We therefore expressed the fluorescent ATP biosensor Perceval in mouse pancreatic islets and loaded them with a Ca2+ indicator. With total internal reflection fluorescence microscopy, we recorded subplasma membrane concentrations of Ca2+ and ATP  in superficial α- and β-cells of intact islets and related signaling to glucagon and insulin secretion by immunoassay. Consistent with ATP’s controlling glucagon and insulin secretion during hypo- and hyperglycemia, respectively, the dose-response relationship for glucose-induced [ATP]pm generation was left shifted in α-cells compared to β-cells. Both cell types showed [Ca2+]pm and [ATP]pm oscillations in opposite phase, probably reflecting energy-consuming Ca2+ transport. Although pulsatile insulin and glucagon release are in opposite phase, [Ca2+]pm synchronized in the same phase between α- and β-cells. This paradox can be explained by the overriding of Ca2+ stimulation by paracrine inhibition, because somatostatin receptor blockade potently stimulated glucagon release with little effect on Ca2+. The data indicate that an α-cell-intrinsic mechanism controls glucagon in hypoglycemia and that paracrine factors shape pulsatile secretion in hyperglycemia.—Li, J., Yu, Q., Ahooghalandari, P., Gribble, F. M., Reimann, F., Tengholm, A., Gylfe, E. Submembrane ATP and Ca2+ kinetics in α-cells: unexpected signaling for glucagon secretion.Cytoplasmic ATP and Ca Insulin and glucagon have central roles in maintaining normal glycemia by lowering and increasing blood glucose, respectively. The circulating concentrations of both hormones oscillate , 2, whic+ (KATP) channels resulting in depolarization with influx of secretion-triggering Ca2+ through voltage-dependent channels  of peripheral cells in mouse pancreatic islets. Supporting a role of α-cell ATP in glucagon-mediated glucose counterregulation, [ATP]pm in α-cells was relatively more sensitive than that in β-cells, in response to the low glucose concentrations that characterize hypoglycemia. Both α- and β-cells showed oscillations of [ATP]pm that were in opposite phase to those of the Ca2+ concentration in the subplasma membrane space ([Ca2+]pm) indicating energy-dependent Ca2+ transport. Although 20 mM glucose induces a pulsatile release of insulin and glucagon in opposite phase  type 2 antagonist potently stimulated glucagon release with little effect on α-cell [Ca2+]pm.In the present study, we used Perceval  and totate phase , 5, thisn pulses , 28, thol-lysine, diazoxide, glutamic acid, and HEPES were from Sigma-Aldrich . Fetal bovine serum (FBS) was from Life Technologies-Gibco . The insulin and SSTR-2 antagonists S961 and PRL2903 were kind gifts from Novo Nordisk, Bagsværd, Denmark, and Dr. D. H. Coy , respectively. Adenovirus encoding the fluorescent ATP biosensor construct Perceval  mM CaCl2, 3 mM glucose, 0.5 mg/ml BSA, and 25 mM HEPES with pH adjusted to 7.4 with NaOH.The primary polyclonal rabbit anti-insulin antibody was from Abcam , and the primary polyclonal rabbit anti-glucagon antibody was from Dako . The secondary antibody Alexa Flour 488 goat anti-rabbit IgG was from Life Technologies . Poly-Perceval  was used2 in humidified air.All animal experimental procedures were approved by the local ethics committee for use of laboratory animals in Uppsala, Sweden. Transgenic mice with homozygous expression of RFP in α-cells activated by proglucagon promoter-driven Cre-mediated excision of a stop codon (GLU-RFP mice) were produced in Cambridge , followe6 plaque-forming units/islet or 20 plaque-forming units/cell for 1–2 h. During infection, the concentration of serum was reduced to 2%. The islets were subsequently washed 3 times with normal RPMI 1640 medium and cultured for at least 18 h before use.For ATP measurements, the islets were infected with the Perceval adenovirus at concentrations of 1–2 × 102+ indicator Fluo-4 (Life Technologies). The islets were allowed to attach onto poly-l-lysine-coated coverslips for 5–10 min. Coverslips with Fluo-4-loaded islets were used as exchangeable bottoms of an open 50 µl chamber that was superfused with experimental medium at a rate of 0.12–0.2 ml/min at 37°C. [Ca2+]pm was then measured with TIRF microscopy (Islets from transgenic GLU-RFP mice were incubated for 20–30 min with 1.2 μM acetoxymethyl ester of the Cacroscopy .2+ indicator Fura Red (Life Technologies). The same experimental conditions were used as when measuring [Ca2+]pm alone. Perceval and Fura Red fluorescence were excited at 491 nm by a diode-pumped solid-state laser , and fluorescence was measured with 530/50 nm  and 620 nm long-pass (Fura Red and RFP) filters . Image pairs were acquired every 5 s, as described above.Because of interference between Perceval and RFP, ATP could not be measured reliably in α-cells from transgenic GLU-RFP mice (see below). Most ATP measurements were therefore made on islets from normal mice. Before experiments Perceval-infected islets were preincubated for 60 min in experimental medium containing 5 μM of the acetoxymethyl ester of the CaImmunostaining was performed at room temperature, unless otherwise stated, with PBS. Isolated islets were thoroughly rinsed and then fixed with 4% paraformaldehyde for 10 min, followed by cell permeabilization by exposure to 0.2% Triton X-100 on ice for 10 min. Nonspecific antigens were blocked by 30 min exposure to 5% FBS (blocking buffer). The islets were then incubated for 2 h with rabbit polyclonal anti-insulin or -glucagon diluted 1:100 in blocking buffer followed by 3 washes. Subsequently, the islets were incubated in darkness for 1 h with Alexa Fluor 488 goat anti-rabbit IgG diluted 1:200 in blocking buffer. The islets were then washed 4 times for 5 min in 0.1% Tween-20 to reduce background, followed by 3 additional washings in PBS.A previously described confocal microscope setup  was usedBatches of 8–10 size-matched islets were preincubated at 37°C for 30 min in experimental medium containing 3 mM glucose followed by incubation for 40 min in 500 μl medium containing 3 or 20 mM glucose and hormone receptor antagonists, as indicated in 2+]pm and [ATP]pm were low was used to compensate for variations in cellular content of the different fluorescent indicators. To facilitate a [Ca2+]pm comparison between α- and β-cells, the Fluo-4 fluorescence was expressed as changes in relation to fluorescence after background subtraction observed in Ca2+-deficient medium containing EGTA at the end of the experiments (F/FCa0). The Fura Red fluorescence, which decayed more rapidly, was instead expressed in relation to the initial fluorescence in the presence of 3 mM glucose when [Ca2+]pm was low (F/F0) in the β-cells. However, in α-cells with considerable [Ca2+]pm activity at different glucose concentrations, Fura Red fluorescence was instead expressed in relation to fluorescence at the lowest [Ca2+]pm values during each recording (F/FCaLow). Since Fura Red fluorescence decreases upon Ca2+ binding, the traces were inverted (mirrored) to show increases in [Ca2+]pm as positive deflections. Perceval and RFP fluorescence data were always expressed as changes in relation to initial fluorescence after subtraction of background (F/F0). The relationships between [Ca2+]pm and [ATP]pm oscillations in α- and β-cells were analyzed with asymmetric sliding-window cross-correlation  or FIJI  softwarerelation  in MATLASupplemental Fig. S1). However, unexpected problems became apparent after infection with the ATP sensor Perceval, which interacted with RFP to produce erratic responses affecting both fluorophores in the α-cells . Because the RFP did not interact with the Ca2+ indicator Fluo-4, we instead used the transgenic mouse islets and TIRF microscopy to establish functional criteria for discriminating between α- and β-cells, based on characteristic [Ca2+]pm responses. Most RFP-negative cells had low, stable [Ca2+]pm levels in 3 mM glucose, and introduction of 20 mM glucose caused a β-cell-characteristic  with [Ca2+]pm activity at 3 mM glucose in islets from 15 mice. Cell footprint area, the [Ca2+]pm patterns in 3 and 20 mM glucose, and glutamate responsiveness were therefore always used to discriminate between α- and β-cells.To facilitate cell identification, islets were isolated from GLU-RFP mice with RFP-expressing α-cells. Confocal imaging of immunostained islets showed that nearly all of the RFP-positive and -negative cells were α- and β-cells, respectively . Considering the implication of ATP in glucose-regulated glucagon and insulin release, we next compared [ATP]pm in the 2 cell types at glucose concentrations covering the glucose control range for both hormones (P < 0.001). Further glucose elevation to 20 mM to stimulate insulin release caused a more pronounced increase in Perceval fluorescence in β- than in α-cells  similar to that in the GLU-RFP mouse β-cells . The simultaneous [ATP]pm and [Ca2+]pm recordings reinforced our previous observations .We next investigated the relationships between [ATP]rvations  that the2+]pm and [ATP]pm oscillations in a single α-cell and after averaging data from 4 α-cells within the same islet as the β-cells in Supplemental Fig. S3. In 3 mM glucose, the antiparallel relationship was obvious only when we analyzed single α-cells; both [Ca2+]pm and [ATP]pm oscillations tended to cancel out when data were averaged from several cells. However, in 20 mM glucose, the antiparallel [Ca2+]pm and [ATP]pm oscillations became sufficiently synchronized to be apparent, also after data were averaged from several α-cells, with identical periodicity but much smaller amplitudes, as compared to that of β-cells. The cross-correlogram showed that the phase relationship between the [Ca2+]pm and [ATP]pm oscillations in α-cells was very similar to that in β-cells. However, glutamate induced a pronounced increase in [Ca2+]pm and a lowering of [ATP]pm levels only in the α-cells  characteristics. Indeed, because of the strong interference between Perceval and RFP, we relied entirely on the functional [Ca2+]i responses for the ATP measurements. We were surprised that no Perceval-RFP interference was reported when seemingly identical GLU-RFP mice were used to record the α-cell ATP response to a 1–6 mM glucose increase with confocal microscopy (2+ or ATP or both, in that K+ depolarization also induced parallel changes in RFP and Perceval fluorescence.The relatively low proportion of α-cells within pancreatic islets hampers study of the signaling mechanisms that mediate glucose inhibition of glucagon secretion. Attempts to circumvent this difficulty by purifying α-cells from rat  and mousc islets . These cc islets  and diffcroscopy . We founpm is more elevated in α-than in β-cells exposed to low glucose concentrations. When expressed as a percentage of the subsequent response to 20 mM, glucose elevation from 1 to 5 mM induced a much stronger [ATP]pm response in α- than in β-cells. The higher α-cell sensitivity at low glucose concentrations is consistent with ATP’s involvement in glucose control of glucagon secretion during recovery from hypoglycemia.Although in prior studies, we calibrated the Perceval fluorescence signal in permeabilized β-cells exposed to different ATP concentrations , 43, it 2+ and amplifying cAMP, which show synchronized oscillations in β-cells (2+ and cAMP, the interplay between the messengers is complex, because ATP consumption increases in response to Ca2+ elevation. The β-cells within islets are coupled by gap junctions (2+ signaling (pm is related to average [Ca2+]pm in the same manner as in single β-cells (2+, but the subsequent oscillations of ATP and Ca2+ were essentially antiparallel. Our analysis of α-cells indicated a similar inverse relationship between changes in the levels of [Ca2+]pm and [ATP]pm. At 3 mM glucose, when the α-cells, in accordance with previous observations (2+]pm oscillations, the inverse relation to [ATP]pm was evident only in individual α-cells. However, at 20 mM glucose, the [Ca2+]pm oscillations tended to synchronize among the α-cells. This synchronization was not as unmistakable as between the electrically coupled β-cells but became distinct after [Ca2+]pm of several α-cells within an islet was averaged, probably explaining why it has previously escaped detection (2+]pm and [ATP]pm oscillations in the α cells seemed identical to that in the β-cells and probably reflects energy-dependent Ca2+ extrusion.Glucose-stimulated insulin release is pulsatile , 5, and  β-cells , 46. Altunctions , leadingignaling , 42, whi β-cells . Glucose2+ is the major trigger of insulin secretion (2+]pm oscillations would be in opposite phase in α- and β-cells. We were therefore perplexed that the highly coherent [Ca2+]pm oscillations in β-cells synchronized with the average [Ca2+]pm oscillations in α-cells. This synchronization was consistent in islets from several preparations studied with different Ca2+ indicators and probably involves paracrine coordinating factors. In a previous study, it was noticed that although Ca2+ oscillations are not synchronized among α-cells, oscillations in individual α-cells exposed to 8–12 mM glucose sometimes approximately follow the surrounding coordinated β-cell waves of Ca2+ (2+ synchrony dominates at glucose concentrations that stimulate insulin secretion. Therefore, glucose may act downstream of Ca2+, which is consistent with proposed Ca2+-independent inhibition by hyperglycemic concentrations of the sugar (At 20 mM glucose, glucagon release from isolated islets also is pulsatile and in opposite phase to the insulin pulses , 5, whicecretion  and is gecretion –21. As a of Ca2+ . Our dathe sugar .2+]pm, as is expected if somatostatin acts downstream of Ca2+ (2+]pm oscillations were found to be in the same phase in α- and β-cells. However, somatostatin cannot explain all inhibition; present and previous (The present data support a recent suggestion that glucose inhibition of glucagon release is regulated by α-cell-intrinsic mechanisms during recovery from hypoglycemia and that paracrine factors become important when the glucose concentration exceeds the thresholds for stimulation of δ- and β-cell secretion . The obs of Ca2+ , 51. Pul of Ca2+ , 5 as a previous  data shoprevious , 53. No


