Ca2' Homeostasis in Permeabilized Human Neutrophils - Journal of

Vol. 259,No. 22,Issue of November 25, pp. 13777-13782,1984 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Ca2' Homeostasis in PermeabilizedHuman Neutrophils CHARACTERIZATION OF Ca2+-SEQUESTERING POOLS AND THE ACTION OF INOSITOL 1,4,5-TRISPHOSPHATE* (Received for publication, June 7,1984)

Marc PrentkiS, Claes B. WollheimS,and P.Daniel Lewgn From the Slnstitut de Biochimie Clinique, University of Geneva, Sentier de la Roseraie, 1211 Geneva 4,Switzerland and the SDiuision of Infectious Diseases, University Cantonal Hospital, CH-12114,Switzerland

The regulation ofCa" transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca"+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca"'] to a steady state of around 0.2 pM. A vesicular ATP-dependent and vanadate-sensitive nonmitochondrial pool maintained this low [Ca"+]level. In the absence of ATP, a higher Ca2+steady state of 0.6 p~ was seen, exhibiting the characteristics of a mitochondrial Ca"+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca"+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca"'] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca"+]to thebasal level. The action of inositol 1,4,6-trisphosphate (IPS)which has been inferred to mediate Ca2+mobilization in a few cell types was studied. IPSreleased (detectable within 2 s) Ca"+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IPS additions. Under experimental conditions in which the ATP-dependent Ca"' influx was blocked, the addition of IPS resulted in a very large Ca"+ release fromnonmitochondrial pool. The results strongly suggest that IPS is a second messenger mediating intracellular Ca2+mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways forCa"+ transport, a Ca2+ATPase (the influx component) and an IPS-sensitive efflux component activated duringCaa+mobilization.

of events which occurs upon their interaction with extracellular stimuli. In recent studies using the fluorescent indicator, quin 2, it was demonstrated that a variety of agonists elevate the cytosolic free Ca2+concentration [Ca2+Iiof neutrophils in two phases (2, 3). A first rapid rise in [Ca2+Iithat is independent of extracellular Ca2+and is, therefore, attributed to Ca2+mobilization from intracellular stores is followed by a secondary elevation in [Ca2+Iithat is dependent on the presence of extracellular Ca2+(2,3). Although a MgATP-dependent Ca2+pump has been observed in the neutrophil plasma membrane (4,5), little isknown about the intracellular structures that participate in the regulation of the cytosolic Ca2+ homeostasis. In particular, neutrophils may provide an interesting model for the study of nonmitochondrial intracellular Ca2+regulation, since they contain very few mitochondria (6). In thepresent study we report experiments carried out with neutrophils whose plasma membrane has been permeabilized with digitonin. The regulation of ambient free [Ca"] by the intracellular structures was investigated using a Ca2+selective electrode at Ca2+concentrations similar to those reported for [Ca2+Iiin intact neutrophils (2). The data demonstrate that ambient [Ca"] is buffered by a MgATP-dependent nonmitochondrial pool whichcan maintain asteady state of around 0.2 PM Ca2+. To investigate the link between changes in cytosolic Ca2+ and polyphosphoinositide breakdown (7-15), an event also occurring in response to certain neutrophil activators (16), we tested the effect of one of the direct breakdown products (14, 17, 18), IPS.' This putative second messenger of the Ca2+ agonists (14, 19-22) caused Ca2+mobilization from a nonmitochondrial pool by activation of a specific efflux pathway. EXPERIMENTAL PROCEDURES

* This work wassupported by Grants 3.246-0.82 SR and 3.986-0.82 SR of the Swiss National Science Foundation. The costs of publica-

Isolation and Permeabilization of Human Neutrophils-Neutrophils were isolated from fresh blood samples (usually 96 ml) obtained from healthy volunteers. Neutrophils were purified by dextran sedimentation followed by centrifugation through a layer of Ficoll-Hypaque as described previously (23, 24). The neutrophil pellet was washed in isotonic NaCl, and the contaminating erythrocytes were eliminated by hypotonic shock (23, 24). The preparations thus obtained contained 95% neutrophils. The purified neutrophils were resuspended in 10 ml of ice-cold isotonic NaCl and incubated for 7 min in the presence of the proteolysis inhibitor diisopropyl fluorophosphate (3 mM), followed by two washes (100 X g for 10 min) in isotonic NaCl. Thereafter, cells were suspended (at a concentration of 1 X lo7 cells/ml) in about 8.0 ml of a medium containing NaCl (125 mM), KH~POI(2 mM), MgATP (0.5 mM),EGTA (0.25 mM),and Hepes (25 mM) that was adjusted to pH 7.0 with NaOH. After

tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Recipient of a Max Cloetta Career Development Award.

