The pathogenetic relationship between the two disorders is unclear. Here, we characterised Ca2+ release in fibroblasts from type I GD and PD patients together with age-matched, asymptomatic carriers, all with the common N370S mutation in β-glucocerebrosidase. We show that endoplasmic reticulum (ER) Ca2+ release was potentiated in GD and PD patient fibroblasts but not in cells from asymptomatic carriers. ER Ca2+ signalling was also potentiated in fibroblasts from aged healthy subjects relative to younger individuals but not further increased in aged PD patient cells. Chemical or molecular inhibition of β-glucocerebrosidase in fibroblasts and a neuronal cell line did not affect ER Ca2+ signalling suggesting defects are independent of enzymatic activity loss. Conversely, lysosomal Ca2+ store content was reduced in PD fibroblasts and associated with age-dependent alterations in lysosomal morphology. Accelerated remodelling of Ca2+ stores by pathogenic GBA1 mutations may therefore feature in PD.Mutations in β-glucocerebrosidase (encoded by  The latter are activated by the second messenger cyclic ADP-ribose 2+ pumps (such as SERCA), exchangers and buffers act to temper Ca2+ increases in a highly regulated Ca2+ network 2+2+ signals by providing a “trigger” release of Ca2+ which is then amplified by Ca2+ channels on the ER, possibly through recently described membrane-contact sites between the two organelles 2+ stores are thus functionally and physically coupled similar to coupling between the ER Ca2+ stores and mitochondria/plasma membrane Changes in the concentration of cytosolic CaGBA1 which encodes the lysosomal enzyme β-glucocerebrosidase responsible for hydrolysis of glucocerebroside to glucose and ceramide. Type I GD (often associated with the N370S mutation) is traditionally considered non-neuronopathic whereas types II and III are associated with neurodegeneration. But both type I GD sufferers and carriers of GBA1 mutations are up to 20 times more likely to develop Parkinson disease (PD). Mutations in GBA1 are therefore one of the highest known risk factors for this neurodegenerative disorder GBA1 mutations mediate PD pathogenesis remains undefined. It may involve the unfolded protein response and ER stress as a consequence of mutant protein trapping or interactions with α-synuclein metabolism leading to Lewy body formation GBA1 carriers develop PD suggesting additional pathogenic mechanisms are involved.Gaucher disease (GD) is the most common of the lysosomal storage disorders 2+ signalling is established in a number of pathologies and has been implicated in both GD and PD as well as ageing, a major risk factor for neurodegenerative disease 2+ stores appear to be hypersensitive to ryanodine receptor activation in a pharmacological neuronal model of GD resulting in sensitisation to cell death 2+ stores are affected in the disease is not known, although lysosomal Ca2+ content is reduced in Niemann–Pick type C1 disease 2+ influx since the affected dopaminergic neurons of the substantia nigra pars compacta exhibit unusual pace-making activity associated with influx of Ca2+ through L-type voltage-sensitive Ca2+ channels 2+ are thought to impose metabolic stress on the mitochondria 2+ stores in PD is largely unexplored.De-regulated Ca2+ homeostasis in patient fibroblasts from GD and GBA1-linked PD sufferers. These data point to altered Ca2+ signalling in GBA1-disease and in ageing as possible contributors to PD pathology.In the present study, we identify age-dependent reciprocal changes in ER and lysosomal Ca22.1GBA1mut/mut GD and GBA1wt/mut PD, respectively. Cultures were also established from asymptomatic (ASX) non-manifesting N370S GBA1 carriers (GBA1wt/mut ASX). Thus, all lines had the same mutant allele to facilitate comparison. For control purposes, fibroblasts were acquired from age-matched, apparently healthy individuals (GBA1wt/wt). The fibroblasts were categorised according to age. The “young” cohort were obtained from individuals under the age of 60 whereas the “aged” cohort were derived from individuals over 70 years old (exact age denoted in subscripts). A summary of fibroblasts used in this study is provided in Primary fibroblast cultures were generated from skin biopsies as described in 2.22. In some experiments, cells were cultured for 7–11 days with the irreversible β-glucocerebrosidase inhibitor, conduritol B epoxide . Media, containing CBE, was replenished every 5 (fibroblasts) or 2–3 (SH-SY5Y) days. All cultures were analysed in parallel and fibroblast cultures differed by no more than 2 passages. Prior to experimentation, cells were plated onto glass coverslips (for Ca2+ imaging and immunocytochemistry) or directly into tissue culture flasks (for western blotting). For SH-SY5Y cells, glass coverslips were coated with 20 μg/mL poly-l-lysine.Fibroblasts were maintained in DMEM. SH-SY5Y cells were maintained in a 1:1 mixture of DMEM:Ham's F12 media and 1% (v/v) non-essential amino acids . SH-SY5Y cells with stable knock down of β-glucocerebrosidase were described in 2.32+ imaging was performed using the fluorescent Ca2+ indicator Fura-2 as described in 4, 156 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1.25 mM KH2PO4 and 10 mM glucose (pH 7.4). Cells were stimulated with thapsigargin (Merck), cADPR-AM, synthesised as described previously l-phenylalanine 2-naphthylamide, SantaCruz Biotech). Where indicated, extracellular Ca2+ was replaced with 1 mM EGTA.Ca2.4Western blotting was performed as described in 2.5d-glucopyranoside and 4-methylumbelliferyl-N-acetyl-glucosaminide, respectively as described in β-Glucocerebrosidase and β-hexosaminidase enzyme activities were measured using 4-methylumbelliferyl-β-2.62+ release was calculated by subtracting the basal Fura-2 fluorescence ratio prior to stimulation (60 s of data acquisition) from the peak response. The area under the curve was estimated by summating the increases in fluorescence ratio following stimulation over a given period. For thapsigargin, the periods were 750 s and 400 s for fibroblasts and SH-SY5Y cells, respectively. For GPN, the period was 400 s. These analyses were done at the individual cell level over the entire field of view . Data were derived from the number of passages stated in the figure legends, averaged over multiple fields of view  and presented as mean ± standard error of the mean. Statistical analyses were performed using Minitab 17. Independent-samples t-tests were applied and in the case of multiple comparisons, ANOVA analysis followed by a post hoc Tukey test. p < 0.05 was considered statistically significant.The magnitude of Ca33.12+ signalling, cytosolic Ca2+ levels were measured in age-segregated, passage-matched patient fibroblasts carrying the N370S mutation  in Ca2+-free medium. Thapsigargin-evoked Ca2+ release was significantly elevated in GD (GBA1mut/mut55GD) and PD (GBA1wt/mut55PD) cells when compared to cells from an age-matched (55 year old) healthy individual (GBA1wt/wt55)  2+ signals in a proportion of fibroblasts  A. These wt/wt55) B or the wt/wt55) . To furtroblasts C. The pedividual D. These GBA1 never develop neurological conditions 2+ release was therefore assessed in asymptomatic individuals with heterozygotic mutations in GBA1. Although thapsigargin-evoked Ca2+ release appeared more heterogeneous in GBA1wt/mut58ASX when compared with control GBA1wt/wt55 fibroblasts   individuals. Thapsigargin responses in the oldest fibroblasts examined (GBA1wt/wt82), closely resembled those from the younger GBA1wt/mut55PD fibroblasts (ER Cawt/wt78) . Howeverroblasts G. Such f3.3GBA1 are due to loss of enzymatic function or gain of toxic function is debated GBA1 disrupts ER Ca2+ release, the effects of thapsigargin were examined in fibroblasts from healthy controls by reducing the activity of β-glucocerebrosidase using pharmacological and molecular means. Fibroblasts were chronically treated with conduritol B epoxide , an inhibitor of β-glucocerebrosidase, which reduced β-glucocerebrosidase activity to 6 ± 0.03%. Thapsigargin-induced Ca2+ release after exposure to CBE was unchanged  but to a lesser extent than in GD and PD fibroblasts  which induces leak of low molecular weight solutes (<10 kDa) in fibroblasts upon hydrolysis by the lysosomal protease, cathepsin C 2+ increases, as reported previously GBA1wt/wt55, GBA1mut/mut55GD and GBA1wt/mut55PD fibroblast cultures  PARK8) for which evidence of an endolysosomal locus of action continues to accrue Reduced lysosomal CaGBA1 disposes to PD is unclear. Both loss- and gain-of function models have been proposed GBA1 carrier-PD patients GBA1. However, we cannot rule out that residual β-glucocerebrosidase activity  is sufficient to maintain homeostasis.How mutant GBA1 substantially increase risk of developing PD not all carriers succumb. These data strongly suggest that pathology is not a sole consequence of the mutant GBA1 allele. We show here that ER Ca2+ and lysosomal morphology defects identified in PD cells are not present in asymptomatic GBA1 carriers. Thus, defects correlate with pathology despite similar GBA1 status. It remains to be established whether these phenotypes are contributing causal factors for the disease or a consequence. Nevertheless, disruptions in fibroblast Ca2+ store homeostasis and lysosomal morphology described here might serve as biomarkers for GBA1-linked PD given incomplete penetrance in GBA1 carriers. Further work, however, is required using additional patient cell lines to validate our findings.PD has a complex aetiopathogenesis, which likely results from interplay between genetic and environmental cues. Although it is established that mutations in 2+ stores of potential relevance to the pathology of PD and GD.In summary, we identify age-dependent disturbances in both ER and lysosomal Ca2+ and Lysotracker imaging, immunocytochemistry and Western blotting. MEG and BSK performed enzyme activity measurements, JM and MJWC provided the SH-SY5Y cells. MSB and AM obtained the fibroblasts. DB and GCC synthesised the cADPR-AM. MRD, AHS and SP conceived the study. BSK and SP wrote the paper with input from all authors.BSK performed the CaUCL (to BSK), Parkinson's UK grants K-1107, K-1412 and H-1202 , Wellcome Trust/MRC Joint Call in Neurodegeneration award (WT089698 to AHS and MRD), an MRC CoEN award (to AHS). AHS is a NIHR Senior Investigator and is supported by the NIHR UCLH BRC.This work was supported by an IMPACT studentship from


In the present study, we tested the hypothesis that cigarette smoke enhances the expression and function of Ca2+ regulatory proteins leading to increased store operated Ca2+ entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca2+]i) responses and expression of relevant proteins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca2+]i responses to agonist and SOCE: effects mediated by increased expression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of patients with long-term smoking history, TRPC3 and CD38 expression were significantly increased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregulation of PCNA and Cyclin E. These results support a critical role for Ca2+ regulatory proteins and enhanced SOCE to alter airway structure and function in smoking-related airway disease.Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhancing agonist-induced intracellular Ca Cigarette smoke contributes to or exacerbates airway diseases, such as asthma and COPD, where airway hyperresponsiveness (AHR) and airway smooth muscle (ASM) proliferation are key features. Importantly, even brief exposure to secondhand cigarette smoke affects lung function and inflammatory cytokine synthesis in humans . However2+ concentration ([Ca2+]i) regulate contraction and cell proliferation, and enhancement of both may contribute to the key features of asthma or COPD. Animal data indicates that sidestream cigarette smoke exposure increases ASM responsiveness to ACh and high K+ depolarization in rats: an effect that may be due to augmented RhoA-mediated Ca2+ sensitization [2+]i responses of hASM to agonist [2+]I, mediated by agonist  triggered by inositol 1,4,5-triphosphate (IP3) or activation of ryanodine receptor (RyR) channels possibly by cADPR via CD38 activation i responses by enhanced Ca2+ regulatory protein expression (particularly SOCE), and furthermore increases hASM proliferation.In ASM, changes in intracellular Ca agonist . In thisistamine ) stimulativation ,10,11 orDMEM, antibiotic/antimycotic mixture and fura-2 were obtained from Invitrogen, Carlsbad, CA. All fine chemicals were purchased from Sigma-Aldrich, St. Louis, MO. Cyclopiazonic acid was purchased from Calbiochem, La Jolla, CA.The techniques for isolation of hASM cells have been previously described ,13. Brierd to 6th generation bronchi were dissected, immersed in ice-cold Hanks balanced salt solution  aerated with 100% O2 and the epithelium was removed by abrasion. The ASM cells were isolated using enzymatic dissocation as previously described i measurements in hASM using fluorescent indicators have also been previously described [2+]i calculated from the 340/380 ratios based on an in vitro calibration i levels recorded. Extracellular Ca2+ was then removed using nominally Ca2+-free HBSS, followed by SR Ca2+ depletion using 10 μM cyclopiazonic acid , and rapid reintroduction of extracellular Ca2+ in the continued presence of CPA to trigger SOCE.The techniques for SOCE measurements in ASM cells have also been previously described ,19. ASM hASM cells were pre-exposed to vehicle or 3 mM N-acetylcysteine and then to 1% CSE for 4h. Cells were then loaded with 5μM of dichlorodihydrofluorescein diacetate  for 30 min, washed, and immediately analyzed for fluorescence intensity using a FlexStation3 microplate reader (Molecular Devices).hASM cells were treated with 1% CSE for 24h in the presence or absence of NAC in 96-well plates. The supernatants were collected and analyzed for secreted TNF-α using ELISA kits  according to the manufacturer's instructions.ASM cells were grown to approximately 50% confluence in 96-well culture plates, and were exposed to serum- and antibiotic-free medium for 24 h. Proliferation of ASM cells over 48h was assayed using the CyQuant NF dye (Invitrogen) as described previously . Dye calStatistically significant differences between cell groups were analyzed using one- or two-way ANOVA as appropriate . Multiple comparison tests (Tukey or Scheffe) were employed as appropriate. Experiments involved hASM cells obtained from at least 6 different patients. A value of P< 0.05 was considered significant. All results are expressed as mean ± SEM.Western blot analysis of lysates from epithelium-denuded whole hASM tissue of human bronchi showed significant increases in protein expression  of both TRPC3  and CD382+]i regulatory proteins TRPC3, CD38, STIM1, and Orai1, compared to vehicle treated control cells . Acute exposure to 10 μM bradykinin  produced a typical transient [Ca2+]i elevation followed by a lower plateau . This effect was significantly reduced in cells transfected with siRNAs against either TRPC3 , CD38 F, STIM1 , or OraihASM cell proliferation was determined using fluorescent CyQuant assay. Cells treated with 1% or 2% CSE showed significant proliferation compared to control P<0.05; . This pr2O2 (5mM) in control cells  expression [2+]i levels. Overall, these data support a role for [Ca2+]I in airway diseases such as asthma and COPD that are linked to cigarette smoke exposure. However, the role of cigarette smoke per se has not been established.Recently, several studies suggest that Caic force , as wellogenesis  and thoupression  which ma2+]i regulation in hASM cells. Specifically, we find that a central target of cigarette smoke exposure is enhanced SOCE. In this regard, first, we found increased expression of TRPC3 and CD38 in hASM tissue of bronchial airways obtained from humans with significant smoking history. The ectoenzyme CD38, the second messenger cyclic ADP ribose (cADPR), and ryanodine receptor channels play an important role in smooth muscle Ca2+ homeostasis. Previously, we i responses to cholinergic agonists in airway smooth muscle. The importance of CD38 for airway responsiveness has been shown in CD38 KO mice where methacholine responsiveness is significantly attenuated compared with the wild-type control mice i responses by inducing sarcoplasmic reticulum Ca2+ release from ryanodine receptor channels, such emptying of the SR would likely promote SR refilling via mechanisms such as SOCE. In addition, CD38 may also increase SOCE via mutual co-localization within caveolae i mobilization pathways and proliferation pathways. This finding is consistent with many studies which have demonstrated regulation of Ca2+ influx and Ca2+ currents by thiol modifying agents. Recently, others i. Thus, it is likely that increased SOCE would elicit AHR in clinical states of COPD and asthma. For example, YM-58483/BTP-2, a SOCE inhibitor significantly suppresses AHR in a guinea pig ovalbumin-induced bronchoconstriction model i regulation, including enhanced basal [Ca2+]i, agonist-elicited [Ca2+]i, and SOCE. These findings correlate with CSE-elicited increase in expression of CD38, TRPC3, Orai1 and STIM1. siRNAs directed against these calcium regulation proteins attenuated the CSE effect on [Ca2+]i mobilization. Moreover, CSE increases hASM SOCE in an ROS-dependent manner. CSE also increases hASM proliferation and autocrine TNFα secretion. Taken together, these calcium regulating proteins may be targets to attenuate AHR and airway smooth muscle proliferation in airway disorders such as asthma and COPD. We propose a model in which CSE, via ROS, enhances ASM SR Ca2+ release and activation of SOCE, as well as proliferation and cytokine synthesis leading to a phenotype of increased AHR.In summary, we have demonstrated that CSE exposure of hASM cells alters [Ca


Bovine and human plasma albumin were used. We observed that low albumin concentration decreases [Ca2+]c, while higher concentration, induces increase in [Ca2+]c. It was shown that increase in [Ca2+]c was mediated by inositol 1,4,5-trisphosphate and released from internal stores. Increase in [Ca2+]c was reduced to 19% by blocking the transforming growth factor-beta (TGF-βR) receptor. Albumin induces DNA synthesis in a dose-response manner. Finally, induction of DNA synthesis can be partially blocked by heparin and block of TGF-β; however, the combination of both incompletely inhibits DNA synthesis. Therefore, results suggest that mechanisms other than Ca2+ signals and TGF-β receptor activation might induce DNA synthesis in a lesser degree. These results may be important to further understand the mechanisms involved in the transition from normal to focal epileptic brain.So far, a little is known about transition from normal to focal epileptic brain, although disruption in blood-brain barrier and albumin had recently involved. The main objective of this work is to characterize the response of cultured human astrocytes to plasma albumin, including induction of DNA synthesis. Cortical tissue was obtained from 9 patients operated from temporal lobe epilepsy. Astrocytes were cultured for 3-4 weeks and cytosolic calcium concentration ([Ca Epilepsy is a major neurological disorder, affecting about 0.5–2% of the global population and approximately 20–30% of these patients are resistant to drug therapy –3. DespiβR) c through an Alb receptor, while higher p-Alb concentrations increased the [Ca2+]c in a dose-dependent manner, eliciting calcium-waves that travelled by gap-junctions, probably involving inositol 1,4,5-trisphosphate , could activate several types of cells, including xenopus oocytes, PC12, and fibroblasts microglia , 16–19. d slices . Indeed,te . These te  and operated to control their seizures. The patient's consent was obtained in all cases according to the Declaration of Helsinki and all protocols were approved by the Institutional Ethical Committee . A mass of brain tissue (approximately 3 g) containing grey and white mater, obtained from a nonspiking region of temporal lateral cortex, was obtained from 9 patients  suffering from drug-resistant temporal lobe epilepsy, all with a well-documented medical history.99mTc-HmPAO, magnetic resonance imaging (MRI) 1.5 T, and video-electroencephalography (v-EEG) using 19 scalp electrodes according to the international 10–20 system. In some patients, six-contact platinum foramen ovale electrodes (FO), with 1 cm center-to-center spacing , were inserted bilaterally under general anesthesia, as previously published c) was measured at room temperature from several regions of interest . Cells R) and basal values (R0) were subtracted (ΔR = R − R0 c in cortical rat astrocytes in a dose-dependent manner [2+]c decreased to low dose of BPA (0.1 mg/mL) but the maximum amplitude for [Ca2+]c increased with BPA concentration between 1 mg/mL and 80 mg/mL.It has been shown that bovine plasma albumin (BPA) induces oscillations in the c, as we can observe from Consecutive applications of BPA failed to induce any significant decrease in the maximum amplitude in [Ca2+]c, decreasing at low BPA concentration and increasing at higher concentrations. These responses do not show adaptation.Taken together, these results show that plasma albumin obtained from patients, such as commercially available BPA, induced modifications in c increase even in a calcium free-medium containing 0 Ca2+ + 10−3 mol/L EGTA. However, a second trial failed to induce any change in [Ca2+]c, likely because the internal stores were emptied by the previous pulse.In fact, as we can observe from −6 mol/L   did not modify the maximum peak of cytosolic calcium concentration   at a concentration high enough to block the receptor , decreased a 19.0% .The TGF-6 mol/L, , 34). As2+]c in human astrocytes are due to release from internal stores and are mediated through IP3 as second messenger, but not by calcium-induced-calcium release. Probably, heparin acts extracellularly, blocking the interaction between Alb and its receptor. The rise in [Ca2+]c, nevertheless, depended in a little degree (<20%) on TGF-β receptor. Therefore, we can rule out that the main effect of BPA onto [Ca2+]c is through the TGF-β receptor.These results suggest that increases in [Ca−4 mol/L) and albumin (20 mg/mL). Both glutamate and Alb are able to release calcium from internal stores. As we can observe from 2+]c increase. Then, we perfused heparin (1 mg/mL) during 24 hours. Eight minutes after heparin, glutamate was able to induce a response similar to the control one, but albumin failed to modify the [Ca2+]c. At 12 hours and 23 hours and 45 minutes in heparin, we obtained the same result. Nevertheless, at 24 hours and 10 minutes, that is, 10 minutes after having washed out heparin, both agents induced a [Ca2+]c increase similar to the control , a dose-response relationship between DNA synthesis and BPA concentration was observed .2+]c and DNA synthesis induced by BPA. As we can observe from 2+]c and DNA synthesis with BPA are rather different. In fact, the half maximal effective concentration (EC50) of albumin for [Ca2+]c is 14.5 mg/mL and 10.3 mg/mL for DNA synthesis. Furthermore, both curves are clearly different.We assessed the relationship between increases in c increase are involved in DNA synthesis.So, both mechanisms are responsible approximately for the same amount of DNA synthesis induced by 20 mg/mL BPA, but is there any other mechanisms? To answer this question, astrocytes were incubated with BPA (20 mg/mL) + heparin (1 mg/mL) + LY-364947  it seems unlikely that this effect would be due to an intracellular mechanism and, on other hand, there is no notice to authors about membrane mechanisms able to cross the membrane for a so big molecule. In fact, heparin acting in a selective way through IP3 receptors has to be introduced inside the cell [We have found that heparin can block the rise in [Cathe cell . PrelimiIn summary, it would be extremely important to assess what of the mechanisms effectively applies in humans, in order to design pharmacological strategies. On the other hand, it is conceivable that more than one mechanism acts synergistically.Any case, there is a real and exciting possibility to envisage a pathway running from the normal human brain to focal epilepsy after BBB disruption induced by a local insult . This po


YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a Kd value of approximately 0.79 μM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation.Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor,  The oxidative metabolism of campesterol in plant tissues leads to the production of a group of bioactive polyhydroxylated steroids, collectively called brassinosteroids (BRs). BRs are important plant hormones that play critical roles in regulating broad aspects of plant growth and development . BRs actArabidopsis thaliana (hereafter Arabidopsis) . Wi. WiYCZ-11 (DWF4) . The sec1 (DWF4) . To idenYCZ-18 activity in BR biosynthesis inhibition, we cloned the genes of CYP90C1 and CYP90D1 into the pCold-GST expression vector. Using an E. coli expression system, we attempted to purify these recombinant proteins. Despite substantial efforts to optimize the expression conditions of these genes, we could not obtain the purified recombinant CYP90C1 proteins but had poor expression in soluble fractions. However, we successfully expressed and purified CYP90D1. Thus, we determined the binding affinity of YCZ-18 to CYP90D1.To characterize the binding target responsible for YCZ-18 to CYP90D1 was determined by measuring optical difference spectra upon the addition of YCZ-18 to recombinant CYP90D1. CYP90D1 exhibited a Soret absorption peak at 421 nm, which is characteristic of low-spin P450s  versus the concentration of YCZ-18 -deficient mutants.It has been reported that e plants : the mut mutants . These rcontrols . AlthougYCZ-18 between BR biosynthesis and GA biosynthesis, we have previously shown that the dwarf phenotype of Arabidopsis seedlings induced with YCZ-18 in the dark could be rescued by the application of BR but not by GA . Da. Da26]. YCZ-18 exhibits potent biological activity upon inducing BR-deficient-like phenotypes in Arabidopsis. In addition, YCZ-18 and brassinozole target different enzymes. Therefore, further experiments with YCZ-18 to explore the biological processes related to BRs may provide new insights into the detailed mechanism of BR biosynthesis and its regulation.Until now, brassinazole has been widely used for BR research, both for the elucidation of BR functions in plant physiology and for the identification of BR signal transduction components . The useYCZ-18 is a potent inhibitor of BR biosynthesis with a wide range of applicability for altering BR levels in Arabidopsis. (1) Under three culture methods, YCZ-18 induces an Arabidopsis phenotype that is similar to that of BR-deficient mutants. (2) YCZ-18 regulates the expression of BR-responsive genes, which was shown in both BR-deficient mutants and brassinazole-treated plants. (3) Quantitative analysis on BR levels in YCZ-18 treated Arabidopsis indicated that YCZ-18 significantly decreased the endogenous levels of BRs. (4) YCZ-18 shows high binding affinity to recombinant CYP90D1 protein, suggesting that one of the primary sites of action of YCZ-18 is the C-23 hydroxylation of the side chain of BRs. Combining these results with our previously reported observations [YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis.We present three independent lines of evidence indicating that rvations , we conc


Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters  were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.The type 1 ryanodine receptor (RyR1) is a Ca Cells were treated initially with 10 mM caffeine to deplete Ca2+ and then equilibrated in PSS for 10 min. Fluorescence images were acquired at >420 nm using an inverted microscope equipped with a cooled CCD camera at a rate of one frame every 2 s. Excitation wavelengths were 345 nm and 380 nm. Ca2+ imaging experiments were conducted at RT. Image analysis was carried out using IPLab software . ROIs corresponding to individual cells were selected and the average fluorescence intensity (F) of each ROI minus the background intensity was calculated for each frame. We used the F345/F380 ratio  to estimate [Ca2+]i, as described previously i and R-CEPIA1er [2+ ([Ca2+]ER). Doxycycline was added to the culture medium at the time of transfection. At 24–30 h after transfection, cells were washed with the HEPES-buffered Krebs solution and placed on the stage of the microscope. G-GECO1.1 and R-CEPIA1er were excited by 488 nm and 568 nm laser light, respectively, and fluorescence images at 525 and 620 nm were simultaneously captured, side-by-side on the same camera, using the W-view system . At the end of each experiment, the high Ca2+ Krebs solution was applied to obtain the Fmax of the Ca2+ indicators in individual cells. The fraction of the Ca2+-independent basal fluorescence signal was determined separately in the presence of 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) plus 20 μM ionomycin and subtracted from the total fluorescence signal. [Ca2+]ER was calculated using parameters obtained by an in situ titration  [For simultaneous recording of ER and cytoplasmic Ca-GECO1.1  for [Ca2CEPIA1er  for ER C3H]ryanodine for 5 h at 25°C in a 100 μl solution containing 0.17 M NaCl, 20 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), pH 6.8, 2 mM dithiothreitol, 1 mM AMP and various concentrations of free Ca2+ buffered with 10 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). For [3H]ryanodine binding experiments at 37°C, the incubation period was shortened to 3 h. Ca2+ concentrations were calculated using MaxChelator (http://maxchelator.stanford.edu) [3H]ryanodine was separated by filtering through polyethyleneimine-treated Whatman GF/B glass filters. Nonspecific binding was determined in the presence of 20 μM unlabeled ryanodine. The [3H]ryanodine binding data (B) were normalized by the maximum number of functional channels (Bmax), which was separately determined by Scatchard plot analysis using varied concentrations (3–20 nM) of [3H]ryanodine in a high-salt buffer containing 1 M NaCl ryanodine binding, the data were fitted to the following equation:A is the activity at the specified Ca2+, Amax is the gain that determines the maximal attainable activity, and fA and fI are fractions of the activating Ca2+ site (A-site) and inactivating Ca2+ site (I-site) occupied by Ca2+, respectively [fA and fI at the specified Ca2+ concentration ([Ca2+]) are expressed as:KA and KI are dissociation constants, and nA and nI are Hill coefficients for Ca2+ of A- and I-sites, respectively. From the shape of the Ca2+-dependent curves for WT and mutant channels, we assumed that the Hill coefficients are not largely dissimilar between the WT and mutant channels. We therefore used fixed values for nA (1.2) and nI (1.5) for WT and all the mutants at both 25 and 37°C. These values were chosen so as to maximize the sum of R2 values for curve fitting of WT and all mutant channels. Curve fitting was performed using the Prism 6 software . The curves for fA, 1–fI, and A are shown in To obtain the parameters of Caectively . fA and2+]i, A at pCa 7 for each mutant was calculated by eqs KA, KI and Amax).For estimation of ryanodine binding at resting i) level was higher in some disease-associated mutations than in WT   .2+ transients and corresponding increased resting [Ca2+]i suggest a reduction of Ca2+ in the ER Ca2+ store ([Ca2+]ER). To test this possibility, we simultaneously measured [Ca2+]i and [Ca2+]ER in HEK293 cells . After removal of the caffeine, [Ca2+]ER gradually recovered and achieved a steady-state level. Resting [Ca2+]ER was measured as F/Fmax, in which fluorescence intensity of the steady-state level (F) was normalized by the maximum fluorescence intensity (Fmax) of the indicator, which was determined by treating cells with high Ca2+ plus ionomycin at the end of experiments (filled bars). R164L and Y523S showed markedly lower [Ca2+]ER than WT, whereas R615C exhibited a [Ca2+]ER level similar to that of WT  (2+]i (R2 = 0.82)   and rest = 0.82) , indicat2+ oscillations in HEK cells expressing WT RyR1 and that a MH-associated mutation (R615C) reduced a threshold level of luminal Ca2+ for the spontaneous Ca2+ release, which is referred to as store-overload-induced Ca2+ release (SOICR) level [2+]ER  (2+ oscillations (16 out of 36 cells) with higher frequency than that of WT  level . We therl [2+]ER . Unlike 2 cells) . R615C aat of WT . These r2+ oscillations; instead, they exhibited the constant [Ca2+]ER level during the observations  from 2 mM to 5 mM did not induce Ca2+ oscillations  and dissociation constants for activating Ca2+ (KA) and inactivating Ca2+ (KI) (Amax increased (1.6–10.8-fold) for all the mutations relative to the WT channel. The KA greatly decreased (0.08–0.18-fold) in two MH/CCD mutations (R164C and Y523S) and three MH mutations  when compared with the results of the WT, whereas moderate decreases (0.31–0.57-fold) in KA were observed for the other MH mutations, except for R615C. The KI increased (1.5–2.8-fold) in four mutants  when compared with the results of the WT, but no significant change in the other mutants was observed.The results of the ryanodine binding with the [Ca2+]ER of HEK293 cells, which reflects Ca2+ leakage via Ca2+ release. A good inverse correlation between the two was observed  . It is h2+-dependent [3H]ryanodine binding of WT and mutant channels. Since the rate of [3H]ryanodine binding at 37°C was faster than at 25°C i estimated from the obtained parameters was highly variable among mutations, (3) the channel activity at resting [Ca2+]i correlated well with [Ca2+]ER, and (4) temperature dependences of cellular Ca2+ homeostasis and activity profiles were similar between mutations. These results suggest that the disease-associated mutations in the N-terminal region divergently affect the activity profiles of the RyR1 channel to cause different disease phenotypes.In this study, we generated HEK293 cells stably expressing 11 disease-associated mutations at 7 different positions in the N-terminal region. Pathological phenotype of the mutations were examined by cellular Ca2+ transients is a useful measure to detect MH. Caffeine makes RyR1 sensitive to activating Ca2+ ER  or after treatment with Ca2+-pump inhibitors  [2+]ER and [Ca2+]i. Therefore, we directly measured [Ca2+]ER using a novel genetically-encoded fluorescent Ca2+ indicator, R-CEPIA1er [2+]ER and [3H]ryanodine binding at resting Ca2+  , 15, 31.CEPIA1er  Fig 2)..2+ homeoing Ca2+ . [Ca2+]E2+]i is increased in cells expressing mutant channels  pathway is accelerated in muscle cells from knock-in mice carrying R163C [2+]i in HEK cell expressing RyR1 mutants.We found that resting i and a depletion in [Ca2+]ER. This is consistent with previous studies with heterologous expression systems i and a decrease in [Ca2+]ER. This is the first report to examine caffeine sensitivity and cellular [Ca2+] measurements for these mutations, although sensitivity to 4-chloro-m-cresol has been performed for R402C with HEK293 cells [2+.R402C and R402H showed enhanced caffeine sensitivity with a moderate increase in [Ca93 cells . These m2+]ER and an increase in [Ca2+]i. These properties are consistent with the previous findings with heterologous expression systems [2+. We found that Y523S exhibited a more severe phenotype than R164C  exhibited a high channel activity and severe depletion of [Ca2+]ER, which is comparable to that seen for MH/CCD mutations (R164C and Y523S) (The combination of [d Y523S) . It is e2+ greatly contributes to Ca2+ leakage from Ca2+ stores in MH/CCD mutations . Thus, Most human MH and CCD patients are heterozygous for WT and mutant alleles . This is2+ homeostasis measurements and determination of activity profiles, provides useful information about phenotype and disease severity caused by individual mutations. Construction of a database for this information should aid diagnosis and predict prognosis of the diseases.Among the reported MH and MH/CCD mutations, many have been discovered in one or small numbers of families . In suchS1 FigA. Ca2+-dependent channel activity (bold line) and fractions of A-site occupied by Ca2+  and of I-site free from Ca2+  were simulated by eqs Amax = 0.05, KA = 10 μM, nA = 1.2, KI = 0.15 mM, and nI = 1.5. B. Effect of individual parameters on channel activity. Parameters for the original curve (black line) are the same as those in A. Channel activity was simulated with either 4-fold decreased KA , 4-fold increased Amax , or 4-fold increased KI . C. The activity profiles of WT and mutant channels in B. The three parameters, Amax, 1/KA and KI, were plotted on the radar charts relative to WT. 1/KA was used as the parameter for activating Ca2+ dissociation constants, in which a larger value represents higher sensitivity.(PDF)Click here for additional data file.S2 FigWestern blot analysis of RyR1 in microsomes from HEK293 cells expressing WT or disease-associated mutants. The mutant RyR1s showed gel mobility similar to that of the WT. Calnexin was used as a loading control.(PDF)Click here for additional data file.S1 Tablen = 3–5). Numbers in parentheses indicate fold change of the parameters relative to WT. *P < 0.05 vs. WT.Data are mean ± SE ((DOC)Click here for additional data file.


