Cytosolic Calcium Flux Research Articles

Major trauma enhances store-operated calcium influx in human neutrophils.

Chemotaxins from inflammatory sites prime or activate neutrophils (PMN) by using cytosolic calcium ([Ca2+]i) fluxes as second messengers. [Ca2+]i can be mobilized rapidly by receptor-mediated entry or store-release, or more slowly by store-operated calcium influx (SOCI). We studied [Ca2+]i mobilization by chemotaxins and how trauma impacts the calcium entry mechanisms used by chemotaxins. [Ca2+]i flux was studied by spectrofluorometry. The contributions of early and late [Ca2+]i currents to net calcium flux were compared after stimulation by more potent (fMLP, C5a, PAF) or less potent (IL-8, GRO-alpha, and LTB4) agonists. Store operated [Ca2+]i mobilization was reflected by the ratio of area under the [Ca2+]i efflux curve to peak [Ca2+]i (efflux curve). PMN from trauma patients (ISS > 25) and pair-matched volunteer (n = 7 pairs) were then primed and stimulated with thapsigargin to compare cell calcium stores and SOCI. Late [Ca2+]i mobilization made more important contributions to fMLP, PAF, and C5a signals than to IL-8, GRO-alpha, or LTB4 (p < 0.01 all comparisons). Calcium stores and store release were only marginally lower after injury (p = not significant), but trauma PMN showed far higher [Ca2+]i influx after thapsigargin (p = 0.007), and greater net SOCI (p = 0.034). SOCI may play an important role in PMN activation, and trauma increases PMN SOCI. Prolonged elevations of [Ca2+]i due to enhanced SOCI may alter stimulus-response coupling to chemotaxins and contribute to PMN dysfunction after injury.

Potentiation of Fractional Sarcoplasmic Reticulum Calcium Release by Total and Free Intra-Sarcoplasmic Reticulum Calcium Concentration

Our aim was to measure the influence of sarcoplasmic reticulum (SR) calcium content ([Ca] SRT) and free SR [Ca] ([Ca] SR) on the fraction of SR calcium released during voltage clamp steps in isolated rabbit ventricular myocytes. [Ca] SRT, as measured by caffeine application, was progressively increased by conditioning pulses. Sodium was absent in both the intracellular and in the extracellular solutions to block sodium/calcium exchange. Total cytosolic calcium flux during the transient was inferred from I Ca, [Ca] SRT, [Ca] i, and cellular buffering characteristics. Fluxes via the calcium current ( I Ca), the SR calcium pump, and passive leak from the SR were evaluated to determine SR calcium release flux ( J rel). Excitation-contraction (EC) coupling was characterized with respect to both gain ( ʃ J rel /ʃ I Ca) and fractional SR calcium release. Both parameters were virtually zero for a small, but measurable [Ca] SRT. Gain and fractional SR calcium release increased steeply and nonlinearly with both [Ca] SRT and [Ca] SR. We conclude that potentiation of EC coupling can be correlated with both [Ca] SRT and [Ca] SR. While fractional SR calcium release was not linearly dependent upon [Ca] SR, intra-SR calcium may play a crucial role in regulating the SR calcium release process.

Lack of specificity of [35S]-ATP?S and [35S]-ADP?S as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine-5'-O-3-thio[ 35 S]triphosphate ([ 35 S]-ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X 1 , P2X 2 , P2X 3 , and P2X 4 . Similarly, adenosine-5'-O-2-thio[ 35 S]diphosphate ([ 35 S]-ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [ 35 S]-ATPγS and [ 35 S]-ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X 4 transfected 1321N1 astrocytoma cells. [ 35 S]-ATPγS (0.2 nM) and [ 35 S]-ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [ 35 S]-ATPγS and [ 35 S]-ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y 1 and P2Y 2 receptor subtypes, to 1321N1 cells stably transfected with the human P2X 4 receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X 4 receptor, as measured by cytosolic calcium flux. These data indicate that [ 35 S]-ATPγS and [ 35 S]-ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone.

