cover slips mounted with Vectashield  and viewed under inverted confocal microscope  with 63×/1.4 Plan-Apochromat objective.CPC-derived cardiomyocytes were transiently transfected with NCX siRNA  using HiPerFect Transfection Reagent  according to the manufacturer’s instructions. Meanwhile, cells were transfected with scrambled siRNA as control; these were used for experiments after transfection for 48 hours.6 cells) were harvested, washed with PBS, and then centrifuged at 500×g for 5 min. Cell pellet was homogenized in 200 ul of buffer C plus protease inhibitors, rotated at 4°C for 20 min, then centrifuged at 4°C, 12000×g for 20 min. Cytoplasmic proteins in the supernatant were collected, pellet washed with 400 ul of buffer W and resuspended in 100 ul of buffer N at 4°C for 20 min. Nuclear proteins were extracted and recovered in the supernatant after centrifuge at 4°C, 12000×g for 20 min. Finally, to obtain membrane proteins, the cell pellet containing cell debris was extracted with 100 ul of buffer M and rotated at 4°C for 20 min. Supernatant was centrifuged at 4°C, 12000×g for 20 min. Membrane proteins in supernatant were collected, samples from these fractions denatured and subjected to SDS-PAGE.Cytoplasmic and membrane fractions of human CPC-derived cardiomycytes were prepared with a CNM compartment protein extraction kit  according to manufacturer's instructions. Briefly, transfected cells , as per manufacturer’s instructions. Proteins (30 ug) were run on a 10% SDS-PAGE gel, transferred onto PVDF membranes, and blocked. Membranes were incubated with primary antibodies: anti-NCX (1∶50000.), anti-GRP94 , anti-GRP78 , anti-caspase-12 , anti-GAPDH , anti-pan-cadherin  and anti-β-actin  overnight at 4°C. Membranes were extensively washed and incubated with horseradish peroxidase-labeled immunoglobulin G , immunoreactive bands detected by chemiluminescence methods (Millipore) and visualized on X-ray films . Densitometric analysis was performed by Image J software  p<0.05 was considered statistically significant.Statistical analyses used SPSS version 13.0 software . Data were presented as mean ± standard deviation (S.D.), intergroup comparisons performed with Student's t-test or one-way ANOVA followed by Tukey’s post-hoc test. Effect of levosimendan on apoptosis of CPC-derived cardiomyocytes subjected to A/R was tested, with hydrogen peroxide at 10 mM as positive control. Adding levosimendan at reoxygenation reduced apoptotic CPC-derived cardiomyocytes from 27.6±2.4% to 8.2±2.1% (P<0.05) .2+ transported into cells than controls , and attendant deleterious effects can only be reduced by keeping the duration of ischemia as short as possible. The start of reperfusion is more or less under the control of clinicians, but there is no effective countermeasure against reperfusion injury. Reperfusion induced cardiomyocyte death has become a significant limiting factor to the achievable result in treating patients with myocardial infarction Several groups have isolated cardiac stem cells relying on expression of certain markers  or particular biological behavior of cells (Hoechst dye efflux or formation of cardiosphere) 2+ influx during I/R. Inhibition of reverse mode NCX activity, with reduced cellular Ca2+ accumulation, proves cardioprotective and reduces infarct size 2+ overload during the initial phase of reoxygenation and ensuing changes causing cell death. Addition of levosimendan during reoxygenation period abolished this heightened NCX activity and reduced [Ca2+]i accumulation, ERS response, and cellular apoptosis. Translocation of NCX away from plasma membrane of CPC-derived cardiomyocytes with leveosimendan present may partly explain decreased NCX activity. The mechanism of levosimendan-induced NCX translocation is not clear and merits further investigation.NCX, working in reverse mode, is the main channel for Ca+]i, with ensuant water influx and cell swelling. Reoxygenation aggravates cellular swelling due to rapid normalization (decrease) of extracellular osmolarity and pH (which intensifies intracellular Na+ overload through the action of Na+/H+ exchanger). Cells after anoxia-reoxygenation appeared larger than controls in our experiments. Lighter-stained nuclei might result from dilutional effect, as these cells were swollen. Levosimendan will contribute to [Na+]i accrual in reoxygenation, as it inhibits outward Na+ transport via reverse mode NCX activity.Intracellular osmolarity rises in anoxic period due to accumulated anaerobic metabolic product and [Na2+]i in cellular A/R plays a crucial role in occurrence of