(Received for publication, February 21, 1995; and in revised form, May 9, 1995)
From the
Basic fibroblast growth factor (bFGF) is a potent mitogen for
many cell lineages including fetal cardiomyocytes. Furthermore, bFGF
has been shown to modify gene expression, in vitro, in adult
nonproliferative ventricular myocytes. This effect is suspected to be
partly responsible for the genetic modifications that occur in vivo under pathophysiological conditions such as ischemia or pressure
overload and that lead to myocardial hypertrophy. However, little is
known about the first steps of the molecular mechanisms that take place
soon after cell activation by bFGF. In this study, using biochemical
and electrophysiological approaches, we have established, on
cardiomyocytes cultured from neonatal rat ventricles, that (i)
differentiated beating cells express at least two classes of
bFGF-receptors having high and low affinity (K
Basic fibroblast growth factor (bFGF),
Such changes in gene expression
induced by growth factors have become the subject of many
investigations in cardiac cells, particularly concerning bFGF. Indeed,
in the heart of vertebrates, bFGF is known to play fundamental roles in
the normal and the pathological organ development. At the embryonic
stage, bFGF, synthesized by different cardiac fetal cell lineages,
participates in the proliferation and the differentiation of vascular
and contractile cells(11) . In the adult heart, bFGF is
detected at a relatively high level in the atria and the
ventricles(12) , but its role in the adult cell metabolism
remains unclear(13, 14) . An interesting hypothesis
states that, in adult heart, bFGF may be implicated in the cardiac
response to different physiopathological situations (hemodynamic
overload or genetic diseases) leading to the development of
hypertrophy(15, 16) . In particular, bFGF has been
shown to activate isolated cardiomyocytes and to promote the
reexpression of fetal proteins similar to those present in cells of
hypertrophied heart(17) . Although a number of gene expression
studies have been carried out, the total picture of bFGF-mediated cell
activation remains to be built.
The objectives of this work were to
investigate the presence of FGFRs on cardiomyocytes isolated from
neonatal rat ventricles and to study the action of bFGF on both the
cell contractility and the membrane permeability. Our experiments show
that cultured cardiomyocytes express at least two classes of bFGF
receptors. Their activation generates an increase in the spontaneous
cell beating rate. Patch-clamp experiments allowed us to isolate a
novel Ca
In both cases, pipettes, having resistances of 4-8
megaohms, were filled with high barium concentration solution (110
mM Ba(CH
Figure 1:
Scatchard analysis of the binding of
Figure 2:
Covalent cross-linking of
Figure 3:
Effect of
bFGF on the beating rate of spontaneously active cardiomyocytes.
Contractions were recorded as described under ``Experimental
Procedures'' section, and beating rates were calculated every 7 s.
This plot, representative of 12 experiments, shows that addition of
bFGF (10 nM) in the medium rapidly provokes an increase of the
base-line contraction frequency. The increase (40 ± 10%; n = 12) was maintained at the top level for 30-90 min.
Arrhythmia, as well as an increase in the contraction amplitude, were
also observed in response to bFGF. The beating frequency at base-line
was 120 ± 15 (n = 30) contractions/min.
Isoproterenol (1 µM) was used as a positive control,
leading to a 100 ± 20% of increase of the basal rate (n = 30).
Figure 4:
Effect of basic FGF on
Ca
Figure 5:
Effect of IP
Since their localization in the heart, it has been strongly
hypothesized that fibroblast growth factors, produced by cardiac
myocytes or endothelial cells, induce numerous biological effects
relevant to the embryonic heart development and to the response of the
mature organ to stimuli such as ischemia or pressure or volume overload (11, 12, 13) . Possible roles of bFGF in the
regulation of proliferation, differentiation, and hypertrophy of
cardiomyocytes have been suggested (15, 16, 17) . Since only little is known
about the expression of fibroblast growth factor receptors and the
precise mechanisms that link FGFR activation and cell response, we
examined the mechanical and electrical effects of bFGF on cultured
neonatal rat cardiomyocytes.
