This study utilized 116 methanol serum extract samples from AML (n= 38), nHL (n= 26), CLL (n= 21) and HC (n= 31). Multivariate and univariate data analyses were performed to identify the most abundant changes among the studied groups. Complex and detailed VIP-PLS-DA models were calculated to highlight possible changes in terms of biochemical pathways and discrimination ability. Chemometric model prediction properties were validated by receiver operating characteristic (ROC) curves and statistical analysis. Two sets of eight important metabolites in HC/AML/CLL/nHL comparisons and five in AML/CLL/nHL comparisons were selected to form complex models to represent the most significant changes that occurred.Haematological malignancies are a frequently diagnosed group of neoplasms and a significant cause of cancer deaths. The successful treatment of these diseases relies on early and accurate detection. Specific small molecular compounds released by malignant cells and the simultaneous response by the organism towards the pathological state may serve as diagnostic/prognostic biomarkers or as a tool with relevance for cancer therapy management. To identify the most important metabolites required for differentiation, an  Haematological malignancies are clonal diseases. Neoplasms of the haematopoietic system are derived from myeloid lineage stem or progenitor cells, while the tumours of the lymphatic system originate from lymphoid lineage precursor or mature cells . The facThe purpose of the study was to determine the metabolic profiles of the three common haematological malignancies in adults, acute myeloid leukaemia (AML), non-Hodgkin's lymphoma (nHL) and chronic lymphocytic leukaemia (CLL), using an metabolomics approach, for highlighting potential important influence on organism metabolism caused by haematological malignancies and indication possible biomarkers.Altogether, 50 metabolites were assigned Figure  and 18 rThe obtained chemometric models, which were based on VIP-PLS-DA, could classify patients’ haematological malignancy samples into individual groups versus the healthy control group. Comparisons between the individual haematologic malignancies were also based on VIP-PLS-DA models  and showed lower model parameter values than comparisons with healthy individuals Table . In geneO-phosphocholine, glutamine, phenylalanine, tryptophan, glutamate, histidine, formate, 2-hydroxyisovalerate, citrate, sn-glycero-3-phosphocholine, ornithine, 2-oxoisocaproate, 2-hydroxybutyrate, alanine, 2-methylglutarate, betaine, sarcosine, creatine, acetate, proline, and choline.The analysis of the VIP-PLS-DA models calculated for three malignancies units and control group using a set of metabolites with the greatest discriminatory potential Figure . This po2X = 0.401, Q2 = 0.218). Hence, the following sets of identified compounds were chosen: (1) for HC – histidine, O-phosphocholine, and glutamine; (2) for AML – phenylalanine, glutamate and formate; (3) for CLL – choline, alanine, and proline and (4) for nHL– creatine, sarcosine, and acetate. Among the unidentified metabolites, unk_4 was important for HC, unk_16 for AML, and unk_2 for nHL, while no unidentified metabolites were important for CLL. Moreover, using a single metabolite in multiple groups was avoided. The introduced general comparisons shown in Figure training 1.00; AUCtest 0.975) and basic parameter values among the models  among models based on the HC vs. haematologic malignancy group comparisons. However, the separation between the selected groups was still observed.The loading plot Figure  of comprformate;  for CLL ls Table . In cont2X = 0.512, Q2 = 0.744)    Figure  - 2-hydrP values . ConsideP values , nine re) Figure , four of) Figure  based ont-test) in each of three comparisons between HC and the haematological malignancies  Figure .O-phosphocholine, glutamine, 2-oxoisocaproate, citrate, sarcosine, phenylalanine, ornithine, taurine, tryptophan, 2-hydroxyisovalerate, choline, lysine, glutamate, 3-methyl-2-oxovalerate, sn-glycero-3-phosphocholine, threonine, glucose, and proline. The unknowns resonance signals that were important in this comparison were unk_10, unk_4, unk_16, unk_11, unk_15, and unk_13. Among the metabolites, three based on loading plots were selected for each group. The following metabolites were important for the differentiation between the studied groups  nHL – sarcosine, 2-hydroxyisovalerate, and sn-glycero-3-phosphocholine  AMLe Figure .Three separate chemometric models were calculated to determine accurate differences between the specific cancer groups. The metabolites that were statistically significant and present in the VIP-PLS-DA model after the first iteration are shown in 2X = 0.459, Q2 = 0.383); phenylalanine, citrate, glutamine, O-phosphocholine, dimethyl sulfone, histidne, glutamate, choline, unk_11, unk_10, unk_7, and unk_18 are sorted in a descending order based on their VIP values , glutamine, 2-oxoisocaproate, citrate, proline, sn-glycero-3-phosphocholine, 2-hydroxyisovalerate, unk_10, unk_11, and unk_16   .T tests (dependent on Shapiro Wilk test results) are presented in Figure Among the differentiating metabolites, compounds that were statistically significant in at least two comparisons between the haematological cancers groups in M-W or Pathway analysis was performed on the overall data, which contained the HC, AML, CLL, and nHL groups, and indicated the importance of the most changed biochemical pathways. The determined major changes are associated with the following pathways : (1) D-glutamine and D-glutamate metabolism, (2) histidine metabolism, (3) alanine aspartate and glutamate metabolism, (4) arginine and proline metabolism, (5) glyoxylate and dicarboxylate metabolism, (6) aminoacyl-tRNA biosynthesis, (7) methane metabolism, (8) phenylalanine metabolism, (9) glycine serine and threonine metabolism, and (10) tryptophan metabolism.Haematological malignancies are a frequently diagnosed group of neoplasms and a significant cause of death induced by cancer , 14. TheIn AML cytogenetic and molecular abnormalities are included in European Leukemia Net (ELN) prognostic classification . The isoBoth in AML and CLL another important tools for measurement of minimal residual disease (MRD) is flow cytometry with high sensitivity. There is several studies showing that progression-free survival (PFS) and overall survival (OS) in patients MRD(-) is longer compared to MRD(+).Most of methods mentioned need to be validated in prospective clinical trials. Moreover, introduction of new drugs - FLT3 inhibitors, BCR inhibitors, bcl2 inhibitors may abrogate prognostic value of cytogenetic and molecular abnormalities. Therefore more comprehensive approach including molecular and metabolomics tools may lead to personally tailoring cancer management.Moreover, information about disease development could be valuable and supportive. The evaluation of changes in metabolite composition could be important for diagnosis, cancer stratification and as prognostic compounds with relevance for managing cancer therapies –20. The 1H NMR spectrum. This is reflected in the multivariate data analysis and statistics tests  were adopted, even when the model parameters were not as good as in the detailed comparisons (between two groups) . The groO-phosphocholine appears to be one of most important metabolite, as it is widely considered to be a compound associated with the carcinogenic processes. O-phosphocholine is involved in glycerophospholipid metabolism, which is coupled to cancer cell metabolism and development , 26O-phoAccording to the list of the most influential metabolites examined in this study, glutamine was at the second position. In the literature, glutamine is reported as an essential compound for cancer development . One of Among amino acids, a decrease in tryptophan levels (HC> CLL> AML> nHL) could indicate increased tryptophan catabolism, which might be connected to degradation via the kynurenine pathway. This has been linked to local shutdown of the immune system response by indoleamine 2,3-dioxygenase (IDO) to promote malignant cell proliferation , 33, 34.Another remarkable low molecular weight compound from the amino acids group in the haematological malignancies was phenylalanine. Its relative integral increased in all three haematological disorders groups AML>nHL>CLL>HC and it could be related to protein breakdown. The BCAAs typically associated with this process are not statistically significant and do not cover the same order. Moreover, phenylalanine could amplify the requirement for fuelling tricarboxylic acid cycle for more aggressive haematological malignance through phenylalanine, tyrosine and tryptophan metabolism .Our results showed that histidine negatively correlated with increased hematological diseases aggressiveness levels (CLL>HC>nHL>AML), which could be connected with inflammation processes , 39. TheThe identification of 2-hydroxyisovalerate levels (nHL>CLL>HC>AML) in serum extracts of the haematological units was an unexpected observation, as it is most frequently assigned in urine as a potential indicator of lactic acidosis . It was Considering the data originating from the general comparisons between the diseases (AML vs. CLL vs. nHL) Figures  and besi2-oxoisocaproate (ketoleucine) is a short-chain keto acid, it along with other keto acids may be connected to kynurenine aminotransferase functioning as an amino group acceptor. However, its levels (AML>CLL>nHL>HC) were greatly increased in both of the leukaemia groups, though not in the lymphoma group  43], wh, wh43], The tricarboxylic acid cycle essential metabolite citrate (nHL>HC>CLL>AML) had the lowest relative integral in AML among investigated groups, indicating the ability of increased TCA cycle functioning to be related to the increased activation of carcinogenesis processes. Despite the different values in the CLL and nHL groups, neither displayed the considerable citrate reduction observed in the AML group. This suggests that differences in the levels of this compound could be associated with the increased energy demand correlated with the rapid development of AML .The metabolism of sarcosine (nHL>HC>CLL>AML) leads to the rapid degradation to glycine, which demonstrates that serine and glycine metabolism may have an important impact on CLL and AML progression in contrast to nHL, it could also lead to betaine metabolism , 45.Ornithine (HC>AML>CLL>nHL) is one of the main compounds in the urea cycle. The relative integral of this metabolite was lower in all three studied haematological cancers compared to the healthy control group. However, in the nHL samples, the ornithine levels differences were significantly higher than in the AML and CLL groups. Ornithine is mostly produced from arginine in the urea cycle, but could be also biosynthesized from proline and glutamine , 47. In The nHL group also had the lowest levels of taurine (CLL>HC>AML>nHL), and displayed significant differences from the other two studied groups. It has been reported that taurine could be early biomarker of tumour formation in breast cancer . The litThe described changes can be responses amplified by tumour cells as well as global reactions by the entire organism to pathological conditions influenced by a variety of metabolites. The overall interpretation of the changes in the compound levels in serum samples could be difficult due to fact that metabolites within their biochemical pathways may intertwine and overlap during homeostasis disturbances and changes directly caused by cancer cell metabolism.Summing up discrimination of the studied cancers was possible based on the detailed chemometric calculation between healthy