The transmembrane peptide DWORF activates SERCA2a via dual mechanisms.

The Ca-ATPase isoform 2a (SERCA2a) pumps cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a. However, establishing DWORF's precise physiological role requires further investigation. In the present study, we developed cell-based FRET biosensor systems that can report on protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the SERCA2a-binding site, we transiently transfected DWORF into a stable HEK cell line expressing SERCA2a labeled with a FRET donor and PLB labeled with a FRET acceptor. We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Surprisingly, we also found that DWORF also activates SERCA's enzymatic activity directly in the absence of PLB at subsaturating calcium levels. Using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues P15 and W22 as necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

Involvement of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in mPRα (PAQR7)-mediated progesterone induction of vascular smooth muscle relaxation.

Progesterone acts directly on vascular smooth muscle cells (VSMCs) through activation of membrane progesterone receptor α (mPRα)-dependent signaling to rapidly decrease cytosolic Ca2+ concentrations and induce muscle relaxation. However, it is not known whether this progesterone action involves uptake of Ca2+ by the sarco/endoplasmic reticulum (SR) and increased sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. The present results show that treatment of cultured human VSMCs with progesterone and the selective mPR agonist Org OD-02-0 (OD 02-0) but not with the nuclear PR agonist R5020 increased SERCA protein expression, which was blocked by knockdown of mPRα with siRNA. Moreover, treatments with progesterone and OD 02-0, but not with R5020, increased phospholamban (PLB) phosphorylation, which would result in disinhibition of SERCA function. Progesterone and OD 02-0 significantly increased Ca2+ levels in the SR and caused VSMC relaxation. These effects were blocked by pretreatment with cyclopiazonic acid (CPA), a SERCA inhibitor, and by knockdown of SERCA2 with siRNA, suggesting that SERCA2 plays a critical role in progesterone induction of VSMC relaxation. Treatment with inhibitors of inhibitory G proteins (Gi, NF023), MAP kinase (AZD 6244), Akt/Pi3k (wortmannin), and a Rho activator (calpeptin) blocked the progesterone- and OD 02-0-induced increase in Ca2+ levels in the SR and SERCA expressions. These results suggest that the rapid effects of progesterone on cytosolic Ca2+ levels and relaxation of VSMCs through mPRα involve regulation of the functions of SERCA2 and PLB through Gi, MAP kinase, and Akt signaling pathways and downregulation of RhoA activity.NEW & NOTEWORTHY The rapid effects of progesterone on cytosolic Ca2+ levels and relaxation of VSMCs through mPRα involve regulation of the functions of SERCA2 and PLB through Gi, MAP kinase, and Akt signaling pathways and downregulation of RhoA activity.

The vasodilatory effect of gemigliptin via activation of voltage-dependent K+ channels and SERCA pumps in aortic smooth muscle.

This study investigated the vasodilatory effects and acting mechanism of gemigliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor. Tests were conducted in aortic rings pre-contracted with phenylephrine. Gemigliptin induced dose-dependent vasodilation of the aortic smooth muscle. Several pre-treatment groups were used to investigate the mechanism of action. While pre-treatment with paxilline, a large-conductance Ca2+-activated K+ channel inhibitor, glibenclamide, an ATP-sensitive K+ channel inhibitor, and Ba2+, an inwardly rectifying K+ channel inhibitor, had no impact on the vasodilatory effect of gemigliptin, pre-treatment with 4-aminopyridine, a voltage-dependent K+ (Kv) channel inhibitor, effectively attenuated the vasodilatory action of gemigliptin. In addition, pre-treatment with sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid significantly reduced the vasodilatory effect of gemigliptin. cAMP/PKA-related or cGMP/PKG-related signaling pathway inhibitors, including adenylyl cyclase inhibitor SQ 22536, PKA inhibitor KT 5720, guanylyl cyclase inhibitor ODQ, and PKG inhibitor KT 5823 did not alter the vasodilatory effect of gemigliptin. Similarly, elimination of the endothelium and pre-treatment with a nitric oxide (NO) synthase inhibitor (L-NAME) or small- and intermediate-conductance Ca2+-activated K+ channels (apamin and TRAM-34, respectively) did not change the gemigliptin effect. These findings suggested that gemigliptin induces vasodilation through the activation of Kv channels and SERCA pumps independent of cAMP/PKA-related or cGMP/PKG-related signaling pathways and the endothelium. Therefore, caution is required when prescribing gemigliptin to the patients with hypotension and diabetes.