The abbreviations used are: IPI, inositol 1,4,5-trisphosphate; acid; IPz, inoHepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic sitol 1,4-bisphosphate; EGTA, ethylene glycol his@-aminoethyl ether)-N,N,N'JV-tetraacetic acid.

Circulating human neutrophils are highly motile cells that respond to contact with suitable surfaces by undergoing shape changes, spreading, and active locomotion. When they interact with invading microorganisms, which they recognize through specific cell surface receptors, a series of events are initiated at theplasma membrane which lead to phagocytosis, degranulation, and a respiratory burst; these responses characterize an intense antimicrobial activity (1). There is strong evidence that themobilization of Ca2+from intracellular compartment(s) is anearly step in the sequence

13777

Ca2+Transport by Permeabilized Neutrophils

13778

warming at 37 “C for 5 min, a 100-fold concentrated solution of digitonin dissolved in water was added a t a final concentration of 10 p ~The . incubation was continued at 37 “C for 8 min, and thereaction was stopped by the addition of a 5-fold excess volume of the same ice-cold buffer. Under these conditions and as shown for other cells (25-28), the neutrophil plasma membrane was selectively permeabilized. This treatment resulted in 62 f 3% depletion of total cellular lactate dehydrogenase (a cytoplasmic enzyme), whereas the values were 26 rt 3% for vitamin B12-binding protein (a secondary granule marker) and 9.8 f 1.1% for @-glucuronidase (a primary granule marker)(mean f S.E.of 6 experiments). The methods for the measurements of the threemarkers are described elsewhere (24). The “leaky’heutrophils were then washed twice (100 X g for 10 min) at 4 “C in the same medium without EGTA and finally resuspended at a concentration of 3 X 108 cells/ml and kept on ice until use. This preparation contained 77 & 1%of cells that did not exclude trypan blue (0.2%) (n = 13). Measurements of Free Ca2+Concentration and Incubation of Digitonin-permeabilized Neutrophils-The medium free Caz+concentration was measured with a Ca2+-selectiveelectrode. The experimental set-up and the manufacture and calibration of the Ca2+electrodes have been described in aprevious report (29). None of the compounds tested interfered with the Ca2+electrode. Since in the presence of biological material a progressive impairment in sensitivity and response time of the electrode was observed, electrodes were replaced after 3-4 tracesin order to maintainafast response time and sensitivity (27-29.5 mVper log [Ca2+])a t low ambient [Ca”] (between 10”-10-6 M Ca2+).The traces shown in the figures are taken from representative experiments which have been repeated a t least three times. Materials-Analytical grade chemicals were obtained from Sigma (Munich) Chemical Co. (FRG), Fluka AG, Buchs, Switzerland, and Merck AG, Darmstadt (FRG). IPS and IPz were produced by the alkaline hydrolysis of ox brain phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and purified by preparative paper chromatography (30). Inositol 2-monophosphate was purchased from Sigma. RESULTS

Characterization of the Ca2+Transporting Activities of Permeabilized Neutrophils-Ca2+ sequestration by the leaky neutrophils started immediately after their addition to the me-

’

