ATP Na+

Formyl peptides and ATP stimulate Ca2+ and Na+ inward currents

through non-selective cation channels via G-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells

Involvement of Ca2+ and Na+ in the activation of f-glucuronidase release and superoxide production

Dietmar KRAUTWURST, Roland SEIFERT,

Jurgen

HESCHELER and

Gunter SCHULTZt

Institut furPharmakologie, Freie Universitat Berlin, Thielallee 69/73, D-1000 Berlin33, Federal Republicof Germany

In humanneutrophils, thechemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces increases in theintracellular free Ca2+ concentration([Ca2+]1) with subsequent activation of /8-glucuronidaserelease and superoxide (02) production. Results from several laboratoriessuggestthat the increasein[Ca2+]1 is duetoactivation of non-selective cation (NSC) channels. We studied the biophysical characteristics, pharmacological modulationand functional role of NSC channels in dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60cells. fMLP increased [Ca2+] by release of Ca2+

from intracellular stores and influx of Ca2+ from the extracellularspace.fMLP alsoinduced Mn2+ influx. Ca2+ and Mn2+

influxeswereinhibited by

1-{,8-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-lH-imidazole

hydrochloride(SK&F 96365). Underwhole-cell voltage-clamp conditions, fMLP and ATP (a purinoceptor agonist)activated inwardcurrents

characterized by a linear current-voltage relationship and a reversal potential ^near 0mV. NSC channels were

substantiallymore permeabletoNa+thanto Ca2+. SK&F 96365 inhibited fMLP-and ATP-stimulatedcurrentswitha

half-maximal effectatabout 3 ^M. Pertussis toxin prevented stimulationby fMLP of NSCcurrentsandreduced ATP- stimulated currents by about 80%. Intracellular application of the stable GDP analogue, guanosine 5'-O-[2-

thio]diphosphate,

completely blocked stimulation by agonists of NSC currents. In excised inside-out patches, single channelopenings withanamplitude of 0.24 pAwereobserved in thepresenceoffMLP and the GTPanalogue,guanosine 5'-O-[3-thio]triphosphate. The bath solution contained neither Ca2+norATP.The current/voltage relationshipwaslinear with aconductanceof 4-5 pS and reversedatabout 0 mV. fMLP-induced ,-glucuronidase release and 2-production

weresubstantially reduced by replacement ofextracellular CaCl2 orNaCl byethylenebis(oxyethylenenitrilo)tetra-acetic acid andcholinechloride respectively. In the absence of Ca2+ and Na+, fMLPwas ineffective. SK&F 96365 inhibited fMLP-induced

fl-glucuronidase

release and 2-production in thepresenceof both Ca2+ and Na+, and in thepresenceof Ca2+ orNa+ alone. NaCl (25-50 mM) enhanced the basal and absolute extentoffMLP-stimulated GTP hydrolysis of heterotrimeric regulatory G-proteins in HL-60 membranes. The order of effectiveness of salts in enhancing GTP hydrolysiswasLiCl>KC1>NaCl>choline chloride. Our resultssuggestthat in Bt2cAMP-differentiated HL-60 cells, (i) fMLP and ATP activate NSCchannels permeable for Ca2 ^, Mn2+andNa+; (ii) activation of NSC channels involves

G-proteins and is independent of intracellular Ca2+ and protein kinases; (iii) Ca2+ and Na+ influxes are involved in activation of,-glucuronidase release and

O2-

production; (iv) anincrease in intracellular free Na+concentration may

enhance activation ofG-proteins, leading, amongother possible mechanisms, tosignal amplification.

INTRODUCTION

Neutrophils playamajor role in host defenceagainst bacterial infections and in the pathogenesis ofvarious human diseases such^asmyocardial infarction, rheumatoid arthritisandnephritis (Malech & Gallin, 1987). Dibutyryl cyclic AMP (Bt2cAMP)- and dimethylsulphoxide (Me2SO)-differentiated HL-60 leukaemic cells are widely used as model systems for cell culture of neutrophilsastheycanbeobtainedinsufficiently largeamounts for functional studies and for the purification ofsignal trans- ductioncomponents (Niedeletal., 1980; Chaplinski &Niedel, 1982; Uhingetal., 1987; Dubyaketal., 1988; Gierschiketal., 1989; Pittetetal., 1989; Seifertetal., 1989a;Cowenetal., 1990;

Wenzel-Seifert & Seifert, 1990;Tohkin etal., 1991; Mizuno et

al., 1992). Inaddition,Bt2cAMP-differentiatedHL-60 cellsare a

particularly useful system for the identification ofcDNAs of

signaltransduction components (Murphy etal., 1987; Murphy

&McDermott, 1991; Schultzetal., 1992).

NeutrophilsandHL-60 cellspossessreceptorsfor the chemo- tactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), and for ATP both of which couple to pertussis-toxin- sensitive heterotrimeric regulatory G-proteins (Chaplinski &

Niedel, 1982; Dubyaketal., 1988;Gierschiketal., 1989;Seifert etal., 1989a,b, 1992a;Wenzel-Seifert &Seifert, 1990;Cowenet

al., 1990).Inaddition,ATPreceptorscoupletopertussis-toxin- insensitiveG-proteins (Dubyaketal., 1988;Seifertetal.,1989a;

Wenzel-Seifert &Seifert, 1990;Cowenetal., 1990).Activationof

neutrophiliccells results in therelease of,-glucuronidase from azurophilicgranulesandNADPHoxidase-catalysedsuperoxide (02-) production (Chaplinski & Niedel, 1982; Rossi, 1986;

Sandborg&Smolen, 1988; Baggiolini&Wymann, 1990;Wenzel- Seifert & Seifert, 1990; Seifert & Schultz, 1991). It should be

Abbreviations used: [Ca2+]I, intracellular free-Ca2+ concentration; BtcAMP, dibutyryl cyclic AMP; fMLP, N-formyl-L-methionyl-L-leucyl-L- phenylalanine; GDP,8S, guanosine

5'-O-[thio]diphosphate;

GTPyS,

guanosine 5'-O-[thio]triphosphate; Me2SO, dimethylsulphoxide; [Na+]1,

intracellular free-Na+ concentration; NSC, non-selective cation;

02-, superoxide;

SK&F 96365,

1-{fi-[3-(4.methoxyphenyl)propoxy]-4-

methoxyphenethyl}-IH-imidazole

hydrochloride.

t

Towhom

correspondence

should be addressed.

noted, however, that there are also differences in transmembrane signal transduction between neutrophils and

Me2SO-differen-

tiated HL-60 cells on the one hand and Bt2cAMP-differentiated HL-60 cells on the other (Seifert et

al.,

1989c, 1992a; Wenzel- Seifert & Seifert, 1990; Tohkin et

al.,

^1991).

