Ciliary dysfunction leads to a number of human pathologies, including primary ciliary dyskinesia, nephronophthisis, situs inversus pathology or infertility. The mechanism of cilia beating regulation is complex and despite extensive experimental characterization remains poorly understood. We develop a detailed systems model for calcium, membrane potential and cyclic nucleotide-dependent ciliary motility regulation.2+ spike with amplitude proportional to the degree of membrane depolarization, the ability to maintain stable oscillations, monostable multivibrator regimes, all of which are initiated by variability in ionic concentrations that translate into altered membrane voltage.The model describes the intimate relationship between calcium and potassium ionic concentrations inside and outside of cilia with membrane voltage and, for the first time, describes a novel type of ciliary excitability which plays the major role in ciliary movement regulation. Our model describes a mechanism that allows ciliary excitation to be robust over a wide physiological range of extracellular ionic concentrations. The model predicts the existence of several dynamic modes of ciliary regulation, such as the generation of intraciliary CaComputational investigation of the model offers several new insights into the underlying molecular mechanisms of ciliary pathologies. According to our analysis, the reported dynamic regulatory modes can be a physiological reaction to alterations in the extracellular environment. However, modification of the dynamic modes, as a result of genetic mutations or environmental conditions, can cause a life threatening pathology. Cilia are cellular protrusions which have been conserved in a wide range of organisms ranging from protozoa to the digestive, reproductive and respiratory systems of vertebrates . Mobile μm in diameter and approximately 5-7 μm in length, cilia covering human airways can propel mucus with trapped particles of length up to 1 mm at a speed of 0.5 mm/second  is the total concentration of the Ca2+ binding protein and where [2+ binding protein interaction with the Ca2+ channels is given by:The CaC is the number of channels in the open conductive state, and C0 is the total number of channels. The steady-state solution of equation (23) is given by:where 2+ binding protein-Ca2+ channels interaction. By combing equations (22) and (24) one obtains the dependence of the open Ca2+ channels on the Ca2+ concentration:where where Equation (23) can then be represented in the following non dimensional form:where 2+ channels in the open conductive state in response to a Ca2+ shift from u0 to u1 Ca2+ level is given by:The solution of equation (26) that describes the dynamics of Cawhere 2+ surge. The characteristic time for such an exponential change is given by:The solution (27) suggests that the number of open channels would change exponentially in response to a Cacac0 > > 1 and u > > 1, the characteristic time approximately equals For cases when 2+ channel conductivity, the channel conductivity as a function of Ca2+ concentration can be represented as follows:Under the assumption that the alteration of the transmembrane potential difference influences the Caψ0 to ψ1, and the alteration of the intraciliary Ca2+ concentration is delayed, the conductivity changes from one value to another exponentially with the characteristic time Vτ:When membrane potential changes from 2+ transport channels are present. For such a model, the alteration of Ca2+ concentration in a cilium is given by:Here we consider a simplified scenario when only passive and active Ca2+ currents into equation (31), one obtains:By substituting formulas for the passive and active Ca2+ channels due to the assumption that the dynamics of currents via the active Ca2+ channels is much faster than the dynamics of currents through the passive Ca2+ transport.In this equation, we include the kinetics for the active CaThe non dimensional representation of Equations (32) and (23) is given by:where 2+ current (21) into (33) the equation for the intraciliary Ca2+ dynamics (33) take the following form:By substituting the non dimensional representation of the Cawhere + current activation by membrane potential. The full K+ current in a cilium is given by:Here we consider the mechanism of K+ channels, + potential.where + channel conductance is dependent on the membrane potential (the opposite of the case of Ca2+ channel conductance). The K+ channel conductance dependence on the membrane potential is given by:We assume that the Kψ0 to ψ1, the K+ conductivity is changing exponentially with a characteristic time We further assume that in response to the fast membrane potential alteration from + channels' conductivity dependence on the membrane potential can be estimated from experimental measurements of the fast K+ current (10 msec) as a function of membrane potential. In such a short time K+ current does not reach the maximum value and the Ca2+ current is almost equal to zero. Under such conditions, the equation for the K+ current is given by:The parameters for the KThe full current measured under the voltage clamp conditions is then given by:where 2+ as well as K+ currents can alter the membrane potential. In order to account for this effect, we considered the membrane potential dynamics in the absence of voltage clamp. Having established that the main contributors to the registered currents are the Ca2+ and K+ currents, we analyse the membrane potential dynamics as a function of Ca2+ and K+ current contributions:All the models described above were specifically developed under the voltage clamp conditions. However, CaIn the non dimensional form the equation for the membrane potential dynamics is given by:2+ and K+ channel conductivities, respectively, where 2+ and K+ currents, conductivities 2+ concentration. We also use the fact that Ca2+ and K+ channel conductivities change on a much faster time scale in comparison with membrane potential or Ca2+ concentration. Such an approximation allows us to employ the steady-state solutions for the channel conductivities as a function of membrane potential and Ca2+ concentration. The described assumptions and considerations lead to the following system of two nonlinear coupled equations for intraciliary Ca2+ concentration and membrane potential:In this equation, we take into consideration the contribution of the independent parts of the CaI0 is the non dimensional inward current.where The membrane hyperpolarisation current is given by:2+ channels located on the cell body, 2+ ions.where where By combining the contribution from different types of currents, one can derive the dynamics of the membrane potential alteration as follows:2+ and membrane potential dynamics can be further formulated as:The coupled system of differential equations for the Cahv(ψ) is the Ca2+ current contribution, activated by membrane depolarization, and 2+-dependent K+ current contribution.where 2+ current from cilia to cell into the equations for the intraciliary Ca2+ and membrane potential system leads to an additional term being added to the equations (46):The incorporation of the Ca2+ current from cilia to the cell body depends on the membrane potential:In the general case the conductivity of the protein complexes governing the Ca2+ concentration and membrane potential are given in Table All parameter values used in the above equations are given in Table NVK, ANG, BG, MZQC, ID, CH and AA developed and implemented the project under the supervision of DGB, RNK, YU and NVV. XY, SKS and CMS contributed to the analysis of the model. All authors contributed to the writing of the final manuscript. All authors read and approved the final manuscript.


Termination of the odor response requires removal of this Ca2+, and prior evidence suggests that both Na+/Ca2+ exchange and plasma membrane Ca2+-ATPase (PMCA) contribute to this removal.Within the cilia of vertebrate olfactory receptor neurons, Ca+ with Li+, which reduces the ability of Na+/Ca2+ exchange to expel Ca2+, prolonged the termination as expected. However, treating the epithelium with the specific PMCA inhibitor caloxin 1b1 caused no significant increase in the time course of response termination.In intact mouse olfactory epithelium, we measured the time course of termination of the odor-induced field potential. Replacement of mucosal NaUnder these experimental conditions, PMCA does not contribute detectably to the termination of the odor response. During initiation, a transduction cascade causes a depolarizing influx of Ca2+ and Na+ into the cilium through cyclic-nucleotide-gated (CNG) channels. Intraciliary Ca2+ then gates anoctamin-2 Cl− channels . Other studies in toad 2+ is also pumped out of the cilium by a plasma membrane Ca2+-ATPase (PMCA). Much but not all of the evidence that indicates a role for PMCA in response recovery derives from the use of PMCA inhibitors +, K+-ATPase. Na+, K+-ATPase is expressed in olfactory cilia +, K+-ATPase during the odor response may allow intraciliary Na+ to accumulate, which in turn could prevent Na+/Ca2+ exchange from expelling Ca2+2+ transport via an indirect effect on Na+/Ca2+ exchange. A further complication is that the PMCA inhibitor carboxyeosin modulates the ciliary CNG channels Evidence has suggested a range of mechanisms by which Ca2+ extrusion following the odor response.More selective inhibitors of PMCA are now available All experiments were approved by the University of Cincinnati's Institutional Animal Care and Use Committee (protocol 04-11-05-01) and conducted in accordance with the recommendations in the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health.2 and decapitated, and the lower jaw was removed. After trimming skin and muscle, the caudal 25% of the brain was removed with scissors, the dorsal aspect of the nasal septum cut with spring scissors, the nasal bone removed, and the skull divided along the midline with a scalpel such that the nasal septum remained with one half of the skull. The septum was carefully removed and the exposed olfactory turbinates surrounded by gauze threads so that solutions applied were retained covering the turbinates. The half head was glued into an organ culture dish with water filling the trough surrounding but not contacting the tissue and placed in a Faraday cage for recording. The other side of the head was kept on ice for subsequent use. A silver-silver chloride reference electrode inserted into the posterior aspect of the remaining brain was connected to ground. The dissection and recording area was continuously humidified.All efforts were made to minimize animal suffering and to reduce the number of animals used. Experiments were performed on adult mice from the inbred FVB/N genetic background. Animals were euthanized with CO2, 2 MgCl2, 2 Na pyruvate, 5 HEPES, 9.4 D-glucose, adjusted to pH 7.4 with NaOH. For the Li+-replaced Ringer, LiCl was substituted for NaCl and the pH was adjusted with LiOH. Caloxin 1b1 , 167 µM (PMCA2), 274 µM (PMCA3), and 45 µM (PMCA4) The Ringer solution contained (in mM): 140 NaCl, 5 KCl, 2 CaClWe measured the electroolfactogram (EOG), an odor-induced field potential at the surface of the olfactory epithelium After locating an area of a turbinate that produced an acceptable EOG, the recording electrode was withdrawn. Ringer was applied to the turbinates, left for about 5 min covering the turbinates, and then wicked away. The electrode was repositioned in contact with the same area and several control EOGs were recorded. After applying and wicking a solution from a turbinate, the amplitude of the EOG was initially reduced but recovered