In the heart exists at least three isoforms of NBC, one that promotes the co-influx of 1 molecule of Na+ per 1molecule of HCO3- and two others that generates the co-influx of 1 molecule of Na+ per 2 molecules of HCO3- . In addition, the eNBC generates an anionic repolarizing current that modulate the cardiac action potential (CAP), adding to such isoforms the relevance to modulate the electrophysiological function of the heart. Angiotensin II (Ang II) is one of the main hormones that regulate cardiac physiology. The alkalinizing mechanisms (NHE and NBC) are stimulated by Ang II, increasing pHi and intracellular Na+ concentration, which indirectly, due to the stimulation of the Na+/Ca2+ exchanger (NCX) operating in the reverse form, leads to an increase in the intracellular Ca2+ concentration. Interestingly, it has been shown that Ang II exhibits an opposite effect on NBC isoforms: it activates the nNBC and inhibits the eNBC. This inhibition generates a CAP prolongation, which could directly increase the intracellular Ca2+ concentration. The regulation of the intracellular Na+ and Ca2+ concentrations is crucial for the cardiac cellular physiology, but these ions are also involved in the development of cardiac hypertrophy and the damage produced by ischemia-reperfusion, suggesting a potential role of NBC in cardiac diseases.The sodium/ bicarbonate cotransporter (NBC) is, with the Na Fluctuations of pHi occur physiologically in cardiac myocytes, as during changes in heart rate i) and Ca2+ concentrations ([Ca2+]i), emphasizing the potential relevance of NBC in structural and electrical cardiac disorders. In the present review we will specifically outline the importance of NBC in the maintenance of the cardiomyocytes pH3- free-buffered solutions. Another issue is that until few years ago, there was not available a selective NBC inhibitor. Fortunately in 2008 the group of Dr. Vaughan-Jones presented and characterized a novel and selective NBC inhibitor i, being responsible for 30% of this increase at pHi 6.8 [+]i stimulates the reverse mode of Na+/Ca2+ exchanger (NCX), leading to an increase in [Ca2+]i i -17. This [Ca2+]i  and card [Ca2+]i ,20. Furt [Ca2+]i .et al. as an electroneutral transporter (nNBC), with a stoichiometry of 1 Na+/1 HCO3-i activates hypertrophic pathways, such as the one of calcineurin [2+ regulation is closely linked to [Na+]i because one of the routes for Ca2+ influx into the myocytes is via the reverse mode of NCX. When [Na+]i increases, NCX is shifted to less forward mode activity (Ca2+- efflux) and/or to reverse operation mode, leading to [Ca2+]i overload i and [Ca2+]i [+]i overload, prevented or reverted cardiac hypertrophy i -90. Furtertrophy -94. On tertrophy .+ influx into the myocyte at pHi 6.8 i and [Ca2+]i overload. As it was demonstrated that NBC is responsible for 30% of Na pHi 6.8 , it may  pHi 6.8 , while e pHi 6.8 . Taking +/K+ ATPase [+]i overload and cardiac arrhythmias. The proposed mechanism is the following: [Na+]i overload reduces Ca2+ extrusion and/or increases Ca2+ influx through the NCX. The increase in [Ca2+]i enhance the sarcoplasmic reticulum (SR) calcium load, exceeding the ryanodine receptor channel (RyR) threshold necessary to be opened and finally leading to spontaneous diastolic calcium release. The transient increase in citosolic Ca2+ (waves) activates an inward (depolarizing) current (Iti), mediated by the forward mode of NCX i i , it is a+]i  might bet manner .2+ load is not sufficient to promote diastolic spontaneous SR Ca2+ release i by total NBC activity [+]i overload, respectively . The relationship between NBC and EADs is currently unknown, but keeping in mind the relevance of eNBC for CAP configuration , and theactivity , it mighet al. have reported that CAP prolongation promotes an increase in [Ca2+]i, which activate a hypertrophic signaling pathway, that might be a cause and not a consequence of cardiac hypertrophy i and [Ca2+]i overload and arrhythmias generation [et al. reported that blockade of rat cardiac eNBC during reperfusion results in cardioprotection [The double participation of eNBC in cardiac physiology makes it difficult to call as a “beneficial” or “detrimental” mechanism  against eNBC, which would induce CAP shortening, could be useful to investigate the potential protective effect of eNBC activation during the development of cardiac hypertrophy or the damage during reperfusion after ischemia. Nevertheless, besides the fact that the amount of pharmacological tools has been growing up, we are still in debt, and the more precise knowledge about development of cardiac diseases that we can elucidate, the more close we will be to find the specific treatment for them.


The Renin-Angiotensin System (RAS) acts at multiple targets and has its synthesis machinery present in different tissues, including the heart. Actually, it is well known that besides Ang II, the RAS has other active peptides. Of particular interest is the heptapeptide Ang-(1-7) that has been shown to exert cardioprotective effects. In this way, great compilations about Ang-(1-7) actions in the heart have been presented in the literature. However, much less information is available concerning the Ang-(1-7) actions directly in cardiomyocytes. In this paper, we show the actual knowledge about Ang-(1-7)-mediated signaling in cardiac cells more specifically we provide a brief overview of ACE2/Ang-(1-7)/Mas axis; and highlight the discoveries made in cardiomyocyte physiology through the use of genetic approaches. Finally, we discuss the protective signaling induced by Ang-(1-7) in cardiomyocytes and point molecular determinants of these effects. Arterial hypertension is an important cardiovascular risk factor and contributes to the development of cardiovascular event. Despite the substantial advances in antihypertensive drug therapy, the number of patients with uncontrolled hypertension remains high around the world .1R) and the angiotensin II type 2 receptor (AT2R) [1R plays a critical role in Ang II-mediated actions in the cardiovascular system [2R antagonizes the effects of AT1R preventing, between other effects, its hypertrophic and angiogenic effects [The renin-angiotensin system (RAS) is an important classical player that directly contributes to the development and maintenance of essential hypertension . This syr (AT2R) , 4. Under system , 4. On t effects , 6. Although Ang II is the major effector of this system, several other peptides are now recognized as being biologically important. Of particular importance is the heptapeptide Ang-1-7) that decades ago emerged as a new metabolite of the RAS. Ang-(1-7) was initially detected as an Ang I metabolite in canine brain homogenates  that dec and in cAng-(1-7) can be formed directly from Ang I or Ang II and indirectly from Ang I having as an intermediate step the formation of Ang-(1-9) , 15. AngIn cardiomyocytes the expression of ACE2, the main enzyme involved in Ang-(1-7) formation, was already demonstrated , 22, as The identification of ACE2 as an important Ang-(1-7)-forming enzyme , 29, and1R and blocked by inhibitors of mitogen-activated protein kinase kinase 1 (MAPKK1). Considering that differences in experimental conditions can explain the contrasting results regarding Ang II modulation of ACE2, further investigation will be necessary to elucidate the specific mechanism involved in ACE2 downregulation in cardiomyocytes. Endothelin-1 (ET-1) also significantly reduced myocyte ACE2 mRNA via MAPKK1 activation [A variety of vasoactive peptides and hormones can regulate ACE2 mRNA levels in cardiomyocytes. Modulation of ACE2 mRNA levels by aldosterone has been demonstrated in neonatal cardiomyocytes treated with this mineralocorticoid . Accorditivation . Apparentivation . Collect2+ handling in cardiomyocytes. Initial studies were performed by Tallant et al. [−/−Mas (Mas knockout) mice, indicating an important relationship between NOS3 activity and Ang-(1-7)/Mas axis. Initial investigation into the cellular mechanisms underlying protective effects of Ang-(1-7) against Ang II signaling was recently performed by our laboratory. Gomes et al. [β (GSK3β), in particular, is considered a potent inhibitor of this pathway downstream of calcineurin. In the nucleus, GSK3β phosphorylates NFAT, thereby promoting its nuclear export [β has been shown to regulate hypertrophy development by restraining gene expression [β, by preventing its inactivation by Ang II. The modulation of these two proteins, NFAT and GSK3- β, supports the anti-hypertrophic effect of Ang-(1-7) observed in the heart [2R) antagonist, HOE140, suggesting a cross-talk between Mas and B2R in response to Ang-(1-7).  In the past two decades, since the detection of the Ang-(1-7) as a product of the metabolism of Ang I, the physiological actions of Ang-(1-7) have been extensively investigated, and Ang-(1-7) was finally recognized as a putative biologically active component of the RAS –14. Howet et al. , who cont et al. . Continut et al.  showed tt et al.  demonstrt et al. , 43. Thet et al.  have shos et al.  have dems et al. . Stimulas et al. –46. Glycr export . Moreovepression . Gomes epression  have shohe heart  and in che heart , 39. Corhe heart  reportedα (TNF-α) and interleukin-6 (IL-6) and increased gene expression of ACE2, bradykinin type 2 receptor, and interleukin-10 (IL-10). Taken together, these data show that Ang-(1-7) regulates cytokine responses, which could contribute to its cardioprotective effects.Recently, Qi et al. have provided evidence for an anti-inflammatory role of angiotensin-(1-7) at the cardiomyocyte level . By usin2+ ions for cardiomyocyte contraction [2+ handling in ventricular cardiomyocytes. Recent work by our group has shown that acute Ang-(1-7) treatment does not significantly alter Ca2+ transient amplitude or kinetics of decay [in vivo conditions and showed that cardiomyocytes from transgenic (TG) rats with chronic elevated plasmatic Ang-(1-7) do not show alteration in cytosolic Ca2+ transient parameters [−/−Mas) presented a Ca2+ signaling dysfunction represented by a smaller peak Ca2+ transient and slower Ca2+ uptake. This Ca2+ signaling dysfunction was accompanied by decreased protein levels of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) [2+ reuptake by the sarcoplasmic reticulum (SR), thereby setting SR Ca2+ load, which is an important determinant of Ca2+ release in cardiomyocytes [2+ transient is consistent with the depression of contractility that was previously observed in Mas −/− hearts [2+ handling in the cardiac cell. However, there was still the possibility that the alterations in Ca2+ handling found in −/−Mas cardiomyocytes were secondary to the cardiac dysfunction observed in these hearts.Considering the critical role of Catraction , some stof decay . We exterameters . Interes(SERCA2) . SERCA2 myocytes . The red− hearts . This fi2+  signaling, it was also shown that cardiomyocytes from TG rats with cardiac specific overexpression of Ang-(1-7) presented higher Ca2+ transient amplitude, faster Ca2+ uptake, and increased levels of SERCA2 [2+ handling. Are these changes in Ca2+ handling a direct consequence of local Ang-(1-7) increase in the heart? These findings contrasted with the lack of effect on Ca2+ signaling found in cardiomyocytes from TG rats with chronic elevated plasmatic Ang-(1-7) levels. As it stands, the relationship between Ang-(1-7) and Ca2+ signaling is more complex than one may have anticipated. It is also plausible, that Ang-(1-7) effects on Ca2+ handling observed in an in vivo model of chronic Ang-(1-7) overexpression in the heart are consequences of long-term changes in expression levels of Ca2+ handling proteins. Future studies are needed to demonstrate whether Ang-(1-7) prevents Ca2+ signaling dysfunction in ventricular myocytes from animal models of heart failure. Adding further complexity to the understanding of Ang-(1-7)/Mas modulation of cardiomyocyte Caf SERCA2 , suggest2+ handling and regulate cardiac contractility. The question remains whether long-term Ang-(1-7) effects on Ca2+ handling are mediated by NO or other signaling molecules in cardiomyocytes. In this way, the regulation of some key proteins involved in cardiomyocyte Ca2+ handling, such as ryanodine receptor (RyR), phospholamban (PLN), Na+/Ca2+ exchanger (NCX), and troponins by Ang-(1-7) must be investigated in order to provide a deeper understanding of