However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known.p,p′-DDE concentrations for 3 days at 37°C and 5% CO2in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p′-DDE incubation periods were also performed in order to monitor sperm rapid Ca2+ responses. All experiments were repeated on a minimum of five sperm samples from different individuals.Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several 2+ concentration ([Ca2+]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively.All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Cap,p′-DDE rapidly increased [Ca2+]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca2+ medium demonstrated that extracellular Ca2+ influx was responsible for this Ca2+ increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p′-DDE-induced [Ca2+]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p′-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p′-DDE concentrations, suggesting that [Ca2+]i rise may cause premature acrosome reaction (P < 0.05).in vitro study, and caution must be taken when extrapolating the results.This is an p,p′-DDE mechanism specific to sperm is shown in this study. p,p′-DDE was able to induce [Ca2+]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors.A novel non-genomic The study was supported by both the Portuguese National Science Foundation  and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare. A 99.1% chemically pure Fresh normozoospermic sperm samples from both human healthy donors recruited at the Biosciences School, University of Birmingham, and Medical Research Institute, University of Dundee (Ethics number 08/S1402); as well as healthy patients undergoing routine semen analysis or fertility treatments in the Human Reproduction Service at University Hospitals of Coimbra were used accordingly to the proper ethical and Internal Review Board of the participating Institutions. All individuals signed informed consent forms. Samples were obtained by masturbation after 3–5 days of sexual abstinence and seminal analysis was performed according to the World Health Organization guidelines . All sam2, 5.4 mM KCl, 0.81 mM MgSO4, 25.0 mM NaHCO3, 1.0 mM NaH2PO4, 116.4 mM NaCl, 5.5 mM d-glucose, 2.5 mM Na–pyruvate, 41.8 mM Na–lactate and 0.3% (w/v) BSA, for at least 3 h at 37°C under 5% CO2/95% air before starting imaging. [Ca2+]i measurements were performed after loading 4 million/ml sperm with the Ca2+ fluorescent marker Oregon Green BAPTA-1AM  for 1 h at 37°C under 5% CO2/95% air, as described elsewhere /Fbasal] × 100%, where ▵F is % change in intensity at time t, F is fluorescence intensity at time t and Fbasal is the mean basal F established in the beginning of each experiment before application of any stimulus. Each cell was considered to respond when the mean of 10 determinations of normalized F during the exposure period differed significantly from the mean of 10 determinations of normalized F during control (or inhibitor) treatment (P < 0.05). Mean amplitudes and percent responsive cells were calculated for each concentration in each sperm sample analyzed.After liquefaction, spermatozoa were prepared by direct swim-up and allowed to capacitate in a supplemented Earle's balanced salt solution (sEBSS) containing 1.8 mM CaClp,p′-DDE on [Ca2+]i, spermatozoa were exposed to a wide range of concentrations (1 pM–50 µM) diluted in standard sEBSS. To further assess the contribution of the internal Ca2+ stores, similar experiments were performed in a low-Ca2+ sEBSS medium . Finally, inhibition studies were performed using 30 µM mibefradil and 10 µM NNC 55-0396 . Solvent controls were carried out with 0.3% (v/v) DMSO.To evaluate the effect of +-based medium  was used, thus allowing proper recordings of CatSper monovalent currents. 5 µM p,p′-DDE was added at specific time points. All experiments were performed at 25°C.Cells were prepared by swim-up and capacitated as described by ® Sperm Separation Medium, Irvine Scientific, CA, USA) and allowed to capacitate for at least 3 h at 37°C under 5% CO2/95% air. Spermatozoa (10 million/ml) were then exposed to several p,p′-DDE concentrations  for 3 days at 37°C under 5% CO2/95% air in order to mimic the putative continuous exposure to toxicants in the female reproductive tract in vivo. Cells were maintained in a phosphate buffered saline  containing 0.9 mM CaCl2, 0.5 mM MgCl2, 5 mM d-glucose, 1.0 mM Na-pyruvate, 10.0 mM Na-lactate, 0.3% (w/v) BSA and 1% (v/v) penicillin/streptomycin, pH 7.2–7.4, according to our formerly described long-standing culture system (g. Solvent controls were performed by adding 0.3% (v/v) DMSO.After liquefaction, spermatozoa were isolated by density gradient centrifugation  as previously described . Two hunPisum Sativum agglutinin coupled to fluorescein isothiocyanate (PSA-FITC), as described elsewhere . Correlations were performed by the Spearman non-parametric test. Results are expressed as mean% ± SEM. P < 0.05 was considered significant.Statistical analysis was carried out using the SPSS version 19.0 software for Windows . All variables were checked for normal distribution and multiple comparisons were performed by paired in vivo conditions we determined that p,p′-DDE did not affect sperm viability, except after 3 days of continuous exposure to the highest p,p′-DDE concentration tested  caused a clear increase in Ca2+ levels within seconds of exposure (when compared with the control), reversible upon sEBSS media washout  caused an immediate decrease in sperm [Ca2+]i that stabilized at a new level within 3 min of exposure and remained unaltered when p,p′-DDE was added . When we analyzed the distribution of the single-cell response we found that 72.2 ± 14.7% of cells responded with an increase in fluorescence intensity of up to 20%, resembling the response observed in spermatozoa exposed from 1 pM to 10 µM p,p′-DDE in standard sEBSS medium. In all these experiments performed in a low-Ca2+ medium, when standard sEBSS was returned to the chamber [Ca2+]i levels increased as expected, and responded normally to the progesterone stimulus  to establish Ca2+-influx and after a delay of 2.5 min the inhibitors were added in separate experiments .10 µM NNC 55-0396 also reversed the p,p′-DDE on [Ca2+]i is mediated by Ca2+ influx and can be reversed by CatSper antagonistic drugs, suggesting that this DDT metabolite activates CatSper. To confirm this, we investigated the effect of 5 µM p,p′-DDE  without changing reversal potential or the characteristic outward rectification of the current , 25 and 50 µM p,p′-DDE significantly reduced acrosomal integrity after 2 days of exposure . This effect was further observed at Day 3 for both 25 and 10 µM p,p′-DDE (P < 0.05). No differences were observed at 1 µM p,p′-DDE in this 3-day long approach. Differences between concentrations were only found at day 3 for 1 and 25 µM p,p′-DDE  were involved, a pharmacological approach was used. In mouse and human sperm, CatSper is believed to be the principal plasma membrane Ca2+ channel  and affect sperm viability long before they reach the oocyte, thus adversely affecting male fertility. p,p′-DDE concentrations in follicular fluid have already been correlated with failed fertilization fertility.In this study, prolonged p,p′-DDE was able to induce a rise in [Ca2+]i in human sperm through a novel non-genomic mechanism involving the opening of the sperm-specific cation channel CatSper and consequently affected acrosome status and sperm survival, ultimately compromising male fertility.Even at concentrations found in reproductive fluids, Supplementary data are available at http://humrep.oxfordjournals.org/.J.R.S., R.S.T., S.J.P., S.M.W. and C.L.B. established the concept and design. R.S.T. and S.M. acquired data. R.S.T., S.J.P. and J.R.S. wrote the paper and all authors contributed to the analysis and interpretation of the results, drafting, revising and approving the article.the Portuguese National Science Foundation (FCT) through a grant attributed to the CNC Institution (PEst-C/SAU/LA0001/2011) and a Wellcome Trust Grant #86470 provided to S.J.P. and C.L.R.B. S.M. was supported by the Infertility Research Trust. R.S.T. is the recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). Funding to pay the Open Access publication charges for this article was provided by The Wellcome Trust.This work was supported by None declared.