substantially see . Wicking+-replaced Ringer or Ringer containing 100 µM or 500 µM caloxin, was applied and left for about 5 min covering the turbinates and then wicked. The electrode was repositioned again in contact with the same area and EOGs recorded as above. The recording electrode was withdrawn again and the turbinates were rinsed 3 times with Ringer and then additional Ringer applied for 5 min, wicked, and the EOG recorded again. For a given test solution, only one such experimental series was done on a given turbinate. In some experiments, the second side of the head was also used. The control EOG in every run met the criteria given above for amplitude and time constant.The electrode was withdrawn again, and the test solution, either LiRegardless of the treatment, the recovery of the EOG amplitude from its peak after application of a solution was somewhat variable. One likely source of variability was the exact position of the recording electrode on a turbinate. For example, when recording from a more caudal position on a turbinate, it may have been more difficult to get rid of excess fluid. This might have led to a decrease in the amplitude of the EOG. Based on visual inspection, the rostral portions of turbinates tended to dry out sooner than the caudal portions.e−t/τ+c to the 4.5 s of recording following the peak (minimum) of the EOG using SigmaPlot . Amplitudes and time constants measured in the control (Ringer) condition and the experimental condition were compared using a paired t-test; p values less than 0.05 were considered significant. Results of repeated experiments are reported as mean ± SD.The time constant τ of the recovery from the peak (minimum) of the EOG was calculated by fitting an exponential function y = Ae−t/τ+c to that recovery. The time constant τ of the recovery was 617 ms. For all experimental runs (n = 20), the control amplitudes ranged from 2.5 to 8.0 mV with a mean of 4.5±1.3 mV, and τ ranged from 397 to 910 ms with a mean of 637±171 ms. Control EOGs meeting the criteria described above often could be recorded for at least 5 hr after the mouse was euthanized. These EOG amplitudes were not maximal, since increasing the pressure on the Picospritzer and therefore the amount of odorant delivered to the tissue could more than double the amplitude.EOGs were recorded from 17 different turbinates from 5 mice, with both sides of the head used in 2 of the mice. A typical control EOG with an amplitude of 4.4 mV is shown in +-replaced Ringer, and then after rinsing again with normal Ringer. After Ringer treatment, τ did not change much . On average, τ increased to 153% of control (range 131% to 173%). Treatment with 100 µM caloxin did not significantly change the amplitude or τ relative to control. The mean amplitude in caloxin was 4.0±1.4 mV compared to 4.0±1.2 mV in control. The mean τ in caloxin was 682±136 ms compared to 755±141 ms in Ringer. In caloxin, τ decreased to 90.3% of control (range 65.7% to 123.2%), but the change was not significant . We also tested a higher concentration of caloxin . This decreased τ to 371±17 ms compared to 431±24 ms in Ringer. The decrease was significant .2+ from the olfactory cilia; this relation has been directly established in isolated olfactory receptor neurons + in the solution bathing the epithelium was replaced with Li+, the recovery time increased significantly. This is explained by a reduction in Na+/Ca2+ exchange activity and is consistent with previous results in isolated ORNs 2+ clearance by PMCA had contributed to termination of the odor response, then inhibition of PMCA would have increased the recovery time. However, application of the inhibitor caloxin caused no significant increase in the recovery time. In fact, a high dose of caloxin (500 µM) caused a small but significant decrease in the recovery time. It thus seems unlikely that PMCA plays a major role in termination of the odor response in intact mouse olfactory epithelium.To investigate whether PMCA contributes to termination of the odor response, we tested one of the caloxins, a class of selective inhibitors of PMCA 2+ during termination of the odor response. All four principal isoforms of PMCA are expressed in mouse ORNs, and some isoforms are found in the cilia 2+ transport has been demonstrated in membrane vesicles from a preparation enriched in olfactory cilia Substantial prior evidence indicates that PMCA may facilitate the expulsion of ciliary Ca50 = 20 nM 2+ that accumulates during the response to be more slowly expelled. The retained Ca2+ would then continue to gate the Cl− channels, accounting for the prolonged receptor current. However, one must consider whether a prolonged response might in part reflect effects of carboxyeosin on other transduction proteins. Concentrations of carboxyeosin as low as 0.1 µM significantly increase current through the ciliary CNG transduction channels near the resting potential +/Ca2+ exchange by carboxyeosin would likely do the same. Saidu et al. +/Ca2+ exchange at concentrations up to 20 µM Additional evidence has come from pharmacological studies using carboxyeosin. In isolated ORNs +/Ca2+ exchange could also explain the effect of carboxyeosin +, K+-ATPase, and it is likely that the closely related compound carboxyeosin does as well. The EC50 for inhibition of the Na+, K+-ATPase by eosin is 19 µM +/Ca2+ exchange allows the cilium to expel Ca2+ and take up Na+. Unless Na+, K+-ATPase can concurrently expel Na+ from the cilium, it is predicted that Na+ will accumulate, preventing further expulsion of Ca2+ via Na+/Ca2+ exchange +, K+-ATPase (and indirectly Na+/Ca2+ exchange) rather than by inhibiting PMCA. Other pharmacological designs must also consider this ambiguity. Vanadate +, K+-ATPase. The ambiguities in these pharmacological approaches were our motive for testing the selective PMCA inhibitor caloxin.In theory, an indirect effect on Na+/Ca2+ exchange + prolonged the response recovery, and the extent of this change was taken to reflect the maximum possible contribution of Na+/Ca2+ exchange. Carboxyeosin caused an even greater prolongation, and the additional slowing of the recovery by carboxyeosin could not have arisen from blocking Na+/Ca2+ exchange . Carboxyeosin was applied at a point when the CNG channels are closed, so a non-specific effect on the channels was also excluded. It could therefore be concluded that the effect of carboxyeosin in part reflected an inhibition of PMCA. Two studies have now failed to detect an involvement of PMCA in response recovery. In frog olfactory cilia, no ATP-dependent Ca2+ transport was detectable as Ca2+ entered olfactory cilia There is now evidence both to favor and oppose a significant role for PMCA in termination of the odor response. Evidence in favor includes the clear slowing of the odor response recovery in the neurons of mice lacking PMCA2 +/Ca2+ exchange on the olfactory field potential (EOG) in intact epithelium. As expected, replacing mucosal Na+ with Li+ prolonged the recovery phase of the odor response. However, the effect of Na+ replacement in isolated ORNs is usually greater than the effect we observed in epithelium (refs. + applied to the epithelial surface. Second, epithelial perfusion should primarily affect the membranes exposed to the mucus, whereas the entire cytoplasmic membrane of an isolated ORN is usually exposed to the bath solution provided. (When suction pipette recording is applied to isolated neurons, only the sensory endings are exposed − current itself may depend greatly on experimental conditions − channel anoctamin-2 caused no reduction in the EOG when the odor stimulus was provided as a vapor. When the epithelium was perfused with a Na+-containing Ringer, the amplitude of the EOG was reduced by ∼40% after deleting anoctamin-2. In isolated ORNs, deletion of anoctamin-2 reduced a simulated odor response by ∼90%. It has therefore been suggested that Cl− currents during the odor response may be large only in extracellular solutions with concentrations of monovalent ions that are higher than those in the mucus − via NKCC1 transport. In the native tissue, it has never been clear whether the ionic concentrations measured at the neuronal ending could support Cl− uptake via NKCC1 or Ca2+ extrusion via Na+/Ca2+ exchange 2+ accumulation and clearance are less prominent in intact epithelium. Most physiological evidence of a significant role for PMCA in the odor response has been obtained in isolated neurons When comparing these various studies, it is important to consider the different preparations used. In particular, there are probably significant functional differences between isolated neurons and intact epithelium. This report is the first to assess the effects of chemical modulators of PMCA or Naum refs. . Three e2+ influx was detectable 2+. To what extent it facilitates rapid termination of the odor response now appears to depend significantly on the particular species and preparation examined.Another difficulty in comparing the existing PMCA studies is the diversity of species chosen. As discussed by Antolin et al.


This signaling cascade releases Ca2+ from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate receptor and stimulates phosphorylation of ERK1/2 and CaMKII. A point mutation at Tyr1015 on RET or small interfering RNA gene silencing of PLCγ block the GDNF-induced signaling cascade. Delivery of the RET mutation to neuronal progenitors in the embryonic ventricular zone using in utero electroporation reveal that Tyr1015 is necessary for GDNF-stimulated migration of neurons to the cortical plate. These findings demonstrate a novel RET mediated signaling pathway that elevates cytosolic Ca2+ and modulates neuronal migration in the developing neocortex through the PLCγ binding domain Tyr1015.The receptor tyrosine kinase RET plays an essential role during embryogenesis in regulating cell proliferation, differentiation, and migration. Upon glial cell line-derived neurotrophic factor (GDNF) stimulation, RET can trigger multiple intracellular signaling pathways that in concert activate various downstream effectors. Here we report that the RET receptor induces calcium (Ca RET (REarranged during Transfection) was initially identified as an oncogene 2+) signaling.The intracellular domain of the RET protein has several tyrosine residues that become auto-phosphorylated upon ligand interaction and mediate activation of various downstream signaling targets, including the mitogen-activated protein kinase (MAPK) 2+ ion serves as a universal cytosolic messenger to control a diverse range of cellular processes in both disease and development 2+ handle the temporal and spatial distribution of cytosolic Ca2+ by regulating influx and efflux from the extracellular milieu or release from the endoplasmic reticulum (ER) stores 2+ from ER mainly occurs through the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). The InsP3R is activated by Ca2+ itself or by InsP3 that is produced when PLC cleaves phosphatidylinositol 4,5-bisphosphate. An elevated cytosolic Ca2+ concentration triggers various downstream effectors such as MAPK and CaMKII, which subsequently modulate cellular processes including neuronal migration, axon and dendrite development and regeneration, and synaptic plasticity The Ca2+ signaling through a PLCγ phosphotyrosine binding site at Tyr1015. This GDNF/RET/PLCγ/InsP3R signaling cascade triggers release of Ca2+ from internal ER stores that subsequently phosphorylates p42/44 of MAPK (ERK1/2) and CaMKII. Additionally, we report that RET is present in the neocortex of the developing brain and that overexpressing a RET Tyr1015 point mutation perturbs