Subcellular localization and physiological analyses in neurons reveal that Eag1 may participate in Ca2+‐signaling processes in the synapse. Here, we searched for rat Eag1 (rEag1)‐binding proteins that may contribute to Ca2+ regulation of the K+ channel. Yeast two‐hybrid screening identified centrin 4, a member of the centrin family of Ca2+‐binding proteins. GST pull‐down and immunoprecipitation assays in brain and retina lysates confirm the interaction of centrin with rEag1 in neurons. Centrin 4 binds to rEag1 in the absence of Ca2+. Raising Ca2+ concentration enhances the association efficiency of centrin 4 and rEag1, and is required for the suppression of rEag1 currents by centrin 4. Altogether, our data suggest that centrin 4 is a novel binding partner that may contribute to Ca2+ regulation of rEag1 in neurons.Eag1 is neuron‐specific K CADcarboxyl assembly domainCaMcalmodulinCetncentrinCNBHDcyclic nucleotide‐binding homology domainDMSOdimethyl sulfoxideEagether‐à‐go‐goGFPgreen fluorescent proteinGSTS‐transferaseglutathione HEKhuman embryonic kidneyPMSFphenylmethylsulfonyl fluoridePSDpostsynaptic densityrEagrat EagSPMsynaptosomal+) channel family, a member of the voltage‐gated K+ (Kv) channel superfamily, comprises three subfamilies: Eag (Kv10), Erg (Kv11), and Elk (Kv12) + channel that encompasses two isoforms, Eag1 (Kv10.1) and Eag2 (Kv10.2), both of which are expressed in a wide variety of different regions in the brain + channel at presynaptic axon terminals and postsynaptic dendritic spines The ether‐à‐go‐go potassium  influxes play a pivotal role in regulating neurotransmitter release and synaptic signaling 2+ signaling processes in the synapse. In line with this notion, Eag1 appears to modulate presynaptic Ca2+ influx and neurotransmitter release during high‐frequency neuronal firing 2+/CaM‐dependent protein kinase II, two Ca2+‐binding/activated molecules widely implicated in synaptic signaling mechanisms Drosophila Eag channels, respectively 2+‐dependent manner. Furthermore, the acidic Ca2+‐binding protein S100B seems to interact with human Eag1 channel via a similar Ca2+‐dependent mechanism Activity‐dependent calcium  was employed to screen a rat brain cDNA library as previously reported The cDNA clones used for transfection include rEag1 in pcDNA3  or pEGFP , rEag2 in pEGFP, rat centrin 3 and 4 in pcDNA3.1‐Myc, rat calmodulin in pcDNA3.1‐Myc, and human centrin 2 in pEGFP  or p‐CMV‐Tag3 . Transient transfection of human embryonic kidney (HEK) 293T cells was performed as previously described m Tris–HCl, pH 7.4, 150 mm NaCl, 10 mm Na2HPO4, 1% Triton X‐100, 0.5% Na‐deoxycholate, 0.1% SDS, 1 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride (PMSF)]. Where indicated, 2 mm EGTA or 2 mm CaCl2 was added in lieu of EDTA. Solubilized lysates were incubated for 16 h at 4 °C with protein A and G sepharose beads  precoated with the indicated rabbit and mouse antibodies, respectively. Protein samples were separated on 7.5–15% SDS/PAGE, transferred to nitrocellulose membranes, followed by immunoblotting. For detecting centrin signal, membranes were fixed with 0.2% glutaraldehyde prior to primary antibody incubation. Input represents 5% of the total protein used for immunoprecipitation. The antibodies include mouse anti‐β‐actin , mouse anti‐centrin , rabbit anti‐rEag1 , rabbit anti‐GFP , mouse anti‐GST (Sigma), mouse IgG (Sigma), mouse anti‐Myc (clone 9E10), mouse anti‐PSD‐95 , and mouse anti‐synaptophysin. Results shown are representative of at least three independent experiments.Immunoprecipitation and immunoblotting were performed as described previously S‐transferase (GST) fusion proteins were produced and purified by following the manufacturer's instruction (Stratagene) as previously reported E. coli strain BL21. The lysates of IPTG‐induced bacteria were incubated with glutathione‐agarose beads (Sigma) that bind GST fusion proteins. GST fusion protein‐coated beads (4–8 μg) were subsequently incubated overnight with appropriate HEK293T cell lysates at 4 °C, and eluted by boiling for 5 min in the Laemmli sample buffer.Glutathione m) 320 sucrose, 1 NaHCO3, 0.5 CaCl2, 0.1 PMSF] containing a cocktail of protease inhibitors (Roche) and centrifuged at 1400 g to remove nuclei and other large debris (P1). The S1 fraction was subject to centrifugation at 13 800 g to obtain a crude synaptosome fraction (P2). The pellet was resuspended in buffer H2  and layered onto the top of the discontinuous sucrose density gradient by using 0.85, 1.0, and 1.2 m sucrose layers. The gradient was centrifuged at 65 000 g for 2 h in a Beckman Instruments SW‐28 rotor. The synaptosomal (SPM) fraction was recovered from the 1.0–1.2 m sucrose interface, followed by extraction with ice‐cold 0.5% Triton X‐100/50 mm Tris–HCl (pH 7.9) for 15 min and centrifugation at 32 000 g for 45 min to obtain the postsynaptic density (PSD) I pellet. The pellet was resuspended and further extracted with 0.5% Triton X‐100/50 mm Tris–HCl (pH 7.9), followed by centrifugation at 200 000 g for 45 min to obtain the PSD II pellet. Protein concentration was determined by the BCA protein assay kit .Rat brain homogenates were prepared as described previously m NaCl, 20 mm Tris, pH7.4, 0.1% sodium dodecyl sulfate, 1% Triton X‐100, 0.25% deoxycholate, 1 mm EDTA) with protease inhibitors and 1 mm PMSF. Retinas were homogenized by dounce homogenizer and centrifuged at 15 000 g for 15 min at 4 °C. The supernatants were used for GST pull‐down experiments.For retinal lysate preparation, rat retinas were dissected and collected in the lysis buffer (150 mm) 140 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, pH 7.2. External bath solution comprised (in mm) 140 NaCl, 5 KCl, 1 CaCl2, and 10 HEPES, pH 7.2. Conventional two‐electrode voltage‐clamp recordings were performed in Xenopus oocytes as described previously in vitro from linearized cDNA, and 41.4 nl of cRNA (0.1 μg·μL−1) was injected into each oocyte. Two days after cRNA injection, oocytes were functionally assayed in Ringer solution . Niflumic acid (0.5 mm) was added to minimize the contribution of endogenous Ca2+‐activated Cl− currents. A23187 was dissolved in dimethyl sulfoxide (DMSO) as stock solution for storage at −20 °C, which was diluted to 1 μM in the Ringer solution on the day of experiment. Alternatively, identical amount of DMSO was added to the Ringer solution as the control solution. Voltage‐clamp protocols were applied with the pclamp 8.2/9.0 software . Data were acquired with OC‐725C oocyte clamp (Warner) (for Xenopus oocytes) or Axopatch 200B amplifier (Molecular Devices) (for HEK293T cells), followed by digitization at 10 kHz with the Digidata 1320A/1322A system (Molecular Devices). Data were filtered at 1 kHz and passive membrane properties were compensated with the ‐P/4 leak subtraction method by using the pclamp 8.2/9.0 software. All recordings were performed at room temperature (20–22 °C).Whole‐cell patch clamp in HEK293T cells was performed at 24–48 h post‐transfection as previously reported 2+‐binding proteins, we carried out yeast two‐hybrid screening of a rat brain cDNA library by using the intracellular carboxyl‐terminal region of the K+ channel as the bait. Among the 102 positive hits, 5 clones are CaM, and 7 clones correspond to centrin 4, which is an isoform of the centrin protein family that, like CaM, belongs to the superfamily of EF‐hand Ca2+‐binding proteins To search for rEag1‐interacting CaTo confirm the association of centrin 4 with rEag1, we performed immunoprecipitation experiments by transiently coexpressing Myc‐tagged centrin 4 with rEag1 in HEK293T cells. As illustrated in Fig. The centrin protein family comprises four isoforms: centrin 1‐4  within rEag1, we generated GST fusion proteins encoding either the amino‐terminal (GST‐N207) or the carboxyl‐terminal (GST‐C0) region of the KrEag1 contains a long intracellular carboxyl‐terminal region spanning over 470 amino acids that can be divided into three structural domains: the carboxyl linker (C‐linker), the cyclic nucleotide‐binding homology domain (CNBHD), and the post‐CNBHD region Fig. A. To fur2+‐binding motifs that can cooperatively bind four Ca2+2+‐binding homology motifs, only the fourth EF‐hand motif in centrin 4 may effectively bind Ca2+2+‐dependent manner 2+. As shown in the left panel of Fig. 2+ concentration change was verified by the prominent electrophoretic mobility shift between Ca2+‐free and Ca2+‐bound CaM; as expected, coimmunoprecipitation of rEag1 with Myc‐CaM was only observed when 2 mm CaCl2 was present. The Ca2+‐induced alteration in electrophoretic mobility for Myc‐centrin 4, however, is markedly smaller . We, therefore, attempted to increase intracellular Ca2+ concentration by employing the Ca2+‐ionophore A23187 that was dissolved in the extracellular Ringer solution containing 1.8 mm CaCl2. As demonstrated in Fig. Xenopus oocytes. By contrast, in the presence of centrin 4 or CaM, application of 1 μM A23187, but not the DMSO control solution, leads to a notable reduction in rEag1 K+ current amplitude, indicating that both centrin 4 and CaM inhibit the function of rEag1 in a Ca2+‐dependent manner. Interestingly, in the presence of enhanced intracellular Ca2+ concentration, coexpression with centrin 4 or CaM also notably slows the activation kinetics of rEag1 currents  fails to discernibly affect the functional expression of rEag1 in HEK293T cells  and two in the carboxyl‐terminal (amino acids 674–683 and 711–721) regions 2+‐free centrin 4 may associate with rEag1. Similar lack of Ca2+ requirement has also been reported for CaM binding to small‐conductance Ca2+‐activated K+ (SK) channels 2+ (CaV) channels + (Kv7) channels 2+‐dependent self‐association that resembles actin polymerization 2+, consistent with the idea that centrin 4 may go through certain Ca2+‐dependent conformational change that in turn augments its association with rEag1. Indeed, using the Ca2+‐ionophore A23187, we demonstrate that, similar to SK, CaV, and Kv7 channels, Ca2+ is required for the modulation of rEag1 channel function by centrin 4. In other words, despite their striking difference in Ca2+ requirement for physical association with the K+ channel, both centrin 4 and CaM suppress Eag1 current level in a Ca2+‐dependent manner. If we assume that centrin 4 and CaM indeed compete for the same binding site in the carboxyl‐terminal region of rEag1, then it will be important to ascertain how intracellular Ca2+ concentration may adjust rEag1 function via this dual Ca2+‐senor system.In direct contrast with CaM, however, Ca2+ regulation of the K+ channel. Moreover, our biochemical analyses in neurons highlight the notion that multiple centrin isoforms may contribute to Ca2+‐dependent modulation of rEag1 signaling in photoreceptors, as well as in various regions in the brain.In conclusion, this study identifies centrin 4 as a novel binding partner of rEag1, which may shed new light on the dynamics of CaPHH performed yeast two‐hybrid screening, mutagenesis and subcloning, and biochemical analyses. YCC conducted biochemical and subcellular fractionation analyses. TFL carried out electrophysiological experiments and data analyses. CJJ wrote the manuscript with inputs from the co‐authors.


