Regulation of inositol phosphate metabolism in chemoattractant ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biolopy, Inc.

Vol. 262, No. 24, Issue of August 25, pp. 11546-11552,1987 Printed in U.S.A.

Regulation of Inositol Phosphate Metabolismin Chemoattractantstimulated Human Polymorphonuclear Leukocytes DEFINITIONOFDISTINCTDEPHOSPHORYLATIONPATHWAYSFOR

IPS ISOMERS* (Received for publication, March 3, 1987)

Susan B. Dillon& John J. Murray#, MargrithW.Verghese, and Ralph Snydermann From the Howard Hughes Medical Institute and the Division of Rheumatology and Immunology, Departmentof Medicine, Duke University Medical Center, Durham, NorthCarolina 27710

The metabolism of the calcium mobilizing inositol1,4,5-trisphosphate (IP3)isomer was studied in myo[3H]inositol labeled, chemoattractant-stimulated human polymorphonuclear neutrophils (PMNs), and in PMN lysates. It was determined that 1,4,5-IP3is metabolized in vitro by two distinct pathways: 1) by sequential dephosphorylation to 1,4-IP2,4-IP1, and inositol or 2) by ATP dependent conversion to 1,3,4,5IP,, followed bydephosphorylationtoform1,3,4IP3, 3,4-IPz, 3-IP1, and inositol. In PMNs stimulated with 0.1 PM N-formyl-methionyl-leucyl-phenylalanine (met-Leu-Phe), 1,4-IP2, 1,4,5-IP3, and IP,, were elevated by 5 s; whereas production of 1,3,4-IP3, 3,4-IPz, and IPI occurred only after an initial lag(-15 8). The predominant IP1isomer formedin met-Leu-Phe-stimulatedcells was4-IP1. Production of 1,3,4-IP3and 3,4IP2 was markedly reduced (17 and 35% of control, respectively)inmet-Leu-Phe-stimulatedcellsprea riseinintracellular calcium treatedtoprevent ([Ca2+Ii).PMNs were also stimulated with leukotriene B, (LTB,) since this agent is a poor activator of the respiratoryburstcomparedtoMet-Leu-Phe.Peak levels (5 s) of 1,4,5-IP3 were equivalent after stimulation with 0.1 p~ met-Leu-Phe versus 0.1 p~ LTB, (320 f 38%versus 378 2 38%of control values, respectively; n = 5);however, at 30 s, 1,4,5-IP3 remained elevated only in Met-Leu-Phe-stimulatedcells. Similarly, elevation of [Ca2+Iiwas more prolonged in response to 0.1 p~ met-Leu-Phe (>3min) versus LTB, (1 min). Thus, signal transduction in PMNs may be modulated by boththe durationof the initial1,4,5-IP3 signal andby the metabolic pathway(s) utilized to convert thisIP3isomer to other, potentially active inositol phosphate products.

Stimulation of polymorphonuclear leukocytes (PMNs)’ by

chemoattractants results in the rapid hydrolysis of the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP,)by a phospholipase C(1-7). The products formed by the phosphodiesteric cleavage of PIPz are inositol 1,4,5trisphosphate (IPJ and1,2-diacylglycerol which act assecond messengers by raising intracellular calcium (8) and by promoting activation of protein kinase C (9), respectively. Since PIP, hydrolysis is centrally important for receptor-mediated activation in numerouscell types, the mechanism of phospholipase C activation has been studied in some detail (10-12; reviewed in Refs. 13-15). Studies with PMN plasma membranes showed that agonist binding to chemoattractant receptors activates a guanine nucleotide regulatory protein (G) by enhancing itsability to bind GTP (16); the activatedG protein is postulated to associate witha PIP,-specific phospholipase C and lower its Ca2+ requirement for activation to ambient intracellular levels (12). Thismodel is in concert with studies Nof intactPMNsstimulatedwiththechemoattractant formyl-methionyl-leucyl-phenylalanine(fMet-Leu-Phe), where IP3production occurredevenwhen the associated increase in cytosolic Ca2+was prevented (6, 17). The metabolismof IP3 in intact cells is more complex than originally thought,since isomeric forms of IP3 (1,4,5 and 1,3,4), IP2 (1,4 and 3,4), and IP, (1 and 4) are formed after agonist stimulation in variouscell types (18-28). Mixtures of the isomeric forms of IP1, IP,, and Ips are not resolved by batch-elution on Dowex formate columns, but the isomers canbe resolved by high pressure liquid chromatography (HPLC) with anion exchange systems (20, 27, 29). In some cell types, the 1,3,4-IPS isomer can be derived from inositol 1,3,4,5-tetrakisphosphate(IP,) (22, 24, 25, 30), a n intermediate formed by phosphorylation of 1,4,5-IP3 by an ATP-dependent kinase (31). Although functional roles for 1,3,4-IP3 and IP, have not yet been demonstrated in PMNs, recent studies in othercells have provided evidence that these compounds can either act to mobilize calcium from intracellular stores (1,3,4-IPS)(32) or from the externalmedium (IP4) (33). In order to study the relationship of these compounds to cellular activation in human PMNs,we performed a detailed analysis of inositol phosphate production in cells stimulated with the chemoattractants N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or leukotriene B, (LTB,). These agents are equipotent for initiating chemotaxis but the former is far more potent and active than the latter in stimulating a respiratory burst (34-36). In addition, since the fate of the IPSisomers has not been directly determined in PMNs, dis-

