Arbuscular mycorrhizal (AM) symbiosis increases the absorption of Ca2+ for host plants. Here, we analyzed the physiological and transcriptional changes in the roots of Arachis hypogaea L. colonized by Funneliformismosseae under Ca2+-deficient and -sufficient conditions. The results showed that exogenous Ca2+ application increased arbuscular mycorrhizal fungi (AMF) colonization, plant dry weight, and Ca content of AM plants. Simultaneously, transcriptome analysis showed that Ca2+ application further induced 74.5% of differentially expressed gene transcripts in roots of AM peanut seedlings. These genes are involved in AM symbiosis development, hormone biosynthesis and signal transduction, and carotenoid and flavonoid biosynthesis. The transcripts of AM-specific marker genes in AM plants with Ca2+ deprivation were further up-regulated by Ca2+ application. Gibberellic acid (GA3) and flavonoid contents were higher in roots of AM- and Ca2+-treated plants, but salicylic acid (SA) and carotenoid contents specifically increased in roots of the AM plants. Thus, these results suggest that the synergy of AM symbiosis and Ca2+ improves plant growth due to the shared GA- and flavonoid-mediated pathway, whereas SA and carotenoid biosynthesis in peanut roots are specific to AM symbiosis.Peanut yield is severely affected by exchangeable calcium ion (Ca Arachis hypogaea L.) is an important oil crop and protein source for humans that contributes 20% to oil production and 11% of the human protein supply per year. The yield is often limited by exchangeable Ca2+ deficiency in soil, which causes early embryo abortion in peanut2. Therefore, Ca2+ plays a crucial role in the growth and development of peanut. Calcium is an important macronutrient required for plant growth and development and represents 0.1 to 5% of all plant dry biomass3. Additionally, as a second messenger, Ca2+ has been shown to mediate various aspects of cell and plant development, such as cell division, cell polarity, cell elongation, photomorphogenesis, and biotic and abiotic stress responses6.Peanut  and auxin contents1. However, Ca2+ is largely confined to uptake via the young root tips and can only be taken up by the young root system from the soil and delivered to the shoot via the xylem, and it is not remobilized from old to young tissues8. Thus, Ca2+ deficiency commonly affects plant growth and development if the soil cannot be supplemented with exogenous Ca2+. Fortunately, most plants have coped with limited Ca2+ availability via the establishment of symbiotic associations with microbes, more specifically known as AM association. The fungi forming AM symbiosis belong to the subphylum Glomeromycotina9.In peanut, Ca10. To establish the symbiosis, plant roots recognize chemical signals from AMF, e.g. lipochitooligosaccharides and chitooligosaccharides, which trigger coordinated differentiation and form the symbiotic state11. In turn, AMF require signal communication from the plants that produce strigolactones (which are derived from the carotenoid synthesis pathway), flavonoids, and other diffusible signals exuded by plant roots that induce the germination of AMF spores and branched fungal hyphae12. Then, the AM symbiosis is established by the common symbiosis signaling pathway induced by calcium oscillation after perception of diffusible signals from the symbionts13. In the process of establishing the AM symbiosis, many AM-specific marker genes must be initiated by Ca2+ concentration change14, such as RAM1 (REDUCED ARBUSCULAR MYCORRHIZA 1), RAM2 (glycerol-3-phosphate acetyltransferase), CCD1 (carotenoid cleavage dioxygenase), PT1 (phosphate transporter), and DELLA17. These findings suggest that Ca2+ plays an important role in AM development.This symbiosis plays a significant role in the uptake of nutrients and the carbon cycle, and consequently impacts ecosystem sustainability20. Some of the changes associated with plant hormones were considered to play important roles in this symbiosis21, such as auxin, cytokinins (CKs), gibberellins (GAs), and strigolactones, and were also altered in the roots of AM plants22. In addition, increases in flavonoid and anthocyanin were considered to be indispensable in regulating the establishment of AM symbiosis23.From recognition of the fungi to establishment of the symbiosis, complicated transcriptional reprogramming occurs in plant roots, and many specifically expressed genes involved in development of the symbiosis have been identified in legumes25, the role of plant uptake of Ca2+ needs further study. Several reports showed that a moderate level of Ca2+ supply enhanced the colonization of AMF27, and Ca2+ benefited the maintenance of a functioning mycorrhiza28. However, the transcriptional changes in plant roots colonized by an AMF accompanied with sufficient Ca2+ are still unknown.Even though the molecular basis of the improvement of plant nutrient acquisitions have been well characterized for phosphorus, nitrogen, sulfur, and potassiumet al. (2019) demonstrated that AM symbiosis increased the Ca2+ content in peanut seedlings, and Ca2+ application can also promote the development of AM symbiosis29. However, the molecular mechanism of how AMF and Ca2+ application synergistically promote the growth of peanut seedlings is unclear. In this study, we investigated a combination of transcriptional changes, hormone and metabolomic analyses in roots of peanut seedlings inoculated by AMF and Ca2+ application and compared the observed changes with those in AM plants or Ca2+-treated plants. We observed that changes in secondary metabolites in roots of AM and Ca2+-treated plants coincide with the transcriptional regulation of related biosynthesis pathways. These alternations, such as the increases in GA3 and flavonoid content, were considered to be involved in the growth enhancement of peanut seedlings by the synergy of AMF with Ca2+ application.Cui F. mosseae under both Ca2+-deficient and Ca2+-sufficient conditions, respectively  plants and Caght Fig. . Moreoveght Fig. . Additioots Fig. .0-AM as the control, there were 510, 1483, and 1795 significantly differentially expressed genes (DEGs) from roots of the Ca0 + AM, Ca6 − AM, and Ca6 + AM plants, respectively  and Ca6 + AM plants (1795), respectively. The expression levels of 380 DEGs in Ca0 + AM plants could be further regulated by Ca2+ application  ten of which were up-regulated and two down-regulated. However, only 12 AM-specific marker genes were induced in Ca0 + AM plants and the transcripts of ten up-regulated genes were further increased by Ca2+ application  involved in the biosynthesis of SA was specifically up-regulated by Ca2+ application, and was further up-regulated by AM symbiosis.DEGs involved in plant hormone biosynthesis were screened, including auxin, CKs, GA, and SA Table . One gen3, and SA. IAA content significantly increased in AM plants with Ca0 treatment, but decreased in Ca6 treatment , GAent Fig. . Changesnts Fig. . Additioent Fig. , which went Fig. .Figure 4CCD7, Araip.RJ87T) involved in carotenoid biosynthesis were only up-regulated in AM plants, and were further up-regulated by Ca6 treatment (Table DWARF27) were specifically up-regulated in Ca6 + AM plants.We found an increase in transcripts of DEGs involved in carotenoid biosynthesis. The genes encoding 3-oxoacyl-[acyl-carrier protein] reductase (BGI_novel_G000088), 15-cis-phytoene/all-trans-phytoene synthase (Araip.40 × 13), and 9-cis-beta-carotene 9′,10′-cleaving dioxygenase  encoding shikimate O-hydroxycinnamoyltransferase was up-regulated in the Ca0 and Ca6 treatments, and the expression level was the highest in AM plants treated with Ca6. However, the other gene encoding shikimate O-hydroxycinnamoyltransferase was specifically up-regulated in Ca6 + AM plants. In addition, the gene (Araip.6PA6C) encoding flavonol synthase responsible for the biosynthesis of flavanol, was specifically up-regulated in AM plants with Ca6 treatment . In addition, the establishment of AM symbiosis requires the expression of AM-specific marker genes33. In this study, Ca2+ further up-regulated and specifically induced the transcripts of AM-specific marker genes in AM plants. It is possible that the Ca2+-calmodulin association with CCaMK induces the phosphorylation of CYCLOPS and forms a complex in the presence of calcium, which acts in concert with GRAS  TFs such as DELLA proteins to initiate the expression of AM-specific marker genes that are necessary to establish the AM symbiosis14. These results suggest that Ca2+ plays a vital role in the formation of AM symbiosis.Our previous study reported that AM symbiosis combined with exogenous Ca34, and is also involved in GA biosynthesis as a negative regulator of GA signaling35. AM symbiosis up-regulation of GA-related genes and GA content in roots has been reported in M. truncatula and tomato37. Hence, the observed increase of GA3 content may be the factor involved in Ca2+ further up-regulating the transcripts of DELLAs and the genes encoding gibberellins 20-oxidase, which is a key enzyme that catalyzes the penultimate steps in GA biosynthesis. This result implied that AM symbiosis positively regulated the transcriptional changes involved in GA biosynthesis, and that Ca2+ strengthens this effect.GRAS family TF encoding DELLA protein is a positive regulator in the formation of AM associations38. However, it has been reported that CKs act as a negative regulator in lateral root initiation, because overproduction of CKs inhibited lateral root initiation40. In this study, the lower tZR in roots of AM plants suggested that the genes encoding cytokinin dehydrogenase, which catalyze the irreversible degradation of cytokinin, were down-regulated by Ca2+ application. Reduced tZR content may be beneficial to the initiation of AM symbiotic roots. This result supports the finding in some studies that CKs might not be involved in the regulation of AM symbiosis development16. In addition, SA and carotenoid have been