Multiple Signaling Pathways of Histamine HZ Receptors

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 264,No. 31, Issue of November 5, pp. 18356-18362 198s Printed in 6 , S . A .

Multiple Signaling Pathways of Histamine HZ Receptors IDENTIFICATIONOF AN HZ RECEPTOR-DEPENDENT Ca2+ MOBILIZATION PATHWAY IN HUMAN HL-60 PROMYELOCYTIC LEUKEMIA CELLS* (Received for publication, March 16, 1989)

Masato Mitsuhashi$#ll, Takako Mitsuhashig, and Donald G. Payan$# From the $Howard Hughes Medical Institute Laboratories and §Department of Medicine, University of California Medical Center, San Francisco, California 94143

In order to analyze the complex activities of hista- tors are known to be coupled to phosphatidylinositol hydrolmine HZreceptor activation on neutrophils, human HL- ysis pathways with histamine binding resulting in the mobi60 promyelocytic leukemia cells were differentiated lization of intracellular Ca2+ ([Ca”]i) (1, 5, 6 ) , whereas Hz into neutrophilsby incubation with dimethyl sufoxide, receptors are coupled to adenylate cyclase with histamine loaded with the Caz+-sensitive indicator dyes, indo-1 stimulating increases in intracellular cyclic adenosine monoor fura-2, and thelevels of intracellular Caz+([CaZ+li) phosphate(CAMP) (7). Both H1 and Hz receptors are comeasured in a fluorescent-activated cell sorter and localized in various cells/tissues, and regulate histamine refluorimeter, respectively. Histamine increased [CaZ+li sponses via secondary Ca2+and cAMP pathways, respectively in a dose-dependentmanner with a half-maximal concentration (ECeo) of approximately lo-‘ to lo-’ M, (8). In neutrophils, the actions of histamine are not well charwhich exhibitedHz receptor specificity. Prostaglandin acterized. For example, histamine Hz-specific stimulation Ez and isoproterenol also induced [Caz+]imobilization in HL-60 cells, whereas the cell permeable form of consistently enhances the chemokinetic responses of neutrocAMP and forskolin failed to increase [CaZ+li.Since phils, while the chemotactic response of neutrophils was Hz-receptor mediated [Caz+]imobilization was not in- inhibited in an HZ receptor-dependent manner (9, 10). Alhibited by reducing the concentrationof extracellular though the inhibition of chemotaxis is due to theelevation of Caz+nor by the addition of Caz+ channel antagonists, CAMP,the mechanism of the Hz receptor-mediated stimulaLaC13 and nifedipine, [Caz+]imobilization is due to the tion of chemokinesis is not explained by the activation of release of Caz+from intracellularstores. Furthermore, secondary cAMP pathways. This suggests that Hz receptors both low4M histamine and lo-’ M met-Leu-Phe in- could have multiple independent effects on steps early in creased thelevels of 1,4,5-inositol trisphosphate.How- neutrophil activation. ever, histamine-induced mobilization of [Caz+]iwas inHuman HL-60 promyelocytic leukemia cells can be differhibited by cholera toxin but not by pertussis toxin, entiated into neutrophils by incubation with dimethyl sulfwhereas the action of met-Leu-Phe was inhibitedby oxide (MezSO)’in culture medium for more than 5 days (11). pertussis toxin but not by cholera toxin. These data Furthermore, histamine has been shown to increase cAMP in suggest that HP receptors on HL-60 cells are coupled HL-60 cells in an Hzreceptor-specific manner (12,13). In the to two different cholera toxin-sensitive G-proteins and present study, we found that histamine also induced mobiliactivate adenylate cyclase andphospholipase C simul- zation of [Ca2+]iin HL-60 cells in an HZ receptor-specific taneously. manner. Hz receptor-induced mobilization of [CaZ+liwas significantly inhibited by cholera toxin, whereas cholera toxin enhancedadenylate cyclase activation.Furthermore, Hz receptor stimulation also increased the intracellular levels of Histamine is an important chemical mediator of allergic inositol trisphosphate. These data suggest that HZreceptors and inflammatory reactions involving the stimulation of are coupled to two different cholera toxin-sensitive G-proteins smooth muscle contractility (I), alterations invascular perme- in HL-60cells, resulting in the possibility that histamine can ability, and modification of various leukocyte subset activities activateadenylate cyclase and phospholipase Cpathways (2). Histamine is released from tissue mast cells or blood simultaneously. basophilic leukocytes in conjunction with other potent chemical mediators in response to specific IgE-mediated or nonEXPERIMENTALPROCEDURES specific mechanisms (3, 4), and the action of the released Materials-HL-60 cells were obtained from the American Type histamine is believed to be mediated by specific cell surface Culture Collection (Rockville, MD). Media for tissue culture were receptors. Pharmacological analyses have demonstrated that obtained from the Cell Culture Facility (University of California, San at least two different subclasses of histamine receptors are Francisco, CA). Indo-l-AM, fura-2-AM, NBD-phallacidin (Molecular involved in inflammatory reactions (2). Histamine H1 recep-

* This work was supported in part by Grant NS21710 from the National Institutes of Health, and by the Howard Hughes Medical Institute. The costs of publication 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 solelyto indicate this fact. ll To whom correspondence should be addressed: 533 Parnassus Ave., U-426, University of California Medical Center, San Francisco, CA 94143-0724. Tel.: 415-476-4996.

