Ca2 transients and Mn2 entry in human ne

Cdl caldm (1989) 10, 47740
Q LongmanGroup UK Ltd 19.39

Ca*+ transients and Mn*+ entry in human
neutrophils induced by thapsigargin
B. FODER’, 0. SCHARFF’ and 0. THASTRU?*’
’ Departmenf
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
of Clinical Physiology and Nuclear Me&ine and 2Depattment of Clink& Chemistry,

UniversityHospital,Copenhagen,Denmark
3Thrombosis Group, CopenhagenScience Park Symbion, Copenhagen,Denmark
Abstract - Human neutrophils,preloadedwlth the fluorescent probe, Fura-2, were exposed
to Ca2+-releasing agents. The monitored traces of ffuoresoenoe were transformed by
computer to cytosolic Ca2+concentration([C$?i). Due to quenchingof Fura-2, the addWon
of Mn2+enabled us to compute the cytosollc conoentrabonof total manganese ([mnj#. The
agents used were the novel Ca2+-moblllringagent, thapslgnrgln (Tg), the chem&oUc
peptide, formyl-methlonyl-leucyl-phenylalanlne(FMLPj, and the dlvalent catlon lonophore,
A23187. The agents caused transient rises of [Ca +Ji and monotonous rlses of M]I,
suggesting influx but no efflux of Mn2+. The rise time of [C& and the time constants and
magnitudeof the apparent Mn2+influx were strongly dependenton the sequence of addttlon

of the agonist and Ca2+. Contrary to FMLP, Tg needed several minutes to exert lts full
effect on the rise of [Ca2’]i and on the influx of Mn2+,the latter being dependent on two
phases, activation and partial inactivatfon. Pretreatment with phorbol 12-myrlatate
13acetate (PMA) inhibitedthe responses of Tg, FMLP and A23187. For comparison,human
red blood cells were tested. Contrary to A23187, Tg did not induce Ca2’ uptake In
ATPdepleted red cells but increased the Ca2’ pump flux in Intact red cells by la.
The
experimental data and computer slmulatlons of the granulocyte data suggest that
time-dependent changes of both passive Ca2+flux Into the cytosol and Ca2+ flux of the
plasma membrane pump are Involved in the transient [Ca2”Jiresponse.

Several natural and artificial agonists, for instance steady zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK
st at e level, in some respects similar to the
the
chemotactic peptide
formyl-methiollyl- effect of a Ca2+ ionophore. However, earlier
leucyl-phenylalanine (FMLP), have been reported to findings showed that thapsigsugin did not facilitate
provoke a transient increase of the cytosolic Ca2’ the transport of 4sCa2+from aqneous into organic
concentration ([Ca’+]i)in humau neutmphils [l-4]. phase, nor did the agent cause a Ca2t-mediated
This response seems to be implicated in the release of potassium from human erytbrocytes [9],

initiation of a variety of cellular responses (for suggesting that thapsigargin is not an ionophom.
lUthermore, thapsigargin indnced a transient
review see [5]).
Another type of CaT+-mobilizing agent, rise of [Ca2+]iin the nenronal cell line NGlS4OlL
thapsigargin, increased [Ca‘+]i in platelets [6], without generating inositol phosphate or activating
protein kinase C [lo].
A direct effect of
lymphocytes [71, and hepatocytes [8] to an elevated zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

477

c33LLcALcNM

4%

thapsigargin on an intracellular signaIling pool was
substantiated by the recent finding that thapsigargin
inhibited the Ca2+ATPase of endoplasmic reticulum
in hepatocytes 181.
The different [Ca2+]iresponses of cells caused

by various additions reflect a complex interaction of
passive and active Ca2’ fluxes into and from the
cytosol. In an attempt to evaluate how the single
fluxes contribute to the Ca2+homeostasis in human
neutrophils we have tested the effects of FMLP,
thapsigargin and the Ca2’ ionophore A23187. Since
phorbol 12-myristate 13-acetate (PMA) was shown
to interfere with the Ca2’ transport in neutrophils
[ll-131, we also tested the effect of PMA on the
[Ca2+]iresponses. For comparison, the effects of
thapsigargin and PMA on the Ca2’ pump in human
erythrocytes were explored.
The [Ca2+]iresponses obtained with neutrophils
were compared with computer simulations
performed with an extension of an earlier described
pump-leak model for Ca2’ transients in human
erythrocytes [14,15].

(60-90 MBq/mg Ca2’), 5 mM glucose, and pH was
adjusted to 7.1. The cell suspension was incubated

at 37°C in the presence of 20 pM CCCP that
secured a rapid equilibration of protons across the
membrane and Ca2’ uptake was initiated by
addition of A23187 [15].
The cellular calcium concentration was
determined as described earlier [14, 151. Aliquots
from the suspension were transferred to centrifuge
tubes containing a stop buffer and dibutylphthalate
oil. The cell pellet was isolated below the oil layer
by centrifugation, and the &ellet+wasprocessed for
scintillation counting of Ca zyxwvutsrqponmlkjihgfedcbaZ
. Since the low
intracellular calcium concentration in untreated
human erythrocytes can be neglected, the cellular
calcium (pmol/l packed cells) was calculated from
the measured cellular and total radioactivities and
from the total concentration of calcium in the cell
suspension measured by atomic absorption, using
the haematocrit values.
For some experiments the cells were depleted of

ATP before the incubation with A23187 by preincubation with inosine, iodoacetamide, and sodium
glycolate for 2.5 h at 37°C and rewashing [15].

Materialsand Methods zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Reagents

Percoll was from Pharmacia and Fura-2-acetoxymethylester from Molecular Probes. Thapsigargin
(Tg) was isolated as previously described [9].
Formyl-methionyl-leucyl-phenyl- alanine (FMLP),
phorbol 12-my&ate 13-acetate(PMA) (Sigma), Tg,
the divalent cation ionophores A23187 and
ionomycin (Calbiochem), and the protonophore
carbonyl cyanide m-chloro- phenylhydrazone
(CCCP, Sigma) were administered dissolved in
dimethylsulphoxide (DMSO, final concentration
about 1%). All other chemicals were, unless
otherwise stated, from Merck (pro analysis).

