Vol. 182, No. 1,1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 372-378
Janua~ 15,1992
ACTIVATION OF K + CURRENT IN MACROPHAGES BY PLATELET ACTIVATING FACTOR
M.
Ichinose,
Department
N. Hara,
M. S a w a d a
and T. M a c r o
of P h y s i o l o g y , S h i m a n e M e d i c a l I z u m o 693, J a p a n
University
Received December 2, 1991
P u f f a p p l i c a t i o n of p l a t e l e t a c t i v a t i n g f a c t o r (10 -8 M) o n t o peritoneal macrophages from thioglycollate-stimulated mice i n d u c e d an o u t w a r d c u r r e n t at a h o l d i n g p o t e n t i a l of -63 mY. The current was suppressed by, an a n t a g o n i s t Y - 2 4 1 8 0 b u t n o t b y CV3988. Charybdotoxin (i0 -b M) s u p p r e s s e d the current. Reversal potentials were dependent on e~ternal K + concentrations. The c u r r e n t w a s not s u p p r e s s e d in C a ~ + - f r e e E G T A - c o n t a i n i n g solution but was completely abolished in B A P T A - A M containing solution. T h ~ s e r e s u l t s s u g g e s t that p l a t e l e t a c t i v a t i n g f a c t o r a c t i v a t e s a C a ~ + - d e p e n d e n t K T channel. © 1992AcademicPress, Inc.
Platelet activity
activating
in l e u k o c y t e s
such
as a n a p h y l a x i s
(2),
chemotaxis
(2,4),
and
monocytes to
PAF
which
and
(3),
release
(3),
of e x o g e n o u s
spreading
Ca2+-gated
Cl--channels
ionic
actions
of
valuable
to
present
study
by m e a n s
was
from
intracellular
of w h o l e
have been
methods
cation are
effects
of
in
polyphosstores
and
of
remains well PAF
0006-291X/92 $1.50 372
method.
in m a c r o p h a g e s
are 4
channel.
to a n a l y z e
patch-clamp
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
There
including
in l e u k o c y t e s
undertaken
demonstrated
(8-12).
macrophages
cell
B 2 (4)
(3,6,7).
channels,
the
production macrophages
of Ca 2+
channels
examine
of
release currents
in
responses break-down
ionic
PAF
anion
thromboxane
(5),
and a n o n s e l e c t i v e
of t h o s e
E and
process
aggregation
glucose
by electrophysiological and
including
superoxide
Intracellular
biological
to p a t h o l o g i c a l
(i),
(4),
potent
of
Ca 2+
ionic
possesses
is r e l a t e d
of p r o s t a g l a n d i n
consumption
Several
(PAF)
inflammation
or m a c r o p h a g e s .
are
phoinositides influx
factor
However,
on
3
the r o l e
to be c l a r i f i e d .
studied,
the
voltage-gated
K+-channels,
ionic
it
As
is
very
channels.
The
PAF-induced
current
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS
AND
METHODS
Cells Elicited macrophages were produced from C3H/Heslc mice ( S h i z u o k a Dobutsu, Shizuoka, Japan) of e i t h e r sex (5-10 wk old) by intraperitoneal injection of 3 ml of 3% t h i o g l y c o l l a t e m e d i u m (Nissui. Pharmac. Tokyo, Japan). The cells were p l a t e d on 1 5 - m m cover slips in 35-mm culture dishes (Becton D i c k i n s o n NJ, U.S.A. ) a n d c u l t u r e d [ in m e d i u m 199 ( N i s s u i ) containing 10% FBS (Boehringer, Mannheim, Germany) and antibiotics (100~g/ml s t r e p t o m y c i n and i00 u n i t s / m l p e n i c i l l i n G) at 37 °C. The cells were studied 3-20 days after plating. E1 ec trophysiol ogy Cover slips with adherent cells were transferred into a r e c o r d i n g chamber (0.2 ml). The chamber was superfused at a rate of 0.3 m l / m i n w i t h n o r m a l b a t h s o l u t i o n c o n t a i n i n g (in mM); 140 NaCI, 5 KCI, 2.5 CaCI2, 1 MgCI2, i0 HEPES, pH 7.4. Pipettes (0.5-1.5 M~ resistance in 3 M K C I ) w e r e f i l l e d w i t h h i g h K solution c o n t a i n i n g (in mM); 145 K-aspartate, 1 MgCI2, 0.i EGTA, i0 HEPES, pH 7.4. Whole-cell currents were recorded using a List EPC-7 patch-clamp amplifier (Darmstad, Germany) at r o o m t e m p e r a t u r e (20-23 °C). Access resistance to the cell's interior was 10-20 M~. PAF was a p p l i e d by ~ i f f u s i o n from a puff pipette. The puff pipette, filled w i t h i0 -u to 10-±UM PAF, was p l a c e d at 2 0 - 4 0 ~ m from the cell r e c o r d e d from. The l i q u i d j u n c t i o n p o t e n tial b e t w e e n the pipette and the bath solution was +12.6 mV. Materials PAF was purchased from Avanti Polar Lipids, AL, U.S.A.; CV3 9 8 8 f r o m B i o m o l Res. Lab. , P l y m o u t h M e e t i n g , PA, U . S . A . ; c h a r y b d o t o x i n (ChTX) from P e p t i d e Inst., O s a k a Japan; B A P T A - A M and H E P E S from Dojin, Kumamoto, Japan; t e t r a e t h y l a m m o n i u m (TEA) from Wako, Osaka, Japan; EGTA and p e n i c i l l i n G from Sigma, St. Louis, MO, U.S.A.; s t r e p t o m y c i n from Meiji Seika, Tokyo, Japan. Y - 2 4 1 8 0 w a s k i n d l y p r o v i d e d by Y o s h i t o m i P h a r m a c . , F u k u o k a , Japan.
