Extra Ca2 Research Articles

Effects of a low-Ca2+environment on branchial chloride cell morphology in chum salmon fry and immunolocalization of Ca2+-ATPase in chloride cells

To clarify the involvement of branchial chloride cells in Ca2+uptake in fresh water (FW), chloride-cell morphology was compared in chum salmon (Oncorhynchus keta) fry acclimated to defined FWs with different Ca2+concentrations (0, 0.1, and 0.5 mM). Using immunocytochemical staining with an antiserum specific for Na+,K+-ATPase, chloride cells were detected in both filament and lamellar epithelia. The numbers and sizes of chloride cells in the lamellar epithelia were greater in the low-Ca2+groups (0 and 0.1 mM Ca2+) than in the normal-Ca2+groups (0.5 mM Ca2+and normal FW), whereas filament chloride cells were not affected in number or size by the environmental Ca2+concentration. Electron-microscope observations also revealed that enlarged lamellar chloride cells were more frequently observed in the 0 mM Ca2+group than in the 0.5 mM Ca2+group. To obtain morphological evidence for Ca2+uptake through the branchial epithelia, cellular localization of Ca2+-ATPase was examined with a monoclonal antibody specific for human erythrocyte Ca2+-ATPase. Ca2+-ATPase immunoreactivity was detected in Na+,K+-ATPase-immunoreactive chloride cells in both filament and lamellar epithelia. Using electron-microscope immunocytochemistry, Ca2+-ATPase was found to be localized in the tubular system, which is continuous with the basolateral membrane of chloride cells. These findings indicate that chloride cells in the lamellar epithelia activated by a low Ca2+concentration may constitute the extra Ca2+and NaCl uptake capacity required to maintain homeostasis in soft water.

Vibration spectroscopy study of hydrolyzed precursors for sintering calcium phosphate bio-ceramics

Diffuse reflectance infrared Fourier transform and Raman spectroscopy is applied to investigate the structure of hydrolyzed CaHPO4 · 2H2O (DCPD) processed to non-stoichiometric apatite to be used as starting material for preparing sintered calcium phosphate materials. The spectra of hydrolyzed DCPD samples, obtained under differing experimental conditions, are analyzed to check how the sintered phase composition correlates with the structure of the hydrolyzed precursor. It is shown that the addition of extra Ca2+ ions in the alkaline solution during the process of hydrolyzing affects strongly the degree of disorder in the structure of the hydrolyzed materials, thus increasing the relative amount of hydroxyapatite in the final sintered product. The addition of F− ions to the synthesis mixture strengthens slightly the P-O bonds in the precursors and increases the content of hydroxyapatite phase in the sintered material. As a result bi-phase ceramics of highest content of hydroxyapatite can be produced using both fluorine additives and calcium compensators in the synthesis suspension.

Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P(1) site, such as valine, alanine or phenylalanine. The K(m) and k(cat) of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca(2+) ions. At 70 degrees C, a 10-fold increase in Ca(2+) concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca(2+) to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca(2+)-binding site in Ak.1 protease. Other metal ions, such as Sr(2+), increase the thermostability of the enzyme, but to a significantly lower degree than does Ca(2+). The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

Effect of Ca2+-independent Mechanisms on the Hypoxic Relaxation of Guinea-pig Tracheal Rings

