Imbalance Of Calcium Homeostasis Research Articles

Misregulation of calcium-handling proteins promotes hyperactivation of calcineurin-NFAT signaling in skeletal muscle of DM1 mice.

Myotonic Dystrophy type 1 (DM1) is caused by an expansion of CUG repeats in DMPK mRNAs. This mutation affects alternative splicing through misregulation of RNA-binding proteins. Amongst pre-mRNAs that are mis-spliced, several code for proteins involved in calcium homeostasis suggesting that calcium-handling and signaling are perturbed in DM1. Here, we analyzed expression of such proteins in DM1 mouse muscle. We found that the levels of several sarcoplasmic reticulum proteins (SERCA1, sarcolipin and calsequestrin) are altered, likely contributing to an imbalance in calcium homeostasis. We also observed that calcineurin (CnA) signaling is hyperactivated in DM1 muscle. Indeed, CnA expression and phosphatase activity are both markedly increased in DM1 muscle. Coherent with this, we found that activators of the CnA pathway (MLP, FHL1) are also elevated. Consequently, NFATc1 expression is increased in DM1 muscle and becomes relocalized to myonuclei, together with an up-regulation of its transcriptional targets (RCAN1.4 and myoglobin). Accordingly, DM1 mouse muscles display an increase in oxidative metabolism and fiber hypertrophy. To determine the functional consequences of this CnA hyperactivation, we administered cyclosporine A, an inhibitor of CnA, to DM1 mice. Muscles of treated DM1 mice showed an increase in CUGBP1 levels, and an exacerbation of key alternative splicing events associated with DM1. Finally, inhibition of CnA in cultured human DM1 myoblasts also resulted in a splicing exacerbation of the insulin receptor. Together, these findings show for the first time that calcium-CnA signaling is hyperactivated in DM1 muscle and that such hyperactivation represents a beneficial compensatory adaptation to the disease.

P707 Paediatric IBD patients do not meet the daily recommendations of vitamin D and calcium intake: survey based analysis in a tertiary centre

Background: Achieving optimal levels of vitamin D (VitD) and calcium (Ca) is essential for developing children, especially in patients with inflammatory bowel disease (IBD). VitD and Ca play a major role in bone health and recently VitD has shown to potentiate the effect of anti-inflammatory treatments. However, achieving a sufficient oral intake is difficult in this group taking into account young age, modern eating habits and the nature of IBD itself. The purpose of this study was to evaluate if children with IBD seen in our centre achieve optimal Vit D and Ca intake according to recommendations made by the British Scientific Advisory Committee on Nutrition and the UK Department of Health. Methods: A prospective dietetic survey was conducted among sequential IBD children seen in clinics over a 12 month period. Ca and VitD intakes were assessed through a 24-hour recall of dietary intake questionnaire. Children who had been placed on restricted diets for allergic disease were excluded as well as children under 4 years. Included patients were classified according to age into 2 groups: 4–10 and 11–18 years. Sources of VitD were divided into dairy, oily fish, fortified cereals and egg. Analysis was performed using absolute values, percentages and means in Microsoft Excel. Results: Survey was conducted in 151 patients, this represents 68.3% of all IBD patients under follow-up. 94 patients were included for analysis and 57 were excluded. 43/94 (45.7%) were females. Overall, only 26.6% and 21.3% of the surveyed population achieved the current recommended intake for Ca and VitD respectively. In the younger group, only 7/31 (22.6%) met the current VitD recommendations, the same figure repeats with regards Ca intake. In the older group, only 13/63 (20.6%) and 18/63 (28.6%) met the Ca and VitD recommendations respectively. In both groups dairy was the main source of vitamin D (61.3% young ones and 58.7% older ones). Less than 1/3 of the patients have an optimal intake of oily fish and egg (sufficient intake 19%, 9% for children and 30%, 26% for adolescents). Conclusions: Paediatric IBD patients living in the UK do not meet the minimum requirements of VitD and Ca intake and therefore are at risk of having poor bone health, calcium homeostasis imbalance and VitD deficiency. In the great majority, Ca and VitD sources come from diary whereas the contribution of oily fish and egg as a VitD source is minimal. We recommend that paediatric IBD patients receive frequent counseling on healthy eating habits and proactive intake monitoring. Routine VitD supplementation recommended by local authorities must be followed as there is an insufficient vitD oral intake among these population.

