Maintaining Calcium Homeostasis Research Articles

Calcium-Sensing Receptor Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1: Epitopes, Specificity, Functional Affinity, IgG Subclass, and Effects on Receptor Activity.

A major manifestation of autoimmune polyendocrine syndrome type 1 (APS1) is hypoparathyroidism, which is suggested to result from aberrant immune responses against the parathyroid glands. The calcium-sensing receptor (CaSR), which plays a pivotal role in maintaining calcium homeostasis by sensing blood calcium levels and regulating release of parathyroid hormone (PTH), is an autoantibody target in APS1. In this study, the aim was to characterize the binding sites, specificity, functional affinity, IgG subclass, and functional effects of CaSR autoantibodies using phage-display technology, ELISA, and bioassays. The results indicated that CaSR autoantibody binding sites were at aa 41-69, 114-126, 171-195, and 260-340 in the extracellular domain of the receptor. Autoantibodies against CaSR epitopes 41-69, 171-195, and 260-340 were exclusively of the IgG1 subclass. Autoantibody responses against CaSR epitope 114-126 were predominantly of the IgG1 with a minority of the IgG3 subclass. Only autoantibodies recognizing CaSR epitopes 114-126 and 171-195 affected receptor activity; inositol-phosphate accumulation was increased significantly in HEK293-CaSR cells, and PTH secretion from PTH-C1 cells was reduced significantly when either were incubated with purified Ab and Ca2+ compared with Ca2+ alone. In conclusion, although the majority of APS1 patients do not have CaSR-stimulating autoantibodies, the hypoparathyroid state in a small minority of patients is the result of functional suppression of the parathyroid glands.

Calcium dysregulation mediates mitochondrial and neurite outgrowth abnormalities in SOD2 deficient embryonic cerebral cortical neurons.

Mitochondrial superoxide dismutase 2 (SOD2) is a major antioxidant defense enzyme. Here we provide evidence that SOD2 plays critical roles in maintaining calcium homeostasis in newly generated embryonic cerebral cortical neurons, which is essential for normal mitochondrial function and subcellular distribution, and neurite outgrowth. Primary cortical neurons in cultures established from embryonic day 15 SOD2+/+ and SOD2-/- mice appear similar during the first 24 h in culture. During the ensuing two days in culture, SOD2-/- neurons exhibit a profound reduction of neurite outgrowth and their mitochondria become fragmented and accumulate in the cell body. The structural abnormalities of the mitochondria are associated with reduced levels of phosphorylated (S637) dynamin related protein 1 (Drp1), a major mitochondrial fission-regulating protein, whereas mitochondrial fusion regulating proteins (OPA1 and MFN2) are relatively unaffected. Mitochondrial fission and Drp1 dephosphorylation coincide with impaired mitochondrial Ca2+ buffering capacity and an elevation of cytosolic Ca2+ levels. Treatment of SOD2-/- neurons with the Ca2+ chelator BAPTA-AM significantly increases levels of phosphorylated Drp1, reduces mitochondrial fragmentation and enables neurite outgrowth.

The (Sun)Light and Dark of 25-Hydroxyvitamin D Testing.

Vitamin D is obtained by the body via sunlight on the skin, from the diet, or from supplementation. The primary function of vitamin D is to maintain calcium homeostasis and bone health, but in the past decade, numerous other health benefits have been proposed. With the increased awareness of the potential benefits of maintaining sufficient concentrations of 25-hydroxyvitamin D, clinicians began ordering this test for their patients much more frequently. The number of available methods increased, but with that came a larger focus on the challenges of measuring 25-hydroxyvitamin D accurately due to binding to vitamin D-binding protein and the presence of other vitamin D metabolites. Further, standardization of these assays became a focus for several organizations so that clinical guidelines can be applicable to every patient regardless of what methodology is used in 25-hydroxyvitamin D measurement. Improvements are being made in the specificity, accuracy, and standardization of the measurement of 25-hydroxyvitamin D, and the future of this testing is looking brighter.

Research Progress on the Relationship Between Acute Pancreatitis and Calcium Overload in Acinar Cells.