control and cancer groups as well as between them. The found list of compounds may be key to understand the metabolite biochemical changes occurring in selected cancers, but also potentially allow for the classification and discrimination of haematological malignancies, based on differences in small molecular compounds composition.Obtained models parameters were connected with the clinical symptoms assumptions and increased aggressiveness of the haematological units , and amplified by the changes in the biochemistry of metabolites. The discrimination based on the metabolomics approach was greater for the more aggressive units, which seems to be logical. This assumption was confirmed by the visible correlation between the quality and significance of the chemometric models compared to the average survival time in the groups (AML>nHL>CLL).Hypothetically, the obtained results showed that this metabolomics approach could allow for the additional verification of diagnosis or even faster assignment of individuals to specific cancers in the future. However more accurate and detailed biochemistry studies are required to understand the changes in metabolism and their functions in the selected haematological cancers.Peripheral venous blood samples were drawn from all the participants after overnight fasting for at least 8 hours. Blood samples were collected using Sarstedt S-Monovette system serum tubes  that were centrifuged at 1000 x g for 15 minutes at 4°C. The serum samples serum were stored in 1,5 ml Eppendorf safe-lock tubes and maintained at −80°C until analysis.The study group included 116 patients with an established diagnosis of one of three different haematological malignancies. There were 38 patients with acute myeloid leukaemia (AML), 26 patients with non-Hodgkin's lymphoma  and 21 patients with chronic lymphocytic leukaemia (CLL). All the subjects were patients at the Haematology Clinic of Wroclaw Medical University in Poland. All recruited patients were in the active phase of the disease. All of the subjects had never been treated for their cancers. The serum samples were taken prior to the initiation of chemotherapy.The control group consisted of 31 volunteers who were mainly recruited from familial doctors and from the Internal Diseases Clinic of Wroclaw Medical University in Poland. The volunteers were matched for age, sex, co-morbidities  and smoking habits to the haematological groups. Baseline demographic and medical characteristics for the haematological groups and controls are presented in The study protocol was approved by the Bioethical Committee of Wroclaw Medical University (KB - 41/2011) and each subject gave written informed consent.2O, and 330 μM TSP) and then 550 μl of the mixture was transferred to an NMR tube . Samples were maintained at 4°C before the measurements were taken.The collected serum samples were thawed at room temperature and vortexed. Then, 300 μl of serum was transferred to a new Eppendorf tube and mixed with 700 μl of cold methanol for protein precipitation. Next, the samples were homogenized  for 10 min at 50 Hz and then incubated for 20 min in −20°C. The homogenization step and incubation were then repeated. Subsequently, mixtures of serum-methanol were centrifuged for 30 min at 15 000 rpm at 4°C. Afterwards, 700 μl of supernatant was transferred to a new Eppendorf tube and then evaporated to dryness in a vacuum centrifuge (JWElectronic WP-03) for 5 h at 1500 rpm at 40°C. The dry precipitate was dissolved in 600 μl PBS  were recorded and processed for selected samples. Experiments performed included 1H−1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and 1H−13C heteronuclear single quantum correlation (HSQC). For the metabolomics workflow, the 1D spectra were processed with a line broadening of 0.3 Hz, manually phased, baseline-corrected using the MestReNova software (Mestrelab Research v 11.0) and referenced to a TSP signal δ = 0.0 ppm for the serum samples. Methanol and water resonance signals were removed from the data matrix. All the spectra were normalized to a TSP resonance signal. The alignment of the resonance signals was completed via a correlation optimized warping algorithm (COW) [icoshift algorithm and implemented in MATLAB  [The NMR spectra of the serum were recorded at 300 K using an Avance III spectrometer  operating at a proton frequency of 600.58 MHz. The NMR spectra of the were recorded by using a hm (COW)  and the 1H NMR spectra were assigned. The metabolite resonances were identified based on assignments published in the literature, the Chenomx software (v 8.2 Chenomx Inc.) and on-line databases  and Human Metabolome Data Base, (www.hmdb.ca). The NMR-measured metabolites were obtained as relative signal integrals of the non-overlapping resonance signals.A total 50 metabolites and 18 unknown resonance signals from the serum sample perfcurve function from the Matlab statistical tool-box  was adopted. Specificity and sensitivity were determined according to sample class prediction using the 7-fold cross-validated predicted values from the fitted Y-predcv (implemented in SIMCA-14 software) for observations in the model.Multivariate data analysis was performed using the SIMCA software . The sample order in the dataset was randomized. All the variables were scaled to unit variance (UV), and the samples for model calculation were split into two sample sets (training and test) based on the Kennard and Stone algorithm. The discriminant version of the Partial Least Squares regression (PLS-DA) with a default k-fold cross validation procedure was utilized to identify differences between the subgroups. To improve the obtained models, variable selection was conducted using VIP-plots with a confidence interval 0.95. Variables having VIP values below 0.8 were removed from the analysis. A single iteration was used to minimalize the overfitting models. New models were re-built based on selected variables, and their reliability were tested with a CV-ANOVA at a level of significance of p<0.05. The prediction performance of the VIP-PLS-DA models was estimated based on receiver operating characteristic (ROC) curves and area under curve (AUC) values. For this purpose, a The MetaboAnalyst 3.0 platform with selected features was used for metabolite data analysis. Pathway Analysis was performed on a relative integral matrix with only identified metabolites. All the data were scaled with Auto Scaling; the Pathway Enrichment Analysis was set on Global Test and the Pathway Topology Analysis was set on Relative-betweenness Centrality .t test using STATISTICA 12 (Statsoft Inc.). The K-W test was calculated for limitation to most influential metabolites in discus section. K-W test was performed on the metaboanalyst platform for this purpose and all data were auto-scaled  [The percent difference (PD) and relative standard deviation (RSD) for each assigned resonance signal were calculated. The percent difference was calculated based on the mean values of the metabolite's relative integral in each group. Data set with a relative integral were tested for the type of distribution based on the Shapiro-Wilk test. All assigned resonance signals were tested for statistical significance using an appropriate disturbance type test, either the Mann–Whitney–Wilcoxon or Student


However, cytokine-mediated changes in ion channel activity that alter β-cell Ca2+ handling remain unknown. Here we investigated the role of K+ currents in cytokine-mediated β-cell dysfunction. Kslow currents, which control the termination of intracellular Ca2+ ([Ca2+]i) oscillations, were reduced following cytokine exposure. As a consequence, [Ca2+]i and electrical oscillations were accelerated. Cytokine exposure also increased basal islet [Ca2+]i and decreased GSCI. The effect of cytokines on TALK-1 K+ currents were also examined as TALK-1 mediates Kslow by facilitating [Ca2+]ER release. Cytokine exposure decreased KCNK16 transcript abundance and associated TALK-1 protein expression, increasing [Ca2+]ER storage while maintaining 2nd phase GSCI and GSIS. This adaptive Ca2+ response was absent in TALK-1 KO islets, which exhibited decreased 2nd phase GSCI and diminished GSIS. These findings suggest that Kslow and TALK-1 currents play important roles in altered β-cell Ca2+ handling and electrical activity during low-grade inflammation. These results also reveal that a cytokine-mediated reduction in TALK-1 serves an acute protective role in β-cells by facilitating increased Ca2+ content to maintain GSIS.Cytokines present during low-grade inflammation contribute to β-cell dysfunction and diabetes. Cytokine signaling disrupts β-cell glucose-stimulated Ca As the incidence of T2DM is rapidly increasing, it is important to identify better therapeutic options for reducing β-cell failure during the pathogenesis of the disease. Low-grade inflammation is a key contributor to β-cell dysfunction in T2DM8. Conditions of over-nutrition and inactivity result in low-grade systemic inflammation during which pro-inflammatory cytokine concentrations (e.g. tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ)) increase several fold over basal levels10. For example, in a rat model of T2DM pancreatic cytokine levels were all elevated above nontreated controls (e.g. TNF-α increased from 24.3 ± 3.6 pg/mg protein to 47.9 ± 3.5 pg/mg protein (P < 0.05), IL-1β increased from 25.5 ± 2.7 pg/mg protein to 29.2 ± 1.7 pg/mg protein (P < 0.05), and IFN-γ increased from 49.4 ± 4.2 pg/mg protein to 65.1 ± 6.7 pg/mg protein (P < 0.05))11. The presence of these cytokines contributes to insulin resistance and diminished β-cell function5. Under stressful conditions (e.g. glucolipotoxicity) β-cells are also capable of secreting pro-inflammatory cytokines, which damage islet function12. Cytokine-mediated islet dysfunction correlates with increased basal intracellular Ca2+ ([Ca2+]i), reduced glucose-stimulated Ca2+ influx (GSCI), increased [Ca2+]i oscillation frequency, altered endoplasmic reticulum (ER) Ca2+ ([Ca2+]ER) storage, and increased apoptotic signaling13. While chronic low-grade inflammation leads to β-cell dysfunction in T2DM, the mechanisms responsible remain unresolved. Understanding how cytokines disrupt islet Ca2+ handling may illuminate therapeutic targets for preventing β-cell failure during T2DM.Failure of β-cells to secrete sufficient insulin precedes the onset of type 2 diabetes mellitus (T2DM)2+ channels (VDCCs) that are controlled by ion channel-mediated changes in plasma membrane potential (Vm)15. Thus, cytokine-mediated perturbations in Ca2+ handling would be predicted to result from changes in ion channel activity or expression. For example, accelerated islet [Ca2+]i oscillations may be influenced by changes in K+ currents that control [Ca2+]i oscillations such as Ca2+-activated K+ (KCa), ATP-sensitive K+ (KATP) channels, and/or the two-pore domain K+ (K2P) channel TALK-117. Kslow is a KCa current controlled in part by [Ca2+]ER release that senses and controls [Ca2+]i by hyperpolarizing β-cell Vm22. In β-cells it is believed to be composed of intermediate conductance (IK) KCa, apamin-insensitive small conductance (SK) KCa, and/or KATP channels22. TALK-1, while not sensitive to Ca2+, tunes islet [Ca2+]i oscillation frequency, partly by facilitating [Ca2+]ER release and activating Kslow27. Interestingly, the transcript abundance of some of these K+ channels is decreased following cytokine exposure, including KCNK16 (gene encoding TALK-1) and ABCC8 (gene encoding sulfonylurea receptor 1 (SUR1))28. Likewise, mitochondrial function is reduced following cytokine exposure, which decreases ATP production and would be predicted to activate KATP channels29. This suggests