Effects of deoxynivalenol on calcium homeostasis of concanavalin A—Stimulated splenic lymphocytes of chickens in vitro

In this study, the in vitro effects of the treatment of concanavalin A (Con A)—stimulated splenic lymphocytes with DON were examined. Splenic lymphocytes isolated from chickens were stimulated with 12.5μg/mL Con A and exposed to deoxynivalenol (DON) (0–50μg/mL) for 48h. The intracellular calcium concentration ([Ca2+]i), pH, calmodulin (CaM) mRNA levels, and Na+,K+-ATPase and Ca2+-ATPase activities were detected. With the DON exposure concentrations increased, the [Ca2+]i and CaM mRNA levels gradually increased in a dose-dependent manner, and all the evaluated conconcentrations affected ATPase activity to the same extent. There were significant differences (P<0.05 or P<0.01) between the treatment groups and the control group. These results indicate that an imbalance in calcium homeostasis and intracellular acidification are components of DON cytotoxicity in chicken lymphocytes.

Arsenic trioxide triggered calcium homeostasis imbalance and induced endoplasmic reticulum stress-mediated apoptosis in adult rat ventricular myocytes.

Arsenic trioxide (ATO) is a potent anticancer drug agent but its clinical use is often limited by severe cardiotoxicity. However, its exact mechanism remains poorly understood. In this study, we simultaneously explored the direct effect of ATO on cardiac contraction in adult rat ventricular myocytes and its effects on Ca2+ transient in real time by using an IonOptix MyoCam system. The results showed that ATO increased the amplitude of sarcomere shortening, the maximal velocity of relengthening and shortening (-dL/dtmax and +dL/dtmax), time-to-90% relengthening (TR90), and time-to-peak shortening (TPS), resulting in abnormal cardiomyocyte contraction. Meanwhile, ATO markedly increased the resting Ca2+ ratio, amplitude/resting calcium, the maximal velocity of Ca2+ shortening and relaxation (+d[Ca2+]/dtmax and -d[Ca2+]/dtmax), time-to-50% peak [Ca2+] i and the decay rate of [Ca2+] i transients, suggesting that ATO leads to intracellular imbalance of calcium homeostasis. ATO also inhibited sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) activity in a time-dependent manner and activated the endoplasmic reticulum (ER) stress reaction. These results revealed that ATO dramatically aggravates Ca2+ overload and promotes ER stress, eventually causing abnormal cardiomyocyte contraction in a dose-dependent and time-dependent manner.

A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells

Ultraviolet (UV) radiation–induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future.

1,25-Dihydroxyvitamin D-mediated hypercalcemia in a patient with malignancy without lymphoma

Introduction: Hypercalcemia resulting from calcium homeostasis imbalance is seen in one-third of malignancies. Solid malignancies cause hypercalcemia from high parathyroid hormone-related peptide (PTHrP) and, infrequently, from osteolytic cytokines. Excessive 1, 25-dihydroxyvitamin D, seen mostly in lymphoma, causes hypercalcemia by 1-alphahydroxylation of 25-hydroxyvitamin D. This mechanism has also been reported in a few cases of dysgerminoma, myofibroblastic tumors, and gastrointestinal stromal tumors. Case Report: We present an unusual case of 1, 25-dihydroxyvitamin D mediated hypercalcemia in a 62-year-old male with colonic adenocarcinoma and mesothelioma. Conclusion: This case emphasizes that elevated 1, 25-dihydroxyvitamin D may be a hypercalcemia etiology in non-lymphoma malignancies and should to be considered when workup for elevated PTHrP and osteolytic hypercalcemia is negative.

