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(Journal of Leukocyte Biology. 2001;69:63-68.)
© 2001 by Society for Leukocyte Biology

PAF-mediated Ca2+ influx in human neutrophils occurs via store-operated mechanisms

Carl J. Hauser, Zoltan Fekete, John M. Adams, Matthew Garced, David H. Livingston and Edwin A. Deitch

Department of Surgery, Division of Trauma, University of Medicine and Dentistry of New Jersey/New Jersey Medical School, Newark, New Jersey

Correspondence: Carl J. Hauser, M.D., FACS, UMDNJ/New Jersey Medical School, Department of Surgery, MSB G-524, 185 South Orange Avenue, Newark, NJ 07103. E-mail: hausercj{at}UMDNJ.edu


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ABSTRACT
 
Many inflammatory mediators activate neutrophils (PMN) partly by increasing cytosolic calcium concentration ([Ca2+]i). Modulation of PMN [Ca2+]i might therefore be useful in regulating inflammation after shock or sepsis. The hemodynamic effects of traditional Ca2+ channel blockade, however, could endanger unstable patients. Store-operated calcium influx (SOCI) is known now to contribute to Ca2+ flux in "nonexcitable" cells. Therefore, we studied the role of SOCI in human PMN responses to the proinflammatory ligand PAF. PMN [Ca2+]i was studied by spectrofluorometry with and without external calcium. We studied the effects of PAF on Mn2+ entry into and on Ca2+ efflux from thapsigargin (Tg)-treated cells. Influx was assessed in the presence and absence of the blockers SKF-96365 (SKF), TMB-8, and 2-APB. Half of PAF [Ca2+]i mobilization occurs via calcium influx. The kinetics of calcium entry were typical of SOCI rather than receptor-mediated calcium entry (RMCE). SKF had multiple nonspecific effects on [Ca2+]i. Inhibition of store emptying by TMB-8 and 2-APB blocked all calcium entry, demonstrating influx was store depletion-dependent. PAF has no direct effect on calcium efflux. Where SOCI is maximal, PAF has no further effect on calcium-channel traffic. PAF-induced calcium signals are highly dependent on SOCI and independent of RMCE. SOCI-specific blockade might modulate PMN-mediated inflammation and spare cardiovascular function in shock and sepsis.

Key Words: platelet-activating factor • calcium channels • store-operated influx • G protein-coupled receptors • neutrophils • inflammation


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INTRODUCTION
 
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a lipid autocoid, which plays a role in polymorphonuclear neutrophil (PMN) adhesion, chemotaxis, granule release, and oxidative burst [reviewed in ref. 1 ]. PMN and endothelial cells (EC) synthesize PAF after inflammatory stimuli. PAF stimulates the synthesis of interleukin (IL)-8 and leukotrienes [2 , 3 ], which may then stimulate further production of PAF [4 ]. Because PAF can elicit and perpetuate inflammatory PMN-EC interactions, clinical trials of PAF antagonism in sepsis and the Systemic Inflammatory Response Syndrome are now under way [5 , 6 ].

PAF mobilizes cytosolic-free calcium ([Ca2+]i) from inositol 1,4,5 triphosphate (InsP3)-sensitive endoplasmic reticulum (ER) calcium stores via a G protein-coupled, phospholipase C-InsP3 pathway. PAF initiates Ca2+ entry also into cells via channels [7 ]. Where early studies suggested PAF-calcium entry was a delayed event, subsequent studies suggest that receptor-mediated calcium entry (RMCE) was dominant in PAF signaling [8 9 10 ]. Thus, the specific Ca2+ entry mechanisms involved in PMN-PAF responses are unclear. Because myeloid cells lack voltage-operated channels, calcium entry can occur only via RMCE or store-operated calcium influx (SOCI) [11 , 12 ].

This distinction has important clinical implications: PMN Ca2+ signaling is altered in infection and inflammation [13 , 14 ]. Ca2+ signaling and SOCI are altered by human injury [15 16 17 ]. In animals, calcium blockade decreases mediator production and mortality in sepsis and hemorrhage [18 , 19 ]. Calcium-channel blockade with clinically available agents, however, is associated with diminished cardiac contractility and vasomotor suppression. A precise understanding of Ca2+ signaling mechanisms in PMN is therefore needed to optimize pharmacologic strategies targeting PAF in inflammatory states.

