Various Concentrations Of Ca2 Research Articles

High monosilicic acid supply rapidly increases Na accumulation in maize roots by decreasing external Ca2+ activity

AbstractBoth calcium (Ca2+) and silicon (Si) improve plant performance under salt (NaCl) stress. Although these two mineral elements share numerous similarities, the information on how their extracellular interactions in the root apoplast affect uptake of sodium (Na+) is still lacking. Here, we investigated the effect of high Si supply in the bioavailable form of monosilicic acid (H4SiO4) on the activity of Ca2+ in the external root solution, and subsequent root uptake and compartmentation of Na in maize (Zea mays L.). In the short‐term experiments (6 h), 14‐d‐old maize plants were exposed to various concentrations of Ca2+ at three different pH‐values (6.5, 7.5, and 8.5) and two Si concentrations, i.e., low (1 mM) and high (4 mM) supply of H4SiO4. The activity of Ca2+ and Na+ in the external solution as well as the root concentrations of total and cell sap and BaCl2‐exchangeble apoplastic fractions of both elements were analyzed. The pH of the nutrient solution affected neither the ion activities nor the root accumulation of both Ca2+ and Na+. At higher pH values (7.5 and 8.5) the interactions of Ca2+ and Si at high Si supply led to a decrease of Ca2+ activity and, hence, an increase of Na+ : Ca2+ activity ratio in the external root solution. Concomitantly, despite the elevated exchangeable apoplastic fraction of both Ca2+ and Na+, the total and cell sap concentrations were remarkably decreased for Ca2+ and increased for Na+ by the addition of 4 mM H4SiO4. This work demonstrates that at high Si supply extracellular Ca‐Si interactions leading to lowered activity of Ca2+ might rapidly compromise the ameliorative effect of Ca2+ on Na+ accumulation in roots. Practically, Si over‐fertilization of saline and, in particular, sodic soils may further promote the accumulation of Na+ in root tissues hours after Si application and, hence, increase a potential risk of Na+ toxicity.

Simultaneous assessment of blood coagulation and hematocrit levels in dielectric blood coagulometry.

Background:In a whole blood coagulation test, the concentration of any in vitro diagnostic agent in plasma is dependent on the hematocrit level but its impact on the test result is unknown.Objective:The aim of this work was to clarify the effects of reagent concentration, particularly Ca2+, and to find a method for hematocrit estimation compatible with the coagulation test.Methods:Whole blood coagulation tests by dielectric blood coagulometry (DBCM) and rotational thromboelastometry were performed with various concentrations of Ca2+ or on samples with different hematocrit levels. DBCM data from a previous clinical study of patients who underwent total knee arthroplasty were re-analyzed.Results:Clear Ca2+ concentration and hematocrit level dependences of the characteristic times of blood coagulation were observed. Rouleau formation made hematocrit estimation difficult in DBCM, but use of permittivity at around 3 MHz made it possible. The re-analyzed clinical data showed a good correlation between permittivity at 3 MHz and hematocrit level ().Conclusions:Changes in the hematocrit level may affect whole blood coagulation tests. DBCM has the potential to overcome this effect with some automated correction using results from simultaneous evaluations of the hematocrit level and blood coagulability.

Ca2+ detection utilising AlGaN/GaN transistors with ion-selective polymer membranes

We demonstrate highly selective and sensitive potentiometric ion sensors for calcium ion detection, operated without the use of a reference electrode. The sensors consist of AlGaN/GaN heterostructure-based transistor devices with chemical functionalisation of the gate area using poly (vinylchloride)-based (PVC) membranes having high selectivity towards calcium ions, Ca2+. The sensors exhibited stable and rapid responses when introduced to various concentrations of Ca2+. In both 0.01 M KCl and 0.01 M NaCl ionic strength buffer solutions, the sensors exhibited near Nernstian responses with detection limits of less than 10−7 M, and a linear response range between 10−7–10−2 M. Also, detection limits of less than 10−6 M were achieved for the sensors in both 0.01 M MgCl2 and 0.01 M LiCl buffer solutions. AlGaN/GaN-based devices for Ca2+ detection demonstrate excellent selectivity and response range for a wide variety of applications. This work represents an important step towards multi-ion sensing using arrays of ion-selective field effect transistor (ISFET) devices.