We hypothesized that resveratrol stimulates NO production by thick ascending limbs via a Ca2+/calmodulin-dependent mechanism. To test this, the effect of resveratrol on NO bioavailability was measured in thick ascending limb suspensions. Cai was measured in single perfused thick ascending limbs. SIRT1 activity and expression were measured in thick ascending limb lysates. Resveratrol (100 µM) increased NO bioavailability in thick ascending limb suspensions by 1.3±0.2 AFU/mg/min (p<0.03). The NOS inhibitor L-NAME blunted resveratrol-stimulated NO bioavailability by 96±11% (p<0.03). The superoxide scavenger tempol had no effect. Resveratrol elevated Cai from 48±7 to 135±24 nM (p<0.01) in single tubules. In Ca2+-free media, the resveratrol-induced increase in NO was blunted by 60±20% (p<0.05) and the rise in Cai reduced by 80%. Calmodulin inhibition prevented the resveratrol-induced increase in NO (p<0.002). AMPK inhibition had no effect. Resveratrol did not increase SIRT1 activity. We conclude that resveratrol increases NO production in thick ascending limbs via a Ca2+/calmodulin dependent mechanism, and SIRT1 and AMPK do not participate. Resveratrol-stimulated NO production in thick ascending limbs may account for part of its beneficial effects.The thick ascending limb of the loop of Henle reabsorbs 30% of the NaCl filtered through the glomerulus. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl absorption by this segment. Resveratrol, a polyphenol, has beneficial cardiovascular and renal effects, many of which are mediated by NO. Resveratrol increases intracellular Ca Defective NO signaling in thick ascending limbs contributes to several forms of salt-sensitive hypertension 2+ (Cai) which cause the Ca2+/calmodulin complex to associate with the enzyme. Binding of Ca2+/calmodulin to NOS3 facilitates the electron flux through its domains and increases NO production i levels, such that normal basal Cai is sufficient to increase NO production. Other kinases such as AMP kinase (AMPK) also phosphorylate NOS3 at S1177, activating it independently of increases in CaiOriginally NOS3 was reported to be activated by increases in intracellular CaVitis vinifera grapes, and remains present at high concentrations in red wines The polyphenol resveratrol is abundant in the skin of ivia a Ca2+/Calmodulin-dependent mechanism.Although NO mediates many of the effects of resveratrol, the mechanisms by which it activates NOS3 and increases NO are not fully understood, and may be tissue dependent. Resveratrol has been reported to stimulate AMPK This study was approved by the Case Western Reserve University and the Henry Ford Hospital Institutional Animal Care and Use Committees. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats  weighing between 220–260 g were anesthetized with ketamine (100 mg/kg bw IP) and xylazine (20 mg/kg bw IP), and given 2 IU heparin (IP). All efforts were made to minimize suffering. Animals were sacrificed while still under anesthesia.2+-sensitive dye FURA 2-AM, and the Ca2+ ionophore 4-Bromo A-23187 (4Br-A23187) were obtained from Invitrogen . Coomasie Plus Protein Assay Reagent was obtained from Thermo-Scientific, .Unless specified, all drugs and reagents were obtained from Sigma-Aldrich . The cell-permeable NO-selective fluorescent dye DAF-FM-diacetate, the Ca2+ media) contained [in mmol/l]: 10 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.5), 130 NaCl, 4 KCl, 2.5 NaH2PO4, 1.2 MgSO4, 5.5 glucose, 6.0 DL-alanine, 2.0 Ca(lactate)2, and 1.0 Na3citrate. Ca2+-free media contained [in mmol/l]: 10 HEPES (pH 7.5), 130 NaCl, 4 KCl, 1.2 MgSO4, and either 100 or 200 µmol/l ethylene glycol tetraacetic acid (EGTA). Osmolalities of both solutions were adjusted to 300±5 mOsmol/l with mannitol.HEPES-buffered physiological saline , and the resulting pellet of tubules resuspended in fresh HEPES-buffered physiological saline and stirred on ice for 30 min. After stirring, the sample was filtered through a 250 µm nylon mesh, and the filtered tubules were collected and rinsed at 4°C. This preparation resulted in a 95% pure suspension of thick ascending limbs.Suspensions were prepared as follows: kidneys were perfused retrograde 2+ media with 100 µmol/l L-arginine and gassed with 100% O2 at 37°C. They were then incubated and rinsed repeatedly every 5 min at 37°C for 15 min to cleave the dye and wash out the acetylated form. After the last rinse, the suspension was split into two 1500-µl aliquots, loaded into different cuvettes, and incubated for 5 min at 37°C before acquiring measurements. Then, vehicle or resveratrol was added and the increase in fluorescence over time was measured for 20 sec, at 1-min intervals, over 5 min. Dye was excited at 485 nm and fluorescence collected at 515 nm. After these measurements, the suspensions were transferred to 2-ml tubes, cooled on ice, and the tubules were recovered by centrifugation for protein measurements. Data from both vehicle and resveratrol treatments were analyzed by linear regression and expressed as resveratrol-induced arbitrary fluorescence units (AFU) per mg protein per min. The DMSO concentration never exceeded 0.1%. In these experiments, when the effects of different drugs were assessed, the drugs were added just after aliquots were separated and kept in the media until the end of the experiment.Intracellular NO bioavailability in thick ascending limb suspensions was measured using the dye DAF-FM. A 5 mmol/l stock solution was prepared daily in dimethyl sulfoxide (DMSO). Tubules were loaded by adding 5 µmol/l DAF-FM to the medium while being stirred on ice during suspension preparation In the second method, resveratrol-induced NO production was continuously measured over time on a single aliquot. For this purpose, thick ascending limb suspensions were loaded with DAF-FM and washed as described above. Then the whole suspension was transferred to a cuvette and stirred in the spectrofluorometer for 5 min with the shutter closed for stabilization, followed by a 2-min equilibration period with the shutter open. Basal fluorescence was collected for 4 additional min, resveratrol was added, and the fluorescence was collected again for 4 min. In these experiments, drugs other than resveratrol were added during the 5-min stabilization period.Data depicted in 2+ on resveratrol-induced NO were measured, 2.0 mM Ca2+ media was substituted with Ca2+-free media. This medium was supplemented with either 100 µmol/l or 200 µmol/l EGTA. Results with either 100 or 200 µmol/l EGTA were not different so the data were pooled together.In the experiments in which the effect of removing extracellular CaSingle medullary thick ascending limbs were isolated and perfused as previously described 2+ media. To study the effects of resveratrol in the absence of extracellular calcium, the 30-min washing/cleavage period was performed using Ca2+-free media with 200 µmol/l EGTA in both luminal and basolateral sides.Individually perfused thick ascending limbs were loaded for 30 min with 1 µmol/l FURA 2-AM dissolved in 2 mM Ca media, followed by a 30-min washing/cleavage period using dye-free 2 mM Cai levels were measured for 5 min taking images every 30 sec. Then, 100 µmol/l resveratrol was added to the basolateral bath dissolved in either Ca2+-containing or Ca2+-free media, and Cai was measured again. At the end of each individual experiment, a 2-point calibration was performed using Ca2+-free media with 5 mmol/l EGTA and 2 mmol/l Ca2+ media added with 10 µmol/l 4-Br-A23187. Cai was calculated using the following equation as previously described:d is the dissociation constant of FURA 2 for Ca2+ (224 nmol/l); R is the fluorescence ratio following 340-nm and 380-nm excitation respectively; Fmin is the minimum fluorescence intensity at 380-nm excitation during EGTA treatment; Fmax is the maximum fluorescence intensity at 380-nm excitation during 4-Br-A23187 treatment; Rmin is the minimum fluorescence ratio following 340-nm and 380-nm excitation respectively during EGTA and Rmax is the maximum fluorescence ratio following 340-nm and 380-nm excitation respectively during 4-Br-A23187 treatment.For measurements, the dye was alternatively excited at 340 nm and 380 nm and fluorescence collected at 510 nm. Thick ascending limbs were digitally imaged using an image intensifier adapted to a charge-coupled device camera, and data were recorded using MetaFluor 7 Fluorescence Ratio Imaging Software . Resting CaTubules were dissolved in lysis buffer and 25, 50 and 100 µg protein were loaded onto a 6% polyacrylamide gel. Electrophoresis was performed and membranes were incubated with a primary mouse monoclonal anti-SIRT1 antibody  (diluted 1∶1000) for 2 hr; washed, and reincubated for 1 hr with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (diluted 1∶1000) (Amersham Pharmacia Biotech). The membranes were incubated with a luminol-based chemiluminescent HRP substrate  and exposed to a radiographic film for 5 min. Films were scanned and densitometry of the blots was performed using a customized program. As a loading control, tubulin was detected using rabbit polyclonal anti-β tubulin antibody  (diluted 1∶10000) and a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (diluted 1∶5000) (Amersham Pharmacia Biotech).2+-dependent deacetylases inhibitor) and kept on ice. Protein concentration was measured and samples were diluted to 2 µg/µl. SIRT1 activity was assessed using 20 µg protein according to the manufacturer recommendations. Basal and resveratrol-induced SITR1 activity was assayed by adding either vehicle or 100 µmol/l resveratrol. Lysates supplemented with human-recombinant SIRT1 (hrSIRT1) were run as positive controls.The SIRT1 Direct Fluorescent Screening Assay Kit  was used as follows: tubules were dissolved in lysis buffer supplemented with 100 µM trichostatin A  or vehicle (0.1% DMSO) on DAF-FM fluorescence in thick ascending limb suspensions. We found that 100 µmol/l resveratrol increased DAF-FM signal by 1.3±0.2 AFU/mg/min as compared to vehicle p<0.03;  while lo2−) scavenges NO, thus reductions in O2− by resveratrol could augment DAF-FM fluorescence. To test this, we measured resveratrol-induced DAF-FM fluorescence in the presence of either NOS inhibitor L-NAME or the O2− scavenger tempol (100 µmol/l). We found that L-NAME blunted resveratrol-stimulated NO by 96±11% , from 2.5±0.5 AFU/mg/min (n = 9) to 1.0±0.5 AFU/mg/min (n = 11) , a response 80% lower than in a Ca2+-containing media. These results indicate that the NO-response to resveratrol largely depends on Ca2+ influx.To examine whether the resveratrol-induced increase in Ca(n = 11) . In sepai increase NO production is via calmodulin. Thus, we tested whether calmodulin participates in the activation of NOS by resveratrol in the thick ascending limb. To do this, we measured resveratrol-induced NO production in the presence of 100 µmol/l calmodulin inhibitor W-7. We found that inhibition of calmodulin completely blocked the NO response to resveratrol; −0.8±0.2 AFU/mg/min (n = 4) vs. 2.2±0.5 AFU/mg/min in the controls . Under these conditions, addition of resveratrol increased NO production by 2.9±0.8 AFU/mg/min