Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation.

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca2+-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca2+]i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca2+-ATPases (SERCAs), "thapsigargin." Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca2+-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.

Homologous cardiac calcium pump regulators phospholamban and sarcolipin adopt distinct oligomeric states in the membrane.

Phospholamban (PLN) and Sarcolipin (SLN) are homologous membrane proteins that belong to the family of proteins that regulate the activity of the cardiac calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA). PLN and SLN share highly conserved leucine zipper motifs that control self-association; consequently, it has been proposed that both PLN and SLN assemble into stable pentamers in the membrane. In this study, we used molecular dynamics (MD) simulations and Western blot analysis to investigate the precise molecular architecture of the PLN and SLN oligomers. Analysis showed that the PLN pentamer is the predominant oligomer present in mouse ventricles and ventricle-like human iPSC-derived cardiomyocytes, in agreement with the MD simulations showing stable leucine zipper interactions across all protomer-protomer interfaces and MD replicates. Interestingly, we found that the PLN pentamer populates an asymmetric structure of the transmembrane region, which is likely an intrinsic feature of the oligomer in a lipid bilayer. The SLN pentamer is not favorably formed across MD replicates and species of origin; instead, SLN from human and mouse atria primarily populate coexisting dimeric and trimeric states. In contrast to previous studies, our findings indicate that the SLN pentamer is not the predominant oligomeric state populated in the membrane. We conclude that despite their structural homology, PLN and SLN adopt distinct oligomeric states in the membrane. We propose that the distinct oligomeric states populated by PLN and SLN may contribute to tissue-specific SERCA regulation via differences in protomer-oligomer exchange, oligomer-SERCA dynamics, and noise filtering during ß-adrenergic stimulation in the heart.

Calcium and Heart Failure: How Did We Get Here and Where Are We Going?

The occurrence and prevalence of heart failure remain high in the United States as well as globally. One person dies every 30 s from heart disease. Recognizing the importance of heart failure, clinicians and scientists have sought better therapeutic strategies and even cures for end-stage heart failure. This exploration has resulted in many failed clinical trials testing novel classes of pharmaceutical drugs and even gene therapy. As a result, along the way, there have been paradigm shifts toward and away from differing therapeutic approaches. The continued prevalence of death from heart failure, however, clearly demonstrates that the heart is not simply a pump and instead forces us to consider the complexity of simplicity in the pathophysiology of heart failure and reinforces the need to discover new therapeutic approaches.

Intermedin protects thapsigargin­induced endoplasmic reticulum stress in cardiomyocytes by modulating protein kinase A and sarco/endoplasmic reticulum Ca2+­ATPase.

Intermedin (IMD) is a calcitonin/calcitonin­related peptide that elicits cardioprotective effects in a variety of heart diseases, such as cardiac hypertrophy and heart failure. However, the molecular mechanism of IMD remains unclear. The present study investigated the effects of IMD on neonatal rat ventricular myocytes treated with thapsigargin. The results of the present study demonstrated that thapsigargin induced apoptosis in cardiomyocytes in a dose­ and time­dependent manner. Thapsigargin induced endoplasmic reticulum stress, as determined by increased expression levels of 78­kDa glucose­regulated protein, C/EBP­homologous protein and caspase­12, which were dose­dependently attenuated by pretreatment with IMD. In addition, IMD treatment counteracted the thapsigargin­induced suppression of sarco/endoplasmic reticulum Ca2+­ATPase (SERCA) activity and protein expression levels, and cytoplasmic Ca2+ overload. IMD treatment also augmented the phosphorylation of phospholamban, which is a crucial regulator of SERCA. Additionally, treatment with the protein kinase A antagonist H­89 inhibited the IMD­mediated cardioprotective effects, including SERCA activity restoration, anti­Ca2+ overload, endoplasmic reticulum stress inhibition and antiapoptosis effects. In conclusion, the results of the present study suggested that IMD may protect cardiomyocytes against thapsigargin­induced endoplasmic reticulum stress and the associated apoptosis at least partly by activating the protein kinase A/SERCA pathway.

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases.

