Differential regulation of Ca2+ influx by fMLP and PAF in human neutrophils: possible involvement of store-operated Ca2+ channel

Calcium (Ca2+) influx into human polymorphonuclear cells (PMNs) in response to N-formyl-Met-Leu-Phe (fMLP) and platelet-activating factor (PAF) stimulation was studied. Whole blood was taken by venous puncture from healthy human volunteers. PMNs were isolated, diluted, and incubated with 2 microM fura-2 AM. The cytosolic free calcium concentration, [Ca2+]i, in human neutrophils was determined by microfluorometry. We found that the net area under the fMLP- or PAF-induced [Ca2+]i rise curve in Ca2+-free medium decreased to 75% or 30% of the area under the curve in Ca2+ medium. Treatment of PMNs with phorbol myristate acetate (PMA), a protein kinase C activator, completely abolished the intracellular Ca2+ level stimulated by PAF, but not the intracellular Ca2+ level stimulated by fMLP. Treatment of PMNs with PAF did not abolish the intracellular Ca2+ level elevation stimulated by fMLP. In addition, treatment of PMNs with fMLP did not abolish intracellular Ca2+ level elevation stimulated by PAF. Loperamide, a positive modulator for store-operated calcium (SOC) channels, elicited an increase in intracellular calcium after the activation of SOC channels stimulated by fMLP or PAF. After the addition of guanosine 3',5'-cyclic monophosphate, N2,2'-O-Dibutyryl-, sodium salt (db-cGMP), the initial increase of PAF- or fMLP-induced PMNs intracellular Ca2+ fluorescences was well preserved, but the slope and the peak height of fluorescence curves declined compared with the curves without db-cGMP. In conclusion, we found that PAF and fMLP regulate the Ca2+ influx of PMNs in different ways. Most of the PAF-induced [Ca2+]i rise resulted from Ca2+ influx, and most of the fMLP-induced [Ca2+]i elevation resulted from intracellular stores release. The initial mobilization of intracellular Ca2+ stores in PAF-stimulated signals is mediated by protein kinase C (PKC) phosphorylation, but not in fMLP-stimulated route. SOC channels are present and important in the fMLP- or PAF-induced PMNs Ca2+ influx. There was no apparent cross-regulation between PAF- and fMLP-stimulated intracellular Ca2+ influx.     

Calcium ionophore, phorbol ester, and chemotactic peptide-induced cytoskeleton reorganization in human neutrophils

Formyl-methionylleucylphenylalanine (fMLP) activation of neutrophils causes an increase in intracellular Ca2+, activation of protein kinase C and an increase in F-actin content. To examine the role of Ca2+ and protein kinase C activation as determinants of change in F-actin content of neutrophils, we used the NBD-phallacidin extraction assay to compare the kinetics and extent of change in F-actin content of cells activated with fMLP, the calcium ionophore A23187 or phorbol myristate acetate (PMA). All stimuli increase the F-actin content in a dose-dependent manner; however, the rate of increase is slower and the maximum F-actin content is less for calcium ionophore and PMA than for fMLP-activated cells. The A23187-induced increase in F-actin content, but not that of fMLP, depends upon external free [Ca2+]. In A23187-activated cells, F-actin content increases at [Ca2+]free greater than or equal to 5 microM, is maximal at [Ca2+]free greater than or equal to 10 microM and is negligible at physiologic free [Ca2+] (10(-7)-10(-6) M). Combinations of PMA with A23187 or fMLP inhibit the A23187, but not the fMLP, activated actin polymerization. Comparison and combination of these activators shows that neither Ca2+-dependent activation with A23187 nor activation with PMA alone or in combination mimic the fMLP-induced changes in cytoskeleton organization of neutrophils.     