Acute pancreatitis is a human disease with multiple causes that leads to autodigestion of the pancreas. There is sufficient evidence to support the key role of sustained increase in cytosolic calcium concentrations in the early pathogenesis of the disease. To clarify the mechanism of maintaining calcium homeostasis in the cell and pathological processes caused by calcium overload would help to research directly targeted therapeutic agents. We will specifically review the following: intracellular calcium homeostasis and regulation, the occurrence of calcium overload in acinar cells, the role of calcium overload in the pathogenesis of AP, the treatment strategy proposed for calcium overload.

Expression of prolactin receptors in the duodenum, kidneys and skeletal system during physiological and sulpiride-induced hyperprolactinaemia.

Hyperprolactinaemia in pregnancy leads to mild and reversible changes in the maternal skeletal system, and medicamentous hyperprolactinemia causes more detrimental effects. We conducted an experimental study to evaluate differences between Prlr gene expression in the duodenum, vertebrae and kidneys during physiological and medicamentous hyperprolactinaemia, which could influence calcium homeostasis. Experimental animals (18 weeks old, Wistar female rats) were divided as follows: group P (nine rats that were 3 weeks pregnant), group M (ten rats that were intramuscularly administrated sulpiride (10 mg/kg) twice daily for 3 weeks), and the control group (C, ten age-matched nulliparous rats, 18-week-old). Laboratory investigations included measurements of serum ionized calcium, phosphorus, urinary calcium and phosphorus excretion, osteocalcin (OC), serum procollagen type 1 N-terminal propeptide (P1NP), vitamin D, parathyroid hormone (PTH) and prolactin (PRL). Relative quantification of gene expression for prolactin receptors in the duodenum, vertebrae and kidneys was determined using real-time PCR. Expression of the Prlr gene was significantly higher in the duodenum (p < 0.001) and lower in vertebrae (p < 0.001) and kidneys (p < 0.01) in rats with physiological hyperprolactinaemia (PHP) than in the control group. Significantly lower Prlr expression in the duodenum was verified (p < 0.001), along with increased Prlr gene expression in vertebrae (p < 0.001) and kidneys (p < 0.01), in rats with medicamentous hyperprolactinaemia (MHP) than in the C group. Downregulation of Prlr gene expression in the duodenum may explain the diminished intestinal calcium absorption in medicamentous hyperprolactinaemia. Prolactin takes calcium from the skeletal system following increased Prlr gene expression in the vertebrae to maintain calcium homeostasis, which increases the harmful effect on bone metabolism compared to that of physiological hyperprolactinaemia.

Altered sarco(endo)plasmic reticulum calcium adenosine triphosphatase 2a content: Targets for heart failure therapy.

Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is responsible for transporting cytosolic calcium into the sarcoplasmic reticulum and endoplasmic reticulum to maintain calcium homeostasis. Sarco(endo)plasmic reticulum calcium adenosine triphosphatase is the dominant isoform expressed in cardiac tissue, which is regulated by endogenous protein inhibitors, post-translational modifications, hormones as well as microRNAs. Dysfunction of sarco(endo)plasmic reticulum calcium adenosine triphosphatase is associated with heart failure, which makes sarco(endo)plasmic reticulum calcium adenosine triphosphatase a promising target for heart failure therapy. This review summarizes current approaches to ameliorate sarco(endo)plasmic reticulum calcium adenosine triphosphatase function and focuses on phospholamban, an endogenous inhibitor of sarco(endo)plasmic reticulum calcium adenosine triphosphatase, pharmacological tools and gene therapies.

Gushukang exerts osteopreserve effects by regulating vitamin D and calcium metabolism in ovariectomized mice.

Calcium homeostasis plays vital roles in the management of bone health. Traditional herbal formula Gushukang (GSK) was clinically applied to treat primary osteoporosis. This study aimed to explore the osteoprotective effects of GSK and its roles in maintaining calcium homeostasis in ovariectomized (OVX) mice. The OVX mice were orally treated with low (0.38g/kg), middle (0.76g/kg) and high (1.52g/kg) dose of GSK for 8weeks. GSK treatment dramatically increased serum calcium level and decreased urinary calcium excretion as well as enhanced calcium content in bone of OVX mice. Serum level of 25-hydroxyvitamin D was significantly increased in OVX mice with exposure to GSK. Treatment with GSK improved bone mass and micro-structure of trabecular bone at distal metaphysis of femur and proximal metaphysis of tibia in OVX mice shown by safranin O staining and micro-CT measurement. GSK treatment at all doses up-regulated mRNA expression of calcium-binding protein-28k and vitamin D receptor in kidney of OVX mice, and dose-dependently decreased mRNA expression of claudin-14 and elevated mRNA expression of claudin-16 in duodenum of OVX mice. Taken together, GSK exerted beneficial effects on trabecular bone of OVX mice by improving calcium homeostasis via regulating paracellular calcium absorption in duodenum and transcellular calcium reabsorption in kidney.