Hypoxia induces bronchodilation in vivo and in vitro, but the mechanisms are still unclear. To evaluate whether an extra- or intracellular free Ca2+ion is involved in the mechanisms of hypoxic relaxation, we simultaneously measured cytosolic Ca2+levels and tensions in both intact and denuded guinea-pig tracheal strips precontracted with histamine (100 μM), and assessed the effect of hypoxia on guinea-pig tracheal rings precontracted with okadaic acid (10 μM) and calyculin-A (0.1≈10 μM) under an extracellular Ca2+-free state. The exposure of tracheal rings to hypoxia induced an immediate decrease of tracheal tension without decrease in intracellular free Ca2+levels. In the presence of okadaic acid but not calyculin-A, hypoxic air exposure caused significant transient reductions in tracheal tone. Further, thapsigargin (5 μM or 10 μM) did not affect hypoxic bronchodilation, suggesting that the release of intracellular Ca2+does not take a role in hypoxic bronchodilation. Hypoxic dilation decreased ATP content in epithelium-intact rings but not epithelium-denuded rings, indicating a relationship between hypoxic dilation and change of adenine nucleotide in epithelium-intact rings. Our findings indicate that the epithelium dependent mechanisms of hypoxic relaxation of guinea pig tracheal rings preconstricted with histamine may not be related to the mobilization of extra and intra-cellular Ca2+.

Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.

In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.

Alterations in contractile properties and Ca2+ handling in streptozotocin-induced diabetic rat myocardium.

The mechanisms of the slower time courses of Ca2+ transients (CaT) and contraction in diabetic (diabetes mellitus, DM) myocardium were studied. The aequorin method was applied to papillary muscles of streptozotocin-induced DM and control rats. The time courses of CaT and tension of twitch in DM were slower than those in control, although the magnitudes of the CaT and contraction were identical. The dependence of CaT decay time and relaxation time on developed tension in DM and control rats differed. The length-tension relation in twitch and the pCa-tension relation in tetanus were identical in the two groups. The magnitude of extra Ca2+ (transient increase in intracellular Ca2+ concentration induced by a quick release in tetanus) was identical in both groups. pCa-tension relations of skinned trabeculae at different sarcomere lengths were nearly identical. The cross-bridge cycling rate (CCR) in DM was slower than that in control. These results indicate that the tension-dependent change in the Ca2+ affinity of troponin C in DM myocardium functions as in control myocardium. The slower time courses of CaT and tension in DM myocardium are caused by slower Ca2+ uptake by the sarcoplasmic reticulum and the slower CCR.

Investigation of HEU-type zeolite crystals after interaction with Sr2+ cations in aqueous solution using nuclear and surface analytical techniques

Pure HEU-type zeolite (heulandite) crystals were allowed to interact with Sr2+ cations in aqueous solution. The powdered solid experimental products obtained from batch-type sorption experiments, using solutions of Sr2+-concentrations between 10 and 1000 mg/l, were investigated using INAA, RI-XRF and SEM-EDS. The Sr uptake by the mineral which can adequately be described with a Freundlich-type isotherm, varies from 3.14 to 6.22 mg/g. The distribution coefficients increase progressively by decreasing the solutions concentration reaching a value of 1800 ml/g. The investigated zeolite interacts with Sr2+ cations through ion exchange reactions and initial exchangeable Ca2+ cations are replaced into the structural micropores. However, in the best case, only ca. 43% of the theoretical CEC can be covered because of the limited availability of the extra framework Ca2+ cations that can be removed from the lattice under ambient treatment conditions. The XPS investigation of Sr-loaded single crystals indicated that adsorption of Sr2+ cations on the outer surface also occurs while surface precipitation phenomena must be excluded. Similar surface analyses by means of12C-RBS showed that the Sr depth-distribution at near-surface layers is quite homogenous in contrast to a previous relevant study revealing an intense surface Sr-accumulation on a natural Ca-zeolite of different structural characteristics (scolecite).

The role of luminal Ca2+ in the generation of Ca2+ waves in rat ventricular myocytes