that cytokine-induced changes in K+ channel function modulate [Ca2+]i oscillation frequency during the pathogenesis of T2DM.Calcium enters β-cells through voltage-dependent Ca2+]i we investigated the electrophysiological mechanisms responsible for defective β-cell Ca2+ handling during low-grade inflammation. A cytokine-mediated increase in β-cell electrical oscillations was identified, which resulted from Vm depolarization and shorter interburst interval duration. Furthermore, these results demonstrated that Kslow and TALK-1 currents modify β-cell Ca2+ handling to preserve GSIS during acute low-grade inflammation representative of T2DM.To further reveal how cytokines dysregulate β-cell [CaKCNK16 (encodes TALK-1) and ATP2A2 (encodes sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b)) transcript in mouse islets following cytokine exposure . Cytokine exposure increased basal [Ca2+]i in WT islets  and stimulatory conditions  and plateau Vm ) in whole islets. Cytokines depolarized WT β-cell resting Vm . The resulting Kslow currents were biphasic, composed of a rapidly decaying fast phase followed by a gradually declining slow phase  transcript levels were indistinguishable in WT and TALK-1 KO islets. The insulin content of WT islets decreased to a level similar to TALK-1 KO islets following cytokine exposure . Insulin secretion at 2 mM glucose was indistinguishable in WT and TALK-1 KO islets (WT 7.26 ± 1.19 pg/islet/hr and TALK-1 KO: 7.67 ± 0.84 pg/islet/hr) and cytokine exposure increased basal secretion equivalently in WT and TALK-1 KO islets  tests were used to determine statistical significance between test groups as appropriate. P < 0.05 was considered statistically significant and P < 0.10 was considered to be trending toward significance.slow and TALK-1 currents are important contributors to cytokine-mediated changes in β-cell electrical activity and Ca2+ handling. Cytokine exposure reduced Kslow, which was accompanied by increased electrical excitability and [Ca2+]i oscillation frequency. These cytokine-induced changes were caused in part by reduced [Ca2+]ER release, which resulted from diminished TALK-1 expression. Moreover, during low-grade inflammation, downregulation of TALK-1 preserved GSIS presumably by maintaining 2nd phase GSCI. However, β-cells without functional TALK-1 channels failed to undergo cytokine-induced compensatory Ca2+ changes leading to defective GSCI and GSIS. Together these data suggest that TALK-1 and Kslow play important roles in β-cell adaptation to acute low-grade inflammation.Here we demonstrated that both K2+ handling, the electrical signaling mechanisms responsible for these changes have not been characterized13. Many ion channels contribute to physiological control of β-cell electrical excitability and some of these channels become perturbed during conditions of low-grade inflammation31. For example, TALK-1 K+ flux plays a key role in modulating β-cell electrical excitability and expression of the gene that encodes TALK-1 (KCNK16) is reduced during inflammation28. As predicted based on knockout of the channel, cytokine-mediated loss of TALK-1 currents depolarized β-cell Vm, which was accompanied by elevated electrical and [Ca2+]i oscillation frequency. TALK-1 KO β-cells displayed a similar cytokine-induced increase in excitability, but Vm was not changed and 2nd phase GSCI was decreased. The frequency of β-cell [Ca2+]i oscillations controls insulin secretion32. Therefore, by lowering TALK-1 expression β-cells were able to increase [Ca2+]i oscillation frequency and acutely maintain physiological [Ca2+]i levels during 2nd phase GSCI in response to low-grade inflammation34. However, when [Ca2+]i oscillations become too rapid (e.g. in TALK-1 KO islets following cytokine exposure) the amplitude of [Ca2+]i oscillations begins to decrease35. Thus, in TALK-1 KO islets [Ca2+]i levels declined during 2nd phase GSCI along with [Ca2+]i oscillation amplitude. Disruption of islet [Ca2+]i oscillations is a hallmark of metabolic stress in obese rodent models like those on a high fat diet (HFD) or without leptin receptors37. The deterioration of orderly [Ca2+]i oscillations in islets is also believed to contribute to the loss of pulsatile insulin secretion observed in T2DM, which leads to insulin resistance and hyperglycemia39. Thus, β-cells accelerated [Ca2+]i oscillations to maintain GSCI during acute low-grade inflammation; however, with chronic inflammation the Ca2+ response continued to change, eventually resulting in reduced islet [Ca2+]i and GSIS. Although TALK-1 plays a role in the adaptation of β-cells to low-grade inflammation, [Ca2+]i oscillations were still accelerated in TALK-1 KO islets following cytokine exposure, which implicates other ion channels in these cytokine-induced β-cell effects.Although it has been established that low-grade inflammation perturbs β-cell Caslow current is the primary contributor to β-cell Vm repolarization following the slow waves of depolarization that mediate [Ca2+]i oscillations22. Following cytokine exposure β-cell Vm became depolarized during the electrically silent periods between plateau potentials and the frequency of electrical oscillations increased. This suggests that cytokines affect one or more KCa channels that make up the Kslow current. Indeed, pharmacological inhibition of Kslow accelerates islet [Ca2+]i oscillations similar to cytokine exposure22. As anticipated, our results showed that low level cytokine exposure was sufficient to significantly reduce the magnitude and duration of Kslow in WT β-cells. The Kslow current was biphasic in nature, likely because the current is comprised of multiple K+ channels with varying degrees of Ca2+ sensitivity as well as differing activation and inactivation kinetics. Furthermore, it has been established that [Ca2+]ER release contributes significantly to Kslow activation21. Therefore, Kslow kinetics may be influenced by the temporal profile of ER Ca2+-induced Ca2+ release (CICR) or the proximity of KCa channels to [Ca2+]ER release sites40. TALK-1 has been shown to act as a counter-current for Ca2+ release from the ER of β-cells and indeed genetic ablation of TALK-1 increases β-cell [Ca2+]ER storage and reduces the magnitude of Kslow27. Thus, cytokine-mediated reduction in ER TALK-1 likely contributes to reduced Kslow currents by preventing efficient release of Ca2+ from ER stores. Likewise, under conditions of β-cell stress (e.g. low-grade inflammation) SERCA expression is reduced, which would be expected to perturb [Ca2+]ER and further diminish Kslow currents41. Cytokine-induced reduction of Kslow caused more frequent and shorter interburst silent phases in β-cell electrical activity during glucose stimulation. Due to these effects on electrical activity, islet [Ca2+]i oscillations were accelerated, amplitude was reduced, and [Ca2+]i was elevated at the nadir between oscillations. In the future, it will be important to identify the KCa channel(s) responsible for the reduction in Kslow currents following cytokine exposure and to determine if these channels can be manipulated to improve β-cell function during low-grade inflammation present during the pathogenesis of T2DM.Activation of a K2+]ER homeostasis is essential for many biological processes including protein expression, folding, and transport, dysregulation of this equilibrium could also lead to unfolded protein response (UPR) and ER stress42. In fact, pro-inflammatory cytokine exposure has been shown to cause ER stress, hence it is plausible that cytokine-induced disruption of [Ca2+]ER handling contributes to β-cell dysfunction and destruction43. Interestingly, the islets of TALK-1 KO mice on a long-term (20 week) HFD displayed reduced ER stress markers27. Cytokine exposure increased [Ca2+]ER levels to those observed in TALK-1 deficient β-cells, which may help mitigate β-cell ER stress during acute low-grade inflammation. However, it is well-established that prolonged cytokine exposure depletes β-cell [Ca2+]ER levels44. This results in part from diminished islet SERCA expression observed under low-grade inflammatory conditions and in T2DM patients46. Reduced SERCA expression should reduce [Ca2+]ER refilling and thus lower [Ca2+]ER stores. These data suggest that loss of TALK-1 expression preserves [Ca2+]ER levels during acute inflammation, but may not compensate for the loss of [Ca2+]ER uptake during chronic low-grade inflammation. Further studies are required to fully determine the importance of TALK-1-mediated elevation of β-cell [Ca2+]ER levels in response to acute and chronic low-grade inflammation.Because [Ca2+]i and electrical oscillations, low-grade inflammation resulted in increased basal [Ca2+]i at 2 mM glucose and decreased 1st phase GSCI at 11 mM glucose. While these changes in β-cell Ca2+ handling could be partly due to impaired [Ca2+]ER uptake (e.g. reduced SERCA activity), other mechanisms are anticipated to play a significant role7. For example, cytokine exposure diminishes mitochondrial ATP production, which could decrease 1st phase GSCI by enhancing KATP channel activity and thus limiting β-cell Vm depolarization47. Cytokine exposure also activates voltage-dependent Ca2+ channels (VDCCs), which would be predicted to elevate basal β-cell [Ca2+]i levels31. This suggests that the global changes in ion channel activity (e.g. KATP and VDCC activation) only modestly depolarize β-cell Vm. However, cytokine-induced depolarization of β-cells at rest (2 mM glucose) diminishes glucose-stimulated Vm depolarization. This decreased glucose-stimulated ΔVm leads to reduced GSCI following cytokine exposure. Interestingly, islets of T2DM patients display elevated basal insulin secretion and a defective 1st phase GSIS response, which would be projected to result from increased basal islet [Ca2+]i and decreased 1st phase GSCI49. In the future, it will be important to determine how these cytokine-mediated changes to islet Ca2+ handling contribute to perturbations in GSIS during the pathogenesis of T2DM.In addition to accelerating islet i resulting in part from VDCC activation31. GSIS from WT islets was unaffected by acute cytokine exposure, but GSIS from TALK-1 KO islets was decreased by approximately 50% at 11 mM glucose. This suggests that acute augmentation of β-cell [Ca2+]i by reducing TALK-1 expression maintains physiological GSIS following cytokine exposure. However, this adaptive GSIS response becomes less robust when TALK-1 is absent for an extended period of time (i.e. TALK-1 KO), which impairs 2nd phase GSCI and GSIS. TALK-1 KO β-cells may compensate for channel loss over time, and as a result they become more sensitive to low-grade inflammation. TALK-1 KO β-cells are also more sensitive to glucose, displaying higher GSIS at lower glucose concentrations14. Thus, it is possible that the readily releasable insulin granule pool was depleted under these conditions, resulting in decreased GSIS. Interestingly, without cytokines TALK-1 KO islets contained less insulin protein and Ins2 transcript than WT islets even though their GSIS was greater. The enhanced secretory ability of TALK-1 KO islets likely feeds back and limits insulin expression. As insulin content becomes equivalent in WT and TALK-1 KO islets following cytokine exposure, decreased GSIS from TALK-1 KO islets was not likely due to defective insulin expression. While WT islets responded to cytokines with increased secretory capacity, TALK-1 KO islets may have been maximally active and thus not capable of further amplifying GSIS. It is probable that changes in islet GSIS under acute low-grade inflammatory conditions are related to the Ca2+-modulatory function of islet TALK-1 and Kslow currents. By