The presence of specific bFGF membrane
receptors in cardiomyocytes is commonly admitted. Some authors even
state, that modifications in the isoform expression of the different
FGFR families explain the diverse roles of bFGF on proliferation,
differentiation, and size growth of these cells. However no direct
experiment has been done to control these statements. In the present
work, performed on cardiomyocytes purified and cultured from newborn
rat ventricles, we show the binding of bFGF to both low and high
affinity receptors. The results of biochemical assays and cross-linking
experiments indicate that these two binding sites are proteoglycan
heparan sulfate and tyrosine kinase receptors described on other cell
lineages(22, 23, 24) . Different isoforms of
high affinity tyrosine kinase receptors are classically observed, but
the molecular mass of the major complex determined in our cross-linking
experiments (102 kDa) is in lower range of data presented in literature
(about 145 kDa(25, 26, 27, 28) ). It
is interesting to note that, very recently, the transfection and the
expression of the tyrosine kinase FGFR1 gene (flg), cloned
from mouse heart at adult stage, generated three isoform proteins. The
major isoform of the adult cells was shown to have a molecular mass of
102 kDa, which is similar to the value observed in our
model(29) . It is clear that more studies are required to
determine the roles and the evolution of the expression level of the
different classes and isoforms of FGFRs in cardiac myocytes under
normal or pathological development. However, the obtained data provide
evidence that FGFRs are expressed in the cardiomyocytes taken after 2
or 3 days of growth and placed for 1 day in a serum-free medium.
The
activation of the FGFRs leads to an increase in the beating frequency
and also in contraction amplitude of spontaneously active
cardiomyocytes (Fig. 3). These observations corroborate previous
experiments, showing that epidermal growth factor, insulin-like growth
factor, and transforming growth factor
The major finding of these studies is
the presence in cardiomyocyte membranes of channels activated in
presence of bFGF and which generate, under patch-clamp experiment
conditions, calcium influxes. These channels displayed low conductance
and reversal potential, no voltage-dependent openings, and no
dihydropyridine blocker sensitivity. Moreover, as it is mentioned in
the result session, some other growth factors (epidermal growth factor
and insulin-like growth factor) seems to stimulate some comparable
currents, proving that the channel activation would not be specific to
bFGF. These characteristics define a novel kind of channel, permeable
to calcium and activated in the presence of bFGF, different from the
other Ca
In conclusion, the results
presented in this article support the hypothesis that bFGF, by its
binding to specific receptors, promotes, in part via phospholipid
metabolism, the opening of calcium-permeable channels. The fact that
some other growth factors could stimulate comparable currents may
indicate that the channel activation could be a general mechanism
implicated in the growth factor receptor activation. This raises the
question of the implications of the recorded currents that, according
to their activity (fast bursts and multiple step events), could
modulate the rise in cytosolic calcium due to the release of calcium
from intracellular stores(40) . The implication of such ionic
fluxes are known to be a critical step in the cascade activation
leading to oncogene expression in mitotic, quiescent, and
differentiated cells(8, 40, 41) . Recently,
it has been shown in neurons that the oncogene induction by bFGF was
dependent on an increase in
[Ca
= 10 ± 2 pM and 1 ± 0.5
nM); (ii) the stimulation of these bFGF receptors promotes an
increase in the beating frequencies of cultured cardiomyocytes (40
± 10%); (iii) bFGF provokes the activation of poorly specific
and voltage-independent calcium channels (12pS); (iv) inositol
1,4,5-trisphosphate enhances similar bFGF-induced Ca
currents and is therefore suspected to be a second messenger
triggering this activation. These results support the presence, in
cultured cardiomyocytes, of new calcium channels whose activation after
bFGF binding may be partly responsible for the cell response to this
growth factor.
(
)the second member of the heparin-binding growth
factor family(1) , has been found to be a powerful mitogen for
a wide variety of cells(2) . Moreover, bFGF has been shown to
be implicated in the maturation and activation of many differentiated
cells, thus conferring to this growth factor a broad spectrum of
activities(3) . All of these effects are known to be mediated
by interaction with different types of receptors (FGFRs), among which
some belong to a family of transmembrane high affinity binding proteins
possessing a cytoplasmic tyrosine kinase activity (FGFR1 to
FGFR4)(4, 5) . Via different activation pathways,
these receptors initiate cascades of reactions that lead to the
expression of nuclear oncogenes and finally regulate either DNA
replication or gene expression(6) . One of the tyrosine kinase
activation pathways has been shown, on different cell models, to affect
ionic distribution that includes a transient increase of cytoplasmic
calcium ([Ca
]
) from
internal stores or influxes through channels of the plasma
membrane(7, 8) . In many cases, this
[Ca
]
increase appears
to be an important step in the signal transduction that, in association
with biochemical events, leads to the activation of
oncogenes(9, 10) .
channel activated in the presence of bFGF.
