Dibutyl phthalate rapidly alters calcium homeostasis in the gills of Danio rerio.

This study investigates the impacts of exposure to an environment Ca2+ challenge and the mechanism of action of dibutyl phthalate (DBP) on Ca2+ influx in the gills of Danio rerio. In vitro profile of 45Ca2+ influx in gills was verified through the basal time-course. Fish were exposed to low, normal and high Ca2+ concentrations (0.02, 0.7 and 2 mM) for 12 h. So, gills were morphologically analysed and ex vivo45Ca2+ influx at 30 and 60 min was determined. For the in vitro studies, gills were treated for 60 min with DBP (1 pM, 1 nM and 1 µM) with/without blockers/activators of ionic channels, Ca2+ chelator, inhibitors of ATPases, ionic exchangers and protein kinase C to study the mechanism of DBP-induced 45Ca2+ influx. Exposure to high environmental Ca2+ augmented 45Ca2+ influx when compared to fish exposed to normal and low Ca2+ concentrations. Additionally, histopathological changes were observed in the gills of fish maintained for 12 h in low and high Ca2+. In vitro exposure of gills to DBP (1 pM) disturbed Ca2+ homeostasis. DBP stimulated 45Ca2+ influx in gills through the transitory receptor potential vanilloid 1 (TRPV1), and reverse-mode Na+/Ca2+ exchanger (NCX) activation, protein kinase C and K+ channels and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). These data suggest that in vivo short-term exposure of gills to low and high Ca2+ leads to 45Ca2+ influx and histopathological changes. Additionally, the DBP-induced rapid 45Ca2+ influx is mediated by TRPV1, NCX activation with the involvement of PKC, K+-channels and SERCA, thereby altering Ca2+ homeostasis.

Absorption and distribution of root, fruit, and foliar-applied 45 Ca in 'Clemenules' mandarin trees.

BACKGROUND: The mechanisms of calcium (Ca) absorption and transport in plants are still poorly understood. This study focused on assessing the absorption and distribution of Ca in different plant organs after root (soil), foliar, or fruit application to 6-year-old 'Clemenules' mandarin trees, grown in pots, using 45 Ca as a tracer. RESULTS: The rate of 45 Ca absorption and transportation in plant tissues varied according to the treatment method. The fruit and shoot Ca supply led to a rate of 97% to 98% 45 Ca retention in such organs. In Ca-treated fruits, 22% of the applied 45 Ca moved to the pulp and 78% remained in the flavedo and albedo. The fruit peel was examined by scanning electron microscopy and transmission electron microscopy (SEM and TEM) and variations were observed during fruit development. Following 45 Ca soil treatment, approximately 56% of 45 Ca activity was measured in the soil, with 19.5% determined in the roots, 14.6% in the trunks (90% in bark and sapwood and only 10% in heartwood), 9.6% in shoots, and 0.3% in fruits. CONCLUSION: Calcium mobility in 'Clemenules' mandarin trees is limited and depends on the mode of Ca fertilizer application. The distribution of Ca to and within the fruits may be limited during development because of structural and functional constraints. © 2020 Society of Chemical Industry.

Ocean acidification: effects of pH on 45Ca uptake by lobster branchiostegites.

