(Received for publication, April 27, 1995; and in revised form, July 27, 1995)
From the
Superoxide anion and arachidonic acid were produced in guinea
pig neutrophils in response to a chemotactic peptide
formyl-methionyl-leucyl-phenylalanine (fMLP). Both responses were
markedly, but the former response to a phorbol ester was not at all,
inhibited when the cellular cAMP level was raised by prostaglandin
E combined with a cAMP phosphodiesterase inhibitor.
Increasing cAMP was also inhibitory to fMLP-induced activation of
phosphatidylinositol (PI) 3-kinase and Ca
influx
without any effect on the cation mobilization from intracellular
stores. The fMLP-induced respiratory burst was abolished when PI
3-kinase was inhibited by wortmannin or LY294002, but was not affected
when Ca
influx was inhibited. On the contrary, fMLP
released arachidonic acid from the cells treated with the PI 3-kinase
inhibitors as well as from nontreated cells, but it did not so when
cellular Ca
uptake was prevented. The chemotactic
peptide activated PI 3-kinase even in cells in which the
receptor-mediated intracellular Ca
mobilization and
respiratory burst were both abolished by exposure of the cells to a
permeable Ca
-chelating agent. Thus, stimulation of
fMLP receptors gave rise to dual effects, activation of PI 3-kinase and
intracellular Ca
mobilization; both effects were
necessary for the fMLP-induced respiratory burst. Increasing cellular
cAMP inhibited the respiratory burst and arachidonic acid release as a
result of the inhibitions of PI 3-kinase and Ca
influx, respectively, in fMLP-treated neutrophils.
The intracellular signaling pathways responsible for the
formyl-methionyl-leucyl-phenylalanine (fMLP)-induced
respiratory burst in phagocytes remain to be fully understood as yet,
although the activation mechanism of the respiratory burst oxidase is
currently elucidated as an assembly of a number of membrane-bound and
cytosolic components, including gp91
,
p22
, p47
,
p67
, and Rac (see (1) for review).
In general, one of the best strategies for identification of essential cellular signals involved in a cell response to a receptor stimulation is to search for the target with which an inhibitor interacts to abolish the response efficiently. Pertussis toxin was a good inhibitor and was successfully applied to the fMLP-induced respiratory burst. Prior exposure of guinea pig neutrophils to low concentrations of pertussis toxin for several hours prevented the cells from producing superoxide anion in response to the subsequent addition of fMLP(2) . The prevention resulted from the toxin-induced ADP-ribosylation of G protein (Gi-2), the chemical modification by which the modified G protein is uncoupled from receptors. Evidence has been thus provided for involvement of the G protein in the fMLP-induced respiratory burst via phospholipase C activation(2, 3, 4) .
An additional
example of useful inhibitors of the phagocytic respiratory burst is
wortmannin, a fungal metabolite with hydrophobic sterol structure.
Baggiolini and his colleagues (5, 6) first reported
that incubation of human neutrophils with wortmannin or
17-hydroxywortmannin for 5 min inhibited fMLP-induced
O generation and degranulation without
affecting the chemoattractant-induced Ca
mobilization. The inhibition induced by wortmannin was so
selective that it did not inhibit similar responses of neutrophils to a
phorbol ester, a direct inhibitor of protein kinase C. Wortmannin has
lately been proven to abolish the cellular signaling as a result of its
direct interaction with phosphatidylinositol (PI) 3-kinase, providing
convincing evidence for essential roles of this enzyme or the products
of the enzyme in signaling pathways connecting G protein-coupled
receptors and the respiratory burst(7, 8) .
The
purpose of the present paper is further application of this useful
strategy to identification of important signaling pathways responsible
for the fMLP-induced respiratory burst in guinea pig neutrophils.
Increases in cAMP within cells have long been known to inhibit
receptor-mediated respiratory bursts in reticulocytes and macrophages (e.g.(9) and (10) and references cited
therein). We have identified intracellular signals that were impaired
in parallel with the inhibition of fMLP-induced
O generation upon the addition of
cAMP-increasing agents to the cells.
Figure 1:
Inhibition of fMLP-induced
O generation by cAMP-generating
agents. Freshly prepared (noncultured) guinea pig neutrophils were
incubated with cAMP-increasing agents such as 10 µM PGE
(
), 1 mM Bt
cAMP
(
), 0.3 mM IBMX (
), 10 µM PGE
plus 0.3 mM IBMX (
) or vehicle alone
(
) for 10 min, before the subsequent 15-min incubation with
increasing concentrations of fMLP (A) or PMA (B) and
were then submitted for analyses of O
generation as described under ``Experimental
Procedures.'' Essentially the same results were obtained in three
similarly designed experiments.