Partial autolysis of μ/m-calpain during post mortem aging of chicken muscle.

The objective of this study was to investigate changes occurring in μ/m-calpain in post mortem chicken muscles and to determine the origin of the unknown bands found in calpain casein zymography. The unknown bands were reported with slightly greater mobility compared to conventional μ/m-calpain bands in casein zymography. Identification of these bands was accomplished using native polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry and with protein phosphatase treatment. Results showed that the unknown bands were corresponding to μ/m-calpain, and dephosphorylation by protein phosphatase did not change their appearance. The calpain samples were then incubated with various concentrations of Ca2+ to determine the relationship between changes in μ/m-calpain and the appearance of the unknown bands. The products of μ/m-calpain partial autolysis were found to be consistent with the appearance of the unknown bands. Therefore, the appearance of these bands did not result from phosphorylation of μ/m-calpain as previously hypothesized, but from partial autolysis of μ/m-calpain. Also their presence suggests that μ/m-calpain undergoes partial autolysis during aging which may play certain roles in meat quality improvement.

Calcineurin Selectively Docks with the Dynamin Ixb Splice Variant to Regulate Activity-dependent Bulk Endocytosis

Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Fetuin-A (α2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells

Attenuation of Colistin Bactericidal Activity by High Inoculum of Pseudomonas aeruginosa Characterized by a New Mechanism-Based Population Pharmacodynamic Model

Colistin is increasingly being utilized against Gram-negative pathogens, including Pseudomonas aeruginosa, resistant to all other antibiotics. Since limited data exist regarding killing by colistin at different initial inocula (CFUo), we evaluated killing of Pseudomonas aeruginosa by colistin at several CFUo and developed a mechanism-based mathematical model accommodating a range of CFUo. In vitro time-kill experiments were performed using >or=8 concentrations up to 64 x the MIC of colistin against P. aeruginosa PAO1 and two clinical P. aeruginosa isolates at CFUo of 10(6), 10(8), and 10(9) CFU/ml. Serial samples up to 24 h were simultaneously modeled in the NONMEM VI (results shown) and S-ADAPT software programs. The mathematical model was prospectively "validated" by additional time-kill studies assessing the effect of Ca(2+) and Mg(2+) on killing of PAO1 by colistin. Against PAO1, killing of the susceptible population was 23-fold slower at the 10(9) CFUo and 6-fold slower at the 10(8) CFUo than at the 10(6) CFUo. The model comprised three populations with different second-order killing rate constants (5.72, 0.369, and 0.00210 liters/h/mg). Bacteria were assumed to release signal molecules stimulating a phenotypic change that inhibits killing. The proposed mechanism-based model had a good predictive performance, could describe killing by colistin for all three studied strains and for two literature studies, and performed well in a prospective validation with various concentrations of Ca(2+) and Mg(2+). The extent and rate of killing of P. aeruginosa by colistin were markedly decreased at high CFUo compared to those at low CFUo. This was well described by a mechanism-based mathematical model, which should be further validated using dynamic in vitro models.

Mitochondrial oxidative phosphorylation in hearts subjected to Ca2+ depletion and Ca2+ repletionThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Repletion of Ca2+ in the Ca2+-depleted heart has been shown to produce cardiac dysfunction, myocardial cell damage, intracellular Ca2+ overload, and defects in sarcolemmal and sarcoplasmic reticulum function (Ca2+ paradox). Although these alterations in the Ca2+-paradox heart are associated with a depression in the high-energy phosphate stores, little information regarding changes in mitochondrial oxidative phosphorylation is available. Perfusion of rat hearts with Ca2+-free medium for 5 min followed by reperfusion with a medium containing 1.25 mmol/L Ca2+ for 10 min depressed mitochondrial state 3 respiration, respiratory control index, ADP/O ratio, and rate of oxidative phosphorylation without any change in state 4 respiration. These alterations were partially prevented when the reperfusion was carried out with a medium containing low Ca2+ (0.10-0.50 mmol/L). Treatment of heart with inhibitors of sarcolemmal Ca2+ channels (verapamil and diltiazem) or inhibitors of Na+/Ca2+ exchange (KB-R7943) and Na+/H+ exchange (amiloride) failed to modify changes in mitochondrial function due to Ca2+ paradox. Likewise, antioxidants N-acetylcysteine and N-(2-mercaptopropionyl)-glycine and an oxyradical-scavenging mixture of superoxide dismutase and catalase were ineffective in preventing the mitochondrial alterations in the Ca2+-paradox heart. Incubation of mitochondria with various concentrations of Ca2+ inhibited oxidative phosphorylation; this Ca2+-induced change in mitochondrial function was not affected by different oxyradical-scavenging systems. These observations suggest that defects in mitochondrial function in the Ca2+-paradox heart may be due to the occurrence of intracellular Ca2+ overload rather than the development of oxidative stress.