* This work was supported by Grants DE03738 and CA29589 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Supported by National Institutes of Health Postdoctoral Training Grant 5T32CA09058. Recipient of Pfizer Postdoctoral Fellowship and FUR Nabisco Awards. 7 To whom correspondence should be addressed Genentech, Inc. 460 Point San Bruno Blvd., S. San Francisco, CA 94080 ’ The abbreviations used are: PMNs, polymorphonuclear leukocytes; PIP,, phosphatidylinositol 4,5-bisphosphate; HPLC, high pres- sitol monophosphate; [Ca*+]i,intracellular calcium; 2,3-DPG, 2,3sure liquid chromatography; Ips, inositol trisphosphate; IP,, inositol disphosphoglycerate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetratetrakisphosphate; Met-Leu-Phe, N-formyl-methionyl-leucyl-phen- acetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic ylalanine; LTB,, leukotriene B4; IP2, inositol bisphosphate; IP,, ino- acid; HBSS, Hanks’ balanced salt solution.

11546

IP, Metabolism in Human PMNs

11547

hydrolysis of [3H]1,3,4-IP3,we previously determined that the1- and 3-IP1 isomers coelute in this system; and theretention times for 1,3Ip2 uersus 3,4-IP2 are 95 and 113 min, respectively (see Ref. 28 and Fig. 6A ) EXPERIMENTALPROCEDURES Determination of Intrucelluhr Free Calcium Levels-PMNs (2 x M u ~ e r ~ u ~ - M e t - ~ u - P hATP, e, 2,3-diphosphoglycerate (2,3- 107/ml) in assaybuffer were loaded with 6.25 p~ Quin-2 AM or 1 pM DPG), EGTA, 1-phenylmethylsulfonyl fluoride, dithiothreitol, and Fura-2 AM for 40 min at 37 "C. Measurement of intracellular calcium +in-2 AM were obtained from Sigma. LTB. and ionomycin were was performed as previously described (17,38-40). Metabolism of Radiolabeled Inositol Phosphates by PMN Sonifrom Behring Diagnostics; ammonium formate was from Sigma or Aldrich. Fura-2 AM was from Molecular Probes, Eugene, OR, Hanks' cates-PMN suspensions were pelleted at 50 X g for 10 min to remove balanced salt solution (HBSS) was from Gibco and HPLC grade contaminating platelets. The cells were then resuspended at 2 x lo8/ phosphoric acid (85%)was from Fisher Chemical Co. Met-Leu-Phe ml ( 4 0 % platelets) in ice-cold buffer containing 0.32 M sucrose, 25 and ionomycin were stored as 0.01 M stock solutions in dimethyl mM Hepes/Tris, 1mM EGTA, 2 mM dithiothreitol, 2 mMMgC12, and sulfoxide at -20 "C. Stock solutions of LTB, were stored in ethanol 1 mM phenylmethylsulfonyl fluoride (pH 7.5). Cells in an ice slurry at -70 "C; aliquots were dried under N, gas before resuspending in were sonicated six times for 10 s a t a setting of 35% with an Artek assay buffer (see below). my0-[2-~H]Inositol-l-P(8.4 Ci/mmol) and sonic 300 dismembranator (Artek Systems Corp., Farmingdaie, NY). my0-[2-~H]inositol1,4-P2(2 Ci/mmol) were from Du Pont-New Eng- Nuclei and n o n d i s r u p ~cells were removed by centri~gation(250 land Nuclear; my0-[2-~H]inositol1,4,5-P~(15 Ci/mmol) was from X g for 5 min), and PMN sonicates were frozen at -70 "C until use. Amersham Corp. my0-[2-~H]Inositol(15 Ci/mmol) was from either Enzyme assays were performed in a final volume of 0.1 ml at 37 "C. Du Pont-New England Nuclear or American Radiolabeled Chemicals, Cell sonicates (50 pl) were prewarmed for 10 min in the presence of Inc. my0-[2~~H]Inositol 1,3,4,5-P4(1Ci/mmol) and my0-[2-~H]inosi- CaC& (25 pl) to give a final concentration of 10 p~ (calculated as tol-4-P were generously provided by Du Pont-New EnglandNuclear. described, Ref.41). To test for 1,4,5-IP3 kinase activity, reactions Cell Preparation-PMNs (295% purity)were isolated as described were started by adding 25 p l 4 X assay buffer (pH 7.5) containing 40 previously (37) from heparinized (10 units/ml) blood collected from mM ATP, 40 mMMgCL,, 4 mM dithiothreitol, 12 mM 2,3-DPG, and healthy volunteers. The cells were culturedfor 24 h in medium 2.0 p~ 13H]1,4,5-IP3(1 Ci/mmol). To analyze metabolism of 1,4-IPz containing 20 #Ci/ml my0-(~H]inosito1(12). Prior to assay, the PMNs or LP,, reactions were started with 25 g14 X assay buffer containing were washed twice and then resuspended in assay buffer (Hepes- 440 mM KCl, 40 mM NaCl, 4 mM KHpPOd, 12 mM MgCL, 80 mM K+/ buffered HBSS; pH 7.4) with 20 mM LiC1, at 6 X lo7cells/ml. Aliquots Hepes (pH 7.4) and 2.0 [3H]IP4(1 Cilmmol) or [3H]1,4-IP2(2 Ci/ of cells (0.18 ml) were prewarmed at 37 "C for 5-10 min before adding mmol). Reactions were terminated with ice-cold trichloroacetic acid. 10 X concentrated stimulants (0.02 ml) or assay buffer control with an equivalent concentration of dimethyl sulfoxide where appropriate. RESULTS Reactions were terminated a t the times noted with ice-cold 10%(final Formation of IPS Isomers and IP4 in Stimulated PMNconcentration) trichloroacetic acid, and samples were prepared for HPLC analysis as described (29). Briefly, after trichloroacetic acid Formation of the IP, isomers and IP, was analyzed for5 min precipitation, supernatants were washed four times with diethyl ether, after stimulation with t h e