Measurements of ICl(Ca) confirmed that endogenous Ca2+ buffering is equivalent to ˜0.05 mmol/L EGTA in rod and cone terminals. Confocal imaging showed that with such buffering, depolarization stimulated large, spatially unconstrained [Ca2+] increases that spread throughout photoreceptor terminals. We calculated immediately releasable pool (IRP) size and release efficiency in rods by deconvolving excitatory postsynaptic currents and presynaptic Ca2+ currents. Peak efficiency of ˜0.2 vesicles/channel was similar to that of cones (˜0.3 vesicles/channel). Efficiency in both cell types was not significantly affected by using weak endogenous Ca2+ buffering. However, weak Ca2+ buffering speeded Ca2+/calmodulin (CaM)-dependent replenishment of vesicles to ribbons in both rods and cones, thereby enhancing sustained release. In rods, weak Ca2+ buffering also amplified sustained release by enhancing CICR and CICR-stimulated release of vesicles at nonribbon sites. By contrast, elevating [Ca2+] at nonribbon sites in cones with weak Ca2+ buffering and by inhibiting Ca2+ extrusion did not trigger additional release, consistent with the notion that exocytosis from cones occurs exclusively at ribbons. The presence of weak endogenous Ca2+ buffering in rods and cones facilitates slow, sustained exocytosis by enhancing Ca2+/CaM-dependent replenishment of ribbons in both rods and cones and by stimulating nonribbon release triggered by CICR in rods.Differences in synaptic transmission between rod and cone photoreceptors contribute to different response kinetics in rod- versus cone-dominated visual pathways. We examined Ca This Ca2+ binding ratio is equivalent to approximately 0.05–0.1 mmol/L EGTA. For comparison, buffering at the synapses of ribbon-bearing hair cells and rod bipolar cells has been measured to be equivalent to approximately 1–2 mmol/L EGTA or BAPTA , followed by slower release of a second reserve pool  accelerates this resupply process by acting on ribbon-associated proteins to enhance the likelihood of vesicle attachment , 18–25 cm in length . Animal housing and protocols were approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Center. Euthanasia was conducted in accordance with AVMA Guidelines for the Euthanasia of Animals.All experiments were performed using vertical slices from retinas of aquatic tiger salamanders  and cut into 125 μm vertical slices using a razor blade tissue chopper (Stoelting), as described previously  and adjusted to 245 mOsm. Experiments were performed ˜2–8 h after the beginning of the dark cycle. Previous studies have indicated that photoreceptor ribbon morphology changes throughout the day in both mouse and turtle, with ribbons maintaining their classical plate-like morphology during the hours over which we conducted experiments  for 15 min, 1–2 h after the beginning of the dark cycle. They were then decapitated, quickly pithed, and enucleated. Retinas were mounted on a nitrocellulose membrane  patch pipette contained (in mmol/L) 90 Cs-gluconate, 10 TEA-Cl, 3.5 NaCl, 1 MgCl2, 1 CaCl2, 0.5 GTP-Na, 9.6 ATP-Mg, and 10 HEPES. The photoreceptor (presynaptic) pipette solution contained (in mmol/L) 50 Cs-gluconate, 40 Cs-glutamate, 10 TEA-Cl, 3.5 NaCl, 1 MgCl2, 10 ATP-Mg, 0.5 GTP-Na, 10 HEPES, and 0.05 EGTA. The use of 0.05 mmol/L EGTA was intended to approximate the endogenous Ca2+ buffering present in photoreceptor terminals  in the photoreceptor pipette solution to inhibit the plasma membrane Ca2+ ATPase (PMCA) ], the photoreceptor pipette solution was as above, except that it contained 5, 0.5, or 0.05 mmol/L EGTA and 0 added Ca2+ in all experiments. For perforated patch recordings, we used a pipette solution containing 200 μmol/L amphotericin-B and 5 μg/mL gramicidin with 0.1 mmol/L EGTA and no added Ca2+. Amphotericin-B was diluted 1000× from a stock made in DMSO and gramicidin 1000× from a stock made in 95% ethanol. Fresh antibiotic solutions were made every 3 h and stored in the dark. We also included Lucifer yellow (2 mg/mL) or Sulforhodamine-B (1 mg/mL) in the perforated patch pipette solution. To establish perforated patch recordings, we waited for the appearance of whole cell capacitance transients after establishing a giga-ohm seal. Cells were typically fully perforated for 5–10 min. At the end of the recording, we confirmed that the patch had not ruptured by taking images showing that fluorescent dye did not fill the cell. We then applied suction to rupture the patch, which caused an increase in the amplitude of the whole cell capacitance transients and diffusion of dye into the cell.When measuring the voltage dependence of CaIn paired rod-horizontal cell (HC) recordings, a clear fast component of the excitatory postsynaptic current (EPSC), representing exocytosis of ribbon-associated vesicles is followed by two distinguishable slow components. These are illustrated in the examples of rod- and cone-driven EPSCs shown in ICa, measured with a P/8 protocol) and EPSC was performed using OriginPro8. ICa was deconvolved using a single channel probability density function with a peak amplitude of 0.31 pA and a decay time constant of 1.1 msec describing the properties of L-type Ca2+ channels in photoreceptor terminals  contributing to evoked EPSCs – was measured using a deconvolution approach, as described previously  included in the patch pipette solution (500 μmol/L). OGB-5N at this concentration contributes a Ca2+ binding ratio (κ’) of ˜20. When included with a pipette solution containing 0.05 mmol/L EGTA, this produces a total Ca2+κ’ of ˜50, which is within the range of measurements we previously reported for both rods and cones  in response to depolarizing steps in voltage clamp (–79 to –19 mV) of varying duration (20–500 msec).Ca2+ imaging data were analyzed with PerkinElmer Ultraview Imaging Suite software. Data are presented as mean ± standard error of the mean (SEM) of individual measurements from single cells or single pre- and postsynaptic recordings. Unless otherwise noted, differences were considered statistically significant with P < 0.05 measured with a two-tailed independent Student’s t-test.Electrophysiology data were analyzed using Microsoft Excel, Clampfit 10.4, and GraphPad Prism 4.0 software. Ca2+ signals in the photoreceptor terminal are determined by a combination of Ca2+ influx, buffering, and extrusion and these dynamics shape the relative contributions of each of the Ca2+-dependent phases of exocytosis ) in rod and cone photoreceptors (Barnes and Deschênes ICl(Ca) differ between rods and cones, which might be attributable to differences in ICl(Ca) properties between each photoreceptor type such as channel distance relative to Ca2+ channels and/or Ca2+ cooperativity and sensitivity  channels are located within several hundred nanometers of Ca2+ channels and the current has an EC50 for Ca2+ of approximately 550 nmol/L in rods and 380 nmol/L in cones  activation by buffering progressively more Ca2+ near the ICl(Ca) channels. In this experiment, therefore, the meaningful comparison is between the voltage of half-maximal activation (V50) of ICl(Ca) at different levels of Ca2+ buffering within the population of rods or cones rather than a comparison of ICl(Ca) properties between rods and cones. A similar approach, which involved recording Ca2+-activated potassium currents, has been used to measure the endogenous buffering in retinal bipolar cells  was evoked with a 1-sec long depolarization to test potentials ranging from –59 to –19 mV. The V50 shifted to more depolarized potentials as the concentration of EGTA in the pipette solution was increased in both rods  and cones . Using perforated patch recordings to preserve endogenous buffering, we found that the V50 was –44.5 ± 1.5 mV (n = 8) for rods and –34.3 ± 2.0 (n = 8) for cones. These values are quite similar to the V50 measurements obtained with the 0.05 mmol/L EGTA solution, indicating that this is a good approximation for the endogenous buffering in rod and cone synaptic terminals and supporting measurements made by the added buffer approach  through the patch pipette using solutions containing either 0.05 mmol/L or 5 mmol/L EGTA. Ca2+ influx was evoked by step depolarizations (to –19 mV) of 20, 50, 100, or 500 msec, which mimics the response to a transition to darkness following a strong flash of light in photoreceptors. Under both buffering conditions, the amplitude of the Ca2+ signal increased with increasing step duration and, when mimicking endogenous buffering by using 0.05 mmol/L EGTA, the Ca2+ signals were greater in amplitude, as expected. With 100 msec steps in rods (F/F0 was increased from 0.06 ± 0.018 (n = 6) with 5 mmol/L EGTA to 0.197 ± 0.025 with 0.05 mmol/L EGTA . In cones (F/F0 of 0.034 ± 0.005 (n = 8) with 5 mmol/L EGTA to 0.155 ± 0.020 (n = 6) with 0.05 mmol/L EGTA (P = 0.0016). Increased Ca2+ buffering also constrained the Ca2+ signal to a hotspot, reflecting influx through Ca2+ channels located beneath a single ribbon or a few ribbons. With 0.05 mmol/L EGTA, in contrast, the fluorescent Ca2+ signal spread throughout the terminal. We quantified this by measuring the ratio of ΔF/F0 in the terminal to ΔF/F0 at the hotspot . In rods, Ca2+ was largely restricted to sites of entry in the terminal with 5 mmol/L EGTA  and nearly uniformly filled the terminal with 0.05 mmol/L EGTA . The pattern was similar in cones, with Ca2+ largely restricted to near the entry site with 5 mmol/L EGTA  and spreading throughout the terminal when the EGTA concentration was lowered to 0.05 mmol/L .We next performed Cas in rod  and cones in rod  synaptic in rods , for insIn cones , respons2+ concentration and extends the time course of Ca2+ signals  in the patch pipette  in the presence of dantrolene (P = 0.02). Additionally, the decay to 50% (T50) was 2.02 ± 0.31 sec (n = 7) with 0.05 mmol/L EGTA alone and 0.76 ± 0.22 sec  when dantrolene was included. The spread of Ca2+ throughout the terminal was also significantly inhibited, with Caterminal/Cahotspot being reduced to 0.56 ± 0.09 . Thus, a portion of the Ca2+ signal measured with 0.05 mmol/L EGTA reflects a boosting in amplitude, duration, and spatial extent of [Ca2+] due to enhanced CICR in rod terminals.In rods, CICR boosts the average intraterminal Ca pipette  and D. T2+ buffer in cones suggested that cone terminals do not exhibit CICR . Dantrolene also had no effect on the time course of the Ca2+ signal in cones; the T50 was 0.22 ± 0.02 sec (n = 6) with 0.05 mmol/L EGTA alone and 0.24 ± 0.02 sec  when dantrolene was included in the patch pipette. Likewise, Ca2+ spread was unchanged by dantrolene; Caterminal/Cahotspot was 0.83 ± 0.06 (n = 8) with dantrolene, not significantly different from cones with 0.05 mmol/L EGTA alone (P = 0.83). Thus, while endogenous-like Ca2+ buffering conditions enhance the amplitude and spread of Ca2+ in the cone terminal, this process does not involve