1. We used confocal Ca2+ imaging and fluo-3 to investigate the transition of localized Ca2+ releases induced by focal caffeine stimulation into propagating Ca2+ waves in isolated rat ventricular myocytes. 2. Self-sustaining Ca2+ waves could be initiated when the cellular Ca2+ load was increased by elevating the extracellular [Ca2+] ([Ca2+]o) and they could also be initiated at normal Ca2+ loads when the sensitivity of the release sites to cytosolic Ca2+ was enhanced by low doses of caffeine. When we prevented the accumulation of extra Ca2+ in the luminal compartment of the sarcoplasmic reticulum (SR) with thapsigargin, focal caffeine pulses failed to trigger self-sustaining Ca2+ waves on elevation of [Ca2+]o. Inhibition of SR Ca2+ uptake by thapsigargin in cells already preloaded with Ca2+ above normal levels did not prevent local Ca2+ elevations from triggering propagating waves. Moreover, wave velocity increased by 20 %. Tetracaine (0.75 mM) caused transient complete inhibition of both local and propagating Ca2+ signals, followed by full recovery of the responses due to increased SR Ca2+ accumulation. 3. Computer simulations using a numerical model with spatially distinct Ca2+ release sites suggested that increased amounts of releasable Ca2+ might not be sufficient to generate self-sustaining Ca2+ waves under conditions of Ca2+ overload unless the threshold of release site Ca2+ activation was set at relatively low levels (< 1.5 microM). 4. We conclude that the potentiation of SR Ca2+ release channels by luminal Ca2+ is an important factor in Ca2+ wave generation. Wave propagation does not require the translocation of Ca2+ from the spreading wave front into the SR. Instead, it relies on luminal Ca2+ sensitizing Ca2+ release channels to cytosolic Ca2+.

Potentiation of Calcium‐Mediated Stimulation of DNA Synthesis by Ethanol in Human and Mouse Fibroblasts

Alcohol abuse is a risk factor for cancers of the gastrointestinal tract, and it also can precipitate psoriasis characterized by hyperproliferation of epidermal cells. Because these effects of alcohol may involve stimulation of cell growth, and ethanol (EtOH) was shown to enhance DNA synthesis in mouse fibroblasts and epidermal cells, we conducted a study to determine whether EtOH can also stimulate mitogenesis in human fibroblasts and keratinocytes. In keratinocytes, EtOH had no effects on mitogenesis after shorter (17-hr) treatments, but it partially prevented inhibition of DNA synthesis elicited by longer treatments (3-4 days) with 2 mM calcium (Ca2+), a differentiation-inducing agent. In contrast, treatment of serum-starved zinc-treated (40 microM) human skin fibroblasts with 50-60 mM EtOH for 17 hr resulted in increased DNA synthesis. EtOH-induced DNA synthesis was blocked by 1 mM EGTA, a specific Ca2+ chelator. Despite the presence of 1.8 mM Ca2+ in the cell culture medium, the addition of 1 mM extra Ca2+ (final concentration, 2.8 mM) for 17 hr induced DNA synthesis, presumably mediated by Ca2+ receptors. In eight independent human skin fibroblast lines examined, treatment with EtOH for 46 hr, but not for 17 hr, invariably enhanced the effects of Ca2+ on DNA synthesis, consistent with synergistic stimulation of cell proliferation by EtOH and Ca2+. Neomycin, a Ca2+ receptor agonist, and EtOH also exerted synergistic effects on DNA synthesis after longer (46-hr) treatments. In mouse NIH 3T3 fibroblasts, both EtOH- and Ca2+-enhanced DNA synthesis after 17-hr treatment, but they stimulated cell proliferation only in combination. The results indicate that in human fibroblasts, EtOH can potentiate the longer-term effects of high concentrations of Ca2+ on DNA synthesis whereas, in keratinocytes, EtOH may inhibit Ca2+-induced differentiation.

