Simvastatin significantly potentiated the inotropic response to β2-, but not β1-, adrenoceptor stimulation. Under conditions of β2-adrenoceptor stimulation, phosphorylation of phospholamban at Ser16 and troponin I at Ser23/24 was enhanced with statin treatment. Simvastatin increased NO production without significant effects on eNOS expression or phosphorylation (Ser1177), consistent with the reduced expression of caveolin 3, its constitutive inhibitor. In conclusion, statin treatment can reduce caveolin 3 expression, with functional consequences consistent with the known role of caveolae in the cardiac cell. These data are likely to be of significance, particularly during the early phases of statin treatment, and in patients with heart failure who have altered β-adrenoceptor signalling. In addition, as caveolin is ubiquitously expressed and has myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae.The number of people taking statins is increasing across the globe, highlighting the importance of fully understanding statins' effects on the cardiovascular system. The beneficial impact of statins extends well beyond regression of atherosclerosis to include direct effects on tissues of the cardiovascular system (‘pleiotropic effects’). Pleiotropic effects on the cardiac myocyte are often overlooked. Here we consider the contribution of the caveolin protein, whose expression and cellular distribution is dependent on cholesterol, to statin effects on the cardiac myocyte. Caveolin is a structural and regulatory component of caveolae, and is a key regulator of cardiac contractile function and adrenergic responsiveness. We employed an experimental model in which inhibition of myocyte HMG CoA reductase could be studied in the absence of paracrine influences from non-myocyte cells. Adult rat ventricular myocytes were treated with 10 µM simvastatin for 2 days. Simvastatin treatment reduced myocyte cholesterol, caveolin 3 and caveolar density. Negative inotropic and positive lusitropic effects (with corresponding changes in [Ca The number of people taking statins will increase as the threshold for statin prescription in cardiovascular disease prevention continues to decrease across the globe. For example, recent amendments to ACC/AHA guidelines for statin prescription Pleiotropic effects of statins have often been linked with depletion of isoprenoid intermediates of the HMG CoA reductase pathway, particularly farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) which modulate signalling by facilitating membrane targeting of elements including small and heterotrimeric G proteins. Endothelial dysfunction, characterised by reduced bioavailability of NO, is a common finding in cardiovascular disease and increased NO production with statin treatment provides an excellent illustration of the breadth of statins' pleiotropic effects. Statins alter NO bioavailability by a number of different isoprenoid-dependent mechanisms including increased eNOS expression , of proteins involved in the synthesis (HMG CoA reductase), uptake (LDL receptors), and efflux  of cholesterol However, in jumping on the ‘isoprenoid bandwagon’, one factor which is often overlooked is that alterations in As well as its contribution to cholesterol homeostasis, caveolin is of fundamental importance in most cells as a structural component of caveolae, invaginated lipid rafts, and as a regulatory scaffold This work shows for the first time that inhibition of cholesterol synthesis has the capacity to modestly depress basal cardiac inotropy and enhance lusitropy, whilst increasing β-AR responsiveness. Our data suggest that these functional changes are due in part to altered caveolin expression and distribution. However, a contribution from isoprenoid-dependent effects cannot be excluded.de novo myocyte cholesterol synthesis? In order to select a concentration of simvastatin which is of clinical relevance, we considered serum levels of drug, tissue accumulation and IC50. In man, an oral dose of 40 mg simvastatin gives peak serum levels of up to 0.1 µM 50 of simvastatin for HMG CoA reductase is 0.1 µM in rat hepatocytes Our study employed a simple experimental model to look directly at the acute effects of statin treatment on the adult cardiac myocyte. Our first question was whether culturing myocytes in the presence of simvastatin reduces cellular cholesterol i.e. does simvastatin reduce http://www.cbrc.jp/research/db/TFSEARCH.html), providing some support for the concept of SRE control of Cav3 transcription in this species.Next we addressed the question of whether simvastatin affects the expression of Cav3 - the muscle-specific caveolin isoform. Cav3 staining in fixed permeabilised cells was reduced by ≈30% (P<0.01) following statin treatment ; depletiManipulation of cellular cholesterol also affects Cav3 distribution in the cell Although the focus of our work is the muscle specific Cav3 isoform, some Cav1 is also found in the cardiac myocyte. We measured levels of Cav1 in cell lysates. There was significant variability in Cav1 expression between cell populations in the absence of statin treatment, and a trend (P>0.05) for lower Cav1 expression  in statin treated cells vs. controls .−1 membrane in controls to 0.63±0.08 µm−1 in treated myocytes (P<0.05). Interestingly, we also recorded a significant reduction in cell capacitance in statin-treated cells compared with controls  relaxation which was accompanied by a corresponding hastening of [Ca2+]i transient decay  function. Indeed, 2+ load and fractional SR Ca2+ release indexed using caffeine. Simvastatin's effects on excitation-contraction coupling are similar to those reported following caveolae disruption with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD); reduced [Ca2+]i transient amplitude and shortening in MBCD-treated cells was ascribed to reduced fractional SR Ca2+ release without any change in ICa,LAre statin-induced changes in cholesterol and Cav3 associated with changes in basal cardiac myocyte function? As shown in nt decay . Simvast  significantly enhanced (P<0.001) basal shortening compared with simvastatin alone  and shortening in myocytes cultured with simvastatin and mevalonate was not different (P>0.05) to that in control cells .in vitro; effects were ascribed to isoprenylation-dependent effects on Gαs 2+]i transient amplitude to β1-AR stimulation with 10 or 100 nM isoproterenol  by 80% in statin-treated cells  was significantly reduced (P<0.001) compared with cells cultured with simvastatin alone . These data are consistent with enhanced β2-AR responsiveness being mediated through simvastatin inhibition of HMG CoA reductase.16, the PKA site 16 phosphorylated PLB (pPLB) in control and statin-treated samples under basal conditions and following selective β2-AR stimulation with 100 nM zinterol. Interestingly, under basal conditions (i.e. in the absence of β2-AR stimulation) we saw a significant 300% increase (P<0.05) of pPLB in statin-treated cells compared with controls, which could explain the faster transient decay and relaxation kinetics in these cells  in pTnI over basal levels (We have previously shown that increased β2-AR responsiveness following caveolae disruption is due to enhanced phosphorylation of the SR protein phospholamban (PLB) at Serells see . This wal levels . TogetheIt is worth highlighting that ventricular myocytes cultured for 2 days show differences in their response to β2-AR stimulation  1177 Akt site (p-eNOS) in myocyte lysates. We were unable to detect nNOS expression in our myocyte lysate, likely because of very low expression of this protein. There was no significant difference (P>0.05) in eNOS expression or in the amount of p-eNOS  between control and statin-treated cells  pathways. However, given the body of data that show isoprenoid-dependent statin effects in neonatal cardiac myocyte and non-cardiac cells, we looked at the possible contribution of isoprenoids to the observed functional effects. Basal shortening and the inotropic response to β2-AR stimulation were measured in cells cultured with statins in the presence of FPP and/or GGPP. Isoprenoid supplementation did not increase (P>0.05) basal shortening in statin-treated cells , therebyin vitro and shows no evidence of toxicity. Our chosen regime results in an identical depletion of cellular cholesterol to that seen following a 5 day treatment with 1 µM simvastatin, yet allows us to best preserve the adult myocyte phenotype.Statin treatment of cultured myocytes is an excellent experimental model in which the concentration of drug and duration of treatment can be exactly controlled, and contribution from non-myocytes excluded. In the present study we have treated cells for 2 days with 10 µM simvastatin, a concentration which is widely used de novo cholesterol synthesis by simvastatin reduced cellular and caveolar cholesterol in the adult cardiac myocyte and this was, in turn, associated with a significant depletion of cellular and caveolar Cav3. Ultimately this change could act to preserve cellular cholesterol by reducing efflux. However, decreased caveolin expression has many additional consequences for cardiac cell function.Until recently, very little was known about cholesterol homeostasis in the cardiac muscle cell in vivo, we predict that the maintained decrease in cellular caveolin required to effect this normalisation of cholesterol would be sufficient to reduce the number of caveolae even in the presence of normal cellular cholesterol, since caveolin is required for the presence of caveolae e.g. Caveolar density was reduced in simvastatin-treated myocytes, as we would predict from reduced cellular and caveolar levels of cholesterol and Cav3. Although myocyte cholesterol levels would tend to normalise with statin treatment 2+]i transient. Our data suggest that the reduction in amplitude of [Ca2+]i transient is due to reduced Ca2+ -induced Ca2+ release secondary to decreased SR Ca2+ load. The hastening of Ca2+ transient decay is consistent with the 3-fold increase in pPLB  measured in statin-treated cells. However, in the absence of other changes in Ca2+ handling processes, the predicted effect of enhancing SERCA activity by increased pPLB would be an increase in SR Ca2+ content, [Ca2+]i transient amplitude and contractility 2+ load at a lower level . A previous study has reported increased SERCA, but not PLB, expression using a similar in vitro treatment regime with 10 µM simvastatin, however these experiments were performed with neonatal ventricular myocytes which differ structurally and functionally from their adult counterparts Simvastatin-disruption of caveolae was associated with 2 fundamental changes in cardiac myocyte contractility: a decrease in the degree of shortening and an increase in the rate of relaxation. Both could be explained by corresponding changes in the i transient responses to 10 nM isoproterenol. This is consistent with enhanced sensitivity of functional responses to β1-AR stimulation, comparable with data obtained using MBCD to disrupt myocyte caveolae ICa,L and contraction suggests there may be some selective effect of statin treatment on extra-junctional L type Ca2+ channels which are not involved in excitation-contraction coupling  ICa,L has been identified as one mechanism which subtends impaired contractility as a result of nNOS-derived NO ICa,L following statin treatment. Furthermore, it is necessary to account for the observed decrease in SR Ca2+ load with simvastatin treatment, seen despite elevated pPLB. One possibility is an increase in SR Ca2+ leak via NO-dependent S-nitrosylation of RyR It is difficult to interpret the consequences of statin treatment for cardiac myocyte function observed in the present study