In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca2+ releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na+-Ca2+ exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).Mitochondria dynamically buffer cytosolic Ca2+ (Ca2+m), cytosolic Ca2+ (Ca2+c)-SR-Ca2+ crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.To determine if mitochondrial Ca2+ influx into (Ru360) or Ca2+ efflux from (CGP-37157) decreased [Ca2+]m to 80±8% control or increased [Ca2+]m to 119±7% control, respectively. Concurrent with inhibition of mitochondrial Ca2+ influx or efflux, the SR Ca2+ load, and LCR size, duration, amplitude and period  significantly increased or decreased, respectively. Changes in total ensemble LCR Ca2+ signal were highly correlated with the change in the SR Ca2+ load (r2 = 0.97). Changes in the spontaneous AP cycle length  in response to changes in [Ca2+]m were predicted by concurrent changes in LCR period (r2 = 0.84).Inhibition of mitochondrial Ca2+m flux translates into a change in the AP firing rate by effecting changes in Ca2+c and SR Ca2+ loading, which affects the characteristics of spontaneous SR Ca2+ release.A change in SANC Ca Unlike VM, even in the absence of β-adrenergic stimulation in SANC, high levels of basal cAMP and cAMP-mediated protein kinase A-dependent (PKA) and Ca2+/calmodulin-dependent protein kinase II phosphorylation of Ca2+ cycling-proteins of both clocks enables the SR (Ca2+ clock) to generate spontaneous rhythmic local, subsarcolemmal Ca2+releases (LCRs) via SR ryanodine receptors. LCRs activate an inward Na+-Ca2+ exchange current that accelerates the rate of diastolic depolarization, prompting the membrane clock to generate the next action potential (AP).Sinoatrial nodal cells (SANC) initiate each heart beat by generating spontaneous APs that emanate from the sinoatrial node. Numerous studies over the last decade indicate a crucial role for SR cycling in SANC normal automaticity, giving rise to a 2+ flux into and out of mitochondria in SANC will directly affect SR Ca2+ loading, and thus indirectly affect SR Ca2+ release, and therefore that a change in mitochondrial Ca2+ (Ca2+m) cycling will modulate the normal automaticity of the coupled-clock system.We hypothesized that changes in Ca2+ cycling. But, this method can not be explored in SANC, which fire spontaneous AP’s. Therefore, to test our hypothesis that mitochondrial-SR Ca2+ crosstalk exists and links to SANC AP firing, we measured [Ca2+]c, [Ca2+]m, SR Ca2+ load and Ca2+ releases, and the spontaneous AP firing rate in intact, single, isolated SANC in response to specific inhibitors of Ca2+ flux into (Ru360) and Ca2+ flux from (CGP-37157) mitochondria.Changing the external electrical stimulation rate in VM is a convenient approach to induce changes in cross-talk between mitochondrial and SR Ca2+m was indexed by selective Mn2+ quenching of the cytosolic fluorescence of the Ca2+ probe, Indo-1 2 to Indo-1-AM loaded SANC selectively quenched the cytosolic Indo-1 fluorescence ratio 410/490 in a time dependent manner (11±2 min), as evidenced by the disappearance of the AP-induced cytosolic Ca2+ transient . Saponin (25 µg/ml) reduced the fluorescence to 64±5% (n = 5), i.e. to the same level as permeabilizing of sarcolemma with the lower concentration of digitonin. Thus, when the SANC plasma membrane was selectively permeablized, the Indo fluorescence decreased to the same level as it did in cells with an intact surface membrane in the presence of Mn2+. In contrast, a higher concentration of digitonin, which also permeabilizes mitochondrial membranes, decreased the fluorescence to 10±4% control . These results provide evidence that the Mn2+-resistant fluorescence of Indo-1 loaded cells is derived from Indo-1 within the mitochondrial compartment. Note, that in the time-control experiment protocol without Mn2+ application, Indo-1 fluorescence was only slightly quenched (−7±3%).To define the compartmental Indo-quenching, we quenched Indo-1 fluorescence using varying concentrations of digitonin: a low concentration of digitonin (5 µM) or saponin (25 µg/ml), which permeabilizes only the sarcolemma ; and a h2+]m. 2+]m to 80±8% of control (from 210±19 to 171±19 nM) of the Mn2+ quenched control; conversely, CGP-37157 (1 µM) increased [Ca2+]m to 119±7% of control (from 210±19 to 270±13 nM)  caused, over a 10-min period, a progressive increase in the spontaneous SANC AP firing rate to 111±1% control (from 2.7±0.25 to 3±0.3 Hz). Conversely, inhibition of Ca2+ efflux from mitochondria by CGP-37157 (1 µM) progressively reduced the spontaneous AP firing rate to 89±2% control (from 2.65±0.2 to 2.3±0.2 Hz) over a 10-min period. Note that both effects evolved and saturated with roughly the same time course. These drug effects were not reversible even after 10-min wash. The changes in other AP parameters are summarized in The main question of our study is whether a change in Ca2+ buffering by the mitochondria affects [Ca2+]c we loaded cells with Indo-1AM. Changes in spontaneous AP firing rate by inhibition of mitochondrial Ca2+ influx or efflux were determined in Indo-1/AM loaded cells (see representative examples in 2+c transients measured by Indo-1 and 2+c transients in the presence of Ru360 or CGP-37157. Inhibition of Ca2+m influx significantly increased peak systolic Ca2+ from 380±23 to 415±28 nM (p = 0.001) and decreased the 90% decay time of cytosolic [Ca2+] (T-90c) from 302±13 to 285±11 ms (p = 0.04). The diastolic Indo fluorescence (p = 0.6), time to peak Ca2+ , and 50% decay time of cytosolic Ca2+  were not significantly altered. Conversely, inhibition of Ca2+m efflux significantly decreased peak systolic [Ca2+] from 369±13 to 342±13 nM (p = 0.02) and increased T-90c from 301±14 to 325±12 ms (p = 0.04). The diastolic Indo fluorescence (p = 0.12), T-Pc (p = 0.3), and T-50c (p = 0.2) were not significantly altered. Therefore, the effects of inhibition of [Ca2+]m influx or efflux on [Ca2+]c in SANC are similar to their effects in prior studies in guinea pig ventricular cardiac myocytes To determine how Ca2+ cycling are determined, in part, by the SR Ca2+ load. Perturbations of mitochondrial Ca2+ flux might affect SR Ca2+ loading if these two organelles were located substantially close to each other. To determine the proximity of mitochondria to SR we employed dual immunolabeling to SANC with anti-SERCA2 antibody and Mitotracker orange to visualize the SR and mitochondrial membrane, respectively. SANC of varying size exhibit robust uniform SERCA2 immunolabeling  onto the cell following drug application to perturb Ca2+m flux. Representative examples and average data are presented in 2+ influx into mitochondria increased the SR Ca2+ load (assessed by change in amplitude of the Ca2+ transient induced by a brief rapid application of caffeine) by 17±5% of control ; conversely inhibition of Ca2+ efflux from mitochondria reduced the SR Ca2+ load by 13±2% of control  , these effects were not statistically significant , and the LCR duration from 35±0.8 to 41±0.8ms (p<0.001). In contrast to Ru360, CGP-37157 decreased the average LCR size from 4.3±0.1 to 2.6±0.1 µm (p<0.001), the average LCR amplitude from 1.22±0.02 to 1.14±0.01F/Fo (p = 0.02), and the average LCR duration from 35±0.5 to 29±0.6ms (p<0.001).Prior studies have shown that changes in SR Caexamples  show tha2+ of the LCR ensemble shifted to lower values in response to CGP-37157, and to higher values in response to Ru360 (2+ from 38±6 to 61±7 ms*µm*F/F0 (p<0.001) and CGP-37157 decreased it from 35±3 to 17±2 ms*µm*F/F0 (p = 0.003). The Ru360-induced increase, and the CGP-37157-induced decrease in the total LCR ensemble Ca2+ paralleled the effects of Ru360 and CGP-37157 on the amplitude of caffeine induced Ca2+ release into the cytosol  to an LCR onset . The relationship between the change in LCR period and AP cycle length by perturbations of Ca2+m flux is quantitatively strikingly similar to that which occurs in response to other interventions which affect the LCR period and AP cycle length of SANC m, the spontaneous AP firing rate, nor LCR characteristics  (5 µM) 2+m decreased  decreased the spontaneous AP firing rate by 15±3%  and reduced the SR Ca2+ load by 17±3% . This decrease in SR load by CPA is comparable to decreases in both spontaneous AP firing rate and SR Ca2+ load effected by CGP-37157. These results are consistent with our interpretation that the changes in AP cycle length due to manipulation of [Ca2+]m content are linked to changes in the SR Ca2+ load.To prove that a change in SR load is a necessary component in the feedback mechanism between m, under basal conditions, and when mitochondrial Ca2+ flux is inhibited (2+ influx by Ru360 decreased the maximum diastolic [Ca2+]m (from 89.8 to 60 nM) and the systolic [Ca2+]m (from 222.7 to 160 nM). Inhibition of mitochondrial Ca2+ efflux by CGP-37157, however, increased both diastolic [Ca2+]m (to 137.2 nM) and systolic [Ca2+]m (to 308 nM). Note, that manipulation of Ca2+m affected the cycle length without affecting If kinetics, which is also consistent with our experimental result. Moreover, the model predicts that sarcolemmal Na+-Ca2+ exchanger current kinetics are altered indirectly due to change in LCR characteristics  to measure the kinetics of the changes in intracellular Caing rate . Simulat2+ cycling proteins and surface membrane electrogenic molecules that regulates SANC normal automaticity includes SR-mitochondrial Ca2+ cross-talk.In summary, we conclude that the coupled clock system of intracellular Ca2+ cross talk, previously demonstrated in ventricular myocytes 2+ cycling into and out of mitochondria in SANC, effected by using Ru360 and CGP-37157, modulate basal coupled-clock system automaticity via an impact on Ca2+c, SR loading and indirectly on Ca2+ release characteristics: blocking mitochondrial Ca2+ efflux increases [Ca2+]m, decreases the SR load, decreases the amplitude of the AP triggered cytosolic Ca2+ transient and total Ca2+ released spontaneously by the LCR ensemble, and reduces the AP firing rate. Conversely, reducing mitochondrial Ca2+ influx decreases [Ca2+]m, increases SR Ca2+ loading, increases the amplitude of the AP triggered cytosolic Ca2+ transient and total Ca2+ released spontaneously by the LCR ensemble, and increases the AP firing spontaneous rate . External stimulation in SANC is not feasible, however, because SANC beat spontaneously, and it is challenging to over-drive the spontaneous SANC AP firing rate, while maintaining a normal rhythm . Beat-to-beat Ca2+m flux into or out of the mitochondria of SANC in the present study can be roughly estimated, however assuming that Ru360 or CGP-37157 reduces the influx or efflux by 30%, respectively , and assuming a linear time-course of drug effects to inhibit their targets. Given the experimentally determined time course of the drug-induced changes in [Ca2+]m, and the average number of spontaneous AP cycles over this time, a rough estimation of drug-induced changes in Ca2+ flux from or into the mitochondria per AP cycle is on the order of 0.5 nM/beat.Whether mitochondrial Ca2+m does not substantially affect [Ca2+]m itself, or affect other functions within SANC that are related to the AP firing rate. Because our major goal was to explore whether perturbations of mitochondrial Ca2+ cycling modulates the basal AP firing rate of SANC, neither cell permeabilization 2+]m that requires acute rupture of a membrane patch, often creating a small leak current, that notably affects the balance of ionic currents during diastolic depolarization and spontaneous AP frequency of SANC 2+ quench approach to assess [Ca2+]m in SANC because measurements of [Ca2+]m in VM by cell permeabilization and Mn2+ quench are in close agreement 2+ to selectively quench Ca2+c fluorescence, without causing marked changes in Ca2+m, is based on its slower quenching of the mitochondrial fluorescence 2+ that rapidly quenches cytosolic Ca2+but has minimal effects on sarcolemmal or mitochondrial Ca2+ ion transport be employed and 2) that during Ca2+c quenching the temperature be maintained at a near physiological level. We employed 50 µM Mn2+ to quench Ca2+c fluorescence, which at this concentration has no effect on our major functional endpoint, the spontaneous AP firing rate. Specifically, since we previously have shown 2+ and AP firing rate, the lack of a sustained effect of Mn2+ on the SANC spontaneous AP firing rate strongly suggests that there is no functionally significant inhibition of sarcolemmal Ca2+ channels (i.e. L-type channels) by 50 µM Mn2+. Another concern about the Mn2+ quench technique is that Mn2+ can inhibit oxidative phosphorylation. The lack of a sustained effect of Mn2+ on the basal spontaneous AP firing rate again suggests that Mn2+ does not perturb the matching of ATP supply to demand, since we have previously shown