Impaired neutrophil store-mediated calcium entry in Type 2 diabetes

BACKGROUND: In Type 2 diabetes impaired neutrophil function leads to increased bacterial infection and cardiovascular disease. Many neutrophil functions depend on calcium signalling, which involves release of calcium from intracellular stores and subsequently translocation of stores via the cytoskeleton to the plasma membrane, causing store-mediated calcium entry (SMCE) into the cell. We hypothesized that in Type 2 diabetes there would be a defect in SMCE. MATERIALS AND METHODS: Neutrophils were prepared from patients with Type 2 diabetes (DM, n=15) and controls (NC, n=15). Free cytosolic calcium [Ca2+]i was measured with Fura-2 in resting cells and after stimulation of calcium release with fMLP and thapsigargin. RESULTS: Baseline [Ca2+]i was higher in neutrophils from the patients than the controls (NC 65 +/- 5 nm, DM 80 +/- 4 nm, P<0.05). However, after fMLP-treatment [Ca2+]i was significantly lower in the patients (NC 301 +/- 28 nm, DM 210 +/- 20 nm, P<0.01). The greater increase in controls was not observed when cells were treated with fMLP in the absence of extracellular calcium (-fold increase NC 2.9 +/- 0.5, DM 2.7 +/- 0.3). Treatment of cells with thapsigargin caused a similar greater increase in [Ca2+]i in the controls than in the patients that was not seen in the absence of extracellular calcium (-fold increase with Ca2+ NC 5.2 +/- 1.0, DM 3.0 +/- 0.4, P<0.05; fold increase without Ca2+ NC 2.5 +/- 0.4, DM 2.2 +/- 0.2). CONCLUSIONS: In Type 2 diabetes there is a defect in neutrophil calcium signalling which results in a lesser increase in free cytosolic calcium owing to impaired influx across the plasma membrane. Abnormal calcium signalling is likely to be important in the pathogenesis of diabetic complications.     

Relationship between membrane depolarization and extracellular calcium influx during neutrophil activation

To better define the relationship between membrane depolarization and extracellular Ca2+ influx during neutrophil activation, we compared stimulation by elevating the extracellular K+ concentration, [K+]o, with stimulation by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Elevation of [K+]o resulted in uniform depolarization of the entire population of cells. This was associated with an influx of Ca2+ that was temporally delayed and quantitatively less than that induced by fMLP. K+ depolarization also caused increased expression of type 1 (C3b/C4b) complement receptor (CR1) and type 3 (C3bi) complement receptor (CR3), but the increments were less than with fMLP. We then used pertussis toxin to determine if guanosine triphosphate (GTP)-binding proteins were involved in these responses. Toxin inhibited the fMLP-induced membrane depolarization as well as the uptake of extracellular Ca2+ and the expression of both CR1 and CR3 induced by the chemoattractant. This indicates that the fMLP receptor is not directly coupled to an ion channel. The membrane depolarization induced by elevating [K+]o was not inhibited by toxin, but the uptake of Ca2+ and the increased expression of CR1 and CR3 were all significantly inhibited. The toxin failed to block increased CR1 and CR3 expression induced by ionomycin, demonstrating that its effects were not attributable to general toxicity. The results suggest that voltage gating is not the major mechanism by which polymorphonuclear leukocytes (PMNs) increase their permeability to extracellular Ca2+. Initial signals, whether generated by chemoattractants binding to their receptors or by small initial influxes of extracellular Ca2+, must be amplified by pertussis toxin-sensitive steps to fully increase the Ca2+ permeability and optimally activate the cell.     

The effect of atrial natriuretic peptide on cytosolic free calcium in cultured vascular smooth muscle cells