Adenomatous Human Parathyroid Cells Exhibit Impaired Sensitivity tol-Amino Acids

Primary hyperparathyroidism, which occurs most commonly in patients with adenomatous disease of a single parathyroid gland, arises as a result of impaired extracellular Ca(2+) (Ca(2+)(o))-dependent feedback on PTH secretion, a process mediated by the calcium-sensing receptor (CaR). Because the Ca(2+)(o) sensitivity of the CaR is positively modulated by L-amino acids, we decided to investigate whether the impaired feedback of PTH secretion in adenomatous parathyroid cells might arise from decreased sensitivity to L-amino acids. Samples of normal and adenomatous human parathyroid cells were prepared by collagenase treatment and then exposed in vitro to various concentrations of Ca(2+)(o) or the CaR-active amino acid, L-phenylalanine (L-Phe). Excess normal parathyroid tissue was obtained from parathyroid autotransplants at the time of thyroid surgery. Samples of adenomatous tissue were obtained from histologically confirmed parathyroid adenomas. The primary measure was sensitivity of Ca(2+)(o)-dependent PTH secretion to the amino acid L-Phe. The secondary measure was sensitivity of Ca(2+)(o)-dependent intracellular Ca(2+) mobilization to L-Phe. Parathyroid adenomas exhibited reduced sensitivity to the CaR-active amino acid L-Phe, which affected both Ca(2+)(o)-dependent PTH secretion and Ca(2+)(o)-dependent intracellular Ca(2+) mobilization as a measure of CaR-dependent signaling in parathyroid cells. Impaired L-amino acid sensing by calcium-sensing receptors in adenomatous parathyroid cells contributes to the loss of feedback control of PTH secretion in primary hyperparathyroidism. The CaR's amino acid binding site may be exploited as a target in the medical treatment of primary and perhaps other forms of hyperparathyroidism.

A two-gate model for the ryanodine receptor with allosteric modulation by caffeine and quercetin

We have developed a model of the tetrameric ryanodine receptor--the calcium channel of the sarcoplasmic reticulum. The model accurately describes published experimental data on channel activity at various concentrations of Ca2+, caffeine and quercetin. The proposed mechanisms involve allosteric regulation of Ca2+ affinity by both caffeine and quercetin, and the existence of two independent, A- and I-gates controlled by Ca2+ binding to an activating and an inhibitory module of the receptor. There are four different configurations of the receptor that affect ligand binding to the activation module, but not to the inhibition module. Consequently, there are four kinetic modes for the A-gate and one mode for the I-gate. At a certain moment, the receptor can be in any of the four possible conformations with equal probability. By fitting the data we are able to derive ligand affinities and Hill coefficients, to describe the observation that quercetin is an activating agent stronger than caffeine, and that caffeine and quercetin activate the channel at very low Ca2+ concentration (approximately 10(-11) M). We predict that the activation regime at saturating caffeine or quercetin should present four distinct regions at increasing Ca2+, corresponding to the four different gating modes. Another interesting prediction is the enlargement of the activity domain toward higher Ca2+ concentrations in the presence of caffeine or quercetin.

Functional Characterisation of Homomeric Ionotropic Glutamate Receptors GluR1-GluR6 in a Fluorescence-Based High Throughput Screening Assay

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.