1-

1

dium and resulted in a decrease in ambientfree [Ca2+].When incubated in a medium containing MgATP and an ATPregenerating system, the permeabilized cells lowered [Ca’+] within 5 min from 2 p M to about 0.2 p M (Fig. 1). Several sequential pulse additions of Ca2+could be taken up by the “leaky” cells, indicating that the Ca2+pool(s) were not easily saturable. The mitochondrial inhibitors, ruthenium red and antimycin, when added at low ambient [Ca”] (around 0.2 p ~ did not induce Ca2+release from the cells. This demonstrates that the low [Ca”] level was not maintained by the mitochondria and that these were largely depleted of their exchangeable Ca2+under this condition. The Ca2+accumulated by this nonmitochondrial pool could be rapidly released by the combined addition of glucose plus hexokinase to lower the ATP present in themedium or by adding the Ca2+ionophore A23187 (Fig. 1).These data indicate that Ca2+was accumulated against a gradient in a vesicular ATP-dependent nonmitochondrial pool. Saturation of the nonmitochondrial pool was obtained after several pulse additions of Ca2+;in total, 3.06 f 0.45 nmol of Ca2+/106 cells wereadded to the medium containing antimycin (not shown). In order to further characterize this nonmitochondrial pool, leaky cells were incubated in the absence of the respiratory mitochondrial substratesandinthe presence of the mitochondrial poison, antimycin, with various concentrations of the Ca2+ATPase inhibitor, vanadate. Vanadate inhibited in a dose-dependent manner the nonmitochondrial Ca2+transporting activity. A total inhibition of Ca2+transport was obtained with 1 mM vanadate (Fig. 2). Experiments were carried out to demonstrate that themitochondria in the leaky cells werepreserved following the digitonin treatment. Thepermeabilized neutrophils were incubated in thepresence of respiratory substrates and ADP instead of ATP. Vanadate was included at a maximal concentration to block the nonmitochondrial pool. Under these conditions, the leaky cells werefound to buffer ambient [Ca”] around 0.6 p~ (Fig. 3); whenever a pulse addition of Ca2+was made or a small amount of the Ca2+chelator EGTA was added, the ambient free [Ca”] returned to the same steady-state level. Thus, a true bidirectional Ca2+buffering was observed, and the cell maintained a “set point” at 0.6 f 0.05 PM Ca2+ (n = 3). When the mitochondrial Ca2+influx blocker (ruthenium red) was added, the leaky cells immediately released Ca2+(Fig. 3). The datashow that themitochondria contained in theleaky cells were preserved and capable, under some experimental conditions, of buffering ambient 2.4 2.0

-

1.6

E

I

I

N

m “

’

0.: FIG. 1. Ambient free Ca2+concentration maintained by per-

meabilized neutrophils. Cells were incubated a t 30 “C, pH 7.0, in 0.2mlof a medium containing 110 mM KCl, 10 mM NaCl, 2 mM KHzP04,25 mM Hepes, 1 mM MgCl,, 1 mM MgATP, 10 mM creatine phosphate, 50 pg/ml creatine kinase, 2.5 mM succinate, 2.5 mM pyruvate, 2.5 mM malate, 1 pg/ml oligomycin, and 0.5 mg/ml of bovine serum albumin. At the points indicated, cells (7 X lo‘), CaCIZ (2 nmol), 5p~ ruthenium red (RR),0.5 pM antimycin (ANT), or the calcium ionophore A23187 (1 pg/ml) were added. Hk indicates the addition of the “ATP trap,” hexokinase (10 units/ml) plus glucose (15 mM).

1.2 0.8 0.4

50 n

0

0

FIG. 2. Effect of various concentrations of vanadate on the ATP-dependent nonmitochondrial Ca2+ transport. Cells were incubated as described in the legend to Fig. 1, in the absence of the mitochondrial substrates, i.e. succinate, pyruvate, and malate, and in the presence of the mitochondrial poison, antimycin (0.5 p ~ ) Cells . (7 X 10‘) were added to the medium already containing various amounts of vanadate.

)

13779

Ca2+ Transportby Permeabilized Neutrophils

2.0

-

9

1.4

-

1.2

-

1.0

-

0.8

-

0.6

-

0.4

'

N

"

I

FIG. 3. Maintenance of a steady-state ambient free [Ca"] by the mitochondrial pool of permeabilized neutrophils. Cells were incubated as described in the legend to Fig. 1, except for omission of ATP and theATP-regenerating system and theaddition of MgADP (1 mM), the "ATP trap" consisting of hexokinase (10 units/ml) plus glucose (15 mM), and vanadate (1mM). At the points indicated cells (7 X lo'),CaC12 (1 nmol), IPS (2.5 p M ) , EGTA (1 nmol), or 5 pM ruthenium red (RR),were added.