GDNF-stimulated migration of neocortical neuronal progenitor cells.We here demonstrate that RET receptor activation by GDNF stimulates cytosolic Ca2+ imaging in HeLa cells was used to determine whether the RET receptor was involved in cytosolic Ca2+ signaling. Cells were transfected with green fluorescent protein (GFP)-tagged wild-type RET (RETWT) 24 h prior to loading with the Ca2+-sensitive dye Fura-2/AM (2+ concentration was exclusively examined in GFP positive cells. Treatment with GDNF and GFRα1 in RETWT expressing cells resulted in a rapid cytosolic Ca2+ increase in 58% of the cells whereas an oscillatory Ca2+ response was observed in 25% of the cells (Single-cell live Caura-2/AM . The cythe cells .1062), 1015 (RET1015) or both (RET1062/1015), were used to identify a tyrosine residue that mediated the Ca2+ response . Tyr1015 is known to bind PLCγ to the RET receptor, suggesting that PLC played a role in this signaling pathway. The effect of the PLC-inhibitor U73122 was therefore examined. GDNF failed to elevate cytosolic Ca2+ in RETWT expressing cells that were pre-treated with U73122 (2+ increase in cells expressing RETWT (2+ response (The mechanism by which GDNF stimulated Cah U73122 . A siRNAh U73122  and blocng RETWT . The mocresponse .2+ release from the ER are the InsP3R and the Ryanodine receptor (RyR). Exposing RETWT expressing cells to the InsP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) blocked the cytosolic Ca2+ increase triggered by GDNF  pump depletes the ER of Ca2+, so that cytosolic Ca2+ can no longer be elevated from ER Ca2+ stores. Pre-treatment of RETWT expressing cells with the SERCA pump inhibitor Thapsigargin triggered a typical cytosolic transient Ca2+ increase as the ER stores depleted. Subsequent GDNF exposure failed to elevate free cytosolic Ca2+  of the cells. Expressing RETWT in primary cortical neurons produced a rapid Ca2+ response in 12.9% (n = 31) of the cells (1015 responded to GDNF (n = 34). Ex utero electroporation was then performed to determine whether the Tyr1015 of RET played a role for neocortical neuronal migration. RETWT or RET1015 constructs were injected into the lateral ventricles of E14.5 embryonic forebrains and electroporated ex utero (WT expressing cells showed a significant 6.3±0.7-fold (n = 5) increase in migration towards the GDNF-beads in the CP, as compared to control regions and vehicle (n = 6) of RETWT expressing cells (n = 6). These results indicated that PLC-dependent GDNF/RET-signaling played a role in the migratory movement of neuronal progenitor cells overexpressing RET in the VZ.To explore the biological relevance of GDNF/RET-induced Caeocortex . The neueocortex . Westerneocortex  and reveeocortex , in accoeocortex . Primaryhe cells  whereas ex utero . Organotex utero . Confoca vehicle . Blockinng cells . Treatin1015 construct was thereafter delivered into the embryonic VZ progenitor cells to further test the influence of RET Tyr1015 on neuronal migration. GDNF-beads in the CP failed to stimulate migration  of neocortical neuronal progenitors expressing the RET1015 construct  were shown to have perturbed enteric neuronal development as a result of hypersensitivity to RET signaling −/− mice. Neurite outgrowth in human neuroblastoma cells stimulated by RET was shown to be regulated through downstream activation of Ras/ERK 2+ signaling 2+ signaling 2+ signaling. Such a Ca2+ signaling link between RET and Slit2/Robo2 could, at least in part, be involved in the GDNF-directed neuronal migration in neocortex of the developing brain.GDNF/RET has previously been reported to modulate differentiation and migration through multiple cell signaling pathways. For example, migration of enteric nervous system progenitor cells and cortical GABAergic neurons has been linked to the pathways of Ras/ERK and PI3K/Akt 2+ signaling through the PLCγ phosphotyrosine binding site Tyr1015 of RET. This GDNF/RET/PLCγ/InsP3R signaling cascade elevates the cytosolic Ca2+ concentration by releasing Ca2+ from internal ER stores. The cytosolic Ca2+ response mediated through Tyr1015 of RET subsequently phosphorylates the downstream effectors ERK1/2 and CaMKII. Our data also show that RET is homogenously expressed in the cortex of the developing brain. Mutating Tyr1015 and delivering the DNA to neuronal progenitors in the VZ of mouse embryos impairs GDNF-stimulated migration in the developing neocortex. These data further the understanding of the multifactorial RET receptor in regulating multiple signaling pathways and biological processes.Our results demonstrate a novel RET signaling pathway where GDNF stimulates cytosolic CaHuman cervical carcinoma HeLa cells and mouse embryonic NIH3T3 fibroblasts (obtained from the American Type Culture Collection), were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Embryonic brain slices were obtained from wild type CD1 pregnant mice euthanized at 14.5 days postcoitum. Cerebral cortical neurons in primary culture were prepared from CD1 mouse fetuses at E15.5 as described elsewhere Reagents and concentrations, unless otherwise stated, were as follows: GDNF , GFRα1/FC chimera , U73122 , U73343 , 2-aminoethoxydiphenyl borate , Ryanodine , Dantrolene , Thapsigargin , and bis(2-aminophenoxy)ethane tetraacetic acid .2+-sensitive fluorescence indicator Fura-2/AM  in cell culture medium at 37°C with 5% CO2 for 30 min. The Ca2+ measurements were conducted at 37°C in a heat-controlled chamber (Warner Instruments) with a cooled back-illuminated EMCCD camera Cascade II:512 (Photometrics) mounted on an inverted microscope Axiovert 100M (Carl Zeiss) equipped with a LCI Plan-Neofluar 25×/0.8NA water immersion lens (Carl Zeiss). Excitation at 340, 380 and 495 nm took place using a Lambda LS xenon-arc lamp (Sutter Instrument) equipped with a Lambda 10-3 filter-wheel (Sutter Instrument) and a SmartShutter (Sutter Instrument). Emission wavelengths were detected at 510 nm, and the sampling frequency was set to 0.2 to 1 Hz. MetaFluor software (Molecular Devices) was used to control all devices and to analyze the acquired images. The experiments were performed in Krebs-Ringer's buffer containing 119.0 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1.0 mM NaH2PO4, 20.0 mM Hepes (pH 7.4), and 11.0 mM dextrose. Drugs were bath-applied.Cells were loaded with the CaCells were lysed by sonication and protein concentrations were determined using a BCA protein assay (Pierce). Equal amounts of cellular protein were separated by sodium dodecyl sulphate gel electrophoresis, followed by wet transfer to PVDF membranes. Membranes were blocked in 5% skim milk in Tris-buffered saline solution plus 0.5% Tween-20 for 1 h before being incubated with primary antibodies  overnight at 4°C and re-incubated with horseradish peroxidase-conjugated secondary antibody  for 1 h. Immunoreactive bands were visualized using an enhanced chemiluminescence kit .Mouse brain slices were cut (30–100 µm) with a vibratome (Leica), fixed with 4% PFA overnight at 4°C and then incubated in a blocking solution  for 1 h at 24°C. Blocking solution was replaced by washing solution  containing the appropriate dilution of the primary antibody overnight at 4°C. Primary antibodies used were anti-RET  or anti-Tuj1 . Alexa Fluor 488 and 555 secondary antibodies (Invitrogen) were used to reveal the primary antibodies . TO-PRO-3 (Invitrogen) and only secondary antibody staining were used as control . Slices WT and RET1015 were seeded in plastic culture dishes and grown to confluence. A 1.2-mm wide region devoid of cells was made in the dish using a 200-µl plastic pipette tip held against a caliper. Cells were starved overnight and then pretreated with the Ca2+ inhibitors for 30 min before GDNF and GFRα1 stimulation for 6–8 h. Quantification of cells moving into the empty region were performed by counting the number of cells within equal areas using bright-field light microscopy. Images were captured using a digital camera (Olympus C-7070) and processed in the Photoshop Lightroom software (Adobe).Cells transfected with RETCells were transfected with 80 nM specific small interfering RNA (siRNA) against PLC  or non-targeting siRNA (Mock-siRNA) as a control  by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Efficiency was determined by Western Blot towards PLCγ.WT or RET1015 plasmids with 0.01% Fast Green FCF (Sigma-Aldrich) in phosphate buffered saline (PBS) were injected (Transfection of HeLa cells was conducted using Lipofectamine 2000 (Invitrogen) in Opti-MEM (Invitrogen) according to the manufacturer's protocol. Electroporation of mouse embryos and organotypic brain slice culture were conducted as described previously injected . After i3 in a spherical region with diameter twice of the GDNF-bead  of the forebrain were obtained using a vibratome (Leica) and cultured in Neurobasal medium supplemented with B27. A maximum of three beads  of diameter ∼100 µm soaked  in GDNF (500 ng/ml) or in PBS were placed in the cortical plate (CP) as described DNF-bead . A regioGTACACAAACACACTCCTCTCAGG, reverse CAGGCTCCTGTTGAGAATCAG, TATA-box binding protein (TBP) forward GGGGAGCTGTGATGTGAAGT, reverse CCAGGAAATAATTCTGGCTCA. The thermal cycling conditions were: 94°C for 4 min, 35 cycles of 94°C (30 s), 60°C (30 s), and 72°C (30 s) and at the end 72°C for 4 min. The PCR products were separated on 2% agarose gel and visualized under ChemiDoc XRS+ (Biorad) after staining with GelRed (Biotium). Real-time PCR was performed in triplicates using SYBR green PCR master mix according to the manufacture's instruction (Applied Biosystems) in a 7900HT Fast real-time PCR system (Applied Biosystems). Products were analyzed with ABI 7900HT Sequence Detection System (Applied Biosystems). 2−ΔCt values were used to calculate the relative expression levels and were given as mean ± SEM.Total RNA was extracted from cultured cortical neurons using RNeasy kit (Qiagen). RNA quality and quantity were measured with Nanodrop 2000 (Thermo Scientific). RNA was then treated with DNaseI (Biolabs) and reverse transcription was conducted with Superscript II reverse transcriptase (Invitrogen) according to manufacturer's protocol. cDNA from NIH3T3 cells and mouse E15.5 cerebellum served as negative and positive control, respectively. Reverse transcription-PCR analysis was performed using Taq Polymerase (Invitrogen), and primers were as follows: RET forward 2+ recording data were normalized and cells were considered responsive to a treatment if the mean fluorescence was increased by at least 25% over the baseline. The data were presented as means ± SEM. Student's t-test was used and significance was accepted at P<0.05.The CaFigure S1WT or RET1015.Western blotting of HeLa cells transfected with RET (A) Cells expressing RETWT or RET1015 treated with GDNF (100 ng/ml) show normal phosphorylation of RET Tyrosine residues. (B) Small interfering RNA (siRNA) against PLCγ (PLCγ-siRNA) knocked-down the PLCγ protein level in HeLa cells expressing RETWT.(PDF)Click here for additional data file.Figure S22+ signalling is inhibited by BAPTA.GDNF/RET-induced Ca Representative single-cell Ca2+ recording of a Fura-2/AM-loaded HeLa cell transfected with RETWT and treated with GDNF (100 ng/ml). Quenching intracellular Ca2+ with BAPTA (10 µM) abolishes the GDNF/RET-triggered Ca2+ response.(PDF)Click here for additional data file.Figure S3Immunohistochemistry control of embryonic cortex with only secondary antibodies. Immunohistochemistry of an E14.5 mouse forebrain cortex coronal slice  and cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ) regions  with only secondary antibodies Alexa488 and Alexa555.(PDF)Click here for additional data file.