In neutrophilic cells, fMLP and ATP induce an increase in intracellular

free-Ca2+

concentration

([Ca2+]1)

(von

Tscharner

et

al.,

1986; Andersson et

al.,

1986;

Nasmith

& Grinstein, 1987;

Dubyak et

al.,

1988; Pittet et

al.,

1989; Wenzel-Seifert & Seifert, 1990; Seifert & Schultz, 1991). In neutrophils and

Me2SO-

differentiated HL-60 cells, fMLP mobilizes

Ca2+

from intra- cellular stores and induces a sustained influx of

Ca2+

from the extracellular space through channels referred to as non-selective cation (NSC) channels (von

Tscharner

et

al.,

1986; Andersson et

al.,

1986; Nasmith & Grinstein, 1987; Simchowitz & Cragoe, 1988; Pittet et

al.,

1989; Merritt et

al.,

1989, 1990; Meldolesi et

al.,

1991; Montero et

al.,

1991 ; Demaurex et

al.,

1992; Schumann et

al.,

1992).

1-{p[3-(4-Methoxyphenyl)propoxy]-4-methoxy-

phenethyl}-1H-imidazole

hydrochloride (SK&F 96365) inhibits fMLP-induced Ca2' and

Mn2+

influxes in human neutrophils (Merritt et

al.,

1990). There isa debate as to whether or not NSC- channel opening in human neutrophils and

Me2SO-differentiated

HL-60 cells depends on a rise in

[Ca2+]I

(von Tscharner et

al.,

1986; Nasmith & Grinstein, 1987; Demaurex et

al.,

^1992;

Schumann et

a.,

1992). Preliminary results point to thepresence of NSC channels in Bt2cAMP-differentiated HL-60 cells as well (Aviram & Shaklai, 1990; Gusovsky et

al.,

1990). However, the biophysical and pharmacological properties and the functional role of NSC channels in Bt2cAMP-differentiated HL-60 cells are unknown.

In 1979 Simchowitz & Spilberg suggested that Na+ influx is involved in the activation

of 02-

production in human neutro- phils. More recently, Nordmann & Stuenkel (1991) put forward the hypothesis that an increase in intracellular free-Na+ con- centration

([Na+],)

plays a key role in the activation of exocytosis in neuronal cells. By contrast, the results of another

study

indicate that

Na+

plays an inhibitory role in the regulation of,- glucuronidase release and

02-

production in human neutrophils (Della Bianca et

al.,

1983).

Na+

(20-100

mM)

inhibits basal GTPase activity of G-proteins in membranes from

Me2S0-

differentiated HL-60 cells, but enhances the absolute extent of GTP hydrolysis caused by fMLP (Gierschik et al., 1989). As

[Na'],

in stimulated human neutrophils and platelets may be as high as 60mm (Simchowitz, 1985; Borin & Siffert, 1991; Sage et

al.,

1991), the question arises of whetherNa+ could modulate the activity of G-proteins in vivo. Most intriguingly, SK&F 96365 inhibits the receptor-agonist-stimulated increase in

[Na+]i

in platelets which suggests the involvement of NSC channels inNa+

influx

(Borin

& Siffert, 1991; Sage et

al.,

1991).

All these findings prompted us to study the properties of NSC channels in Bt2cAMP-differentiated HL-60 cells with the fluor- escent dye fura-2, and in whole-cell and single-channel voltage- clamp experiments, and to assess their functional role in the regulation of

f8-glucuronidase

release and 02- production. We show that fMLP and ATP activate NSC channels permeable for Ca2+,

Mn2+

and

Na+

and that these channels are involved in the activation of

f8-glucuronidase

release and 2- production.

MATERIALS

AND METHODS Materials

SK&F 96365 was kindly provided by Dr. J. E.Merritt, SmithKline Beecham (Welwyn, Herts., U.K.). Stock solutionsof SK&F 96365 (30

mM)

were prepared in

Me2SO

and were stored at -20 'C. Dilutions of SK&F 96365 were made immediately

before experiments were performed. Pertussis toxin was a gift fromDr.M.Yajima (Kyoto, Japan). Isradipine was from Sandoz (Basel, Switzerland). Sources of other materials have been described elsewhere (Seifert & Schultz, 1987; Seifert et al., 1989a-c, 1992a,b; Wenzel-Seifert & Seifert, 1990).

Cell

culture

HL-60cells were grownin suspension culture in RPMI-1640 medium supplemented with 10 % (v/v) horse serum as described (Seifert et

al.,

1989a). To inducedifferentiation, cells were seeded at1 x

106

cells/ml and were cultured for 48 h in the presence of 0.2

mM-Bt2cAMP,

or for 120 h in the presence of 160mM-Me2SO (Seifert & Schultz, 1987; Seifert et al., 1989a). In some experi- ments pertussis toxin (100 ng/ml) or carrier was added to cells 24h before experiments. Concentrations of SK&F 96365 up to 100

/M

did not cause damage of Bt2cAMP-differentiated HL-60 cells as assessed by Trypan Blue exclusion and lactate de- hydrogenase release (results not shown). The release oflactate dehydrogenase from HL-60 cells generally amounted to < 5 % of cellular content (results not shown). SK&F 96365 concen- trations up to 30

uM

did not stimulate /-glucuronidaserelease or 02- production (results not shown).

Measurement of

[Ca

l1

[Ca2+J,

was determined with the dye, fura-2/acetoxymethyl ester, as described in detail (Grynkiewicz et al., 1985; Seifertet

al.,

1992a). In brief, HL-60 cells were suspended at 5 x 106 cells/mil in a buffer consisting of 138mM-NaCl, 6.0mM-KCl, 1.0

mM-MgSO4,

1.0

mM-Na2HPO4,

5.0mM-NaHCO3, 5.5 mm- glucose, and 20

mM-Hepes/NaOH,

pH 7.4, supplemented with

0.1

% (w/v) BSA and fura-2/acetoxymethyl ester (2/M). Cells were incubated for1 h at 37 'C. Subsequently, cells werediluted with the above buffer to a cell density of 0.5 x 106 cells/ml and were centrifuged at 250 g for 10min at 20 'C. Cells were re- suspended at 1.0 x

106

cells/ml in the above buffer, andwerekept at 20 'C until

[Ca2+]1

had been measured. Cellswere used up to 4 h after loading without substantial leakage of fura-2from the cells, increase in basal

[Ca2+]1

or decrease in responsiveness to fMLP (Dubyak et al., 1988; Seifert etal., 1992a). Fluorescence of HL-60 cells (1.0x 106 cells) was determined at 37 'C with constant stirring, using a RatioII spectrofluorimeter (Aminco, Silver Spring, MD, U.S.A.). Cells were incubated for 3min at 37 'C in the presence of various substances before addition of fMLP; basal fluorescence was measured for1 min. Excitation and emission wavelengths were 340 nm and 500nmrespectively.