The respiratory
burst induced by fMLP was likewise inhibited by wortmannin (Fig. 2A) and staurosporine (Fig. 2B)
in concentration-dependent manners. LY294002, a wortmannin-like
inhibitor of PI 3-kinase(14, 15) , gave rise to
effects, just like wortmannin(8) , very similar to those caused
by cAMP-increasing agents; fMLP-induced O generation was, but the PMA-induced generation was not at all,
inhibited by the inhibitor (Fig. 2C). It is conceivable
that the increase in cAMP may interact with the step that functions,
like PI 3-kinase, upstream of protein kinase C in G protein-initiated
signaling pathways. Bisindolylmaleimide, an inhibitor of protein kinase
C much more specific than staurosporine(16) , inhibited, in
concentration-dependent manners, the fMLP-induced respiratory burst as
well as PMA-induced one (Fig. 2D). Thus, activation of
protein kinase C, which was not susceptible to cAMP increases within
cells or PI 3-kinase inhibitors such as wortmannin and LY294002, was
essential for the G protein-initiated respiratory burst. In fact,
staurosporine did, but wortmannin did not, inhibit PMA-induced
O
generation at all the concentrations of
the phorbol ester employed (Fig. 2E).
Figure 2:
Effect of inhibitors on fMLP-induced
O generation in neutrophils. Fresh guinea
pig neutrophils were first exposed for 10 min to increasing
concentrations of wortmannin (A), staurosporine (B),
LY294002 (C), or bisindolylmaleimide (D) before
further 15-min incubation with 0.1 µM fMLP (
), 0.1
µM PMA (
) or without addition (
). In E, the first exposure was done to 0.1 µM wortmannin (
), 0.1 µM staurosporine (
)
or vehicle (
), and the further incubation was with increasing
concentrations of PMA. These cells were then assayed for
O
generation as described under
``Experimental Procedures.'' The same results were reproduced
in additional two or three experiments.
Figure 3:
Effects of cAMP-increasing agents and
inhibitors on fMLP-induced
[Ca]
increases. Guinea
pig neutrophils were cultured for 4 h to be loaded with Fura2 as
described under ``Experimental Procedures.'' In C and D, the culture medium was supplemented with 100 ng/ml
of pertussis toxin. The loaded cells were incubated with 10 µM PGE
plus 0.3 mM IBMX (E and F), 1 µM wortmannin (G and H),
1 µM staurosporine (I and J), or vehicle (A, B, C, and D) for 10 min before being submitted to
fluorescence analysis. The incubation medium used was the regular
(Ca
-containing) Krebs-Ringer-Hepes medium (A, C,
E, G, and I) or the nominally Ca
-free
medium (B, D, F, H, and J). The addition of 0.1
µM fMLP was shown by the first arrowhead in each
panel. In B, D, F, H, and J, CaCl
was
added as shown by the second arrowhead to make the final
concentration of 2.5 mM. These tracings are representative of
three experiments with similar results.
Figure 4:
Failure of the cAMP-increasing agent to
inhibit thapsigargin-induced Ca influx. Fura2-loaded
neutrophils, after first 10-min exposure to vehicle (A) or 10
µM PGE
plus 0.3 mM IBMX (B), were submitted to Ca
fluorescence
analysis in the Ca
-free medium as described for Fig. 3. In each panel, the first and the second
arrowheads show the addition of thapsigargin (10 µM)
and Ca
(2.5 mM), respectively. Dotted
lines show [Ca
]
observed without addition of thapsigargin (with the later
Ca
addition only). Similar results were obtained in
three separate experiments.