ERS, and NCX activity was responsible for this increase in [Ca2+]i. Concomitant to this ERS response alleviation, there was also reduced cardiomycyte apoptosis, as ascertained by FACS analysis. In short, results suggest levosimendan inhibiting reverse-mode NCX activity to protect CPC-derived cardiomyocytes from A/R-induced ER stress and cell death. Further studies must confirm this result to enhance understanding of I/R injury mechanisms.Intense ERS obviously emanates from A/R injury, with its attendant deprivation of substrate and energy source and perturbation of protein synthesis process. Greater expression of GRP78, GRP94 and activation of caspase 12 in results clearly indicated this reaction. Surprisingly, levosimendan could return GRP78, GRP94 expression and caspase 12 activation to control levels, a mitigation of ERS response. NCX knockdown by siRNA could achieve a similar effect. Adding levosimendan to NCX knockdown yielded no synergistic effects. This indicated increased [Ca


BIRC6 is associated with unfavorable clinical features and negatively impacts relapse-free survival in childhood acute myeloid leukemia (AML). Currently, BIRC6 levels in adult primary AML have not been compared to the expression in normal myeloid cells. Thus, we compared for the first time BIRC6 levels in adult primary AML patient samples to normal myeloid cells and studied its regulation and function during neutrophil differentiation.Inhibitors of apoptosis (IAPs) were intensively investigated in the context of cancer where they promote tumor growth and chemoresistence. Overexpression of the IAP BIRC6 levels in particular AML subtypes as compared to granulocytes from healthy donors. The lowest BIRC6 expression was found in CD34+ progenitor cells. Moreover, BIRC6 expression significantly increased during neutrophil differentiation of AML cell lines and knocking down BIRC6 in NB4 acute promyelocytic leukemia (APL) cells significantly impaired neutrophil differentiation, but not cell viability.We found significantly lower BIRC6 levels with an immature myeloid phenotype and describe a function for BIRC6 in neutrophil differentiation of APL cells.Together, we found an association of low  BIRC6  is an exceptionally large protein of 528 kDa belonging to the family of inhibitor of apoptosis (IAP). BIRC6 contains one bacalovirus IAP repeat (BIR) domain that shows homology to the IAP Survivin. Furthermore, BIRC6 is the only IAP with an ubiquitin-conjugating domain further pointing to a particular function of this protein in the IAP family [BIRC6 executes its function via inhibition of Smac and Caspase-9[BIRC6 inhibits cell death by p53 destabilization and inactivation of caspase-3[p53-dependent BIRC6 effector functions was confirmed by investigations of Ren et al. in mice and human lung cancer cells [P family . SeveralCaspase-9. Moreovecaspase-3. The rolSurvivin, for example, one of the best studied IAPs is up regulated in most tumors and has been associated with their chemoresistence [BIRC6 overexpression in colon cancer stem cells when compared to more differentiated tumor cells. BIRC6 expression protected colon cancer stem cells from the cytotoxic effects of oxaliplatin and cisplatin. Furthermore, knocking-down BIRC6 led to growth inhibition in several cancer cell lines and xenografted mice and rendered the tumor cells more sensitive to 5-fluoruracil treatment in vivo and in vitro[Survivin or XIAP expression in adult AML samples, whereas BIRC6 overexpression is associated with unfavorable clinical features and negatively impacts on the 3-year relapse-free survival in childhood acute myeloid leukemia (AML) [de novo AML samples.Due to their anti-apoptotic function it was hypothesized that overexpression of IAPs might contribute to tumorigenesis. sistence ,7. Recensistence  observed in vitro. The sig in vitro did not ia (AML) . Similaria (AML) . Abe et ia (AML)  found loBIRC6 levels in primary AML have never been compared to the respective expression in normal myeloid cells. Thus, we aimed at comparing BIRC6 levels in a large cohort (n = 98) of adult AML patient samples and mature neutrophils from healthy donors. Since a hallmark of AML is a differentiation block of hematopoietic precursors at different developmental stages and since this block can be overcome by treating acute promylocytic leukemia (APL) patients with all-trans retinoic acid (ATRA) and low dose chemotherapy, we also analyzed the role of BIRC6 during neutrophil differentiation of AML cells.To our knowledge, http://www.hovon.nl) between 1987 and 2006 [In vitro differentiation of