3

Ca2'

more rapidly in the absence of mitochondrial inhibitors as in their presence (Fig. 4). Effect of IP,on the Ambient Free [Ca2+]-When added to leaky cells incubated in the presence of antimycin and maintaining thelower ATP-dependent Ca2+steady state of around 0.2 p~ Ca", a pulse addition of IP, resulted in anincrease in medium [Ca"] to about 0.45 p~ followedby a decrease, representing Ca2+reuptake (Fig. 5c). A second addition of IP3 barely altered medium [Ca2+], indicating that the previous elevation in [Ca"], following the first IP, addition, was not due to Caz+ contaminationof the compound. If IP, was added to cells incubated inthe presence of mitochondrial substrates and without antimycin, similar observations were made. The source of the released Ca2+was truly from an intracellular vesicular pool since IP, had noeffect either after depletion of the stores with the Ca2+ionophore A23187 (Fig. 5b) or when added to "nonleaky" neutrophils (not treated with digitonin) that were not capable of lowering medium [Ca"] (Fig. 5a). To further test the organelle specificity of the response, IP3 was added to cells under conditions where a mitochondrial Ca2+set point was maintained. It was observed that IP, did not promote Ca2+release from the mitochondrial pool (Fig. 3). The Ca2+ response was specific for the trisphosphate derivative (IP,) since either IP, (5 p ~ or) inositol 2-monophosphate (15 p ~ had ) no effect (not shown). The concentration dependence for the IP3 effect is shown

I

n

n

U t t

cel Is

2

(non leaky)

c

f

+

N

"

1 rnin n

1

0 -

;pi c"+

6-

0

'1

FIG. 4. Handling of a large Cas+ pulse by permeabilized cells in the presence and absence of mitochondrial inhibitors. Cells were incubated as described in the legend to Fig. 1 in the absence (solid line) or in the presence (brokenline) of the mitochondrial inhibitors antimycin(ANT) (0.5 phi) plus ruthenium red (RR)(5 p M ) . At the points indicated cells (7 X 10') and CaC12 (3.6 nmol) were added. Both traces shown in the figure were performed under strictly identical experimental conditions using the same cell preparation.

[Ca2+],albeit at a higher level than the nonmitochondrial pool. An experiment was carried out to gain insight into the respective roles and relationship between the mitochondrial and nonmitochondrial pools in the regulation of cytosolic Ca2+homeostasis. The permeabilized neutrophils were incubated in the presence of both the respiratory substrates and ATP, with or without the mitochondrial poisons, antimycin plus ruthenium red (Fig. 4). It was of interest to study how the cells under these two situations would handle a single large pulse addition of Ca2+.Such an approach was used to mimic an early stimulated state of the neutrophils where cytosolic Caz+ has been shown to be rapidly increased above 2 p~ (3). It was observed that although the same [Ca"] was eventually reached (0.2 p ~ )ambient , [Ca"] was loweredfrom a peak Ca2+of 2.6 p~ to thesubmicromolar level about twice

b

4 -

2-

1 rnin n

0

C

'

0.1

'

p3

n

I

FIG. 5. Effect of IPSon the ambient free [Ca"] maintained by the nonmitochondrial pool. Cells were incubated as described in the legend to Fig. 1, except for omission of the mitochondrial substrates (succinate, pyruvate, malate) and the presence of antimy. the points indicated cells (7 x lo6), IP3 (2.5 p M ) , cin (0.5 p ~ ) At A23187 (1pg/ml), and CaCI2 (1 nmol in a and b, and 0.5 nmol in c) were added. q nonleaky cells, i.e. not permeabilized with digitonin; b and c, permeabilized cells.