In addition, inside-out patch-clamp experiments provide evidence
indicating that the bFGF-induced Ca
channel
activation could be mediated by the second messenger inositol
1,4,5-trisphosphate (IP
).
Materials
Bovine recombinant bFGF was purchased
from Boehringer Mannheim (Meylan, France); trypsin was from Difco;
NaI was from Amersham Corp.; Ham's F-10 and fetal
calf serum were from Life Technologies, Inc. Genistein, tyrphostin 51,
IP
, and the other chemical products were from Sigma. Cell
observations were achieved under a phase-contrast microscope, Nikon TMS
(Nikon, Inc., NY). Patch-clamp signals were measured with an RK-300
amplifier and stored using a digital audio tape recorder 1200
(Biologic, Claix, France). After acquisition with an IEEE-N interface,
data were analyzed with the Bio-Patch software (Biologic, Claix,
France).
Cell Culture
Neonatal rat cardiac myocytes were
trypsin-dissociated and grown according to an established technique
(adapted from (18) ). Briefly, myocardial cells were isolated
from ventricles of 1-2-day-old newborn Wistar rats by enzymatic
digestion (0.1% trypsin 1/250) and gentle mechanical desegregation
(stirred water bath at 37 °C). Cardiomyocytes were separated from
the total cell population by centrifugation (at 1800 rpm for 10 min) on
a discontinuous Percoll gradient (1.06/1.086 g/ml) followed by a 90-min
preplating (in order to eliminate the few non-myocyte cells remaining
after Percoll purification), which yielded cell cultures with >95%
myocytes(19) . Culture medium (Ham's F-10, 20%
heat-inactivated fetal calf serum) was changed every 48 h. After
2-3 days of culture under a 5% CO, 95% O
atmosphere at 37 °C, myocytes reached a near confluent state
corresponding to a terminal differentiation stage, after which myocyte
proliferation no longer occurs. Electrophysiological properties of
these spontaneously beating cardiomyocytes have been shown to be close
to those of adult cells in vivo(20) . All experiments
were performed 3 or 4 days after the cell preparation, when the degree
of purity is maximum and 24 h after removal of serum from the medium,
in order to avoid any interaction with growth factors from the fetal
calf serum.
Electrical Recording
Membrane currents were
recorded using the patch-clamp technique. For the cell-attached
configuration, cells were placed in Tyrode's solution containing
the following: 125 mM NaCl; 5.6 mM KCl; 2.4 mM CaCl; 1.2 mM MgCl
; 10 mM
HEPES; 11 mM glucose, pH 7.4. Before external application of
bFGF (10 nM), bovine serum albumin (0.1%) was added in the
medium to mask the nonspecific binding sites. For experiments with the
inside-out configuration, IP
(5 µM) was
directly added to bath solution with the following composition: 150
mM KCl; 0.55 mM CaCl
; 2 mM
MgCl
; 1.1 mM EGTA; 10 mM HEPES, pH 7.35
with KOH.
COO)
; 10 mM HEPES, pH
7.4 adjusted with Ba(OH)
). For some experiments, CaCl
and BaCl
were also used instead of
Ba(CH
COO)
at the same concentration.
Recording of Contraction Frequencies
Beating rates
were determined by the means of a basic monitoring system using a
photoconductive element placed on the screen of a TV monitor on which
pictures of cultured cells were projected (adapted from (21) ).
Changes in light intensity caused by contractions were locally detected
(at the edge of the cells). Beating rates were computerized every 7 s,
before and after the addition of drugs into Tyrode's solution,
under thermostable conditions (at 35 °C). Tyrosine kinase
inhibitors were dissolved in dimethyl sulfoxide so that the final
concentration of the solvent was 0.1%. Cells were incubated with
the corresponding inhibitor concentration for at least 20 min for
genistein and 1 h for tyrphostin. All results are presented as mean
± S.E.