compared to vehicle  or 10, 50 and 100 µmol/l resveratrol on DAF-FM fluorescence. In these experiments, only 100 µmol/l resveratrol significantly increased NO bioavailability as compared to vehicle. Similar to our results, the cGMP levels in vasculature derived cells were reported to be elevated after a 2-min treatment with 100 µmol/l resveratrol, while lower doses had no significant effect Our results regarding resveratrol-induced NO in renal tissue are consistent with a previous report, in which intravenous infusion of resveratrol increased renal NO in rats 2−) scavenges NO, thus reductions in O2− levels would augment NO bioavailability. The renal medulla is the primary source of O2− in the kidney. In addition NADPH oxidase is the main source of O2− in the thick ascending limb An increase in NO bioavailability can be either due to enhanced production or decreased degradation. Superoxide anion (O2− levels, or both; we measured resveratrol-induced NO in the presence of either L-NAME or the O2− scavenger tempol. L-NAME blunted resveratrol-stimulated NO by 96%. In contrast, scavenging O2− with tempol has not a significant effect on the ability of resveratrol to enhance NO bioavailability.To test whether the increase in DAF-FM signal was due to elevated NO production, decreased O2− or inhibiting NADPH oxidase. If resveratrol would have increased NO simply by reducing O2−, one would have expected the effect of resveratrol to be blunted in the presence of tempol. Our results show just the opposite, a trend to a higher response in the absence of O2−. Thus our data suggest that resveratrol does not increase NO bioavailability by scavenging O2−. Furthermore, since there was a trend for tempol to enhance the effect of resveratrol on NO bioavailability, our data suggest that endogenous O2− scavenges part of the resveratrol-induced NO. Our results are similar to those of a previous report in which systemic infusion of resveratrol produced an endothelium-dependent dilation of the renal vasculature mediated mainly by NO These data suggest that the increase in NO levels elicited by resveratrol in thick ascending limbs is due to stimulation of NOS activity rather than scavenging Ovia signaling cascades. In several cell types, resveratrol increases Caiii in isolated perfused thick ascending limbs. We found that basolateral addition of resveratrol produced a sustained elevation in Cai. This increase in Cai parallels the increase in NO production measured separately in thick ascending limb suspensions.Since our data show that resveratrol stimulates NOS activity, we next investigated the mechanism involved. Originally it was proposed that resveratrol might enhance NO production by binding directly to NOS; however, it had no effect on NO production by purified NOS3 in solution or in rat aortic homogenates i was due to release from intracellular stores or influx of extracellular Ca2+, we measured resveratrol-induced increases in Cai and NO production in a Ca2+-free media. Under these conditions, the increases in Cai and NO production were blunted by 80% and 60% respectively, suggesting that the NO-response to resveratrol largely depends on Ca2+ influx.Then, to examine whether the resveratrol-induced increase in Cai increases NO production is via calmodulin. The Ca2+/calmodulin complex binds to NOS, facilitating the electron flux throughout its domains and subsequently increasing NO production via Ca2+/calmodulin upon Ca2+ influx from the extracellular medium.The most likely mechanism by which Ca2+ are consistent with previous reports in epithelial 2+-dependent fashion; however, none of those reports evaluated the effects of calmodulin inhibition.Our results concerning Cai have been reported in several cell-lines including human prostate cancer cells i occur remain elusive. For instance, in vascular cells i seems to be due to activation of several types of transmembrane Ca2+-permeable channels, while in a cancer cell-line, Ca2+ apparently is released from the endoplasmic reticulum i due to release from intracellular stores followed by a sustained increase due to Ca2+ influx from the extracellular space. In 50% of the cells, resveratrol produced a slow and sustained rise in Cai similar to that observed by us in isolated perfused thick ascending limbs. The explanation for these apparently disparate data remains elusive; however, it is possible that resveratrol acts to elevate Cai through different simultaneous mechanisms.Resveratrol-dependent increases in Cai, some enzymes that can mediate increases in NO such as SIRT1 and AMPK, have been reported to be stimulated by resveratrol In addition to CaFinally, to study the involvement of AMPK in the resveratrol-induced NO production in the thick ascending limb, we measured resveratrol-induced NO in the presence of AMPK inhibitor compound C. We found that AMPK inhibition does not blunt the acute response to resveratrol . These ri levels 2+/calmodulin-dependent protein kinase are activated, AMPK is unlikely to play a major role in NOS3 regulation As opposed to the thick ascending limb, on human umbilical vein endothelial cells, AMPK inhibition exerts a mild inhibitory effect in acute resveratrol-induced NO production independently of CaAMPK and SIRT1 could be important though in the long-term effects of resveratrol which are mediated by activation of several different genes The renal medulla is the main site for NO production in the kidney, where NOS activity is fine-tuned by several mechanisms acting simultaneously Our current work provides a mechanistic explanation of how resveratrol acutely increases NO production in renal tissue obtained from healthy animals. Future studies should better characterize the pharmacological properties of resveratrol and evaluate the feasibility of resveratrol supplementation.2+ influxes from the extracellular environment. AMPK and SIRT1 are not involved.In summary, our experiments show that resveratrol acutely increases NO production in the thick ascending limbs of healthy rats. This increase depends on the activation of NOS by calmodulin upon Ca


Circular Dichroism (CD) measurements suggest a major rearrangement of the protein secondary structure, which reversibly switches from disordered at low Ca2+ concentrations to predominantly alpha-helical when Ca2+ is added. SAXS experiments confirm the transition from an unfolded to a compact structure, which matches the structural prediction of a trilobal fold. Overall our experiments suggest that calumenin is a Ca2+ sensor, which folds into a compact structure, capable of interacting with its molecular partners, when Ca2+ concentration within the ER reaches the millimolar range.Human calumenin (hCALU) is a six EF-hand protein belonging to the CREC family. As other members of the family, it is localized in the secretory pathway and regulates the activity of SERCA2a and of the ryanodine receptor in the endoplasmic reticulum (ER). We have studied the effects of Ca CalumenSERCA2a)  and the SERCA2a) , therebyiewed in ).2+ concentration. In this study we have analysed the Ca2+ -dependent structural behaviour of calumenin and propose a folding model that provides a structural explanation of Ca2+ sensing–a feature likely to be shared across the CREC family.So far very little is known about the structure-function relationship of calumenin in response to variation of CaE. coli cells (Novagen) were used for expression starting from a saturated overnight culture in LB-Miller broth supplemented with 0.1% glucose. The protein was expressed from 1 litre of LB-Miller broth supplemented with autoinduction additives [The region encompassing the 6 EF-hand repeats of human calumenin isoform 1  was cloned in the vector pOPINS as previously described . Rosettadditives , inocula2, 0.1% α-monothyoglycerol) supplemented with mini EDTA-free protease inhibitors (Roche). Cells were lysed by 2 passes on a Constant Systems TS Cell Disruptor at 4°C and a pressure of 30 kPsi. Lysate was clarified by centrifugation (45 minutes at approx. 39000 xg) and applied on a 5 ml HisTrapFF 5ml column . The peak from the gradient elution with 100% of buffer B  was concentrated on 10 kDa cut-off Amicon Ultra filter units (Millipore) to approx. 3 ml. This sample was applied on a size-exclusion (SEC) column Superdex75 HR16/60  equilibrated with buffer C . The central fractions of His-SUMO-calumenin were pooled and treated with SUMO-protease (1:10 molar ratio) overnight at 4°C.Approximately 40 g of frozen bacterial cell pellet were suspended in buffer A  to approx. 8.82 mg/ml. 15 μl aliquots were flash frozen in liquid nitrogen and stored at -80°C.The cleaved calumenin  equilibrated with buffer E  and F  respectively. The same column was equilibrated with buffer G  to evaluate the effect of Sr2+.Analytical gel-filtration profiles of calumenin were obtained in the presence (+ 2.5 mM) or in the absence of CaClElution peaks were also submitted to multi-angle light scattering (MALS), dynamic light scattering (DLS) and refractive index (Ri) analysis  and data were processed through the software package ATLAS 5 to estimate the molecular weight and hydrodynamic radius.2+ from the protein. Both Ca2+ and the chelating agent were removed from solution by loading 200 μl of the protein solution on a 5 ml HiTrap desalting column  equilibrated in buffer I.The concentrated protein solution, stored at -80°C in 300 μM aliquots, was thawed on the day of the experiments and diluted to 100 μM using a buffer H  to strip the CaThe apo-protein was further diluted to 5 μM with buffer I or J  to a final NaCl concentration of 25 mM (for low ionic strength experiments) or 150 mM .2+ concentration points. These were achieved by small additions of concentrated CaCl2 to the original solution, using three stock solutions obtained by serial dilution of a CaCl2 standard (Fluka 21049). All CD spectra were obtained from quadruplicate measurements, averaged and buffer subtracted.Circular dichroism spectra were collected in the far-UV region (185–260 nm) using a 0.05 mm pathlength quartz cuvette kept at 20°C at different CaThe CD titration experiments  were per2 at pH 4.5 according to the manufacturer’s protocol to obtain a response of approximately 2000 a. u. during the binding phase.Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 instrument . A sensor-chip CM5 was functionalised with calumenin in the presence of 1 mM CaClThe binding experiments were performed by flowing solutions containing different concentration of the cations in 1x HBS-N buffer . At the end of each cycle the residual metals were stripped from the chip with a wash of HBS-N buffer supplemented with 5 mM EDTA followed by a re-equilibration in HBS-N.All binding experiments, performed in quadruplicate, were averaged and analysed with the Biacore analysis software.2+ at the beamline I22 (Diamond Light Source). Averaging of repeated scans and background subtraction were performed with the DAWN software package.Preliminary small-angle X-ray scattering (SAXS) data were collected on samples at different concentration of calumenin both in the presence and in the absence of CaSAXS data collection coupled to SEC was performed at beamline B21 (Diamond Light Source) using a Shodex PROTEIN KW-803 size-exclusion column loaded with buffer D from the purification. The scattering curves from 40 data points within the chromatography peak were scaled and averaged to obtain the I(q) function and the same region from a blank run (no protein loaded), was used to estimate the background scattering.Data reduction and preliminary analysis were performed with the software ScÅtter .2+-free calumenin was compatible with that of an unfolded protein (pure scattering); further analysis was not performed on the apo-protein datasets.Since the radial scattering profile of Ca2+-bound calumenin, estimation of Rg was obtained by the AutoRg module of the ATSAS suite [2 plot of 180 points at low q. The shape of the protein was reconstructed using DAMMIF [For the CaAS suite  through g DAMMIF  to proviSecondary structure prediction was obtained through the Xtalpred server .et. al [Manual alignment of the EF-hand domains was performed according to Rogstam et. al .Three dimensional structure prediction was performed by submitting the amino acid sequence of calumenin (68–315) to the servers iTasser , Phyre2 g from the coordinate files of the different predictors was performed using the software Hydropro [Calculation of RHydropro .The highest ranking models from the predictors above were superposed to the SAXS envelope using the software Supcomb .2+ binding motifs, bearing a canonical loop sequence (DxDxDGxxxxEE) within two alpha-helices. We also