Gill chambers of the Atlantic lobster, Homarus americanus, possess three structures that are involved with respiration and ion regulation: gill filaments, epipodites, and branchiostegites. This paper describes ion transport mechanisms present in the plasma membranes of branchiostegite epithelial cells and the effects of pH on the uptake of 45Ca by these processes. Partially purified membrane vesicles (PPMV) of branchiostegite cells were produced by a homogenization/centrifugation method that has previously been used to define ion transport processes in both crab and lobster gill tissues. In the present study, lobster branchiostegite PPMV 45Ca uptake was highest at pH 8.5 and lowest at pH values between 6.0 and 7.0 (p < 0.02). At pH 8.0, 45Ca uptake was a biphasic process consisting of a saturable process at low [Ca] and a linear process at higher [Ca]. At pH 6.0, 45Ca uptake was only a linear process and paralleled linear uptake at pH 8.0. A valinomycin/K+-induced membrane potential (PD, inside negative) doubled 45Ca uptake at pH 7.0 above that in the absence of a PD (p < 0.05). An induced PD at pH 8.0 did not significantly (p > 0.05) affect 45Ca uptake observed in the absence of a PD, but was threefold greater than uptake at pH 7.0 in the absence of a PD (p < 0.05). Amiloride (2 mM) did not affect 45Ca uptake at pH 8.0, but 2 mM amiloride + 100 µM verapamil reduced uptake by approximately 50%. In the presence of both 2 mM amiloride + 100 µM verapamil, 15 s 45Ca influx at pH 8.5 was a hyperbolic function of [Ca] (0.1-5 mM) (Km = 4.2 ± 0.3 mM; Jmax = 9792 ± 439 pmol/mg protein × 15 s). 45Ca influxes at pH 7.5 under the same conditions were also hyperbolic with Km = 8.3 ± 1.4 mM; Jmax = 10732 ± 1250 pmol/mg protein × 15 s. Km values were significantly different (p < 0.05), but Jmax values were not (p > 0.05). These results suggest that 45Ca uptake by lobster branchiostegites may have occurred by the combination of diffusion through a verapamil-inhibited calcium channel and carrier-mediated transport by amiloride-insensitive, electroneutral, 1Ca2+/2H+ antiporters. Decreased pH, as might occur during ocean acidification, did not appear to modify calcium diffusion through the channels, but protons acted as competitive inhibitors of calcium transport by carrier-mediated antiport. Decreased calcium uptake with continued ocean acidification may significantly affect calcification processes during periodic molting, potentially influencing mortality.

Palliative care of bone pain due to skeletal metastases: Exploring newer avenues using neutron activated (45)Ca.

INTRODUCTION: With an objective to develop a cost-effective radiochemical formulation for palliation of pain due to skeletal metastases, we have demonstrated a viable method for large-scale production of (45)Ca (t½=163 days, Eßmax=0.3MeV) using moderate flux research reactor, its purification from radionuclidic impurities adopting electrochemical approach and preclinical evaluation of (45)CaCl2. METHODS: Irradiation parameters were optimized by theoretical calculations for production of (45)Ca with highest possible specific activity along with minimum radionuclidic impurity burden. Based on this, the radioisotope was produced in reactor by irradiation of isotopically enriched (98% in (44)Ca) CaO target at a thermal neutron flux of ~1 × 10(14) n.cm(-2).s(-1) for 4 months. Scandium-46 impurity co-produced along with (45)Ca was efficiently removed adopting an electrochemical separation approach. The bone specificity of (45)CaCl2 was established by in vitro studies involving its uptake in hydroxyapatite (HA) particles and also evaluating its biodistribution pattern over a period of 2 weeks after in vivo administration in Wistar rats. RESULTS: Thermal neutron irradiation of 100mg of enriched (98% in (44)Ca) CaO target followed by radiochemical processing and electrochemical purification procedure yielded ~37 GBq of (45)Ca with a specific activity of ~370 MBq/mg and radionuclidic purity>99.99%. The reliability and reproducibility of this approach were amply demonstrated by process demonstration in several batches. In vitro studies indicated significant uptake of (45)CaCl2 (up to 65%) in HA particles. In vivo biodistribution studies in Wistar rats showed specific skeletal accumulation (40-46%ID) with good retention over a period of 2 weeks. CONCLUSIONS: To the best of our knowledge, this is the first study on utilization of (45)CaCl2 in the context of nuclear medicine. The results obtained in this study hold promise and warrant further investigations for future translation of (45)CaCl2 to the clinics, thereby potentially enabling a cost-effective approach for metastatic bone pain palliation especially in developing countries.