Ang-(1-7) actions on Ca2+ signaling. A summary of current knowledge regarding Ang-(1-7)/Mas modulation of cardiomyocyte Ca2+ signaling is shown in NO has been attributed as a key mediator of Ang-(1-7) effects on different cell types, including cardiomyocytes, and it is known to interact with proteins involved in Cain vitro findings correlated with in vivo data showing that treatment with rhACE2 prevented Ang II–induced hypertrophy and myocardial fibrosis. Thus, these findings give further support to the fact that enhanced Ang-(1-7) signaling at the cardiomyocyte level prevents Ang II pathological effects and highlight Ang-(1-7) anti-oxidative actions on cardiomyocytes.Evidence for a direct role of ACE2/Ang-(1-7)/Mas axis against oxidative stress in cardiomyocytes was also obtained . Experimin vivo ACE2 overexpression in the myocardium of stroke-prone spontaneously hypertensive rats resulted in profound cardiac dysfunction. The authors have argued that the detrimental effects of ACE2 overexpression were possibly due to higher amounts of protein expressed using this approach. Using similar overexpression strategy, neonatal rat cardiomyocytes were infected with adenovirus encoding the human Mas receptor. Intriguingly, overexpression of Mas induced a significant increase in IP3 accumulation and cellular hypertrophy. These responses were due to enhanced Gq-mediated signaling via Mas receptor [Contrasting to the view of a protective role of ACE2/Ang-(1-7)/Mas axis some reports have shown hypertrophic effects of ACE2 and Mas overexpression in cardiomyocytes. Masson et al.  reportedreceptor . Whether2+ signaling in cardiomyocytes seems unlikely. However, there still are many ways by which Ang-(1-7) may regulate Ca2+ signaling in ventricular myocytes. Chronic increase in local Ang-(1-7) levels could be a mechanism by which Ang-(1-7) enhances Ca2+ handling, as observed in cardiomyocytes from transgenic rats with cardiac specific overexpression of Ang-(1-7). To understand how Ang-(1-7) regulates Ca2+ handling in ventricular myocytes is of fundamental importance in light of Ang-(1-7) therapeutic potential in several disease conditions. The literature shows a tight control of ACE2 synthesis and activity in cardiomyocytes. How this enzyme expression is modulated, the signaling pathways involved in this regulation, and whether this occurs in vivo are questions that remain to be answered, considering the pivotal role of ACE2 as modulator of Ang II/Ang-(1-7) levels. The actions of Ang-(1-7) on cardiomyocytes are just beginning to unravel, dissecting the signaling pathways, and the conditions under which Ang-(1-7) signaling is turned on will be a major issue to be addressed in the future. In cardiomyocytes, the actual knowledge of Ang-(1-7) protective effects was mainly focused on the modulation of Ang II signaling with emphasis on anti-hypertrophic actions. This effect was dependent on Mas, indicating that important cardioprotective aspects of Ang-(1-7) signaling are mediated through Mas receptor, and involved NO and cGMP generation. It remains to be elucidated whether antioxidative and anti-inflammatory responses of Ang-(1-7) also depend on NO/cGMP production. A direct acute effect of Ang-(1-7) on Ca


On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes  whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17  lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the  Homologous recombination is essential for the accurate segregation of homologous chromosomes during meiosis The mechanism for controlling meiosis-specific DSB formation has been extensively characterized using budding yeast as a model organism. Meiotic DSBs are formed by the Spo11 protein, a meiosis-specific endonuclease that is homologous to type II topoisomerases Initiation of meiotic recombination needs to be coordinated with other events along the meiotic cell cycle. DSBs are most efficiently formed at the early stage of meiotic prophase I, and these DSBs finish being repaired toward the end of prophase I Budding yeast has two major DNA damage checkpoint pathways that involve Mec1 and Tel1 respectively. Mec1 is the ortholog of ATR and is involved mainly in recognizing and responding to exposed ssDNA, whereas Tel1, the ortholog of ATM, responds to unprocessed DSB ends sae2 mutant background. On the other hand, the absence of Rad17, a major component of the Mec1-dependent pathway, lead to an increase in DSB formation in the rad51 dmc1 mutant background. Thus we propose that the Tel1 pathway facilitates DSB formation, especially in larger chromosomes, while the Mec1 pathway negatively regulates DSB formation. Furthermore, our results identified the transition from prophase I to metaphase I to be a critical event in down-regulating DSB formation.With budding yeast as a model organism, we investigated the possible involvement of DNA damage checkpoint mechanisms in the regulation of meiotic DSB formation. We found that DSB formation was reduced in larger chromosomes in the sae2 mutant has often been used for evaluating the amount and distribution of meiotic DSBs. Under this condition, DSBs are not processed and the Tel1-dependent pathway is predominantly used for DNA damage responses tel1 mutation on DSB formation was examined in the sae2 mutant background. The efficiency of DSB formation was evaluated per chromosome using pulsed-field gel electrophoresis (PFGE) followed by Southern blotting, with probes specifically recognizing an end of a particular chromosome pch2 mutant, although the effect caused by the tel1 mutation is much milder  hereafter. Unlike the rad17 null mutant, rad17-mn showed resistance to methyl methanesulfonate, a DNA damaging agent, to levels comparable to that of wild type . This level of spore viability is slightly higher than that of the rad17 null mutant (∼35%) but much lower than the ∼100% spore viability seen in wild type  complex sae2 mutant background in which DSB ends are not resected, thus it is possible that such an effect is restricted to the sae2 mutant or similar mutant backgrounds. Another possibility is that Tel1 (and possibly Mec1) also affects chromosome conformation or the rate of DSB processing, which can potentially lead to the differential accumulation of DSB markers such as histone H2AV or Spo11-linked oligonucleotides.In ATM deficient mice, the total level of Spo11-oligonucleotide complexes is elevated ndt80 mutant background made it possible to investigate the role of Rad17 in DSB formation independently of the cell cycle progression effect that is usually associated with a defect in the DNA damage checkpoint. In the rad51 dmc1 double mutant background, DSB formation was pronounced in the absence of Rad17. A similar effect was seen when pch2 or spo11-HA was combined with rad17-mn in the ndt80 background. These results demonstrate the role of Rad17 in negatively regulating DSB formation. Rad17 is an indispensible component of the Mec1(ATR)-dependent DNA damage checkpoint pathway. ATR is recruited to ssDNA through its interaction with RPA and activated by interacting with the PCNA-like 9-1-1 complex consisting of Ddc1, Mec3 and Rad17 Employing the The difference in the usage of the ATM and ATR pathways in mice and budding yeast is interesting, given that ATM is primarily used for down-regulating DSB formation in mice. Once DSBs are formed, ATM is primarily used in responding to DSBs in mice whereas ATR (Mec1) is the major pathway in budding yeast. Thus, the apparent bias toward ATR utilization in budding yeast might reflect the overall usage preference to ATR in choosing a damage response pathway. After all, if meiotic DSBs, once formed, are processed to expose ssDNA in a relatively prompt manner, ATR would almost inevitably become the pathway of choice because ATM is less likely to be retained on processed DSB ends. On the other hand, in mice, it is possible that meiotic DSB ends are kept unprocessed for some time, which might allow ATM to respond to them, sending a negative feedback signal to the DSB forming mechanism.ndt80 mutants in general, in both sae2 and rad51 dmc1 mutant backgrounds, than their NDT80 positive counterparts. Second, when DSB formation is compromised , the reduction in DSB formation is further exacerbated by a DNA damage checkpoint defect , which is completely suppressed by arresting the cell cycle at prophase I . Thus, the synergistic effect between DSB formation inefficiency and a DNA damage checkpoint defect in the NDT80 positive background is due to the Ndt80-dependent cell cycle progression.Our results further highlighted the importance of Ndt80 as a negative controller of DSB formation. First, more DSBs are formed in the Ndt80 is the master regulator that controls exit from prophase I and entry into metaphase I. Ndt80 is a downstream target of the DNA damage checkpoint mechanism during meiosis (recombination/pachytene checkpoint), which functions to coordinate homologous recombination (DSB repair) and cell cycle progression The presence of unrepaired DSBs is sensed by DNA damage checkpoint mechanisms. In this work, we showed that Rad17 (and most likely the ATR pathway) is in charge of repressing DSB formation once DSBs are formed. Our results also suggest that DSB formation is shut off when cells exit prophase I. However, cells exit prophase I when the previously formed DSBs are repaired. This is contradictory because, as DSBs are repaired, the ATR-pathway becomes less active, possibly leading to reactivation of DSB formation. It is therefore likely that an unknown mechanism is responsible for gradually diminishing the DSB formation activity towards the end of prophase I. This mechanism may utilize the progress of homologous recombination as a temporal marker for prophase I. For example, the loading of proteins involved in later stages of homologous recombination, such as resolvases, and the formation of the synaptonemal complex can be exploited to serve such roles.RAD51, DMC1, PCH2, SAE2, TEL1 were replaced with drug resistant markers by PCR mediated gene disruption rad17-mn allele, the promoter of CLB2 was inserted immediately before the start codon of the RAD17 ORF by PCR rad51::URA3, spo11-HA, ndt80::LEU2 were previously described Genotypes of yeast strains are given in Strains used are: TBR5514, 5515, 5188, 6618, 6619 and 6620 in SK1 strains were introduced into meiosis as described previously with minor modifications Meiotic DSBs were detected as described previously with minor modifications Calculations to obtain the estimated DSBs on chromosomes used in rad51 dmc1 double mutant strains in which DSB formation is much more efficient than the sae2 strains, we employed only one third of a chromosome for analysis, from the end of a chromosome that a probe recognizes to one third of the total lane length. The value equivalent to the ratio of unbroken chromosomes at this position is given by the signal ratio of the signal corresponding to the other two thirds of the lane including unbroken chromosomes per total lane signal. This way all the DSB numbers are widely dispersed within the range of 2.5 between the variety of the rad51 dmc1 mutant strains , the calculation is more easily affected from other factors such as a fraction of cells that did not go into meiosis and the quality of Southern blot. Thus, in the  strains .Figure S1The rad17-mn allele reduces functionality of RAD17 in a meiosis-specific manner. (A) Vegetatively growing cultures of indicated strains were serially diluted and spotted on complete medium with or without methylmethane sulfonate (MMS). Two independent cultures were tested per genotype. (B) Diploid strains as indicated were sporulated and spore viability was measured by tetrad dissection. 40 tetrads were dissected per strain. (C) rad17-mn diploid cells before and after introduction into meiosis were examined for the production of the Rad17 protein by western blotting. Rad17 in the rad17-mn strain is tagged with the HA epitope, and thus can be detected with anti-HA antibodies. veg., vegetatively growing cells. (D) Indicated diploid strains were introduced into meiosis and the level of the Cdc5 protein, a marker for exit from the pachytene stage of prophase I, was examined by western blotting.(TIF)Click here for additional data file.Table S1Yeast strains.(PDF)Click here for additional data file.Table S2Numbers used to calculate DSB amount in .(PDF)Click here for additional data file.