Icilin-, WS3-, or WS12-induced [Ca2+]i increases were inhibited by capsazepine or 5-benzyloxytriptamine. The increase in [Ca2+]i elicited by allyl isothiocyanate (AITC) was inhibited by HC030031. WS12 and AITC exerted a desensitizing effect on [Ca2+]i increase. Low-temperature stimuli elicited [Ca2+]i increases that are sensitive to both 5-benzyloxytriptamine and HC030031. Hypotonic stimulation-induced membrane stretch increased [Ca2+]i; HC030031 but not 5-benzyloxytriptamine inhibited the effect. The results suggest that TRPM8 channels in rat odontoblasts play a role in detecting low-temperature stimulation of the dentin surface and that TRPA1 channels are involved in sensing membrane stretching and low-temperature stimulation. The results also indicate that odontoblasts act as mechanical and thermal receptor cells, detecting the stimulation of exposed dentin to drive multiple cellular functions, such as sensory transduction.Odontoblasts produce dentin during development, throughout life, and in response to pathological conditions by sensing stimulation of exposed dentin. The functional properties and localization patterns of transient receptor potential (TRP) melastatin subfamily member 8 (TRPM8) and ankyrin subfamily member 1 (TRPA1) channels in odontoblasts remain to be clarified. We investigated the localization and the pharmacological, biophysical, and mechano-sensitive properties of TRPM8 and TRPA1 channels in rat odontoblasts. Menthol and icilin increased the intracellular free Ca Odontoblasts are tall columnar cells that are arranged along the junction between the dentin and dental pulp  and possess cellular processes that lie inside the dentinal tubules, which are tubular microstructures of dentin. These cells are responsible for dentin formation and mineralization (dentinogenesis) during physiological and developmental processes Transient receptor potential (TRP) channels, nonselective cation channels constituting 6 main subfamilies, play an important role in nociception and thermo-, osmo-, and mechano-sensation El Karim et al. (2011) reported that long-term cultured human odontoblast-like dental pulp cells express TRPM8 and TRPA1 channels. However, the expression of these channels in acutely isolated odontoblasts from rat or mouse has not yet been demonstrated TRPM8 channels are activated by temperatures of 8–28°C or by cooling compounds such as icilin, menthol, eucalyptol, WS3, and WS12 The purpose of this study was to investigate the expression patterns and the pharmacological, biophysical, and mechano-sensitive properties of TRPM8 and TRPA1 channels in acutely isolated rat odontoblasts to clarify whether TRPM8- and/or TRPA1-mediated signal transduction mechanisms are involved in the physiological or pathological stimulation of odontoblasts.All animals were treated in agreement with the guidelines established by the National Institutes of Health, USA regarding the care and use of animals for experimental procedures. The study was approved by the Ethics Committee of our institute (No. 230303 and No. 240301).Male Wistar rats (150–200 g) were perfused through the left ventricle with 4% paraformaldehyde diluted in 0.1 M phosphate buffer under anesthesia . Dissected mandibles were immersed in 4% paraformaldehyde for an additional 12 hr at 4°C. After decalcification with 10% EDTA-2Na solution, selected specimens were dehydrated through a graded ethanol series, embedded in paraffin, and sagittally sectioned to 5 µm in thickness. Dewaxed sections were treated with 0.1% hydrogen peroxide for 15 min and 1 µg/mL trypsin for 30 min. After pre-incubation with 10% goat serum for 30 min at room temperature, sections were incubated with rabbit polyclonal antibody against TRPM8  or TRPA1 channels   overnight at 4°C. For immunohistochemistry, sections were incubated with HRP-conjugated goat anti-rabbit IgG  and rinsed with PBS. Immunoreactivity was detected with 3,3′-diaminobenzidine tetrahydrochloride . Paraffin-embedded sections were counterstained with methyl green. For immunofluorescence, sections treated with antibody against TRPM8 or TRPA1 channels were incubated with secondary antibody  at room temperature for 1 hr. To stain nuclei, 4′,6-diamino-2-phenylindole was used at room temperature for 5 min with subsequent washing.2+]i measurement, enzymatically treated and isolated odontoblasts in the dental pulp slice were plated onto a culture dish, immersed in alpha-minimum essential medium (Invitrogen) containing 10% fetal bovine serum and 5% horse serum, and maintained at 37°C in a 5% CO2 incubator. The primary cultured odontoblasts in the dental pulp slice were used for [Ca2+]i measurement within 24 hr after isolation. These cells were positive for the odontoblast markers dentin matrix protein-1, dentin sialoprotein, and nestin Dental pulp slice preparations were obtained from newborn Wistar rats (3- to 10-day-old) using a previously described method 2+]i was expressed as the fluorescence ratio (RF340/F380) at these two excitation wavelengths, and then expressed as 0F/F units; the RF340/F380 value (F) was normalized to the resting value (0F/F = 1.0) in the presence of extracellular Ca2+ (0F).Cells in dental pulp slices were loaded for 30 min at 37°C in standard solution containing 10 µM fura-2-acetoxymethyl ester (Dojindo Laboratories) and 0.1% (w/v) pluronic acid F-127 (Invitrogen). They were then rinsed with fresh standard solution. A dish with fura-2-loaded odontoblasts was mounted on the stage of a microscope , which was equipped with an Aquacosmos system , excitation wavelength selector, and intensified charge-coupled device camera system. Fura-2 fluorescence emission was measured at 510 nm under alternating excitation wavelengths of 380 nm (F380) and 340 nm (F340). i was measured 2+]i to peak values of 1.03±0.01 0F/F units (N = 4) and 1.07±0.003 0F/F units (N = 3), respectively  (2+]i increases were inhibited by 10 µM capsazepine (CPZ), a non-specific TRPM8 channel antagonist . However, 5-benzyloxytriptamine hydrochloride, a specific TRPM8 channel antagonist, reversibly inhibited this increase  during the activation of TRPM8 channels. In the presence of extracellular Ca2+, WS3 (10 µM) increased [Ca2+]i to 1.09±0.03 0F/F units (N = 3). In the absence of extracellular Ca2+, however, no WS3-induced increase in [Ca2+]i was observed. The peak value of [Ca2+]i in the application of WS3 was 0.96±0.01 0F/F units (N = 3)  . Both ictagonist . In addiincrease . To exam (N = 3) .Immunoreactivity against TRPA1 channels was observed in odontoblasts; it localized to the distal membrane and the cellular processes of odontoblasts inside dentinal tubules in sections prepared from incisors brown in  and mola2+, 100 µM AITC, a TRPA1 channel agonist, transiently increased [Ca2+]i to a peak value of 1.10±0.01 0F/F units (N = 9). The increase was reversibly inhibited by 100 µM HC030031, a TRPA1 channel-specific antagonist, to 1.03±0.004 0F/F units (N = 9) (In the presence of extracellular Ca (N = 9) .2+-dependent desensitization 2+ entry by the fourth application  or 87.4±9.2% (N = 3), respectively, of the peak value of increase in [Ca2+]i after the first application. Additionally, the increase in [Ca2+]i after the fourth application of WS12 or AITC was 47.1±2.5% (N = 4) or 34.3±1.3% (N = 3), respectively, of the increase in [Ca2+]i after the first application  (2+]i increased (N = 8). The increase was inhibited by 100 µM HC030031 (N = 8)  . To acti (N = 8) .2+]i increase in rat odontoblasts by applying hypotonic solution (200 mOsm/L) 2+, the application of hypotonic solution increased [Ca2+]i (N = 7) (N = 5)  ; no effe (N = 5) .2+]i; the increase was inhibited by non-specific (capsazepine) or selective (5-BOT) antagonists of TRPM8 channels. The WS3-induced increase in [Ca2+]i was not observed in the absence of extracellular Ca2+, indicating that the increase in [Ca2+]i upon activation of TRPM8 channels was mediated by Ca2+ influx from the extracellular medium. The results also showed that rat odontoblasts were sensitive to AITC and that the AITC-induced [Ca2+]i increase was inhibited by the selective TRPA1 antagonist HC030031. These results clearly indicate that rat odontoblasts functionally express TRPM8 and TRPA1 channels.We investigated the expression, localization, and the pharmacological, biophysical, and mechano-sensitive properties of TRPM8 and TRPA1 channels in odontoblasts. Selective TRPM8 channel agonists WS3 or WS12 increased [Ca2+]i was sensitive to HC030031 but not 5-BOT. The results suggest that TRPA1 channels contribute to thermal- and mechano-sensitivity; TRPM8 channels, however, are not involved in the mechano-sensitive process in odontoblasts. The results are also consistent with data showing intense immunoreactivity for TRPM8 across the cell body and immunoreactivity for TRPA1 on the distal membrane and cellular processes of odontoblasts inside dentinal tubules.In the present study, TRPM8 and TRPA1 channels showed sensitivity to low-temperature stimuli . Meanwhi2+ entry via TRPV1, TRPV2, and TRPV4 channels According to the hydrodynamic theory The latencies of dentinal sensory and pulpal neuron responses to various stimulations, such as thermal stimulation applied to the dentinal surface, are too short. This indicates that the sensory transduction mechanism in dentin is unlikely to be sensitive to temperature changes inside the dental pulp or at the pulp/dentin interface, where odontoblasts and free nerve endings are located However, the results from a recent study in humans suggested that pain produced by cold stimuli was due not to a hydrodynamic mechanism, but to something another mechanism such as cold-sensitive molecular sensors located on the odontoblast membrane The intradental afferent and its nerve endings reach the odontoblast region and form a dense network of sensory axons (known as the plexus of Raschkow). It was recently suggested that chemical substance(s), such as nitric oxide, ATP, or galanin, may participate in the interaction between odontoblasts and sensory neurons as neurotransmitter(s) In conclusion, we demonstrated the expression of TRPM8 and TRPA1 channels in odontoblasts. The results indicate that TRPM8 channels play an important role in detecting low-temperature stimuli, while TRPA1 channels are involved in sensing low-temperature stimulation and plasma membrane mechanical deformation. The results also indicate that odontoblasts act as mechanoreceptor cells and thermal receptor cells, detecting the stimulation of exposed dentin to drive various cellular functions, such as sensory transduction.