‘The abbreviations used are: Me2S0, dimethyl sulfoxide; BSA, bovine serum albumin; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; CT, cholera toxin; FACS; fluorescent-activated cell sorter; FCS, fetal calf serum; fMLP, f-Met-Leu-Phe; AM, acetoxymethyl ester; NBD, N-[7-nitrobenz-2-0xa-1,3-diazole-4-y1]; HBSS, Hanks’ balanced salt solution; 4m-HA, 4-methylhistamine; HPLC, high performance liquid chromatography; IPS,inositol trisphosphate; PGE,; prostaglandin E,; PMA, phorbol 12-myristate 13-acetate; PT, pertussis toxin; H-8, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride.

18356

H2Receptor-induced

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18357

zation, polymerized actin was stained with NBD-phallacidin and Probes, Junction City, OR), [3H]myoinositol (40-60 Ci/mmol), ['HI 1,4,5-IP3 (1-5 Ci/mmol (Amersham Corp.) were obtained from the analyzed in a FACS. These experiments are based on the evidence designated suppliers. The HIand Hz-specificagonists and H,-specific that phallacidin binds to polymerized actin butnot tononpolymerized antagonists ~metiamide~ were kind gifts from Dr. J. Skidmore (Smith actin (18).MezSO-differentiated HL-60 cells were suspended in BSAKline & French Laboratory, Hertsordshire, United Kingdom). The HBSS at 2 X IO6 cells/ml and aliquoted into 0.9 ml/tube. After M &methylhistamine (4m-HA, HI! agonist) or stereoisomer, d-chlorpheniramine was kindly provided byDr. W. stimulation with M M L P , 0.1 ml of a 37% formalin solution was added into each Kreutner (Schering Corp.). The &-specific antagonist, thioperamide was a kind gift from Dr. Arrang (INSERM, Paris). Other chemicals tube and incubated for 15 min at room temperature. Fifty p1 of a 2 mg/ml solution of lysophosphatidylcholine and 50 pl of NBD-phalwere purchased from Sigma. Cell Culture-HL-60 cells were grown in RPMI 1640 containing lacidin (100 units/ml) were added and incubated at 37 "Cfor another 100 units/ml penicillin, 100 pg/ml streptomycin, and 10% FCS (FCS- 10 min. Cells were centrifuged at 400 X g for 5 min to remove excess RPMI) at 37 "C in 5% CO,, 95% air. Cells were subcultured twice dye, resuspended in fresh HBSS, and analyzed in a FACS at 488 nm weekly at a ratio of 1:4-6. For the granulocytic differentiation of HL- excitation and 522 nm emission. 60 cells, the culture medium was replaced with fresh FCS-RPMI Statistical Anulysis-The Student's t test was performed for the containing 1.2% Me&O and the cultures were continued for 7 days comparison of matched pairs or independent two groups after the test (11).After Me,SO treatment, thegranulocyte population was approx- of the similarity of variance between groups. imately 80% by morphological analysis. Cell viability was more than 90% as assessed by the exclusion of trypan blue. RESULTS suspended in Measurement of Zndo-1 Ca" Signals-Cellswere Histamine HzReceptor Specificity-In order to analyze the fresh RPMI 1640 containing 0.1% bovine serum albumin (BSA) at 0.5-2 X lo' cells/ml, and loaded with the acetoxymethyl ester of indo- stereospecificity of the action of histamine, undifferentiated 1 (3 p ~ for ) 20 min at 37 "C in the dark as described previously for and MezSO-differentiatedHL-60 cellswere loaded withindothe analysis of HI receptor activation on cultured smooth muscle cells 1, and the changes in [Caz+];were analyzed in a FACS using (14). The cells were then diluted 10 times with BSA-RPMI and various histamine agonists and antagonists. The results of incubated for a further 20 min at 37 "C. After washing once with BSA-RPMI, cell pellets were resuspended in fresh BSA-RPMI at 5 FACS experiments were the sum of the signals from 4000 to 5000 cellsanalyzed during 20-25 s after stimulation with X 106/ml and analyzed in a fluorescent-activated cell sorter (FACS kinetic IV, Becton Dickinson, Sunnyvale, CA) equipped with an argon ion ligand, based on the peak response observed from laser emitting 600 milliwatts at 351-364 nm. In order to analyze the studies in a fluorimeter (Figs. 2-6). changes in [Ca'+Ii in individual cells, blue (486 nm) and violet (404 Histamine ( 1 0 - 5 ~increased ) [Ca2+Ijin MezSO-differennm) band pass filters were used to collect indo-1 fluorescence emis- tiated HL-60 cells (Fig. L4, inset). In order to quantify the sion. The ratio of violet to blue fluorescence is directly proportional FACS data, the window measuring positive responders was to thelevels of [Ca'"];. cell population, Measurement of Furu-2 Ca" Signals-HL-60 cells were suspended set at the right margin of the unst~m~lated in phenol red-free Hanks' balanced salt solution (HBSS) containing andthe percentage of cellscrossing the window (% re0.1% BSA at 2 X lo7 cellsfml, and loaded with the acetoxymethyl sponders) were counted. Multiple separate experiments indiester of fura-2 (2.5 ptM) for 60 min at 37 "C in the dark as described cated that thepercentage of responders following stimulation previously for the analysis of HI receptor activation (1). After washing M histamine and 4m-HAwere 32.17 C 5.42 (mean three times with BSA-HBSS, cell pellets were resuspended in fresh with BSA-HBSS at 5 X 10' cells/ml. The intracellular fura-2 signals ( F ) & S.E., n = 10) and 18.81 f 1.87 (n = 8), respectively. The were measured at 340 nm excitation and 510 nm emission by a Hz-specific antagonists metiamide and cimetidine inhibited fluorimeter (model 650-40, Perkin Elmer, Norwalk, CT). The values histamine-induced [Ca2+];mobilization in MenSO-differenof [Ca2+Iiwere calibrated by exposing cell suspensions to 0.1% Triton tiated HL-60 cells at M (Fig. L4, MET and CIM). The X-100 (Fmax) followed by addition of 10 mM EGTA, pH 10 (Fmi,,). percentage of responders to M histamine after pretreat[Caa+Iivalues are then derived from the following equation (15): ment with M cimetidine (6.43 f 2.08, n = 3) was significantly (p < 0.05) less than that of control (32.17 -+ 5.42, n = [Caz+14 (nM)= K M p n d / ( p m a x - F)

