(Received for publication, May 22, 1995; and in revised form, July 14, 1995)
From the
The role of nitric oxide (NO) in the phosphatidylinositol
4,5-bisphosphate (PIP) hydrolysis and intracellular
Ca
release responses induced by epidermal,
platelet-derived, and fibroblast growth factors was investigated in
three cell lines, a clone of NIH-3T3 fibroblasts overexpressing
epidermal growth factor receptors and the tumoral epithelial cells A431
and KB. In all three cell types, pretreatment with NO donors decreased
growth factor-induced PIP
and Ca
responses, whereas pretreatment with NO synthase inhibitors
increased them. The Ca
-dependent PIP
hydroysis induced by micromolar concentrations of the
Ca
ionophore, ionomycin, was also modulated
negatively and positively by NO donors and synthase inhibitors,
respectively. In contrast, the Ca
content of the
intracellular stores was unaffected by the various pretreatments
employed. NO donors and synthase inhibitors induced an increase and
decrease, respectively, of the intracellular cGMP formation in all
three cell lines investigated. All of the effects of the NO donors were
mimicked by 8-bromo-cGMP administration and abolished by pretreatment
with the specific blocker of the cGMP-dependent protein kinase I,
KT5823, which by itself mimicked the effects of the synthase
inhibitors. Together with previous observations on G protein-coupled
receptors, the present results demonstrate that PIP
hydrolysis and Ca
release occur under the
feedback control of NO, independently of the phospholipase C (
,
, or
type) involved and of the mechanism of activation. Such
a control, which appears to be effected by the cGMP-dependent protein
kinase I acting at the level of the phospholipases C themselves, might
ultimately contribute to the inhibitory role of NO on growth previously
observed with various cell types.
Individual molecules of the signal transduction cascades turned
on by receptor agonist binding can play important roles not only in the
intracellular activation process but also in the fine feedback
regulation of signaling itself. In this respect particular attention
has been devoted to nitric oxide (NO). ()In the cells
competent for the Ca
-dependent, constitutive forms of
NO synthases (NOSs), this highly reactive radical gas, generated in
response to appropriate increases of the cytosolic Ca
concentration
([Ca
]
), works as the
controller of a number of enzymes including guanylyl cyclase (1) . The ensuing increase of cGMP formation, with activation
of cGMP-dependent protein kinase I (G kinase), yields responses that
may be variable from cell to cell(2, 3) . In the case
of receptors coupled to phosphatidylinositol 4,5-bisphosphate
(PIP
) hydrolysis via the activation of heterotrimeric G
proteins of the Gq family(4) , negative modulations by NO have
been described, with decreased generation of the two second messengers,
inositol 1,4,5-trisphosphate (IP
) and diacylglycerol, and
ensuing blunting of Ca
release from intracellular
stores (3) . With these receptors the modulation was shown to
depend upon G kinase activation, and the site of action was proposed at
the G protein/phospholipase C (PLC) interface(5) .
PIP hydrolysis is induced not only by G protein-coupled receptors but
also by growth factor receptors, working, however, on different PLCs
and by a different activation process, i.e. by direct tyrosine
phosphorylation of the PLCs of the
family rather than by G
protein activation of those of the
family(4, 6) . Because of these distinct molecular and
functional differences, we thought it worth investigating whether NO
and cGMP had any effect on the growth factor-induced PIP
hydrolysis and [Ca
]
responses.
The results reported here indicate in three
types of cells that the above responses induced by growth factors are
indeed inhibited by NO working through the cGMP/G kinase I pathway.
Similar inhibition by NO was observed also when the same responses were
induced by persistent [Ca]
increases triggered by the Ca
ionophore,
ionomycin. Under the latter condition activation is not restricted to a
single family of PLCs but affects them all:
,
, and
families altogether(7, 8, 9, 10) .
We conclude therefore that the G kinase-sustained negative modulation
is a [Ca
]
and
NO-induced feedback regulation process occurring most probably at the
level of PLC.
All the results shown are from experiments in which L-NIO was used as a NOS inhibitor, and SNP as a NO donor.
Qualitatively similar results were obtained using the NOS inhibitor, N-nitro-L-arginine methylester, and
the NO donor, S-nitroso-N-acetylpenicillamine. With
the less active enantiomer of N
-nitro-L-arginine methylester, N
-nitro-D-arginine methylester, the
results did not differ significantly from those obtained with
untreated, control cells.