Cytoplasmic Sodium, Calcium and Free Energy Change of the Na+/Ca2+-Exchanger in Rat Ventricular Myocytes

The relationship between changing driving force of the Na+/Ca2+-exchanger (ΔGexch) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. ΔGexchwas abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of ΔGexchwas calculated. Following abrupt reversal of ΔGexchfrom +4.1 to −9.2 kJ/mol [Na+]iexponentially decreased from 9.6–2.5 mmol/l (t1/2about 30 s) and [Ca2+]itransiently increased to a peak value after about 30 s. Negative values of ΔGexchwere associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (ΔGexch=0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s ΔGexchreached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]itransient and shifted its peak to 80 s, but did not affect the time course of [Na+]Ichanges. In the presence of ryanodine or thapsigargin the time required for ΔGexchto recover to equilibrium was also shifted to 80 s. When we changed the ΔGexchto the same extent by the reduction of [Na+]oin combination with a sustained depolarization, [Na+]idecreased less and the amplitude of [Ca2+]itransient was much enhanced. This increase of [Ca2+]iwas completely abolished by verapamil. ΔGexchonly recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]iand caused a much larger increase of [Ca2+]i, which remained elevated; ΔGexchrecovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to ΔGexch, despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]ocauses reversal of the Na+/Ca2+-exchanger and its driving force ΔGexch, a transient increase of [Ca2+]iand a decrease of [Na+]i. The influx of calcium associated with reversed ΔGexchtriggers the release of calcium from SR. Both the decrease of [Na+]iand the increase of [Ca2+]icontribute to the recovery of ΔGexchto equilibrium. The time at which ΔGexchreaches equilibrium always coincides with the time to peak of [Ca2+]itransient. Activation of the Na+/K+-ATPase is required to reduce [Na+]iand recover ΔGexchto positive values in order to reduce [Ca2+]i. We conclude that ΔGexchis a major regulator of cytosolic calcium by interaction with SR.

Prostaglandin E1 attenuates cytotoxic mechanisms of primed neutrophils.

In a recent clinical trial, liposomal prostaglandin E1 (PGE1) improved oxygenation, increased compliance, and decreased ventilator dependency in patients with adult respiratory distress syndrome (ARDS), thus renewing interest in PGE1 as a potential modulator of inflammation. The neutrophil (PMN) is believed to play a key role in the development of ARDS. Consequently, we investigated the effects of PGE1 on three components of the neutrophil inflammatory response: reactive oxygen species (ROS) generation, protease release, and surface expression of adhesion molecules. Human neutrophils were incubated with PGE1 and then primed with platelet-activating factor (PAF) and activation with N-formyl-methionyl-leucylphenylalanine (fMLP). PGE1 at a dose range of (10[-8] to 10[-5] M) attenuated primed/activated (PAF/fMLP) PMN superoxide anion generation and elastase release. In contrast, PGE1 doses > or =10[-5] M were required to attenuate PAF-stimulated neutrophil upregulation of CD11b/CD18 adhesion molecules. PGE1 also diminished the duration of the PAF-induced cytosolic calcium (Ca2+) flux. Our results suggest that plasma levels of PGE1 attained in patients with ARDS may attenuate ROS and protease neutrophil cytotoxicity but may not effectively block PMN-endothelial cell (EC) adhesion. This attenuation may occur through abrogation of the Ca2+ influx.

Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin

The following study was undertaken to determine if calcium ions move from the plasma membrane to the nucleus of Trypanosoma brucei. Nuclear and cytosolic calcium flux was measured with the calcium sensitive photoprotein, aequorin which was targeted to various locations in stably transformed procyclic cells. Immunoblots revealed that the recombinant proteins, CYT-AEQ and NUC-AEQ were translated in transformants, and that CYT-AEQ was contained in a soluble fraction. Immunolocalization demonstrated that NUC-AEQ was contained within the trypanosome nucleus. To evaluate calcium movement from the plasma membrane to the nucleus in live trypanosomes, aequorin was reconstituted in vivo with coelenterazine and luminescence was recorded. The resting levels of [Ca 2+] cyt and [Ca 2+] nuc were similar (314 ± 43 and 287 ± 28 nM, respectively). When calcium influx across the plasma membrane was initiated with 2 μM ionomycin, [Ca 2+] cyt and [Ca 2+] nuc each became elevated in parallel to a new steady state which was approximately 2-fold above the resting level. Compound 48 80 initiated a calcium flux across the plasma membrane by a different mechanism from ionomycin, and in a manner that was inhibited by the calcium channel antagonist, La 3+. Compound 48 80 (8 μg/ml) transiently elevated [Ca 2+] cyt to 1.73 ± 0.3 μM over the course of 20 s, and also generated a transient rise in [Ca 2+] nuc which peaked at 1.32 + 0.29 μM over the same time course. Overall, these data demonstrate that calcium moves into and out of the trypanosome nucleus in a manner which closely parallels changes in [Ca 2+] cyt. A small calcium ion gradient between nucleus and cytoplasm was also observed.