downregulating TALK-1 channels and inhibiting KCa channel activity, normal GSIS is maintained in the short-term.Not only do cytokines affect β-cell electrical excitability and Caslow currents are reduced during low-grade inflammation resulting in increased β-cell electrical and [Ca2+]i oscillation frequency. Cytokine-induced downregulation of TALK-1 contributes to decreased Kslow activity by reducing [Ca2+]ER release. Importantly, these changes acutely preserve normal β-cell [Ca2+]i levels during 2nd phase GSCI and thus help maintain GSIS during low-grade inflammation. These results also predict that during chronic inflammation loss of TALK-1 impairs β-cell Ca2+ handling resulting in defective GSIS.In conclusion, KUnless otherwise stated all research materials were obtained from Thermo Fisher Scientific and Sigma-Aldrich.Guide for the care and use of laboratory animals, Eighth edition (2011)  and in line with guidelines approved by the Vanderbilt University Animal Care and Use Committee (protocol # M1600063-00). Wild type (WT) and TALK-1 knockout (KO) islets were isolated from the pancreata of age-matched 6- to 12-week old C57B16/J mice as previously described52. Islets were dispersed using 0.0075% trypsin-EDTA and cultured overnight on poly-d-lysine coated 35 mm glass-bottom dishes in RPMI 1640 supplemented with 15% FBS, 100 IU∙ml−1 penicillin, and 100 mg∙ml−1 streptomycin (islet media) at 37 °C, 5% CO2. Alternatively, whole islets were cultured overnight on poly-d-lysine coated glass-bottom dishes in islet media. Unless otherwise specified, after overnight incubation all samples were supplemented with either fresh islet media or islet media with cytokines  and used for testing within 48 hrs.Animals were handled in compliance with the 2 in 6-cm petri dishes in islet media or in islet media with cytokines. RNA was isolated with TRIzol Reagent as per manufacturer’s instructions. Islet cDNA was prepared from RNA with SuperScript IV Reverse Transcriptase using an S1000 Thermal Cycler (Bio-Rad). Transcript abundance relative to GAPDH was determined by qRT-PCR using primers specific to TALK-1 , SERCA2b , preproinsulin 1 , preproinsulin 2 , proprotein convertase 1 , and GAPDH  with a CFX Real time PCR Instrument (Bio-Rad).WT or TALK-1 KO mouse islets were cultured for 24 hrs at 37 °C, 5% COhttps://iidp.coh.org/). The IIDP obtained informed consent for deceased donors in accordance with NIH guidelines prior to reception of human islets for our studies. Work detailed here was approved by the Vanderbilt University Health Sciences Committee Institutional Review Board (IRB# 110164). Islet donor information is provided in Table Human islets were provided through an approved protocol by the Integrated Islet Distribution Program (IIDP) , then the PFA was removed and the gels were transferred to 3 mL of cold PFA in a 12 well plate for 15 min (Corning). After fixation the gels were embedded in paraffin and processed into 5 µm slices. Rehydrated slices were stained with rabbit anti-TALK-1 (1:175) and guinea pig anti-insulin  followed by secondary antibodies (1:500 anti-rabbit Alexa Fluor 488 and 1:500 anti-guinea pig Alexa Fluor 647 (Jackson ImmunoResearch Laboratories)). Immunofluorescence was imaged using a Nikon Eclipse TE2000-U microscope equipped with an epifluorescence illuminator (Sutter Instrument Company), a CCD camera , and Nikon Elements software . TALK-1 immunofluorescence was quantified using the ImageJ Fiji image processing pack.WT mouse islets or human islets were cultured overnight in islet media or islet media with cytokines. The islets were washed with cold PBS and resuspended in 150 µL of 2.25 mg/mL rat tail collagen I supplemented with 1x DMEM, 200 mM HEPES, and 75 mg/mL sodium bicarbonate on ice. The samples were incubated for 40 min in a 96 well-plate at 37 °C, 5% CO2. The islets were washed twice with Krebs-Ringer–HEPES buffer (KRHB) with (in mmol/L) 119.0 NaCl, 2.0 CaCl2, 4.7 KCl, 25.0 HEPES, 1.2 MgSO4, 1.2 KH2PO4 (adjusted to pH 7.4 with NaOH) then incubated 20 min in KRHB with 2.0 mM glucose (KRHB-2mM) at 37 °C, 5% CO2. Islet [Ca2+]i was measured every 5 sec as a ratio of Fura-2 AM fluorescence at 340 and 380 nm (F340/F380) with a Nikon Eclipse TE2000-U microscope and the data was analyzed using Nikon Elements software. The islets were perifused at 37 °C with a flow of 2 mL/min KRHB-2mM for 2 min then perifused under identical conditions with KRHB with 11.0 mM glucose (KRHB-11mM) for 30 min.WT and TALK-1 KO mouse islets were loaded with 2 µM Fura-2-acetoxymethyl ester (AM) for 25 min at 37 °C, 5% CO2+-free KRHB-11mM with 125 µM diazoxide (Enzo), then incubated 10 min in Ca2+-free KRHB-11mM with 125 µM diazoxide. Islet [Ca2+]i was measured every 5 sec as a ratio of Fura-2 AM fluorescence at 340 and 380 nm (F340/F380) with a Nikon Eclipse TE2000-U microscope and the data was analyzed using Nikon Elements software. The cells were perifused at 37 °C with a flow of 2 mL/min KRHB-11mM with 100 µM diazoxide for 2 min then perifused with KRHB-11mM with 100 µM diazoxide and 50 µM cyclopiazonic acid (CPA) for 3 min to release [Ca2+]ER into the cytoplasm. Flow was then stopped and the Fura-2 AM imaging allowed to continue for 15 min.WT and TALK-1 KO mouse islet cells were loaded with Fura-2 AM, washed twice with Ca2+ probe D4ER then transferred to islet media in poly-D-lysine coated glass-bottom dishes for 48 hrs at 37 °C, 5% CO2. After 48 hrs, the islet media was replaced with fresh islet media or islet media with cytokines and the islets were cultured an additional 24 hrs at 37 °C, 5% CO2. The islets were washed twice with KRHB-11mM with 100 µM diazoxide then incubated 10 min in KRHB-11mM with 100 µM diazoxide. [Ca2+]ER concentrations were measured every minute as a ratio of D4ER fluorescence at 535 and 475 nm (F535/F475) using a Zeiss Confocal Laser Scanning Microscope equipped with Zeiss ZEN software.WT and TALK-1 KO mouse islets were cultured for 2 hrs with a 1000x multiplicity of inflection (MOI) of the ER-targeted Ca2+-free KRHB-11mM with (in mmol/L) 0.2 tolbutamide , 10.0 tetraethylammonium chloride hydrate (TEA), and 1.0 ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) (adjusted to pH 7.4 with NaOH). Patch electrodes (6–12 MΩ) were filled with voltage clamp intracellular solution (IC) with (in mmol/L) 140.0 KCl, 1.0 MgCl2, 10.0 EGTA, 10.0 HEPES, and 8.0 Mg-ATP (adjusted to pH 7.2 with KOH). For all voltage-clamp recordings the command voltage was held at −80 mV for 15 sec then increased from −120 mV to +60 mV over a period of 1 sec.WT and TALK-1 KO mouse islet cells were washed twice with KRHB-11mM. A whole-cell patch clamp technique was employed to record TALK-1-like K2P currents in single β-cells using an Axopatch 200B amplifier in voltage clamp mode with pCLAMP10 software (Molecular Devices). Cells were patched in KRBS-11mM, then in order to isolate K2P channel currents flow was switched to CaVm IC with (in mmol/L) 140.0 KCl, 1.0 MgCl2, and 5.0 HEPES (adjusted to pH 7.2 with KOH) supplemented with 20 µg/mL amphotericin B. The Vm of individual β-cells within islet clusters (10–20 cells) was recorded in current clamp mode using an Axopatch 200B amplifier with pCLAMP10 software. The electrical activity of patched β-cells was recorded for at least 10 min in KRHB-11mM then flow was changed to KRHB-2mM until action potential (AP) firing ceased for at least 3 min and baseline Vm stabilized. Cells were identified as β-cells if electrical activity ceased with 2 mM glucose.WT and TALK-1 KO mouse islet clusters were washed twice with KRHB-11mM then incubated in KRHB-11mM for 20 min. Patch electrodes (4–6 MΩ) were filled with slow currents using an Axopatch 200B amplifier with pCLAMP10 software. Patch electrodes (6–12 MΩ) were filled with Kslow IC with (in mmol/L) 76.0 K2SO4, 10.0 KCl, 10.0 NaCl, 1.0 MgCl2, and 5.0 HEPES (adjusted to pH 7.35 with KOH). For all Kslow electrophysiological recordings the command voltage was held at −40 mV for 7 sec, ramped between −40 and 20 mV for 5.2 sec , and returned to −40 mV for 20 sec.Mouse islet cells were washed twice with KRHB-11mM. Single cells were patched in KRBS-11mM and a perforated patch clamp technique was employed to record β-cell K2. Islets were cultured in equilibration media (DMEM (no glucose) with 10% FBS, 0.5 mg/mL BSA, 10 mM HEPES, and 0.5 mM CaCl2) supplemented with 5.6 mM glucose for 1 hr at 37 °C, 5% CO2. For insulin secretion, 20 islets were picked on ice into a 24-well plate (Corning) containing 500 µL of secretion media (DMEM (no glucose) with 0.5 mg/mL BSA, 10 mM HEPES, and 0.5 mM CaCl2) supplemented with 2 mM or 11 mM glucose, then insulin secretion was initiated for 1 hr at 37 °C, 5% CO2. After 1 hr, the plate was transferred to ice for 10 min to halt secretion and supernatants were collected in low retention 1.6 mL centrifuge tubes, which were stored at −20 °C until analyzed. Supernatants were supplemented with 1:100 mammalian protease inhibitor cocktail (MPIC). Whole islet insulin was extracted overnight at 4 °C into 400 µL of acid ethanol. Islet secretion supernatants and protein extracts were analyzed by the Vanderbilt Hormone Assay and Analytical Services Core (supported by NIH grants DK059637 and DK020593).WT and TALK-1 KO mouse islets were cultured overnight in islet media (supplemented with 0.5 mg/mL BSA) or islet media with cytokines at 37 °C, 5% COAll data are available upon request from the corresponding author.Supplemental Information


Here, we demonstrated that multiple H+ sensors, such as ASICs, TRPV1 and proton-sensing G protein coupled receptors (GPCRs) are involved in extracellular acidification-induced intracellular calcium ([Ca2+]i) elevation. By using calcium imaging measures, we observed that both ASIC and TRPV1 channels inhibitors suppressed the [Ca2+]i elevation induced by extracellular acidosis in cultured rat cardiac myocytes. Then, both channels mRNA and proteins were identified by RT-PCR, western blotting and immunofluorescence. ASIC-like and TRPV1-like currents were induced by extracellular acidification, suggesting that functional ASIC and TRPV1 channels jointly mediated extracellular calcium entry. Furthermore, either pre-exhaustion of sarcoplasmic reticulum (SR) Ca2+ with thapsigargin or IP3 receptor blocker 2-APB or PLC inhibitor U73122 significantly attenuated the elevation of [Ca2+]i, indicating that the intracellular Ca2+ stores and the PLC-IP3 signaling also contributed to the acidosis-induced elevation of [Ca2+]i. By using genetic and pharmacological approaches, we identified that ovarian cancer G protein-coupled receptor 1 (OGR1) might be another main component in acidosis-induced release of [Ca2+]i. These results suggest that multiple H+-sensitive receptors are involved in “Ca2+ transients” induced by acidosis in the heart.Acidosis has been known to cause “Ca Several studies have shown that the increase of [Ca2+]i is induced by the influx of extracellular Ca2+ via membrane Ca2+ channels or Na+/Ca2+exchangers32+ also can be resulted from the mobilization of sarcoplasmic reticulum (SR) during acidosis5+-sensitive ion channels or receptors, such as acid-sensing ion channels (ASICs), transient receptor potentialvanilloid-1 (TRPV1) and a group of proton sensing G protein coupled receptors (GPCRs). All of them could be activated by acidosis and mediate “Ca2+ transients”.Acidosis is a noxious stimulus that often comes from inflammation, ischemia or other pathological conditions. It is known to cause “CaC. elegans. Until now, the proton-sensitive members have found to be encoded by four different genes (ASIC1-ASIC4) with six protein subunits cloned, which are ASIC1a (ASIC or BNaC2), ASIC1b (ASIC1β), ASIC2a , ASIC2b (MDEG2), ASIC3 (DRASIC) and