identified a seventh loop sequence upstream of the first EF-hand domain. This loop (aa. 46–67) resembles a canonical EF-hand motif including aspartate and glutamate residues, but lacking the fundamental glycine residue in position 6 that confers high flexibility to the EF-hand by allowing major rearrangements upon Ca2+ binding. The loop also lacks the flanking helical pattern characteristic of the EF-hand domains and was predicted to be in a disordered conformation. We thus defined it an “orphan” loop  using different algorithms identified six Helix-Loop-Helix (HLH)-motifs downstream of the N-terminal signal peptide. All six motifs contain the hallmarks of the EF-hand Caan” loop  which isoop aa. 4–67 resemFor this study we have focused exclusively on the six canonical EF-domains and we have cloned and expressed the C-terminus of calumenin in bacteria (aa. 68–315).E. coli using the pOPINS vector [6-SUMO tag, was purified through subsequent steps of IMAC, size exclusion chromatography and tag cleavage. Initial attempts to purify the protein in the absence of Ca2+ (supplementing most purification buffers with 1 mM EDTA), resulted in very low purity of final sample fractions, which we attribute to the intrinsic “stickiness” of the protein to the bacterial ones as well as degradation issues. To test this hypothesis, we added 2.5 mM CaCl2 to all the buffers and performed the purification as described in the Material and Methods section. This approach resulted in much higher purity compared to initial purifications, allowing us to isolate a few pure fractions  profiles of CaTo investigate this behaviour in greater detail, we loaded a concentrated purified sample of calumenin(68–315) on an analytical Superdex 200 Increase 10/300 GL column coupled to a setup for measuring MALS/DLS/Ri.2+ (2.5 mM CaCl2 and 2.5 mM EGTA in the column buffer respectively), and the system was equilibrated thoroughly before each run to allow precise Ri measurements.We analysed calumenin with and without Ca2+-bound form.The chromatograms, shown in The elution volumes are calculated by fitting the whole peaks to a Gaussian profile over 100 data points (n = 100) and are given in the figure as mean values and standard deviations. A t-test confirms the difference between the two populations is extremely statistically significant (P≤0.0001)2+. The values of RH, Dt and molecular weight (MW) reported in MALS measurements on the two peaks confirm that in both cases the protein is in its monomeric state, which rules out multimerisation/aggregation, in contrast with calsequestrin, which oligomerises in the presence of mM concentrations of Ca2, subsequently stripped with 2.5 mM EGTA and injected in a column equilibrated with the buffer containing 2.5 mM CaCl2. The profile  of the proteins in the two peaks. The RH value of 25.1 ± 0.1 Å found for the Ca2+-bound calumenin(68–315) is compatible with a slightly elongated protein of approx. 30 kDa. For the apo-calumenin, the much higher value of RH (37.7 ± 0.1 Å) suggests that a highly disordered/unfolded structure is present in the absence of Ca2+.DLS analysis allows the calculation of the hydrodynamic radius  in the absence of Caeriments .2+, treated with EGTA and finally re-buffered through a desalting column  to eliminate any traces of Ca2+and EGTA.To further elucidate the mechanism, we performed CD-titration experiments with calumenin which had been purified in the presence of Ca2 solutions and the data were analysed with the dedicated software CDApps [2+, the spectrum changes to that of a typical alpha-helical protein and displays a positive peak at 192 nm, a negative doublet at 208 nm and 219 nm and ellipticity = 0 at 200 nm . Based on the graphs in 2+ concentration in plots d,Ca of 21 ± 1 μM (n = 14) at buffer salt concentration of 25 mM NaCl. Increasing Ca2+ concentration leads to a marked loss in turns/disorder content (from 58% to 40%) and a corresponding increase of alpha helix content (from 17% to 50%).The formation of secondary structure was monitored as molar ellipticity at 219 nm in the case of the spectra collected at 150 mM NaCl. For the experiments at 25 mM NaCl, the peak at 192 nm was considered. Based on the graphs in i.e. the percentage of alpha-helical structure was obtained considering all 71 CD data points at a given Ca2+ concentration  and the increase of alpha helix content is slightly reduced (~35%) compared to the low salt condition.Secondary structure estimation from CD spectra was calculated using the CONTIN/LL algorithm  in CDAppntration . When th2+ to form a secondary structure either at high or low NaCl concentrations, but the final folded states are different in the two cases  was investigated using SPR. The protein was immobilised on a CM5 chip  either in the presence or in the absence of 2.5 mM Ca2+ in the immobilisation buffer.A further confirmation on the Ca2+ precipitation, we used the 1xHBS-N buffer provided by the manufacturer. Serial dilutions of Ca2+, Sr2+, Mg2+ and Mn2+ were prepared and used on the calumenin(68–315)-immobilised chip to quantify the binding constant of the ions. An equilibrium analysis confirms that Ca2+ is the only ion in the panel binding the protein in the sub millimolar range. The kon and koff for Ca2+ were extremely fast which, combined with the low signal and the anomalous post-injection peak shape, did not allow us to make a full kinetics analysis , which presents features for around q~0.1Å-1 which are indicative of the presence of small structured objects in the range of the 10s of Å  in its Cag) applying the Guinier law [2 curve  agrees with the RH value of 25.1 ± 0.1 Å (n = 40) estimated from the Ri analysis described above. The calculated Rg of 2.28 for the Ca2+-bound protein gives an Rg/RH ratio = 0.908 compatible with an elongated structure (0.775 < Rg/RH < 1).However, a detailed analysis of the diffraction images showed a severe radiation damage effect most likely due to a combined effect of the high flux delivered by the insertion device beamline and the long exposure time over the same portion of sample. To minimise both effects to the sample, we repeated the measurements using the HPLC-SAXS setup available at the B21 beamline of Diamond Light Source. SAXS images were obtained from the eluted peak so that each image was collected from protein not previously exposed to X-rays. This procedure enabled us to obtain high quality and highly redundant data (scatter profile averaged on 40 images). We calculated the radius of gyration of the molecule  function, the distribution of the lengths of interatomic vector lengths obtained by Fourier transform of the scattering curve . We obtaD. rerio, a neuronal cytosolic protein involved in stress hormone release which shares only 21% homology with calumenin [To interpret the SAXS envelope described above we searched for possible models by submitting the protein sequence of calumenin(68–315) to online servers dedicated to the prediction of 3D structures of proteins. We tested results obtained using Swiss-Model , Phyre2 alumenin ,29.g from the corresponding coordinate files using the software Hydropro [All the models proposed arranged twelve helices in non-globular structures, those based on secretagogin (PDB 2q4u) being the most elongated. To rank the protein predictions, we estimated the RHydropro .g values of 19.7 Å, 21.0 Å and 22.0 Å respectively, values comparable to the experimental value of 22.3 Å obtained from our HPLC-SAXS experiments. We fitted the protein envelopes calculated for our SAXS experiments on Ca2+-bound calumenin(68–315) with the models generated using the above servers using the software Supcomb [The highest ranking predictions from Phyre2, iTasser and SwissModel, displayed R Supcomb .The structure predicted by Phyre2 was too compact to fit the experimental envelope, failing to occupy the most peripheral zones, while bulging out of the central zone in portions of the space for which no electron density was observed. Manually fitting the model to the experimental data was not successful either: while one portion of the model could be forced to overlap the envelope, the orientation of the remaining part was unsatisfactory We obtained much more convincing fits with the structure predicted by iTasser and SwissModel which overlapped almost perfectly to the SAXS model . Each of2+ signal via structural rearrangements of helices around a Ca2+-chelating loop. A prototypical member of the EF-hand containing protein superfamily is calmodulin (CaM), a protein that undergoes major structural changes in response to variations in cytosolic Ca2+ concentration. This Ca2+-sensing protein selectively activates numerous biological processes, including Ca2+ homeostasis through interactions with the Plasma Membrane Ca2+ATPase (PMCA). Binding of Ca2+-loaded calmodulin to PMCA release autoinhibition and activates the pump. Calumenin has also been found to regulate a primary Ca2+ transporter, albeit with a different functional outcome. Binding of Ca2+-loaded calumenin to SERCA2a reduces the apparent Ca2+ activity of the transporter. Findings that calumenin also binds the ryanodine receptor which releases Ca2+ from the SR to induce muscle contraction, puts calumenin as a key player in the regulation of the SR and cellular Ca2+ homeostasis.The EF-hand domain is a fundamental functional unit designed by nature to transduce CaE. coli, crystallisation has been hampered by a number of factors such as (a) its propensity to co-elute with other bacterial proteins and to degrade (b) its low pI and (c) its intrinsic flexibility. The structures of cytosolic hexa EF-hand proteins have been determined [2+-affinities compared to the EF-hand proteins located in the secretory pathway, suggests that cytosolic EF-hand proteins may have a different structural arrangement.Very little is known about the organisation of the domains of the hexa-EF hand proteins, and no high-resolution structure is available so far for any of the CREC family members, to which calumenin belongs. Despite the high yields of calumenin obtained from heterologous expression in termined ,30, but 2+ concentrations, so the authors concluded that this SERCA2a-calumenin interaction occurred when the pump was in its E1 conformation. The structural alignment of the close homologue SERCA1a in all the states of the catalytic cycle does not explain the preference for the E1 state over the E2 since the L4 loop, although highly flexible, always appears in similar conformations. This large solvent-exposed loop, one of the most flexible regions of SERCA1a, as reflected by the high B-factor values, cannot completely justify the binding, unless calumenin can provide such type of conformational selection. As all EF-hands domains, calumenin was thought to change the arrangement of alpha-helices in response to local Ca2+ concentration changes and, as such the Ca2+-bound form of calumenin could stabilise the L4 of SERCA2a in one of its E1 conformations.Here we have focused on characterising calumenin, which has previously been reported to bind the L4 loop of SERCA2a through hydrophobic interactions with the domains EF3 and EF4 . This in2+-binding to calumenin provides the structural determinant essential to interact with Serca L4 loop in possibly any conformation (E1 or E2 sub-states).Overall our results suggest that Ca2+, calumenin exists in a random coiled apo-form. We have shown through CD titration that this converts into a predominantly alpha-helical structure by increasing the concentration of Ca2+ to the high μM range . Further SPR and SR-CD experiments at physiological ionic strength account for slightly higher values (201 ± 13 and 109 ± 1 μM respectively). These values are lower than those previously found with different techniques, but still consistent with the knowledge that CREC proteins have sub-mM Ca2+ affinities [2+ should not surprise, since Ca2+ concentrations within the ER can be in the mM range [2+ sensor, capable of regulating molecular partners when the ER is loaded with Ca2+, i.e. when the amount pumped into the ER by SERCA2a reaches the mM range.Differently from other EF-hand rich proteins, which only rearrange their helical content upon binding Cafinities . This remM range . Calumen2+-sensing proteins such as calsequestrin which can bind other cations such as Mn2+ and Mg2+ [2+ (or the very close mimic Sr2+). In the case of calmodulin, for instance, Sr2+ binds to Ca2+ sites, but the resulting structure is locked in a transition intermediate between the apo- and the Ca2+-bound state [Differently from other Caand Mg2+ , EF-handnd state .2+ appears even higher, since mM concentrations of Sr2+ only induced a partial alpha-helical