that this matching is closely linked to the AP firing rate The validity of our interpretations of the present results assumes that the method used to measure Ca2+m flux is another important issue that merits consideration in interpreting the present results. We employed Ru360 at concentration of 2 µM to selectively block Ca2+ influx into the mitochondria, i.e. a concentration similar to that used in prior studies in VM 2+ influx to the mitochondria and to decrease [Ca2+]m. In rat VM, in which Ca2+c is not substantially affected by changes in Ca2+m, this concentration of Ru360 does not affect SR Ca2+ uptake or L-type current 2+m is higher during ouabain application than during control conditions The selectivity of drugs employed to perturb Ca+-Ca2+ exchanger (IC50 = 0.5 µM) 2+]m, but has only minor effects on sarolemmal Na+-Ca2+ exchanger (IC50 = 13 µM), or Ca2+ uptake by SR (IC50 = 10 µM) or on ryanodine receptor open probability (IC50 = 9.5 µM) 2+ current is a crucial element of SANC automaticity 50 = 0.3 µM) We employed CGP-37157 at concentration of 1 µM because it selectively blocks the mitochondrial Na2+ flux affect the spontaneous AP firing rate of SANC via changes in SR Ca2+ loading and release. Furthermore, while our experimental method to measure [Ca2+]m does not permit evaluation of systolic and diastolic [Ca2+]m, our model predicts that under basal conditions both diastolic and systolic [Ca2+]m are lower than the diastolic and systolic [Ca2+]c, respectively. However, the systolic [Ca2+]m is higher than diastolic [Ca2+]c.Our novel numerical model simulations reproduce our experimental findings and validate our interpretation of these findings: changes in mitochondrial Ca2+ cycling into and out of the mitochondria acts as a dynamic buffer of cytosolic Ca2+, thus affecting Ca2+ availability for SR Ca2+ loading and release. When mitochondrial Ca2+ influx or Ca2+ efflux is selectively blocked, SR Ca2+ cycling and Ca2+ load are altered. This leads to changes in spontaneous AP triggered SR Ca2+ release that produce changes in both global AP induced cytosolic Ca2+ transients and to spontaneous, localized submembrane diastolic SR Ca2+ release. Therefore, under basal conditions mitochondrial Ca2+ fluxes modulate SR Ca2+ cycling via an effect on SR Ca2+ loading, and on this basis mitochondrial Ca2+ flux is indirectly linked to the SANC spontaneous AP firing rate.In summary, the present study shows, for the first time, that SANC mitochondria and SR are closely associated, i.e., that Ca2+ cycling were studied in Tyrode solution at 35±0.5°C, with the following composition (in mM): 140 NaCl, 5.4 KCl, 2 MgCl2, 5 HEPES, 1.8 CaCl2, and 5.5 Glucose, and was titrated to pH 7.4 with NaOH. Cells were imaged with an LSM-510 inverted confocal microscope using a 63×/1.4 N.A. oil immersion lens (Carl Zeiss). Linescan images , were recorded with a scan line oriented along the short axis of the cell to quantify the spontaneous AP induced contraction rate. Cell contraction measurements were recorded for 15 min under control conditions and following drug application for times specified. Spontaneous action potentials were measured via a perforated patch-clamp technique with 35 µM β-escin (Sigma) added to the pipette solution that contained (in mM): 120 K-gluconate, 5 NaCl, 5 MgATP, 5 NaATP, 5 HEPES and 20 KCl, pH 7.2 with KOH. Action potentials were recorded using an Axopatch-200 B patch-clamp amplifier . For both cell contraction and electrophysiological recordings cells from at least 5 rabbits were used.Single, spontaneously beating SANC were isolated from New Zealand White rabbit hearts as previously described 2+ was measured with calibrated Indo-1 fluorescence to assess cytosolic Ca2+ signals. SANC were placed in a chamber on the inverted fluorescence microscope stage (Zeiss IM-35) and were loaded with 14 µM Indo-1 AM (Molecular Probes) for 15-min at room temperature, and subsequently superfused with Tyrode’s solution for 20-min to remove excess indicator and allow full de-esterification as the bath temperature slowly was increased to 35±0.5°C. The apparatus to detect Indo-1 fluorescence is as described previously 2+]i was calculated according to the equation [Ca2+]i = βxKdx(R-Rmin)/(Rmax-R), using a Kd of 844 nM. The average Rmin , Rmax , and β  for the fluorescence system were determined by sequential exposure of SANC to a high potassium, zero- Ca2+ solution  containing metabolic inhibitors  (2), the same solution with 1 mM EGTA and 20 µM ionmycin (for Rmin). Measurements were taken when the fluorescence at both wavelengths reached stable values, and (3) high Ca2+ Tyrode’s solution (5 mM Ca2+ instead of EGTA) was used for determining Rmax. Average Rmax, Rmin and β were 1.9±0.16, 0.9±0.01, and 2.1±0.6, respectively (n = 10). The method limitation derived from: 1) a change in pH from the calibrated solution (pH = 7.4 in bath solution compare to 7.2 see above), 2) imprecision of Kd of Indo-1 and 3) partial quenching of mitochondrial Ca2+ by Mn2+ affect the calculated [Ca2+]m. For Ca2+ recordings cells from at least 5 rabbits were used.Intracellular Ca2+ cycling into and out of the cytosol was measured with Flou-4 AM (Molecular Probes) to assess AP triggered Ca2+ release, spontaneous LCRs, and caffeine induced SR Ca2+ release. SANC were loaded with 5 µM Flou-4 AM for 20 min at room temperature, and subsequently superfused with Tyrode’s solution at 35±0.5°C. The Ca2+ fluorescence was imaged by a LSM510 confocal microscope (see above) using a 40x/1.3 N.A. oil immersion lens. Cells were excited with the 488 nm laser line of an Ar laser, and fluorescence emission was collected with LP 505 nm, with the pinhole set to obtain no more than 5 µm optical slice (512×1 pixels at 14.9 pixel/µm and 2 ms/line). All images were recorded with a scan line oriented along the long axis of the cell, close to the sarcolemmal membrane, and processed with IDL software or Matlab.Ca2+ transient was expressed as a peak value (F) normalized to minimal fluorescence (F0). The amplitude of individual LCRs was also expressed as a peak value (F) normalized to minimal fluorescence (F0). LCR spatial size (FWHM) was indexed as the full width at half-maximum amplitude. LCR duration (FDHM) was characterized as the full duration at half-maximum amplitude. The number of LCRs was normalized per 100 µm of the linescan image and during a 1s time interval. The integral of Ca2+ of an individual LCR was estimated as follows: FWHMxFDHMx(F/F0-1)/2. The total Ca2+ released by LCR ensemble was calculated as the sum of the signal masses of diastolic LCRs that occurred between AP-induced Ca2+ transients. The SR Ca2+ content was estimated by the amplitude of the Ca2+ transient induced by a brief rapid application of caffeine  onto the cell by pressure-ejection via a nearby pipette, as described previously 2+ recordings cells from at least 5 rabbits were used.The AP induced Ca2+] ∼1 mM), 20 HEPES, 0.5 EGTA, 10 phosphocreatine, and 5 U/ml creatine phosphokinase; pH 7.2 with KOH.SANC were permeabilized with 5 or 25 µM digitonin at 35±0.5°C with solution containing (in mM): 100 K aspartate, 25 KCl, 10 NaCl, 3 MgATP, 0.8 MgCl2,  at 4°C overnight. This was followed by labeling with a Cy5-conjugated secondary antibody  incubation. The fluorescence was imaged by a LSM510 confocal microscope (see above) using a 40x/1.3 N.A oil immersion lens. The Cy5 and MT fluorophores were excited with 633 and 543 nm He-Ne lasers, respectively.SANC were incubated with 500 nM Mitotracker Orange , for 1 h at 37°C, fixed with 4% formaldehyde in phosphate buffer (PBS) and permeabilized with 1% Triton X-100/PBS. Nonspecific cross-reactivity was blocked by incubating the samples overnight in solution containing: 2% BSA/PBS, 5% donkey serum, 0.01% NaN3VO4. Total protein concentration was determined using a standard, commercially available protein quant kit . The accuracy of protein quantification was verified on the 4–12% Bis-Tris polyacrylamide SDS-PAGE gel (Invitrogen) labeled with the fluorescent Sypro Ruby gel stain (Invitrogen). The total integral of fluorescence intensities for each protein lane was calculated using ImageQuant5 software . 5-15 µg of proteins from each aliquot were mixed with the NuPage sample buffer and DTT , and resolved on 4–12% Bis-Tris polyacrylamide SDS-PAGE gel using standard MES/SDS running buffer. Proteins were transferred for 25 min to the activated PVDF membrane  using standard semidry transfer unit (Bio-Rad) in NuPage transfer buffer . PVDF membranes with transferred proteins were blocked overnight in 5% nonfat milk (Bio-Rad), and resuspended in TBS-T buffer .SANC suspensions were divided equally into 4 aliquots: the first aliquot was treated with 2 µM Ru360, the second with 1 µM CGP-37157, the third with 1 µM isoproterenol; and the forth was used as a control. Cells were incubated at 36°C for 10 min. After the treatment, samples were centrifuged and the pellets were frozen in liquid nitrogen. Pelleted cells were resuspended in RIPA lysis buffer  that also included a standard mammalian protease inhibitor cocktail (Sigma) and phosphatase inhibitors 1 mM NaF, 2 mM NaTo detect total phospholamban the first membrane was incubated over-night with a total anti-PLB monoclonal phospholamban antibody  and incubated 1 h at room temperature with goat anti-mouse conjugated antibodies . To detect phospholamban phosphorylation, the second and the third membranes were incubated over-night with anti-P-Ser-16 rabbit polyclonal antibody  or the anti- P-Thr-17 rabbit antibody , and incubated for 1 h at room temperature with polyclonal goat anti-rabbit secondary antibody . To detect actin, the membranes were incubated over-night with anti-actin goat polyclonal antibodies , and incubated for 1 h at room temperature with donkey anti-goat-IgG-HRP secondary antibody . Membranes were developed for 5 min using SuperSignal West Pico (or Dura) Chemiluminescent Substrate (Thermo Fisher Scientific) and visualized on the HyBlot CL autoradiography film using a series of sequential exposure times (Denville Scientific Inc.) to obtain an optimum signal intensity.+-Ca2+ exchanger inhibitor, and Ru360, an inhibitor of Ca2+ flux into the mitochondria, were purchased from EMD Chemicals; Isoproterenol, digitonin, saponin, ionmycin, cesium chloride, cyclopiazonic acid, caffeine and MnCl2 were purchased from Sigma.CGP-37157, a mitochondrial NaP<0.05 was taken to indicate statistical significance.Data are presented as mean±SEM. When the means of paired samples were to be compared, a paired t-test was employed. For multiple pharmacologic treatments, a linear mixed-effects model with Dunnett’s method to adjust p-values was used. This model accounts for repeated measurements on the same preparation while allowing testing for differences among pharmacological perturbations. Figure S1Validation of compartmentatal indo-quenching. (A–B) Low concentration of digitonin (5 µmol/L) does not permeabilize mitochondrial membrane, however high concentration of digitonin (25 µmol/L) does permeabilize mitochondrial membrane (C–D) visualized by 125 nmol/L tetramethylrhodamine methyl ester (TMRM).(TIF)Click here for additional data file.Figure S2Representative examples of Indo-1 fluorescence. Mn2+ (50 µmol/L) quenching of the cytosolic indo-1 fluorescence ratio 410/490 in the presence of Ru360 (upper panel) and CGP-37157 (lower panel).(TIF)Click here for additional data file.Figure S3Co-immunoelabeling of SERCA2 and part of the mitochondrial mass in SANC, visualized by anti-SERCA2 antibody and Mitotracker orange (MT) staining, respectively.(TIF)Click here for additional data file.Figure S4Western blots of phospholamban phosphorylation. (A) Representative examples of phospholamban phosphorylated at serine-16 site (PKA site) and total phospholamban in the basal state and following Ru360 (2 µmol/L), CGP-37157 (1 µmol/L) or isoproterenol (1 µmol/L). Actin is used as a protein loading control. (B) Fluorescent Sypro Ruby gel stain to validate the accuracy of protein loading. (C) Average phosphorylation ratio (n = 5) of phospholamban phosphorylated at serine-16 site to protein loading. *p<0.05 vs. control.(TIF)Click here for additional data file.Figure S5Average time-dependent change in the rate of AP induced contractions in the presence of CsCl and CGP-37157 (n = 7) or CsCl and Ru360 (n = 7).(TIF)Click here for additional data file.Figure S6Mitochondrial-SR numerical model. The extended coupled clock numerical model simulations of the effect of specific inhibition of Ca influx into or efflux from mitochondria in intact SANC on (A) Ca2+ in network SR, (B) Ca2+ in junctional SR, (C) Ca2+ release flux from the SR, and (D) Ca2+ in the sub membrane space.(TIF)Click here for additional data file.Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.Text S1(DOC)Click here for additional data file.