simply in terms of increased NO production, in part because the concentration and location of NO are key factors which determine its effects (eNOS and nNOS show different subcellular distribution) and in part because there is no clear consensus from different laboratories about the effect of NO derived from different sources. Data from NOS knockout mice suggest that eNOS derived NO has little effect on basal myocyte contractility, whereas nNOS-derived NO decreases or increases contractility whilst hastening relaxation see . The latIn terms of NO effects on β-AR responsiveness, it is generally agreed that eNOS-derived NO limits the response to non-selective β-AR stimulation whereas nNOS-derived NO effects are more diverse see . The marin vivo; our aim was to isolate the direct effects of statins on the cardiac myocyte. In vivo, contributions from statin effects on other cells of the myocardium  will be of significance, as well as the long-term compensatory changes in cholesterol homeostasis. So how do our data relate to what is known about myocardial function in people taking statins? There are several reports of improved cardiac function (measured using echocardiography) following statin treatment normocholesterolaemic subjects, statin treatment improves myocardial function indexed by strain imaging We are aware of the limitations of this work to the clinical situation where statins are taken long-term On balance, the effects of statins that we observe in this study are unlikely to compromise cardiac function in healthy individuals. In patients with heart failure, the observed direct effects of statin treatment on the cardiac myocyte are likely to be of benefit. A mild reduction in basal contractility may, similar to β-blockers, improve cardiac function by reducing myocardial energy expenditure. Increased myocardial relaxation will improve diastolic filling. In heart failure, the ratio of β1/β2-AR decreases, and Gi protein increases in vitro. This approach has allowed us to show for the first time that statins can modulate myocyte function via cholesterol-dependent effects on caveolin expression. Furthermore, as caveolin isoforms are ubiquitously expressed and have myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae.Our study presents a fundamental view of the effects of statins on the cardiac myocyte by treating myocytes All animal experimentation was carried out in accordance with the Directive 2010/63/EU of the European Parliament. Adult male Wistar rats were group housed with 12 h light/dark cycles and free access to food and water. Animals were used to harvest tissue only and were sacrificed according to a Schedule 1 method of the Animal (Scientific Procedures) Act of 1986 at a designated establishment.Ventricular myocytes were isolated enzymatically from hearts of rats (250–280 g) according to the method described by Calaghan et al. 2CO3 (pH 11.0) containing 0.5 mM EDTA and 1% protease inhibitor cocktail (Sigma). Samples were homogenised , then sonicated  3 times each for 20 s at full power. Approximately 2 ml of homogenate was mixed with an equal volume of 90% sucrose in MES-buffered saline  to form a 45% sucrose solution. A discontinuous sucrose gradient was created by layering on to this a further 4 ml each of 35% and 5% sucrose solution . Gradients were centrifuged for 17 h at 280,000 g (Beckman SW40Ti rotor) at 4°C. A total of 12 fractions (each 1 ml) were collected following fractionation.Myocytes were fractionated using detergent-free methods as described previously −1 with 4% donkey serum in phosphate-buffered saline) for 1 h at 37°C  in fixed, triton-permeabilised myocytes was imaged by immunocytochemistry as described in Protein expression in cell lysates before and after fractionation was measured by Western blotting as described in Myocytes were fixed overnight with 2.5% glutaraldehyde, post-fixed using 1% osmium tetroxide and dehydrated through an ethanol series. Myocytes were embedded in araldite, sectioned and stained with 20% uranyl acetate (30 min) followed by Reynolds lead citrate (30 min). Sections were visualised using a JEOL 1200 transmission electron microscope. Caveolae were defined as either 50–150 nm flask-shaped invaginations in the surface membrane or sealed circular vesicles of the same size within 300 nm of the sarcolemma 2+]i were measured in fura-2 loaded myocytes field-stimulated at 0.5 Hz (22–24°C) in HEPES-based physiological solution containing (mM): NaCl 137; KCl 5.4; NaH2PO4 0.33; MgCl2 0.5; HEPES 5; glucose 5.5; CaCl2 1 (pH 7.4) . Parameters were measured under basal conditions and following selective stimulation of β1 or β2 ARs. Recordings were taken at the peak steady-state response (≈5 min) following application of agonist.Shortening and [Ca2+ load and fractional SR Ca2+ release, electrically-stimulated Ca2+ transients were recorded at steady state, stimulation was switched off and caffeine (10 mM) was applied 10 s later via a local perfusion device. SR Ca2+ load was indexed as the amplitude of the caffeine-stimulated Ca2+ transient. Fractional SR Ca2+ release was calculated from the ratio of electrically-stimulated to caffeine-stimulated Ca2+ transient amplitude.In order to assess sarcoplasmic reticular (SR) CaICa,L was measured by voltage clamp, as described by INa, followed by 100 ms test pulse to 0 mV to activate ICa,L. Changes in ICa,L magnitude were monitored by repeating this protocol once every 5 s.β1-AR stimulation was achieved with 10 and 100 nM isoproterenol in the presence of the β2 antagonist ICI 118,551 . Selective β2-AR stimulation was achieved with 50 and 100 nM zinterol (Tocris) in the presence of the β1 antagonist CGP20712A . Myocyte populations used to measure phospho-protein responses were field-stimulated during β-AR stimulation. At 5 min, perfusion buffer was rapidly aspirated and replaced with Laemmli sample buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Thermo Fisher Scientific).−1 PTX for 3 h at 37°C. To determine the contribution of HMG CoA reductase inhibition and depletion of isoprenoids to basal and β-AR responses, mevalonate (100 µM), FPP and/or GGPP (10 µM) were added to statin-containing medium at the start of the 48 h culture period. For these protocols, shortening was measured in the absence of fura-loading.For experiments using pertussis toxin (PTX) to disable Gαi, cells were treated with 1.5 µg.mlet al. Quantification of nitrite and nitrate levels in culture medium was achieved using a method outlined by Nussler 1177 phosphorylated eNOS # 612392 (1∶1000) (BD Biosciences), phospholamban #A010-14 (1∶5000), Ser16 phosphorylated PLB #A010-12 (1∶2000), SERCA #A010-23 (1∶2000) (Badrilla), TnI #4002 (1∶1500), Ser22,23 phosphorylated TnI #4004 (1∶1500) , desmin #ab8592 (1∶1000) (Abcam), GAPDH #G9545  (Sigma).All reagents were from Sigma Chemical Co. unless otherwise stated. Antibodies used for Western blotting/immunocytochemistry: Cav 3 #610420 (1∶2500/1∶100), Cav1 #610406 (1∶2500), eNOS #610296 (1∶1000), Sern observations. Where experiments were performed on individual cells, these were from at least 3 different hearts. Statistical analysis was performed using the Student's t-test, Mann-Whitney Rank test, one way- or two way-ANOVA , as appropriate.Results are expressed as mean ± S.E.M of


The pathogenesis of chronic urticaria is not well delineated and the treatment is palliative as it is not tied to the pathomechanism. The centrality of mast cells and their inappropriate activation and degranulation as the key pathophysiological event are well established. The triggering stimuli and the complexity of effector mechanisms remain speculative. Autoimmune origin of chronic urticaria, albeit controversial, is well documented. Numerical and behavioral alterations in basophils accompanied by changes in signaling molecule expression and function as well as aberrant activation of extrinsic pathway of coagulation are other alternative hypotheses. It is also probable that mast cells are involved in the pathogenesis through mechanisms that extend beyond high affinity IgE receptor stimulation. An increasing recognition of chronic urticaria as an immune mediated inflammatory disorder related to altered cytokine-chemokine network consequent to immune dysregulation resulting from disturbed innate immunity is emerging as yet another pathogenic explanation. It is likely that these different pathomechanisms are interlinked rather than independent cascades, acting either synergistically or sequentially to produce clinical expression of chronic urticaria. Insights into the complexities of pathogenesis may provide an impetus to develop safer, efficacious, and targeted immunomodulators and biological treatment for severe, refractory chronic urticaria. Chronic urticaria is a distressing disorder that adversely impacts the quality of life; yet its pathogenesis is not well delineated and, accordingly, the treatment is often palliative and therapeutic outcome is suboptimal. This necessitates an understanding of the pathogenesis to facilitate development of improved therapies. In the recent past rapid strides in understanding the pathomechanism of chronic urticaria have been recorded; yet, most of the evidence based, seemingly impregnable and conclusive hypotheses have been countered by alternative, equally authentic, convincing and logistic counter explanations.The knowledge of molecular immunopathogenesis and complexities of effector mechanisms in chronic urticaria has been enhanced by immunohistologic studies performed on sequential biopsies of urticarial wheals and focused on infiltrating cell immunophenotypes and related cytokines, chemokines/chemokine receptors, and adhesion molecules .The urticarial wheal is characterized by dermal edema, vasodilatation, and perivascular nonnecrotizing infiltrate comprising primarily of mononuclear cells, predominantly CD4+ lymphocytes, with variable numbers of monocytes, neutrophils, eosinophils, and basophils –4. The dChronic urticaria is initiated by inappropriate activation and degranulation of dermal mast cells. This key pathophysiological event is predominant at the very onset and the released cellular contents prime the immediate phase of inflammation, which progresses to a complex interplay of varied proinflammatory mediators, cytokines, chemokines, chemokine receptors, and adhesion molecules that regulate vasoactivity and specific kinetics of cellular infiltration, ultimately evolving into a lymphocyte and granulocyte mediated hypersensitivity reaction, evident as urticarial wheals. The incoming inflammatory cells, in turn, release more proinflammatory mediators that serve to recruit and activate other cell types, thereby amplifying and extending the host response. The upregulation of inflammatory molecules, almost comparable expression of chemokines and adhesion molecules, and higher T-cell numbers in uninvolved skin is indicative of widespread immunologic activation, representing a low level priming of the cutaneous inflammatory and immunologic response apparatus, reconfirming the hypothesis of latent, minimal persistent inflammation in apparently uninvolved skin , 5. ThisHigher prevalence of thyroid autoantibodies in chronic urticaria .A wheal-and-flare reaction on intradermal injection of autologous serum in a subpopulation of patients (positive autologous serum skin test) and reproducibility on passive transfer of serum to normal healthy control subjects .Subsequent identification of IgG antibody directed to the alpha subunit of the IgE receptor, capable of inducing positive autologous serum skin test as well as histamine release from basophils . The incPositive association with HLA subtypes DRB*04 (DR4) and DQB 1*0302 (DQ8) .Therapeutic response