Effect of atrial natriuretic peptide (ANP) on cytosolic free calcium [( Ca2+]i) was studied in monolayers of cultured vascular smooth muscle (VSM) cells loaded with a fluorescent calcium indicator, fura-2. Vasoconstrictive hormones, angiotensin II (AII) and Arg8-vasopressin (AVP) induced initial rapid rises in [Ca2+]i, followed by sustained elevation of [Ca2+]i. ANP (Atriopeptin III 10(-8) M) decreased both the resting level and the sustained elevation of [Ca2+] i induced by AII and AVP. ANP also decreased the rise in [Ca2+]i induced by high potassium (K+) depolarization. AVP-induced initial rapid rise in [Ca2+]i was not inhibited by ANP in the presence or absence of the phosphodiesterase inhibitor, isobutylmethylxanthine 0.1 mM, which has been shown to fully enhance ANP-induced cyclic GMP accumulation. On the other hand, a calcium antagonist, nicardipine, inhibited the high K+-induced rise in [Ca2+]i, whereas it had no effect on not only initial but also sustained rises in [Ca2+]i induced by AVP or AII. These results suggest that ANP has an ability to decrease [Ca2+]i not through inhibition of voltage-sensitive calcium channels, and that neither ANP nor ANP-induced cyclic GMP may affect initial hormone-induced rise in [Ca2+]i. In conclusion, an ability to decrease [Ca2+]i is implicated in ANP-induced relaxation of VSM.     

Chemotactic peptide activation of human neutrophils and HL-60 cells. Pertussis toxin reveals correlation between inositol trisphosphate generation, calcium ion transients, and cellular activation.

The mechanism of neutrophil activation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) has been studied by pretreatment of human neutrophils with pertussis toxin. Upon stimulation with FMLP, the cytosolic-free calcium concentration, [Ca2+]i, is increased both by stimulation of calcium influx and mobilization of cellular calcium. We have measured [Ca2+]i as well as the generation of the phospholipid breakdown product inositol trisphosphate (IP3), which is thought to mediate Ca2+ mobilization. As the phosphoinositide pool in human neutrophils is difficult to prelabel with [3H]myoinositol, experiments were also carried out in the cultured human promyelocytic leukemia cell line HL-60 after differentiation with dimethylsulfoxide. Pertussis toxin pretreatment of both cell types inhibited FMLP stimulated membrane depolarization, exocytosis, and superoxide production in a dose-dependent manner. This toxin effect was selective for the receptor agonist, since stimulation of these parameters by two substances bypassing the transduction mechanism, the calcium ionophore ionomycin and the phorbolester phorbol myristate acetate, were unaffected. Rises in [Ca2+]i, as well as generation of IP3 in response to FMLP, were inhibited in parallel; for the inhibition of functional responses, slightly lower toxin concentrations were required. The attentuation of the [Ca2+]i rise was more marked in the absence of extracellular calcium, i.e., when the rise is due only to calcium mobilization. The results provide evidence that phospholipase C stimulation by FMLP resulting in IP3 generation is involved in the signal transduction mechanism. Coupling of FMLP receptor occupancy to phospholipase C activation is sensitive to pertussis toxin, suggesting the involvement of a GTP binding protein (N protein), which has been shown to be a pertussis toxin substrate. The parallel changes in [Ca2+]i and IP3 further support the hypothesis that IP3 is the calcium-mobilizing mediator in FMLP-activated cells.     

Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.

Many mammalian cell types exhibit Ca2+-dependent K+ channels, and activation of these channels by increasing intracellular calcium generally leads to a hyperpolarization of the plasma membrane. Their presence in B lymphocytes is as yet uncertain. Crosslinking Ig on the surface of B lymphocytes is known to increase the level of free cytoplasmic calcium ([Ca2+]i). However, rather than hyperpolarization, a depolarization has been reported to occur after treatment of B lymphocytes with anti-Ig. To determine if Ca2+-dependent K+ channels are present in B lymphocytes, and to examine the relationship between intracellular free calcium and membrane potential, we monitored [Ca2+]i by means of indo-1 and transmembrane potential using bis(1,3-diethylthiobarbituric)trimethine oxonol in human tonsillar B cells activated by anti-IgM. Treatment with anti-IgM induced a biphasic increase in [Ca2+]i and a simultaneous hyperpolarization. A similar hyperpolarization was induced by ionomycin, a Ca2+ ionophore. Delaying the development of the [Ca2+]i response by increasing the cytoplasmic Ca2+-buffering power delayed the hyperpolarization. Conversely, eliminating the sustained phase of the [Ca2+]i response by omission of external Ca2+ abolished the prolonged hyperpolarization. In fact, a sizable Na+-dependent depolarization was unmasked. This study demonstrates that in human B lymphocytes, Ca2+-dependent K+ channels can be activated by crosslinking of surface IgM. Moreover, it is likely that, by analogy with voltage-sensitive Ca2+ channels, Na+ can permeate through these ligand-gated Ca2+ "channels" in the absence of extracellular Ca2+.     