Transport Ca2+

13780 150

T+

I

by Permeabilized Neutrophils

T

mately 7 min following celladdition, medium [Ca"] was about 0.25 pM (Fig. 7). At this point either IP3or vanadate (1mM) or glucose plus hexokinase were added. Vanadate was used at a maximal concentration which blocksCa2+influx totally (see alsoFig. 2). The combination of glucose and hexokinase, which markedly reduces the ATP level, was used as another approach to inhibit the influx component of the nonmitochondrial pool(see also Fig. 1). It wasobserved that IP3 released Ca2+from the store(s) at a rate about three times that caused by blockade of Ca2+influx with either vanadate or glucose plus hexokinase (Fig. 7). Furthermore, when IP3 was added after vanadate or glucose plus hexokinase, a dramatic Ca2+release ensued (Fig. 7). DISCUSSION

The cellular activation of human neutrophils following I I I I I I 0 1 2 3 4 5 6 agonist stimulation is largely independent of the presence of extracellular Caz+ (31, 32). In contrast to excitable tissues 'P3 [PHI such as muscle, nerves, endocrine pancreas, and adrenal gloFIG. 6. Concentration dependence of IPs-induced Caz+re- merulosa cells, neutrophils appear to lack voltage-dependent lease. Cells were incubated as described in the legend to Fig. 5. The responses to IP3 were calibrated by additions of known aliquots of Caz+channels (33). Furthermore, information on the presence Ca2+to give equivalent peak heights. Mean f S.E.of 3-4 separate of receptor-activated Ca2+channels is lacking. It is very likely that intracellular Ca2+ mobilization by agonists acting via experiments. receptors is a much more critical event in neutrophil stimulation than Ca2+influx from the extracellular medium. Indeed, I all physiological agonists tested so far release Ca2+ from ,cells intracellular stores (2, 34-36). Therefore, it is of importance to understand the respective role and interaction of the different organelles in the regulation of cytosolic Ca2+and to try to define the link between the membrane receptors and the organelle(s) fromwhich Caz+ is mobilized.We approached these questions by studying, at Ca2+concentrations occurring in the cytosol, the Caz+ homeostasis of human neutrophils whose plasma membranes had been permeabilized with digitonin. Permeabilization of cells with saponin or digitonin (2528) offers the advantage that the cell membrane is bypassed and the internal Ca'+-transporting devices can be, therefore, lkec studied in an environment which is much closerto physiological conditions than isolated organelles. When incubated in a medium containing respiratory substrates and MgATP, human neutrophils rapidly lowered medium [Ca"] to the low submicromolar range. They were able to maintain an ambient [Ca"] steady state of 170-300 nM v'P3 Ca2+,depending on the preparation. These values compare favorably with the resting cytosolic [Ca"] in intact human 0.2 neutrophils whichwas reported recently to be 120 nM, as vanadate FIG. 7. IP8-induced stimulation of a Caz* efflux component determined with the indicator quin 2 (2) or 100-300 nM with of the nonmitochondrial pool. Cells (7 X lo6) were incubated as the photoprotein obelin (37). This suggests that intracellular described in the legend to Fig. 5 in a medium containing ATP (1 mM) structure(s), in addition to theplasma membrane, are capable and creatine phosphate (2 mM). At the points indicated, either IP3 (5 of regulating the resting [Ca"] level in intact neutrophils, as NM)or vanadate (1 mM) or the "ATP trap" consisting of hexokinase (Hk, 20 units/ml) plus glucose (15 mM) were added. The three traces proposed in other tissues (25, 27, 28, 38). We could exclude shown in the figure were performed under strictly identical experi- that this[Ca"] steady state was maintained by the mitochonmental conditions with cells from the same preparation. a, IP, added dria since it was insensitive to mitochondrial inhibitors. FurIP3 addition wherenoted; c, thermore, this low[Ca"] steady state was maintained by a only; b, vanadateaddedfollowedby hexokinase plus glucose added followed by IPSaddition where noted. vesicular and MgATP-dependent pool(s), sensitive only to large concentrations of vanadate. Several lines of evidence suggestthat theinternal structure in Fig. 6. The half-maximal concentration of IPSwas 0.75 pM, and the maximal effect was reached around 2 p~ IPS. The that buffers ambient Ca2+in permeabilized cells could bethe maximal Ca2+ released, calibrated with a pulse addition of endoplasmic reticulum. Firstly, it was sensitive only to very Ca2+giving similar peak height, was 0.12 nmol of Ca2+/106 large (millimolar) concentrations of vanadate. This excludes the possibility that endocytotic vesicles originating from the cells. Experiments were carried out to clarify the mechanism of plasma membrane were responsible for the Ca'+-transporting IP3 action. Thus, IP3 may act either by inhibiting the influx activity, since the ATP-dependent Ca2+pump of neutrophils 80% with only 2 p~ vanadate (5). Furthermore, component of the Ca2+ transport of the nonmitochondrial is inhibited by pool orby stimulating an independent efflux component. Cells in other tissues it was shown that endoplasmic reticulum were incubated in the presence of antimycin, and approxi- vesicles are sensitive to much higher concentrations of vana-