Since LCRs are generated by Ca2+ release channel (i.e. ryanodine receptor) openings, they exhibit a degree of stochastic behavior, manifested as notable cycle-to-cycle variations in the time of their occurrence.Spontaneous, submembrane local CaThe present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells.We imaged single rabbit sinoatrial nodal cells using a 2D-camera to capture LCRs over the entire cell, and, in selected cells, simultaneously measured action potentials by perforated patch clamp.2+ transient, at about the time of the maximum diastolic potential. Shortly after the maximum diastolic potential , the ensemble of waxing LCR activity converts the decay of the global Ca2+ transient into a rise, resulting in a late, whole-cell diastolic Ca2+ elevation, accompanied by a notable acceleration in diastolic depolarization rate. On average, cells (n = 9) generate 13.2±3.7 LCRs per cycle (mean±SEM), varying in size (7.1±4.2 µm) and duration (44.2±27.1 ms), with both size and duration being greater for later-occurring LCRs. While the timing of each LCR occurrence also varies, the LCR period (i.e. the time from the preceding Ca2+ transient peak to an LCR’s subsequent occurrence) averaged for all LCRs in a given cycle closely predicts the time of occurrence of the next action potential, i.e. the cycle length.LCRs begin to occur on the descending part of the action potential-induced whole-cell CaIntrinsic cycle length variability in single sinoatrial nodal cells is linked to beat-to-beat variations in the average period of individual LCRs each cycle. A loss of this complexity is associated with diverse pathological conditions 2+ releases (dubbed “Ca2+ sparks”) via ryanodine receptors  2+ releases (LCRs) in various cell loci in the absence of APs, which are larger in size compared to sparks in ventricular myocytes and importantly, are roughly periodic in occurrence 2+ transient and en masse Ca2+ depletion of the SR 2+ by its ATPase, ‘SERCA’, generates the appearance of partially synchronized LCRs. These LCRs activate an inward diastolic Na+/Ca2+ exchanger (NCX) current In the absence of APs, the SR in cardiac ventricular cells generates spontaneous, stochastic local Caen masse RyR Ca2+ release (i.e. the Ca2+ transient (CaT) peak) and the subsequent appearance of a diastolic LCR in SANC is termed the ‘LCR period’. Since LCRs are generated by Ca2+ release channel (RyR) openings, individual LCRs exhibit a degree of stochasticity, manifested as notable cycle-to-cycle variations in their time of occurrence The time between the preceding AP-induced Single, spontaneously beating, spindle-shaped SANC were isolated from 8–12 week old male New Zealand White rabbit hearts as previously described The study conformed to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health. The experimental protocols were approved by the Animal Care and Use Committee of the National Institutes of Health (protocol # 034 LCS 2013). The rabbits weighed 1.8–2.5 kg and were deeply anaesthetized with sodium pentobarbital (50–90 mg/kg) injected to the central ear vein. The adequacy of anesthesia was monitored until reflexes to ear pinch and jaw tone were lost.2+ dynamics within isolated single SANC were measured by 2D imaging of fluorescence emitted by the Ca2+ indicator Fluo-4 using a high-speed Hamamatsu C9100-12 CCD camera . This was mounted on a Zeiss Axiovert 100 inverted microscope  with a x63 oil immersion lens and a fluorescence excitation light source  housing a 175W xenon lamp. The light from the lamp was transferred to the fluorescence microscope via a liquid light guide  to avoid vibrations from the light source. Fluo-4 fluorescence excitation  and emission light collection  were performed using the Zeiss filter set 38 HE. Cells were loaded with 1.5 µM Fluo-4AM (Sigma-Aldrich) for 10 minutes at room temperature. Fluo-4AM was subsequently washed out of the chamber, and Ca2+ signals were measured within the ensuing 60 minutes at 35°C ±0.1°C. To avoid phototoxicity, Fluo-4 was excited only for short periods of time . Data acquisition was performed using SimplePCI . Only cells with a spontaneous cycle length of less than 500 ms  were considered for further analysis. Movie S1 shows a representative high-speed camera recording of typical Ca2+ signal dynamics in SANC.Ca2+ buffering) and also by Fluo-4 excitation (via Fluo-4 degradation resulting in photo-damage), we performed control experiments to ensure that our chosen experimental protocol did not substantially alter the intrinsic spontaneous CL and CL variability (the subject of the study). An example of such an experiment performed in one cell is illustrated in Since SANC spontaneous beating rate and rhythm can be influenced by Fluo-4 loading (via its recognized effect on Cabefore cell loading with Fluo-4AMafter cell loading with Fluo-4AM in a low intensity transmitted red light (to prevent excitation and photo-damage by Fluo-4)after cell loading with Fluo-4AM when fluo-4 was excited by blue light of 470/40 nm2+ transients (when Fluo-4 was excited by blue light), the CL further slightly increased to 404.9±16.6 ms (P<0.005). However, the coefficient of variation of CL (defined as 100%×(CL standard deviation)/(CL mean)) before loading in red light, after loading in red light, and after loading in blue light did not significantly differ and was 5.29%, 5.24%, and 5.14%, respectively  to 392.0±13.8 ms (P<0.005). During measurements of Caectively . Thus, uIn longer recordings the sensitivity of cells to phototoxicity notably varied on a cell-to-cell basis. The effect of phototoxicity (i.e. increase in CL and CL variability) becomes notable on average after about 20 seconds of excitation of Fluo-4 (not shown). However, some cells were able to keep their 10 second moving average of CL and CL variability up to 40 seconds. On the other hand, in the worst case scenario, phototoxicity was clearly observed after about 12 seconds of excitation. We did not further examine the basis for different cell sensitivity to phototoxicity, but rather used all available data limited by 10 seconds of recording, which was sufficient to address the specific aims of our study.2+ signals. Membrane patch perforation was achieved using β-escin added to the patch pipette solution, as described elsewhere 2ATP (pH adjusted to 7.2 with NaOH). Axopatch 200B and pCLAMP software  were used for data acquisition. The liquid junction potential was corrected by tuning “the pipette offset” each time before touching the cell surface by setting the patch pipette current to zero, with the “command” voltage being set to “zero”. AP measurements were performed in a standard zero-current-clamp (fast mode) using a gap-free acquisition protocol, with a sampling interval of 0.1 ms. Ca2+ and AP recordings were synchronized by application of a short (20 ms) voltage pulse that was simultaneously recorded by the high-speed Hamamatsu camera (via an LED flash) and by pCLAMP (in a separate acquisition channel on the Digidata).A perforated patch-clamp technique was used in selected experiments to simultaneously measure APs and 2D Ca2+ and AP measurements were performed at 35°±0.1°C (500 µl chamber volume). Temperature was controlled by an Analog TC2BIP 2/3Ch bipolar temperature controller from CellMicroControls (USA). This heated both the glass bottom of the perfusion chamber and the solution entering the chamber (via a pre-heater). The physiological (bathing) solution contained (in mM): NaCl 140; KCl 5.4; MgCl2 2; HEPES 5; CaCl2 1.8; pH 7.3 (adjusted with NaOH).All Ca2+ waveforms were analyzed by a custom-made  program built with Delphi-7 developing software. DD rate was calculated as the difference between Maximum Diastolic Potential (MDP) and an AP threshold (set to −40 mV), divided by the time span required for the membrane to depolarize from the MDP to −40 mV. Ca2+ signal waveforms  were generated from movies (i.e. stacks of consecutive images) of Ca2+-dependent fluorescence of Fluo-4 by calculating the spatial average fluorescence of each image within a user defined ROI (Region Of Interest). ROIs were chosen to cover either the entire cell perimeter  or a specific region within SANC .Both APs and CaIn a comprehensive study involving hundreds of isolated SA node cells, Lyashkov et al. 2+ releases to maximize their visibility. 2+ signal was calculated. The cycle of interest was defined as the period of time from one peak Ca2+ transient to another. The frame with minimum fluorescence within that cycle was considered as the Ca2+ signal nadir (F0) and used further for normalization of all the other images in the stack. After normalization and contrasting maximum intensity (max F/F0) with a color scheme, the LCRs (events) could be observed as discrete, temporary and local increases in fluorescence intensity within the cell perimeter (2+ transient peak) - see 2+ transient; where this was the case, the LCR maximum amplitude was taken to be that reached at the end of the diastolic period (end of image stack).We used a semi-automated method to detect LCRs in a given spontaneous cycle using a custom-made image analysis program . erimeter . To sepaerimeter . We meas2+ signals exhibit two discrete phases within each spontaneous cycle , which appear relatively synchronously in geographically diverse locations within the cell, and begin to propagate locally. Some LCRs terminate and disappear, while others continue to grow in size, merging into the second (systolic) phase of Ca2+ release, observed as a strong, high-amplitude global cytosolic Ca2+ transient (CaT) induced by the occurrence of the subsequent AP. Thus, the diastolic LCR ensemble develops progressively until interrupted by the subsequent AP-induced CaT’s upstroke. In turn, as the CaT decays, a new cycle of diastolic LCR ensemble emerges once more, recapitulating the events of the preceding cycle.Ca2+ signal (black curve).We explored how LCR activity during diastole is linked to pacemaker APs via simultaneous perforated patch-clamp and 2D camera recording. An example of a typical simultaneous recording from a selected cell is shown in Movie S2 and 2+ release signal . In a representative short cycle dV/dt increase closely follows dF/dt increase (inset), reflecting diastolic Ca2+ release flux and its effect on membrane potential .To illustrate the timing of the onset of DD increase with respect to diastolic Caivatives : for mem2+ signal  of the short and long cycles in 2D images , shortly thereafter heralding LDCaE occurrence.It is important to note that the observed cycle-to-cycle variation in LCR synchronization results not only in variation in the prominence of LDCaE, but also in variation of the timing of LDCaE onset. In the example shown in 2+–dependent fluorescence nadir represents the impending onset of LDCaE that is associated with DD acceleration, we further explored the importance of this parameter with regards to beat-to-beat variations in DD rate and CL. m and Ca2+ fluorescence (left-hand column shows the raw data from these cells). It can easily be seen that the DD rate reproducibly predicts CL in all of these cells  in each of the individual cells); similarly DD rate is predicted by the ‘time-to-nadir’ (i.e. the time period between the occurrence of the prior peak Ca2+ transient and the following minimum (nadir) of the spatially averaged whole cell Ca2+ signal; statistical significance (p<0.05) for this relationship is present in only two of the five represented cells, though the numbers of cycles studied in the cells in which statistical significance is absent is relatively few).Since the timing of the global CaWe further explored the characteristics of the entire LCR ensemble and determined:the total number of LCRs per cycle2+ signal peakthe ‘LCR period’, i.e. the specific time of each LCR occurrence with respect to the prior global Ca‘LCR duration’, i.e. how long each LCR lasts for.descending part of the prior CaT, at about 50% of each spontaneous cycle duration (if measured from peak-to-peak of consecutive CaTs), i.e. relatively long before the subsequent CaT. 2+ signal  for 3 consecutive diastolic periods. The plot shows LCRs once more as ‘span lines’, delineating their duration from beginning to end, superimposed on the whole cell Ca2+ transient. 