Measurement of cytoplasmic free Ca2+ concentration in rabbit aorta using the photoprotein, aequorin. Effect of atrial natriuretic peptide on agonist-induced Ca2+ signal generation.

Addition of norepinephrine, angiotensin II, or histamine leads to a transient rise in the cytoplasmic Ca2+ concentration ([Ca2+]i), as measured with aequorin, in rabbit aortic strips. Each induces a [Ca2+]i transient which peaks in 2 min and then falls either back to baseline (angiotensin II) or to a plateau (norepinephrine and histamine). The [Ca2+]i transient is due to the mobilization of Ca2+ from a caffeine-sensitive, intracellular pool. An elevation of [K+] to 35 mM leads to a monotonic sustained rise in [Ca2+]i which depends entirely on extracellular Ca2+, but an increase to 100 mM leads to a [Ca2+]i transient from the mobilization of intracellular Ca2+. Atrial natriuretic peptide does not alter basal [Ca2+]i nor inhibit the [Ca2+]i transient induced by either histamine or angiotensin II, but blocks that induced by norepinephrine, and blocks the plateau phase induced by either histamine or norepinephrine. The peptide inhibits the contractile response to all three agonists and to K+.     

Corticotropin-releasing hormone excites adrenocorticotropin-secreting human pituitary adenoma cells by activating a nonselective cation current.

The mechanisms of corticotropin-releasing hormone (CRH) induced excitation of ACTH-secreting adenoma cells were investigated using the perforated whole-cell clamp technique and intracellular Ca2+ concentration ([Ca2+]i) measurement. CRH depolarized ACTH-secreting adenoma cells by activating a nonselective cation current that showed slight inward rectification. This channel did not seem to be a member of the Ca(2+)-activated cation currents because it was activated even when the [Ca2+]i was chelated below 50 nM. The activation of the current was induced by protein kinase A-mediated pathways. By [Ca2+]i measurement, CRH increased [Ca2+]i of these cells dependently on voltage-gated Ca2+ current. This CRH-induced [Ca2+]i increase was abolished in Na(+)-free extracellular solution, but was not abolished by the addition of 5 microM tetrodotoxin to the extracellular solution. CRH-induced ACTH secretion from the cultured adenoma cells was also abolished in Na(+)-free extracellular solution, but not in tetrodotoxin-containing extracellular solution. These data indicate that a Na+ current (maybe the nonselective cation current) other than voltage-gated Na+ current plays an important role in CRH-induced [Ca2+]i increase and ACTH secretion. CRH also activated a nonselective cation current in nonadenoma human corticotrophs, suggesting that the activation of a nonselective cation current is a physiological mechanism of CRH-induced excitation in human corticotrophs.     

Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation

The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti- CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.     

Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes.

We evaluated CD2 (E rosette) and CD3 (T3)-triggered activation of resting lymphocytes by measuring the intracellular free calcium concentration ([Ca2+]i) of individual cells. The [Ca2+]i of indo-1-loaded cells was measured by flow cytometry and responses were correlated with cell surface phenotype. Stimulation with anti-CD3 antibody caused an increase in [Ca2+]i in greater than 90% of CD3+ cells within 1 min, and furthermore, the response was restricted to cells bearing the CD3 marker. In contrast, stimulation of cells with anti-CD2 antibodies produced a biphasic response pattern with an early component in CD3- cells and a late component in CD3+ cells. Thus, the CD2 response does not require cell surface expression of CD3. In addition, stimulation of a single CD2 epitope was sufficient for activation of CD3- cells, whereas stimulation of two CD2 epitopes was required for activation of CD3+ cells. Both the CD2 and CD3 responses were diminished in magnitude and duration by EGTA. However, approximately 50% of T cells still had a brief response in the presence of EGTA, indicating that the increased [Ca2+]i results in part from intracellular calcium mobilization, and furthermore demonstrates that extracellular calcium is required for a full and sustained response. Our results support the concept that CD2 represents the trigger for a distinct pathway of activation both for T cells that express the CD3 molecular complex and for large granular lymphocytes that do not.     

