Peak Cytosolic Ca2 Research Articles

Lithium protects against lipopolysaccharide‐induced cytotoxicity in SH‐SY5Y cells expressing Alzheimer’s presenilin 1 mutation by ameliorating calcium dysregulation

AbstractBackgroundLipopolysaccharide is a crucial mediator of neuroinflammation in sepsis. Neuroinflammation is implicated in normal brain aging and neurodegeneration in various neurodegenerative disorders including Alzheimer's disease. We examined the effect and mechanisms of lithium protection against lipopolysaccharide‐induced cytotoxicity in human neuroblastoma SH‐SY5Y cells expressing familial Alzheimer’s disease mutant presenilin‐1 (PS1) and compared to wide type control.MethodHuman neuroblastoma SH‐SY5Y cells transfected with human wild type or point mutant PS1 (M146L) were pretreated with lithium chloride (1 mM) for 1 week, and then challenged with lipopolysaccharide for 24 h. Cell viability was determined by the 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction assay. Alterations of cytosolic Ca2+ concentrations were measured using jellyfish specific photo protein aequorin‐based probes.ResultLipopolysaccharide resulted in a dose‐dependent cytotoxic effects (starting from the concentration of 400 μg/ml) in both types of cells. Moreover, lipopolysaccharide (400 μg/ml) caused significantly more cytotoxicity in M146L than its corresponding wild type control cells, which could be inhibited significantly by lithium (1 mM) pretreatment. Lipopolysaccharide induced significantly more elevation of peak cytosolic Ca2+ concentrations in M146L cells than in wide type control cells, which could be abolished by lithium (1 mM) pretreatment.ConclusionsOur results suggest that lithium pretreatment at clinically relevant concentrations could effectively mitigates lipopolysaccharide mediated cytotoxicity, especially in Alzheimer’s PS 1 mutated cells, possibly by ameliorating the abnormally elevated cytosolic Ca2+ concentration.

Incubation with sodium nitrite attenuates fatigue development in intact single mouse fibres at physiological .

Dietary nitrate supplementation increases plasma nitrite concentration, which provides an oxygen-independent source of nitric oxide and can delay skeletal muscle fatigue. Nitrate supplementation has been shown to increase myofibre calcium release and force production in mouse skeletal muscle during contractions at a supra-physiological oxygen tension, but it is unclear whether nitrite exposure can delay fatigue development and improve myofibre calcium handling at a near-physiological oxygen tension. Single mouse muscle fibres acutely treated with nitrite had a lower force and cytosolic calcium concentration during single non-fatiguing contractions at a near-physiological oxygen tension. Nitrite treatment delayed fatigue development during repeated fatiguing isometric contractions at near-physiological, but not at supra-physiological, oxygen tension in combination with better maintenance of myofilament calcium sensitivity and sarcoplasmic reticulum calcium pumping. These findings improve understanding of the mechanisms by which increased skeletal muscle nitrite exposure might be ergogenic and imply that this is related to improved calcium handling. Dietary nitrate (NO3- ) supplementation, which increases plasma nitrite (NO2- ) concentration, has been reported to attenuate skeletal muscle fatigue development. Sarcoplasmic reticulum (SR) calcium (Ca2+ ) release is enhanced in isolated single skeletal muscle fibres following NO3- supplementation or NO2- incubation at a supra-physiological but it is unclear whether NO2- incubation can alter Ca2+ handling and fatigue development at a near-physiological . We hypothesised that NO2- treatment would improve Ca2+ handling and delay fatigue at a physiological in intact single mouse skeletal muscle fibres. Each muscle fibre was perfused with Tyrode solution pre-equilibrated with either 20% ( ∼150Torr) or 2% O2 ( =15.6Torr) in the absence and presence of 100µM NaNO2 . At supra-physiological (i.e. 20% O2 ), time to fatigue was lowered by 34% with NaNO2 (control: 257±94 vs. NaNO2 : 159±46s, Cohen's d=1.63, P<0.05), but extended by 21% with NaNO2 at 2% O2 (control: 308±217 vs. NaNO2 : 368±242s, d=1.14, P<0.01). During the fatiguing contraction protocol completed with NaNO2 at 2% O2 , peak cytosolic Ca2+ concentration ([Ca2+ ]c ) was not different (P>0.05) but [Ca2+ ]c accumulation between contractions was lower, concomitant with a greater SR Ca2+ pumping rate (P<0.05) compared to the control condition. These results demonstrate that increased exposure to NO2- blunts fatigue development at near-physiological, but not at supra-physiological, through enhancing SR Ca2+ pumping rate in single skeletal muscle fibres. These findings extend our understanding of the mechanisms by which increased NO2- exposure can mitigate skeletal muscle fatigue development.