Mn2+

influx in HL-60 cells was measured byquenching of fura- 2 fluorescence as described (Merritt etal., 1989).Basal [Ca2+]1in Bt2cAMP-differentiated HL-60 cells was 105 +15nm (mean+S.D.

of five different preparations of HL-60 cells). With each prep- aration, up to 40 separate experiments were performed.

Electrophysiology

HL-60cells (8 x106 cells) werecentrifuged at 250 gfor 5 min at 20 'C, and were resuspended in standardextracellular Tyrode's solution (El) containing 140 mM-NaCl, 1.8mM-CaCl2, 1.0mm- MgCl2, 5.4mM-KCl, 10mM-glucose, 10mM-Hepes/NaOH, pH 7.4, at 37 'C. Cells were transferred into aperfusion chamber (0.2 ml) mounted on an inverted microscope(Zeiss,Oberkochen, Germany). Cells settled to the bottom ofthe chamber within 3min. Patch pipettes were prepared from glass capillaries (Jencons, Leighton Buzzard, U.K.) according to Hamill et a!.

(1981).

With an open diameter of1-2,um,theaverage resistance of the electrodes was 4-6

MQ.

Whole-cell

recordings. Pipettes werefilled witha solution

(II)

containing 90mM-potassium aspartate, 50

mM-KCl,

1.0mm-

MgCl2, 3.0mM-sodium ATP, 0.1

mM-EGTA,

10

mM-Hepes/

KOH, pH 7.4, at 37 °C. Electrical approachto HL-60 cells was gained by suction for access resistance below 50MO. Mem- brane potentials and whole-cell membrane currents were measured according to Hamill etal. (1981), using a List LM/

EPC7 patch-clamp amplifier (List Electronics, Darmstadt, Germany). Resting potentials in HL-60 cells ranged between -60 and -30 mV and cell capacitance between 4 and 8 pF.

Currents and conductances were referred to membrane ca- pacitance. HL-60 cells weresuperfusedat 2ml/min with El. In someexperimentsElwasmodifiedasfollows.ForCa2+depletion, CaCl2 (1.8 mM)was replaced bycholinechloride (1.8mM), and the solution in addition containedEGTA(1 mM)(E2).In-some experiments all permeant cations(i.e. Na+, K+ andMg2+) with theexception ofCa2+(1.8mm, E3, or 100mM,E4) were replaced bycholine. For Na+depletion, NaCl (140mM) was replaced by choline chloride (140mM) (E5). In solution E6, all permeant cations werereplaced bycholine.

Single-channel recordings. Excised inside-out patches were obtained from the on-cell configuration with GQ-seal re- sistance. Single-channel currents were recorded at 25 'C. The pipette solution (12) contained 135mM-NaCl, 2.0mM-MgCl2, 100nM-fMLP and 20mM-Hepes/NaOH, pH7.4, at 25 'C. In- side-outpatcheswere faced to abath solution (E7) containing 65mi-caesiumaspartate, 13mM-CsCl,50mM-potassiumaspar- tate, 2.0mM-MgCl2, 1.0mM-EGTA, 0.01mM-guanosine 5'-O-

[thio]triphosphate

(GTPyS) and 20mM-Hepes/CsOH, pH 7.4, at 25'C. Cs' was added to block K+ currents. The unfiltered currents at variouspotentials were continuously recordedon a Sony DTR 1200 DAT-recorder (Biologic, Echirolles, France).

Foroff-line analysis, data were filtered at a frequency of 800 Hz using a three-pole Bessel filter (Frequency Devices, Haverhill, MA, U.S.A.)and weresampledat 3 kHzusingthe AxonAD/DA converter(Axon, FosterCity, CA,U.S.A.).

fi-Glucuronidase

release

Enzyme release was assessed as described (Seifert et al., 1989a,b; Wenzel-Seifert & Seifert, 1990). HL-60 cells were centrifugedat 250gfor10min at 20 'C. Cells weresuspendedin bufferconsisting of138mM-NaCl, 3.0mM-KCl, 1.0mM-MgCl2, 5.5mM-glucose,20mM-Hepes/NaOH,pH 7.4, or inbuffercon- sisting of138mM-cholinechloride,3.0mM-KCl, 1.0mM-MgCl2, 5.5mM-glucose, 20mM-Hepes/NaOH, pH 7.4. Cells were re- centrifuged andweresuspendedinthe above buffers.Prior tothe addition ofstimuli, HL-60cells (5.0x106 cells in 0.5ml) were incubated for 5 min at 37 'C inthe presence of

cytochalasin

B (5,ug/ml) with or without SK&F 96365. Reaction mixtures containedeither 1.0mM-CaCl2or1.0mM-EGTA.Reactionswere conductedfor 10minand were terminatedby

placing

the tubes on to meltingice. Reaction mixtureswere

centrifuged

at 1000g for10minat4'C. Thedeterminations of theactivitiesoflactate dehydrogenase and

fl-glucuronidase

in supernatant fluids of reactionmixturesandofcelllysateswere

performed

asdescribed

(Seifert

et al.,

1989b;

Wenzel-Seifert &

Seifert, 1990).

Basal

,8-glucuronidase

release in HL-60 cells

generally

amounted to

<5% ofcellularcontent (resultsnot

shown).

02-

productionin intact HL-60 cells

02-

productionwasmonitored

by

continuousmeasurementof

ferricytochrome

creduction inhibitable

by superoxide dismutase,

using a Uvikon 810 dual-beam

spectrophotometer (Kontron,

Eching, Germany)

(Seifert

et al.,

1989a).

Reaction mixtures (0.5

ml)

contained 2.5x106

Bt2cAMP-differentiated

HL-60cells and 100

1M-ferricytochrome

c in the buffers described above supplemented with 1.0

mM-CaCl2

or 1.0mM-EGTA. Reaction mixtures were incubated for 3 min at 37

°C

with or without SK&F 96365 beforeaddition of stimuli.

02-

productionincell-free systems

Reaction mixtures (0.5ml) contained 50 ,g of membrane protein from Me2SO-differentiated HL-60cells, 150 ,gofcyto- solic protein from Me2SO-differentiated HL-60 cells, 10,lM- FAD, 500,tM-NADPH,100

/LM-ferricytochrome

c,2mM-MgCl2, 20mM-KH2PO4,40mM-KCl, and 20

mM-triethanolamine/HCl,

pH7.0. Reaction mixtures were incubated for 2 min at 25°C with orwithoutSK&F 96365.