E.coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+-sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca2+-dependent activation of KCa3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis.The pore-forming exotoxin α-hemolysin from  E.coli does not only cause swelling and lysis of the erythrocytes. The toxin inflicts distinct biphasic volume changes, where the volume increase that leads to lysis is preceded by marked erythrocyte shrinkage. This shrinkage is triggered by the influx of Ca2+, which happens very quickly after insertion of HlyA into the membrane. This Ca2+ influx activates Ca2+-sensitive K+ channels (KCa3.1) and Cl− channels (TMEM16A) i. Erythrocytes were incubated for 10 min at 37 °C with a HlyA concentration, which results in 50% hemolysis after 60 min of incubation. This way reassured that our assay reports about the phagocytosis of erythrocytes, we tested whether HlyA would make the erythrocytes more prone to phagocytosis. In these experiments, the erythrocytes were loaded with calcein-AM to prevent the fluorescence intensity of the THP-1 cells to depend on changes in i, which is likely to be mainly mediated via Ca2+ permeation through the pore itself. Consequently, the [Ca2+]i increases and activates the Ca2+-dependent K+ channels (KCa3.1) and Cl− channels (TMEM16A) i results in KCa3.1-dependent cell shrinkage and PS exposure i observed after addition of HlyA. Our very recent data show that this sharp increase in [Ca2+]i is likely to be caused by the pore insertion itself, and that the P2X receptor activation is likely to account for the following slower [Ca2+]i increment induced by HlyA [Ca2+]i (data not shown). Determination of whether P2X receptor blockage on the erythrocytes also reduces the phagocytosis is not completely straightforward. In this context, it should be emphasized that monocytes express a variety of P2 receptors, at least P2Y1, P2Y2, P2Y6 P2Y11–13, P2X1, P2X4, and P2X7 [2+]i in the THP-1 cells upon addition of extracellular ATP, resembling a P2Y-type of [Ca2+]i response. P2 receptor activation is essential in many monocyte/macrophage functions, such as the ATP concentration gradient responsible for the monocytic chemotaxis towards the infected cell, by stimulating the formation of pseudopodia i measurements, erythrocytes were incubated with fluo 4-AM  and attached to glass coverslips by BD Cell-Tak™ and placed in an incubation chamber on a IMIC stage . The fluorophore was excited at 488 nm by a monochromator , and the emission was collected >520 nm. The preparation was imaged with a 60×, 1.45 NA Plan Apo  objective, and a charge coupled device camera . The entire set-up was delivered from Bio-Science ApS, Gilleleje, Denmark. The experiments were conducted at 37 °C.For  138.0, [Cl−] 132.9, [K+] 5.3, [Ca2+] 1.8, [Mg2+] 0.8, [SO42−] 0.8, [HEPES] 14, [glucose] 5.6, pH 7.4 at 37 °C. ATP-2',3'-dialdehyde (oxidized ATP), PPADS, TRAM-34, and MRS2159 were obtained from Sigma-Aldrich. Brilliant blue G (BBG) was purchased from INC Biomedicals Inc. . Fluo 4-AM and calcein-AM was from Invitrogen . Standard reagents were dissolved in distilled water and pH adjusted to 7.4 at 37 °C. BBG and TRAM-34 were both dissolved in DMSO to a final concentration of maximal 0.5% DMSO. Control experiments for possible DMSO effects alone were included.The HEPES-buffered salt solution (HBS) constituted of (in mM): [Nat-test or one-way Anova (Tukey post test) for multiple comparisons as appropriate. In both cases a p-value less than 0.05 was considered statistically significant.Data are presented as mean ± SEM. The n value indicates number of experiments. In the hemolysis and phagocytosis experiments, the erythrocytes were isolated from a new donor for each experiment. The data were tested for normality by the Kolmogorov-Smirnov test. Significant differences were determined by paired or unpaired Student’s E.coli toxin HlyA efficiently marks erythrocytes for recognition and phagocytosis through monocytes. The initial erythrocyte shrinkage inflicted by HlyA is associated with PS exposure, which appears to be essential to the erythrocyte removal. PS exposure is hindered if either the HlyA-induced cell damage is prevented by P2X receptor inhibition or if cell shrinkage is prohibited. Our data are consistent with the notion that the erythrocyte shrinkage, which is the initial consequence of HlyA exposure, prevents acute lysis of the erythrocytes and is a strong signal for removal of the damaged erythrocytes from the blood stream, thereby reducing the risk of intravascular hemolysis.In conclusion, the early cell damage inflicted by the pore-forming


CYP2E1 mRNA levels correlated with CBFβ-MYH11 transcript levels and with bone marrow blast counts in all cases. CYP2E1 over-expression correlated positively with NQO1 mRNA levels . By immunohistochemistry, CYP2E1 protein was more frequently expressed in AML with inv(16) compared with other types of AML (p < 0.001). We obtained serial bone marrow samples from two patients with AML with inv(16) before and after treatment. CYP2E1 mRNA expression levels decreased in parallel with CBFβ-MYH11 transcript levels and blast counts following chemotherapy. In contrast, CYP1A2 transcript levels did not change in either patient. This is the first study to demonstrate concurrent over-expression of CYP2E1 and NQO1 mRNA in AML with inv(16). These findings also suggest that a balance between CYP2E1 and NQO1 may be important in the pathogenesis of AML with inv(16). Environmental exposure to benzene occurs through cigarette smoke, unleaded gasoline and certain types of plastic. Benzene is converted to hematotoxic metabolites by the hepatic phase-I enzyme CYP2E1, and these metabolites are detoxified by the phase-II enzyme NQO1. The genes encoding these enzymes are highly polymorphic and studies of these polymorphisms have shown different pathogenic and prognostic features in various hematological malignancies. The potential role of different cytochrome p450 metabolizing enzymes in the pathogenesis of acute myeloid leukemia (AML) in an area of active interest. In this study, we demonstrate aberrant CYP2E1 mRNA over-expression by quantitative real-time polymerase chain reaction in 11 cases of  Benzene is a long established leukemogen implicated in the pathogenesis of myelodysplastic syndromes (MDS) and acute leukemias . AlthougCYP2E1 is a hepatic monooxygenase involved in xenobiotic metabolism. The CYP2E1 gene is associated with the metabolism of many carcinogens. Polymorphisms of CYP2E1 and their possible effects have been reported by others. The CYP2E1*5 allele is associated with a higher risk of de novo acute myeloid leukemia (AML) and acute lymphoblastic leukemia . InThis study provides corroborating evidence implicating benzene in the pathogenesis of AML with inv(16), confirming the findings of others . High leThe strong association between CYP2E1 and AML with inv(16) has implications for pathogenesis. First, this association allows one to infer that AML with inv(16) may be related to exposure to environmental toxins. The elevated expression of CYP2E1 alone would enhance the carcinogenic potential of environmental toxins. The general population is exposed to benzene and its toxic metabolites through gasoline, cigarette smoking, air pollution and dietary intestinal breakdown of proteins . ElevateThe explanation for the elevated levels of CYP2E1 specifically in most cases of AML with inv(16) remains unclear. AML with inv(16) is associated with a favorable outcome with long periods of complete remission ,18. WhilNQO1 gene is located on chromosome 16q22.1, some authors have proposed that inversion (16) may be a primary event leading to disruption of this gene, thereby increasing the likelihood of leukemia [There are limitations to this study, the most important being the lack of NQO1 protein levels to reinforce the mRNA levels in the myeloid system. The results, therefore, need to be confirmed in a larger subset of patients with well characterized polymorphisms in the CYP2E1 and NQO1 genes. Since the leukemia . Furtherin vitro experiments also provide direct evidence of induction of cytochrome p450 and NQO1 by benzene and its analogues/metabolites. Based on this data, we suggest that cases of AML, especially those types associated with recurrent genetic abnormalities, are uniquely associated with an increase in specific types of cytochrome p450 enzymes. This increase appears to be more than just a generalized increase in cytochrome p450 enzyme expression. The increase in specific types of cytochrome enzyme also appears to be unique to each type of leukemia as shown by others, such as specific induction of CYP1A1 in AML with t [The results from our  t  as well  t . It alsoIn conclusion, we have shown simultaneously increased mRNA expression levels of the benzene metabolizing enzymes, CYP2E1 and NQO1, in AML with inv(16). Elevated levels of CYP2E1 mRNA and protein occur more commonly in AML with inv(16) compared with other types of AML. We further suggest that the balance between CYP2E1 (toxin producing) and NQO1 (detoxifying) enzymes may determines the overall carcinogenic potential of the xenotoxins.