Mitochondrial free Ca2+-concentration ([Ca2+]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca2+ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca2+]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca2+ concentration. The rise in [Ca2+]mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s−1. Accordingly, frequency-dependent increase in [Ca2+]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca2+]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca2+]mito transients were increased by inhibition of mitochondrial Na+/Ca2+ exchanger (mNCX). These results provide direct evidence for fast Ca2+ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca2+-handling and a dependence of mitochondrial Ca2+-handling on intracellular (SR) and external Ca2+ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca2+-leakage.Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca This Ca2+ transport is crucial for the regulation of mitochondrial activity 2+ concentration inside the mitochondrial matrix ([Ca2+]mito) In all excitable and non-excitable cells, mitochondria take up and release Ca2+ transport through the mitochondrial membranes has been the object of several studies in the last ten years, which have demonstrated that mitochondrial free Ca2+ concentration undergoes rapid variations in parallel with the cytosolic Ca2+ transients 2+ uniporter (MCU) which has recently been identified as a 40-kDa protein located in the inner mitochondrial membrane +/Ca2+ exchanger (mNCX), e.g. 2+ uptake and extrusion, respectively.Ca2+ concentration is high during the initial phase of the SR Ca2+-release process, see for a review 2+ between the mitochondria and the SR during contraction and relaxation in skeletal muscle.Evidence has been presented that microdomains are present near the Ryanodine receptor (RyR) where the local Ca2+ available inside the SR becomes insufficient for sustained contractile activity and muscle fibers become dependent on extracellular Ca2+ which might enter the cells via store-operated Ca2+ channels (SOCE) [17)]. Indeed, increased SOCE has been reported in skeletal muscle with reduced calsequestrin-1 expression 2+ handling is affected by a depletion of the SR calcium store and by the lowering of extracellular Ca2+ concentration.Our previous studies 2+ concentration [Ca2+]mito was studied in adult skeletal muscle fibers using a ratiometric FRET-based probe (4mtD3cpv) which is specifically targeted to the mitochondrial matrix 2+ concentration in dCASQ-null (lacking both CASQ isoforms) and wild type fibers both at rest and during contractile activity induced by electrical stimulation. In addition we used electron microscopy to study the differences in mitochondrial density and in the local arrangement of the mitochondria and the SR between the dCASQ-null and wild type fibers.The intramitochondrial free Ca2+ concentration was found to be higher at rest but lower during contraction in dCASQ-null fibers compared to wild type fibers. Detailed analysis of the kinetics of the changes in mitochondrial free Ca2+ concentration during contraction revealed significant differences between dCASQ-null and WT fibers and significant changes therein upon removal of extracellular Ca2+. This indicates that mitochondrial Ca2+-handling in fast skeletal muscle fibers is sensitive to the calcium available in both intracellular and extracellular Ca2+-stores. The importance of these findings for altered muscle function in malignant hyperthermia will be discussed.The mitochondrial free CaExperiments were carried out on 4 months old dCASQ-null and wild type (WT) mice. Double (d)CASQ-null mice c oxidase (COX), and replicated 4 times 4mtD3cpv cameleon targeted to the mitochondrial matrix via a targeting sequence, derived from subunit VIII of human cytochrome 2 1, MgSO4 1, KH2PO4 1, glucose 5, HEPES 20, pH 7.2) and equipped with platinum field electrodes. The zero external calcium solution used in part of the experiments had an identical composition except that CaCl2 was omitted and 50 µM EGTA was added to scavenge any remaining Ca2+-ions. Fibers were incubated for 1 hour, prior to the measurements. As in our previous experiments we did not add MgCl2 to replace CaCl2. Temperature during the measurements was kept at 25−26°C, in order to slow the kinetics of Ca2+ release and reuptake into the mitochondria, and to facilitate comparison with previous studies Enzymatic isolation of single skeletal muscle fibers from FDB muscle was performed using collagenase treatment Fibers were fixed with 4% formalin in PBS for 30 minutes, permeabilized with 1% Triton and blocked in 10% goat serum for 1 hour to avoid non-specific signal. Fibers were incubated overnight at 4°C with primary antibodies specific for mitochondrial outer membrane protein Tom20  and ryanodine receptor  and with appropriate fluorescent secondary antibodies for 2 h at room temperature. The fibers were viewed with a confocal microscope .Electron-micrographs were obtained from FDB muscles as described previously 2+-concentration ([Ca2+]mito) were carried out using an inverted fluorescence microscope  at 20X magnification equipped with the perfect focus system (Nikon Instruments) and a cooled CCD camera , essentially as described previously 2+-indicator was studied in fibers stimulated at 0.1 Hz and occasionally at 0.033 Hz. At such low frequency of stimulation the recovery in the YFP/CFP ratio after the contraction is almost complete before the next stimulus starts and fibers, in addition, do not fatigue during the measurements. The results and the method to correct for bleaching are shown in the Supporting Information .Measurements of the intramitochondrial free Ca2+ inside the mitochondria. In these latter experiments the effect of the mitochondrial Na+/Ca2+ exchanger blocker  was assessed.In a separate set of experiments, the time resolution of the recordings was reduced to 900 ms by increasing the integration time of the CCD camera and reducing the illumination intensity. In this way bleaching of the probe was negligible and the duration of the recordings could be increased to assess very slow changes in Ca2+-concentration in quiescent fibers. Additional information on the correction for bleaching of the probe is given in the Supporting Information The correction for bleaching and the subsequent analysis of the recordings was performed using a computer program written in MatLab . A third order polynomial fit to the initial and final 5-s periods of the recordings was calculated and subsequently subtracted from the data points to correct for the change in baseline as a result of bleaching. The intercept at t = 0 s of the polynomial fit to the baseline of the first fiber recorded in each dish was used to estimate the basal level of the free mitochondrial Ca2+]mito. The rise time (t10–90%) of the increase in free [Ca2+]mito from 10 to 90% of the peak amplitude was calculated from the interpolated recording after correction for bleaching. The decline in free [Ca2+]mito during 1 Hz stimulation was fitted with a double exponential: y(t) = a0+a1exp (−k1⋅t) + a2exp (−k2⋅t). The value of this equation at t = 1 second was used as the minimum value of the averaged recording in the determination of the rise time in order to minimize the effect of noise. The amplitude a1 was calculated by extrapolating the double exponential to t = 0 seconds, the moment at which the maximum value was reached. The decline in free [Ca2+]mito after the train of stimuli was fitted to a single exponential: y(t) = a3⋅exp (−k3⋅t), in which the parameter a3 reflected the decrease in free [Ca2+]mito after the train of stimuli as well as the (quasi steady-state) increase in free [Ca2+]mito during the train of stimuli.The time averaged steady-state responses during a train of stimuli at 1 Hz were used for the analysis of the early rapid alterations in free [Ca2+]mito, we extended the recording of the decay phase after a train of 5 Hz stimulation to 10 minutes. In these recordings the integration time of the camera was increased to 900 ms and the illumination intensity was reduced accordingly in order to reduce bleaching of the probe. The final decline in free [Ca2+]mito in these recordings was fitted to a single exponential: y(t) = a4⋅exp (−k4⋅t).To resolve very slow components of the decline in free [Ca2+ sensor was performed as describes previously 2+]  = β·K'd·[(R-Rmin)/(Rmax-R)]1/n was used to estimate the free [Ca2+]mito in quiescent fibers as well as the levels reached at the end of stimulation, using a K'd of the 4mtD3cpv sensor of 0.76 μM and n value of 0.74 in vitro and in situ K'd value Calibration of the Ca2+ signals were recorded using a dual-beam excitation fluorescence photometry setup  at 25–26°C. Calibration of the Fura-2 signals and calculation of the free cytosolic Ca2+ concentration were as reported previously Fibers were loaded with 5 µM Fura-2 acetoxymethyl ester  in incubation buffer as described previously 2+ or Ca2+ x Stimulation Frequency) or three-way ANOVA (Type x Ca2+ x Stimulation Frequency (repeated measures), respectively) followed by Bonferroni post hoc test, if appropriate, to assess differences between WT vs. dCASQ-null, 1 mM vs. 0 external Ca2+, at the various stimulation frequencies used. P<0.05 was considered statistically significant.Data are expressed as mean ± SEM. Comparisons between groups were made using two-way ANOVA  showed a striated sarcomeric pattern Fig. 1. 2 i.e. by 55±10%. The percentage of mitochondria coupled to Ca2+ release units (CRUs) was already high in WT fibers (70.4±0.8%) and showed a further significant increase (+12.0±0.2% P<0.0001) up to 78.9±0.7% in dCASQ-null. In addition, the width of the terminal cisternae of the SR in dCASQ-null fibers was reduced  and 4.7±0.5% in dCASQ-null (n = 157). Subsarcolemmal mitochondria were counted in a layer of 1 μm in width and the typical radius of the fibers studied was 20 μm. The subsarcolemmal volume fraction thus represents 1-(19/20)2+]mito was assessed from the ratio (R) of the YFP and CFP intensities in single WT and dCASQ-null fibers both in the presence and absence of external Ca2+. The initial recordings from each fiber were used to determine the basal values of the YFP/CFP ratios in order to eliminate any possible effect of electrical stimulation and bleaching of the Ca2+-sensor. The basal values were calculated from the intercept at time  = 0 of the polynomial fit of the baseline (dotted line in 2+]mito in dCASQ-null fibers relative to WT fibers (P<0.001), whereas no significant differences were observed between the measurements in the presence and absence of external Ca2+. The average values of the pooled data amounted to 1.83±0.02 in WT fibers (n = 96) and to 1.94±0.03 in dCASQ-null fibers (n = 54).Free mito were observed during repetitive electrical stimulation at 1 Hz. The rising and decay phases of the mitochondrial Ca2+ transients were highly asymmetric and this resulted in incomplete recovery of free [Ca2+]mito during the contractions even at low stimulation frequency. During the first contractions, the transients were accompanied by a gradual increase relative to the baseline, reflecting an accumulation of Ca2+ inside the mitochondrial matrix, reaching a dynamic steady state that was maintained during the remainder of the train of stimuli. At the stimulation rate of 5 Hz mitochondrial Ca2+ accumulated more rapidly and [Ca2+]mito reached a higher final level than in those at 1 Hz, while during the recordings at 20 and 60 Hz the rates and the levels attained were only slightly higher than those at 5 Hz. Whereas in WT fibers the accumulation of calcium in mitochondria continued during the stimulation period even when cytosolic calcium concentration was stable and constant, in dCASQ-null fibers, the initial fast rise in [Ca2+]mito was followed by a slow increase or, in many instances, by a clear interruption of calcium accumulation, see for example the 60 Hz recording shown in Fig. S4 in File S1. This behavior may be linked to the decline in cytosolic free [Ca2+] and in contraction strength observed previously  in