Identification of bFGF Receptors
The presence of
bFGF-receptors on neonatal rat cardiomyocytes was detected according to
the standard radioreceptor assay method(22) . Radioactive
iodine was incorporated into basic FGF using the chloramine-T method
(as described in (23) ). I-bFGF with a Specific
Activity of
50,000 cpm/ng was routinely obtained. The binding
assay, based on the experimental protocol of Moscatelli(24) ,
allowed the segregation of
I-bFGF bound to different
types of binding sites. Increasing concentrations of labeled bFGF were
incubated (at least 2 h at 4 °C) with cardiomyocyte cultures. After
removal of unbound
I-bFGF, a first fraction of growth
factor bound to low affinity receptors was collected by rinsing cells
with a 2 M NaCl solution. The fraction of
I-bFGF
linked to high affinity receptors was extracted by the addition of 0.5%
Triton X-100 and cell scrapping. The amount of labeled bFGF was
determined by
counting each fraction. These experiments were
completed by cross-linking analysis(25) . Cardiomyocytes were
incubated with radiolabeled bFGF (at least 2 h at 4 °C) in the
absence or presence of different concentrations of unlabeled acidic or
basic FGF. After phosphate-buffered saline washing, disuccinimidyl
suberate was added (0.25 mM for 15 min at 4 °C) in order
to covalently link bound
I-bFGF to its high affinity
receptors. Glycine (2 M) was added to stop the reaction, and
cells were lysed (Triton X-100 and scrapping in phosphate-buffered
saline containing protease inhibitors). The supernatants of the cell
lysates were electrophoresed on a 7.5% SDS-polyacrylamide gel. The
positions of the
I-bFGF/high affinity receptor complexes
were revealed either by autoradiography or by
-imaging on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Identification and Characterization of Basic FGF
Binding Sites on Neonatal Rat Cardiomyocytes
In order to
demonstrate the presence of bFGF receptors, cultures of cardiomyocytes,
isolated from newborn rat ventricles, were incubated with increasing
concentrations of I-bFGF. A Scatchard analysis of the
data obtained in a representative experiment is shown in Fig. 1.
The shape of this curve typically matches with a two binding site
system. The low affinity receptors presented an affinity constant of
about 1 nM (K
= 1
± 0.5 nM) and 50,000-160,000 sites/cell (n = 4). These values are similar to those described for the
heparan sulfate proteoglycan receptors on other cell
types(23, 24) . The K
of
the high affinity binding system (10 ± 2 pM) was also
similar to that of tyrosine kinase receptors characterized in a variety
of cell types(22, 23, 24) . However, the
number of high affinity binding sites detected per cardiomyocyte (750
± 200) is in lower range of the number of high affinity
receptors in other cell lineages (1,000-80,000
sites/cell(24, 26) ). This seems to indicate that
cardiomyocytes, under standard culture conditions, expressed FGFRs at a
low level. Nonetheless, it is worth noticing that the real number of
sites per cell may be more important than the calculated value. Indeed,
the evaluation of the cell density was done by counting nuclei after
crystal violet staining. Now, even under standard culture conditions, a
certain percentage of cardiomyocytes are known to be
binucleated(27) . This could lead to an overestimation of the
number of cells/dish and thereby to an underestimation of the number of
receptors/cell. The other technique of cell counting by trypsin
dissociation was not used since cardiomyocytes are known to remain
associated in groups of about 10 (because of their numerous cell to
cell junctions).
I-basic FGF to cardiomyocytes. Cardiomyocytes, isolated
as described under ``Experimental Procedures,'' were
incubated for 2 h at 4 °C with increasing concentrations of
radiolabeled bFGF. The unbound ligand was removed, and cells were
washed with a 2 M NaCl solution in order to release
I-bFGF bound to low affinity receptors. The labeled bFGF
bound to high affinity sites was extracted by membrane disruption in
Triton X-100. The Scatchard analysis of data, representative of five
independent experiments, showed the presence of two affinity binding
systems (high affinity binding site: K = 12
pM, N
= 700 sites/cell; low affinity
binding site: K = 1.0 nM, N
= 130,000 sites/cell). Cell density (5
10
cells/dish) was evaluated by nuclei counting, which leads to an
underestimation of the site numbers since cardiomyocytes could present
binucleation(27) . F, free
I-bFGF; B, total bound
I-bFGF.
In order to better characterize the presence of
high affinity sites on cardiomyocytes, we performed cross-linking
experiments. The molecular complexes formed between the I-bFGF and the high affinity receptors were analyzed by
SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography. The results showed (Fig. 2, lane1) the presence of one major radiolabeled complex
corresponding to a 102-kDa receptor and two minor complexes
corresponding to 72- and a 152-kDa forms of receptors. Whether these
receptors are distinct proteins or whether the smaller forms just
represent degradation products from the larger one has not been
determined. Competition experiments in which unlabeled basic or acidic
FGF were added to the incubation medium showed a significant decrease
of the detected radioactivity in every distinct form of receptor.