chemoattractants met-Leu-Phe and then neutralized with 1 M Tris (pH8).Prior tochromatographic analysis, a mixture of unlabeled ATP, ADP, and AMPwas added to (0.1 p M ) or LTB4 (0.1 p M ) or with the calciumionophore ) 1). In the c h e m o a t t r a c ~ n t - s t i m u " each sample and thesamples were passed through a 0.45 p~ HV filter ionomycin (1.0 p ~ (Fig. lated cells, both 1,4,5-IP3 and IP, were elevated above back(Millipore Corp., Bedford, MA). Analysis of Radiolabeled Inositol Phosphates by HPLC-Inositol ground b y 5 s, but there was a definite lag periodbefore 1,3,4phosphates (10' cells/sampb) were separated ona Whatman Partisal IP, levels began to increase. As the rapid risein the 1,4,5-IP3 SAX 10 column (0.46 X 25 cm) with a Guard-Pak silica precolumn isomer diminished, the 1,3,4-IP3 isomer continued to accu(Millipore Corp., Milford, MA) and column inlet filter (Beckman Instruments) using slight modifications of two previously described mulate until both isomers comprised approximately 50% of anion exchange systems (20,27,29). In the first system, the column the total IP, (60s for met-Leu-Phe and 15 s for LTB,). The was initially washed with water for 8 min to elute free inositol, and levels of 1,4,5-IPS fell more rapidlyin LTB,-stimulated cells, over the next 32 min a linear gradient of 0-1 M ammonium formate although the mean peak levels of 1,4,5-IP3 produced in re(pH 3.7 with phosphoric acid) was passed through the column and sponse to 0.1 p~ Met-Leu-Phe uersus the same dose of LTB, then held for an additional 5 min. A lineargradient up to 2 M were not significantlydifferent (p > 0.05). The relatively ammonium formate was then run over the next 5 min and held at this concentration for an additional 5 min. The flow rate was 1.25 prolonged elevation of 1,4,5-IP3 in ~ e t - L e u - P h e - s ~ m u l a ~ d ml/min throughout and 0.5-min fractions were collected. Peak elution PMN was also evidentwhen cells were stimulated with higher times for radiolabeled inositol phosphate standards were 3,22, 29,47, doses of chemoattractants (1.0 p ~ ) since , by 15 s the 1,4,5and 61 min for inositol, 1- and 4-IP,, 1,4-IP2, 1,4,5-IP3,and 1,3,4,5- IPZ levels had declined to 85% uersus 39% of peak values (5 IPI, respectively. The inositol-labeled compound eluting at 45 min s) in response to Met-Leu-Phe uersus LTB.,, respectively was identified as 1,3,4-IP3 by its position relative to 1,4,5-IP3 and ATP (29);although with our system the 1,3,4-IP3 isomer eluted (not shown). Since 1,4,5-IP3 levels disappeared more rapidly slightly later than ATP. A second IP2 isomer, identified as 3,4-IP2, in cells stimulated withLTB, uersus Met-Leu-Phe, it was of the patterns of calcium mobilization after eluted at 31.5 min (28). The absorbance at 254 nm was monitored interest to compare with each sample to allow comparison of radioactive peak elution stimulation with each of these agents. The results i n Fig. 2 times with that of AMP, ADP, and ATP. Fractions were diluted with show that both of these chemoattractants (0.1 PM) elicited a or fluid 0.5 ml H,O and mixed with 3.5 ml of ~ ~ o f l u scintiliation peaked by 10(Research ProductsInternational Corp., Mount Prospect, IL) for rapid risein intracellular calcium [Ca2+Ii which scintillation counting. Background radioactivity (15 cpm) was sub- 15 s. However,whereas the et-Leu-Phe-induced rise i n tracted from each fraction before calculating peak counts/min. Re- [Ca2+Ii remained elevated for over 3 min, the [Ca2+Iilevels covery of radioactive standards andradioactivity in cell preparations had returned to baseline in LTB,-stimulated cells by 60 s. was at least 85% of the injected counts/min. In the second system, The pattern of 1,3,4- and 1,4,5-IP3 production in response consecutive linear ammonium phosphate gradients (1 ml/min) were to ionomycin (1 pM) was similar to that seen after chemoatrun as follows: 0.04-0.075 M ammonium phosphate (pH 3.8) over 65 min; 0.075-0.15 M over 30 min; and 0.15-0.54 M over 25 min. The tractant stimulation in that the levels of 1,4,5-IP3 were into the appearanceof the 1,3,4column effluent was mixed with Tru-count scintillation fluid (5:l creased over background prior ratio) (IN-US Corp., Fairfield, NJ), and radioactivity was monitored IPSisomer (Fig. 1C). However, the levels of both IP3and IP4 by an on-line radioactivity detector (Ramona LS-4, IN-US Corp.) formed in response to the calcium ionophore were relatively equipped with a Roland DG computer and software version 5.3e for peak integration. The counting efficiency was set at60%. The radio- low, and both IP3 isomers accumulated more slowly in ionoactive 1-IP,, 4-IP1, and 1,4-IP2 standards eluted at 20, 23, and 100 mycin uersus chemoattractant-stimulated cells. A lower dose min, respectively, in this system. Using a mixture of radiolabeled I-, of ionomycin (0.2 p M ) did not elicit significantIPSproduction 3-, and 4-IP1 and L3-, 1,4-, and 3,4-IPZ isomers prepared from base (not shown).