P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca2+-ATPase, mammalian Cu+-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

A hallmark of phospholamban functional divergence is located in the N-terminal phosphorylation domain.

Sarcoplasmic reticulum Ca2+ pump (SERCA) is a critical component of the Ca2+ transport machinery in myocytes. There is clear evidence for regulation of SERCA activity by PLB, whose activity is modulated by phosphorylation of its N-terminal domain (residues 1-25), but there is less clear evidence for the role of this domain in PLB's functional divergence. It is widely accepted that only sarcolipin (SLN), a protein that shares substantial homology with PLB, uncouples SERCA Ca2+ transport from ATP hydrolysis by inducing a structural change of its energy-transduction domain; yet, experimental evidence shows that the transmembrane domain of PLB (residues 26-52, PLB26-52) partially uncouples SERCA in vitro. These apparently conflicting mechanisms suggest that PLB's uncoupling activity is encoded in its transmembrane domain, and that it is controlled by the N-terminal phosphorylation domain. To test this hypothesis, we performed molecular dynamics simulations (MDS) of the binary complex between PLB26-52 and SERCA. Comparison between PLB26-52 and wild-type PLB (PLBWT) showed no significant changes in the stability and orientation of the transmembrane helix, indicating that PLB26-52 forms a native-like complex with SERCA. MDS showed that PLB26-52 produces key intermolecular contacts and structural changes required for inhibition, in agreement with studies showing that PLB26-52 inhibits SERCA. However, deletion of the N-terminal phosphorylation domain facilitates an order-to-disorder shift in the energy-transduction domain associated with uncoupling of SERCA, albeit weaker than that induced by SLN. This mechanistic evidence reveals that the N-terminal phosphorylation domain of PLB is a primary contributor to the functional divergence among homologous SERCA regulators.

Effects of a high-fat diet on intracellular calcium (Ca2+) handling and cardiac remodeling in Wistar rats without hyperlipidemia.

A high-fat diet is often associated with cardiovascular diseases. Research has suggested that consumption of a high-fat diet for 10 weeks is associated with cardiac dysfunction, including arrhythmias, through alterations in cardiac remodeling and myocardial intracellular calcium (Ca2+) handling. In this study, rats were randomly divided into two groups: the standard diet (N = 5) and high-fat diet (N = 5) groups. To evaluate the effects of a high-fat diet on cardiac remodeling, we investigated the myocardium obtained from male Wistar rats fed a high-fat diet or standard diet for ten weeks via scanning electron microscopy, polarization microscopy, and RT-PCR. We found that compared with the standard diet cohort, the high-fat diet cohort exhibited increased levels of SERCA2a and SERCA2b mRNA and a decreased level of PLB mRNA (P < .05). These findings showed that a high-fat diet may lead to cardiac upregulation of Ca2+ transport-related genes in the sarcoplasmic reticulum. Additionally, we observed endocardial injury accompanied by focal dense layered collagen, increased spacing between endocardial cells that was often filled with collagen debris, and increased amounts of collagen fibers among enlarged cardiomyocytes in the high-fat diet cohort. The abnormal intracellular calcium (Ca2+) handling and cardiac remodeling may be contributing factors in arrhythmias and sudden cardiac death in high-fat diet-fed rats.

Inhibition of endotoxin on the function of cardiac sarcoplasmic reticulum in rats / 解放军医学杂志

Objective To observe the enzymatic changes of myocardial sarcoplasmic reticulum in rats after injecting endotoxin (LPS), and provide basic research results for the future study of myocardial sarcoplasmic reticulum dysfunction caused by LPS in rats. Methods Ten SD rats were randomly divided into blank control group and LPS injection group with 5 rats in each group. In the LPS injection group, endotoxin was injected into the tail vessels of the rats. Results The heart rate (HR) of the LPS injection group increased and was faster than that in the blank control group [(204±18) beat per min vs. (139±10) beat per min on the first day, and (199±22) beat per min vs. (143±17) beat per min on the next day, both P values were less than 0.05]. The mean arterial pressure (MAP) was lower than that of the blank control group on the first day [(87±12) mmHg vs. (102±7) mmHg, P<0.05]. Under light and electron microscope, the myocardial cells of rats with LPS injection were loosely arranged, with intercellular infiltration with inflammatory cells, muscle fibers broken, and difficult to identify the morphology of mitochondria and sarcoplasmic reticulum. Quantitative PCR results showed that after endotoxin injection, troponin (CASQ1), sodium-calcium exchanger (NCX), calmodulin phosphatase 1 (ppplCa), phospholipid protein (PLN), sarcoplasmic reticulum Ca2+-ATPase (SERCA2) increased significantly (P<0.05). Conclusion Endotoxin can inhibit cardiomyocyte function by affecting the activity of sarcoplasmic reticulum calcium regulatory protein-related enzymes through various mechanisms.

Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2.

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.

Inotropes in Acute Heart Failure: From Guidelines to Practical Use: Therapeutic Options and Clinical Practice.

Inotropes are pharmacological agents that are indicated for the treatment of patients presenting with acute heart failure (AHF) with concomitant hypoperfusion due to decreased cardiac output. They are usually administered for a short period during the initial management of AHF until haemodynamic stabilisation and restoration of peripheral perfusion occur. They can be used for longer periods to support patients as a bridge to a more definite treatment, such as transplant of left ventricular assist devices, or as part of a palliative care regimen. The currently available inotropic agents in clinical practice fall into three main categories: beta-agonists, phosphodiesterase III inhibitors and calcium sensitisers. However, due to the well-documented potential for adverse events and their association with increased long-term mortality, physicians should be aware of the indications and dosing strategies suitable for different types of patients. Novel inotropes that use alternative intracellular pathways are under investigation, in an effort to minimise the drawbacks that conventional inotropes exhibit.

Schekwanglupaside C, a new lupane saponin from Schefflera kwangsiensis, is a potent activator of sarcoplasmic reticulum Ca2+-ATPase.

Schefflera kwangsiensis Merr. ex H.L. Li (Araliaceae) is a widely used traditional Chinese medicine for pain management in the clinic. In the present study, we isolated a previously undescribed lupane saponin, designated as schekwanglupaside C (Sch C) from the ethanolic extract of S. kwangsiensis. The structure of Sch C was determined by comprehensive spectroscopic and spectrometric analyses and chemical degradation. In primary cultured cortical neurons, Sch C altered the pattern of spontaneous Ca2+ oscillation (SCO) with a slight increase in the frequency of SCO right after addition and a gradual decrease in the frequency and amplitude of SCO, that dynamic change mimicked by an activator of sarcoplasmic reticulum Ca2+-ATPase (SERCA). The IC50 values for Sch C suppression of the frequency and amplitude of SCO were 1.75 and 2.51 µM, respectively. Furthermore, we demonstrated that Sch C is a potent SERCA activator (EC50 = 1.20 µM). Given the pivotal role of SERCA in the progression of neuropathic pain and neurodegenerative diseases, Sch C represents a new drug lead compound to develop the treatment of neuropathic pain and Alzheimer's disease.

Unbalance Between Sarcoplasmic Reticulum Ca2 + Uptake and Release: A First Step Toward Ca2 + Triggered Arrhythmias and Cardiac Damage.

The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners' proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.

Beta-cell hubs maintain Ca2+ oscillations in human and mouse islet simulations.

Islet ß-cells are responsible for secreting all circulating insulin in response to rising plasma glucose concentrations. These cells are a phenotypically diverse population that express great functional heterogeneity. In mice, certain ß-cells (termed 'hubs') have been shown to be crucial for dictating the islet response to high glucose, with inhibition of these hub cells abolishing the coordinated Ca2+ oscillations necessary for driving insulin secretion. These ß-cell hubs were found to be highly metabolic and susceptible to pro-inflammatory and glucolipotoxic insults. In this study, we explored the importance of hub cells in human by constructing mathematical models of Ca2+ activity in human islets. Our simulations revealed that hubs dictate the coordinated Ca2+ response in both mouse and human islets; silencing a small proportion of hubs abolished whole-islet Ca2+ activity. We also observed that if hubs are assumed to be preferentially gap junction coupled, then the simulations better adhere to the available experimental data. Our simulations of 16 size-matched mouse and human islet architectures revealed that there are species differences in the role of hubs; Ca2+ activity in human islets was more vulnerable to hub inhibition than mouse islets. These simulation results not only substantiate the existence of ß-cell hubs, but also suggest that hubs may be favorably coupled in the electrical and metabolic network of the islet, and that targeted destruction of these cells would greatly impair human islet function.