RESULTS
PAF
induced
voltage clamped
an o u t w a r d at -63 mV
(Fig.
observed upon PAF application; 3Bc) Some
IA).
(Io(PAF))
when
Two t y p i c a l
cells
responses
one a single outward current
were were (Fig.
and the o t h e r an o s c i l l a t i n g o u t w a r d c u r r e n t (Fig. 3Ba). c a s e s w e r e a m i x e d t y p e (Fig. IAa), i.e. a l a r g e o u t w a r d
current
f o l l o w e d by an o s c i l l a t i n g
different
responses
(i0 -I0 M)
induced
concentrations cells
current
(319/341
responses
(Fig.
were
induced
current. by PAF
The r e a s o n that two
is not yet
I o ( P A F ) in 6 out of 9 cells.
than
5x10 -I0
cells).
M,
PAF
Repetitive
induced
I o ( P A F ) in
application
IA) and prolonged a p p l i c a t i o n
gradually
application
To c h a r a c t e r i z e examined (Fig.
of C V - 3 9 8 8
(10 -6 M)
373
the IB).
alone
PAF
higher 93%
of
reduced
(30-60 sec) induced
s i n g l e r e s p o n s e w h i c h r e c o v e r e d to b a s e l i n e l e v e l application (12 o u t of 12 c e l l s ) , suggesting desensitization. antagonists were
clear.
At m u c h
d u r i n g the receptor
receptor, specific Unexpectedly, puff
induced
PAF-like
action,
V o l . 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B control
10-8 M PAF
"•k..__ 10'
a
.
.
.
.
.
~
-p
P
10-9 M PAF
CV-3988
5 ' ~
b
j'~ 10'
. . . . . . .
P 10 lo M PAF c --~'---~---
5'
Y-24180 I C ---I 2O0 pA p 20 sec
I 200 pA
~
10S~C
Fiaure i. A. Dose response of outward currents to PAF. a. 10 -8 M PAF was a p p l i e d at 5 min interval 3 times, the first r e s p o n s e o s c i l l a t e d , the s e c o n d w a s a s i n g l e r e s p o n s e and the t h i r d a p p l i c a t i o n f a i l e d to i n d u c e an o u t w a r d c u r r e n t . N u m b e r s on second and third traces show the time after first application. In this and subsequent figures, the duration of PAF application is indicated by the bar with "P" below each record, b. i0 -~ M PAF i n d u c e d o u t w a r d c u r r e n t 5 min i n t e r v a l . N o t e t h a t d e l a y s of responses to PAF were Gradually prolonged in the second and third applications, c. i0 - I ~ M PAF induced smaller amplitude response and second application did not induce any current. 1 ~2 E f f e c t s of antagonists, a. Control outward current by PAF 8 M. b. PAF induced larger outward current during application of the CV-3988 containing solution, c. During perfusion of Y-24180 containing solution, PAF failed to induce an outward current.
i.e., When
inducing oscillating another
antagonist,
agonist-like perfusion
of Y - 2 4 1 8 0
712
(10 -7 M),
( m e a n ± S.E.,
± 130 p A
(n=8).
To
characterize
dependency outward the
of
current
at
hyperpolarized
level
current
(n=10).
Io(PAF),
the reversal
estimated was
to
mainly
By
be
and
did
potential ±
5.3
by
K +.
to
2.5,
external
K+
concentrations from
25
-84.5
same
value
±
as
Io(PAF), 2A).
When
(n=5),
the
PAF
but
in n o r m a l
To
confirm
this,
mM
(Fig.
2.5
to
(n=ll)
to
the
2B).
i0 m M -50.4
increase
is
assuming
that an internal K + concentration
theoretical
374
no
to
the
to
an
-83 mY, more
an
inward of
solution was that
Io(PAF)
external
K + was
An
raised ±
voltage
dependency
indicating
50
had
induced
at
changed
voltage
Io(PAF)
the
clamped
(n=21),
from
to
amplitude
(10 -7 M)
mV
and 3.4
of
the current
of
the
suppressed
to a c o n t r o l
(Fig.
change
-93 mY,
During
no
n=10).
(n=6).
extrapolating
changed potential
i0,
not
of
-82.1
carried
examined -73 m V
applied,
ii c e l l s ) .
CV-3988
mechanisms
was
-53
level
compared
(735 ± 339 pA,
Io(PAF)
current
puff
Io(PAF ) was potently
n=10),
ionic
was
(ii o u t of
On the contrary,
such effect on Io(PAF)
(12 o u t of 23 c e l l s ) .
(10 -6 M),
effect was observed
129 ± 126 p A of
outward current Y-24180
1.5
increase the
(n=8).
difference is 128 m M
in
reversal
of
(13).