Early studies suggesting RMCE is important in PMN-PAF responses used nickel ion (Ni2+) to inhibit RMCE [20 ], but it is now clear that Ni2+ inhibits SOCI also [21 22 23 ]. Thus, recent studies [10 ] have used SKF-96365 (SKF) as a specific inhibitor of RMCE, but other studies suggest SKF inhibits SOCI also [24 , 25 ]. Because available studies do not differentiate between PAF-activated, calcium-influx pathways, we studied PAF-activated, calcium-influx pathways by novel means.


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MATERIALS AND METHODS
 
Neutrophil isolation
Whole-blood samples were obtained from healthy volunteers. PMN were isolated immediately by a one-step gradient centrifugation method using polymorphoprep (PMP; Robbins Scientific Corp., Sunnyvale, CA). Briefly, heparinized (10 U/ml) whole blood was centrifuged at 150 g for 10 min. The upper platelet-rich plasma layer was aspirated and discarded. The buffy coat and top 2 cm of red blood cells (RBC) were then collected, layered onto 5 ml PMP, and centrifuged at 300 g for 30 min. Supernatants and the mononuclear cell layer were discarded. The neutrophil layer was aspirated, diluted with an equal volume of 0.45% NaCl solution, and allowed to rest 5 min so as to restore normal osmolarity. Suspensions were diluted with sufficient RPMI (Mediatech, Herndon, VA) to give a final vol of 15 ml and centrifuged at 150 g for 10 min. Neutrophil pellets were then resuspended in 2 mL buffer solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 0.1% fatty acid-free bovine serum albumin (pH=7.4; HEPES buffer). PMN were counted on a flow cytometer and kept on ice until dye-loaded for study.

Dye loading and preincubation
Our methods for studying PMN [Ca2+] have been published previously [26 , 27 ] and are described here only briefly. PMN were incubated in 2 µM fura-2-acetoxymethyl ester (fura-2AM; Molecular Probes Inc., Eugene, OR) for 30 min at 37°C in the dark. Cells were then divided into 150 µl aliquots, returned to the dark, and put on ice. Just prior to each experiment, individual aliquots were returned to a 37°C bath to incubate in buffer under the conditions discussed below. Prior to use, cells were centrifuged 5 sec at 4500 RPM in a programmable microcentrifuge. The supernatants are removed, and the cells are resuspended in 100 µl HEPES buffer with or without 1 mM CaCl2 and other agents used for that experiment. All experiments done in nominally calcium-free media contain 0.3 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA). Resuspended aliquots are injected finally into cuvettes containing 2.9 ml of the same buffer for study.

Spectrofluorometry
Intracellular Ca2+ was monitored by measuring fura fluorescence at 505 nm, using 340/380 nm excitation in a Fluoromax-2 spectrofluorometer (Jobin-Spex, Edison, NJ) with constant stirring at 37°C. Calibration is performed at the end of every experiment by treating PMN with 100 µM digitonin (Molecular Probes) and then measuring the fura fluorescence in 1 mM (Rmax) and zero calcium (15 mM EGTA) solutions (Rmin). The fluorescence of a sample cell suspension treated with 100 µM digitonin and 2 mM MnCl2 was subtracted from total fluorescence. [Ca2+]i was calculated from the 340/380 nm fluorescence ratio using our modifications [28 ] of the methods of Grynkiewicz et al. [29 ]. Using our terminal resuspension technique, dye leakage has minimal influence on [Ca2+]i calculations, but dye leakage was nonetheless measured routinely and corrected for. The order in which PMN isolates were studied was alternated to avoid bias related to the duration of dye loading or the time of cell study.

Quantification of calcium flux
Peak [Ca2+]i responses to PAF as well as net [Ca2+]i flux over 2 min were recorded for each experiment performed. Net calcium flux was defined as the area (in nM · s) above the mean baseline [Ca2+]i and under the [Ca2+]i response curve for 120 sec (AUC120) after stimulation (Fig. 1 ). This reflects the net influence of free cytosolic calcium over time. All calculations were performed using an automated software package (GRAMS/32, Galactic Industries, Salem, NH). PAF [Ca2+]i flux in human PMN is characterized by a prolonged post-peak plateau [16 ], which generates a substantial portion of the net [Ca2+]i response. Thus, shorter studies do not represent the total effect of PAF on PMN [Ca2+]i. Conversely, PMN specimens deteriorate over time after fura loading. The 120-sec study period was chosen as a compromise, studying each cell aliquot long enough to observe late-calcium mobilization and completing study of each PMN isolate within 30–45 min.



