H. pylori in the pathogenesis of urticaria has also been re-reviewed. Urticaria could sometimes mimic erythema multiforme and is termed urticaria multiforme. Skin biopsy showed features of vasculitis in good number of urticaria irrespective of clinical features. Contact sensitization showed positive results in certain cases thus proving contact urticaria. Topical clobetasol, systemic omalizumab and NB UVB have shown promising results in certain forms of urticaria.Urticaria, a perplexing disease of ever-changing explanations, is being renovated almost everyday by newer facts and findings accumulated from different parts of the globe. Cost of the urticaria treatment gradually grows higher and higher whereas the ailment disturbs the quality of life very adversely. Disorder of coagulation cascade has recently thrown some new light into its mechanism. Non-allergic angioedema induced by bradykinin caused by genetic defects and ACE-inhibitors has also been noted. Role of  Urticaria was first described by Hippocrates. Since thA study in the JA national database of hospitalizations in the United States was queried for hospitalizations with a principal diagnosis of angioedema and other major acute allergic disorders . The angioedema hospitalization rate was 3.3 in 100,000, in 1998, and rose to 4.0 in 100,000, in 2005. In contrast, the combined hospitalization rate for non-angioedema allergic disorders showed an overall decline, and was exceeded by angioedema hospitalization rates after 2000. 24% of the hospitalizations for angioedema were accounted for by adverse effects due to cardiovascular or antihypertensive agents.[CIU is associated with severely impaired quality of life (QoL). Physical health and psychological health were found to be the areas of the QoL most affected in CIU patients. CIU patients frequently suffer from depression and anxiety. The severity of these parameters was found to be positively correlated with the extent of the decrease in QoL.Studies carried out on chronic urticaria (CU) during the last two decades have depicted an autoimmune pathogenesis mediated by functionally active autoantibodies to the high affinity IgE receptor (FcɛRI) or to IgE, which are able to induce histamine release from basophils and mast cells. However, such a mechanism can explain the disease in less than 50% of the patients only. Recent findings[Many cases of angioedema are nonallergic reactions, for example, bradykinin-induced angioedema caused by genetic defects and angiotensin-converting enzyme (ACE) inhibitors. This difference is crucial for a successful therapy, particularly when complete emergency care is not available. Five important forms of nonallergic angioedema can be distinguished: hereditary (HAE), acquired (AAE), renin-angiotensin-aldosterone system (RAAS)-blocker-induced (RAE), pseudoallergic angioedema (PAE), and idiopathic angioedema (IAE). A vast majority of nonallergic angioedema are RAE, particularly those caused by ACE inhibitors. The current pharmacotherapy of nonallergic angioedema is not satisfactory. Of late, several new drugs have been developed: A recombinant C1-INH, a kallikrein inhibitor , and a specific bradykinin-B2-receptor antagonist (icatibant), which may improve the treatment of kinin-induced angioedema.The underlying pathophysiology of urticaria is reported to be mast cell activation, with release of mast cell mediators, predominantly histamine. Substance P is a neuropeptide and has the capacity to provoke histamine release from skin mast cells. Angiotensin-converting enzyme (ACE), widely expressed in skin, is one of the major peptidase for the degradation of substance P. An insertion/deletion polymorphism (I/D) in the ACE gene has been reported to be related to the levels of enzyme. A statistically significant association has not been found between the ACE I/D polymorphism and chronic ordinary urticaria. Further analyses of chronic ordinary urticaria patients have shown that the ACE I/D polymorphism is not associated with the autologous serum skin test status of the patients. However, the frequency of II genotype and I allele are statistically, significantly higher in chronic ordinary urticaria patients with accompanying angioedema, as compared to angioedema-negative patients. Thus, this study shows noHelicobacter pylori eradication on chronic urticaria patients, with and without the positive autologous serum skin test (ASST). Eradication of H. pylori infection by triple therapy significantly and equally reduces the urticaria activity score (UAS) in CU patients with positive and negative ASST. Another Indian study[H. pylori eradication therapy in infected patients of CIU is significant. It recommended that H. pylori should be included in the diagnostic workup of patients with CIU.An Israeli study has showian study supporteAcute annular urticaria is a common and benign cutaneous hypersensitivity reaction seen in children, which manifests with characteristic annular, arcuate, and polycyclic urticarial lesions in association with acral edema. It is mistaken most often for erythema multiforme and occasionally, for a serum-sickness-like reaction. Although these three entities may present in a similar manner, specific clinical features help to distinguish them, and it is important for the clinician to be able to differentiate among them. Because of the frequency of its clinical confusion with erythema multiforme, the term “urticaria multiforme” has been proposed as a more apt description, to highlight the distinctive clinical features of this urticaria variant.purpura or bruising. It is often responsive to oral corticosteroids and poorly to oral antihistamines. Hypocomplementemia and systemic involvement are also commonly reported. An Italian study[Urticarial vasculitis (UV), an uncommon type of chronic urticaria (CU), exhibits histopathologically leucocytoclastic vasculitis and clinically painful and long lasting (>24 hours) wheals associated with ian study has receContact allergy plays a role in chronic urticaria. In a study, out of 121 patients with chronic urticaria, 50 (41%) tested positive to contact allergens. In all patients, avoidance measures led to a complete remission within one month. The authors[Clobetasol propionate 0.05% in a novel foam formulation is safe and effective in the short-term treatment of patients with delayed pressure urticaria, as seen in a randomized, double-blind, placebo-controlled trial.Three patients with CU refractory to conventional treatment responded to omalizumab therapy. They hadP < 0.01) at three months post treatment. The study concluded that NB-UVB may be an effective complementary treatment for patients with chronic urticaria.Data regarding narrowband ultraviolet B (NB-UVB) phototherapy in patients with chronic urticaria is limited. An open, controlled study in Turkey was perf


We examined the effects of FFA, a tool that can activate TRPC6, on [Ca2+]i in human conditionally immortalised glomerular endothelial cells (ciGEnC) and human podocytes (ciPod) that form the GFB. Changes in [Ca2+]i stimulated by FFA were measured in Fura 2-AM loaded cells. In GEnC, cell activation by FFA was dependent on external Ca2+, yet in ciPod it was not. Depletion of internal Ca2+ stores with thapsigargin did not affect cell activation by FFA in ciGEnC, but inhibited it in ciPod in a nephrin-dependent manner, demonstrated using nephrin deficient (ND) ciPod in conjunction with nephrin rescue experiments. FFA induced [Ca2+]i store release in ciPod, but not in ciGEnC or ND ciPod. In parallel, there were differences in the localisation of overexpressed TRPC6 between ciGEnC and ciPod. Furthermore, co-transfection of nephrin with TRPC6 in HEK293 cells reduced the FFA-induced increase in [Ca2+]i and nephrin clustering altered TRPC6 distribution. In conclusion, cell activation by FFA in podocytes stimulates the opening of a Ca2+ channel, probably TRPC6, in a nephrin-dependent manner with a different activation profile to GEnC.The importance of intracellular calcium ([Ca It has been used historically to block CaTRPC6 gene was identified in a large family with a high incidence of late onset autosomal dominant hereditary focal and segmental glomerulosclerosis (FSGS), a renal specific pathology TRPC6. Overall 3 of the 6 described hereditary TRPC6 mutations caused increased Ca2+ influx when transfected into HEK293 cells In 2005, a missense mutation in exon 2 of the 2+ is a secondary messenger commonly involved in a number of cellular signalling pathways, such as differentiation, apoptosis, proliferation and cell contraction. Recently, the focus on Ca2+ signalling in podocytes has been directed to TRPC6 signalling, which is thought to act as a mechanosensor. Entry of Ca2+ through TRPC6 modulates the actin cytoskeleton resulting in foot process “effacement”, leading to loss of size selectivity signalling has important implications for both cell types of the GBF in the progression of FSGS.Intracellular Ca2+ enhancing properties of FFA on TRPC6, specifically in cultured podocytes 2+ in a TRPC6 dependent manner, in contrast to other fenamates. Furthermore TRPC3 and TRPC7, which can form heterotetramers with TRPC6 did not enhance FFA induced Ca2+ activation. Since both endothelial cells and podocytes form the filtration barrier and both potentially use TRPC6 to regulate Ca2+ entry, we went on to compare and contrast FFA Ca2+ activation within podocytes and glomerular endothelial cells, in addition to TRPC6 protein analysis. Our results suggest that the pattern of FFA induced cytosolic Ca2+ influx in podocytes is distinct from GEnC and other described cell types, and is at least partially controlled by the podocyte specific protein nephrin.We have recently further explored the Ca22.1All chemicals and solutions and antibodies were obtained from Sigma Chemical Co.  unless otherwise stated.2.2A rabbit polyclonal human anti-TRPC6 antibody  was used for immunofluorescence and Western blotting. A mouse monoclonal human anti β-actin antibody was also used for Western blotting.2.3in vivo arborised podocytes. To assess the significance of nephrin in the functional responses investigated another cell line was studied. This was a ciPod cell line isolated from a patient with congenital Finish nephrotic syndrome (Finmajor)—nephrin deficient (ND) ciPod Normal human podocytes were conditionally immortalised with a temperature sensitive mutant of SV-40 large T-antigen (ciPod). These cells have been previously characterised in detail elsewhere Normal human GEnC were also conditionally transformed (ciGEnC), using the same technique as that described above. These cells have been previously characterised in detail elsewhere 2.4Differentiated ciPod, and ciGEnC, platelets and glomeruli were lysed on ice in Triton X-100 lysis buffer  containing a proteinase inhibitor cocktail (1:100 dilution). The samples were cleared by centrifugation at 13,000 rpm for 3 min at 4 °C and the pellet discarded. Total protein was then quantified by bicinchoninic acid assay according to manufacturer's instructions . Protein samples were separated by SDS-PAGE under reducing conditions and were transferred to nitrocellulose membranes. The membranes were blocked in 10% fat-free milk before incubation with antibodies described above. After incubation with horseradish peroxidase-conjugated secondary antibodies , bands were detected using the SuperSignal West Femto Maximum Sensitivity Substrate  and imaged using the Chemidoc-IT system .2.5Full-length wild type (WT) TRPC6 cDNA incorporated in a pcDNA3 vector was a kind gift of Professor Thomas Gudermann . Twenty to 100 μg/ml WT TRPC6 plasmid  was microinjected into podocytes, using an Eppendorf AG semi-automatic microinjection system . After microinjection, the cells were incubated in RPMI media, containing supplements described previously, for 24 h prior to immunofluorescence. Cells were then fixed in 4% paraformaldehyde, permeabilised in 0.3% Triton X-100/PBS for 5 min and blocked in 5% FCS/0.05% Tween for 30 min. Cells were then incubated in 4 μg/ml primary anti-TRPC6 antibody for 1 h, washed and then incubated in 1:200 dilution Alexa Flour 488 conjugated anti-rabbit secondary antibody (Santa Cruz) for 30 min. Coverslips were washed and mounted using Vectashield mounting medium . Confocal microscopy was performed using a Leica SP2 confocal microscope .2.6Cells on 22 mm diameter glass coverslips were incubated with Fura 2-AM (10 μM) with 0.006% pluronic  for ∼90 min in RPMI/EGM-2 containing serum at 37 °C. Changes in fluorescent intensity were analysed as described previously 2, 10 mM d-glucose and 10 mM HEPES) containing normal Ca2+ (1.5 mM CaCl2) or minimal CaCl2 , thereby reducing the external Ca2+ concentration gradient. We chose not to fully chelate Ca2+ because this would create a Ca2+ gradient out of the cell, which may affect Ca2+ kinetics. Where minimal Ca2+ was used, 10 μM ionomycin (IM) only induced a minimal increase in [Ca2+]i following stimulation with 200 nM thapsigargin , demonstrating that the external Ca2+ concentration was successfully reduced (data not shown).Experiments were conducted in Krebs-Ringer phosphate buffer  for 10 min. Cells were then perfused continuously with buffer containing stimulus or vehicle for 500 s. Each condition was applied to a separate coverslip to avoid the introduction of extraneous variables. Two hundred nanomolar TG was used to passively deplete stores. This concentration was chosen since it induced maximal store depletion in all cell lines (data not shown). To ensure that [Ca2+]i could be measured in each cell population 10 μM IM was added, stimulating Ca2+ entry into the cells. 5 mM manganese chloride (MnCl2), in the continued presence of 10 μM IM was then used to quench the Ca2+ sensitive Fura to determine the (Ca2+-independent) background fluorescence signal. The normalised ratio (Rnorm), proportional to the Ca2+ concentration, was calculated as described previously Rnorm traces between experiments the Rnorm was expressed as fold change from baseline for that experiment (change in Ca2+). Data are expressed as the area under the curve after 500 s (AUC500), calculated as shown below:n = 1) and the 500th (n = 500) time point (T) at 0.25 s intervals after the start (T = 0) for the mean fold change in baseline Rnorm (R) between each time point ((Rn + Rn−1)/2). This approximates to the area under the curve when the average rate of change of the ratios is linear between time points [i.e. ((Rn + Rn−1)/2) approximates to the average ratio between time points n and n − 1]. Where TG was used to deplete stores in the presence of normal external Ca2+, [Ca2+]i levels were much higher due to capacitative Ca2+ entry, which can inhibit channel activity. Under these circumstances only the positive integers of the area under the curve were measured (+AUC500).Cells were perfused with buffer  using Genejuice (Invitrogen) as instructed by the manufacturers. HEK293 cells were seeded on coverslips at 60–70% confluency and were transfected with 2.5 μg WT TRPC6 with 2.5 μg pcDNA3.1, 2.5 μg CD16-nephrin (wild type) containing a GFP tag  for 10 min at 37 °C or left untreated, then incubated with 1 μg/ml anti-mouse secondary conjugated to AF488 for 10 min at 37 °C. These time courses were chosen because CD16-nephrin is phosphorylated yet still remains predominantly at the cell surface 2.9Transfected HEK293 cells, loaded with Fura-2 as above, were incubated with 1 μg/ml mouse anti-human CD16 in Krebs’ buffer for 10 min or left untreated, then incubated with 1 μg/ml normal mouse IgG (Santa Cruz) for 10 min in Krebs’ buffer. Cells were then stimulated with 200 μM FFA as described above.2.10t-tests) and Prism  was used for ANOVA. Data are presented as mean ± S.E.M and p < 0.05 was taken to indicate statistical significance.Microsoft Office Excel  was used for simple statistics (paired and unpaired 33.12+]i (Rnorm) were used as a measure of channel activity. Firstly we confirmed that ciGEnC were activated by FFA. ciGEnC were incubated in Fura-2AM, and perfused with buffer containing either vehicle or FFA ), confirming that FFA induces an increase in [Ca2+]i in ciGEnC. This Ca2+ influx was reduced in limited extracellular Ca2+ . Yet in minimal Ca2+ the response was surprisingly significantly larger . To investigate whether it is possible that the differences seen in FFA induced Ca2+ activation involve the depletion of internal stores in either ciGEnC or ciPod, the effects of FFA were examined after the depletion of Ca2+ stores in normal external Ca2+. When stores were depleted using 200 nM thapsigargin (TG), FFA continued to induce an increase in [Ca2+]i in ciGEnC .Typically, TRPCs either induce Can ciGEnC A and B bn ciGEnC C (+AUC502+]i in ciGEnC are dependent on the entry of extracellular Ca2+ and not the release of Ca2+ from intracellular stores (as expected for the activation of TRPC6), whereas those in ciPods depend on the release of Ca2+ from intracellular stores.These results also suggest that the FFA-induced rises in [Ca3.32+ and stimulated with FFA with or without pre-incubation with TG. We observed that the overall increase in [Ca2+]i induced by FFA was much greater in ND ciPod than WT ciPod , suggesting that the absence of nephrin expression enhanced FFA induced signalling. In contrast to WT ciPod this increase in [Ca2+]i was blocked by reducing external Ca2+ . The protein expression of nephrin at 180 kDa was confirmed in transfected proliferating ND ciPod by Western blotting, in To confirm that the results seen in ND ciPods in response to FFA were specific to nephrin we carried out nephrin rescue experiments. Transfection of differentiated ciPods is technically difficult, but proliferating ciPods (at the permissive temperature of 33 °C) are readily transfectable using conventional techniques (as described in methods) and show a similar amplitude in increased [Ca2+ and stimulated with FFA. Transfected nephrin inhibited the [Ca2+]i response to FFA in ND ciPods  which was not significantly different to cell activation by FFA in normal Ca2+ either with or without nephrin  and, again, was consistent with the nephrin rescue experiments in ND ciPod but to a much lesser degree (2+ from intracellular stores induced by FFA (seen in ciPod), even when expressed in endothelial cells.To understand whether the i store. To test these hypotheses the effect of cell activation by FFA on store depletion was examined. Cells were loaded in Fura-2AM and perfused with buffer containing normal Ca2+. Cells were stimulated with FFA, or vehicle for 8 min or until the response started to diminish, then cells were stimulated with TG to inhibit the endoplasmic reticulum Ca2+ ATPase (SERCA), and release Ca2+ from intracellular stores, as demonstrated in ciGEnC in 2+ entry (CCE) because FFA blocks the store-dependent TRPC channels at the plasma membrane 2+ released by TG after FFA stimulation, results were compared to cells perfused in minimal Ca2+ and stimulated with TG alone . In ciGEnC FFA minimally affected store release by TG . In TRPC6/pcDNA3 transfected HEK293 cells, FFA stimulated an increase in [Ca2+]i (AUC500 106 ± 20) which was significantly reduced in untreated TRPC6/CD16-nephrin transfected cells  and reduced further in anti-CD16 treated (nephrin clustered) TRPC6/CD16-nephrin transfected cells (AUC500 46 ± 8, one way ANOVA, p < 0.01, p < 0.05 post hoc test, 2+]i activation by FFA and that nephrin clustering may have an additional effect on FFA Ca2+ activation.To examine whether clustering of CD16-nephrin had an effect on FFA induced Ca42+ release, conversely FFA stimulation results in the release of Ca2+ from stores. Both of these characteristics are lost in the absence of nephrin, which is presumably why they are not observed in ciGEnC. TRPC6 expression in ciGEnC is restricted to the plasma membrane and is diffuse whereas in ciPod it is distributed throughout the plasma membrane and cytoplasm in punctate vesicular-like bodies and FFA-induced Ca2+ release is reduced when TRPC6 is redistributed by CD16-nephrin clustering. It is therefore clear from our findings that the FFA-induced changes in [Ca2+]i, presumably through TRPC6 activation, is markedly different in the two cell types forming the glomerular filtration barrier.We have shown that, in contrast to GEnC, store-depletion in ciPod inhibits FFA-induced Ca2+, FFA curiously induced a marked increase in overall [Ca2+]i in ciPod (2+ from 2 to 0.05 mM causes an increase in whole cell current through TRPC6 due to a relieved block by Ca2+, whereas an increase in extracellular Ca2+ from 2 to 100 mM attenuates this 2+]i simultaneously in TRPC6 transfected HEK293 cells in 20 μM extracellular Ca2+, OAG stimulation only affected the TRPC6 current, and did not stimulate Ca2+ entry. This suggests that even though the current through TRPC6 had increased, there was no effect on Ca2+ because the extracellular source was too low and there was no secondary source. In ciPod however FFA stimulation, which increases whole cell current through TRPC6 in low extracellular Ca2+, did appear to result in an increase in [Ca2+]i due to a secondary source other than extracellular Ca2+.In the absence of significant extracellular Cain ciPod C. It is 2+]i responses do appear inconsistent in their activation pattern between cell types and conditions. This is probably due to the native channel configurations upon which FFA is acting i.e. native TRPC6 heterotetramers may well differ between GEnC and ciPod therefore altering its activation kinetics The FFA induced i through a non-selective cation channel, which was eliminated by pre-treatment with TG. When this work was carried out it was not known that FFA activated TRPC6. Interestingly, TRPC6 is highly expressed in neurons 2+ sensor, STIM1 in combination with the plasma membrane Ca2+ channel Orai 1, but acted as a SOC in association with TRPC3 2+ entry is plastic.At closer inspection of the literature, the FFA-induced release of Ca2+ release from stores induced by FFA as the one we describe in ciPod has been described for TRPM8, a temperature sensitive branch of the TRP family. Tsuzuki et al show that the menthol-induced increase in [Ca2+]i through TRPM8 in dorsal root ganglion cells was eliminated by TG pre-treatment. The authors also demonstrate that menthol directly induced the release of Ca2+ from TG sensitive stores through TRPM8 2+ release channel” on stores in prostate carcinoma cell lines 2+ from stores such as the endoplasmic reticulum or Ca2+ containing microsomes.In recent years a similar mechanism of Ca2+ signalling, the use of FFA to activate TRPC6 coems with a number of assumptions. Recent work carried out by Tu et al 2+ from mitochondria in