Figure 1:
Effects of L-NIO, SNP, 8-Br-cGMP, and 8-Br-cAMP on EGF-evoked
Ca release. Fura-2-loaded EGFR-T17 (A), A431 (B), and KB (C) cell suspensions were incubated for
15 min at 37 °C in KRH medium, alone (control) or supplemented with L-NIO (200 µM), SNP (30 µM),
8-Br-cGMP (200 µM), or 8-Br-cAMP (200 µM).
Cell aliquots were then challenged in Ca
-free KRH
medium with increasing concentrations of EGF. Values are expressed as
percent peak increase over basal, resting
[Ca
]
levels. Basal
[Ca
]
values were, on
average, 141 ± 15, 112 ± 28, and 126 ± 16 nM for EGFR-T17, A431, and KB cell lines, respectively, and were not
modified by the various pretreatments. The graphs show the results of
8-10 experiments (mean values ±
S.D.).
EGFR-T17 cells are known to exhibit
[Ca]
responses not only to EGF
but to other growth factors, i.e. PDGF and FGF(19) .
The effects of L-NIO, SNP, 8-Br-cGMP, and 8-Br-cAMP on
Ca
release induced by the latter agonists were
therefore investigated, with results qualitatively similar to those
induced by EGF. Ca
release elicited by PDGF and FGF
was potentiated when cells were preincubated with L-NIO,
inhibited after treatment with SNP or 8-Br-cGMP, and almost unaffected
by 8-Br-cAMP (Fig. 2, A and B). The effects of
NO-modulating drugs on Ca
release induced by growth
factors were then compared to those exerted on the responses elicited
by activation of a G protein-coupled receptor. Fig. 2C shows the results obtained with UTP, a receptor agonist specific
for the purinergic P
receptor(20) . As already
demonstrated in PC12 cells (15) , preincubation with L-NIO increased, that with SNP or 8-Br-cGMP decreased, the
UTP-induced Ca
release to extents similar to those
observed with growth factor-induced
[Ca
]
responses.
Figure 2:
Effects of L-NIO, SNP, 8-Br-cGMP,
and 8-Br-cAMP on agonist-evoked Ca release.
Fura-2-loaded EGFR-T17 cell suspensions were incubated for 15 min at 37
°C in KRH medium, alone (control) or supplemented with L-NIO (200 µM), SNP (30 µM),
8-Br-cGMP (200 µM), or 8-Br-cAMP (200 µM).
Cell aliquots were then challenged in Ca
-free KRH
medium with increasing concentrations of either PDGF (A), FGF (B), or UTP (C). Values are expressed as percent peak
increase over basal, resting [Ca
]
levels. The graphs show the results of 8-10
experiments (mean values ± S.D.).
Figure 3:
Effects of L-NIO and SNP on the
release of Ca from the various intracellular
Ca
pools. A, EGFR-T17 cells were loaded to
equilibrium with
Ca
, then incubated for
15 min at 37 °C in KRH medium alone (control) or supplemented with L-NIO (200 µM) or SNP (30 µM). After
addition of excess EGTA (Ca
-free medium), basal
Ca
leak was analyzed before challenging
the cells by sequential addition of EGF (100 nM), thapsigargin (Tg) (100 nM), and ionomycin (Iono) (1
µM) where indicated. Results illustrated are the averages
of four experiments ± S.D. B, fura-2-loaded EGFR-T17
cells, preincubated for 15 min with KRH medium, alone or supplemented
with L-NIO or SNP as in Fig. 1, were challenged in
Ca
-free KRH medium with increasing concentrations of
thapsigargin. The graph shows the mean percent
[Ca
]
increases over
basal (± S.D.) in eight experiments.
Figure 4:
Effects of L-NIO, SNP, and
8-Br-cGMP on total IP accumulation and IP generation.