conformation to the protein . Similarly to what is observed for calmodulin [2+ is only partially capable of inducing folding in calumenin. However, since this protein lacks pre-formed alpha-helical structures, Sr2+ is probably insufficient to induce the formation of an alpha-helical backbone  than in a low ionic strength buffer (25 mM NaCl). This not only accounts for the aforementioned differences in Kd values, but also for the different degree of cooperativity observed. The Hill coefficient indicates high positive cooperativity (n ~ 3.2) for calumenin titration conducted at physiological NaCl concentrations, similarly to what Iida and Potter observed for calmodulin [The existence of two flavours of EF-hand proteins, pre-formed and binding induced, possibly provides an extra regulatory level. Extensive molecular-dynamic studies of the potential underlying mechanisms would be very interesting and we believe our findings on calumenin could provide an excellent case study to define the parameters involved in ion-assisted folding of proteins further. Our CD titration studies on calumenin show that the alpha helical content of the Calmodulin .d,Ca, whose value also changes from 109 μM to 21 μM.When the NaCl concentration is lowered to 25 mM, the Hill coefficient decreases (n ~ 1.2) as does the K2+ to calumenin) and ~ 0.8  in 25 mM and 150 mM NaCl buffer respectively show the availability of different sets of Ca2+ sites  and to induce reversible folding.Further CD and SPR experiments have shown how this process is CaH values for a protein of this size and a pure solution X-ray scattering profile, together with the disordered CD results confirm that the protein is intrinsically unstructured in its Ca2+-free conformation. Upon Ca2+-binding, calumenin(68–315) converts into a more compact protein in which secondary structure elements are arranged in an elongated shape that is compatible with 3D structure predictions.The conformational switch determines not only a major rearrangement of the secondary structure, but also the formation of a compact three-dimensional shape. This has been shown both by our analytical SEC analysis and by SAXS measurements. The apo-form of calumenin(68–315) gives extremely high R2+-binding protein, which in the absence of Ca2+ retains the secondary structure elements which are rearranged in space upon Ca2+ binding [2+-binding protein also has a preformed core, held together by hydrophobic interaction, and rearranges in space at mM concentration of the ion to form multimeric structures [This is very different from calmodulin, another EF-hand Ca binding ,38,39. Cructures ,41,42.2+ homeostasis regulators of the ER as proposed. The presence of a third module (EF-5 and EF-6), which has not yet been fully characterised, opens the possibility that besides its Ca2+ sensor function, calumenin might act as a docking platform for more complex Ca2+ signalling within the ER.In the model best matching the experimental SAXS envelope, adjacent pairs of EF-hand domains are arranged in three separate lobes in a modular fashion. Such a model is in accordance with the observation that different proteins bind to different regions of calumenin. In particular the ryanodine receptor recognizes directly the EF-2 domain, whereasS1 Fig2+-binding domains of calumenin. The purple box represents the “orphan” EF-hand, while the green boxes indicate the EF1-6 (a). Logo diagram of the EF-hand loop consensus for the CREC family members (b).Primary structure alignment of the members of the CREC family with highlight on the Ca(EPS)Click here for additional data file.S2 FigQuantitation of the purity of each band from fractions 72, 73 and 74 reported in (EPS)Click here for additional data file.S3 Fig2 (a). SR-CD spectra of the apo-, the Ca2+-bound and Sr2+-bound forms of calumenin (b).SEC-MALS profiles from (EPS)Click here for additional data file.S4 Fig2+ up to 1:150 molar ratio (B). Each point in (b) was calculated from the deconvolution over all 71 data points of a single CD curve (a) and represents the α-helix or unordered structure contents at a specific calumenin:Ca2+ molar ratio.CD spectra of calumenin in 25 mM NaCl (a) obtained in the absence (A) or in the presence of increasing concentrations of Ca(EPS)Click here for additional data file.S5 Fig2+, Sr2+, Mn2+, Mg2+ and Cd2+ calculated as in d values in the M range, associated with a different ion-dependency profile to that of Ca2+, indicate the binding of the other ions is non-specific.The figure reports the affinity fit for Ca(EPS)Click here for additional data file.S6 FigVisual description of the process adopted to select structure simulation targets to overlap to the SAXS envelope.(EPS)Click here for additional data file.S7 FigJob plots for the titration of calumenin in the presence of 25 mM NaCl (a) and 150 mM NaCl (b).(EPS)Click here for additional data file.S1 Videoin silico model obtained.Rotation in 3D of the SAXS envelope superposed to the best fitting (ZIP)Click here for additional data file.


Area CA2 is emerging as an important region for hippocampal memory formation. However, how CA2 pyramidal neurons (PNs) are engaged by intrahippocampal inputs remains unclear. Excitatory transmission between CA3 and CA2 is strongly inhibited and is not plastic. We show in mice that different patterns of activity can in fact increase the excitatory drive between CA3 and CA2. We provide evidence that this effect is mediated by a long-term depression at inhibitory synapses (iLTD), as it is evoked by the same protocols and shares the same pharmacology. In addition, we show that the net excitatory drive of distal inputs is also increased after iLTD induction. The disinhibitory increase in excitatory drive is sufficient to allow CA3 inputs to evoke action potential firing in CA2 PNs. Thus, these data reveal that the output of CA2 PNs can be gated by the unique activity-dependent plasticity of inhibitory neurons in area CA2. Long overlooked, recent work has demonstrated that area CA2 of the hippocampus is a critical region for social memory and aggression. How area CA2 integrates into the hippocampal circuit is not understood. While CA2 pyramidal neurons (PNs) receive excitatory input from CA3 PNs, these inputs cannot drive action potentials in CA2 PNs because of a large feedforward inhibition. Furthermore, CA2 PNs do not express long-term potentiation, so it is unclear whether CA2 PNs can be engaged by CA3. We demonstrate that a unique activity-dependent plasticity of CA2 interneurons can increase the excitatory/inhibitory balance between CA3 and CA2 PNs, and allow the recruitment of CA2 PNs. Therefore, our results reveal a mechanism by which CA2 PNs can be engaged by CA3.Recent findings have revealed that hippocampal area CA2 plays an important role in learning and memory. Area CA2 has been shown to be necessary for social memory formation , and mayAxons from CA3 PNs [i.e. the Schaffer collaterals (SCs)] connect both CA2 and CA1 PNs. However, unlike the well described SC–CA1 synapses, the SC–CA2 connection does not display activity-dependent long-term potentiation LTP;  due to t+) interneurons in area CA2 undergoes a long-term depression at inhibitory synapses (iLTD) mediated by δ opioid receptor (DOR) activation : 10 NaCl, 195 sucrose, 2.5 KCl, 15 glucose, 26 NaHCO3, 1.25 NaH2PO4, 1 CaCl2, and 2 MgCl2. A cut was made between CA3 and CA2 with a scalpel in some of the slices before being transferred to 30°C ACSF  for 30 min and kept at room temperature for at least 1.5 h before recording. All experiments were performed at 33°C. Cutting and recording solutions were both saturated with 95% O2 and 5% CO2, pH 7.4.All animal procedures were performed in accordance with the regulations of the animal care committee of the Université Paris Descartes. The 400 µm transverse hippocampal slices were prepared from 6- to 9-week-old C57BL6 male mice. Animals were killed under anesthesia with isoflurane. Hippocampi were removed, placed upright into an agar mold, and cut with a vibratome  in an ice-cold extracellular solution containing the following (in mm NaCl and positioned in the middle of stratum radiatum (SR) or stratum pyramidale in CA1 or CA2. Whole-cell recordings were obtained from CA2 PNs in current-clamp mode held at −73 mV with a patch pipette (3–5 MΩ) containing the following (in mm): 135 K methyl sulfate, 5 KCl, 0.1 EGTA-Na, 10 HEPES, 2 NaCl, 5 ATP, 0.4 GTP, 10 phosphocreatine, and 5 µm biocytin, pH 7.2 (280–290 mOsm). Inhibitory currents were recorded with pipette solution containing 135 Cs methyl sulfate instead of K methyl sulfate. The liquid junction potential was ∼1.2 mV, and membrane potentials were corrected for this junction potential. Some recordings were performed with the perforated patch technique. For these experiments, 75 μg/ml gramicidin was added to the intracellular solution along with an additional 4 mm calcium to ensure that recordings were acquired in perforated configuration only. Series resistance  was monitored throughout each experiment; cells with a >15% change in series resistance were excluded from analysis. Synaptic potentials were evoked by monopolar stimulation with a patch pipette filled with ACSF and positioned in the middle of CA1 SR or stratum lacunosum-moleculare (SLM). When evoking PSPs in both SR and SLM in the same experiment, we tested the independence of the inputs prior to the experiment. Using the same stimulation intensity used for the experiment, we compared the PSP amplitude evoked by SR stimulation with the PSP amplitude evoked by SR stimulation 100 ms after SLM stimulation. We also performed the reverse measurement, comparing the PSP amplitude evoked by SLM stimulation to a PSP evoked with SLM stimulation after SR stimulation by 100 ms. The stimulation of the SR and SLM inputs was considered to be independent if a preceding evoked PSP in the separate pathway had no effect on the amplitude of the second evoked PSP. When axons of CA2 pyramidal neurons were directly recruited by the stimulation pipette, as observed with a back-propagating AP in the recorded CA2 neuron, the stimulating pipette was moved until the direct activation of the axon disappeared.Field recordings of PSPs were performed in current-clamp mode with a recording patch pipette (3–5 MΩ) containing 1 2,5]Enkephalin) was applied after a stable baseline of 15–20 min duration. Before beginning whole-cell experiments, we identified the CA2 PNs by somatic location and size. Furthermore, the cell type was confirmed by several electrophysiological properties . For several experiments, particularly when Cs+ was in the pipette solution, the slices were fixed after the recording with 4% paraformaldehyde, and the neurons were identified by biocytin-streptavidin labeling and immunohistochemistry to label the CA2-specific protein RGS-14.High-frequency stimulation , 10 Hz stimulation (100 pulses at 10 Hz repeated twice), or DPDPE . We expected a smaller change in the fPSP when measuring the slope, as most of the inhibition evoked by the stimulation is recruited by the SC. The extrasynaptic delay of this feedforward inhibition onto CA2 PNs (compared with the direct SC transmission) will result in a larger control of the peak rather than of the slope of the fPSP. Therefore, the amplitude of the PSP was used for the analysis of the subsequent experiments.Inhibition in area CA2 has been shown to play a very prominent role in controlling the size of the depolarizing component of the compound EPSP–IPSP after stimulation of the SC . Further, n = 10 A; with 4, n = 8 B]. We ap = 0.06) or after 10 Hz stimulation . In addition, we also applied HFS in slices that had a detached CA3. In this condition, we found a similar increase of the compound fPSP amplitude . These data indicate that the long-term increase in the amplitude of the compound fPSP in area CA2 is not due to recurrent activation of CA3 neurons.To address whether a change in the excitability of CA3 PNs was responsible for the increase in the fEPSP in CA2, we measured the amplitude of the fiber volley . As shown in n = 9; 195.5 ± 26.3% after 10 Hz stimulation, n = 6). The larger increase in PSP amplitude observed in whole-cell recording compared to extracellular recording is not surprising as DORs have been reported to be expressed in interneurons that target PN soma and proximal dendrites . Thus, our data show that different patterns of activity can increase the excitatory drive between CA3 and CA2.We also tested the effect of HFS and 10 Hz stimulation in whole-cell current-clamp recordings of CA2 PNs to ascertain that the increased amplitude of the fPSP was a result of an increased excitatory/inhibitory ratio onto