CD34+ progenitor cells was done as previously described [A cohort of 98 samples from patients with a diagnosis of primary AML (FAB M0-M4) were enrolled on HOVON/SAKK  protocols −04, -04A, -29, and −42 (available at and 2006 -18). Allall-trans retinoic acid (ATRA) was added to the cells that were seeded at a density of 0.2x106 cells/ml. Successful neutrophil differentiation was assessed by CD11b surface and by increased granulocyte colony-stimulating factor receptor  mRNA expression.The acute myeloid leukemia (AML) cell lines HL60, FAB M2, the ATRA-resistant subline HL60-R, NB4, FAB M3 , the ATRA-resistant subline NB4-R2 and HT93 were kept in RPIMI-1640 culture media  containing 10 % foetal bovine serum (FBS). In order to differentiate the AML cell lines towards granulocytes 1 μM BIRC6 and G-CSFR mRNA was performed using the TaqMan® Gene Expression assays Hs00212288_m1 and Hs00167918_m1, respectively . LDA measurements as well as data analysis were done as described [HMBS primers and probes have been described previously [For RNA extraction the miRCURY RNA Isolation Kit from Exiqon was used. RT-PCR has been described elsewhere . Quantitescribed . HMBS prBIRC6 mRNA . Lentivirus production has been described previously [NB4 cells were transduced with a lentiviral vector (pLKO.1) expressing a small hairpin (sh)RNA targeting the eviously . As a no5 cells were washed in 500 μl binding buffer (PBS with 0.33 g/L Ca2+) and resuspended in a final volume of 100 μl. 5 μl AnnexinV-PE were added  and the samples were incubated for 15 minutes at room temperature, before FACS analysis.For Trypan blue exclusion assay cells were washed with PBS before diluting 1:10 in 0.4 % Trypan blue solution . For AnnexinV staining 1x10Differences between two groups were assessed using the non-parametric Mann–Whitney-U test. P-values <0.05 were considered to be statistically significant.BIRC6 expression patterns in normal versus leukemic myeloid cells, we measured BIRC6 levels in a large cohort of 98 primary AML patients (FAB M0-M4), in 24 granulocyte preparations from healthy donors and in 3 CD34+ progenitor cell samples. We were able to detect BIRC6 in 95/98 AML patient, in 14/24 granulocytes and in 3/3 CD34+ progenitor cell samples. Surprisingly, we found significantly lower BIRC6 levels in AML patients with the translocations t and t as well as in AML patients with a complex karyotype, whereas no significant differences in BIRC6 expression was found in AML patients with inv(16) or normal karyotype as compared to its expression levels in granulocytes from healthy donors . In a further experiment, we determined BIRC6 expression in HL60 and ATRA-resistant HL60-R AML cells during neutrophil differentiation. We observed a significant 1.6- and 2.8-fold increase of BIRC6 transcript expression at day 4 and 6, respectively. No significant change in BIRC6 expression was seen in HL60-R cells upon 4 days of ATRA treatment, whereas at day 6 a minor but significant increase was seen .In order to assess a possible role of y Figure A. In ordBIRC6 expression with neutrophil development of AML and CD34+ progenitor cells.Overall, our findings clearly show an association of increased BIRC6 depletion inhibits neutrophil differentiation of APL cells, we generated NB4 BIRC6 knockdown cells. BIRC6 knockdown efficiency was confirmed in control and ATRA-treated shBIRC6 expressing NB4 cells , the t or a complex karyotype express BIRC6 significantly lower than normal human granulocytes. Subtype-specific BIRC6 expression in AML is supported by earlier findings published by Ismail et al. . Low exp samples . Furtherid cells . Consistas found . In cont-subtype . This maBIRC6 mRNA expression with neutrophil differentiation and inhibiting BIRC6 resulted in attenuated neutrophil differentiation but did not alter cell survival. In summary, we established a new role for BIRC6 in neutrophil differentiation of AML cells.In conclusion, we were able to link increased IAP, Inhibitor of apoptosis; AML, Acute myeloid leukemia; APL, Acute promyelocytic leukemia; ATRA, All-trans retinoic acid; BIR, Bacalovirus IAP repeat; ALL, Acute lymphoblastic leukemia.No competing interest to be declared.AS performed the experimental research, interpreted the data and drafted the article. BET provided primary cells and essential reagents, analyzed patient data and revised the article. MFF investigated the initial concept and experimental design and revised the drafted article. MPT designed the project and gave final approval of the submitted manuscript. All authors read and approved the final manuscript.