demonstrated to be activated by AM colonization41, and these activations were specific to AM symbiosis but not Ca2+ ; the other half were not colonized by F. mosseae. The peanut seedlings were grown in a greenhouse at 24 °C/18 °C with a 16/8 h photoperiod, at a photosynthetic photo flux density of 700 µmol·m−2·s−1, and 60% relative humidity. Each seedling was watered regularly with 80 ml of modified Hoagland’s solution  supplemented with 6 mM Ca(NO3)2·4H2O (Ca2+ sufficient) or 6 mM NH4·NO3 2). There were four treatments: Ca0-AM, Ca0 + AM, Ca6 − AM, and Ca6 + AM, where 0 and 6 represent the Ca2+ concentrations (mM), + and − represent with or without inoculation of F. mosseae spores. In this study, 6 mM of Ca(NO3)2 was chosen according to our previous report6. Six weeks later, the shoots and roots of AM and NM plants were harvested and further analyzed.Peanut cultivar ‘Huayu 22’ seeds were surface sterilized with 70% alcohol for 3 min and rinsed six times with sterile water. They were then germinated in the dark at 25 °C for 3 days. The germinated seeds were transferred to pots filled with quartz sand which was rinsed with deionized water 10 times to remove as much Ca2+-deficient or Ca2+-sufficient conditions were harvested separately. Young roots of AM plants were examined by light microscopy  to estimate the extent to which the roots had been colonized by hyphae and arbuscules46. The fresh shoots and roots were dried at 105 °C for 30 min, and then dried at 80 °C until a constant weight. The Ca2+ contents in the roots from the different treatments were determined according to Yang et al.6.After six weeks, shoots and roots of the AM and NM plants under Ca1. The cDNA from three biological replicates composed of four plants in each treatment were sequenced using an Illumina HiSeq. 2000 Platform. After filtering, high quality clean reads were aligned with a reference genome (https://peanutbase.org/organism/Arachis/ipaensis) using HISAT47; on average 70.61% reads were mapped, indicating that the samples were comparable.Total RNA was isolated from roots of AM and NM plants, and then enrichment of mRNA and synthesis of cDNA were conducted47, then identified novel transcripts using Cuffcompare and predicted the coding ability of those new transcripts using CPC software49. After novel transcript detection, the gene expression level was calculated for each sample with RSEM50. Based on the gene expression level, we used DEseq. 2 algorithms to detect differentially expressed genes (DEGs). A threshold of 1 for transcript ratio (log2FoldChange) in treatments versus control (Ca0-AM), and Padj  ≤0.05 were set as criteria for the selection of DEGs in NM plants and AM plants under Ca2+-deficient and Ca2+-sufficient conditions. With DEGs, Gene Ontology (GO) classification and functional enrichment were performed using WEGO software51, and the pathway analyses were obtained using the KEGG database (https://www.genome.jp/kegg/pathway.html).After genome mapping, we used StringTie software to reconstruct transcripts with genome annotation information52. At least three replicates were tested per sample. Relative mRNA (fold) differences were assessed with the 2− ΔΔCt formula53, the values were subsequently transformed to the log2 scale.To verify the RNA-Seq results, the expression levels of 15 selected genes were determined by quantitative RT-PCR. mRNA was isolated from the same samples sequenced by RNA-Seq, and the first-strand cDNAs were synthesized for qRT-PCR analyses using SYBR Premix Ex Taq polymerase (Takara) according to the manufacturer’s protocol. The designed primers are shown in Supplementary Table 54, including IAA, tZR, and GA3. The SA content was measured according to a previous method55. Three independent replicates per sample were statistically analyzed.The roots (fresh weight) were ground into a powder in liquid nitrogen, and 1.0 g of powder was used to determine the concentration of endogenous hormones by high performance liquid chromatography (HPLC)56. Total carotenoid content in roots was calculated using absorbance at 450 nm. Flavonoids in roots were measured by chloride colorimetric assay57, and total flavonoid content was determined according to the standard curve of quercetin at an absorbance of 510 nm.Carotenoids were extracted from the roots of AM and NM plantsP value ≤ 0.05 was considered to be significant.Analysis of variance was performed using SSPS software version 16.0 for Windows. One-way analysis of variance (ANOVA) was used, followed by Duncan's test for multiple comparisons. The values obtained are the mean ± SE for the three replicates in each treatment. A Supplemental Figure S1Supplemental Tables


However, the exact mechanisms involved in the communication among these transporters are still unclear. In the present study, thapsigargin, an irreversible inhibitor of SERCA, and ML-9, a broadly used SOCE inhibitor, were applied in human neutrophils to better understand their effects on Ca2+ pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca2+ flux were evaluated in freshly isolated human neutrophils, using a microplate reader for monitoring fluorimetric kinetic readings. The obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca2+ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca2+ levels due to its influx by SOCE. Moreover, it was obvious that ML-9 enhanced the thapsigargin-induced emptying of the internal stores. Indeed, ML-9 does not have this effect by itself, which indicates that, in neutrophils, thapsigargin does not act only on the influx by SOCE, but also by other Ca2+ pathways, that, in the future, should be further explored.In neutrophils, intracellular Ca Cell viability and cell yield were evaluated by the trypan blue exclusion method as previously reported , from 8.1 μM (37.4 ± 4.7%) to 32.5 μM (93.8 ± 4.2%).Thapsigargin produced a clear fluctuation in the intracellular free Ca pattern A. At tim2+ flux, a broadly described inhibitor of SOCE, ML-9, was used. In a first stage, thapsigargin (16.2 μM) and ML-9 (0–117 µM) were simultaneously added to the cells and their combined effect in the fluctuation of intracellular Ca2+ concentration, was monitored during 1 h. The pattern of Ca2+ concentration fluctuation along the time was very similar to the one found when thapsigargin was tested alone. However, the intensity of the fluorescence was higher with increasing concentrations of ML-9, indicating an escalation in the intracellular Ca2+ concentration  with the complete disappearance of the second fluorescence escalation, in all the tested conditions, suggesting a clear decrease in the intracellular Ca2+ concentration. Interestingly, and contrary to what happened in the absence of EGTA, ML-9 did not induce the increase of the fluorescence intensity of the first peak when compared to thapsigargin alone (When EGTA (800 µM) was added B to the in alone B.2+ concentration, except for the first 300 s  for 30 min. After this time, SOCE was inhibited by ML-9 (0–117 µM). Contrary to the results obtained when thapsigargin and ML-9 were added simultaneously, in this case, the addition of ML-9 after thapsigargin exerted its effects and did not affect the fluorescence increase produced by thapsigargin A. The adst 300 s B.p ≤ 0.001, compared with the control assay (without thapsigargin and ML-9)] fluorescence increase, in the presence of ML-9 or not  was incubated with the cells during 30 min, after which thapsigargin (16.2 μM) was added for 30 min more. Immediately after the addition of ML-9, time 0, a small tendency for an increase of the fluorescence was observed. However, this increment did not achieve statistical significance, either in the presence or absence of EGTA . After this peak, the Ca2+ levels start to decrease until 1800 s (30 min) of exposure. The second pattern of increase starts to appear after this time and it is a sustained signal, even after 60 min. Previous studies also showed a transient intracellular Ca2+ concentration increase to a peak value attained in 1 to 2 min that then decline to a lower level , both at the beginning of the experiment and at the second phase of Ca2+ increase. Considering that ML-9 inhibits SOCE, it was expected that the second increase (after the first 30 min) would not appear, because it is believed to be due to the influx of Ca2+ from the extracellular medium. However, such effect was not observed. In fact, the characteristic thapsigargin effect is still observed and it seems that ML-9, in some way, has a synergic effect, because ML-9 alone does not alter the Ca2+ levels when compared to the control (data not shown). In the presence of EGTA, i.e. without extracellular Ca2+, the first peak is preserved, meaning that this Ca2+ comes from the intracellular stores. The second peak completely disappears, indicating that the second peak was caused by the entry of Ca2+ from the extracellular medium to neutrophils cytoplasm. This behaviour was observed for all the conditions, so even in the presence of ML-9, the origin of the Ca2+ was the same, the intracellular stores.Beginning with experiment (a), where thapsigargin and ML-9 were simultaneously added to the cells, and their effect in the Ca2+ levels were exactly the same with or without ML-9; contrary to what we registered in experiment (a), where these values were increased with higher ML-9 concentrations. Moreover, in the presence of EGTA, the first fluorescence peak was maintained, as expected, and the second increase disappeared, meaning once more the participation of extracellular Ca2+ in this last effect. This leaves a doubt, ML-9 which inhibits Ca2+ entry into cells by SOCE, does not alter the signal, but EGTA does. We may argument that ML-9 concentrations were not high enough and in this sense did not inhibit SOCE, as expected. However, ML-9 has been used in some other similar works in concentrations of 50 and 100 µM . As above mentioned, Ca2+ has an extremely relevant role in cellular processes, namely in cell death. In