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  		Figure 1. Illustration of the area under the [Ca2+]i signaling curve as assessed for 120 sec in response to 10 nM PAF stimulation (AUC120). This figure shows the [Ca2+]i signal trace from a single human PMN isolate with the area to be integrated hatch-marked. Note that the area above baseline [Ca2+]i is used rather than the area above the x-axis, because the aim is to assess net response to the agonist rather than total calcium activity.

Cell stimulation
Fura-loaded PMN pretreated as described below were resuspended in the appropriate media. Basal [Ca2+]i was recorded for 20 sec (Fig. 1) . The PMN were then exposed to 10 nM PAF (Calbiochem Corp., San Diego, CA) in the cuvette using constant stirring. Preliminary data demonstrated that 10 nM PAF was an EC90 dose for PMN [Ca2+]i responses in our system.

Strategies used to evaluate calcium flux

Kinetic studies of [Ca2+]i
AUC120 was compared in PMN stimulated by PAF in 1 mM calcium (Ca+) or in calcium-free (Ca-) media (0.3 mM EGTA added). The response to PAF in Ca- conditions reflects only cell calcium-store release. By mathematically subtracting the kinetic calcium curves of PMN studied in Ca- conditions from the response of identical PMN aliquots stimulated in Ca+ conditions, we can derive the kinetic curves of net calcium entry into the cells (Fig. 2 ). The size, timing, and morphology of these derived calcium-entry curves can then be studied.



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  		Figure 2. Combined curves (n=6 paired experiments) for human PMN [Ca2+]i responses to PAF in control (Ca+, 1 mM Ca2+) and calcium-free (Ca-, EGTA added) conditions. Responses in Ca+ show net calcium flux in response to PAF. Results in Ca- conditions reflect only the release of intracellular stores after PAF. The third (lowest) curve was derived by arithmetically subtracting the Ca- from the Ca+ curve first for each of the six paired experiments. The resulting {triangleup}[Ca2+]i curves were then combined into a single mean influx curve. This curve reflects influx of extracellular calcium in response to PAF. Peak influx occurs 20–30 sec after peak [Ca2+]i, typical for SOCI. Note that in this diagram, the error bars are + SE for ease of reading the figure only.

Store-operated calcium entry can be isolated also and observed as a discrete event by stimulating cell aliquots in a Ca- environment, waiting for [Ca2+]i to return to baseline, and then adding calcium to the medium [12 , 30 ]. This maneuver (Fig. 3 ) separates stimulatory events into rapid, receptor-linked calcium currents and later calcium-entry currents that depend on store depletion.



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  		Figure 3. Responses of normal human PMN stimulated by 10 nM PAF in calcium-free media with later recalcification compared with their responses when stimulated in Ca+ media (representative traces, n=4 paired experiments). In all cases, delayed and immediate calcium influx (heavy arrows) was of the same magnitude. This suggests calcium influx from the medium is independent of the presence of calcium in the medium in the early time period when RMCE would be expected to occur.

Because enhanced PMN calcium efflux as a result of PAF exposure could obscure the presence of a PAF-related RMCE current, it was important to show that PAF had no direct effect on calcium efflux. Efflux was studied by using Ca2+ adenosinetriphosphatase (ATPase)-inhibitor thapsigargin (Tg; 100 nM) to block ER calcium reuptake, thus also elevating [Ca2+]i and depleting ER calcium stores. Tg-treated PMN suspensions are then treated with excess EGTA (Fig. 4 ). Under these conditions, external Ca2+ falls to zero instantaneously, and a kinetic [Ca2+]i curve is generated that reflects calcium efflux. Simultaneous addition of PAF along with the EGTA will reveal enhanced efflux if PAF stimulation is linked directly to the opening of an efflux channel.



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  		Figure 4. Calcium efflux from normal human PMN. In this representative experiment (n=3 paired studies), Tg-treated PMN in Ca+ media were treated with EGTA (to initiate efflux) or EGTA plus 10 nM PAF. Under these conditions, differences in calcium efflux attributable to PAF will be observed as a divergence of the efflux curves.