Potentiation of Calcium-Mediated Stimulation of DNA Synthesis by Ethanol in Human and Mouse Fibroblasts

Alcohol abuse is a risk factor for cancers of the gastrointestinal tract, and it also can precipitate psoriasis characterized by hyperproliferation of epidermal cells. Because these effects of alcohol may involve stimulation of cell growth, and ethanol (EtOH) was shown to enhance DNA synthesis in mouse fibroblasts and epidermal cells, we conducted a study to determine whether EtOH can also stimulate mitogenesis in human fibroblasts and keratinocytes. In keratinocytes, EtOH had no effects on mitogenesis after shorter (17-hr) treatments, but it partially prevented inhibition of DNA synthesis elicited by longer treatments (3-4 days) with 2 mM calcium (Ca2+), a differentiation-inducing agent. In contrast, treatment of serum-starved zinc-treated (40 μM) human skin fibroblasts with 50-60 mM EtOH for 17 hr resulted in increased DNA synthesis. EtOH-induced DNA synthesis was blocked by 1 mM EGTA, a specific Ca2+ chelator. Despite the presence of 1.8 mM Ca2+ in the cell culture medium, the addition of 1 mM extra Ca2+ (final concentration, 2.8 mM) for 17 hr induced DNA synthesis, presumably mediated by Ca2+ receptors. In eight independent human skin fibroblast lines examined, treatment with EtOH for 46 hr, but not for 17 hr, invariably enhanced the effects of Ca2+ on DNA synthesis, consistent with synergistic stimulation of cell proliferation by EtOH and Ca2+. Neomycin, a Ca2+ receptor agonist, and EtOH also exerted synergistic effects on DNA synthesis after longer (46-hr) treatments. In mouse NIH 3T3 fibroblasts, both EtOH- and Ca2+ -enhanced DNA synthesis after 17-hr treatment, but they stimulated cell proliferation only in combination. The results indicate that in human fibroblasts, EtOH can potentiate the longer-term effects of high concentrations of Ca2+ on DNA synthesis whereas, in keratinocytes, EtOH may inhibit Ca2+ -induced differentiation.

Effects of rainfall acidity and ozone on foliar leaching in red spruce (Picea rubens)

The effects of acid rain and ozone on the leaching of chemicals from the canopy of red spruce (Picea rubens Sarg.) saplings was measured over a 4-year treatment period. The saplings were exposed to various levels of ozone and to acid rain (pH 3.1, 4.1, and 5.1) using open-top chambers. No effects of ozone treatments on canopy leaching were observed. Significant effects of rainfall pH on canopy leaching of Ca2+ and Mg2+ were observed, and post-hoc analysis indicated that these effects were associated only with the most acidic treatment (pH 3.1). In the low pH treatment (high NO3- and SO42-) significant canopy retention of both NO3- and SO42- were observed. The observed increase in base cation leaching flux from the red spruce canopy in the low pH treatment was particularly prominent early in the growing season (June) probably reflecting incomplete cuticle formation in the young foliage. Although the annual magnitude of extra Ca2+ leaching from the canopy of red spruce trees in the low pH treatment was small relative to the foliar calcium pool (about 10%), if this leaching preferentially depletes a physiologically important pool, it may influence tree health.

Effects of ramp shortening during linear phase of relaxation on [Ca2+]i in intact skeletal muscle fibers.

The effects of shortening distance at Vu, the unloaded shortening speed, and filament overlap on the amount of extra Ca2+ released during relaxation in muscle, as indicated by the bump area, were studied. Single, intact frog skeletal muscle fibers at 3 degreesC were used. The myoplasmic free Ca2+ concentration ([Ca2+]i) was estimated by using fura 2 salt injected into the myoplasm. Ramps were applied, either at full overlap with different sizes or at varying overlaps with a fixed size, in the linear phase of relaxation. At full overlap, a plot of bump area vs. ramp size was fit by using a sigmoidal curve with one-half of the bump area equal to 25.9 nm. With a fixed ramp size of 100 nm/half-sarcomere, the plot of bump area vs. mean sarcomere length (SLm) was fit by a straight line intersecting the SLm axis at approximately 3.5 micrometers, close to just no overlap. The results suggest that the transition in the distribution of attached cross bridges from the isometric case to one appropriate for unloaded shortening at Vu is completed within 50 nm/half-sarcomere and support the view that attached cross bridges in the overlap zone influence the affinity of Ca2+ for troponin C in the thin filament.