Truncation and Activation of Calcineurin A by Calpain I in Alzheimer Disease Brain

A disturbance of calcium homeostasis is believed to play an important role in the neurodegeneration of the brains of Alzheimer disease (AD) patients, but the molecular pathways by which it contributes to the disease are not well understood. Here we studied the activation of two major Ca(2+)-regulated brain proteins, calpain and calcineurin, in AD brain. We found that calpain I is activated, which in turn cleaves and activates calcineurin in AD brain. Mass spectrometric analysis indicated that the cleavage of calcineurin by calpain I is at lysine 501, a position C-terminal to the autoinhibitory domain, which produces a 57-kDa truncated form. The 57-kDa calcineurin maintains its Ca(2+)/calmodulin dependence of the phosphatase activity, but the phosphatase activity is remarkably activated upon truncation. The cleavage and activation of calcineurin correlate to the number of neurofibrillary tangles in human brains. These findings suggest that the overactivation of calpain I and calcineurin may mediate the role of calcium homeostatic disturbance in the neurodegeneration of AD.

Annexin 2 Binding to Phosphatidylinositol 4,5-Bisphosphate on Endocytic Vesicles Is Regulated by the Stress Response Pathway

Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.

Modulation of recombinant transient-receptor-potential-like (TRPL) channels by cytosolic Ca2+.

Whole-cell current recordings were used to examine the involvement of intracellular Ca2+ in the modulation of recombinant transient-receptor-potential like (TRPL) channels of Drosophila photoreceptor cells. TRPL was stably transfected in Chinese hamster ovary (CHO) cells and the expression of a calmodulin-binding protein with a molecular mass that corresponded to TRPL was demonstrated using calmodulin overlays. In cells expressing TRPL, ionic currents that were prominently outwardly rectifying were detected prior to activation of intracellular signalling pathways. The outwardly rectifying currents reversed close to 0 mV and did not occur after removal of permeant cations from the intracellular space. This suggests that TRPL forms non-selective cationic channels that appear to be constitutively active in mammalian cell lines. The TRPL channel currents were enhanced by manoeuvres that activate the phospholipase C (PLC) signalling pathway. These included activation of membrane receptors by thrombin, activation of G proteins by cell dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and release of Ca2+ from intracellular stores by dialysis with inositol 1,4,5-trisphosphate (IP3). After complete depletion of Ca2+ stores, IP3 had no effect on TRPL currents, suggesting that IP3 does not activate recombinant TRPL channels directly. However, thapsigargin, which induces a rise of cytosolic Ca2+, increased TRPL channel currents. Cell dialysis with solutions containing various concentrations of Ca2+ enhanced TRPL currents in a dose-dependent manner (EC50=450 nM Ca2+). Conversely, chelation of cytosolic Ca2+ abolished TRPL channel currents. The present results indicate that the activity of recombinant TRPL channels expressed in mammalian cell lines is up-regulated by a rise of cytosolic Ca2+.

Distinct Ca2+ Binding Properties of Novel C2 Domains of Plant Phospholipase Dα and β

Of the isoforms of plant phospholipase D (PLD) that have been cloned and characterized, PLDalpha requires millimolar levels of Ca(2+) for optimal activity, whereas PLDbeta is most active at micromolar concentrations of Ca(2+). Multiple amino acid sequence alignments suggest that PLDalpha and PLDbeta both contain a Ca(2+)-dependent phospholipid-binding C2 domain near their N termini. In the present study, we expressed and characterized the putative C2 domains of PLDalpha and PLDbeta, designated PLDalpha C2 and PLDbeta C2, by CD spectroscopy, isothermal titration calorimetry, and phospholipid binding assay. Both PLD C2 domains displayed CD spectra consistent with anticipated major beta-sheet structures but underwent spectral changes upon binding Ca(2+); the magnitude was larger for PLDbeta C2. These conformational changes, not shown by any of the previously characterized C2 domains of animal origin, occurred at micromolar Ca(2+) concentrations for PLDbeta C2 but at millimolar levels of the cation for PLDalpha C2. PLDbeta C2 exhibited three Ca(2+)-binding sites: one with a dissociation constant (K(d)) of 0.8 microm and the other two with a K(d) of 24 micrometer. In contrast, isothermal titration calorimetry data of PLDalpha C2 were consistent with 1-3 low affinity Ca(2+)-binding sites with K(d) in the range of 590-470 micrometer. The thermodynamics of Ca(2+) binding markedly differed for the two C2 domains. Likewise, PLDbeta C2 bound phosphatidylcholine (PC), the substrate of PLD, in the presence of submillimolar Ca(2+) concentrations, whereas PLDalpha C2 did so only in the presence of millimolar levels of the metal ion. Both C2 domains bound phosphatidylinoistol 4,5-bisphosphate, a regulator of PC hydrolysis by PLD. However, added Ca(2+) displaced the bound phosphatidylinoistol 4,5-bisphosphate. Ca(2+) and PC binding properties of PLDalpha C2 and PLDbeta C2 follow a trend similar to the Ca(2+) requirements of the whole enzymes, PLDalpha and PLDbeta, for PC hydrolysis. Taken together, the results suggest that the C2 domains of PLDalpha and PLDbeta have novel structural features and serve as handles by which Ca(2+) differentially regulates the activities of the isoforms.