Manganese (Mn2+) influx
SOCI channels were activated maximally by pretreating fura-loaded PMN with the Ca2+ ATPase-inhibitor Tg (100 nM) for 10 min in Ca2+-free media [12 ]. Cell suspensions were then placed in cuvettes as below. After a baseline period, the cells were treated with 200 µM Mn2+ or 200 µM Mn2+, given simultaneously with 10 nM PAF (Fig. 5 ). In these experiments, cells were illuminated at 360 nm, removing any influence of the [Ca2+]i on observed fluorescence. Mn2+ entry through Ca2+ channels was detected as the quenching of fura fluorescence at 505 nm. When Tg depletes calcium stores, SOCI occurs in a functionally complete "all-or-none" fashion [31 32 33 ]. If Mn2+ enters Tg-treated PMN more rapidly in the presence of PAF, the difference represents the opening of PAF receptor-operated channels [22 ].



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  		Figure 5. Mn2+ influx in response to PAF. In this representative experiment (n=3 paired studies) fura-loaded human PMN were pretreated with 100 nM Tg for 10 min in calcium-free medium. After equilibration in the cuvette for 20 sec, 200 µM MnCl2 is added with or without 10 nM PAF. Mn2+ enters PMN via channels used by Ca2+ normally but quenches fura fluorescence. Note that quenching of fura fluorescence by Mn2+ entering the cells proceeds at the same rate with or without PAF. FI, fluorescence intensity.

Inhibitor studies
Three different blocking strategies were used to try to isolate the sources of PMN calcium influx. SKF (Calbiochem Corp.) was used (30 µM/5 min) because of its common use as an inhibitor of RMCE [34 , 35 ]. However, SKF has been shown to affect calcium reuptake [36 ] and SOCI [24 , 25 ] also. We studied PMN responses also after preincubation (100 µM, 8 min) in 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; Calbiochem Corp.). Rather than targeting calcium channels, TMB-8 stabilizes Ca2+ binding to ER-storage proteins, thus inhibiting store release in response to InsP3 [37 ]. TMB-8 prevents depletion of InsP3-sensitive stores, thus inhibiting SOCI. After TMB-8, any [Ca2+]i responses to PAF in Ca+ media should represent RMCE, and the absence of [Ca2+]i influx after TMB-8 will infer the absence of RMCE. Some studies have suggested that TMB-8 has other nonspecific metabolic effects [38 ], but TMB-8 has not been shown to interfere with calcium-channel traffic [34 ]. Last, we used 2-aminoethoxydiphenyl borate (2-APB; Calbiochem Corp.), a cell-permeant inhibitor of the InsP3 receptor (100 nM for 3 min). Currently, this agent is believed to act specifically on the InsP3 receptor [39 , 40 ]. As with TMB-8, any [Ca2+]i response to PAF in the presence of 2-APB should represent RMCE, and the absence of a response should infer the absence of any direct receptor-mediated entry.

Statistical analysis
Responses seen under the various study conditions were always compared in paired PMN aliquots isolated from the same donation. The grouped observations were analyzed using paired t-tests. Statistical significance was accepted at p-values <= 0.05. Individual [Ca2+]i response curves were combined into mean response curves using Sigma Plot and Sigma Stat software. Combined response curves are portrayed in the figures as mean ± SE, with the exception of Figure 2 , where overlapping graphed data are presented as mean + SE for clarity.


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RESULTS
 
Kinetic [Ca2+]i studies
Mean PMN responses to 10 nM PAF in Ca+ (upper-most curve) and Ca- conditions (middle curve) are shown in Figure 2 . When the responses in Ca+ and Ca- conditions are compared, the slope of the initial calcium upstroke is noted to be identical. [Ca2+]i rose longer typically in Ca+ conditions, however, with peak [Ca2+]i values taking longer to reach and being higher than those observed in the absence of external calcium. Net 2 min calcium flux (AUC120) in response to PAF in Ca+ media was 15,844 ± 1223 nM · s. It was 6909 ± 1517 nM · s in Ca- conditions or only 44% of the [Ca2+]i flux seen in Ca+ medium (p=0.00004). [Ca2+]i had almost returned to baseline after 2 min in Ca- conditions, whereas it was still elevated in Ca+. Thus, about half of total PMN calcium flux in response to PAF is generated from influx mechanisms, and Ca2+ influx was responsible for sustaining PAF responses.