Neutrophils

The neutrophil leukocytes were obtained from
titrated blood from healthy donors. Fresh huffy
coat from about 250 ml of human blood was mixed
with 2% methylcellulose in saline (final
concentration 0.2%) and centrifuged at 20 g for 14
min in order to sediment most of the erythrocytes.
The supematant was centrifuged at 150 g for 5 min.
the plasma layer containing the platelets was
removed, and the cells were centrifuged further in a
discontinuous Percoll gradient containing 0.12 M
NaCl and 20% celI free plasma. The cells were
resuspended in 2 ml of 55% Percoll (about 10’
cells/ml) and layered on the top of 3 ml of 70%
Percoll and 5 ml of 80% Percoll[161. The densities
Ery throcy tes
of the three layers were 1.073, 1.090, and 1.101.
After centrifugation at 1600 g for 20 min the
Human red blood cells were isolated from healthy neutrophils accumulated in the middle of the
donors, washed and resuspended to a haematocrit of gradient whereas the mononuclear cells and some
4% in a salt solution containing 1 mM KCl, 155 neutrophils stayed near the top and the erythrocytes

mM NaCl, 0.15 mM MgClz, 0.010 mM 45CaC12 were found near the bottom

479

THAPSIGARGIN INDUCED Ca TRANSIENTS & Mn JiNTRY

Table 1 Parametersof Fura- fluorescence in cell suspension (Total) and in exkaceUar phase (Medhun). The
additions were 1 m.M Ca”, 0.2 mM M2’, 1 @i4 ionomycin, or 10 mM EGTA. The excitation wavekngths wae 340 zyxwvutsrqp
or
360 nm zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Additions

36onnl

34Onm

Ionomycln
Medfwn
Cation zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
EGTA

Totd
ca2+

0

0

F

Ca2+

t

0

Fm,

Fek,

Ca2’

t

t

Fmin

Fexmi,,

c2+ t Ml?+

0

0

FUI

ca2+ t Ml?’

t

0

FW

The neutrophils were resuspended in 10 ml of
buffer A containing 10 mM HEPES (pH 7.4 at
37-C), 140 mM NaCl, and 5 mM glucose,
centrifuged at 1500 g for 5 min and washed once in
10 ml of buffer A before the loading with Fura-2.
The neutrophils were loaded with Fura- by
incubation for 30 min at 37°C in buffer A
containing, in addition, 5 @I Fura-Zacetoxymethylester and lo7 cells/ml. After centrifugation
the cells were resuspended to 2.5 x lo6 cells/ml in
buffer A including 5 mM KC1 and 1 mM MgC12.
The cell suspension was stored at room temperature
with gentle shaking until the measurement of
fluorescence. Addition of CaClz was postponed
until the start of the experiment in order to minimke
the tendency to cell aggregation during storage.

Determination

of

calcium

and

manganese in

neutrophils

The concentration of cytosok free ca2’ ([Ca2’]i)
was determined by fluorescence measurements (F)
at 37°C with continuous stirring in a Perkin Elmer
LS-3B spectrofluorometer with monochromator
settings of 340 run excitation and 500 nm emission.
The time dependent fluorescence signal from the
fluorometer was transmitted to an IBM PC XT 286
computer equipped with a data acquisition card
allowing sampling of fluorescence data with a
frequency of 3 Hz for later calculations (see below).
The Fmax and Fmin fluorescences were obtained by
addition of 1 pM ionomyciu in the presence of 1
mM Ca2’ and further addition of 10 mM EGTA,

Total

Meakn

F’

FeX’

Fm’
Fmcxo

Fmcxo’

FmO’

respectively. The occtmence of extracellular Furaduring the experiments was unavoidable, and the
extracelhtlar Fura- fluorescence (Fexand
Fexmia) was measured in the supernatant from
centrifuge aliquots of cell suspension . Fexminwas
obtained by the addition of 10 mM EGTA. A
survey of these and other fluorescence pammekrs is
given in Table 1.
The granulocy teexperiments w ere perfomed in
the presence of either Ca2’ or Ca2+ and Mn2’. In

the latter experiments the fluorescence traces F, Fm,
F, and Fm’ (see Fig, 5A) were determined in four
different measurements. This allows computer
calculations of [Ca2+]iin the absence or presence of
manganese, and of cytosolic concentrations of fine
and total manganese, &ln2+]i and m]i, all in a
time dependent manner (see below and Fig. 5B).
The shown curves, depicting [Ca2+]i or m]i
versus time, were representative of at least three
experiments of each type.

Calculations of
concentrations

calcium

and

manganese

In the experiments without manganese [Ca2+]iwas
calculated from :
[Ca2’]i - KG, . (F - Fmin - Vexmax - Fexmin))/(F,,,= - F)

in which Kca = 224 nM is the apparent dissociation
constant for Ca2+-Fura- [17] and F, F-, Fmin,
Fexmaxand Fexti were dekxmined as mentioned
above (see also Table 1 and Fig. 5A, upper trace).

cm‘LcALcxuM

480

In the experiments with both calchrm and
manganese, [Ca2+]iand lMn2+]iwere calculated by
iteration from

eXjWIerimenti zyxwvutsrqponmlkjihgfedcbaZYXWVUT
C UIVe S,
yA had t0 be raised to the

second power (suggesting co-operativity between at
least two molecules of the agonist). Rurthermore,
the differential equation had to be supplied with a
[Ca2+]ip Kca. ((Fm- IGin- 0%cxo - Fexr,,in~)
fourth tern analogous to the third, representing an
- (Fmo- Fm) ([Mn2+l#K&/
inactive fraction, yr, of the agonist that has lost its
- Fmexo))
ability to increase the transport of M.n2’ or ca2’
(Fmax- Fm - TFexmax
(e.g., by binding to unspecific sites, by emptying a
metabolic pool, or by desensitization of a putative
W2+]i - Ka . (1 + [Ca2+]i/Ka) . (F’ - Fm’ fFex’ - Fmexo’~)/(Fm’- Fmo’)
receptor). If LA exceeds LI part Of the activating
effect on the transport persists (irreversibility)
in which I& = 224l42 = 5.33 nM is the apparent whereas if LA equals LI the effect of the agonist
dissociation constant for Mn2+-Rura-2 [l?]. The disappears with time (reversibility).
The model for computer simulations of [Ca2+]*
paran&ers of Fura- fluorescence at the excitation
wavelengths 340 (F values) or 360 nm (F’ values) vs time (t) implies three ca2’ compartments: thi
were determined analogously to F, Fma~, Fmin, cytosol, an intracellular calcium pool (for
Fexmq and Fexti, as described above (see also convenience named endoplasmic reticulum, ER),
Table i and Fig. 5A). At 360 nm free Fura- and and the extracellular medium (ca2’0). This model
Cazt-Fura- show identical fluorescences, and the affords the iterative solution of four simultaneous
decrease in fluorescence is entirely dependent on the differential equations:
Mn2+,concentration.
The total concentration of cytosolic manganese d[Ca2Ti/dt- LPM - VPM t fw (LER - VER) ....Eq. 2
(free Mn2’ and Mn2’-Fura- complex), @&Ii, was
d[Ca”]ER/dt- LPMER t VER - LER ...................Q. 3
found from :
l