When fMLP receptors were uncoupled from G
proteins by prior exposure of cells to pertussis toxin, the agonist
failed to increase [Ca]
significantly in either the presence (Fig. 3C) or
the absence (Fig. 3D) of extracellular
Ca
. No significant
[Ca
]
increase occurred upon
further addition of Ca
into the
Ca
-free medium bathing pertussis toxin- and
fMLP-treated neutrophils, providing evidence for involvement of the
toxin-sensitive G protein in the Ca
influx (Fig. 3D). In the cells pre-exposed to the
cAMP-increasing agents (PGE
plus IBMX), the peak
value of fMLP-induced [Ca
]
increase was slightly smaller than the non-exposed cells and was
followed by the return to the level much lower than that observed for
control (Fig. 3E as compared with 3A),
suggesting that receptor-coupled Ca
influx was
inhibited upon increases in the cellular cAMP concentration. In fact,
addition of fMLP into Ca
-free medium caused the same
increase in [Ca
]
in the cells
treated with cAMP-increasing agents as in control cells, whereas the
subsequent addition of Ca
elicited much smaller
increase in [Ca
]
in the former
cells than in the latter cells (Fig. 3F). Thus,
elevation of intracellular cAMP attenuated fMLP receptor-operated
Ca
influx without affecting the receptor-mediated
mobilization of Ca
from the internal stores. There
was only 20-30% decrease in fMLP-induced generation of inositol
1,4,5-trisphosphate, when guinea pig neutrophils had been exposed to
the cAMP-generating agents (data not shown). Conceivably, such slightly
attenuated generation of inositol 1,4,5-trisphosphate is still enough
for the maximal mobilization of Ca
from the stores.
The inhibition of receptor-operated Ca influx was
very unique to the action of cAMP-generating agents, in the sense that
other inhibitors of fMLP-induced superoxide anion release, such as
wortmannin and staurosporine, did not inhibit but rather enhanced the
receptor-coupled Ca
influx (Fig. 3, G-J). Staurosporine may enhance Ca
entry by antagonizing protein kinase C-induced phosphorylation of
proteins that is possibly involved in inhibition of capacitative
Ca
influx in this cell type(17) , although
the mechanism for wortmannin-induced enhancement (Fig. 3H) remains to be clarified.
The addition of
Ca following thapsigargin into neutrophil suspensions
in the Ca
-free medium gave rise to a marked increase
in [Ca
]
, despite lack of the
increase without preaddition of the endoplasmic Ca
pump inhibitor, due to the capacitated Ca
entry
following emptying of the intracellular stores but not mediated by
receptor stimulation (Fig. 4). This capacitated Ca
entry was not affected significantly, or inhibited only slightly,
by exposure of cells to cAMP-generating agents (Fig. 4).
Emptying the Ca
stores after the thapsigargin
treatment was evidenced by the failure of the treated cells to respond
to fMLP by increasing [Ca
]
(data not shown). Thus, the major target of increased cAMP or
cAMP-dependent protein kinase appears to be the protein(s) involved in
receptor-operated, rather than store-operated, Ca
channels.
Figure 5:
fMLP-induced O generation and its susceptibility to cAMP-increasing agents in
the presence or absence of extracellular Ca
. Guinea
pig neutrophils were first exposed for 10 min to 0.3 mM IBMX,
10 µM PGE
plus 0.3 mM IBMX,
1 mM Bt
cAMP or vehicle (none), as described in the top panel, and then incubated for 15 min with 0.1 µM fMLP, before being submitted to O
assay as described under ``Experimental Procedures.''
The incubations were performed in the regular
(Ca
-containing) Krebs-Ringer-Hepes medium (A) or in the Ca
-omitted medium (B). Similar results were obtained in two separate
experiments.
Figure 6:
Arachidonic acid release from neutrophils
in the presence of cAMP-increasing agents or inhibitors. The
[H]arachidonate-labeled cells were first exposed
for 10 min, as described in the left-hand panel, to 0.1
µM wortmannin, 10 µM PGE
plus 0.3 mM IBMX, 5 mM NiCl
, 5
mM CoCl
, 0.1 µM staurosporine, or
vehicle (control) and further incubated for 15 min with 0.1 µM fMLP, 10 µM A23187, or vehicle (None), as
shown at the bottom of columns, before being finally submitted
to the arachidonic acid release assay according to the procedure
described under ``Experimental Procedures.'' The incubations
were conducted in the regular Krebs-Ringer-Hepes medium (A) or
in the Ca
-omitted medium (B). The data are
representative ones from experiments performed more than three times
with similar results.
Again, wortmannin and
cAMP-increasing agents exerted different effects on the response of
neutrophils. The fungal metabolite did not inhibit arachidonic acid
release under any condition tested, whereas PGEplus IBMX was an inhibitor of the fMLP-induced release as strong as
Ni
or Co
, antagonists of
transmembrane Ca
inflow (Fig. 6A).