Effect of 5-AZA-2'-dC on angiotensin Ⅱ-induced cardiomyocyte hypertrophy / 中华急诊医学杂志

Objective To investigate the effect of 5-AZA-2'-dC on Angiotensin Ⅱ (Ang Ⅱ)-induced cardiomyocyte hypertrophy.Methods Cultured cells derived from neonatal heart of rat were divided into 5 groups:normal control,hypertrophic group,5-AZA-2'-dC treatment group,and 5-AZA-2'-dC pretreatment group.Neonatal rat cardiomyocyte hypertrophic response was assayed by the size of cardiomyocytes and atrial natriuretic polypeptide (ANP) expressive level.The level of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a),total calmodulin kinase Ⅱ (CaMK Ⅱ) and phospho-CaMK Ⅱ (p-CaMK Ⅱ) detected by Western blot.The intracellular calcium changes of cardiomyocytes were imaged by confocal fluorescent microscopy.Results Cells treated with Ang Ⅱ at 10-6 mol/L for 48 h were chosen as hypertrophic cardiomyocyte model.The mRNA expression and protein level of ANP were significantly decreased in the treatment and pretreatment groups compared with hypertrophic group.The protein level of SERCA2a was significantly decreased in the hypertrophic group,and increased in the treatment and pretreatment group compared with hypertrophic group.The protein level of SERCA2a was significantly decreased in the hypertrophic group,and increased in the treatment and pretreatment group compared with hypertrophic group,whereas phospho-CaMK Ⅱ showed an opposite change tendency.The time required for increasing and declining to half of the intracellular calcium peak value were both delayed in hypertrophic group,as the treatment and pretreatment groups showed shorter time required compared with hypertrophic group.Conclusion 5-AZA-2'-dC could inhibit Ang Ⅱ-induced cardiomyocyte hypertrophy which might be related to regulate SERCA2a expression.Increased SERCA2a expression may maintain the calcium homeostasis through shortening the time of transfer Ca2+ from the cytosol of the cell to the lumen of the sarcoplasmic reticulum.

Cardioprotective Effects of Serca2a Overexpression Against Ischemiareperfusion- induced Injuries in Rats.

AIMS: The aim of the present study was to assess how genetically increased Sarcoplasmic reticulum Ca2+-ATPase (Serca2a) expression affects cardiac injury after Ischemia/Reperfusion (I/R) exposure and the related mechanisms involved. METHODS AND RESULTS: Rats were subjected to Left Anterior Descending coronary artery (LAD) occlusion for 30 min followed by a 24-hour reperfusion. Cardiac function analysis revealed that cardiac function dramatically improved in Serca2a transgenic rats, (Serca2aTG) rats, compared to Wild Type (WT) rats. Serca2aTG rats developed a significantly smaller myocardial infarction size compared to those in WT group. The expression of the Bcl-2 was lower in Serca2aTG rats compared with WT rats; but, Bcl-2 expression was markedly increased in Serca2aTG rats compared with WT after I/R. In addition, Bax was markedly downregulated in Serca2aTG rats compared to WT group after I/R. Meanwhile, autophagy marker LC-3B was increased in Serca2aTG group, and p62 was only increased in WT group but not in Serca2aTG group in response to I/R. Electron microscope observation confirmed that there were more autophagosomes in Serca2aTG group than in WT rats after I/R. CONCLUSION: our findings demonstrated that the overexpression of Serca2a plays an important role in myocardial protection from I/R injury and postischemic functional recovery, which may be via antinecrotic, anti-apoptotic and pro-autophagy signal pathways. Our research provides solid basic data and new perspective on clinical treatment in heart failure patients with long-term over-expression of Serca2a.