l

[Mn]i k (1 + Ft/(Km {I + [Ca2’]i/K,) t [Mn2’]i))
l

l

dw/dt = kt (1 - w) . zyxwvutsrqponmlkjihgfedcbaZYXW
Z -h
w ...................Eq. 4
l

l

[Mn2+]i
duldt-b*(l-u)=Z-k.+*u

where Ft is the concentration of cellular Furacalculated by assuming that the cell water traction
of the cell suspension was l/1000.
h4Qdelcalculations

The experimental curves depicting [Mn]i vs. tirrkz(t)
could be modelled by solving the differential
equation :
d[llln]ildt= Lo1- h
(1 - ex&t})2 t LA .
(1 - exp{-kAt))2- Lx (I - exxp{-ti})2 ....... Eq. 1
l

l

.....................Eq.5

Eq. 2 describes the time dependence of [ca2+]1 zyxwvuts
as a function of two passive Ca2+ flares into the
cytosol, LPM through the plasma membrane (PM)
and LER through the ‘ER’ membrane, and two
oppositely directed Ca2+ pump fluxes, WM and
VER, fm (chosen to 0.02 1 ER/l cells) being the
cellular volume fraction of the ER pool.
Analogously, Eq. 3 describes the time dependence
of the concentration of ionized calcium, [Ca2+]~,
inside the ER pool, LPMER being the passive Ca2+
flux through a hypothetical ‘gate’ between the
extracellular and the ‘ER’ compartments (cf. [ 181).
LPM is calculated as the right side of Rq. 1.
The ground fluxes Lo1 and I..ozare typically chosen
to 9.6/(1 + 1000/[Ca2+l,)and 3.2/(1 + 1000/[Ca2+],)
pmol/min/l cells. The agonist-dependent fluxes LA
and Lr are calculated from

in which t is time, and Lo1 and Io2 represent a
ground flux. The bracket of the third term expresses
the active fraction, y.4, Of the ago&t that &WiSeS
transport of Mn2’ (or Ca2’) dependent on time with
the maximum rate LA (at high t), assuming a first
order reaction, dyA/dt = k~ 0.(1 - yA) that leads to zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED
L = -P*C*([Ca2+]0-exp{C~[Ch2+]iY(l-Cxp{C~)
= (I --exp{-kAt}). In order to fit the
YA

481
THAPSIGARGIN
INDUCEDCa TRANSIENTS & Mn ENTRY zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML
The Ca2+ pump activity, VPM, is calculated as
described in detail earlier for a rmxlel of the Ca2+
pump in human red cells [14, 151. In the model
VPM is dependent on the fractions (Eqs 4,5) of the
pump (w) and competing targets (u) that bind
calmodulin, Z. The xate constants for association,
kl and ks, and dissociation of Z, IQ and k4, are
functions Of [Ca2+]i(see [14]). The maximum value
of VPM is 490 pmol/min/I cells. The ER pump
acti;ityl VER, is equal ;o 2vwmax’(K*X +
2% lX )/(2*(1 + K*X + K lX )) in which X is
[Ca2+]w, l/K (= 0.5 pM) is the Ca2+concentration
that
activates the pump half ma&rally, and VERman
OL
0
is
1
mmol/minil
ER.
2 min zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
4
6 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
2min4
6

0

Fig. 1 Fhuxem
of Fun-2 (A) and cymolic
Ca2+
concentmtion ([Ca2+li) (B) in human neutmphila dcpcdmt on
Cxmmtlntion of thapsigafgin (Tg). The scale of flW_
ia
arbitrary. 2.5 x IO6 cellshI were mpemied in buf% A
conmining 10 mM HEPEB (pH 7.4 at 3TC), 140 mM NaCl, 5
mMglucoseand,inadditi~,5mMKcIend1mMMgc12.
The
cytosol contained about 100 pM Fura-2. [C& was cahhted as
described in Materials and Methods.
Tg in DMSO was
administaed at 1 min together with 1 mM Cal’. ‘Be Tg
concentrations (nM) were (a) 100, (b) 25, (c) 10, (d) 1, and (e) 0

Resnlts zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ
Dependence on thapsigarginconcentration

of the human neutrophils with FWa-2
and resuspension in buffer A with no added Cam
the extracellular Ca2+ coruzntration was about 25
p.M and the cytosolic Ca’+ concentration ([Ca2+])
wasdeterminedtobe68*17nM(meanfSD,n:
13). The addition of CaCl2 to a final concemration
using appropriate values of P, for imtance PA =
Of 1 mM caused an increase Of [Ca2+]ito 15O-200
0.028 and PI = 0.005, respectively (see Figs 9, lo), nM (see Fig. 1B).
and setting C = 2EF/RT in which E is the membrane
The simultaneous addition of 1 mM CaCla and
potential (chosen to be -30 mV), F and R the thapsigargin in final concentrations tiom 1 to 100
Faraday and gas constants, and T the temperature nM increased [Ca2+]ifintber to levels from about
0.2 to 1 pM (Fig. 1B). It appears that [Ca2+]i
(OK).
LER and LPMER are chosen to be independent increased transiently to a peak value attained in l-2
of the membrane potential. LER is calculated as the min and then declined to a lower level.
right side of Eq. 1. The ground fluxes Lo1 and Loz
A comparison of the traces of fluorescence (Fii
are chosen to 120 and 0 pmol/min/l ER The IA) and [Ca2+]i(Fig. 1B) SLOWStht, at 1 p.MCa2 ,
agonist-dependent fhtxes LA and LI am calculated small changes in fluorescence carzespond to large
from L’ = P*( [Ca2+]uu - zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
[caZ+]i),
inserting changes in [ca2’]i (trace a and b) and the peak
appropriate values of P’, for instance zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC
PA’ = 20*P~ values were therefore quite variable in this region.
and pr’ = 20*P1(see Figs 9, 10). In the calculation
of LPh4ER the ground flux is incorporated in the
expression for the agonist-dependent fhtx, i.e., L” = Comparison of thapsigargin with FlULP and
(Pground” + P”)*([Ca2+], - [Ca2+lER>,calculated A23187
analogously to L’. Pground” is chosen appropriately
to be the LPM ground flux multiplied with 0.05 (L” Apart from the height of the [Ca2+]ipeaks, the most
expressed as pmoI/min/l ER). In the shown pronounced difference between the Ca2+ transients
simulations (Figs 9, 10) the P” values are chosen to evoked by thapsigargin and FMLP was the rise time
zero, in other calculations PA” = 50 PA and FY’= of [Ca2f]i subsequent to addition of Ca2’ together
with the Ca2+ releasing reagent (see Fig. 2). The
50 Pr were inserted for P”.
l