to plasmapheresis  and intrThe evidence favouring autoimmune pathomechanism, although persuasively convincing, is incomplete. Certain issues, elucidated below, need to be addressed for unequivocal acceptance of the proposed hypothesis.The cutaneous response to intradermal injection of autologous serum may be due to the presence of nonimmunoglobulin vasoactive histamine releasing factors . MoreoveAnimal model, mandatory to establish autoimmune status of the disorder, is yet to be developed for chronic urticaria . de novo. Thus the concept of conditional autoimmunity has evolved [The autoantibodies of similar specificity have been detected in sera of healthy persons and may belong to the natural repertoire. Such natural autoantibodies may become pathogenic under certain circumstances and this occurrence is dependent on the state of occupancy of the FceRI receptor by its natural ligand IgE. The urticaria results from alteration in the tissue binding of preexisting autoantibody in susceptible individuals rather than its production evolved  in chronIt has been proposed that anti-FceRI and anti-IgE autoantibodies are not actually pathogenic but are secondary to the presence of urticaria in individuals with a predisposition to develop autoimmunity .The autoimmune origin is the most accepted hypothesis advanced to explain inappropriate activation and degranulation of mast cells in urticaria. Immune tolerance is maintained by a balance between autoreactive lymphocytes and regulatory mechanisms that counteract them. An increase in number and/or function of naturally occurring autoreactive T-cells or diminished regulator mechanism manifests as autoimmunity. Regulatory T-cells (T(REG)), particularly the naturally occurring CD4(+) CD25(+) subset of T (REG), provide a substantial component of autoimmune counterbalance. The identification of forkhead box P3 (FOX P3) as a critical determinant of CD4(+) CD25(+) T(REG) cell development and function has provided insights into the delicate balance between autoreactive and regulatory mechanisms in autoimmune disorders including chronic autoimmune urticaria. Functional assays and phenotype analysis have revealed that T(REG) isolated from patients of autoimmune disorder exhibit reduced regulatory function as opposed to those from healthy controls. It may be concluded that reduced percentage of CD4(+) CD25(+) FOX P3(+) regulator T-cells contributes to the autoimmune pathogenic process of chronic urticaria . The autTC, the subtype dominant in the skin, and participates in mast cell activation [The complex pathway involved in triggering, maintaining, and controlling autoantibody formation against FceRI and/or IgE remains unexplained. The autoantibodies relevant to chronic urticaria belong to complement fixing subtypes IgG1 and IgG3 . The vastivation . This trtivation  and the tivation . The dowtivation .The autoantibodies to alpha chain of FceRI and/or IgE have been detected in only 35 to 40 percent and 5 to 10 percent of patients, respectively . MoreoveNonimmunologic agonists including substance P, endorphins, enkephalins, endogenous peptides, and somatostatin may induce regulated degranulation and liberation of proinflammatory molecules from mast cells especially when the products of activated immune system lower the cutaneous mast cell release threshold .The primary abnormality in some patients of chronic urticaria might be cellular/subcellular rather than immunologically mediated autoimmune mechanism. in vivo to further FceRI induced activation. However, autoantibody mediated desensitization of IgE receptor seems unlikely as similar response of basophils has been observed in patients lacking autoimmune antibodies [There is increasing evidence of altered number, structure, function, and trafficking defects in basophils. Basopenia is well documented and basophil numbers are inversely related to urticaria severity . Other etibodies .A more complex picture has emerged from insight into the dysregulated expression of molecules that are critical to signal propagation or its inhibition after IgE receptor activation . Spleen  ex vivo activation of basophils by optimal concentration of polyclonal anti-IgE, it has been confirmed that distinct basophil degranulation phenotypes exist in chronic urticaria and a bimodal stratification of basophils has been proposed [In addition to these observations, based on the profile ofproposed . Fifty pThis pattern of basophil functional phenotypes (CIU-R and CIU-NR) appears to be independent of the existence and/or levels of autoantibodies  and remaThe levels and/or expression of regulatory proteins is functional and normalizes during remission . Such shThis profiling of signal proteins in basophils in conjunction with their stratification into distinct phenotypes has reaffirmed that abnormal basophil function may be a key factor in disease pathogenesis.A direct role of mast cells in CU is speculated . The act in vitro study performed to evaluate permeabilizing activity of CU serum, a similar pattern of mast cell degranulation and increased endothelial monolayer permeability was observed after exposure of two distinct mast cell lines (LAD-2 and HMC-1) [In and HMC-1) , 37 to Cd HMC-1) . It was d HMC-1) .It is likely that varied membrane receptors expressed on mast cells are selectively triggered by ligands such as IgG, peptides, microbial derivatives, and fragments of activated complement  and mastThe concept of chronic urticaria being an immune mediated inflammatory disorder evolved from incidental observation that recommendation of targeted immunomodulator biologic therapy, directed at a particular cytokine/cell receptor, administered for some other inflammatory disorder ameliorated coexisting chronic urticaria . It was The investigation of early events of innate immunity through the study of dendritic cells that link innate and adaptive immune system has provided insights into the dysregulated immune response in chronic urticaria . PlasmacAccordingly, a study of early events of the immune response, through the activation of pDC by TLR sensing, was undertaken to evaluate immune dysregulation in chronic urticaria .Plasmacytoid dendritic cells are unremarkably scattered amongst cellular infiltrate in the cutaneous lesion and their percentage in the peripheral blood mononuclear cells is unaltered and is similar to healthy controls. A normal degree of activation of pDC by the expression of costimulatory molecules and an increased constitutive STAT 1 phosphorylation on nonstimulated lymphocytes was observed. However, IFN-alpha secretion by pDC upon stimulation by CpGA was impaired and it was associated with altered IRF-7 and downregulation of TLR 9 expression, indicating a functional impairment of pDC, and was supportive of immune dysregulation in CU .Several hypotheses have been proposed to explain the downregulation of TLR9 in pDC after CpGA stimulation. It is probable that IgE or anti-FceRI autoantibodies cross-link FceRI on immature pDC impairing immune function by suppressing IFN-alpha production . AlternaThe dysfunctional innate immune response in CU consequent to functional impairment of pDC to TLR9 activation disturbs the cytokine production by T-cells, mainly of IL-17A and IL-10 . BesidesSignificantly higher serum levels of chemokines (C-C and C-X-C) including CXCL8, CXCL9, CXCL10, and CCL2 have been observed in CU and are not correlated with either the clinical parameters of the disorder or the outcome of basophil histamine release (BHR) and/or ASST assays . These pCXCL8/IL-8 is chemotactic for neutrophils, T-lymphocytes, and monocytes .CXCL9/Mig, a monokine induced by interferon- (IFN-) gamma induced protein 1 (IP-10), is a type 1 C-X-C chemokine and displays strong chemoattraction for T-helper type 1 (Th 1) lymphocytes , 58.C-C ligand 2 (CCL2) (monocyte chemoattractant protein 1), prototype of C-C chemokine, is secreted mainly by monocytes, as evidenced by increased mRNA expression in CD14 + cells in CU. CCL2 activates a variety of cells including monocytes, macrophages, lymphocytes, eosinophils, and basophils and is a crucial factor for the development of Th 2 responses . in vivo priming with potent basophil activating factor such as CCL2 [The upregulation of chemokines in CU contributes to the maintenance of activated status of inflammatory cell subsets. Basophils in CU display an upregulation of activation/degranulation markers, CD203c and CD63, and high responsiveness to interleukin-3 (IL-3) stimulation. It is likely that activated profile of basophils is triggered by as CCL2 .CCL2 induces degranulation of mast cell and has a potent basophil histamine releasing activity. Furthermore, it has been documented that an assembly of circulating chemokines CCL2, CCL5, and CXCL8 play an important role in mast cell activation and generation of histamine and serotonin .It may be inferred that immunologic dysregulation consequent to disturbed innate immune response alters the cytokine-chemokine network that triggers the inflammatory status contributing to the pathogenesis of CU.It has been observed that autologous plasma, anticoagulated with substances other than heparin, generates positive autoreactive responses in a higher percentage of patients than autologous serum skin test (ASST) , 63. As  In vitro studies have confirmed that plasma of urticaria patients has significantly higher levels of prothrombin fragment F1+2, a polypeptide of 34 kDa that is released into circulation during the activation of prothrombin to thrombin by factor X [The extrinsic pathway of coagulation is activated and thrombin is generated from prothrombin by activated factor X, in the presence of activated factor V and calcium ions . In vitrfactor X , and sevThrombin is a serine protease that enhances vascular permeability, activates and degranulates mast cells, and induces generation of anaphylatoxin C5a. The activation of extrinsic pathway of coagulation is thus proposed as yet another explanation.It is likely that different pathomechanistic pathways, namely, seroimmunologic autoimmune, inflammatory, cellular defects, coagulative, and complement system, are interlinked rather than separate independent cascades and there is extensive cross talk amongst them with mutual regulation of activation. They act synergistically or sequentially as either independent or interlinked pathomechanisms to activate mast cells with release of preformed mediators and/or secretion of newly synthesized vasoactive molecules to produce final clinical expression of urticaria , 69 Fig.The various mast cell mediators, preformed and newly synthesized, relevant to chronic urticaria, are summarized in Histamine is the principal vasoactive mediator and combined histamine-1 and -2 receptor responses are required for the full expression of histamine vasoactivity, including immediate vasodilation, alteration in vasopermeability, plasma extravasation, and dermal sensory nerve stimulation –71. Its It may be concluded that the pathogenesis of chronic urticaria is as perplexing as the disorder is intriguing and a concise pathomechanism has, as yet, not been identified that may provide a rational explanation for all cases. An incomplete understanding has hampered the search for novel, efficacious, low toxicity drugs that may be offered as alternatives in severe, unremitting urticaria, unresponsive to standard first line of management. It is, however, likely that newer insights into the complexities of pathogenesis may pave the way for evolving therapies that are more specifically tied to the pathomechanics of the disorder and provide an impetus to develop targeted immunomodulators and biological therapies that may be favorably included in therapeutic armamentarium.


B receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent  transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABA The magnitude of GHB-induced elevations was not different in magnitude to those evoked by the selective GABABR agonist baclofen . These [Ca2+]i elevations were present in the soma and in the fine astrocytic processes , and sometimes seen in processes without an apparent clear somatic response. Moreover, the GHB- and baclofen-elicited [Ca2+]i transients could be observed in the mouse VTA (figure 1c) as well as in the rat nucleus accumbens (not shown) and in the mouse and rat VB thalamus (figure 1c). The astrocytic effect of GHB and baclofen was concentration dependent with an ED50 of 1.6 mM and 1.3 µM, respectively (figure 1b), with 10 mM GHB and 10 µM baclofen evoking [Ca2+]i transients in 6.82 ± 0.39 astrocytes (n = 11 slices) and 7.14 ± 1.14 astrocytes (n = 14 slices), respectively.In the continuous presence of TTX (1 µM), relatively brief (2 min) application of GHB to rat VTA slices bulk-loaded with the CaBRs were indeed mediating the astrocytic GHB and baclofen responses described above, experiments were conducted using pharmacological and transgenic interventions. Both GHB and baclofen effects were virtually abolished by two structurally different GABABR antagonists, CGP5426 and SCH50911 . Interestingly, GHB responses were also markedly inhibited by the putative GHB receptor antagonists NCS382 , which, however, had no effect on the number of astrocytes responding to baclofen application . Finally, astrocytes in VTA slices from wild-type littermate mice  readily responded with [Ca2+]i elevations to GHB and baclofen applications, whereas both agonists failed to elicit astrocytic transients in slices from GABABR knockout  mice . However, astrocytes in GABABR KO and WT littermate mice responded similarly to glutamate , thus excluding the possibility that the mechanism underlying [Ca2+]i transients in astrocytes from the GABABR KO mice was compromised. Identical results were obtained in the VB thalamus of GABABR KO and WT littermate mice .To confirm that GABABRs that resulted in glutamate release and an indirect effect mediated by the activation of metabotropic glutamate receptors (mGluRs), which comprise a major signalling pathway in astrocytes . To deplete intracellular stores, we used the SERCA pump inhibitor cyclopiazonic acid , whereas in separate experiments we tested the action of GHB and baclofen in slices bathed with a nominal extracellular Ca2+-free solution. Responses to baclofen were significantly reduced by both interventions  as were those to GHB , showing that the effects of both agonists are dependent on Ca2+ release from intracellular stores and an extracellular Ca2+ supply, as previously described for similar [Ca2+]i transients observed spontaneously and evoked by other neurotransmitter agonists i oscillations which is associated with a [Ca2+]i-dependent increase in the frequency of astrocytic glutamate release events, i.e. neuronal SICs i elevations . Exposure to GHB also resulted in a reduction that approached statistical significance . To test whether these changes in spontaneous [Ca2+]i signalling were translated to spontaneous astrocyte–neuron glutamatergic signalling recorded as SICs, we therefore treated VTA slices with baclofen or GHB for extended periods of 1–3 h and compared these results with those of untreated slices. Patch-clamp recordings made from VTA neurons in the presence of TTX showed the presence of spontaneous SICs in the VTA, confirming the existence of spontaneous astrocyte–neuron signalling in this brain area (figure 4b). Short (3 min) pre-incubation of the slice with baclofen application, however, did not induce astrocytic glutamate release as measured by SIC recording . Nevertheless, in agreement with the observed decrease in astrocytic [Ca2+]i elevations observed during prolonged GABABR activation (1–3 h), SIC frequency was significantly reduced by sustained pre-exposure to GHB and baclofen .We have previously shown that the sustained stimulation of G-protein coupled mGluRs either by synaptic glutamate release or by agonist exposure results in an increase in the frequency of spontaneous astrocyte i transients in astrocytes of both the VTA and the VB thalamus of young rodents, two brain areas that are involved in the reward properties and the pro-absence effect of this drug, respectively. This astrocytic GHB action is mediated by GABABRs and is comparable to that evoked in astrocytes of the same regions by the selective GABABR agonist baclofen. Moreover, prolonged GHB and baclofen exposure causes a reduction in spontaneous glutamate release from VTA astrocytes.The main findings of this study are that GHB consistently elicits robust i transients were (i) abolished by two highly selective but structurally dissimilar GABABR antagonists, (ii) absent in GABABR KO mice and (iii) unaffected by the block of ionotropic and metabotropic glutamate receptors, indicating that in astrocytes, as occurs in neurons, the action of GHB is mediated by GABABRs. Indeed, the 1000-fold difference in the ED50 of the two drugs on astrocytic responses is very similar to that reported for their activation of pre- and post-synaptic neuronal GABABRs, including hyperpolarization of the membrane potential and inhibition of synaptic potentials [2+]i transients indicates that these responses are not mediated by activation of astrocytes by glutamate, secondary to neuronal activation, and suggests that they results from direct activation of astrocytic GABABRs.Both GHB- and baclofen-elicited i transients were blocked following perfusion of VTA slices with either a nominal extracellular Ca2+-free solution or with CPA. Thus, our results indicate that both GHB and baclofen astrocytic responses in VTA involve intracellular Ca2+ stores and their refilling by extracellular Ca2+. Whether GHB activity requires Gi/o proteins  or Gq proteins (which have been strongly linked to Ca2+ release from intracellular stores) remains to be elucidated, as is indeed the case for the astrocytic response to baclofen in VTA or any other brain region.GHB- (and baclofen)-elicited [Ca2+]i transients (see control grey bar in figure 4a), but decreased their frequency when applied for periods of 15 min or longer. A similar picture was obtained when looking at spontaneous SICs, the neuronal counterpart of astrocytic released glutamate, suggesting that long-term activation of GABABR lead to a reduction in the glutamate-dependent astrocyte to neuron signalling.Finally, in contrast to the activation of astrocytes, brief application of GHB and baclofen had no effect on spontaneous [Ca(b)2+]i transients [2+]i transients by NCS382 provide a potential explanation for the anti-absence effect of this putative GHB receptor antagonist, the action of which had so far been difficult to reconcile with its lack of action on neuronal response in thalamus i elevations. The mechanism underlying this increase is unclear but may involve some activation of Gq-coupled pathways, or novel interactions between GABAB auxiliary subunits and intracellular Ca2+ release pathways. Whatever the physiological role of the GABABR-induced [Ca2+]i elevations, it does not seem to invoke glutamate gliotransmitter release. It does, however, provide an experimental reporter of astrocyte GABABR activation, and our observations are consistent with dominant activation of non-Gq pathways, because we found a reduction in spontaneous gliotransmitter glutamate release (i.e. SICs). Interestingly, this observation is analogous to the effect of GABABR activation on inhibiting spontaneous presynaptic neurotransmitter release [Drugs that cause addiction act in the VTA to increase dopamine release in target areas, notably the nucleus accumbens. It has been suggested that GHB achieves this by inhibiting GABAergic interneuron activity which results in disinhibition of dopaminergic neurons in the VTA, and so increased dopamine release ,57. Our eceptors : these evia Gi/o . We foun release , and ind