2+ transient .On average (n = 9 cells), under basal conditions, SANC generated 13.2±3.7 LCRs per cycle. Note that the distribution of LCR number per diastole is not Gaussian, but rather is skewed towards larger values . In cont2+ signal. Contrastingly, many late LCRs do not terminate as such, but rather continue to propagate until they are engulfed by the overwhelming effect of the on-rushing AP-induced CaT.About half of all LCRs 53%,  occur at2) and duration (44.2±27.1 ms)  within a given cycle predicts the length of that cycle (2+ nadir (2+ release (generated in diastole by LCRs) overcomes the general prior trend of decreasing, or ‘waning’, global cell Ca2+ signal due to fading of the prior AP-induced Ca2+ transient. In other words, the finding that the time at which the LCR ensemble Ca2+ release causes a nadir and initiates the LDCaE is related to cycle length represents further evidence that the timing of LCRs contributes to CL determination.The importance of waxing LCR activity in determining CL is also evidenced by the close and proportional relationship between CL and the timing of the global Ca2+ nadir . The nad2+ signals arising from the entire cell . Our results provide direct, novel evidence of the cycle-to-cycle variations in local cell Ca2+ regulation, and the importance of these Ca2+ variations in cycle-to-cycle length variation in isolated SANC. Specifically, using a fast 2D camera to capture the total cell Ca2+ dynamics , we have, for the first time, directly linked the characteristics of the entire ensemble of LCRs to DD rate and CL on a beat-to-beat basis.We have developed a new fast 2D imaging technique in SANC that allows for monitoring of local Ca2+ signals was substantially less than estimated from line scans in previous studies , so that the observed global late diastolic Ca2+ elevation and its importance have been logically associated with “late” LCR activity. Our study demonstrates, however, that many LCRs occur close to the MDP . While logically it is tempting to assign the role of early LCRs to that of priming the subspace for further stronger LCR activity, their exact role, mechanisms and importance of the discovered dynamics of LCR characteristics need to be further examined. With respect to late LCRs, these larger and longer LCRs would be expected to form a stronger integrated signal  is crucial with respect to understanding how their integration or ensemble behavior is coupled to the AP on a beat-to-beat basis. We observed that CL fluctuations are linked, in large part, to the mean time at which stochastic LCRs emerge in each cycle, ultimately forming the LDCaE, which in turn affects the DD rate . On the other hand, SR Ca2+ refilling depends upon the amount of Ca2+ available for pumping (i.e. Ca2+ influx via ICaL), along with the state of phosphorylation of Ca2+ cycling proteins, which may also have local and time-dependent variations. These complex (“coupled-clock”) systems 2+ control Previous studies from our group have demonstrated a crucial role for SR Cain vivo mandates further study. One approach that has been recently developed is to directly measure Ca2+ signals within individual cells of intact heart tissue 2+ elevation, LDCaE  Ca2+ signal previously measured in SAN cells and tissue. Intrinsic cycle length variability in single SANC is linked to beat-to-beat variations in the average period of individual LCRs each cycle. Thus, these new results together with our recent results using photo-flash-induced Ca2+ release 2+ regulation of cardiac pacemaker function on a beat-to-beat basis.This is the first study to quantify the fine spatiotemporal structure of CaFigure S12+ release in representative short (2) and long (10) cycles identified in panels B,C in main text Illustration of the timing of the onset of diastolic depolarization increase with respect to Ca. Panels show two derivatives: for membrane potential (dV/dt) and for the global Ca2+ signal (dF/dt). In the short cycle diastolic dV/dt increase follows dF/dt increase (inset), indicating that diastolic Ca2+ release accelerates diastolic depolarization. In contrast, a longer cycle showed “flat” time course for both derivatives during almost entire diastolic depolarization span, i.e. with no evidence of Ca2+ release and dV/dt remaining relatively small (close to zero line).(JPG)Click here for additional data file.Figure S22+ transient in the short and long cycles identified in panels B,C in main text A comparison of the number of overlapping LCR events relative to time from the peak of the prior AP-induced Ca. The number of overlapping LCR events in the shorter cycle (2) is illustrated by the red bars, while the number of overlapping LCR events in the longer cycle (10) is shown by the green bars.(JPG)Click here for additional data file.Movie S12+ dynamics  for twenty pacemaker cycles in an isolated single rabbit SA node cell.A representative example of original 2D recording of intracellular Ca The movie consists of 575 frames (images) consequently recorded at a physiological temperature of 35°C by a high-speed Hamamatsu C9100-12 CCD camera with an approximate rate of 100 frames/second . The movie is played 3 times slower vs. real time recording. Images have size 512×208 pixels (or 128×52 µm). The cell shown in this movie is also presented in (MP4)Click here for additional data file.Movie S22+ dynamics  by a high-speed Hamamatsu C9100-12 CCD camera and action potentials (bottom panel) by perforated patch clamp in an isolated single rabbit SA node cell.A representative example of simultaneous recording of 2D intracellular Ca Middle panel shows simultaneous dynamics of the total Ca2+ signal . The movie shows 14 pacemaker cycles  recorded at a physiological temperature of 35°C. The movie is played about 4.6 times slower vs. real time recording. Images have size 512×160 pixels (or 128×40 µm). The cell shown in this movie is also presented in (WMV)Click here for additional data file.


Chlamydomonas, has long been used as a model system to understand flagella structure and function. In addition to the well characterised roles of cilia and flagella in motility, cell biologists are becoming increasingly aware of their importance as cellular sensors. Ca2+-dependent signalling mechanisms are associated with many flagellar processes, although in many cases the underlying mechanisms remain unclear. Whilst Chlamydomonas possesses many genetic and biochemical advantages for the study of flagella, imaging Ca2+ in this alga has proven to be problematic. We have developed techniques to introduce Ca2+ responsive fluorescent dyes into Chlamydomonas cells via biolistics, which enables us to routinely and robustly image Ca2+ in both the cytosol and in the flagella. To visualise Ca2+ elevations within the flagella, we have developed imaging techniques using Total Internal Reflectance Fluorescence (TIRF) microscopy. This approach allows us to specifically image fluorescence from the flagella at high spatial and temporal resolution in the absence of interfering fluorescence from the cell body. We have found that Chlamydomonas flagella exhibit a range of dynamic Ca2+ elevations in response to different stimuli. Each flagellum demonstrates the ability to generate Ca2+ elevations independently from each other, suggesting a level of control in Chlamydomonas flagella signalling processes not previously demonstrated. We are currently using RNAi approaches to assess the contribution of different flagella-localised ion channels to these Ca2+ signalling processes.The motile green alga,


NN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger.The antifungal protein AFPNN5353 hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the α-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFPNN5353. The activation of the CWIP by AFPNN5353, however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi.We determined an AFP2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFPNN5353 increased about 2-fold the cytosolic free Ca2+ ([Ca2+]c) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFPNN5353 toxicity, ameliorated the perturbation of the [Ca2+]c resting level and prevented protein uptake into Aspergillus sp. cells.Furthermore, we provide evidence that calcium (CaA. giganteus antifungal protein AFPNN5353. We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.The present study contributes new insights into the molecular mechanisms of action of the  Aspergillus giganteus, ANAFP from Aspergillus niger, PAF from Penicillium chrysogenum and NAF from Penicillium nalgiovense c resting level c resting level in response to AFPNN5353 c within the first 10 min of protein treatment is not the result of uncontrolled Ca2+ influx due to plasma membrane permeabilization.To exclude the possibility that the AFPin the Ca+c restin2+]c in response to AFPNN5353 treatment could originate from extracellular and/or from intracellular Ca2+ stores, such as mitochondria, vacuoles, endoplasmic reticulum or the Golgi apparatus. To discriminate between the extracellular and intracellular source of the [Ca2+]c increase, we tested the influence of the Ca2+-selective membrane impermeable chelator BAPTA. On its own, BAPTA did not influence the resting level of [Ca2+]c in twelve h old A. niger cultures . The values of the [Ca2+]c resting levels of the last 10 min (50 to 60 min) measurement of AFPNN5353 treatment in the presence or absence of high Ca2+ concentration (20 mM versus 0.7 mM) are summarized in Table 2+]c of the controls which were not exposed to AFPNN5353 was 0.039 μM in the presence of 0.7 μM CaCl2 (standard condition) and 0.062 μM in the presence of 20 mM CaCl2. When AFPNN5353 was added, there was no significant elevation of the [Ca2+]c in high-Ca2+ medium (20 mM) (0.057 μM) whereas the [Ca2+]c rised to 0.146 μM at standard CaCl2 concentration (0.7 mM). These results suggest that Ca2+ externally added prior to the addition of AFPNN5353 counteracts the AFPNN5353 induced perturbation of the [Ca2+]c and growth inhibitory effect, at least partly, by controlling the [Ca2+]c resting level.To decipher the observation that high external CaCl2+]c levels in Aspergilli and other fungi c resting level, kinetics and amplitude, were determined and compared with controls that were not exposed to the protein but also subjected to mechanical perturbation. As shown in Figure NN5353 provoked a less pronounced [Ca2+]c amplitude; however, the [Ca2+]c level remained elevated even after the stimulus specific response had stopped.It is known that a range of external stimuli transiently increase c resting level depended on a sustained influx of Ca2+ ions from the external medium. Moreover, the AFPNN5353 induced changes in the Ca2+ signature of mechanically perturbed A. niger cells further underlines the disruption of the Ca2+ response and homeostasis by AFPNN5353. The addition of CaCl2 to the growth medium reduced the susceptibility of A. niger towards the antifungal protein and decreased the AFPNN5353 specific rise in the [Ca2+]c resting level. Both observations point towards an adaptive response which is mediated most probably via Ca2+ signalling. First, high extracellular Ca2+ concentrations trigger chitin synthesis in A. niger and thereby confer increased protection against antifungal proteins as shown for AFP [2+ homeostatic machinery to better maintain a low [Ca2+]c resting level when challenged with the antifungal protein, e.g. by (i) the increase of the activity of existing Ca2+ pumps/transporters to counteract the AFPNN5353-specific intracellular Ca2+ perturbation, or (ii) the modulation of the expression of Ca2+ channels/pumps/exchangers [2 only 10 min before A. niger was challenged with AFPNN5353 restored the low [Ca2+]c