CA2 PNs. We found that both the HFS and 10 Hz protocols triggered a large increase in the PSP recorded in this condition (endrites . A potenWe hypothesize that the increase in the amplitude of the PSP we measured is the consequence of the previously described DOR-mediated iLTD at inhibitory synapses . That isA and GABAB receptor antagonists (1 µm SR 95531 and 2 µm CGP 55845). In area CA1, the HFS induced a small increase in the fPSP amplitude when inhibition was intact . As expected, this increase was significantly enhanced when the recordings were performed in the continuous presence of GABA receptor antagonists . In area CA2, a robust and lasting increase of the fPSP was induced with inhibition intact . Furthermore, unlike what was observed in area CA1, the HFS did not evoke any lasting change in the fPSP amplitude when inhibitory transmission was blocked .To test this hypothesis, we recorded extracellular compound fPSPs in areas CA1 and CA2 in response to HFS of SC inputs either in the presence or absence of GABAp < 0.00001, n = 10), but not in the presence of GABA receptor antagonists . Altogether, these results show that in area CA2, intact inhibitory transmission is a requirement for the long-term increase of the compound PSP amplitude.We also performed the same experiment using whole-cell current-clamp recordings of CA2 PNs. Consistent with the extracellular recordings, the HFS induced an increase in the amplitude of the depolarizing response when inhibition was intact , confirming that GABAergic transmission exerts a strong negative control on the PSP amplitude in CA2 PNs (p < 0.00001 (p = 0.0074 compared with blockers without HFS), n = 6]. However, the final PSP amplitude after the HFS and GABA blocker application was identical to the amplitude of the PSP after GABA blocker application alone . Altogether, these results indicate that the HFS-induced increase of the PSP amplitude is mediated by a decrease in GABAergic transmission.If the increase in the PSP after HFS indeed results from a decrease in inhibition, then it is possible to make the following prediction: pharmacological block of inhibition should increase the amplitude of the PSP, and part of this increase should be occluded by a previous HFS. In order to test this, we performed current-clamp recordings of CA2 PNs and applied GABA receptor blockers after an HFS of SC inputs, or in control experiments with no tetanus stimulation. We found that when the GABA receptor blockers were applied without the HFS, the PSP amplitude was strongly increased  of −77 mV. The previous study  n = 5]. Finally, in order to directly monitor the compound EPSP/IPSP in pyramidal neurons without affecting intracellular [Cl−], we performed perforated patch recordings with gramicidin in the pipette. In these conditions, we found that HFS evoked a large increase in the amplitude of the PSP that was not different than the one observed with whole-cell recordings . Altogether, these data strongly indicate that the disinhibitory increase in PSP amplitude can occur during physiological conditions.It was previously reported that the SC–CA2 synapse does not express LTP when inhibition is pharmacologically blocked, but also when inhibition is kept intact . One potus study  used 16 o et al. , we perfOur results show that the potentiation of the PSP amplitude in area CA2 induced by an HFS results from a decrease in inhibition, rather than from a direct potentiation of excitatory inputs. If iLTD in area CA2 indeed mediates the increase in the compound PSP, both iLTD and the increase in PSP amplitude should share similar pharmacology. Because the induction of iLTD in area CA2 requires the activation of DORs , we testn = 7, p = 0.135; interleaved control: 215.5 ± 20.6% of the PSP amplitude, p = 0.005, n = 5). Similarly, using extracellular recordings, we found no lasting change in the amplitude of the fPSPs after HFS in the presence of DOR antagonists ICI 74864 or naltrindole . For these experiments, we used two structurally distinct DOR antagonists . Because the increase in PSP amplitude was blocked with both antagonists, data were pooled.First, we performed extracellular and whole-cell current-clamp recordings of CA2 PNs in response to SC stimulation and applied an HFS in the presence of a DOR competitive antagonist, ICI 74864. We found that the HFS did not significantly change the CA2 PN PSP amplitude if DORs were not activated during the tetanus stimulation . To make sure that this long-term potentiation is due to a DOR-dependent decrease in inhibition and not to a potential direct increase in SC–CA2 excitatory transmission, we performed the same experiment in the presence of GABA receptor blockers. We found that when inhibitory transmission was blocked, DOR activation did not induce any lasting increase in the PSP amplitude at SC–CA2 synapses . Altogether, these data show that DOR activation is necessary and sufficient to trigger a lasting increase in the amplitude of the PSP between SC and CA2 PNs.Together, these data show that DOR activation is needed for induction of the lasting increase in CA2 PN PSP amplitude. We then asked whether the activation of DORs would be sufficient to mediate this plasticity. To address this question, we performed extracellular recordings in the SR of area CA2 and applied the specific DOR agonist DPDPE for 15 min. We found that the application of DPDPE induced a long-term increase in the fPSP amplitude . In contrast to SC inputs, SLM inputs have been described to express an LTP that is independent of inhibitory transmission , which is consistent with a disinhibitory mechanism. We then tested whether this disinhibitory mechanism at the SLM pathway also depends on DOR activation by applying the SR tetanus stimulation in the presence of naltrindole. We found that the increase in the PSP amplitude of SLM input was completely blocked under these conditions .CA2 PNs are strongly activated by distal inputs in SLM. Thus, we wondered whether the stimulation of proximal SC inputs, via a disinhibitory mechanism, could also influence distal inputs in SLM. We used two stimulating electrodes to evoke PSPs in SC and SLM inputs, as diagrammed in smission . Thus, wp = 0.005 for SR IPSCs; 87.0 ± 2.5% of baseline, p = 0.003 for SLM IPSCs; n = 6).We postulate that this increase in the strength of SLM inputs is a result of the same DOR-dependent disinhibition that increases the strength of SC inputs. To test this hypothesis, we directly monitored IPSCs evoked by stimulation in SR and SLM. We found that tetanus stimulation in SR evokes a lasting and significant iLTD in both SC and SLM inputs into CA2 PNs . This result shows that the plasticity induced by the HFS is sufficient to allow CA3 inputs to evoke the firing of cells in CA2.To test this hypothesis, we monitored the magnitude of the population spike (PS) in the somatic layer of area CA2 before and after HFS. We found that before HFS there was no PS or a PS of very small amplitude that was measurable only at the highest intensity stimulation (30 V). This observation confirms that the stimulation of CA3 SC axons was not capable of driving the firing of PNs in the CA2 region. Interestingly, a large PS was revealed in CA2 after HFS . To ensure that this increase in firing results from the DOR-mediated plasticity at inhibitory synapses evoked by HFS, we performed the same experiment in the presence of the DOR antagonist ICI 174864 (2 µm). When HFS was applied in the presence of the drug, no significant change in action potential firing was evoked . These results show that the DOR-dependent disinhibitory increase in PSP is sufficient to allow CA3 inputs to drive AP firing in CA2 PNs.To determine whether the pyramidal cells in area CA2 experience an increased firing probability after HFS of SC, we performed whole-cell current-clamp recordings of CA2 PNs and measured the probability of AP firing in response to a series of five pulses at 100 Hz at different stimulus intensities before and after HFS. Before HFS, no APs were evoked in CA2 PNs. However, after HFS, CA2 PNs were able to generate APs in response to SC stimulation. Both the number of APs per train and the proportion of cells firing at least one AP during the train were significantly increased after HFS . And finally, the application of a DOR agonist, which induces a lasting decrease in inhibition but does not alter isolated excitatory transmission, was sufficient to evoke the lasting increase in PSP amplitude.There are several examples of disinhibitory plasticity in the hippocampus. For instance, a pairing protocol induces a shift in GABA reversal potential, thereby increasing synaptic strength at CA3–CA1 synapses . Similar− in the recording solution (16 mm), we used 7 mm Cl−. In accordance with this idea, we found no increase in PSP amplitude with 16 mm Cl− in the recording pipette. The precise physiological concentration of intracellular Cl− in CA2 PNs is unknown. However, the fact that we observe an increase in PSP amplitude when intracellular [Cl−] is not perturbed (using gramicidin perforated patch recordings), and when both intracellular ionic composition and resting membrane potential are not affected (using extracellular recordings), strongly indicates that the disinhibition-mediated increase in PSP amplitude can occur in an intact system. Furthermore, we are confident that the fPSP we recorded in CA2 is not contaminated by PSP in CA1 because we did not observe LTP in CA2 in the presence of GABA blockers, while a large potentiation was observed with simultaneous fPSP recordings in CA1.Our results are in agreement with those of several studies showing a lack of plasticity at SC–CA2 synapses when inhibition was blocked. Nonetheless, they seem to differ from the results of a previous study  in whichOur results strongly indicate that the DOR-mediated iLTD recently described  is involOpioids have long been known to increase excitability in the hippocampus and to facilitate the induction of plasticity at excitatory synapses. For instance, the activation of μ opioid receptors (MORs) and DORs is required for LTP induction at the perforant path–granual cell synapse in the dentate gyrus , and theOur results differ in several ways from the previously reported action of opioids. First, in contrast to the transient effect of DORs on GABAergic transmission in CA1, the activation of DORs was shown to induce a lasting depression of inhibition in area CA2 . In addi+ basket cells in area CA2 have dendrites that traverse SR and SLM (+ interneurons in area CA2 is depressed during DOR-mediated iLTD (Our results show that stimulation in SR increases the synaptic strength of both proximal and distal inputs. The increase in the strength of excitatory transmission was dependent on GABAergic transmission. There have been several reports of heterosynaptic plasticity between proximal and distal inputs onto hippocampal PNs; for example, the potentiation of SC inputs onto CA1 PNs after theta-burst stimulation of distal dendritic inputs , and the and SLM . Furtherted iLTD . TogetheWhen inhibition is intact, SC EPSPs are very small and fail to induce AP in CA2 PNs. Our results show that, after the induction of DOR-mediated iLTD, the stimulation of SC inputs can evoke APs in CA2 PNs. This was observed both in whole-cell and extracellular recordings, indicating that it can occur when the resting potential of CA2 PNs is not affected. This result is consistent with those of a previous study  using vo+ interneurons has been reported (+ cells is considerably reduced in CA2 during epilepsy (While our results suggest that an activity-dependent decrease in inhibition could be relevant for information transfer by allowing SC inputs to activate CA2 PNs, it is likely that a more global decrease in inhibition in CA2 is detrimental for the hippocampus. For instance, a decrease in the density of PVreported  to occurepilepsy , and phyepilepsy  or a comepilepsy . Our resepilepsy , and whyepilepsy  and in hepilepsy .In summary, our results show that while there is no LTP at the SC–CA2 PN synapse, the excitatory/inhibitory balance can be persistently shifted toward excitation in an activity-dependent manner through a disinhibition mechanism. Interestingly, this disinhibition results in an increase in net excitatory drive at both SC inputs as well as distal dendritic inputs. Furthermore, our results reveal that the decrease in inhibitory transmission sufficiently increases the excitatory drive from SC inputs to allow recruitment of CA2 by CA3 PNs. Thus, our data add complexity to the hippocampal circuitry and reveal how CA2 PNs can be engaged by intrahippocampal inputs after activity.