recent years it has become obvious that Ca2+ can activate distinct paths of cell death mechanisms. There are several theories [2+ fluxes. Even that induction of apoptosis also occurs in the absence of Ca2+ fluxes. It is commonly accepted that severe dysregulation of Ca2+ fluxes lead to necrosis and that more controlled increases lead to apoptosis. So, subtler changes of Ca2+ may have different effects on cell death modulation, as in cell proliferation and differentiation, for example. It is now known that Ca2+ related processes are intimately connected to caspases, the apoptosis effectors. Under stress response, ER can trigger apoptosis. The ER stress may be triggered by changes in the protein folding due to Ca2+ overload or depletion of the Ca2+ pool. ATP levels and mitochondrial function are also vital to influence the death mode [2+-related processes can also trigger the elimination of death cells by phagocytosis or by their lysis. This process of cell death is even more important when we talk about neutrophils [2+ is also described, but it is not conclusively identified [The cell viability was evaluated after an hour of incubation in the already described conditions. All the tested conditions led to a statistically significant loss of viability [** theories  about thath mode ,23. Furttrophils . Indeed,trophils . Curiousentified .2+ level leads to the accumulation of mitochondrial Ca2+. Another hypothesis is that thapsigargin inhibits mitochondrial Ca2+ influx, via Ca2+ uniporter, and in this way Ca2+ efflux is not unimpeded. Indeed, these two mechanisms: cytosolic Ca2+ increase and MPT induction may be responsible for the known thapsigargin-induced cell death [2+. Finally, one should keep in mind the intrinsic correlation between Ca2+ variations and cell viability [It was obvious that thapsigargin led to a more drastic loss of cell viability even when ML-9 was present. However, it was clear that during the first 30 min of incubation with just ML-9, and only after the addition of thapsigargin, this loss was lower. The thapsigargin-induced neutrophils death is known . High coll death . It is all death ,23. Noteiability , which m2+ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca2+ levels due to the influx by SOCE. Moreover, it was obvious that ML-9 enhanced the emptying of the internal stores. Indeed, ML-9 does not have this effect by itself, which indicates that, in human neutrophils, thapsigargin does not act only on the influx by SOCE, but also by other Ca2+ pathways; this should be further explored in the future.In conclusion, the obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca


One fundamental question is how LTD is selectively induced by specific numbers of Ca2+ pulses and which are the frequency and duration of this train of pulses required for LTD induction. The molecular mechanism which leads the integration of postsynaptic Ca2+ pulses in the LTD signaling network has not been elucidated either. Recent publications have shown that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is required for LTD induction. Additionally, protein kinase C (PKC), CaMKII, and MAPK play an important role to transduce the frequency of Ca2+ pulses into their enzymatic activity levels; however, it is still unknown which enzymes are involved in decoding Ca2+ pulses in LTD. We have extended a stochastic model of LTD by adding the molecular network regulating CaMKII activity and its activation. We solved this model with stochastic engine for pathway simulation to include the effect of biochemical noise in LTD. We systematically investigated the dependence of LTD induction on stimulus frequencies, and we found that LTD is selectively induced by a specific number of Ca2+ spikes at different frequencies. We observed that CaMKII is essential to induce LTD, and LTD is only weakly induced when its Thr286 phosphorylation site has been deleted. We found that CaMKII decodes the frequency of Ca2+ spikes into different amounts of kinase activity during LTD induction. In addition, PKC and ERK enzyme activity is highly sensitive to the frequency and the number of Ca2+ pulses and this sensitivity has an important effect on LTD activation. This research predicts the postsynaptic Ca2+ requirements to induce LTD using a typical synaptic activation sequence and explains how LTD is selectively induced by specific number of Ca2+ pulses at different frequencies.Cerebellar long-term depression (LTD) is a form of synaptic plasticity dependent on postsynaptic Ca Cerebellar long-term depression (LTD) is a form of synaptic plasticity involved in motor learning . It is c2+ concentration in the Purkinje dendritic spine via AMPA receptor and metabotropic glutamate receptor pathways. It has been shown that PF and climbing fiber (CF) coactivation causes a supralinear Ca2+ response in mammalian PCs  and calcium influx entry into the spine . PF inpulian PCs . The supthe IP3R .2 positive feedback loop and a mechanism of AMPAR trafficking  are thought to influence its ability to decode Ca2+ spikes. However, the molecular mechanism by which this sensitivity contributes to LTD is not fully understood.The LTD is expressed as a reduction in the number of synaptic AMPA receptors (AMPAR) and depends on a protein kinase C (PKC)-ERK-cPLAfficking . Experimfficking . Additiollations . The actCaMKII is notoriously difficult to simulate in detail because of its multi-subunit nature, which produces a combinatorial explosion in the number of states and chemical species that must be simulated. Several models of CaMKII activation have been developed, but most of them have avoided the mechanistic, detailed modeling of every chemical reaction . Develop2 feedback loop, Raf-RKIP-MEK interactions, and AMPAR trafficking. We have extended this model by adding the molecular network regulating CaMKII activity and its activation. The CaMKII molecular network is based on a published model by 2+ input requirements to induce LTD in PCs using local uncaging of Ca2+. These experiments used a single and prolonged pulse, and varied the amount of Ca2+ by changing the peak light intensity to show the relationship between Ca2+ concentration and LTD amplitude CaMKII inactive.(2)CaM is bound to CaMKII and CaMKII becomes active.(3)Phosphorylation at Thr286 when CaM is bound to a subunit.(4)CaM is unbound from autophosphorylated subunit.(5)Second phosphorylation at Thr305.We developed a detailed molecular model of CaMKII activation and its dephosphorylation by PP1 and PP2. It is based on a previous model developed by We have chosen PP1 and PP2A as phosphatases in our model based on previous experimental information that PP1 enhances the apparent cooperativity for Thr286 autophosphorylation of CaMKII  and deph2+/CaM binding, intersubunit autophosphorylation at threonine residue Thr286, Ca2+-independent activation state through autophosphorylation and secondary intersubunit autophosphorylation at threonine residue Thr305/306.The reactions included in the CaMKII model are: activation of the enzyme by CaNoise in the signaling networks plays an important role in cellular processes . We have2+ input stimulus at the starting time of the simulation. The input calcium signal was designed as a consecutive train of pulses, each with a duration of 200 ms  and an amplitude of 1.8 μM on average and 0.18 μM of SD. We calculated the response of the system for every 10 number of calcium pulses, from 0 to 100 pulses at frequencies of 0.1, 1, 4, and 10 Hz.We computed the mean response of the LTD model over 100 iterations with different random seeds. The LTD response was calculated as the proportion of synaptic AMPARs 30 min after a CaWe calculated the probability to get LTD at a specific number of calcium pulses for every frequency. LTD was considered successful when a single trial reached the equilibrium state with the mean of AMPA receptors in the last 3 min of the simulation less than the 70% from the initial value. We counted the number of successful events over the total of simulation experiments . LTD probability values range from 0 to 1, the maximum probability is 1 when all the trials showed LTD. The curves are fitted by Hill equation, as follows:2+ pulses required to achieve the half maximum magnitude of LTD (K1/2), and the maximum probability LTDmax.Sigmoidal curves were obtained by the Curve fitting toolbox of Origin . LTD means the probability to get LTD over 100 trials. The parameters of the curves are Hill coefficient (nHill), the number of CaWe changed the width of calcium pulses to 90 ms and 500 ms at 1 and 0.1 Hz to show the effect of stimulation width on LTD probability, and we changed the amplitude of calcium influx per pulse to 4–5 μM and 10–11 μM at 1 Hz to emulate a supralinear increase of calcium influx and its effect on LTD probability.2+ requirements to induce LTD using a typical synaptic activation procedure  and to explain how LTD is selectively induced by specific numbers of Ca2+ pulses. In addition, we studied which kinases are involved in transducing the repeated Ca2+ pulses into their enzymatic activity in the induction of cerebellar LTD.Through stochastic modeling we built a detailed mechanistic model of CaMKII activation and we included this model into the LTD molecular network in order to study the postsynaptic CaTo understand how bistability arises in the LTD molecular network, we analyzed the activation of PKC and CaMKII in single trials in the model at 1 and 4 Hz Figure . For LTD2+ pulses reduces the number of failures of LTD induction and PKC activation. We analyzed the bistability in PKC activity over time at 1 and 4 Hz. PKC activation is bistable when the stimulus is a low number of Ca2+ pulses, for 1 Hz we observe bistability from 20 to 50 pulses and for 4 Hz from 10 to 30 pulses  is induced after 50 pulses at 1 Hz and after 30 pulses at 4 Hz . This dependency is congruent with the previous experimental results  and consequently the LTD probability increases as a function of integrated