rupted cells were used to study the metabolic pathways by which 1,4,5- and 1,3,4-IP3 are converted to free inositol.

.

11548

IP3 Metabolism in Human PMNs

-

A

FMet - LOU Phe

w

4001

i 2

u B

4001

I*-.'

I

L I

I

0

1

I

2

I

3

,

4

5

,

l

6

7

#

8

~

9

j

1

,

0

TIME (rnin) FIG.2. Comparison of chemoattractant-induced changes in [Ca2'Ii measured in Fura-2-loaded PMN. Cells (5 X 106/ml)were prewarmed in HHBSS for 5 min (37 'C) before adding stimulants (0.1 p ~ (arrows). ) Tracings shown are representative of at least three additional experiments with different donors.

= 400t

30 0Y

t

,,?

500 400

? I I I

,

I , , I

,

I

a

C 400

100

200

0 5 15 30 45 60 180

b " A d

100

0

0

5 15

30

45

60

180

300

TIME (Sec) FIG.1. Kinetics of IPS isomer and IP, production by chemoattractant- or ionomycin-stimulated human PMN.Extracts of my~-[~H]inositol-labeled PMN were analyzed by HPLC. Net counts/ min (stimulated minus buffer control) are shown for 1,4,5-IP3 (0); 1,3,4-IP3(0), and IP, (A)peaks in cells stimulated with 0.1 p~ m e t Leu-Phe ( A ) , 0.1 pM LTB, ( B ) ,or 1.0 pM ionomycin (c).Results shown are the mean counts/min f S.E. for three to six separate experiments with different donors; data points without error bars are the mean of two experiments only.

TIME ( s e c )

FIG.3. Kinetics of IP2 isomer production in met-Leu-Phestimulated PMN. Inositol trisphosphate isomers ( A ) ;the IP, isomers ( B ) ,and total IP1, IP,, and IPS (C) were measured by HPLC my~-[~H]inositol-labeled PMN (ammonium formate gradient)in after stimulation with 0.1 p~ fMet-Leu-Phe. Results from one donor are shown; comparable results were also obtained with separate donors in three additional experiments.