Measurement of calcium uptake in yeast using 45Ca.

This protocol describes how to measure calcium uptake in yeast by (45)Ca radioactive isotope incorporation.

Measuring Ca2+ pump activity in overexpression systems and cardiac muscle preparations.

Sarco-/endoplasmic reticulum (SR/ER) Ca(2+) pumps (SERCAs) build up vital Ca(2+) gradients across the intracellular SR/ER membrane, helping to control cell function, proliferation, growth, differentiation, and death. We describe two techniques to measure the SERCA activity either in mammalian culture cells overexpressing SERCAs or in muscle tissue containing high levels of endogenous SERCAs. As Ca(2+) transport is tightly coupled to ATP hydrolysis, it is possible to determine the rate of Ca(2+)-dependent ATP hydrolysis and use it as a measure for SERCA activity or, in a second approach, to quantify ATP-stimulated uptake of radioactive (45)Ca(2+). Here, we first provide an overview of the mechanism of Ca(2+)-transport ATPases and show how this can be taken advantage of in protocols for measuring Ca(2+) pump activity.

Promising prospects for 44Sc-/47Sc-based theragnostics: application of 47Sc for radionuclide tumor therapy in mice.

UNLABELLED: In recent years, (47)Sc has attracted attention because of its favorable decay characteristics (half-life, 3.35 d; average energy, 162 keV; Eγ, 159 keV) for therapeutic application and for SPECT imaging. The aim of the present study was to investigate the suitability of (47)Sc for radionuclide therapy in a preclinical setting. For this purpose a novel DOTA-folate conjugate (cm10) with an albumin-binding entity was used. METHODS: (47)Sc was produced via the (46)Ca(n,γ)(47)Ca[Formula: see text](47)Sc nuclear reaction at the high-flux reactor at the Institut Laue-Langevin. Separation of the (47)Sc from the target material was performed by a semi-automated process using extraction chromatography and cation exchange chromatography. (47)Sc-labeled cm10 was tested on folate receptor-positive KB tumor cells in vitro. Biodistribution and SPECT imaging experiments were performed in KB tumor-bearing mice. Radionuclide therapy was conducted with two groups of mice, which received either (47)Sc-cm10 (10 MBq) or only saline. Tumor growth and survival time were compared between the two groups of mice. RESULTS: Irradiation of (46)Ca resulted in approximately 1.8 GBq of (47)Ca, which subsequently decayed to (47)Sc. Separation of (47)Sc from (47)Ca was obtained with 80% yield in only 10 min. The (47)Sc was then available in a small volume (∼500 µL) of an ammonium acetate/HCl (pH 4.5) solution suitable for direct radiolabeling. (47)Sc-cm10 was prepared with a radiochemical yield of more than 96% at a specific activity of up to 13 MBq/nmol. In vitro (47)Sc-cm10 showed folate receptor-specific binding and uptake into KB tumor cells. In vivo SPECT/CT images allowed the visualization of accumulated radioactivity in KB tumors and in the kidneys. The therapy study showed a significantly delayed tumor growth in mice, which received (47)Sc-cm10 (10 MBq, 10 Gy) resulting in a more than 50% increase in survival time, compared with untreated control mice. CONCLUSION: With this study, we demonstrated the suitability of using (47)Sc for therapeutic purposes. On the basis of our recent results obtained with (44)Sc-folate, the present work confirms the applicability of (44)Sc/(47)Sc as an excellent matched pair of nuclides for PET imaging and radionuclide therapy.

Soil-to-plant transfer factors of radioactive Ca, Sm and Pd isotopes: critical assessment of the use of analogies to derive best-estimates from existing non-specific data.