To further investigate the potential involvement of SOCs in the generation of [Ca2+]i signals in human sperm, we have used cell-penetrating peptides containing the important basic sequence KIKKK, part of the STIM–Orai activating region/CRAC activating domain (SOAR/CAD) of the regulatory protein stromal interaction molecule 1. SOAR/CAD plays a key role in controlling the opening of SOCs, which occurs upon mobilization of stored Ca2+. Resting [Ca2+]i temporarily decreased upon application of KIKKK peptide (3–4 min), but scrambled KIKKK peptide had a similar effect, indicating that this action was not sequence-specific. However, in cells pretreated with KIKKK, the transient [Ca2+]i elevation induced by stimulation with progesterone decayed significantly more slowly than in parallel controls and in cells pretreated with scrambled KIKKK peptide. Examination of single-cell responses showed that this effect was due, at least in part, to an increase in the proportion of cells in which the initial transient was maintained for an extended period, lasting up to 10 min in a subpopulation of cells. We hypothesize that SOCs contribute to the progesterone-induced [Ca2+]i transient, and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca2+]i transient.Previous work has provided evidence for involvement of store-operated channels (SOCs) in [Ca Maturation of the ejaculated sperm to acquire competence to fertilize (capacitation), the regulation of motility pattern (behaviour and chemotaxis) and control of the secretion of the acrosomal vesicle (acrosome reaction), which releases hydrolytic enzymes and exposes proteins required for fertilization at the sperm surface, are all controlled separately through [Ca2+]i signals. Ca2+ channels and Ca2+ stores are present in mammalian sperm  mediated by store-operated channels (SOCs) , causing SOCs to open  from the store at the sperm neck  or SOAR (STIM–Orai activating region) is clear  that mimics the basic patch on the SOAR region of STIM. KIKKK may act directly to stimulate CCE by binding Orai or may interact with the acidic patch on STIM, thus interfering with the refolding that would normally render STIM quiescent upon store refilling.Use of cell-penetrating peptides (CPPs) to traffic targeted peptides and small proteins across the plasma membrane offers an alternative potentially more specific technique than pharmacological intervention. Rab3A and GST, both ∼25 kDa, are delivered into sperm when fused to a polyarginine CPP (RRRQRRKRRRQ) at the C terminus (2). Cells were harvested by direct swim-up into supplemented Earle's balanced salt solution (sEBSS), as described previously  for 4–5 h.Healthy donors were recruited in accordance with the Human Fertilisation and Embryology Authority Code of Practice (Version 8) and gave informed consent. Protocols were approved by the University Ethical Committee . Semen collected by masturbation after 2–3 days of sexual abstinence was allowed to liquefy for ≈30 min COONa, 1.8 CaCl2·2H2O and 15 HEPES , supplemented with 0.3% (w/v) fatty acid-free bovine serum albumin (BSA).sEBSS contained (mM) 118.4 NaCl, 5.4 KCl, 0.81 MgSOInitial experiments with complete sEBSS showed no effect of the peptides used here, but when peptides were applied in BSA-free medium, clear effects of peptide treatment occurred. We therefore completed preparation of spermatozoa and transfer to the recording chamber in complete medium, but the cells were then superfused with BSA-free EBSS for ∼5 min before recording commenced. No peptide controls were run under similar conditions.371-392; KQLLVAKEGAEKIKKKRNTLFG) [KIKKK] and a scrambled homologue (LKNKFKGVKLAEIEKQALKGTR) [scrambled KIKKK] were synthesized using microwave-assisted solid-phase peptide synthesis. Syntheses  were performed using a Discover SPS Microwave Peptide Synthesizer  with fibre optic temperature control employing an N-α-Fmoc protection strategy with O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) activation on Rink amide methylbenzhydrylamine resins pre-loaded with the first amino acid . Deprotection with 7 ml of 20% piperidine was performed for 3 min at 50 W/75°C. A majority of AA coupling reactions were accomplished with a 4-fold molar excess of Fmoc-protected AA with HCTU and diisopropylethylamine (DIPEA), molar ratio of 1 : 1 : 2 (AA/HCTU/DIPEA), in 4 ml for 10 min at 25 W/75°C. Arg coupling was performed in two stages: 30 min 0 W/∼25°C followed by 5 min at 17 W/75°C. N-terminal acylation with 6-carboxy-tetramethylrhodamine  yielded fluorescent peptides to confirm efficient uptake efficacy (A polycationic sequence derived from human STIM (STIM+) were confirmed to an accuracy of +1 by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry , probably reflecting specific binding of KIKKK after membrane translocation.Crude peptides were purified to apparent homogeneity using semipreparative scale, reversed-phase high-performance liquid chromatography. The predicted masses of all peptides used (average M+HLoading of cells with Oregon Green BAPTA 1 (Invitrogen) and time-lapse fluorescence imaging was done as described previously  in the experiment.Analysis of images, background correction and normalization of data were performed using IQ  and Excel (Microsoft) as described previously. Unless stated otherwise, the region of interest was drawn around the posterior head and neck (PHN) region of each cell. Raw intensity values were imported into Microsoft Excel and normalized using the following equation:2+]i transient was calculated from the three points spanning the peak of the ΔFmean trace. Amplitude at 3 min after progesterone application was calculated using six consecutive points spanning 2.5–3.5 min after application of progesterone.Amplitude of the progesterone-induced i was monitored during application. Exposure of human sperm to 5 µM KIKKK induced a transient decrease in [Ca2+]i that was detectable within <30 s, was maximal at ≈90 s  and recovered to control level within ∼3–4 min  were spontaneously active, generating [Ca2+]i oscillations under control conditions. In ∼40% of these cells, application of CPPs briefly arrested and reset this activity , but oscillation amplitude and kinetics were maintained, indicating that basal Ca2+ influx and Ca2+ clearance mechanisms were not affected by KIKKK or scrambled KIKKK peptide treatment  of control experiments , the rate of decay was significantly reduced in KIKKK-pretreatment experiments compared with parallel controls  was significantly greater in cells pretreated with KIKKK . This difference between control and pretreated cells persisted into the plateau phase of the [Ca2+]i signal in some sets of experiments, but this was inconsistent and at 7.5 min after progesterone addition the three conditions did not differ statistically (P > 0.2).We then investigated whether KKKK could affect Ca2+]i responses to progesterone occur in ≈98% of human sperm , a similar effect occurring with scrambled KIKK . However, inspection of single-cell traces showed that there was a marked increase in a subpopulation of ‘atypical’ responses where the decay of [Ca2+]i transient was greatly delayed, such that the [Ca2+]i response appeared monophasic rather than the normal biphasic pattern  gave values of 8.8% in control experiments with no pretreatment , 6.3% after pretreatment with scrambled peptide  and 15.2% after pretreatment with KIKKK peptide . To further quantify the occurrence of prolonged [Ca2+]i transients in KIKKK-pretreated cells, we calculated for each cell the mean amplitude  of the early sustained [Ca2+]i signal for the period from 3 to 5 min after progesterone application  and normalized this value to the amplitude of the preceding transient in that cell (S/T ratio). For control cells, the S/T ratio was 0.36 ± 0.03  and this was not significantly altered in cells pretreated with scrambled peptide . However, in cells pretreated with KIKKK, this ratio was significantly increased (P < 0.003) to 0.55 ± 0.05 . The frequency distribution of S/T ratios was clearly bi-modal. In control and scrambled peptide pretreated cells, most S/T ratios formed a peak with a mode of ≈0.4, but there was also a clear shoulder at 1.0, reflecting cells in which the initial [Ca2+]i ‘transient’ had not decayed within 3 min . We conclude that pretreatment with KIKKK increases the proportion of cells in which the progesterone-induced [Ca2+]i transient persists for several minutes before decaying.Though [Ca2+]i signal, we observed strong enhancement of the sustained Ca2+ response that was localized specifically to the midpiece, possibly reflecting enhanced accumulation of Ca2+ in the mitochondria due to the effects of the drug on mitochondrial Ca2+ export , it binds electrostatically to the net negative charge on the membrane surface. Until equilibration of the peptide across the membrane is reached, this binding is asymmetric, making the extracellular surface more positive and increasing the voltage gradient within the membrane. This effect, termed charge screening, functionally hyperpolarizes the membrane and can reduce the open probability of voltage-sensitive channels (such as CatSper) sufficiently to inhibit passive Ca2+ influx and lower resting [Ca2+]i  2-APB, which sensitizes SOC activation but does not affect CatSper currents, enhanced [Ca2+]i transient, apparently inhibiting a ‘late’ component of the transient which activates 10–20 s after initiation of the response  of the progesterone-induced [Ca2+]i oscillations, consistent with cyclical mobilization of stored Ca2+, occur after the initial [Ca2+]i transient. These are dependent on a low level of Ca2+-influx and are sensitive to manipulation of ryanodine receptors  decay of the [Ca2+]i. If this model is correct, pharmacological blockade of SOCs should have effects on the progesterone-induced [Ca2+]i signal. Indeed at high doses of 2-APB [100 µM, which inhibits SOC function . Though the effects of these drugs on the response to progesterone are consistent with activation of SOCs, the effects of SKF 96365 alone are such that they cannot be interpreted.These observations suggest that SOCs at the sperm neck . J.M. was supported by a University of Birmingham Teaching Assistantship. Funding to pay the Open Access publication charges for this article was provided by the Welcome Trust.L.L. was supported by the There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.