resting level. However, the perturbation of the Ca2+ homeostasis by a sustained elevation of the [Ca2+]c resting level indicates that A. niger is not able to restore the low [Ca2+]c resting level after exposure to AFPNN5353 and this might trigger programmed cell death (PCD) on the long term as it was shown to occur in A. nidulans in response to the P. chrysogenum PAF c resting level reaches its maximum. This result is further corroborated by the fact that higher external concentrations of Ca2+ reduced the AFPNN5353 specific rise in [Ca2+]c resting level which - in our opinion - would not occur with leaky membranes. However, we do not exclude changes in membrane permeability at longer exposure times to this antifungal protein and more studies are needed to answer this question.Since AFP was shown to cause membrane permeabilization , the infNN5353 is characteristic for sensitive but not resistant moulds. A lack of binding of AFPNN5353 to insensitive fungi might point towards the absence or inaccessibility of a putative interacting molecule at the cell surface. AFPNN5353 localized to the cytoplasm of target fungi only when actin filaments were formed. This is in agreement with the endocytotic uptake and intracellular localization of the P. chrysogenum antifungal protein PAF in sensitive filamentous fungi c resting level in the cell. This sustained perturbation of the Ca2+ homeostasis could lead to PCD [2+ neutralizes the toxic effects of AFPNN5353 and improves the resistance of the target organism possibly by decreasing the elevated [Ca2+]c resting level and stimulating the fortification of the cell wall by the induction of chsD expression as shown for AFP c resting level and mechanical perturbation experiments and the calibration of [Ca2+]c were performed as described in [10ribed in .2+ measurements and the calculations to convert the luminescence units into the [Ca2+]c levels. She also performed the statistical analysis and helped to draft the manuscript. MB contributed the A. niger A533 strain, helped with the Ca2+ measurements and participated in the design of the study. AE contributed to the indirect immunofluorescence stainings. VM contributed the A. niger RD6.47 strain and performed the agsA induction assays. FM conceived of the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.UB carried out the growth inhibition assays, the indirect immunofluorescence stainings, the CaagsA promoter in A. niger in response to cell wall interfering substancesThe expression of nucleus-targeted GFP under the control of the . Differential interfering contrast images and corresponding fluorescence images of A. niger RD6.47 indicate the expression of a nucleus-targeted GFP under the control of the A. niger agsA promoter. Five h old germlings were (A) left untreated (negative control), (B) treated with 50 μg/ml AFPNN5353 and (C) with 10 μg/ml caspofungin (positive control) as described in Materials and Methods. Scale bar, 20 μm.Click here for fileA. niger germlings after AFPNN5353 exposureViability staining of . Twelve h old A. niger germlings were stained with fluorescein diacetate  and propidium iodide (right pannels). The left panels show the respective light micrographs. All samples were pretreated with the dyes for 15 min before 20 μg/ml AFPNN5353 was added (B). Controls remained untreated (A) or were exposed to 70% ethanol (C). Scale bar, 50 μm.Click here for file


Background: Mitochondria may utilize different proteins to decode high and low cytosolic Ca2+.Results: Inhibition of SERCA shifts mitochondrial Ca2+ uptake from being UCP3-dependent to Letm1-dependent.Conclusion: Depending on the mode of intracellular Ca2+ release, two different mitochondrial Ca2+ uptake pathways are engaged.Significance: The dissection of two molecularly distinct mitochondrial Ca2+ uptake routes depending on SERCA activity points to the complexity of the mitochondrial Ca2+ uptake machinery. 2+ across the inner mitochondrial membrane is an important physiological process linked to the regulation of metabolism, signal transduction, and cell death. While the definite molecular composition of mitochondrial Ca2+ uptake sites remains unknown, several proteins of the inner mitochondrial membrane, that are likely to accomplish mitochondrial Ca2+ fluxes, have been described: the novel uncoupling proteins 2 and 3, the leucine zipper-EF-hand containing transmembrane protein 1 and the mitochondrial calcium uniporter. It is unclear whether these proteins contribute to one unique mitochondrial Ca2+ uptake pathway or establish distinct routes for mitochondrial Ca2+ sequestration. In this study, we show that a modulation of Ca2+ release from the endoplasmic reticulum by inhibition of the sarco/endoplasmatic reticulum ATPase modifies cytosolic Ca2+ signals and consequently switches mitochondrial Ca2+ uptake from an uncoupling protein 3- and mitochondrial calcium uniporter-dependent, but leucine zipper-EF-hand containing transmembrane protein 1-independent to a leucine zipper-EF-hand containing transmembrane protein 1- and mitochondrial calcium uniporter-mediated, but uncoupling protein 3-independent pathway. Thus, the activity of sarco/endoplasmatic reticulum ATPase is significant for the mode of mitochondrial Ca2+ sequestration and determines which mitochondrial proteins might actually accomplish the transfer of Ca2+ across the inner mitochondrial membrane. Moreover, our findings herein support the existence of distinct mitochondrial Ca2+ uptake routes that might be essential to ensure an efficient ion transfer into mitochondria despite heterogeneous cytosolic Ca2+ rises.The transfer of Ca Moreover, though early patch-clamp studies revealed one Ca2+ current in mitoplasts 2+ uniporter. In agreement with these findings several proteins of the IMM have been identified as putative mitochondrial Ca2+ carriers and/or components of mitochondrial Ca2+ uptake sites , which was initially described as a K+/H+ exchanger in the IMM  pathway in endothelial and HeLa cells  and evaluated the contribution of UCP3, Letm1 and MCU to mitochondrial and cytosolic Ca+ sensor  and furam): 138 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 1 HEPES, 2.6 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4, 10 d-glucose, 0.1% vitamins, 0.2% essential amino acids, and 1% penicillin/streptomycin; pH adjusted to 7.4 with NaOH. During the experiments cells were continuously perfused with a Ca2+ containing buffer, which consisted of (in mm): 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 d-glucose, and 10 HEPES; pH adjusted to 7.4 with NaOH. In experiments where a Ca2+-free solution was applied to the cells, the CaCl2 was replaced with 1 mm EGTA.Cell culture materials were obtained from PAA laboratories . Thapsigargin was purchased from Abcam® , Histamine and EGTA were from Sigma . Prior to experiments cells were washed and maintained for 20 min in a HEPES buffered solution composed of  and their nucleotide sequences (5′-3′) were as follows: si1-hMCU: GCCAGAGACAGACAAUACUtt; si2-hMCU: GGAAAGGGAGCUUAUUGAAtt; si1-hLetm1: UCCACAUUUGAGACUCAGUtt; si2-hLetm1: AUGUUCCAUUUGGCUGCUGtt; si-hUCP2: GCACCGUCAAUGCCUACAAtt; si-hUCP3: GGAACUUUGCCCAACAUCAtt.For controls, a scrambled siRNA was used: UUCUCCGAACGUGUCACGUtt. Although all siRNAs used in this study have been previously approved to exhibit reliable gene knock-down efficiency , their eTotal RNA was isolated from control and target siRNA treated HeLa cells using a RNA isolation kit . Reverse transcription was carried out using a cDNA synthesis kit from Applied Biosystems. The efficiency of siRNA was validated by performing Real time PCR using QuantiFast SYBR Green RT-PCR kit  on LightCycler 480 . RNA polymerase II (RPOL2) was used as housekeeping control. Primers for RPOL2, UCP2, UCP3, MCU, and LETM1 were obtained from Invitrogen  and there sequences (5′-3′) are as follows: RPOL2 for: CATTGACTTGCGTTTCCACC, RPOL2 rev: ACATTTTGTGCAGAGTTGGC, UCP2 for: TCCTGAAAGCCAACCTCATG, UCP2 rev: GGCAGAGTTCATGTATCTCGTC, UCP3 for: AGAAAATACAGCGGGACTATGG, UCP3 rev: CTTGAGGATGTCGTAGGTCAC, MCU for: TTCCTGGCAGAATTTGGGAG, MCU rev: AGAGATAGGCTTGAGTGTGAAC, Letm1 for: TGTTCTTCAAGGCCATCTCC, Letm1 rev: TGTTGCTGTGAAGCTCTTCC. The expression data were analyzed by ΔΔCt method as described previously . Knock-dm siRNA using 4 μg/well TransFastTM transfection reagent  in 0.5 ml of serum and antibiotic-free transfection medium. Cells were maintained in a humidified incubator  for 16–20 h before changing back to complete RPMI 1640 medium. All experiments were performed either 48 h or 72 h after transfection.HeLa cells were cultured as described previously . Brieflym fura-2/AM  for 45 min prior to the experiments. Co-imaging of fura-2 and the 4mtD1GO-Cam was achieved with a digital wide field imaging system, the Till iMIC  using a 40× objective . For illumination of fura-2 and the 4mtD1GO-Cam an ultra fast switching monochromator, the Polychrome V (Till Photonics) equipped with an excitation filter  and a dichroic filter  was used. fura-2 was excited alternatively at 340 nm and 380 nm and the red-shifted mitochondrial-targeted cameleon was excited at 477 nm, respectively. Emitted light was simultaneously collected at 510 nm (fura-2 and GFP of GO-Cam) and at 560 nm (FRET-channel of GO-Cam) using a single beam splitter design  that was equipped with a dual band emission filter  and a second dichroic filter . Images were recorded with a charged-coupled device (CCD) camera . For the data acquisition and the control of the digital fluorescence microscope the live acquisition software (LA) version 2.0.0.12 (Till Photonics) was used. Post-acquisition image analysis was performed on MetaMorph 7.7.0.0 .4mtD1GO-Cam  transfecn reflects the number of cells. Statistical analyses were performed with unpaired Student's t test, and p < 0.05 was considered to be significant.Data shown represent the mean ± S.E., where 2+]cyto and [Ca2+]mito were simultaneously measured using Fura-2/AM-loaded cells that transiently expressed 4mtD1GO-Cam, a recently developed red shifted genetically encoded Ca2+ probe targeted to the mitochondrial matrix (A). This approach allowed an accurate temporal correlation of changes in [Ca2+]cyto with [Ca2+]mito. Stimulation with the IP3-generating agonist histamine in the absence of extracellular Ca2+ induced a fast increase of both cytosolic and mitochondrial Ca2+ levels , indicating an efficient transfer of Ca2+ from the endoplasmic reticulum (ER) into mitochondria. The Ca2+ signal in the cytosol occurred slightly faster than the respective Ca2+ elevation within mitochondria of same single individual cells . Pretreating the cells with the SERCA inhibitor thapsigargin 40 s prior to the addition of histamine, enhanced the cytosolic Ca2+ elevation . Notably, in the presence of thapsigargin, [Ca2+]cyto started to increase slowly, indicating Ca2+ leakage from the ER. This weak thaspsigargin-induced cytosolic Ca2+ signal was not accompanied by a significant elevation in [Ca2+]mito . Subsequent addition of histamine evoked a further pronounced rise of both [Ca2+]cyto and [Ca2+]mito. However, these signals increased with a slower kinetics compared with respective Ca2+ elevations in the absence of the SERCA inhibitor (B & C). In addition, the time gap between the histamine induced rise of [Ca2+]cyto and the respective mitochondrial Ca2+ signal was considerably extended in the presence of thapsigargin (B & C), indicating that SERCA inhibition decelerates the transfer of Ca2+ into mitochondria upon IP3-mediated Ca2+ release. A correlation between changes of [Ca2+]cyto and respective Ca2+ signals within mitochondria showed that in the presence of thapsigargin almost twice as much cytosolic Ca2+ was elevated, until mitochondrial Ca2+ uptake was activated .