calcium.The calcium sensitivity of LTD depends on the frequency of calcium input. In order to know the effect of the frequency of the calcium stimulus on LTD over time, we calculated the mean of 100 trials of AMPAR dynamic for frequencies at 0.1, 1, 4, and 10 Hz using 100 pulses of calcium Figure . At thisUnderstanding the molecular mechanisms involved in synaptic plasticity, specifically how a postsynaptic calcium signal is transduced into a long-lasting change of synapses is an important question. To elucidate the molecular decoders of calcium signal into enzymatic activity in LTD molecular network we calculated the enzymatic activity of PKC, CaMKII, and ERK as a function of calcium pulses number at 1 and 4 Hz Figure .2+ spikes into different amounts of kinase activity during LTD induction. This result is congruent with previous studies of CaMKII sensitivity to Ca2+ oscillations . This CaMKII model may be useful to build other simulations of other systems, which require activation of CaMKII. We added this model to the LTD molecular network and we studied systematically the calcium requirements to induce LTD at postsynaptic density showing that cerebellar LTD is sensitive to the Ca2+ input requirements to induce LTD at postsynaptic neurons using local Ca2+ uncaging. These experiments showed a sigmoidal relationship between the amplitude of a single calcium pulse and LTD. In those experiments, the Ca2+ stimulus duration influences the calcium requirements for LTD induction, long elevation of calcium caused a leftward shift in the sigmoidal relationship. However, typically protocols to induce LTD use repeated synaptic activation leading to many postsynaptic Ca2+ pulses. We emulated the trains of synaptic activity typically used to induce LTD to explain how LTD is related to the number of Ca2+ pulses at the postsynaptic density. We found a sigmoidal relationship between the number of Ca2+ pulses and LTD amount with a similar relation as that in 2+ stimulus frequency influences the calcium requirements for LTD induction. This dependency is closely related with the response of CaMKII, ERK, and PKC activity to the Ca2+ number pulses at specific frequencies.2+-induced Ca2+ release via IP3R, and they can reach values above 10 μM in spiny dendrites (2+ measurements between the Parallel fibers and CF coactivation causes supralinear calcium signals in PC . Supraliendrites . We showendrites , 6 basedendrites . PKC bis2+ signals (2+ pulses into their enzymatic activity levels (2+ oscillations on activation on Ras and the ERK/MAPK pathway, and it has been suggested that synaptic signaling network may function as a temporal decoder (2+ pulses in cerebellar LTD has not been studied. The molecular network of LTD induction includes several molecular players that transduce Ca2+ spikes frequency into their enzymatic activity. CaMKII, PKC, and ERK enzyme activities are highly sensitive to the frequency and this is strongly related with the amount of LTD. PKC and ERK responses over calcium pulses are different when the stimuli are at different frequency. These responses are shifted to the left at 4 Hz in comparison with 1 Hz. Also, the slope of the sigmoidal of PKC and ERK changes with the frequency, at higher frequency the steepness is higher than at low frequency stimulation, this means that the enzyme reaches faster the maximum level of activity at 4 Hz than at 1 Hz. At the same frequency, the slope of ERK is more sensitive to the Ca2+ input because the enzyme activity reaches the 90% at 30 pulses, meanwhile PKC reaches the same activity at 40 pulses. This means that the threshold of ERK and PKC activation is different at specific number of Ca2+ pulses. CaMKII activity responses faster to Ca2+ input than ERK and PKC, its activity reaches around 20% when the enzyme is stimulated just by 10 pulses. However, the slope of the curve increases slowly for further increases in the number of Ca2+ pulses.Several theoretical and experimental works have studied the role of kinases to transduce calcium signals at specific frequency into their activity level. CaMKII has been studied as a decoder of Ca signals . Some sty levels . Theorety levels . Kupzig  decoder . However2+-independent activity (2+ pulses in dendritic spines and phosphorylation at Thr286 is necessary for optimal integration of Ca2+ pulses in hippocampal LTP (2+ signal input. Further experimental studies may focus on cerebellar LTD to test the hypothesis about CaMKII threonine phosphorylation sites as an important integrator of Ca2+ pulses at different frequencies.The importance of the Thr286 site on CaMKII activity has been studied extensively . Coultraactivity . Throughmpal LTP . This ishttp://senselab.med.yale.edu/ModelDB/showModel.cshtml?model=245412.The updated model of LTD signaling pathway is available at ModelDB: URL: CZC contributed to the LTD model, ran simulations, analyzed data, and wrote the manuscript. EDS supervised the project and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We show that GPN evokes a sustained increase in lysosomal pH (pHly), and transient increases in cytosolic pH (pHcyt) and Ca2+ concentration ([Ca2+]c). None of these effects require cathepsin C, nor are they accompanied by rupture of lysosomes, but they are mimicked by structurally unrelated weak bases. GPN-evoked increases in [Ca2+]c require Ca2+ within the endoplasmic reticulum (ER), but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. We conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER in a manner that is independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes.The dipeptide glycyl- Highlighted Article: GPN causes an increase in the cytosolic pH, evoking Ca2+ release from the ER that is independent of cathepsin C. More thly ∼4.5) . Lysosomly ∼4.5) . They ally ∼4.5) . Lysosom (P2RX4) . These pgnalling . There assipated , and glyembranes A.ly they are protonated and trapped in the lysosome lumen, where they can become highly concentrated , disrupts lysosomes and, thereby, triggers apoptosis. The likely mechanism involves an initial lysosomotropic accumulation of LLOMe within lysosomes, causing pHly to increase; cathepsin C then catalyses polymerisation of the de-esterified dipeptide and, as the hydrophobic polymer accumulates, it perturbs the lysosomal membrane, rendering it permeable to small molecules (molecular mass<10 kDa)  is widely supposed to be expressed mainly within lysosomes , where i<10 kDa) .ly increases.GPN A, anothe2+ content of lysosomes and the lack of evidence that these effects are mediated by cathepsin C encouraged us to assess both the mechanism of action of GPN and its selectivity for lysosomes. We show that GPN evokes a rapid and sustained increase in pHly, and transient increases in cytosolic pH (pHcyt) and cytosolic free Ca2+ concentration ([Ca2+]c). None of these responses requires cathepsin C activity; neither are they accompanied by detectable rupture of lysosome membranes. Rather than selectively stimulating Ca2+ release form lysosomes, GPN increases [Ca2+]c by causing an increase in pHcyt that then stimulates Ca2+ release from the ER by a mechanism that requires neither inositol 1,4,5-trisphosphate (IP3) receptors nor ryanodine receptors.The widespread use of GPN to acutely assess the Ca2+ from lysosomes (see Introduction) . The Ca2+ signals evoked by GPN were slower than those evoked by carbachol, which stimulates IP3 formation and Ca2+ release through IP3 receptors (IP3Rs), but faster than those evoked by inhibiting the ER Ca2+ pumps  with cyclopiazonic acid (CPA) or thapsigargin . While these results are consistent with the reported actions of GPN, additional observations are not consistent with its presumed mode of action  A. In HEKf action A.Fig. 1cyt)  triggers loss of H+ and cathepsin D from lysosomes during the early stages of apoptosis . Dissipation of the lysosomal pH gradient by inhibiting the V-ATPase with bafilomycin A1 had no effect on basal pHcyt or the subsequent GPN-evoked increase in pHcyt , indicating that the GPN-evoked increases in fluo 8 fluorescence are due to an increase in [Ca2+]c  H. Rupturpoptosis . Similarin pHcyt I,J. Thestive see , we conf [Ca2+]c D-F.ly and pHcyt were not accompanied by loss of endocytosed fluorescent molecules with molecular masses ranging from 447 (Lucifer Yellow), through ∼3 kDa (fluorescein-dextran) to ∼10 kDa (dextran conjugates of Alexa Fluor 488 and Oregon Green) . These results are not consistent with the effects of GPN on pH or [Ca2+]c arising from rupture of lysosomes. More prolonged incubations with GPN (>10 min) caused some loss of lysosomal Alexa Fluor 488-dextran but, even after 25 min, most was retained by lysosomes Second, the acute effects of GPN on pH2+]c evoked by GPN was abolished by depleting the ER of Ca2+ by using CPA or thapsigargin to inhibit SERCAs . Although no SERCAs were detected in the proteome of lysosomal membranes . This confirms that loss of Ca2+ from the ER, rather than inhibition of SERCAs themselves, rapidly inhibits GPN-evoked increases in [Ca2+]c.Third, the increase in c evoked by GPN (3R and TMCO1) that can mediate CICR from the ER contributed to the GPN-evoked increase in [Ca2+]c. The results, so far, demonstrate that GPN increases pHly and [Ca2+]c, but they challenge the conventional explanations of these phenomena.Transmembrane coiled-coil domain 1 (TMCO1) is an ER membrane protein that oligomerises to form a Caith Ca2+ A , in which the l-phenylalanine of l-GPN (hitherto described as GPN) is replaced by d-phenylalanine, because d-GPN, unlike l-GPN, is not a substrate for cathepsin C . We confirmed that LLOMe caused lysis of lysosomes only in cells expressing cathepsin C , which is consistent with published work . These results demonstrate that the effects of GPN on pHly, pHcyt and [Ca2+]c do not require cathepsin C, whereas LLOMe causes cathepsin C-mediated lysis of lysosomes without increasing [Ca2+]c.LLOMe is a substrate of cathepsin C and, like GPN, it is a weak