EGFR-T17 cells, labeled with 3 µCi/ml myo-[2-
H]inositol, were incubated for 15
min at 37 °C in KRH medium, alone (control) or supplemented with L-NIO (200 µM), SNP (30 µM), or
8-Br-cGMP (200 µM) as described under ``Experimental
Procedures.'' A, total IP accumulation measured in cells
challenged with increasing concentrations of EGF for 15 min in the
presence of 20 mM LiCl. B, IP
generation
induced by 30 nM EGF, followed up to 120 s after EGF addition. C, total IP accumulation measured in cells challenged with the
indicated concentrations of ionomycin (Iono). The average
basal radioactivity of total IPs and IP
, here and in the
experiments of Fig. 5and Fig. 6, were 6.1 ± 2.3
10
and 1.3 ± 0.9
10
cpm/mg of protein, respectively. No appreciable difference in
basal radioactivity was observed between cell preparations treated with L-NIO, SNP, or 8-Br-cGMP. Results are expressed as percent
increase of radioactivity over basal. Graphs show the results obtained
in six independent experiments (averages ±
S.D.).
Figure 5:
Effects of the G kinase inhibitor KT5823
on SNP-induced variations in
[Ca]
and
total IP production elicited by EGF and ionomycin (Iono). A, fura-2-loaded EGFR-T17 cells, preincubated for 15 min at 37
°C with KRH medium, alone or supplemented with SNP (30
µM) or SNP plus KT5823 (10 µM), were
challenged in Ca
-free KRH medium with increasing
concentrations of EGF. The graph shows the mean percent increases in
peak [Ca
]
over basal
± S.D. in nine experiments. B and C, EGFR-T17
cells, labeled with 3 µCi/ml myo-[2-
H]inositol, were incubated for 15
min at 37 °C in KRH medium, alone or supplemented with SNP or SNP
plus KT5823 as above, then challenged with increasing concentrations of
EGF or the indicated concentrations of ionomycin. Results shown are the
mean percent increases of radioactivity over basal ± S.D. in six
experiments. Experimental details as in Fig. 4.
Figure 6:
Effects of the G kinase inhibitor KT5823
on [Ca]
variations and
total IP production elicited by EGF and ionomycin (Iono) in
the presence or absence of L-NIO. A, fura-2-loaded
EGFR-T17 cells, preincubated for 15 min at 37 °C with KRH medium,
alone or supplemented with L-NIO (200 µM), KT5823
(10 µM), or a combination of the two, were challenged in
Ca
-free KRH medium with increasing concentration of
EGF. The graph shows the mean percent increases in peak
[Ca
]
over basal
± S.D. in four experiments. B and C, EGFR-T17
cells, labeled with 3 µCi/ml myo-[2-
H]inositol, were incubated for 15
min at 37 °C with KRH medium, alone or supplemented with L-NIO, KT5823, or both as above, then challenged with
increasing concentrations of EGF or the indicated concentrations of
ionomycin. Results shown are the mean percent increases of
radioactivity over basal ± S.D. in four experiments.
Experimental details as in Fig. 4.
In further experiments SNP preincubation of
EGFR-T17 cell suspensions was carried out with or without KT5823, a
widely employed inhibitor of the G kinase I
activity(15, 24, 25) . Fig. 5A shows that in the presence of KT5823 (10 µM) the
inhibitory effect of SNP on EGF-induced Ca release
was almost completely abolished. Similarly, SNP inhibition of total IP
accumulation triggered by either EGF or ionomycin was largely prevented
by the kinase blocker (Fig. 5, B and C).
KT5823 was also administered alone or in combination with L-NIO. Fig. 6shows the results obtained in EGFR-T17
cells challenged with EGF (A and B) or ionomycin (C). The effects of the kinase blocker on
[Ca
]
variations and total IP
accumulation induced by either stimulant resembled those induced by L-NIO; when KT5823 and L-NIO were administered
together, no additive effect was measured. Similar results were found
when the effects of KT5823 were investigated in A431 and KB cells (not
shown).
The results reported here demonstrate that NO has a role in
the chain of intracellular events elicited by activation of growth
factor receptors. While the Ca storage machinery is
unaffected by cell treatment with NO, the gaseous messenger is shown to
modulate negatively PIP
hydrolysis and the ensuing
generation of IP
. An important consequence is the reduction
of the growth factor-induced release of Ca
from the
intracellular stores. Although obtained not by direct application of NO
but by a pharmacological approach, our results appear unambiguous
because of the contrast between the effects of NO donors, which induce
release of the gas within the cells, with those of NOS blockers, which
preclude the synthesis of endogenous NO. The fact that NO-induced
negative signal modulations were observed in all three cell types
investigated, NIH-3T3, A431, and KB lines, strongly suggests that they
have a general significance. Moreover, their appearance with all the
growth factors we have employed, i.e. EGF, PDGF, and FGF,
suggests these effects to be generated at the level of the common
signal cascade activated after receptor binding rather than at a level
of receptors themselves. Indeed, it has been recently reported that EGF
binding to its receptor is unaffected by NO(26) .