(45)Ca, (151)Sm and (107)Pd are three radionuclides present in low to intermediate in activity radioactive wastes for which no soil-to-plant Transfer Factors (TF) values are available to be used in biosphere models for Ecological Risk Assessment. In the absence of specific radioecological studies, this work reviews and analyzes the existing literature for stable isotopes of Pd, Sm and Ca in order to derive best estimates for TF values that could be used as Transfer Factors. Alternative methods of extrapolation are also critically assessed. The values have been classified according to climatic zone, plant class and soil type for each element. The overall geometric mean TF values (for all plants and conditions) was calculated as 8.4E-02 for Pd, for which the value of radioRu in TRS-472 is also available. The mean TF for Sm was 4.2E-04. This value was lower than the TF values for radioactive Ce that are proposed as alternative values for Sm in TRS-472. The former may be relevant for long term assessments and the latter could possibly used to describe the short term (151)Sm post-release behaviour. The mean value for Ca is 2.3E-01 but varies considerably among plants of a given class due to the variety of plant Ca uptake behaviors. Alternatively, to limit this variability, Ca data content for dry plant matter, as analyzed using the phylogenetic method, could be used to derive TF values if the conservation of isotopic ratio of (45)Ca to stable Ca in soils and in plants hypothesis is taken into account. The TF for Ca in sub-tropical zones is 10-fold lower than in temperate zones. There is a lot of data available about exchangeable Ca in soil, which mean that we could calculate an available TF. The analysis shows that Ca bioavailability is also a key factor within transfer.

Measurement of intracellular Ca2+ release in intact and permeabilized cells using 45Ca2+.

Ca(2+) is an important ion that controls almost every function in a cell. Activator Ca(2+) can be released from intracellular Ca(2+) stores, and there are various ways to study this release. Here, we introduce a technique that uses radioactive (45)Ca(2+) to quantitatively measure the unidirectional release of Ca(2+) from the nonmitochondrial Ca(2+) stores in monolayers of cultured cells. This technique can be used in cells with an intact plasma membrane as well as in cells in which the plasma membrane has been permeabilized.

Measurement of intracellular Ca2+ release in permeabilized cells using 45Ca2+.

This protocol describes a technique to measure Ca(2+) release from the nonmitochondrial intracellular Ca(2+) stores in monolayers of saponin-permeabilized cells cultured in 12-well 4-cm(2) clusters. The (45)Ca(2+)-flux technique described here can only be applied to cell types that still adhere to the plastic after exposing them to saponin. We describe the permeabilization procedure, the loading of the nonmitochondrial Ca(2+) stores with (45)Ca(2+), and the subsequent (45)Ca(2+) efflux.

Measurement of intracellular Ca2+ release in intact cells using 45Ca2+.

This protocol describes a technique using (45)Ca(2+) to measure the release of Ca(2+) from the intracellular stores in monolayers of intact cells cultured in 12-well 4-cm(2) clusters. The (45)Ca(2+)-flux technique described here can only be applied to cell types that adhere to plastic. We describe the loading of the stores with (45)Ca(2+), and the subsequent (45)Ca(2+) efflux.

The influence of therapeutic radiation on the patterns of bone remodeling in ovary-intact and ovariectomized mice.

Our purpose was to characterize changes in bone remodeling associated with localized radiation that models therapeutic cancer treatment in ovary-intact (I) and ovariectomized (OVX) mice and to evaluate the influence of radiation on the pattern of bone mineral remodeling. Young adult, female BALB/c mice, I and OVX, were used (n = 71). All mice were intravenously injected with 15 µCi (45)Ca. Thirty days post-(45)Ca administration, the hind limbs of 17 mice were exposed to a single dose of 16 Gy radiation (R). The time course of (45)Ca excretion, serum CTx and osteocalcin markers, and cancellous bone volume fraction (BV/TV) and cortical thickness (Ct.Th) of the distal femur were assayed. Cellular activity and dynamic histomorphometry were performed. Irradiation resulted in rapid increases in fecal (45)Ca excretion compared to control groups, indicating increased bone remodeling. CTx increased rapidly after irradiation, followed by an increase in osteocalcin concentration. BV/TV decreased in the I mice following irradiation. Ct.Th increased in the OVX groups following irradiation. I+R mice exhibited diminished osteoblast surface, osteoclast number, and mineral apposition. Our murine model showed the systemic effects (via (45)Ca excretion) and local effects (via bone microarchitecture and surface activity) of clinically relevant, therapeutic radiation exposure. The I and OVX murine models have similar (45)Ca excretion but different bone microarchitectural responses. The (45)Ca assay effectively indicates the onset and rate of systemic bone mineral remodeling, providing real-time assessment of changes in bone histomorphometric parameters. Monitoring bone health via a bone mineral marker may help to identify the appropriate time for clinical intervention to preserve skeletal integrity.

