Multiple Calcium Research Articles

Klotho-Dependent Cellular Transport Regulation.

Klotho is a transmembrane protein that in humans is encoded by the hKL gene. This protein is known to have aging suppressor effects and is predominantly expressed in the distal convoluted tubule of the kidney, parathyroid glands, and choroid plexus of the brain. The Klotho protein exists in both full-length membrane form and a soluble secreted form, which exerts numerous distinct functions. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it functions as β-glucuronidase and a hormone. Soluble Klotho is a multifunction protein present in the biological fluids including blood, urine, and cerebrospinal fluid of mammals. Klotho deficiency leads to multiple organ failure accompanied by early appearance of multiple age-related disorders and early death, whereas overexpression of Klotho results in the opposite effects. Klotho, an enzyme and hormone, has been reported to participate in the regulation of cellular transport processes across the plasma membrane either indirectly through inhibiting calcitriol (1,25(OH)2D3) formation or other mechanism, or by directly affecting transporter proteins, including ion channels, cellular carriers, and Na(+)/K(+)-ATPase. Accordingly, Klotho protein serves as a powerful regulator of cellular transport across the plasma membrane. Importantly, Klotho-dependent cellular transport regulation implies stimulatory or inhibitory effects. Klotho has been shown to play a key role in the regulation of multiple calcium and potassium ion channels, and various cellular carriers including the Na(+)-coupled cotransporters such as NaPi-IIa, NaPi-IIb, EAAT3, and EAAT4, CreaT1 as well as Na(+)/K(+)-ATPase. These regulations are parts of the antiaging function of Klotho, which will be discussing throughout this chapter. Clearly, further experimental efforts are required to investigate the effect of Klotho on other transport proteins and underlying molecular mechanisms by which Klotho exerts its effect.

What have we learned from the Psychiatric Genomics Consortium.

Decades of research in the pre-molecular genetics era firmly established that major psychiatric disorders are highly to moderately heritable, but only with the emergence of molecular genetic technology about 35 years ago was it possible to envisage identifying the specific pathogenic genes responsible. In psychiatry, the opportunity to probe pathophysiology using DNA seemed particularly attractive given that other biomedical approaches had been frustrated by the complexity of the brain, the challenges in obtaining access to fresh tissue, and the extensive potential for reverse causal associations due to the many environmental and behavioural consequences of the disorders. Simple in concept, translating heritability to pathophysiology has proven arduous. This paper provides a perspective on that process, how some of the obstacles have been overcome, and some of the implications of the current findings. The focus is the work of the Psychiatric Genomics Consortium (PGC), whose main impacts relate to common rather than rare genetic variation. This reflects the data available rather than any ideological position that rare genetic variation is unimportant in psychiatry.

High Throughput Measurement of Ca++ Dynamics in Human Stem Cell-Derived Cardiomyocytes by Kinetic Image Cytometery: A Cardiac Risk Assessment Characterization Using a Large Panel of Cardioactive and Inactive Compounds.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as a powerful invitro model for cardiac safety assessment which may allow for better identification of compounds with poor arrhythmogenic liability profiles early in the drug discovery process. Here, we describe our examination of the Kinetic Image Cytometer (KIC) system's ability to predict adverse compound effects using hiPS-CMs and a library of 53 compounds, the majority of which are known to be cardioactive compounds, and several negative controls. The KIC provides a high throughput method for analyzing intracellular calcium transients. In the cardiomyocyte, intracellular calcium transients integrate the electrochemical signals of the action potential (AP) with the molecular signaling pathways regulating contraction. Drug-induced alterations in the shape and duration of AP result in changes to the shape and duration of the intracellular calcium transient. By examining calcium transient dynamics in hiPS-CMs, KIC can be used as a phenotypic screen to assess compound effects across multiple ion channel types (MITs), detecting MITs, calcium handling and signaling effects. The results of this blinded study indicate that using hiPS-CMs, KIC is able to accurately detect drug-induced changes in Ca(2+) transient dynamics (ie, duration and beat rate) and therefore, may be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.