neuronal and HEK293 cells, which depresses SOC activity. Although there is no evidence that FFA can induce the release of Ca2+ from mitochondria in podocytes, we cannot rule it out entirely. However, it would have to occur in ciPod alone and not ciGEnC or HEK293 cells since FFA had no effect when HEK293 (including TRPC6 over expressing HEK293 2+, suggesting that there was no mitochondrial release. In our experiments we demonstrated that depletion of TG sensitive stores inhibited subsequent FFA-induced Ca2+ entry, whereas Tu et al. show that the effects of FFA were independent of TG-sensitive stores which led them to investigate mitochondrial Ca2+ release. Therefore, whether FFA stimulates TRPC6 activation directly or indirectly it certainly stimulates a different Ca2+ activation pathway in ciPod from GEnC, and does so only in the presence of the podocyte-specific protein nephrin.Although we and many other groups have described an effect of FFA on TRPC6 Ca2+ activation and nephrin clustering reduced the Ca2+ activation by FFA further still. Therefore, it appears that nephrin activation may enhance its negative regulatory effects on FFA-induced Ca2+ signalling and its effects on TRPC6 distribution may also be responsible for communication of TRPC6 with internal Ca2+ stores.It appears that nephrin negatively regulates the amplitude of cell signalling by FFA in ciPod and is at least partially responsible for the FFA-induced depletion of stores. Interestingly, nephrin also appears to reduce the amplitude of cell signalling by FFA in GEnC. There is also a suggestion that it may be involved in GEnC store-release, however these results are not very dramatic, which suggests that nephrin is only partially responsible for the FFA-induced store-release in ciPod and that other podocyte factors are likely to play a role. This area will need further research to elucidate the other factors involved. We demonstrated that TRPC6 and CD16-nephrin colocalised at cell-cell junctions in an artificial expression system, suggesting that TRPC6 interacts with the cytoplasmic tail of nephrin. Phosphorylation of the cytoplasmic tail of CD16-nephrin by clustering (as described before 2+ channel that is functionally different in human podocytes than in human glomerular endothelial cells. We have previously shown that FFA can activate TRPC6 in ciPod 2+ entry by FFA stimulation. Although not definitive, together these results strongly suggest that FFA stimulates TRPC6 activation in both ciPod and GEnC and the cell differences seen are due to differences in TRPC6 activation.The results from this manuscript suggest that FFA activates a CaPatients described by Winn et al. 2+ activation pattern in ciPod, which suggests that TRPC6 may be functionally different between human glomerular endothelial cells and podocytes through nephrin regulation, which in light of recent findings may play a role in the progression of glomerulosclerosis such as in FSGS.In conclusion, we have demonstrated that FFA stimulates a different Ca


P=0.0003). Acute myeloid leukaemia samples showed heterogeneous BRCA1 mRNA levels, which were lower than those of normal bone marrows (P=0.0001). We found a high frequency of hypermethylation of the BRCA1 promoter region in AML , in particular in patients with karyotypic aberrations (P=0.026), and in t-AML, as compared to de novo AML . Examining eight primary tumour samples from hypermethylated t-AML patients, BRCA1 was hypermethylated in three of four breast cancer samples, whereas it was unmethylated in the other four tumours. BRCA1 hypermethylation correlated to reduced BRCA1 mRNA (P=0.0004), and to increased DNA methyltransferase DNMT3A (P=0.003) expression. Our data show that reduced BRCA1 expression owing to promoter hypermethylation is frequent in t-AML and that this could contribute to secondary leukaemogenesis.BRCA1 plays a pivotal role in the repair of DNA damage, especially following chemotherapy and ionising radiation. We were interested in the regulation of BRCA1 expression in acute myeloid leukaemia (AML), in particular in therapy-related forms (t-AML). Using real-time PCR and Western blot, we found that BRCA1 mRNA was expressed at barely detectable levels by normal peripheral blood granulocytes, monocytes and lymphocytes, whereas control BM-mononuclear cells and selected CD34+ progenitor cells displayed significantly higher BRCA1 expression ( BRCA1 occur in at least 10% of inherited breast cancers, but are very rare in sporadic breast cancer. BRCA1 expression is repressed through promoter hypermethylation in 11–31% of breast, 5–15% of ovarian cancer and about 60% of pancreatic ductal carcinoma , as drugs and radiation have been shown to induce DNA hypermethylation, and BRCA1 has an important role in the repair process of DNA damage, induced by these agents  and peripheral blood (PB) haematopoietic cells, and in AMLs. In particular, we studied BRCA1 regulation by promoter hypermethylation in Our analysis included 133 AML patients , diagnosed between April 1995 and October 2004. The diagnosis of AML was established according to morphology and immunophenotype.Twenty-one patients had a leukaemia secondary to treatment for a previous malignancy  and were defined ‘therapy-related AML’ . Karyotyn=8, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Mature granulocytes were separated from the PB of four normal donors, following 2-h of incubation with Voluven , and density-gradient centrifugation on Lympholyte-H . Peripheral blood-monocytes (n=7) were separated from lymphocytes (n=9) following 1-h adherence in culture dishes.We studied BRCA1 mRNA expression by real-time PCR in BM samples of 56 AML patients and in several normal blood cell subsets. Mononuclear progenitor cells were separated from 16 normal BM aspirates using Ficoll density centrifugation and CD34+ cells were isolated using immunomagnetic beads (Mononuclear cells (MNC) were separated from the BM of AML patients at the time of initial diagnosis, using Ficoll and were frozen in RLT Buffer  for RNA extraction and at −80°C as pellets for DNA and protein extraction. The haematopoietic cell lines HL-60, TF-1, KG1 and the lymphoblastic cell lines Raji and Jurkat  were also examined.BRCA1  and the iQ™ SYBR Green SuperMIX (Biorad). Oligonucleotides used are described in RNA was extracted using the RNeasy kit (Qiagen) and was reverse transcribed using random hexamers as reaction primers. Quantitative assessments of cDNA amplification for β-tubulin antibodies  in 5% bovine serum albumin/1 × TBST/0.1% sodium azide, overnight at 4°C and 1 h at room temperature, respectively.BRCA1 protein expression was analysed by Western blot in 19 AML patients. Proteins were detected by hybridisation with the primary mouse monoclonal anti-BRCA1  and anti-BRCA1 promoter hypermethylation in the BM of 133 AML patients, at the time of diagnosis.We then studied regulation of The PB of 25 donors  and 15 normal BMs  were analysed as control. The cell lines HL-60, TF-1, KG1, Raji and Jurkat (LGC-ATCC) were also studied. Patients' and controls' DNA was extracted using DNAzol , following the manufacturers' instructions. DNA samples extracted from paraffin-embedded tissue from the primary tumour were available for eight patients . Six of these patients had a t-AML, two patients received surgery alone for the primary tumour.BRCA1 promoter was determined by methylation-specific PCR (MSP), following sodium bisulphite treatment. Methylation-specific PCR, specific for the promoter region of BRCA1, was performed with the oligonucleotides in BRCA1 hypermethylation was confirmed in two independent experiments. The sensitivity of this MSP, obtained by diluting a completely methylated sample into a nonmethylated sample, was 10% (data not shown).DNA methylation pattern in the CpG island of the U Test. The strength of the association between the expression levels of each DNMT and BRCA1 according to the presence of BRCA1 hypermethylation was calculated by the Spearman rank-correlation coefficient. The Fisher's exact test was used to examine for differences in patients' characteristics according to BRCA1 hypermethylation. A multivariate analysis using the Cox regression model was also performed. Odds ratios (OR) are given within 95% confidence intervals (CI). All computations were performed using the Stata 7.0 software .Differences in BRCA1 expression levels between two sample groups were analysed using the Mann–Whitney We studied BRCA1 expression in normal haematopoietic cells and AML samples by real-time PCR and by Western blot.vs 0.03±0.01, vs 0.08±0.02, respectively). In contrast, control BM-MNCs and selected CD34+ progenitor cells expressed BRCA1 mRNA at high levels  resulted hypermethylated. Fifteen BM and 25 PB samples from healthy individuals resulted unmethylated. BRCA1 hypermethylation, we found that methylation (n=32 AML samples) correlated to reduced BRCA1 expression, when compared to unmethylated samples . Median expression of DNMT3A was higher in AML samples with hypermethylated BRCA1 in comparison to unmethylated samples (P=0.003), whereas expression levels of DNMT1 and 3B did not differ according to BRCA1 methylation . There was a moderate negative correlation between DNMT3A and BRCA1 expression .We next analysed whether dysregulation of  samples , P=0.003BRCA1 hypermethylation, the presence of chromosomal abnormalities was associated to BRCA1 hypermethylation  .Furthermore, a significantly higher frequency of .4–20.7) . The CoxBRCA1 hypermethylation and t-AML. Three tumour samples (three of four breast cancers) were hypermethylated for BRCA-1, whereas in four patients hypermethylation was present in the t-AML sample only.DNA from the primary tumour sample was available for eight patients with We studied expression of BRCA1, which plays a pivotal role in DNA repair, particularly in response to DNA-damaging agents, in AML and in normal haematopoiesis. We found that BRCA1 is expressed at high levels in normal BM progenitor and in CD34+ cells, whereas its expression is reduced in mature PB cells, such as granulocytes, monocytes and lymphocytes. The variable BRCA1 expression may be related to different proliferation rates in the mature compartment when compared to progenitor cells, but also point to a functional role of BRCA1 during the early phases of haematopoiesis, to maintain genomic integrity.BRCA1 correlated to reduced mRNA and protein expression, when compared to unmethylated samples, and to high levels of DNA-methyltransferases 3A. High DNMT expression in AML has been reported also by other authors, and is the basis for new therapeutic approaches using demethylating agents .BRCA1 is of particular importance as a regulator of chemo/radiotherapy-induced damage: it accumulates in nuclear foci during S-phase and reassembles into DNA repair-associated foci after DNA damage. The loss of BRCA1 function results in the accumulation of genomic alterations after exposure to DNA-damaging agents. In the same line, we observed a higher frequency of chromosomal translocations and other aberrations in AML patients with The results of our study lead to the hypothesis that loss of BRCA1 function could be a pathogenetic mechanism in t-AML. Further experimental studies will have to examine the consequences of reduced BRCA1 gene expression in AML on DNA repair and genomic stability.