In view of conflicting reports, we have re-examined the issue by manipulating luminal Ca2+ with the membrane-permeant, low affinity Ca2+ buffer, TPEN, and monitoring NAADP-induced Ca2+ release in sea urchin egg homogenate. NAADP-induced Ca2+ release was almost entirely blocked by TPEN (IC50 17–25 μM) which suppressed the maximal extent of Ca2+ release without altering NAADP sensitivity. In contrast, Ca2+ release via IP3 receptors was 3- to 30-fold less sensitive to TPEN whereas that evoked by ionomycin was essentially unaffected. The effect of TPEN on NAADP-induced Ca2+ release was not due to an increase in the luminal pH or chelation of trace metals since it could not be mimicked by NH4Cl or phenanthroline. The fact that TPEN had no effect upon ionophore-induced Ca2+ release also argued against a substantial reduction in the driving force for Ca2+ efflux. We propose that, in the sea urchin egg, luminal Ca2+ is important for gating native NAADP-regulated two-pore channels.The regulation of Ca Eggs were then washed four times in Ca2+-free ASW (the first two washes containing 1 mM EGTA) and then washed in intracellular-like medium (GluIM (mM): 250 potassium gluconate; 250 N-methylglucamine (NMDG); 20 HEPES and 1 MgCl2, pH 7.2). Homogenization was effected with a glass Dounce tissue homogenizer in ice-cold GluIM supplemented with 2 mM MgATP; 20 U/ml creatine phosphokinase; 20 mM phosphocreatine; Complete™ EDTA-free Protease Inhibitor tablets (Roche). Homogenate  was centrifuged at 13,000 × g, 4 °C for 10 s and the supernatant stored at −80 °C. On the day of use, an aliquot of homogenate was sequentially diluted in equal volumes of GluIM containing the ATP regenerating system over a period of 3 h at 17 °C to give a 2.5% (v/v) final concentration.Sea urchin egg homogenate was prepared as detailed 2.22.2.12+ release was measured in homogenate with 3 μM fluo-3 (excitation/emission: 506/526 nm) which was calibrated using the standard equation [Ca2+] = Kd × (F − Fmin)/(Fmax − F), using a Kd of 0.4 μM; Fmin and Fmax were determined by addition of 0.5 mM EGTA and 10 mM Ca2+ respectively at the end of each run. TPEN (dissolved in ethanol) had no effect upon dye calibration . The upstroke kinetics were determined by linear regression of the raw fluorescence (in units (U)/s) normalized to the resting fluorescence (F0) to account for machine variability and therefore expressed as units•F0/s (U•F0/s).All fluorimetry was conducted at 17 °C in a microcuvette containing a magnetic stir bar mounted in a Perkin Elmer LS-50B fluorimeter. Ca2.2.2L) as before L. Data were expressed as a percentage of the maximum minus minimum fluorescence , P < 0.01 (**), P < 0.001 (***). Curve fitting was conducted using Graphpad Prism.Representative traces are plotted as raw fluorescence  against time. Data are expressed as the mean ± SEM. Two data sets were compared using Student's 3 was from LC Laboratories . Acridine orange and fluo-3  were from Invitrogen . Nigericin, TPEN, phenanthroline and potassium oxalate were from Sigma–Aldrich whilst ionomycin (free acid) was from Calbiochem-Novabiochem . All other reagents were of analytical grade.NAADP was enzymatically synthesised 33.12+ release (NICR) in sea urchin egg homogenate was monitored fluorescently in the presence of ethanol vehicle or the membrane-permeant, low affinity  because the response to the Ca2+ ionophore, ionomycin, was essentially unaffected  – amplitude: ethanol 32 nM (9–113 nM), TPEN 56 nM (7–47 nM); kinetics: ethanol 79 nM (17–370 nM), TPEN 218 nM (33–1460 nM)).NAADP-induced Caaffected A–C, as w3.22+, the IP3 receptor. Similar to NICR, IP3-induced Ca2+ release was also inhibited by TPEN but a major difference was that it required 3- to 30-fold higher concentrations of TPEN  – amplitude: ethanol 217 nM (46–1028 nM), TPEN 143 nM (50–413 nM); kinetics: ethanol 1303 nM (450–3774 nM), TPEN 953 nM (11–8106 nM)). The data are consistent with TPEN altering ER channel gating by chelating luminal Ca2+ as it does in other systems To ascertain whether the effect of TPEN was unique to NICR, we examined the effect of TPEN upon another channel regulated by luminal Ca release D–F. As wmplitude G–I: IP3 3.33 releases Ca2+ from the neutral ER, NAADP mobilizes Ca2+ from acidic Ca2+ stores which, in the sea urchin egg, appear to be the lysosome-related organelles, yolk platelets 2+ store loading L), we investigated whether the inhibition by TPEN was due to changes in pHL rather than Ca2+. First, we monitored pHL in NAADP-sensitive vesicles using acridine orange as reported previously L slightly as judged by the increase in acridine orange fluorescence but with a lower potency than its effect upon NICR  with an EC50 of ∼1 mM  and Ca2+ release in the presence of TPEN or NH4Cl (L to inhibit NICR (as seen with NH4Cl), it is clear that the effect of TPEN is greater than would be expected from an effect on pHL alone. We conclude that TPEN does not act via pHL.Whilst IPof ∼1 mM B and C bpon NICR D and E ol stores D and F. or NH4Cl G and H: 2+ and Fe2+ with high affinity Kds in the nanomolar to low micromolar range) but its Kd for Ca2+  facilitates Ca2+ uptake 2+ uptake. Note that sea urchin egg acidic vesicles are not very H+ “leaky” +-ATPase inhibitor, bafilomycin A1, to passively collapse ΔpH. Instead, we used nigericin  to rapidly dissipate the ΔpH of acidic vesicles without substantially altering their Δψ2+ release via the basal leak pathway can be revealed.The rate and extent of Ca2+ from the NAADP-sensitive acidic vesicles. Increasing concentrations of nigericin promptly released Ca2+ and the size of the NAADP-sensitive store was subsequently assessed by application of a high concentration of messenger: we observed a reciprocal relationship between the extent of nigericin- and NAADP-induced Ca2+ release . The reduction of the response to NAADP was not simply a consequence of nigericin raising the baseline because thapsigargin, which also elevates Ca2+ by mobilizing the ER Ca2+ stores, has no effect upon NICR We have previously shown that nigericin collapses ΔpH in sea urchin egg acidic vesicles  release A and B c2+ store, the response to nigericin was small when added after NAADP  gating.We then directly assessed the effect of TPEN upon nigericin-induced Ca release F and G.  release G. This i42+ store filling  modulates resident receptor channels in the store membrane has been with us for many years, applied first to RyRs and then later to IP3Rs 2+ homeostatic proteins have emerged that tailor their activity to the luminal Ca2+ content such as SERCA 2+ singled them out as unique 2+ stores and the luminal Ca2+ was manipulated with ionomycin 2+ stores 2+ chelator.The idea that the degree of Ca4.12+ release from any acidic store. The sea urchin egg homogenate has proven an excellent system in which to investigate fundamental properties of NICR owing to its ease of use and access to the cell ‘cytosol’ 2+  2+] in the ER and given insights into RyR and IP3R gating 2+ entry To the best of our knowledge, TPEN has not been previously used to investigate NICR or indeed CaL (acridine orange fluorescence)  it does not alter basal [Ca2+]; (ii) it does not affect ionomycin- or nigericin-induced Ca2+ release; (iii) the Ca2+ calibration parameters Fmin and Fmax are unaffected. Therefore, the inhibition of NICR is not merely an artefact of “cytosolic” Ca2+ buffering.First, does TPEN actually enter the acidic vesicle lumen? The fact that it promptly and persistently increases pHescence)  is consi2+ release. Dealing with the latter, if the components of the electrochemical potential  are substantially altered by TPEN, this would abrogate Ca2+ egress from the store. Our data suggest that this is not the case because Ca2+ release by ionophores would also be subject to the same thermodynamic constraints and yet the responses to ionomycin  and nigericin (acidic stores) were not reduced by TPEN.Logically, it follows that TPEN must be interfering with the NAADP-regulated TPC itself or with the electrochemical driving force for Ca4.2L or heavy metals, but TPEN has also been reported to inhibit SERCA We presented evidence that TPEN does not act by altering pH2+-binding agent, oxalic acid, which also loads into the lumen of Ca2+ stores 2+ release and Ca2+-dye calibration (data not shown) and so we could not differentiate between potentially real luminal effects upon NICR and increased ‘cytosolic’ Ca2+ buffering by high concentrations of oxalate.To offset concerns about TPEN pharmacology, we attempted to use a chemically dissimilar Ca3-induced Ca2+ release  indicated that the effect is not peculiar to NAADP-regulated channels. Since TPEN does not directly block IP3Rs 2+2+ upon IP3 receptor gating remain unclear , there would have to be a steep relationship between luminal Ca2+ and channel (IP3R or TPC) opening, i.e. chelating Ca2+ over a narrow range alters channel function. Furthermore, modulation by luminal Ca2+ would have to be different between sea urchin and human because NAADP affinity was shifted by luminal Ca2+ in human TPC2 2+  priming NAADP-sensitive stores.In summary, we show that the luminal Ca


In normotensive anesthetized rats, oligopeptides induced an immediate and transient hypotensive activity. In rat aortic rings, oligopeptides induced a concentration-dependent vasorelaxation in vessels precontracted with both KCl and phenylephrine (PE) with endothelium-intact or endothelium-denuded rings. In endothelium-intact rings, pretreatment with Nω-Nitro-L-arginine methyl ester hydrochloride , an inhibitor of the NO synthase (NOS) or 1H-oxadiazoloquinoxalin-1-one , a selective inhibitor of the guanylyl cyclase enzyme, significantly reduced the relaxant effect of oligopeptides. However, indomethacin, an inhibitor of the cyclooxygenase, had no effect on oligopeptides-induced relaxation. In addition, pretreatment with tetraethylammonium  reduced the maximal relaxant effect induced by oligopeptides. By contrast, relaxation was not affected by 4-aminopyridine , glibenclamide (10 µM), or barium chloride . In depolarization Ca2+-free solution, oligopeptides inhibited calcium chloride- (CaCl2-) induced contraction in endothelium-denuded rings in a concentration-dependent manner. Nevertheless, oligopeptides attenuated transient contractions in Ca2+-free medium containing EGTA (1 mM) induced by 1 µM PE, but they were not affected by 20 mM caffeine. It is obvious that potent vasodilation effect of oligopeptides is mediated through both the endothelium and the vascular smooth muscle.Sericin-derived oligopeptides obtained from silk cocoons were investigated for the  Oligopeptides at the concentrations of 0.001–10 mg/mL were cumulatively added. The ability of vasorelaxation was compared in absence (control) and presence of ODQ.To