35
This mV,
V o l . 182, N o . 1, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
A -53/-73 mV
a
(mV)
_~~
-p
o
~~ -20
/
•/
06
-40 t8
b
-60
3 mV
/-
-80
/'8
-100
c
] 200 pA External K concentration
p
(mM)
10 sec
Figure 21 A. The cells were voltage-clamped from -53 mV to -73 (a), -83 (b) and -93 mV (c) by command pulses (i sec duration, 3 sec interval), a. Current levels at voltage of -53 and -73 mV were outwardly shifted by PAF. b. Current level of -83 mV was not changed after PAF application, but level at -53 mV was greatly outward shifted, c. Current level at -93 mV was inwardly shifted by PAF a p p l i c a t i o n . B. R e v e r s a l p o t e n t i a l s on d i f f e r e n t K + concentrations. Numbers at e a c h p o i n t were numbers of experiments. Standard deviation at 50 mM K + was smaller than the circle.
Next, (TEA,
the
ChTX)
accelerated TEA
Io(PAF)
application C a 2+ 329
pA,
(17
on
n=14)
blocked
of
solution,
the
it
to
compared
ChTX
examined that
solution within
with
the
to
not
any
and
(Fig.
3B).
induced
an
(570
+ 279
min
control
(n=6), sup-
after
its
intracellular In
Ca2+-free
I o ( P A F ) (399
pA,
n=6)
EGTA
PAF d i d n o t after
M)
(n=7) .
markedly
immediately
PAF
Ca 2+,
i0
(10 -2
effect
(10 -6 M)
containing
blockers
TEA
baseline
have
baseline
(I mM),
intracellular
cells)
did
(Kca)
3A).
of e x t r a c e l l u l a r
were than
(Fig.
Io(PAF)
Io(PAF).
EGTA
smaller
17
of
The e f f e c t s
Io(PAF)
K+-channel
Io(PAF)
solution
In C a 2 + - f r e e
chelates
out
on
returning
containing
solution. which
bath
(n=8).
levels
solution
Ca-dependent
recovery
of t h e
partially
pressed
of
examined
the
Application so
effects
were
and
in
amplitude
of
596
activates
the
normal
BAPTA-AM,
induce
exchanging
-+
Io(PAF ) the
bath
+ 227
pA
(n=8).
DISCUSSION
Present K + current of
results~uggest (IK,Ca)
Ca2+-free,
cnntaining
that
PAF
in a m a c r o p h a g e .
EGTA
solution,
I o ( P A F ). P A F p r o d u c e s
solution
and
intracellular inositol
Because
of
the
sensitivity Ca 2+
store
small to
is
1,4,5-trisphosphate
375
Ca-dependent effect
BAPTA-AM
important
for
(3),
may
which
Vol. 182, No. 1, 1 9 9 2
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
A control
b -
B
~
1200 pA a __
b __
_......... = 12oopA
P P
2 0 se--~c
T
ChTX
~ ~
Ca-free, EGTA, BAPTA-AM
(50 pM)
7 rain
C ----J~
P
I100pAC -P
d
--
7 min
--
- .......
~
e
__
P
--
--
p
c 10 sec
F i a u r e 3. A. E f f e c t s of C a 2 + - d e p e n d e n t K + c h a n n e l blockers, a. control response to PAF (10 -8 M). Bar-B shows that a p p l i c a t i o ~ of the b a t h s o l u t i o n did not p r o d u c e any e f f e c t , b. TEA (i0M) accelerated the ~ecovery p h a s e , a p p l i e d at t h e b a r - T , c. C h a r y b d o t o x i n (i0 - u M) markedly suppressed o~tward current, shown in the bar-C. B. E x t e r n a l and i n t e r n a l Ca z+ d e p e n d e n c y of PAF responses, a. Control PAF response, b. Even in Ca-free with EGTA (i mM) s o l u t i o n , PAF i n d u c e d an o u t w a r d c u r r e n t , f o l l o w i n g an i n w a r d shift. B e c a u s e it is i m p o s s i b l e to r e c o r d r e p e t i t i v e and reproducible r e s p o n s e s in s i n g l e c e l l , P A F - i n d u c e d outward c u r r e n t in a and b w e r e r e c o r d e d from cells in the same c u l t u r e dish at least in same cultural condition, c. Control response to PAF. d. PAF response was completely suppressed in Ca2+-free with EGTA (i mM) and BAPTA-AM (50 ~M) solution 7 min after perfusion. e. Same c o n d i t i o n as d but 7 min after i m m e r s i n g the cell into the chamber, c, d and e were recorded from cells in same culture dish.
induce
Ca 2+
release
intracellular quin-2
free
(3,6,7).
intracellular an
Two (KL,Ca) ATP
channels,
and
36
( > 4 0 mV) be
(8).
suggests
by that
by
by
single
KL,Ca
is
K+
efflux
the K + efflux
pS,
of
activated
study.
of
is
by
(9).