Modulation of Ca2+ transient decay by tension and Ca2+ removal in hyperthyroid myocardium.

We investigated the contribution of sarcoplasmic reticulum (SR) and Na+/Ca2+ exchanger in the tension-dependent change in the decay of the Ca2+ transients (CaT) in euthyroid (Eu) and hyperthyroid (Hy) myocardium. Hy was induced by thyroxine treatment to enhance the rate of SR Ca2+ uptake. With the use of the aequorin method, CaT and tension in twitch contraction were simultaneously measured under various conditions (changing muscle length and Ca2+ concentration in solution). In both groups, the decay time of CaT (DT) showed a significant dependence on the developed tension, but the tension dependence of DT in Hy was significantly less than in Eu. In the presence of caffeine (3 mM), the tension dependence of DT in Hy became apparent as in Eu. Inhibition of Na+/Ca2+ exchanger by replacing Na+ with Li+ did not affect the dependence in Hy. The normalized extra Ca2+, which is the Ca2+ concentration change in response to a quick length change, in Hy was similar to that in Eu. pCa-tension relations of skinned trabeculae measured at different lengths (1.9 and 2.3 micrometer) were nearly identical in both groups. These results indicate that the tension-dependent change in the affinity of troponin C for Ca2+ works in both Eu and Hy myocardium and that the tension-dependent change in DT is influenced by the Ca2+ uptake rate of SR.

Effects of acidosis on Ca2+ sensitivity of contractile elements in intact ferret myocardium.

We investigated the effects of acidosis on the intracellular Ca2+ concentration ([Ca2+]i) and contractile properties of intact mammalian cardiac muscle during tetanic and twitch contractions. Aequorin was injected into ferret papillary muscles, and the [Ca2+]i and tension were simultaneously measured. Acidosis was attained by increasing the CO2 concentration in the bicarbonate (20 mM)-buffered Tyrode solution from 5% (pH 7.35, control) to 15% (pH 6.89, acidosis). Tetanic contraction was produced by repetitive stimulation of the preparation following treatment with 5 microM ryanodine. The relationship between [Ca2+]i and tension was measured 6 s after the onset of the stimulation and was fitted using the Hill equation. Acidosis decreased the maximal tension to 81 +/- 2% of the control and shifted the [Ca2+]i-tension relationship to the right by 0.18 +/- 0.01 pCa units. During twitch contraction, a quick shortening of muscle length from the length at which developed tension became maximal (Lmax) to 92% Lmax produced a transient change in the [Ca2+]i (extra Ca2+). The magnitude of the extra Ca2+ was dependent on the [Ca2+]i immediately before the length change, suggesting that the extra Ca2+ is related to the amount of troponin-Ca complex. Acidosis decreased the normalized extra Ca2+ to [Ca2+]i immediately before the length change, which indicates that the amount of Ca2+ bound to troponin C is less when [Ca2+]i is the same as in the control. The decrease in the Ca2+ binding to troponin C explains the decrease in tetanic and twitch contraction, and mechanical stress applied to the preparation induced less [Ca2+]i change in acidosis.

The effect of tetracaine on spontaneous Ca2+ release and sarcoplasmic reticulum calcium content in rat ventricular myocytes.

1. The effects of tetracaine were studied on voltage-clamped rat ventricular myocytes, which exhibited Ca2+ overload as identified by spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) as shown by the associated contractions. This Ca2+ release was initially abolished by tetracaine before returning at a lower frequency, but greater amplitude, than the control. On removal of tetracaine, there was a burst of spontaneous Ca2+ release activity. All these effects were dose dependent, from 25 to 200 microM tetracaine. 2. The spontaneous Ca2+ release activated an inward Na(+)-Ca2+ exchange current as Ca2+ was pumped out of the cell. The integral of this current (i.e. the Ca2+ efflux) was increased in the presence of tetracaine. The calcium efflux per unit time was unaffected by tetracaine. 3. The SR Ca2+ content was increased by tetracaine, as shown by the integral of the caffeine-evoked Na(+)-Ca2+ exchange current. The increase of SR Ca2+ content was equal to the extra Ca2+ lost from the cell during the burst on removal of tetracaine, and to estimates of the extra calcium gained over the quiescent period following addition of tetracaine. 4. It is concluded that partial inhibition of calcium-induced calcium release increases SR Ca2+ content. In the steady state, cell Ca2+ balance is maintained as the lower frequency of spontaneous release (that activates efflux) is compensated for by their greater size.