Collapsin Response Mediator Protein 2 (CRMP2) Interacts with N-Methyl-d-aspartate (NMDA) Receptor and Na+/Ca2+ Exchanger and Regulates Their Functional Activity

Collapsin response mediator protein 2 (CRMP2) is traditionally viewed as an axonal growth protein involved in axon/dendrite specification. Here, we describe novel functions of CRMP2. A 15-amino acid peptide from CRMP2, fused to the TAT cell-penetrating motif of the HIV-1 protein, TAT-CBD3, but not CBD3 without TAT, attenuated N-methyl-d-aspartate receptor (NMDAR) activity and protected neurons against glutamate-induced Ca(2+) dysregulation, suggesting the key contribution of CRMP2 in these processes. In addition, TAT-CBD3, but not CBD3 without TAT or TAT-scramble peptide, inhibited increases in cytosolic Ca(2+) mediated by the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) operating in the reverse mode. Co-immunoprecipitation experiments revealed an interaction between CRMP2 and NMDAR as well as NCX3 but not NCX1. TAT-CBD3 disrupted CRMP2-NMDAR interaction without change in NMDAR localization. In contrast, TAT-CBD3 augmented the CRMP2-NCX3 co-immunoprecipitation, indicating increased interaction or stabilization of a complex between these proteins. Immunostaining with an anti-NCX3 antibody revealed that TAT-CBD3 induced NCX3 internalization, suggesting that both reverse and forward modes of NCX might be affected. Indeed, the forward mode of NCX, evaluated in experiments with ionomycin-induced Ca(2+) influx into neurons, was strongly suppressed by TAT-CBD3. Knockdown of CRMP2 with short interfering RNA (siRNA) prevented NCX3 internalization in response to TAT-CBD3 exposure. Moreover, CRMP2 down-regulation strongly attenuated TAT-CBD3-induced inhibition of reverse NCX. Overall, our results demonstrate that CRMP2 interacts with NCX and NMDAR and that TAT-CBD3 protects against glutamate-induced Ca(2+) dysregulation most likely via suppression of both NMDAR and NCX activities. Our results further clarify the mechanism of action of TAT-CBD3 and identify a novel regulatory checkpoint for NMDAR and NCX function based on CRMP2 interaction with these proteins.

Increased ER–mitochondrial coupling promotes mitochondrial respiration and bioenergetics during early phases of ER stress