HRT could also be more effective in the perimenopausal period than in older postmenopausal women, and may prevent the development, while worsening preexisting CVD. Lastly, progesterone may modify the vascular effects of estrogen, and modulators of estrogen/testosterone ratio could provide alternative HRT combinations. Thus, the type, dose, route of administration and the timing/duration of HRT should be customized depending on the subject’s age and preexisting cardiovascular condition, and thereby make it possible to translate the beneficial vascular effects of sex hormones to the outcome of HRT in postmenopausal CVD.Cardiovascular disease (CVD) is more common in men and postmenopausal women than premenopausal women, suggesting vascular benefits of female sex hormones. Experimental data have shown beneficial vascular effects of estrogen including stimulation of endothelium-dependent nitric oxide, prostacyclin and hyperpolarizing factor-mediated vascular relaxation. However, the experimental evidence did not translate into vascular benefits of hormone replacement therapy (HRT) in postmenopausal women, and HERS, HERS-II and WHI clinical trials demonstrated adverse cardiovascular events with HRT. The lack of vascular benefits of HRT could be related to the hormone used, the vascular estrogen receptor (ER), and the subject’s age and preexisting cardiovascular condition. Natural and phytoestrogens in small doses may be more beneficial than synthetic estrogen. Specific estrogen receptor modulators (SERMs) could maximize the vascular benefits, with little side effects on breast cancer. Transdermal estrogens avoid the first-pass liver metabolism associated with the oral route. Postmenopausal decrease and genetic polymorphism in vascular ER and post-receptor signaling mechanisms could also modify the effects of HRT. Variants of cytosolic/nuclear ER mediate transcriptional genomic effects that stimulate endothelial cell growth, but inhibit vascular smooth muscle (VSM) proliferation. Also, plasma membrane ERs trigger not only non-genomic stimulation of endothelium-dependent vascular relaxation, but also inhibition of [Ca The HERS trial enrolled postmenopausal women with established coronary artery disease and failed to demonstrate the benefits of estrogen on secondary cardiovascular prevention. Instead, there was an increase in cardiac events within the HRT group in the first year of the study I, NOS activity and NO release from human endothelial cells. E2 also promotes the association of heat shock protein 90 (Hsp90) with eNOS and reduces the Ca2+ requirement for its activation [2+]i causes transient translocation of eNOS from endothelial cell plasma membrane to intracellular sites close to the nucleus, while prolonged exposure to E2 induces the return of eNOS to the plasma membrane for its full activation i due to Ca2+ release from the sarcoplasmic reticulum and Ca2+ entry from the extracellular space. Activation of myosin light chain (MLC) kinase, Rho kinase and MAPK also contribute to VSM contraction. Also, the agonist-receptor interaction is coupled to increased production of diacylglycerol, which activates protein kinase C (PKC) i mainly due to Ca2+ release from the intracellular stores, and a maintained [Ca2+]i due to Ca2+ entry from the extracellular space. In Ca2+-free solution, Phe or caffeine causes transient cell contraction and [Ca2+]i that are not different between intact and gonadectomized male and female rats, suggesting that the gender differences in VSM contraction do not involve Ca2+ release from the intracellular stores. In contrast, the maintained Phe-induced VSM [Ca2+]i is greater in intact male than female rats, suggesting gender differences in the Ca2+ entry mechanism of VSM contraction. The maintained Phe-induced [Ca2+]i is greater in OVX than intact females, but not different between E2-replaced OVX and intact females, or between castrated and intact males, suggesting that the gender differences are likely related to estrogen [2+ entry may be related to the plasmalemmal density and/or permeability of VSM Ca2+ channels.In isolated VSM cells incubated in the presence of external Caestrogen . The cau2+ mobilization and fluxes. E2 does not inhibit caffeine- or carbachol-induced VSM contraction or [Ca2+]i in Ca2+-free solution, suggesting that it does not inhibit intracellular Ca2+ release. On the other hand, E2 inhibits maintained agonist- and KCl-induced contraction, Ca2+ influx and [Ca2+]I, suggesting inhibition of Ca2+ entry through Ca2+ channels i are not clear, but acute application of progesterone decreases Ca2+ influx and [Ca2+]i in rabbit and porcine coronary VSM i in VSM I. Also, dihydrotestosterone decreases TNF-α and LPS-induced inflammatory response in human endothelial cells I, PKC and Rho kinase. Other signaling pathways such as MLC kinase and phosphatase, and tyrosine kinase could regulate VSM contraction, and the changes in the expression/activity of these protein kinases and phosphatases with gender and gonadal hormones need to be further examined.In addition to nuclear ERs that mediate the classic genomic effects of estrogen, estrogen binds to cell membrane ERs and induces rapid nongenomic effects on the mechanisms of vascular relaxation/contraction. These effects include increased endothelium-derived vasodilators as well as inhibition of VSM [CaThe lack of vascular benefits of HRT in postmenopausal women could also be related to its timing/duration and the subjects’ age or preexisting cardiovascular condition. Studies have shown that ovariectomy augments hypertension in aging female Dahl salt-sensitive rats . Also, aSimilar to estrogen, progesterone appears to confer cardiovascular effects and could modify the vascular effects of estrogen, and therefore, the benefits/risks of including progesterone in the HRT combination should be further examined. Although androgens may be involved in some forms of hypertension by up-regulating the renal renin-angiotensin system, data suggest an effect of testosterone on the vascular control mechanisms of blood pressure. The effects of testosterone on the mechanisms of vascular relaxation/contraction warrant further examination of its role in coronary artery disease and hypertension. However, it is important to note that sex steroids have different sexual effects, and their vascular effects may be different in males and females. Gender differences in the inhibitory effects of estrogen on vascular contraction have also been observed . Althoug


Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Gαq/11-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP2. In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCδ1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C12 domain of protein kinase C-γ  allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca2+ concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca2+ up to 100–200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca2+ concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP2 in hippocampal neurons.Phosphatidylinositol 4,5-bisphosphate (PIP The related see . A numbefor PIP2  has accr2-binding domain (2 biosensor. A GFP-labelled version of the full-length Tubby protein was found to enrich to the plasma membrane when recombinantly expressed in a variety of cell backgrounds (q/11-coupled receptors GFP-Tubby rapidly translocated from membrane to cytosol and ultimately (within 2 h) to the nucleus, where it has been proposed to act as a transcriptional regulator  of Tubby [R332H-Tubby (248–505)-yellow fluorescent protein] to visualize changes in PIP2 levels in human embryonic kidney 293 cells ; 2 biosensor. We have established that GFP-Tubby translocation on PLC activation reflects dynamic changes in plasma membrane PIP2, in both neuroblastoma cells and cultured rat hippocampal neurons. In contrast, the translocation of eGFP-PH predominantly reports changes in cytosolic IP3, at least in the cell systems investigated in this study. GFP-Tubby is therefore a potential real-time fluorescent biosensor, suitable for the visualization of changes in PIP2 levels in live cells and we have used it here to evaluate the Ca2+-sensitivity of agonist-mediated PLC activity in SH-SY5Y cells.Therefore, we set out to investigate further the acute translocation of (full-length) GFP-Tubby on Gαd-lysine, cytosine arabinoside and methacholine (MCh) were provided by Sigma-Aldrich . Tocris Bioscience  supplied wortmannin (Wort) and LY294002, while Fluo-4 AM and Fura-Red-AM were obtained from Molecular Probes .Cell culture supplies and lipofection reagents were obtained from Invitrogen . Thermolysin, pronase, Dnase I, poly-l-glutamine and 10% newborn calf serum. Cells were maintained at 37°C in a humidified atmosphere of O2/CO2 (19 : 1) and were routinely split 1 : 5 every 3–4 days, using trypsin–EDTA. For experiments, cells were seeded onto 25 mm glass coverslips for 24 h prior to transient transfection (where appropriate) with 0.5 μg of eGFP-PH, full-length GFP-Tubby or eGFP-C1(2) plasmid DNA, using Lipofectamine2000 (1 : 3 ratio). For co-expression experiments, cells were transfected as above, but with 1.0 μg of dsRed-IP3 3-kinase plasmid DNA in addition to 0.5 μg of GFP-labelled biosensor plasmid DNA. Experiments were performed 24–48 h post-transfection.SH-SY5Y cells were maintained in minimum essential medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B, 2 mM 4 1.0, glucose 25 and CaCl2 1.8, pH 7.2) for 30 min. The tissue was re-suspended in Hank’s balanced salt solution supplemented with 40 μg/mL Dnase I and triturated through a fire-polished glass pipette. Following centrifugation  and further trituration, cells were re-suspended in Neurobasal medium containing B27 supplement, 10% heat-inactivated foetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), l-glutamine (0.5 mM), sodium pyruvate (1 mM) and l-serine (1 mM) and plated onto poly-d-lysine-coated 25 mm glass coverslips. After 24 h, cytosine arabinoside (5 μM) was added to inhibit glial cell proliferation and a further 48 h later, cells were transferred to serum-free Neurobasal medium. Cells were transfected after 11 days in vitro (DIV) using Lipofectamine2000, as described above. Neurons were routinely imaged at 12–15 DIV.Hippocampal neurons were prepared from 1-day-old Lister Hooded rats as described previously . Cells were imaged at a rate of 1–2 Hz using an Olympus FV500 confocal microscope  fitted with a 60× oil immersion objective lens. Fluo-4 was excited using the 488 nm line of an argon ion laser, and emissions over 505 nm were