base but it is less membrane-permeable than GPN , which, in common with other lysosomotropic agents, accumulate within lysosomes  to GPN (Table S1).GPN, but not ysosomes A . By using carbachol or thapsigargin to estimate the residual Ca2+ content of the ER , we confirmed that both GPN and NH4Cl caused a concentration-dependent decrease in the ER Ca2+ content . These results establish that four different weak bases  have similar effects. Each causes a sustained increase in pHly, and transient increases in pHcyt and [Ca2+]c. The latter invariably requires Ca2+ within the ER, but requires neither IP3Rs nor RyRs.As expected NH in pHly B,C, but d by GPN D-K. Furtsigargin F-K. The 2+ signals evoked by GPN , the requirement for ER Ca2+ cannot arise from it amplifying an initial GPN-evoked Ca2+ release from lysosomes. However, we  argues against this proposal, and subsequent experiments confirm that it is untenable.Since no known CICR mechanism contributes to the Caever, we  and otheever, we  have sugysosomes L. We, th1, the GPN-evoked increase in [Ca2+]c was exaggerated .After dissipation of the lysosomal pH gradient by sustained treatment with bafilomycin Aggerated A,B, justmycin A1 . Similarggerated C,D. Hencate Ca2+ , GPN evom the ER L2.Fig. cyt evoked by GPN. NaP caused an initial drop in pHcyt that slowly recovered, and subsequent addition of GPN caused an attenuated increase in pHcyt , was almost abolished by NaP , a weak acid, to buffer changes in pHin pHcyt E. The im by >60% F. NaP diing pHly G,H. The d by NaP I,J. NaP onomycin K,L. Thesffective .Fig. 7.2+ signalling has grown, has been widely used to release Ca2+ from lysosomes (see Introduction). It has been universally assumed that GPN achieves selectivity for lysosomes because its cleavage by the lysosomal enzyme cathepsin C causes osmotic stress that ruptures lysosomal membranes  and that it causes a transient increase in pHcyt . GPN also increases pHly and [Ca2+]c. None of these effects of GPN require cathepsin C , amplified when lysosomes are no longer acidified .We have shown that GPN does not acutely rupture lysosomes , depletion of the ER of Ca2+ abolishes the GPN-evoked increase in [Ca2+]c  without affecting the pHcyt increase , and an increase in pHly is not required for GPN to release Ca2+ . Finally, GPN-evoked Ca2+ release is almost abolished when the pHcyt changes are buffered under conditions where GPN still causes an increase in pHly  through protein kinase C  is assumed to equilibrate rapidly with pHcyt . SERCAs, which transport Ca2+ in exchange for H+, are inhibited by increased pH . Hence, modest increases in pHcyt, similar to those evoked by many physiological stimuli, trigger a substantial Ca2+ release from the ER that requires neither RyRs nor IP3Rs.Cells tightly control their pHsporters , but mankinase C  and signkinase C . Furtherncreased . In many2+ entry  but in my the ER . The mecth pHcyt , the effimulated  and IP3 to IP3Rs , but GPNeased pH . However2+ release from lysosomes through its cathepsin C-mediated proteolysis. Instead, GPN, in common with other membrane-permeant weak bases and many physiological stimuli, transiently increases pHcyt, and that directly stimulates Ca2+ release from the ER by a mechanism that is independent of known ER Ca2+ release channels.We conclude that GPN does not, as hitherto supposed, evoke CaMr 10,000), Alexa Fluor-488  or fluorescein (Mr 3000), LysoTracker Red DND-99, Lucifer Yellow, Platinum Pfx DNA polymerase, Tris-acetate SDS running buffer, 4–12% Bis-Tris polyacrylamide gels, iBLOT transfer kit, custom primers, EDTA-free Pierce protease inhibitor mini-tablets, Rapid DNA ligation kit, and Spectra multicolour broad range protein ladder were from ThermoFisher . Glycyl-l-phenylalanine 2-naphthylamide (GPN) was from Bachem  and Santa Cruz Biotechnology ; most experiments used GPN from Bachem . Glycyl-d-phenylalanine 2-naphthylamide (d-GPN) was custom-synthesised by LifeTein . Gly-Arg-7-amino-4-methylcoumarin (Gly-Arg-AMC) was from Bachem. Gly-Phe-diazomethylketone (Gly-Phe-DMK) was from MP Bio . Fibronectin was from Merck Millipore . ECL prime western blotting detection reagent was from GE Life Sciences . Thapsigargin and cyclopiazonic acid (CPA) were from Bio-Techne . Fluo 8-AM was from Stratech Scientific . 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) was from Phion . Ionomycin was from Apollo Scientific . Ryanodine, Pluronic F-127, sodium propionate (NaP), fluoxetine hydrochloride, Leu-Leu methyl ester hydrobromide (Leu-Leu-OMe) and carbamylcholine chloride  were from Sigma .SNARF-5F, dextran-conjugates of Oregon Green 488  were from Kerafast   (2. Cells were passaged every 3–4 days using GibcoTrypLE Express (ThermoFisher). All cell lines were confirmed to be free of mycoplasma.HEK and HeLa cell lines were cultured in Dulbecco's modified Eagle’s medium (DMEM)/F-12 with GlutaMAX (ThermoFisher) supplemented with foetal bovine serum  at 37°C in humidified air with 5% COton, MA) . HAP1 cedge, UK) . HAP1 ce3 muscarinic acetylcholine receptors (M3Rs) was produced as described .For imaging, cells were grown on glass-bottomed dishes  coated with human fibronectin (10 µg/ml). BacMam baculovirus encoding human Mescribed . HEK cel48138) (CRISPR/Cas9 was used to disrupt the genes encoding cathepsin C (CTSC). Guide sequences were selected using E-crispr.org  and zitf48138) . The cod48138) A-C.The methods used to generate cells deficient in TMCO1 were the same as those used to generate HEK-CTSC-KO cells. The only effective sgRNA (5′-GAAGCGGAAGTGCGATCTTC-3′) targeted exon 1. Neither different sgRNAs, targeting exon 1 (5′-GTGCACGGCTCTGCTCGCAG-3′) or exon 3 (5′-GAAACAATAACAGAGTCAGC-3′ and 5′-GTTTACAGTGGAAAAGAAGA-3′), nor repeated transfections with multiple sgRNAs succeeded in yielding cells with both TMCO1 alleles disrupted .2, 1.5 mM CaCl2, 11.5 mM glucose, 11.6 mM HEPES pH 7.3. Cells were washed in HBS, and fluorescence was recorded from cells in HBS at 20°C at intervals of 3.8 s  using a FlexStation 3 fluorescence plate-reader with SoftMaxPro software . Fluorescence was calibrated to pHcyt fromaK is the negative log of the acid-base dissociation constant (aK); R is the fluorescence emission ratio (F580:F640); Ra (Fa) and Rb (Fb) are the fluorescence ratios (or fluorescence intensities at 640 nm) for the fully protonated and de-protonated forms of the indicator.Confluent cultures of cells grown in 96-well plates  were loaded with the ratiometric pH indicator SNARF-5F  by incubaK of SNARF-5F, cells loaded with SNARF-5F were treated for 30 min in Ca2+-free cytosol-like medium (CLM pH 7.4) with the H+/K+ antiporter nigericin (50 µM). CLM was then replaced by CLM supplemented with nigericin (50 µM) and buffered at different pH values (pH 5.0–8.5), and SNARF-5F fluorescence was measured. Ca2+-free CLM had the following composition: KCl 140 mM, NaCl 4 mM, MgCl2 1.4 mM, HEPES 10 mM, EGTA 1 mM. The resulting calibration plot of pH versus Ka (7.09) used in all further calibrations.To determine the p2+] ([Ca2+]c), confluent cell monolayers grown in 96-well plates were loaded with fluo 8 by incubation for 1 h at 20°C in HBS (100 μl) containing fluo 8-AM and 0.02% Pluronic F-127. Cells were then washed and incubated in HBS for 1 h at 20°C before use. CaCl2 was omitted from nominally Ca2+-free HBS, and in some experiments (see figure legends) BAPTA  was added to the HBS immediately before stimulation to reduce the free [Ca2+] of the HBS to <20 nM. Fluorescence was recorded using a FlexStation 3 fluorescence plate-reader. Fluorescence was recorded at 1.44-s intervals, with excitation at 485 nm and emission at 525 nm. Data were collected and analyzed using SoftMax Pro software. Maximal (Fmax) and minimal (Fmin) fluorescence values were determined from parallel wells after addition of Triton X-100 (0.1%) with either 10 mM CaCl2 (Fmax) or 10 mM BAPTA (Fmin). Fluorescence values (F) were calibrated to [Ca2+]c using a Kd=389 nM (fluo 8) fromFor measurements of cytosolic free [Cal-lysine-coated, glass-bottomed 35-mm dishes were incubated  in culture medium supplemented with either a dextran-conjugated indicator (0.1 mg/ml) or Lucifer Yellow (0.2 mg/ml). After a further 4–6 h in the same medium without indicator, cells were used for experiments. For HEK cells expressing LAMP1-mCherry  . For labelling with LysoTracker Red, cells were incubated in HBS with LysoTracker Red DND-99 . After 20 min, cells were washed three times with HBS and used immediately.To load lysosomes by endocytosis with fluorescent dyes, cells grown on poly-Fig. S3) , conflue2 targeted laser illumination system . Excitation light was transmitted through a quad dichroic beam-splitter (TRF89902-QUAD), and emitted light was passed through appropriate filters (Cairn Optospin). Wide-field images were collected with either an iXon Ultra 897 EMCCD camera  or a 95B Scientific CMOS camera  and MetaMorph microscopy automation and image analysis software (Molecular Devices). Bright-field images were acquired using a Cairn MonoLED illuminator.Fluorescence microscopy used inverted Olympus IX83 microscopes with 100× objectives , a multi-line laser bank (488 and 561 nm) and an iLasFluorescence from Oregon Green, fluorescein and Alexa Fluor 488 was excited at 488 nm and captured using a 525/50-nm filter (peak/bandwidth). LysoTracker Red and mCherry were excited at 561 nm and captured at 630/75 nm. All fluorescence images were corrected for background by subtraction of fluorescence from a region outside the cell.0, where F0 and F denote the average fluorescence within the ROI at the start of the experiment (F0) and at each subsequent time point (F).Time-lapse recordings of cells loaded with dextran-conjugated indicators were analyzed by taking randomly selected regions of interest (ROI) large enough for each to include several lysosomes . Fluorescence changes were then expressed as F/FTo analyse the number of lysosomes in cells before and after GPN treatment L, the Fi488:F561). The F488:F561 values