Phospholipase C activation by Na+/Ca2+ exchange is essential for monensin-induced Ca2+ influx and arachidonic acid release in FRTL-5 thyroid cells.

BACKGROUND: Monensin, a Na+ ionophore, can increase cytosolic Ca2+ ([Ca2+]i) by reversing the Na+/Ca2+ exchange mechanism. This study provided additional insights into the mechanism of this Na+ ionophore-induced increase in [Ca2+]i, and emphasized the critical role of phospholipase C (PLC) in amplifying Na+/Ca2+ exchange-induced Ca2+ influx and subsequent arachidonic acid (AA) release in FRTL-5 thyroid cells. The possible involvement of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and GTP-binding (G) protein in mediating monensin-induced AA release was also explored. METHODS: FRTL-5 thyroid cells were maintained in Coon's modified Ham's F-12 medium supplemented with a 6-hormone (6H) mixture. Cytosolic Ca2+ was measured by using indo-1 AM and a dual-wave-length spectrofluorometer. Release of 3H-labeled inositol trisphosphates and arachidonic acid were determined by a scintillation counter. RESULTS: In Hank's balanced salt solution with Ca2+ (HBSS+), monensin (100 mumol/L) induced a 2.3-fold sustained Ca2+ increase associated with IP3 generation and a 6-fold increase in AA release. Deletion of extracellular Ca2+, or replacement of Na+ by choline chloride in the medium, reduced the [Ca2+]i increase by 77% and completely prevented the monensin-induced rise in AA release. Similar inhibitory effects were observed in cells pretreated with a Na+ channel blocker, or Na+/Ca2+ exchange inhibitors. In HBSS without Ca2+ (HBSS-), monensin induced a 1-fold transient [Ca2+]i increase but did not increase the AA. This Ca2+ increase was not suppressed by U-73122, a PLC inhibitor. In HBSS+, U-73122 did not affect the monensin-induced initial transient peak increase of [Ca2+]i, but reduced the sustained second phase of [Ca2+]i from 400 nmol/L to 250 nmol/L, and completely blocked AA release. A phospholipase A2 (PLA2) inhibitor blocked the monensin-induced AA release without affecting the [Ca2+]i increase. Inhibition of PKC prevented 87% to 94% of the monesin-stimulated AA release. The monensin-induced AA release was also inhibited 94% by pertussis and 51% by a MAP kinase cascade inhibitor. CONCLUSIONS: The results suggest that monensin initiates an increase in [Ca2+]i via a Na+/Ca2+ exchange mechanism that triggers more pronounced and sustained [Ca2+]i increase via activation of PLC and Ca2+ influx. The PLC activation, followed by sustained Ca2+ influx and PKC activation, is a prerequisite for PLA2-mediated processes in monensin-challenged FRTL-5 thyroid cells.     

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.     

Synergism of platelet-aggregating agents. Role of elevation of cytoplasmic calcium.

When a pair of platelet agonists, each in subthreshold concentration, is added together or in sequence to a platelet suspension, the platelet response is enhanced. Addition of two agonists to platelets loaded with aequorin also enhanced the observed rise in cytoplasmic ionized calcium ([Ca2+]i) in response to the second agonist if the agonists were added within 20 s of each other. Enhancement of aggregation and secretion required that an increase in [Ca2+]i (as indicated by aequorin but not necessarily indo-1) followed the first agonist, but not that the [Ca2+]i remain elevated until addition of the second agonist. Enhancement was not prevented by aspirin, ADP scavengers, or chelators of extracellular Ca2+. We conclude that a rise in [Ca2+]i induced by a first agonist "primes" platelets for an augmented functional response to a second agonist, which is not, however, determined by the [Ca2+]i at the time of addition of the second agonist.     