Addition of 50 ng/ml of unlabeled bFGF in the previous incubation
medium partially decreased the binding of
I-bFGF (Fig. 2, lane2). The displacement was total
at a concentration of 500 ng/ml bFGF (Fig. 2, lane 3).
Similarly, unlabeled acidic FGF competed for the binding of
I-bFGF (Fig. 2, lanes4 and 5), but some binding was still observed in the presence of 500
ng/ml aFGF. This indicates that the affinity of the cardiomyocyte FGFRs
was stronger for bFGF than for aFGF. It is worth noting that
cross-linking experiments have also been done with cell cultures having
different degrees of purity (by reincorporating non-myocyte cells after
Percoll separation). The results (data not shown) demonstrated that
high affinity sites were detected in non-myocyte or poorly purified
cell cultures but also in very pure cell preparations in which the
presence of high affinity receptors could not be due to fibroblasts or
any other non-myocyte cells.
I-bFGF to high affinity receptors in the presence or
absence of unlabeled basic or acid FGF. Cardiomyocytes were incubated
at 4 °C for at least 2 h with 20 ng/ml of
I-bFGF (lane1). Cells were washed, and labeled FGF was
covalently linked to the high affinity receptors by disuccinimidyl
suberate as described under ``Experimental Procedures.''
Triton X-100 extracts of the cell lysates were electrophoresed on 7.5%
SDS-polyacrylamide gels. For competition experiments, cells were
incubated with the same concentration of
I-bFGF in the
presence of 50 ng/ml (lane2) or 500 ng/ml (lane3) of unlabeled basic FGF. Similarly, 50 and 500 ng/ml (lanes4 and 5) of unlabeled acidic FGF were
added during the incubation period. After autoradiographic detection,
the relative molecular weight of the different binding complexes was
calculated by comparison with that of prestained size markers.
Molecular mass of each isoform of the high affinity receptor could then
be calculated after subtraction of the 18-kDa mass of the growth factor
.
Effect of bFGF on the Mechanical Activity of
Cardiomyocytes
The ability of bFGF to modify the contractile
activity of cultured cardiomyocytes has been studied as described under
``Experimental Procedures.'' It can be seen from Fig. 3that, at a base line level, cells exhibited a constant and
synchronized beating rate, even in absence of any electrical
stimulation. Addition of bFGF (10 nM) to the medium led to a
significant increase (40 ± 10%, n = 12) of the
beating rate. After an average period of 5-15 min, contraction
frequencies reached a maximum and remained at this top level for
30-90 min. This positive chronotropic effect caused by bFGF also
led to arrhythmia and to a significant increase of the contraction
amplitude. Complementary experiments have been achieved, using two
tyrosine kinase inhibitors, genistein and tyrphostin 51. The obtained
data are summarized in Table 1. When cells were incubated with
low concentrations of genistein and tyrphostin (2 and 4
µM, respectively) a subtotal inhibition of the bFGF effect
was observed since the bFGF-induced increase of the basal frequency was
limited to 5 ± 2 and 7 ± 3% (n = 4). A
total inhibition of the bFGF effect was obtained with 20 µM genistein and 40 µM tyrphostin. Under these
conditions, the basal beating frequency (120 ± 15) was not
modified during the incubation period and remained at the same basal
level after the addition of bFGF in the medium (n = 12
and 10). Higher concentrations of inhibitors (200 and 400
µM) were found to have a certain cytotoxicity, which led
to alterations of the basal beating rate, to unexpected beating stop
and, at the end of the incubation period, to the presence of cytosolic
granulations and alterations in cell shapes. These last doses were not
used any more, since lower concentrations were revealed efficient to
block the bFGF-induced effects. Isoproterenol (1 µM) was
used as positive control, leading to an increase of the beating
frequency of 100 ± 20% (n = 30).