Ca2+ and its substitutes have two different binding sites and roles in soluble, quinoprotein (pyrroloquinoline-quinone-containing) glucose dehydrogenase.

To investigate the mode of binding and the role of Ca2+ in soluble, pyrroloquinoline-quinone (PQQ)-containing glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH), the following enzyme species were prepared and their interconversions studied: monomeric apoenzyme (M); monomer with one firmly bound Ca2+ ion (M*); dimer consisting of 2 M* (D); dimer consisting of 2 M and 2 PQQ (Holo-Y); dimer consisting of D with 2 PQQ (Holo-X); fully reconstituted enzyme consisting of Holo-X with two extra Ca2+ ions (Holo) or substitutes for Ca2+ (hybrid Holo-enzymes). D and Holo are very stable enzyme species regarding monomerization and inactivation by chelator, respectively, the bound Ca2+ being locked up in such a way that it is not accessible to chelator. D can be converted into M* by heat treatment and the tightly bound Ca2+ can be removed from M* with chelator, transforming it into M. Reassociation of M* to D occurs spontaneously at 20 degrees C; reassociation of M to D occurs by adding a stoichiometric amount of Ca2+. Synergistic effects were exerted by bound Ca2+ and PQQ, each increasing the affinity of the protein for the other component. Dimerization of M to D occurred with Ca2+, Cd2+, Mn2+, and Sr2+ (in decreasing order of effectiveness), but not with Mg2+, Ba2+, Co2+, Ni2+, Zn2+, or monovalent cations. Conversion of inactive Holo-X into active Holo, was achieved with Ca2+ or metal ions effective in dimerization. Although it is likely that activation of Holo-X involves binding of metal ion to PQQ, the spectral and enzymatic activity differences between normal Holo- and hybrid Holo-enzymes are relatively small. Titration experiments revealed that the two Ca2+ ions required for activation of Holo-X are even more firmly bound than the two required for dimerization of M and anchoring of PQQ. Although the two binding sites related with the dual function of Ca2+ show similar metal ion specificity, they are not identical. The presence of two different sites in sGDH appears to be unique because in other PQQ-containing dehydrogenases, the PQQ-containing subunit has only one site. Given the broad spectrum of bivalent metal ions effective in reconstituting quinoprotein dehydrogenase apoenzymes to active holoenzymes, but the limited spectrum for an individual enzyme, the specificity is not so much determined by PQQ but by the variable metal-ion-binding sites.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

Measurement of sarcoplasmic reticulum Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release.