Treatment dose and the elimination rates of electrolytes, vitamins, and trace elements during continuous veno-venous hemodialysis (CVVHD).

During continuous renal replacement therapy, achievement of recommended treatment dose is important. However, relevant substrate loss may occur and recommended nutrition during critical illness could not be sufficient for higher dialysis doses. We investigated the correlation of dialysis dose and substrate loss for a broad range of dialysis doses. Forty critically ill patients with acute kidney injury undergoing citrate CVVHD were included in this prospective study. Three different corresponding blood flow (BF) and dialysate flow (DF) rates were applied (BF/DF: 100ml/min, 2000ml/h; 80ml/min, 1500ml/h; 120ml/min, 2500ml/h). Delivered effluent flow rate (DEFR) was calculated and correlated with losses of vitamins, electrolytes, and trace elements during recommended nutritional supplementation. For folic acid, vitamin B12, zinc, inorganic phosphate, and magnesium, no correlation of losses and DEFR was detected. For ionized calcium, a correlation was observed and additional substitution was required. Clinically relevant loss of folic acid, vitamin B12, zinc, inorganic phosphate, and magnesium was not observed for differently used dialysis doses of CVVHD, and the loss was covered sufficiently by daily recommended nutritional supplementation. Increased loss of ionized calcium for higher dialysis doses occurred during citrate CVVHD. Therefore, a strict protocol must maintain calcium homeostasis to avoid calcium depletion.

Insights into calcium-sensing receptor trafficking and biased signalling by studies of calcium homeostasis.

The calcium-sensing receptor (CASR) is a class C G-protein-coupled receptor (GPCR) that detects extracellular calcium concentrations, and modulates parathyroid hormone secretion and urinary calcium excretion to maintain calcium homeostasis. The CASR utilises multiple heterotrimeric G-proteins to mediate signalling effects including activation of intracellular calcium release; mitogen-activated protein kinase (MAPK) pathways; membrane ruffling; and inhibition of cAMP production. By studying germline mutations in the CASR and proteins within its signalling pathway that cause hyper- and hypocalcaemic disorders, novel mechanisms governing GPCR signalling and trafficking have been elucidated. This review focusses on two recently described pathways that provide novel insights into CASR signalling and trafficking mechanisms. The first, identified by studying a CASR gain-of-function mutation that causes autosomal dominant hypocalcaemia (ADH), demonstrated a structural motif located between the third transmembrane domain and the second extracellular loop of the CASR that mediates biased signalling by activating a novel β-arrestin-mediated G-protein-independent pathway. The second, in which the mechanism by which adaptor protein-2 σ-subunit (AP2σ) mutations cause familial hypocalciuric hypercalcaemia (FHH) was investigated, demonstrated that AP2σ mutations impair CASR internalisation and reduce multiple CASR-mediated signalling pathways. Furthermore, these studies showed that the CASR can signal from the cell surface using multiple G-protein pathways, whilst sustained signalling is mediated only by the Gq/11 pathway. Thus, studies of FHH- and ADH-associated mutations have revealed novel steps by which CASR mediates signalling and compartmental bias, and these pathways could provide new targets for therapies for patients with calcaemic disorders.

Myocardin regulates mitochondrial calcium homeostasis and prevents permeability transition

Myocardin is a transcriptional co-activator required for cardiovascular development, but also promotes cardiomyocyte survival through an unclear molecular mechanism. Mitochondrial permeability transition is implicated in necrosis, while pore closure is required for mitochondrial maturation during cardiac development. We show that loss of myocardin function leads to subendocardial necrosis at E9.5, concurrent with elevated expression of the death gene Nix. Mechanistically, we demonstrate that myocardin knockdown reduces microRNA-133a levels to allow Nix accumulation, leading to mitochondrial permeability transition, reduced mitochondrial respiration, and necrosis. Myocardin knockdown elicits calcium release from the endo/sarcoplasmic reticulum with mitochondrial calcium accumulation, while restoration of microRNA-133a function, or knockdown of Nix rescues calcium perturbations. We observed reduced myocardin and elevated Nix expression within the infarct border-zone following coronary ligation. These findings identify a myocardin-regulated pathway that maintains calcium homeostasis and mitochondrial function during development, and is attenuated during ischemic heart disease. Given the diverse role of Nix and microRNA-133a, these findings may have broader implications to metabolic disease and cancer.