Peptidyl-Prolyl Isomerase 1 Regulates Ca2+ Handling by Modulating Sarco(Endo)Plasmic Reticulum Calcium ATPase and Na2+/Ca2+ Exchanger 1 Protein Levels and Function.

BACKGROUND: Aberrant Ca2+ handling is a prominent feature of heart failure. Elucidation of the molecular mechanisms responsible for aberrant Ca2+ handling is essential for the development of strategies to blunt pathological changes in calcium dynamics. The peptidyl-prolyl cis-trans isomerase peptidyl-prolyl isomerase 1 (Pin1) is a critical mediator of myocardial hypertrophy development and cardiac progenitor cell cycle. However, the influence of Pin1 on calcium cycling regulation has not been explored. On the basis of these findings, the aim of this study is to define Pin1 as a novel modulator of Ca2+ handling, with implications for improving myocardial contractility and potential for ameliorating development of heart failure. METHODS AND RESULTS: Pin1 gene deletion or pharmacological inhibition delays cytosolic Ca2+ decay in isolated cardiomyocytes. Paradoxically, reduced Pin1 activity correlates with increased sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) and Na2+/Ca2+ exchanger 1 protein levels. However, SERCA2a ATPase activity and calcium reuptake were reduced in sarcoplasmic reticulum membranes isolated from Pin1-deficient hearts, suggesting that Pin1 influences SERCA2a function. SERCA2a and Na2+/Ca2+ exchanger 1 associated with Pin1, as revealed by proximity ligation assay in myocardial tissue sections, indicating that regulation of Ca2+ handling within cardiomyocytes is likely influenced through Pin1 interaction with SERCA2a and Na2+/Ca2+ exchanger 1 proteins. CONCLUSIONS: Pin1 serves as a modulator of SERCA2a and Na2+/Ca2+ exchanger 1 Ca2+ handling proteins, with loss of function resulting in impaired cardiomyocyte relaxation, setting the stage for subsequent investigations to assess Pin1 dysregulation and modulation in the progression of heart failure.

Effects of Nitrosyl Iron Complexes with Thiocarbamide and Its Aliphatic Derivatives on Activities of Ca2+-ATPase of Sarcoplasmic Reticulum and cGMP Phosphodiesterase.

We studied the effects of water-soluble cationic dinitrosyl iron complexes with thiocarbamide and its aliphatic derivatives, new synthetic analogs of natural NO donors, active centers of nitrosyl [1Fe-2S]proteins, on activities of Ca2+-ATPase of sarcoplasmic reticulum and cGMP phosphodiesterase. Nitrosyl iron complexes [Fe(C3N2H8S)Cl(NO)2]0[Fe(NO)2(C3N2H8S)2]+Cl- (I), [Fe(SC(N(CH3)2)2(NO)2]Cl (II), [Fe(SC(NH2)2)2(NO)2Cl×H2O (III), and [Fe(SC(NH2)2)2(NO)2]2SO4×H2O (IV) in a concentration of 10-4 M completely inhibited the transporting and hydrolytic functions of Ca2+-ATPase. In a concentration of 10-5 M, they inhibited active Ca2+ transport by 57±6, 75±8, 80±8, and 85±9% and ATP hydrolysis by 0, 40±4, 48±5, and 38±4%, respectively. Complex II reversibly and noncompetitively inhibited the hydrolytic function of Ca2+-ATPase (Ki=1.7×10-6 M). All the studied iron-sulphur complexes in a concentration of 10-4 M inhibited cGMP phosphodiesterase function. These data suggest that the studied complexes can exhibit antimetastatic, antiaggregation, vasodilatatory, and antihypertensive activities.

Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells.