Human umbilical vein endothelial cells: Specific binding of platelet-activating factor and cytosolic calcium flux

An interaction of the platelet-activating factor (Paf) with endothelial cells was investigated using human umbilical vein endothelial cells. Confluent endothelial cells bound [ 3H]Paf in the presence of 0.25% fatty acid-free serum albumin after culture in media containing either heat-inactivated foetal calf serum or serum substitute. The Scatchard analysis of the saturated specific [ 3H]Paf binding showed a B max of 2.5 fmol indicating 2800 binding sites per endothelial cell. [ 3H]Paf binding was partially reversible at 20 ° and 4 ° and endothelial cells partially metabolized [ 3H]Paf at 20 ° but not at 4 °. [ 3H]Paf binding and Paf-mediated increase of cytosolic free calcium were inhibited by specific Paf receptor antagonists which do not interfere with Paf metabolism. Immortalized umbilical vein endothelial cells bound [ 3H]Paf specifically after culture in the presence of insulin (20 hr, 0.4 U/mL) with non-specific binding in the absence of insulin. The results show that specific Paf binding mediated calcium flux in human endothelial cells.

Interleukin 4 receptor signaling in human monocytes and U937 cells involves the activation of a phosphatidylcholine-specific phospholipase C: a comparison with chemotactic peptide, FMLP, phospholipase D, and sphingomyelinase.

Interleukin 4 (IL-4) diminishes cytokine activation of human macrophage. IL-4 binding to monocyte IL-4R is associated with protein kinase C (PKC) translocation to a nuclear fraction. The cleavage of diacyglycerol (DAG), an activator of PKC, from membrane phospholipids was investigated to define the proximal events of IL-4R signaling. IL-4 induced a statistically significant time-and dose-dependent generation of DAG. The IL-4-triggered production of DAG was not derived from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, since neither cytosolic calcium flux nor liberation of inositol phosphates was detected in response to IL-4. Experiments were performed using [14C-methyl]choline-labeled U937 cells and monocytes to determine whether IL-4R activated phospholipase C (PLC), PLD, or PLA2 to use membrane phosphatidylcholine (PC) to form DAG. IL-4 induced a time- and dose-dependent increase of phosphocholine (pchol) with concomitant degradation of membrane PC (p < 0.05 compared with control). The finding that the peak reduction of PC was equivalent to peak production of pchol suggested that IL-4R signaling involved the activation of a PC-specific PLC. Changes in choline (chol) or lyso-PC and glycerolphosphocholine, the respective products of PC cleavage by PLD or PLA2, were not detected in IL-4-treated cells. In contrast, exogenous PLD induced an increase in chol and concomitant loss of membrane PC. Additional investigation suggested that IL-4R signaling does not involve PLD. In cells labeled with L-lyso-3-PC 1-[1-14C]palmitoyl, PLD but not IL-4, increased the production of phosphatidic acid (PA) and phosphatidyl-ethanol when pretreated with ethanol. Propranolol, an inhibitor of phosphatidate phosphohydrolase, and calyculin A, a phosphatase 1 and 2A inhibitor, blocked DAG production in response to FMLP but not to IL-4. In propranolol pretreated cells, PMA but not IL-4 triggered the production of PA and lowered the amount of DAG. Evidence that PLA2 is not coupled to IL-4R is the detection of arachidonate production in response to FMLP but not to IL-4. Furthermore, IL-4R is not coupled to sphingomyelinase (SMase) since IL-4, unlike exogenous SMase, did not generate ceramide but induced the hydrolysis of PC to pchol that was comparable to exogenous PLC. In summary, IL-4R signaling in monocytes and U937 cells involves PLC and not PLD, PLA2, or SMase, and it uses PC and not PIP2 to form DAG.