There were errors published in J. Cell Sci. 124, 2143-2152.In the section given below, PtdIns(3,4,5)P3 was on four occasions incorrectly printed instead of the correct Ins(1,4,5)P3.We apologise for this mistake.Increased mitochondrial Ca2+ drives the adaptive metabolic boost observed during early phases of ER stressIncreases in mitochondrial respiration and ATP production are often consequences of increases in mitochondrial Ca2+ (Green and Wang, 2010). In order to determine whether early phases of ER stress induced by tunicamycin increased mitochondrial Ca2+, we treated cells expressing cytosolic or mitochondrial aequorins with histamine [which evokes Ins(1,4,5)P3-dependent Ca2+ release] and compared their mitochondrial Ca2+ uptake. We observed that histamine led to a mitochondrial Ca2+ uptake that was significantly higher in tunicamycin-pretreated cells (P<0.05; 4 hours) than in untreated cells (Fig. 6A). Cytosolic Ca2+ increased similarly in tunicamycin-treated and untreated cells (Fig. 6B). These results indicate that the differences in mitochondrial Ca2+ levels are not due to altered Ca2+ release mediated by the Ins(1,4,5)P3 receptor but to an enhanced mitochondrial Ca2+ uptake, presumably due to the increased apposition of ER and mitochondrial Ca2+ channels. By using a different dye, Fura-2, we monitored the peak cytosolic Ca2+ levels after thapsigargin addition, reflecting the kinetics of Ca2+ release after sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition. After 4 hours of tunicamycin treatment, the thapsigargin-induced Ca2+ peak was increased, and it was further elevated by inhibition of mitochondrial Ca2+ uptake using Ru360 (Fig. 6C). These results suggest that, besides the Ins(1,4,5)P3-receptor-mediated direct Ca2+ transfer from the ER to neighboring mitochondria, an additional phenomenon associated with the early phases of ER stress involves Ca2+ leak from the ER, also resulting in mitochondrial Ca2+ uptake. Indeed, no mitochondrial Ca2+ uptake following the thapsigargin-induced Ca2+ leak was observed in Mfn2-knockout cells (Fig. 6D), which is reflected by the lack of effect of Ru360. This result further indicates that juxtaposition of mitochondria with the ER is necessary for the mitochondrial Ca2+ uptake evoked by Ca2+ leak during early phases of ER stress.Finally, to test whether mitochondrial Ca2+ levels control the metabolic mitochondrial boost, we measured oxygen consumption rates resulting from OXPHOS in the presence of the Ins(1,4,5)P3 receptor inhibitor xestospongin B or the mitochondrial Ca2+ uptake inhibitor RuRed. We observed that both xestospongin B and RuRed decreased the rate of oxygen consumption after tunicamycin treatment (Fig. 7A,B), which confirms that increased mitochondrial Ca2+ uptake, resulting from ER–mitochondrial coupling, is necessary for the metabolic response observed during early phases of ER stress. Therefore, in order to evaluate whether the early metabolic boost forms part of an adaptive response triggered by ER stress, we inhibited mitochondrial Ca2+ uptake and measured cell viability [through propidium iodide (PI) incorporation] and Δψm. We observed that the inhibition of mitochondrial Ca2+ uptake during the early phase of ER stress increased cell death (PI-positive cells) and also decreased Δψm at 48 hours (Fig. 7C).In total, the results presented in this study suggest strongly that Ca2+ transfer resulting from enhanced ER–mitochondrial coupling leads to a localized increase in mitochondrial metabolism, thus providing energetic substrates key for a cellular adaptive response in face of ER stress. Further experiments will determine whether this bioenergetic response is necessary for improving the energetic state of the ER, and therefore its folding capacity, or, as it is restricted to perinuclear zones, for the activation of a specific nuclear transcriptional program that participates in the cellular adaptation to stress.

Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

BackgroundThe role of protein kinase C (PKC) in regulating the activity of phospholipase C (PLC) in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM), was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM) and staurosporine (400 nM).MethodsAlterations in cytosolic Ca2+, Ca2+ influx, inositol triphosphate (IP3), and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively.ResultsActivation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase.Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils.ConclusionAlthough generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.

Mitochondrial Sensitivity and Altered Calcium Handling Underlie Enhanced NMDA-Induced Apoptosis in YAC128 Model of Huntington's Disease

Expansion of a CAG repeat in the Huntington's disease (HD) gene results in progressive neuronal loss, particularly of striatal medium-sized spiny neurons (MSNs). Studies in human HD autopsy brain tissue, as well as cellular and animal models of HD, suggest that increased activity of NMDA-type glutamate receptors and altered mitochondrial function contribute to selective neuronal degeneration. In this regard, the YAC128 mouse model, expressing full-length human huntingtin with 128 glutamine repeats, has been the focus of much interest. Although NMDA-induced apoptosis is enhanced in YAC128 MSNs, here we report that the initial steps in the death signaling pathway, including NMDA receptor (NMDAR) current and cytosolic Ca2+ loading, are similar to those observed in wild-type MSNs. In contrast, we found that the NMDAR-mediated Ca2+ load triggered a strikingly enhanced loss of mitochondrial membrane potential in YAC128 MSNs, suggesting that NMDAR signaling via the mitochondrial apoptotic pathway is altered. This effect was accompanied by impaired cytosolic Ca2+ clearance after removal of NMDA, a difference that was not apparent after high potassium-evoked depolarization-mediated Ca2+ entry. Inhibition of the mitochondrial permeability transition (mPT) reduced peak cytosolic Ca2+ and mitochondrial depolarization evoked by NMDA in YAC128 MSNs but not wild-type MSNs. Hence, in contrast to YAC models with moderate CAG expansions, the enhanced NMDA-induced apoptosis in YAC128 MSNs is predominantly determined by augmented mitochondrial sensitivity to Ca2+-induced activation of the mPT. These results suggest that the CAG repeat length influences the mechanism by which mHtt enhances NMDAR-mediated excitotoxicity.