O-galloyl-beta-d-glucose from several medicinal herbs triggers apoptosis and has, thus, been proposed for treatment of malignancy. The substance is at least partially effective through caspase activation. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The sensitivity to [Ca2+]i is enhanced by ceramide. The present study explored whether penta-O-galloyl-β-d-glucose stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin-release, [Ca2+]i from Fluo3-fluorescence and ceramide abundance from fluorescent antibodies. A 48-h exposure of human erythrocytes to penta-O-galloyl-β-d-glucose significantly decreased forward scatter (50 µM) and significantly increased annexin V binding (10 µM). Up to 50 µM penta-O-galloyl-β-d-glucose did not significantly modify [Ca2+]i. However, the effect of penta-O-galloyl-β-d-glucose (25 µM) induced annexin V binding was slightly, but significantly, blunted by removal of extracellular Ca2+, pointing to sensitization of erythrocytes to the scrambling effect of Ca2+. Penta-O-galloyl-β-d-glucose (25 µM) further increased ceramide formation. In conclusion, penta-O-galloyl-β-d-glucose stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of ceramide formation with subsequent sensitization of erythrocytes to Ca2+.The polyphenolic 1,2,3,4,6-penta- O-galloyl-beta-d-glucose, a polyphenolic compound from medicinal herbs i), resulting from Ca2+ entry through Ca2+-permeable cation channels [2+]i activates Ca2+-sensitive K+ channels i further triggers phospholipid scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface i). To this end, human erythrocytes were loaded with Fluo3-AM and the Fluo3 fluorescence determined by flow cytometry. Prior to determination of Fluo3-fluorescence erythrocytes were incubated in Ringer solution without or with penta-O-galloyl-β-d-glucose (1–50 µM). As a result, a 48-h exposure of erythrocytes to penta-O-galloyl-β-d-glucose did not significantly modify [Ca2+]i. The respective arbitrary units (a.u.) approached (n = 8 each) 20.29 ± 1.19 a.u., 18.96 ± 1.05 a.u., 17.81 ± 0.93 a.u., 17.61 ± 0.83 a.u., and 22.58 ± 1.37 a.u. after a 48-h exposure to 0, 1, 10, 25, and 50 µM, respectively, of penta-O-galloyl-β-d-glucose. Fluo3 fluorescence was employed in order to test, whether penta-O-galloyl-β-d-glucose did not significantly modify cytosolic Ca2+ activity, the effect of the substance could still be dependent on the presence of Ca2+. In order to test this possibility, erythrocytes were exposed to 25 µM penta-O-galloyl-β-d-glucose for 48 h in the presence and in the nominal absence of extracellular Ca2+. As illustrated in O-galloyl-β-d-glucose on annexin V binding was slightly but significantly blunted in the nominal absence of Ca2+. Thus, the effect of penta-O-galloyl-β-d-glucose required in part the presence of Ca2+. Even though penta-2+ could be enhanced by ceramide, which is known to trigger eryptosis even at constant [Ca2+]i. In order to test, whether ceramide is enhanced by penta-O-galloyl-β-d-glucose, ceramide abundance at the cell surface was elucidated utilizing FITC-labeled anti-ceramide antibodies. As shown in O-galloyl-β-d-glucose significantly increased ceramide-dependent fluorescence.The sensitivity of cell membrane scrambling to cytosolic CaO-galloyl-β-d-glucose is followed by erythrocyte shrinkage and erythrocyte cell membrane scrambling, the two hallmarks of eryptosis. The concentrations required (10–50 µM) are similar to those inhibiting hepatocyte apoptosis [The present study discloses a novel xenobiotic triggering suicidal erythrocyte death or eryptosis. Treatment of human erythrocytes with penta-poptosis . O-galloyl-β-d-glucose is not paralleled by and thus not due to increase of cytosolic Ca2+ activity. Most triggers of eryptosis exert their effect on annexin V binding and cell volume by activation of Ca2+ permeable non-selective cation channels in erythrocytes, which involve the transient receptor potential channel TRPC6 [O-galloyl-β-d-glucose treatment did not significantly modify cytosolic Ca2+ activity, the full effect of penta-O-galloyl-β-d-glucose was dependent on the presence of extracellular Ca2+. Thus, penta-O-galloyl-β-d-glucose appeared to sensitize the erythrocytes to the effects of cytosolic Ca2+. A substance known to sensitize erythrocytes to the scrambling effect of enhanced cytosolic Ca2+ activity is ceramide [O-galloyl-β-d-glucose did stimulate ceramide formation.Penta-el TRPC6 . Even thceramide . As a maO-galloyl-β-d-glucose has been shown to either stimulate [O-galloyl-β-d-glucose has never been shown before to stimulate the formation of ceramide. Thus, the present observations disclose a mechanism, which could well contribute to triggering of apoptosis. In nucleated cells, penta-timulate ,11,12,13timulate ,7,10,14.timulate . To the O-galloyl-β-d-glucose limiting its therapeutic use. Phosphatidylserine exposing erythrocytes adhere to CXCL16/SR-PSO expressed at the luminal cell membrane of endothelial cells [O-galloyl-β-d-glucose may, thus, be limited by triggering of eryptosis with subsequent impairment of microcirculation and development of anemia. It should be kept in mind that the sensitivity of erythrocytes to penta-O-galloyl-β-d-glucose may be increased by other xenobiotics [The present study may further disclose an effect of penta-al cells . The adhal cells ,34,35,36al cells ,37,38. Pal cells . To the al cells . The theobiotics ,48,49,50obiotics ,22,51, robiotics , hemolytobiotics , sepsis obiotics , sickle obiotics , malariaobiotics ,56,57, Wobiotics , phosphaobiotics , and, probiotics .in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to penta-O-galloyl-β-d-glucose  at the indicated concentrations. In Ca2+-free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Leukocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The volunteers (age range 18–68 years) providing blood were tested for HIV, syphilis, and Hepatitis A, B, and C before donation. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated 2. Then, the erythrocytes were washed twice and finally resuspended in 100 μL Ringer solution containing 5 mM CaCl2. Forty microliters were placed with Prolong Gold antifade reagent  onto a glass slide, covered with a coverslip, and images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser-scanning microscope  with a water immersion Plan-Neofluar 40/1.3 NA DIC.For the visualization of eryptotic erythrocytes, 20 μL erythrocytes were incubated under the respective experimental conditions and then stained with FITC-conjugated Annexin V  in 200 μL Ringer solution containing 5 mM CaCl2 and then stained with Annexin-V-FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl2 and 5 µM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5 mM CaClFor the determination of hemolysis the samples were centrifuged  after incubation, and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.For the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 µg/mL anti-ceramide antibody  in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 min with polyclonal fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse IgG and IgM specific antibody  diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .2. Then, the erythrocytes were washed twice and finally resuspended in 100 μL Ringer solution containing 5 mM CaCl2. Forty microliters were placed with Prolong Gold antifade reagent  onto a glass slide, covered with a coverslip, and images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser-scanning microscope  with a water immersion Plan-Neofluar 40/1.3 NA DIC.For the visualization of eryptotic erythrocytes, 20 μL erythrocytes were incubated under the respective experimental conditions and then stained with FITC-conjugated Annexin V  in 200 μL Ringer solution containing 5 mM CaClData are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and t test as appropriate. n denotes the number of different erythrocyte specimens studied. As different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.O-galloyl-β-d-glucose triggers cell shrinkage and cell membrane scrambling of human erythrocytes and thus eryptosis, the suicidal death of erythrocytes. The effect is in part dependent on the presence of extracellular Ca2+ and is at least in part due to stimulation of ceramide formation. Penta-


To better characterize Ca2+ and Mg2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations  were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca2+ or Sr2+. This activation was interfered by Mg2+ and Ba2+ acting at low affinity M2+-unspecific binding sites. When testing the effects of luminal M2+ as current carriers, all M2+ increased maximal RyR2 open probability (compared to Cs+), suggesting the existence of low affinity activating M2+-unspecific sites at the luminal surface. Responses to M2+ vary from channel to channel (heterogeneity). However, with luminal Ba2+or Mg2+, RyR2 were less sensitive to cytosolic Ca2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked . Kinetics of RyR2 with mixtures of luminal Ba2+/Ca2+ and additive action of luminal plus cytosolic Ba2+ or Mg2+ suggest luminal M2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca2+/Sr2+-specific sites, which stabilize high Po mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M2+ binding sites (specific for Ca2+ and unspecific for Ca2+/Mg2+) that dynamically modulate channel activity and gating status, depending on SR voltage.Cardiac ryanodine receptor (RyR2) function is modulated by Ca RyR2 are also sensitive to cytosolic Mg2+2+ is inhibitory. It is thought that Mg2+ inhibition of RyR2 function involves both competition of Mg2+ with Ca2+ binding to cytosolic activating sites and Mg2+ binding to additional inhibitory cytosolic Mg2+ binding sites 2+ with cytosolic Ca2+-mediated activation of RyR2 has also been reported, although the presence of one or multiple binding sites has not been elucidated RyR2 display a biphasic response to cytosolic Ca2+) at the luminal surface of the RyR2 2+ has previously been measured for ATP-activated RyR2 and the reported values range from ∼50 µM 2+ on Ca2+-activated 2+ is still unclear although a combination of luminal M2+ effects on cytosolic Ca2+ and ATP modulation and the “trans effect” of lumen-to-cytosol M2+ flux acting on cytosolic M2+ sites of single RyR2 has been proposed to play a role Current evidence supports the existence of additional binding sites for alkaline earth divalent ions . Moreover, the binding of M2+ to luminal sites differentially affected RyR2 gating kinetics and voltage-dependence as well as RyR2 sensitivity to cytosolic Ca2+ and cytosolic caffeine. Some of the results have been presented in a preliminary form The data presented here suggest that RyR2 channel behavior can be modified by M2 standard for calibration was from Word Precision Instruments Inc . Phospholipids were obtained from Avanti , and decane from Aldrich . BAPTA  ethane-N,N,N',N'-tetraacetic acid), dibromoBAPTA ethane-N,N,N',N'-tetraacetic acid), Ba(OH)2, Ca(OH)2, Mg(OH)2, Sr(OH)2, CsOH, CsCl, and HEPES were obtained from Fluka . All other drugs and chemicals were from Sigma or were reagent grade.CaClAll procedures with animals were designed to minimize pain and suffering and conformed to the guidelines of the National Institutes of Health. SIUMED animal research procedures have AAALAC accreditation and PHS assurances numbers 000551 and A3209-01 respectively. The committee on the Use and Care of Laboratory Animals of Southern Illinois University School of Medicine reviewed and approved the protocols for animal use in our laboratory (196-05-021 and 196-08-003). Sarcoplasmic reticulum (SR) microsomes were obtained from pig heart ventricle using heart homogenization and ultracentrifugation steps that follow the procedures published by Chamberlain et al. trans compartment was filled with HEPES-M2+ solution containing HEPES 250 mM and M(OH)2 53 mM, pH 7.4 . The trans compartment was clamped at 0 mV using an Axopatch 200B patch-clamp amplifier . The cis compartment (ground) was filled with HEPES-Tris solution containing HEPES 250 mM and TrisOH 118 mM, pH 7.4. Bilayers of a 5∶4∶1 mixture of bovine brain phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine (45–50 mg/ml in decane) were painted onto the holes of teflon septa from the cis side. Sarcoplasmic reticulum microsomes (5–15 µg) were then added to the cis solution followed by 500–1000 mM CsCl and 1 mM CaCl2 to promote vesicle fusion. After RyR currents (or Cl− currents >100 pA at 0 mV) were observed, the cis chamber was perfused with HEPES-TRIS solution for 5 min at 4 ml/min. A mixture of BAPTA and dibromo-BAPTA was used to buffer free [Ca2+] on the cytosolic surface of the channel ([Ca2+]cyt) trans - cis) and response to diagnostic ligands . RyR channel currents are depicted as positive (upward deflections of the current) in figures and reflect cation flux from the trans  to the cis (cytosolic) compartment. Membrane voltages always represent the difference between trans - cis compartments (in mV).Reconstitution of RyR2 in planar lipid bilayers was performed as previously described o), which were determined by half-amplitude threshold analysis of single-channel recordings as done before o) was estimated. In the figures we show the Po (for single channels) or nPo/x .Channel currents were first filtered through the Axopatch 200B low-pass Bessel filter at 2 kHz, digitized at 20 kHz with an analog to digital converter  and stored on DVD. Recordings were analyzed using pClamp9 software (Axon Instruments). Analysis with this program included open times, closed times and open probabilities  added either to the cytosolic or luminal channel surface. In all cases, we measured the activity of RyR2 (from pig heart ventricular microsomes) reconstituted in planar lipid bilayers. Unless explicitly stated, recordings were done at 0 mV (transmembrane voltage).In this work, we studied the modulation of RyR2 by alkaline earth cations (M2+ (left panel) or Sr2+ (middle panel) added to the cytosolic compartment. All recordings were made at a holding potential (Vm) of 0 mV with luminal Ca2+ (50 mM) as current carrier. As previously reported (reviewed in 2+ increased to micromolar levels. o) data from n = 10 RyR2 experiments (open circles). From these experiments, we estimated that the effective concentration of Ca2+ that induces half maximal Po (EC50) was 2.3±0.1 µM. Channel activation had a Hill coefficient (nH) of 2.4±0.1. RyR2 were also activated with increasing Sr2+ levels, as shown in the recordings (50 for Sr2+ was 20.2±1.0 µM (∼10 times higher than EC50 for Ca2+). Similar as with Ca2+, nH with Sr2+ was 2.2±0.2. These nH >1 suggest that multiple interacting M2+ binding sites specific for Ca2+>Sr2+ are involved in Ca2+ or Sr2+-induced RyR2 activation. As shown in 2+ or Ba2+ levels were increased (from 0.1 to 500 µM). This confirms that cytosolic M2+ activating sites are selective for Ca2+ and Sr2+.2+ is biphasic. Micromolar Ca2+ levels activate RyR2 while millimolar Ca2+ levels inhibit the channel  and subsequently, added cumulative doses of either Ca2+, Sr2+, Ba2+ or Mg2+. As shown in 2+ tested had a similar inhibitory action on RyR2 Po. The concentrations for half maximal inhibition (IC50) were also similar, ranging from 3.5 to 5.7 mM . Still, the Mg2+ concentrations required to induce equivalent degrees of inhibition are the same, regardless of the presence/absence of caffeine. However, much less Ca2+ was required to counteract the effect of Mg2+ . In these experiments, the luminal cations were also the charge carriers (the current flows in lumen-to-cytosol direction). With luminal Cs+, Ca2+ or Sr2+, the channels were activated by cytosolic Ca2+ with an EC50 ∼ 3 µM . At high cytosolic Ca2+ levels (>100 µM), the maximal Po (plateau) value was similar in the presence of luminal Ca2+, Sr2+, Mg2+ or Ba2+. However, this value was significantly reduced (from Po ∼0.8 to Po ∼0.4) when we used Cs+ as charge carrier . Open probabilities were much lower with luminal Cs+ solutions (where only micromolar contaminant Ca2+ was present) , but Po at 0 mV cannot be accurately determined using this approach because the channels are prone to inactivation (results not shown). In Supporting Information  is observed when RyR2 are bathed with luminal Sr2+, Mg2+ and Ba2+.It has been reported that RyR2 channel function can also be regulated by luminal Ca0 ∼ 3 µM . In contormation  we show 2+ on the sensitivity of single RyR2 to caffeine. These experiments were conducted in the presence of 100 nM cytosolic Ca2+. As shown in the examples with luminal Ca2+ or Ba2+  and high Po, which is denominated modal gating As shown in 50 for cytosolic Ca2+ activation and the changes in RyR2 gating kinetics observed with different luminal M2+ (2+ and one selective for Ca2+ and Sr2+) or that luminal M2+ can affect cytosolic sites when flowing through the channel (feed-through) 2+or Sr2+ flowing through the channels could bind to the high affinity activating cytosolic M2+ binding sites and increase RyR2 activity. On the other hand, any M2+ coming from the lumen could interact with low affinity inhibitory cytosolic M2+ binding sites and decrease RyR2 activity. If feed-through produces further activation of RyR2, increasing SR membrane voltage (which increases the magnitude of lumen-to cytosol M2+ flux) should induce an increase in the Po of RyR2 exposed to luminal Ca2+ or Sr2+ . In contrast, if feed-through negatively modulates RyR2 function, the voltage-dependence curve for any M2+ should have a negative slope.As shown in inal M2+ . This su2+ flux from ∼1 to 8 pA. As reported before for various aspects of channel function o mode with abundance of short lived gating events lasting from 1 to a few ms and the other with higher Po and slower kinetics . In o mode RyR2 with either luminal Ca2+ (2+ (2+ flux (by making Vm more positive) decreases Po regardless of the identity of the luminal M2+. Notice, however, that Po values at comparable cytosolic Ca2+ levels are higher with luminal Ca2+ than with luminal Ba2+. The differences in Po correlate with more abundant and longer openings and shorter closed times in luminal Ca2+ versus Ba2+ , estimated as number of openings divided by (1 – Po) recording time, also decreases with voltage but it is more marked with luminal Ba2+ (not shown).We tested the effect of changing membrane voltage on RyR2 activity. Tested voltages ranged from −20 to +40 mV, which changed the magnitude of Canal Ca2+  or luminCa2+ (2+  as curreo gating mode (slow kinetics). For this type of channel we detected little (if any) voltage-induced change in Po when Ca2+ was the current carrier (2+ was replaced with Ba2+ (2+ (2+ levels was observed in most RyR2 (both populations). Increasing cytosolic Ca2+ levels from 2 µM to 4 µM in the presence of luminal Ba2+ increased Po to values similar to those observed in the presence of luminal Ca2+ with 2 µM cytosolic Ca2+. However, openings are still shorter with luminal Ba2+ compared to luminal Ca2+ and the increase in Po is mainly due to a shortening of closed events. Additionally, the decrease in Po at more positive SR voltage observed with luminal Ba2+ results from a decrease in open times and an increase in closed times .Paired recordings showed in  carrier . Howeverith Ba2+  the chanBa2+  RyR2 bathed with luminal 50 mM Ba2+ before and after adding increasing concentrations of Ca2+ to the luminal chamber. Subsequently, the luminal Ba2+ was completely replaced by 50 mM Ca2+. As shown in 2+ suffices to increase Po to values observed with 50 mM luminal Ca2+. As show in 2+ ]cyt = 100 nM). Indeed, addition of 5 mM Ca2+ to the luminal chamber, increased the Po to the same levels observed with 50 mM luminal Ca2+. Notice that 0.5 mM luminal Ca2+ induced a significant increase in the Po of caffeine-activated channels while it did not affect Ca2+-activated channels. This would suggest that the interplay caffeine - luminal Ca2+ may produce a more robust change in RyR2 activity than luminal Ca2+ alone 2+ fluxes through the open RyR2 would be substantially different with luminal 5 mM Ca2+/45 mM Ba2+ versus 50 mM Ca2+  with 50 mM luminal Ca2+  interference of flowing Ba2+/Mg2+ with cytosolic Ca2+-mediated activation; ii) interaction of flowing M2+ with cytosolic inhibitory low affinity M2+ binding sites or iii) a combination of both effects . In any case, very high levels of the M2+ flowing through the channels should be reached at the cytosolic surface to produce inhibition. In previous experiments, we found that 1 mM cytosolic Mg2+ produces an increase in RyR2 EC50 for cytosolic Ca2+ from ∼2 µM to ∼10-20 µM 50 for cytosolic Ca2+ was found when using luminal Mg2+ as charge carrier with no cytosolic Mg2+ added  produce high frequency of short lived events, even when the channels are exposed to fully activating cytosolic Ca2+ levels (2+ (or Mg2+) feeding through the channel and transiently inhibiting RyR2 by binding to cytosolic sites 2+ flux) on channel kinetics. However, it has been reported that in the absence of luminal Mg2+ , cytosolic Mg2+ increases the length of closures but does not significantly affect open times 2+ and Ba2+ on channels bathed with luminal Ca2+ (results not shown). Indeed, in our paired experiments, the same RyR2 now bathed with luminal Ba2+, still displayed longer closures when exposed to 250 µM cytosolic Ba2+, but open event distribution did not change (2+ (or Mg2+) would not be due to Ba2+ feed-through acting on a cytosolic site but it would be a luminal phenomenon.An important observation in this paper is that luminal Ba+ levels . This cot change . Thus, p2+ reaching cytosolic binding sites. For that, we increased the buffering power of the cytosolic solution keeping [Ca2+]cyt constant by adding mixtures containing Ca2+ and fast buffers (BAPTA/di-Bromo-BAPTA). If feed-through positively modulates RyR2 function, when using Ca2+ as charge carrier, higher levels of buffer would be expected to chelate the Ca2+ flowing from the lumen and decrease Po by preventing Ca2+ interaction with cytosolic activating sites. Conversely, if inhibitory feed-through occurs, increasing buffering should only increase the activity of RyR2 exposed to luminal Ca2+ because BAPTA and di-Bromo-BAPTA are less effective as Ba2+ chelators and they do not significantly bind to Mg2+o, regardless of the current carrier identity  (Results not shown). Some RyR2 activation is also found with EGTA or EDTA, which bind much more slowly to RyR2, suggesting that the buffering effect does not depend on the on-rate or affinity of the chelator for binding to the flowing M2+. This may suggests that BAPTA at high concentrations may have some Ca2+ buffering independent effects 2+, such as 50 mM sulfate (for Ba2+), or 10 mM fluoride (for Ca2+), as alternative chelators to sequester luminal M2+ flowing into the cytosol and prevent feed-through. However, we did not find significant changes in activity .Additional experiments were carried out with the purpose of preventing the putative effects of luminal M+ and Na+), which are more electronegative than Tris+ or Cs+, could compete with M2+ flux effects in the RyR2 cytosolic surface or the vestibule. In this regard, it has been reported that K+ and Na+ may affect Ca2+ sensitivity of [3H]ryanodine binding 50 for Ca2+ using variable levels (100–250 mM) of different cytosolic cations such as Cs+ or Tris+o of RyR2 channels bathed with partially activating cytosolic Ca2+  or maximally activating cytosolic Ca2+ (10–100 µM) was not affected by Na+ or K+ .We tested the possibility that physiological monovalent ions (K2+). On RyR2 cytosolic surface there are two different types of interacting M2+ binding sites: selective (Ca2+ > Sr2+) high affinity activating sites and nonselective (Ca2+ ∼ Mg2+ ∼ Sr2+ ∼ Ba2+) low affinity inhibitory sites. On the luminal surface of the channels, there are nonselective and Ca2+-selective binding sites (both activating and low affinity).In this study, we found that at least four different types