l

After loading

CELLCALlClUM zyxwvuts

482 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

800- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM
z
5

a-bOOcc
d

a

UWHI-

b zyxwvutsrqp

OL,
0

Fig . 2

IF-

,

,

I

I

II

I

I

I

I

2

4

6

8

100
min

2

4

6

8

Bkct

of thapsigargin (Tg) lWLP

I

‘0I

,
2I

4I

6I

d
10
I

8I
min

A23187

Ca2’ s and

PMA on [Ca2+]iin neutrophils. &&kms’as in I&e 1. The
additions at 1 min were (A) 5 nM Tg, (B) 5 nM A23187, (C) 5
nh4 FMLP (D) DMSO added together with 1 &I

Ca2’. 40 nM

PMAwas;ddedat(a);minor(b)lminbefonstsrt

F@. 3

Effect of thapsigargin fig) cmmlhed with various Ca2+

mcenhations

and PMA on [&I,

in n.mophils.

Conditions as

Ca?+and25nMTgwexeaddcdatlmin.
PMA(40
&f) was ad&d at 6.5 min. The Ca” additions (u&I) wm (a)
1.0, (b) 0.5, (c) 0.1, (d) 0 zyxwvutsrqponmlkjihgfedcbaZYXWVUT
iuFigure1.

Table 2 Rise time of [Ca2’]i and Mn2’ fluxes dependent on sequence of additions and dose of thapsigargin ng). Rise
time is the time interval between the addition of Ca2’ (1 mM) or agonist and the attainment of the [Ca2+]ipeak value that
is caused by the last addition. The additions were 1 mM Ca2’,5-10nMFMLparTg(nM),andintheMn2’fl~
experiments in addition 0.2 mM Mn2’. The Mn2’ fluxes were calculated Tom the slope of curves like those in Fims
6A and 6B, kd the valuk represent the flux caused by last addition. Mean f SD (n). The difkences between the rise
time of agonist + Ca2” and that of the other treatments were all significant (P 3 mln

3.2 f 0.6

0.8 f 0.6

0.4 f 0.3 (3)

2.7 i 0.4 (3)

(min)
0.3 fO.l (5)

3.9 f 0.6 (6)

Tg (10)
ca2+
FMLP

ca2+

0.8 f 0.2 (5)

‘b (10)

ca2+

0.7 f 0.1 (5)

21.7 f 5.1

9.8 f 2.4

0.5 f 0.1 (8)

14.5 f 1.3

3.2 f 1.1

1.1 f 0.3 (4)

FMLPtca2+
Tg (5) t Ca2+

Tg (10) t Ca”

7.8 f 1.1

1.9 f 0.3 (4)

3.4 f 2.1

8.7 f 5.1

3.3 f 0.3 (4)

10.7 f 5.4

12.4 f 4.0

7.6 f 2.0 (3)

5.1 f 0.9
1

1.7 f 0.3 (18)

Tg (25) t Ca2+
Ca2+

FMLP

Ca2+

Tg (2.5)

Caz”

Tg (10)

Cc??+

Tg (100)

12
I

IO zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0.3 f 0.1 (9)
2.6 f 1.1 (13)

1.1 f 0.6

3.4 f 3.2

1.8 f 0.2 (4)

1.2 f 0.1

2.7 f 0.5

2.4 f 0.5 (3)

1.5 f 0.5

3.7 f 0.7

7.0 f 2.4 (3)

THAPSIGARGIN INDUCED Ca l’R4NSENTS & h4n JZNTRY

483 zyxwvutsrqp

time interval between addition and peak value of

[Ca2+]i SIllOUI&d t0 1.7 min for 5-10 t&I
thapsigargin and 0.5 min for 5-10 nM FMLP (Table
2). Furthermore, the return of [Ca2+]ito a lower
level proceeded more slowly with tbapsigargin than
with FMLP. The rise time of [Ca2+]ievoked by 5
nM A23187 was 1.2 + 0.2 mitt (SD, n = 3). i.e.,
intermediate to those of thapsigargiu and FMLP
(Table 2).
The addition of PMA rior to ca2’ alone or
2P
Ca2’ together with a Ca -releasing agent had a
dramatic effect on the [Ca2+]iresponses, especially
by suppressing the initial peaks of [Ca2+]i(Pig. 2). zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI

Dependence on extracellularCa2’ concentration

T&e addition of 25 nM thapsigargin together with
CaC12to final concentrations of Ca2+ranging from
the initial 25 pM to 1 mM caused raised levels of
[Ca2’]ifrom about 0.2 to 1 p.M,as shown in Figure
4 zyxwvutsrqponmlkjihgfedcbaZYX
8
12
3. This indicates that the tbapsigargin response iu
min
this cell preparation was dependent mainly on Fig. 4 Effect of sequence of additions on [Ca*+k in neutmphils.
extracellular Ca2+ rather than on Ca2+ from Conditions as in Figure 1. The additions wm 1 mM Ca*‘, 5 nM
intracellular stores.
FMLP or 10 IM thapsigargin (Tg). First and mxmd addition
When PMA was added at a high level of [Ca2’]i wmatland6mia.
Thesequemceswere(Aa)TgbefonCaz+
that was induced by thapsigargin, [Ca2’]i decreased (A,b) Ca*’ before Tg. (B,a) Ca2’ before FIkP. (B,b) FI&
to a lower level (Fig. 3). PMA lowered [Ca2+]ito a before Ca*+
new steady level, but at high [Ca2+lothis level was after last addition increased from 1.7 to 2.6 min
preceded by an ‘overshoot’, i.e., a transient (Table 2). If, on the contrary, thapsigargin was
minimum of [Ca2+]i(Fig. 3).
added 5 min before the addition of 1 mM CaCl2 the
The ‘overshoot’ was diminished when sodium rise time of [Ca2+]iafter last addition was reduced
chloride in the medium was replaced by choline to 0.7 min (Fig. 4A, Table 2).
chloride (data not shown). This suggests that the
The latter value was similar to the rise time
combined action of a Na+/Ca2’ exchange mechan- inducedbyFMLPadded5minbefore1mMCa2+
ism and a PMA-stimulated Nat/Et]- exchange but the rise of [Ca2+]iafter last addition was much
mechanism [19] contributes to the ‘overshoot’ lower than in the case of thapsigargin and no [Ca2+]i
phenomenon.
peak appeared. FMLP added 5 min after or
simultaneously with 1 mM Ca2+caused even lower
rise tune (Table 2) and a pronounced peak of
Sequence of additionsof Ca2+ and agonist
[Ca2’]i(Fig. 4B).
The initial addition of either FMLP or
In the experiments above the agonist was added thapsigargin to the neutropbil suspension containing
simultaneously with CaCl2. Figure 4A shows that 25 pM of extracellular ca2’ zyxwvutsrqponmlkjihgfedcbaZ
only ~J .I CI ESU &[c a 2 ’]i
the effect of thapsigargin on the cellulsr Ca2+ to lOO-200nM (Fig. 4). This iudicates that only a
trausient depended strongly on the sequence of minor part of the [Ca2’]i rise caused by FIvlLP or
additions. If 1 mM Ca2+was added 2-3 min before tbapsigargiu (see also Fig. 3) originated from
the addition of thapsigargin the rise time of [Ca2+]i intracellularstores.

facilitate Ca2+influx (cf.
cm.
Figure 5A shows four fluorescence traces
obtained under different conditions (cf. Table 1). It
appears that the fluorescence was quenched by
Mn2’ whether Fura- was excited by W-light of
340 or 360 nm, whereas the fluorescence was not
aft&ted by Ca2’ when Fnra-2 was excited at 360
nm [17, 211. From these fluorescencetracesthe
concentration curves of cytosolic Ca2+, Mn2’ and
total Mn could be calculated (Fig. 5B, see also
Materials and Methods). Note that the rise of
[Ca2+li was inhibited in the presence of 0.2 mM
Mn2+.
Fignre 6A shows the timedependent cytosolic
concentration of total Mn in response to
40
tha..!igargin addition in various combinations wit$
E
Ca (cf. Fig. 4A). Provided that the cellular Mn
.S
was not removed fkom the cytosol by C!aztpumps
20 r;or other transport mechanisms, the increase of total
z
cytosolic Mn delivered a measure of the Mn2+ flux
into the cell and, by analogy, a relative measure of
0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB
the Ca2+ flux into the cell at the different
0
2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
4
6
8 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
experimental conditions. The Mn2+ fluxes were
min zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
detemrined as the slope of the curve tangents from
Ng. SA ESfect of Mn*+on fluonscence of Fum-2 in neuq&ils,
curves such as those in Figure 6A, and some of the
sampled at various wavelengths. Addition at 1 min of 1 mM Ca”
and 5 nM thapsigargin, either without MI? (traces F and Fq OT resulting Mn2’ fluxes ate shown in Table 2.
with 0.2 mM MI?’ added 5 s before Ca*’ (traqer Fm and Pm’).
It appears from Table 2 that when thapsigargin
Traces F and Fm were sampled (see Materialsand Methods) at the was added before Mn2’ and Ca2’ the initial Mn2’
excitation wavelength 340 mn. traces P and Fm’ at 360 ma The flux (zero to 1 min) was high and the flux then
levels of fluorescencearearbitraq.OtherconditkmaasinFigurel
decreasedwithtime. Thisisinaccordancewiththe
short rise time of [Ca2+]1when thapsigargin was
Fig. 5B
Cytosolic co-tions
of c2+ ([CayI),Ml?
added before Ca2’ (Fig. 4A and Table 2). indicating
(&d+li)andtotal Mn (LMuli)cahlated fkom the horeacence
a high initial cat’ net flux into the cytosol. At the
traces in Fim 5A (see Materiels and Met&h). (a) [ti%]t in
reversed
sequence of additions, i.e., Mn2’ and Ca2’
absence of Mu*’ (&I), (b) [Ca”]~ in presence of MI? (nM), (c)
added
before
thapsigargin, the initial Mn2+flux was
IMnli(PM), (d)m*‘li (m) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
low and the flux increased with time (Table 2).
Analogously, the rise time of [Ca2’]iwas long when
Eflect of ad&d manganese ions
Ca2’ was added before thapsigargin (Table 2).
In order to analyse the components of the Ca2+ indicating a low initial Ca2+net influx.
At the simultaneous addition of Mn2’, Ca2’ and
fluxes we repeated the experiments above with a
single extension, namely the addition of 0.2 mM thapsigar$n the h%n2+fluxes as well as the rise time
Ii
were intermediate to those of the
hhCl2
together with 1 mM CaCla. The addition of Of [Ca zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO
Mu” caused an immediate drop in fluorescence, sequential additions above (see Table 2). In this
due to quenching induced by the binding of M.u2’to case the Mn2’ flux showed a maxhnum in the l-2
extracellular fira- 2.
Subsequently, the cellular min interval after the combined addition (see Fig.
Fura- fluorescence decreased with thne (Fig. 5A), 6A and Table 2). indicating a biphasic time course
probably due to influx of hJn2+ via the same of the thapsigargin effect,
trallspolt mechanismsthat

I

THAPSIffARt3IN INDUCED Ca TRANSlENB & Mn ENTRY

48s

counteracted a Ca2’-ionophoric effect of thapB zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Sigargin.

“0

2

4

60
min

2

Inordertomakethetestmomsensitiveweused
a red cell pmparation which was depIeted of ATP in
order to prevent Ca2’ etfhrx due to ATP=depe&m
Ca2+ pumping. The addition of 1 pM A23187
caused a substantial uptake of calcium in a short
thne, i.e., total cell Ca rose from 5 to 500 pmol/l
cells in 5 min. In com~~&son.the effect zyxwvutsrqponm
of l-20
p.IvIthapsigargin on Ca uptake was negligibIe and
similar to that of 0.1% ethanol, even durhrg
prolonged incubation, i.e., total cell Ca was only
4
6 about 7 pmobl cells after incubation for 2 h. We
therefore conclude that thapsigargin is not a Ca2+
neutn~Phila ionophore.