ASIC4 (SPASIC)78+ ions. Some of ASIC subunits, such as homomeric ASIC1a2+.ASICs belong to amiloride-sensitive epithelial sodium channel family in vertebrates and the degenerin family of 2+ TRPV1 is a polymorphic sensor to sense various stimuli, such as pain, temperature, tension and acid12Ovarian cancer G protein-coupled receptor 1 (OGR1), T cell death-associated gene 8 (TDAG8), G protein coupled receptor 4 (GPR4) and G2 accumulation (G2A) are all belong to proton-sensing GPCRs family, they share 40–50% homology with different action modes and signaling pathways2+ transients” and above H+-sensitive ion channels or receptors. During the process of diseases that cause acidification, ASICs, TRPV1 and proton-sensing GPCRs would sense external changes of pH value, then induce the opening of ion channels and influx of calcium ion, resulting in the activation of various intracellular signaling cascades. Therefore, cardiac H+ sensors will have an important significance in cardiac diseases.Here, we investigate the relationship between cardiac “Ca2+ imaging measurements in cultured rat cardiac myocytes. As shown in 2+]i) was observed when the pH value of extracellular solution was rapidly lowered from 7.4 to 6.0 or 5.0. This elevation is reversible and could be stably induced when another acidosis solution was given again. The peak of ΔF/F was 1.37 ± 0.08 at pH 6.0 (n = 15 cells) and 2.93 ± 0.22 at pH 5.0  inhibited the increase of [Ca2+]i by 50.07 ± 5.70%  and 46.67 ± 4.25% , respectively. The inhibitory effect was mostly recovered after washout  with 100 μM amiloride, and recovered mostly after washout. Thus, ASIC currents in cardiomyocytes displayed similar electrophysiological property as that in nervous system. Local decrease in extracellular solution to pH 7.0 was sufficient to induce an ASIC-like current, and the amplitudes of ASIC current were increased gradually along with the decrease in pH from 7.4 to 7.0, 6.0, 5.0 and 4.0. Fitting with Hill equations, the curve of pH-current density was “S” shape, and the pH50  was 5.73 ± 0.18  stimulation and CPZ (20 μM) blocked it largely , a neuronal TRPV1-specific inhibitor  to 1.03 ± 0.12  is the inhibitor of SR Ca2+-ATPase that can exhaust the Ca2+ content, so TG was used here to examine the role of SR. The results showed that the extracellular acidification-induced [Ca2+]i elevation in the absence of extracellular Ca2+ was almost totally abolished by TG (3 μM) with the inhibition ratio of 90.59 ± 1.46%  and U73122, the inhibitor of PLC in cytoplasm were measured here. As shown in 2+ elevation evoked by extracellular acidification in the absence of extracellular Ca2+, the inhibitory ratios were 49.80 ± 4.85% (n = 15 cell) and 53.93 ± 5.14% , respectively. To rule out the influence of DMSO on responses of cardiomyocytes to acidification, we measured the acidic solution (pH 5.0)-triggered Ca2+ signals with incubation of 0.1% DMSO, and no significant decay of Ca2+ signals was observed after DMSO exposure , and Cu2+, the inhibitor of the protonation of extracellular histidines residues in OGR1, could inhibit this elevation to 0.46 ± 0.05  of Ca2+ transient induced by pH 5.0 solution  in rat ventricular cardiomyocytes, only OGR1 was considered to be the primary protein that transferred extracellular proton signals to Gq proteinssolution . Taken t2+]i in cultured rat cardiac myocytes; We then identified that ASICs and TRPV1 were expressed in the cardiac myocytes and jointly mediated extracellular calcium entry; Thirdly, another sensor of extracellular protons OGR1 was also found in cardiomyocytes, it mediated the IP3R-gated mobilization of intracellular Ca2+ in SR via OGR1-PLC-IP3-IP3R signaling pathway.In the present study, we first found that extracellular acidification can induce an elevation of [Ca2+]i followed by various functional changes in cardiomyocytes2+]i in a pH-dependent manner. Since H+-sensitive ion channels or receptors may be one of the mediators for the influx of extracellular Ca2+, the effects of ASICs and TRPV1 were investigated. The antagonists of ASIC and TRPV1 channels inhibited in part the augmentation of [Ca2+]i, respectively. Meanwhile, blockade of both ASIC and TRPV1 channels inhibited the elevation of [Ca2+]i more significantly, and the inhibition ratio was similar to the effect of EGTA, where extracellular Ca2+ was absent. Therefore, ASIC and TRPV1 channels contributed prominently to the acid-evoked Ca2+ influx.Acidosis is a detrimental condition accompanied with some cardiac disease, for example, myocardial ischemia. The outcome of acidosis is the increase of i that triggered by extracellular acidification, suggesting the possible contributions of intracellular Ca2+ stores in acidosis-induced elevation of [Ca2+]i. Under absence of extracellular Ca2+ conditions, when the Ca2+ contents in SR were exhausted by using TG, the elevation of [Ca2+]i was almost totally abolished, demonstrating that acidosis elevated [Ca2+]i via prompting the release of Ca2+ from SR in cardiomyocytes. Although it is well accepted that SR can be mobilized by ryanodine receptor activation3 receptor has attracted more and more attentions recently, especially for their functions in cardiomyocytes. The activity of IP3 receptors can be directly potentiated by intracellular IP3, triggering the release of Ca2+ that elicits excitation-contraction coupling or cardiac hypertrophy, and predisposes ventricle to arrhythmia272829303 receptor with 2-APB significantly attenuated the acidification-induced [Ca2+]i elevation in cardiomyocytes, which was also observed in the specific inhibitor of PLC, U73122-treated groups, thus, IP3 receptors should mediate the extracellular acidification-induced [Ca2+]i elevation via IP3-PLC signaling. However, the possible contributions of RyRs to the [Ca2+]i elevation could not be completely ruled out. In fact, there is a clear potential for cross-talking between RyRs and IP3R-mediated Ca2+ signaling pathways in heart muscle2+ stores to acidification-induced [Ca2+]i elevation would be further investigated in cardiomyocytes.In the present study, the depletion of extracellular Ca3-generated PLC is mediated by Gq protein that are usually coupled with GPCRs33192+ mobilization from SR by acidification. Using specific antibody for OGR1, we also examined the existence of OGR1 in ventricular cardiomyocytes, and calcium imaging found that OGR1 had a relative high sensitivity to protons, when extracellular pH was lowered from 7.6 to 7.0, it has already been activated. Furthermore, the possible involvement of OGR1 in the proton-induced activation of PLC and the following mobilization of intracellular Ca2+ were also tested by application of Cu2+. Pre-incubation of Cu2+ significantly attenuated the elevation of [Ca2+]i via binding to essential histidine residues in OGR12+ was properly non-specific, the specificity of Cu2+ for proton sensing GPCRs other than OGR1 has not been reported. Hence, we concluded that OGR1 mediated the IP3R-gated mobilization of intracellular Ca2+ in SR via sensing extracellular protons.The activation of IP2+ transients” in cultured rat cardiac myocytes. As H+-sensitive ion channels, ASICs and TRPV1 can be activated by acidosis solution and mediate extracellular Ca2+ entry. On the other hand, OGR1 mediates the mobilization of intracellular Ca2+ from SR via OGR1-PLC-IP3-IP3R signaling pathway  and approved by the Committee of Animal Care of Huazhong University of Science and Technology.In these studies, all experimental protocols were conducted in accordance with Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health . Every 3–5 minutes, the digest was sucked into the prepared Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (1:1)  with 10% fetal bovine serum , and 100 U/ml penicillin – streptomycin to terminate digesting. About repeating 5–6 times, the shivers could be digested completely. The dissociated cells were collected by centrifugation at 118 × g for 6 minutes and suspended again in DMEM/F-12. Bromodeoxyuridine  at a final concentration of 0.1 mM was added during the first 36 hours to prevent proliferation of cardiac fibroblasts. Myocyte purity was about 95% 48 hours after plating. The cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere incubator. Experiments were performed on days of 3–5.Firstly, neonatal Sprague-Dawley rats (1–2 days old) were euthanized by decapitation, ventricular tissues were excised and washed three times in phosphate-buffered saline (PBS), and then were cut into shivers with the size less than 1 mm−1 intraperitoneally) before decapitation, and then the heart was removed rapidly and retrogradely perfused with normal Tyrode solution containing (mM): NaCl 135, KCl 5.4, MgCl2 1, CaCl2 1.8, NaH2PO4 0.33, HEPES 10, Glucose 10 and pH adjusted to 7.2 with NaOH, and then with nominally Ca2+-free Tyrode solution for 5 minutes. Subsequently, Ca2+-free Tyrode solution with 0.25 mg/ml collagenase , 0.15 mg/ml protease  and bovine serum albumin  was perfused through the heart for 8–9 minutes. The perfusate was oxygenated with 100% oxygen and kept constant at 36–37 °C. The digested ventricular myocardium were excised and stored in normal Tyrode solution at room temperature for later experiments.Rat ventricular myocytes were prepared by enzymatic dissociationTM FirstStrand cDNA Synthesis system for RT-PCR kits . Methods were similar to our previous report with slightly modified36Total RNA of cultured ventricular myocytes were isolated and cDNA was synthesized with the RevertAid3VO4, 1 PMSF, and with 1% Nonidet P-40, and protease inhibitor cocktail . The lysates were centrifuged at 12000 × g for 15 minutes, and the supernatant was recovered. After denatured, equal amounts of lysate proteins were separated on 10% SDS/PAGE gels, followed by transferred to nitrocellulose membranes . After blocking, the proteins were probed with the appropriate primary antibodies against ASIC1, ASIC2a, ASIC3, TRPV1  and OGR1 . Membrane-bound primary antibodies were detected using proper secondary antibodies conjugated with horseradish peroxidase. Immunoblots were developed on films using the enhanced chemiluminescence technique37Cultured cardiomyocytes were lysed on ice for 30 min inlyses buffer containing (in mM) 50 Tris - HCl (pH 7.4), 1 EDTA, 100 NaCl, 20 NaF, 3 NaVentricular myocytes were fixed with 4% paraformaldehyde in 0.01 M PBS, pH 7.4 for 30 minutes and then rinsed three times with PBS for 10 minutes. Followed by permeabilized with PBS/0.3% TritonX-100 for 30 minutes, cells were blocked with 2% goat serum and 1% BSA in PBS for 1 hour, and then incubated with 1:50 ASIC1, ASIC2a, ASIC3, TRPV1 or OGR1 antibody in PBS/0.3% TritonX - 100/1% BSA/2% goat serum overnight at 4 °C. After rinsed three times in PBS, cells were incubated with 1:100 goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated  IgG in PBS containing 0.3% TritonX-100, 2% goat serum and 1% BSA for 1 hour at room temperature. After washed three times in PBS, cells were mounted on glassslides with 50% glycerin and imaged using a confocal laser scanning microscope . For double-label immunofluorescence, a mouse monoclonal antibody Troponin I (C-4)  was used to mark myocardium with goat anti-mouse Rodamine conjugated  IgG, or Hoechst33258 was used as nucleus marker.2·6H2O, 5 EGTA, 2 MgATP, 10 HEPES, pH 7.4 with Tris-OH. Cardiac myocytes were voltage-clamped at −80 mV throughout the experiments. A multibarrel perfusion system was used to achieve a rapid exchange of extracellular Tyrode solution (see above), pH adjusted to 7.4, 7.0 and 6.0 with Tris-OH. MES was used instead of HEPES to buffer bath solution pH ranging from 6.0 to 5.0 and 4.0. All experiments were performed at room temperature (22–25 °C).The ASIC current was induced by rapidly lowering pH for 6 seconds and TRPV1 was for 10 seconds.For cultured rat ventricular myocytes, the beating cells during 3–5 days cultured were selected; for acute isolating cells, quiescent, rod-shape cardiomyocytes showing clear striations were selected to perform patch-clamp recording after 15–20 minutes rest at room temperature. The whole-cell patch-clamp techniques were performed in a voltage-clamp mode with HEKA EPC-10  as our previous describe36382·6H2O, 2 mM CaCl2, 10 mM Glucose, 10 mM HEPES, pH 7.4 with Tris-OH) and incubated with 1 μM Fura-2/AM for 20 minutes at 37 °C. Followed by three washes and additional incubation in standard extracellular solutions for 30 minutes, the coverslips were then transferred to a chamber fixed on the movable stage of an inverted microscope . Fura-2/AM loaded cells were illuminated at 340 nm for 150 milliseconds and 380 nm for 50 milliseconds at 1-second intervals using a TILL Polychrome monochromator . Fura-2 fluorescence emission was imaged at 510 nm by an intensified cooled charge coupled device  through an IX-70 fluorescence oil immersion lens  and a 460 nm long-pass barrier filter. Paired F340/F380 fluorescence ratio images were acquired every second for [Ca2+]i. Ratio images (340/380 nm) were analyzed by TILLVISION 4.0 software. The amplitude of [Ca2+]i transient (∆F) represented the difference between baseline concentration (F) and the transient peak response to the stimulation, and [Ca2+]i response amplitude was defined as the normalized variations of [Ca2+]i (∆F/F)37Cultured cardiac myocytes grown on glass coverslips were washed three times with standard extracellular solutions : y = a∙ xb/(cb + xb); a, maximum current density; b, Hill coefficient; c, pH50.Data are expressed as the mean ± s.e.m. Comparisons were made using Student’s How to cite this article: Hu, Y.-L. et al. Multiple H+ sensors mediate the extracellular acidification-induced [Ca2+]i elevation in cultured rat ventricular cardiomyocytes. Sci. Rep.7, 44951; doi: 10.1038/srep44951 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


However, whilst scavenging H2O2 can abolish endothelial dependent hyperpolarization (EDH) and the associated vascular relaxation in some arteries, EDH-dependent vasorelaxation can often be mimicked only by using relatively high concentrations of H2O2. We have examined the role of H2O2 in EDH-dependent vasodilatation by simultaneously measuring vascular diameter and changes in endothelial cell (EC) [Ca2+]i during the application of H2O2 or carbachol, which triggers EDH. Carbachol (10 µM) induced dilatation of phenylephrine-preconstricted rat cremaster arterioles was largely (73%) preserved in the presence of indomethacin (3 µM) and l-NAME (300 µM). This residual NO- and prostacyclin-independent dilatation was reduced by 89% upon addition of apamin (0.5 µM) and TRAM-34 (10 µM), and by 74% when an extracellular ROS scavenging mixture of SOD and catalase  was present. S&C also reduced the carbachol-induced EC [Ca2+]i increase by 74%. When applied in Ca2+-free external medium, carbachol caused a transient increase in EC [Ca2+]i. This was reduced by catalase, and was enhanced when 1 µM H2O2 was present in the bath. H2O2 -induced dilatation, which occurred only at concentrations ≥100 µM, was reduced by a blocking antibody to TRPM2, which had no effect on carbachol-induced responses. Similarly, iberotoxin and Rp-8bromo cGMP reduced the vasodilatation induced by H2O2, but not by carbachol. Inhibiting PLC, PLA2 or CYP450 2C9 each greatly reduced the carbachol-induced increase in EC [Ca2+]i and vasodilatation, but adding 10 µM H2O2 during PLA2 or CYP450 2C9 inhibition completely restored both responses. The nature of the effective ROS species was investigated by using Fe2+ chelators to block the formation of ∙OH. A cell permeant chelator was able to inhibit EC Ca2+ store release, but cell impermeant chelators reduced both the vasodilatation and EC Ca2+ influx, implying that ∙OH is required for these responses. The results indicate that rather than mediating EDH by acting directly on smooth muscle, H2O2 promotes EDH by acting within EC to enhance Ca2+ release.There is abundant evidence that H  •2+]i rises in rat arterioles.We recorded carbachol- evoked endothelial cell (EC) [Ca•2+ rises were reduced by catalase, cPLA2 and CYP29C blockers, and iron chelators.Ca•2+ release was potentiated by 30 pM IL-1β or 1 μM exogenous H2O2.EC Ca•2O2 and carbachol evoked vasodilation, but by dissimilar mechanisms.H•2O2 promotes hyperpolarization-dependent dilatation by increasing EC Ca2+ release.H The possibility that H2O2 acts as an EDHF is also consistent with observations by other groups that application of exogenous H2O2 opens VSMC BKCa channels 2O2 as the primary contributor to the EDHR evoked by bradykinin is also compelling for cerebral arterioles, in which it appears that cyclooxygenase-dependent production of H2O2 causes vasodilation by acting on BKCa channels 2O2. However, one concern with many of the studies in which the effect of exogenous H2O2 was examined is that the EC50 for H2O2 – induced vasodilatation of endothelium-denuded arteries was above 10 μM e.g.2O2, vasodilatation of endothelium-denuded arteries by exogenous H2O2 was negligible at a concentration of 10 μM.Pomposiello et al. 2O2 dilated endothelium-intact human submucosal intestinal microvessels, it caused constriction following endothelial denudation, a finding which led to the suggestion that H2O2 promotes the release of a separate EDHF instead of itself acting as an EDHF 2O2, or the oxidizing agent thimerosal, to rabbit aortic valve endothelial cells (EC) enhanced intracellular Ca2+ release evoked by the SERCA inhibitor cyclopiazonic acid (CPA). Moreover, CPA-induced vasodilatation of rabbit ileac arteries was attenuated by application of catalase. They proposed that H2O2 could potentiate EDH-dependent vasodilatation by increasing IP3 - dependent Ca2+ release 2O2 concentrations of 10–30 μM also potentiated the EDH-associated vasodilation caused by acetylcholine in rabbit ileac arteries Intriguingly, although H2O2 acts to enhance rather than mediate the EDHR, and pose a number of important questions which are currently unanswered. As the ability of H2O2 to facilitate the EDHR has not been compared to its direct vasodilator effects in any single preparation, it remains unknown which of these effects are likely to be of predominant importance during the EDHR. Also, the pathways leading to H2O2 production within EC which would facilitate increases in EC [Ca2+]i during the EDH response have not been defined. In addition, it is not clear whether physiological stimuli which have been shown to increase ROS production in EC lead to increases in the EDHR as would be predicted by this mechanism.These studies suggest strongly that H2O2, and permits endothelial Ca2+ concentration changes to be measured simultaneously. We characterized the effects of anti-oxidants and blockers of potential ROS-producing pathways on increases in EC [Ca2+]i and the associated NO- and PGI2-independent vasodilation evoked by the muscarinic receptor agonist carbachol. In addition, we carried out a comparison of the effects of carbachol and exogenously applied H2O2 in order to determine whether H2O2 might be acting as an EDHF. We also assessed whether EDH and ROS contribute to the vasodilatation induced by the inflammatory mediator cytokine IL-1β. Our results show that under basal conditions, endogenous ROS production involving CYP92C, phospholipase A2 and hydroxyl radical greatly facilitates EC Ca2+ release and EDH-associated vasodilation. A brief pre-incubation with IL-1β further enhances the responses to carbachol. Although H2O2 also dilated these arterioles directly, it only did so at high concentrations, and via mechanisms which differ markedly from those evoked by carbachol.We have investigated these issues in arterioles in the intact rat cremaster circulation, as this preparation exhibits robust, sustained and consistent dilatations to both carbachol and H2Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and is in accordance with UK Home Office regulations  Act, 1986.This study conforms with the 2 followed by cervical dislocation. A midline incision was made along the abdomen and the skin and muscle layer were retracted to expose the underlying organs. The inferior vena cava was tied off above the bifurcation into the common iliac veins and below that an incision was made to allow blood to drain from the lower extremities. A 0.61 mm fine bore polythene cannula was inserted down to the bifurcation into the common iliac arteries via the abdominal aorta and secured with thread. The left common iliac and the right femoral and internal iliac arteries were ligated to ensure that perfusion was only to the right external iliac artery supplying the cremaster artery and the cremaster muscle. This was then perfused through the cannula with a stabilizing solution  containing heparin (30 U/ml) with isoproterenol 10 µM buffered to pH 7.0±0.05 for 10 min to remove all blood.4–6 week old male Wistar rats  weighing 80–100 g were killed by exposure to an increasing concentration of CO+ channel blocker lidocaine (20 mg l−1) to block neural activity and keep the cremaster muscle from contracting.The cremaster muscle was prepared by a midline incision along the scrotum to expose the right testes. Skin and connective tissue were carefully removed from the underlying cremaster muscle, which was then cut longitudinally along the anterior surface from the apex to the inguinal canal while keeping the vascular supply and drainage at the base intact. The testis was separated from the cremaster muscle and was pulled through the inguinal canal so as not to obstruct the preparation. The muscle was spread out flat onto a clear Sylgard disc using forceps and secured using micro- histology pins, and the cardioplegic solution perfusing the circulation was then replaced for the duration of the experiment with PSS containing 0.1% albumin delivered by a gravity controlled reservoir. The preparation was moved to the stage of a Leitz Intravital microscope equipped with Orthoplan optics, and was superfused with PSS at 37 °C at a flow rate of 2.5 ml/min. PSS contained the Nal-arginine methyl (l-NAME) and 3 µM indomethacin to inhibit NO and PGI2 mediated vasodilatation. Constriction was maintained for 20 min to ensure its stability, and the muscarinic receptor agonist carbachol (10 µM) was then added for 2 min to the superfusate to elicit vasodilation. Arterioles were used for experiments only if a vasodilatation of >50% (measured as described below) was observed.Following a 30 min equilibration period an appropriate 2nd order arteriole (diameter 50–100 µm) was constricted by superfusion with 30 µM phenylephrine (PE) in combination with 300 µM N(G)-nitro-l-NAME and indomethacin were present throughout. Drugs used to characterize the EDHR were added to the superfusate for 2–20 min before, and during, the application of carbachol. In experiments examining the effect of IL-1β, the preparation was superfused for 15 min with PSS containing the cytokine at a concentration of 100 pM. IL-1β was then washed out briefly and carbachol was applied.During the experiments PE, 2.12+ concentration (hereafter termed EC [Ca2+]i) was made by subtracting the average background (adjacent to the arterioles) signal from the image, normalizing the signals from the vessel during the 360 and 380 nm excitation intervals, and calculating the 360/380 ratio at each pixel. PE had no effect on the 360/380 ratio, which indicates that FuraPE3 was confined to the endothelium. Furthermore, carbachol