Ni
has been reported to be an inhibitor of
receptor-operated Ca
entry(18) .
Staurosporine, like wortmannin, did not inhibit fMLP-induced
arachidonic acid release. Failure of cAMP-increasing agents to inhibit
Ca
ionophore-induced arachidonate production (Fig. 6A) is in agreement with the view that elevation
of intracellular cAMP inhibits the fatty acid production as a result of
the inhibition of receptor-coupled Ca
entry.
Figure 7:
fMLP-induced PIP production
and O
generation in neutrophils and its
progressive inhibition by increasing concentrations of PGE
in the presence of IBMX. A,
P-labeled
neutrophils were first incubated with increasing concentrations of
PGE
for 10 min in the presence (+) or absence(-)
of 0.3 mM IBMX and then incubated for 15 s with 0.1 µM fMLP or vehicle (None), as shown at the bottom of the panel, before being submitted to thin-layer chromatographic
separation of phospholipids as described under ``Experimental
Procedures.'' The incubations were done in the regular
Krebs-Ringer-Hepes medium and the autoradiogram of the thin-layer plate
is shown, in which the position of spots corresponding to PIP
is indicated on the left. B, fresh neutrophils
were first incubated with increasing concentrations of PGE
in the presence of 0.3 mM IBMX and then stimulated by
0.1 µM fMLP (
) or not (
) under the same
conditions as used for A, except for the stimulation time of
15 min instead of 15 s. Superoxide anion production was determined, as
described under ``Experimental Procedures,'' and plotted as a
function of PGE
concentrations. The cellular content of
cAMP after the 15-min incubation is also shown (
). The results
were reproduced in two or three separate
experiments.
Figure 8:
Differential effects of two respiratory
burst inhibitors, an intracellular Ca-chelating agent
and a cAMP-increasing agent, on fMLP-induced PI 3-kinase activation.
Guinea pig neutrophils, Fura2-loaded (A and B),
P-labeled (C), or nontreated (D), had
been exposed for 15 min to 0.3 mM IBMX plus 10
µM PGE
or 20 µM BAPTA/AM as
described in these panels before further incubation with or without 0.1
µM fMLP in the Ca
-containing regular
medium. Tracings of
[Ca
]
, with the
addition of fMLP as indicated by arrows, are shown in A and B. The incorporation of
P into the
PIP
spots on the thin-layer plate during 15-s incubation
with (hatched columns) or without (open columns) fMLP
was determined as in Fig. 7and is illustrated in C.
O
generation during 15-min incubation
with or without fMLP is likewise shown in D. Typical data are
taken from experiments repeated twice with essentially the same
results.
It is very likely, therefore, that fMLP receptor
stimulation triggered two signaling pathways each, with different
susceptibility to certain inhibitors, separately leading to
superoxide-generating and arachidonate-producing responses of
neutrophils. fMLP is one of the chemokine families whose receptors
possessing seven membrane-spanning regions release, upon being occupied
by the ligand, the -subunits from coupled pertussis
toxin-sensitive G proteins. The signaling pathways may bifurcate at a
point distal to the G protein, as evidenced by the fact that both
responses are totally inhibited by treatment of the cells with
pertussis toxin.
Thus,
it is very reasonable to assume that cAMP-increasing agents inhibit
fMLP-induced arachidonic acid release as a result of their unique
action to suppress Ca influx following the
mobilization of Ca
from internal stores (Fig. 3F), in accordance with previous
reports(9, 10) . It is tempting to speculate that
receptor-operated Ca
channel is one of the targets of
cAMP-dependent protein kinase, since cAMP-increasing agents were
incapable of inhibiting thapsigargin-induced Ca
influx (Fig. 4) as well as Ca
ionophore-induced arachidonic acid release (Fig. 6A). Cyclic AMP has recently been reported to
inhibit thrombin receptor-operated Ca
influx into
platelets directly (25) or indirectly(26) .
Thus, a
readily acceptable possibility is that internal Ca mobilization primes, permits, or supports the signaling pathways
responsible for O
generation. In other
words, the Ca
mobilization and the tyrosine
kinase-related signalings including activation of PI 3-kinase are both
indispensable for fMLP to provoke O
generation. The small amount of Ca
mobilized in
the Ca
-free medium (e.g.Fig. 3B) would be enough to support the
respiratory burst.