Neurotoxic effects of indocyanine green -cerebellar granule cell culture viability study

The aim of this study was to examine neurotoxicity indocyanine green (ICG). We assessed viability of primary cerebellar granule cell culture (CGC) exposed to ICG to test two mechanisms that could be the first triggers causing neuronal toxicity: imbalance in calcium homeostasis and the degree of oligomerization of ICG molecules. We have observed this imbalance in CGC after exposure to 75-125μΜ ICG and dose and application sequence dependent protective effect of Gadovist on surviving neurons in vitro when used with ICG. Spectroscopic studies suggest the major cause of toxicity of the ICG is connected with oligomers formation. ICG at concentration of 25 μM (which is about 4 times higher than the highest concentration of ICG in the brain applied in in-vivo human studies) is not neurotoxic in the cell culture.

Effects of zearalenone on calcium homeostasis of splenic lymphocytes of chickens in vitro

Zearalenone (ZEA) is an estrogenic mycotoxin. It is produced by several Fusarium species and can contaminate food and feed. To investigate the role of calcium homeostasis in ZEA-induced toxicity of poultry and elucidate its cytotoxic mechanism, splenic lymphocytes isolated from chickens were exposed to ZEA (0-25 μg/mL) for 48 h. The intracellular calcium concentration ([Ca2+]i), pH, calmodulin (CaM) mRNA levels, and Na+/K+-ATPase activities and Ca2+-ATPase activities were detected by the fluorescent dyes Fluo-3/AM and BCECF/AM, quantitative real-time PCR, and chromatometry. Supernatant CaM concentrations were simultaneously detected by ELISA. As the ZEA exposure concentration increased, the [Ca2+]i and CaM mRNA levels gradually increased, while intracellular pH, CaM concentrations of supernatants, and intracellular Na+,K+-ATPase and Ca2+-ATPase activities gradually decreased in a dose-dependent manner. There were significant differences (P<0.05 or P<0.01) between the treatment groups and the control group. These results indicate that ZEA cytotoxicity arises by causing an imbalance in calcium homeostasis and intracellular acidification in lymphocytes.

Lower blood calcium associates with unfavorable prognosis and predicts for bone metastasis in NSCLC.

Ionized calcium was involved in various cellular signal pathways,and regulates many cellular processes, including those relevant to tumorigenesis. We hypothesis that imbalance of calcium homeostasis is correlated with development of lung carcinomas. We collected the clinical data of 1084 patients with non small cell lung cancer (NSCLC) treated in Shandong Provincial Hospital, Shandong University. Logistic regression was used to determine the association between calcium levels and clinical characteristics, and COX regression and Kaplan-Meier model were applied to analyze risk factors on overall survival. Blood electrolytes were tested before treatment; and nearly 16% patients with NSCLC were complained with decreased blood calcium, which is more frequent than that in other electrolytes. Further, Multivariate logistic regression analysis disclosed that there were significant correlation between blood calcium decrease and moderate and poor differentiation (P = 0.012, OR = 1.926 (1.203–4.219)), squamous cell carcinoma (P = 0.024, OR = 1.968(1.094–3.540)), and bone metastasis (P = 0.032, OR = 0.396(0.235–0.669)). In multivariate COX regression analysis, advanced lymph node stage and decreased blood calcium were significantly and independent, unfavorable prognostic factors (P<0.001). Finally, the Kaplan-Meier Survival curve revealed that blood calcium decrease was associated with shorter survival (Log-rank; χ2 = 26.172,P<0.001). Our finding indicates that lower blood calcium levels are associated with a higher risk of unfavorable prognosis and bone metastasis of NSCLC.

Total-to-ionized calcium ratio predicts mortality in continuous renal replacement therapy with citrate anticoagulation in critically ill patients