(i0).
the
A
same single
in 8 p a t c h e s
of human
depolarized
voltage
at
in
BN52021
240
macrophages
macrophages
IK,Ca
enhances
measured
The
study.
which
of PAF
PAF
376
oscillation
Ca2+-activated
active
is c a u s e d
or
conductance
of voltage,
KCa.
antagonist
to b e
in human
36
application
the
in
fura-2
present
peritoneal
of
increase
using
sometimes
channel
known
is i n d e p e n d e n t
PAF
PAF
the
ionomycin-induced
conductance
the
in
in mouse
The by
reported PAF,
Io(PAF ) remains
having
Because
and Ki,ca
qunidine-sensitive inhibited
of them
(Ki,ca) , are
the
sites. observed
current
(25 pS)
observed
a small
was
conductance
as
was
monocytes may
pS
channel
conductance channel,
none
of oscillating KCa
KCa
storage PAF
concentration
outward
activates
single
by
Although
C a 2+
oscillating
mechanisms
from
C a 2+
observed
macrophages,
(14). IK,Ca.
here
A23187-induced The
and
which
present
is
study
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
When macrophages exhibit
periodic
following
are p l a c e d
increases
cell spreading,
on a g l a s s
surface,
of i n t r a c e l l u l a r
the c e l l s
Ca 2+ c o n c e n t r a t i o n s
w h i c h m a y be r e l a t e d
to p h a g o c y t o s i s
(15). Such o s c i l l a t i o n of Ca 2+ c o n c e n t r a t i o n s are c o n s i s t e n t with the
present
aggregation release
IK,Ca (2),
chemotaxis
(2,4),
related
present
to m a c r o p h a g e
inhibits
the
phagocytosis) suggesting mediators
induced
by (3),
PAF.
spreading
(4),
suggest
that
results
activation.
Quinine
chemi-luminescence and
leukotriene
CV-3988,
a structural
PAF
activates
superoxide
reduces
response
B 4 release
that IK, Ca is r e l a t e d from m a c r o p h a g e s
Because
IK,Ca
anion
might
be
IK,Ca and also (parameter
(mediator
of
of
asthma),
to the r e l e a s e of i n f l a m m a t i o n
(12). analog,
which inhibits
aggregation
of
platelets by PAF (16) and inhibits the specific binding of PAF to platelets
(17),
had an a g o n i s t i c
o t h e r hand, a n o t h e r a n t a g o n i s t related to the a n t i - a n x i e t y drug effect.
Because
allergic
and
an
role
the
target
effect
tissues,
of
the
care
PAF
has
in
been
various
acute
suggested
and
(i), the present study suggests
individual
requiring
of
reactions
various antagonists were reported that
On the
( Y - 2 1 4 8 0 ) w h o s e s t r u c t u r e is (18, 19), etizolam, had no such
important
inflammatory
a c t i o n on m a c r o p h a g e s .
when
antagonist used
may
depend
in t r e a t i n g
on
disorders
causally related to PAF.
This s t u d y was s u p p o r t e d by g r a n t s p r o v i d e d by the I c h i r o K a n e h a r a Foundation, the U e h a r a M e m o r i a l F o u n d a t i o n , the Science and Technology Agency and the M i n i s t r y of Education, Science and Culture of Japan (03454130, 03770072). We thank Drs. D.J. M c A d o o and S . D . C r i t z for c r i t i c a l r e a d i n g of the m a n u s c r i p t , Ms. Y. T a k e d a for t e c h n i c a l a s s i s t a n c e and Dr. Y. T e r a s a w a for the generous gift of Y-24180. ACIfNOWLEDGMENTS:
REFERENCES
i,
Braquet, P., Touqui, L., Shen, T.Y., and Vargaftig, B.B. (1987) Pharmac. Rev. 39, 97-145. 2. Yasaka, T., Boxer, L.A., and Baehner, R.L. (1982) J.Immuno. 128, 1939-1944. 3, Prpic, V., Uhing, R.J., Weiel J.E., Jakoi, L., Gawdi, G., Herman, B., and Adams, D.O. (1988) J.Cell Biol. 107, 363-372. 4, Hartung, H.-P. (1983) FEBS Lett. 160, 209-212. 5, Hayashi, H., Kudo, I., Inoue, K., Onozaki, K., Tsushima, S., Nomura, H., and Nojima, S. (1985) J. Biochem. 97, 1737-1745. 6, Conrad, G.W., and Rink, T.J. (1986) J.Cell Biol. 103,439-450. 7. Randriamampita, C., and Trautmann, A. (1989) FEBS Lett. 249, 199-206. 8. Gallin, E.K. (1989) Am. J. Physiol. 257, C77-C85. 9, Gallin, E.K. (1991) Physiol. Rev. 71, 775-811. i0. Hara, N., Ichinose, M., Imai, K., Sawada, M., and Maeno, T. (1990) FEBS Lett. 267, 281-284. 377
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ii. Hara, N., Ichinose, M., Sawada, M., and Maeno, T. (1990) Comp. Biochem. Physiol. 97A, 417-421. 12, Kakuta, Y., Okayama, H., Aikawa, T., Kanno, T., Ohyama, T., Sasaki, H., Kato, T. and Takishima, T. (1988) J.Allergy Clin. Immunol. 81, 460-468. 13 Ince, C., Thio, B., Duijn, B., Dissel, J.T., Ypey, D.L. and Leijh, P.C.J. (1987) Biochim. Biophys. Acta 905, 195-204. 14 Garay, R., and Braquet, P. (1986) Biochem. Pharmcol. 35, 2811-2915. 15 Kruskal, B.A., and Maxfield, F.R. (1987) J. Cell Biol. 105, 2685-2693.