-

where Kd = 224. Measurement ofthe Levels ofIP3-Cells were suspended in inositolfree FCS-RPMI and incubated with 200pCi of ~3H]myoinositol overnight. Cells were then washed with BSA-RPMI threetimes, resuspended in BSA-RPMI at 1 X 10' cells/ml, and incubated with 10 mM Licl at 37 "cfor 15min in siliconizedglass tubes. Cell aliquots (1ml/tube) were then stimulated with either M histamine(HA) M met-Leu-Phe (MLP)for 8 s at 37 "C. To stop thereaction, or an equal volume of 30% trichloroacetic acid was added and incubated at 4 "C for 20 min. In order to increase the recovery of IPS,25 pg of phytic acid was also added into each tube (16). After centrifugation at 2000 X g for 10 min at 4 "C, supernatants were extracted with an equal volume of water-saturated ethylether six times to remove cellular lipid components. After ethylether was evaporated, samples were neutralized and separated by a Partisil SAX 10 anion-exchange column using a Beckman HPLC system (450 data system/controller, 114M solvent delivery module, 165 variable wavelength detector, Beckman, Berkeley, CA). The column was first washed with water for 5 min to elute unbound materials and theneluted with a gradient Of 0-750 mM ammonium formate, pH 3.7over 10 min to collect inositol phosphates and inositol bisphosphates. The column was then eluted with a shallow gradient of 750-1000 mM ammonium formate Over 10 min at a flow rate of 1 ml/min to collect 1,3,4-IP3and 1,4,51% separately as described previously (17). Fractions were collected at 0.5-min intervals, and the @-radioactivitywas determined in a liquid scintillat~oncounter (LS 5801, Beckman Instruments, Imine, CA). In order to identify the peak of 1,3,4-IP3and 1,4,5-IP3,ATP and [3H]1,4,5-IP3were also chromatographed, respectively. Actin Polymerization-In order to analyze whether histamineinduced [Ca"], mobilization is effectively coupled to actin polymeri-

10). Three determinations using independent preparations indicated that the concentration of cimetidine for the 50% inhibition (IC&)of histamine-in~uced{Ca2+'Iimobi~ization was approximately M (data not shown). In contrast, three different H1-specificantagonists, pyrilamine, triploridine and the d-form of chkorpheniramine, and the Hs-specific antagonist thioperamide failed to inhibit histamine-induced [Ca2+]; mobilizationeven at doses as high as M (Fig. 1, PYR, TRI, CHL, and T H I ) . The action of histamine was also dose-dependent (Fig. 1B). Three determinations using independent preparations indicated that the half-maximal concentration (EC,) of histamine and 4m-HA were approximately M in Me,SOdifferentiated HL-60 cells (Fig. 1B)and in undifferentiated HL-60 cells (data not shown). Another H, agonist dimaprit also increased [Ca2+];in a dose-dependent manner with a similar EC5o value (10" M) to that of histamine (data not shown). On the other hand, the H1-specificagonist, 2-pyridylethylamine failed to mobilize [CaZ+Jj at concentrations as high as M (Fig. 1s). Effect of CAMPon HZReceptor-mediated fCaZ'Ji Mobilization-As shownin Fig. lB, prostaglandin Ez (PGE,) and isoproterenol,which havebeen reported to increase CAMP in HL-60 cells,also mobilized [Ca2+Iiin a dose-dependent manner with EC50 values of approximately and loq8 M, respectively. The percentage of responders for IO-:" M PGE,

I

i

A %

v

.r

+N 4

u

U

MET

CIM

" " "

1 PM

PYR

TRI

CHL THi

" " " " " " "