We then derived PMN calcium-influx curves for each individual PMN sample by mathematically subtracting the [Ca2+]i curve observed in Ca- conditions (i.e., store release) from the curve obtained using the same cells in Ca+ conditions (i.e., total [Ca2+]i mobilization). The derived influx curves were then used to model a single mean response curve (Fig. 2 , lowest tracing). As seen, PMN calcium influx in response to PAF is slow compared with net [Ca2+]i flux, lagging about 10 sec behind maximal store release and continuing for more than 20 sec after store release has ceased.

In the next studies, identical PMN aliquots were stimulated by PAF in Ca+ media or in Ca- media, where Ca2+ was reintroduced after complete resolution of the initial PAF transient (Fig. 3) . Under these conditions, the magnitude of the [Ca2+]i flux seen on late recalcification was identical essentially to the difference seen in immediate [Ca2+]i flux in the Ca+ and Ca- conditions (<10% variation in all cases, n=4 isolates from different volunteers).

When calcium efflux was assessed by rapidly chelating external calcium after Tg treatment (Fig. 4) , we found that the observed calcium-efflux curves were indistinguishable whether or not PAF was added at the same time as the chelating agent (n=3 isolates from different volunteers).

Mn2+ influx
Mn2+ influx studies were performed in fura-loaded, Tg-treated PMN (n=3 isolates from different volunteers) in Ca2+-free media. These revealed that the quenching of fura fluorescence by addition of Mn2+ alone or by Mn2+ added simultaneously with PAF was indistinguishable in all cases (Fig. 5) . The difference in percent decline of observed fluorescence in the paired samples (Mn2+ alone or Mn2+ plus PAF) was <5% at 30 and 60 sec in all cases.

Inhibitor studies
PMN blocked with SKF in Ca+ medium (Fig. 6 ) demonstrated an AUC120 of 7074 ± 1291 nM · s after PAF. This was 45% of the AUC120 measured in control Ca+ conditions (Fig. 2 p=0.005). Thus, the AUC120 for PAF in Ca+/SKF+ was very similar to the AUC120 after PAF in Ca-/SKF- (p=0.92, shown in Figs. 2 and 6 ). Nonetheless, despite very similar net mobilization of calcium, there were obvious differences in calcium-flux morphology under these two conditions. First, PMN treated with SKF in Ca+ media (Fig. 6) have a delayed and depressed [Ca2+]i upstroke compared with untreated PMN in Ca- media (Figs. 2 3 and 6) . Second, SKF prolonged the elevation of PMN [Ca2+]i by PAF markedly, with an absence of measurable Ca2+ efflux observed during the second minute after PAF stimulation (Fig. 6) . These findings suggested unexpected effects of SKF on Ca2+ store-release. Therefore, we evaluated a separate series of PMN isolates (n=5) stimulated in Ca- media in the presence or absence of SKF (Fig. 7 ). We found SKF suppressed (p<0.05) and delayed (p<0.03) PMN [Ca2+]i store-release responses to PAF.



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  		Figure 6. Responses of normal human PMN (n=6 paired experiments) to 10 nM PAF in Ca- conditions contrasted to their responses to PAF in Ca+ conditions after incubation in SKF. Note that although the two conditions yield similar AUC120, their morphology is very different. Even in Ca+ conditions, SKF delays and flattens the PMN [Ca2+]i transient. [Ca2+]i decay appears markedly impaired by SKF also. The Ca-/SKF- data here are presented in Figure 2 also.



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  		Figure 7. Responses of normal human PMN (n=5 PMN isolates) to 10 nM PAF in calcium-free media in the presence or absence of SKF, which suppressed [Ca2+]i responses even in the absence of extracellular calcium, indicating it suppresses calcium-store release. Responses in Ca-/SKF- conditions appear similar to those seen in Figures 2 and 6 but are from different isolates.

PMN, pretreated with TMB-8 to block calcium release from ER stores and then exposed to 10 nM PAF in Ca+ medium, demonstrated that their [Ca2+]i responses to PAF were attenuated markedly (Fig. 8 ). Similarly, PMN pretreated with 2-APB (Fig. 9 ) to inhibit ER InsP3 receptors and then exposed to 10 nM PAF in Ca+ medium demonstrated essentially absent [Ca2+]i responses (4±3 nM, n=3 isolates from different volunteers) to PAF.