Ng. 6 Entry of manganese Mu (W]i va time) in
caused by additiona of agonist and 01% in verioua sequencea.
Thecurv~waecrrlculated~mtheFm,F~dPm’tncsr(sse
Fig. 5). In all the sequencee 0.2 mM MD* waa added 5 I befon
thelestadditionwhichwasappliedatzm,orl~opltbsahown
timeaxia. Otbera1nditionsasinFigme1.
(A) The xeqwnccs were (a) 10 nM tha@gargin (Tg) followed
after 2 min by Ca2+,(b) 10 nM Tg simultaueoualy with cl%‘, (c) 5
nM Tg simultaaeously with C!a2’t (d) Ca* followed efter 2 min
by 1OnMTg
(B) 5 nM l%iLP or 5 nM Tg were added simultaneously with
Ca2+ (and Mn2+). If present, 40 nM PMA wea added 2 min
before the combined addition. The addition wem (a) FMLP, (b)
Tg, (c) Ph4A and FMLP, (d) PMA and Tg

EJj% ctof thapsigarginon ety throcy teC2+ pump

not a Ca2+ ionophore (cf.
above), one explanation of its effect on [Ca2+]iin
neutrophils and other cell types couki be that
thapsigargin inhibited the ATPdep&att
Cla2’
extrusion through the plasma nrmbmne. In order to
test this we applied human eq&ocy& with an
active Ca2+pump.
We used a non-invasive technique [15 allowing
determination of the pump-media&I Ca2J* ef&x in
cytoplasmic
Figure 6B demonstrates that 5 nM FMLP given intact erythrocytes with all the native zyxwvutsrqponmlkjihgf
together with 1 mM Ca2’ induced a high initial constituents preset& including calmodulin. The
Mn2+ h&x compared to that induced by 5 nM A23187-induced uptake of calcium was intemrpted
thapsigargin together with 1 mM Ca2’ but the after various time of exposure to the ionophore (15
responses of both additions were inhibited by 40 nM to 30 s) by addition of CoCla which has been shown
PMA. The apparent Mn2+ influx was reduced by to block completely the A23187~media&d Ca2’
PMA to 2.3 f 0.4 (O-l min) and 1.7 + 0.8 (>3 min) influx without disturbing the pump-me&ted Ca2+
efflux [15,22].
pmol/min/l cells (SD, n = 5, cf. Table 2).
Thedecreaseofcell~calcinmwasalinear
Manganese experiments with the use of A23187
were not conducted due to the Mn2+ ionophoric fUrctionoftimewithin15saftertheadditionof
CoCl2 to the red cell suspension, and this decrease
effect of this agent. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
was used for de&mu&ion of the pumpmdillted
Ca2+ efflux (see [151). The measured vahres of
Ionophoric effect of thapsigargin tested on Ca2’ efflux were plotted in Figure 7 as a function of
the cellular concentration of total Ca (fCa]i) at the
ery throcy tes
time of CoC% addition. For data tmatment Ca2*
As mentioned above thapsigargin was unable to efflux vs [Cali could be described satisfactorily by
cause a Ca2+-mediated release of potassium from linear regressions (not shown).
Prior to the A23187 addition, we added
human erythmcytes 191. However, the efficient
Ca2+ pump present in the erythmcyte may have thapsigargin, PMA, DMSO, or nothing. It appears
Since thapsigargin is

486 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0
0

10

30
20
Ca cont. (pmol/l cetts)

40
0

2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTS
4
6
min

IQ. 7 Pump-mediated Ca” efflux vs concentration of total Ca in
human red cells dependent on presence of thap&argin (Tg) and Fig. 8 Siiulation of Mn% entry in human neutrophils caused by
simulated additions. The ‘additiona mimicked various seqtumces
PMA (dissolved in DMSO). The cells were suspended in a salt
of
1 mM ca2+, 10 nM thapsigargin (Tg), or 5 nM PMLP. The
solution containing 1 mM KCl, 155 mM NaCl, 0.15 mM MgCla,
curvea were calculated from Eq. 1 in Materials and Methods. Tk
0.010 r&l 4sCaCl~ 5 mM glucose, pH wan initially 7.1, and the
khlitions’ and parameter values of Lox, La LA and 4 (@&nin),
haematocrit was 4%. Celhdar Ca” uptake was hxked by
and hi and h (Umin) were varied as follows: (a) ‘Tg before Ca”‘,
adding A23187 at zero time (10 pmol/l cells), and after 15 s the
@) ‘Tg simuhaneonaly with Ca%‘, 3.2,
A23187-mediated Ca” flux was blocked zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
by adding 0.25 mM 3,2,75.0,73.3,5.0,2,5;
Coc12, allowing detemination of the pump flux as described 75.0.73.3, 1.00.0.85; (c) ‘FMLP simttltaneously with Ca%‘, 3.2,
50.0,50.0,5.0,2.5; (d) Y!a2+before Tg’, 1,0,75.0,73.3,1.0,1.0
previously [15]. Symbols refer to 1 pM Tg (filled chcles), 40
nM PMA (open circles), control including 1% DMSO (triangles),
and control (aquares)

from Figure 7 that there were no or only small Discussion
effects of these additions. Thapsigargin (1 p&4)
increased the pump-mediated Ca2+ efflux The characteristic celhrlar response to the addition
significantly by about 10% at a [Cali of 15 pmol/l of a Ca2+-releasingagonist: a peak value of [Ca2+]i
cells (P ~0.01) whereas neither DMSO nor PMA followed by a fall to a lower level (cf. Figs. l-4).
had any significant effects on the Ca2+ pump suggests that time-dependent mechanisms are
involved in the regulation of [Ca2+]i,for instance,
activity.
The small stimulatory effect of thapsigargin on (a) a transient increase of net flux of Ca2+into the
the Ca2’ pump could be explained as an effect of cytosol and/or (b) a delayed activation of the Ca2+
this agent on the rate by which the Ca2’ pump pump located in the plasma membrane. zyxwvutsrqponmlkji
ATPase was stimulated by calmodulin.
We
therefore tested the effect of thapsigargin on the
cahnoduhn
activation of
membrane-bound Ca2+jlux into cy tosol
erythrocyte Ca2+-ATPase in vitro by a technique
described earlier [23].
The activity of the A transient increase of Ca2’ flux requires that the
calmoduhn-activated ATPase was a little higher in effect of the agonist is dependent on time. The W e
the presence of 1 @4 thapsigargin compared to dependence could be achieved by: (a) a temporary
DMSO (10 f 218, SD, n = 12) but the difference
presence of the agonist itself, (b) a temporary course
was not significant (P >O.l), and thapsigargin did of metabolic events started by the agonist; or (c) a
not affect the rate of cahnodulin activation of the ‘&sensitization’ of an agonist target (receptor or
Ca2’-ATPase. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
channel) with time.