[Cal matrix A. This a2+ rises and coupling between [Ca2+]cyto and [Ca2+]mito were subsequently used to investigate the contribution of the individual proteins that have been proposed to be involved in mitochondrial Ca2+ uptake .These protocols that illustrate the distinct kinetics of the compartmental Ca2+ signals probably reflect the involvement of distinct mitochondrial Ca2+ uptake routes. Therefore, we performed experiments, in which the contribution of the mitochondrial proteins UCP2/3, Letm1 and MCU to mitochondrial Ca2+ uptake in various protocols was investigated by diminution of these proteins with a transient transfection of the respective siRNA. The siRNAs against UCP2/3, Letm1, and MCU have already been validated to specifically and significantly reduce mRNA level of the respective proteins . As recently demonstrated .In line with previous studies , 18, 22,C & D). Mitochondrial Ca2+ sequestration upon IP3-mediated Ca2+ mobilization in the absence of thapsigargin was not affected in cells that were treated with siRNA against Letm1 . In contrast, if SERCA activity prior to the addition of the agonist was blocked, diminution of Letm1 strongly reduced the histamine-induced mitochondrial Ca2+ signal . Notably, the siRNA-mediated knock-down of UCP2/3, Letm1, and MCU neither affected the mitochondrial membrane potential , nor the capacity of mitochondria to extrude Ca2+ . These data indicate that SERCA inhibition switches the mode of mitochondrial Ca2+ uptake from a Letm1-independent to a Letm1-dependent one and, hence, explain the lacking contribution of UCP2/3 to mitochondrial Ca2+ uptake under conditions of SERCA inhibition.Next we performed analogous experiments with cells, in which Letm1 was silenced , C & D. 2+ into mitochondria, respective experiments were performed with MCU-depleted cells. The knock-down of MCU negligibly altered cytosolic Ca2+ signals in response to cell stimulation . However, respective mitochondrial Ca2+ signals were strongly diminished in cells depleted of MCU (A & B). Notably, the inhibitory effect of MCU knock-down on mitochondrial Ca2+ accumulation was independent of the absence or presence of thapsigargin , thus, pointing to a functional interaction between these putative contributors of mitochondrial Ca2+ uptake.A simultaneous knock-down of either MCU and UCP2/3 or MCU and Letm1 did not further reduce mitochondrial Ca2+ store that results in a decelerated, but higher cytosolic Ca2+ signal and a greater delay of the Ca2+ transfer into the mitochondria  suggesting a shift in the mode/route of mitochondrial Ca2+ uptake.SERCA inhibition prior to cell stimulation with histamine partially emptied the internal Ca2+ uptake mode/route also occurs under condition in which the cell is already stimulated by an IP3-generating agonist, we performed different experimental protocols, in which thapsigargin was added after histamine. If cells were continuously exposed to the IP3 generating agonist in the absence of extracellular Ca2+, the subsequent SERCA inhibition transiently elevated [Ca2+]cyto (A). This transient thapsigargin-induced increase of cytsolic Ca2+ levels evoked only small changes of [Ca2+]mito. In contrast, SERCA inhibition following IP3-mediated Ca2+ release in the presence of extracellular Ca2+-induced prominent, longer-lasting elevations of both [Ca2+]cyto and [Ca2+]mito (B), thus, highlighting the involvement of SOCE that promotes mitochondrial Ca2+ accumulation. The latter protocol was further used to test the contribution of UCP3, Letm1, and MCU to mitochondrial Ca2+ sequestration under these conditions of Ca2+ mobilization.To test whether or not this change in mitochondrial Caa2+]cyto A. This ta2+]mito B, thus, 2+ (B and D) cells depleted of UCP3 showed greatly reduced mitochondrial Ca2+ accumulation in response to histamine (A). The subsequent addition of thapsigargin evoked a substantial rise of [Ca2+]mito that was not affected by the diminution of UCP3.Consistent with our previous work and the experiments in the absence of extracellular Ca2+ , B and Distamine A. The su2+ uptake that was elicited by histamine, whereas [Ca2+]mito was significantly reduced in response to a subsequent SERCA inhibition (A). These findings demonstrated that in protocols in which SERCA was blocked after the initiation of IP3-mediated Ca2+ release, mitochondrial Ca2+ uptake also switched from an UCP3-reliant to a Letm1-dependent mode.In contrast, knock-down of Letm1 negligibly influenced mitochondrial Cahibition A. These 2+ signals in response to histamine and to the subsequent addition of thapsigargin (A). Notably, the initial cytosolic Ca2+ peak and the thapsigargin induced rise remained unaffected under all conditions (B).Consistent with previous experiments, cells that were treated with siRNA against MCU showed attenuated mitochondrial Casigargin A. Notablnditions B.3-mediated, rapid Ca2+ rises, that are associated with the generation of high Ca2+ micro-domains at the surface of mitochondria (A). However, if the mode of Ca2+ mobilization was decelerated by SERCA inhibition, which increased ER Ca2+ leakage and, thus, probably attenuated the inter-organelle Ca2+ micro-domains, mitochondria slowly accumulated Ca2+ via an UCP3-independent, but Letm1- and MCU-reliant route (B).Our present data reveal that IPchondria  were trachondria A. Howevent route B.3-mediated Ca2+ release by SERCA inhibition significantly alters the molecular characteristics of mitochondrial Ca2+ uptake. At first glance, these findings are not surprising, considering the central role of ER Ca2+ pumps in the control of the cellular Ca2+ homeostasis  than th μm, , the misitivity  is inact2+ leak from the ER upon SERCA inhibition might per se impact the transfer of Ca2+ into mitochondria. Such a scenario is feasible, as it was shown that an increased ER Ca2+ leakage in cells expressing an inactive truncated version of SERCA1 inhibited mitochondrial movements and increased ER-mitochondria contact sites, which consequently led to mitochondrial Ca2+ overload     that, in in HeLa  and clon in HeLa  but not  in HeLa . Howeverunveiled , thus, i (MCUR1)  might ex2+ uptake in HeLa cells was recently interpreted as an indication that UCP3 do not accomplish mitochondrial Ca2+ uniport or directly modulate a mitochondrial Ca2+ channel  However, the present findings confirm the assumption of Demaurex's group and demonstrate that SERCA activity affects mitochondrial Ca2+ uptake.The findings that SERCA inhibition abrogates the contribution of UCP3 to mitochondrial Ca channel . The autf UCP2/3 , findingrevented .This is 3-triggered intracellular Ca2+ release, and, subsequently, shifts the mode of mitochondrial Ca2+ uptake from an UCP3- and MCU-dependent and Letm1-independent toward a Letm1- and MCU-dependent but UCP3-independent route (2+ uptake and appoints which proteins of the IMM actually contribute to the transfer of Ca2+ into mitochondria.Overall the present study demonstrates that the inhibition of SERCA affects the kinetics of IPnt route . These o


Here, we investigate the content, distribution and demand for Zn2+ in gastric mucosa under baseline conditions and its regulation during secretory stimulation.Recent work has suggested that Zn2+ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, 70Zn2+, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of 70Zn2+ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn2+ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn2+ {[Zn2+]i} during exposure to standard extracellular concentrations of Zn2+ (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn2+ increased with exposure to secretagogues  and under conditions associated with increased intracellular Ca2+ {[Ca2+]i}. Uptake of Zn2+ was abolished following removal of extracellular Ca2+ or depletion of intracellular Ca2+ stores, suggesting that demand for extracellular Zn2+ increases and depends on influx of extracellular Ca2+.Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn2+ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca2+ integrates basolateral demand for Zn2+ with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues.This study is the first to characterize the content and distribution of Zn Our results indicate that there is variation in content and distribution of Zn2+ within the gastric wall and mucosa. We find that, in vivo and in vitro, the mucosa and individual gastric glands are capable of extracting Zn2+ from extracellular sources even when its concentration may be in the nanomolar range. In the isolated gastric gland, a multicellular model of epithelial secretion, we find that basolateral uptake of Zn2+ is modulated by [Ca2+]i during stimulation with agonists of apical secretion of acid. Our findings thus suggest a novel role for classical second messenger pathways in integrating secretory functions of the apical membrane with supply functions across the basolateral membrane, and may be applicable to a variety of epithelial systems.In this study, we utilized complimentary approaches to characterize content and distribution, acquisition and demand for ZnAnesthesia and euthanasia for New Zealand White rabbits were approved according to policies of Harvard Medical School  Standing Committee on Animals; Protocol 03359). As described previously 2) of each element, with a different sensitivity for each element.To survey the distribution of metals in the gastric mucosa, we utilized micro X-Ray Fluorescence microscopy (μXRF) 2, and incident beam size 16×7 µm2 FWHM. For the finer map in 2. and incident beam size 7×7 µm2 FWHM. Fluorescence counts were recorded in energy bands corresponding to K, Ca, Fe, Cu, Ni and Zn, though no significant Ni was found. Since the signals from some elements appear in more than one band, e.g. the Kβ fluorescence of Cu in the Zn Kα band, certain fractions of the signal from such interfering channels were subtracted from the channels interfered with. The coefficients for this subtraction were derived by mapping standards containing some but not all relevant elements, e.g. Cu with no Zn.For full thickness samples, a novel fast-freeze method was developed for near instantaneous tissue vitrification. Fixation and mounting of oriented specimens was performed on silicon wafers . Silicon substrates were used because they exhibit low background signals in μXRF and have high thermal conductivity. Sequential cryosections (50 µm) were stained with hematoxylin/eosin and then analyzed by X-ray fluorescence microscopy on Beamline 10.3.2, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA. 64Zn, 66Zn, 66Zn, 67Zn and 70Zn. 70Zn is the most commonly isotopic tracer due to its low natural abundance and was used for all described experiments. The natural relative abundance for 70Zn and 68Zn are 0.6% and 18.8% respectively with an isotopic ratio of 0.0319 and any increase in this isotopic ratio is indicative of 70Zn uptake in biospecimens. Unless otherwise noted, all reagent and stock solutions were prepared in ultra-trace metal grade nitric acid  made from de-ionized water  in order to match the acid matrix resulting from the sample preparation procedure. Zinc enriched in 70Zn (99%) was obtained  and was dissolved in HCl and diluted with de-ionized water to prepare an 70Zn-enriched spiking solution of 1000 mg/L in 1% HCl/citrate. This stock solution was used to prepare standard and sample solutions for all experiments. Standard-sized tissues samples (∼25 mm3) or volumes of isolated glands  were acid digested in aliquots of concentrated nitric acid and made up to a total volume 2 ml using de-ionized water. Analysis for isotopes 68Zn and 70Zn were performed concurrently on 1 ml samples, at the ICP-MS facility of the Institute of Marine and Coastal Sciences, Rutgers the State University, New Brunswick, NJ. An analytical methodology was developed to scan zinc isotopes using a double-focusing, single-collector Thermo Element 2 ICP-MS . Settings are shown in 70Zn/68Zn was calculated as an index of enrichment Among the five stable zinc isotopes, 0) Concentrated preparations of freshly isolated gastric glands were diluted in DMEM to a final concentration 1.875% and loaded for 30 min to 40 min with fluozin-3AM (8 µM) 2PO4, 1.0 MgSO4 or 1.0 MgCl2, 