Effects of Panax ginseng, zearalenol, and estradiol on sperm function

BackgroundEstrogen signaling pathways are modulated by exogenous factors. Panax ginseng exerts multiple activities in biological systems and is classified as an adaptogen. Zearalenol is a potent mycoestrogen that may be present in herbs and crops arising from contamination or endophytic association. The goal of this study was to investigate the impact of P. ginseng, zearalenol and estradiol in tests on spermatozoal function. MethodsThe affinity of these compounds for estrogen receptor (ER)—alpha and beta (ERα and ERβ)—was assessed in receptor binding assays. Functional tests on boar spermatozoa motility, movement and kinematic parameters were conducted using a computer-assisted sperm analyzer. Tests for capacitation, acrosome reaction (AR), and chromatin decondensation in spermatozoa were performed using microscopic analysis. ResultsZearalenol—but not estradiol (E2)- or ginseng-treated spermatozoa—decreased the percentage of overall, progressive, and rapid motile cells. Zearalenol also decreased spontaneous AR and increased chromatin decondensation. Ginseng decreased chromatin decondensation in response to calcium ionophore and decreased AR in response to progesterone (P4) and ionophore. ConclusionZearalenol has adverse effects on sperm motility and function by targeting multiple signaling cascades, including P4, E2, and calcium pathways. Ginseng protects against chromatin damage and thus may be beneficial to reproductive fitness.

Analgesic effect of a broad-spectrum dihydropyridine inhibitor of voltage-gated calcium channels

Voltage-activated calcium channels are important facilitators of nociceptive transmission in the primary afferent pathway. Consequently, molecules that block these channels are of potential use as pain therapeutics. Our group has recently reported on the identification of a novel class of dihydropyridines (DHPs) that included compounds with preferential selectivity for T-type over L-type channels. Among those compounds, M4 was found to be an equipotent inhibitor of both Cav1.2 L- and Cav3.2 T-type calcium channels. Here, we have further characterized the effects of this compound on other types of calcium channels and examined its analgesic effect when delivered either spinally (i.t.) or systemically (i.p.) to mice. Both delivery routes resulted in antinociception in a model of acute pain. Furthermore, M4 was able to reverse mechanical hyperalgesia produced by nerve injury when delivered intrathecally. M4 retained partial activity when delivered to Cav3.2 null mice, indicating that this compound acts on multiple targets. Additional whole-cell patch clamp experiments in transfected tsA-201 cells revealed that M4 also effectively blocks Cav3.3 (T-type) and Cav2.2 (N-type) currents. Altogether, our data indicate that broad-spectrum inhibition of multiple calcium channel subtypes can lead to potent analgesia in rodents.

Differential CaMKII regulation by voltage-gated calcium channels in the striatum.

Calcium signaling regulates synaptic plasticity and many other functions in striatal medium spiny neurons to modulate basal ganglia function. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a major calcium-dependent signaling protein that couples calcium entry to diverse cellular changes. CaMKII activation results in autophosphorylation at Thr286 and sustained calcium-independent CaMKII activity after calcium signals dissipate. However, little is known about the mechanisms regulating striatal CaMKII. To address this, mouse brain slices were treated with pharmacological modulators of calcium channels and punches of dorsal striatum were immunoblotted for CaMKII Thr286 autophosphorylation as an index of CaMKII activation. KCl depolarization increased levels of CaMKII autophosphorylation ~2-fold; this increase was blocked by an LTCC antagonist and was mimicked by treatment with pharmacological LTCC activators. The chelation of extracellular calcium robustly decreased basal CaMKII autophosphorylation within 5min and increased levels of total CaMKII in cytosolic fractions, in addition to decreasing the phosphorylation of CaMKII sites in the GluN2B subunit of NMDA receptors and the GluA1 subunit of AMPA receptors. We also found that the maintenance of basal levels of CaMKII autophosphorylation requires low-voltage gated T-type calcium channels, but not LTCCs or R-type calcium channels. Our findings indicate that CaMKII activity is dynamically regulated by multiple calcium channels in the striatum thus coupling calcium entry to key downstream substrates.

The synthesis of calcium carbonate microparticles with multiple morphologies through self-assembly method

Taking advantage of the biocompatibility and biodegradability of calcium carbonate, a simple method was introduced to synthesize the calcium carbonate microparticles with different morphologies, including hollow (H-calcium), flower-like (P-calcium) and peanut-like (P-calcium). The possible formation mechanisms of these kinds of calcium carbonate microparticles were discussed in this study. Initially, the aminopropyltriethoxysilane (APTES) was possibly hydrolyzed, and octa(amino-propylsilsesquioxane) (R-NH3+) was obtained. The R-NH3+ might guide the CaCO3 nanoparticles to form multiple calcium carbonate microparticles in aqueous phase because of their positive potential. Furthermore, the final treatments were very essential to obtain the calcium carbonate microparticles with satisfactory shape with this method. The XRD and FTIR results revealed the components and crystalline phase of these products. The morphologies were characterized by SEM and TEM. Furthermore, the potential value of these kinds of microparticles was discussed.