evaluate whether oligopeptides possessed vasorelaxation via the sGC activation, the endothelium-intact ring was incubated with 1H-oxadiazolo[4,3-2+ channel, the endothelium-denuded aortic ring was placed under Ca2+-free Krebs' solution for 20 min and then exposed for an additional 10 min to K+ (80 mM), Ca2+-free solution for complete smooth muscle cell depolarization to open a voltage-operated Ca2+ channel (VOCC). The cumulative concentration-response curve of CaCl2 (ranging from 10 μM to 10 mM) was obtained. After the maximal response was performed, the rings were washed out and replaced with Ca2+-free solution for 20 min. The Ca2+-free 80 mM K+ was reexposed to flowing with preincubation of either oligopeptides  or nifedipine, L-type Ca2+ channel blocker (1 μM) for 20 min. The maximal contraction obtained with the control concentration response curve to CaCl2 was taken as 100%, and all values were calculated as a percentage of the maximal response.To investigate the effects of oligopeptides on Ca2+-free Kreb's solution containing 1 mM EGTA after incubation with 80 mM KCl for successful Ca2+ loading into SR. The transient contractions were obtained in aortic rings by PE (1 μM) or caffeine (20 mM) in Ca2+-free solution before and after being incubated with oligopeptides . The results were expressed as percentage of the response induced by PE or caffeine alone.To investigate the effect of oligopeptides on PE- or caffeine-sensitive intracellular calcium stores, endothelium-denuded rings were bathed in Cat-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. The P < 0.05 was considered to be significant. Concentration-response curves were plotted, and experimental data were obtained by using nonlinear curves fit program (GraphPad Prism 5).All data were expressed as mean ± SEM. Statistical analysis was performed using Student's We investigated the effect of oligopeptides on blood pressure in normotensive rats. The baselines of SBP, DBP, and HR were 122.8 ± 1.64 mmHg, 107 ± 3.74 mmHg, and 366 ± 2.67 BPM, respectively. Intravenous administration of oligopeptides dose dependently decreased SBP and DBP in rats Figures . The hypEmax = 95.46 ± 1.86%) induced contractions in the aortic rings and partially relaxed in KCl-  induced contractions  induced contractions with an EC50 value of 1.86 ± 0.16 mg/mL (μM) induced contraction was markedly attenuated with an EC50 value of 13.49 ± 0.18 and 8.71 ± 0.15 mg/mL, respectively , 4-aminopyridine (1 mM), glibenclamide (10 μM), or BaCl2 (1 mM) before the ring was contracted with PE. All inhibitors did not alter the concentration-response curve for oligopeptides as shown in Figures Emax = 54.82 ± 2.63%) compared to the control (Emax = 83.74 ± 5.61%).To verify the role of KμM) significantly abolished the oligopeptides-induced vasorelaxation in the endothelium-intact ring (50 = 11.22 ± 0.91 mg/mL) and maximal relaxation (Emax = 12.09 ± 4.63%) when compared to untreated control values .ODQ (1 act ring . It mark2 (10 μM–10 mM) were performed in the 80 mM KCl Ca2+-free solution. The percentage of maximal response (Emax) of CaCl2 alone (100%) was attenuated in the presence of oligopeptides  in a concentration-dependent manner , while EC50 values were similar (μM) also abolished the contraction of CaCl2. This suggested that Ca2+ influx was probably reduced by oligopeptides.The concentration-response curves of CaCl similar . In addiμM) in Ca2+-free Krebs' solution containing EGTA (1 mM)  . By contA (1 mM) .Sericin is a protein derived from the silk cocoon that appears as waste in silk processing. Sericin is a mixture of polypeptides of molecular mass varying between 10 and 300 kDa  and has 2+ influx. ODQ, a soluble guanyly cyclase inhibitor, was used to confirm the relationship of the vasorelaxant response of oligopeptides. This finding showed the significant inhibitory effects of ODQ in oligopeptides-induced endothelium-dependent vasorelaxation, which confirmed that the vasorelaxation of the aorta elicited by oligopeptides was mediated in the endothelium-dependent NO/sGC/cGMP pathway. However, the vasorelaxation effect of oligopeptides was still observed in aortic endothelium-denuded rings, suggesting that oligopeptides have a direct effect on vascular smooth muscle cells. The opening of K+ channels or Ca2+ channel blocker in the vascular smooth muscle cells provides an important mechanism to dilate arteries. We further investigated the mechanisms of vasorelaxation that are independent of the endothelium. The vasorelaxant effect of oligopeptides was partially inhibited by the TEA (Ca2+-activated K+ channel blocker). Other K+ blockers, glibenclamide (ATP-sensitive K+ channel blocker), 4-AP (voltage-activated K+ channel blocker), and BaCl2 (inwardly rectifying K+ channel blocker) had no effects, suggesting that the relaxant response of oligopeptides was involved in the role of K+ channel opening of Ca2+-activated K+ channel in vascular smooth muscle cells. Influx of extracellular Ca2+ through voltage and/or receptor-operated calcium channels (VOCCs and/or ROCCs) plays an important role in vascular smooth muscle contraction. We note that oligopeptides induced vasorelaxation in aortic rings precontracted with KCl or phenylephrine. From this result, it can be concluded that oligopeptides induced vasorelaxation via different pathways. It is well known that KCl induces smooth muscle contraction through the activation of voltage-dependent calcium channels and subsequent release of calcium from sarcoplasmic reticulum, whereas phenylephrine-induced vasoconstriction is mediated by the stimulation of G-proteins coupled to alpha-adrenoceptors. We then investigated the mechanism for the vasodilator action of oligopeptides that could directly inhibit the Ca2+ influx in the vascular smooth muscle cells. Oligopeptides significantly reduced the contractile responseinduced by CaCl2 in the endothelium-denuded ring, in concentration-dependent manner under a depolarizing solution. The contraction was abolished by nifedipine, a typical L-type voltage-operated calcium channel blocker, confirming the involvement of L-type voltage-operated calcium channels in the contractile response. The concentration-response curve of CaCl2 decreased in Emax, while the EC50 values were unchanged, suggesting that oligopeptides could act as a calcium channel blocker that interferes with Ca2+ influx through L-type Ca2+ channel of aorta smooth muscle membrane [2+ released from intracellular stores were sensitive to phenylephrine, and caffeine was also determined. The oligopeptides markedly decreased the contractions induced by phenylephrine, which stimulates IP3-dependent Ca2+ release from the intracellular store [2+ from intracellular stores by IP3-independence, were not altered. Thus, it seems likely that the vascular effects of oligopeptides involved a reduction of IP3-dependent Ca2+ releases form SR sensitive to phenylephrine [It is well known that the NO/sGC pathway is one of the most common potential mechanisms that induce vascular smooth muscle relaxation through activation of guanylyl cyclase, leading to the accumulation of cyclic GMP that inhibits Camembrane . The infar store . By contlephrine , 28. Our results demonstrated that bioactive oligopeptides (≤5 kDa) fractions obtained from silk sericin lower blood pressure by a direct effect on both endothelium and vascular smooth muscle leading to vasodilation. Results from this study have also supported us to further identify and characterize bioactive peptides. 2+ antagonism and the NO/sGC/cGMP pathway, which possibly explains the fall in BP. These findings provide scientific evidence supporting the therapeutic uses of sericin-derived oligopeptides as vascular modulators. This study demonstrated the hypotensive effect and vasorelaxant effect of silk sericin-derived oligopeptides on isolated rat aorta and the possible mechanisms. The results suggested that oligopeptides have a dose-dependent relaxing effect on the isolated rat aorta. The relaxing effect of oligopeptides is mediated through Ca


Ca2+ fluxes across the plasma membrane and release from intracellular stores have both been reported to underlie the Ca2+ fluxes induced by Aβ42. Here, we investigated the contribution of Ca2+ release from the endoplasmic reticulum (ER) to the effects of Aβ42 upon Ca2+ homeostasis and the mechanism by which Aβ42 elicited these effects. Consistent with previous reports, application of soluble oligomeric forms of Aβ42 induced an elevation in intracellular Ca2+. The Aβ42-stimulated Ca2+ signals persisted in the absence of extracellular Ca2+ indicating a significant contribution of Ca2+ release from the ER Ca2+ store to the generation of these signals. Moreover, inositol 1,4,5-trisphosphate (InsP3) signaling contributed to Aβ42-stimulated Ca2+ release. The Ca2+ mobilizing effect of Aβ42 was also observed when applied to permeabilized cells deficient in InsP3 receptors, revealing an additional direct effect of Aβ42 upon the ER, and a mechanism for induction of toxicity by intracellular Aβ42.Dysregulation of Ca While the most common type of AD remains idiopathic in origin, with age the most significant risk factor for disease onset , ~5% of cases show a Mendelian pattern of inheritance . The amyloid β-peptide (Aβ) is hypothesized to be central to the pathogenesis of both sporadic and familial AD  receptors coupled to Ca2+ influx  the mechanism (s) by which Aβ42 elicited this effect, (3) the capacity for Aβ42 to mobilize Ca2+ directly from the ER. To allow isolation of effects on the ER from other plasma membrane targets of Aβ42, model cells systems were used that allowed fundamental aspects of ER Ca2+ regulation to be studied. We determined that Ca2+ release from the intracellular ER substantially contributed to the increase in intracellular Ca2+ concentration induced by oligomeric Aβ42. The Aβ42-induced Ca2+ elevation comprised InsP3 dependent and independent components. Using DT40 cells deficient in the three InsP3R isoforms that were permeabilized to allow direct access of Aβ42 to the ER, we also demonstrated that it had the capacity to release Ca2+ from the ER independent of InsP3Rs. Together, these data place the ER and Ca2+ released from it as central to the actions of both extracellular Aβ and Aβ that has reached an intracellular location.In this study, we investigated (1) the contribution of CaPeptides were purchased from The American Peptide Company and rPeptide. Cell culture reagents and chemicals were from Invitrogen or Sigma, unless otherwise stated.2+ imaging experiments, cells were plated onto poly-L-lysine-coated coverslips at a density of 3.2 × 104 cell/cm2. For the MTT reduction assay, cells were plated at a density of 9 × 103 cells/cm2. To overexpress GFP-tagged type 1 InsP3 