Dietary nitrate does not alter cardiac function, calcium handling proteins, or SERCA activity in the left ventricle of healthy rats.

Dietary nitrate has been shown to increase cytosolic calcium concentrations within the heart, which would necessitate greater calcium sequestration for relaxation. In the present study we demonstrate that while nitrate supplementation reduced blood pressure, calcium-handling protein content, sarco(endo)plasmic reticulum Ca-ATPase 2a (SERCA) enzymatic properties, and left ventricular function were not altered. In addition, nitrite did not alter in vitro SERCA activity. Combined, these data suggest that in healthy rats, dietary nitrate does not increase left ventricle SERCA-related calcium-handling properties. Novelty Dietary nitrate decreases blood pressure but does not alter left ventricular calcium-handling protein content or SERCA activity in healthy rats.

Venous Vasomotion.

Veins exhibit spontaneous contractile activity, a phenomenon generally termed vasomotion. This is mediated by spontaneous rhythmical contractions of mural cells (i.e. smooth muscle cells (SMCs) or pericytes) in the wall of the vessel. Vasomotion occurs through interconnected oscillators within and between mural cells, entraining their cycles. Pharmacological studies indicate that a key oscillator underlying vasomotion is the rhythmical calcium ion (Ca2+) release-refill cycle of Ca2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP3R)- and/or ryanodine receptor (RyR)-operated Ca2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca2+ATPase (SERCA). Released Ca2+ from stores near the plasma membrane diffuse through the cytosol to open Ca2+-activated chloride (Cl-) channels, this generating inward current through an efflux of Cl-. The resultant depolarisation leads to the opening of voltage-dependent Ca2+ channels and possibly increased production of IP3, which through Ca2+-induced Ca2+ release (CICR) of IP3Rs and/or RyRs and IP3R-mediated Ca2+ release provide a means by which store oscillators entrain their activity. Intercellular entrainment normally involves current flow through gap junctions that interconnect mural cells and in many cases this is aided by additional connectivity through the endothelium. Once entrainment has occurred the substantial Ca2+ entry that results from the near-synchronous depolarisations leads to rhythmical contractions of the mural cells, this often leading to vessel constriction. The basis for venous/venular vasomotion has yet to be fully delineated but could improve both venous drainage and capillary/venular absorption of blood plasma-associated fluids.

Glutamate signaling at cytoneme synapses.

We investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII. Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by l-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.

Metformin attenuates cardiovascular and renal injury in uninephrectomized rats on DOCA-salt: Involvement of AMPK and miRNAs in cardioprotection.

Hypertension is associated with major cardiovascular (CV) and renal complications. The molecular intricacy by which hypertension leads to end organ damage is not known. To address this, we aimed to determine the effect of deoxycorticosterone acetate (DOCA) -salt (sodium chloride)-induced hypertension on the alterations in renin-angiotensin system, leading to CV and renal dysfunction in uninephrectomized male Sprague Dawley rats. MicroRNAs involved in this were also not yet explored. Metformin was used to delineate the role of AMPK in mitigating the hypertension-induced CV and renal dysfunction. Administering DOCA and offering saline to uninephrectomized rats, induced hypertension and associated abnormalities of diastolic dysfunction, CV and renal hypertrophy and fibrosis via activating local renin angiotensin system. Western blotting and RT-qPCR analysis of diseased heart revealed decreased SERCA2, p-AMPK, miR-146a, miR-99b and increased miR-155 and metformin administered, at dose of 300 mg/kg/day, for a period of 8 weeks prevented CV and renal damage. To our knowledge, we are the first to show that involvement of epigenetic alterations at microRNA level might be responsible for hypertension-induced cardiac dysfunction and metformin reverses these alterations.

LncRNA ZFAS1 as a SERCA2a Inhibitor to Cause Intracellular Ca2+ Overload and Contractile Dysfunction in a Mouse Model of Myocardial Infarction.