Functional Coupling between TRPC3 and RyR1 Regulates the Expressions of Key Triadic Proteins

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 +/- 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 +/- 1.91-, 1.42 +/- 0.08-, 2.99 +/- 0.32-, and 1.91 +/- 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load.

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes.

The effects of endurance run training on Na+-dependent Ca2+ regulation in rat left ventricular myocytes were examined. Myocytes were isolated from sedentary and trained rats and loaded with fura 2. Contractile dynamics and fluorescence ratio transients were recorded during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degreesC. Resting and peak cytosolic Ca2+ concentration ([Ca2+]c) did not change with exercise training. However, resting and peak [Ca2+]c increased significantly in both groups during 5 min of continuous pacing, although diastolic [Ca2+]c in the trained group was less susceptible to this elevation of intracellular Ca2+. Run training also significantly reduced the rate of [Ca2+]c decay during relaxation. Myocytes were then exposed to 10 mM caffeine in the absence of external Na+ or Ca2+ to trigger sarcoplasmic reticular Ca2+ release and to suppress cellular Ca2+ efflux. This maneuver elicited an elevated steady-state [Ca2+]c. External Na+ was then added, and the rate of [Ca2+]c clearance was determined. Run training significantly reduced the rate of Na+-dependent clearance of [Ca2+]c during the caffeine-induced contractures. These data demonstrate that the removal of cytosolic Ca2+ was depressed with exercise training under these experimental conditions and may be specifically reflective of a training-induced decrease in the rate of cytosolic Ca2+ removal via Na+/Ca2+ exchange and/or in the amount of Ca2+ moved across the sarcolemma during a contraction.

Impaired cytosolic calcium mobilization and aggregation in response to collagen in platelets from Japanese Black cattle with Chediak-Higashi syndrome

Abstract Objective To examine whether impaired aggregation of platelets from Japanese Black cattle with Chediak-Higashi syndrome (CHS) was attributable to mobilization of cytosolic Ca2+. Animals 4 healthy Japanese Black cattle and 3 Japanese Black cattle with CHS. Procedure Aggregation and mobilization of cytosolic Ca2+ in response to various receptor agonists was measured in platelets from healthy cattle and cattle with CHS. Involvement of endogenous ADP and arachidonic acid in collagen-induced responses was examined. Cytosolic Ca2+ concentration was measured after platelets were loaded with the Ca2+ indicator fura-PE3. Platelet aggregation was measured with an aggregometer. Results Collagen (3 to 15 μg/ml)-induced increases in cytosolic Ca2+ concentration and aggregation were markedly impaired for platelets from cattle with CHS, compared with values for platelets from healthy cattle. Although aggregation and the sustained phase of the cytosolic Ca2+ response to ADP were also decreased in platelets from cattle with CHS, these decreases were small, compared with those in response to collagen. A cyclooxygenase inhibitor and a phospholipase A2 inhibitor did not have any effect on peak cytosolic Ca2+ concentration or collagen-induced aggregation of platelets from healthy cattle. Responses to a P2T-purinoceptor antagonist suggested that decreased release of endogenous ADP was only partially involved in the impaired response to collagen among platelets from cattle with CHS. Conclusions and Clinical Relevance Marked inhibition of collagen-induced Ca2+ mobilization, rather than decreased release of endogenous substances, appeared to be the major cause of impaired platelet response to collagen and the hemorrhagic tendency in cattle with CHS. (Am J Vet Res 1998;59:744-749)

The effect of insulin-like growth factor-1 on adult rat cardiac contractility.