5′-Phosphatase (GFP-5′P) or GFP  was performed as previously described  containing FBS (10%), penicillin (100 units/ml), streptomycin (100 μg/ml), non-essential amino acids (0.1 mM), and L-glutamine (2 mM). Prior to all experiments, SH-SY5Y cells were cultured overnight in Opti-MEM Reduced Serum Medium, containing FBS (1.5%), penicillin (100 units/ml), streptomycin (1.0 μg/ml), non-essential amino acids (0.1 mM), and L-glutamine (2 mM). For live-cell Ca42 were obtained at a purity of >95%. Peptide mass was verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptides from the same batch were used throughout. Samples of synthetic Aβ42 oligomers were prepared as previously described  were prepared in the same way as Aβ42 oligomers. All Aβ samples were stored at 4°C and were used within 10–15 days of preparation. Toxicity of Aβ42 preparations was confirmed by MTT assay before use in Ca2+ imaging experiments  spectroscopy using an antibody specific to oligomeric Aβ42  or Ca2+ free (as for Ca2+ containing with 1.8 mM CaCl2 replaced with 1 mM EGTA) buffer as indicated. Fura-2 imaging was carried out using an imaging system configured around a Nikon TE300 inverted epi-fluorescence microscope equipped with a 20× 0.75 NA multi-immersion objective. Samples were illuminated by alternate excitation at 340 and 380 nm using a Sutter filter changer  and emitted light was filtered at >460 nm (1 ratio pair per 2 s). Images were captured using a Hamamatsu ORCA ER CCD camera. The imaging system was controlled with Ultraview software . Acquired images were processed with Ultraview software and analyzed in MATLAB. Background subtracted fura-2 ratios were calibrated according to standard procedures  and the percentage of responding cells.Methods for single cell analysis of intracellular Ca3-induced Ca2+ release (IICR) from permeabilized wild type and InsP3R null DT40 cells  .InsP42 on SH-SY5Y cells and was performed according to manufacturer's instructions. Briefly, cells were incubated with Aβ42 (n = 4) for 24 h prior to the addition of the MTT dye solution and a further 4 h incubation at 37°C, 5% CO2. Thereafter, the solubilization/stop solution was added and incubated overnight at room temperature. Absorbances were read at 570 nm with a reference wavelength of 650 nm using a fluorescence plate reader . The data is expressed as the percentage of MTT reduction relative to both live- and dead-cell controls and thus represents the percentage of viable cells. Aβ42 samples were considered to be toxic if 25–40% of cells remained metabolically healthy at an Aβ42 concentration of 1 μ M and if more than 50% remained metabolically healthy at a concentration of 100 nM.The Cell Titer 96 Non-Radioactive Cell Proliferation Assay (Promega) was used to validate the cytotoxic effect of Aβt-test (two-tailed). Data was accepted as significant when p < 0.05 and is denoted by *p < 0.05, **p < 0.01, or ***p < 0.001.Data is presented as the mean value of the combined datasets ± SEM. Statistical significance was determined by Student's 2+ mobilizing properties of oligomeric Aβ42 over the concentration range of its toxicity. Application of Aβ42 spanning its cytotoxic range  caused an elevation in intracellular Ca2+ . Between 1 μM and 10 μM Aβ, the number of responding cells, the peak amplitude and the integral of the Ca2+ responses increased in a concentration-dependent manner. The number of responding cells reached 100% at 5 μM Aβ42  was applied subsequent to Aβ. CCH elicited Ca2+ responses in 100% of cells pre-exposed to 1 or 5 μM oligomeric Aβ42 or to a vehicle control (10%) . F42. Although significantly less toxic than the wild type sequence  than when Aβ42 oligomers were previously applied  prior to the application of Aβ42. In the absence of replete ER Ca2+ stores, Aβ42-induced Ca2+ transients were completely abrogated  Ca2+ release channels localized to this organelle.Having determined that Aβ2+ response in the SH-SY5Y cells used in this study , an agonist of the three RyR isoforms (10 mM) did not elicit a Cay Figure . Furthery Figure . Based o3Rs and elicit robust Ca2+ responses to InsP3-generating agonists including CCH  , allows the requirement for InsP3Rs for Ca2+ release to be tested  and DT40 TKO cells (p = 0.0195) exposed to Aβ42 oligomers compared to the passive Ca2+ leak detected in each cell type  or in DT40 TKO cells (p = 0.0993) compared to the passive Ca2+ leak observed in each cell type  and DT40 TKO cells (p = 0.0045) was observed, indicating that Aβ-induced Ca2+ leak from the ER is dependent and specific to the amino acid sequence of Aβ42. Taken together, these results suggest that Aβ42 oligomers trigger a Ca2+ leak from the ER, which does not depend upon a direct interaction with InsP3Rs.Aβ scramble did not increase the rate of the Cae Figure , and thu42 has potent Ca2+ mobilizing properties and we identify mechanisms responsible for its action. Using both intact and permeabilized cell assays to investigate the effects of extracellular and internalized Aβ42, respectively, we establish that Ca2+ release from the ER makes the greatest contribution to the Ca2+ mobilizing effects of Aβ42. The InsP3 signaling pathway also contributes to the Ca2+ mobilizing properties of oligomeric Aβ42 in intact cells. InsP3Rs were not required for Aβ42-stimulated Ca2+ flux in permeabilized cells ruling out a direct regulation of InsP3Rs by Aβ42.Here we show that the oligomeric form of the AD-associated peptide Aβ2+ hypothesis of amyloid toxicity is the property of Aβ to induce Ca2+ elevations in its target cells. This sets in motion a cascade of events, which culminates in neuronal death. Ever since this hypothesis was put forward more than 20 years ago , its importance is increased.Analysis of the mechanisms underlying Ca3 signaling in Aβ42-induced Ca2+ responses provides robust evidence in support of this pathway in Aβ42-mediated Ca2+ signals thus far. In particular, the use of InsP3 5′phosphatase overexpression to suppress InsP3 signaling is a highly selective strategy, overcoming issues regarding incomplete knockdown of InsP3Rs and contribution of the isoforms not targeted when using siRNA approaches. The inhibition of Ca2+ signals by caffeine is also consistent with a role for the InsP3 signaling pathway in the Ca2+ mobilizing effects of Aβ . Indeed, a number of different GPCRs, including metabotropic glutamate receptors, are activated by Aβ42, contributing to modulation of LTP, Aβ42 synthesis and processing and cytotoxicity (Wang et al., Our demonstration of the participation of InsP3Rs or to alter InsP3 generation/metabolism. Since significant intracellular Aβ42 accumulation would require up to 1 h (Bucciantini et al., 42 contributes to the acute modulation of Ca2+ fluxes observed in this study and elsewhere in intact cells. Endocytosis of Aβ42 may, however, contribute to the more chronic effects on Ca2+ homeostasis as well as cytotoxicity previously reported (Ferreiro et al., 42 could directly affect ER Ca2+ homeostasis from an intracellular location was therefore also considered. Using a permeabilized cell assay to allow control of cytosolic conditions and access of Aβ42 to the ER, an Aβ42-stimulated Ca2+ efflux from the ER was observed. Unlike that observed for intact cells, the difference between Aβ42 and Aβ42 scrambled was dramatic, revealing a highly specific effect of Aβ42 upon ER Ca2+ mobilization. These effects were observed in the absence of exogenous InsP3 suggesting that the effects were InsP3R-independent. The extensive dilution of cytosol following permeabilization of the DT40 cells would also likely preclude a contribution of Aβ42-stimulated InsP3 generation. More significantly, InsP3Rs were not required for the Ca2+ mobilizing properties of Aβ42, since deficiency in all three InsP3R isoforms did not affect the Ca2+ mobilizing properties of Aβ42. The absence of a requirement for InsP3Rs for Aβ42-stimulated Ca2+ flux in the permeabilized cell system does not rule out the possibility that IICR contributes to Ca2+ fluxes and toxicity mediated by intracellular Aβ42. Indeed, by activating Ca2+-sensitive PLC and generation of InsP3, Ca2+ mobilized by Aβ42 could promote IICR. Consistent with this notion, microinjected Aβ42 was recently shown to promote Ca2+ signals in Xenopus oocytes in a manner that involved InsP3 generation (Demuro and Parker, The internalization of Aβ from the extracellular space (Bucciantini et al., 2+ store by Aβ42 has important implications for the mechanisms of its toxicity. Depletion of ER Ca2+ stores results in the accumulation of unfolded proteins and activation of the ER stress response, which via caspase 12 activation and Bap31 cleavage can subsequently induce mitochondrial apoptotic cascades (Verkhratsky, 3Rs during Aβ42-stimulated depletion of ER Ca2+ may be of greater consequence. Specifically, InsP3R-induced Ca2+ release from the ER and its subsequent sequestration by neighboring mitochondria could lead to mitochondrial Ca2+ overload, permeability transition and death (Csordas et al., The depletion of the ER Ca2+ signaling to Aβ-induced Ca2+ signals independent from Ca2+ fluxes that may arise in neurons as a result of electrical or synaptic activity. Moreover, using this cell line, contributions from other Aβ targets described in neurons such as NMDA receptors are excluded. Analogous to a number of other studies in electrically non-excitable primary and cultured cells including Xenopus oocytes (Demuro and Parker, 2+ mobilizing properties of Aβ42 are neuron-independent and do not require the expression of any other of its reported targets. Fundamental aspects of the Ca2+ mobilizing properties of Aβ42 were further revealed and exemplified by the Aβ42-stimulated Ca2+ flux from the InsP3R-deficient ER of permeabilized DT40 B-lymphocytes. These latter data demonstrate for the first time that Aβ42 has the capacity to directly induce Ca2+ flux from the ER. Given the importance of the ER and InsP3Rs in neuronal functions, future studies will be required to test whether InsP3Rs contribute to Aβ-mediated neuronal pathology.The use of SH-SY5Y neuroblastoma cell line and permeabilized DT40 B-lymphocytes in this study, rather than primary neurons allowed careful dissection of the role of ER CaLaura E. Jensen: substantial contributions to conception and design, acquisition, analysis and interpretation of data as well as writing of manuscript. H. Llewelyn Roderick: substantial contributions to conception and design, interpretation of data as well as writing of manuscript. Geert Bultynck and Tomas Luyten: designed, acquired, analysed and interpreted data of Figure Some of this work was supported by a grant from Senexis, Babraham Research Campus and Hozefa Amijee was employed by Senexis at the time this work was carried out.