For example, loss of calpain-3 (CAPN3) activity leads to limb-girdle muscular dystrophy 2A (LGMD2A). In neuronal excitotoxicity, the cleavage of the Na+-Ca2+ exchanger isoform 3 (NCX3) has been associated with an increase in activity and elevation of the Ca2+ content in the endoplasmic reticulum (ER). Since NCX3 is expressed in skeletal muscle, we evaluated the cleavage of different NCX3 splice variants by CAPN1 and CAPN3. Using Fura-2-based cellular Ca2+ imaging, we showed for the first time that CAPN3 increases NCX3 activity and that only NCX3-AC, the variant predominantly expressed in skeletal muscle, is sensitive to calpain. The silencing of the endogenous CAPN1 and the expression of the inactive form of CAPN3 (C129S CAPN3) confirmed the specificity for CAPN1 and CAPN3. Functional studies revealed that cellular Ca2+ uptake through the reverse mode of NCX3 was significantly increased independently of the mode of activation of the exchanger by either a rise in intracellular Ca2+ ([Ca2+]i) or Na+ ([Na+]i). Subsequently, the sensitivity to CAPN1 and CAPN3 could be abrogated by removal of the six residues coded in exon C of NCX3-AC. Additionally, mutation of the Leu-600 and Leu-601 suggested the presence of a cleavage site at Leu-602. The increased Ca2+ uptake of NCX3 might participate in the Ca2+ refilling of the sarcoplasmic reticulum (SR) after the excitation-contraction uncoupling following exercise and therefore be implicated in the impaired reticular Ca2+ storage observed in LGMD2A.Ca A compromised function of one of the calpain family members can lead to diverse pathologies such as embryonic lethality, gastropathy, muscular dystrophy, or platelet dysfunction [2+ levels ([Ca2+]i). This phenomenon triggers the autolysis of the protease. The cytosolic concentration of Ca2+ required for this activation differs between isoforms. The targets of its proteolysis are difficult to predict as the calpain family recognizes a tertiary structure and not the primary sequence unlike many conventional proteases i and by [Na+]i, together with the use of site-directed mutagenesis, enabled a full characterization of the transport capacities of the exchanger upon regulation by calpain and provided a new insight into the molecular determinants responsible for this sensitivity.In the present study, we investigated for the first time the sensitivity of the different NCX3 variants to calpain using Fura-2-based CaFour C57BL/6 mice were sacrificed, and subsequently, the skeletal muscles from the left and right hind limbs were dissected and directly frozen in liquid nitrogen in order to obtain the gastrocnemius (Gast), the tibialis anterior (TA), the extensor digitorum longus (EDL), and the soleus. The heart sample used as a negative control was removed from four C57BL/6 mice.v/v) fetal calf serum and 2 mM l-glutamine at 37 °C with 5 % (v/v) CO2. Forty-eight hours prior to experiments, cells were seeded on a six-well plate and transiently transfected. For double transfections, cells received 1 μg of the each constructs for a total of 2 μg/well, using polyethylenimine cationic polymer PEI  in accordance with the manufacturer’s instructions.The human embryonic kidney cells HEK293T were cultured in a Dulbecco’s modified Eagle’s medium  containing 10 %  1.0 prediction tool i was monitored by exciting Fura-2 with monochromatic light of wavelength 340 and 380 nm . Fluorescence emission was directed by a 415DCLP dichroic mirror  through a 510WB40 emission filter  onto a CoolSNAP HQ monochrome charge-coupled device (CCD) camera . The integration time of the CCD camera was set at 200 ms with a sampling interval of 3 s. All hardware was controlled with Metafluor software . For each wavelength, the mean fluorescence intensity was monitored in an intracellular region and, for purpose of background correction, in an extracellular region of identical size. After background correction, the fluorescence emission ratio (340 nm/380 nm) was calculated to determine the Fura-2 ratios. Ten to 19 individual GFP-positive cells were selected and monitored simultaneously from each coverslip. After double transfection of CAPN3 and NCX3, only cells positive for both GFP and mCherry were selected. The reverse mode of NCX activity was evaluated using the Ca2+ uptake that follows the removal of extracellular Na+ using a NMDG solution  [2+]i using the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (1 μM) simultaneously with the Na+ removal or raising the [Na+]i with a 60-min incubation in presence of the inhibitor of the Na+-K+ ATPase ouabain (1 μM) i or [Ca2+]i. In order to elevate the Ca2+ levels, the cells were perfused with a Na+-free medium containing thapsigargin. Thapsigargin inhibits the SERCA ATPase and therefore prevents the re-uptake of Ca2+ in the endoplasmic reticulum (ER). As the ER constitutively leaks Ca2+, the Ca2+ stocks were emptied in a slow manner. [Na+]i was elevated by incubating cells with ouabain in a Na+-rich medium prior to the measurements. By blocking the Na+-K+-ATPase, ouabain causes a gradual rise in intracellular Na+ until similar concentrations across the plasma membrane are reached. The activity of both variants of NCX3 was recorded in HEK293T cells expressing an NCX3 variant together with an empty vector (Mock) or the calpain inhibitor calpastatin (CAST). Importantly, our group previously determined the plasma membrane localization of NCX3 in transfected HEK293T cells by cell surface biotinylation. Importantly, NCX3-AC and NCX3-B were both expressed at the plasma membrane in similar amount i to micromolar levels such as that following eccentric exercise. In these conditions, a disruption of the excitation-contraction (EC) coupling can occur, caused by the decrease in Ca2+ release from the SR that could lead to post-exercise fatigue and contribute to the muscle weakness observed in several muscular pathologies [2+ content and release capacity leading to a cardiac contractile dysfunction [2+ stores in skeletal muscle and to delay the E-C uncoupling or to compete with the Ca2+ re-uptake into the SR by an enhanced Ca2+ extrusion. The low rate of [Ca2+]i increase during these specific uncoupling events greatly favors the activation of the reverse mode. Furthermore, the capacity of NCX to refill the SR Ca2+ levels has been observed in several excitable tissues such as cardiomyocytes i. This might well explain the activation of CAPN3 in our experiments despite the specific requirements for its activation, described by others, in terms of [Ca2+]i and exercise. Our study has been performed in HEK cells, as a well-used cell model for the investigation of the capacity of exchange and the ionic regulations of the NCX family [2+ disturbances of LGMD2A.Regarding CAPN3, in the myofiber, the protease is tightly bound at the myofibril, and at the triad, a privileged region for Caexercise , a phaseexercise . The incurbances , 23, succ action , the proX family , 20. The2+ transporters in the Ca2+ refilling of the SR, subsequent to the EC uncoupling. The loss of regulation of NCX3 by CAPN3 could therefore be implicated in diverse muscular pathologies such as the LGMD2A.In conclusion, a novel target sensitive to the muscle-specific CAPN3 was identified. This sensitivity was found to be restricted to the skeletal muscle variant NCX3-AC. The site located in the exon C was identified as implicated in the regulation of the exchanger by CAPN1 and CAPN3, possibly by a direct cleavage and might be involved with other Ca


Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca2+ content, diminished their Ca2+-ATPase activity, and kept their Mg2+ content unchanged. Membranes preincubated with different concentrations of Ca2+ increased their Ca2+ content without changes in their Mg2+ content. There is a direct relationship between Ca2+ content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca2+ content and Ca2+-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg2+ showed a higher Mg2+ content without changing their lipid peroxidation and Ca2+-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca2+-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca2+ content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg2+ might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals.In the current study the possible relationship between the Ca Preeclampsia is a clinical syndrome characterized by vascular endothelial damage, hypertension, proteinuria, edema, generalized arteriolar vasospasm, and a state of oxidative stress . One of 2+-ATPase (PMCA) is dependent upon lipid-protein interactions, and its activity is greatly affected by the level of lipid peroxidation in its environment [Interaction of ROS with lipids, proteins, and carbohydrates of the plasma membranes can increase their level of lipid peroxidation, thus decreasing their fluidity and the activity of membrane enzymes . Particuironment –12.Incubation of placental tissue under hypoxic conditions, which occurs with preeclampsia, induces oxidative stress, the release of proinflammatory cytokines, and trophoblast cell death –16. ConsInterestingly, tissues maintained under periods of hypoxia increase their intracellular calcium , an effe2+ and a higher content of Ca2+ in membranes of red blood cells from preeclamptic pregnant women, as compared with the red blood cells of uncomplicated pregnant women. These alterations could lead to interactions of these ions with membrane components, resulting in modifications of the lipid microenvironment that interacts with membrane transporters. It is well known that the interaction of metal cations and lipids has a significant impact on membrane properties, such as the area per lipid or chain ordering [Kisters and collaborators  observedordering .2 and MgSO4, in order to modify their Ca2+/Mg2+ ratio. In addition, syncytiotrophoblast plasma membranes were prepared from placental explants cultured under either normoxic or hypoxic conditions, in order to evaluate possible changes in the Ca2+/Mg2+ ratio of their cell membranes. The results were used to try to establish a relationship between the Ca2+/Mg2+ ratio, the lipid peroxidation, and the PMCA activity of the syncytiotrophoblast plasma membranes.In the current study, we incubated syncytiotrophoblast plasma membranes with different concentrations of CaCl2+ metabolism disorders or any other chronic medical illness, or over 1 g of supplemental Ca2+ per day during the pregnancy.Term placentas obtained