Adrenal Cortisol Hyper-Responsiveness as a Possible Cause of Obesity, Insulin Resistance and Cardiovascular Risk

Introduction: Increased cortisol concentrations due to endogenous over-secretion or exogenous administration result in obesity, insulin resistance, and increased risk of cardiovascular disease (CVD). We wanted to assess whether non pathological adrenal hyper responsiveness to ACTH could reproduce these changes and thus explain how stress, via the pituitary-adrenal axis, can also cause such abnormalities. Methods: ACTH stimulation test was performed on 1072 non diabetic healthy participants in the St. Francis Heart Study and multiple blood tests, anthropometric measurements and coronary calcium scores were also obtained. Results: Post stimulation cortisol concentrations were correlated with obesity, insulin resistance, lipid abnormalities, Framingham scores (all p ≤ 0.01), but not with blood pressure and not with coronary calcium scores. In multivariate models, increased cortisol secretion was significantly predictive of abdominal fat even after controlling for insulin resistance, and of insulin resistance even after controlling for abdominal fat. Conclusions: Adrenal hyper responsiveness may explain some of the observation of the relationship between stress and the metabolic abnormalities which can lead to CVD. Increased cortisol secretion predicts insulin resistance and obesity , independently of each other.

Low-Flow Aortic Stenosis and Reduced Ejection Fraction: New Insights.

Low-Flow Aortic Stenosis and Reduced Ejection Fraction: New Insights.

Calreticulin Gene Mutations in Suspicion of Myeloproliferative Neoplasms: A Single Institution Experience

Recently, Klampfl et al. and Nangalia et al. reported recurrent somatic mutations in the calreticulin (CALR) gene in patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. All CALR mutations described to date are either deletions or insertions, and occur in exon 9 and result in a frameshift leading to a loss of KDEL sequence (endoplasmic reticulum retention motif) and multiple calcium biding sites with a new basic (instead of acidic) C terminal region.The aim of this work is to know how far the CALR mutations can fill the molecular diagnostic gap for myeloproliferative neoplasms (MPNs) left by JAK2 and MPL mutations.The study was approved by the local ethics committee.Our study population consists of 155 patients with platelet counts > 500 G/L sampled between 2012 and 2013 for suspicion of MPNs. Only JAK2 V617F negative patients were included.Analyses were performed using genomic DNA isolated from peripheral blood. Patients were screened for CALR mutations using a High Resolution Melting (HRM) and then the precise mutations were characterized by Sanger sequencing which is a new approach for this test.A total of 21 (13.5%) patients with CALR mutations were detected with 8 distinct variants.Among CALR mutations, type 1 (52 basepair deletion, c.1092_1143del) and type 2 (5 basepair insertion, c.1154_1155insTTGTC) were the most common (respectively 7 (33%) and 7 (33%) cases). Three patients had 2 concurrent mutations (insertion+substitution / Deletion+substitution / deletion +insertion). We found a deletion that has not been reported so far to our knowledge (c.1125_1147del). Sequencing is ongoing for 4 patients.Compared with negative patients, CALR-mutated patients demonstrated markedly higher platelet counts (median count: 820 vs 543 G/L). DisclosuresNo relevant conflicts of interest to declare.

Aggregation of Calcium Phosphate and Oxalate Phases in the Formation of Renal Stones.

The majority of human kidney stones are comprised of multiple calcium oxalate monohydrate (COM) crystals encasing a calcium phosphate nucleus. The physiochemical mechanism of nephrolithiasis has not been well determined on the molecular level; this is crucial to the control and prevention of renal stone formation. This work investigates the role of phosphate ions on the formation of calcium oxalate stones; recent work has identified amorphous calcium phosphate (ACP) as a rapidly forming initial precursor to the formation of calcium phosphate minerals in vivo. The effect of phosphate on the nucleation of COM has been investigated using the constant composition (CC) method in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our findings indicate COM nucleation is strongly promoted by the presence of phosphate; this occurs at relatively low phosphate concentrations, undersaturated with respect to brushite (dicalcium phosphate dehydrate, DCPD) formation. The results show that ACP plays a crucial role in the nucleation of calcium oxalate stones by promoting the aggregation of amorphous calcium oxalate (ACO) precursors at early induction times. The coaggregations of ACP and ACO precursors induce the multiple-point nucleation of COM. These novel findings expand our knowledge of urinary stone development, providing potential targets for treating the condition at the molecular level.