Myocytes were isolated from anesthetized C57BL/6 female mice. Animals received an ovariectomy or sham-operation at one month and were aged to ~24 months. Excitation-contraction coupling parameters were compared in fura-2 loaded myocytes (37°C). While Ca2+ transients were larger and faster in field-stimulated myocytes from ovariectomized mice, ovariectomy had no effect on peak fractional shortening. Similarly, ovariectomy had no effect on fractional shortening measured in vivo by echocardiography . Ovariectomy did decrease myofilament Ca2+ sensitivity, as evidenced by a 26% increase in the Ca2+ required to activate actomyosin MgATPase in ovariectomized hearts. Larger Ca2+ transients were attributable to a 48% increase in peak Ca2+ current, along with an increase in the amplitude, width and frequency of Ca2+ sparks measured in fluo-4 loaded myocytes. These changes in Ca2+ handling were not due to increased expression of Ca2+ channels (Cav1.2), sarcoplasmic reticulum Ca2+ ATPase (SERCA2) or Na+-Ca2+ exchanger in ovariectomized hearts. However, ovariectomy increased sarcoplasmic reticulum Ca2+ stores by ~90% and promoted spontaneous Ca2+ release from the sarcoplasmic reticulum when compared to sham controls. These observations demonstrate that long-term ovariectomy promotes intracellular Ca2+ dysregulation, reduces myofilament Ca2+ sensitivity and increases spontaneous Ca2+ release in the aging female heart.This study determined whether deficiency of ovarian estrogen starting very early in life promoted age-associated Ca Studies in humans have shown that cardiac contractile function declines with age, even in the absence of overt cardiovascular disease ,2. Altho2+ transient required to initiate contraction [2+ handling. However, little is known about the influence of sex steroid hormones such as estrogen on cardiac Ca2+ homeostasis in the setting of aging.Most previous studies of the impact of age on cardiac contractile function in animal models have used hearts, tissues and myocytes from male animals. However, there is evidence that the effect of age on cardiac contractile function differs between the sexes. Studies have shown that contractile force, fractional shortening and left ventricular function deteriorate with age in male rats and non-human primates, but are unaffected by age in female animals -9. Previtraction . These f2+ homeostasis. Studies have shown that Ca2+ transients and contractions are smaller and slower in ventricular myocytes from young adult female rats when compared to age-matched males [2+ transients and contractions compared to sham-operated controls [2+ current, but arises from increased sarcoplasmic reticulum (SR) Ca2+ release as a consequence of increased SR stores and larger Ca2+ sparks [2+ release and leads to Ca2+ transients and contractions that are similar to those seen in myocytes from young adult males. It is possible that long term estrogen deprivation, starting early in life, may lead to age-associated Ca2+ dysregulation and contractile dysfunction as seen in myocytes from aged males. However, whether long-term OVX alters Ca2+ homeostasis and causes deterioration in cardiac contractile function in the aging female heart has not been investigated.Cardiac myocytes possess estrogen receptors  and evided males ,13,14. Hcontrols -19 but ccontrols ,21. We h+ sparks . These f2+ homeostasis and disrupts contractile function in the aging mouse heart. Studies used very old (e.g. ~24 month old) female C57BL6/J mice that received either a bilateral OVX or sham surgery at an early age (e.g. one month of age). Ventricular myocytes were loaded with Ca2+-sensitive fluorophores to investigate specific Ca2+ handling mechanisms. Contractions, Ca2+ transients, Ca2+ currents, sarcoplasmic reticulum (SR) Ca2+ content and Ca2+ sparks were compared in myocytes from aged sham and OVX mice. In vivo cardiac function was evaluated with echocardiography and myofilament Ca2+ sensitivity was assessed by measurement of actomyosin MgATPase activity. Ca2+ handling proteins were assessed by Western blot analysis. Our results showed that long-term OVX reduced myofilament Ca2+ sensitivity, promoted cardiomyocyte Ca2+ dysregulation and increased spontaneous SR Ca2+ release in the aging female heart. The overall aim of this study was to determine whether long-term OVX modifies myocardial CaFor full details of Methods, please refer to Protocols were approved by the Dalhousie University Committee on Laboratory Animals (No. 12-022) and followed Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals . Sodium pentobarbital anesthesia was used and all efforts were made to reduce suffering.2+ transients (fura-2 AM) were measured simultaneously as described in our previous studies [+ current was inhibited by 4-aminopyridine (4 mM) and steps were made from -40 mV to inactivate Na+ current. SR Ca2+ load was measured by rapid application of 10 mM caffeine in 0 Na+/0 Ca2+ buffer to inhibit Ca2+ extrusion via Na+-Ca2+ exchange [2+ sparks were measured in fluo-4 AM-loaded myocytes as described previously [Myocytes were isolated from 20-24 mos female C57BL/6 mice that had either sham surgery or OVX at 1 mos. Ventricular myocytes were isolated by enzymatic digestion as described in detail previously . OVX was studies -25. In vexchange ,27. Actieviously  and analeviously .Two-dimensional guided M-mode echocardiography was performed in anesthetized mice (2% isoflurane). ECG electrodes were placed subcutaneously and mice were assessed with a high-resolution linear transducer connected to a Vivid 7 imaging system.2+ as described earlier [Myofilaments were isolated from the ventricles and frozen until use as described previously . Actomyo earlier .v1.2 polyclonal antibody , mouse anti-NCX monoclonal antibody , mouse anti-SERCA2 monoclonal antibody  and rabbit anti-Na/KATPase polyclonal antibody . Secondary antibodies were goat anti-mouse HRP-conjugated polyclonal antibody  and goat anti-rabbit HRP-conjugated polyclonal antibody . Protein was resolved on an 8% polyacrylamide gel, transferred to nitrocellulose membrane and blocked. Loading controls were anti-Na/K ATPase antibody (Cav1.2 and NCX) or amido black (SERCA2). Signals were visualized with Immuno-Star™ WesternC™ Kit (BioRad) and quantified with Quantity One software.Ventricles were homogenized (60 s) in buffer . Tissues were sonicated 3 times and flash frozen in liquid nitrogen after each sonication step. Samples were then heated , cooled on ice (5 mins), centrifuged  and the supernatant was stored (-80°C). Protein concentration was determined with a DC Protein Assay (BioRad). Antibodies used were rabbit anti- CaStatistical analyses were performed with SigmaStat . Differences between means were evaluated by a t-test or two-way repeated-measures analysis of variance. Graphs were constructed with Sigmaplot . Data are presented as means ± SEM and differences were considered significant if p<0.05. Selected physical characteristics of the sham and OVX mice used in this study were compared as shown in To determine whether OVX affected cardiomyocyte size, myocyte capacitance and myocyte volume were compared in sham and OVX animals. Cell capacitance was 24% lower in OVX myocytes compared to sham cells . Cell vo2+ homeostasis and contractile function, contractions and Ca2+ transients were simultaneously recorded in sham and OVX myocytes that were field-stimulated at 2 Hz. 2+ transients and contractions recorded from sham and OVX myocytes paced at 2 Hz. Mean peak contractions, normalized to cell resting length, did not differ between sham and OVX cells  was not different between the two groups  increased by 26% in OVX when compared to sham controls  also were similar in the two groups  was not affected by OVX  were compared in the two groups. The time constants were similar in sham and OVX myocytes  in an OVX myocyte. Long-term OVX did not significantly increase the incidence of spontaneous Ca2+ release when compared to sham controls . Intere females -19 but c females ,21, whic females ,11, it ie levels  that canadectomy ,37.2+ release is augmented in cardiomyocytes from young adult mice, 3-26 weeks after OVX [2+ release was not due to an increase in Ca2+ current. Instead, this was the result of higher gain due to elevated SR Ca2+ content and an increase in the amplitude of Ca2+ sparks [2+ handling parameters in young adult OVX mice as reported in our previous study [2+ release persists in the aging heart, even almost two years after OVX. A key observation in this study is that effect of OVX on specific mechanisms involved in cardiomyocyte homoeostasis differed markedly in the aging heart when compared to young adult hearts, as shown in 2+ content was similar regardless of age, Ca2+ transients increased in parallel with Ca2+ current in aged OVX myocytes, with no effect on the gain of SR Ca2+ release. As the magnitude of SR Ca2+ release is proportional to the size of the Ca2+ current [2+ current is a key mechanism responsible for augmenting SR Ca2+ release following long-term OVX in the aging heart. We also demonstrated that the increase in peak Ca2+ current was not due to an increase in the expression of Cav1.2, which has been reported in hearts from younger animals subjected to short term OVX [v1.2 expression was actually reduced following long-term OVX in the aging heart. The increase in the activity and expression of protein kinase A (PKA) that occurs following OVX [2+ current, as discussed in more detail below.We and others have previously shown that SR Cafter OVX . In youn+ sparks . Table 3us study . The pre current , our resterm OVX . Indeed,wing OVX  may be i2+ spark properties than short-term OVX, as shown in 2+ transient and shorter time-to-peak contraction observed in OVX myocytes in this study. Together, these observations suggest that a marked increase in the size of individual Ca2+ release events contributes to enhanced SR Ca2+ release following long-term ovarian estrogen deprivation in the aging heart.The results of the present study also demonstrate that that long-term OVX had much more dramatic effects on Ca2+ release, it would be expected to increase the magnitude of cardiac contraction, although this was not explored in our earlier study  OVX increases myofilament Ca2+ sensitivity [2+ transients observed in our study [2+ dysregulation in the estrogen-deprived heart could be informative.There is recent evidence that the production of reactive oxygen species (ROS) is increased in the aged OVX heart  and thisur study . Increase models  and coulhe heart , it is psitivity , so prolur study ,61. AddiPrevious studies have provided evidence that OVX can modify the structure of the heart. While heart weight-to-body weight ratios are similar ,51,62,632+ handling [2+ homeostasis were dramatic. This also produced a model that was characterized by the lack of exposure of the heart to normal pubertal systemic estrogen modeling. This model of early ovarian steroid withdrawal has been used previously in other studies [There are limitations to the experimental approaches used in this study. Our studies did not investigate OVX mice treated with estrogen replacement, although this would be an interesting area for additional investigation in future studies. In addition, we removed the ovaries early in life to determine whether long-term estrogen withdrawal would result in an aging phenotype characteristic of that seen in aging males with respect to Cahandling -11. In c studies ,15,63, b studies . These n studies ,66 and i studies . It is p studies . Whether2+ homeostasis and contractile function in the aging female heart. Although Ca2+ transients were larger in OVX myocytes, in vitro and in vivo fractional shortening were similar in sham and OVX mice. The underlying mechanism involved a decrease in myofilament Ca2+ sensitivity in the aging OVX heart. The increase in peak Ca2+ transients in OVX myocytes was mediated by an increase in both Ca2+ current and the size of unitary Ca2+ release events. Higher intracellular Ca2+ led to an increase in SR Ca2+ load, an increase in spark frequency and spontaneous SR Ca2+ release. These results demonstrate that long-term ovarian estrogen deprivation reduces myofilament Ca2+ sensitivity, promotes Ca2+ dysregulation, and increases spontaneous Ca2+ release in the aging female heart.In summary, our study showed that long-term deprivation of ovarian estrogen disrupted myocyte CaMethods S1Supplemental methods.(DOCX)Click here for additional data file.