CASQ1-null and dCASQ-null fibers and suggests that the decline in cytosolic free [Ca2+] limited further increase in [Ca2+]mito in dCASQ-null fibers.Representative examples of the recordings obtained during contractions elicited by trains of electrical stimulation at 1, 5, 20 and 60 Hz in fibers from WT and dCASQ-null animals are shown in 2+ accumulation during the train of stimuli was quantified by measuring the drop in the YFP/CFP ratio at the end of the train of stimuli (ΔR), after correction for bleaching of the probe as described in the Supporting Information . The averaged data obtained in WT and dCASQ-null fibers are shown in 2+]mito in dCASQ-null fibers were significantly smaller than in WT fibers (P<0.01) and somewhat blunted in the absence of extracellular Ca2+.The overall mitochondrial Ca2+]mito (t10–90%) for the pooled data amounted to 27.8±1.6 ms (n = 179).Information on the kinetics of the rising phase of the mitochondrial calcium transient was obtained in twitches or the first contractions during stimulation trains at 0.1 and 1 Hz Fig. 5A.10–90% during repetitive 1 Hz stimulation, when the transient responses had reached a steady state. The average t10–90% from all groups amounted to 18.5±0.4 ms (n = 94). This value is significantly smaller (−33%) than the value obtained during the initial response in rested fibers.The analysis of averaged responses at 1 Hz allowed us to estimate t2+ (2+ (P<0.05).There were no significant differences between WT and dCASQ-null but small, highly significant differences  were observed in the presence and absence of external Ca2+ Fig. 5B.2+], we studied the averaged steady state recordings obtained during 1 Hz stimulation. At this stimulation rate, cytosolic calcium transients and contractile responses are well separated by periods at resting calcium concentration, while the free [Ca2+]mito remained well above the resting initial values . Averaging of the steady state responses as well as pooling the results obtained in different fibers from the same experimental group allowed us resolve changes in the sub-millisecond range. A typical example is shown in 2+]mito during the 1 Hz stimulation period was fitted to a double exponential as indicated in Methods. The decay in free [Ca2+]mito appeared to be dominated by two exponentials with a very fast rate constant (k1) of about 40 s−1 and a slower rate (k2) of about 1.5–2 s−1.To obtain more information on the kinetics of the intial phase of the decay in mitochondrial [Ca2+]mito after the end of a train of stimuli was dominated by a single exponential with a rate constant (k3) of approximately 0.20–0.25 s−1  and the rate constant (k4) of this component amounted to 12±4% and 0.060±0.005 s−1 and 9±2% and 0.014±0.010 s−1, in WT + Ca and dCASQ-null + Ca fibers, respectively. In these experiments, the effects of CGP37157 a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger were also tested. It can be seen in 2+]mito and an increase in the final level reached, during the 10 s period of 5 Hz stimulation.To quantitate possible very slow components of the mito were derived from the equation: [Ca2+]  = β·K'd·[(R-Rmin)/(Rmax-R)]1/nmax and Rmin values of 5.83 and 1.50, respectively, an in vitro K'd of the 4mtD3cpv sensor of 0.76 μM, an n value of 0.74 2+ in quiescent fibers amounted to 0.16 μM (WT) and 0.23 μM (dCASQ-null) and to a maximum free [Ca2+]mito at the end of the train of stimuli at 20 or 60 Hz of 1.10 μM (WT) and 0.73 μM (dCASQ-null).The actual values of the free [Ca2+] (or [Ca2+]cyto) and the free [Ca2+]mito, we determined free [Ca2+]cyto with the fluorescent indicator Fura-2. In quiescent fibers, no significant differences were observed in the free [Ca2+]cyto between WT and dCASQ-null fibers both in the absence and presence of extracellular Ca2+. The free [Ca2+]cyto of the pooled data amounted to 101±2 nM (n = 90).To assess the relation between free cytosolic mito in dCASQ-null fibers is significantly higher than in WT fibers, as indicated by the difference in the basal YFP/CFP ratios. Our estimates of the free [Ca2+]mito are 0.16 μM in WT and 0.23 μM in dCASQ-null fibers. These values are very similar to previous estimates of 0.2 μM in WT, see e.g. 2+]mito are rather crude because of uncertainties in the (in situ) values of K'd and β of the probe used in our experiments.In quiescent fibers free mito (t10–90%) amounted to 27.8±1.6 ms during the first contraction in a rested fiber and to 18.5±0.4 ms during steady state contractions at 1 Hz. Since the rise time is influenced by the intrinsic response time of the probe, the actual rise time of mitochondrial Ca2+-uptake might even be faster. The agreement between values obtained in WT and dCASQ-null fibers indicates that it is not significantly influenced by a reduction in the SR storage capacity, induced by ablation of both CASQ isoforms, possibly because the local cytosolic calcium concentrations during the initial contrations in WT and dCASQ-null are similar.No differences were observed in the kinetics of the rising phase of the mitochondrial Ca2+ transients during the initial contraction and the steady state contractions at 1 Hz are very similar , the difference in the rise time in rested fibers and during steady state at 1 Hz is in fact opposite to what would be expected in a situation where substantial Ca2+-accumulation in the mitochondria would have taken place. However, the speed of mitochondrial Ca2+-uptake process could be increased as a result of a rise the local Ca2+-concentration in the microdomains between CRUs and mitochondria during repetitive stimulation at 1 Hz. In order to be consistent with Ca2+-uptake through the low affinity MCU, this local Ca2+-concentration would need to be increased considerably above the cytosolic free Ca2+-concentration at rest (∼100 nM). Evidence suggests that the Ca2+ uniporter can be sensitized by the locally elevated [Ca2+], see e.g. 2+] in cardiac muscle cells, up to 10 μM Since the cytosolic Ca2+ transient during 1 Hz stimulation was observed in dCASQ-null in the absence of external Ca2+. Under these conditions the intracellular stores are almost exhausted and unable to support a prolonged contractile activity 2+ availability in the cytosol rather than a direct specific effect of extracellular Ca2+ on mitochondrial Ca2+-uptake kinetics per se.A small but significant prolongation of the rising phase of mitochondrial Ca2+]mito during and after stimulation trains is governed by 4 exponentials with rate constants of approximately 40, 1.5, 0.2 and 0.03 s−1 (at 26°C). The amplitude of the very fast component amounted to 25–30% of total Ca2+ release both in WT as in dCASQ-null fibers . Assuming that the free [Ca2+]mito is the result of a balance between Ca2+ influx and efflux across mitochondrial inner membrane +/Ca2+ exchanger promotes Ca2+ efflux already during the stimulation period. Since the effect was visible on a 10 s time scale we consider it likely that the mNCX may contribute to the intermediate (1.5 and 0.3 s−1) phases of the mitochondrial Ca2+-extrusion process.Selective blockade of the mNCX by CGP37157 Fig. 6 e2+]mito at rest imply that the total amount of Ca2+ stored in mitochondria is increased, as discussed above, and that the exchange of Ca2+ between the SR and mitochondria could be facilitated by an improved coupling and a shortened Ca2+ diffusion distance between the sites of release and the mitochondria 2+ leakage from the SR results in an elevation of the Ca2+ concentration near the mitochondria relative to the concentration in the bulk of the cytosol, the improved coupling with SR and the increased Ca2+ leakage from SR could explain the increase in free [Ca2+]mito in quiescent dCASQ-null fibers relative to WT fibers, whereas global free cytosolic Ca2+ concentration remained the same. Moreover, it can be noted that the intermyofibrillar mitochondria are located close to the regions of the SR where SERCA is localized  and 2+-accumulation is predominantly reflected during the decline phase in a reduction of the amplitude of the slow recovery process (a3). Thus, the amplitude of this latter phase appears to be particularly sensitive to the reduced mitochondrial Ca2+-content of dCASQ-null fibers in comparison with WT fibers. This observation is compatible with the idea that the speed of this phase is determined at least partly by the Ca2+-off rate(s) of mitochondrial Ca2+ buffer(s).The steady levels of the free mitochondrial Ca2+, indicating that sarcolemmal Ca2+-influx possibly via SOCE occurred during stimulation and was required to support the activation of the contractile process in those fibers. Our present results indicated that the elevated free mitochondrial [Ca2+] in quiescent dCASQ-null fibers was maintained in the absence of external Ca2+. We attribute this to enhanced coupling between the SR and the mitochondria because the cytosolic Ca2+ concentration at rest: 1) did not differ between WT and dCASQ-null fibers and 2) was not significantly affected by external Ca2+ removal.Our previous study 2) of the fast Ca2+ extrusion phase is reduced in dCASQ-null fibers when extracellular Ca2+ is removed, back to WT values. This suggests that in addition to improved coupling between CRUs and mitochondria in dCASQ-null fibers, the activity of the mNCX could be reduced in these fibers in the absence of extracellular Ca2+, e.g. by a rise in mitochondrial Na+ concentration Interestingly, the rate constant  in mitochondrial Ca2+ extrusion process, relevant at the intermediate time scale (seconds or tens of seconds).This study represents the first detailed quantitative analysis of the kinetics of mitochondrial Ca2+ uptake and release by the mitochondria play an important but still under-illuminated role in the regulation mitochondrial metabolic activity. Our study indicates that mitochondrial Ca2+-uptake and release are fast enough to allow the rapid adjustment of metabolic activity to increased demand for instance during exercise. Moreover, Ca2+ uptake by the mitochondria is crucial in skeletal and cardiac dysfunction and in programmed cell death, e.g. Rapid Ca2+ concentration inside the mitochondria were increased in calsequestrin-null muscle and the maximum free Ca2+ levels reached during electrical stimulation were sensitive to the presence of extracellular Ca2+. These alterations in mitochondrial Ca2+ handling may represent a compensatory response when SR Ca2+-content is reduced, e.g. by calsequestrin ablation and/or increased SR Ca2+ leakage, but also may render the tissue more prone to the development of contractures.Calsequestrin-null mice represent a model for malignant hyperthermia File S1min (left) and Rmax (right) of the cameleon (4mtD3cpv).Supporting text and figures. Figure S1. In situ determination of R The 4mtD3cpv responses  are shown. Aliquots of ionomycin (Iono) were added up to a final concentration of 5 μM (left) and 10 μM (right); 50 μM N-benzyl-ptoluene sulphonamide (BTS) was added in order to reduce movement artifacts during the Rmax determination. The minimum YFP/CFP value reached (Rmin) amounted to 1.5. Addition of 5 mM CaCl2 caused an abrupt increase in the YFP/CFP ratio up to a maximum value of 6.3. Upon the addition of 5 mM CaCl2, the fiber started to contract despite of the presence of BTS (for 7 minutes), resulting in an increase in the CFP (and YFP) intensity and moved out of the focal plane of the microscope near the end of the recording (resulting in a decline in the YFP and CFP intensity). Figure S2. Correction for bleaching. Top: during a train of stimuli at 0.1 Hz, the amplitude of the increase in the YFP/CFP ratio declined as a result of bleaching of the probe. Bottom: a linear relation was observed between peak amplitude and the concurrent baseline value, yielding a straightforward way to correct the ΔR values for bleaching of the probe (see Results). 2+]mito during and after a single twitch.Figure S3. Recording of the change in mito after an early rapid increase accumulates gradually in WT but remains rather constant in dCASQ-null fibers (Panels C and D).(DOC)Click here for additional data file.