9

13781

ea2+Transport by Permeabilized Neutrophils

date than are plasma membrane vesicles (39, 40). Secondly, this Ca2+ pool was nonmitochondrial since it was not influenced by mitochondrial poisons. Thirdly, it was sensitive to the second messenger IPS which has been reported to specifically mobilize Ca2+from the endoplasmic reticulum but not from the mitochondria or the secretory granules of a rat insulinoma (22). Alternatively, the nonmitochondrial pool could be of granular origin in view of the fact that electron microscopic observations have documented that neutrophils are rich in granules while the endoplasmic reticulum is clearly less abundant (1, 6). However, in other tissues, secretory granules apparently do not participate inthe short-term regulation of cytosolic Caz+ (38, 41). To distinguish between these possibilities which are notexclusive, future experiments with well-characterized highly purified subcellular fractions enriched in the various organelles isolated from human neutrophils are necessary. Although this tissue is poor in mitochondria we could demonstrate that the mitochondria of the digitonin-permeabilized neutrophils were preserved and operative for Ca2+ transport. Thus, in the absence of ATP, the leaky cells maintained a Caz+steady state of 0.6 p~ exhibiting all the characteristics of a mitochondrial Ca2+set point (25,29). In order to know whether the mitochondria could play some role in the regulation of cytosolic Ca2+in the presence of ATP, we studied how the leaky cells were handling a single large pulse addition of Caz+in the presence or absence of mitochondrial inhibitors. This approach was used to mimic the early stimulation of intact human neutrophils. In experiments performed in neutrophils loaded with very low concentrations of quin 2 (3), it has been demonstrated that upon chemotactic peptide-mediated receptor activation, cytosolicfree [Ca"] rises to at least 2 PM. We observed that in the absence of mitochondrial inhibitors, ambient [Ca'+] was lowered twice as rapidly to thesubmicromolar range than in their presence. This finding extended to the intact cell indicates that mitochondria participate in lowering cytosolic Ca2+to the submicromolar range. Nonmitochondrial internal structure(s) and the plasma membrane would then further decrease cytosolic Caz+to the resting state. It should be noted that under basal conditions the neutrophil mitochondria are likely to be depleted of Ca2+since we found that themitochondrial poisons when added at low [Ca'+] (around 0.2 PM) did not alter, even transiently, the Ca2+steady state maintained by the leaky cells. This finding appears to exclude the possibility that mitochondria in the neutrophils function as a Ca2+-mobilizing organelle. IPS mobilized Caz+ from a vesicular vanadate-sensitive ATP-dependent nonmitochondrial pool. This finding is at variance with the suggestion that receptor activation in neutrophils leads to mobilization of membrane-bound Ca2+,as assessed by decreased chlorotetracycline fluorescence (42). It was found that thecells wereinsensitive to a second challenge of IPS.This observation is in accord with the one described in rat insulinoma microsomes (22) but contrasts with the one described in permeabilized hepatocytes (20, 21) and insulinoma cells (43). The reason for this phenomenon is notclear. It could reflect an interesting process of desensitization to IP, due to a slow degradation of the molecule in this cell type. Alternatively, a sensitive pool might be depleted of Ca2+by IPS, and the Ca2+ releasedwouldbe taken up by an IPSinsensitive pool (44). Further studies, especially on IPS degradation, are required to evaluate these possibilities. The concentration dependence of the IPSeffect was similar to that reported for other cell types (19-21, 43). At present, direct determinations of the IPSlevels in intact cells are not