02-

productionwasinitiated by the addition of arachidonic acid (0.2mM) (Seifert & Schultz, 1987).

Measurementof GTPaseactivity

GTP

hydrolysis

wasmeasuredasdescribed

previously (Seifert

et al., 1992b). Reaction mixtures (100

,cdl)

^contained HL-60 membranes (7.5 ,tg of protein/tube), 0.5

tM-[y-32P]GTP

(0.1

uCi/tube),

0.5mM-MgCl2, 0.1 mM-EGTA, 0.1mM-ATP, 1 mM-adenosine

5'-[/J,y-imido]triphosphate,

SmM-creatine phosphate, 40 ,ug of creatine kinase, 1 mM-dithiothreitol and 0.2% (w/v) BSA in 50

mM-triethanolamine/HCl,

pH7.4. Re- action mixtures additionally contained NaCl, KCl, LiCl or choline chlorideatvariousconcentrationswithorwithoutfMLP.

Reactionswereinitiatedbytheadditionof

[y-32P]GTP

andwere conducted for15 minat25'C.Low-affinity GTPase activitywas determined in the presence of 50

fM-GTP

and amounted to

<5% ofGTP

hydrolysis

in thepresenceof0.5' M-GTP.

Miscellaneous

Membranesfrom Bt2cAMP-or Me2SO-differentiated HL-60 cells and

cytosol

from

Me2SO-differentiated

HL-60 cells were preparedasdescribed(Seifert&Schultz, 1987).

[y-32P]GTP

was

prepared according

to Johnson &Walseth (1979). Proteinwas determined

according

to Lowry et al. (1951), using BSA as standard.

RESULTS

Theeffects offMLPonthe release ofCa2+from intracellular storesandon

Ca2+

influx in Bt2cAMP-differentiated HL-60 cells areshown in

Fig.

1. Inthe presence ofextracellular Ca2 , fMLP induced a rapid and large increase in

[Ca2+]1

which slowly declinedto asustainedplateau abovebasal values. SK&F 96365 decreased

peak [Ca2+]i

values as well as the

magnitude

and duration of the

plateau phase.

SK&F 96365

(10 /tM

and30

/SM)

inhibited fMLP-stimulated

peak [Ca2+]1

values

by

about

40%

and 55%

respectively (Table 1). Isradipine (1 #M),

a blockerof

voltage-dependent

Ca2+

channels,

didnotinhibitfMLP-induced increases in

[Ca2+]i

(results not

shown).

^In the absence of extracellular Ca2 , the

magnitude

and duration of the rise in

[Ca2+]i

induced

by

fMLPwere

greatly

reduced

(see Fig.

lb and Table

1).

Underthese conditionsneitherSK&F 96365

(see Fig.

lband Table1)nor

isradipine (1 /tM) (results

not

shown)

affected the increase in

[Ca2+]i.

These results suggest that the fMLP- induced increase in

[Ca2+]i

ⁱⁿ

Bt2cAMP-differentiated

HL-60 cells

depended largely

on Ca2+influx.

Toanswerthe

question

whether fMLP stimulated the influx of bivalent

cations, quenching

of fura-2 fluorescence

by

Mn2+was

studied. At anexcitation

wavelength

of 340 nm, fluorescence is increased

by

Ca2+ and is reduced

by

Mn2+; at an excitation

wavelength

of 360 nm, fluorescence is

quenched by

Mn2+aswell but is insensitive to

changes

in

[Ca2+]i

(Merritt etal.,

1989).

At both excitation

wavelengths

Mn2+ induced a small decrease in

fluorescence, indicating

basalMn2+influx

(Fig. 2).

Atexcitation

wavelengths

of 340 nm, but not of 360 nm, fMLP

transiently

increased fluorescence, reflecting a release of

Ca2+ from

intracellular stores. At excitation

wavelengths

^{of 340nm and}

360nm fMLP

substantially

enhanced fluorescence

quenching,

1000-

300- I

C4

M1000-

300 -

100-

(a)

2~~~~~~~~~~

(b)

.J~-~-.v--

¹

0

c 0)

0

4-i

0 7

Time(min)

14

Fig. 1. Effect of extraceliularCa2" andSK&F 96365onthetimecourseof the fNILP-induced increase inICa2+i inBt2cAMP-differentiated HL-60 cells

HL-60 cellswereloaded withfura-2/acetoxymethyl ester,and the increasesin[Ca2+]1 induced by fMLP (1

#M)

wereassessed. Arrows indicatetheadditionof stimulus. Varioussubstanceswereaddedto

cells 3min before fMLP.(a) Experiments performed in the^presence ofCa2+ (1mM). Trace 1, solvent (control); trace2, SK&F 96365

(10#M);

trace 3, SK&F 96365 (30,UM). Superimposed original fluorescence tracings are shown. (b) Experiments performed in

presence of EGTA (1 mM). Trace 1, solvent (control). Traces of fMLP-inducedincreasesin

[Ca2+],

in thepresenceofSK&F 96365 (10/ZMor30/LM)werevirtually superimposableontrace1.Similar resultswere obtained four times with each preparationof HL-60 cellsand inthree experiments with different preparationsof HL-60 cells.

Table 1. Effect of SK&F96365 on fMLP-induced increases in

ICa2+li

in Bt2cAMP-differentiatedHL-60 cells in thepresence and absence ofextraceliularCa2+

HL-60cells wereloaded withfura-2/acetoxymethyl ester. Basal and peak

[Ca21]i

values stimulated by fMLP(1 M) were determined.

Basal

[Ca21]i

values weresubtracted from the corresponding peak

[Ca21]i

values tocalculate the increase in

[Ca21]i

induced byfMLP.

Experiments were performed in the presence ofCa21 or EGTA (1mM each). SK&F 96365 or solvent (control) was added to cells 3 min beforefMLPtreatment. Results shown are the means + S.D.of fourexperiments performed with one preparation of HL-60 cells.

Similar results were obtained in three experiments with different preparations of HL-60 cells.

Increasein

[Ca2+]i

(nM)

Addition Ca2` (1 mM) EGTA(1mM)

Fig.2.Effect of fMLP on Mn2" quenching of fura-2 fluorescence in Bt2cAMP-differentiated HL-60cells

HL-60cellswereloadedwith fura-2/acetoxymethylester,and fura- 2fluorescencewasmonitoredatexcitation wavelengths of340nm

(traces1)or360nm(traces 2). The emission wavelength^was500nm.

Experimentswereperformedin the absence of Ca2+. Arrowsindicate theaddition of MnCl2 (100 uM) andfMLP (1

#M).

(a)Experiments performedinthe absence of SK&F96365(control). (b) Experiments performedinthe^presenceofSK&F96365 (30

/iM).