CICR, in contrast to rods. Although ryanodine receptors are present in cone inner segments, strong Ca2+ extrusion mechanisms appear to prevent substantial CICR-evoked elevation in intraterminal [Ca2+] under most conditions , slower than measurements of IRP release from cones   or 25 ± 2 vesicles/ribbon (15.5 fC/vesicle) . This is lower than the number of ribbons counted in cone terminals using a similar approach (13 ribbons) . This contrasts with spherules in fish and mammals, which commonly have only a single large synaptic ribbon  of the fast component of the rod-driven EPSC when the rod pipette solution contained either 5 or 0.05 mmol/L EGTA. Based on the measurements of rod ribbon number above, we calculated the contribution of Ca2+ channel openings at each ribbon by scaling the Ca2+ current by seven ribbons per rod. We also scaled the EPSC amplitude by assuming that each ribbon contributed 36 pA to the fast component of the EPSC recorded in each horizontal cell, based on the multiple Gaussian fit of the rod-driven EPSC amplitude histogram. With 5 mmol/L EGTA, efficiency of release at rod synapses averaged 0.17 ± 0.03 vesicles/channel opening, or approximately six channel openings per vesicle fusion event (n = 16 rod-HC pairs). When we instead dialyzed the rod with 0.05 mmol/L EGTA to mimic the endogenous buffering conditions, efficiency was 0.21 ± 0.3 vesicles/channel opening, or ˜5 channel openings per vesicle fusion (n = 11). Although release efficiency was slightly higher with 0.05 mmol/L EGTA than 5 mmol/L EGTA, the difference was not significant (P = 0.33), suggesting that this 100-fold change in Ca2+ buffer concentration does little to alter Ca2+ domains responsible for triggering fast exocytosis.In cones, deconvolution analysis of voltage-gated Can = 15 cone-HC pairs), or ˜4 channel openings per fusion event, similar to previous measurements in cones  when the EGTA concentration was reduced to 0.05 mmol/L to mimic endogenous buffering. This agrees with previous results, where efficiency was unchanged whether the cone was recorded under perforated patch configuration or dialyzed with exogenous Ca2+ buffers .We performed a similar analysis of release efficiency with cones . With 5 j) between adjacent rods with paired whole cell recordings. In these experiments, the Gj was 1228 ± 1188 pS with 5 mmol/L EGTA (n = 7 rod–rod pairs) and 1206 ± 196 pS with 0.05 mmol/L EGTA . These values are more than double the previously reported rod–rod Gj in salamander retinal slices (˜500 pS)  while simultaneously recording postsynaptic responses from voltage-clamped horizontal cells . We mini2+-imaging experiments revealed that CICR activation in rods was favored with 0.05 mmol/L EGTA, we tested the contribution of CICR to this enhanced synaptic output by bath-applying 10 μmol/L dantrolene. Dantrolene has been shown to block CICR in rods . With 5 mmol/L EGTA, in contrast, the responses began to diverge from the dantrolene-treated condition only with very long stimuli (>500 msec), suggesting that the strong buffering prevented average intraterminal [Ca2+] from reaching sufficiently high concentrations to trigger CICR and nonribbon release. These data indicate that introducing exogenous Ca2+ buffers designed to mimic the endogenous conditions boosts sustained components of synaptic transmission by enhancing CICR in rods.As Ca2+ imaging experiments above, we showed that CICR does not contribute to the depolarization-induced elevation of intraterminal Ca2+ concentration in cones. However, in bipolar cells that also do not exhibit CICR, nonribbon release can nevertheless be revealed after saturating intracellular Ca2+ buffering  with durations of 2–1000 msec while simultaneously recording postsynaptic responses in voltage-clamped horizontal cells  was found to unmask strong caffeine-evoked Ca2+ signals suggesting that elevation of Ca2+ within the cone synaptic terminal due to CICR can be counterbalanced by strong Ca2+ extrusion and sequestration  from cones. Using a CsCl-based pipette solution (ECl = –2.7 mV) to enhance ICl(Ca) at the holding potential by increasing Cl– driving force, the inclusion of orthovanadate slowed the time to 50% recovery (t50) of the ICl(Ca) following 1 sec steps to –19 mV from 191 ± 27 msec (n = 9) to 639 ± 166 msec  indicating that inhibition of extrusion dramatically enhances the duration of Ca2+ signals in cones . This indicates that, in contrast to rods . When the cone pipette solution instead contained 0.05 mmol/L EGTA, the change in the slope was significantly greater than with 5 mmol/L EGTA, increasing by a factor of 2.59 ± 0.21 . When the cone pipette contained 0.05 mmol/L EGTA and slices were treated with the CaM inhibitor calmidazolium (20 μmol/L), the slope only increased by a factor of 1.28 ± 0.06 .Replenishment of vesicles to the synaptic ribbon in cones is accelerated by Canishment . Cones wnishment . With thn = 13; calmidazolium PPR = 0.38 ± 0.04, n = 10; P < 0.05) and the 10 sec interval . Synaptic transmission was strongly depressed at the 500 msec interval and was not significantly different between control and calmidazolium-treated conditions . This appears somewhat slower than similar measurements in cones , the properties of fast ribbon-mediated release are generally similar in rods and cones. Fast phasic transmission from rods and cones was also not significantly altered by conditions that mimic native Ca2+ buffering in the synaptic terminal, which is equivalent to ˜0.05 mmol/L EGTA  and pool size (N) . A second, slower phase is the result of the ribbon-associated reserve pool moving down the ribbon and being released with τ = 100–500 msec  by acting on synaptic ribbon sites to increase the probability that a colliding vesicle will attach , consistent with a greater proportion of Ca2+ ions contributing to free [Ca2+] and thereby triggering CICR-mediated nonribbon exocytosis during shorter stimuli  which do not exhibit CICR are nevertheless capable of sustained nonribbon release triggered by high [Ca2+] following buffer saturation (Zenisek et al. 2+ buffering and the resultant enhancement of [Ca2+] in the cone terminal did not recruit additional slow components of the postsynaptic response, even when PMCA-dependent extrusion was dramatically inhibited. Thus, unlike rods or bipolar cells, cones do not appear to be capable of employing nonribbon release.One key question we sought to address in this study was whether cones are capable of nonribbon sustained synaptic transmission. Previous work has shown that CICR generally does not contribute to intraterminal Ca2+ buffering by knocking out key Ca2+ binding proteins enhances exocytosis measured with capacitance recordings, but has no detectable effects downstream in the auditory pathway (Pangršič et al. 2+ buffering enhances sustained release in hair cells, the additional glutamate was not detected postsynaptically. It is unlikely, however, that nonribbon release by cones would go similarly undetected. In the retina, residual levels of cleft glutamate derived from rod release can make a substantial contribution to horizontal and bipolar cell responses (Cadetti et al. An alternative interpretation of these results is that nonribbon release may occur in cones but that it went undetected in our experiments. In hair cells, reducing intracellular CaThe involvement of CICR-driven nonribbon release therefore appears to represent a fundamental difference in synaptic transmission between the two photoreceptor types. What cellular processes enable rods but not cones to release vesicles at nonribbon sites? One hypothesis suggests that in rods, nonribbon release occurs at random, rather than fixed release sites, in contrast to bipolar cells (Midorikawa et al. 2+ channels and reduces the rate of glutamate release. With a transition back to darkness, as simulated by the depolarizing steps employed in this study, Ca2+ channel openings trigger a burst of glutamate release that is important in allowing the synapse to encode contrast (Jackman et al. 2+ buffering can help restrict Ca2+ to regions near the channels (Nowycky and Pinter Rod and cone photoreceptors typically have resting potentials of –35 to –40 mV in the dark and are hyperpolarized by a flash of light. This hyperpolarization closes Ca2+] is high around the ribbon and throughout the synaptic terminal, the ribbon itself, by slowing the movement of vesicles toward release sites at the ribbon base, effectively restricts fast exocytosis to nanodomain control so that only vesicles positioned near the membrane and Ca2+ domains can be quickly released following Ca2+ influx. Later stages of exocytosis are facilitated by low endogenous buffering, which promotes CICR-mediated nonribbon release in rods and CaM-dependent replenishment of ribbons in both rods and cones evoked by spread of Ca2+ ions to sites some distance from the channels.This division of release regimes appears to be promoted by the synaptic ribbon, which slows the delivery of vesicles to release sites (Jackman et al. 2+] combined with nanodomain-driven fast and efficient release permits each photoreceptor type to report both luminance and temporally precise changes in luminance to postsynaptic neurons, respectively (Jackman et al. 2+ via diffusion through the endoplasmic reticulum, which extends from the soma to the terminal (Chen et al. 2+ to enhance vesicle replenishment and sustained release by ribbons might similarly enhance positive contrast encoding in cone-driven circuits. Additionally, the regulation of IRP replenishment kinetics by Ca2+/CaM adds an additional facet to the computations performed at the synapse; by making the size of the IRP dependent on time, the Ca2+/CaM-dependent replenishment process also sets the temporal response properties of the synapse and light responses of second-order retinal neurons (Van Hook et al. 2+ channel number and voltage dependence appear to vary from one ribbon to the next within single hair cells (Frank et al. 2+ buffering and resultant spatially unconstrained Ca2+ signals would have a normalizing effect across individual ribbons in a given terminal, so that release at neighboring ribbons would be similar during sustained darkness. Finally, expression of Ca2+ channels in chick retina is known to vary with circadian time (Ko et al. 2+ influx in salamander photoreceptors (Stella and Thoreson 2+ influx and exocytosis (Hull et al. 2+ influx and ribbon structure could represent an additional means by which photoreceptor Ca2+ signals are regulated to match synaptic signaling requirements. Thus, the exact nature of Ca2+ buffering and the means by which it differentially influences each phase of rod and cone synaptic output are likely to play important roles in shaping early stage visual processing at the photoreceptor synapse.Functionally, sustained release driven by average intraterminal [CaNone declared.