of binding sites are involved in the modulation of cardiac RyR2 by earth alkaline cations . Luminal M2+ differentially affected the apparent affinity to Ca2+ of cytosolic sites ("trans effect"). This effect could be due to the ability of luminal Ca2+/Sr2+ to stabilize RyR2 high Po gating mode . Our results suggest that the differential effects of luminal M2+ mainly result from them acting in different luminal sites and would not be due to the luminal M2+ flowing through the pore and interacting with cytosolic binding sites.RyR2 were heterogeneous in their response to M2+, both Ca2+ and Sr2+ bind to M2+ activating sites in the cytosolic surface of RyR and activate the channels in a cooperative fashion (nH >1) to high Po levels (>0.80) with different relative affinities (EC50 = 1.5 and 15 µM respectively). Selectivity of skeletal and cardiac RyRs for Ca2+ and Sr2+ as activators has been previously reported 2+ versus Sr2+. This could reflect differences between isoforms (RyR1 versus RyR2) and/or experimental conditions (e.g. presence/absence of Mg2+/ATP).In the presence of 50 mM luminal Ca2+ and Ba2+ do not induce channel openings, instead, they interfered with Ca2+ with Sr2+ activation. Many studies have previously defined the modulation of RyRs by Mg2+2+, the alkaline earth cation with the smallest radius (0.078 nm) and Ba2+ (the biggest one with 0.143 nm) bind to inhibitory cytosolic sites with equivalent affinity. The characteristics of selectivity usually described for M2+ binding sites 2+ and Sr2+  would be able to bind to the same inhibitory sites. Mg2+ competition with Ca2+ binding to the activating cytosolic M2+ binding site has been proposed 2+ and Ba2+ were more effective inhibitors at lower cytosolic Ca2+ concentrations. However, in the presence of caffeine, Ca2+ and Sr2+ activated RyRs more efficiently, while Mg2+ inhibitory efficiency was unchanged (2+ and Sr2+ bind to different cytosolic sites (referred as “Site A” 2+ and Ba2+ (which bind to “Inhibitory M2+ Site 1”).Mgnchanged . This su2+ binding sites responsible for the bell-shaped response to cytosolic Ca2+ are non-selective (2+ Site 2”) than the sites interfering with Ca2+ activation (Site 1) 2+ Sites 1 and 2 cannot be distinguished based on M2+ selectivity. In our opinion, functional studies of RyR2 sensitivity to M2+ are also inconclusive. As shown here and as previously reported, occupation of Site 1 or 2 results in different kinetics 2+ and Ba2+ increase closed times of partially activated RyRs whereas addition of millimolar cytosolic M2+ correlates with a decrease in open times. In principle, these differences in response to M2+ may suggest two different cytosolic inhibitory M2+ sites. However, RyR2 gating status, which is heavily influenced by Ca2+ may also play a role o mode is more sensitive to Mg2+ inhibition 2+, may mask flickery block during long openings. In contrast, at >100 µM cytosolic Ca2+, long openings are much more frequent Our data also suggests that the low affinity inhibitory Melective . These s2+<5 µM) are sensitive to inhibition by 1 mM Mg2+2+ ∼ 200 µM) are not inhibited by increasing cytosolic Ca2+ to 5 mM 2+ Site 1 present in all RyR2 and Site 2 present in ∼50% of the channels. Still, the observed differences may also reflect variability in RyR2 sensitivity to M2+. Indeed, we found that IC50 for RyR2 inhibition by Mg2+ (Site 1) range from 0.2 to 1 mM 2+ from 1 to 10 mM could generate additional unspecific electrostatic effects on the cytosolic RyR2 surface. Still, to confirm if the cytosolic inhibitory M2+ binding sites encompass one or more domains (Site 1 and Site 2) in the RyR2 molecule further molecular/structural data are required.All partially activated RyR2  may produce an increase in maximal Po. Calsequestrin (CSQ), a regulatory protein closely associated to RyR2, has been proposed to have a role in sensing luminal Ca2+2+ levels greater than 5 mM dissociate CSQ from RyR 2+, RyR2-CSQ association may have been disrupted in our experiments. Thus, the luminal M2+ activation observed here would more likely involve M2+ binding sites located on the RyR2 protein itself.We found that the maximal Po reached by RyR2 in the presence of luminal M2+ is the closest substitute for luminal Ca2+ (regarding the responses to cytosolic Ca2+ and caffeine). Notice, however, that saturating Sr2+ levels did not produce the same stabilization of high Po mode in caffeine-activated RyR2 as observed with luminal Ca2+  than that reported in previous studies for cytosolic Sr2+ activation in presence of luminal Sr2+ (EC50 ∼50 µM) 2+ and Sr2+ differ in their ability to generate sparks In our hands, Srnal Ca2+ . Further2+ versus luminal Ba2+ or Mg2+. We estimate (paired comparisons) the probability of a RyR2 to open from the closed state (PC→O). At equivalent cytosolic Ca2+, PC→O was 2–20 times higher in RyR2 bathed with luminal Ca2+ versus luminal Ba2+ (the differences were more marked at positive SR holding voltages). This suggests that luminal Ba2+ decreases the on-rate and increases the off-rate of Ca2+ binding to activating cytosolic sites. A previous report found that at 0 mV, the effects of luminal Ba2+ on Po are only noticeable in caffeine-activated RyR2 2+-activated RyR2, equivalent Po  was maintained by increasing the frequency of openings, which compensated the decrease in open times 2+ levels of RyR2 bathed with luminal Ba2+  found that RyR function is largely affected by changes in luminal Ca2+ levels, but there were discrepancies regarding the sensitivity of luminal Ca2+ binding sites, with reported values ranging from micromolar to millimolar 2+ to millimolar levels was reported to have no effect on maximal Po 2+ significantly increased maximal Po compared to that in the presence of luminal Cs+.Large differences in RyR2 behavior were found with luminal Ca2+ decreases the activity of RyR2 both at negative (no significant Mg2+ flux) and positive SR voltages. The effects of luminal Mg2+ were attributed to competition with luminal Ca2+ sites and with cytosolic Ca2+ sites (feed-through modulation) 2+ fluxes larger than 1 pA are required to observe voltage-dependence 2+ and absence of ATP, these previous reports did not describe an increase in maximal Po compared with luminal Cs+ alone.Using ATP-activated channels Laver et al. (2008) found that 50 µM Mg2+ moving through the pore with activating and inhibitory sites in the cytosolic side of the channel Voltage dependence, observed with high flux of luminal divalent ions has been attributed to the interaction of the M2+ which should maximize feed-through effects. All RyR2 bathed with luminal Ba2+ or Mg2+ display modal gating and exhibit voltage-dependence, even in the presence of caffeine. In contrast, when RyR2 are bathed with luminal Ca2+ a population displaying long openings (high Po – slow kinetics gating mode) is not sensitive to voltage. Our results differ from a previous study where inhibitory effects of flux were found to be nearly equivalent with both Mg2+ and Ca2+2+ only the subset displaying modal gating was voltage-dependent but addition of caffeine switched them to high Po mode and abolished their voltage-dependence. These observations suggest that modal gating (ability to switch between high and low Po modes) is required to observe voltage-dependence.In our experiments we used saturated luminal Mo decreased with increased M2+ flux . It has been suggested that activating and/or inhibitory cytosolic Ca2+ binding sites of cardiac RyR2 and skeletal RyR1 can sense the Ca2+ flux through the open channel pore (feed-through M2+ regulation) 50 for cytosolic Ca2+ is higher with luminal Mg2+/Ba2+ versus Ca2+/Sr2+ flowing through the channel  feeding through the channel would be much less than 0.25 mM, which could not explain the magnitude of the effect of luminal Ba2+ (or Mg2+) versus luminal Ca2+  and of cytosolic Ba2+ being more effective to block RyR displaying “low Po” mode o” mode is promoted by positive voltages; i.e., by increased flux of luminal M2+  indistinguishable from that obtained with 100% (50 mM) luminal Ca2+. As RyR2 are equally permeable to all M2+2+ would only account for 10% of the M2+ feeding through the pore, which may not explain the magnitude of the effect on channel properties. A possible explanation would be that 5 mM Ca2+ saturates the Ca2+-selective luminal M2+ binding sites, as suggested by previous studies Replacement of 5 mM of the luminal Mg2+ feeding-through the channel as proposed by others in the absence of ATP and stimulating cofactors 2+ using EGTA (n = 16) versus BAPTA/diBromoBAPTA (n = 37) and found no significant differences in Po . Analogous conclusions were obtained from similar experiments performed using even larger skeletal RyR1 populations (data not shown). If there were any activating effects of Ca2+ flux, we would expect large differences in Po among experiments with different buffers, as it is estimated that Ca2+ levels at the cytosolic surface would reach much higher levels with EGTA (thousand times slower kon for binding Ca2+) than with BAPTA 2+ fluxes at positive voltages would have increased cytosolic [Ca2+] to micromolar levels 2+ should have activated RyR2  and this effect should have been more evident for partially activated RyR2 (bathed with 100 nM Ca2+) in the presence of caffeine, where RyR2 sensitivity to cytosolic Ca2+ increases ∼20 times and full activation only requires cytosolic [Ca2+] ∼ 500 nM. As indicated, much higher levels of Ca2+ accumulate at the cytosolic surface of an open RyR2 with Ca2+ currents of up to 10 pA 2+ feeding through the pore may not be observed even in the presence of the stimulating cofactor caffeine.Our experiments also support the idea of open RyR2 being insensitive to activation by Ca2+ levels. This RyR2 behavior mirrors that of BK channels, which are also voltage- and Ca2+-gated channels + channels seems to result from dynamic interactions between various channel structures, including the pore helix, selectivity filter and external vestibule + channels As stated above, our results suggest that voltage-dependence requires modal gating. It is apparent that SR voltage depolarization destabilizes long openings and then short openings and long closures become more and more abundant. As shown, the effect of voltage can be counteracted in part by increasing cytosolic Ca2+ release by Ca2+ will depend on luminal and cytosolic resting levels of Ca2+ and Mg2+ as well as on the SR – cytosol membrane voltage. During the Ca2+ release event , luminal Ca2+ levels will decrease and cytosolic Ca2+ levels will increase. In contrast, cytosolic and luminal Mg2+ levels are expected to remain relatively constant . Consequently, when intra-SR Ca2+ levels fall, Mg2+ would maintain occupancy of luminal non-selective M2+ binding sites and keep the channels active  while cytosolic Ca2+ remain higher than 10 µM, which are the estimated Ca2+ levels on the RyR2 cytosolic surface 2+ levels, which in concomitance with a decrease in the driving force for Ca2+ flux, could greatly decrease SR Ca2+ release. On the other hand, the increase in cytosolic Ca2+ levels would tend to maintain RyR2 channels active even with lower levels of luminal Ca2+. How RyR2 close in cells for the termination of Ca2+ release is a process of still unknown nature Our current and previously published data o mode and high Po mode 2+-selective luminal sites may be important to stabilize RyR2 in high Po mode. This suggests that a switch from high to low Po mode may have physiological significance for RyR2 deactivation following SR Ca2+ depletion while the cytosolic Ca2+ levels remain elevated. Additionally, our results and previous reports suggest that open RyR2 do not sense the local increase in cytosolic Ca2+ they produce via luminal-to-cytosol flux. This may imply that if multiple RyR2 open simultaneously 2+ spark 2+ after activation, they might still be able to close in some synchrony, even when cytosolic Ca2+ levels remain high. For this, the RyR2 should remain refractory to activation by cytosolic Ca2+ for some time after they close (few milliseconds), as to allow for the dissipation of the local Ca2+ levels.RyR2 are known to alternate between two gating modes: low P2+  is a complex process which involves several interacting M2+ binding sites  that may dynamically modulate the function of heterogeneous RyR2 during local and global Ca2+ release events.In conclusion, our studies indicate that the regulation of single RyR2 by MFigure S12 on RyR2 kinetics.Effect of cytosolic CaTop: Open probability  and Probability of transition from close to open  of RyR2 bathed with luminal Ca2+ (50 mM) as a function of cytosolic Ca2+. Bottom: Time constants for openings as a function of cytosolic Ca2+. Open time distributions were fitted with 2 components (τ1 and τ2).