A new approach for measuring real-time calcium pumping and SR function in muscle fibers.

Different muscles are designed to perform a wide variety of motor activities that extend over a large range of frequencies. To increase our understanding of the molecular basis for these different designs, we are attempting to model the activation-relaxation cycle of different muscles by obtaining kinetic information about each of the constituent processes (1, 2). An important component in muscle relaxation is Ca*+ sequestration by sarcoplasmic reticulum (SR)-Ca*’ pumps, but the pump turnover rate in different fiber types is not known precisely. Previous measurements of SR function have generally involved one of two approaches. One approach involves homogenizing muscle, making SR preparations and measuring the rate of Ca*+ accumulation in the presence of oxalate (e.g., 3). It is unclear, however, whether homogenization alters the pumping rate compared to that of intact SR in muscle fibers. Further, the presence of oxalate probably increases the rate of accumulation above that in normal fibers by reducing the free Ca*+ concentration inside the SR. An alternate approach involves physiological measurements of intracellular free [Ca*+] using calcium sensitive dyes (e.g., 1, 4). However, this approach provides direct information only about the small amount of Ca*+ that is actually free in the myoplasm. The total movements of Ca*+ can only be estimated by modelling the large quantities of other Ca*+ buffers in the cell (e.g., parvalbumin and troponin; 4), but the amounts and the kinetics of these calcium buffers are not always known in detail. We have developed a technique and an instrument that permit direct measurements of Ca*+ sequestration by intact SR of muscle fibers as a function of [Ca2+rree]. Single muscle fibers are exposed to 50 wg/ml saponin (20 min), which permeablizes the cell membrane, but leaves the SR membrane intact (5). The “skinned” muscle fibers are then bathed in solutions that contain various concentrations of Ca2+rree; and calcium sequestration (i.e., Ca *+ leaving the bath) is monitored by using a Ca*+ sensitive dye, FURA-2. This approach solves two important problems. First, during the “skinning” procedure, most of the intracellular Ca*+-buffer (the soluble parvalbumin) leaks out of the cell membrane and is washed away. Second, the amounts of remaining intracellular buffers (e.g., troponin), become very small compared to the total buffer in the bath, which is well defined in terms of amounts and kinetics, and can be accurately calibrated. Hence Ca*+ sequestration by the SR can be accurately measured as a removal of Ca*+ from the bathing solution.

Increased Postharvest Survival of Young White Bass and Sunshine Bass by Addition of Calcium and Sodium Chloride to Soft Water