Vitamin D: part I; from plankton and calcified skeletons (500 million years ago) to rickets.

The vitamin D history started early in the evolution of life (billion years ago) as a photochemical reaction producing an inert molecule. During the early evolution of vertebrates, this molecule became essential for calcium and bone homeostasis of terrestrial animals and arrived to the status of hormone. Phytoplankton, zooplankton, and most plants and animals that are exposed to sunlight have the capacity to make vitamin D. Vitamin D is critically important for the development, growth, and maintenance of a healthy skeleton from birth until death. The major function of vitamin D is to maintain calcium homeostasis. It accomplishes this by increasing the efficiency of the intestine to absorb dietary calcium. When there is inadequate calcium in the diet to satisfy the body's calcium requirement, vitamin D communicates to the osteoblasts that signal osteoclast precursors to mature and dissolve the calcium stored in the bone. The typical "vitamin D-deficiency" disorder was observed for growing children in the west and south of England in the early 1600s. This disease was described by Glisson and named "rickets" (known also as "the English disease") and was observed with epidemic proportions in northern Europe and North America. The corrections of deformities of rickets were at the origin of the name "orthopedia" and of the technique of osteotomies.

Kinetics of inhibitory effect of hydrogen peroxide on activity of plasma membrane transporting Cа2+, Mg2+-ATPase of sperm cells

Decreased potential of spermatozoa fertility is closely associated with the development of oxidative stress and dysfunction of ion-transporting ATPases. Oxidative stress may have negative impact on the activity of membrane-bound enzymes, such as Са 2+ ,Мg 2+ -АТPase that is involved in maintaining calcium homeostasis in sperm cells. The aim of present work was to evaluate the exogenous H 2 O 2 effect on the main kinetic parameters of ATP hydrolysis by plasma membrane Са 2+ ,Мg 2+ -АТPase of spermatozoa of fertile (normozoospermia) and infertility (asthenozoospermia) men. Since Са 2+ ,Mg 2+ -АТPase is one of the targets for the reactive oxygen species and is directly involved in oxidative stress, spermatozoa obtained from normo- and asthenozoospermic samples were subjected to oxidative stress in the form of exogenous H 2 O 2 . Then ATP hydrolysis by thapsigargin-resistant Ca 2+ ,Mg 2+ -ATPase in media with different Ca 2+ concentrations was measured. An effective inhibitory effect of H 2 O 2 on the activity of the thapsigargin-resistant component of Са 2+ ,Мg 2+ -АТPase of sperm cells was demonstrated. In order to elucidate possible mechanisms of change in Ca 2+ ,Mg 2+ -ATPase activity under H 2 O 2 -induced oxidative stress, the concentration curves were linearized using Hanes–Woolf plot {[S]/V; [S]}. The apparent activation constant for Ca 2+ (K Ca2+ ) in sperm cell obtained from men with proven fertility was not changed, whereas in the asthenozoospermic samples, it was decreased almost twice under H 2 O 2 -induced oxidative stress. These results indicate that in normozoospermic samples H 2 O 2 implements its inhibitory action through the mechanism of uncompetitive inhibition of plasma membrane Ca 2+ ,Mg 2+ -ATPase activity. According to formal features of kinetics in the asthenozoospermic samples a mixed type of enzyme inhibition occurs under the oxidative stress induced by H 2 O 2 . Strategies to protect against a loss in Cа 2+ ,Mg 2+ -ATPase activity may be useful to prevent the harmful biochemical cascades leading to Ca 2+ overload and dysfunction of spermatozoa as a result of the oxidative stress.

Disorders of Mechanisms of Calcium Metabolism Control as Potential Risk Factors of Prostate Cancer.