Streptococcus pneumoniae. S. pneumoniae is one of the leading causes of bacterial endophthalmitis. The aim of this study is to determine whether passive immunization with the 23-valent pneumococcal polysaccharide vaccine  or PLY protects against pneumococcal endophthalmitis.Capsule and pneumolysin (PLY) are two major virulence factors of S. pneumoniae. In a separate group of experiments, vancomycin was injected 4 hours post-infection (PI) for each passively immunized group. Severity of infection, bacterial recovery, myeloperoxidase (MPO) activity and percent loss of retinal function were determined.New Zealand white rabbits were passively immunized with antiserum to PLY, PPSV23, a mixture of PPSV23/PLY, or PBS (mock). Vitreous was infected with a clinical strain of 10 CFU) compared to controls . Retinas from immunized rabbits were more intact. Vitreous of PLY (2.88 MPO untis/mL) and PPSV23/PLY (2.17) passively immunized rabbits had less MPO activity compared to controls , and both passive immunizations  helped to significantly preserve retinal function compared to controls . When vancomycin was administered 4 hours PI, all eyes were sterile at 24 hours PI. A significantly lower clinical severity was observed for rabbits administered the combination immunization (5.29) or PPSV23 (5.29) with vancomycin treatment compared to controls .Passive immunization with each antiserum significantly lowered clinical severity compared to mock immunization . A significantly higher bacterial load was recovered from the vitreous of PLY passively immunized rabbits 24 hours PI (7.87 logPassive immunization with antisera to these antigens is effective in reducing clinical severity of pneumococcal endophthalmitis in rabbits. Addition of vancomycin to immunization is effective at eliminating the bacteria. Streptococcus pneumoniae (pneumococcus) is a gram-positive coccus that is a normal resident of the human nasopharynx. This bacterium is an opportunistic cause of many human diseases such as bacteremia, meningitis, otitis media, peritonitis, pneumonia, and sinusitis [S. pneumoniae has been reported to be a common cause of bacterial endophthalmitis, often as a result of ocular surgery complications [S. pneumoniae was one of the top 3 causes of infection in cases that caused pathology in the first 3 days [inusitis -4. Likewications -7. One st 3 days . Intraoct 3 days ,10.S. pneumoniae is the polysaccharide capsule [S. pneumoniae, and purified capsules from several types have been used as immunogens in vaccines for protection against invasive pneumococcal diseases. Two pneumococcal vaccines are currently licensed for use in the United States and are based on the polysaccharide capsule, the 13-valent conjugate vaccine , New York, NY, USA) and the 23-valent polysaccharide vaccine  [One of the major virulence factors of  capsule -16. TherNJ, USA) -19.® 23 contains purified polysaccharides of the 23 most commonly found serotypes in pneumococcal infections . In studies thus far, immune protection has been tested for non-ocular diseases. A study by Jeurissen et al. [S. pneumoniae with these serotypes. Regarding ocular studies, this laboratory previously showed that capsule is important for pneumococcal endophthalmitis [Pneumovaxhalmitis . The patS. pneumoniae is pneumolysin (PLY), a cholesterol-dependent cytolysin [S. pneumoniae cause less severe pathology than their parent strains in rabbit models [Another virulence factor of ytolysin . PLY binytolysin -25. Pureytolysin . PLY mutt models ,27. A prt models . HoweverSpecific pathogen-free (SPF) New Zealand white rabbits  were used in these studies and maintained according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.S. pneumoniae[Antisera were produced by active primary immunization of rabbits as previously described ,29. The neumoniae; therefo410).Serum anti-PPSV23, anti-PLY, and anti-19 F IgG titers were determined by ELISA . As a coFor passive immunization studies, rabbits were administered 1 mL of antiserum against PPSV23, PPSV23/PLY, PLY, or mock antiserum in the ear vein at the same time as infection. Mock antiserum had no titer. All other sera, after pre-adsorption with cell-wall polysaccharide where applicable, had specific titers ≥ 12,800.S. pneumoniae E335, a serotype 19 F clinical endophthalmitis strain, was provided by Regis Kowalski  and was grown to approximately 108 colony-forming units (CFU) per mL as previously described [escribed . Accurac2 CFU in 10 μL as previously described [Each rabbit was anesthetized and infected intravitreously with approximately 10escribed .At 4 hours PI, 100 μL of vitreous was aspirated from passively immunized rabbits using a 30-gauge needle. Each eye was then injected with 100 uL of vancomycin  using a 30-gauge needle.SLE for endophthalmitis was previously described . In shor2 at 37°C overnight for quantitation of log10 CFU per mL recovered.Vitreous was removed from each eye at 24 hours PI for all experiments using a 22-gauge needle. The vitreous samples were serially diluted, cultured in triplicate on blood agar, and incubated in 5% COS. pneumoniae in Todd Hewitt broth containing 0.5% yeast extract (THY). Each sample was placed in a shaker incubator at 37°C for 3 hours, serially diluted, plated in triplicate, and incubated overnight to determine bacterial survival.A bacterial survival assay was performed on blood from the rabbits that were actively immunized to generate the antisera used in the passive immunization experiments using a previously published technique . In shorWhole eyes were removed, and histologic sectioning and staining were performed . Hematoxylin and eosin staining was used to stain eosinophils and general ocular architecture.The MPO activity of neutrophils in infected vitreous of passively immunized rabbits was determined using a colorimetric assay as described previously . Uninfec410).The presence of PPSV23 or PLY specific IgG in the vitreous was determined using ELISA . SamplesERG was used to quantify the retinal response, as previously described . PercentU test [Clinical SLE scores and bacterial CFU data were analyzed as dependent variables using a two level factorial model in the analysis of variance. The main factor was treatment . The model was analyzed as a repeated measures model where within subject correlation (eyes within rabbit) was taken into account . The errU test . The alpAntisera to PPSV23 had specific anti-PPSV23 IgG titers ≥ 12,800. PLY-specific serum IgG titers were ≥ 51,200. Antisera to PPSV23/PLY had anti-PPSV23 IgG titers ≥ 12,800 and anti-PLY IgG titers ≥ 51,200. No IgG titer was observed for control (mock) antisera (< 100). A low titer (~ 800) for the histidine tag of the unrelated recombinant protein was observed in antisera to PLY and PPSV23/PLY. Anti-19 F IgG titers were between 6,400 and 12,800 for PPSV23/PLY antisera and 1,600 and 12,800 for PPSV23 antisera.Passive administration of antisera from all experimental groups was able to reduce SLE scores significantly as compared to rabbits administered mock serum at 24 hours PI . No significant difference was observed among any other groups tested at 24 hours PI . Also, whole blood from PPSV23/PLY actively immunized rabbits was significantly better at killing bacteria than whole blood from mock immunized rabbits . No significant difference was observed between PPSV23 and PPSV23/PLY whole blood (P = 0.8475).Less destruction of the retina was observed in infected eyes of PLY, PPSV23 and PPSV23/PLY passively immunized rabbits as compared to mock passively immunized rabbits . No significant difference was observed among any other group of passively immunized rabbits P = 0.1165; Table ; Table 5Anti-PLY IgG was detected in undiluted vitreous of rabbits passively immunized with PLY or PPSV23/PLY antiserum. Anti-PPSV23 IgG was not detected in the vitreous of rabbits passively immunized with PPSV23 or PPSV23/PLY antiserum. Neither anti-PLY IgG nor anti-PPSV23 IgG was detected in the vitreous of mock passively immunized rabbits (data not shown).A significantly lower percent loss of retinal function was observed in rabbits passively immunized with PLY or PPSV23/PLY antiserum as compared to mock passively immunized rabbits at 24 hours PI (P = 0.0323). Passive immunization with PPSV23 antiserum lowered the percent loss of retinal function when compared to mock immunized rabbits at 24 hours PI, although not significantly P = 0.1309). No significant difference was observed among any other treatment groups . One explanation could be that complement is involved in the clearing of bacteria. The interior of the eye is an immune privileged area. Far lower amounts of complement proteins are found inside the eye, even when the eye is inflamed due to infection [For the current study, a capsule type 19 F strain was used for infection because capsule type 19 F is included in the polyvalent pneumococcal vaccine. Whereas whole blood from PPSV23 and PPSV23/PLY actively immunized rabbits was able to kill nfection ,40. Alsonfection . Alternain vivo compared to bacterial killing in vitro is due to disparities in capsule type antibodies. One study showed that the antibody titers to PPSV23 vaccination are not equal for each capsule type [Another explanation for the differences observed in bacterial recovery ule type . Also, aule type . The capIt is of note that less neutrophil activity was observed in the infected vitreous of rabbits passively immunized with PPSV23/PLY or PLY compared to rabbits passively immunized with mock serum. It is not surprising that these immunized animals had higher bacterial loads recovered from the vitreous than mock-immunized animals since fewer neutrophils were present to kill the bacteria. Since anti-PLY IgG but not anti-PPSV23 IgG was detected in the vitreous of the immunized rabbits, it is a likelihood that anti-PLY antibodies may account for the reduction in neutrophil infiltration and thus an increase in bacterial loads.The lack of bacterial clearance could be circumvented by administration of an antibiotic, which is normally given to patients affected by bacterial endophthalmitis. Vancomycin is a commonly used treatment for gram-positive bacterial endophthalmitis ,44. SincS. pneumoniae is commonly isolated as the cause of post-surgical endophthalmitis [Immunization strategies to provide some protection against pneumococcal endophthalmitis could be useful in clinical situations. Since ocular surgery, especially cataract surgery, has been documented to be a major cause of bacterial endophthalmitis ,6,45, anhalmitis , passivehalmitis . These pTaken together, the results indicate that passive immunization with PPSV23 and/or PLY is significantly more effective at preserving ocular integrity during pneumococcal endophthalmitis than negative controls. It appears that retinal function fared better in rabbits that were administered PLY or PPSV23/PLY antisera than in those given PPSV23 antisera, although there was some preservation of function in the PPSV23 group. Immunization alone was not able to reduce the bacterial burden in the vitreous, however, the addition of vancomycin provided a synergistic effect of sterilizing the eye while the combination PPSV23/PLY antiserum reduced clinical severity and loss of retinal function.The authors declare that they have no competing interests.MS and MM conceived and designed the study and contributed in preparation of the study protocol. MS, ST, NT, EN, QM and LK were involved in data collection supervision. MS and HT performed the statistical analyses. MS drafted the manuscript. All authors read and critically revised the manuscript and approved the final draft.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/13/8/prepub