available for comparison with the Ca" release.However, indirect measurements using [3H]myoinositol labeling indicate that the calculated IPSlevels in hepatocytes lie roughly in the same range as theones which mobilizeCaz+in permeabilized cells (45). The present study extends our knowledge of the mode of action of IPS.Two lines of evidence suggestthat IPSmobilizes Ca"by activating Ca2+ effluxfrom the nonmitochondrial pool(s) rather than by inhibiting Ca2+influx. Firstly, the rate of Ca2+release from the nonmitochondrial pool in the presence of IP, was 2-3 times more rapid than therelease observed after blocking Ca2+ influx with vanadate or glucose plus hexokinase. Secondly, and more importantly, IPS was still active when added after blocking the influx component with vanadate or glucose plus hexokinase. Hence, an important conclusion is that the nonmitochondrial pool(s) of human neutrophils possesses independent influx and efflux pathways for Caz+transport, aCaz+ATPase that allows Ca2+sequestration (influx component) and a separate IPS-stimulated efflux component which is responsible for Caz+mobilization. As IPSprobably mediates Ca2+ mobilization (14, 19-22) it is of interest to estimate the quantity of Ca2+that itreleases. A way which has been used to calibrate the Ca2+released by IPS is the addition of known aliquots ofCa" giving similar peak heights of[Ca"] (19, 21,22, 43). However, the data represented in Fig. 7 suggest that with such a calibration the amount of Ca2+released from the store is grossly underestimated. Indeed, if the influx component is not blocked, it is expected that a certain amount of the Caz+mobilized is rapidly resequestered by the stores. In particular, under non-steadystate conditions, such as after IPS addition, a diffusion gradient may exist between the interior of the leaky cellsand the surrounding medium, so that partof the Ca2+released escapes detection by the electrode. In conclusion, human neutrophils possess intracellular Ca2+ sequestering organelles capable of maintaining a free [Ca"] in the range of resting cytosolic Ca2+levels. Atleast two pools participate in this regulation. In concert with other structure(s), the mitochondria are effective in the lowering of [Ca'+] to the submicromolar range. ATP-dependent nonmitochondrial pool(s) determine the low resting [Ca"] and are able to deplete the mitochondria of their exchangeable Ca2+. The nonmitochondrial pool(s) is the source of the Ca2+released by IP, which specificallyactivates an independent Ca2+ efflux component. Acknowledgments-We thank Dr. R. F. Irvine for his generous gift of IP3 and IP2 and for helpful discussions of the present manuscript. The authors are indebted to Antoinette Monod for her skilled technical assistance. REFERENCES 1. Stossel, T. P. (1974) N.Engl. J. Med. 2 9 1 , 717-723,774-780, 833-839 2. Pozzan, T., J A W , P. D., Wollheim, C. B., and Tsien, R. Y.(1983) Science (Wash. D.C.) 221,1413-1415 3. Lew, P. D., Wollheim, C. B., Waldvogel, F. A., and Pozzan, T. (1984) J. Cell Bid., in press 4. Volpi, M., Naccache, P. H., andSha'afi, R. I. (1983) J. BioL Chenz. 258,4153-4158 5. Lagast, H.,Lew, P. D., andWaldvogel, F. A. (1984) J. Clin. Invest. 73,107-115 6. Bainton, D. F., Ullyot, J. L., and Farquhar, M. G . (1971) J. Exp. Med. 134,907-922 7. Michell, R. H.,Kirk, C. J., Jones, L. M., Downes, C. P., and Creba, J. A. (1981) Philos. Trans. R. Soc. Lond. B Biol. Sci. B296,123-133 8. Weiss, S. J., McKinney, J. S., and Putney, J. W. (1982) Biochem. J. 206,555-560