Dear Editor,Mycobacterium tuberculosis  is a formidable challenge to global health and is caused by an ancient pathogen, sis WHO, . In the sis WHO, . Since aM. tuberculosis has evolved elegant strategies to evade a host’s immune defensive system. For example, D’Arcy Hart and colleagues observed that M. tuberculosis-containing phagosomes do not fuse with the lysosome inside murine macrophages , one of the classical innate defense mechanisms against infection. Additionally, during latency, M. tuberculosis continues to be under oxidative stress; thus, the accumulation of mutations caused by oxidative DNA damage was predicted to be a potential risk for drug resistance  resistance and growth benefits in mycobacteria under stress, we developed a model for evolutionary screening of H2O2-resistant mutations in a pathogenic mycobacteria model, M. smegmatis, using a clinically important stressor, H2O2  of H2O2 in different mutants selected under different H2O2 concentrations were different, indicating that gradually increasing H2O2 resistance was due to the accumulation of mutated genes rather than a key mutated gene that caused the resistance to H2O2 . After 54 days of selection, the MIC of the mutant strains with the maximum resistance to H2O2, selected at an H2O2 concentration of 1.5 mmol/L, was 3.125 mmol/L, while that of the wild type strain mc2155 under the same conditions (7H9 medium with 10% ADS) was 0.039 mmol/L. We named the mutant strain mc251. As evolutionary cross-stress protection was observed under various stresses, we compared the growth advantage between mc251 and mc2155 in corresponding stresses. We first measured the doubling time of both wild type mc2155 and mutant strain mc251. A significant difference in doubling time was detected: the doubling time of wild type mc2155 was 2.734 ± 0.051 (n = 30), while the doubling time of mc251 was 3.448 ± 0.087 (n = 30)  . To provide insight into the mechanisms involved, we sequenced the whole genomes of both mutant strain mc251 and wild type mc2155. Compared with mc2155, there are 22 single nucleotide polymorphisms (SNPs) in the mc251 genome, of which 19 are located in coding regions, and 12 of these 19 SNPs are nonsynonymous mutations (Table S2). The data showed that G:C > T:A transversion and G:C > A:T transition were the major mutation types  and introduced into mc251 via electroporation. Surprisingly, there are no changes in the MIC of H2O2 for most detected transformed strains, except for one strain of mc251 with pMV361-fur(msmeg_3460) (Table S3 and data not shown). Furthermore, because fur is located upstream of msmeg_3461, which encodes KatG, the mutated Fur (Ferric uptake regulation protein) with substituted A28V would influence KatG expression. Increased peroxidase and catalase activities were found in mc251 . Furthermore, the mass spectrometry results indicated that the mutation of fur changed the expression of katG, causing high resistance to H2O2. Our results showed that the development of H2O2 resistance in mycobacteria led to the gain of growth benefits in starvation conditions and within macrophages.As an intracellular pathogen, the success of M. tuberculosis isolated from macaques with latent infection . In addition, MS data showed that mc251 had increased enzyme activity of KatG Supplementary material 2 (PDF 152 kb)Below is the link to the electronic supplementary material.


The LGs were processed for laser capture microdissection (LCM), real time RT–PCR, western blot, and immunofluorescence for the detection of mRNA and proteins of the α1, α2, β1, β2, and β3 subunits of NaIn the rabbits with IAD, mRNA levels of α1, β1, and β3 from whole LGs were significantly lower. In samples of acini and epithelial cells from various duct segments, collected by LCM, mRNA levels of α1, β1, β2, and β3 were significantly lower in the rabbits with IAD, although mRNA for α2 could not be detected. However, western blots demonstrated that all five subunits were significantly higher in the rabbits with IAD, although their distribution patterns were similar to those of the control rabbits, as demonstrated by immunofluorescence.+/K+-ATPase subunits in rabbits with IAD, suggesting that these changes may play a role in the pathogenesis of Sjögren’s syndrome and altered LG secretion, as observed in these animals.The data presented herein demonstrated significant changes in mRNA and protein expressions of Na Sjögren’s syndrome is an autoimmune disease that causes functional deficiency of the lacrimal and salivary glands; it is one of the most common causes of dry eye . AlthougAmong many animal models that have been used to study Sjögren’s syndrome, rabbits with induced autoimmune dacryoadenitis (IAD) have been shown to demonstrate many of the ocular surface symptoms and lacrimal gland (LG) pathologies characteristic of Sjögren’s syndrome, and they have been used extensively to study its pathophysiology -5.Like other exocrine gland secretions, LG fluid secretion is an osmotic process mediated by many ion transporters, channels, and aquaporins -13. It i+/K+-ATPase, an enzyme located in the plasma membranes of all animals, has been detected in the LGs of rabbits [+/K+-ATPase is a heterotetramer that comprises two α subunits and two β subunits, of which two α subunit isoforms (α1 and α2) and three β subunit isoforms  have been identified so far, with the β subunit being essential for its normal function [+/K+-ATPase uses the energy released from ATP hydrolysis to move three Na+ out of and two K+ into the cells, both against electrochemical potential gradients. Depending on the asymmetric localizations of other transport proteins, Na+/K+-ATPase can power either net absorption or secretion [Na rabbits ,13,15-22 rabbits ,16,23-26function . Na+/K+-ecretion .+/K+-ATPase may play in LG function, little is known about the expression patterns of its subunits in rabbit LG and its potential contribution to LG deficiency in Sjögren’s syndrome. A recent study showed that Na+/K+-ATPase was one of the main targets of immunoglobulin G (IgG) autoantibodies that interact with the subtype 3 of muscarinic acetylcholine receptors (M3 AChR) in the salivary glands of patients with primary Sjögren’s syndrome, and it was suggested that it may play a role in the pathogenesis of dry mouth [+/K+-ATPase subunits and their potential changes in rabbits with IAD.Although some studies have investigated the role that Nary mouth . TherefoTwo groups of adult female New Zealand White rabbits  were used. One group consisted of six rabbits with IAD, and the other consisted of six age- and sex-matched normal controls. The rabbits were narcotized with a mixture of ketamine (40 mg/ml) and xylazine (10 mg/ml) and given an overdose of Nembutal (80 mg/kg) for euthanasia. Inferior LGs were removed and embedded in OCT, frozen in liquid nitrogen, and stored at −80 °C until use. This study conformed to the standards and procedures for the proper care and use of animals by the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.To generate the IAD model, the epithelial cells from one LG were first co-cultured with the same rabbit’s ex vivo-activated lymphocytes for two days, and then the cell mixture was injected into the same rabbit’s remaining LG. Typically, the rabbits fully developed IAD within two weeks. The detailed procedures have been published previously -5.Frozen sections were collected with PEN membrane-coated slides  and stained with cresyl violet in RNase-free conditions with an LCM Staining Kit . Acinar cells and epithelial cells from various duct segments were then laser captured using a PixCell II LCM System . Approximately 100 cells were collected for each sample for isolation of total mRNA, and six replicates of each acinus and duct segment were collected from each animal .® Midi Kit  plus on-column DNase digestion. Detailed procedures were described in our previous report [Total cellular RNA was isolated from RNAlater-treated samples with an RNeasys report . BrieflyThe sequences of primers and probes used in this study are listed in GAPDH) in each sample was used to calculate the level of target mRNA relative to that of GAPDH mRNA in the same sample.For the real-time RT-PCR step, amplification was performed on a sequence-detection system  containing the internal dye ROX as a passive reference, in accordance with the procedures described. The PCR reaction volume was 10 μl. It contained 1× master mix, 900 nM forward and reverse primers, 250 nM probes, and 2.5 μl of 1× TE–diluted cDNA template. The FAM signal was measured against the ROX signal to normalize for non-PCR-related fluorescence fluctuations. The cycle threshold (CT) value represented the refraction cycle number at which a positive amplification reaction was measured and was set at 10× the standard deviation from the mean baseline emission calculated for PCR cycles 3 through 15. Each sample was measured in triplicate. The difference between the CT for each target mRNA and the internal housekeeping gene glyceraldehyde 3-phosphate dehydrogenase . The dilution for α1  was 1:400; for α2 , 1:400; for β1 , 1:100; for β2 , 1:100; and for β3 , 1:250. Secondary antibodies used were fluorescein isothiocyanate (FITC)-conjugated AffiniPure donkey anti-goat and anti-mouse IgG , at a dilution of 1:200. Rhodamine conjugated phalloidin , at a dilution of 1:200, was also used to stain F-actin to show the morphological profiles of the LGs.The samples frozen in Optimal Cutting Temperature (OCT) compound were cut 8 μm thick and placed on slides then fixed with ready-to-use formaldehyde/zinc fixative  for 15 min. They were then washed in phosphate-buffered solution (PBS) 3× for 10 min each and blocked with donkey normal serum (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. The slides were then incubated with primary antibodies at respective dilutions overnight at 4 °C. On the next day, slides were again washed 3× for 10 min in PBS and incubated with secondary antibody for 1 h at room temperature then washed 3× for 10 min in PBS and 1× for 15 min in 4 mM sodium bicarbonate. Finally, one drop of aqueous mounting medium  was placed on slides and covered with coverslips. Slides were observed with a Leica epifluorescence microscope  and a Zeiss LSM 710 confocal laser scanning microscope . FITC-conjugated secondary antibodies were visualized by excitation at 488 nm using an argon laser. Images were analyzed with LSM image browser and PhotoShop .LGs were homogenized in RIPA buffer  and centrifuged at 2,000× g for 20 min. The supernatant was denatured in SDS–PAGE sample buffer for 20 min at 60 °C, resolved on a 7.5% or 4%–20% gradient SDS–PAGE gel , and then transferred onto PVDF membrane . To assess transporter proteins, a constant quantity of proteins from each sample was analyzed. Membrane blots were probed with α1 at a dilution of 1:1,000, α2 at 1:500, β1 at 1:2,000, and β2 and β3 at 1:500. All blots were incubated with Alexa 680-labeled donkey anti-goat or goat anti-mouse secondary antibody (Invitrogen) and detected with an Odyssey Infrared Imaging System . Densitometry analysis of the resulting gel was performed by the manufacturer’s software.t-test and ANOVA were used to evaluate the significance of the differences; a p<0.05 was considered significant.Data were presented as mean±SEM. Student’s The mRNA levels for α1 from whole LG were significantly lower in the animals with IAD (0.63±0.04) compared with the control group (0.89±0.06), with a 29.1% difference p<0.05; . Data fr+/K+-ATPase subunits  than in the control group (2.411±0.125), with a 28.8% difference p<0.05; . Data frNo significant difference of β2 mRNA from whole LG was detected (p>0.05) between the control rabbits (0.02±0.003) and the rabbits with IAD  than in the control group (0.062±0.003), with a 27.7% difference p<0.05; . Data frWe studied the expressions of α  and β F subunits+/K+-ATPase α1 immunoreactivity (IR) was detected in all acinar cells, most prominently on the basolateral membranes  and prolactin treatment both partially restored its activity [+/K+-ATPase activity by 29%, while the synthetic estrogen diethylstilbestrol (DES) decreased total Na+/K+-ATPase by 12% [It has been shown that Naactivity . Howevere by 12% .+/K+-ATPase from intracellular pools to basolateral membranes [+/K+-ATPase, suggestive of its increased flux to lysosomes, where it was degraded [+/K+-ATPase cytoplasmic reserves were recruited to the basolateral membrane [+/K+-ATPase are available for rapid recruitment to the basolateral membranes. It has also been suggested that the carbachol-induced redistribution of Na+/K+-ATPase to the basolateral membrane represents a mechanism by which the cell compensates for the increased Na+ influx [In rat LGs, cholinergic stimulation caused translocation of Naembranes . Carbachdegraded . Cholinemembrane -17, sugg+ influx .