from uncomplicated  pregnant women were collected immediately after delivery from the “Concepción Palacios” Maternity Hospital, Caracas, Venezuela, and transported to our laboratory on ice. All the women participated in this study in accordance with the ethical standards established by the Declaration of Helsinki. The study protocol was approved by the Institutional Review Board of “Concepción Palacios” Maternity and the Bioethics Committee of IVIC and all women gave signed informed consent. Patients were followed from the time of admission and then before and after delivery. All of the pregnant women enrolled in the study were nulliparous, gave birth by vaginal delivery, belonged to urban population of Caracas, and had similar demographic backgrounds. They had no history of hypertension or proteinuria during the pregnancy and gestational age was estimated from the date of the last menstrual period and confirmed by ultrasonography. The characteristic averages of the participating pregnant women are shown in μg/mL, and amphotericin B 3 μg/mL. The preparation of placental explants was carried out on ice at 4°C and completed in approximately 30 min. The explants prepared in this way, without further incubations, were identified as freshly prepared placental explants.After removal of the chorionic plate and about 0.25 cm of decidua, explants were prepared using only tissue from the intermediate region of the placenta. In brief, randomly sampled villous tissue fragments, of roughly 0.5 cm × 0.5 cm, were cleaned of large vessels and blood clots, rinsed 5 times in cold sterile saline, placed in 12-well plates (Nunclon TM Surface) containing 2 mL of DMEM-F12 medium and 10% fetal calf serum as culture medium, and enriched by addition of crystalline penicillin 100,000 UI/mL, gentamicin 48 2 incubator (Shel Lab Model IR2424) with a gas mixture that composed of 8 percent O2, +87 percent N2+5 percent CO2 (normoxia), with constant gas pressure. The culture medium was then removed and replaced with new culture medium. The explants were divided in two groups: one group was kept under normoxia for 18 h at 37°C and the other group was cultured under hypoxia  for 18 h at 37°C. At the end of the culture period, the explants were carefully removed and rinsed 5 times in cold sterile saline. The explants were then used for the isolation of syncytiotrophoblast plasma membranes.The tissue explants were incubated for 4 h at 37°C in 4 mL medium (DMEM-F12 with 10% FCS) in a sterile COβ-adrenergic receptor , as well as cytochrome-c oxidase/succinate dehydrogenase and glucose-6-phosphatase and alkaline phosphatase activities [The membranes were prepared from placental explants following a method previously described , 23. In tivities .2+ (as CaCl2) and Mg2+ (as MgSO4), in order to modify their Ca2+ and Mg2+ content. At the end of the preincubation period, the suspension was diluted with a solution containing 250 mM sucrose and 10 mM Tris-Hepes (pH 7.2) and washed 6 times by centrifugation-suspension at 47,500 ×g in order to remove traces of Ca2+ and Mg2+. The membranes were resuspended with the same washing solution  and kept at −70°C until use. The membranes were used to determine their PMCA activity, TBARS, and Ca2+ and Mg2+ content.Aliquots of BM and MVM (1 mL of a 1 mg protein/mL suspension) were preincubated for 24 h at 0–4°C with a solution of 150 mM NaCl and, according to the experimental design, different concentrations of CaμL aliquot of the membrane suspension was mixed with 500 μL of 70% nitric acid and placed in a heating block (Fisher Scientific Dry Bath incubator) at 80°C for 24 h. After 24 h digestion, 200 μL of 60% perchloric acid was added, and the digestion was continued for 24 h at 80°C. Finally, the whole mixture was suspended with 2 mL of MQ water. The Ca2+ and Mg2+ contents of the sample were measured by inductively coupled plasma spectroscopy (Perkin Elmer Optima 3000 DV ICP system).Red blood cell ghosts were prepared with a modification of a previously described method . AliquotμL of the incubation medium was preincubated for 2 min at 37°C, and the reaction was started by the addition of 20 μL of membrane suspension. After 10 min of incubation, the reaction was stopped by the addition of 300 μL of a cold solution containing 2.85% ascorbic acid, 1.76% HCl, 0.48% ammonium molybdate, and 2.85% SDS. The samples were shaken and kept at 0°C for 10 min. Then, 500 μL of a solution of 2% sodium citrate, 2% sodium arsenite, and 2% glacial acetic acid was added to each tube. The tubes were shaken and then incubated for 10 min at 37°C. The absorbance of each tube was determined in a Sunrise (Tecan) spectrophotometer at 705 nm. The PMCA activity was calculated as the difference in the phosphate liberated in a medium containing 250 mM sucrose, 5 mM ATP, 5 mM MgCl2, 1 mM ouabain, 2 mM EGTA, 2 mM EDTA, 30 mM Tris-HCl (pH 7.2 at 37°C), 55 mM KCl, 2 μg/mL calmodulin, 1 mM thapsigargin, and 2 μM free calcium, minus the one liberated in the same medium, but in the absence of calcium. A blank control was run in parallel under the same conditions, except for the fact that the membrane suspension was added only after the inclusion of ascorbic acid solution. The ATPase activity was expressed as nmol Pi/mg protein per min, after subtraction of the value obtained with the blank. The protein concentrations were determined using the Bio-Rad microassay [μg SDS/μg protein.The ATPase activity was determined by measuring the quantity of inorganic phosphate liberated from the hydrolysis of ATP, following a modification of the method described elsewhere . Brieflycroassay . In ordecroassay . The optThe amount of lipid peroxidation of the plasma membranes was estimated by measuring the thiobarbituric acid-reactive substances (TBARS). The TBARS was determined according to the method described by Feix et al. . The val n represents the number of experiments performed with different preparations. ATPase activities were calculated from paired data. A P  value < 0.05 was accepted as statistically significant.Comparisons between the different conditions were assessed by one-way ANOVA with the post hoc analysis with the Student-Newman-Keuls test. All results are expressed as means ± standard error (S.E.) and2+ and Mg2+ content of syncytiotrophoblast plasma membranes (BM and MVM) from freshly prepared placental explants are shown in 2+ content, as well as an important diminution in PMCA activity. Hypoxia did not produce any change in the Mg2+ content of either BM or MVM. These results suggest that hypoxia, by increasing the Ca2+ content of the syncytiotrophoblast plasma membranes, stimulates lipid peroxidation, which in turn inhibits PMCA activity.Level of lipid peroxidation (determined as TBARS), PMCA activity, and Ca2, in order to try to alter their Ca2+ content. The membranes were then analyzed for Ca2+ and Mg2+ content, level of lipid peroxidation, and PMCA activity. The results in Figures 2 increased their Ca2+ content, without affecting their Mg2+ content. In addition, the level of lipid peroxidation of the membranes increased  and then analyzed for Ca2+ and Mg2+ content, level of lipid peroxidation, and PMCA activity. The results of this experiment are shown in The variations shown in 4 in the preincubation medium had a minimal effect on the Mg2+ content of the syncytiotrophoblast plasma membranes, a concentration of 4 mM MgSO4 was able to significantly increase their Mg2+ content. None of the tested concentrations of MgSO4 altered the Ca2+ content of the membranes  used for incubation of the placental explants adjusted to a final concentration of MgSO4. The results of these experiments showed that the presence of 4 mM MgSO4 prevented both the lipid peroxidation of syncytiotrophoblast plasma membranes  of placental explants cultured under hypoxic conditions showed a significant increase in Cat Tables . Similarables 2+ . The Mg22+ content of the syncytiotrophoblast plasma membranes, stimulates their level of lipid peroxidation which, in turn, inhibits their PMCA activity. How can changes in the membrane Ca2+ content modify the degree of lipid peroxidation? Cations have been proposed to penetrate deeply into the head group region of the membrane, probably binding between the carbonyl oxygen and the phosphate group [2+ ions interact with plasma membranes to produce important changes in their lipid profile; that is, phospholipid scrambling in the human erythrocyte membrane involves the synergistic action of phosphatidylinositol 4,5-bisphosphate and Ca2+ [These results may suggest that hypoxia, by increasing the Cate group . Consequand Ca2+ .2+ and low levels of membrane Mg2+ [A relationship between lipid peroxidation and PMCA activity has been established for different cell membranes –12, 32. ane Mg2+  and alsoane Mg2+ .4 administration is the medical treatment for preeclamptic women to prevent the recurrent seizures of eclampsia and for tocolysis in preterm labor [4 therapy, both the level of lipid peroxidation and PMCA activity of red blood cell membranes from preeclamptic women show values similar to those of normotensive pregnant women [ in vitro with 4 mM MgSO4 for 24 h at 0°C [4 increases their Mg2+ content. Taken together, it may be concluded that the MgSO4 treatment raises the Mg2+ content of the red cell ghosts and thereby reduces the level of lipid peroxidation and increases the PMCA activity of these membranes. Furthermore, the results presented in 2 and 0.8 mM MgSO4 (Dulbecco's Modified Eagle Medium) show a significant increase in their Ca2+ content, as compared to results from explants cultured under normoxia, with no variations in their Mg2+ content. However, increasing the concentration of the culture medium to 4 mM MgSO4 protected the membranes against the effects of hypoxia (2+ is required to protect the membranes and to circumvent the rise of lipid peroxidation and the inhibition of the PMCA activity of the syncytiotrophoblast plasma membranes seen during preeclampsia [Parenteral MgSOrm labor . In prevnt women . A similh at 0°C . In addi hypoxia . These rclampsia .2+ and Mg2+ within the plasma membranes of preeclamptic women remain to be determined. However, our study points out the importance of these interactions for the control of the level of lipid peroxidation of the plasma membranes and for the activity of transporting proteins such as the PMCA during preeclampsia.The exact molecular mechanisms explaining the interactions of Ca