(Received for publication, June 13, 1995; and in revised form, February 1, 1996)
From the
The aim of this study was to evaluate the effect of
protein-tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP)
inhibitors on Ca channels in GH
cells.
The activity of Ca
channels was monitored either by
single-cell microfluorometry or by the whole-cell configuration of the
patch-clamp technique. Genistein (20-200 µM) and
herbimycin A (1-15 µM) inhibited
[Ca
]
rise induced
either by 55 mM K
or 10 µM Bay K
8644. In addition, genistein and lavendustin A inhibited whole-cell
Ba
currents. By contrast, daidzein, a genistein
analogue devoid of PTK inhibitory properties, did not modify
Ca
channel activity. The inhibitory action of
genistein on the [Ca
]
increase was completely counteracted by the PTP inhibitor
vanadate (100 µM). Furthermore, vanadate alone potentiated
[Ca
]
response to both
55 mM K
and 10 µM Bay K 8644.
The possibility that genistein could decrease the
[Ca
]
elevation by
enhancing Ca
removal from the cytosol seems unlikely
since genistein also reduced the increase in fura-2 fluorescence ratio
induced by Ba
, a cation that enters into the cells
through Ca
channels but cannot be pumped out by
Ca
extrusion mechanisms. Finally, in unstimulated
GH
cells, genistein caused a decline of
[Ca
]
and the
disappearance of [Ca
]
oscillations, whereas vanadate induced an increase of
[Ca
]
and the
appearance of [Ca
]
oscillations in otherwise non-oscillating cells. The present
results suggest that in GH
cells PTK activation causes an
increase of L-type Ca
channel function, whereas PTPs
exert an inhibitory role.
It has been largely demonstrated that the activity of L-type
Ca channels can be regulated by different types of
kinases, such as protein kinase A (PKA) (
)(1, 2) and protein kinase C
(PKC)(3, 4) . These two kinases phosphorylate serine
(Ser) and threonine (Thr) residues on the
- and
-subunits of
these channel proteins(5, 6) . Recently, a great deal
of interest in the literature has been devoted to another class of
kinases, the protein-tyrosine kinases
(PTKs)(7, 8, 9) . These enzymes, which exist
both in transmembrane receptor-linked (7) or non-transmembrane
forms(8, 9) , phosphorylate tyrosine (Tyr) residues on
several cellular proteins. Since it has been recently reported that in
non-excitable cells such as T-lymphocytes the overexpression of PTK
activity, obtained transfecting these cells with the PTK-encoding
oncogene v-src, induces a remarkable increase of basal and
stimulated [Ca
]
levels(10) , it appeared of interest to explore the
possibility that PTKs could modulate the activity of L-type
Ca
channels. For this purpose, the effect of the
specific PTK inhibitors genistein(11, 12) , herbimycin
A (13) , and lavendustin A (14) on the function of
L-type Ca
channels was evaluated in pituitary
GH
cells (15) by single-cell microfluorometry and
patch-clamp electrophysiology.
On the other hand, since PTK activity
is functionally counteracted by protein-tyrosine phosphatases
(PTPs)(16, 17) , the possible effect of the PTP
inhibitor vanadate (18) on L-type Ca channels
was also investigated.
Figure 1:
Effect of
genistein and herbimycin A on K-induced
[Ca
]
increase in GH
cells. Panel A shows the
effect on [Ca
]
of two
consecutive 55 mM K
pulses delivered with a
10-min interval. During the interval between the pulses, cells were
perfused with Krebs-Ringer saline solution. Panel B shows the
effect of genistein (200 µM), added to the perfusion 7 min
before and throughout the whole second K
pulse. The
mean peak after the second 55 mM K
pulse was
significantly lower than the first one (p < 0.01). In
addition, genistein significantly reduced basal
[Ca
]
(124 ± 4 versus 104 ± 3 nM Ca
; p < 0.01). In panel C the concentration dependence of
the inhibitory effect of genistein on the
[Ca
]
increase induced
by 55 mM K
is represented. Each point is the
mean of 10-30 single-cell recordings. The solid line is
the fit of the experimental points to the equation y =
max/(1+(x/K
)
,
where K
is the K
for the block and n is the Hill coefficient. Panel D shows the effect of different concentrations of
herbimycin A on the 55 mM K
-induced
[Ca
]
increase. *
= p < 0.01 versus control
group.