Elevation in intracellular Ca2+ concentration is dependent either on Ca2+ influx from the extracellular space through the plasma membrane, or on Ca2+ release from intracellular Ca2+ stores, such as the endoplasmic/sarcoplasmic reticulum (ER/SR). Mitochondria are also major components of calcium signalling, capable of modulating both the amplitude and the spatio-temporal patterns of Ca2+ signals. Recent studies revealed zones of close contact between the ER and mitochondria called MAMs (Mitochondria Associated Membranes) crucial for a correct communication between the two organelles, including the selective transmission of physiological and pathological Ca2+ signals from the ER to mitochondria. In this review, we summarize the most up-to-date findings on the modulation of intracellular Ca2+ release and Ca2+ uptake mechanisms. We also explore the tight interplay between ER- and mitochondria-mediated Ca2+ signalling, covering the structural and molecular properties of the zones of close contact between these two networks.Calcium (Ca Mitochondria are equally important in physiological Ca2+ signalling through a process unraveled by a series of works demonstrating that Ca2+ release from the ER results in cytosolic Ca2+ increases that are paralleled by similar or even larger cycles of mitochondrial calcium uptake, and subsequent release ) is controlled by the simultaneous interplay of multiple counteracting processes, which can be split into "ON mechanisms" and "OFF mechanisms". With these, cells maintain a rigid control over the cytosolic level of Ca2+. In resting conditions, cells maintain a low Ca2+ concentration in the cytoplasm (around 100 nM). Ca2+ influx from the extracellular space (that posses a concentration around 1-2 mM), or Ca2+ release from intracellular Ca2+ stores, such as the ER (with concentrations of 250-600 μM) generate the intracellular calcium signals  rises sharply to about 10 μM m leads to the opening of the mitochondrial permeability transition pore (PTP), a critical event driving to cell death by apoptosis m signal evoked by an IP3-linked agonist. Silencing MICU1 does not impair mitochondrial respiration or membrane potential but abolishes Ca2+ entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic Ca2+ transients and activation of matrix dehydrogenases.In spite of repeated efforts by different researchers, the molecular identity of the MCU has remained elusive. Among the early candidates proposed for the MCU were the uncoupling proteins UCP2/3 , but exp+ uptake . Recentl+ uptake  demonstr2+ uptake and sensitizes cells to apoptotic stimuli, and the employment of short interfering RNA (siRNA) silencing of MCU strongly reduced mitochondrial Ca2+ uptake. This reduction is specific for mitochondria , does not induce impairment of the electrochemical gradient or change in mitochondrial morphology and the induction of specific mutations at the level of the putative pore-forming region reduce the mitochondrial calcium uptake and blocks the channel activity of the protein : Ca2+ concentration; [Ca2+]c: cytosolic Ca2+ concentration; [Ca2+]m: mitochondrial Ca2+ concentration; CaM: calmodulin; CaMKII: calmodulin-dependent protein kinase II; DAG: diacylclycerol; DRP1: dynamin-related protein 1; ER: endoplasmic reticulum; FACL4: long-chain fatty acid-CoA ligase type 4; grp75: glucose-regulated protein 75; IMM: inner mitochondrial membrane; IMS: intermembrane space; IP3: inositol 1,4,5-trisphosphate; IP3R: inositol 1,4,5-trisphosphate receptor; Letm1: leucine zipper-EF-hand containing transmembrane protein 1; MAMs: mitochondria-associated membranes; MCU: mitochondrial Ca2+ uniporter; MICU1: mitochondrial calcium uptake 1; MFN-1/-2: mitofusin-1: -2; NCX: Na2+/Ca2+ exchanger; NE: nuclear envelope; OMM: outer mitochondrial membrane; p66Shc: 66-kDa isoform of the growth factor adapter shc; PKA: protein kinase A; PKC: protein kinase C; PKG: cGMP-dependent protein kinase; PLC: phospholipase C; PMCA: plasma membrane Ca2+ ATPase; PML: promyelocytic leukemia protein; PP2a: protein phosphatase 2a; PSS-1: phosphatidylserine synthase-1; PTP: permeability transition pore; ROCs: receptor operated Ca2+ channels; ROS: reactive oxygen species; SERCA: sarco-endoplasmic reticulum Ca2+ ATPase; Sig-1R: Sigma-1 receptor; SMOCs: second messenger operated Ca2+ channels; SPCA: secretory-pathway Ca2+-ATPase; SR: sarcoplasmic reticulum; UCP: uncoupling protein; VDAC: voltage-dependent anion channel; VOCs: voltage operated Ca2+ channels.ψThe authors declare that they have no competing interests.SP, JMS, CA, AB, MB, EDM, CG, SM, SM, FP, AR, JD, MRW and PP contributed to the preparation of the manuscript and approval of its final version.


Nitric oxide (NO) in guard cells plays a very important role as a secondary messenger during stomatal closure induced by effectors, including hormones. For example, exposure to abscisic acid (ABA) triggers a marked increase in NO of guard cells, well before stomatal closure. In guard cells of multiple species, like Arabidopsis, Vicia and pea, exposure to ABA or methyl jasmonate or even microbial elicitors  induces production of NO as well as reactive oxygen species (ROS). The role of NO in stomatal closure has been confirmed by using NO donors  and NO scavengers (like cPTIO) and inhibitors of NOS (L-NAME) or NR (tungstate). Two enzymes: a L-NAME-sensitive, nitric oxide synthase (NOS)-like enzyme and a tungstate-sensitive nitrate reductase (NR), can mediate ABA-induced NO rise in guard cells. However, the existence of true NOS in plant tissues and its role in guard cell NO-production are still a matter of intense debate. Guard cell signal transduction leading to stomatal closure involves the participation of several components, besides NO, such as cytosolic pH, ROS, free Ca2+, and phospholipids. Use of fluorescent dyes has revealed that the rise in NO of guard cells occurs after the increase in cytoplasmic pH and ROS. The rise in NO causes an elevation in cytosolic free Ca2+ and promotes the efflux of cations as well as anions from guard cells. Stomatal guard cells have become a model system to study the signaling cascade mechanisms in plants, particularly with NO as a dominant component. The interrelationships and interactions of NO with cytosolic pH, ROS, and free Ca2+ are quite complex and need further detailed examination. While assessing critically the available literature, the present review projects possible areas of further work related to NO-action in stomatal guard cells.The modulation of guard cell function is the basis of stomatal closure, essential for optimizing water use and CO Due to such dual role, the regulation of stomatal aperture, and yet maintenance of opening are essential to keep up the water balance and at the same time make CO2 available for photosynthesis. Stomatal opening and closure are mediated by the changes in turgor pressure of guard cells. Stomata open when guard cells are turgid and close when the guard cells are flaccid. As closed stomata restrict pathogen entry into leaves, stomata become key players also in defense response against several pathogens . Some of the plant hormones , salicylic acid, polyamines and even elicitors  cause stomatal closure, while auxins and cytokinins promote stomatal opening , cytosolic Ca2+, cytoplasmic pH, G-proteins, protein kinases as CDPK and MAPK, protein phosphatases, phospholipases and sphingolipids  or 1-aminocyclopropane-1-carboxylic acid (the precursor of ethylene) induced stomatal closure in a dose-dependent manner in V. faba guard cells   and diacylglycerol (DAG) regulate a wide range of growth and developmental processes including ABA signaling, programmed cell death and defense response  roots, has been proposed to be involved in the reduction of apoplastic nitrite to NO  aleurone layers further promoted by phenolic compounds  Reduction of nitrite to NO occurred under the acidic and highly reduced conditions, and such NO formation was not impaired by typical NOS inhibitors  Employing inhibitors or scavengers, (ii) Monitoring the components by suitable fluorescent dyes; and finally (iii) Validation by using mutants deficient in a given component of signal transduction chain Table . The inhA. thaliana guard cells  and by inhibitors like DPI, confirmed the strong association between ROS and NO  silenced plants of N. benthamiana prove that G-proteins facilitate NO production, before stomatal closure  and activates Cl− ion channels (increasing anion currents), and both events lead to stomatal closure (Garcia-Mata et al., A direct well-known effect of NO is it's up-regulation of Ca2+ and PA, and to some extent, with pH. For e.g., Ca2+ stimulates NO production and NO in turn can rise Ca2+ levels (Garcia-Mata and Lamattina, 2+ is extremely interesting and warrants detailed examination. Similarly, the production of NO and PA promote the levels of each other (Zhang et al., Besides their key roles during the rise in NO and subsequent effects, several signaling components tend to interact Table . The bes2+ mobilized from intracellular sources, could induce stomatal closure. Similarly, Beguerisse-Díaz et al. (The marked interactions between signaling components, involving NO, constitute a dynamic and complex regulatory network. Because of the complicated nature of signaling network and strong interactions among them, only a few attempts have been made to model these events. Li et al.  presentez et al.  proposedThe available literature amply demonstrates that NO is a common signaling component and a converging step for events initiated by ABA, MJ, or elicitors. The upstream components of NO, which rise during ABA action, are broadly understood. For example, ABA binds to PYR/PYL/RCAR proteins and then to PP2C forming a trimeric complex. Due to the non-availability of PP2C, protein kinases are activated to trigger several downstream elements (Cutler et al., 2S and CO, termed gasotransmitters (García-Mata and Lamattina, A range of highly interesting topics are emerging, studies on which can be quite useful. Among these are: modulation of NO by endogenous plant hormones, such as ABA (Lozano-Juste and León, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.