1.0 CaCl2, 10 HEPES, and 10 glucose, pH 7.4; concentration of Zn2+ measured by atomic absorption is 1.2 µM, kindly determined by Dr. Shannon Kelleher, Pennsylvania State University, University Park, PA) or modified Ringer's solutions. Readout of fluozin-3 fluorescence was performed (Excitation 485 nm/Emission 520 nm) after correction for background fluorescence of solutions and unlabelled glands 0) For high-throughput fluorometry, aliquots of glands  were transferred to 96 well plates  following loading of dye in media and experimental manipulations in standard .Thapsigargin and TPEN {tetrakis-(2-pyridylmethyl)ethylenediamine} were obtained from Molecular Probes  and were dissolved in Ringer's solutions directly. Nigericin, DTPA (diethylenetriaminepentaacetic acid) and pyrithione were obtained from Sigma-Aldrich and was dissolved under alkaline conditions, then added to Ringer's solutions for final dilutions of 1∶1000. All solutions were checked for changes in pH and adjusted if necessary to baseline pH. For Ringer's, calculations of free and bound concentrations of CaIn the microscopy imaging system, fluorescence intensities were monitored continuously throughout each experiment (SimplePCI software). At discrete time intervals, measurements were summarized as means ± SE. For comparison between treatments, unless stated otherwise, measurements in individual parietal cells (4 to 8 cells identified in each isolated gland) were combined to provide a single integrated value at each time point for each gland. Unless stated otherwise, comparisons were performed using analysis of variance for sequential or multiple measurements , using purchased software .Panel 1A provides a standard tissue frozen section, stained with hematoxylin and eosin, corresponding to a neighboring section that was used to generate metal maps . In these maps, white represents the maximum intensity while black areas show little or none of the relevant element. Of note, consistently strong signals for the Fe and Zn are detected in region of the surface epithelium, near the luminal interface; signals for Cu are very minimal throughout the section although perhaps more concentrated in the surface epithelium. Relatively homogeneous signals for Fe are observed throughout the glandular mucosa, deep to the surface epithelium, in areas known to be populated by mitochondria-rich parietal cells Shown in 2+ is homogeneous throughout the glandular region.To further define distribution of Zn within the glandular region of the mucosa Figure 22+ from the circulation, in vivo, three pairs of rabbits were injected intravenously with 70Zn2+ in sterile saline  to one animal and saline alone to the other. Gastric mucosa was harvested 24 hr later, some preserved for analysis and the rest processed for isolation of individual gastric glands. The relative abundance in nature for 70Zn and 68Zn are 0.6% and 18.8%; and the ratio of 70Zn/68Zn is 0.0319. Any enrichment of 70Zn reflected in a higher ratio would reflect uptake in tissues and individual glands. 70Zn∶68Zn in saline injected animals, along with significant uptake of 70Zn2+ in whole gastric mucosa and isolated gastric glands. Avidity of gastric mucosa for 70Zn2+ is higher than that of peripheral tissues such as skin, retina, cornea and much higher than the avascular lens.To demonstrate that gastric mucosa acquires Znin vitro studies, we evaluated potential threshold levels of extracellular [Zn2+] which would permit detection of 70Zn2+ uptake by gastric mucosa. Isolated gastric glands were exposed to increasing concentrations of 70Zn2+ in Ringer's solutions , with total content of added Zn2+ calculated to provide a free [Zn2+] of 1 nm, 10 nM or 100 nM. After 1 hour, glands were processed for ICP-MS. In unstimulated glands, evidence of accumulation started to become evident during exposure to extracellular [Zn2+] at 10 nM  in order to monitor changes in intracellular concentration of Zn2+ {[Zn2+]i} in individual parietal cells 2+] of 10 µM to 50 µM. As shown in 2+ leads immediately to increases in [Zn2+]i, reaching plateaus within 10 minutes. These responses were abolished during exposure to low concentrations of a membrane-permeable chelator, TPEN (10 µM), indicating that Zn2+ is the source of the fluorescence signal within the cell Flux measurements of intracellular accumulation of labile Zn2+ in isolated gastric glands when extracellular Zn2+ was at least 50 µM, and this was observed in the presence or absence of extracellular Ca2+. We also observed an increase in the rate of Zn2+ uptake when glands were exposed to the secretagogue, forskolin (10 µM), a finding consistent with the idea that stimulation of acid secretion across the apical membrane leads to increased demand for Zn2+ across the basolateral membrane. These observations were corroborated by preliminary studies indicating that exposure to forskolin (10 µM) enhances influx of isotopic 70Zn2+, more than three-fold over baseline influx . In these in vitro studies, when extracellular Ca2+ was removed, responses became less consistent, particularly if extracellular Zn2+ concentrations were 10 µM or lower. These observations led us to hypothesize that uptake of Zn2+ into the parietal cell might be connected to intracellular Ca2+ homeostasis.Utilizing fluozin-3 measurements, previous studies in our laboratory 2+ requires the presence of Ca2+, a protocol was devised to totally deplete Ca2+ from extracellular solutions and intracellular stores. Glands pre-loaded with fluozin-3 were perfused with Ca2+-depleted Ringer's (0.3 mM EGTA) containing 2 µM thapsigargin to deplete the intracellular Ca2+ store, followed by exposure to 10 µM Zn in Ca2+-depleted Ringer's. As shown in a representative recording from 5 parietal cells in a single gland in 2+ accumulation was abrogated under these conditions, indicating a requirement for intracellular Ca2+. Exposure to thapsigargin depletes intracellular, membrane-bound stores of Ca2+, but activates store-operated channels at the plasma membrane 2+ lead to increases in [Ca2+]i without any re-filling of stores 2+ can occur when intracellular, membrane–bound stores are depleted but store-operated entry of Ca2+ is present (2+ to deplete intracellular and extracellular sources of Ca2+. Under these conditions no accumulation was observed in the presence of extracellular Zn2+ (10 µM). When Ca2+ was subsequently restored to the perfusate (1 mM) in the presence of thapsigargin, increases in [Zn2+]i were observed  and no added Ca2+, in order to deplete intracellular stores. These solutions also contained DTPA (10 µM), a chelator of Zn2+ that is membrane-impermeable and therefore depletes free Zn2+ in the extracellular perfusate but not within the cell. Under these conditions, no increase in [Zn2+] was observed. Glands were then exposed to solutions containing Zn2+ (10 µM) and Ca2+ (1 mM) and, as expected, increases in intracellular Zn2+ were observed. These increases were, however, arrested when the chelator DTPA (10 µM) was added .In fluorescence microscopy, there is a possibility of inadvertent bias in selection of individual glands for study. To be confident of the overall characteristics of the response to depletion and restoration of i were decreased and very little uptake was detected when extracellular and intracellular sources of Ca2+ had been depleted.A first set of studies  and then 0CaR with TG (30 min). Glands were then exposed to solutions in which Ca2+ was restored  for 30 min, with replacement by fresh solution at 30 min. As in studies of individual glands . Glands were then exposed to solutions in which Ca2+ had been restored  for 30 min, followed by exposure to solutions in which Ca2+ was again depleted  for 30 min. As previously observed, when Ca2+ was restored to the system, uptake of Zn2+ was significantly enhanced; however, when Ca2+ was again removed accumulation of Zn2+ was impaired and actually decreased. These findings suggest that Zn2+ accumulation is responsive to Ca2+ availability in the cytoplasm, by a mechanism that is consistent with exchange of intracellular Ca2+ for extracellular Zn2+.A third set of studies  and the cAMP/PKA agonist forskolin  2+. Responses were read 10 minutes after placement in these solutions, in order to obtain immediate estimates of labile Zn2+ accumulation within the cell.In a final series of studies, we evaluated demand for extracellular Zn2+ was not influenced by the absence of extracellular Ca2+. During exposure to secretory agonists (CCh/FSK), accumulation of Zn2+ was significantly enhanced if Ca2+ was present in the Ringer's solution, but significantly impaired if Ca2+ was absent. These findings confirm that, during secretory stimulation, intracellular content of Zn2+ depends on influx of extracellular Ca2+. In an additional experiment, also shown in 2+ during exposure to CCh/FSK, if Ca2+ was present in the Ringer's solution, indicating that functional intracellular stores of Ca2+ are not, per se, required for the ability of the gland to take up Zn2+ during secretory stimulation. In contrast, pretreatment with TG in Ca2+-free Ringer's enhanced the failure of the glands to preserve content of Zn2+, a finding consistent with the hypothesis that accumulation of Zn2+ is even more completely impaired by profound depletion of Ca2+ from the extracellular compartment, intracellular stores and the cytoplasm.As shown in 2+ by the parietal cell is a critical feature of its ability to regulate acidity within its secretory compartment, which includes the tubulovesicles of the parietal cell and the lumen of the gland. Chelation of Zn2+ within these compartments led to marked alkalization, suggesting that the presence of Zn2+ insulates these compartments against leakage of H+ ions 2+, accumulating it in response to proton gradients and failing to accumulate it when secretion is blocked by proton pump inhibition Work reported previously from our laboratory indicates that the transport of Znin vivo intravenous injection, the non-radioactive isotope 70Zn2+ accumulates readily within both gastric mucosa and in the acid-secreting gastric glands; and third, as suggested by in vitro studies of isolated gastric glands, that exposure to well-recognized secretory agonists accelerates the uptake of Zn2+ from the nutrient side of the epithelium and requires intact intracellular stores of Ca2+. The studies reported here refine and extend the concept that demand for Zn2+ from the circulation and nutrient compartment may be regulated acutely by alterations in H+ secretion to the lumen.The studies reported here provide three sets of observations with respect to utilization of zinc within the gastric mucosa: first, as demonstrated by μXRF, that zinc accumulates differently in distinct micro-anatomic regions of the mucosa; second, as observed following 2+ and other cations in the mucosa of a specific region of the gastrointestinal tract. The basis of the method is fluorescence produced by synchrotron-derived x-rays, which can be focused to localize elemental content within isolated cells In vitro, quantification within individual cells and resolution of individual isotopes of different metals is feasible To our knowledge, this is the first report of the use of μXRF to evaluate the distribution of Zn+ ions. Within the gastric gland, the region toward the surface is populated with mucus neck cells; the middle region is dominated by the acid-secreting parietal cells, and the deepest regions harbor pepsinogen-secreting chief cells The gastric mucosa is divided into two spatially and functionally distinct regions 2+ present at the interface of the lumen and the surface epithelium  that are more likely to be free and labile than bound. This observation offers the possibility that Zn2+ is secreted with the mucus by the surface epithelium or, possibly, is selectively trapped within the mucus layer after discharge into the gastric lumen with secretion. It is tempting to speculate that associations of Zn2+ or other metals