respectively); however, by 5 min after stimulation, IP1 continued to accumulate in met-Leu-Phe- but not in LTB4-stimulated cells (524 f 127 uersus 180 k 32%of buffer control values, respectively). Formation of IP, isomers in PMNs was analyzed by HPLC using an ammonium phosphate gradient system which in the 1-and 3-IP1isomers coelutedwith AMP, and the 4-IP1 isomer eluted several minutes later (see Ref. 28 and Fig. 6A). In Kinetics of IP2 and IPl Isomer Formation in Chemoattract- resting cells, 70 f 4% ( n = 4) of the total IPl was present in ant-stimulated PMN-Inmet-Leu-Phe-stimulatedPMN, the peak containing 1- and/or 3-IP1; the remainder was rethe 1,4-IP2isomer was formed without an apparentlag (by 5 covered as 4-IP1. In fMet-Leu-Phe- (1.0 p M ) stimulated cells, s afterstimulation),whereasthe3,4-IP2 isomer appeared 4-IP1 accumulated more rapidly and comprised the majority concomitantly with 1,3,4-IP3 at 15 s after stimulation (Fig. of the total IP, at all times tested (Fig. 4).However, the 3). LTB, induced a similar pattern of IP, isomer formation; relative proportion of 4-IP1 declined from 89 f 6% t o only 65 at s uersus 10 min,respectively. however, 1,4-IP2levels peaked earlier ( 5 uersus 15 s) and fell f 3% ( n= 4) of the total IP, 30 more rapidly (28 -t- 11% uersus 81 f 10% of peak levels at 30 Route of Formation of IP4,1,3,4-IP3,and 3,4-IP2in PMNss; n = 3) in cells stimulated with 0.1 F M LTB, uersus M e t Recent studies have identified an ATP-dependent kinase in Leu-Phe, respectively. Measurement of total IP1 production several tissues that phosphorylates 1,4,5-IP3to yield 1,3,4,5(Fig. 3C) confirmed what had been shown previously in fMet- IP, (31).Furthermore, 1,3,4-IP3 can be derived by the phosLeu-Phe-stimulated PMNs (6, 17) or differentiated HL-60 phomonoesteric cleavage of the 5-phosphatefrom IP4 (20,42). cells (3); namely that the accumulation of IP, is slower but To determine whether these pathways are utilized in PMNs, more prolonged than either IP2 or IP3. IP, levels were com- [3H]1,4,5-IP3 or t3H]IP4 were incubated with cell sonicates in parable in fMet-Leu-Phe uersus LTB4-stimulated PMNs at order to measure the metabolic products formed. In initial 30 s (180 f 31% uersus 170 f 37% of buffer control values, experiments, it was confirmed that 2,3-DPG (3 mM), a com-

IP, Metabolism in Human PMNs

~

O

O

O

I

~,

,# ,

,

,

o 0 1 2 3 4 5 6 7 8 9 1 0 TlME [min)

FIG. 4. Multiple IP1 isomers are formed in met-Leu-Phestimulated PMNs. Extracts from ~~o-[3H]inositol-labeled PMNs were analyzed by an HPLC system (ammonium phosphate gradient) that resolved two IP, peaks corresponding to 1- or 3-IP1 (o"--o) and 4-1P, (0-- 4)(see Fig. 6). Basal levels of radioactivity (0-10 min) in the first peak (1-or 3-IPJ equaled 1133 k 78 cpm and in the second peak (4-IP1)equaled 698k 63 cpm. Data represent net counts/ min (1.0 p~ et-Leu-Phe-stimulated minus assay buffer control) in radioactive peaks measured with an on-line radioactivity detector (see "Experimental Procedures") for one of three comparable experiments.

11549

same retention time as the 3,4-IP2 isomer which was formed in met-Leu-Phe-stimulated intact PMNs. Degradation of IF, Isomers by Disrupted PMNs-When PMN sonicates were incubated with [3H]1,4-IP2(0.5 pM) for 60 min, the major IP, isomer formed was 4-IP1 (>95% of total counts/min in IPI peaks; Fig. 6B ). To determine which IP, is produced via degradation of 3,4-IP2 in PMNs, cell sonicates 3,4were incubated with ["H]1P4for 60 min to allow 1,3,4-IP~, IP2, and IP, to accumulate. The results in Fig. 6C show that only one IP, isomer, coeluting with AMP, was produced under these conditions. Since the only IP2 isomer formed was 3,4IPn, theIPI peak should represent 3-IP,. To confirm that 3,4IP, is notdegraded to 4-IP1, an aliquot of a [3H]4-IP1 standard was added to the cell extract shown in Fig. 6C. The results confirmed that the IP, product formed via 3,4-IP2degradation did not coelute with 4-IP1 (Fig. 6 D ) . These results therefore show that inhuman PMNs, 1,4-IPzis preferentially degraded by a 1-phosphatase, whereas 3,4 IP, is preferentially degraded by a 4-phosphatase. Calcium Dependencyof the IP4 Pathway in Human PMNsPrevious studies have shown that inositol phosphate production occurs in et-~u-Phe-stimulated PMNs in the absence of extracellular calcium (6) and in cells pretreated to prevent a rise in [Ca2+Ji(6, 17). To assess the dependency on [Ca2+Ii for production of the IP2and IP3 isomers, and for IP4, PMNs were pretreated by stimulating with ionomycin (0.2 PM) in the presence of EGTA (2 mM) to prevent the fMet-Leu-Pheinduced [Ca2+],rise. Studies in Quin-2 loaded cells confirmed (17, 39) that this treatment entirely abrogated the agonistinduced rise in [ea2+]; (not shown). Pretreatment of cells with EGTA and ionomycin did not affect the levels of 3,4-IP2, 1,3,4-IP3,or IP4 in unstimulated PMN; however, both 1,4,5, IP3 and 1,4-IP2 levels were reduced (250%) after this treat: ment(Table I) or after incubation with EGTA only (not /A.