There is increasing evidence that insulin-like growth factor-1 (IGF-1) may play a role in both physiological and pathophysiological events in the mammalian myocardium. The present study investigated the acute effects of IGF-1 on isometric force development in isolated rat cardiac muscle and on intracellular calcium (Ca2+) handling in isolated cardiac myocytes. IGF-1 had a positive inotropic effect on rat ventricular papillary muscles increasing force development by 17.8 +/- 4.6%, 18.5 +/- 5.8% and 11.9 +/- 4.9% (n = 12-20) at concentrations of 1, 10 and 100 ng/ml respectively. Isoprenaline increased tension in these papillary muscles by 56.7 +/- 7.7% at a concentration of 100 nM (n = 22). In comparison, insulin increased papillary muscle force development by 11.6 +/- 3.2%, 17.7 +/- 4.1% and 19.7 +/- 5.6% at concentrations of 1, 10 and 100 nM respectively (n = 16-20). In the single cardiac myocyte IGF-1 increased, the peak cytosolic free Ca2+ concentration, the amplitude of the Ca2+ transient and the time to peak Ca2+ as measured with the fluorescent bioprobe Indo-1 AM. The positive inotropic response to IGF-1 by rat ventricular muscle is therefore associated with a rise in free, peak cytosolic Ca2+ in isolated cardiac myocytes. Increasing insulin concentrations (1-1000 nM) elicited a progressive elevation in isometric force and free, cytosolic Ca2+. In contrast, in the presence of IGF-1, the maximal rise in isometric force and free cytosolic Ca2+ were both observed at 10 ng/ml. Recent reports have suggested that IGF-1 may act on the mammalian myocardium when administered chronically, but this study is amongst the first to demonstrate an acute effect of IGF-I on the mammalian heart. IGF-1 may prove then to be a novel cardioactive agent in both normal and pathophysiological states.

Calcium currents in postinfarction rat cardiac myocytes.

Myocytes isolated from rat hearts that have suffered 35% myocardial infarction (MI) 3 wk prior have lower peak cytosolic Ca2+ concentration ([Ca2+]i) during contraction compared with Sham myocytes, a difference that is amplified by isoproterenol or high extracellular Ca2+ concentration ([Ca2+]o). To evaluate whether reduced [Ca2+]i in MI myocytes is due to decreased Ca2+ entry, we measured [3H]PN-200-110 [dihydropyridine (DHP)] binding and whole cell Ca2+ current (ICa). DHP binding decreased in both sarcolemmal vesicles and intact myocytes from hearts 3 wk after MI. In contrast, ICa was not different between Sham and MI myocytes incubated at 1.8 mM [Ca2+]o. At 5.0 mM [Ca2+]o, ICa increased similarly in Sham and MI myocytes. Steady-state voltage dependence of activation and inactivation were similar between Sham and MI myocytes, as were the fast- and slow-inactivation time constants. Isoproterenol (1 microM) significantly increased ICa in Sham but not in MI myocytes. Forskolin (10 microM) dibutyryl adenosine 3',5'-cyclic monophosphate (5 mM) significantly increased ICa in MI myocytes; the magnitude of ICa increase was similar to that observed in Sham myocytes. We conclude that 1) decreased systolic [Ca2+]i in MI myocytes was not due to reduced Ca2+ entry via L-type Ca2+ channels; 2) discrepancy between DHP binding (decrease) and ICa (no change) results may be explained by higher channel availability and/or increased long-opening modes (mode 2) in MI myocytes; 3) reduction in isoproterenol-induced [Ca2+]i increase in MI myocytes was partly due to decreased ICa, resulting in less Ca2+ release from sarcoplasmic reticulum; 4) the adenylate cyclase-protein kinase A signal-transduction pathway functioned normally in MI myocytes; and 5) decreased beta-adrenergic responsiveness in MI myocytes was likely due to altered coupling by G proteins.

Correlation of Real-time Catecholamine Release and Cytosolic Ca2+ at Single Bovine Chromaffin Cells

Previous investigations of the role of Ca2+ in stimulus-secretion coupling have been undertaken in populations of adrenal chromaffin cells. In the present study, the simultaneous detection of intracellular Ca2+, with the fluorescent probe fura-2, and catecholamine release, using a carbon-fiber microelectrode, are examined at single chromaffin cells in culture. Results from classic depolarizing stimuli, high potassium (30-140 mM) and 1,1-dimethyl-4-phenylpiperazinium (3-50 microM), show a dependence of peak cytosolic Ca2+ concentration and catecholamine release on secretagogue concentration. Catecholamine release induced by transient high K+ stimulation increases logarithmically with K+ concentration. Continuous exposure to veratridine (50 microM) induces oscillations in intracellular Ca2+ and at higher concentrations (100 microM) concomitant fluctuation of cytosolic Ca2+ and catecholamine secretion. Mobilization of both caffeine- and inositol trisphosphate-sensitive intracellular Ca2+ stores is found to elicit secretion with or without extracellular Ca2+. Caffeine-sensitive intracellular Ca2+ stores can be depleted, refilled, and cause exocytosis in medium without Ca2+. Single cell measurement of exocytosis and the increase in cytosolic Ca2+ induced by bradykinin-activated intracellular stores reveal cell to cell variability in exocytotic responses which is masked in populations of cells. Taken together, these results show that exocytosis of catecholamines can be induced by an increase in cytosolic Ca2+ either as a result of transmembrane entry or by release of internal stores.