Prostate cancer significantly affects the overall morbidity and mortality of malignant tumours in highly developed countries. Important risk factors include family predisposition and regional, racial and dietary determinants. The scientific literature contains a great deal of data on the role of calcium and dairy products in general in the process of neoplastic transformation of the prostate. This is most likely linked to the fact that changes in the concentration of calcium ions control such varied life processes as secretion of hormones and neurotransmitters, the level of cyclic nucleotides, and cell growth, division and differentiation. Research is conducted to demonstrate that disorders of cell cycle control due to differences in calcium ion concentrations may be crucial for the development and prevention of cancer. Disturbances of calcium homeostasis in the body can be caused by various mechanisms, such as excessive calcium intake in the diet, vitamin D deficiency, structural and functional changes in vitamin D receptor (VDR), Calcium-Sensing Receptor (CaSR), and parathyroid hormone receptor (PTH-1-R), changes in calcium ion channels, phosphate metabolism disorders (phosphatonin and the Klotho protein), changes in the level of parathyroid hormone-related protein (PTHrP), and others. The article presents data on the mechanisms maintaining calcium homeostasis at the molecular level and genetic aspects playing a role in the pathogenesis of prostate cancer. The data cited on the occurrence of abnormal mechanisms of calcium metabolism in prostate cancer suggest the need for individualized intake of this element in the diet, especially in the case of patients with a family history of PCa.

Ca2+ mediates axotomy-induced necrosis and apoptosis of satellite glial cells remote from the transection site in the isolated crayfish mechanoreceptor

Severe nerve injury such as axotomy induces neuron degeneration and death of surrounding glial cells. Using a crayfish stretch receptor that consists of a single mechanoreceptor neuron enveloped by satellite glia, we showed that axotomy not only mechanically injures glial cells at the transection location, but also induces necrosis or apoptosis of satellite glial cells remote from the transection site. We studied Ca2+role in spontaneous or axotomy-induced death of remote glial cells. Stretch receptors were isolated using the original technique that kept the neuron connected to the ventral cord ganglion (control preparations). Using Ca2+-sensitive fluorescence probe fluo-4, we showed Ca2+ accumulation in neuronal perikarion and glial envelope. Ca2+ gradually accumulated in glial cells after axotomy. In saline with triple Ca2+ concentration the axotomy-induced apoptosis of glial cells increased, but spontaneous or axotomy-induced necrosis was unexpectedly reduced. Saline with 1/3[Ca2+], oppositely, enhanced glial necrosis. Application of ionomycin, CdCl2, thapsigargin, and ryanodine showed the involvement of Ca2+ influx through ionic channels in the plasma membrane, inhibition of endoplasmic reticulum Ca2+-ATPase, and Ca2+ release from endoplasmic reticulum through ryanodine receptors in axotomy-induced glial necrosis. Apoptosis of glial cells surrounding axotomized neurons was promoted by ionomycin and thapsigargin. Possibly, other Ca2+ sources such as penetration through the plasma membrane contributed to axotomy-induced apoptosis and necrosis of remote glial cells. Thus, modulating different pathways that maintain calcium homeostasis, one can modulate axotomy-induced death of glial cells remote from the transection site.

Reduced serum vitamin D concentrations in healthy early-lactation dairy cattle

Cattle obtain vitamin D by ingestion or cutaneous exposure to UV light. Dairy cattle diets are frequently supplemented with vitamin D to compensate for limited sun exposure or during times of increased metabolic demands, such as the periparturient period, to maintain calcium homeostasis. Whether housing and supplemental vitamin D practices supply adequate amounts of vitamin D to optimally support the transition from gestation to lactation in dairy cattle is unknown. Our objective was to determine how serum vitamin D concentrations of dairy cows change with season, age, parity, and stage of lactation. Clinically healthy cows (n = 183) from 5 commercial dairies were enrolled in the study. Serum samples were collected at dry off, within 7 d of entering the close-up group, and within 7 d after calving (calving+7). Vitamin D status was determined by measuring serum 25-hydroxyvitamin D [25(OH)D] by radioimmunoassay. We performed repeated-measures mixed-effects linear regression to determine the effects of season, age, parity, and lactation stage (dry off, close-up, and calving+7) on 25(OH)D concentrations in serum. Bivariable analysis indicated that parity, age, and season were not associated with serum 25(OH)D concentrations. Sample period affected 25(OH)D concentrations, with the highest 25(OH)D levels at dry off (99.7 ± 1.9 ng/mL) followed by close up (93.8 ± 2.1 ng/mL), with the lowest levels at calving+7 (82.6 ± 1.7 ng/mL). These data showed a large depletion of 25(OH)D in dairy cattle postpartum compared with late prepartum, although the biological significance of this change in these healthy cattle is unclear. Consumption of serum 25(OH)D by immune system functions and calcium homeostasis in early lactation likely caused the reduction in serum 25(OH)D concentrations after calving. These results suggest that determining whether serum 25(OH)D concentrations are associated with the incidence of transition period disease is an appropriate next step. Assessing the effects of enhanced vitamin D supplementation of cows in early lactation on postpartum diseases may be warranted.