collected. Increases in intracellular Ca2+ were defined as F/F0 where F was the fluorescence at any given time, and F0 was the initial basal level of Ca2+.Cells were loaded with Fluo-4 AM  before mounting on the stage of an Olympus IX70 inverted epifluorescence microscope. Cells were incubated at 37°C using a temperature controller and microincubator  and were continuously perfused with Krebs–Henseleit buffer  and excited by the 488 nm line of an argon ion laser. Emissions from GFP-labelled biosensors and Fura-Red were collected at 505–560 and > 660 nm respectively.Cells expressing GFP-labelled biosensors  were imaged using an Olympus FV500 laser scanning confocal IX70 inverted microscope and continuously perfused with Krebs–Henseleit buffer at 37°C. Cells were excited via the 488 nm line of the argon laser and GFP emissions were collected at 505–560 nm. Increases in signal were calculated as the graphpad Prism 4.0 . Data were presented throughout as mean ± SEM from three or more coverslips and statistical comparisons were made using Student’s unpaired t-test or one way anova followed by Bonferroni’s or Dunnett’s post-test .Data were analysed using 2, we transiently expressed GFP-Tubby  and eGFP-C1(2) (4.89 ± 0.23) were not significantly different to one another, but MCh was significantly less potent in translocating GFP-Tubby (pEC50 = 4.53 ± 0.21) than eGFP-PH  than either of the other two probes, with a mean t10–90 of 59 s , as has previously been suggested for eGFP-PH (2+ release in response to MCh (using Fura-Red as a Ca2+ indicator) in cells expressing each of the GFP-labelled biosensors, compared with untransfected control cells. Ca2+ responses to 1 μM MCh (given as 1 − F/F0 self-ratios) were significantly lower in cells expressing eGFP-PH  compared with untransfected control cells  (P<0.01). In contrast, neither GFP-Tubby [0.33 ± 0.03 (+ GFP−Tubby) vs. 0.39 ± 0.02 (control)] nor eGFP-C1(2) [0.32 ± 0.02 (+ eGFP−C1(2)) vs. 0.32 ± 0.03 (control)] significantly affected agonist-induced intracellular Ca2+ responses in SH-SY5Y cells. These data indicate that expression of eGFP-PH, but not GFP-Tubby, can attenuate agonist-mediated PLC activity.One limitation of the use of protein domains with high affinity for inositol phospholipids (such as the PH domain of PLCδ1) is that their over-expression might bind to and thereby preclude hydrolysis , the cell expressing eGFP-PH alone exhibited a robust elevation in cytosolic fluorescence  had no influence on the translocation of the DAG sensor to either 10 or 100 μM MCh  were similar in SH-SY5Y cells regardless of whether the probe was expressed alone or co-expressed with either ‘kinase-active’ or ‘kinase-dead’ dsRed2-IP3 3-kinase  alone and then again following incubation with Wort, either at 1 μM  and GFP-Tubby in response to MCh. Removal of extracellular Ca2+ and depletion of intracellular Ca2+ stores with thapsigargin (5 μM) completely abolished Ca2+ responses, measured using Fluo-4 AM, to MCh (100 μM) (data not shown). MCh-mediated GFP-Tubby translocation was substantially reduced under Ca2+-depletion conditions, while eGFP-PH translocation in response to MCh was significantly, but much less markedly attenuated , cells were perfused with Ca2+-free buffer and treated with thapsigargin (5 μM) to deplete intracellular Ca2+ stores. A second MCh, response was obtained and on washout, extracellular [Ca2+] was subsequently raised to 0.03, 0.1 and finally 1.3 mM, with further responses to MCh (100 μM) assessed by GFP-Tubby translocation under each condition , Fluo-4 AM-loaded SH-SY5Y cells were subjected to the protocol described above (without the MCh treatments). A typical trace is shown in 2+ concentration under these conditions were obtained using the method described by 2+ concentrations from single excitation Ca2+-indicators such as Fluo-4. Mean resting [Ca2+]i levels in SH-SY5Y cells were ∼53 nM, while following treatment with thapsigargin in the presence of Ca2+-free buffer, intracellular Ca2+ levels fell to as low as 4 nM. Mean [Ca2+]i following ‘add-back’ of different levels of extracellular Ca2+ were determined and plotted against % control response, to generate an estimated mean [Ca2+]i concentration–response curve for MCh-stimulated GFP-Tubby translocation [2+]i required for half-maximal rescue of MCh-stimulated PLC activation can be estimated to be around 20 nM, indicating that low levels of intracellular Ca2+ are sufficient to facilitate the activation of PLC by Gαq/11-coupled receptor systems in SH-SY5Y neuroblastoma cells. These data were consistent with the observation that IP3 3-kinase over-expression had no effect on GFP-Tubby/eGFP-C1(2) responses  for 3 min, GFP-Tubby cytosolic fluorescence decreased to baseline following agonist washout in control cells, while in cells pre-incubated with Wort (10 μM), the response was maintained at a plateau level until Wort was washed out (see n = 5), while those in cells pre-treated with Wort (10 μM) were significantly higher . In contrast, GFP-Tubby responses in cells pre-treated with 1 μM Wort exhibited plateau values not significantly different to control [20 ± 8% (n = 5)]. Overall, these data suggest that, as in SH-SY5Y neuroblastoma cells, the translocation of GFP-Tubby reports dynamic changes in plasma membrane PIP2 in hippocampal neurons.Although the above data were qualitatively consistent with our findings in SH-SY5Y cells see , the eff out see . Over a 2 and the products of PLC-mediated PIP2 hydrolysis, in intact living cells. Use of eGFP-PH, eGFP-C1(2) and GFP-Tubby has also allowed us to clarify the specificity of these probes and to address questions regarding the regulation of PLC activity in human neuroblastoma cells and primary neurons.We have used GFP-labelled biosensors to study, in real-time, dynamic changes in the levels of PIP3. Over-expression of IP3 3-kinase, to attenuate the IP3 response to MCh, substantially reduced eGFP-PH translocation, indicating a predominant role for IP3 in facilitating translocation of this biosensor. This is in agreement with the findings of previous studies in a variety of cell backgrounds, including Madin-Darby canine kidney cells ], indicating that PLC activity was not compromised by IP3 3-kinase over-expression. This strongly suggests that the diminished eGFP-PH responses observed under these conditions reflect the IP3-dependence of the translocation. However, it should be noted that if DAG metabolism is also Ca2+-dependent  suggest that PIP2 re-synthesis is essential for the full re-association of eGFP-PH with the plasma membrane. However, as the eGFP-PH domain of PLCδ1 associates with PIP2 at the membrane, inhibition of PIP2 re-synthesis would be expected to impair the re-localization of the probe. Similarly, eGFP-PH translocation may be initiated by a rapid and profound depletion of PIP2 following the activation of a PIP2 5-phosphatase, in the absence of a rise in [IP3]  level, competing with IP3 for binding to eGFP-PH. In addition, 3 was sufficient to induce a marked eGFP-PH translocation in Madin-Darby canine kidney cells. 3 was predicted to be highly sensitive to eGFP-PH expression levels, over only a 10-fold range, suggesting that such conflicting results might be because of differences in the expression levels of eGFP-PH biosensor. The relative contribution of PIP2 to the translocation of eGFP-PH may be greater in some cell types than others and may be influenced by a variety of experimental conditions (2.The observation that only very high (≥ 10 μM) levels of IPnditions , but it 2 in live cells, leading to the plasma membrane localization of a GFP-labelled form of the protein  that GFP-Tubby was able to re-associate with the plasma membrane. Although the inhibitors used in these experiments (Wort and LY294002) are not specific for PI4-kinase, the consistency between the data obtained with each inhibitor  strongly supports the notion that PI4-kinase activity is crucial for the re-association of GFP-Tubby to the plasma membrane. We therefore believe that GFP-Tubby translocation provides a selective means of visualizing changes in cellular PIP2, without the confounding influence of changes in IP3 concentration.Tubby protein has previously been shown to bind with high affinity and selectivity to PIP3, DAG and PIP2, allowed us to perform a comprehensive investigation of PLC signalling in SH-SY5Y cells. Comparison of the kinetics and potencies of translocation for each biosensor highlighted some interesting differences. First, in response to the same agonist stimulus, GFP-Tubby translocated substantially more slowly than eGFP-PH, which in turn was marginally slower than eGFP-C1(2). Earlier biochemical measurements in SH-SY5Y cells indicate that the decrease in PIP2 observed on MCh stimulation reaches a peak at around 60 s (t10–90 = 59 s), while IP3 mass responses peak more quickly  competes with translocation of the probe into the cytosol, causing a rightward shift in the concentration–response curve relative to that reported by eGFP-PH . In addition, the translocation of GFP-Tubby reports the net change in concentration of its target molecule and will therefore reflect the influence of both synthesis and metabolism of this target. Given that PIP2 synthesis may be stimulated alongside PLC activation in some cases  in rat superior cervical ganglion cells, this was considered to be because of the absence of a Ca2+ response to M1 mACh receptor activation in ganglion cells. As we observed robust Ca2+ responses to MCh in SH-SY5Y cells, it is possible that in this system mACh receptor activation could stimulate PIP2 synthesis and this requires further investigation.The availability of selective fluorescent biosensors for IP2+ (2) and both products (DAG and IP3) of PLC allowed us directly to visualize the Ca2+-sensitivity of PLC activity in neuroblastoma cells. Although direct activation of various PLC isoenzymes by Ca2+ in the micromolar range has been demonstrated (2+ (data not shown). In contrast, 2+. However, removal of extracellular Ca2+ and depletion of intracellular Ca2+ stores (reducing intracellular Ca2+ concentrations to the low nanomolar range) significantly attenuated MCh-stimulated translocation of eGFP-PH, GFP-Tubby and eGFP-C1(2). Although both eGFP-C1(2) and eGFP-PH responses were reduced by a similar amount following Ca2+ depletion/removal, GFP-Tubby responses were more substantially attenuated. This suggests that, in addition to inhibiting agonist-mediated PLC activity, lowering intracellular Ca2+ may have additional effects on the metabolism/synthesis of one or more of the