Effect of bFGF on Calcium Channel
Activation
Patch-clamp experiments were performed in order to
record possible calcium membrane permeability modifications at a single
cell level. Fig. 4a shows a typical record obtained
from a cell-attached patch held on cardiomyocytes, before and after
bFGF (10 nM) addition to the bath solution, with a high
concentration barium pipette solution (110 mM) and a holding
potential of -30 mV. The current amplitude distribution of such
bFGF-induced channels is shown on Fig. 4b, displaying a
single inward current of 0.25 pA amplitude. This value varies linearly
with holding potentials, which were converted to membrane potentials
according to the diastolic resting potential of the cells assumed to
-60 ± 5 mV (n = 10). The current-potential
curve (Fig. 4c) displays a slope conductance of 12
picosiemens and a reversal potential of +20 mV. Since the reversal
potential is lower than the theoretical value (calculated from the
Nernst equation), the specificity of the observed channel either to
calcium or to barium seems to be relatively weak. Barium was used as
charge carrier since calcium channels are generally more permeable to
this ion, which often permits a better resolution of differences in
single-channel amplitudes. However, when barium was substituted for
calcium ions, no significant modification of bFGF-induced current
amplitude occurred. Moreover, current amplitudes were the same whether
chloride or acetate was the anion of the pipette solution, which
confirmed that the currents were carried by inward influx of cations
(Ca or Ba
), rather than by outward
anion fluxes (which anyway would display, in the experimental
conditions, negative reversal potentials). Potassium cannot be taken
into account since this ion was not in the pipette solution and since
high concentration of barium is known to inhibit K
permeability. The histograms of distribution for the mean open
and closed times were computerized as shown on Fig. 4, d and e; the open time constants were 1.5 and 14 ms and the
closed time constants were 0.6 and 7 ms.
channel activity. a, representative
example of bFGF-induced Ca
currents obtained from a
cultured cardiomyocyte using the cell-attached patch-clamp
configuration and a barium pipette solution. For an holding potential
of -30 mV, no current was detected at a base-line level, but in
minutes after the addition of basic FGF (10 nM, at the time
pointed by the arrow) specific openings were observed. b, the probability density histogram of current amplitudes
derived for the experiment shown in a reveals multiple step
events corresponding to an inward unitary current of 0.25 pA. c, the current-voltage relation of single Ca
channel current was plotted after conversion of holding
potentials into membrane potentials (MP), the average
diastolic resting potential being estimated at -60 mV. Each point
represents the mean of three to six experiments. The resulting straightline defines a single channel conductance of
12 picosiemens with a reversal potential at +20 mV. d, distribution histogram of open times of Ca
channel was fitted with the sum of two exponential curves. The
corresponding time constants are 14 (slow constant) and 1.5 ms (fast
constant). e, distribution histogram of closed times of
Ca
channel was fitted with the sum of two exponential
curves, with time constants of 7 (slow constant) and 0.6 ms (fast
constant).
No voltage dependence was
detected, and inhibitors of voltage-dependent calcium channels such as
dihydropyridines (nicardipine or nifedipine) or verapamil were without
effect on the bFGF-activated currents. Besides, preliminary experiments
have shown that epidermal growth factor and insulin-like growth factor
1 led to an increase of the calcium permeability of patched membranes,
indicating that other growth factors could also prompt channel
activations (data not shown). Finally, the fact that the presence of
bFGF in the pipette was not necessary to stimulate the ion flux proves
that a second messenger is required to convey the signal from FGFRs to
calcium channels.
Role of IP
With a success rate of around 50%, we found
that inositol 1,4,5-trisphosphate promote an increase in channel
openings of excised patches. As it can be seen in Fig. 5a, the addition of IPin the bFGF-induced
Ca
Channel Activation, in Inside-out
Configuration
(5
µM) to the bath of an inside-out patch (held at +30
mV, corresponding to -30 mV of membrane potential) induced an
activation of inward currents with elementary amplitude of 0.25 pA (Fig. 5b). Reversal potential (+15 mV) and
conductance (12 pS) were deduced from the linear correlation of the
current-potential curve (Fig. 5c). The mean open and
closed time constants (Fig. 5, d and e) were
also computed. When compared with the previous bFGF-induced currents (Table 2), these IP
-activated openings displayed
similar characteristics concerning the conductance, the reversal
potential, and the kinetics constants. Thus, it could be hypothesized
that the same type of channels was isolated under the patch pipette in
both cases. This observation suggests that IP
could be a
second messenger participating in bFGF-activation of Ca
channels. Other second messengers (cAMP, cGMP), used under the
same conditions, failed to activate similar currents. Nevertheless, we
cannot formally exclude the possibility that other molecular mechanisms
would act in the regulation of the observed current. Failure to show
opening inhibition by addition of calcium channel blockers (verapamil
or nifedipine) suggest that the observed channels were insensitive to
these agents. However, lanthanum has been found to reversibly inhibit
the observed openings, which indicates that the currents were indeed
carried by calcium (or barium) influxes (data not shown).