The endoplasmic reticulum, calcium signaling and junction turnover in Sertoli cells

The endoplasmic reticulum (ER) forms a continuous network throughout morphologically differentiated Sertoli cells. It is an integral component of intercellular adhesion junctions in this cell type, as well as forming membrane contact sites with the plasma membrane and intracellular organelles. One of the major functions of the ER in cells generally is maintaining calcium homeostasis and generating calcium signals. In this review, we discuss what is currently known about the overall pattern of distribution of the ER in Sertoli cells and the location of calcium regulatory machinery in the various subdomains of the organelle. Current data are consistent with the hypothesis that calcium signaling by the ER of Sertoli cells may play a significant role in events related to junction remodeling that occur in the seminiferous epithelium during spermatogenesis.

Calcium sensing receptor protects high glucose-induced energy metabolism disorder via blocking gp78-ubiquitin proteasome pathway

Diabetic cardiomyopathy (DCM) is a major complication and fatal cause of the patients with diabetes. The calcium sensing receptor (CaSR) is a G protein-coupled receptor, which is involved in maintaining calcium homeostasis, regulating cell proliferation and apoptosis, and so on. In our previous study, we found that CaSR expression, intracellular calcium levels and cardiac function were all significantly decreased in DCM rats; however, the exact mechanism are not clear yet. The present study revealed the protective role of CaSR in myocardial energy metabolism disorder induced by high glucose (HG) as well as the underlying mechanism. Here, we demonstrated that HG decreased the expression of CaSR, mitochondrial fusion proteins (Mfn1, Mfn2), cell gap junction related proteins (Cx43, β-catenin, N-cadherin), and intracellular ATP concentration. In contrast, HG increased extracellular ATP concentration, the expression of gp78, mitochondrial fission proteins (Fis1, Drp1), and the ubiquitination levels of Mfn1, Mfn2 and Cx43. Moreover, CaSR agonist and gp78-siRNA significantly reduced the above changes. Taken together, these results suggest that HG induces myocardial energy metabolism disorder via decrease of CaSR expression, and activation of gp78-ubiquitin proteasome system. In turn, these effects disrupt the structure and function of the mitochondria and the cell gap junction, result in the reduced ATP synthesis and the increased ATP leakage. Stimulation of CaSR significantly attenuates HG-induced abnormal myocardial energy metabolism, suggesting CaSR would be a promising potential therapeutic target for DCM.

Acetylcholine attenuated TNF-α-induced intracellular Ca2 + overload by inhibiting the formation of the NCX1-TRPC3-IP3R1 complex in human umbilical vein endothelial cells

The endoplasmic reticulum (ER) forms discrete junctions with the plasma membrane (PM) that play a critical role in the regulation of Ca2+ signaling during cellular bioenergetics, apoptosis and autophagy. We have previously confirmed that acetylcholine can inhibit ER stress and apoptosis after inflammatory injury. However, limited research has focused on the effects of acetylcholine on ER-PM junctions. In this work, we evaluated the structure and function of the supramolecular sodium-calcium exchanger 1 (NCX1)-transient receptor potential canonical 3 (TRPC3)-inositol 1,4,5-trisphosphate receptor 1 (IP3R1) complex, which is involved in regulating Ca2+ homeostasis during inflammatory injury. The width of the ER-PM junctions of human umbilical vein endothelial cells (HUVECs) was measured in nanometres using transmission electron microscopy and a fluorescent probe for Ca2+. Protein-protein interactions were assessed by immunoprecipitation. Ca2+ concentration was measured using a confocal microscope. An siRNA assay was employed to silence specific proteins. Our results demonstrated that the peripheral ER was translocated to PM junction sites when induced by tumour necrosis factor-alpha (TNF-α) and that NCX1-TRPC3-IP3R1 complexes formed at these sites. After down-regulating the protein expression of NCX1 or IP3R1, we found that the NCX1-mediated inflow of Ca2+ and the release of intracellular Ca2+ stores were reduced in TNF-α-treated cells. We also observed that acetylcholine attenuated the formation of NCX1-TRPC3-IP3R1 complexes and maintained calcium homeostasis in cells treated with TNF-α. Interestingly, the positive effects of acetylcholine were abolished by the selective M3AChR antagonist darifenacin and by AMPK siRNAs. These results indicate that acetylcholine protects endothelial cells from TNF-alpha-induced injury, [Ca2+]cyt overload and ER-PM interactions, which depend on the muscarinic 3 receptor/AMPK pathway, and that acetylcholine may be a new inhibitor for suppressing [Ca2+]cyt overload.