signalling intermediates (PIP2/DAG/IP3) being detected by these biosensors. It is interesting to note that 2+-buffered conditions, while eGFP-PH was unaffected, providing further evidence that Tubby is more sensitive to the effects of Ca2+ on PLC activity.Agonist-stimulated PLC activity has been known for some time to be dependent on intracellular Ca2+  and the 2+ to demonstrate that agonist-mediated PLC activity appears to be highly sensitive to Ca2+ in SH-SY5Y cells. By adding Ca2+ back to the bath solution in a graded manner, we were able to demonstrate that maximal recovery was obtained by raising intracellular Ca2+ to between 100 and 200 nM, indicating the exquisite sensitivity of agonist-mediated PLC activity around the physiological resting Ca2+ concentration. Although this suggests a greater sensitivity than reported for carbachol-mediated stimulation of PLC-β1 in re-constituted vesicles  was highly dependent on the free Ca2+ concentration in the range of 20–100 nM  mediating agonist-stimulated PIP5Y cells . Howeverells  generated a more robust plateau in the response. This suggests that GFP-Tubby translocation reflects changes in plasma membrane PIP2, but that a more sustained stimulus may be required in this system sufficiently to deplete PIP2 (even in the presence of Wort) to prevent the probe re-associating with the membrane. In contrast, eGFP-PH translocation in response to MCh was unaffected by Wort, consistent with the probe detecting changes in IP3 levels in hippocampal neurons as reported by us earlier  and DAG [eGFP-C1(2)], have provided a comprehensive analysis of the Ca2+-sensitivity of PLC activity in live cells. Depolarization-induced Ca2+ entry has been shown to potentiate G protein-coupled receptor-driven PLC activity in neurons, with PLC-β1 acting as the ‘coincidence detector’ (q/11-coupled receptor-mediated IP3 responses in single hippocampal neurons may be enhanced by coincident glutamate-mediated synaptic activity and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-activated Ca2+ entry (2, IP3 and DAG will provide crucial new insights into the regulation of PLC activity at the single cell and subcellular level, facilitating the further study of this ubiquitous signalling pathway.In summary, we have demonstrated the utility of GFP-Tubby as a fluorescent ‘biosensor’ for PIP


Regulation of insulin release, membrane potential, transmembrane 45Ca fluxes and cytoplasmic free Ca2+ concentration, [Ca2+]i, was examined using suspensions of transplantable NEDH rat insulinoma cells previously cultured for 2-3 days to eliminate necrotic tumour cells and counter prior hypoglycaemia. Insulinoma cells displayed a resting [Ca2+]i of 94 +/- 8 nM (n = 17) and released 104 +/- 15 ng insulin 10(-6) cells (n = 7) during 60 min incubations with uptake of 2.7 +/- 0.2 nmol 45Ca 10(-6) cells (n = 7). High concentrations of glucose did not affect membrane potential, transmembrane 45Ca fluxes, [Ca2+]i or insulin release by insulinoma cells. K+ at 25 mM depolarised the plasma membrane, induced a small increase in 45Ca efflux and increased [Ca2+]i by 65%. This modest action was not associated with demonstrable effects on 45Ca uptake and insulin release. The effect of 25 mMK+ on [Ca2+]i was counteracted by D-600, but this blocker of voltage-activated Ca2+ channels and verapamil lacked effects on transmembrane 45Ca fluxes and insulin release. The Ca2+-calmodulin antagonist, trifluoroperazine, was also without effect on 45Ca fluxes and insulin release. Ca2+ ionophore ionomycin increased [Ca2+]i, whereas A23187 and X537A did not affect transmembrane 45Ca fluxes. Moreover, insulin release was independent of extracellular Ca2+ over the range 0-20.4 mM despite marked affects on transmembrane 45Ca fluxes and a greater than 4-fold change of [Ca2+]i. Dibutyryl cyclic AMP increased insulin release by 55% without affecting transmembrane 45Ca fluxes or [Ca2+]i. The phosphodiesterase inhibitor, theophylline, also enhanced insulin release by 10-36% with no change of 45Ca uptake. The effectiveness of theophylline was independent of extracellular Ca2+ over the range 0-10.2 mM. These results indicate that inappropriate Ca2+ regulation is a key pathogenic feature underlying the inappropriate insulin secretion of rat insulinoma cells.


The effect of E2 was investigated on rat detrusor muscle strips stimulated with carbachol, KCl and electrically, in the absence or presence of a selective oestrogen receptor antagonist  and, by using confocal Ca2+ imaging technique, measuring the amplitude (ΔF/F0) and the frequency of spontaneous whole cell Ca2+ flashes. Moreover, the effect of 1 and 2 h of anoxia–glucopenia and reperfusion (A-G/R), in the absence or presence of the hormone, was evaluated in rat detrusor strips perfused with Krebs solution which underwent electrical field stimulation to stimulate intrinsic nerves; the amplitude and the frequency of Ca2+ flashes were also measured. 17β-Oestradiol exhibited antispasmogenic activity assessed on detrusor strips depolarized with 60 mm KCl at two different Ca2+ concentrations. 17β-Oestradiol at the highest concentration tested (30 μm) significantly decreased detrusor contractions induced by all the stimuli applied. In addition, the amplitude and the frequency of spontaneous Ca2+ flashes were significantly decreased in the presence of E2 (10 and 30 μm) compared with control detrusor strips. In strips subjected to A-G/R, a significant increase in the amplitude of both spontaneous and evoked flashes was observed. 17β-Oestradiol was found to increase the recovery of detrusor strips subjected to A-G/R. The ability of E2 to suppress contraction in control conditions may explain its ability to aid recovery following A-G/R.The aim of this study was to investigate the effect of 17β-oestradiol (E Oestrogens have widespread biological actions. They stimulate growth, blood flow and water retention in sexual organs and they also influence differentiation, maturation and function of various tissues throughout the body, including the peripheral and central nervous systems. Furthermore, oestrogens have been shown to have beneficial effects in cellular and molecular systems relevant to neurodegenerative disorders  phosphorylation and contraction of smooth muscle  increasing urethral resistance; (b) raising the sensory threshold of the bladder; (c) increasing α-adrenoreceptor sensitivity in the urethral smooth muscle; and (d) promoting β-adrenoceptor-mediated relaxation of the detrusor muscle  and xylazine hydrochloride  and killed by cervical dislocation. The bladders were isolated, cleaned of external fat and connective tissue, and opened along the ventral surface. Strips of detrusor muscle measuring approximately 1.0 mm × 0.5 mm × 8 mm were dissected following the direction of the muscle bundles. Fine silk ligatures were tied to each end of the strips, which were mounted in small (0.2 ml) superfusion organ baths between two platinum electrodes 1 cm apart. Strips were continuously superfused with Krebs solution  pumped by a peristaltic pump  at a constant rate of 1.5 ml min−1. Strips were placed under an initial tension of 10 mN and allowed to equilibrate for at least 60 min. Contractions were measured isometrically using mechanoelectrical transducers  and recorded using a PowerLab 8/30 data acquisition system  connected to a notebook computer running Chart 5 software (ADInstruments). Electrical field stimulation  was delivered via a digital stimulator  every 30 min. From preliminary experiments, when tissues were pre-incubated with 3 μm TTX for 20 min, EFS responses were 4.7 ± 1.6% of control values (n= 4), demonstrating their neurogenic origin.All experiments were performed in strict compliance with the recommendations of the EEC (86/609/CEE) for the care and use of laboratory animals and were approved by the Animal Care and Ethics Committee of the University of Siena, Italy. Sixty Wistar male rats  were anaesthetized with a mixture of ketamine hydrochloride (30 mg kg2 and 5% CO2 (anoxia). After this A-G period, initial conditions were restored (reperfusion).In order to mimic ischaemic conditions, a number of modifications were carried out to the organ bath apparatus. The Krebs solution at 37°C was replaced by a glucose-free Krebs solution  and the solution was gassed with 95% N2–95%N2–5%CO2, air or 95%O2–5%CO2). The equivalent O2 saturation of the hypoxic–glucopenic solution in the contraction bath was 0.7%. Assuming an atmospheric pressure of 760 mmHg, this implies a partial pressure of O2 under anoxic–glucopenic conditions of 5 mmHg.To test the oxygen tension in the bath during A-G conditions, oxygen was measured with a galvanic oxygen electrode . The electrode was calibrated using a three-point calibration with glucose-free Krebs solution  were added to the superfusing solution 60 min before applying A-G , during A-G and during the first 30 min of R, while stimulating the strips every 30 min, as described in the previous subsection.In a first set of experiments, after a 60 min equilibration period, in which control responses to various stimuli were obtained, strips were subjected to 60 min of A-G conditions followed by 120 min of R. In a second set of experiments, the length of the A-G phase was extended to 120 min, followed by 180 min of R. Drugs , with 10 μm carbachol (CCh) or with high-potassium (60 mm) solution with 30 min intervals between each stimulus. To examine the concentration-dependent effects of E2 on muscle contractility, each strip was exposed to a different concentration of the hormone  or to the solvent (ethanol), taken as control. After 20 min of incubation, strips were stimulated again in the presence of E2 or ethanol.Detrusor strips, placed in the organ bath with standard Krebs solution at 37°C and bubbled with 95% O2 was assessed by incubating the strips for 20 min with the selective ER antagonist ICI 182,780 (at the same concentration of E2) before addition of E2 to the organ bath.The involvement of oestrogen receptors (ERs) in the effects of E2 or nifedipine was assessed in strips in which contractions were elicited by depolarization with 60 mm K+ in the presence of 0.5 or 5 mm Ca2+. Krebs solution containing 60 mm K+ was prepared by replacing NaCl with equimolar KCl. When the Ca2+ concentration was changed, Ca2+ was replaced isosmotically with NaCl. After a 60 min equilibration period with Krebs solution containing either 0.5 or 5 mm Ca2+, strips were exposed to high K+ for 4 min every 20 min, until responses were reproducible, and these were taken as control values. Drugs at