Consistent with a role of Ca2+-permeable plasma membrane channels of the TRPC family, the inhibitor SKF96365 prevented the BDNF-induced increase in mEPSC frequency. Furthermore, labeling presynaptic terminals with amphipathic styryl dyes and then monitoring their post-BDNF destaining in slice cultures by multiphoton excitation microscopy revealed that the increase in frequency of mEPSCs reflects vesicular fusion events. Indeed, BDNF application to CA3-CA1 synapses in TTX rapidly enhanced FM1-43 or FM2-10 destaining with a time course that paralleled the phase of increased mEPSC frequency. We conclude that BDNF increases mEPSC frequency by boosting vesicular fusion through a presynaptic, Ca2+-dependent mechanism involving TrkB receptors, Ca2+ stores, and TRPC channels.Brain-derived neurotrophic factor (BDNF) is well known as a survival factor during brain development as well as a regulator of adult synaptic plasticity. One potential mechanism to initiate BDNF actions is through its modulation of quantal presynaptic transmitter release. In response to local BDNF application to CA1 pyramidal neurons, the frequency of miniature excitatory postsynaptic currents (mEPSC) increased significantly within 30 seconds; mEPSC amplitude and kinetics were unchanged. This effect was mediated via TrkB receptor activation and required both full intracellular Ca Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic factor family and is well known as a survival factor and chemoattractant during development of the central nervous system . In receAcute application of BDNF enhances spontaneous and evoked glutamatergic excitatory postsynaptic currents in cultured neurons –17. Longγ activation leading to Ca2+ mobilization from IP3-sensitive stores [2+ influx following Ca2+ store depletion may contribute to spontaneous vesicular release [2+ release from intracellular stores only [2+ mobilization from intracellular stores, a signal further amplified by Ca2+ entry through TRPC plasma membrane channels. Additionally, BDNF causes immediate FM dye destaining in the absence of action potentials, directly proving its presynaptic effect and the vesicular origin of postsynaptic quantal responses. The rate of BDNF-induced destaining was similar for two FM dyes with different membrane departition rates, suggesting that BDNF increases mEPSC frequency by either full-fusion events or through fusion pores with limited FM dye permeability.Two potential mechanisms mediating the BDNF-induced increase in transmitter release include synapsin-I phosphorylation  and TrkBe stores . Previou release . Howeverres only . We hereμm thick slices using a custom-made wire slicer fitted with 20 μm thick gold-coated platinum wire [2, 98% relative humidity incubators . Slices were maintained in culture media , containing 20% equine serum for the first 4 days in vitro (div). To avoid the confounding effects of hormones and growth factors in the serum, its concentration was gradually reduced over a period of 48 h starting at 4 div (24 h each in 10 and then 5% serum). After a period of 24 h in serum-free media , 7–10 div slices were used for electrophysiology [All animal procedures strictly adhered to national and international guidelines for the ethical use of research animals. The Institutional Animal Care and Use Committee (IACUC) of The University of Alabama at Birmingham (UAB) reviews and approves all animal procedures described in the present paper on an annual basis. Briefly, hippocampi were dissected from anesthetized postnatal days 7–11 Sprague-Dawley rats  and cut transversely into 400 num wire . Hippocaysiology .2PO4, 1.3 MgSO4, 17.6 NaHCO3, 2.5 CaCl2, 10 glucose, and 29.2 sucrose (310–320 mOsm). aCSF was bubbled with 95% O2/5% CO2, pH 7.4. Superficial CA1 pyramidal neurons were visualized with a water-immersion 63x objective ) using infrared differential interference contrast (IR-DIC) microscopy. Whole-cell intracellular recordings were performed as previously described [Individual 7–10 div slices were transferred to a recording chamber mounted on a fixed-stage upright microscope  and continuously perfused (2mL/min) with artificial CSF (aCSF) at room temperature 24°C) containing the following (in mM): 124 NaCl, 2 KCl, 1.24 KH°C contaiHuman recombinant mature BDNF  was pressure applied to hippocampal slices from glass pipettes (~5 MΩ) using a Picospritzer III  as described , 22.+ aCSF containing 0.5 μM TTX and either 15 μM FM1-43 or 100 μM FM2-10; the solution had been prewarmed to 37°C and was under constant aeration (95% O2/5% CO2) throughout the 15 min incubation. Individual slices were then placed in standard aCSF containing 0.5 μM TTX and either 5 μM FM1-43 or 100 μM FM2-10 for 3 minutes, then in dye-free, nominally zero Ca2+ aCSF containing 0.5 μM TTX for a 30-minute washout. Imaging was conducted in standard aCSF containing 0.5 μM TTX, and FM dye fluorescence intensity was monitored during pressure application of BDNF from glass pipettes (~5 MΩ) using a Picospritzer-III .Hippocampal slices, maintained in serum-free medium for 10 to 12 days, were labeled with either FM1-43 or FM2-10 dyes. The total recycling pool of vesicles was loaded via endocytosis by incubating slices in 40 mM K FM dye fluorescence images were collected with a 60x, 0.9 NA water-immersion objective , using a custom-built multiphoton excitation laser scanning microscope consisting of an upright BX50WI microscope and a modified FV300 scanhead (Olympus). A Ti-sapphire laser pumped by a 12 W diode Ar laser  was tuned to 840 nm center wavelength. Infrared laser intensity was controlled by an external Pockels cell  before entering the scanhead. Infrared-filtered FM dye fluorescence emission was detected by a GaASP photomultiplier (PMT)  in nondescanned mode. The lowest intensity necessary for adequate signal-to-noise ratio was used to avoid photodamage and FM dye bleaching. Average power in the back focal plane of the objective never exceeded 50 mW. IR-filtered FM dye fluorescence was detected by laser-scanning microscopy and time-lapse image acquisitions were controlled by FluoView-Tiempo software (Olympus).