for all ROIs within each cell  were averaged for each image, and then expressed relative to the value determined before treatment . This averaging within a cell was required because lysosomes move during the 25-min recording interval, making it impossible to track individual ROIs in recordings that were collected at 5-min intervals (to minimise photobleaching).For analysis of Alexa Fluor 488-dextran distribution in cells expressing LAMP1-mCherry, the FFT bandpass filter and thresholding functions in Fiji  were useg, 15 min, 4°C) was used for analysis.Cells in 6-well plates were washed, lysed in RIPA buffer  by using a syringe needle and sonication, and the supernatant  membrane using an iBLOT system, and blocked by incubation in TBST with gentle shaking . Tris-buffered saline (TBS) comprised: 150 mM NaCl, 50 mM Tris pH 7.5. For TBST, TBS was supplemented with 0.1% Tween-20 and 5% BSA. The membrane was washed with TBST, incubated with primary antibody , washed with TBST , incubated with HRP-conjugated secondary antibody in TBST with 1% BSA , and washed with TBST . Enhanced chemiluminescence (ECL) primer western blotting detection reagent  and a Syngene PXi chemiluminescence detection system were used to detect HRP. The antibodies used were mouse anti-cathepsin C , rabbit anti-TMCO1 , mouse anti β-actin , donkey anti-mouse IgG-HRP  and donkey anti-rabbit IgG-HRP .Cathepsin C activity was measured by using a substrate, Gly-Arg-AMC, that becomes fluorescent after its proteolytic cleavage . HEK celn describing the number of independent analyses. Student's t-test, one-way or two-way ANOVA with Tukey's or Bonferroni test were used to determine statistical significance. P<0.05 was considered statistically significant.Results are presented as means±s.e.m. or ±s.d., with


The increase in the levels of reactive oxygen species (ROS) in acute myeloid leukemia (AML) patients has been previously described; thus, it is important to regulate ROS levels in AML.Flow cytometry were used to assess the in vitro effect of compound kushen injection (CKI). Quantitative proteomics were used to analyse the mechanism. The AML patient-derived xenograft (PDX) model were used to evaluate the in vivo effect of CKI.We found that intracellular ROS levels in AML cells were decreased, the antioxidant capacity were increased when treated with CKI. CKI inhibited the proliferation of AML cells and enhanced the cytotoxicity of AML cells, which has few toxic effects on haematopoietic stem cells (HSCs) and T cells. At the single-cell level, individual AML cells died gradually by CKI treatment on optofluidic chips. CKI promoted apoptosis and arrested cell cycle at G1/G0 phase in U937 cells. Furthermore, higher peroxiredoxin-3 (Prdx3) expression levels were identified in CKI-treated U937 cells through quantitative proteomics detection. Mechanically, the expression of Prdx3 and peroxiredoxin-2 (Prdx2) was up-regulated in CKI-treated AML cells, while thioredoxin 1 (Trx1) was reduced. Laser confocal microscopy showed that the proteins Prdx2 could be Interacted with Trx1 by CKI treatment. In vivo, the survival was longer and the disease was partially alleviated by decreased CD45+ immunophenotyping in peripheral blood in the CKI-treated group in the AML PDX model.Antioxidant CKI possess better clinical application against AML through the Prdxs/ROS/Trx1 signalling pathway.The online version of this article (10.1186/s13046-018-0948-3) contains supplementary material, which is available to authorized users. Acute myeloid leukaemia (AML) is a haematological malignant hyperplastic disease that originates in haematopoietic stem cells. A large number of abnormal cells in bone marrow accumulate and infiltrate other tissues due to out-of-control proliferation and differentiation, resulting in the inhibition of normal haematopoietic function and poor prognosis in AML patients. Inducing chemotherapy to inhibit AML cell proliferation and further strengthening chemotherapy to keep AML patients in a remission state is the main method for treating AML in the clinic. However, chemotherapy can lead to bone marrow failure in AML patients because of the many toxic side effects and chemotherapy complications, even due to bleeding or severe infection resulting in death, which are difficult challenges for the clinical treatment of AML.Jab1) and thioredoxin 1 (Trx1) expression; moreover, Jab1 binding to Trx1 affected Trx1 [Reactive oxygen species (ROS) are a second messenger that regulate normal physiological processes and maintain the intracellular REDOX balance. An ROS imbalance causes hydroxyl radicals to attack the DNA skeleton, causing DNA oxidation damage and increasing DNA mutation, leading to genome instability and cancer . Hole etted Trx1 . The higIn view of these results, we believe that reducing ROS levels and regulating the ROS pathway with high efficiency and low toxicity antioxidants will effectively improve survival in AML patients . We haveIn the clinic, our team has reported that CKI combined chemotherapy could effectively treat acute leukaemia, and 81.8% of AML patients could achieve complete remission; importantly, CKI could decrease the toxic side effects associated with chemotherapy . However2.Acute myeloid leukaemia THP-1, U937, HL60 and Molm-13 cells were obtained from the cell resource centre from Shanghai Institutes for Biological Sciences  and maintained in PRMI 1640 media containing 10% foetal bovine serum  with 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C and 5% COHuman cells were collected from 36 AML patients and 15 healthy controls; information on the collected clinical samples is provided in Additional file The haematopoietic stem cells (HSCs) and hyperleukocytic acute myeloid leukaemia cells were collected using a Fresenius COM.TEC machine from Zhongnan Hospital; the procedures were described in our previous reports . The perAfter AML cells were treated with CKI for 24 h, the collected AML cells were incubated with dichlorofluorescein diacetate (DCFH-DA) dye  for 25 min at 37 °C and then intracellular ROS levels were detected by flow cytometry . Cultured AML cells were collected to test T-AOC (No. A105), GSH (No. A006–1) and XOD (No. A002) with kits from the Nanjing Jiancheng Bioengineering Institute .2O2 concentration  and CAT activity  in mice blood were tested according to the manufacturer’s instructions.The T-AOC vitality, GSH content, H4 AML cells per well were seeded into a 96-well plate and subsequently cultured with a different dose of CKI for 48 h. Then, 10 μl CCK-8 reagent was added to each well and the plate was incubated for the appropriate amount of time. Then, the absorbance was measured at 450 nm using a microplate reader .Cell Counting Kit-8 (CCK8) assays were used to determine cell proliferation according to the manufacturer’s instructions . Briefly, 1 × 10Cell apoptosis was measured using an Annexin V-FITC/PI apoptosis kit (MultiSciences (Lianke) Biotech Co., Ltd.) according to the manufacturer’s instructions. The detailed methods for the apoptosis, JC-1 and cell cycle assays are shown in the Additional file Apoptotic cell morphology was detected by transmission electron microscopy (TEM). U937 cells were prepared as described previously . BrieflyFor drug toxic assays, the U937 cells were treated with eight different CKI dosages  for 48 h, and the inhibition rates were detected by MTT assay. The IC50 was calculated from a plotted graph with X representing the dosage and Y representing the inhibition rate in Excel. The cytotoxic effects on HSCs and T cells were examined by MTT assays to determine the cell death rate after CKI or Ara-c treatment for 48 h, respectively.The cytotoxicity and cell pore-forming activities  were meaTo detect the cytotoxicity of CKI at the single-cell level, microfluidics chips were designed to perform the injection and capture of single cells with optical technology –22. Brie5 Molm-13 GFP+ cells per well were treated with CKI for 48 h at 37 °C. Then, the fluorescence intensity changes of the cells were observed under a fluorescence microscope (OLYMPUS IX73) with intensity of 50 and a 121 ms exposure time.Molm-13 GFP+ cells were treated with 110 μl/ml CKI on optofluidics chips and the single-cell morphology were observed under a laser scanning confocal microscope . The images were captured using NIS-Elements (AR 4.50.00). In a 6-well plate, 5 × 10The proteins were degraded to peptides by tryptic digestion as previously described.  Then, thFor Western blot, the AML cells were lysed with RIPA containing PMSF to make cell lysates. In total, 30 μg of proteins measured using a BCA protein assay kit  were subjected to 15% SDS-PAGE for Western blot. After electrophoresis, the gels were transferred onto 0.22-μm PVDF membranes (Millipore Corp.) and the membranes were blocked with 5% BSA in 0.1% TBST (0.1% Tween-20 in 1 × TBS). The blot was probed with rabbit polyclonal antibodies against Prdx3  and Trx1 , and mouse monoclonal antibodies against Prdx2 , as primary antibodies, overnight at 4 °C. The membranes were incubated with HRP-conjugated goat anti-rabbit IgG  and goat anti-mouse IgG  as secondary antibodies for 1 h at RT and detected with the Immobilon™ Western Chemiluminescent HRP Substrate  using a Western Chemiluminescent Imaging System (Tanon 5200).Molm-13 and U937 cells were separately seeded into 6-well culture plates. After CKI treatment for 48 h, the cells were fixed in 4% paraformaldehyde (PFA) in 1 × PBS for 15 min at room temperature. The cells were first pre-treated and then blocked in 1 × PBS with 0.5% Triton X-100 and 5% BSA for 1 h. Next, the samples were incubated with anti-Prdx3 (1:100), Prdx2 (1:100), or Trx1 (1:100) antibodies overnight at 4 °C, followed by incubation with secondary antibodies conjugated to AF647 or CF568 (1:400 dilution). The samples were then analysed with a laser confocal microscope  with a 640 nm or 560 nm pulse. The images were collected with the Volivity software containing Acquisition, Quantitation, and Visualization modules.Five-week-old female B-NSG mice were obtained from Beijing Biocytogen Co., Ltd. (permission number: SCXK 2016–0004) and maintained in a pathogen-free animal facility in laminar airflow cabinets with a 12-h light/12-h dark schedule. The animals were fed an autoclaved rodent diet ad libitum. All procedures followed the institutional and national guidelines for the care and use of laboratory animals.