with the mucin layer might be important in its structure or ability to resist back-diffusion of H+ ions. The development of a method for evaluating distribution of metal ion species in a complex and highly hydrated mucosal surface provides opportunities to evaluate alterations in content and distribution of metal ion species under pathologically relevant conditions, for example, in response to systemic stress or during infestation with pathogenic organisms such as Helicobacter pylori.Curiously, we find higher levels of Znithelium 2, more 70Zn2+, uptake is demonstrated within the mucosa as a whole and in individual glands. Such movements can be tracked within hours of injection into the circulation and provide evidence that demands of the tissues are readily replenished by movement of Zn2+ from the circulation into the mucosa.The concept of “demand” for any nutrient is based on the need of a cell, tissue or organism to maintain homeostasis. In studies utilizing sector field ICP-MS, following injection of a non-radioactive and naturally scarce isotope, in vitro assay to monitor uptake of 70Zn2+ by isolated glands, observing that it can be detected within minutes when extracellular concentrations are in the 10 nanomolar range and very consistently when concentrations are 100 nM. Moreover, rates of uptake are accelerated during stimulation with a powerful secretory agonist, forskolin. The use of rare, non-radioactive isotopes such as 70Zn2+ permits investigation of Zn2+ distribution into whole tissues and cell preparations, without the restrictions required when radioactive isotopes  would be used. The specificity of these methods for individual metal species not only permits quantification of rates of entry into cells and tissues, but also increases the confidence in designing studies utilizing potentially less specific reporters of Zn2+ movements, such as the fluorescent reporter fluozin-3.To more precisely characterize the avidity of individual glands, we developed an 70Zn2+, we were able to design experiments to study conditions of uptake under baseline conditions and during exposure to well-recognized secretory agonists, using utilizing relatively inexpensive and rapidly responsive fluorescence imaging and fluorometric methods. Utilizing these methods, we were able to confirm enhanced uptake of Zn2+ across the basolateral membrane during secretory stimulation. In addition, we found that manipulation of [Ca2+]i homeostasis leads to alterations in the ability of the parietal cell to take up and preserve [Zn2+]i. Thus, intracellular accumulation of Zn2+ is nearly abolished when intracellular stores of Ca2+ are depleted by exposure to thapsigargin and Ca2+-depleted Ringer's. Following exposure to thapsigargin, uptake of Zn2+ was observed—albeit at a reduced level—if Ca2+ is present in the extracellular solution. This uptake was arrested when the chelator DTPA (10 µM) was added. These observations indicate that the increases in [Zn2+]i are indeed due to influx from extracellular sources and not release from intracellular stores. They argue that, under baseline conditions, uptake of Zn2+ across the basolateral membrane depends on adequate stores of intracellular Ca2+. With stimulation by powerful agonists such as forskolin and carbachol, demand for extracellular Zn2+ increases and depends on influx of extracellular Ca2+. In addition, these studies illustrate physiologically relevant approaches for using both real-time imaging of individual glands, which is time-intensive, and 96-well platforms, which would be amenable to rapid through-put approaches for small molecule screens.Based on studies with 2+ from insulin-secreting beta cells 2+ [Zn2+] in different cell types 2+ under a number of experimentally relevant conditions 2+ from the reported dK and from maximum and minimum fluorescence responses, as was originally proposed for Ca2+ reporters 2+]i, based on fluozin-3 measurements.The fluorescent reporter utilized here was fluozin-3, which has been utilized in its free acid form for monitoring extracellular secretion of Zn2+ facilitates optimal uptake of Zn2+ across the cell membrane, implying that it is either a counter-ion in exchange or it is acting as a regulatory second-messenger. Membrane proteins that facilitate Zn2+ transport constitute the SLC30A (ZnT) and SLC39A (Zip) gene families 2+ to the cytoplasm (either from extracellular sources or possibly intracellular compartments) have been identified in mammals 2+ transport does not implicate Ca2+ in the former role and emerging information for ZIP, ZnT and related transporters in yeast suggest that counter exchanging ions are likely to be protons 2+ as a second messenger in responses to secretagogues 2+ is a second messenger that can match the basolateral demand for Zn2+ with the secretory response to physiologic stimulation. It seems likely that similar connections will be found in other secretory cells and tissues—neural, endocrine and exocrine.In the current set of studies, we find that Ca2+, the amount of free Zn2+ within the cytoplasm is even more tightly controlled. Our studies have suggested that, in different epithelial cells, the baseline concentration of [Zn2+]i is in the nanomolar range 2+ and its disposal from the cytoplasm. Observations in other cell types such as neurons suggest that exposure to elevated levels of Zn2+ can be cytotoxic, within minutes 2+]i2+ homeostasis is also a consequence of such oxidant-related stress 2+ could be amplified by dysregulation of intracellular Ca2+ homeostasis. Further studies will help to clarify this potential relationship, not only in gastric mucosa but in other secretory cell types or tissues.Compared to levels of free Ca


Many syts play roles in neurotransmitter release or hormone secretion or both, and a growing body of work supports a role for some syts as Ca2+ sensors of exocytosis. Work in many types of endocrine cells has documented the presence of a number of syt isoforms on dense-core vesicles containing various hormones. Syts can influence the kinetics of exocytotic fusion pores and the choice of release mode between kiss-and-run and full-fusion. Vesicles harboring different syt isoforms can preferentially undergo distinct modes of exocytosis with different forms of stimulation. The diverse properties of syt isoforms enable these proteins to shape Ca2+ sensing in endocrine cells, thus contributing to the regulation of hormone release and the organization of complex endocrine functions.Synaptotagmins (syts) are abundant, evolutionarily conserved integral membrane proteins that play essential roles in regulated exocytosis in nervous and endocrine systems. There are at least 17 syt isoforms in mammals, all with tandem C-terminal C2 domains with highly variable capacities for Ca One can hope to gain a better understanding of these problems by studying functional variations within the diverse families of exocytotic proteins.Nature employs the same basic molecular machinery for the release of both hormones and neurotransmitters –4. SeverExocytosis proceeds through a sequence of distinct steps. Release can start once a fusion pore has formed to create a continuous fluid pathway from the vesicle interior to the extracellular space. The fusion pore is initially very small and can allow only small molecules such as norepinephrine to pass, but after it expands larger molecules such as chromogranins, insulin, and glucagon can escape. It is well established that DCVs of endocrine cells undergo two modes of exocytosis, kiss-and-run, and full-fusion –19. In k2+ triggers exocytosis by binding to syt  Ca2+ can rise to >100 μM, which is substantially higher than the average bulk cytoplasmic level, even under conditions of intense stimulation (2+ domains around a Ca2+ channel can form and collapse rapidly (within a few ms) so that the activation of a Ca2+ sensor will depend critically on the kinetics of Ca2+ association and dissociation. A low-affinity sensor can detect this high local Ca2+ as long as it binds with rapid kinetics. A high affinity Ca2+ sensor may fail to respond if it binds too slowly. Syt 1 has properties suited for responding to transient domains with high Ca2+. Syt 7 has properties suited for responding to modest but prolonged rises in bulk Ca2+ concentration  have been shown to trigger norepinephrine release from chromaffin cells without triggering chromogranin release (2+ concentration will rise to moderate levels (well under 50 μM) for longer times on the order of seconds  It is important to address the expression of multiple isoforms, either by ablating endogenous proteins or varying each isoform individually with careful assessment of protein levels. Overexpression of proteins can result in high protein concentrations, possibly leading to mis-targeting and artificial functions. (2) Experiments need to address the different Ca2+ sensitivities of syt isoforms. This requires measurement and control of Ca2+ concentration. (3) Biophysical techniques for measuring exocytosis are very sensitive but often measure surrogates of release and detect events on different time scales. Amperometry measures release and can detect rapid processes but its greatest sensitivity is realized with biogenic amines, and is not nearly as powerful in the measurement of peptides. Furthermore, after content expulsion the closure of a fusion pore can no longer be detected, and the mode of release as kiss-and-run or full-fusion can no longer be ascertained. Capacitance measures membrane area, and single-vesicle capacitance steps provide strong evidence for kiss-and-run. Total internal reflectance microscopy of fluorescent tracers follows the fate of the vesicle content or membrane (Studies of how different syt isoforms influence the kinetics of exocytosis are starting to resolve functionally relevant distinctions in the mechanisms by which Camembrane . Like camembrane . Amperommembrane .2+ signals. The syt isoforms present on a vesicle will determine what form of Ca2+ signal will trigger fusion, how different Ca2+ signals direct the choice between kiss-and-run and full-fusion, and what proportion of small and large molecules are released.Given the functional versatility within the syt protein family, the studies to date probably have only scratched the surface in addressing the roles of syt isoforms in the complex and varied forms of hormone release. By selecting among the different syt isoforms and regulating their expression, and by sorting to different vesicles containing different hormones, cells can regulate the response to diverse forms of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pasteurella multocida was undertaken. Twelve commercially produced rabbits of different ages and respiratory health status were divided into four groups: healthy from 0 to 21 days ; healthy from 23 to 49 days ; healthy from 51 to 69 days ; diseased rabbits with septicemia and the rhinitic form of P. multocida infection . The main ultrastructural changes observed were a widening of the interepithelial spaces, increased activity and number of goblet cells, the formation of two types of vacuoles in epithelial cells, the degranulation and migration of heterophils between the epithelial cells, and the association of this migration with some of the other changes. No bacteria were observed adhering to the epithelium, and very few were observed free in the mucus. Scant inter-epithelial spaces were found in healthy rabbits, but they were not as large and numerous as those found in diseased animals. We discuss the origin and meaning of these changes but, we focus on the significance of the inter-epithelial spaces and goblet cells for the defense of the upper respiratory airways against the bacterium and its lipopolysaccharide.An ultrastructural comparison between the nasal cavities of healthy rabbits and those suffering from two forms of spontaneous infection with  P. multocida is commonly associated with different entities of rabbits [ rabbits –7. Clini