Until now,
negative effects of NO on PIP hydrolysis and Ca
release had been reported only with receptors coupled to PLC via
heterotrimeric G proteins. In the latter case, the site of NO action
was proposed to occur at the G protein/PLC interface(5) . The
PLCs activated by G proteins, however, are a family of isoenzymes
(defined as PLC
) molecularly and functionally different from those
activated by growth factors, the PLC
, which are activated at the
receptor level by direct tyrosine
phosphorylation(4, 6) . Taken together, the present
and previous results indicate therefore that inhibition by NO is a
widespread regulatory process that involves many (possibly all) types
of transductive PLCs. In fact, also PIP
hydrolysis induced
by administration of a Ca
ionophore, ionomycin, a
mechanism effective with all types of the enzyme known so far (see
Refs. 7, 8, 9, and 10, for PLC
,
PLC
, PLC
, and PLC
,
respectively), was inhibited by NO. The inhibition by NO of PLC
activity appears to be mediated by accumulation of cGMP and activation
of G kinase I. Whether this kinase phosphorylates PLC(s) directly, or
whether its effect is mediated through the phosphorylation of
regulatory, yet unidentified protein(s), remains to be established.
Also to be elucidated is the mechanism of inhibition of the PLC
activity we have now observed. Various possibilities are open:
decreased complex formation of the enzyme with growth factor receptors,
of its degree of tyrosine phosphorylation, or of its activation level.
Until now the results of preliminary experiments failed to reveal clear
evidence supporting either one of the first two mechanisms (not shown).
Under unstimulated conditions the role of cGMP in the control of
PIP hydrolysis and [Ca
]
does not appear important, inasmuch as the decrease of the
nucleotide level following preincubation with a NOS blocker was not
accompanied by any appreciable changes of basal IPs and
[Ca
]
values. Only after
stimulation with growth factors (and also with UTP and ionomycin) did
the inhibition by NO become clear. NO appears therefore to work as a
feedback controller. In NOS-competent cells, any increase in
[Ca
]
is in fact expected to
activate the enzyme. The NO thus produced would then modulate
negatively all PLCs, via cGMP and the cognate kinase, thus contributing
to the control of intracellular Ca
homeostasis.
NO- and cGMP-induced inhibitory modulation of growth factor receptor
function may account for a number of cell growth effects that up to now
had remained without an adequate explanation. Inhibition of mitogenesis
by the gas and G kinase have been reported in various cell systems
including vascular smooth muscle(3) , rat
hepatocytes(27) , and retinal pigmented epithelial
cells(28) . Moreover, proliferation and development of bone
marrow were reported to be inhibited(29) , and neuronal and
muscular differentiation to be stimulated by
NO(30, 31, 32) . In the array of
intracellular signals elicited by growth factor receptor activation,
PIP hydrolysis and [Ca
]
responses are now recognized to promote mitogenesis and
differentiation in various cell systems(33, 34) ,
while impaired Ca
homeostasis or altered
Ca
release exert an inhibitory effect on growth (see e.g. Refs. 20, 35, and 36). The possibility can therefore be
considered that the effects of NO and cGMP on cell growth are mediated,
at least in part, by their negative modulatory actions here described.
Whether these actions are accompanied by others as yet still unknown,
also induced by NO and cGMP, remains to be investigated.
In
conclusion, our results expand the importance of the NO-cGMP-mediated
modulation in transmembrane signaling, demonstrating that it covers the
entire PIP hydrolysis field, independent of the PLC isoform
families involved and the mechanisms of their activation. In addition
to the inhibition of cell growth discussed above, an important role of
the NO modulation of PIP
hydrolysis could be in the control
of Ca
homeostasis, with prevention of excess
Ca
release. This possibility is supported by the
recently recognized, NO-induced positive modulation of the surface
Ca
channels of the type activated by intracellular
Ca
release(13, 14) , which are
believed to be responsible for the replenishment of discharged
Ca
stores.