(TIF)Click here for additional data file.Figure S22 on RyR2 kinetics.Effect of cytosolic Ca Representative traces of consecutive 4 minute-recordings (at 0 mV) of a RyR2 bathed with luminal Mg2+ (50 mM) under various conditions as indicated. Notice that in presence of caffeine, channels reach Po ∼0.5 with 0.3 µM cytosolic Ca2+ but openings are short.(TIF)Click here for additional data file.Figure S32+/Ba2+ on the kinetics of RyR2 exposed to the same cytosolic Ca2+ levels.Effect of luminal Ca Dwell time distribution histograms for openings (left panels) and closures (right) on the same single RyR2 (paired measurements) in the presence of luminal Ca2+ (black outlines) or luminal Ba2+ (grey outlines). Cytosolic [Ca2+] was 4 µM. All four-minute recordings were taken at −20 mV (top panels), 0 mV (middle) and +20 mV (bottom).(TIF)Click here for additional data file.Figure S42+/Ba2+ on the kinetics of RyR2 displaying similar open probabilities.Effect of luminal Ca Dwell time distribution histograms for openings (left panels) and closures (right) on the same single RyR2 (paired measurements) with luminal Ca2+ (black outlines) or luminal Ba2+ (grey outlines). Cytosolic [Ca2+] concentration was adjusted so that the open probabilities at 0 mV were similar in the presence of luminal Ca2+ compared with luminal Ba2+ . All four-minute recordings were taken at the indicated holding voltages.(TIF)Click here for additional data file.Figure S5Effect of holding voltage on caffeine-activated RyR2. Mean open probabilities (±S.E.M.) as a function of holding voltage of RyR2 bathed with luminal Ca2+. All channels were tested in the presence of 20 mM caffeine and at the indicated cytosolic Ca2+ levels.(TIF)Click here for additional data file.Table S1Kinetic parameters calculated from the dwell time distribution histograms depicted in Supporting Information, (DOCX)Click here for additional data file.Table S2Kinetic parameters calculated from the dwell time distribution histograms depicted in Supporting Information, (DOCX)Click here for additional data file.


Wnt signaling in the colon cancer tumor microenvironment (TME) may affect cancer biologic properties including invasion and metastatic dissemination. Prior reports have suggested that the expression of select frizzled (Fz) receptors may be altered in cancers and in the TME.Colon cancer, colonic adenoma and normal colonic mucosal specimens were obtained under institutional review board approval and analyzed for the expression of Fz1 and Fz2 by confocal fluorescent immunohistochemistry and Wnt-specific membrane array. In vitro, the effect of Wnt3a on Fz1 expression was examined in normal-derived NCM460 cells by qRT-PCR and immunohistochemistry.Fz1 was expressed in colon cancer and villous adenomas but not in more benign tubular adenomas. Fz1 expression was seen in normal colonic mucosa in close proximity to colon cancer, but not villous or tubular adenomas. Normal colonic mucosa distant from colon cancer did not express Fz1. Fz2 was expressed ubiquitously in cancer, adenomas and normal colonic mucosa. Fz1 expression was induced by Wnt3a in a normal colon mucosa-derived cell line in vitro.Fz1 is a Wnt responsive gene in colon-derived tissues. Fz1 expression exhibited increased expression in normal mucosa only in close proximity to colon cancer. This field effect was not seen with pre-malignant adenomas and may be due to Wnt/β-catenin signaling within the TME. Fz1 may represent a new TME-directed therapeutic target for patients with colon cancer. There is increasing recognition that tumor biology is modulated by signals derived from, and received by cells within the tumor microenvironment (TME). InteracWnt activity in colon cancer stem cells is modulated by myofibroblast-secreted factors in the TME and otheFrizzled receptors are 7-transmembrane proteins which serve, in conjunction with LRP5/6 co-receptors as the binding partners for extracellular Wnt ligands to initiate the Wnt signal. Conceptually, the signals transduced progress along one of two intracellular pathways – canonical, resulting ultimately in the accumulation of β-catenin in the nucleus where it promotes transcriptional activation in conjunction with the LEF/TCF family of DNA binding proteins and non-canonical, resulting in activation of protein kinase C, c-Jun-N-terminal kinases or calcium/calmodulin-dependent kinases. However, Wnt signaling is significantly more complex as there is crosstalk between canonical and non-canonical pathways, and ligands and receptors may result in differential signaling in a context-dependent fashion.Few studies have examined the role of distinct frizzled receptors in the TME. Factors in the TME have been implicated in the regulation of Fz7 on colorectal cancer cells and Fz7 Over 20 distinct de-identified colon cancer and corresponding normal colonic mucosa was obtained from archived specimens at UCIrvine under a non-human subjects exemption by the UCIrvine institutional review board. Adenoma specimens with biopsies of corresponding normal mucosa were obtained from 12 different patients following informed consent under a UCIrvine institutional review board approved protocol from patients undergoing elective colonoscopic screening as described previously. All resThe NCM460 cell line was purchased from INCELL Corporation  and cultured in M3 media supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a 95% humidified atmosphere with 5% CO2. This epithelial cell line was derived from normal colonic mucosa and is not tumorigenic though, over time in culture it has acquired some transformation-associated characteristics. NCM460 http://www.stanford.edu/group/nusselab/cgi-bin/wnt/. Cells were analyzed after 24 h of Wnt3a treatment for Fz1 expression.Wnt3a CM and control CM were prepared using the LWnt3A cell line (ATCC#CRL-2647) and the parental cell line (ATCC #CRL-2648) according to procedures perfected in the laboratory of Dr. Roel Nusse; for a pImmunohistochemistry for Fz1 and Fz2 were performed with specific anti-Fz1 antibodies  and anti-Fz2 antibodies, , 1 mg/ml, at 1:200 dilution. Paraffin-imbedded slides were deparaffinized and sodium citrate buffer pH 6 20 min at 99°C was used as an antigen retrieval agent. Santa Cruz Biotechnology rabbit ABC Staining System (SC-2018) was used according to the manufacturer’s protocol, and the signal was amplified by TSA™ Fluorescein Tyramide Reagent from Perkin Elmer (SAT701001EA). Slides were stained with DAPI and covered with anti-fade embedding medium . Confocal microscopy was performed with a Carl Zeiss LSM 510 and 40x fluorescent objective.Fz1 expression in NCM460 was determined by qRT-PCR with Fz1 specific primers. The efficiency of the Fz1 and actin primers was equivalent. Primer pairs were obtained from Qiagen  with cycling parameters as defined by the manufacturer. For qRT-PCR experiments, the relative RNA expression was calculated by the comparative threshold cycle method.Fz1 mRNA expression in adenomas and normal mucosa was defined with cDNA generated from tissue-derived RNA and analyzed with a GEArray Q Series Human Wnt Signaling Pathway Gene Array . GEArray expression analysis software was utilized for background normalization, correction for different degrees of exposure, and normalization with multiple housekeeping gene controls on each membrane.t-test between experimental (Wnt3a CM) and control (l-Cell CM) groups. Expression of Fz1 in adenomas, was analyzed across the 3 groups by 1-way ANOVA with a post-hoc Newman Kuels test. Individual subgroups were also compared by unpaired t-test.Fz1 mRNA and protein expression in NCM460 cells was compared with an unpaired Fluorescent immunohistochemistry with specific anti-Fz1 and anti-Fz2 antibodies was performed on tissue sections from colon cancer, adjacent normal colonic mucosa and distant normal colonic mucosa. All experiments included secondary antibody only controls on sequential tissue sections. Fz1 and Fz2 were both expressed in colon cancer tissues. Fz1 was detected in histologically normal mucosa in close proximity to the neoplastic cells . Villous adenomas have a substantially higher malignant potential than tubular adenomas.At the protein level, villous adenomas had abundant Fz1 expression (Figure Colonic adenomas were evaluated for Fz1 expression in order to define the stage in the neoplastic progression where Fz1 expression begins and whether the field effect for Fz1 noted in normal mucosa adjacent to colon cancer is specific for invasive carcinoma. Using a Wnt specific membrane array, Fz1 mRNA could be detected at low levels in both normal colonic mucosa and tubular adenomas. However, Fz1 mRNA was markedly increased in villous adenomas in comparison to normal mucosa and tubular adenomas (p=0.0176 1 way ANOVA, p<0.05 Newman-Kuels for both comparisons; p=0.024 villous vs. tubular by unpaired  Figure , panel IThe expression of the Wnt pathway receptors Fz1 and Fz2 has been previously implicated in cancer development and progression. Their expression is reported to be increased in breast cancer in comparison to normal breast epithelium. There iWhat is clear from this report is that Fz1 expression is tightly regulated in the tumor microenvironment. As both an effector molecule for Wnt signaling and also, as we have shown here, as a Wnt responsive gene, Fz1 may function as part of a positive autocrine feedback loop in colon cancer cells. Whether this effect is limited to Wnt3a which promotes Fz1 transcription and to which Fz1 also binds requires further study. In the tumor microenvironment, a field effect of Fz1 expression exists suggesting paracrine regulation of this receptor. Fz1 expression on normal colonic mucosa within the TME is induced by cancer but not by non-invasive adenomas, including villous adenomas which themselves express Fz1. We speculate that this is related to the degree of Wnt, and particularly Wnt3a ligand production by the colon cancer cells. Increased nuclear localization of β-catenin at the invasion front is a characteristic of colon cancer and coloIn addition to its expression in colon cancer, Fz1 expression appears to be confined to pre-malignant adenomas with villous features which represent the histologic type of adenoma with greatest risk for malignant transformation. DevelopThe significance of Fz1 on normal colonic mucosa in the TME requires further study. Given the known functions of Wnt/β-catinin signaling, it may promote proliferation and block differentiation of colonic mucosal cells, promoting tumor invasion by accelerating mucosal turnover. Fz1 expression in the TME may modulate colon cancer progression and dissemination and therefore may represent a new TME-directed therapeutic target for patients with colon cancer.KP, MP, AVN and RFH have no conflicts of interest to disclose. MPM is owner of INCELL Corporation which markets the NCM460 cell line utilized in this study.KP and MP conducted the majority of studies outlined in this study. AVN conducted membrane array studies related to adenomas. MPM provided the cell line for in vitro studies and reviewed the data and manuscript. RFH designed the studies and provided oversight for the project overall. All authors approved the final manuscript.