RATIONALE: Ca2+ homeostasis-a critical determinant of cardiac contractile function-is critically regulated by SERCA2a (sarcoplasmic reticulum Ca2+-ATPase 2a). Our previous study has identified ZFAS1 as a new lncRNA biomarker of acute myocardial infarction (MI). OBJECTIVE: To evaluate the effects of ZFAS1 on SERCA2a and the associated Ca2+ homeostasis and cardiac contractile function in the setting of MI. METHODS AND RESULTS: ZFAS1 expression was robustly increased in cytoplasm and sarcoplasmic reticulum in a mouse model of MI and a cellular model of hypoxia. Knockdown of endogenous ZFAS1 by virus-mediated silencing shRNA partially abrogated the ischemia-induced contractile dysfunction. Overexpression of ZFAS1 in otherwise normal mice created similar impairment of cardiac function as that observed in MI mice. Moreover, at the cellular level, ZFAS1 overexpression weakened the contractility of cardiac muscles. At the subcellular level, ZFAS1 deleteriously altered the Ca2+ transient leading to intracellular Ca2+ overload in cardiomyocytes. At the molecular level, ZFAS1 was found to directly bind SERCA2a protein and to limit its activity, as well as to repress its expression. The effects of ZFAS1 were readily reversible on knockdown of this lncRNA. Notably, a sequence domain of ZFAS1 gene that is conserved across species mimicked the effects of the full-length ZFAS1. Mutation of this domain or application of an antisense fragment to this conserved region efficiently canceled out the deleterious actions of ZFAS1. ZFAS1 had no significant effects on other Ca2+-handling regulatory proteins. CONCLUSIONS: ZFAS1 is an endogenous SERCA2a inhibitor, acting by binding to SERCA2a protein to limit its intracellular level and inhibit its activity, and a contributor to the impairment of cardiac contractile function in MI. Therefore, anti-ZFAS1 might be considered as a new therapeutic strategy for preserving SERCA2a activity and cardiac function under pathological conditions of the heart.

VDAC1 and SERCA3 Mediate Progesterone-Triggered Ca2+ Signaling in Breast Cancer Cells.

Progesterone is a biphasic hormone whose confounding role in breast cancer cells involves an initial proliferative surge, followed by sustained growth arrest. Recently we reported that progesterone induces a time- and concentration-dependent release of reactive oxygen species and thus regulates the antiproliferative activity in the breast cancer cell line. Furthermore, the expression of p27, a crucial cell cycle control protein, was regulated by binding of progesterone on progesterone receptor B, thus leading to antiproliferative signaling via multiple signaling pathways including p53, PTEN, and antioxidant systems. Here, we performed an LC-MS/MS analysis of three different breast cancer cell lines. Bioinformatics data analysis and functional classification of proteins revealed a role of progesterone in calcium signaling in MCF-7 cells, and the major differentially expressed calcium regulators were S100A11, S100A10, calreticulin, VDAC1, SERCA3, and SERCA1. Later on we confirmed it by a cell-line-based system having a calcium cameleon sensor targeted at endoplasmic reticulum and found moderate calcium efflux from endoplasmic reticulum upon progesterone treatment. Real-time PCR, Western blot, and TMRM staining confirmed the role of calcium signaling regulators VDAC1 and SERCA3 in progesterone response. Taking together all of these results with our previous studies, we suggest that progesterone, by regulating important proteins involved in calcium signaling and transport, can modulate cell proliferation and cell death. Furthermore, our research may open new avenues for the hypothesis that surgery conducted during the luteal phase of the menstrual cycle might facilitate improved patient survival.

UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis.

Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or ß3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

The vasorelaxant effect of mitiglinide via activation of voltage-dependent K+ channels and SERCA pump in aortic smooth muscle.