Halothane increases cytosolic Ca2+ and inhibits Na+/H+ exchange in L6 muscle cells

The effects of the general anesthetic halothane on the concentration of cytosolic free calcium ([Ca2+]i) and cytosolic pH (pHi), were investigated in L6 rat skeletal muscle cells. Basal [Ca2+]i was 169 +/- 8 nM, measured with the fluorescent Ca2(+)-indicator 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5- methylphenoxy)ethane-N,N,N',N'-tetra-acetate. Halothane (5.7 mM) increased [Ca2+]i to 225 +/- 15 nM in the presence of extracellular Ca2+, and from 137 +/- 6 nM to 179 +/- 9 nM in Ca2+ absence. This increase was dose-dependent. The anesthetic released about 50% of the releasable Ca2+ from intracellular stores. The resting pHi of L6 cells was 7.24 +/- 0.04, measured with the fluorescent pH indicator bis-carboxyethylcarboxyfluorescein. Halothane did not affect resting pHi, but inhibited cytoplasmic alkalinization by hypertonicity or cytoplasmic acidification: (1) The hypertonicity-induced alkalinization via activation of Na+/H+ exchange (to 7.50 +/- 0.08, initial rate 0.10 +/- 0.02 pH U/min) was inhibited with 5.7 mM halothane by 67%. (2) Acid-loaded cells (pHi 6.43 +/- 0.01 in cells) recovered towards neutrality via activation of Na+/H+ exchange (rate 0.47 pH U/min), and halothane inhibited the rate of pHi recovery by 50%. The halothane-mediated inhibition of alkalinizations after hypertonic exposure or acid-loading was also observed in bis-(o-amino-phenoxy)ethane-N,N,N',N'-tetra-acetate-loaded cells in Ca2(+)-free medium. Therefore, halothane increases [Ca2+]i and in parallel inhibits Na+/H+ exchange, compromising the ability of muscle cells to recover from imposed acidification.     

Interactions between alkylglycerols and human neutrophil granulocytes

We evaluated whether various alkylglycerols would initiate a functional response of human neutrophils or modify responses induced by a formyl peptide (fMLP) in vitro. We found that platelet activating factor (PAF) was the most potent with regard to the ability to produce an oxidative response (assessed by cytochrome c reduction and/or chemiluminescence), followed by ET-16-OCH3. Lyso-PAF, ET-18-OCH3, batyl- and chimyl alcohols exhibited only a weak activity. PAF was also the most efficient lipid conferring a rise of intracellular calcium concentrations ([Ca2+]i). ET-16-OCH3, ET-18-OCH3 and lysoPAF were less potent, although maximal [Ca2+]i levels were similar to that of 0.1 mumol/l fMLP. The kinetics of the calcium responses were highly specific for each ether lipid. When neutrophils had been treated with PAF or ET-18-OCH3 and were subsequently stimulated by fMLP, enhancement of the oxidative response was noted. Thus, this study shows that there was an association between the ability of an alkylglycerol to initiate oxidative and calcium responses, indicating strict structure-activity relationships for these lipids.     

Simultaneous measurements of cytosolic free calcium level and prostaglandin synthesis reveal a correlation between them in perfused monolayer of cultured rat vascular smooth muscle cells: effects of bradykinin and angiotensin II.