Transport and distribution of (45)Ca(2+) in the perfused rat liver and the influence of adjuvant-induced arthritis.

The purpose of the present work was to investigate Ca(2+) transport and distribution under the conditions of the intact rat liver in health and disease (adjuvant-induced arthritis). The multiple-indicator dilution technique was used with the simultaneous injection of (45)Ca(2+) and indicators into the portal vein under defined conditions and analysis of the outflow profiles by means of a space-distributed variable transit time model. The best description of the (45)Ca(2+) outflow profiles corresponds to a model that assumes rapid distribution of (45)Ca(2+) between the vascular space and the cell surface and a slower transfer into the hepatocytes. In kinetic terms two distinct cellular pools were distinguishable, the cytosol and the endoplasmic reticulum. The concentration of Ca(2+) in the cytosol was much lower than in the vascular space and in the endoplasmic reticulum. The most prominent modification observed in the livers of arthritic rats was the increased Ca(2+) concentration in the hormone-sensitive cellular pool. Furthermore, reduced rates of Ca(2+) influx and efflux between the hormone-sensitive cellular pool and the cytosolic space were also detected in combination with a significantly reduced expression of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA2) protein. All these observations mean that in livers from arthritic rats more time is required to replenish the hormone sensitive Ca(2+) stores.

Circadian rhythm leaf movement of Phaseolus vulgaris and the role of calcium ions.

Legume plants, due to their distinctive botanical characteristics, such as leaf movements, physiological characteristics, such as nitrogen fixation, and their abilities to endure environmental stresses, have important roles in sustainable pastures development. Leaf movement of legume plants is turgor regulated and osmotically active fluxes of ions between extensor and flexor of pulvinus cause this movement. To determine the role of calcium ions in circadian leaf movements of Phaseolus vulgaris L., a radiotracer technique experiment using 45Ca ions were employed. Measurements were taken during circadian leaf movements, and samples were taken from different parts of the leaflet. The 45Ca beta-particle activity reduced from leaflet base pulvinus to leaf tip. The pulvinus had the highest activity, while the leaf tip had the lowest. By increase of the ratio of 45Ca beta-particle activity within flexor to extensor (Fl/Ex) the midrib-petiole angle, as an indicator of leaf movement, increased linearly during circadian leaf movement (r = 0.86). The 45Ca beta-particle activity of Flex/Ext ratio reduced linearly (r = -0.88) toward midnight. In conclusion, it was found that calcium ions accumulation is opposite to the fluxes of osmatically active ions and water movement. Calcium ions accumulate at less negative water potential side of the pulivnus.

Cation regulation by the terrestrial isopod Armadillidium vulgare (Crustacea: Isopoda: Oniscidea) during dehydration in air.