AIMS: The vasorelaxant effects of the anti-diabetic drug, mitiglinide in phenylephrine (Phe)-pre-contracted aortic rings were examined. MATERIALS AND METHODS: Arterial tone measurement was performed in aortic smooth muscle cells. KEY FINDINGS: Mitiglinide dose-dependently induced vasorelaxation. Application of the large-conductance Ca2+-activated K+ (BKCa) channel blocker paxilline, inwardly rectifying K+ (Kir) channel blocker Ba2+, and ATP-sensitive K+ (KATP) channel blocker glibenclamide did not affect the vasorelaxant effect of mitiglinide. However, application of the voltage-dependent K+ (Kv) channel blocker 4-AP, effectively inhibited mitiglinide-induced vasorelaxation. Although pretreatment with the Ca2+ channel blocker nifedipine did not alter the mitiglinide-induced vasorelaxation, pretreatment with the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin and cyclopiazonic acid reduced the vasorelaxant effect of mitiglinide. In addition, the vasorelaxant effect of mitiglinide was not affected by the inhibitors of adenylyl cyclase, protein kinase A, guanylyl cyclase, or protein kinase G. Elimination of the endothelium and inhibition of endothelium-dependent vasorelaxant mechanisms also did not change the vasorelaxant effect of mitiglinide. SIGNIFICANCE: We proposed that mitiglinide induces vasorelaxation via activation of Kv channels and SERCA pump. However, the vasorelaxant effects of mitiglinide did not involve other K+ channels, Ca2+ channels, PKA/PKG signaling pathways, or the endothelium.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats.

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Differences in the Force-Interval. Relationship of Isolated Human Myocardium with Chronic Coronary Artery Diseasein Patients with and without Type 2 Diabetes Mellitus and the Role of Ca²âº-ATPase of Sarcoplasmic Reticulum.

We studied the force--interval relationship of human myocardium in coronary artery d isease (CAD) and CAD with concomitant diabetes mellitus and evaluated its dependence on the level of expression of Ca²âº-AT Pase of the sarcoplasmic reticulum (SR). We used the myocardial biopsies obtained during coronary artery bypass grafting with the use of cardiopulmonary bypass techniques. The study included patients with chronic CAD and patients with CAD and concomitant type II diabetes mellitus. It was found that patients with CAD with and without diabetes with similar clinical signs had both negative and positive dynamics of the force-interval relationship. The positive. force-interval relationship was associated with the ''high level" of Ca²âº-ATPase, while the negative force-interval relationship was associated with the "low level" of this protein. In cases of small CAD and diabetes duration, the positive "force-interval" relationship is more pronounced and corresponds to a higher level of expression Ca²âº- ATPase in the SR than in case of CAD without diabetes mellitus.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Multiscale Modeling Indicates That Temperature Dependent [Ca2+]i Spiking in Astrocytes Is Quantitatively Consistent with Modulated SERCA Activity.

Changes in the cytosolic Ca(2+) concentration ([Ca(2+)]i) are the most predominant active signaling mechanism in astrocytes that can modulate neuronal activity and is assumed to influence neuronal plasticity. Although Ca(2+) signaling in astrocytes has been intensively studied in the past, our understanding of the signaling mechanism and its impact on tissue level is still incomplete. Here we revisit our previously published data on the strong temperature dependence of Ca(2+) signals in both cultured primary astrocytes and astrocytes in acute brain slices of mice. We apply multiscale modeling to test the hypothesis that the temperature dependent [Ca(2+)]i spiking is mainly caused by the increased activity of the sarcoendoplasmic reticulum ATPases (SERCAs) that remove Ca(2+) from the cytosol into the endoplasmic reticulum. Quantitative comparison of experimental data with multiscale simulations supports the SERCA activity hypothesis. Further analysis of multiscale modeling and traditional rate equations indicates that the experimental observations are a spatial phenomenon where increasing pump strength leads to a decoupling of Ca(2+) release sites and subsequently to vanishing [Ca(2+)]i spikes.

Aerobic interval training reduces inducible ventricular arrhythmias in diabetic mice after myocardial infarction.