increasing concentrations  were tested on successive responses to high K+ . Drugs were applied 10 min before as well throughout the depolarizing period  ethane-N,N,N′,N′-tetraacetic acid-1 acetoxymethyl ester (BAPTA-1 AM) in 1% dimethyl sulphoxide and 0.2% pluronic F-127 in standard Krebs solution for 90 min at 36°C. Each strip was then rinsed in Krebs solution, and bubbled with 95% O2 and 5% CO2 for at least 10 min. Tissues were then pinned flat, serosal side up, in a Sylgard®-lined organ bath and mounted on the stage of an upright confocal microscope.After subjecting tissues to ischaemic-like conditions, as previously described, each detrusor strip was exposed to 10 μThe detrusor strips were continuously superfused with Krebs solution (bath temperature 33–34°C). Images were acquired with a Leica SP2 upright confocal microscope . Oregon Green-488 BAPTA-1 AM was excited with 488 nm laser light, and emission was collected through a prism and shutters set to pass wavelengths longer than 510 nm. A series of 100 frames was captured at approximately 5 Hz, to generate one image set. Such sets were acquired once every minute. Ten sets were generated for each region of the preparation, with at least three regions sampled per preparation (http://www.liv.ac.uk/~sdb/ImageSXM/); to correct for lateral movements (particularly the movement generated by contraction), all images were automatically aligned to a template image using the ‘Autoregister’ function of Image SXM and custom-written macros. A region of interest was established which encompassed the portion of a smooth muscle cell that was consistently within the field of view. The fluorescence signal in this region was measured over time throughout the image set. Data were exported to Chart 5 software for measurement of spikes in the Ca2+ signal. The threshold for spike detection  was manually chosen to match the sensitivity of manual detection for that cell  plug-in written by R. J. Amos  to detect increases in fluorescence of the Ca2+ indicator. All images were automatically aligned to a template image using the ‘Stack reg’ plugin of Image J. In the first frame of the image series, a region of interest was established which encompassed the portion of a smooth muscle cell visible within the confocal plane. The fluorescence signal in this region was measured over time throughout the image set. Data were exported to Chart 5 software to measure calcium spikes and then to Excel for further analysis.In a second set of experiments, image analysis was performed with an Image J (s.e.m. The area under the response–time curve (AUC) was calculated by the trapezoidal rule with the software GraphPad Prism 4 . Statistical analysis of the data was performed using Student's t test for paired or unpaired samples, or by one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test for multiple comparisons. Values of P < 0.05 were considered significant. Values of IC50 were estimated by linear regression analysis. The number of the strips used corresponds to the number of the animals, unless stated otherwise.Results are expressed as means ±17β-Oestradiol, carbamylcholine chloride (carbachol), nifedipine, TTX and pluronic F-127 were purchased from Sigma-Aldrich . Oregon Green-488 BAPTA-1 AM was purchased from Invitrogen  and ICI 182,780 from Tocris Bioscience .m) were prepared by dissolving 17β-oestradiol and nifedipine in absolute ethanol, kept refrigerated at −20°C and used within 1 week. Stock solutions of ICI 182,780 (10−2m) were made by dissolving the drug in DMSO. Aliquots of 10 μm Oregon Green-488 BAPTA-1 AM were obtained by dissolving the powder in 40 μl DMSO and 20% pluronic acid F-127, then adding 460 μl Krebs solution, shielded from light and stored at −20°C. Subsequent dilution, on the day of the experiment, yielded a 10 μm Oregon Green-488 BAPTA-1 AM solution in 1% DMSO and 0.2% pluronic acid. The DMSO and ethanol exerted no significant effects at the maximal concentration used in all the experiments.Stock solutions (10 mm carbachol (CCh) and 60 mm KCl in the absence (CTRL) or in the presence of E2 at various concentrations are shown in m, E2 significantly decreased detrusor contraction induced by all the stimuli applied , 10 μbout 37% .m did not modify the effects of E2 on detrusor muscle contractility evoked either by EFS or by the two pharmacological stimuli , which increased to 20.2 ± 2.7 mN  at a Ca2+ concentration of 5 mm. The Ca2+ antagonist activity of E2 was assessed by comparing its antispasmogenic activity at the two Ca2+ concentrations. 17β-Oestradiol exhibited antispasmogenic activity, since the hormone showed the same behaviour as the well-known Ca2+ antagonist nifedipine; its pIC50 value significantly decreased as Ca2+ concentration increased  strips were imaged with a laser scanning confocal microscope. Urinary bladder strips, loaded with Oregon Green-488 BAPTA-1 AM, appeared as light green, homogeneous bundles of cells when examined with laser scanning confocal microscopy. In a single field, several smooth muscle cells were often seen lying in parallel (m) was added to the superfusing Krebs buffer on the confocal microscope stage for 1 h.In order to gain a better understanding of the effect of Eparallel . 17β-Oes2+ flashes were observed. Smooth muscle cells displayed repetitive, large and rapid increase in the Ca2+ fluorescence that rose almost instantly in a single smooth muscle cell and spread quickly throughout bundle  was calculated. In the presence of 30 μm E2, a gradual decrease of the amplitude of spontaneous Ca2+ transient in UBSM strips . In 30 μm E2-treated strips the amplitude gradually decreased, reaching approximately 50% of the initial value; however, after washing out the hormone, the amplitude fully recovered. Similarly, in 10 μm E2-treated strips the amplitude declined gradually to 60% of the control value. In contrast, at the lowest hormone concentrations tested (1 and 3 μm), the amplitude of evoked Ca2+ transients did not differ from the initial amplitude  at the end of A-G phase. Moreover, 0.1 μm E2-treated strips showed a significantly higher recovery during R, compared with control strips. On the contrary, responses to EFS of 10 and 30 μm E2-treated strips were significantly lower than those of control strips. At 1 and 3 μm, E2 did not exert any significant effect (m E2-treated strips during the R phase was markedly increased (by 11.4%) compared with the control AUC, while the AUCs of 10 and 30 μm E2-treated strips were significantly lower  compared with control tissues. Moreover, E2 exerted some significant effects by itself on the response to EFS. In fact, at the highest E2 concentrations used (30 μm) the AUC of the pre-incubation phase was significantly lower than that of control strips.During A-G, the response to EFS in control strips gradually decreased, being abolished within an hour. On the contrary, the response to EFS of 0.1 μt effect . Accordit effect , inset o2 on EFS-induced contractile responses, when the length of exposure to A-G conditions was extended to 120 min. As described in the previous subsection, at the highest E2 concentration tested (30 μm), the response to EFS was significantly decreased. However, during the R phase, only 1 μm E2-treated strips showed a significantly higher recovery of response to EFS (about 22% higher than that of control strips) while in 30 μm E2-treated strips, EFS recovery in the R phase was poor, being around 34% of the initial value.2+ flashes (2 concentrations tested (10 and 30 μm), both in spontaneous and evoked whole Ca2+ flashes, the amplitude was significantly lower compared with control values.After subjecting tissues to the contraction studies, as shown above, each detrusor strip was imaged with a laser scanning confocal microscope. In the tissues subjected to A-G/R (CTRL) the amplitude of the spontaneous A and evo flashes B was sig2+ transients .The frequency of spontaneous Caansients A and theransient B were al flashes A increas2 used (10 and 30 μm), reaching about 68 and 71% of the control values, respectively.The probability of evoking a flash B, howeve2+ flashes nor significant changes with E2 treatments were seen. Similarly, there were no significant effects either on the frequency of Ca2+ transients or on the probability that EFS evoked them at all the concentrations of E2 used (data not shown).Using the protocol of 2 h of A-G and 3 h of R, neither consistent effects on spontaneous and evoked Ca2 on detrusor smooth muscle contractility was investigated, by using different excitatory stimuli to activate different pathways. To test the possible role of E2 as a neuroprotective agent, urinary bladder smooth muscle strips were subjected to A-G/R conditions and the functionality of intrinsic nerves was assessed through EFS. Moreover, since the relaxing effect of E2 seems to depend upon the relative contribution of Ca2+ influx through voltage-gated Ca2+ channels, the effects of E2 on global calcium flashes, spontaneous and evoked, were studied.In this study, the effect of E2 at high concentrations reduces the contractility of male rat urinary bladders in response to either EFS or pharmacological stimuli (CCh and KCl). The effect of E2 on the response to CCh, which acts through muscarinic receptors by activating IP3-mediated release of calcium from intracellular stores  and the fact that ICI 182,780 did not inhibit the relaxant effect of E2 are also not compatible with the genomic pathway. Furthermore, the lowest concentrations used were in the micromolar range, much higher than physiological plasma levels of E2, which are in the nanomolar range. Moreover, 2+ channels. In the present study, the Ca2+ antagonist effect of E2 on rat UBSM is also likely. Firstly, E2 caused a concentration-dependent decrease in the KCl-induced contractions, with an IC50 for contractile inhibition of 4.0 μm, in agreement with a previous study by 2 had an IC50 of 1.7 μm. Secondly, whole cell Ca2+ flashes in UBSM, which are diltiazem sensitive and thus require Ca2+ influx through voltage-dependent Ca2+ channels  E2 reduces the amplitude of the Ca2+ flashes, and since these Ca2+ flashes depend on the opening of L-type Ca2+ channels during smooth muscle action potentials in this tissue  the ability of E2 to reduce KCl-induced contractions also argues against an obligate action on intracellular stores. However, we cannot be sure whether E2 decreases electrical excitability, or more directly inhibits L-type Ca2+ channels.It seems unlikely that an effect of E2+ events, compared with the effect on their frequency, suggests that the mechanism driving action potential frequency is separate from that determining the amplitude, but that they are both affected by E2.In the present work, the slower recovery time of the amplitude of Ca2 were subjected to A-G/R, and whole cell Ca 2+ flashes of the same strips were investigated. These cell flashes represent a synchronous increase in the fluorescence of the indicator, hence of Ca2+, throughout the visible portion of the smooth muscle cell. In strips subjected to A-G/R, a significant increase in the amplitude of both spontaneous and evoked flashes was observed. At higher concentrations of E2, Ca2+ antagonist activity seemed to predominate, directly causing a decrease in amplitude of both spontaneous and evoked flashes. Moreover, a A-G/R-induced increase in the frequency of spontaneous Ca2+ transients was observed; the cause of this has not been identified. Furthermore, in strips subjected to A-G/R and incubated with high concentrations of E2, there was a fall in the probability that a field stimulus evoked a response. 