100 pM

TIME

-

1 min FIG. 2. Effect of cAMP on 4m-HA-mediated [Ca2+lr mobilization. MezSO-differentiated HL-60 cells were suspended in phenol

LOG (LIGAND CONCENTRATION),

M

FIG. 1. Agonist and antagonist specificity of histamine-inHL-60 cells. MezSO-differentiated duced increases in [Ca2+Ji on HL-60 cells were suspended in RPMI 1640 containing 0.1% BSA a t 0.5-2 X 107/ml,and loaded with indo-1 (3 NM) for 20 min a t 37 "C in the dark. Cells were then diluted 10-fold and incubated for a further 20 min a t 37 "C. After washing once with BSA-RPMI, cell pellets were resuspended in fresh BSA-RPMI at 5 X 106/ml and analyzed in a FACS equipped with an argon ion laser emitting 600 milliwatts at 364 nm. Blue (486 nm) and violet (404 nm) band pass filters were used to collect indo-1 fluorescence emission. The ratio of violet to blue fluorescence is directly proportional to the levels of [Ca2+],. Results were displayed as a function of relative cell number and the levels of[Ca'+]; (404/486nm emission) in a logarithmic scale as shown in the inset. A, indo-1-loaded MezSO-differentiated HL-60 M cimetidine cells were pretreated with metiamide (MET), (CIM), M pyrilamine (PYA?),zo-* M triploridine (TRI), M d-chlorpheniramine (CHL), or M thioperamide ( T H I ) for 2-3 M histamine was analyzed in a FACS. min, and then the effect of In order to quantify the FACS data, the window measuring positive responders was set at the right margin of the unstimulated cell population, and the percent of cells crossing the window (% OF ~ ~ ~ were counted. ~ O Each bur~ indicates ~ the mean E value ~ of the percent inhibition compared to that of histamine stimulation alone from 2 to 3 separate experiments. B,indo-1-loaded HL-60 cells were stimu~atedwith various concentrations of histamine (O), 4mH A (e),2-pyridylethylamine (A), PGEz (A), and isoproterenol (0). Each graph represents a mean value of the percentage of responders 20-25 s after stimulation from 2 to 3 separate experiments.

red-free HBSS containing 0.1% BSA at 2 X lo7 cells/mi, and loaded with fura-2 (2.5 ,UM)for 60 min at 37 "C in the dark. After washing three times with BSA-HBSS, cell pellets were resuspended in fresh BSA-HBSS at 5 X IO6 cells/ml. The intracellular fura-2 signals ( F ) were measured at 340 nm excitation and 510 emission by a fluorimeter. The levels of [Ca2+Iiwere calibrated by exposing cell suspension to 0.1% Triton X-100 followed by addition of 10 m M EGTA, pH 10 (F,in). [Ca2+Iivalues are thenderived from the following equation (15): [Ca2+li(nM) = Kd(F - F m d / ( F m a x

- F)

where Kd = 224.The solid lines represent the recordings of untreated M 4m-HA, andthe broken lines represent cells stimulated with the recordings of the identical cell preparations pretreated with 0.5 mM 8-Br-CAMP ( A ) , 0.5 mM 8-Br-cGMP ( B ) , and 0.1 m M forskolin ( C ) .Shown is a typical paired experiment reproduced twice.

mM) failed to increase [Ca2+li,and had no effect on 4m-HAinduced increases in [Caz+];(Fig. 2). Forskolin (0.1 mM) has been shown to increase CAMP, but failed to modulate 4mHA-induced [Ca*+Iimobi~ization(Fig. 2). Furthermore, another cell-permeable form of CAMP, &butyryl-CAMP (0.25 mM), 3-isobutyl-1-methylxanthine(0.5 mM), which inhibits the activity of phosphodiesterase, and the inhibitor of cyclic nucleotide-dependent protein kinase, N-[2-(methyIamino)ethyl]-5-isoquinolinesulfonamidedihy~ochloride(H-8) (80 GM) all failed to change the levels in [Ca*+]i,and also showed no effect on the 4m-HA-induced increase in [Ca2+]i(data not ~shown). ) These data were reproduced twice onseparate preparations, and also confirmed by two separate FACS analyses (data not shown). Effect of Extracellular Cu2+and Ca2+Channel AntagonistsIn order to evaluate whether histamine-induced increases in [Ca2+liwere due to the influx of extracellular Ca2+ or the release of Ca2+from intracellular stores, fura-%loaded HL-60 cellswere suspended in Caz+-free HBSS containing 1 mM and M isoproterenol were 18.43 +- 1.9 ( n = 3) and 25.76 EGTA, and the changes in [Ca2+Iiwere analyzed in a fiuo& 1.65 ( n -- 4), respectively. rimeter. As shown in Fig. 3A, lo-* M 4m-HAmobilized In order to further analyze the kinetics of [Ca"]i mobili- [Ca2+Iiinthe absence of extracellular Ca2+. The percent zation, HL-60 cells were loaded with fura-2 and the effects of maximal increase of [Ca2+]iafter stimulation with M 4mvarious drugs which modulate the action of cAMP were ana- HA in the absence of extracellular Ca2+ (256It 7.6%, n = 3) lyzed in a fluorimeter. The basal levels of [Ca2+];in HL-60 was not significantly different from that in the presence of cells were 80-250 nM ( n = go), with M 4m-HA transiently 1.4 mM extracellular Ca2+ (280 +- 29.9%, n = 3) in identical increasing [Ca*+']ito 300-400 nM which returned to basal paired preparations. Moreover, the Ca2+channel antagonists levels within 1-2 min (Fig. 2 ) . Because of the wide variation 40 FM LaC13 {Fig. 3B)and 4 PM nifedipine (Fig. 3CJalso had of the peak values of [Ca2+jiamong different preparations, no effect onthe 4m-HA-induced increase in [Ca2+Iiin HL-60 each experiment was performed using paired identical prepa- cells. These data were reproduced three times on separate rations with or without drug treatments. As shown in Fig. 2, preparations in the fluorimeter and also confirmed by two the cell-permeableform of cyclic nucleotides, 8-bromo-CAMP separate FACS analyses (data not shown). Mechanism of H2 R ~ c e ~ t o r - m ~ dMobil~zatwn ~te~ of (8-Br-cAMP, 0.5 mM) and 8-bromo-cGMP (8-Br-cGMP, 0.5