SK&F96365or

solvent(control) were added to cells 3min before Mn2". Super- imposed originalfluorescence tracings are shown. Similar results

wereobtained fourtimes with each preparation ofHL-60cells andin threeexperiments withdifferentpreparations of HL-60 cells.

(a) pA 0-

m'^VV ^0.-0- ' -20- -60-

100pA 20ms

(b)

pA 0.-

3

mV 0 2

-60 -

-100

100pA 100ms

Fig.3. Current-voltagerelationship of fMLP-induced inward currentsin Bt2cAMP-differentiatedHL-60^cells

Inwhole-cellvoltage-clampexperimentscurrentsweremeasured in the samecell. (a) Depolarizing pulses. (b)Ramppulses. Traces 1, currentsin theabsence of fMLP;traces2,currentsaftersuperfusion ofcell with fMLP(100 nM)for 6s; traces3,currentsaftersuperfusion offMLP-stimulatedcell withSK&F 96365 (10

pM)

for 6s.Broken linesrepresentthezero-currentlevel. Similarcurrentswereobserved in 16experiments.

Solvent

SK&F 96365(10

/tM)

SK&F 96365(30/tM)

852+63 517+ 52 395+36

192+23 204+ 13 186+ 15

reflecting Mn2+ influx. SK&F 96365 (30

#M)

abolished fMLP- induced Mn2+ influx without affecting release of Ca2+ from intracellularstores (see Fig. 2b). Isradipine (1 ,sM)didnot alter fMLP-induced changes in fluorescence quenching (results not

shown).

The results shown in Figs. ¹ and2 and Table 1 suggest that fMLP inducedCa2l andMn2+influxes inBt2cAMP-differentiated HL-60 cells via NSC channels.Therefore,thepropertiesof NSC channelswerestudiedin^moredetail in whole-cellvoltage-clamp experiments under physiological ionic conditions (El). HL-60 cells showed membranepotentials rangingfrom-60to -30 mV.

Within thisvoltagerange,theinputconductancewaslinear and

amounted to 45+9pS/pF (mean+S.D., n=25). In a manner

similarto humanneutrophils, atpotentials more positive than -20 mVapprox.40%of theexamined cellsadditionallyshowed

an outward-rectifying K+ current (results not shown) (von Tscharneretal., 1986;Krause&Welsh, 1990).Thepropertiesof thisK+current werenotfurtherinvestigated.

Uponstepdepolarizations from -60to -20mV and 0mV, fMLP stimulated inward currents (Fig. 3). Under these con-

ditions, currents showed no time-dependent activation or in- activation. Currents at -60mVwere approx. 3-fold greaterin amplitudethancurrents at -20mV,and at0 mVnonet currents

wereapparent. Whenramppulses (400msfrom ^-100to0mV)

were usedas voltage-clamp protocol, currentswerelinear and showed areversalpotential closeto0mV(see Fig. 3b). fMLP- stimulated currents resulted in membrane depolarization as

revealed under current-clamp conditions (results not shown).

1992 (a)

1 min

1 ~~~~~~~~~~~~~~~~

Mn2+ fMLP 2

(b)

_ _ -, 1

2

Mn~

^fMLP

ATP

SK&F ATP+fMLP

0 .- IL

ki 1.5

(U c

o 1.0

0 0cW

D 0.5 100pA

10s

Fig. 4. Time course of agonist-stimulated inward currents in Bt2cAMP- differentiated HL-60cells

Currents were measured in a cell continuously voltage-clamped to a holdingpotential of -60 mV. Application periods of ATP (30

JiM)

andfMLP (100nM) and of SK&F 96365 (10

gm)

areindicated by the horizontal bars. The broken line represents the zero-current level.

Similar resultswereobtained in three experiments.

100

0 c 0

-6.

0 1-0

c n 50

0 (U

CD 0 0l

._4

n

.c

I

l

8 7 6 5 4

-log{[SK&F963651(M)}

Fig.5. Concentration-inhibitioncurvesforSK&:

inBt2cAMP-differentiatedHL-60cells F

3 3

96365on inward currents Currents werestimulatedwithfMLP (100nM)(-)orATP(30/tM) (M) in the presence ofvarious concentrations of SK&F 96365.

Currents were measuredat aholding potential of -60mV. Each point represents themean of3-8experiments; the S.D. of results generallyamounted to less than 15% of themeans.

fMLP-stimulated currents were present in about

600%

of the examined cells(resultsnot

shown).

Thisdifferential

responsive-

ness tofMLP may be dueto the fact that differentiated HL-60 cellsarenot ahomogeneouspopulationand mayshowdifferences inexpression offormyl peptidereceptors (Niedeletal., 1980).

ATPstimulated inwardcurrents aswell

(Fig.

4).fMLPdidnot further enhance ATP-stimulated currents, indicating that both agonists activated the same current. The effects of ATP and fMLPoninwardcurrentswere

rapid

in onset, i.e. withina few seconds, andwerereversible upon

washing

out

agonists.

Theeffects of SK&F 96365oninwardcurrentsin HL-60cells werestudied. SK&F 96365

(10 /tM)

reduced the effect of fMLP

by

about 60%,

independently

ofthe membrane

potential (see Fig.

3). No change in current

linearity

was observed. Blockade of currentsbySK&F96365occurred withinafew seconds

(see Fig.

Con. PTX GDP,BS

_

_

0.0L

(9)

(16)

(25) (15) (8 (13)(6) (7)

fMLP - + - - + - - + -

ATP - - + - - + ^- +

Fig. 6. Effects of pertussis toxin and GDPp8S on fMLP- and ATP- stimulated inward currents inBt2cAMP-differentiated HL-60 ceUs Currents stimulated by fMLP (100nM) and ATP (30

/SM)

were measured incontrol cells (Con.), inpertussis-toxin-pretreated cells (PTX) and in

GDP,/S-infused

cells

(GDPp?S).

These cells were infused with

GDP,/S

(0.5mM) for 5 min. Results shown represent the means + S.D. of linear conductances measured at -60 mV referredtothe membrane capacitance. The number of experiments isgiveninparentheses.

4). Onwashing, the effect of SK&F 96365 was reversible. The concentration-inhibition curve for SK&F 96365 on inward currentsisshown in Fig. 5. SK&F 96365 inhibited both fMLP- and ATP-stimulated currents with IC50 values of about 3

/sM,

againsupporting the view that both agonists activated thesame current(seeFig. 4). Isradipine (1

/aM)

did not inhibit fMLP- and ATP-stimulated inward currents (results not shown).