17β-Oestradiol may have decreased transmitter release from the nerves or, alternatively, the high frequency of spontaneous smooth muscle action potentials may have suppressed postjunctional excitability, through, for example, activation of BK channels. 2 possesses K+ channel opening activity in guinea-pig urinary bladder smooth muscle, activating the Ca2+-dependent large-conductance K+ channels. Moreover, E2, opening BK channels, significantly diminished action potential generation and spontaneous activity, providing negative feedback to limit Ca2+ influx (When an obstruction is present or when there is overactivity of the bladder wall, a drop in blood flow and subsequent reduction of substrate and oxygen could occur, resulting in an ischaemic environment within the detrusor . The uri2, at concentrations of 3 μm and above, suppresses the contractility of urinary bladder smooth muscle to nerve stimulation, consistent with a decrease in the amplitude of the Ca2+ response in the smooth muscles cells in conjunction with a decrease in the frequency of spontaneous smooth muscle action potentials. The ability of E2 to suppress contraction in control conditions may explain its ability to aid recovery following anoxia–glucopenia, by reducing the metabolic load.In summary, E


Limonium sinense extracts (LSE) was studied in lipopolysaccharide and D-galactosamine  intoxicated mice. It was found that increased activities of serum aspartate aminotransferase and alanine aminotransferase induced by LPS/D-GalN were significantly inhibited by pretreatment with LSE. The obvious disruption of membrane potential, intramitochondrial Ca 2+ overload and suppression in mitochondrial Ca 2+ -ATPase activity induced by LPS/D-GalN were significantly blocked by pretreatment with LSE. It was concluded that mechanisms underlying protection of LSE against liver mitochondria damage might be related to the preservation on mitochondrial Ca 2+ homeostasis through the preservation on mitochondrial Ca 2+ -ATPase activity.Mechanisms underlying the mitochondrial protection of  Limonium sinense (Girard) Ktze is a plant belonging to the Plumbaginaceae family and is mainly distributed along seashores and salts marshes in southern China, Ryukyus (Japan) and western Taiwan. Historically, both the roots and whole plants have been used as a folk medicine for the treatment of fever, hemorrhage, and menstrual disorders.in/[Rh123]out, assuming that the distribution of Rh123 between mitochondria and the medium follows the Nernst equation.[Mitochondrial membrane potential (MMP) was evaluated according to the method of Emaus, from theequation.2+ level was assayed by the change in fluorescent intensity (F) of the Ca2+ indicator dye fura-2. To load mitochondria with the fluorescent Ca2+ indicator fura-2, 0.5 mg protein/ml of mitochondria isolated from various groups’ mice livers was incubated for 30 min at 30°C in a suspension medium containing 125 mmol/l of sucrose, 65 mmol/l of KCl, 5 mmol/l of HEPES and 1 μmol/l of fura-2/AM, pH 7.4, and then washed twice with the medium without the dye to eliminate free fura-2/AM. The final mitochondrial pellet was diluted in the suspension medium to obtain a protein concentration of 0.5 mg/ml. For every sample, the F of fura-2-loaded mitochondria was recorded on a Hitachi 850 fluorescence spectrometer at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. Fmax was determined by adding 0.4% Triton X-100 and 1 mmol/l of CaCl 2 to the mitochondrial suspension; Fmin was measured by adding 10 mmol/l of ethylene glycolbis(2-aminoethyl ether)-n,n,n′,n′ tetraacetic acid (EGTA) to the above system. The intramitochondrial Ca2+ content was calculated as follows:Kd (F – Fmin)/(Fmax – F).[The intramitochondrial Camax – F).2, pH 7.4. Ca2+ -ATPase activity was assayed by measuring phosphate release according to the protocol in the ATPase kit (Jiancheng Bioengineering Institute). One unit of the specific activity of the ATPase was defined as one micromole of inorganic phosphorus released from 1 mg of protein within 1 h (μmol Pi/mg protein/h).Liver mitochondria (0.5 mg protein/ml) were prepared in the assay buffer containing 50 mmol/l of Tris-HCl, 75 mmol/l of KCl, 0.4 mmol/l of EDTA and 6.0 mmol/l of MgCl q-test using SPSS 10 software.Differences among all the groups were analyzed by one-way analysis of variance, followed by SNK-As shown in Compared with the normal group, obvious structure changes such as massive fatty change, gross necrosis, broad infiltration of the lymphocytes and kupffer cells around the central vein and loss of cellular boundary were observed in LPS/d-GalN-insulted mice. However, the histological pattern of the livers of the mice treated with LSE showed only mild degrees of fatty change, necrosis and lymphocyte infiltration .P < 0.01) when mice were intraperitoneally injected with LPS/d-GalN [Under the present experimental condition, the MMP of normal mice was –188.6 ± 5.9 mV, which dropped to –160.7 ± 7.8 mV  than that in normal mice. However, the rise in Ca2+ handling level induced by LPS/D-GalN was effectively inhibited by pretreatment with various concentration of LSE, and the inhibitory rates in the 100, 200 and 400 mg/kg LSE groups reached 31.6, 89.9 and 90.5%, respectively Measurement of Ca2+ -ATPase activity is shown in 2+ -ATPase activity in LPS/D-GalN-intoxicated mice (3.8 ± 0.6 μmol Pi/mg protein/h) was obviously lower than that in normal mice (5.2 ± 0.5 μmol Pi/mg protein/h). However, the LSE of various concentrations obviously blocked the defect in mitochondrial Ca2+ -ATPase activity. The inhibitory rates of 100, 200 and 400 mg/kg of LSE reached 14.3, 64.3 and 78.6%, respectively.The effect of LSE on mitochondrial CaL. sinense (Girard) Ktze is a folk medicine popularly used as a remedy for bleeding, piles, fever, hepatitis, diarrhea, bronchitis and other disorders.[L. sinense are flavonoids.[4 and LPS/d-GalN-induced liver damage and that the hepatoprotection is related to its protection of liver mitochondria though stabilizing the expression of mitochondrial VDAC.[2+ handling involved in the mitochondrial protection of LSE is still not known.isorders. As reporavonoids. Our previal VDAC.8 HoweverD-GalN is a well-characterized system of acute hepatic failure and usually used for screening of anti-hepatotoxic and/or hepatoprotective activity of drugs.[D-GalN-induced liver injury was used to study the mechanisms underlying the mitochondrial protection of LSE. It was found that increases in the activities of serum AST and ALT induced by LPS/D-GalN were significantly inhibited by oral pretreatment with 100, 200 or 400 mg/kg LSE. Morphological observation further confirmed the hepatoprotective effects of LSE. Meanwhile, the obvious disruption of membrane potential in LPS/D-GalN-intoxicated mice was siginificantly blocked by pretreatment with LSE, which coincided with our previous results[D-GalN.Liver injury induced by LPS/of drugs.26 In this results and demo2+ homeostasis in damaged hepatocytes is one of the most important steps. Moreover, the hepatocellular Ca2+ overload can activate the mitochondrial Ca2+ uniporter in the mitochondrial inner membrane and eventually cause swelling of the mitochondrial matrix, dissipation of mitochondrial membrane potential, release of Ca2+ and proapoptotic factors, and finally induce cell death, which has been considered as one of the important mechanisms in liver injury.[Previous reports have suggested that there are several steps in the mechanism by which GalN induces hepatocytes’ death, and loss of Car injury.2+ homeostasis was evaluated by measuring intramitochondrial Ca2+ content in LPS/D-GalN-intoxicated mice with or without pretreatment with LSE. The present results show that 200 and 400mg/kg LSE effectively suppressed the intramitochondrial Ca2+ overload induced by LPS/D-GalN, which suggests that LSE could protect liver mitochondria against the toxicity of LPS/d-GalN by preserving the mitochondrial Ca2+ homeostasis. We can speculate that the suppression of LSE on LPS/D-GalN-induced intramitochondrial Ca2+ overload might result in a blockage of mitochondrial calcium influx, which in turn inhibits the swelling of the mitochondrial matrix, dissipation of mitochondrial membrane potential and release of proapoptotic factors through prohibiting PTP opening, and finally blocking the hepatocyte death.Ca2+ homeostasis through the action of Ca2+ -ATPase located on the plasma membrane. This enzyme uses the energy of ATP to extrude cytoplasmic Ca2+ against a large concentration gradient into the extracellular space. Also, more and more evidences suggest that mitochondria are particularly important in controlling cytoplasmic Ca2+ levels under pathological conditions. Ca2+ -ATPase located on the mitochondrial membrane can take up and retain large quantities of Ca2+ to buffer cytosolic Ca2+ levels and prevent damage to a cell.[2+ -ATPase activity induced by LPS/D-GalN. We therefore speculate that the maintenance of the mitochondrial Ca2+ homeostasis by LSE may be related to its preservation of mitochondrial Ca2+ -ATPase activity.It was also believed that cells maintain a cytosolic Cao a cell. Our presD-GalN in mice could be related to the preservation on mitochondrial Ca2+ homeostasis through the preservation on mitochondrial Ca2+ -ATPase activity, which reveals a new mechanism of the mitochondrial protective effect of LSE.In summary, the results in the present study demonstrate that mechanisms underlying protective function of LSE on liver mitochondria against damage by LPS/