18359

Hz ~ e ~ e ~ t o r - i nea2' d ~ eMobilization d

E fMLP

P M 10 ngfml

"----

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TIME TIME

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1 min

FIG. 4. Effect of PMA on 4m-HA- or fMLP-induced mobiFura-2-loaded MezSO-differentiated HL-60 ceils FIG. 3. Effect of extracellular Ca2+ on 4m-HA-induced lization of [Ca2+Ji. [Ca2+Iimobilization. A, fura-2-loaded MezSO-differentiated HL-GO resuspended in BSA-HBSSwere preincubated with 10 ng/ml ( B and cells were suspended in Ca2+-free HBSScontaining 1 mM EGTA E ) or 100 ng/ml (C and F ) of PMA for 5 min, and stimulated with M 4m-HA (A, B, and C) or lo-* M fMLP (D, E, and F ) . The ( . . . .), and the changes in 4m-HA-induced increasein [Ca2+]i was compared with that of CaZ+ containing HBSS(-) in a fluorimeter levels of [Ca2+]iwere measured in a fluorimeter as described under as described under "Experimental Procedures" and Fig.2. B and C, "Experimental Procedures" andin Fig. 2. A and Lf represent control fura-&loaded HL-GO cells were suspended in Ca2+containing HBSS, recordings of the identical cell. preparations without PMA pretreatand the effects of Ca" channel antagonistsLacla (40 phi; B, . . .) or ment.Shown is a typical experiment reproduced three (fMLP) to nifedipine (4 p ~ C,; . . .) were analyzed in a fluorimeter. The solid four (4m-HA) times. lines represent the recordings of the identical cell preparations in the absence of Ca2+ channel antagonists.Shown is a typicalpaired experiment reproduced three times. [Ca2+],-The signal transduction mechanism of the chemotactic peptide, fMLP has been extensively studied in MezSOdifferentiated HL-60 cells (19, 20). For example, the fMLP receptor is coupled to cholera toxin (CT)- andpertussis toxin (PT)-sensitive G-proteins and activates phospholipase C following the hydrolysis of phosphatidylinositol. The resulting second messengers, 1,4,5-IP3and diacylglycerol, mobilizeCa2+ and activate protein kinase C, respectively. Therefore, in order to further analyze the mechanism ofH2 receptor-mediated mobilization of [Caz+ljin HL-60 cells, the actions of histamine and 4m-HA were compared to those of fMLP. Me2SO-differentiatedHL-60 cells were loaded with fura-2 and the kinetics of 4m-HA and fMLP-induced mobilization of [Ca2+liwere compared in a fluorimeter. 4m-HA-induced [Ca2+Iimobilization was transient and returned basal to levels within 1-2 min, whereas fMLP-induced [Ca2+]imobilization was sustained for more than 10 min (Fig. 4,A and D).Phorbol 12-myristate 13-acetate (PMA) has been shown to activate protein kinase C resulting in the down regulation of various cell surface receptors (14). As shown in Fig. 4, fMLP-induced [Ca"+li mobilization was reduced by pretreatment with 10100 ng/ml PMA, but 4m-HA-induced [Caz+limobilization was resistant to PMA (Fig. 4). After pretreatment with 100 ng/ml PMA, fMLP-induced increases in [Caz+liwere inhibited to 9.6 rt 2.3% ( n = 3) of the identical paired control response, a significantly (p c 0.01) more complete inhibition than that of 4m-HA (67.4 k 5.6%, n = 4). Furthermore, 4mHA and fMLP desensitized the secondary 4m-HA and fMLP responses, respectively, but failed to desensitize each other (Fig. 5). Since ionomycin has been shown to deplete fMLPsensitive intracellular Ca2+ storeswhen cells weresuspended in a Ca2+-freesolution (17), we have also analyzed whether or not 4m-HA mobilizes [Ca2+Ijfrom ionomycin-sensitive Ca2+ stores. As a result, both 4m-HA and fMLP failed to increase [Ca"'], after ionomycin treatment (data not shown). In order to investigate whether histamine Hz receptorinduced increases in [Ca"Ji are sensitive to PT or CT, Me,SOdifferentiated HL-60 cellswere preincubated with various concentrations of PT or CT from 2 h to overnight at 37 "C.