16 17 18 19
Terashita, Z., Tsushima, S., Yoshioka, Y., Nomura, H., Inada, Y., and Nishikawa, K. (1983) Life Sci. 32, 1975-1982. Terashita, Z, Imura, Y and Nishikawa, K, (1985) Biochem. Pharmacol. 34, 1491-1495. Terasawa, M., Aratani, H., Setoguchi, M., and Tahara, T. (1990) Prostaglangins 40, 553-569. Takehara, S., Mikashima, H., Muramoto, Y., Terasawa, M., Setoguchi, M. and Tahara, T. (1990) Prostaglangins 40, 571-583.
378
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 372-378
Janua~ 15,1992
ACTIVATION OF K + CURRENT IN MACROPHAGES BY PLATELET ACTIVATING FACTOR
M.
Ichinose,
Department
N. Hara,
M. S a w a d a
and T. M a c r o
of P h y s i o l o g y , S h i m a n e M e d i c a l I z u m o 693, J a p a n
University
Received December 2, 1991
P u f f a p p l i c a t i o n of p l a t e l e t a c t i v a t i n g f a c t o r (10 -8 M) o n t o peritoneal macrophages from thioglycollate-stimulated mice i n d u c e d an o u t w a r d c u r r e n t at a h o l d i n g p o t e n t i a l of -63 mY. The current was suppressed by, an a n t a g o n i s t Y - 2 4 1 8 0 b u t n o t b y CV3988. Charybdotoxin (i0 -b M) s u p p r e s s e d the current. Reversal potentials were dependent on e~ternal K + concentrations. The c u r r e n t w a s not s u p p r e s s e d in C a ~ + - f r e e E G T A - c o n t a i n i n g solution but was completely abolished in B A P T A - A M containing solution. T h ~ s e r e s u l t s s u g g e s t that p l a t e l e t a c t i v a t i n g f a c t o r a c t i v a t e s a C a ~ + - d e p e n d e n t K T channel. © 1992AcademicPress, Inc.
Platelet activity
activating
in l e u k o c y t e s
such
as a n a p h y l a x i s
(2),
chemotaxis
(2,4),
and
monocytes to
PAF
which
and
(3),
release
(3),
of e x o g e n o u s
spreading
Ca2+-gated
Cl--channels
ionic
actions
of
valuable
to
present
study
by m e a n s
was
from
intracellular
of w h o l e
have been
methods
cation are
effects
of
in
polyphosstores
and
of
remains well PAF
0006-291X/92 $1.50 372
method.
in m a c r o p h a g e s
are 4
channel.
to a n a l y z e
patch-clamp
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
There
including
in l e u k o c y t e s
undertaken
demonstrated
(8-12).
macrophages
cell
B 2 (4)
(3,6,7).
channels,
the
production macrophages
of Ca 2+
channels
examine
of
release currents
in
responses break-down
ionic
PAF
anion
thromboxane
(5),
and a n o n s e l e c t i v e
of t h o s e
E and
process
aggregation
glucose
by electrophysiological and
including
superoxide
Intracellular
biological
to p a t h o l o g i c a l
(i),
(4),
potent
of
Ca 2+
ionic
possesses
is r e l a t e d
of p r o s t a g l a n d i n
consumption
Several
(PAF)
inflammation
or m a c r o p h a g e s .
are
phoinositides influx
factor
However,
on
3
the r o l e
to be c l a r i f i e d .
studied,
the
voltage-gated
K+-channels,
ionic
it
As
is
very
channels.
The
PAF-induced
current
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS
AND
METHODS
Cells Elicited macrophages were produced from C3H/Heslc mice ( S h i z u o k a Dobutsu, Shizuoka, Japan) of e i t h e r sex (5-10 wk old) by intraperitoneal injection of 3 ml of 3% t h i o g l y c o l l a t e m e d i u m (Nissui. Pharmac. Tokyo, Japan). The cells were p l a t e d on 1 5 - m m cover slips in 35-mm culture dishes (Becton D i c k i n s o n NJ, U.S.A. ) a n d c u l t u r e d [ in m e d i u m 199 ( N i s s u i ) containing 10% FBS (Boehringer, Mannheim, Germany) and antibiotics (100~g/ml s t r e p t o m y c i n and i00 u n i t s / m l p e n i c i l l i n G) at 37 °C. The cells were studied 3-20 days after plating. E1 ec trophysiol ogy Cover slips with adherent cells were transferred into a r e c o r d i n g chamber (0.2 ml). The chamber was superfused at a rate of 0.3 m l / m i n w i t h n o r m a l b a t h s o l u t i o n c o n t a i n i n g (in mM); 140 NaCI, 5 KCI, 2.5 CaCI2, 1 MgCI2, i0 HEPES, pH 7.4. Pipettes (0.5-1.5 M~ resistance in 3 M K C I ) w e r e f i l l e d w i t h h i g h K solution c o n t a i n i n g (in mM); 145 K-aspartate, 1 MgCI2, 0.i EGTA, i0 HEPES, pH 7.4. Whole-cell currents were recorded using a List EPC-7 patch-clamp amplifier (Darmstad, Germany) at r o o m t e m p e r a t u r e (20-23 °C). Access resistance to the cell's interior was 10-20 M~. PAF was a p p l i e d by ~ i f f u s i o n from a puff pipette. The puff pipette, filled w i t h i0 -u to 10-±UM PAF, was p l a c e d at 2 0 - 4 0 ~ m from the cell r e c o r d e d from. The l i q u i d j u n c t i o n p o t e n tial b e t w e e n the pipette and the bath solution was +12.6 mV. Materials PAF was purchased from Avanti Polar Lipids, AL, U.S.A.; CV3 9 8 8 f r o m B i o m o l Res. Lab. , P l y m o u t h M e e t i n g , PA, U . S . A . ; c h a r y b d o t o x i n (ChTX) from P e p t i d e Inst., O s a k a Japan; B A P T A - A M and H E P E S from Dojin, Kumamoto, Japan; t e t r a e t h y l a m m o n i u m (TEA) from Wako, Osaka, Japan; EGTA and p e n i c i l l i n G from Sigma, St. Louis, MO, U.S.A.; s t r e p t o m y c i n from Meiji Seika, Tokyo, Japan. Y - 2 4 1 8 0 w a s k i n d l y p r o v i d e d by Y o s h i t o m i P h a r m a c . , F u k u o k a , Japan.