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  		Figure 8. PMN responses (n=6 paired experiments) to 10 nM PAF in Ca+ medium in the presence of TMB-8, which stabilizes cell calcium stores. Note that PMN [Ca2+]i responses to PAF were abolished almost totally by TMB-8.



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  		Figure 9. Representative trace of PMN responses to 10 nM PAF (n=3 paired experiments) in Ca+ medium in the presence of the InsP3 inhibitor 2-APB. PMN [Ca2+]i responses to PAF were totally abolished by 2-APB.


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DISCUSSION
 
These studies demonstrate that about half of total PMN calcium flux in response to PAF is a result of calcium entry from the environment. Multiple findings, however, show that this calcium influx cannot be attributed to a RMCE mechanism. First, initial rises in cell calcium after PAF occur at identical rates whether calcium is in the environment or not (Fig. 2) . This makes a direct receptor-linked, channel-opening event unlikely. Second, entry of Ca2+ from the medium begins only after store-depletion has begun. Influx peaks 20–30 sec later and then continues for 30–45 sec (Fig. 2) . During the later phases of the PMN response to PAF (i.e., after the first minute), the majority of PMN calcium-flux response is the result of calcium influx. Thus, the time course observed is typical of SOCI not RMCE.

In addition, when store-operated currents are observed in a delayed fashion by recalcifying the media after the resolution of early calcium flux, the magnitude of this delayed calcium-uptake event is found to be equivalent to the magnitude of calcium influx observed concurrent with store release (Fig. 3) . Further, these findings cannot be attributed to any occult calcium-efflux event, which is initiated or inhibited by PAF directly (Fig. 4) .

The inability of PAF to elicit divalent cation channel traffic over and above SOCI elicited by Tg was confirmed separately in Mn2+ influx experiments. Tg depletes the endoplasmic reticulum of Ca2+ and causes maximal SOCI [12 , 22 , 31 32 33 ]. Mn2+ moves freely through calcium channels but quenches rather than augments fura fluorescence. Thus, under the experimental conditions used, if PAF were to open a Ca2+ influx pathways other than the SOCI elicited by Tg, the rate of entry of Mn2+ into the cells (and thus of fura quenching) would be increased. As is appreciated in Figure 5 , PAF fails to augment Mn2+ entry into the cells.

We used a variety of inhibitor strategies to demonstrate that PAF-induced PMN calcium influx occurs through SOCI. Our first strategy was to use SKF. This agent has been used as a "specific" blocker of RMCE and has been used recently as a proof of PAF-induced RMCE in PMN [10 ]. As has been suggested by others [36 ], however, we found that SKF has a variety of nonspecific effects that made its effects on calcium influx here uninterpretable. These included inhibition of calcium reuptake (Fig. 6) and suppression of agonist-mediated Ca2+ release from ER stores (Fig. 7) .

The other inhibitors used do not act on calcium channels [34 ]. TMB-8 exerts its actions via stabilizing the binding of calcium to ER storage proteins [37 ]. Thus, TMB-8 should prevent store-emptying and hence abolish SOCI. Similarly, 2-APB acts through inhibiting the InsP3 receptor. Although it acts at a different site, this agent should prevent store depletion also and thus block SOCI. In effect, therefore, increases in [Ca2+]i in response to PAF in the presence of TMB-8 or 2-APB should represent RMCE. Moreover, because SOCI tends to occur in an "all or none" fashion [31 , 33 ], inhibition of SOCI by these agents should be marked or complete. In fact, we found that PMN calcium flux in response to PAF was abolished almost completely after TMB-8 (Fig. 8) and 2-APB (Fig. 9) . These findings support the kinetic calcium-flux data independently and Mn2+ influx studies in suggesting that RMCE does not occur after PAF stimulation.

Considered together, the data demonstrate that PAF does not elicit measurable RMCE in normal human PMN. Rather, the data suggest that PAF-induced calcium flux into PMN occurs exclusively via SOCI. This suggests that the trp channels now thought to mediate SOCI, reviewed by Putney and McKay [41 ], might be appropriate targets for therapeutic interventions aimed at limiting PMN calcium mobilization in inflammatory diseases. Moreover, such interventions could potentially have fewer hemodynamic side-effects than traditional calcium-channel blockade, a key consideration for patients with critical illnesses.


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ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Foundation of UMD/New Jersey Medical School and by National Institutes of Health grant GM-59179.

Received March 31, 2000; revised August 21, 2000; accepted August 22, 2000.


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