+ 0

5

10

15

I

ADP

AMP

1.d;1P2

I

30

TIME (min) FIG. 5. Metabolism of [3H)1,4,5-fP3 and [3H]IP4 by PMN sonicates. Aliquots of trichloroacetic acid extracts from PMN sonicates incubated for 0-30 min with 0.5 pM [3H]1,4,5-IP3( A ) or 0.5 pM 13H]IP4( S ) (described under "Experimental Procedures") were analyzed by HPLC (ammonium formate gradient). Disappearance of the

A0

2 x

vi

3.0 20 1.0

ao

radiolabeled substrate (insets) is represented as the percent input counts/min in 1,4,5-IP3( A )or IP, ( B )peaks (14,379 and 12,077cpm, respectively, at time 0). (These counts/min equalled the counts/min recovered when the radioactive substrates were incubatedin the reaction buffers without cell sonicates, not shown.)

petitiveinhibitor of the 1,4,5-IP3 5-phosphomonoesterase (431, retarded but did not eliminate the breakdown of ["HI 1,4,5-IP3by PMN sonicates (not shown). Therefore 2,3-DPG was included in experimentsdesigned to detect conversion of ['H]1,4,5-IP3 to IP,. In the presence of ATP, about 7% of the total added 1,4,5-IP3was converted to IP, by 30 min, whereas conversion to 1,4-IP2and IPIaccounted for 9.5 and 40.2% of the initial radiolabel, respectively (Fig. 5A). The remainder was converted to free inositol (not shown). The fact that the levels of 1,3,4-IPS and 3,4-IP2were still only minimal by 30 min could result from the ability of 2,3-DPG to also inhibit the dephosphorylation of IPgby a 5-phosphomonoesterase (20). Theresults shown in Fig. 5B clearly show, however,that ['HjIP, incubated in the absence of 2,3-DPG was converted to 1,3,4-IP3and an apparentsecond IP2isomer which had the

ELUTION TIME

[mln)

FIG.6. Metabolism of 1,4,-1Pz and 3,4-IPp by disrupted PMN. A shows a tracing of the chromatograph from HPLC analysis (ammonium phosphate gradient) of base hydrolyzed [3H]1,3,4-IP3; the 3,4-IP2 peak was identified previously on the basis of the IP1 isomers formed after base hydrolysis (28). The peak eluting before 1,4-IPzis therefore assumed to represent 1,3-IP2.The positions of 1IP,, 4-1P1, and 1,4-IP2 correspond to radioactive standards(not shown). Tracings B-D represent chromatographs of trichloroacetic acid extracts from F M N sonicates that were incubated with 0.5 p~ [3H]1,4-IPz( B ) or 0.5 p~ 13H]IP4(C and D ) for 60 min. In D,an aliquot of the [3H]4-IP, standard was added to a portion of the cell extract shown in C prior to chromatographic analysis. Data shown represent tracings of radioactivity measured with the radioactivity detector; absorbance was monitored at 254 nm to detect unlabeled AMP and ADP.

11550

IP3 Metabolism in HumanPMNs TABLEI Elevated [Ca2+],is required for production of 1,3,4-ZPa and 3,4-IP2 in human PMNs stimulated with fMet-Leu-Phe

1,4,5

Stimulant 3,4

IP, isomers

Group"

IP, isomers

1,4

1%

IP,

cpm (&.E.)

Buffer fMet-Leu-Phe

A B A

B

29 290 f 24 151 f 26b 611 f 49 470 f 71b

k9

38 f 9 91 f 15 47 f 8'

109 f 8 37 f 16' 357 f 46 207 f 4gb

5 f 3 18 f 12 120 f 10 33 f 5'

29 f 10 26 f 7 129 f 18 110 f 9

Net cpm (% of controly

A B

320 f 67 52 f 7 248 f 46 115 f 7 99 f 15 320 f 96 (100) 170 f 13 (69) 18 f 8' (35) 20 & 11' (17) 84 f 4 (85) a PMN in Hepes-buffered HBSS (A) or in Hepes-buffered HBSS with 2 mM EGTA (B) were prewarmed for 5 min before adding either buffer (A) or 0.2 p M ionomycin (B).After an additional 5 min, the cells were stimulated with bufferor 0.1 p M Net-Leu-Phe; reactions were then terminatedat 30 s with trichloroaceticacid. Values shown represent mean counts/min f S.E. from three separate experiments. ' p 5 0.05 uersus control group (A) in Student's pairedt test. ' p 5 0.01 uersus control group (A) in Student's paired t test. * Net counts/min = countdmin in Net-Leu-Phe-stimulatedcells minus counts/min in cells treatedwith assay

buffer only.

shown). After the EGTA/ionomycin pretreatment (group B), 0.1 pM fMet-Leu-Phe still induced an increase in 1,4,5-IPS and IP4 to 69 and 85%, respectively, of that produced by control PMNs (group A) (Table I). The most dramatic change in PMNs pretreatedto prevent an fMet-Leu-Phe-induced rise in [CaZ+liwas in the 1,3,4-IP3and 3,4-IPz peaks,which were reduced to only 17 and 35%, respectively, of values obtained in the control group cells. Formation of 1,4-IPz was not markedly affected when the rise in [CaZ+liwas prevented rise before (Table I). In PMNs pretreatedto prevent a [CaZ+li stimulating with a 10-fold higher dose (1.0 p ~ of) fMet-LeuPhe, the 1,3,4-IP3 and 3,4-IPz levels werecomparably reduced in comparison to control values (not shown). Pretreatment of cells with EGTA alone reduced the fMet-Leu-Phe-induced increase of the 1,3,4-IP3isomer to 55% of the control values (p 5 0.05), but the levels of both IP2 isomers, 1,4,5-IP3 and IP,, were not significantly different from those seen when extracellular calcium was present (data not shown).