RESULTS
PAF
induced
voltage clamped
an o u t w a r d at -63 mV
(Fig.
observed upon PAF application; 3Bc) Some
IA).
(Io(PAF))
when
Two t y p i c a l
cells
responses
one a single outward current
were were (Fig.
and the o t h e r an o s c i l l a t i n g o u t w a r d c u r r e n t (Fig. 3Ba). c a s e s w e r e a m i x e d t y p e (Fig. IAa), i.e. a l a r g e o u t w a r d
current
f o l l o w e d by an o s c i l l a t i n g
different
responses
(i0 -I0 M)
induced
concentrations cells
current
(319/341
responses
(Fig.
were
induced
current. by PAF
The r e a s o n that two
is not yet
I o ( P A F ) in 6 out of 9 cells.
than
5x10 -I0
cells).
M,
PAF
Repetitive
induced
I o ( P A F ) in
application
IA) and prolonged a p p l i c a t i o n
gradually
application
To c h a r a c t e r i z e examined (Fig.
of C V - 3 9 8 8
(10 -6 M)
373
the IB).
alone
PAF
higher 93%
of
reduced
(30-60 sec) induced
s i n g l e r e s p o n s e w h i c h r e c o v e r e d to b a s e l i n e l e v e l application (12 o u t of 12 c e l l s ) , suggesting desensitization. antagonists were
clear.
At m u c h
d u r i n g the receptor
receptor, specific Unexpectedly, puff
induced
PAF-like
action,
V o l . 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B control
10-8 M PAF
"•k..__ 10'
a
.
.
.
.
.
~
-p
P
10-9 M PAF
CV-3988
5 ' ~
b
j'~ 10'
. . . . . . .
P 10 lo M PAF c --~'---~---
5'
Y-24180 I C ---I 2O0 pA p 20 sec
I 200 pA
~
10S~C
Fiaure i. A. Dose response of outward currents to PAF. a. 10 -8 M PAF was a p p l i e d at 5 min interval 3 times, the first r e s p o n s e o s c i l l a t e d , the s e c o n d w a s a s i n g l e r e s p o n s e and the t h i r d a p p l i c a t i o n f a i l e d to i n d u c e an o u t w a r d c u r r e n t . N u m b e r s on second and third traces show the time after first application. In this and subsequent figures, the duration of PAF application is indicated by the bar with "P" below each record, b. i0 -~ M PAF i n d u c e d o u t w a r d c u r r e n t 5 min i n t e r v a l . N o t e t h a t d e l a y s of responses to PAF were Gradually prolonged in the second and third applications, c. i0 - I ~ M PAF induced smaller amplitude response and second application did not induce any current. 1 ~2 E f f e c t s of antagonists, a. Control outward current by PAF 8 M. b. PAF induced larger outward current during application of the CV-3988 containing solution, c. During perfusion of Y-24180 containing solution, PAF failed to induce an outward current.
i.e., When
inducing oscillating another
antagonist,
agonist-like perfusion
of Y - 2 4 1 8 0
712
(10 -7 M),
( m e a n ± S.E.,
± 130 p A
(n=8).
To
characterize
dependency outward the
of
current
at
hyperpolarized
level
current
(n=10).
Io(PAF),
the reversal
estimated was
to
mainly
By
be
and
did
potential ±
5.3
by
K +.
to
2.5,
external
K+
concentrations from
25
-84.5
same
value
±
as
Io(PAF), 2A).
When
(n=5),
the
PAF
but
in n o r m a l
To
confirm
this,
mM
(Fig.
2.5
to
(n=ll)
to
the
2B).
i0 m M -50.4
increase
is
assuming
that an internal K + concentration
theoretical
374
no
to
the
to
an
-83 mY, more
an
inward of
solution was that
Io(PAF)
external
K + was
An
raised ±
voltage
dependency
indicating
50
had
induced
at
changed
voltage
Io(PAF)
the
clamped
(n=21),
from
to
amplitude
(10 -7 M)
mV
and 3.4
of
the current
of
the
suppressed
to a c o n t r o l
(Fig.
change
-93 mY,
During
no
n=10).