Background: TPCs are regulated by NAADP and other factors.Results: NAADP-induced Ca2+ release from acidic stores evokes depolarizing currents in pancreatic β cells. Inhibition of NAADP signaling or TPC knock out attenuates Ca2+ signaling and insulin secretion.Conclusion: NAADP-evoked Ca2+ release enhances β cell excitability and insulin secretion in response to glucose or sulfonylureas.Significance: NAADP signaling pathways offer novel therapeutic targets for diabetes treatment. 2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells.Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca Howignaling –24.2+-mobilizing messengers described, has been shown to mediate local Ca2+-signaling events by releasing Ca2+ from acidic, endolysosomal Ca2+ stores in several vertebrate and invertebrate cells  or bafilomycin, which disrupts acidic store Ca2+ storage implicating lysosomally related organelles as the principal target for NAADP in these cells . Except for the hormone release measurements (for which intact islets were used), clusters of islet β cells and single β cells were prepared by dispersing islets in a Ca2+-free medium and cultured on circular coverslips for 1–4 days in RPMI 1640 culture medium  containing 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10 mm glucose.Islets of Langerhans were aseptically isolated by collagenase digestion of the pancreases of 8–10-week-old male mice of the following strains: CD1, Tpcn2−/− , Tpcn1+/Tpcn1−/− , with Tpm Fura PE3-AM or Fura 2-AM  for 60 min at 37 °C in a bicarbonate-buffered solution containing 10 mm glucose. The coverslip was then used as the bottom of a temperature-controlled perifusion chamber  mounted on the stage of an inverted microscope. The flow rate was 1.5 ml/min, and the temperature within the chamber was 37 °C. [Ca2+]i was measured at dual-wavelength (340 and 380 nm) excitation spectrofluorimetry, using a CCD camera  to capture the emitted fluorescence at 510 nm. When [Ca2+]i was simultaneously measured in a voltage-clamped single cell, the patch pipette contained 100 μm Fura 2 pentapotassium salt, and the emitted fluorescence was captured at 510 nm using a photomultiplier .Cultured clusters of islet cells were loaded with 1 μm glucose and then transferred to the stage of an LSM 510 confocal microscope. After a further 10 min of perfusion by 3 mm glucose, the recording was started. The oxidized form of the FAD was excited at 488 nm. Emitted fluorescence was collected with a 505-nm long-pass filter.Cultured clusters of β cells were preincubated for 60 min at 37 °C in a control medium containing 3 mAll patch clamp measurements were carried out using a multiclamp 700B patch clamp amplifier and the software pClamp 9 . When using the perforated whole-cell mode of the patch clamp technique, the electrical contact was established by adding the pore-forming antibiotic, amphotericin B, to the pipette solution. Amphotericin (stock solution of 60 mg/ml in DMSO) was used at a final concentration of 300 μg/ml. The tip of the pipette was filled with antibiotic-free solution, and the pipette was then back-filled with amphotericin-containing solution. The voltage clamp was considered satisfactory when the access resistance was <30 megohms and stable. In the standard whole-cell configuration, the access resistance was <15 megohms. Patch pipettes were pulled from borosilicate glass capillaries ; they had resistances of 3–5 megohms when filled with intracellular solution.+ current, which is known to be largely inactivated at resting potential in β cells but not in α and δ cells. Thus, cells in which a large Na+ current could be activated by a small depolarizing pulse from a holding potential of −70 mV were discarded. By contrast, cells that displayed a Na+ current only after a hyperpolarizing pulse to −140 mV were considered to be β cells and were used for the experiments.All experiments were carried out on single β cells. Two criteria were used to identify β cells. The capacitance of mouse α-, δ-, and β cells has been reported to be 4.4, 5, and 7.4 picofarads, respectively. Therefore, only large cells with a capacitance of >5 picofarads were chosen for this study. The average capacitance was 7.6 ± 0.2 picofarads. After verification of the capacitance, a depolarizing protocol was applied to identify the properties of the voltage-dependent NaATP channel current (IK(ATP)) was monitored by 100-ms duration pulses of ±20 mV from a holding potential of −70 mV. Whole-cell Ca2+ currents were recorded by depolarizing the plasma membrane with a 100-ms pulse from −80 to 10 mV.The whole-cell Km) the following: 120 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgCl2, and 24 NaHCO3. It was gassed with O2/CO2 (94:6) to maintain pH 7.4 at 37 °C. Except for the electrophysiological experiments, it was supplemented with 1 mg/ml BSA . When the concentration of KCl was increased, the concentration of NaCl was correspondingly decreased to keep the osmolarity of the medium unchanged.The medium used for the isolation of islets and for all experiments was a bicarbonate-buffered solution containing (in mIK (ATP), the standard extracellular solution contained (in mm) the following: 140 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgCl2, 5 HEPES (pH adjusted to 7.40 with NaOH), and 10 mm glucose. These solutions were gassed with O2/CO2 (94:6%). For the perforated patch measurements of membrane currents and potential, the pipette solution contained (in mm) the following: 70 K2SO4, 10 NaCl, 10 KCl, 3.7 MgCl2, and 5 HEPES (pH adjusted to 7.1 with KOH). For whole Ca2+ current, the pipette solution consisted of (in mm) the following: Cs2SO4 substituted for K2SO4. For NAADP infusion experiments, the pipette solution contained (in mm) the following: 125 K+ gluconate, 10 KCl, 10 NaCl, 10 KCl, 1 MgCl2, 3 Mg-ATP, 0.1 Na-GTP, and 5 HEPES (pH adjusted to 7.1 with KOH). In Fig. 3A, 100 μm Fura 2 pentapotassium was added.For electrophysiological measurements of Tpcn2 exons 4–8 amplicon . The following parameters were used for RT: 50 °C (30 min), 94 °C (2 min); PCR, 30 cycles of 94 °C (15 s), 57 °C (30 s), and 68 °C (1 min), followed by a final extension at 68 °C (10 min). For gene expression analysis in β cells, cDNA was produced from total RNA using the High Capacity cDNA reverse transcription kit (Applied Biosystems), and PCR was performed with the following gene-specific primers for Tpcn2 exons 22–25 amplicon . The following parameters were used for PCR: 30 cycles of 95 °C (30 s), 53 °C (30 s), and 72 °C (1 min), followed by a final extension at 72 °C (10 min). Tpcn1 mRNA expression was analyzed as described previously . RT-PCR was performed in a reaction containing extracted RNA, the SuperScriptII One-Step RT-PCR system with Platinum Taq (Invitrogen), and the following gene-specific primers: eviously .m) the following: 18.5 NaCl, 2.54 CaCl2, 1.19 KH2PO4, 4.74 KCl, 25 NaHCO3, 1.19 MgSO4, and 10 HEPES (pH 7.4). Samples of the supernatant were assayed for insulin using a mouse insulin ELISA kit . Where Ned-19 was used, islets were preincubated for 5 min with the drug prior to the addition of secretagogues.Islets were isolated from mice and cultured in RPMI 1640 medium overnight before insulin secretion was assessed. Insulin secretion was measured during 1-h static incubations in Krebs-Ringer Buffer (KRB) containing (in mTpcn1−/− (Tpcn1tm1Dgen) (Tpcn2−/− (Tpcn2Gt(YHD437)Byg)   and Tpcn437)Byg)  mice and2O/HCl, 52:17:1). All samples were then stored at −20 °C. Only experiments with an output rate greater than 200 ml/min were assayed for insulin. The hormone assay was done using a commercially available RIA kit .Pancreatic perfusions were performed within 15 min of cervical dislocation in 80–100-day-old mice essentially as described elsewhere . At the Human islets were prepared from beating heart donors with appropriate ethical permission and consents as described previously . Islets en bloc with uranyl acetate. Sections were further contrasted with Reynold's lead.The islet preparations were fixed in 2% paraformaldehyde and 2% glutaraldehyde in cacodylate buffer in the presence of calcium chloride, washed, post-fixed in 1% osmium tetroxide, and contrast-enhanced by staining Ned-19, Ned-20 , and NAA2+ concentrations ([Ca2+]i) in primary mouse pancreatic β cells using the membrane-permeant NAADP analogue NAADP-AM (A) , in the presence of low glucose (3 mm), evoked delayed [Ca2+]i increases (A). The peak was reached >15 min after application of NAADP-AM (F). To assess the role of the ER in the action of NAADP-AM  . In addition, the membrane-permeant NAADP antagonist Ned-19 (100 μm) (F) (2+ transient evoked by NAADP-AM (D and E). Collectively, these data suggest that NAADP targets acidic Ca2+ stores rather than the ER in mouse pancreatic β cells.We first investigated the effects of NAADP on intracellular CaNAADP-AM . Given tNAADP-AM , differeNAADP-AM A 58). 1. 12+ conncreases A. The peNAADP-AM F. To assNAADP-AM , we trearvations , the NAA, and F) . Previouer cells , 60. Accreatment , C and E(100 μm) F 21) al al2+ conNAADP-AM , D and Em glucose resulted in [Ca2+]i oscillations that were superimposed upon a sustained plateau (A). Acute application of 60 nm NAADP-AM first enhanced Ca2+ spiking from 5.8 ± 0.7 to 11.6 ± 1.3 spikes/min  and then abolished glucose-evoked [Ca2+]i oscillations after about 20–25 min (B). This finding is consistent with the bell-shaped concentration-response curve to NAADP in mammalian cells  . The structurally related analogue Ned-20, which is not an NAADP antagonist (G) (E).We next examined whether NAADP signaling plays a role in glucose-mediated responses in primary β cells as suggested previously in MIN6 cells . Stimula plateau A. Acute 0–25 min B. This fan cells , 51, 61;an cells , 18. They the ER C 62). W. Wm glucbolished D. The sttagonist G 21), w, wm glucnist (G) E.2+ from acidic organelles (C) , as observed previously in the MIN6 β cell line (2+]i oscillations (I). Similar results were obtained with concanamycin, another V-type-H+-ATPase blocker (K). The effect of bafilomycin on the frequency of glucose-evoked [Ca2+]i transients is summarized in Fig. 2J. These observations are consistent with the hypothesis that NAADP-sensitive acidic Ca2+ stores play a key role in sustaining glucose-induced [Ca2+]i oscillations in mouse pancreatic β cells (Because NAADP mobilized Caganelles C 60), w, w2+ fro+]i rise H, as obsell line . When ballations I. Simila blocker K. The ef β cells .2+ release on the excitability of pancreatic β cells in more detail, we infused NAADP (10–100 nm) through a patch pipette in the standard whole-cell configuration while simultaneously measuring the membrane currents. In β cells clamped at −70 mV, intracellular application of 100 nm NAADP evoked intermittent inward currents of varying amplitudes (A). These currents contrast with the periodic outward hyperpolarizing K+ currents evoked by IP3-evoked Ca2+ release in β cells (2+-mobilizing messengers. The inward currents were absent in control cells where no NAADP was added to the intracellular solution (B), as well as when highly desensitizing concentrations (>1 μm) of NAADP were used (C), consistent with the bell-shaped concentration-response curve due to self-desensitization of the NAADP receptor . When N-methyl-d-glucamine replaced extracellular K+ and Na+, no inward currents could be seen in response to 100 nm NAADP . Taken together with the BAPTA studies, these data are consistent with the inward currents being due to opening of Ca2+-activated cation channels. The NAADP antagonist Ned-19 (100 μm) also abolished the NAADP-evoked currents, as shown previously  , suggesting a possible involvement of TRPM4 channels.We have previously shown that intracellular application of NAADP in β cells evokes oscillatory currents , 29. To plitudes A. These  β cells , and thesolution B, as welere used C, consisreceptor , 61. MorFig. 