μm) were acquired every 1.1 second. All image fields were obtained from within ~200 μm of the slice surface, typically ~25–150 μm deep. Ten image frames were acquired before stimulation and used to determine baseline fluorescence (Fb). Once stimulation was delivered, FM dye emission intensity was measured, and changes were expressed as ΔF/Fb. FM dye bleaching was measured by image sequences of similar duration but without stimulation and was less than 5% over the 30-minute long movies. FM dye fluorescence intensity was measured from visually identified puncta or clusters of several puncta that could be individually tracked in a single z-plane  section over time. The fluorescence intensity of each of these regions-of-interest (ROI) was individually measured, normalized, and plotted over time, after background and bleaching subtraction [μm (seven pixels) were used to select individual puncta and ensure consistent analysis over time and regions in a slice. Data from experiments where lateral displacement (x-y) or focal drift in the z-axis of FM dye puncta occurred, were discarded. To avoid imaging nonselective FM staining, only puncta that showed stimulus-dependent destaining were included in the analyses.Full-frame images . P < 0.05 was considered significant. Data are presented as mean ± SEM.Data were analyzed using paired Student's μm above hippocampal slice cultures, a configuration that prevents pressure and mechanical artifacts [Long-term (minutes to hours) exposure to BDNF induces varied effects on hippocampal neurons, ranging from modulation of synaptic transmission and plasticity to structural changes of dendrites, spines, and presynaptic terminals , 6, 30. rtifacts .stratum radiatum, 200 μm away from their cell bodies, to reproduce the spatiotemporal profile of a paracrine neuropeptide released from dense-core vesicles acting on perisynaptic receptors [n = 3, P = 0.0262)   . The inwn = 3, P > 0.05) .The amplitude of individual mEPSCs before and after BDNF exposure was not statistically different  . Similarn = 3, P = 0.51; median: 2.6 Hz versus 3.3 Hz; Kolmogorov-Smirnov Z value: 1.9 ± 1.3) , which was a 6.77 fold increase over the baseline . The persistence of such response—albeit attenuated—reflects the contribution of presynaptic Trk receptors (which are not inhibited in k-252b-loaded neurons) to the overall effect measured under control conditions. The fact that intracellular k-252b application reduced the amplitude and duration of the BDNF effect on mEPSC frequency suggests the intriguing possibility that postsynaptic Trk receptors signal retrogradely to modulate presynaptic transmitter release. Alternatively, k-252b could simply leak out of the postsynaptic cell to act on nearby presynaptic terminals. Both of these pharmacological agents were used at a concentration of 200 nM in 0.01% DMSO, which is specific for receptor tyrosine kinases of the trk gene family [The BDNF-induced increase in mEPSC frequency was mediated by Trk receptors, as the effect did not occur after bath application of k-252a, an inhibitor of tyrosine kinases  (mean: 29 ± 1.3) . On the baseline . The BDNe family . Vehicle2+ and BDNF signaling engages intracellular Ca2+ signaling, we next examined whether the BDNF effect on mEPSC frequency was sensitive to manipulations of Ca2+ levels. Indeed, BDNF failed to increase the mean mEPSC frequency in the absence of extracellular Ca2+  , demonstrating that influx of extracellular Ca2+ is required for the effect of BDNF on mEPSC frequency.Since the frequency of asynchronous synaptic transmission is sensitive to changes in Ca 0.7523) . The med2+ stores were depleted by bath application of the SERCA pump inhibitor thapsigargin (1 μM) [n = 3) was significantly smaller than that observed under control conditions (P < 0.05). The median mEPSC frequency changed from 1.1 Hz to 3.7 Hz   . Under t6 ± 2.6) . The temn = 5, P = 0.0196)  . The med2+ in presynaptic terminals mediated by both Ca2+ influx and mobilization of intracellular stores is necessary for BDNF to increase mEPSC frequency.Taken together, these results suggest that an elevation of intracellular Ca2+ influx and intracellular Ca2+ stores suggested the involvement of TRPC channels, which are activated by the PLC cascade and mediated the activation of postsynaptic currents and dendritic Ca2+ signals by BDNF in CA1 and CA3 pyramidal neurons [n = 3, P = 0.4256) .The requirement of both Ca neurons , 22, 35. neurons  complete 0.4256) . The med+) containing 5 μM FM1-43 (2+ solution (to prevent FM1-43 destaining by spontaneous vesicle fusions), slices were imaged by multiphoton excitation microscopy (840 nm excitation) in Ca2+-containing aCSF with TTX to block Na+-dependent action potentials. BDNF was applied to apical dendrites in area CA1 using a Picospritzer in the same manner as for whole-cell recordings of mEPSCs, and FM1-43 fluorescence intensity was followed over time . n = 6 slices). These