Postharvest survival of white bass Morone chrysops and sunshine bass M. chrysops ♀ × Morone saxatilis ♂ was determined in water containing additional NaCl, CaCl2, MgCl2, or combinations of these salts. Age of fish was near the time of metamorphosis from larval to juvenile stages; white bass were 23 d old and sunshine bass were 30 d old. All white bass confined in 2-L beakers died within 7 h in soft water (2.5 mg Ca2+/L), and addition of NaCl (0.7–5.0 g/L) alone did not increase survival. Addition of 35 mg Mg2+/L to water containing 5.0 g NaCl/L increased survival to 57% after 7 h, but only 7% survived for 24 h. In tests with various concentrations of Ca2+ (2.9–20 mg/L) and 5.0 g NaCl/L, survival rate increased with increasing concentration of Ca2+ up to 85.7% at 14 mg/L, and a higher concentration of Ca2+ did not increase survival. A Ca2+ concentration of 9.1 mg/L (95% confidence interval, 7.5–10.9) was needed to achieve 50% survival for 7 h. Few white bass died after 7 h in the Ca2+ experiments. Amiloride, a sodium transport inhibitor, added to the water did not affect survival. All sunshine bass were dead after 19 h in containers with 10 L of soft water (2.0 mg Ca2+/L), and only 25% survived in water with 5.0 g NaCl/L. Addition of 84 mg Ca2+/L increased survival to 64%, and addition of both 93 mg Ca2+/L and 5.0 g NaCl/L resulted in 91% survival. Results for white bass and sunshine bass were generally similar to those of previous studies with striped bass M. saxatilis at this stage of development.

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor)

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.

Effects Of Ca2+ On Oxidative Phosphorylation In Mitochondria From The Thermogenic Organ Of Marlin

Mitochondria from the muscle-derived thermogenic (heater) organ and oxidative red muscle of the blue marlin (Makaira nigricans) were studied in order to evaluate aspects of the mechanism of thermogenesis in heater tissue. We investigated whether short-term Ca(2+)-induced uncoupling of mitochondria contributes to the thermogenic cycle of the heater organ by enhancing the respiration rate. Specific electrodes were used to obtain simultaneous measurements of oxygen consumption and Ca2+ fluxes on isolated mitochondria, and the effects of various concentrations of Ca2+ on respiration rates and the ADP phosphorylated/atomic oxygen consumed (P/O) ratio were examined. Addition of Ca2+ in excess of 10 mumol l-1 to respiring heater organ or red muscle mitochondria partially inhibited state 3 respiration and reduced the P/O ratio, indicating that the mitochondria were partially uncoupled. These effects were blocked by 2 mumol l-1 Ruthenium Red. In heater organ mitochondria, state 3 respiration rate and the P/O ratio were not significantly reduced by 1 mumol l-1 free Ca2+, a concentration likely to be near the maximum achieved in a stimulated cell. This indicates that transient increases in cytosolic Ca2+ concentration may not significantly reduce the P/O ratio of heater organ mitochondria. The activity of 2-oxoglutarate dehydrogenase in heater organ mitochondria was stimulated by approximately 15% by Ca2+ concentrations between 0.2 and 1 mumol l-1. These results suggest that heater organ mitochondria are able to maintain a normal P/O ratio and should maintain ATP output during transient increases in Ca2+ concentration, supporting a model in which an ATP-consuming process drives thermogenesis. Activation of mitochondrial dehydrogenases by low levels of Ca2+ may also enhance respiration and contribute to thermogenesis.

Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin sperm axonemes.

Active sliding between doublet microtubules of sea urchin sperm axonemes that were demembranated with Triton X-100 in the presence or absence of calcium was induced with ATP and elastase at various concentrations of Ca2+ to examine the effects of Ca2+ on the direction of the power stroke of the dynein arms. Dark-field light microscopy of microtubule sliding revealed that the sliding from the axonemes demembranated with Triton and millimolar calcium and disintegrated with ATP and elastase showed various patterns of sliding disintegration, including loops of doublet microtubules formed near the head or the basal body. These loops were often thicker than the remaining axonemal bundle. In contrast, only thinner loops were found from the axonemes demembranated with Triton in the absence of calcium and disintegrated with ATP and elastase at high Ca2+ concentrations. Electron microscopic examination of the direction of microtubule sliding showed that the doublet microtubules in the axonemes demembranated in the presence of millimolar calcium moved toward the base of the axonemes by the dynein arms on the adjacent doublet microtubule as well as by their own dynein arms. Doublet microtubules in the axonemes demembranated in the absence of calcium moved toward the base of the axonemes only by their own dynein arms. Similar observations have been obtained from the axonemes from which the outer dynein arms were selectively extracted. From these observations, we can conclude that the dynein arms generate force in both directions and this feature of the dynein arms arises from at least the inner dynein arms.