+ cells were cultured in RPMI1640 medium (GIBCO) supplemented with 10% foetal bovine serum . At day 0, B-NSG female mice (five-week old) were given a 2.5 Gy χ-ray dose for total body irradiation, followed by an intravenous injection of harvested Molm-13 GFP+ cells (3.6 × 105 per mouse) or human AML patient cells (1.7 × 107 per mouse) separated from a hyperleukocytic AML patient via tail vein within 24 h of irradiation.The AML animal model , 28 and The leukaemia animal model was successfully established after injection of the cells via routine blood tests and peripheral blood cell smear tests. The mice were randomly distributed into two groups. The CKI group was intraperitoneally injected (i.p.) with CKI  at a dose of 50 mg/kg/mouse  once a day for 10 days, while the control groups received saline with the same volume of 200 μl i.p. once a day. All mice were sacrificed and the harvested tissue samples were used for further analysis.A routine blood examination was performed by a Coulter STKS automated blood cell analyser . The fluorescence intensity of the blood smear and bone marrow smear on slides was observed under a fluorescence microscope (OLYMPUS IX73). The blood smear and bone marrow smear were also stained via Wright’s staining and observed with a microscope under an oil immersion lens (OLYMPUS BX41) to measure the ratio of leukaemia cells and determine whether the AML models were successfully constructed. The immunophenotypes of CD117, CD45, CD33 and CD34 in mouse peripheral blood were also measured by flow cytometry.For the bone marrow biopsy, bone marrow collected from mouse thighs was fixed in formalin and embedded in paraffin for bone marrow examination. The sections were sliced using a slicing machine Leica RM2235). Immunohistochemistry was performed per our previously described method [5. ImmunoThe images were obtained with the Aperio versa 10.3.37 software using an ultra-high-resolution microscopic imaging system  at the Medical Research Institute at Wuhan University.http://biodb.swu.edu.cn/cgi-bin/wego/index.pl). The protein-protein interaction networks were constructed using STRING v9.1 (http://string-db.org/cgi/input.pl). For all statistical comparisons, the data were analysed using the parametric unpaired t test in GraphPad Prism (version 6.0). The survival curve was also constructed by SPSS 24.0. p < 0.05 was considered statistically significant. All results are presented as the mean ± standard error of the mean.The heat map was analysed with R code (V i386 3.0.1). The identified proteins showing changes were annotated by GO terms using BGI WEGO  through dimethyl labelling quantitative proteomics in U937 cells. The proteins from two groups were digested and labelled with two different dimethyl labelling reagents to allow for quantitation and identification by LC-MS/MS. The workflow is shown in Additional file The 288 DEPs were further annotated by Gene Ontology (GO) analysis. All DEPs were classified according to their biological process, cellular component and molecular function Fig. , and mosPrdx2 is epigenetically silenced in AML and causes the generation and accumulation of ROS, which promotes AML cell proliferation [Peroxiredoxin-2 (Prdx2) is another Prdxs family member located in cytoplasm that reduces intracellular ROS level. It has been reported that the tumour suppressor gene feration . Moreoveferation , and PrdIn this study, we detected the Prdx3 and Prdx2 proteins that resisted ROS levels. QPCR results showed that their mRNA levels were significantly increased in CKI-treated AML cell lines and AML patient cells  are shown in Additional file The anti-leukaemic effects of CKI were verified by construction of an AML animal model via a Molm-13 GFP+ cells injection. The construction of the AML model and administration of CKI to irradiated B-NSG mice (n = 19) were injected with human hyperleukocytic AML patient cells. The construction of the PDX model and CKI injection into B-NSG mice are shown in Fig. n = 9). Among the two AML animal models, the PDX AML animal model mice died faster, so they could not be observed for 29 days. At the 20th day, all mice were euthanized and acute myeloid leukaemia cells in the bone marrow smear were observed, which indicated that the PDX model was successfully constructed  model of acute myeloid leukaemia to investigate the anti-leukaemic effects of CKI. The B-NSG mice  is effective for treatment AML by regulating the ROS signalling pathway. CKI inhibited the proliferation of non-hyperleukocytic not only AML cells but also hyperleukocytic AML cells, even at the single-cell level, which indicated that CKI may be used for the treatment of hyperleukocytic AML patients after leukapheresis. Additionally, as we have already known, after chemotherapy, leukaemia stem cells (LSCs) and residual lesions are present. LSCs showed relatively low levels of ROS , but ext2O2environment, Prdx is irreversibly transformed into a highly oxidized derivative form (Prdx-SO3), which cannot maintain homeostasis levels of intracellular oxidative stress [ROS production and the peroxidase defence system are regulated and balanced and closely related to apoptosis and necrosis. The peroxiredoxins (Prdxs) family resists ROS and scavenges free oxygen radicals in the body, protecting intracellular genomic DNA, lipids and proteins from oxidative damage by regulating intracellular ROS levels. In an excessive He stress .2O2 detoxification and is used to regulate ROS levels. In mammals, Prdx-3 only accounts for 1.6% of soluble mitochondrial protein, but it is responsible for the detoxification of 90% of H2O2 [2O2 in the mitochondria [Mitochondria are the main source of intracellular ROS production. In mitochondria, Prdx3 is mainly involved in H of H2O2 . Under o of H2O2 . Studieschondria , and thechondria . We have2O2 or free oxygen radicals and regulate the REDOX state to control AML cell growth. However, whether CKI can regulate Prdx2 methylation and acetylation levels and affect Prdx2 expression is worth further investigation.Prdx2 is a Cys-dependent peroxidase that located in the cytoplasm. Agrawal-Singh et al. proposed that Prdx2 is a newly discovered tumour suppressor gene in AML. Histone H3 acetylation decreased and methylation increased on the Prdx2 promoter, leading to lower Prdx2 expression and the accumulation of ROS, which further promoted the malignant proliferation of AML cells and poor prognosis in AML patients. When Prdx2 expression was up-regulated, ROS levels decreased and AML cell growth was inhibited . In our Trx1 gene activated hypoxic induction factor 1 (HIF-1α) in cancer cells, increasing VEGF expression [Trx1 is a small molecular protein that plays important physiological functions, such as protein binding and transcriptional regulation , 43, andpression , 46 and pression . Overexppression . Followipression . Trx1 ispression . Trx1 ispression . As mentCKI inhibited intracellular ROS levels in AML cells by increasing Prdx2 and Prdx3 expression and decreasing Trx1 expression. The antioxidant CKI inhibits cell proliferation by regulating the ROS signalling pathway in AML and is a promising drug for the treatment of AML in the clinic. We aimed to explore the therapeutic efficacy of low-toxic natural antioxidants against AML by regulating ROS pathways and providing new strategies to improve survival in AML patients.Additional file 1:Figure S1. The component of CKI. (JPG 68 kb)Additional file 2Table S1. The information of new diagnosed AML patients. (XLSX 35 kb)Additional file 3:Supplemental methods. (DOCX 23 kb)Additional file 4:Figure S2. The SOD vitality after CKI treatment. (TIFF 518 kb)Additional file 5:Figure S3. The cell growth. Cell growth was tested by trypan blue staining to analyse the cell count after 110 μl/ml CKI treatment for 48 h. (TIF 882 kb)Additional file 6:Figure S4. Cell apoptosis and cell cycle for HL60 cells and THP-1 cells. (A) Cell apoptosis was tested by flow cytometry after HL60 cells were treated with 110 μl/ml CKI for 48 h. (B) Cell cycle was analysed by flow cytometry after HL60 cells were treated with CKI for 24 h. (C) Early apoptosis was determined by JC-1 assay after THP-1 cells were treated with 110 μl/ml CKI for 24 h. (TIF 2472 kb)Additional file 7:Figure S5. Workflow for the quantitative proteomics with dimethylation labelling after U937 cells were treated with CKI. (JPG 198 kb)Additional file 8:Table S2. The identified proteins by LC-MS/MS. (XLSX 74 kb)Additional file 9:Table S3. The identified 54 differentially expressed proteins. (XLSX 28 kb)Additional file 10:Figure S6. The proteins interacting with Prdx2 by STRING analysis. (TIF 1604 kb)Additional file 11:Figure S7. The mRNA expression levels of Prdx2 and Prdx3 after CKI treatment. (TIFF 785 kb)Additional file 12:Figure S8. The anti-leukaemic effects of CKI on B-NSG mice with Molm-13 GFP+ cell injections. (A) A schematic diagram of the AML animal model. (B) Analysis of the blood and bone marrow smears. At day 8, the blood was collected from the tail vein using a capillary tube at day 8 after injection of the Molm-13 GFP+ cells and blood smears were performed. At day 10, bone marrow smear analysis was performed to detect Molm-13 GFP+ cell targeting. The fluorescence intensity was observed by fluorescence microscopy. Magnification fold: ×20. At day 29, the bone marrow smears were stained and leukaemia cells were observed. Magnification fold: ×100 under an oil immersion lens. (C) The changes in body weight. (D) The survival analysis. (E) The tissue weight index. (TIF 4232 kb)