observations were confirmed with FM2-10, a styryl lipophilic dye with lower membrane-binding affinity (n = 4 slices), which was not significantly different that that observed using FM1-43 (P > 0.05). These results show that BDNF induces FM dye loss by a vesicular fusion mechanism that is independent of the membrane affinity and molecular size of FM1-43 and FM2-10 dyes. Furthermore, these imaging results directly demonstrate that the elevated mEPSC frequency caused by BDNF representS vesicular transmitter release from presynaptic terminals.To directly demonstrate that the elevated mEPSC frequency evoked by BDNF represents vesicular transmitter release, we performed multiphoton imaging of FM dyes in slice cultures. The total pool of recycling synaptic vesicles was labeled by exposing slices to a depolarizing solution  coaffinity . The exp2+-dependent vesicle release from presynaptic terminals. The BDNF-induced increase in mEPSC frequency is prevented by bath application of the tyrosine kinase inhibitor k-252a. Depletion of intracellular Ca2+ stores with thapsigargin also inhibited the BDNF-induced mEPSC frequency increase, as did the removal of extracellular Ca2+. These results demonstrate the critical requirement of TrkB-initiated Ca2+-dependent signaling in the BDNF-induced increase in mEPSC frequency.The present study demonstrates that the BDNF-induced increase in mEPSC frequency recorded in hippocampal CA1 pyramidal neurons represents Ca2 by PLCγ leading to IP3 formation, has long been known to cause intracellular Ca2+ mobilization in neurons [2+ levels within presynaptic terminals of Xenopus neuromuscular junctions in culture, leading to a transient enhancement of transmitter release that was dependent on extracellular Ca2+ levels and presynaptic depolarization [2+ was required for BDNF to increase the frequency of mEPSCs and somatic Ca2+ levels in cultured hippocampal neurons [2+ influx, Ca2+ signals mediated by depletion of intracellular Ca2+ stores are fundamental component of neuronal Ca2+ signaling. In non-excitable cells, the depletion of Ca2+ stores activates plasma membrane channels often called store-operated channels (SOCs), allowing Ca2+ entry for the replenishment of emptied stores; this process is called capacitative Ca2+ entry [2+ entry [2+ signals in CA1 and CA3 pyramidal neurons through the activation of TrkB/PLC and mediated by TRPC3-containing channels [One of the signaling cascades activated by Trk receptors, the hydrolysis of PIP neurons . Indeed,rization , 39. Sim neurons . In addi2+ entry , 42. Rec2+ entry , 43, 44.channels , 22, 35.2+ that contributes to the BDNF-induced increase in mEPSC frequency originates from presynaptic ER stores. The contribution of intracellular ER stores as a source of Ca2+ to asynchronous (spontaneous) quantal transmitter release has been a highly debated issue. Although some reports demonstrate that store depletion in hippocampal pyramidal neurons leads to Ca2+-induced Ca2+ release (CICR) and subsequent paired-pulse facilitation of excitatory postsynaptic potentials [Here, we present evidence consistent with the model that the source of Catentials , other rtentials . These stentials . Still otentials .Our studies have directly demonstrated that the elevated frequency of mEPSCs evoked by brief and local BDNF application indeed reflects vesicular fusion events from presynaptic terminals. We also attempted to determine whether the vesicular fusions underlying FM1-43 loss from terminals represented kiss-and-run or full-fusion events by comparing them to FM2-10 loss, which has a slower departition rate from lipid membranes , 48. How2+-dependent mechanism involving TrkB receptors, Ca2+ stores, and TRPC channels.Taken together, these results demonstrate that BDNF increases mEPSC frequency by enhancing vesicular fusion through a presynaptic Ca


We demonstrate that all the stimuli induce ATP release from model human mast HMC-1 cells, and this release is associated with an intracellular free Ca2+ rise. We hypothesize that ATP released from mast cells supplements the already known release of ATP from keratinocytes and, by acting on P2X receptors, it may serve as initial mediator of acupuncture-induced analgesia.In Chinese medicine acupuncture points are treated by physical stimuli to counteract various diseases. These stimuli include mechanical stress as applied during the needle manipulation or tuina, high temperatures as applied during moxibustion, and red laser light applied during laser acupuncture. This study aimed to investigate cellular responses to stimuli that might occur in the tissue of acupuncture points. Since they have a characteristically high density of mast cells that degranulate in response to acupuncture, we asked whether these processes lead to ATP release. We tested in  The treatment of specific points on the body surface by physical stimuli has been shown to affect various body functions including pain sensation , 2 and tMast cells (MCs) play a significant role in the pathophysiology of many diseases including asthma and allergies, pulmonary fibrosis, and rheumatoid arthritis . In addi2+ concentration ([Ca2+]i), and stimulation of other signaling pathways [2+]i elevation, suggesting the involvement of Ca2+-dependent exocytosis i measurements in the experiments with mechanical stimulation cells were loaded  with 10 μM Fura-2-AM in physiological solution containing 0.02% Pluronic F127 and 2.5 mM Probenecid, followed by 30 min deesterification in PS-containing Probenecid. For fluorescence