3F) . NAADP-eLa cells , and bececretion  along wiecretion –69, we tm) concentrations also evoked spontaneous inward currents in cells voltage-clamped at −70 mV (H). These glucose-induced inward currents were also blocked by Ned-19 (100 μm) (I). The frequencies and amplitudes of the above NAADP- and glucose-evoked currents from A–I, are summarized in J and K, respectively. Taken together, these data raise the exciting possibility that NAADP-mediated Ca2+ release from acidic stores may modulate the glucose-mediated membrane currents that in turn initiate β cell electrical activity and insulin secretion.Importantly, like NAADP, high glucose (15 mt −70 mV H. These (100 μm) I. The frm) alone (A) had no effect on resting membrane potential (around −70 mV). In the presence of 3 mm glucose, NAADP-AM (60 nm) evoked low amplitude voltage oscillations that did reach the threshold for action potential firing (B). At 10 mm glucose, glucose-induced electrical activity consisting of 30–40-mV action potentials was observed. Under these conditions, the blockade of NAADP action with Ned-19 (100 μm) resulted in membrane hyperpolarization and suppression of electrical activity (C).We next examined the effect of intracellular NAADP on the pancreatic β cell plasma membrane potential. A nonstimulatory level of glucose (3 mm) alone A had no l firing B. At 10 activity C.2+ release is involved in the regulation of the membrane potential by glucose, pharmacological manipulation of ion fluxes across the endomembranes involved would be predicted to impact on glucose-mediated changes in membrane excitability. It is therefore of interest that bafilomycin reversibly suppressed glucose-evoked action potentials (D).If NAADP-evoked Catentials D.2+ signaling and electrical changes above were ascribed to antagonism of NAADP and abrogation of acidic organelle Ca2+ storage, it was important to rule out other targets that could potentially account for the effects of these two agents on glucose action.Although the profound effects of Ned-19 and bafilomycin on glucose-mediated CaA). The inhibitor was also without effect on KATP channel activity; in the presence of 10 mm glucose, the whole-cell conductance averaged 0.21 ± 0.04 and 0.20 ± 0.06 nanosiemens in the absence or presence of Ned-19 (B). By contrast, the combination of the KATP channel activator diazoxide and the mitochondrial inhibitor sodium azide (1 mm) resulted in a large increase in KATP channel activity. Similarly, there was no inhibitory effect of Ned-19 on the voltage-gated Ca2+ currents; the peak current during depolarization from −70 to 0 mV averaged 110 ± 1 pA (n = 10) and 114 ± 1 pA (n = 10) in the absence and presence of Ned-19, respectively (C). Thus, the suppression of electrical activity cannot simply be attributed to activation of KATP channels or inhibition of VDCCs. In addition, Ned-19 was without effect on Ca2+ release induced by the stimulation of muscarinic receptors with acetylcholine (100 μm) (D), which leads to the opening of IP3 receptors and discharge of ER stores  D, which R stores . These dE–G), VDCCs (H), or modulation of KATP channels (I). Moreover, in the absence of extracellular Ca2+, bafilomycin increased [Ca2+]i after thapsigargin treatment (J), and vice versa (K), confirming that bafilomycin-sensitive Ca2+ stores are distinct from the ER. The application of the lysomotropic agent GPN, a lysosomotropic agent that abrogates Ca2+ storage by lysosomes (L), again indicating that a lysosome-related organelle is the likely source of Ca2+ release.We next analyzed the effects of bafilomycin on the same functional parameters. The effects of the latter inhibitor were independent of alterations in glucose-evoked changes in mitochondrial membrane potential or cell metabolism , E–G, VD), VDCCs H, or modchannels I. Moreovreatment J, and vice versa K, confirysosomes , 25, exeysosomes L, again Tpcn2 gene, as a critical mediator of the NAADP response (2+ release from acidic stores (Tpcn2 is expressed in mouse islets (A). Affirming the localization of TPC2 to acidic stores in β cells, immunolocalization of the endogenous TPC2 in primary human β cells revealed substantial overlap with immunoreactivity of LAMP1, a major lysosomal marker (B). There was substantially less overlap with immunoreactivity for insulin or EEA1, used as markers for insulin granules (C). A similar lysosomal localization of TPC2-mCherry transfected into this murine β cell line was seen, but some co-localization with insulin granules could also be seen.Turning to the molecular targets for NAADP, we have recently identified TPC2, encoded by the response , and we c stores . This chc stores . RT-PCR e islets A. Affirml marker B. There granules  and endogranules , respectgranules C. A simim) into mouse pancreatic β cells from age-, sex-, and background-matched wild-type and Tpcn2−/− mice via the patch pipette during standard whole-cell recordings while simultaneously measuring intracellular Ca2+ concentrations (by Fura 2) and inward membrane currents. In wild-type β cells, NAADP (100 nm) evoked [Ca2+]i transients, some of which associated with transient inward currents (A). However, both NAADP-evoked Ca2+ transients and currents were absent in cells prepared from Tpcn2−/− mice (B), mirroring the effect of Ned-19 on the wild-type cells (F). Detailed morphological comparison of β cells from Tpcn2−/− and wild-type mice by electron microscopy indicated no substantial differences in morphology, organelle number, or distribution, including those of insulin granules (C), making it unlikely that changes in these parameters were the underlying cause for alterations in NAADP responses observed in Tpcn2−/− null β cells.We next examined the effects of infusing NAADP . In Tpcn2−/− β cells, glucose-evoked Ca2+ transients were either abolished, reduced in amplitude, or delayed (B and C) compared with the robust responses observed in wild-type cells (A and C). The average [Ca2+]i rises evoked by high glucose were substantially reduced (but not abolished) in all Tpcn2−/− β cells studied (C), although activation of VDCCs by membrane depolarization by K+ (45 mm) in the presence of the KATP channel opener, diazoxide (100 μm), still evoked a large [Ca2+]i response (B). Similar results were obtained from Tpcn1−/− β cells (D–F). These studies indicate that the lysosomal TPC2 and endosomal TPC1 channels play a significant role in the generation of glucose-induced Ca2+ signals in pancreatic β cells, likely through their modulation of β cell electrical activity.Next, we examined the Ca−/− mice , A and B delayed , B and Cpe cells , A and C studied C, althouresponse B. Simila β cells , D–F. ThATP inhibitor tolbutamide to mimic the electrical effects of raised glucose levels in mouse β cells (m), tolbutamide (25 μm) treatment failed to increase [Ca2+]i, in contrast to the effect in the presence of 3 mm glucose. However, pretreatment with NAADP-AM (60 nm) partially reconstitutes the Ca2+ signal with tolbutamide in the absence of glucose (G and H). Furthermore, we found that in wild-type mouse β cells exposed to 3 mm glucose, tolbutamide (25 μm) evoked a rise in [Ca2+]i (I), whereas cells from Tpcn2−/− mice failed to respond to tolbutamide.It has been noted that a minimum concentration of glucose “fuel” is required for threshold concentrations of the oral hypoglycemic agent and K β cells . In agre glucose , G and H [Ca2+]i I, wherea2+ stores in Ca2+ signaling and electrical activity, we finally examined the effect of disrupting NAADP signaling upon insulin secretion itself. Insulin secretion was measured from isolated whole islets in response to glucose or 45 mm K+. Prior treatment of islets with Ned-19 (100 μm) for 5 min substantially inhibited insulin secretion induced by 15 mm glucose but not by 45 mm K+ (A). The absence of an effect of Ned-19 on insulin secretion induced by high K+ makes it unlikely that there is a relevant off-target effect of the inhibitor upon the exocytotic machinery.Having implicated a key role for NAADP, TPC2, and lysosomal Ca45 mm K+ A. The abm glucose using the perfused pancreas preparation in wild-type, Tpcn1−/−, and Tpcn2−/− mice. In the Tpcn1−/− and Tpcn2−/− pancreata, both the 1st  and 2nd phase release (insulin secretion once the 1st phase release had ended) were reduced by ∼50% (B–D).We then studied insulin secretion evoked by 20 m by ∼50% , B–D.Tpcn1−/− mice (E) the blood glucose levels peaked at significantly higher levels than WT, and the time course revealed an impaired glucose tolerance capacity. In contrast, Tpcn2−/− mice (F) were less affected.Finally, we performed intraperitoneal blood glucose tests in fasted TPC knock-out mice in comparison with the corresponding strain-matched wild-type animals to examine the effect of perturbing TPC expression upon glucose homeostasis in the whole animal. This revealed that in −/− mice E the blo−/− mice F were le2+ signaling during stimulus-secretion coupling in mouse pancreatic β cells. Since its discovery as a potent Ca2+-mobilizing agent in sea urchin egg homogenates . In contrast, thapsigargin (a SERCA pump inhibitor that blocks Ca2+ uptake into the ER) was found to enhance NAADP-AM-induced Ca2+ release. This suggests that NAADP-evoked Ca2+ release in the β cell does not trigger further Ca2+ release through ER mechanisms. Rather the predominant role of the ER here is to act to buffer Ca2+ rather than as a source for release, and the functional removal of the ER decreases Ca2+ buffering, allowing Ca2+ release from acidic stores to increase further in the cytoplasm. The role of the ER to buffer Ca2+ during signaling has also been noted for glucose-evoked Ca2+ signals where glucose first decreases cytoplasmic Ca2+ due to increased ATP generation and stimulation of SERCA pumps . We found that endogenous TPC2 proteins in human β cells co-localized with lysosomal markers (B). Interestingly, TPC2 did not appear to colocalize with insulin granules, which have also been proposed to function as NAADP-sensitive Ca2+ stores in β cells . Application of higher NAADP (100 μm) gave no response, consistent with the bell-shaped concentration-response curve for NAADP in mammalian systems and paralleling the concentration dependence of NAADP for Ca2+ release in β cells (A) , an agonist that has also been reported to elevate NAADP levels , and neither NAADP-evoked Ca2+ transients nor currents were observed in cells from Tpcn2−/− mice (B). We propose that these currents are due to NAADP-evoked Ca2+ release from endolysosomal stores via TPC2 channels and that this, in turn, via elevation of [Ca2+]i, leads to Ca2+-dependent activation of plasma membrane cation channels, possibly TRPM4 or TRPM5. Activation of these channels would then result in membrane depolarization. The finding that application of NAADP-AM elicited a series of