Theinvolvement of G-proteins in fMLP- and ATP-stimulated inward currentswasstudied. ATP wassubstantially more effec- tive than fMLPatstimulatingcurrents(Fig. 6). Similarly, UTP andastableanalogue of ATP were moreeffective than fMLP at inducingphosphoinositide turnover and an increase in

[Ca2l],

(Wenzel-Seifert & Seifert, 1990; Cowen et al., 1990). Pertussis toxindifferentially inhibited inward currents. In pertussis-toxin- pretreatedcells, fMLP-stimulated currents were abolished, and ATP-stimulated currents were reduced by approx.

800%.

The stable GDP analogue, guanosine 5'-O-[thio]diphosphate

(GDPp3S),

which stabilizes a-subunits of G-proteins in the inactive form(Gilman,1987),wasappliedintracellularly. GDP/?S prevented stimulation of inward currentsbyATP andfMLP.

For determination of ion selectivity of NSCchannels,fMLP- stimulated inward currents were assessed under various extra- cellular ionic conditions (Fig. 7). Choline was employed as a cation whichdoesnotpermeateNSCchannels(Schumannetal., 1992). Withdrawal of Ca2+ (E2) resulted in onlyslightly dimin- ished inward-current amplitudes [cf. Figs. 7(a) and 7(b)]. The reversal potential was unaffected. However, when Na+ was removed(E5),thereversalpotentialshiftedbyapprox. -20 mV and theamplitudewasdiminishedbyapprox. 90%[cf.Figs. 7(a) and 7(d)]. Omission of all permeant cations (E6) resulted in outward-rectifyingcurrents(see

Fig. 7f), presumably

carriedby K+. fMLP did not stimulate inward currents under these con-

ditions,and the reversalpotentialwas

strongly

shiftedto

negative

values (-75mV).

Permeation of

Ca2+

throughNSCchannelswasstudied in the presenceofCa2'asthe

only

permeantcation.Inthe presence of Ca2+ (1.8

mM) (E3)

fMLP stimulated a small but

significant

inwardcurrentwithanamplitudeofabout -50pAat-60 mV (seeFig. 7c).Elevation of Ca2+ concentration upto 100mm

(E4)

resulted in a small increase in currents

(see Fig. 7e).

Under all Vol. 288

2.0r

mVI-100 ^{-60 0} 3

2Va

(a)

mV-100 -60

2

1

(c)

mVI-100

--250

L-500 pA

0

-60 1.

3

2

(b)

mV

75

--150 pA

-60

3 2

(e) 0

--75 (f)