Absence of transitory [Ca2+]i flux during early in vitro metacyclogenesis of Trypanosoma cruzi.

The phorbol ester TPA (phorbol 12-myristate 13-acetate) substitutes for CO2 as an agonist for transforming Trypanosoma cruzi epimastigotes to the metacyclic trypomastigote stage in a starvation medium consisting of phosphate buffered saline + 10 mM proline, 10 mM sodium acetate and 0.035% NaHCO3. Since TPA is thought to stimulate protein kinase C by mimicking the activity of the secondary messenger diacylglycerol, the above result suggested that T. cruzi metacyclogenesis could be activated by a Ca(2+)-dependent protein kinase C signal induction pathway. Accordingly, cytosolic calcium flux ([Ca2+]i) in epimastigotes, activated with 5% CO2 or TPA (10(-7) M), was measured with the Ca2+ molecular probe, fluo-3AM. In addition, [Ca2+]i was measured in cells incubated with putative metacyclogenic agonists (e.g. proline, glutamate, bioamines, ionophores and catecholamines). None of the compounds studies, except for EGTA, affected cytosolic Ca2+ levels. Control assays with 11 microM thapsigargin, which mobilizes noncytoplasmic Ca2+ stores by inhibiting endoplasmic reticulum Ca(2+)-ATPase, validated our fluorometric assay procedure. Although thapsigargin significantly increases cytoplasmic Ca2+ fluorescence, it has no effect on transformation. The protein kinase C inhibitors staurosporine, H-7 and HA 1004 were tested for their effect on T. cruzi metacyclogenesis. Low concentrations of staurosporine and HA 1004 significantly elevated Peru strain transformation while H-7 had no effect on Peru strain metacyclogenesis. Inhibitor H-7 did significantly depress CL transformation. The results indicate that induction of T. cruzi metacyclic trypomastigote formation by CO2 and TPA is not accompanied by changes in cytosolic Ca2+ and do not provide supporting evidence for participation of a protein kinase C-mediated phosphoinositide cascade in metacyclogenesis.

AVP-induced Ca fluxes and contraction of rat glomerular mesangial cells

Arginine vasopressin (AVP) is known to exert Ca mobilization and contraction in glomerular mesangial cells and vascular smooth muscle cells. However, the relationship between changes in intracellular Ca and transmembrane Ca fluxes is not clear at the present time. Therefore, this study was undertaken to determine the effect of AVP on cytosolic calcium ([Ca2+]i) and Ca fluxes as estimated by measurements of 45Ca2+ efflux. Changes of [Ca2+]i in response to AVP were directly measured in monolayers of adherent cultured mesangial cells. AVP induced rapid concentration-dependent increases in [Ca2+]i and Ca2+ efflux. AVP also induced contraction of mesangial cells. This effect was blocked only by the V1 (pressor)-antagonist, d(CH2)5Tyr(Me)AVP. Stimulation of Ca2+ efflux and changes in [Ca2+]i by AVP completely desensitized the mesangial cells to a subsequent identical challenge of AVP with no cross-tachyphylaxis to other hormones. Even in Ca2+-free medium, AVP increased [Ca2+]i and Ca2+ efflux, but to a lesser extent. Under this condition, contraction of mesangial cells induced by AVP was also observed. Readdition of extracellular Ca2+ following the AVP-induced increase in [Ca2+]i caused a second and slower [Ca2+]i increase. In Ca2+-containing conditions, lanthanum ion-reduced AVP evoked [Ca2+]i stimulation to the value observed in Ca2+-free medium. The Ca2+ channel blocker, verapamil, partially inhibited AVP-induced Ca2+ influx but totally blocked the increase in [Ca2+]i induced by high K. Verapamil did not inhibit AVP-stimulated Ca2+ efflux and cell contraction. Dantrolene, a blocker of Ca2+ release from endoplasmic reticulum, inhibited AVP-stimulated Ca2+ efflux and cell contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of 12-0-tetradecanoylphorbol 13-acetate on platelet responses to various agonists in the presence and absence of extracellular ca 2+