On the
other hand, when GH cells were superfused with two 10
µM consecutive pulses of the dihydropyridine activator of
L-type Ca
channels Bay K
8644(20, 21) , two equivalent elevations of
[Ca
]
occurred (Fig. 2A). However, if genistein (200 µM)
was superfused 5 min before the second pulse with the L-type
Ca
channel activator, a 40% reduction of the
[Ca
]
increase was observed (Fig. 2B).
Figure 2:
Effect of genistein on
[Ca]
increase induced by 10 µM Bay K 8644
in GH
cells. Panel A shows the effect on
[Ca
]
of two 10
µM Bay K 8644 pulses delivered with an approximately
25-min interval. During the resting period between the two
stimulations, the cells were perfused with Krebs-Ringer saline
solution. Panel B shows the effect of 200 µM genistein added to the superfusion medium 5 min before and
throughout the second Bay K 8644 pulse. Each trace is the mean of at
least 30 single-cell recordings obtained during a single experiment
representative of at least three other experimental
sessions.
To identify more directly the target of
PTK inhibition, Ba currents through Ca
channels were recorded in GH
cells by means of the
whole-cell configuration of the patch-clamp technique. From the holding
potential of -90 mV, test potentials above -60 mV elicited
large inward Ba
currents, which peaked around
-35 mV (Fig. 3E). At all the test potentials the
currents displayed less than 10% inactivation during the 100-ms pulse
duration (Fig. 3A). These properties suggest the
presence of a large population of L-type Ca
channels.
This was further confirmed by the ability of the selective L-type
Ca
channel blocker nifedipine to inhibit
approximately 80% of the whole-cell Ba
currents (Fig. 4C). Perfusing GH
cells with the PTK
inhibitor genistein (100 µM) caused a 50% reduction of the
currents at all potentials tested (Fig. 3B). Complete
suppression of the currents was achieved with 200 µM Cd
(Fig. 3C). Upon extensive
washout (5 min) Ba
currents recovered (Fig. 3D). The extent of genistein-induced inhibition
of Ba
currents was comparable to that observed in
microfluorometric studies (Fig. 4D). Lavendustin A (25
µM), another PTK inhibitor which could not be studied
microfluorometrically because of its intrinsic fluorescence, also
inhibited Ba
currents (Fig. 4, A and D). By contrast, daidzein, the inactive analogue of
genistein(12) , did not exert any influence on Ba
currents (Fig. 4, C and D). It should be
underlined that although nifedipine inhibition of Ba
currents occurred with a very short latency (10 s), the effect of
genistein required a longer period of time (30 s) (Fig. 4E).
Figure 3:
Genistein inhibits voltage-dependent
Ba currents in GH
cells. The same cell
was recorded in control solution (panel A), 3 min after the
exposure to 100 µM genistein (panel B), 1 min
after the exposure to 200 µM Cd
(panel C), and after 5 min of washout in 10 mM Ba
control extracellular solution (panel
D). The holding potential was -90 mV, and 100-ms
depolarization steps from -80 to +25 in 15-mV increments
were delivered. The data are shown without any leak subtraction
procedure. Panel E shows the current to voltage (I/V) relationship for genistein-induced inhibition
of voltage-dependent Ba
currents. Current values were
taken at the end of the depolarizing steps. Each point is the mean of
three different cells recorded in the same experimental conditions. The
data have been normalized to the peak value of the control I/V (-35 mV) for each cell, to facilitate
comparison.
Figure 4:
Comparison among the effects of genistein,
lavendustin A, daidzein, and nifedipine on Ba currents in GH
cells. Panel A shows
single-current traces obtained from a cell depolarized to -40 mV
from a holding potential of -90 mV. As indicated, the same cell
was subsequently recorded in control solution, after a 2-min exposure
to 25 µM lavendustin A, and after a 1-min exposure to 200
µM Cd
. Panel B shows
single-current traces obtained from a cell depolarized to -30 mV
from a holding potential of -90 mV. As indicated, the same cell
was subsequently recorded in control solution, after a 3-min exposure
to 100 µM daidzein, after a 3-min exposure to 100
µM genistein, and after a 1-min exposure to 200 µM Cd
. Panel C shows single-current traces
obtained from a cell depolarized to -40 mV from a holding
potential of -90 mV. As indicated, the same cell was subsequently
recorded in control solution, after a 2-min exposure to 5 µM nifedipine, and after a 1-min exposure to 200 µM Cd
. Each trace is shown without any leak
subtraction procedure. In panel D is reported the percent of
inhibition of the Ba
currents at -30 mV by 25
µM and 100 µM genistein, 25 µM lavendustin A, 100 µM daidzein, and 5 µM nifedipine. Each point is the mean ± S.E. of at least four
separate experiments. * denotes p < 0.01. Panel E,
time course of nifedipine and genistein inhibition of Ba
currents. Inward Ba
currents were elicited by
depolarizing pulses to -30 mV from a holding potential of
-90 mV every 5 s in two separate cells. After the first three
pulses in control solution, the perfusion solution was changed with the
respective drug-containing one, and the time course of inhibition was
followed for both 5 µM nifedipine and 100 µM genistein. The two cells shown are representative of at least five
experiments, each giving comparable
results.