--150 pA

mV-100

130 2

65 3

1 ~

~~~

^{- o}

0 50

pA Fig. 7. Effects ofvariousextraceliularionic conditions on fMLP-induced inwardcurrents in Bt2cAMP-differentiatedHL-60 cells

Currentvoltage relationsweremeasured during linear-voltage ramp pulsesfrom -100 to -20 mV (0.4 V/s). Traces 1, currents in the absence offMLP; traces 2, currents after superfusion with fMLP(100 nM)for 6s; traces 3,currents after superfusion of fMLP-stimulated cells with SK&F 96365(10

#M)

for6s.Broken lines areextrapolations to the reversal potential. Cells were superfused with buffers(El-E6) described in more detail in the Materials and methods section. (a) El (1.8mM-Ca2' and 140mM-Na').(b) E2 (140mM-Na'without Ca21). (c) E3 (1.8mm-Ca21 inthe absence of otherpermeant cations). (d) E5 (1.8mM-Ca21in the absence of Na+). (e) E4(100mM-Ca21 in the absence of other permeant cations).

(f)E6(nopermeant cations). Similar currents were observed in 6-16experiments.

Table2. Effect of various extraceliular ionic conditions on fMLP- stimulatedinward-current conductances and reversal potentials in Bt2cAMP-differentiatedHL-60cells

Conductances and reversal potentials

(Erev.)

offMLP-stimulated inward currents inBt2cAMP-differentiated HL-60 cells under vari- ousionic conditions(El-E6,seethe Materials andmethodssection) were calculated.

El

(1.8 mM-Ca2" and 140mM-Na'), E2 (140mM- Na+ without Ca2+), E3 (1.8mM-Ca21 in the absence of other permeantcations), E4 (100mM-Ca2+ inthe absence of other per- meantcations), E5 (1.8mM-Ca2+ in theabsence of Na+), E6 (no permeantcations). Results shownarethemeans+S.D. (n=6-16) of linear conductances measured at -60mV referred to membrane capacitance and the approx. reversal potentials.

Extracellular Conductance Erev.

solution (pS/pF) (mV)

El E2 E3 E4 E5 E6

785 +72 730+50 40+ 15 65+20 42+10

2+5 -2+3 -10+5 -7+6

-19+2 -75 +4

SingleNSC channelswerecharacterized further in-inside-out patchesunder voltage-clampconditions. The experiments were performedin the presenceof fMLP

(100

nM) in the patch pipette and GTPyS (10 ,tM)in the bath medium. At -60 mV, inward currentswithanamplitude of0.24+0.01 pA (mean+S.D.,n=6, sampling interval of 4-5min) were observed (Fig. 8a). The open probability was 0.025+0.005 (mean+S.D., n=6). Currents reversed at about 0 mV, indicating similar permeabilities for the cations present on both sides of the channel. At a holding potential of + 60mV, outwardcurrentswithsimilar amplitudes and openprobabilitiesas at -60 mVwereevident (see Fig. 8a).

Thecurrent-voltagerelationship was linear with a conductance of 4-5pS(seeFig. 8b). When SK&F 96365(10

/IM)

waspresent in thebathsolution,nochannelopeningswereobserved(results not shown,n=6).

The above results show thatbothCa2+andNa+permeateNSC channels. Therefore the roles of extracellularCa2+and Na+ inthe

regulation

of

8-glucur3onidas&

release

anzuk)2-

productionwere studied. In the presence of bothcations, fMLP stimulated the release of approx. 20%of the cellularcontentof

/8-glucuronidase

(Table 3).SK&F 96365

(30 /SM) inhibited-fl-glucuronid-ase

release bymorethan 80%. By contrast, isradipine (1

puM)

waswithout effect (resultsnot shown). When Ca2+ was

replaced by EGTA,

and when Na+ was

replaced by choline,

fMLP-stimulated fi-

glucuronidase

releasewasreduced

by

80%and 65

% respectively.

Under these conditions, SK&F 96365

(30 /M)

reduced the respectivestimulatoryeffects of fMLP

by

85% and 65

%.

Inthe 1992 0

- -250

- -500 pA

mV-100

0

-25

L -50 pA

conditions

employed (El-E6),

fMLP-stimulated NSC currents were inhibited by SK&F 96365. Table 2 summarizes conduc- tancesand reversal

potentials

ofcurrentsunder these conditions.

I~~~~~~

...

...s

Table 3.Effects ofextraceliular Ca2l and Na+ and SK&F 96365-on. i glucuronidaserelease inBt2cAMP-differentiated HL-60 cells fl-Glucuronidaserelease in HL-60 cells induced by fMLP(14M)was assessed undervariousconditions. SK&F 96365 (30

/LM)

orsolvent (control) were added to cells 5min beforefMLP treatment. The concentration ofCa2l and EGTA was1 mm, and the concentrations of Na+ and choline were138 mm. Results shown are the means+S.D.

of three experiments performed with one preparation of HL-60 cells.

Similar results were obtained in three experiments with different preparations ofHL-60 cells.

Stimulatedfl-glucuronidase release (% of cellular content) Additions Control SK&F 96365 Ca2"+ Na+

Ca2`+ choline EGTA +Na+

EGTA + choline

21.0+1.0 4.1+0.3 7.3+0.5 0.2+0.1

3.4+0.4 0.6+0.2 2.6+0.2

0

mV

0 30 60

-0.15

-0.30

Fig. 8. Single-channel recordings in inside-out patches of Bt2cAMP- differentiated HL-70cells

Single-channel currents in the ^presence of fMLP (100 nM) ^were measuredininside-out patches. (a) Originalcurrentrecordingsat variousholdingpotentials.Brokenlinesrepresentopen-statelevels ofchannels. Currentswere filtered at800 Hz. Representative seg- mentsofrecordings fromasingle patchareshown.Therecording time for each potential was 4min. No channel openings were

observed at0 mV. Similarcurrentswereobserved in experiments with fivedifferentinside-outpatches. (b) Current-voltage relation- shipofsingle-NSC-channel activityatvarious holding potentials.

Symbolsreferto currentsrecorded in six different inside-outpatches.

Thecurrent-voltage relationship^wasdeterminedbylinearregression analysis (r=0.98).

C')C')

X4)

C'.'

oQ

0- 4 _1

a ( a)

-0

C4)

en.

0 10 20 30

SK&F 96365(pM)

Fig.9. Concentration-inhibition^curveforSK&F96365on/-glucuronidase release inBt2cAMP-differentiatedHL-60cells

,8-Glucuronidaserelease inHL-60cellswasstimulated with fMLP (1/tM) in the presence ofSK&F 96365 at various concentrations under the conditionsdescribed in detail in theMaterials and methods section.Buffercontained1mM-Cal+and138mM-Na'.SK&F 96365

was added to cells 5min before fMLP. Results shown ^are the

means+S.D. of threeexperiments performedwithonepreparation ofHL-60cells. Similar resultswereobtained in threeexperiments withdifferentpreparationsofHL-60 cells.

absence of both Ca2+ and Na+, stimulation by fMLP of ,- glucuronidase release was virtually abolished. Fig. 9 shows a

concentration-inhibition curve for SK&F 96365 on fMLP- stimulated ,-glucuronidase release inthe presenceof Ca2+ and Na+.SK&F 96365 inhibited

,-glucuronidase

release withan

IC,,

ofabout 12

#uM

andamaximumat25 ^1uM.

SK&F 96365 isanimidazolederivative(Merrittetal., 1990), and imidazoles may bind to cytochrome b558, a protein com-

ponent of the

O2--producing

NADPH oxidase (Iizuka et al., 1985). Toexclude thepossibilitythatSK&F96365 inhibits02- production because of interference with cytochrome b558, the effects of SK&F 96365 on NADPH oxidase-catalysed 02- production in a cell-free system with membranes of Me2SO- differentiated HL-60 cellswere studied. In this system, arachi- donic acid (0.2 mM) induced 02- production at a rate of 43.9+1.0nmol/min permgof membrane protein. As reported Vol. 288

previously (Seifert & Schultz, 1987), GTPyS (10 /M) enhanced this rate of0°- production 2.5-fold. SK&F 96365 (10^1uM and 301uM) did not inhibit 02- production under these conditions (results not shown). Additionally, SK&F 96365 (10^/SM and 30,uM)didnotaffect0°-productioninducedby4fl-phorbol 12- myristate 13-acetate (100ng/ml) in intact Bt2cAMP-differen- tiated HL-60 cells(resultsnotshown).Theseresultssuggestthat SK&F 96365 did not interfere with cytochrome b558 or inhibit