Translating neuronal activity at the synapse: presynaptic calcium sensors in short-term plasticity.

The complex manner in which patterns of presynaptic neural activity are translated into short-term plasticity (STP) suggests the existence of multiple presynaptic calcium (Ca2+) sensors, which regulate the amplitude and time-course of STP and are the focus of this review. We describe two canonical Ca2+-binding protein domains (C2 domains and EF-hands) and define criteria that need to be met for a protein to qualify as a Ca2+ sensor mediating STP. With these criteria in mind, we discuss various forms of STP and identify established and putative Ca2+ sensors. We find that despite the multitude of proposed sensors, only three are well established in STP: Munc13, protein kinase C (PKC) and synaptotagmin-7. For putative sensors, we pinpoint open questions and potential pitfalls. Finally, we discuss how the molecular properties and modes of action of Ca2+ sensors can explain their differential involvement in STP and shape net synaptic output.

Hypercalcemic crisis treated with calcium-free hemodialysis with subsequent parathyroidectomy and postsurgical hypocalcemia

Hypercalcemic crisis is a rare but life-threatening condition defined by decompensation of hypercalcemia with significant disturbance to cardiac, renal, gastrointestinal and neurological function. Here we document the case of a 74-year-old gentleman with a parathyroid adenoma who presented to hospital after bloods revealed severe hypercalcemia (5.58 mmol/L). Rehydration with diuretics and bisphosphonate infusions failed to revert the hypercalcemia over 42 hours (5.58 mmol/L). Low calcium hemodialysis rapidly reduced calcium levels and allowed for further investigations. Computerised tomography (CT) scan and magnetic resonance cholangiopancreatography revealed associated acute pancreatitis. A parathyroid adenoma was discovered by ultrasound and removed surgically. Parathyroidectomy led to resistant hypocalcemia requiring multiple calcium infusions. Hypercalcemia may lead to nausea and vomiting, decrease oral fluid intake and nephrogenic diabetes insipidus. This in turn leads to volume depletion and hypovolemic acute kidney injury. In patients severe acute kidney injury leading to renal failure, aggressive fluid replacement may be detrimental and these patients may require correction by dialysis. Literature review returned 13 case reports/case series totalling 68 patients undergoing dialysis for hypercalcemia. These case reports found a rapid decrease in serum calcium levels post calcium free hemodialysis (CFHD). Mortality was observed in only one case report which was attributed to sepsis.1 Hemodialysis reportedly clears 682 mg/hr of calcium vs 82 mg/hr in saline diuresis.2 It offers a safe way of treatment in patients with cardiac and renal co-morbidities and results in rapid reduction in calcium levels and improvement in cognition.1,3,4

Characterization of the lipid binding properties of Otoferlin reveals specific interactions between PI(4,5)P2 and the C2C and C2F domains.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30–50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25–95 μM) and low affinities (Kd = 400–700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B–C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1′-myoinositol-4′,5′-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

Osteopontin binds multiple calcium ions with high affinity and independently of phosphorylation status

Osteopontin (OPN) is an acidic, intrinsically disordered extracellular matrix protein with a capacity to modulate biomineralization in vitro and in vivo. The role of posttranslational modification of osteopontin has been intensively studied. Phosphorylation of OPN has been demonstrated to play a role in inhibition of biomineral formation and growth in vitro. Here, we used isothermal titration calorimetry (ITC) to investigate the ability of OPN to bind the divalent cations Ca2+ and Mg2+, both essential components of inorganic minerals in vivo. We found, that bovine OPN binds ~10 Ca2+ ions with an apparent affinity ~50-fold tighter than Mg2+, both regardless of OPN phosphorylation, and with affinities significantly stronger than previously reported. These results were confirmed using human derived OPN. This implies that a majority of the acidic residues within OPN must be engaged in calcium interaction under physiological conditions.