Figure 5:
Enhancement by 100 µM vanadate of [Ca]
response to two different L-type Ca
channel-activating stimuli. Panels A and B represent the mean traces of
[Ca
]
response to two
consecutive stimuli with 55 mM K
and 10
µM Bay K 8644, respectively. Vanadate (100
µM) was superfused 20 min before and throughout the second
stimulus. Each trace is the mean of at least 30 single-cell recordings
obtained during a single experiment representative of at least three
other experimental sessions. In addition, vanadate significantly (p < 0.01) increased mean basal
[Ca
]
after its
addition to the medium (107.9 ± 2.7 versus 83.8
± 1.9 nM in panel A and 124.3 ± 5.7 versus 93.1 ± 3.3 nM in panel
B).
In addition, the superfusion of GH cells with 100 µM vanadate for 15 min completely
abolished the inhibition of the [Ca
]
response to 55 mM K
which follows the
exposure of these cells to 200 µM genistein for 2 min (Fig. 6, A and B).
Figure 6:
Reversal by 100 µM vanadate
of genistein-induced inhibition of 55 mM K-elicited
[Ca
]
increase. In panel A, genistein (200 µM)
was added 2 min before the second 55 mM K
stimulus. In panel B, 100 µM vanadate was
superfused for 20 min before throughout the second 55 mM K
pulse. Genistein was added to the superfusion
medium 2 min before the second 55 mM K
pulse.
Each trace is the mean of at least 30 single-cell recordings obtained
during a single experiment representative of at least three other
experimental sessions.
Figure 7:
Effect of the PTK inhibitor genistein and
of the PTP inhibitor vanadate on
[Ca]
in non-oscillating and oscillating GH
cells.
Typical response of a non-oscillating (panel A) and an
oscillating (panel B) GH
cell superfused with
genistein (200 µM for 250 s) and vanadate (100 µM for 20 min) (panels C and D). All traces shown
in the figure are single-cell recordings and are representative of the
pattern of 53 cells exposed to vanadate and 57 cells superfused with
genistein, recorded in at least three experimental
sessions.
The results of the present study, obtained by means of
single-cell microfluorometry and whole-cell patch-clamp techniques,
demonstrate that the activity of Ca channels in
GH
cells can be influenced by the interplay between PTK and
PTP activity: PTK activation seems to cause an increase, whereas PTP
activation appears to exert an inhibitory role on this ion channel.
The hypothesis that the L-type Ca channel is the
target of PTK and PTP modulation derives from the results showing that
the increase of [Ca
]
elicited
by the specific L-type Ca
channel activator Bay K
8644 and high K
concentrations was reduced by the PTK
inhibitor genistein and enhanced by the PTP blocker vanadate. A further
support to this idea is the ability of genistein and lavendustin A to
inhibit Ba
currents through Ca
channels that displayed biophysical and pharmacological features
of the L-type. On the other hand, the possibility that the action of
PTK inhibitors is exerted on the T-type Ca
channels,
which have been described in GH
cells, seems unlikely since
this Ca
channel type does not play a significant role
in the [Ca
]
elevation elicited
by strong activating stimuli (55 mM K
or Bay
K 8644)(24, 25) . In addition, the biophysical
features of Ba
currents recorded in GH
cells in the present study do not show the presence of a
significant population of this Ca
channel type.
Furthermore, the remarkable inhibition of Ba
currents
by the L-type blocker nifedipine suggests that the largest population
of Ca
channels is represented by the L-type.