Acute myeloid leukemia (AML) patients with t constitute a small subgroup with a distinct genetic and clinical profile. We present a unique case of a female infant with monocytic AML associated with t, a rarely reported variation of t. The patient presented with leukemia cutis and demonstrated erythrophagocytosis in the diagnostic bone marrow. She responded well to standard AML chemotherapy and is currently in remission. Here, we highlight her case as the youngest AML patient with t described in the literature, discuss the significance and prognostic implications of this genetic variant, and review 8p11.2 fusion proteins in AML. KAT6A (lysine acetyltransferase 6A) with CREBBP (CREB-binding protein), and the gene expression profile of these leukemias resembles that of AML with MLL (mixed lineage leukemia) gene rearrangement [In acute myeloid leukemia (AML), genetic mutations are often associated with specific disease subtypes which have biologic and prognostic implications . While Aangement . Additioangement , 4, 6, 7angement . HoweverAML associated with t is an extremely rare variant of 8p11.2-rearranged acute leukemias. Although infrequently reported in the literature, it is believed to behave similarly to t AML –10. We r3/μL) with a normal differential, hemoglobin (11.1 g/dL) and platelet count (281 × 103/μL). Flow cytometry of the bone marrow aspirate revealed a distinct population of cells totaling 26% of the sample, expressing bright CD4, CD11c, dim CD13, CD15, CD16, and CD33, CD36, CD38, CD58, CD123, HLA-DR, myeloperoxidase (MPO), dim terminal deoxynucleotidyl transferase (TdT), and moderate CD45. These findings confirmed the diagnosis of AML with monocytic differentiation (French-American-British (FAB) M4). Striking erythrophagocytosis was noted in the bone marrow biopsy (in situ hybridization (FISH) was performed on a bone marrow aspirate smear using a KAT6A break-apart probe set , confirming a KAT6A rearrangement in metaphases   .The patient was treated per our institutional standard of care for AML patients. She received induction therapy with ADE which included cytarabine 3.3 mg/kg every 12 hours for days 1–10, daunorubicin 1.7 mg/kg for three doses on days 1, 3, and 5, and etoposide 3.3 mg/kg daily for days 1–5. She had no evidence of minimal residual disease (MRD), defined as <0.1% disease in bone marrow by flow cytometry, at the end of induction. Her subsequent chemotherapy included three more cycles of chemotherapy: Induction II with cytarabine 33 mg/kg every 12 hours on days 1–4 and mitoxantrone 0.4 mg/kg daily on days 3–6, Intensification I with cytarabine 33 mg/kg every 12 hours on days 1–5 and etoposide 85 mg/kg daily on days 1–5, and Intensification II with cytarabine 100 mg/kg every 12 hours on days 1-2 and 8-9 and Erwinia asparaginase 830 units/kg on days 2 and 9. Per institutional standard, she remained hospitalized following chemotherapy through count recovery. She tolerated each course of chemotherapy well with minimal toxicity and continues to remain in remission now two years from initial diagnosis.KAT6A (previously MOZ or MYST3) gene on chromosome 8 with the CREBBP gene on chromosome 16 [KAT6A and CREBBP modulate gene transcription and induce hematopoietic cell transformation [de novo AML [MLL (KMT2A)-rearranged leukemias. Median overall survival in two large cohorts was poor (4.7–8.5 months), which may be related to the high number of therapy-related AML cases most often seen following treatment for solid tumors in these groups [Cytogenetic abnormalities play a prominent role in risk stratification of AML, with tp11.2;p13)-associated AML demonstrating a unique phenotype . This trosome 16 . Both KA.2;p13-asnovo AML , 12. Mosde novo disease (60/62) and presented with M4/M5 FAB AML (97% compared to only 41% in the reference group). Leukemia cutis occurred more commonly in infants and with higher frequency (58% versus 8%) as compared to other AML patients. Selective activation of HOXA (homeobox A cluster) genes without activation of HOXB (homeobox B cluster) genes was noted in gene expression profiling, similar to KMT2A-rearranged AML. Disseminated intravascular coagulation (DIC) at presentation was noted in 15/38 of the t patients at diagnosis. A substantial subgroup of patients (17/62) was diagnosed in the first month of life which was significantly higher than the reference cohort. Seven of these seventeen infants were initially untreated and noted to have a spontaneous remission. Four patients subsequently developed recurrence requiring treatment; however, 5/7 patients were alive at last follow-up. Five-year overall survival rate for the entire cohort was 59% (±9%) similar to the rate for the reference group (62% ± 2%).Similar to adults, pediatric AML with tp11.2;p13) has been characterized as a distinct subgroup commonly associated with leukemia cutis and hemophagocytosis at diagnosis. In the largest pediatric evaluation of this translocation to date, the International Berlin-Frankfurt-Munster (I-BFM) AML study group reviewed 62 patients with t and compared them with a pediatric AML reference cohort [ has beende novo AML (4/5). Other coexisting cytogenetic mutations were rare (1/5), suggesting this is likely an initiating event in leukemogenesis. All patients with de novo disease had a favorable response to standard chemotherapy alone, and only one patient underwent stem cell transplantation. The one patient with therapy-related AML did not respond to initial chemotherapy and died shortly after.While t8;16)-associated AML is described in several studies, AML involving t8;19) has been rarely reported and is considered a subgroup of the t subtype. In 1988, Brizard et al. first noted the t variant in an 8-month-old infant with M5 AML and marked erythrophagocytosis [;16p11.2 [7, 9, 1 was recently identified in a patient with therapy-related AML to be the leucine twenty homeobox (LEUTX) gene located on chromosome 19q13 [CREBBP and LEUTX, four other KAT6A fusion partners have been identified, namely, EP300, NCOA2, NCOA3, and ASXL2 [CREBBP, all except ASXL2 and LEUTX contain a histone acetyltransferase domain. Cases involving KAT6A-CREBBP fusions have demonstrated gain of MYB and overexpression of HOXA9, HOXA10, HOXA11, CEBPA, LMO2, and PTPN6 genes [C-MYB gene is a downstream target of HOXA9 and upregulation of HOXA9, and other HOX genes have been implicated in leukemic transformation via increased self-renewal of leukemic stem cells [KAT6A leukemias are classified with M4/M5 AML with the exception of those having ASXL2 as a partner [The fusion partner of the me 19q13 . It playnd ASXL2 . SimilarN6 genes . C-MYB gem cells . KAT6A lAML associated with t remains a rarely described entity in both pediatric and adult patients. Similar to other infant cases of AML with t, our patient presented with leukemia cutis, demonstrated erythrophagocytosis in the bone marrow, and was able to achieve remission with standard AML therapy supporting the biologic similarities between the two disease subtypes. While there likely are similar underlying pathways for leukemogenesis between the two 8p11.2-translocated subgroups, our case suggests that the t variant is associated with a favorable prognosis in pediatric patients, similar to previously reported cases of t AML.


Ocotea duckei Vattimo (Lauraceae), were studied in rats using combined functional and biochemical approaches. In non-anaesthetized rats, yangambin  induced hypotension  accompanied by tachycardia . In isolated rat atria, yangambin (0.1 µM–1 mM) had very slight negative inotropic (Emax = 35.6% ± 6.4%) and chronotropic effects (Emax = 10.2% ± 2.9%). In endothelium-intact rat mesenteric artery, yangambin (0.1 µM–1 mM) induced concentration-dependent relaxation (pD2 = 4.5 ± 0.06) of contractions induced by phenylephrine and this effect was not affected by removal of the endothelium. Interestingly, like nifedipine, the relaxant effect induced by yangambin was more potent on the contractile response induced by KCl 80 mM (pD2 = 4.8 ± 0.05) when compared to that induced by phenylephrine. Furthermore, yangambin inhibited CaCl2-induced contractions in a concentration-dependent manner. This lignan also induced relaxation (pD2 = 4.0 ± 0.04) of isolated arteries pre-contracted with S(−)-Bay K 8644. In fura-2/AM-loaded myocytes of rat mesenteric arteries, yangambin inhibited the Ca2+ signal evoked by KCl 60 mM. In conclusion, these results suggest that the hypotensive effect of yangambin is probably due to a peripheral vasodilatation that involves, at least, the inhibition the Ca2+ influx through voltage-gated Ca2+ channels.The pharmacological effects on the cardiovascular system of yangambin, a lignan isolated from  Animals were housed under conditions of controlled temperature (21 ± 1 °C) and lighting (lights on: 06:00–18:00 h). In addition, they had free access to food  and tap water Ocotea duckei Vattimo (Lauraceae) according to the procedures previously described by Morais et al. i was measured with the fluorescent dye Fura-2/AM. The vascular smooth muscle cells were incubated with 5 µM Fura-2/AM for 40 min in a humidified atmosphere (5% CO2) at 37 °C. After loading with Fura-2/AM, the cells were immersed in Hank’s solution and placed in recording chambers maintained at 37 °C equipped with stage of a leitzdiavert inverted fluorescence microscope. Fluorescence Fura-2/AM emission was excited alternately at 340-nm and 380-nm bandpass filters mounted in a computer controlled by filter wheel . The cells that showed fluorescence above 106 counts were used. At least three independent experiments were performed using separate cells. To study the effect of yangambin on Ca2+ influx through voltage-gated Ca2+ channels, the vascular smooth muscle cells were stimulated with KCl (60 mM) before and after pre-incubation with yangambin , and changes in [Ca2+]i were recorded. i in vascular smooth muscle cells and consequent peripheral vasodilation.In conclusion, the present study, using combined