47 for reviews). Since 25-50% of fMet-Leu-Phe (48) or LTB, (49) remains intact after 5-min exposure to PMNs at 37 "C, differences in the relative rates of degradation of these chemoattractants are unlikely to account for the differences (560 s ) in 1,4,5-IP3production or calcium mobilization reported here. Other possible explanations for these differences include rates of receptor internalization and/or desensitization which have not been directly compared for the two chemoattractants. It is noteworthy that whereas a reserve population of fMet-Leu-Phe receptors exists inPMN-specific granules (50), LTB, receptors are only detected in the plasma membrane (51). Recruitment of new receptors to the plasma membrane could prolong PIPz hydrolysis in response to met-Leu-Phe; but this hypothesis has not been directly tested. The present results also show that the immediate rise in [CaZ+lideclined more rapidly in LTB, uersus fMet-Leu-Phestimulated PMNs. Recent studies in sea urchin eggs have provided evidence that IP, mediates calcium mobilization across the plasma membrane via a process that requires the presence of a calcium-mobilizing IPS isomer (33). Thus it is DISCUSSION The data herein define the kinetics, metabolic fates, and possible that the rapid dissipation of 1,4,5-IPS, IP4, or other metabolite levels after LTB, stimulation results in failure to relative calcium dependencies for production of inositol phosopen a calcium-dependent plasma membrane cation channel phate isomers in chemoattractant-stimulated human PMNs. (52) and thus theelevated cystosolic free Ca2+concentration PMNs stimulated with fMet-Leu-Phe and LTB, were comis not sustained. This hypothesis is supported by studies in pared since, although both are potent chemoattractants (EGO Fura-2-loaded PMNs in which the magnitude and duration -1 nm), they differ markedly in their ability to activate the of the cytosolic Caz+rise induced in the presence of extracelrespiratory burst in PMNs in terms of both potency (EGO= lular EGTA is similar in met-Leu-Phe- and LTB,- (0.01-1 0.02 p~ for Met-Leu-Phe and >1 p~ for LTB,) and activity p ~ stimulated ) cells? The failure of LTB, to sustain elevated (maximal LTB4-induced responses equaling about half those [CaZ+lilevels mayaccount for its inability to activate asecond induced by fMet-Leu-Phe) (34-36). Stimulation with either pathway (independent of phosphoinositide hydrolysis) for of these chemoattractants (0.1 p ~ results ) in a similar early producing 1,2-diacylglycerol (53). This in turn probably repattern of IP3 and IP4formation which is, in all cases, char- flects the relative inability of LTB, to induce protein kinase acterized by a rapid ( 5 5 s ) rise in 1,4,5-IP3and IP, before any C translocation (54); an event which closely correlates with detectable increase in the 1,3,4-IP3 isomer occurs. Although activation of the respiratory burst in PMNs(55-56). the peak values of 1,4,5-IPSand IP, were equivalent in cells Studies performed with cell sonicates showed that PMNs stimulated with Met-Leu-Phe uersus LTB, (0.1-1 p M ) , the possess two distinct metabolic pathways for removing 1,4,5production of both of these products was substantially more IP3: dephosphorylation to 1,4-IP2 or ATP-dependent phosprolonged in response to fMet-Leu-Phe. The ligand binding phorylation to IP,. Conversion to IP, appears to be unidireccharacteristics and catabolism of met-Leu-Phe versus LTB, tional, as 1,4,5-IP3was not generated when [3H]IP4was added do not appear to explain the different potencies of these to sonicated PMNs; rather themajor metabolic products were chemoattractants for stimulating biochemical responses. For 1,3,4-IP3 and IP2 isomer which was previously identified as example, the number of cell surface receptors on human 3,4-IPz on the basis of the IP, products formed after base PMNs for LTB, is reported to be greater than that for fMetM. Verghese and R. Snyderman, unpublished observations. Leu-Phe (-3 X lo5 versus 5 X lo4) (44, 45; see Refs. 46 and