I

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4m-HA

fMLP

fnLP

fMLP

*A

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FIG. 5. Interaction between 4m-HA- and fMLP-induced [Ca2+Iimobilization. Fura-2-loadedMe~SO-differentiated HL-GO cells were suspended in BSA-HBSS and [Ca2+],was measured in a fluorimeter as described under "Experimental Procedures" and in M fMLP or M 4mFig. 2. Cells were thenstimulatedwith HA at the indicated points. Shownis a typical experiment reproduced three times. Cells were then loaded with fura-2, and [Ca2+jisignals were analyzed in a fluorimeter as described. As a result, HAinduced [Caz+Jimobilization was inhibited by CT, but not by PT (Fig. 6). The inhibitory effect of CT was observed over a range of 0.4-10 pg/ml, a similar dose range reported to increase in CAMPon HL-60 cells (21). The inhibitory effect of CT appeared 2 h after CT pretreatment and continued for at least 16 h (data not shown). In contrast, PT failed to alter HA-induced [Caz+limobilization even after overnight incubation at 100-500 ngfml, in which fMLP-induced mobilization of [Ca2+]iwas totally inhibited (Fig. 6).These data were reproduced twice on separate preparationsin thefluorimeter, and also confirmed by three separate FACS analyses (data not shown). Furthermore, PGE2-induced [Ca2+]imobilization was also sensitive to CT but notto PT(data not shown). As shown in Fig. 7, both M histamine and 1O"j M fMLP increased the levels of 1,3,4-IP3 and 1,4,5-IP~. Three determinations using independent preparations indicated that the level of 1,4,5-IP38 s after stimulation with lo-* M histamine was 106 17.6% of that of loa M fMLP in identical paired preparations, which was not significantly different from each other.

*

18360

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UNSTAINED

fMLP I

W 0

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HA I

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FIG.6. Effect of PT and CT on histamine or NLP-induced increases in [Caa+]i onHL-60 cells. MezSO-differentiated HL-60 cells were treated with 0.5 pg/ml PT at 37 "C overnight or 2 pg/ml CT at37 "C for 2 h. The cells were then loaded with fura-2, and the M fMLP was analyzed in a effect of 10" M histamine and fluorimeter as described under "Experimental Procedures" and in Fig. 2. Shown is a typical experiment reproduced three times. 60

T

A. fMLP 1,3,4-tP3

1,4,5-lP3

RELATIVE FLUORESCENCE INTENSITY

FIG. 8. 4m-HA- and ~ ~ P - i n d actin u ~ d polymerization. MezSO-differentiated HL-60 cells were suspended in BSA-HBSS at 2 X lo6 cells/ml and aliquoted into 0.9 ml/tube. After stimulation M 4m-HA or lo-' M fMLP, 0.1 ml of 37% formalin was with added into each tube and incubated for 15 min at room temperature. Fifty pl of a 2 mg/ml solution of lysophosphatidylcholine and 50 pl of NBD-phallaci~n(100 u n i ~ / m l were ) added and incubated at 37 "C for another 10 min. Cells were centrifuged at 400 X g for 5 min to remove excess dye, resuspended in fresh HBSS, and analyzed in a FACS at 488 nm excitation and 522 nm emission. Each graph represents a sum of the analysis of 4000-5000 cells. Shown is a typical experiment reproduced three (4m-HA) to four ( M L P ) times.

dation. Theeffect of fMLP appeared at 15 s and was maximal at 5-10 min after stimulation (data not shown). However, 4m-HA failed to increase the fluorescence of NBD-phallacjdin in individual cells (Fig. 8). The percentage of responders for M fMLP (31.7 k 5.7, n = 4) was significantly fp C 0.01) higher than that of M 4m-HA(-1.3 k 0.7, n = 3). DISCUSSION