Many terrestrial arthropods display tight osmotic and ionic regulation of the hemolymph during dehydration. In this study, we sought to quantify the level of regulation of the major hemolymph cations in the terrestrial isopod Armadillidium vulgare (Isopoda, Oniscidea). Inulin space measurements showed that the hemolymph comprises 52 ± 2.2% of the hydrated water content but contributes 71 ± 9.8% of water losses during desiccation. Hemolymph concentrations of Na+, K+ and Ca²+ were measured in variably dehydrated animals using ion-selective microelectrodes and compared with predicted concentrations assuming no regulation. Na+ and Ca²+ are quite tightly regulated, showing respective concentration increases of 20.8% and 7.1% following a 50% reduction in hemolymph volume, but K+ showed no measurable regulation. The excreted cation fraction during desiccation is negligible. Sites of ion sequestration were examined by injecting ²²Na and 45Ca into the hemolymph of hydrated animals and assaying tissue-specific activities following dehydration. Na+ is apparently sequestered non-specifically by an unknown mechanism. Ca²+ accumulates in the dorsal somatic tissues, possibly in the calcium pool of the cuticle. How A. vulgare avoids significant disruptions of E(m) and neuromuscular function in the absence of K+ regulation, and how it sequesters Na+, both pose intriguing challenges for future work.

Interpretation of 41Ca data using compartmental modeling in post-menopausal women.

Calcium-41 (t(1/2) = 10(5) years) can be used after a single dose to follow calcium metabolism over a subject's lifetime. The aims of this study were to expand a (41)Ca kinetic model and estimate bone resorption in women with stable bone loss, compare the rates with those calculated with classical isotope studies, and to use the model to simulate dynamic changes in urinary (41)Ca:Ca ratios and bone balance for the design and interpretation of (41)Ca studies. Forty-two women >5 years post-menopause were given (41)Ca intravenously. Bone mineral content and bone mineral density of total body were measured by dual-energy X-ray absorptiometry at the beginning of the study. Urine collections were made periodically for up to ~5 years while subjects were free living. Urinary (41)Ca:Ca ratios were measured using accelerator mass spectrometry. The isotope data were analyzed by compartmental modeling. Four compartments were necessary to fit the urinary tracer data and total bone calcium. The final model included pathways for absorption, distribution, urinary excretion, and endogenous excretion and was used to calculate rates of bone turnover. Estimates of bone resorption in a subset of the women (n = 13), studied previously in a 3-week balance and full kinetic study with (45)Ca, agreed with those using (41)Ca methodology. Thus, rates of bone resorption can be estimated from (41)Ca urinary data in stable post-menopausal women. The model was used to simulate dynamic changes in urinary (41)Ca:Ca ratios and bone balance, as a result of interventions that perturb calcium metabolism to aid in study design and interpretation.

Antiresorptive effects of phytoestrogen supplements compared with estradiol or risedronate in postmenopausal women using (41)Ca methodology.

INTRODUCTION: Reduction of ovarian estrogen secretion at menopause increases net bone resorption and leads to bone loss. Isoflavones have been reported to protect bone from estrogen deficiency, but their modest effects on bone resorption have been difficult to measure with traditional analytical methods. METHODS: In this randomized-order, crossover, blinded trial in 11 healthy postmenopausal women, we compared four commercial sources of isoflavones from soy cotyledon, soy germ, kudzu, and red clover and a positive control of oral 1 mg estradiol combined with 2.5 mg medroxyprogesterone or 5 mg/d oral risedronate (Actonel) for their antiresorptive effects on bone using novel (41)Ca methodology. RESULTS: Risedronate and estrogen plus progesterone decreased net bone resorption measured by urinary (41)Ca by 22 and 24%, respectively (P < 0.0001). Despite serum isoflavone profiles indicating bioavailability of the phytoestrogens, only soy isoflavones from the cotyledon and germ significantly decreased net bone resorption by 9% (P = 0.0002) and 5% (P = 0.03), respectively. Calcium absorption and biochemical markers of bone turnover were not influenced by interventions. CONCLUSIONS: Dietary supplements containing genistein-like isoflavones demonstrated a significant but modest ability to suppress net bone resorption in postmenopausal women at the doses supplied in this study over a 50-d intervention period.

Unique isoform-specific properties of calsequestrin in the heart and skeletal muscle.