The possibility of titanium dioxide nanoparticles (TiO2 NPs), a nanomaterial pervasively used in nanotechnology and pharmaceutical industries, to directly induce histamine secretion without prior allergen sensitization has remained uncertain.Histamine released from mast cells, through complex interactions involving the binding of IgE to FcεRI receptors and the subsequent intracellular Ca2 NP exposure increased both histamine secretion and cytosolic Ca2+ concentration ([Ca2+]C) in a dose dependent manner in rat RBL-2H3 mast cells. The increase in intracellular Ca2+ levels resulted primarily from an extracellular Ca2+ influx via membrane L-type Ca2+ channels. Unspecific Ca2+ entry via TiO2 NP-instigated membrane disruption was demonstrated with the intracellular leakage of a fluorescent calcein dye. Oxidative stress induced by TiO2 NPs also contributed to cytosolic Ca2+ signaling. The PLC-IP3-IP3 receptor pathways and endoplasmic reticulum (ER) were responsible for the sustained elevation of [Ca2+]C and histamine secretion.TiO2 NP exposure and allergen-independent histamine release that can exacerbate manifestations of multiple allergic responses.Our data suggests that systemic circulation of NPs may prompt histamine release at different locales causing abnormal inflammatory diseases. This study provides a novel mechanistic link between environmental TiO Activation of histamine receptors  greatly influences inflammatory responses C increase by loading RBL-2H3 cells with Rhod-2 AM dye and exposing them to 0.1 mg/ml-1 mg/ml of TiO2 NPs. The TiO2 concentration range used in our study is consistent with the concentrations found in ambient, nanotechnology industries and existing literatures [2+]C were measured by monitoring the intracellular fluorescence intensity. Figure 2 NP concentrations (< 0.25 mg/ml) caused a smaller elevation, when compared to untreated cells. The results demonstrated a TiO2 NP concentration dependent increase in [Ca2+]C  failed to induce a significant increase in [Ca2+]C, when compared with the 160% increase observed in normal Hanks' buffer  markedly inhibited the rise in [Ca2+]C by approximately 75%  also hampered the rise in [Ca2+]C elicited by TiO2 NPs , 2-APB or Xestospongin (IP3 receptor blockers), before being exposed to TiO2 NPs. The data revealed a > 75% drop in the [Ca2+]C after TiO2 NP stimulation .ELISA was used to assess the amount of histamine secreted from RBL-2H3 cells when stimulated with TiOn Figure . Incubatn Figure . Our dat2 NPs have previously been shown to trigger mucin secretion, pulmonary inflammatory responses and emphysema-like pathology . We. We2+ inl Figure  and 2E. channels ,39,40. Os Figure  and thats Figure . Supportnditions . Other s) levels ,42. ROS d lipids ,44. Moreproteins . In addi2+ entry ,39. Co-ao Figure . The calchannels .2+]C from extracellular influx is usually relayed by a secondary Ca2+ release from internal organelles C, and when coupled with the IP3-IP3 receptor pathway, can trigger the release of ER resident Ca2+ and subsequent histamine secretion. These results suggest that mast cell degranulation of histamine may be significantly augmented and intensified in NP exposed tissues with or without IgE antibody-based sensitization. This model may also provide a new explanation for chronic inflammatory diseases elicited by airborne PM. The inhaled NPs can circulate and accumulate in various organs thereby elevating the risks of activating mast cell degranulation in other tissues [Our findings revealed a new mechanism depicting how TiO tissues ,50,51. A tissues -54. Fina2. Cell counts were performed using trypan blue  exclusion and a Bright-Line haemocytometer.The RBL-2H3 rat mast cell line  is a widely-used mast cell model that responds to stimuli by secreting histamine and other mediators . RBL-2H32 NPs have a primary crystalline size of 100 nm (maximum), specific surface area of 46.3 m2/g (as determined by BET), equivalent spherical diameter of 100 nm (maximum) and a trace metallic impurities of 1000 ppm (maximum) (information from Sigma). All TiO2 NP samples were reconstituted in Hanks' buffer  and sonicated for approximately 1 minute immediately before usage. The concentrations used were 1 mg/ml, 0.75 mg/ml, 0.5 mg/ml, 0.25 mg/ml, and 0.1 mg/ml. All sizes of NP suspension were independently confirmed using homodyne dynamics laser scattering (DLS) as described in the previous study [2 NP powder were determined by transmission electron microscope  and analyzed using an analysisPRO .A mixture of anatase and rutile forms of ultrafine titanium (IV) dioxide (< 100 nm diameter by Brunauer Emmett Teller (BET) method, purity of 99.5% trace metals basis) (Cat No. 634662)  was used in this study because this form has been shown to result in more severe cellular injuries . The TiOus study . Morphol5 cells per well in a 24-well plate, and incubated for 24 hrs in MEM medium supplemented with 10% FBS. Following 24 hr incubation, the MEM medium was removed from the cells and the culture was rinsed with Hanks' solution twice before use.Cells were seeded at 1 × 10d = 570 nM, λEx = 552 nm and λEm = 581)  for 45 minutes. After the dye loading, the cells were rinsed and incubated with either normal Hanks' or Ca2+-free Hanks' solution, and treated with the appropriate TiO2 NP concentrations. All Ca2+ signaling experiments were carried out on a thermoregulated stage at 37°C mounted on a Nikon microscope . RBL-2H3 cells were then incubated with cadmium chloride  to block the membrane Ca2+ channels [2 NP stimulation. To test the interaction between TiO2 and L-type membrane Ca2+ channels, nifedipine  and verapamil , L-type Ca2+ channel blockers [2. Antioxidant N-acetylcysteine  was also added to RBL-2H3 cells to study the involvement of reactive oxygen species (ROS) [2 stimulation, and the activation of Ca2+ channels. Thapsigargin  was used to deplete the ER Ca2+ content in order to investigate the contribution of the internal ER Ca2+ pool [2+ response, U73122 (PLC blocker) , Aminoethoxydiphenyl borate (2-APB)  and Xestospongin C (IP3 receptor blockers)  were pre-incubated with cells prior to TiO2 NP exposure [All experiments were performed in dark conditions. The cells were loaded with a Rhod-2 AM dye (1 μM)  ,39, possa2+ pool ,56. In o5 cells per well in a 24-well plate and cultured for 24 hrs. TiO2 prepared with calcein fluorescent dye (50 μM)  in Hanks' buffer was incubated with the cells for 5 minutes at 37°C. Calcein is a biological inert green-fluorescent molecule of a molecular mass of 623 Daltons and an estimated molecular radius of 0.6 nm [2 NP solution containing calcein dye was then removed and the cells were rinsed twice with PBS to remove any possible remnants of the dye. Subsequently, the cells were loaded with Hoechst (10 μM) , a fluorescent nucleus dye, for 5 minutes at 37°C, and were then rinsed thoroughly [2 NP concentrations used in the experiment.RBL-2H3 cells were seeded at a density of 1 × 10f 0.6 nm . The TiOoroughly . Fresh H2DCFDA dye . The cells (1 × 105 cells/well) were cultured for 24 hrs before being rinsed with PBS solution. Hanks' buffer, containing TiO2 NPs at concentrations ranging from 0-0.75 mg/ml, was then incubated with the cells for 15 minutes in 37°C followed by PBS washing and loading with 2 μM CM-H2DCFDA dye for 30 minutes. Fluorescent images of ROS generated in cells were captured and analyzed by calculating the ratio of increase in fluorescent intensity between TiO2 NP treatment and control groups.ROS production was evaluated by fluorescence microscopy using oxidation of CM-H5 cells density in a 24-well plate and cultured for 24 hrs. RBL-2H3 cells were then rinsed with PBS and pre-treated with BAPTA-AM (50 μM) , Thapsigargin (SERCA pump inhibitor), U73122, 2-APB, or Xestospongin for 20 minutes in the same manner. Cells were stimulated for 5 minutes with the appropriate TiO2 NP concentrations (0-0.75 mg/ml) which were prepared in PBS. The histamine-containing supernatant was collected and centrifuged to remove any remaining TiO2 NPs. The supernatant was then incubated in a 96-well plate overnight at 4°C. The rest of the assay was carried out in accord to Neogen ELISA instructions pertaining to the histamine kit .The cells were seeded at a density of 1 × 102+ signals of more than 200 cells and the experiments were conducted independently for at least 3 times.After staining the treated cells, image analysis was performed with an inverted Nikon Eclipse TE2000-U fluorescent microscope. Each photo was taken at a magnification of 200 × and analyzed using SimplePCI . The data shown is a representative of CaThe data was presented as means ± SD. Each experiment was performed independently at least three times. Statistical significance was determined using a Student's t-test analysis with p values < 0.05 .The authors declare that they have no competing interests.EYC, MG, YCW: Designed research, conducted experiments, analyzed and interpreted data. EYC, MG, YCW, AJM, CSC and WCC: wrote, reviewed and revised the manuscript. All authors have read and approved the final manuscript.


Astatotilapia latifasciata (Figure Haplochromis obliquidens. This species was described as Haplochromis latifasciatus [Astatotilapia [Haplochromis "zebra obliquidens" in the aquarium trade. Astatotilapia latifasciata has been reported to occur in Lake Nawampasa a small satellite lake of the much larger Lake Kyoga, and in Lake Kyoga located north of Lake Victoria in Uganda [After the publication of our work , we detea Figure , was errasciatus  and lateotilapia . Our misn Uganda .