Diabetes mellitus (DM) increases the risk of heart failure after myocardial infarction (MI), and aggravates ventricular arrhythmias in heart failure patients. Although exercise training improves cardiac function in heart failure, it is still unclear how it benefits the diabetic heart after MI. To study the effects of aerobic interval training on cardiac function, susceptibility to inducible ventricular arrhythmias and cardiomyocyte calcium handling in DM mice after MI (DM-MI). Male type 2 DM mice (C57BLKS/J Lepr (db) /Lepr (db) ) underwent MI or sham surgery. One group of DM-MI mice was submitted to aerobic interval training running sessions during 6 weeks. Cardiac function and structure were assessed by echocardiography and magnetic resonance imaging, respectively. Ventricular arrhythmias were induced by high-frequency cardiac pacing in vivo. Protein expression was measured by Western blot. DM-MI mice displayed increased susceptibility for inducible ventricular arrhythmias and impaired diastolic function when compared to wild type-MI, which was associated with disruption of cardiomyocyte calcium handling and increased calcium leak from the sarcoplasmic reticulum. High-intensity exercise recovered cardiomyocyte function in vitro, reduced sarcoplasmic reticulum diastolic calcium leak and significantly reduced the incidence of inducible ventricular arrhythmias in vivo in DM-MI mice. Exercise training also normalized the expression profile of key proteins involved in cardiomyocyte calcium handling, suggesting a potential molecular mechanism for the benefits of exercise in DM-MI mice. High-intensity aerobic exercise training recovers cardiomyocyte function and reduces inducible ventricular arrhythmias in infarcted diabetic mice.

AMPK Dilates Resistance Arteries via Activation of SERCA and BKCa Channels in Smooth Muscle.

The protective effects of 5'-AMP-activated protein kinase (AMPK) on the metabolic syndrome may include direct effects on resistance artery vasomotor function. However, the precise actions of AMPK on microvessels and their potential interaction are largely unknown. Thus, we set to determine the effects of AMPK activation on vascular smooth muscle tone and the underlying mechanisms. Resistance arteries isolated from hamster and mouse exhibited a pronounced endothelium-independent dilation on direct pharmacological AMPK activation by 2 structurally unrelated compounds (PT1 and A769662). The dilation was associated with a decrease of intracellular-free calcium [Ca(2+)]i in vascular smooth muscle cell. AMPK stimulation induced activation of BKCa channels as assessed by patch clamp studies in freshly isolated hamster vascular smooth muscle cell and confirmed by direct proof of membrane hyperpolarization in intact arteries. The BKCa channel blocker iberiotoxin abolished the hyperpolarization but only partially reduced the dilation and did not affect the decrease of [Ca(2+)]i. By contrast, the sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor thapsigargin largely reduced these effects, whereas combined inhibition of SERCA and BKCa channels virtually abolished them. AMPK stimulation significantly increased the phosphorylation of the SERCA modulator phospholamban at the regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular smooth muscle cell by a decrease of [Ca(2+)]i. This is achieved by calcium sequestration via SERCA activation, as well as activation of BKCa channels. There is in part a mutual compensation of both calcium-lowering mechanisms. However, SERCA activation which involves an AMPK-dependent phosphorylation of phospholamban is the predominant mechanism in resistance vessels.

Novel role of Sarco/endoplasmic reticulum calcium ATPase 2 in development of colorectal cancer and its regulation by F36, a curcumin analog.

Sarco/endoplasmic reticulum calcium ATPase (SERCA) enzymes play important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. Here, we reported that SERCA2 expression was positively correlated with tumor node metastasis (TNM) stages (n=75, P=0.0251) and grades (n=63, P=0.0146) of patients with colorectal cancer. The animal experiments demonstrated that SERCA2 expression was consistent with PCNA staining of intestinal tissues of male C57BL/6J-Apc(Min/)JNju mice. Besides, SERCA2 expression was also increased in undifferentiated HT-29 cells as compared with that in differentiated HT-29gal cells. Moreover, SERCA2 overexpression promoted proliferation and migration of SW480 cells via activating MAPK and AKT signaling pathways, while silence of SERCA2 inhibited the proliferation and migration of SW480 cells. In addition, we identified that a curcumin analog, F36, exhibited more potent inhibitory effect in colorectal cancer cells than curcumin through inhibiting SERCA2 expression. Taken together, our findings indicate that SERCA2 is involved in the malignant progress of colorectal cancer and maybe a therapeutic target for colorectal cancer treatment. Curcumin analog F36 shows enhanced anti-cancer activity in colorectal cancer cells by targeting SERCA2.