and TRAM-34 co-applied with apamin produced changes in the 360/380 ratio that would be expected from selective FuraPE3 endothelium loading.Arterioles were visualized with a x40 water-immersion objective lens  and were perfused for 1 h at room temperature with PSS containing 10 μM Fura-PE 3 AM, 1% DMSO, 0.02% pluronic acid and 1% albumin in order to selectively load the endothelium with furaPE3. The preparation was then perfused with PSS and the superfusate temperature was raised to 37 °C. Following a 15 min equilibration and washout period, arterioles were illuminated alternately at 360 and 380 nm at 1 Hz using a filter wheel  and images were recorded at >530 nm using an Intensified ISIS camera . Images were analyzed using ImageHopper  software. Diameter was measured using the inner margin of the 380 nm signal. A semi-quantitative calculation of the endothelial Ca2.23, 5 glucose, 2 MgSO4, 0.125 NaH2PO4, 2 CaCl2, and was gassed with 5% CO2 - balance air to maintain the pH at 7.40. St. Thomas’ cardioplegic (stabilizing) solution contained (in mM) 110 NaCl, 7.9 KCl 34 MgCl2, 1 CaCl2, 11 HEPES, 0.01 isoprenaline and 0.01 ascorbic acid as an antioxidant. Drugs were made up freshly each day or made from stock solutions stored at −20 °C with final concentrations made up in PSS. All drugs were purchased from Sigma Aldrich UK, other than: TRPM2 antibody (Cambridge Bioscience), arachidonyl trifluoromethyl ketone , heparin , TRAM-34 (Tocris Bioscience) and lidocaine . CP94  and CP85 (Na-1-ethylsulfoxide-2-methyl-3-hyroxypyridin-4-one) were kind gifts from Professor R.C. Hider, King's College London.PSS contained (in mM) 124 NaCl, 5 KCl, 22 NaHCO2.32+]i is represented as the peak % increase in the Fura-PE3 360/380 ratio from the baseline. Vasodilatation is represented as the maximum % reversal of PE-induced constriction. Carbachol responses were recorded under control conditions and then again following an intervention (e.g. application of an inhibitor), so that each arteriole acted as its own control and results could be analyzed using a two-tailed paired t-test. It was possible to do so because the responses evoked by carbachol were stable when repeated . Bar charts represent mean±S.E.M values.Endothelial cell i which was not affected by the presence of l-NAME and indomethacin . All experiments described hereafter were carried out in the presence of l-NAME and indomethacin in order to allow the EDH-associated vasodilatation to be recorded in isolation.The carbachol-induced arteriolar vasodilatation was accompanied by a sustained increase in EC [Ca2+]i and the vasodilatation induced by carbachol were greatly diminished in the presence of S&C. 2+]i was sustained, as shown in 2+]i (right) on each occasion.As shown in 1 and M2 receptors which will result in activating phospholipase C and phospholipase A2 respectively. Inhibiting PLC with 3 μM U73122 resulted in much reduced vasodilatation and Ca2+ responses , whereas the inactive U73122 analogue U73343 (3 μM) had no effect on either response , notably cyclooxygenases, lipoxygenases, xanthine oxidase, and cytochrome P450 isoforms. We used a number of inhibitors to investigate possible sources, namely allopurinol (xanthine oxidase), nordihydroguaiaretic acid (lipoxygenase), and sulfaphenazole (cytochrome P450), while indomethacin was always present to block cyclooxygenase. Of these only sulfaphenazole, a selective cytochrome P450 2C9 inhibitor 2+ response with AACOCF3 and sulfaphenazole was significantly less than with PLC inhibition  .Carbachol stimulates muscarinic Mresponse A. This sesponses B, but to3.22+ by the endothelium was recorded in arterioles perfused and superfused with Ca2+ free PSS containing 0.2 mM EGTA in order to prevent Ca2+ influx. Under these conditions carbachol caused a transient increase in EC [Ca2+]i, which was significantly attenuated by S&C , and was accompanied by a rise in EC [Ca2+]i which was also somewhat smaller than that caused by carbachol .Application of H μM H2O2 A. When e2O2 resulted in opening the TRPM2 channel was tested by using a blocking antibody that had been shown to block H2O2-induced Ca2+ influx into cultured endothelial cells i, which was strongly supressed by the TRPM2 antibody and TRAM-34+apamin. Inhibiting PKG and the BKCa channel, however, had no effect on the carbachol-mediated responses (2O2 (−37±2.8%), to an extent which was which was significantly less than that observed for the response to carbachol .Although both 100 μM Hesponses E and 6F.2O2 . H2O2-induced vasodilation was also unaffected by AACOCF3 .In additional experiments we found that CP94, which inhibited carbachol induced vasodilation (see below), had no affect on dilatation to H3.42+]i in Ca2+ free PSS, while neither desferrioxamine nor CP85 (100 μM) had any effect on this response. This supports the view that ∙OH, rather than H2O2 itself, is the radical responsible for Ca2+ release from stores.Desferrioxamine and the hydroxypyridinones CP94 and CP85 are selective iron chelators, but differ in that CP94 is cell permeable while the other two are not . As show2+, all three iron chelators inhibited the carbachol-induced rise in endothelial [Ca2+]i  was co-applied and washed off with IL-1β. NADPH oxidase assembly requires phosphorylation of p47phox by protein kinase C We have previously shown that a brief application of a low concentration of interleukin-1β (30 pM for 10 min) results in endothelial ROS generation 42O2 released by the endothelium. Instead, the results demonstrate that ROS generated by endothelial cytochrome P450 2C9 make a crucial contribution to the EDHR triggered by muscarinic receptor stimulation in rat cremaster arterioles. They do this by acting on the endothelium to potentiate Ca2+ release from intracellular stores and enhancing Ca2+ entry across the plasma membrane rather than by acting directly as an EDHF. There have been previous indications that this is an important mechanism: thus catalase suppresses cyclopiazonic acid (CPA) induced Ca2+ release in rabbit aortic valve endothelium, as well as EDH-dependent, acetylcholine-induced vasodilatation of rabbit iliac arteries. Furthermore, raising the H2O2 concentration facilitates acetylcholine and CPA induced vasodilatation 2+ release was also shown to be potentiated by the IP3R sensitising agent thimerosal, which implies that H2O2 may be exerting a similar sensitising effect, as described for the IP3R in human aortic endothelial cells 2O2 was acting to promote the EDH response by facilitating IP3R-mediated endothelial Ca2+ release and thereby raising [Ca2+]IThe key finding of this study is that whilst the EDHR triggered by carbachol is largely ROS-dependent, it does not involve an effect on VSMCs of H4.11 and M2 receptors, which are linked to phospholipase C and phospholipase A2 respectively. The present findings indicate that both are required for the full EDHR. We showed that blocking PLC with U73122 inhibits increases in both endothelial [Ca2+]i and vasodilatation  across the EC membrane 2O2 resulted in a significant increase in carbachol-induced Ca2+ release from intracellular stores  using Mag-indo, and found that this was not affected by 100 μM H2O2, although H2O2 at concentrations above 3 μM sensitized these cells to IP3-induced Ca2+ release and 10 μM H2O2 promoted histamine-mediated Ca2+ release in intact HAEC 2O2 had no effect on EC Ca2+ store content, measured using Mag fluo-4, in intact rabbit aortic valve endothelium It has been argued that a relatively high exogenously applied Hores see C. Notabl2 with AACOCF3, or CYP450 2C9 with sulfaphenazole were completely reversed by adding H2O2 (10 μM), a concentration which on its own had no effect  inhibited bradykinin-induced vasodilatation of mouse cerebral arterioles 2 suppressed the responses to carbachol in cremaster arterioles, we examined whether these were also affected by DFO and the iron chelating 3-hydroxypyridin-4-ones CP94 and CP85. These have little direct radical scavenging ability The question of whether the Fenton reaction is required for H2O2 is sensitising the IP3R, it would be predicted that CP94 and DFO but not CP85 should inhibit carbachol-induced Ca2+ release, since hydroxyl radical, which immediately reacts with other molecules in its vicinity as it is formed, is unlikely to cross the cell membrane 2+ release. Notably, it was shown that treatment of aortic EC with H2O2 leads to glutathionylation of the IP3R1 and an increase in its sensitivity to [Ca2+] 3R1 by binding to it in a redox-sensitive manner, such that oxidation of cysteine residues on the intraluminal LV3 domain of the IP3R1 prevents binding and therefore enhances channel opening 2+ release.If hydroxyl radical rather than H2+ release, the cell impermeant CP85 and DFO had no effect (2+, however, all the chelators reduced the response, which indicates that the hydroxyl radical formed outside the cell membrane is involved in the opening of the KCa channels (2O2 crossing the plasmalemma. There is evidence that several ROS-sensitive Ca2+ - permeable channels, including TRPC3, TRPC4 and TRPM2, can contribute to Ca2+ influx in EC during the response to muscarinic agonists Although CP94 profoundly inhibited carbachol-induced endothelial Cao effect A. In thechannels B and 6C,4.4via a prostanoid- and cyclic nucleotide-dependent-mechanism 2+]i in the vascular endothelium, and may be one of the factors that are involved in septic shock.The ability of IL-1β to promote EDH has not, to our knowledge, been reported previously, although it has been reported that this cytokine caused dilatation of new-born pig pial arterioles 52O2 in EDH. In one group, as evidenced by the lack of effect of catalase, H2O2 is probably not involved in this response 2O2 itself seems to act as an important EDHF 2O2 facilitates EDH, and focussed attention on IP3R-mediated Ca2+ release in EC as its likely target. Our results now provide a more detailed picture of how H2O2 acts to promote EDH in the type of arteriole that falls into this category. We find that very small changes in the EC [H2O2] around the basal level can strongly modulate the EDHR via an effect on Ca2+ release. The data demonstrate that EDH triggered by muscarinic receptor activation is greatly facilitated by ROS which are produced when arachidonic acid synthesised by cPLA2 is metabolised by CYP450 2C9. A short pre-treatment with IL-1β is able to further enhance carbachol-induced EDH. An increase in EC Ca2+ release, which would be expected to potentiate SOCE, and thereby also facilitate EC Ca2+ influx, is implicated in these responses. The effects of CP94 and CP85 indicate that hydroxyl radical plays an important role in this facilitation of Ca2+ release, and also demonstrate that ROS also promote EC Ca2+ influx during EDH.We speculate that arteries/arterioles may fall into three categories with regard to the involvement of H2+ release. PLA2 has, however, been shown to be activated by increases in EC [Ca2+]i, e.g.2-dependent ROS production would occur upon the application of carbachol.A limitation of our results is that we could not record changes in [ROS] in the endothelium of these arterioles, possibly because these were very small. We were therefore unable to determine whether muscarinic receptor activation was increasing EC [ROS], or whether basal ROS levels were sufficient to facilitate CaJames Chidgey: Conception and design of experiments; collection, analysis and interpretation of data.Paul Fraser: Conception and design of experiments; analysis and interpretation of data, critical revision of the article.Philip Aaronson: Conception and design of experiments; analysis and interpretation of data: drafting and revising the article.None declared.