In the present study, histamine-induced [Ca"]i mobilization has been determined by two different methods using the Ca2+-sensitiveindicator dyes indo-1 and fura-2. Both dyes were established by the group of Tsien (15) and extensively studied in the analysis of [Ca"Ji mobilization in various cells, principally because the acetoxymethyl ester form of these dyes are cell-permeable and are able to quantify the levels of 10 15 20 25 30 intracell~arfree Ca2+without cellular damage. In the first Fractions (rnin) series of experiments, the levels of[Caz']i in indo-1-loaded FIG. 7. Histamine- and fMLP-induced IPS production. cells were analyzed in a FACS, where the changes in [Ca2+Ji Me2SO-differentiated HL-60 cells were suspended in inositol-free in individual cells can be determined. However, because the FCS-RPMI and incubated with 200 pCi of [3H]myoinositolovernight. results of FACS experiments were the sum of the signals from Cells were then resuspended in BSA-RPMI containing 10 mM LiCl 4000 to 5000 cells during the maximum response period after at 1X lo7cells/ml, and thealiquots (1ml/tube) were then stimulated stimulation, it is difficult to monitor the changes in [Ca2+li with either M fMLP ( A )or lo-* M histamine ( B )for 8 s at 37 "C. The values of 1,3,4-IP$and 1,4,5-IP$were determined by HPLC using continuously. Furthermore, FACS analysis would underestia Partisil SAX-10 anion exchange column as described under "Ex- mate the percentage of responders if fCa"']i oscillations are perimental Procedures." Shown is a typical experiment reproduced occurring (22). Therefore, to confirm and expand the FACS experiments and further analyze the kinetics of [Ca2+li,cells three times. . . . indicates the result of unstimulated cells. wereloaded with fura-2 and analyzed in a fluorimeter as In order to analyze further whether elevated [Ca2+]iis described previouslyfor the analysis of HI receptor activation effectively coupledto actin polymerization pathways, MezSO- on cultured smooth muscle cells(1). Histamine increased [Caz+]i in undifferentiated and differentiated HL-60 cellswere stimulated with M 4mHA or lo-' M fMLP, and actin polymerization of individual MezSO-differentiated HL-60 cells in a dose-dependent manto M (Fig. 11, cells was evaluated by NBD-phallaci~nstaining in a FACS. ner with an ECso of approximately These experiments were based onthe evidence that phallaci- which is equivalent to that observed for the activation of din binds to polymerized actin but not to nonpolymerized CAMP on these cells (12). Hz receptor specificity was demactin (18). As shown in Fig. 8, the fluorescence of NBD- onstrated by the fact that theaction of histamine was inhibphallacidin in individual cells was increased after fMLP stim- ited by the Hz-specificantagonists cimetidine and metiamide

-

H z Receptor-induced Ca2+Mobilization

18361

TABLEI Comparison of the actiuities between histamine Hz and fMLP receptors on MezSO-differentiated HL-60cells [Ca'+I, Receptors

cAMP

Hz fMLP

+-

IP,

Source of Ca2+

+ +

Intracellular CT Intracellular

PMAsenToxin sensitivity

Sustained Transient

+ +

FIG.9. Hypothesis of the functionalheterogeneity of Hz receptor(s). A , two different CT-sensitive G proteins (G. and G?) couple to different domains of a single Hz receptor ( H z R ) , and then activate adenylate cyclase ( A C ) andphospholipase C (PLC), respectively. B, both G. and G? can couple to the same or different domains of HzR, but one molecule of G protein binds toa single HzR. This phenomenon is based on the distributionof relative abundance of G. and G?. C, two different subclasses of Hz receptors (H2R and HZR-2) are present on HL-60 cells and each receptor is coupled to adenylate cyclase and phospholipase C activation, respectively. The functionalconsequences of histamineinduced Caz+release are still unknown.

-

+

HA

j; ;:/ ...... ......

..... ...... ..... .... ....

CT/PT

sitivity

Actin polymerization

-

-

+

+

HA HA

I/

Membrane

cyt0601

(12). These data suggest that but not by the HI- and Hs-specific antagonists (Fig. 1). The afterhistaminestimulation histamine-induced mobilization of [Ca2+Iiis independent of M, which is similar IC5,of cimetidine was approximately to thevalue for cAMP in HL-60 cells (13). Moreover the HZ- cAMP pathwaysin HL-60 cells. Prostaglandin E1 (12), Ez(13), and isoproterenol (25) have specific agonists, 4m-HA and dimaprit also increased [Caz+li in a dose-dependent manner, whereas the HI-specific agonist been shown to increase cAMP in HL-60 cells with EC50 values 2-pyridylethylamine failed to increase [Ca2+Iieven at concen- of 3.2 and 0.49 pM, respectively. @-Adrenergicreceptors have also been identified on HL-60 cells using the radiolabeled pM (Fig. 1).Thesedata suggest that trationsas high as both histamine-induced activation of adenylate cyclase and receptor-specific ligand, (-)-CGP-12177 (25), inwhich the Kd . PGEz and preceptors [Caz+]imobilization are mediated by Hz-specific mechanisms of isoproterenol was 0.058 p ~ Both are coupled to the CT-sensitive 43,000-dalton G , protein in with a similar dose dependence in HL-60 cells. To analyze whether histamine Hz receptor-mediated mo- HL-60 cells (21, 26), andCTtreatment increased cAMP bilization of [Ca2+Iiis dependent or independent of cAMP in through the ribosylation ofG. proteins (21). However, in the HL-60 cells, we have tested the effect of various drugs which present study, both PGE, and isoproterenol also increased modulate the levels of intracellular CAMP.The cell permeable [Caz+liwith a similar dose dependence to that seen in cAMP form of CAMP,8-Br-CAMPand dibutyryl-CAMPare effective assays (Fig. 1).Furthermore, both histamine and PGEz-mesubstitutes for CAMP in HL-60 cells, where they have been diated [CaZ+li mobilization were abolished by pretreatment shown to inhibit interleukin-2receptor expression (23), c-myc with CT, but not by PT (Fig. 6). These data suggest that the oncogene expression (21), and the induction of granulocytic Hz receptors that are coupled to G, protein may also be differentiation (24) inHL-60 cells in a CAMP-dependent coupled to another cholera toxin-sensitive G-protein in HLmechanism. However, both 8-Br-CAMP and dibutyryl-CAMP 60 cells. failed to change the levels of [Ca2+Ii,and had no effect on 4mAlthough Yatani et al. (27) have reported that CT-sensitive HA-induced mobilization of [Ca2+Ii(Fig. 2). Forskolin which G. proteins directly activate mammalian cardiac Ca2+chanhas been shown to increase cAMP in HL-60 cells also had no nels, the mobilization of [Ca2+];in our system is not due to effect on 4m-HA-induced [Caz+limobilization (Fig. 2). The the activation of Ca2+ channels, because [CaZ+liwas not cell-permeable form of cGMP, 8-Br-cGMP(Fig. 2), the inhib- diminished by reducing the concentration of extracellular itor of cyclic nucleotide-dependent protein kinase (H-8), and Ca2+(Fig. 3). Furthermore, both LaC13 and nifedipine failed the inhibitor of phosphodiesterase (3-isobutyl-1-methylxan- to inhibithistamine-induced [Ca2+]; mobilization (Fig. 3). thine) also had no effect on the 4m-HA-induced increase in These data indicate that Hz receptor stimulation mobilizes [Ca2+]i (datanot shown).Furthermore,histamine-induced [CaZ+Iiprincipally from intracellular Ca2+stores. increases in [Ca'+Ii weremaximal at 15-25 s after stimulation The chemotactic peptide fMLP hasbeen shown to activate and returned to the basal levels within 1-2 min (Fig. 2), CT- and PT-sensitive 40,000-dalton G proteins (19), phoswhereas maximal levels of cAMP were attained at 5-10 min pholipase C following increases in [Ca"];, and stimulate cell