The Parathyroid Gland and Heart Disease.

The parathyroid glands are critical to maintaining calcium homeostasis through actions of parathyroid hormone (PTH). Recent clinical and molecular research has shown that direct and indirect actions of PTH also affect the heart and vasculature through downstream actions of G protein-coupled receptors in the myocardium and endothelial cells. Patients with disorders of the parathyroid gland have higher incidences of hypertension, arrhythmias, left ventricular hypertrophy, heart failure, and calcific disease which translate into increased cardiac morbidity and mortality. Importantly, clinical research also suggests that early treatment of parathyroid disorders through medical or surgical management may reverse cardiovascular remodeling and mitigate cardiac risk factors.

Circannual rhythm of plasmatic vitamin D levels and the association with markers of psychophysical stress in a cohort of Italian professional soccer players

ABSTRACTAdequate plasmatic Vitamin D levels are crucial to maintain calcium homeostasis and bone metabolism both in the general population and in athletes. Correct dietary supply and a regular sun exposure are fundamental for allowing the desired and effective fitness level. Past studies highlighted a scenario of Vitamin D insufficiency among professional soccer players in several countries, especially in North Europe, whilst a real deficiency in athletes is rare. The typical seasonal fluctuations of Vitamin D are wrongly described transversally in athletes belonging to teams that play at different latitudes and a chronobiologic approach studying the Vitamin D circannual rhythm in soccer players has not been described yet. Therefore, we studied plasma vitamin D, cortisol, testosterone, and creatin kinase (CK) concentrations in three different Italian professional teams training at the same latitude during a period of two consecutive competitive seasons (2013 and 2014). In this retrospective observational study, 167 professional soccer players were recruited (mean age at sampling 25.1 ± 4.7 years) and a total of 667 blood drawings were carried out to determine plasma 25(OH)D, serum cortisol, serum testosterone and CK levels. Testosterone to cortisol ratio (TC) was calculated based as a surrogate marker of overtraining and psychophysical stress and each athlete was drawn until a maximum of 5 times per season. Data extracted by a subgroup of players that underwent at least 4 sample drawings along a year (N = 45) were processed with the single and population mean cosinor tests to evaluate the presence of circannual rhythms: the amplitude (A), acrophase (Φ) and the MESOR (M) are described. In total, 55 players (32.9%) had an insufficient level of 25(OH)D during the seasons and other 15 athletes (9.0%) showed, at least once, a deficiency status of Vitamin D. The rhythmometric analyses applied to the data of Vitamin D revealed the presence of a significant circannual rhythm (p < 0.001) with the acrophase that occurred in August; the rhythms of Vitamin D levels were not different neither among the three soccer teams nor between competitive seasons. Cortisol, testosterone and TC showed significant circannual rhythms (p < 0.001): cortisol registered an acrophase during winter (February) while testosterone and TC registered their peaks in the summer months (July). On the contrary, CK did not display any seasonal fluctuations. In addition, we observed weak but significant correlations between 25(OH)D versus testosterone (r = 0.29 and p < 0.001), cortisol (r = −0.27 and p < 0.001) and TC (r = 0.37 and p < 0.001). No correlation was detected between Vitamin D and CK. In conclusion, the correct chronobiologic approach in the study of annual variations of Vitamin D, cortisol and testosterone could be decisive in the development of more specific supplementation and injury prevention strategies by athletic trainers and physicians.