1. Intracellular calcium concentration ([Ca2+]i) and Na(+)-Ca2+ exchange currents were measured in calcium-overloaded voltage-clamped rat ventricular myocytes loaded with the Ca(2+)-sensitive fluorescent indicator indo-1. Sarcoplasmic reticulum (SR) Ca2+ content was measured from the integral of the caffeine-evoked current. In cells that had spontaneous SR Ca2+ release in 1 mM external Ca2+ concentration ([Ca2+]o)i raising [Ca2+]o increased the frequency of release with no effect on SR Ca2+ content. In quiescent cells, increased [Ca2+]o produced spontaneous Ca2+ release associated with increased SR Ca2+ content. Further increase of [Ca2+]o had no effect on SR Ca2+ content. The amount of Ca2+ leaving the cell during each release was constant over a wide range of frequencies and [Ca2+]o values. It appears there is a maximum level of SR Ca2+ content, perhaps because spontaneous Ca2+ release results when the content reaches a threshold. 2. From the relationship between [Ca2+]i and Na(+)-Ca2+ exchange current during a caffeine response, it is possible to estimate the changes in Na(+)-Ca2+ exchange current expected from a change of [Ca2+]i. The data show that the calcium oscillations contribute a significant fraction of the total extra Ca2+ efflux induced by increasing [Ca2+]o. Raising [Ca2+]o decreased the rate of calcium removal from the cell as measured from the rate of decay of the caffeine response, suggesting that both inhibition of Ca2+ efflux and increased Ca2+ entry account for the Ca2+ overload at elevated [Ca2+]o. 3. Inhibiting spontaneous SR Ca2+ release increases resting [Ca2+]i. The Ca2+ efflux is identical to that in the presence of release. It is concluded that spontaneous release of calcium, although potentially arrhythmogenic, is an effective way to activate Ca2+ efflux in overloaded conditions and minimizes any increase of diastolic tension.

Overexpression, purification and characterization of Dictyostelium calcineurin A

The catalytic subunit of Ca2+/calmodulin-dependent protein phosphatase (calcineurin A) was overexpressed about 50-fold in Dictyostelium discoideum cells transformed with a vector containing the cDNA for D. discoideum calcineurin A under control of the actin-6 promoter. In crude lysates from the overexpressing cell line, high Ca2+/calmodulin-stimulated phosphatase activity was detected. Calcineurin A was purified by anion exchange chromatography and calmodulin-Sepharose affinity chromatography, and the enzymatic activity of the isolated protein was characterized. Its phosphatase activity was strictly dependent on the addition of divalent metal ions such as Mg2+ or Mn2+. Disulphide-reducing agents increased the activity more than 10-fold. Ca2+/calmodulin stimulated the activity by a factor of 2.5-5. Despite the high extra Ca2+/calmodulin-dependent phosphatase activity, the overexpressing cell line showed no phenotypic aberrations.

Caffeine and micromolar Ca2+ concentrations can release Ca2+ from ryanodine-sensitive stores in crab and lobster striated muscle fibres

Ca2+ release mechanisms were studied in striated muscle from the walking legs of crabs using isometric tension recordings from isolated myofibrillar bundles. Caffeine-induced phasic contractions had properties consistent with Ca2+ release from a sarcoplasmic store, which could be optimally loaded in the presence of ATP at pCa 6.4&shy;6.1. Ryanodine (10 &micro;mol l-1) abolished the caffeine-induced contractions and in solutions with low Ca2+ buffering (0.1 mmol l-1 EGTA) itself caused phasic contractions, indicative of Ca2+ release. Ca2+-induced Ca2+ release (CICR) was observed in a pCa 5.8 solution (buffered by 1 mmol l-1 EGTA) as a phasic contraction of variable nature, inhibited by ryanodine (10 &micro;mol l-1), procaine (10 mmol l-1) or benzocaine (5 mmol l-1). Ca2+ release was measured as a function of releasing pCa by using the force&shy;time integral of the caffeine-induced contraction as an estimate of the Ca2+ remaining in the store. After the Ca2+ store had been loaded for 2 min at pCa 6.6, CICR was measured in the presence of 1 mmol l-1 Mg2+, 1 mmol l-1 EGTA and 5 mmol l-1 ATP. The threshold pCa for CICR was 6.0&shy;6.4 under these conditions and more than 90 % of stored Ca2+ was released in 1 min by pCa values in the range 3.5&shy;5.3. Benzocaine totally inhibited the release and promoted extra Ca2+ loading. Preliminary experiments showed a similar caffeine-releasable store in lobster abdominal muscle, which was slightly less sensitive to free [Ca2+]. It is concluded that in crustacean muscle caffeine and micromolar [Ca2+] can release Ca2+ from a ryanodine-sensitive store, which in many respects is similar to the sarcoplasmic reticulum of vertebrate skeletal and cardiac muscle.