The level of cytosolic free calcium ([Ca2+]i) and the production rate of prostacyclin were simultaneously measured in perfused monolayers of cultured vascular smooth muscle (VSM) cells. After loading of fura-2 (a fluorescent calcium indicator), the monolayer of VSM cells (cultured on a cover glass) was fixed in the perfusion cuvette and the cuvette was placed in a fluorometer to monitor the change in [Ca2+]i. The monolayer was perfused and the fractionated perfusion solution was collected to determine 6-keto-PGF1 alpha (a metabolite of prostacyclin) production found in the solution. Afterwards, the time-dependent changes in [Ca2+]i and 6-keto-PGF1 alpha synthesis were compared. Bradykinin (BK, 10(-6) M), angiotensin (Ang) II (10(-7) M) as well as ionomycin (10(-6) M) induced simultaneous increases in [Ca2+]i and 6-keto-PGF1 alpha production. An inhibitor against prostaglandin synthesis, acetylsalicylic acid (ASA, 10(-6) M) abolished BK-induced 6-keto-PGF1 alpha synthesis, whereas ASA did not affect the increase in [Ca2+]i. BK-induced increases in [Ca2+]i and 6-keto-PGF1 alpha production occurred in a dose-dependent manner and the half-maximal response was observed at the same concentration of BK (10(-7) M). These results indicate that an increase in [Ca2+]i is closely associated with BK as well as AngII-induced prostacyclin synthesis. It is suggested that an increase in [Ca2+]i plays a prior role in prostacyclin synthesis. Thus, an interaction between phospholipase A2 (prostaglandin synthesis) and phospholipase C (inositol trisphosphate-Ca2+ mobilization) is suggested.     

Electrophysiology and calcium signalling in human bronchial smooth muscle

Recently, cells isolated from airways have been used to characterize precisely the electrophysiological properties of this smooth muscle and to describe the changes in cytosolic calcium concentration ([Ca2+]i) occurring upon agonist stimulation. Although most studies have produced consistent results in terms of types of ion channel and pathways of calcium signalling implicated in the mechanical activity of airways, there are differences according to (i) the site along the bronchial tree (trachea vs. bronchi); (ii) the proliferating status of the cells (freshly isolated vs. cultured) and (iii) the species (human vs. animals). With regard to the electrophysiological properties of airway smooth muscle, the contribution to [Ca2+]i rise of Ca2+ influx through L-type voltage-dependent calcium channels depends on the balance between depolarization related to non-specific cation channel and/or chloride channel activation and hyperpolarization related to activation of a variety of potassium channels. Most of the above-mentioned channels appear to be controlled, directly or indirectly, by agonists in human bronchial smooth muscle. With regard to calcium signalling, the pattern of agonist-induced [Ca2+]i responses, the so-called [Ca2+]i oscillations, has been observed recently in freshly isolated airway smooth muscle cells. The role and the calcium sources involved in these oscillations in human bronchial smooth muscle are currently being investigated.     

Altered calcium regulation and function of human neutrophils during multiple trauma

Altered intracellular Ca2+ concentration is a pivotal regulatory mechanism of leukocyte function. Since polymorphonuclear neutrophils (PMN) are involved in traumatic organ dysfunction, we prospectively investigated Ca2+ regulation and function of circulating PMN multiple trauma patients (Group A: ISS < 27; Group B: ISS > or = 27). Circulating PMN were isolated during 12 days, followed by determination of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced PMN-superoxide production (PMN-SOP) by SOD-inhibitable ferricytochrome C reduction, and PMN cytosolic Ca2+ concentration ([Ca2+]i) by fluorescent fura2/AM (340/380 ratio). PMN-SOP was significantly higher in Group B (mean ISS: 39.9 +/- 2; n = 21) at day of admission than in controls and Group A (mean ISS: 18.2 +/- 1; n = 22) (P< 0.05). In Group B, the significant rise of basal [Ca2+]i between Day 2 and Day 4 was associated with significant lower PMN-SOP during that period (P < 0.05). The fMLP-induced [Ca2+]i response was supranormal in both groups. PMN-elastase concentrations were substantially higher in Group B compared with Group A until Day 4. Circulating IL-6, IL-8, and soluble TNF-receptor (55 kD) were significantly increased in Group B compared with Group A at the day of trauma (P < 0.05). Severe trauma is characterized by a biphasic pattern of neutrophil priming characterized by early increase and secondary suppression. The association of depressed neutrophil superoxide production (deactivation) and elevated basal [Ca2+]i suggests Ca2+-mediated disturbance of neutrophil NADPH-oxidase metabolism.