membrane potential spikes (B) is consistent with this scenario and in agreement with a report that NAADP causes membrane depolarization in INS1 cells   and otheells (A) . These cP levels , in the P levels . The ideLa cells . Moreoveell line –68, 89 ativation . The NAAansients A, and ne−/− mice B. We prol spikes B is consS1 cells .2+ signaling in glucose-induced electrical activity and [Ca2+]i, four different approaches were used to block NAADP signaling. These were as follows: (i) abrogation of Ca2+ storage by acidic stores with vacuolar proton pump inhibitors and GPN; (ii) inhibition of the NAADP receptor by Ned-19; (iii) self-desensitization of the NAADP receptor by NAADP; and (iv) knock-out of Tpcn2 and Tpcn1, genes encoding proposed NAADP target channels. Intriguingly, high glucose was also found to evoke small Ned-19-sensitive currents similar to those evoked by pipette application of NAADP (H). Thus, NAADP signaling may contribute, at least partly, to bringing the membrane potential from rest to the threshold for activation of VDCCs . Indeed, it is remarkable that tolbutamide cannot by itself mimic glucose-induced Ca2+ signals but requires NAADP/TPCs. Indeed, tolbutamide will only evoke Ca2+ signals when the acidic vesicle pathway is co-stimulated either with subthreshold concentrations of NAADP/AM or with a permissive subthreshold glucose (3 mm) concentration (G and H).To investigate the role of NAADP-mediated Caof NAADP H. Thus, of VDCCs . As has required . We proppe cells I. Indeedntration , G and H+, which bypasses electrical activity and depolarizes the membrane potential to ∼−10 mV and opens the VDCCs. This finding makes it possible to discard the explanation that Ned-19 inhibits insulin secretion by an off-target effect on the exocytotic machinery. In a more in vivo setting, glucose-evoked insulin secretion from perfused whole pancreata from Tpcn2−/− and Tpcn1−/− mice was investigated. Insulin secretion stimulated by glucose (20 mm) was substantially reduced compared with that from wild-type animals (B). Thus, we provide evidence that NAADP signaling is an important regulator of stimulus-secretion coupling in pancreatic β cells (The final step in the stimulus-secretion coupling is the exocytosis of insulin-containing granules. In isolated islets, Ned-19 completely blocked glucose-evoked insulin secretion. Ned-19, however, had no effect on secretion evoked by depolarizing islet cells with high extracellular K animals B. Thus,  β cells .Tpcn−/− mice are only mildly diabetic as assessed by glucose tolerance tests (Tpcn1−/− mice. However, a recent study has implicated TPC2 as a novel gene for diabetic traits in mice, rats, and humans (Tpcn2−/− mice. The effects of knocking out Tpcn genes in mice may result in complex phenotypes, including compensatory mechanisms, with regard to blood glucose and insulin levels because NAADP-mediated Ca2+ release has been implicated in GLUT4 translocation in murine skeletal muscle (Surprisingly, ce tests , with a d humans , with a l muscle . Furtherl muscle . Future 2+ signaling (Although NAADP levels in β cells have been reported to be increased in response to elevated glucose, and also in response to the incretin hormone GLP-1 , 20, theignaling . NAADP signaling . The lumignaling . We showignaling .CD38 gene (db/db mice, an animal model of type 2 diabetes (Antibodies to CD38  and a miD38 gene  have beediabetes , presuma2+ release from acidic stores via TPC2 or TPC1 channels evokes a small local Ca2+ signal that activates Ca2+-dependent cation currents in the plasma membrane. The finding that the membrane currents evoked by intracellular Ca2+ mobilization are blocked by 9-phenanthrol implicates TRPM4 channels in this process. Additional direct effects of NAADP-evoked Ca2+ release on exocytosis itself cannot be excluded at this stage, with a possible contribution from exocytotic granules themselves (ATP channel closure resulting in VDCC-mediated Ca2+ influx and insulin secretion (We propose that NAADP-evoked Caemselves , 96. Theecretion . This neTpcn2−/− mice. F. C., T. P., and G. S. C. performed some of the [Ca2+]i measurement experiments. K. C. performed the gene expression experiments. R. P., A. M. L., and G. C. C. synthesized and characterized NAADP-AM, Ned-19, and Ned-20. A. J. M. designed experiments and produced A. A. and A. G. designed the experiments, and A. A. conducted the project. A. A., J. P., G. A. R., and A. G. wrote the manuscript. M. R., L. T., K. R., and J. P. produced and characterized the


In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca2+ ([Ca2+]i) via Ca2+ influx, Ca2+ mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca2+ via multiple mechanisms in beta-cells from both diabetic db/db mice and non-diabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca2+]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca2+ concentration in the ER, a compensatory up-regulation of the plasma membrane Na+/Ca2+ exchanger (NCX) and a reduction in depolarization-evoked Ca2+ influx. As a result, the patterns of glucose-stimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.Upon glucose elevation, pancreatic beta-cells secrete insulin in a CaThe online version of this article (doi:10.1007/s13238-014-0075-7) contains supplementary material, which is available to authorized users. This process involves different components of the Ca2+ signaling pathways. For example, depolarization triggered by glucose metabolism opens L-type voltage-gated Ca2+ channels, leading to an initial extracellular Ca2+ influx (Rorsman 2+ stores via pathways such as the IP3 and ryanodine receptors (Islam 2+-ATPase (SERCA), the plasma membrane Ca2+-ATPase (PMCA) and the sodium calcium exchanger (NCX) are activated to reduce elevated [Ca2+]i to the physiological resting level  diabetic rats  was reduced only slightly, to 89% of the control, while the ER mobilization pathways remained unchanged. Overall, these changes led to significant alterations in the glucose-induced calcium oscillations in the beta-cells of the db/db mice relative to the control.To address these questions, we compared calcium clearance in age-matched pancreatic beta-cells that were isolated from both C57BL/6J control mice and db/db mice, a widely used type II diabetic mouse model. The changes can be quantitatively modeled as a 35% reduction in SERCA2 activity, a full inhibition of the SERCA3 pump, a 30% increase in the NCX capacity and a 27% reduction in Ca2+ clearance after depolarization, control and db/db cells were stimulated with 70 mmol/L KCl for 3 s and then switched to normal extracellular solution , an irreversible inhibitor of the SERCA pump, the increase in [Ca2+]i remained smaller, but the clearance was faster in db/db cells than it was in the control cells  or a high pH extracellular solution (Na8.8) after depolarization in cells pretreated with TG . In contrast, [Ca2+]i returned to the basal level with the same kinetics in db/db and control cells when only the PMCA pump was functioning (TG + Li7.4). Taken together, these results indicate the selective down-regulation of depolarization-induced Ca2+ influx and the SERCA pump and the up-regulation of NCX in db/db cells.We have previously dissected the contributions of multiple clearance mechanisms in pancreatic beta-cells isolated from Balb/c mice through pharmacological manipulation  and the initial slope of the [Ca2+]i trace immediately before BHQ application (J1) represented the SERCA-dependent uptake of cytosolic Ca2+, which was correlated with different [Ca2+]i levels  than does SERCA2 (270 nmol/L) . In contrast, the relative contribution of the NCX increased, especially at high [Ca2+]i (>900 nm), indicating that NCX hyper-activity may compensate for the loss of the SERCA3 subtype.Previously, we built a mathematical model to simulate calcium clearance in normal beta-cells that used the Kons Fig. A. This s2+ to ER calcium stores  to block the NCX and Ca2+ influx and were then perfused with BHQ to trigger Ca2+ release from the ER calcium stores  and could be described by a Hill function that correlated with the different [Ca2+]i levels ) were a result of both the release from the ER (Jrelease) and Ca2+ extrusion via the PMCA, the Jrelease(t) was calculated as the difference between JBHQ and JPMCA at each time point  at different time points was calculated based on the following equation:SERCA pump defects combined with an up-regulation of NCX function often result in reduced uptake of Ca Na+ Li7. to block Na+ Li7. to block Na+ Li7. to blockvi and vER are the volumes of the cytoplasm and the ER, respectively, and κi and κER are the calcium buffering ratios of the cytoplasm and the ER, respectively. The minus of the integral . Although the time courses of Jrelease were apparently different in different cell types  in db/db beta-cells was relatively small, in contrast to the severe reduction in [Ca2+]ER from insulin-secreting cell lines treated acutely or chronically with saturated fatty acids  as previously described  at room temperature for 20–25 min. The modified Ringer’s solution was also used as the extracellular solution for basal recording in the glucose-induced calcium oscillation experiments, in which 15 mmol/L glucose replaced 4 mmol/L glucose as the stimulus. For the Ca2+ clearance/[Ca2+]ER evaluation experiment, the cells were perfused with the modified Ringer’s solution containing 15 mmol/L (instead of 4 mmol/L) glucose and 250 µmol/L diazoxide, and various reagents were added to this solution to inhibit clearance mechanisms, as described in the individual experiments. The depolarization solution (KCl) consisted of 70 mmol/L KCl, 67 mmol/L NaCl, 4 mmol/L CaCl2, 1 mmol/L MgCl2, 15 mmol/L glucose, 250 µmol/L diazoxide, and 10 mmol/L HEPES, pH 7.3. To inhibit the NCX, we used a Na+-free solution in which Li+ replaced Na+ (Li7.4) and raised the pH of the solution to 8.8 (Na8.8) to slow the PMCA pump  in modified Ringer’s solution  light source, and the emission at 505 nm was collected by a photodiode. The PULSE software was used to coordinate protocols and collect data, which were analyzed using IGOR Pro. The standard calibration parameters R2+ by the ER via the SERCA pump. The total cellular Ca2+ flux can be calculated as the rate of change of [Ca2+]i (defined as –d[Ca2+]i/dt). Prior to the application of BHQ, the total cellular Ca2+ flux (J1) can be described by the equationFollowing previously published reports  according toJSERCA was calculated as the difference between J1 and J2. In Fig. + solution containing BHQ to obtain Ca2+ clearance by the PMCA pump alone, and JPMCA was fitted to a Hill equation to describe its relationship to different [Ca2+]i levels. Therefore, a different Jrelease(t) was calculated based on Eq. JPMCA was inserted.Therefore, the value of t-test for single Gaussian distributed datasets or the Mann-Whitney rank sum test for non-single Gaussian-distributed datasets. The asterisks *, **, and *** denote statistical significances with P values less than 0.05, 0.01, and 0.001, respectively.All data were analyzed using the Igor Pro software . The averaged results are presented as the mean value ± SEM of the number of experiments indicated. The statistical significance was evaluated using either Student’s Supplementary material 1 (PDF 30 kb)Below is the link to the electronic supplementary material.