In the presence of 1 mM extracellular Ca 2+ (Ca 2+), incubation of washed, quin 2 loaded platelets with a low concentration (1 nM) of 12-0-tetradecanoylphorbol 13-acetate (TPA) inhibited platelet shape change, aggregation, ATP secretion and cytosolic calcium (Ca 2+ i) fluxes in response to thrombin, platelet activating factor (PAF), adenosine diphosphate (ADP), vasopressin (VP) and the thromboxane mimetic U46619, but potentiated platelet activation induced by the calcium ionophores, A23187 and ionomycin, the Ca 2+ flux remaining unaltered. In the presence of <100nM Ca 2+ Q, TPA again inhibited shape change but potentiated ATP secretion induced by all agonists, even those in which the cytosolic calcium flux was inhibited. Addition of TPA 30 seconds after a low dose of agonist, sufficient to elevate [Ca 2+ i] to about 250 nM without causing aggregation, induced substantial aggregation and dense granule secretion without affecbting Ca 2+ flux. These results confirm that very slight elevation of [Ca 2+ i] above resting levels greatly enhances the effect of low concentations of TPA, and suggest that protein kinase C activation may exert a feedback inhibition of receptor-mediated shape change, aggregation, ATP secretion and Ca 2+ mobilization.

Calcium fluxes and calcium buffering in human neutrophils.

Neutrophils loaded with the calcium indicator quin-2 and challenged with the ionophore ionomycin or the chemotactic peptide fMet-Leu-Phe were examined in the light of a theory that relates time-dependent changes in the fluorescence of the indicator to cytosolic calcium fluxes and levels. The cytosolic binding capacity was estimated from the theory to be 1.5 +/- 0.6 X 10(8) sites/cell (0.76 mM based on a cell volume of 330 micron 3, irrespective of water content and the distribution of sites), each site having an apparent average single class dissociation constant of 0.55 +/- 0.2 microM. Some 20% of the total available cytosolic calcium sites of the normal resting cell appear to be occupied when no quin-2 is present. In a calcium-free medium, the amount of calcium released by fMet-Leu-Phe from storage pool locations that are distinct from the cytosolic sites is sufficient to further raise the cytosolic site occupancy level to 50%, at which point the calcium buffering capacity of the cytosol is maximal. In a calcium-containing medium, however, simultaneous influx from the outside appears to supply enough additional calcium to saturate most of the remaining sites. The combined initial rate of storage pool calcium release plus influx through the plasma membrane was roughly twice the initial rate at which calcium was released from storage locations alone, suggesting that stimulus-induced influx from the outside may be comparable in importance to storage pool mobilization in determining physiological calcium levels in stimulated cells.

Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S.

The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at Mr approximately 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/10(5) cells per 24 h and contain 44 fmol/10(5) cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor Va inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by greater than 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 microM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.

The effects of anoxia on cytosolic free calcium, calcium fluxes, and cellular ATP levels in cultured kidney cells.

The effect of anoxia and substrate removal on cytosolic free calcium (Ca2+i), cell calcium, ATP content, and calcium efflux was determined in cultured monkey kidney cells (LLC-MK2) exposed to 95% N2, 5% CO2 for 60 min. In the control period, the basal Ca2+i level was 70.8 +/- 9.4 nM. During 1 h of anoxia without substrate, ATP content decreased 70%, Ca2+i and calcium efflux increased 2.5-fold, while the total cell calcium did not change. When the cells were perfused again with O2 and 5 mM glucose, the ATP concentration, Ca2+i, and calcium efflux returned to control levels within 15-20 min. In the presence of 20 mM glucose, anoxia did not produce any change in ATP, in Ca2+i or in calcium efflux. An important source of calcium contributing to the rise in Ca2+i induced by anoxia appears to be extracellular because the rate of rise in Ca2+i is proportional to the extracellular calcium concentration, and because La3+ which blocks calcium influx greatly reduces the rise in Ca2+i. Mitochondria appear to control Ca2+i as well since the early rise in Ca2+i cannot be blocked by La3+ during the initial phase of anoxia, and since the mitochondrial inhibitor carbonyl cyanide p-trifluoromethoxyphenylhydrazone increases Ca2+i further during reoxygenation and slows the return of Ca2+i to control levels.