13782

Ca2+Transport by Permeabilized Neutrophils

9. Farese, R. V. (1983) Metabolism 32,628-641 10. Thomas, A. P., Marks, J. S., Coll, K. E., and Williamson, J. R. (1983) J. BioL Chem. 258, 5716-5725 11. Agranoff, B. W., Murthy, P., and Seguin, E. B. (1983) J. Bid. Chem. 258,2076-2078 12. Berridge, M. J., Dawson, R. M. C., Downes, C. P., Heslop, J. P., and Imine, R. F. (1983) Biochem. J. 212,473-482 13. Laychock, S. G. (1983) Biochem. J. 216, 101-106 14. Berridge, M.J. (1984) Biochem. J. 220,345-360 15. Schlegel, W., Roduit, C., and Zahnd, G.R. (1984) FEBS Lett. 168,54-59 16. Volpi, M.,Yassin, R., Naccache, P. H., and Sha’afi, R. I. (1983) Bwchem. Bwphys. Res. Commun. 112,957-964 17. Berridge, M.J. (1983) Biochem. J. 212,849-858 18. Rebecchi, M. J., andGershengorn,M. C. (1983) Biochem. J. 216, 287-294 19. Streb, H., Imine, R. F., Berridge, M. J., and Schulz, I. (1983) Nature (Lord.)306,67-69 20. Burgess, G. M., Godfrey, P. P., McKinney, J. S., Berridge, M. J., Imine, R. F., and Putney, J. W. (1984) Nature (Lord.)309, 63-66 21. Joseph, S. K., Thomas, A. P., Williams, R. J., Imine, R. F., and Williamson, J. R. (1984) J. Bid. Chem. 259,3077-3081 22. Prentki, M., Biden, T. J., Janjic, D., Imine, R. F., Berridge, M. J., and Wollheim, C. B. (1984) Nature (Lord.)309, 562-564 23. Lew, P. D., Southwick, S., Stossel, T. P., Whitin, J.C., Simons, E., and Cohen, H.J. (1981) N. Engl. J. Med. 305,1329-1333 24. Dewald, B., Bretz, U., and Baggiolini, M. (1982) J. Clin. Invest. 70,518-525 25. Becker, G. L., Fiskurn, G., and Lehninger, A. L. (1980) J. Bwl. Chem. 256,9009-9012 26. Murphy, E., Coll, K., Rich, T. L., and Williamson, J. R. (1980) J. Bwl. Chem. 255,6600-6608 27. Streb, H., and Schulz, I. (1983) Am. J. Physwl. 245, G347-G357

28. Burgess, G.M.,McKinney, J. S., Fabiato, A., Leslie, B. A., and Putney, J. W. (1983) J. Bwl. Chem. 258, 15336-15345 29. Prentki, M., Janjic, D., and Wollheim, C. B. (1983)J. Bwl. Chem. 258,7597-7602 30. Imine, R. F., Brown, K. D., and Berridge, M. J. (1984) Bwchem. J. 221,269-272 31. Stossel, T. P. (1973) J. CeU Bwl. 58,346-356 32. Smolen, J. E., Korchak, H. M., and Weissmann, G. (1981) Biochim. Biophys. Acta 677,512-520 33. Lew, P. D., Wollheim, C. B., Seger, R. A., and Pozzan, T. (1984) Blood 63,231-233 34. White, J. R., Naccache, P. H., Molski, T. F. P., Borgeat, P., and Sha’afi, R. I. (1983) Biochem. Biophys. Res. Commun. 113,4450 35. Lew, P. D., Dayer, J. M., Wollheim, C. B., and Pozzan, T. (1984) FEBS Lett. 166944-48 36. Gennaro, R., Pozzan, T., and Romeo, D. (1984) Proc. Natl. Acud. Sci. U. S. A. 81, 1416-1420 37. Hallett, M. B., and Campbell, A. K.(1982) Nature (Lord.)295, 155-158 38. Prentki, M., Janjic, D., Biden, T. J., Blondel, B., and Wollheim, C. B. (1984) J. Bid. Chern. 259,10118-10123 39. Famulski, K., and Carafoli, E.(1982) CeU Calcium 3, 263-281 40. Kribben, A., Tyrakowski, T., and Schulz, I. Am. J. Physwl. 244, G480-G490 41. Johnson, R. G., and Scarpa, A. (1976) J. Gen. Physiol. 68,601631 42. Naccache, P. H.,Volpi, M., Showell, H. J., Becker, E. L., and Sha’afl, R. I. (1979) Science (Wash. D. C.) 203,461-463 43. Biden, T. J.,Prentki, M., Imine, R. F., Bemdge, M. J.,and Wollheim, C.B. (1984) Biochem. J., in press 44. Dawson, A. P., and Imine, R. F. (1984) Biochem. Biophys. Res. Commun. 120,858-864 45. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984) J. BwL Chem. 259,5574-5584