imaging, a Fura-2-loaded cells were introduced into an imaging/perfusion chamber  attached to a heated platform  on the stage of an inverted microscope (Nikon TE300). A thin vacuum grease barrier was made at one end of the chamber, close to the perfusion outlet, to trap cells in the chamber and prevent their wash out during perfusion. The imaging chamber was perfused continuously with a warm solution (37°C) via an in-line heater  at ~0.5 mL/min. The cells were illuminated for 100 ms with alternate light wavelengths of 340 and 380 nm, using a high-pressure mercury lamp (100 W) via interference filters  mounted on a filter wheel  and a dichroic mirror . Fluorescence images were recorded at 15 s intervals with a digital camera and stored for later analysis. Fura-2 measurements are presented as the fluorescence F340/F380 ratio. To chelate intracellular Ca2+, cells were preloaded with 25 μM BAPTA-AM for 30 min at room temperature in PS. For [Ca2+]i measurements in the experiments with laser irradiation and heat application, [Ca2+]i measurements were performed as described previously i response showed a time course similar to the ATP release, that is, a peak of [Ca2+]i at 1.5–2 min after stress application followed by a decay phase , suggesting that Ca2+ mobilization from intracellular stores plays a dominant role in this response. For mechanical stimulation of HMC-1 cells the perfusion solution in the flow-through filter chamber was changed at ay phase . Similar2+ in hypotonic stress-induced ATP release, cells were pretreated with a Ca2+ chelator 25 μM BAPTA-AM before they were subjected to hypotonic shock. 2+-free solution ATP release was significantly diminished in BAPTA-loaded cells compared to controls . 2+]i response was almost completely abolished in these cells demonstrating a tight correlation between ATP release and [Ca2+]i elevations induced by the hypotonic stress . To test the role of intracellular CaP = 0.0299, n = 8). The concentration of released ATP for untreated control cells varied between different experiments from 4.5 nM to 8.7 nM. To investigate the effect of red laser light on ATP release, HMC-1 cells were exposed to laser light of 656.7 nm at 35 mW. For cells that had been exposed for 5 min to laser light, the amount of released ATP was higher than in the controls by 10.7 ± 4.0%  and 21.5 ± 1.1%  appeared when HMC-1 cells were exposed to irradiation for 1 min and 5 min, respectively. The presence of 5 mM EGTA partially prevented [Ca2+]i elevation induced by 1 min irradiation.P = 0.0253), and an even more significant increase to 49.5 ± 5.0 nM (P = 1.8 × 10−4) was found at 52°C. In order to exclude that this response was only due to cell lysis, experiments were performed in solution containing 100 μM NEM, an inhibitor of exocytosis [2+]i in HMC-1 cells. The relative fluorescent intensity increased by 4.5 ± 1.1%  and 7.3 ± 1.1%  at 42°C and 52°C, respectively.Similar to the mechanical and laser light stimuli, also heat could enhance [Ca2+ with nearly identical time course. While the intracellular [Ca2+]i returned to the level before stimulation, the amount of ATP release dropped to even lower concentrations than before stimulation indicating partial depletion of ATP stores during the period of elevated release. Neither the release of ATP nor the increase in [Ca2+]i was significantly affected if the experiments were performed in Ca2+-free medium; this observation suggests that the release of Ca2+ from intracellular stores might be involved in the process of ATP release. The hypothesis is supported by the finding that loading of the cells with the Ca2+ chelator BAPTA completely blocked the Ca2+ signal and strongly inhibited the ATP release. Similar observations were reported for hypotonic stress-induced ATP release from A549 cells, a model of human type 2 alveolar cells i. In contrast to hypo-osmotic stress, the 1 min irradiation-induced increase in [Ca2+]i was partially reduced when the cells were kept for 1 min in Ca2+-free medium indicating that also extracellular Ca2+ contributes to this process. This agrees with our previous work, which demonstrated that red laser irradiation activated TRPV2 channels allowing Ca2+ uptake from extracellular space [2+ homeostasis. Involvement of regulated exocytosis in the observed osmotic stress-induced and red laser light-induced ATP release is indicated by the accompanied increase of [Ca2+]i and strong inhibition of the release by NEM. Irradiation with red laser light also stimulated ATP release. This increase in ATP release was also accompanied by an increase in [Caar space . Basal Ain vitro experiments, further animal tests are needed for support. The physical stimuli applied in this investigation to the MCs are used in Chinese medicine to stimulate acupuncture points: mechanical stimulation during the needling, heat during moxibustion, and red light in laser acupuncture. Acupuncture and moxibustion have been demonstrated to be effective in analgesia . Recentl