A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils.

A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.

Responses of Cytosolic Free Calcium to ADP in Platelets of Spontaneously Hypertensive Rats

Abnormalities of Ca2+ handling have been reported in patients with essential hypertension and in spontaneously hypertensive rats (SHR). In this study, responses of cytosolic Ca2+ to ADP in platelets of SHR were examined. Four- and seven-week-old male SHR and age- and sex-matched Wistar-Kyoto rats (WKY) were used. Basal levels of the intracellular Ca2+ concentration in platelets and responses to ADP were estimated using fluorescent indicator fura-2 in the medium containing 1 mmol/L CaCl2 and Ca2(+)-free buffer with 1 mmol/L EGTA. Basal levels of platelet cytosolic Ca2+ of SHR were significantly higher than those of WKY at 4 and 7 weeks of age in the presence of external Ca2+. However, no significant difference was observed in basal levels of platelet cytosolic Ca2+ in the Ca2(+)-free EGTA-containing buffer between SHR and WKY. The peak cytosolic Ca2+ concentration evoked by ADP was significantly diminished in SHR compared with WKY in the absence of external Ca2+, whereas the responses of platelet cytosolic Ca2+ to ADP were similar in SHR and WKY in the presence of external Ca2+. These results suggest that release from intracellular Ca2+ store is reduced in SHR and that the regulation of cytosolic Ca2+ in SHR is more dependent on extracellular Ca2+ compared with WKY.

Cyclic AMP enhances inositol trisphosphate-induced mobilization of intracellular Ca2+ in cultured aortic smooth muscle cells.

The effect of cAMP on ATP-induced intracellular Ca+ mobilization in cultured rat aortic smooth muscle cells was investigated. Treatment of cells for 3 min at 37 degrees C with dibutyryl cAMP, a membrane-permeable analogue of cAMP, at concentration up to 500 microM resulted in 1.5- to 1.7-fold increase in the peak cytosolic Ca2+ concentration when cells were stimulated with 3 to 200 microM ATP either in the presence or absence of extracellular Ca2+. Similar results were obtained when 0.5 mM 8-Br-cAMP or 10 microM forskolin was used instead of dibutyryl cAMP. In contrast to the Ca2+ response, dibutyryl cAMP did not affect ATP-induced formation of inositol trisphosphate (IP3). Furthermore, the dibutyryl cAMP treatment did not affect the size of the Ca2+ response elicited by 10 microM ionomycin. These results suggest that intracellular cAMP potentiates the ATP-induced Ca2+ response by enhancing Ca2+ release from the intracellular Ca2+ store(s), rather than by increasing the ATP-induced production of IP3 or by increasing the size of the intracellular Ca2+ store. Using saponin-permeabilized cells, we have shown directly that cAMP enhances Ca2+ mobilization by potentiating the Ca2+-releasing effect of IP3 from the intracellular Ca2+ store.

Effects of influenza A virus on human neutrophil calcium metabolism.

Bacterial superinfection in influenza A virus-related illness may in part be explained by virus-induced neutrophil dysfunction. We here provide evidence that this effect is related to abnormal calcium metabolism of virus-infected cells. Neutrophils exposed to influenza virus for 0.5 h at 37 degrees C showed depressed O2- generation and release of radiolabeled arachidonic acid upon stimulation with FMLP. The peak cytosolic Ca2+ level achieved by virus-infected neutrophils after FMLP stimulation was significantly depressed as is efflux of 45Ca2+. This deficient Ca2+ mobilization could not be attributed to alterations of inositol phosphate production or Ca2+ influx in response to FMLP, both of which were unaffected by prior virus infection. Given these findings, the immediate effects of influenza virus on neutrophil Ca2+ metabolism were examined. The virus itself caused a rise in cytosolic Ca2+ and an efflux of 45Ca2+ without any corresponding 45Ca2+ influx. Total cell Ca2+ however was not depleted as measured by atomic absorption. Influenza virus, therefore, causes neutrophil activation leading to significant perturbations in Ca2+ metabolism and later to impaired mobilization of Ca2+ stores. This system offers a model for phagocyte deactivation and an opportunity to define control mechanisms of signal transduction.