In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca2+  during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca2+ ([Ca2+]i) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.Cyclic ADP-ribose (cADPR) releases Ca Fura 2 AM and Fura 2 lowaff were obtained from Invitrogen  and TEFLabs , respectively.−1 ketamine and 20 mg kg−1 xylazine hydrochloride. The hearts were removed after deep anesthesia was confirmed by the disappearance of the corneal reflex and the withdrawal of the hindlimb resulting from clamping of the paw. Cardiac myocytes were enzymatically isolated from the ventricle as previously described11 and superfused at 34−36 °C with a solution containing (mM) 8.5 NaCl, 14.5 NaHCO3, 4.2 KCl, 1.18 MgSO4·7H2O, 2.5 CaCl2 and 11.1 glucose .This study was approved by the institutional review committee of Chonbuk National University (Reference Number: CBU 2008-0057). New Zealand white rabbits (1.8–2.4 kg) were anesthetized with an intramuscular injection of 50 mg kgM) through a 60-min incubation. After they were washed, the cells were seeded in a 200-μl well with a glass coverslip on the bottom and incubated on the stage of an inverted fluorescence microscope  that was continuously perfused at 37 °C. The fluorescence was measured at a determined site through a pinhole with alternating excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm using a Ca2+ microspectrofluorometer (PTI). At the end of each recording, the data were calibrated in terms of [Ca2+]i as described by Grynkyewicz et al. based on equation (a)12 and normalized to calculate the % increase of [Ca2+]i (b)15 A Kd value of 229 nM was assumed for the binding of Ca2+ to fura 2-AM. Rmax, Rmin, Sf2 and Sb2 were measured in each experimental cell by the addition of 20 mM CaCl2 (Rmax) and 50 mM EGTA (Rmin).Rabbit ventricular myocytes were loaded with fura 2-AM (5 μ2+-free solution containing (mM) 135 NaCl, 5.4 KCl, 1 MgCl2, 10 glucose, and 5 HEPES. Patch-pipettes were pulled from borosilicate glass capillary tubules by a micropipette puller  and fire-polished. The patch-pipette tip resistance was between 4–6 MΩ. The intracellular solution for filling each patch pipette contained 110 mM KCl, 5 mM K2ATP, 10 mM HEPES (pH 7.2 with KOH). After attachment to the myocytes, the resistance was monitored beginning when the giga seal formation (Axon AxoScope 10). cADPR (100 μM) or IP3 (100 nM) was applied to the cytosol by rupturing the plasma membrane with a patch-pipette containing each reagent. The reagents 8-Bromo cADPR (Br-cADPR), ryanodine, caffeine, dantrolene, xestospongin, tetracaine, the PTP1B inhibitor, the SHP1/2 PTPase inhibitor or sodium stibogluconate were applied to the bath. Thapsigargin was also applied to the cytosol with a patch pipette.At the beginning of each experiment, the cells were washed in a CaM) 150 NaCl, 20 HEPES, 1 EDTA, and 0.1 PMSF, as well as 1% Triton X-100 (in μg ml−1), at pH 7.2, and the lysates were centrifuged at 13,000 g for 10 min. The proteins were then immunoprecipitated with rabbit IgG (Sigma), mouse anti-SERCA (Affinity BioReagents), mouse anti-α-actin (Sigma) or mouse anti-PLB antibodies (Affinity BioReagents) (1:100 dilution). The immune complexes were subsequently collected by adding protein A or G beads , fractionated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The blots were incubated with anti-phosphotyrosine , anti-α-actin, anti-PLB antibodies, anti-phospho-Ser,16 or anti-phospho-Thr17 PLB antibodies  and subsequently incubated with goat anti-mouse or anti-rabbit alkaline phosphatase-conjugated secondary antibodies (Santa Cruz Biotechnology). The proteins were visualized using an enhanced chemiluminescence system  and an LAS 3000 imaging system . To detect the total or tyrosine-phosphorylated α-actin in SR vesicles, we removed the IgG heavy chain band using ImmunoCruzTM IP/WB Optima E  according to the manufacturer’s instructions.The isolated ventricular myocytes were lysed in lysis buffer containing (in m2+ uptake solution containing (mM) 50 KCl, 20 MOPS, 0.01 CaCl2, 5 NaN3, 1 KH2PO4, 5 creatine phosphate, and 10 U creatine phosphokinase. Rabbit SR vesicles (0.5 mg per 1 ml cuvette) suspended in a cuvette were mixed with 1 μM Fura-2 lowAff. The Fura 2 fluorescence outside of the vesicles was monitored over time with alternating excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm, using a Ca2+ microspectrofluorometer (PTI). The extravesicular Ca2+ concentration was expressed as a fluorescence excitation ratio (R340nm/380nm).Rabbit SR vesicles (0.5 mg per 1 ml cuvette) were pre-incubated for 1 min at room temperature in the SR Cahttp://prospector.uscf.edu.The dried samples were analyzed by matrix-assisted laser desorption/ionization-time-of flight (MALDI-TOF) mass spectrometry (Voyager-DE PRO) for peptide mass fingerprinting and by electrospray ionization quadrupole time of flight (ESI-Q-TOF) mass spectrometry for peptide sequencing. Database searches were carried out using MS-Fit, accessed via the World Wide Web at M) with or without a 10 min pretreatment with various inhibitors. The mixture was incubated for 1 min at 37 °C in substrate buffer  with or without Na-orthovanadate (2–20 μM). After incubation, the reaction was stopped by the addition of 50 μl of Gold Mix from the ATPase assay kit. Two minutes later, 20 μl of stabilizer from the ATPase assay kit was added, and the solution was incubated for 20 min at 37 °C in the dark. The enzyme activity was calculated by measuring the Pi-dye complex released via ATP hydrolysis using an ELISA plate reader at 635 nm .The ATPase activity in the SR vesicles was determined using an ATPase assay kit  according to the manufacturer’s instructions with minor modifications. Briefly, SR vesicles (0.1 μg) in a total volume of 100 μl were incubated for 10 min in the presence of various concentrations of cADPR  for 10 min, the intracellular cADPR application had no effect on the resting [Ca2+]i (2+]i. Pretreatment with xestospongin C (2 μM), an IP3 antagonist, did not affect cADPR-induced increase in resting [Ca2+]i  relieves the inhibitory effect on SERCA.19 PLB is regulated via phosphorylation/dephosphorylation of Ser16 and Thr17 by PKA, a Ca2+/calmodulin-dependent protein kinase and protein phosphatases.22 It has been reported that cADPR increases SERCA activity in Xenopus oocytes.23 However, we found that cADPR inhibits SR ATPase activity in a concentration-dependent manner, which is completely blocked by 8-Br-cADPR pretreatment , a serine phosphatase inhibitor, had no effect on cADPR-induced increase in resting [Ca2+]i. In contrast, phenylarsine oxide (PAO), a tyrosine phosphatase inhibitor, blocked cADPR-induced increase in resting [Ca2+]i  coupling in cardiac myocytes is mediated by a mechanism known as calcium-induced calcium release (CICR) where Ca2+ entry via L-type calcium channels induces calcium release from ryanodine-sensitive Ca2+ stores.35 In cardiomyocytes, a cytosolic injection of 8-amino-cADPR reduces Ca2+ transient and contractions, indicating that cADPR increases EC coupling.29 In addition, photoreleased cADPR induces an increase in the magnitude and frequency of whole cell Ca2+ transient. In this study, we focused on the effect of cADPR on resting [Ca2+]i but not Ca2+ transient. We investigated the effect of cADPR on resting [Ca2+]i at higher concentrations than physiological level. We observed that relatively high concentrations of cADPR inhibit rather than activate SERCA activity, resulting in an increase in resting [Ca2+]i in cardiac myocytes. The endogenous level of cADPR in the heart is 1.04–150 pmol mg−1, which corresponds to 30 nM–4.5 μM depending on experimental conditions.37 Hypoxia increases cADPR concentration by twofold in the second-order branches of the pulmonary artery and by 10-fold in the third order branches.38 These results suggest that cADPR level could increase up to approximately 50 μM during specific pathological states in the heart. It has been known that myocardial ischemia, a hypoxic condition induced by blood flow restriction, increases Ca2+ concentration in cardiomyocytes, and it is associated with abnormal cardiac function.13 Cardiac ADPR cyclase, a protein distinct from CD38 or the archetypical ADPR cyclase from A. californica, has not been cloned to date.39 To verify the direct relationships between higher level of cADPR and ischemic injury, the identification of cardiac ADPR cyclase is necessary. The present study suggests that high levels of cADPR under pathophysiological conditions might decrease SR load and inhibit CICR, resulting in the inhibition of excitation-contraction (EC) coupling in cardiac myocytes.Ca2+]i. The disruption of α-actin blocked cADPR- but not IP3- or caffeine-induced increases in resting [Ca2+]i, suggesting that α-actin integrity is required for cADPR action. Interestingly, α-actin was associated with PLB, and their association was decreased by cADPR treatment. The cADPR-mediated dissociation was inhibited by PTP1B inhibition. Our results suggest that cADPR activates PTP1B, which causes tyrosine dephosphorylation of α-actin.Our results show that α-actin is involved in cADPR- but not IP3- or caffeine-induced increase of [Ca3 act on the same calcium stores, only cADPR-mediated calcium response was sensitive to the disruption of α-actin integrity. IP3 mediates calcium release by direct action on IP3 receptor in SR vesicles, whereas cADPR mediates it indirectly via PTP1B, possibly resulting in the sensitivity difference to α-actin disruption.Interestingly, although cADPR and IP