Recent data indicate that NCKX4 plays a critical role in controlling normal Ca2+ signal dynamics in olfactory and other neurons. Synaptic Ca2+ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca2+ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca2+ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII . Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca2+ signalling by activating a signal damping and termination pathway.K +-dependent Na+/Ca2+-exchanger, NCKX4, is stimulated by purinergic signals that depend on dual activation of two protein kinase pathways. This regulation provides a novel mechanism to shape Ca2+ signaling and thus to have important impact on neuronal processes.Activity of the K Since Ca2+ cannot be metabolized, an increase in [Ca2+]i must be balanced by subsequent extrusion processes. In excitable cells where Ca2+ fluxes are high, this extrusion is predominantly mediated by Na+/Ca2+-exchangers, which have a high rate of transport i dynamics [2+ depending on the molecular sub-type of the receptor and the G protein it couples to: Gs/Gi/o or Gq/11 [2+]i, and a slower ‘off’ process that is associated with Ca2+ extrusion to ensure proper [Ca2+]i homoeostasis  cells, and found that NCKX4 activity can be stimulated by ATP through a P2Y-dependent signalling pathway. We demonstrated, using pharmacological interventions, that stimulation of NCKX4 requires the simultaneous activation of CaMKII  or EMD (emdcanada.com), and were analytical grade or higher, except where otherwise indicated. All molecular techniques were performed according to previously established protocols or manufacturer's instructions, unless otherwise specified.L-glutamine, 1% non-essential amino acids, and maintained in 5% (v/v) CO2 at 37°C. Transfection of Qiagen purified expression plasmid into HEK-293 cells was performed according to a previously published standard Ca2+-phosphate precipitation protocol [All cell culture reagents were obtained from Invitrogen (invitrogen.com). HEK-293 cells [HEK-293T)] were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS, 1% (v/v) protocol . Cells tMouse NCKX4 cDNA in the vector pcDNA3.1(+) was used for all experiments described here. This construct was based on GenBank accession AY156046, and included a 72 nucleotide 5′-end extension, as present in GenBank accession AK44368, encoding an additional 17 amino acids at the N-terminus of the NCXK4 protein. In addition, the current clone included a silent C for G replacement at amino acid residue 44, and lacked the exon corresponding to the alternatively spliced region (amino acids 275–293). Site-directed mutagenesis was performed to create T256A, T312A, S325A, T256A/T312A and T256A/T312A/S325A mutants using the QuikChange II Site-Directed Mutagenesis Kit from Agilent Technologies (agilent.com), according to the manufacturer's protocol. Each NCKX4 mutant was sequenced to ensure only the intended change was present in the construct.+  , an inhibitor of the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) , AMPK (AMP-activated protein kinase) and Akt (protein kinase B) families. None of these treatments, however, influenced ATP-induced stimulation of NCKX4 (results not shown).2+ entry to measure NCKX4 activity. This approach allows the isolation of NCKX activity from other plasma membrane or intracellular Ca2+ transport pathways. Since our data indicate an effect of stimulation on the maximal activity of the exchanger rather than individual ion-binding site affinities, it seems likely that purinergic stimulation will also influence the more physiologically relevant forward-mode, Ca2+ extrusion activity of the exchanger. Similar reasoning has been used when examining Ca2+-regulation of the cardiac exchanger, NCX1 [The experiments in this paper all used ionic manipulation to induce reverse-mode Caer, NCX1 .2+-mediated kinase pathways modulating the activity of plasma membrane ion channels. Much work has demonstrated important roles for both PKC and CaMKII in the regulation of activity and membrane insertion of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-receptor ion channels [2+ homoeostasis that also requires control of Ca2+ efflux pathways. Here we have demonstrated that NCKX4 activity, a principle component of Ca2+ extrusion in brain neurons, is stimulated only by dual activation of PKC and CaMKII pathways. Intriguingly, the close family member, NCKX2, is stimulated similarly, but required only activation of PKC [2+ homoeostasis depends on the cellular expression and subcellular localization of both transporter isoforms and regulatory kinase signalling pathways. The regulated activity of NCKX-mediated Ca2+ extrusion, integrated with Ca2+ entry pathways, is therefore critical to understanding molecular pathways of synaptic plasticity.Synaptic plasticity is crucially dependent on Cachannels –39. Howen of PKC . Thus, t2+-mediated synaptic plasticity, regulation of the AMPA-receptor offers a potentially interesting comparison. In this case, stimulation of ion channel activity is mediated largely by recruitment of AMPA-receptor channels from an intracellular storage compartment to the post-synaptic membrane. Redistribution in response to synaptic Ca2+ signals requires the synergistic action of both CaMKII and PKC [It remains unclear if NCKX4 stimulation corresponds to an allosteric increase in the activity of existing exchanger units in the plasma membrane, the recruitment of new units from a sub-membrane reservoir or both. In the context of Ca and PKC –42. In a and PKC ,43. ThesIn summary, our current study reveals a purinergic-dependent stimulation of NCKX4 that requires the simultaneous presence and activation of PKC and CaMKII. While the effect of stimulation can be reduced by mutating a putative PKC phosphorylation site T312, the specific mechanisms still require detailed investigation. At present, our data provide the first evidence of NCKX4 regulation in a physiologically relevant context, and open up the door to understanding the complex regulatory and physiological properties of this protein.