02-productioninanunspecific ^manner.

InthepresenceofextracellularCa2+ andNa+, fMLPrapidly and effectively activated 02- production, although this process ceased after about 8min (Fig. 10).SK&F 96365(30

ItM)

inhibited this 02- formation by about 80%. Isradipine (1 fM) did not inhibitfMLP-induced02-production (resultsnotshown).Inthe absence of Ca2+ or Na+, fMLP-induced 0°- production was

reduced by 70-75%. Similar to the results obtained for f,- glucuronidase release, SK&F 96365 substantially inhibited 02-

(a) mV

60

*---~~~~~~~~~~6

I 0.5pA 300 ms

(b)

pA

0.4

0-

0 4 8 0

Time

(min)

^{4 8}

Fig.

10. Effects of

extracellular

^{Cal' and}

Na+

and SK&F 96365 on

time courses of fMLP-induced

02°

formation in Bt2cAMP- differentiated HL-60cells

022

formation in HL-60 cells induced

by

fMLP(1

#m)

wasassessed under various conditions. Arrows indicate the additionofstimulus.

Various substances wereadded to cells 3

min

^{before stimuli. (a)}

Experiments performed

in thepresenceof

Na+

^{(138mm).Trace 1,}

Ca"+

⁽¹

mm) plus

solvent

(control);

trace 2, EGTA (1 mm) plus

solvent

(control);

trace3,

Ca"+

⁽¹

mm) plus

SK&F96365 (30 tim);

trace4,EGTA

(1 mm) plus

SK&F 96365(30um).(b)Experiments

performed

in presenceof choline

(138

mm)insteadof

Nat.

^{Trace 1,}

Ca2 (1

mm) plus

solvent(control);trace2, Ca21 ( mm) plusSK&F 96365

(30Mtm);

^{trace 3,} EGTA

(1 mm)

plus solvent (control) or

SK&F 96365

(30 /tm). Superimposed

original spectrophotometric

tracings

are shown. Similar results were obtained four times with each

preparation

of HL-60 cells and in three experiments with different preparations of HL-60 cells.

Table4. Effects of chloride salts of monovalent cations ^on basal and

fMLP-stimulated GTP

hydrolysis

in membranes ofBt2cAMP- differentiatedHL-60cells

High-affinity

GTPase

activity

in membranes from Bt2cAMP-

differentiatedHL-60cellswasmeasured inthepresenceof chloride saltsof monovalent cationsatthe indicatedconcentrationswithout

(basal)

orwithfMLP

(10 #M).

Values inparenthesesin the column 'basal' indicateGTPase activities relative tothat measuredin the

absence of salts

(1.00).

Values in

parentheses

in the column'fMLP' indicate absolute stimulations

by

fMLP ofGTP hydrolysis(pmoles of

Pi

releasedin the presenceoffMLPminuspmolesof

Pi

^released

in the absence of

fMLP).

^Numbers in brackets in the column 'fMLP' indicate relative increases

by

fMLP of GTP hydrolysis

(fMLP-stimulated

GTP

hydrolysis/basal

GTPhydrolysis). Results

shownarethemeans+

S.D.

ofassay

quadruplicates.

Similarresults

wereobtainedinthreeseparateexperiments.

GTPhydrolysis

(pmol

of

Pi/min

^permgofprotein)

Salt Basal fMLP

None

13.2+0.4 (1.00) 18.6+0.2

(5.4)[1.41]

NaCl

(25 mM) 15.0+0.5 (1.14) 21.0+0.7

(6.0) [1.40]

NaCl

(50

mM) 16.0+0.2

(1.21)

22.6+0.5

^(6.6)[1.42]

KCI

(50 mM)

16.7+0.7 1.26)

23.4+0.9

^(6.7)[1.40]

LiCl (50 mM) 17.0+1.0 (1.29)

23.8+1.1(6.8) [1.40]

Choline chloride(50

mM) 13.9+0.4 ^(1.05) 19.9+0.8

(6.0)[1.43]

production

under these conditions.Intheabsence of both

Ca2l

and

Na+,

fMLP-induced

O2- ^production

^{wasabolished.}

Finally,

the effects of various chloride salts of monovalent cations on GTPase

activity

^{in membranes from}

Bt2cAMP-

differentiatedHL-60cellswerestudied. NaCl

(25 mm

^{and 50}

mM)

stimulated basal GTP

hydrolysis by

14 and21 respectively

(Table 4).

In the absence and presence of NaCl, fMLP (10 am)

stimulated

high-affinity

GTPase

activity by

approx. 40 %. NaCl

significantly

enhanced the absolute stimulation of GTP hy-

drolysis

caused

by

fMLP.KCl and LiCl

(50

mm) enhancedbasal GTP

hydrolysis

^{to a}

greater

extent than did NaCl and choline chloride.

KCI,

LiCl and choline chloride also enhanced, to

different

degrees,

the absolute extent ofGTPhydrolysis caused

by fMLP.

DISCUSSION

In

Bt2cAMP-differentiated

HL-60

cells,

the fMLP-induced increase in

[Ca

21]

depended largely

^on the presence ofextra-

cellular Ca² and was

partially

inhibited by SK&F 96365 (see

Fig.

andTable

1).

SK&F 96365 at concentrations ^as high as

303

^mdidnotinterfere withCa2 mobilizationfromintracellular stores

(see Figs.

^and2and Table

1).

Moreover, fMLPinduced

Mn2 influxin Bt cAMP-differentiatedHL-60 cells in^an SK&F

96365-sensitive manner

(see Fig. 2).

Results of recent studies

suggested

the presence ofNSCchannels in human neutrophils and

Me2SO-differentiated

^{HL-60 cells}

(von

Tscharner et al.,

1986;

Andersson et

al., 1986;

Nasmith & Grinstein, 1987;

Merritt et

al., 1989, 1990;

Pittet et

al.,

1989; Demaurex ^et al.,

1992;

Schumann et

al., 1992).

These studies showed that fMLP induces transient increases in

[Ca2+],

through Ca2 mobilization from intracellular stores followed by sustained Ca2 influx

(Andersson

et

al., 1986;

Nasmith&Grinstein, 1987;Pittetetat.,

1989). Additionally,

fMLP induces

Mn2+

^{influx in} neutrophils

(Merritt

^et

al., 1989). Ca2+

^and

Mn2+

influxes in neutrophils are

blocked

by

SK&F 96365

(Merritt

^et

al.,

1990). Taken together, these results indicate that

Bt2cAMP-differentiated

HL-60 cells possessNSCchannelswith

properties

similartothoseofneutro-

phils

and

Me2SO-differentiated

HL-60 cells.

fMLP-stimulated inward currents in

Bt2cAMP-differentiated

HL-60cellsshowed

properties

which arecharacteristicof^recep- tor-activated NSC channels

(Partridge

&Swandulla,1988). First, currents were activated

by receptor agonists

but notby voltage

(see Fig. 3). Secondly,

^{currents showed no} time-dependency of

activation or inactivation kinetics

(see Figs.

3 and 4). Thirdly, currents hada linear

current-voltage

relationship and reversed

closeto0 mV

(see Figs. 3,

7and

8;

Table2). Fourthly,currents were inhibited

by

SK&F 96365 butnot

by

blockers ofvoltage-

dependent Ca2+

^channels

(see Figs.

3-5and7).The

IC50

^{value for}

SK&F 96365toinhibit inwardcurrentsin HL-60 cellsamounted toabout 3#M

(see Fig. 5).

^Similar

potencies

for SK&F96365to

inhibit

receptor-stimulated Ca2+

influx have been reported for

neutrophils, platelets,

endothelial cells and aneuroglial cell line

(Merritt

et

al., 1990;

Monteroet

al., 1991;

Masonet

al.,

^1991).

Small NSC-channelcurrents mayresult inlargerisesin

[Ca2+]i (Penner

et

al., 1988). Bt2cAMP-differentiated

HL-60cellspossess

NSC channels with a conductance of 4-5

pS

and ^a reversal

potential

atabout 0

mV

^asassessed

by single-channel

recordings inexcised inside-out

patches (see Fig. 8).

von

Tscharner

et

al.

(1986)

identified

4-6-pS

and

18-25-pS

NSC channels in inside- out

patches

of human

neutrophils

with reversal potentials at

0 mV.

Possibly,

the

18-25-pS

channel isnotpresentinBt2cAMP-

differentiated HL-60 cells

(see Fig. 8).

Although both human

neutrophils

and

Bt2cAMP-differentiated

HL-60 cells possess

NSCchannels with conductances of about 5

pS,

^{thesechannels}

may be different. This view is

supported

by the finding that

opening

^{of the}

4-6-pS

^{NSC channel in}neutrophils obligatorily

depends

^onthepresence of

Ca2+

concentrations of above 10

nm

inthebathsolutionoftheinside-out patch(vonTscharneret

at.,

1986), whereas in our hands the 4-5-pS NSC channel in

Bt2cAMP-differentiated

HL-60 cells

opened

in the absence of 1992

(a) 1 (b)

$~~~~~~~~~~~~~~~~~~~~

2 33

4 2

3