THAPSIGARGIN

INDUCED Ca TRANSIENTS

& h-in FiNTRY

In our experiments the agonists remained present
in the cell suspension. FMLP was highly active
when added together with or after 1 mM Ca2’ (Pigs
2C and 4Ba) but much less active a few minutes
after the FMLP addition when 1 mM Ca2’ was
added (Pig. 4Bb). FMLP activates the hydrolysis of
phosphatidylinositol phosphates (PIP& giving rise
to products that mobilize Ca2’ from internal stores
(e.g. inositoltrisphosphate, see for instance [24]) or,
apparently, from the external medituq cf. the time
course of the FMLP-induced Mn2+ entry (Fig. 6B).
The temporary effect of FMLP may be due to the
observed discharge of a mcmbraneous pool of
phosphatidyliuositol and PR within 1 min [5], i.e.
case (b) above, or to transient openings of cation
channels in the plasma membrane, possibly
Ca2+-gated (see [25]), i.e. case (c). Alternatively,
the small effect of FMLP in the experiment shown
in Figure 4Bb could be due to a low Ca2’
concentration of the internal stores compared to the
parallel experiment in which Ca2’ preloading
preceded the FMLP addition (Fig. 4Ba).
The effect of tha sigargin on the Mn2+flux and
!!t
by analogy the Ca flux, differed from th$ of
FMLP in a characteristic way: the full effect was
achieved only with a delay of l-3 min (Pigs 4A-6),
indicating that thapsigargin required time to exert its
Ca2t-releasing effects. Once established, the effect
of thapsigargin did not vanish with time, as
indicated by the rapid response on addition of 1 mM
Ca2+ five minutes after the addition of agent,
contrary to the effect of FMLP (Figs 4A and 4B).
Apparently, the effect of thapsigargin was
dependent on the time of exposure to 1 mM
extracellular Ca2’. The induced Mn2’ flux was less
when thapsigargin was added 2 min after the
addition of 1 mM Ca2’ than when added before or
simultaueous with 1 mM Ca2+(Fig. 6A, Table 2).
The kinetics of Mn2+ entry induced by
thapsigargin could be mimicked by a model
described in Materials and Methods (see Eq. 1 and
Fig. 8). The model suggests that thapsigargin, once
established in the cell, is maximally active at low
Ca2+ concentrations, i.e., [Ca2+]iless than 100 nM,
but that part of the agent becomes inactive as
[Ca2+]i increases, due to inactivation either of
thapsigargin itself (e.g., by Ca2+-mediatedbinding
of the agent to unspecific cellular sites) or of the

487

targets for the agent. Conformational changes of
putative Ca2* channels induced by Ca2’ and/or
protein kinase C could also contribute to the
inactivation (cf. below).
The lack of effect of thapsigargin on passive
Ca2+ transport across the erythrocyte membrane
suggests that thapsigargin acts on specific trausport
sites that ate not available in human erythrocytes in
arty sign&ant amounts. zyxwvutsrqponmlkjihgfedcbaZYXW

C2+

pump

of pl asma

membr ane

A delayed activation of the plasma membrane Ca2’
pump has been suggested to occur in human
erythrocytes [14, 151 in which the Ca2’ pump has
the potential of being regulated by calmodulin (for
review see [26]). In the red cells [Ca2’]i has been
reported to be lo-30 nM [27] and since G&nod&r
seems to be dissociated from the pump at Ca2+
concentrations below 100 nM (see [23]), the Ca2+
pump is not expected to be activated by &noduhu
at low values of [Ca2+]*
1. Along with increasing
values of [Ca2+]i.the Ca2’ pump seems to be
activated slowly until [Ca2’]i has reached a level (1
to 10 @!I) at which calmodulin binds to the pump
more rapidly [14, 151.
The occurrence of a cahnodulindependent Ca2+
pump in human neutrophils shnilar to that in
erythrocytes has been reported previously 281, and
certain indications of a delayed onset of Ca6+ efflux
subsequent to a transient increase in [Ca2’]i in
human neutrophils induced by FMLP have been
presented by Korchak et al. [l].

Si mul at i on

Of

[&?+]I

changes

by pump-l eak

model

order to investigate the presumed role of the Ca2+
pump in the plasma membrane we have simulated
the time course of [Ca2+]i by the aid of the
pump-leak model described in Materials and
Methods (see Eqs 2-6), using time constants derived
from the Mn2+ entry experiments for simulation of
the Ca2+fhrxes.
In the model calculations possible effects of
thaEigargin or FMLP were tested: (i) increase of
Ca influx via plasma membraue channels; (ii)
In

nln 2

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
4
6 mi” 8
10 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ

“0

0

2

4

6

8

100

2

G

6

8

10

min
Simulations of [Ca2+]i in qxmse
to thapsigargiu
combined with various concentrations of C!a% and addition of
‘PMA’. See also Figure 3. At the simulated PMA addition at 6.5 Fig. 10 Simulations of [Ca2$ iu nqnmse to various sequences of
additions calculated with or without Ca2+ pump &lay. See also
min the Ca2’ fluxes across the plasma membrane were hang&
for calculation wem as in Figure 9, except
the passive influx (LPM) and the active efflux (VPM) were Pigun 4. Pammemultiplied by 0.3 and 1.5, respectively. Parametem used for for the change.3below. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ
calculation (see also Model calculations iu Materials and Methods) (A) PA - 0.033, R - 0.005 (LPM and LER range3, 0.270
were PA - 0.028 and PI - 0.005, leading to LPM values rtmging pmoUmin/l cells and 120-800 pmolhin/l ER), and (A& ‘Tg
before Ca’. kA - kt- 0.45 (lhin), (A,b) ‘Ca before Tg’, 1- h between 0.2 and 60 pm~l/rnirUl cells, P’A - 20 PA and P’r - 20
R, leading to LER values between 120 and 650 cunollmiafl ER, 1.3 (Ihin)
P)A - 50
P& FI - 50 9, kA - 50 (h&l), kJ - 5 (Ihit&
p’,, - P”I - 0 (LPMER values between 0 and 70 pmol/minIl ER). (B) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ
For the different curves Ca2’ addition (mM), kr, (hi@, kI and (B,a) ‘Ca before FMLP’, PA - PI - 0.0065 (LPM and LER
(hi@: (a) 1.0, 1.00, 0.8;; (b) 0.5, 1.00,0.85: (c) O&0.75, 0.60; raoges, 0.3-20 and 12&500), (B,b) ‘Fh&P before Ca’, PA - PI 0.004 (JJ’M and LER mnges, 0.3-6.5 and 120-300)
(d) 0. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
0.10, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
0.10
(C) and (D) as above but with no delay of ce2” pump activation
increase of Ca2+ influx via a gate between the in plasma membrane: (C,a) as (A,a), (C,b) as &a), and (D) as
curve a in Figure 9 (note difihut scales)

Fig .

9

l

l

l

l

extracellular and ‘endoplasmic reticulum (RR)’
compartments (cf. 1181)and from ER to cytosol; and just as the ER pump, was assumed to be activated
(iii) thapsigargin-induced inhibition of the ER Ca2’ by Ca2’ without delay the experimental fmdings
could not be simulated (Fig. 10).
Pump Bl.
Using case (i), we calculated the [Ca2+]icurves
Similar [Ca2+]isimulations could be calculated
shown in Figures 9 and 10, which simulate the (not shown) by assuming that the cells hyperexperimental findings (Figs 3-5A) reasonably well. polarized due to Ca2’-induced opening of potassium
Calculations based on case (ii) alone, for channels (see [15]) instead of assuming a constant
thapsigargin combined with (iii), yielded [C!a2’]i membrane potential.
curves (not shown) with similar relative
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
courses but
with considerable deviations from the absolute
[Ca2+]i values in Figures 3-5A. However, the M odifLig effects of Pith4on Ca2’ j&x
calculations did not exclude that the concerted
response included all three cases (i -iii).
Fmtrealment with PMA, which activateszprotein
Most important, in all the calculations it was kinase C [29], inhibited the increase of [Ca ]i that
necessary to assume a delayed activation of the was caused by additions of FMLP, thapsigargin,
plasma membrane Ca2’ pump in order to simulate A23187, or by raising the external Ca2’
the Ca2+ spikes. If the plasma membrane pump, concentration (Fig. 2). FMA inhibited the Mnz’

489

THAPSIGARGIN INDUCED Ca TRANSIENTS & Mn ENTRY

entry induced by FMLP or thapsigargin (Pig.6B).
This effect of PMA agrees with earlier findings on
PMA inhibition of the changes of Ca2’ pemreability
induced by chemotactic factors in rabbit neutrophils
WI.
Possibly, protein kinase C, when activated
directly or indirectly by various agonists, exerts a
negative feedback on receptors linked to the Ca”
signalbug system (see 1301). This might cause the
desensitization of agonist receptors that is suggested
above. An effect of thapsigargin on protein kiuase
C may be indirect (cf. [lo]).
The effect of PMA on [Ca2+]iin A231874reated
neutrophils (Pig. 2) suggests that the Ca2+pump, in
addition, may be a target for protein kiuase C,
because it is unlike1 that PMA would reduce the
A23 187~induced Ca& flux since our erythrocyte
experiments indicated no effect of PMA on
A231874nduced Ca2’ flux (data not shown). This
is in accordance with the stimulating effect of PMA
on the plasma membrane Ca2’ pump iu neutrophils
suggested earlier [ll, 131. In the ms,
however, we found no effect of PMA addition on
the Ca2’ pump (Fig. 7).
The ‘overshoot’ phenomenon caused by
PMA-addition at high but not at low Ca2+
concentrations (Fig. 2) could be simulated by the
pump-leak model (Fig. 9), assnmirrf that PMA
increased the 2~ump-mediated Ca -efllux and
reduced the Ca influx (cf. Fig. 6B) but with no
effect on the Ca2+ release from the ERcompartment. The simulated ‘overshoot’ was less
pronounced than the experimental ‘overshoot’,
possibly because the model does not include the
mechanisms of Na+/Ca2’ exchange and PMAstimulated Na+/@I+] exchange (cf. 1191).

Conclusions
The results above demonstrate that temporal
changes of passive and/or active Ca*+ fluxes are
essential to the generation of transient Ca2+ peaks in
neutrophils. If an agent (e.g. thapsigargin) induces
passive Ca2+ fluxes that are irreversible then a
time-dependent change of a ca2’ pump flux seems
necessary for a transient [Ca2+]i response. Besides
the more conventional Ca2+-releasing agonists,
thapsigargin seems to be a valuable tool in such

studies. Model simulatious of agonist-induced
changes of the cytosolic Ca2’ couceutration may
contributeessentially to the tdmtadng
of how
the various Ca2+ fluxes coutlibute to the Ca2+
homeostasls but the calarlatious also emphasize the

need for further quantitative characWUtion of the
single parameters.

Acknowledgements zyxwvutsrqponmlkjihgfedcbaZYXWVUT
The authors thank Dr S. Bragger Ch&tenaen, Royal Danish
School of Phatmacy, for the supply of thap&@l.
Bxpert
technical assistance was given by Hanne Jepsen. This work was
supported by the Danish Reseamh Council, the NOVO
Foundationand the Danish CancerSociety.

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1. Komhak HM. Vienne K. Rutksford LE. WiIkenBeldC.

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FinkelsteinMC. Weissmarm0. (1984) Stimuhmmapolme
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HartialaKT. Scott IO. Vi&men MK. &man KEO.
(1987) Lack of correlationbetween calcium mobilization
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Lew PD. Monod A. Waldvogsl FA. Rxam T. (1987) Role
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leukottiene-B4-atimuhuedseomtion in buman ocutmphils.
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fotmyl-methionyl-leucyl-pknylahmioc. Eur. J. Bicchem.,
162, 161-168.
Co&lofts.
(1986) Phoqhohmaitider and neutrophil
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