IntroductionRegional citrate anticoagulation is safe, feasible and increasingly used in critically ill patients on continuous renal replacement therapy (CRRT). However, in patients with hepatic or multi-organ dysfunction, citrate accumulation may lead to an imbalance of calcium homeostasis. The study aimed at evaluating the incidence and prognostic relevance of an increased total to ionized calcium ratio (T/I Ca2+ ratio) and its association to hepatic dysfunction.MethodsWe performed a prospective observational study on n = 208 critically ill patients with acute kidney injury (AKI) and necessity for CRRT with regional citrate anticoagulation (CRRT-citrate) between September 2009 and September 2011. Critical illness was estimated by Simplified Acute Physiology Score II; hepatic function was measured with indocyanine green plasma disappearance rate. After achieving a steady state of calcium homeostasis patients were classified into tertiles according to the T/I Ca2+ ratio (<2.0 versus 2.0 - 2.39 versus ≥2.4).ResultsThe T/I Ca2+ ratio was determined as an independent predictor for 28-day mortality in critically ill patients with AKI on CRRT-citrate confirmed by receiver operating characteristics and multivariate analysis (Area under the curve 0.94 ± 0.02; p<0.001). A T/I Ca2+ ratio ≥2.4 independently predicted a 33.5-fold (p<0.001) increase in 28-day mortality-rate. There was a significant correlation between the T/I Ca2+ ratio and the hepatic clearance (p<0.001) and the severity of critical illness (p<0.001). The efficacy and safety of citrate anticoagulation, determined by blood urea nitrogen, mean filter patency and bleeding episodes, were not significantly different between the tertiles.ConclusionsIn patients on CRRT-citrate T/I Ca2+ ratio is closely related to the clinical outcome and emerged as an independent predictor of 28-day mortality. Larger studies are required to define the cut-off and predictive value for the T/I Ca2+ ratio. This ratio is associated with hepatic and/or multi-organ dysfunction and therefore an important therapeutic target.

Increased expression of annexin A7 in temporal lobe tissue of patients with refractory epilepsy.

Annexin A7 is a member of the family of annexins, which are thought to function in the regulation of calcium homeostasis and the fusion of vesicles. Refractory epilepsy may be related to the imbalance of calcium homeostasis. Our aims are to investigate the expression of Annexin A7 in epileptic brains in comparison with human controls and to explore Annexin A7's possible role in refractory epilepsy. We examined the expression of Annexin A7 via immunohistochemistry, double-label immunofluorescence and western blot. The expression of Annexin A7 was shown to be significantly increased in patients with refractory epilepsy. Double-label immunofluorescence and confocal microscopy disclosed Annexin A7 immunoreactivity in the neurons, which were recognized by the antibody of neuron specific enolase (NSE). The result showed that Annexin A7 may be involved in the pathophysiology of refractory epilepsy and may play a role in developing and maintaining the epilepsy.

Induction of Ca 2+ signal mediated apoptosis and alteration of IP3R1 and SERCA1 expression levels by stress hormone in differentiating C2C12 myoblasts

Glucocorticoid (GC) are stress hormones, whose cytotoxicity has been shown in various cells. The imbalance of calcium homeostasis is believed to be associated with the dexamethasone (DEX, a synthetic GC)-induced apoptosis. Here we show that in C2C12 myoblasts, DEX markedly up-regulated the expression of inositol 1,4,5-triphosphate receptor 1 (IP3R1) and down-regulated the expression of SERCA1 (sarcoendoplasmic reticulum Ca 2+-ATPase 1), leading to calcium overload. Furthermore, the imbalance of calcium homeostasis increased the level of BAX, decreased the level of Bcl-2, induced cytochrome c release and activated caspase-3, leading to intranucleosomal DNA fragmentation and plasma membrane damage, eventually resulting in cell apoptosis. Taken together, by using C2C12 myoblasts as a model system, we demonstrated a novel mechanism for stress hormone-induced apoptosis: it is dependent on the induction of intracellular calcium overload via the alterations of IP3R1 and SERCA1 expressions.