Intracellular calcium (Ca2+) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H2O2) on intracellular Ca2+ accumulation in mouse pancreatic acinar cells. Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels and termination of oscillatory Ca2+ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca2+. Antioxidants, catalase or DTT, completely prevented H2O2-induced additional Ca2+ increase and termination of Ca2+ oscillation. In Ca2+-free medium, H2O2 still enhanced CCh-induced intracellular Ca2+ levels and thapsigargin (TG) mimicked H2O2-induced cytosolic Ca2+ increase. Furthermore, H2O2-induced elevation of intracellular Ca2+ levels was abolished under sarco/endoplasmic reticulum Ca2+ ATPase-inactivated condition by TG pretreatment with CCh. H2O2 at 300 µM failed to affect store-operated Ca2+ entry or Ca2+ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca2+ uniporter blocker, failed to attenuate H2O2-induced intracellular Ca2+ elevation. These results provide evidence that excessive generation of H2O2 in pathological conditions could accumulate intracellular Ca2+ by attenuating refilling of internal Ca2+ stores rather than by inhibiting Ca2+ extrusion to extracellular fluid or enhancing Ca2+ mobilization from extracellular medium in mouse pancreatic acinar cells.

Liraglutide directly protects cardiomyocytes against reperfusion injury possibly via modulation of intracellular calcium homeostasis.

BACKGROUND: Liraglutide is glucagon-like peptide-1 receptor agonist for treating patients with type 2 diabetes mellitus. Our previous studies have demonstrated that liraglutide protects cardiac function through improving endothelial function in patients with acute myocardial infarction undergoing percutaneous coronary intervention. The present study will investigate whether liraglutide can perform direct protective effects on cardiomyocytes against reperfusion injury. METHODS: In vitro experiments were performed using H9C2 cells and neonatal rat ventricular cadiomyocytes undergoing simulative hypoxia/reoxygenation (H/R) induction. Cardiomyocytes apoptosis was detected by fluorescence TUNEL. Mitochondrial membrane potential (ΔΨm) and intracellular reactive oxygen species (ROS) was assessed by JC-1 and DHE, respectively. Fura-2/AM was used to measure intracellular Ca2+ concentration and calcium transient. Immunofluorescence staining was used to assess the expression level of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a). In vivo experiments, myocardial apoptosis and expression of SERCA2a were detected by colorimetric TUNEL and by immunofluorescence staining, respectively. RESULTS: In vitro liraglutide inhibited cardiomyotes apoptosis against H/R. ΔΨm of cardiomyocytes was higher in liraglutide group than H/R group. H/R increased ROS production in H9C2 cells which was attenuated by liraglutide. Liraglutide significantly lowered Ca2+ overload and improved calcium transient compared with H/R group. Immunofluorescence staining results showed liraglutide promoted SERCA2a expression which was decreased in H/R group. In ischemia/reperfusion rat hearts, apoptosis was significantly attenuated and SERCA2a expression was increased by liraglutide compared with H/R group. CONCLUSIONS: Liraglutide can directly protect cardiomyocytes against reperfusion injury which is possibly through modulation of intracellular calcium homeostasis.

Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

Intracellular calcium (Ca²⁺) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (H₂O₂) on intracellular Ca²⁺ accumulation in mouse pancreatic acinar cells. Perfusion of H₂O₂ at 300 µM resulted in additional elevation of intracellular Ca²⁺ levels and termination of oscillatory Ca²⁺ signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca²⁺. Antioxidants, catalase or DTT, completely prevented H₂O₂-induced additional Ca²⁺ increase and termination of Ca²⁺ oscillation. In Ca²⁺-free medium, H₂O₂ still enhanced CCh-induced intracellular Ca²⁺ levels and thapsigargin (TG) mimicked H₂O₂-induced cytosolic Ca²⁺ increase. Furthermore, H₂O₂-induced elevation of intracellular Ca²⁺ levels was abolished under sarco/endoplasmic reticulum Ca²⁺ ATPase-inactivated condition by TG pretreatment with CCh. H₂O₂ at 300 µM failed to affect store-operated Ca²⁺ entry or Ca²⁺ extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca²⁺ uniporter blocker, failed to attenuate H₂O₂-induced intracellular Ca²⁺ elevation. These results provide evidence that excessive generation of H₂O₂ in pathological conditions could accumulate intracellular Ca²⁺ by attenuating refilling of internal Ca²⁺ stores rather than by inhibiting Ca²⁺ extrusion to extracellular fluid or enhancing Ca²⁺ mobilization from extracellular medium in mouse pancreatic acinar cells.