on Ca
channel activity. a, representative example of
IP
-induced Ca
currents obtained under
inside-out configuration conditions. The holding potential of this
trace was +30 mV, and only rare openings were detected. Thirty
seconds after the addition of IP
(5 µM,
located by the arrow) an increase of the current activity was
observed (record sampled at 1 kHz). b, the resulting density
histogram of current amplitudes revealed multiple step events, which
correspond to the sum of inward unitary currents of 0.25 pA. c, the current-voltage relation of single Ca
channel current correspond to a straightline with a slope conductance of 12 picosiemens and a reversal
potential of 15 mV (each point being the mean of five to nine
experiments). d, distribution histogram of open times of
Ca
channel, fitted with the sum of two exponential
curves. The corresponding time constants are 12 (slow constant) and 1.5
ms (fast constant). e, distribution histogram of closed times
of Ca
channel, fitted with the sum of two exponential
curves, with time constants of 5 (slow constant) and 0.5 ms (fast
constant).
have chronotropic or
inotropic effects on
cardiomyocytes(30, 31, 32) . This indicates
that different growth factors, some acting through tyrosine kinase
receptors, positively stimulate mechanical activity of these cells. In
the present work, genistein and tyrphostin, two tyrosine kinase
inhibitors, are found to suppress the bFGF-induced activation of the
beating rate, providing evidence of the implication of the tyrosine
kinase activation pathway. Obviously, such increases in contraction
frequency as those observed in this in vitro model cannot be
immediately extrapolated to the in vivo situation. However,
these modifications of the beating rates and contraction amplitude
reveal changes in the distribution of ions in the cardiomyocyte
cytoplasm, indicating that bFGF triggers modifications in the ionic
equilibriums, which partly results in an intracellular free
Ca
increase.
channels previously identified in the
cardiomyocyte membrane(33) . By contrast, the observed channel
shares many common points with other growth factor-activated channels
identified in the literature. Indeed, most of the currents, recorded on
other cell models, show low specificity, low conductance, fast bursts,
dihydropyridine insensitivity, and voltage
independence(34, 35, 36) . Another point
common to many of the growth factor-activated channels is their
coupling to a second messenger generation system. In the present work,
the possibility of an implication of IP
was confirmed by
inside-out experiments showing that this phosphoinositol could provoke
similar channel activations. This result is consistent with the general
activation pathways of the tyrosine kinase receptors. Indeed, similar
to epidermal growth factor, platelet-derived growth factor, and
insulin-like growth factor, bFGF has been shown to stimulate
phosphorylations of intracellular target proteins, among which is a
subunit of phospholipase C-
(5, 37) . The
activation of this phospholipase C-
, via the phosphatidyl
metabolism, is known to stimulate the syntheses of diacylglycerol and
IP
, which both modify the distribution of several ions,
particularly Ca
and
K
(38, 39) . However, even if the
pathway linking tyrosine kinase activation and inositol phospholipid
synthesis is well known, the complete signal transduction cascade
remains to be determined. In particular, little, if anything, is known
about the mechanism of cell membrane channel opening triggered by
IP
. The present results corroborate previous findings
stating that IP
can directly enhance membrane permeability
without interaction of cytosolic co-factors(34, 36) .
However, the success rate of 50% in the observation of this phenomenon
might be due to the fact that some agents are required together with
the channel in the excised membrane.
]
(10) .
Moreover, some studies have strengthened the hypothesis that calcium
fluxes through the plasma membrane are necessary for the nuclear
activation of oncogene transcription by growth factors(42) .
Consequently, the observed ionic fluxes may be sufficient to directly
or indirectly affect cellular biosyntheses and in turn, may contribute
to cardiomyocyte oncogene expression. Finally, the bFGF-induced channel
activation, described in this work, may help in the understanding of
the mechanism by which, during myocardium-increased work, bFGF and
other growth factors can induce the expression of fetal-like
contractile protein genes.
], intracellular free calcium
concentration; IP
, inositol 1,4,5-trisphosphate;
I-bFGF, radioactive iodine-labeled bFGF.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.