(n=6).
extrapolating
changed potential
i0,
not
of
-82.1
carried
examined -73 m V
applied,
ii c e l l s ) .
CV-3988
mechanisms
was
-53
level
compared
(735 ± 339 pA,
Io(PAF)
current
puff
Io(PAF ) was potently
n=10),
ionic
was
(ii o u t of
On the contrary,
such effect on Io(PAF)
(12 o u t of 23 c e l l s ) .
(10 -6 M),
effect was observed
129 ± 126 p A of
outward current Y-24180
1.5
increase the
(n=8).
difference is 128 m M
in
reversal
of
(13).
35
This mV,
V o l . 182, N o . 1, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
A -53/-73 mV
a
(mV)
_~~
-p
o
~~ -20
/
•/
06
-40 t8
b
-60
3 mV
/-
-80
/'8
-100
c
] 200 pA External K concentration
p
(mM)
10 sec
Figure 21 A. The cells were voltage-clamped from -53 mV to -73 (a), -83 (b) and -93 mV (c) by command pulses (i sec duration, 3 sec interval), a. Current levels at voltage of -53 and -73 mV were outwardly shifted by PAF. b. Current level of -83 mV was not changed after PAF application, but level at -53 mV was greatly outward shifted, c. Current level at -93 mV was inwardly shifted by PAF a p p l i c a t i o n . B. R e v e r s a l p o t e n t i a l s on d i f f e r e n t K + concentrations. Numbers at e a c h p o i n t were numbers of experiments. Standard deviation at 50 mM K + was smaller than the circle.
Next, (TEA,
the
ChTX)
accelerated TEA
Io(PAF)
application C a 2+ 329
pA,
(17
on
n=14)
blocked
of
solution,
the
it
to
compared
ChTX
examined that
solution within
with
the
to
not
any
and
(Fig.
3B).
induced
an
(570
+ 279
min
control
(n=6), sup-
after
its
intracellular In
Ca2+-free
I o ( P A F ) (399
pA,
n=6)
EGTA
PAF d i d n o t after
M)
(n=7) .
markedly
immediately
PAF
Ca 2+,
i0
(10 -2
effect
(10 -6 M)
containing
blockers
TEA
baseline
have
baseline
(I mM),
intracellular
cells)
did
(Kca)
3A).
of e x t r a c e l l u l a r
were than
(Fig.
Io(PAF)
Io(PAF).
EGTA
smaller
17
of
The e f f e c t s
Io(PAF)
K+-channel
Io(PAF)
solution
In C a 2 + - f r e e
chelates
out
on
returning
containing
solution. which
bath
(n=8).
levels
solution
Ca-dependent
recovery
of t h e
partially
pressed
of
examined
the
Application so
effects
were
and
in
amplitude
of
596
activates
the
normal
BAPTA-AM,
induce
exchanging
-+
Io(PAF ) the
bath
+ 227
pA
(n=8).
DISCUSSION
Present K + current of
results~uggest (IK,Ca)
Ca2+-free,
cnntaining
that
PAF
in a m a c r o p h a g e .
EGTA
solution,
I o ( P A F ). P A F p r o d u c e s
solution
and
intracellular inositol
Because
of
the
sensitivity Ca 2+
store
small to
is
1,4,5-trisphosphate
375
Ca-dependent effect
BAPTA-AM
important
for
(3),
may
which
Vol. 182, No. 1, 1 9 9 2
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
A control
b -
B
~
1200 pA a __
b __
_......... = 12oopA
P P
2 0 se--~c
T
ChTX
~ ~
Ca-free, EGTA, BAPTA-AM
(50 pM)
7 rain
C ----J~
P
I100pAC -P
d
--
7 min
--
- .......
~
e
__
P
--
--
p
c 10 sec
F i a u r e 3. A. E f f e c t s of C a 2 + - d e p e n d e n t K + c h a n n e l blockers, a. control response to PAF (10 -8 M). Bar-B shows that a p p l i c a t i o ~ of the b a t h s o l u t i o n did not p r o d u c e any e f f e c t , b. TEA (i0M) accelerated the ~ecovery p h a s e , a p p l i e d at t h e b a r - T , c. C h a r y b d o t o x i n (i0 - u M) markedly suppressed o~tward current, shown in the bar-C. B. E x t e r n a l and i n t e r n a l Ca z+ d e p e n d e n c y of PAF responses, a. Control PAF response, b. Even in Ca-free with EGTA (i mM) s o l u t i o n , PAF i n d u c e d an o u t w a r d c u r r e n t , f o l l o w i n g an i n w a r d shift. B e c a u s e it is i m p o s s i b l e to r e c o r d r e p e t i t i v e and reproducible r e s p o n s e s in s i n g l e c e l l , P A F - i n d u c e d outward c u r r e n t in a and b w e r e r e c o r d e d from cells in the same c u l t u r e dish at least in same cultural condition, c. Control response to PAF. d. PAF response was completely suppressed in Ca2+-free with EGTA (i mM) and BAPTA-AM (50 ~M) solution 7 min after perfusion. e. Same c o n d i t i o n as d but 7 min after i m m e r s i n g the cell into the chamber, c, d and e were recorded from cells in same culture dish.
induce
Ca 2+
release
intracellular quin-2
free
(3,6,7).
intracellular an
Two (KL,Ca) ATP
channels,
and
36
( > 4 0 mV) be
(8).
suggests
by that
by
by
single
KL,Ca
is
K+
efflux
the K + efflux
pS,
of
activated
study.
of
is
by
(9).