motility in Me2S~"differentiatedHL-60cells(20). In the present study, fMLP increased the levels of both IPS and [Ca2+]ifollowed by actin polymerization in Me2SO-differentiated HL-60 cells (Figs. 4-8). Although M histamine increased the levels of 1,4,5-IPS(Fig. 7) and induced [CaZ+li mobilization to a similar degree to that of M M L P , our results show that histamine and fMLPact differently (Table I). First, Ha stimulation did not have a sustained phase of elevated [Ca2+Ii(Fig. 4). Second, Hzagonist-induced [Ca2+]i mobilization was resistant to PMA pretreatment (Fig. 4)and failed to induce actin polymerization (Fig. 8). Third, histamine-induced mobilizationof [Ca2+liwas inhibited by CT but not by PT, whereas the action of M L P was inhibited by PT but not by CT (Fig. 6). Furthermore, 4m-HA and fMLPfailed to desensitize the [Ca2+Jiresponse to each other (Fig. 5). These datasuggest that thepathways or mechanisms regulating histamine-inducedmobilization of [Ca"]i are unique and different from those of fMLP, although both histamine and fMLP mobilized [Caz+]i from ionomycin-sensitiveintracellular stores through phosphatidylinositol hydrolysis pathways. Although Mattera et al. (28) recently reported that a single G. protein a subunit activates both adenylate cyclase and Ca2+ channels, at least two different G proteins maybe involved in N2 receptor activation on HL-60 cells, because CT activates adenylate cyclase but inhibits ICaz*)i mobilization pathways. Imboden et al. (29) reported a unique CTsensitive G protein on Jurkat human T-cell lines, in which CT inhibited T-cell antigen receptor-mediated increases in [Ca2+]iand inositol phosphates. However, the additional G protein in HL-60 cells may be different from that of Jurkat cells,because Ha receptor stimulation activates adenylate cyclase inJurkat cells (30)but failed to mobilize [Ca2+li?The recent analysis of cDNA clones forG, proteins indicates that at least four different subclasses of G, proteins are present and may be functionally different from each other (31), hence our system might be a useful model forthe analysis of these G. proteins. It is still unknown whether a single subset ofH2 receptors is able to activate two different CT-sensitive Ca2*and cAMP pathways simultaneously (Fig.9A) or independent~y(Fig. 9B). Alternatively, two different subclasses of Hz receptors may be present on HL-60 cells and be coupled to Ca2+and cAMP pathways, respectively (Fig. 9C). Further workwill elucidate possible interactions beteween Ca2+-and CAMPmediated activation pathways and the complex mechanismof receptor activation on neutrophils. Acknowledgments-Wewould like to thank Prof. H. R. Bourne (Department o f Pharmacology, University of California, San Francisco) for his helpful comments and discussion, P. F. Dazin for his technical assistance in the FACS analysis, Dr. M. Ebisawa (National Children's Medical Research Center, Tokyo, Japan) for his technical assistance, and Dr. J. Skidmore (Smith Kline & French Laboratory, Hertsordshire, U.K.), Dr. W. Kreutner (Schering Cop., Bloomfield, NJ) and Dr. J. M. Arrang (Centre Paul Broca de l'INSERM, Paris)

M. Mitsuhashi, T. Mitsuhashi, and D. G. Payan, manuscript in preparation.

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