Calcium signaling in myocytes is dependent on the cardiac ryanodine receptor (RyR2) calcium release channel and the calcium buffering protein in the sarcoplasmic reticulum, cardiac calsequestrin (CSQ2). The overall properties of CSQ2 and its regulation of RyR2 have not been explored in detail or directly compared with skeletal CSQ1 and its regulation of the skeletal RyR1, with physiological ionic strength and Ca(2+) concentrations. We find that there are major differences between the two isoforms under these physiological conditions. Ca(2+) binding to CSQ2 is 50% lower than to CSQ1. Only approximately 30% of CSQ2 is bound to cardiac junctional face membrane (JFM), compared with approximately 70% of CSQ1 and the ratio of CSQ2 to RyR2 is only 50% of the CSQ1/RyR1 ratio. Chemical crosslinking shows that CSQ2 is mostly monomer/dimer, while CSQ1 is mostly polymerized. In single channel lipid bilayer experiments, CSQ2 monomers and/or dimers increase the open probability of both RyR1 and RyR2 channels, while CSQ1 polymers decrease the activity of RyR1. We speculate that CSQ2 facilitates high rates of Ca(2+) release through RyR2 during systole, while CSQ1 curtails RyR1 opening in response to a single action potential to maintain Ca(2+) and allow repeated Ca(2+) release and graded activation with increased stimulation frequency.

Modified calcium accumulation after controlled cortical impact under cyclosporin A treatment: a 45Ca autoradiographic study.

OBJECTIVE: As a neuroprotective drug, cyclosporin A (CsA) has been subject of multiple experimental works in traumatic brain injury (TBI) research. It is well known that CsA inhibits calcium (Ca2+) induced mitochondrial permeability transition (mPT). The aim of this study was to investigate the influence of CsA on the alteration of Ca2+ homeostasis after experimental brain injury. METHODS: Sprague-Dawley male rats (n = 36) with a mean weight of 330 g (280-350 g) were general anesthetized with isofluran through gas mask. The anesthetized animals (n = 24) were subjected to a controlled cortical impact (CCI) over the left parietotemporal cortex using round-tip impounder with a 5 mm diameter at a velocity of approximately 3.7 m/s and a penetration depth of 2 mm. The sham group (n = 12) underwent anesthesia and craniotomy without CCI. In the CCI groups, CsA (n = 12) or vehicle (n = 12) was administered 15 minutes post-injury with a subsequent i.p. injection after 24 hours. Thirty-three hours after injury or sham craniotomy, 45calcium (45Ca) suspended in physiologic saline solution was injected in the left femoral vein. Five hours after isotope administration, animals were killed and the brain was quickly removed and placed in powdered dry ice. Coronal plane sections (20 microm thick) taken every 400 microm from the frontal cortex through the occipital cortex, were exposed to cyclotron films for 14 days at -18 degrees C. Relative optical density was utilized to provide a relative measure of 45Ca accumulation within seven different structures. RESULTS: The difference of 45Ca accumulation (measured by relative optical density) in the CsA group was greater by 30-70% in the following structures compared to vehicle treated traumatized animals: temporal cortex, CA1, anteromedial and posteromedial thalamus (p < 0.05). CONCLUSION: Post-traumatic 45Ca accumulation is modified under CsA. The crucial neuroprotective effect of CsA might be unrelated to a reduction of post-traumatic Ca2+ accumulation, especially with regard to the importance of Ca2+ as an intracellular messenger governing a large number of cellular functions.

Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells.

Rotavirus infection of cells in culture induces major changes in Ca(2+) homeostasis. These changes include increases in plasma membrane Ca(2+) permeability, cytosolic Ca(2+) concentration, and total cell Ca(2+) content and a reduction in the amount of Ca(2+) released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca(2+) homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca(2+) permeability of the plasma membrane and cytosolic Ca(2+) concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca(2+) permeability and Ca(2+) pools released by agonist (ATP). In addition, the increase of total Ca(2+) content (as measured by (45)Ca(2+) uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca(2+) homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca(2+) homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.