(i0).
the
A
same single
in 8 p a t c h e s
of human
depolarized
voltage
at
in
BN52021
240
macrophages
macrophages
IK,Ca
enhances
measured
The
study.
which
of PAF
PAF
376
oscillation
Ca2+-activated
active
is c a u s e d
or
conductance
of voltage,
KCa.
antagonist
to b e
in human
36
application
the
in
fura-2
present
peritoneal
of
increase
using
sometimes
channel
known
is i n d e p e n d e n t
PAF
PAF
the
ionomycin-induced
conductance
the
in
in mouse
The by
reported PAF,
Io(PAF ) remains
having
Because
and Ki,ca
qunidine-sensitive inhibited
of them
(Ki,ca) , are
the
sites. observed
current
(25 pS)
observed
a small
was
conductance
as
was
monocytes may
pS
channel
conductance channel,
none
of oscillating KCa
KCa
storage PAF
concentration
outward
activates
single
by
Although
C a 2+
oscillating
mechanisms
from
C a 2+
observed
macrophages,
(14). IK,Ca.
here
A23187-induced The
and
which
present
is
study
Vol. 182, No. 1, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
When macrophages exhibit
periodic
following
are p l a c e d
increases
cell spreading,
on a g l a s s
surface,
of i n t r a c e l l u l a r
the c e l l s
Ca 2+ c o n c e n t r a t i o n s
w h i c h m a y be r e l a t e d
to p h a g o c y t o s i s
(15). Such o s c i l l a t i o n of Ca 2+ c o n c e n t r a t i o n s are c o n s i s t e n t with the
present
aggregation release
IK,Ca (2),
chemotaxis
(2,4),
related
present
to m a c r o p h a g e
inhibits
the
phagocytosis) suggesting mediators
induced
by (3),
PAF.
spreading
(4),
suggest
that
results
activation.
Quinine
chemi-luminescence and
leukotriene
CV-3988,
a structural
PAF
activates
superoxide
reduces
response
B 4 release
that IK, Ca is r e l a t e d from m a c r o p h a g e s
Because
IK,Ca
anion
might
be
IK,Ca and also (parameter
(mediator
of
of
asthma),
to the r e l e a s e of i n f l a m m a t i o n
(12). analog,
which inhibits
aggregation
of
platelets by PAF (16) and inhibits the specific binding of PAF to platelets
(17),
had an a g o n i s t i c
o t h e r hand, a n o t h e r a n t a g o n i s t related to the a n t i - a n x i e t y drug effect.
Because
allergic
and
an
role
the
target
effect
tissues,
of
the
care
PAF
has
in
been
various
acute
suggested
and
(i), the present study suggests
individual
requiring
of
reactions
various antagonists were reported that
On the
( Y - 2 1 4 8 0 ) w h o s e s t r u c t u r e is (18, 19), etizolam, had no such
important
inflammatory
a c t i o n on m a c r o p h a g e s .
when
antagonist used
may
depend
in t r e a t i n g
on
disorders
causally related to PAF.
This s t u d y was s u p p o r t e d by g r a n t s p r o v i d e d by the I c h i r o K a n e h a r a Foundation, the U e h a r a M e m o r i a l F o u n d a t i o n , the Science and Technology Agency and the M i n i s t r y of Education, Science and Culture of Japan (03454130, 03770072). We thank Drs. D.J. M c A d o o and S . D . C r i t z for c r i t i c a l r e a d i n g of the m a n u s c r i p t , Ms. Y. T a k e d a for t e c h n i c a l a s s i s t a n c e and Dr. Y. T e r a s a w a for the generous gift of Y-24180. ACIfNOWLEDGMENTS:
REFERENCES
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ii. Hara, N., Ichinose, M., Sawada, M., and Maeno, T. (1990) Comp. Biochem. Physiol. 97A, 417-421. 12, Kakuta, Y., Okayama, H., Aikawa, T., Kanno, T., Ohyama, T., Sasaki, H., Kato, T. and Takishima, T. (1988) J.Allergy Clin. Immunol. 81, 460-468. 13 Ince, C., Thio, B., Duijn, B., Dissel, J.T., Ypey, D.L. and Leijh, P.C.J. (1987) Biochim. Biophys. Acta 905, 195-204. 14 Garay, R., and Braquet, P. (1986) Biochem. Pharmcol. 35, 2811-2915. 15 Kruskal, B.A., and Maxfield, F.R. (1987) J. Cell Biol. 105, 2685-2693.
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Terashita, Z., Tsushima, S., Yoshioka, Y., Nomura, H., Inada, Y., and Nishikawa, K. (1983) Life Sci. 32, 1975-1982. Terashita, Z, Imura, Y and Nishikawa, K, (1985) Biochem. Pharmacol. 34, 1491-1495. Terasawa, M., Aratani, H., Setoguchi, M., and Tahara, T. (1990) Prostaglangins 40, 553-569. Takehara, S., Mikashima, H., Muramoto, Y., Terasawa, M., Setoguchi, M. and Tahara, T. (1990) Prostaglangins 40, 571-583.
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