A constitutive model for cytoskeletal contractility of smooth muscle cells

The constitutive model presented in this article aims to describe the main bio-chemo-mechanical features involved in the contractile response of smooth muscle cells, in which the biochemical response is modelled by extending the four-state Hai–Murphy model to isotonic contraction of the cells and the mechanical response is mainly modelled based on the phosphorylation-dependent hyperbolic relation between isotonic shortening strain rate and tension. The one-dimensional version of the model is used to simulate shortening-induced deactivation with good agreement with selected experimental measurements. The results suggest that the Hai–Murphy biochemical model neglects the strain rate effect on the kinetics of cross-bridge interactions with actin filaments in the isotonic contractions. The two-dimensional version and three-dimensional versions of the model are developed using the homogenization method under finite strain continuum mechanics framework. The two-dimensional constitutive model is used to simulate swine carotid media strips under electrical field stimulation, experimentally investigated by Singer and Murphy, and contraction of a hollow airway and a hollow arteriole buried in a soft matrix subjected to multiple calcium ion stimulations. It is found that the transverse deformation may have significant influence on the response of the swine carotid medium. In both cases, the orientation of the maximal value of attached myosin is aligned with the orientation of maximum principal stress.

A Case of Acute Renal Failure Following Ethylene Glycol Intoxication in a Dog

The manuscript describes a clinical case of severe oligoanuric acute renal failure in a young dog following ethylene glycol intoxication. Significant deviations were established in urinary renal markers and blood biochemical parameters: severe azotemia, hypocalcaemia, hyperphosphataemia, hyperkalaemia and metabolic acidosis. The ultrasound renal findings demonstrated increased renal cortex echogenicity the typical for ethylene glycol intoxication, with a characteristic halo sign around the medulla. Electrocardiography showed a progressive decrease in amplitudes of P and R peaks, increased sharp edged repolarisation T wave. The performed symptomatic therapy did not lead to favourable outcome due to delayed intervention by the owner and non-administered antidotal therapy. The histopathological finding was consisted in degeneration, necrosis, desquamation of kidney epithelial cells, dilated tubules and multiple calcium oxalate deposits. The described changes, in our opinion, are relevant and could be used for diagnostics of the studied pathology.

Nutritional deficiencies in the pediatric age group in a multicultural developed country, Israel.

Nutrient deficiencies are prevalent worldwide. Diseases and morbid conditions have been described to result from nutritional deficiencies. It is essential to address nutrient deficiencies as these may lead to chronic long-term health problems such as rickets, iron deficiency anemia, goiter, obesity, coronary heart disease, type 2 diabetes, stroke, cancer and osteoporosis. In the present review we surveyed the extent and severity of nutritional deficiencies in Israel through a selective and comprehensive Medline review of previous reports and studies performed during the last 40 years. Israeli populations have multiple nutritional deficiencies, including iron, calcium, zinc, folic acid, and vitamins B12, C, D and E, spanning all age groups, several minorities, and specific regions. In Israel, some of the nutrients are mandatorily implemented and many of them are implemented voluntarily by local industries. We suggest ways to prevent and treat the nutritional deficiencies, as a step to promote food fortification in Israel.

Validation of 1-hour post-thyroidectomy parathyroid hormone level in predicting hypocalcemia

BackgroundPrior work by our group suggested that a single one hour post-thyroidectomy parathyroid hormone (1 hr PTH) level could accurately stratify patients into high and low risk groups for the development of hypocalcemia. This study looks to validate the safety and efficacy of a protocol based on a 1 hr PTH threshold of 12 pg/ml.Study designRetrospective analysis of consecutive cohort treated with standardized protocol.MethodsOne hundred and twenty five consecutive patients underwent total or completion thyroidectomy and their PTH level was drawn 1-hour post operatively. Based on our previous work, patients were stratified into either a low risk group (PTH < 12 pg/ml) or a high risk group (PTH ≥ 12 pg/ml). Patients in the high risk group were immediately started on prophylactic calcium carbonate (5–10 g/d) and calcitriol (0.5-1.0 mcg/d). The outcomes were then reviewed focusing mainly on how many low risk patients developed hypocalcemia (false negative rate), and how many high risk patients failed prophylactic therapy.ResultsThirty one patients (25%) were stratified as high risk, and 94 (75%) as low risk. Five (16%) of the high risk patients became hypocalcemic despite prophylactic therapy. Two of the low risk group became hypocalcemic, (negative predictive value = 98%). None of the hypocalcemic patients had anything more than mild symptoms.ConclusionsA single 1-hour post-thyroidectomy PTH level is a very useful way to stratify thyroidectomy patients into high and low risk groups for development of hypocalcemia. Early implementation of oral prophylactic calcium and vitamin D in the high risk patients is a very effective way to prevent serious hypocalcemia. Complex protocols requiring multiple calcium and PTH measurements are not required to guide post-thyroidectomy management.

Erratum to: Validation of 1-hour post-thyroidectomy parathyroid hormone level in predicting hypocalcemia

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