+/K+-ATPase in the LGs of Sjögren’s syndrome patients showed that the distribution of Na+/K+-ATPase was unchanged in the LGs, although its expression was not quantified [+/K+-ATPase was one of the main targets of anti-M3 IgG. These patients also produced functional IgG autoantibodies that acted as partial muscarinic agonists in the submandibular gland and increased PGE2 and cAMP production, therefore inhibiting Na+/K+-ATPase activity. These autoantibodies could also interfere with the secretory effect of parasympathetic neurotransmitters, and may play a role in Sjögren’s syndrome-related dry mouth [The only literature regarding the presence of Naantified . Howeverry mouth .+/K+-ATPase subunits in rabbits with IAD was a primary or secondary consequence of IAD remains unknown, but given the fact that LG fluid production in these animals was greatly reduced [In LG samples from rabbits with IAD, the mRNA levels from whole LG of α1, β1, and β3 were significantly lower than those of the control animals, while the levels of α2 and β2 remained unchanged. Compared to the results from the control rabbits , mRNA le reduced , it is mIt should be noted that there were discrepancies in mRNA and protein expressions in the data presented herein, which also have been reported in previous studies in other tissues and organs ,34. Vari+/K+-ATPase in the ductal cells, and their significant changes in rabbits with IAD, point to active Na+ and K+ transport in the ductal cells and their potential contribution to LG deficiency during IAD.Despite the fact that up to 15% of all epithelial cells in the LG are ductal cells -38, most+/K+-ATPase subunits in the acinar and ductal cells of rabbits with IAD. These data strongly suggest that changes in this solute transporter and its subunits may contribute to reduced tear secretion in rabbits with IAD, although direct functional studies are needed to provide definitive evidence. Data presented herein also support the notion that acini and ducts play different roles in LG secretion. However, the exact mechanisms of how Na+/K+-ATPase functions in the LG in physiologic and pathological conditions are unknown, and more studies into their roles are certainly needed.In summary, our findings demonstrated that there were significant changes in mRNA and protein expression of Na


Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30−/− mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30−/− mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30−/− CCDs . To further test our hypothesis we performed CD [Ca2+]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca2+]i oscillations in free-flowing CDs of wild type but not Cx30−/− mice. The [Ca2+]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption.ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y Renal epithelial i , whereas no significant changes were detected in CCDs from Cx30−/− mice   form of fluorophores for confocal fluorescence imaging. The calcium-sensitive fluorophore Rhod-2 was employed in the present experiments, as red fluorescent probes are preferred over green dyes due to the low overlap of their emission with the significant renal tissue autofluorescence. Initially, we used the multicell bolus loading (MCBL) technique that we previously described for real-time imaging of intracellular pH and [Ca2+]i in the proximal tubules . In kidneys from wild-type mice a subset of CD cells showed spontaneous oscillations in Rhod-2 fluorescence reflecting changes in [Ca2+]i .To further support our hypothesis that flow-induced [Cai Figure . In cont Figures . To testin vitro microperfused CCDs and ratiometric calcium imaging we first found that the classic phenomenon of tubular flow-induced increases in CD [Ca2+]i was dependent on the expression of Cx30. In addition, spontaneous [Ca2+]i oscillations in free-flowing CDs were observed in the intact living kidney of wild type but not Cx30−/− mice and the [Ca2+]i responses were found to be sensitive to suramin, a P2 purinergic receptor inhibitor. Our results are consistent with the presence of mechanosensitive, ATP releasing Cx30 hemichannels in the distal nephron-CD system and established that CD cell [Ca2+]i responses are an integral element of the intra-tubular autocrine/paracrine purinergic system in the CD that is driven by Cx30-mediated ATP release. The present studies also established the exciting new technical advance of directly and quantitatively visualizing CD [Ca2+]i responses in the intact mouse kidney in vivo.This study is a direct and logical continuation of our recent line of research which aims to characterize ATP releasing mechanisms and their physiological relevance in the kidney. In the present experiments we used confocal fluorescence imaging approaches to further examine the function of one particular ATP conduit, the Cx30 hemichannel in the renal CD. Using 2+]i responses were measured as an indicator of ATP release .The present findings are in line with our previous study showing that Cx30-mediated ATP release in the CD can be triggered by mechanical forces, for example by increases in tubular fluid flow rate  that are known to exist due to renal hemodynamic feedback mechanisms  since in the in vitro dissected and microperfused CD, in which fluorophore loading was very efficient, all cells produced flow-induced [Ca2+]i responses . However, these important questions need to be addressed in future work using more suitable experimental approaches .It should be noted that the present studies detected s Figure . Also, i2+]i signaling could be regulating epithelial transport processes in other tubular segments in other species. In addition, the ATP channel pannexin 1 which was recently localized to the apical plasma membrane of several renal tubule segments including the CD (Hanner et al., These studies were carried out in mice, where the expression of Cx30 is restricted to the intercalated cells of the CNT and CCD (McCulloch et al., 2+]i responses in the CD in the intact kidney in vivo and further characterize the function of ATP releasing Cx30 hemichannels in the mouse CD. Our results established that Cx30-dependent [Ca2+]i responses are an integral element of the intra-tubular autocrine/paracrine purinergic system in the CD that plays an important role in the regulation of renal salt and water reabsorption, the maintenance of body fluid and electrolyte balance and normal blood pressure.In summary, the present studies successfully visualized [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.


BVWM was analyzed to contain a crude protein value of 62.35%. There were comparable feed intake values by rabbits on all the diets although the rabbits in the higher replacement levels of BVWM tended to have consumed more of the feed. There were significant increases (P < 0.05) in body weight gain by rabbits as the BVWM level increased in the diet. Similarly, the feed to gain ratio improved and nitrogen digestibility increased (P < 0.05) with higher levels of BVWM in the diet. This study shows that rabbits can tolerate the 45% BVWM replacement of groundnut cake effectively.A total of seventy-two weaner rabbits of eight weeks of age were used to assess the effects of replacing groundnut cake (GNC) with blood vegetable waste meal (BVWM) in the diets of rabbits. The BVWM was fed to replace dietary GNC at 0, 15, 30, and 45%, with GNC being 15% in the control diet. The four experimental diets were fed  Amaranthus cruentus is a form of vegetable that cannot be consumed by humans and are ready for disposal after 48 hrs of being harvested. Amaranthus cruentus is species of vegetable that is produced abundantly in most African counties. It serves as spices in human diets [Discardedan diets  but begiConventional protein feedstuffs such as soyabean meal, ground nut cake, and fish meal are quite expensive. The high cost of these ingredients has resulted in the inadequate/poor feeding of rabbits and other livestock species. The need to sources for alterative feedstuffs so as to reduce the high cost of livestock production cannot be overemphasized. Blood vegetable waste meal (BVWM) is obtained from processed blood and vegetable; both are waste. The blood is washed away at the abattoir, while the unsold vegetable is discarded at refuse site. Hence, BVWM is obtained free and can be fed to rabbits to reduce production cost, particularly as feed accounts for almost 70% of the cost of production.Rabbit has come under focus as an animal because of its several potentials. It has small body size, short gestation interval, induced ovulator, and an ability to utilize forage. Rabbits do not really compete with humans in terms of feeding. This is because they can feed on vegetative-based diets and kitchen waste. Rabbits are hindgut fermenters, capable of utilizing poor quality feed, and can tolerate up to 25% dietary crude fibre [Improved rabbit production can play important role in increasing animal protein supply to under nourished populace, considering the high cost of chicken, pork, and beef. The study was aimed at investigating the effects of replacing groundnut cake with BVWM on the performance of rabbits and to also ascertain what level of BVWM that can best replace groundnut cake (GNC).Amaranthus cruentus  was collected from the market and processed along with fresh bovine blood at ratio 1 : 2 (w/w), that is, one of blood to two of vegetable. The discarded vegetable and blood were thoroughly mixed and cooked at 100°C for about 1.5 hours. It was stirred regularly to prevent burning. The cooked mixture blood vegetable waste meal (BVWM) was later sun-dried until the moisture content was below 15%. On analysis, BVWM contained 62.35% crude protein, 6.4% crude fibre, and 3.45% ether extract.Discarded A total of 72 eight-week old weaner rabbits of both sexes were used in this study there were four treatments, with each treatment having 3 replicate groups, and there were 6 rabbits housed individually placed in each replicate. There are four experimental diets  which arad libitum to the experimental animal.All rabbits were individually housed in metal cages throughout the feeding trial which lasted for a period of eight (8) weeks. Feed and water were supplied Initial and weekly body weight records and daily feed intake record were kept. The feed to gain ratio was calculated. At the last week of feeding trial, faecal and urine samples were collected for 3 consecutive days by the total collection method to determine nitrogen digestibility. All proximate analysis were conducted by the method of AOAC .P > 0.05) to the body weight gained by rabbit on the 15%. BVWM replacement diet (7.95 g); but significantly lower (P < 0.05) to the body weight gain values for the rabbits on 30 and 45% BVWM replacement diets that gained 9.69 and 9.38 g, respectively. The feed intake values were comparable (P > 0.05) for rabbits on all the diets, but these feed intake values tended to increase as the BVWM replacement level increased in the diets.Results of growth performance of rabbits fed BVW are presented in P < 0.05) affected by the treatment. These ratios decreased as the BVWM replacement level increased in their feed. The rabbits on the 30 and 45% BVWM replacement diet had significantly lower (P < 0.05) feed to gain ratio values  compared with the 0 and 15% BVWM diets that had feed to gain ratios of 7.85 and 7.81, respectively.The feed to gain ratio was significantly (P > 0.05) for rabbits on all the diets, the intake seemed to have increased with the increasing levels of BVWM replacement in the fed. Faecal nitrogen values were highly comparable (P > 0.05) for all treatments except rabbits fed on the 45% BVWM replacement diet that had significantly lower (P < 0.05) faecal nitrogen value of 0.37 g.P > 0.05) nitrogen balance values except for the rabbits on the 45% BVWM replacement diet with a higher (P < 0.05) nitrogen balance of 1.76 g. Nitrogen digestibility increased (P < 0.05) as the level of BVWM replacement also increased in the rabbit diet. The rabbits on the control feed had a digestibility of 60.49% which kept increasing up to 76.86% for the rabbits fed 45% BVWM diet.The rabbits had comparable (The rabbits seemed to have much appetite for the test ingredient (BVWM). The rabbits tended to consume more of the BVWM diets than the groundnut cake control diets. This is contrary to reports of Adeniji , who repThe improved feed to gain ratio at the high levels of BVWM shows that BVWM is a promising feedstuff. BVWM can help to solve the problem of feed scarcity and reduce demand on expensive protein supplements like groundnut cake and soyabean meal. Akinfala et al.  and EkenThe improved digestibility with higher levels of BVWM implies that rabbits are able to effectively digest higher levels of BVWM whose could have been discarded as waste. BVWM which monogastrics might not be able to digest well because of their high fibre level should not be discarded but converted for rabbit feed.Based on the lower feed to gain ratio and higher nitrogen digestibility, rabbits can tolerate the 45% groundnut cake replacement with BVWM. This will reduce the heavy demand for groundnut cake in livestock feeding and its high price reduced. Improved rabbit production can play on important role in increasing animal protein supply to people in the under developed countries, considering the high cost of chicken, pork, and beef.