However, the physiological and molecular mechanisms of Ca2+ distribution within specific cell types in different root zones under hypoxia is poorly understood. In this work, whole-plant physiological and tissue-specific Ca2+ changes were studied using several ACA (Ca2+-ATPase) and CAX (Ca2+/proton exchanger) knock-out Arabidopsis mutants subjected to waterlogging treatment. In the wild-type (WT) plants, several days of hypoxia decreased the expression of ACA8, CAX4, and CAX11 by 33% and 50% compared with the control. The hypoxic treatment also resulted in an up to 11-fold tissue-dependent increase in Ca2+ accumulation in root tissues as revealed by confocal microscopy. The increase was much higher in stelar cells in the mature zone of Arabidopsis mutants with loss of function for ACA8, ACA11, CAX4, and CAX11. In addition, a significantly increased Ca2+ concentration was found in the cytosol of stelar cells in the mature zone after hypoxic treatment. Three weeks of waterlogging resulted in dramatic loss of shoot biomass in cax11 plants (67% loss in shoot dry weight), while in the WT and other transport mutants this decline was only 14–22%. These results were also consistent with a decline in leaf chlorophyll fluorescence (Fv/Fm). It is suggested that CAX11 plays a key role in maintaining cytosolic Ca2+ homeostasis and/or signalling in root cells under hypoxic conditions.Waterlogging is a major abiotic stress that limits the growth of plants. The crucial role of Ca Overall, our results suggest that Ca2+ efflux systems and especially CAX11 are essential to mediate plant Ca2+ homeostasis under hypoxic conditions.Knowledge of hypoxia-induced transport processes is largely restricted to ion exchange at the root surface, which is easily accessible. However, little is known about the character of hypoxic signal transduction in specific cell types and tissues of roots  and loss-of-function mutants  aca8 (SALK_0578770), aca11 (SALK_002747), cax4 (SALK_119863), and cax11 (SALK_013040) were obtained from the Arabidopsis Biological Resource Centre (http://www.arabidopsis.org/abrc/). Seeds were surface sterilized with 1ml of commercial bleach [1% (v/v) NaClO] for 10min, and then washed five times with sterilized distilled water. Seeds were kept at 4 °C for 2 d and sown in Petri dishes containing 1% (w/v) phytogel, half-strength Murashige and Skoog (MS) medium, and 0.5% (w/v) sucrose at pH 5.7. Petri dishes containing seeds were sealed with 3M micro-pore tape and then transferred into a growth chamber with a 16h/8h day/night, 100 µmol m−2 s−1 photon flux density, and 22 °C. The Petri dishes were oriented upright, allowing the roots to grow down along the surface without penetrating into the medium. All chemicals were from Sigma-Aldrich  in analytical grade unless individually specified.Seeds of 2 . For measurement of gene expression levels, WT Arabidopsis seedlings were treated with hypoxia for 1, 24, and 72h; for measurement of K+ and Ca2+ distribution in roots, the WT and the four mutants were treated with hypoxia for 24h. Combined hypoxia and salinity treatment was also imposed on the 10-day-old Arabidopsis seedlings in deoxygenated agar solution with addition of 50mM NaCl for 24h, with measurement of Na+ distribution in roots. The whole seedlings were submerged in the bubbled solution and kept in the growth chamber with a 16h/8h day/light, 100 µmol m−2 s−1 photon flux density, and at 22 °C. Compared with hypoxia treatment or combined hypoxia and salinity treatment, the control condition was normoxic, non-saline, and without agar solution.Hypoxic treatment was imposed on the 10-day-old Arabidopsis seedlings submerged with 0.2% (w/v) agar solution pre-bubbled with high purity Naca8, aca11, cax4, cax11, and WT plants were grown in 0.2 litre pots filled with peat moss, perlite, vermiculite, and coarse sand, with a ratio of 2:1:1:1 (v/v), and watered with half-strength Hoagland’s nutrient solution. Plants were grown in a growth chamber at a 12h/12h light/dark regime and at 21 °C for 3 weeks. Plants were then subjected to waterlogging treatment by immersing pots in water and maintaining the water level 0.5cm above the soil surface for another 3 weeks. The above-ground biomass of individual plants was determined as fresh weight, and dry weight was measured after drying in a Unitherm Drier  for 2 d at 65 °C.In the phenotyping experiments, Fv/Fm) was recorded at a saturating actinic light (660nm) intensity of 1100 µmol m−2 s−1 . Plants were adapted in a dark chamber for 30min before measuring. The maximum quantum efficiency of photosystem II .Total RNA was extracted from 10-day-old Arabidopsis roots with TRIZOL reagent   and 20 µM APG-2. The indicators were dissolved in DMSO (Sigma) to a stock concentration of 1mM. Then, 20 µM CoroNa Green, 20 µM Asante Potassium Green-2, and 15 µM Calcium Green-5N were added to measuring buffers , respectively. Arabidopsis seedlings were incubated in the dye-containing measuring buffers for 3h in the dark. The stained seedlings were washed in distilled water for 3min to remove residual dyes before measuring fluorescence intensity in meristem, elongation epidermal, elongation stelar, mature epidermal, and mature stelar root cells after 24h of hypoxic treatment. All dying steps in this work was done at room temperature.CoroNa Green acetoxymethyl (AM) ester , Calcium Green-5N, AM (Invitrogen), and Asante Potassium Green-2  were employed to measure the Na+, Ca2+, and Na+ concentration data shown in A confocal laser scanning microscope fitted with a TCS SPII confocal head  was used to measure fluorescent signals from roots. We used the 488nm excitation line of an argon multiline laser and the tripledichroic TD 488/543/633nm beam splitter. CoroNa Green fluorescence emission was detected in the photomultiplier at 505–525nm. Calcium Green-5N fluorescence emission was detected in the photomultiplier at 520–550nm. APG-2 fluorescence emission was detected in the photomultiplier at 530–550nm. Images were analysed with Image J software  to calculate cell fluorescence based on integrated density. The background signal was measured from an empty region with a similar size and subtracted from the whole-cell signal to obtain relative total cell fluorescence values . Continuous fluorescence was quantified in arbitrary units by LAS AF software based on intensity and plotted in an Excel file  . All data in the figures are given as means ±SE. The significant differences in relative gene expression level, relative total cell KACA8, ACA11, CAX4, and CAX11 in the roots of Arabidopsis WT after 1, 24, and 72h of hypoxia treatment  and tissues (epidermis and stele) in roots of Arabidopsis WT and four Ca2+ transporter mutants after 24h of hypoxia treatment (+ concentration in the root meristem but a decrease in the mature zone cells in WT Col-0 plants (+ concentration was increased by 1.5- to 2-fold in both apex (elongation) and mature root tissues  and cax mutants (a 1.3- to 1.7-fold increase) and no changes in the aca mutants  with hypoxia caused dramatic increases in the Naure zone . aca11 senotypes . Hypoxiaenotypes , 7A. In ectively ; Table 1 control .2+]cyt have been rarely considered in the context of the tissue-specific expression and regulation of appropriate Ca2+ transporters. Significantly (P<0.01) higher quantities of Ca2+ were accumulated in the cytosol of meristematic cells in aca8 and cax11 after 24h of hypoxia, while no changes were observed in the WT, aca11, and cax4 (2+]cyt increased significantly in the WT (P<0.05) and aca8 (P<0.05) and highly significantly in aca11 (P<0.001), but no changes were found in cax4 and acax11  increase in the [Ca2+]cyt was found in all the five genotypes (Hypoxic stress-induced changes in the [Caand cax4 . In the d acax11 . Epidermpt aca11 . Stelar and aca8 . In stelenotypes .P<0.05) reduced after 3 weeks of waterlogging stress  in leaves of cax11  and low-capacity transporters are responsible for termination of [Ca2+]cyt signalling; while Ca2+ exchangers with low-affinity (Km=10–15 µM) and high-capacity transporters may be involved in the removal of [Ca2+]cyt when its elevations are high  medium and also suppressed its Na+ sensitivity  compared with the control, consistent with the insignificant changes of ACA11 expression in the WT, which suggests that ACA11 is unlikely to play an important role in hypoxic stress responses. In rice, Ca2+ was reported to act as a physiological transducer for the expression of the alternative oxidase 1a (AOX1a) gene under O2 deficiency , the stelar cells in the elongation zone showed no changes in Ca2+ concentration, while in the mature zone they had higher Ca2+ than in normoxic controls  channels. Since the stele is the first root tissue suffering O2 deficiency, the H+-ATPase activities in the xylem would be inhibited, leading to the closure of SKOR channels for a substantial decline of xylem K+ concentration   , and the2 deprivation might result in a prolonged Na+ accumulation even after return to normoxic conditions . This may be suggestive that even under hypoxia there is still Na+ flow in the xylem from the root to the shoot via other pathways. Some studies found that protein kinase SOS2 /CIPK24, involved in salt tolerance , tissues (epidermis and stele), and specific cell types. In this study, unique cell types in different zones regulate concentrations of K+, Ca2+, and Na+ in distinct ways when faced with hypoxia. In the WT and all of the four Ca2+ transporter mutants, the stelar cells in the elongation zone showed no changes in Ca2+ concentration, while in the mature zone they had higher Ca2+ than normoxic controls. We also speculate that more K+ and Na+ accumulation in the stele than in the epidermis in both the elongation zone and mature zone could probably flow via NORC which could open during hypoxia. In addition, Ca2+ efflux systems and in particular CAX11 were shown to mediate plant Ca2+ homeostasis under hypoxic conditions, with an influence on waterlogging tolerance.The results presented here allow a new hypothesis concerning the hypoxic signal transduction in Arabidopsis roots linked with CAX and ACA CaJXB online.Supplementary data are available at Figure S1. Dose dependence of Calcium Green loading into five types of Arabidopsis root cells.Figure S2. Illustration of the quantification procedure for Ca2+ distribution in the cytosol.Table S1. The primers for quantitative RT-PCR experiments.Table S2. Effect of 24h of hypoxia on Na+ relative concentrations in stelar cells of the elongation zone and mature zone in Arabidopsis WT, aca8, aca11, cax4, and cax11 (ratio of Na+ concentration in mature stelar cells relative to elongation stelar cells under control or hypoxic conditions).