Low extracellular Ca2+: a mediator of endothelial inflammation

Recent studies have suggested that vitamin D and an imbalance in calcium homeostasis may have an impact on the cardiovascular system. The aim of this study was to assess the impact of different concentrations of extracellular Ca(2+) on human umbilical vein cord endothelial cells (HUVEC) by measuring its effect on parameters involved in the pathogenesis of vascular inflammatory responses. HUVEC were grown in the 3.5, 4.5 or 5.8 mg/dL concentration of extracellular Ca(2+) for 2-3 weeks. Expression of adhesion molecules was analysed by flow cytometry. Endothelial nitric oxide synthase (eNOS), receptor of advanced glycation end-product (RAGE) and interleukin-6 (IL-6) mRNA expressions were determined by real-time PCR. eNOS, inhibitor kappa Balpha (IkappaBalpha) and phosphorylated IkappaBalpha protein levels by Western blot, eNOS activity by conversion of [(14)C]-arginine to [(14)C]-citrulline, IL-6 and osteoprotegerin (OPG) secretion by ELISA and DNA-binding activity of nuclear factor kappa B (NFkappaB)-p65 were assayed colorimetrically in nuclear extracts. In the presence of low Ca(2+) (3.5 mg/dL), protein expressions and activity of eNOS were diminished, while the protein expressions of intercellular adhesion molecule-1 (ICAM-1), as well as the mRNA expressions of RAGE and IL-6, were elevated. The protein secretions of IL-6 and OPG were also stimulated in low Ca(2+) concentration. At this concentration, the DNA-binding activity of NFkappaB was enhanced, probably due to the decreased level of IkappaBalpha. These results suggest that lower extracellular ionized Ca(2+) may play a relevant role in modifying endothelial cells functions.

Reactive oxygen species and imbalance of calcium homeostasis contributes to curcumin induced programmed cell death in Leishmania donovani

Curcumin, a polyphenol compound, has been recognized as a promising anti-cancer drug. The purpose of the present study was to investigate the cytotoxicity of curcumin to Leishmania donovani, the causative agent for visceral leishmaniasis. Flow cytometric analysis revealed that curcumin induced cell cycle arrest at G2/M phase. Incubation of Leishmania promastigotes with curcumin caused exposure of phosphatidylserine to the outer leaflet of plasma membrane. This event is preceded by curcumin-induced formation of reactive oxygen species (ROS) and elevation of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (DeltaPsim), release of Cytochrome c into the cytosol and concomitant nuclear alterations that included deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) and DNA fragmentation. Taken together, these data indicate that curcumin has promising antileishmanial activity that is mediated by programmed cell death and, accordingly, merits further investigation as a therapeutic option for the treatment of leishmaniasis.

Urinary calcium excretion in the monitoring of bone metastases from prostatic carcinoma.

One of the greatest problems in treating advanced prostate carcinoma is monitoring the therapeutic response of bone metastases. As these metastases are mainly osteosclerotic and lead to a markedly increased bone calcium requirement that may give rise to an imbalance in calcium homeostasis, the authors investigated whether changes in calcium balance may be useful for evaluating the response of bone metastases to treatment. The study involved 268 prostate carcinoma patients: 142 in Stage A-C2 (International Union Against Cancer [UICC] staging system, 1998) and 126 with bone metastases who had failed to respond to hormone therapy and were receiving chemotherapy. Prostate-specific antigen (PSA), calcium and phosphate metabolism, and the main bone formation and resorption markers were all assayed before and after chemotherapy. Of the 126 patients on chemotherapy, 109 were evaluable for response: according to standard criteria, 25 (23%) had improved, 43 (39.5%) were unchanged, and 41 (37.5%) had worsened. All of the improved and 16 unchanged patients had decreased PSA and bone marker levels and an increased urinary calcium/creatinine ratio (UCa/Cr); the worsened patients had increased PSA and bone marker levels, and their UCa/Cr decreased after only six treatment cycles. PSA and UCa/Cr were the biochemical markers whose changes showed the best agreement with treatment response. The UCa/Cr ratio was the most useful marker of clinical response, mainly because it allowed an early decision to continue or to stop chemotherapy. Furthermore, UCa/Cr and PSA together identified a percentage of patients classified as unchanged on the basis of standard criteria but whose condition had actually improved.

Experimental aspects of cardiomyopathy

Heart failure, characterized by myocardial dysfunction, is divided into four etiologic types: 1) myopathy caused by genetic mutation; 2) myopathy due to ischemia; 3) myopathy due to myocarditis; and 4) work-induced myopathy. The end stage of heart failure, irrespective of the etiologic origin, is characterized by an imbalance in calcium homeostasis, vasoconstriction, and cardiac dilatation. Treatment of heart failure during early stages of development depends on the etiologic origin, whereas treatment for end-stage heart failure is universal, ie, vasodilator and positive inotropic therapy.