rabbits . P. multocida in different species, including the rabbit [P. multocida have been described in rabbits. The nasal cavity is considered the natural habitat of e rabbit ; howeverP. multocida A3, P. multocida A3 plus hydrocortisone, and P. multocida serotype D1. The experimental animals were killed 14 and 21 days after inoculation. In general, the gross findings in all three studies were congestion of the nasal mucosa, hemorrhages, and/or mucopurulent exudates. Histologically, these authors observed mild to chronic catarrhal and suppurative rhinitis, with few rabbits displaying ulceration of the mucosal surface. Ultrastructurally, they found severely degenerated epithelial cells that were swollen and had vacuolated cytoplasm; these changes involved the mitochondria and rough endoplasmic reticulum, and loss of cilia was also frequently seen. Necrotic cells had both loss of cilia and breakdown of the cell membrane. Polymorphonuclear heterophils and macrophages infiltrated between the degenerated epithelial cells and the subepithelial layer. Finally, goblet cell hyperplasia and hypertrophy were documented. Al-Haddawi et al. –13 descrP. multocida [P. multocida, though these changes were more severe in the latter. Other findings in diseased rabbits were goblet cell hyperplasia and infiltration of heterophils between epithelial cells, the latter of which was associated with damaged epithelial cells. To our knowledge, ultrastructural descriptions of the nasal cavities of healthy rabbits or rabbits with respiratory diseases have not been reported. On the other hand, histological or ultrastructural studies documenting lesions in the respiratory epithelium of the nasal cavities of rabbits affected with a natural respiratory disease are not frequent. Histologically, we reported various changes in this region in rabbits infected with ultocida  includinP. multocida type A. The main purpose of this study was to describe and compare the transmission electron microscopy (TEM) findings of the respiratory epithelium and the propria of the nasal septum from healthy rabbits of different ages under commercial production conditions with those of rabbits affected with the rhinitic and septicemic forms of P. multocida type A based on their cultural and biochemical characteristics [After the animals were euthanized, nasal and tracheal swabs as well as lung samples were collected for microbiology. The isolates were cultured in brain-heart agar (BHI) at 37°C for 24 h. Isolates were identified as eristics . For theeristics .Twelve New Zealand White rabbits of different ages from a commercial rabbitry located at the Savannah of Bogotá at 2600 m.a.s., Colombia, were divided into 4 groups: 2 healthy rabbits between 0 and 21 days of age (group 1), 2 healthy rabbits between 23 and 49 days (group 2), 2 healthy rabbits between 51 and 69 days of age (group 3), and 6 acute diseased rabbits, 3 with septicemia  and 3 with rhinitis (group 4). The animals were anesthetized i.m. with 35 mg/kg of ketamine and 5 mg/kg of xylazine then instilled with 2.5% glutaraldehyde  in Millonig's buffer (4°C) via the nasal cavity. The animals were euthanized with an overdose of anesthesia. The nasal cavity was cross-sectioned at the level of the first molar, and the nasal septum was taken. The samples were divided into two parts: one was immersed in buffered formaldehyde (3.7%) for paraffin embedding and the other in glutaraldehyde  for TEM. The experimental procedures were approved by the Veterinary School Bioethics Committee of the National University of Colombia. μm. Sections were stained with hematoxylin and eosin (H&E). Tissues were processed by routine paraffin wax embedding and sectioned at 5 2 area of tissue that included the epithelium, propria, and glandular epithelium was selected for ultrastructural evaluation. Ultrafine sections were made, contrasted with uranyl acetate and lead citrate and evaluated in a Phillips 110 TEM.The tissues for TEM were postfixed in osmium tetroxide (1%) and embedded in epoxy resins. Five semithin sections per animal were obtained and stained with toluidine blue. The normal and the pathological morphologies were described, and a 2 mmP. multocida was isolated from the nasal cavities and lungs of the animals in groups 3 and 4. The bacteria were identified by PCR as P. multocida A. Bacteria could not be isolated from the rabbits in groups 1 and 2, but n = 4) did not show major lesions or changes. The most important change was the appearance of intercellular spaces between cells of the respiratory epithelium. These spaces were represented by small dilatations between the cells, without separation at the apical end and were more common on the basolateral side of the cells , they showed different types of lesions in varying degrees. Interepithelial spaces were more obvious in these animals and were not limited to the basal region of the cells, with some cells completely separated, detached, and desquamated. Moderate to severe vacuolation of the epithelial cells was also present in both animals. Vacuoles of different sizes were observed, some of which filled the whole cytoplasm. Ultrastructurally, in one rabbit, the epithelium was lower, with apparent loss of cilia and excessive cytoplasmic vacuolation. The vacuoles were of different sizes and contained a membranous or granular material. In the second animal, the ciliated cells showed large vacuoles with granular content, which was heavily electrodense and resembled a typical phagosome in a phagocytic cell . In someAnimals in this group had the most intense and numerous lesions. In two of the rabbits, copious amounts of detritus, inflammatory cells, and mucus accumulated in the lumen, but this accumulation was moderate in the remaining rabbit. A small number of bacteria were also observed free in the lumen. The intercellular spaces were severely dilated , with soThe lesions differed in quantity but not in quality from those of septicemic rabbits, though some of them were milder in the rhinitic rabbits. Migrating inflammatory cells were associated with widened interepithelial spaces and cytoplasmic vacuolation, with both changes ranging from mild to moderate. More significant in the epithelium was the increased number of goblet cells, which in some cases were the only cells in a large area. Two rabbits were severely affected, while the remaining one was moderately so. Ultrastructurally, the goblet cells displayed high activity. They were larger than and out-numbered the ciliated cells over large areas of tissue. In between, lymphocytes and heterophils migrated through the intercellular spaces. P. multocida is the main pathogen associated with rhinitis, pneumonia, and septicemia in rabbits. The primary ultrastructural changes in some regions of the upper respiratory tract of rabbits have been described in experimentally infected animals [ animals , and a d animals . We did Mannheimia haemolytica infection in cows, one of the hallmark pathological changes is the extensive leakage of vascular products into the lung interstitium and air spaces [M. haemolytica infection, McClenahan et al. [The presence of interepithelial spaces in both healthy and diseased rabbits was one of the most important findings of group three. In healthy animals, however, such spaces were always restricted to the basal region of the epithelium Figures  and werer spaces –26. Lipor spaces . Becausen et al.  postulatn et al. –30. Diffn et al. –34. At least two potential mechanisms would explain the changes in the permeability and morphology of the cell monolayers: changes in the actin cytoskeleton or modifications to the tight junction proteins. It is likely that actin-myosin contraction creates intercellular gaps in both epithelial and endothelial cell monolayers , 30, 35,For the mucociliary apparatus to function properly as the first defense mechanism against different invaders, it is important that the fluid surrounding the cilia as well as the viscoelasticity of the mucus blanket over the cilia maintains a correct hydration and concentration of electrolytes. However, both of these defense responses are altered during infectious and inflammatory reactions. Three mechanisms for water secretion onto the surface of the airway epithelium have been elucidated: by serous cells in the surface epithelium , by serP. multocida, even as soon as two hours of exposure, was the increased number and activity of goblet cells, which makes this response one of the hallmarks of the lesions induced by these bacteria. Despite that the animals of group 3 were taken during the age of most frequent development of the respiratory entity, none of them showed clinical signs of the disease; although it is not possible to state that they would not develop the disease later, there was a significant change in rabbits of group 4 that was negligible or absent in the healthy groups, namely, the number and the activity of goblet cells, making this event unlikely. Recent findings from our group  demonstrHypertrophy, hyperplasia, and metaplasia of the mucous cells in the epithelium and of the mucous and serous glands in the submucosa are associated with oversecretion of mucus and are some of the most prevalent lesions in rhinitis, sinusitis, and tracheobronchitis . The intThe cytoplasmic vacuolation of epithelial cells in the nasal cavity was more severe in diseased animals, particularly, those with septicemia . This phP. multocida LPS. Kang et al. [In our study, three patterns of vacuolation were observed in the epithelial cells: one corresponded to swollen or exploded mitochondria, the most common one had the appearance of digested cellular components, and the final one contained material resembling myelin figures. These changes might be induced by the g et al.  found agg et al. . The accg et al. . In our g et al. , 48. ThiIn addition, there appeared to be a direct relationship between the number and size of vacuoles, the extension of intercellular spaces, and the number of migrating heterophils. The heterophils of the diseased rabbits seemed to be degranulating, which was reflected by the decreased quantity and smaller size of their granules, leaving clear spaces in the cytoplasm. Klut et al.  documentIn asthma, bronchial hyperreactivity, infectious pneumonia, endotoxemia, and so forth, authors have demonstrated that heterophils and eosinophils are responsible for tissue damage , 50–53. P. multocida A3 were obtained by Al-Haddawi et al. [Pasteurella adhering to degenerated cilia, the microvilli, and the mucus [9 UFC) intranasally in rabbits free of P. multocida, our animals developed the disease naturally. Ultrastructural changes similar to those observed here in the upper airways of rabbits inoculated experimentally with i et al. . The prihe mucus . In sevehe mucus , 54, andhe mucus  and ourshe mucus  inoculatP. multocida was the only, or even the main, etiologic factor of the respiratory disease in the rabbits described here because there were few of these cells and they did not adhere to the epithelium. The TEM technique is highly sensitive to detect this host-microorganism interaction. Even more, cell damage was not associated with the presence of microorganisms. In our opinion, more attention should be paid to some of its toxins, such as LPS, rather than to bacterial proliferation alone. Our findings do not guarantee that Endotoxins are commonly found in different environments , 55, andP. multocida by TEM. The importance of plasma transudation through the epithelial cells of the rise in mucus production by goblet cells to defend against the infection is thoroughly discussed. We also discuss the meaning of the small number of bacteria found in this work. Findings such as the migration of heterophils through the epithelium and vacuole formation are also taken into account. In summary, to our knowledge, this is the first description of the nasal cavity of healthy rabbits and of rabbits afflicted with the spontaneous form of the disease caused by