11551 in trace levels. The IPI was most likely a mixture of 4-iP1 (30%) and l-IP, (70%) since 3-IP1 (which coeluted with 1Ip,) would not be expected to be present in the absence of 3,4-IPz formation. In stimulated PMNs, production of both 1,3,4-IP3and 3,4-IPnwas dramatically reduced when the rise in [Ca2+Iiwas prevented, indicating that these products are only formed after receptor-mediated PIPn hydrolysis. These 1,4.5-IP3 1,3,4.5-lP4 results raise the interesting possibility that 1,3,4-IP3 and/or 3,4-IP2 could act as second messengers to regulate calciumdependent PMN activation. Alternatively, if prolonged [Ca2+li increases are infact induced by IP, in PMNs, calcium may in turn actas a feedback signal to promote the metabolic 1,3,4-IP3 1,4,-IP2 breakdown of IP, to 1,3,4-IPs and 3,4-IP2, thus attenuating the calcium signal. In fMet-Leu-Phe-stimulated HL-60 cells, Lew et al, (58)also found that 1,3,4-IP3production is relatively I-phosphatase ca~cium-dependent;but IP, levels were not measured. In hormone-stimulated RINm5F cells, the 1,3,4-IP3 isomer was $. also reduced when the [Ca2+Iirise was prevented (23). From 4-lP, 3,4-IP2 in uitro studies in this system, it appeared that 1,4,5-IP3 3kinase activity could be modulated by calcium (231, although a similar effect was not seen in studies of the kinase activity in brain (31). Several in uitro studies of IP3 5-phosphatase activity have failed to demonstrate a direct calcium dependency of this enzyme for 1,4,5-IP3 hydrolysis (23, 43, 59). In preliminary in uitro studies with PMN lysates, we also could not demonstrate any calcium dependency (0.1-10 p ~ for ) ATP-dependent conversion of 1,4,5-IP3 to IP,; or of degrashown). dation of IP, to 1,3,4-IP2 and3,4-IPn(datanot Therefore,unless calcium-dependent and -independent 5phosphatase enzymes exist in different subce~lu~ar compartments, it appears that the 5-phosphatase is not directly actiFIG. 7. Pathways of t,4,5-IP3 metabolism in human PMNs. vated by calcium in PMNs. Recent studies by Connolly et al. A PIP2-specific phospholipaseC can be activated via a G protein (G,) (42,60) showed that protein kinase C mediates phosphorylacoupled to chemoattractant receptors. The initial products formed are 1,4,5-IP3and 1,2-diacylglycerol(not shown). 1,4,5-IP3 is metabo- tion of the purified 5-phosphatase from platelets and thereby kizedvia the indicated pathways to free inositol. Production of the enhances both 1,4,5-IP3 and IP, degradation.Therefore, ele1,3,4-IPS and 3,4-IP2 isomers is dependent on elevatedlevels of vated cytosolic calcium may increase the 5-phosphatase activ[Ca"],. ity indirectly by promoting protein kinase C-mediated phosphorylation of the enzyme in activated cells (60). hydrolysis (28). The present study shows that the 3,4-IP2 The information described in the presentreport contributes isomer appears only after an initial lag, concomitant with to understanding the fine regulation of inositol phosphate 1,3,4-IP3, infMet-Leu-Phe-stimulatedPMNs. The major formation in chemoattractant-stimulated human PMNs and route of 1,3,4-1P3degradation in intact PMNs thus occurs via provides evidence that the longevity of the 1,4,5-IPZ3 and IP4 a 1-phosphatase to form 3,4-IPn.In disrupted PMN, 295% of response may be important in eliciting certain biological [3H]1,4-IP2was further degraded to 4-IP1, whereas the 3,4- responses in these cells. Metabolism of 1,4,5-IP3can proceed IP, isomer is metabolized only to the 3-IPiisomer. Thus the via two pathways which depend upon the action of an ATPaction of 1-, 3-, 4-, and 5-phosphatases are apparentlyrequired dependent 3' kinase (30) and a series of phosphatases which for full recycling of the higher inositol phosphates to free exhibit relative specificity for the different inositol phosphate inositol in PMNs (Fig. 7). The majority of the IP, formed in isomers. Since the 1,4-IPz and 3,4-IPnisomers are metabolized activated PMNs was the 4-IPIisomer. The importance of this to different isomeric forms of IP, (4-IP1 and 3-IP1, respecobservation lies in the fact this inositol phosphate product tively), the IP, isomers can serve as markers for the two could be derived solely via metabolism of 1,4-IP2 and thusis different degradation pathways. This may be useful in deterreflective of inositol polyphosphate catabolism. Conversely, mining the potential role of each pathway in cellular activaI-IP, can only be derived via phospholipase C-mediated hy- tion. drolysis of phosphatidylinositol or via the 1,2 cyclic IP, phosphatase-mediated conversion of 1,2 cyclic IP, to l-IP1 (57). Acknowledgments-We thank Dr. Ron Uhing for critically reading for excellent secretarial support. From the experiments reported here, we cannot determine the manuscript and Sharon Goodwin the relative amount(s) of 1- andfor 3-IP1 formed in intact Note Addedin Proof-An inositol polyphosphate 1-phosphatase PMNs, since these two IP, isomers coeluted on the gradient activity was recently identified which dephosphorylates 1,4-IP, and system used. or 1-IP, (61). Previous studies have shown that chemoattractant recep- 1,3,4-IPsbut not 1,3,4,5-IP4, 1,4,5-IP3, tor-mediated PIP, hydrolysis and IP3production can occur in REFERENCES PMNs at ambientintracellular calcium levels, in theabsence 1. Volpi, M., Yassin, R., Naccache, P. 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t

11552

IPSMetabolism i:nHuman PMNs

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