Pulmonary adenocarcinoma (PA) is the most common histologic type of primary lung cancer. Generally, adenocarcinoma was composed by five major components. The present study aimed to evaluate changes in the composition of adenocarcinoma components as the tumor grows; in addition, to analyze the correlation between the occupancy rates of histologic components of the tumor in regard to prognosis.Pathologic data were retrospectively evaluated for 206 patients who underwent curative resection of PA. We investigated how histologic component occupancy rates changed as tumor size and N stage increased. To evaluate local invasiveness, the major components of the present group and absent group of pleural invasion, lymphatic invasion, and vascular invasion were compared.P = 0.006, P < 0.001) ; however, the percentage of lepidic components decreased (P < 0.001). In cases with a solid component and a micropapillary component, a gradual increase was found with an increase N stage ; however the percentage of lepidic components decreased (P < 0.001). Average differences of histologic components dependent upon whether pleural, lympathic and vascular invasion were present, the difference of micropapillary and lepidic components were statistically significant. With logistic regression analysis, as the occupancy rate of the lepidic component increased, the probability of pleural invasion, lymphatic invasion, and vascular invasion decreased; in cases with a micropapillary component, as the occupancy rate of increased, the probability of lymphatic invasion and vascular invasion increased. In multivariate analysis using the Cox propotional hazards model, the occupancy rates of acinar, micropapillary and lepidic  components were significantly associated with recurrence.The mean percentages of acinar and solid components significantly increased with an increase in size (The lower the occupancy rate of a lepidic component and the higher the occupancy rates of acinar, solid, and micropapillary components, the likelihood of tumor progression increased. In addition, as the occupancy rate of a lepidic component decreased and a micropapillary component increased, local invasiveness and recurrence rate increased; thus, increasing the probability of a poor prognosis. Pulmonary adenocarcinoma (PA) is the most common histologic type of primary lung cancer . Since NThe majority of primary adenocarcinomas that occur in the peripheral lung have the radiologic appearance of a ground glass opacity nodule (GGN) at the incipient stage; in most cases, the lesion becomes more solid as the tumor grows , 5. MoreThe most important prognostic factor of non-small cell lung cancer is its anatomical stage; this staging is commonly determined by the American Joint Committee on Cancer (AJCC) TNM stage . HoweverFrom March 2011 through September 2013 at Seoul St. Mary’s Hospital in Korea, 407 patients were diagnosed with non-small cell lung cancer and underwent pulmonary resection that entailed more than a lobectomy for an attempt to achieve a radical cure. Among these 407 patients, 275 were diagnosed with adenocarcinoma. The following patients were excluded from the evaluation: treatment with neoadjuvant chemotherapy preoperatively; diagnosed with atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), or minimally invasive adenocarcinoma (MIA); had multiple lesions, staging was not possible because a lymph node dissection was not performed; or the occupancy rates of histologic components were not recorded. Ultimately, 206 patients were included in this retrospective chart review. This study was approved by the Institutional Review Board of Seoul St. Mary's Hospital (The Catholic University of Korea).The five components of adenocarcinoma were recorded in 5% increments. The recording method was followed via the recommendations of IASLC/ATS/ERS . If the P < 0.05.The averages of the respective component occupancy rates in each tumor group classified by size and N stage were compared by ANOVA and the Kruskal Wallis H test; the averages of the respective component occupancy rates of each component classified with pleural invasion, lymphatic invasion, and vascular invasion were comparatively analyzed via the t-test. In addition, logistic regression was used for the analysis of the factors influencing pleural invasion, lymphatic invasion, and vascular invasion. Statistical significance was set at The mean patient age was 63.13 years (range: 33–85 years). A total of 91 patients (44.2%) were men, and 115 patients (55.8%) were women. In regard to the location of tumor onset, it was central in 15 cases (7.3%) and peripheral in 191 cases (92.7%); thus, the tumor onset was primarily in the peripheral area. In regard to stage, it was IA in 114 cases (55.3%), IB in 36 cases (17.5%), IIA in 22 cases (10.7%), IIB in 9 cases (4.4%), IIIA in 21 cases (10.2%), IIIB in 1 case (0.5%), and IV in 3 cases (1.5%).Classification by tumor size was in accordance with T stage criteria; the tumor size was < 2 cm in 80 cases (38.9%), ≥ 2 cm and < 3 cm in 65 cases 31.6%), ≥ 3 cm and < 5 cm in 8 cases (3.8%), and ≥ 7 cm in 2 cases (0.9%). In regard to N stage, it was N0 in 164 cases (79.6%), N1 in 22 cases (10.7%), and N2 in 20 cases (9.7%). Pleural invasion was present in 50 cases (24.3%), lymphatic invasion was present in 95 cases (46.1%), and vascular invasion was present in 40 cases (19.4%) . In cases with a solid component, it also increased as the size increased: < 2 cm, 3.1%; ≥ 2 cm and < 3 cm, 11.8%; and ≥ 3 cm, 13.1% (P < 0.001). However, in cases with a lepidic component, it decreased as the size increased: < 2 cm, 49.6%; ≥ 2 cm and < 3 cm, 30.0%; and ≥ 3 cm, 14.9%. In micropapillary component cases, it increased as the size increased: < 2 cm, 2.3%; ≥ 2 cm and < 3 cm, 4.8%; and ≥ 3 cm, 7.1%; however, the difference was not statistically significant (P = 0.143). In cases with a papillary component, there was no significant change (P = 0.138) ; ≥ 2 cm and < 3 cm, 65 31.6%), and ≥ 3 cm, 61 (29.6%). We compared the change in the average histologic component occupancy rate in accordance with the increase of size. In cases of a predominant acinar component, when the size was < 2 cm, it was 35.6%; it was 35.8% when the size was ≥ 2 cm and < 3 cm. However it increased significantly to 48.52% when the size was ≥ 3 cm . In cases with a micropapillary component, a gradual increase was also found: N0, 3.5%; N1, 6.6%; and N2, 11.0% (P < 0.001). However in cases with a lepidic component, a gradual decrease was found: N0, 39.9%; N1, 7.5%; and N2, 6.3% (P < 0.001); no significant changes were found in cases with an acinar (P = 0.051) or papillary component (P = 0.053) ; micropapillary component, 7.8% (P < 0.001); and solid component, 15.6% (P < 0.001). In cases with a lepidic component, it was 39.8% in the group without pleural invasion and 13.4% in the group with pleural invasion; thus, showing a statistically significant lowering-aspect (P < 0.001) ; and cases with a solid component, 12.2% (P < 0.001). In cases with a lepidic component, it was 44.2% in the group without lymphatic invasion and 20.2% in the group with lymphatic invasion; thus, showing a statistically significant lowering aspect (P < 0.001) , in cases with a micropapillary component, it was 8.5% (P = 0.002), and in cases with a solid component, it was 15.0% (P = 0.027). In cases with a lepidic component, it was 38.5% in the group without vascular invasion and 11.0% in the group with vascular invasion; thus, showing a statistically significant lowering-aspect (P < 0.001) ) and solid components increased ; a similar decrease was found as the occupancy rate of the lepidic increased . In cases of lymphatic invasion and vascular invasion, only the micropapillary and lepidic types showed significant results. As the percentage of the micropapillary component increased, the likelihood of lymphatic invasion  and vascular invasion ) increased. As the percentage of the lepidic component increased, the likelihood of lymphatic invasion ) and vascular invasion ) decreased . During follow up, 35 patients (17%) of all patients n = 206) and 16 patients of stage I patients (n = 150) experienced recurrence. In stage I, 3-year disease free survival was 86.6% . In cases with an acinar component, the growth rate increased as the N stage increased; however, the increase did not reach statistical significance. In summary, we found that as the tumor progressed, the occupancy rate of the lepidic component decreased while that of the solid component increased.Pleural invasion, lymphatic invasion, and vascular invasion are known prognostic factors of lung cancer. Cases with pleural invasion are considered to be one of factors influencing prognosis because pleural invasion is one of the factors included in the TNM stage . HoweverOur data were relatively recent data, so we couldn’t analyzed 5-year overall survival. However, we performed 3-year disease free survival analysis. In multivariate analysis, an increase of micropapillary component occupancy and an decrease of lepidic component occupancy were significantly associated with recurrence. This results showed that the occupancy rates of micropapillary and lepidic components are related with prognosis.All of the foregoing results contributed somewhat to the prediction of the characteristics and changes of the typical histologic components of pulmonary adenocarcinoma. Each component would have different characteristics, which could influence the clinical course. If the occupancy rates of these histologic components were analyzed in detail and the characteristics in accordance with the change in the occupancy rates were revealed, individualization of a treatment protocol for adenocarcinoma could defined; thus, helping to maximize the benefits of the treatment.Our study had several limitations. It is retrospective and single center. The patient sample was relatively small; this particularly impacted categorization by tumor size. Even though the classification was made in compliance with T stage criteria of TNM stage, the number of patients in the group > 5 cm was only 10; thus, the group was combined with the group ≥ 3 cm and < 5 cm. If the patient sample was larger, more size subdivisions could be made and more accurate results could have been attained; furthermore, this would increase the significance of the study. Long-term follow-up was not conducted. Long-term follow-up regarding the influence of each histologic component could provide a more detailed analysis of the recurrence rate and mortality.In most cases, pulmonary adenocarcinoma consists of various histologic components and the occupancy rate of each component changes as the tumor progresses. The lower the occupancy rate of a lepidic component and the higher the occupancy rates of acinar, solid, and micropapillary components, the likelihood of tumor progression increased. In addition, as the occupancy rate of a lepidic component decreased and the occupancy rate of a micropapillary component increased, local invasiveness and recurrence rate increased; thus, increasing the probability of a poor prognosis.