The
possibility that the genistein-induced reduction of the
[Ca]
increase elicited by high
K
concentrations could be due to an increase of
Ca
removal from the cytoplasm to the extracellular
space or into the intracellular Ca
stores seems not
to be compatible with the results of the present study. In fact,
genistein also reduced the entrance of Ba
ions, a
cation that is known to be unable to substitute for Ca
in the extrusion mechanisms. In support of this interpretation,
the entity of the genistein-induced inhibition of the
[Ca
]
rise induced by 55 mM K
and 10 µM Bay K 8644 was
comparable to the inhibition observed in electrophysiological
experiments.
Since it has been reported that genistein, besides
inhibiting PTKs, can also block other protein kinases such as PKA and
PKC(11, 12) , which are known to modulate L-type
Ca channels(1, 2, 3, 4) , the
possibility exists that its effects on the activity of L-type
Ca
channels could occur via PKA or PKC inhibition.
However, this hypothesis seems unlikely since herbimycin A and
lavendustin A, two other specific PTK inhibitors devoid of PKA or PKC
inhibitory action (14, 26) and structurally unrelated
to genistein, effectively inhibited Ca
channel
activity in GH
cells. This evidence strongly suggests that
PKA or PKC inhibition is not involved in the genistein action on
Ca
channels. In addition, the IC
for
genistein inhibition of Ca
channels (30
µM) was very similar to that for PTK inhibition and much
lower than that for PKA and PKC blockade(11) . The specificity
of genistein action on Ca
channels via PTKs was
confirmed further by the inability of the genistein analogue daidzein,
which lacks PTK inhibitory properties, to modify Ca
channel activity in electrophysiological recordings.
The
existence of a PTK regulation of L-type Ca channels
in GH
cells is also supported by the fact that PTPs, which
physiologically counteract the activity of
PTKs(16, 17) , exert an opposite modulation on L-type
Ca
channel activity. In fact, the inhibition of PTPs
by orthovanadate, a well known inhibitor of these enzymes(18) ,
was able to enhance the [Ca
]
increase induced by high K
concentrations and to
counteract the inhibitory effect of genistein on this response.
The
modulation exerted by PTKs and PTPs seems to occur not only when L-type
Ca channels are activated by high depolarizing
stimuli, but also in resting conditions. In fact, the inhibition of
PTKs by genistein caused a decline of
[Ca
]
and a disappearance of
[Ca
]
oscillations in
oscillating GH
cells, whereas the blockade of PTPs by
vanadate induced an increase of [Ca
]
or the appearance of [Ca
]
oscillations. These findings were not unexpected since in
unstimulated conditions, L-type Ca
channels of
GH
cells are spontaneously active, as shown by the fact
that spontaneous action potentials have been detected (27) and
that these potentials are coupled to oscillations of
[Ca
]
, which can be abolished by
the specific L-type Ca
channel blocker nifedipine (15) .
The results of the present study showing that PTKs
exert a stimulatory modulation on L-type Ca channels
are in line with the recent report that genistein induces a
concentration-dependent inhibition of Ca
channel
currents in vascular smooth muscle cells(28) . In addition,
evidence has been provided that the inhibition of PTKs can also reduce
Ca
influx through plasma membrane
``refilling'' channels (29, 30, 31) and that different types of
receptor-operated channels, like the nicotinic, N-methyl-D-aspartic acid, and
-aminobutyric acid
receptor channels, can be modulated by
PTKs(32, 33, 34) .
The results of the
present study could be of interest to explain the Ca dependence of certain biological responses elicited by some
growth factors(35) . In fact, the stimulation of many growth
factor receptors, such as those for the epidermal growth factor,
recognize as a signaling pathway the activation of a receptor-linked
PTK(7) . Since the results of the present study indicated that
PTK activation leads to Ca
entrance through L-type
Ca
channels into the cells, the
Ca
-dependent epidermal growth factor-induced
differentiation of GH
cells toward the lactotroph phenotype (36) could be the consequence of the activation of L-type
Ca
channels, especially if one considers that in a
different pituitary cell line, epidermal growth factor induces an
increase of [Ca
]
which is
independent of phospholipase C
1-dependent inositol
1,4,5-trisphosphate generation(37) .
In conclusion, all of
these results suggest that L-type Ca channels are
modulated by the PTK/PTP system in GH
cells. The molecular
mechanism of this modulation remains to be clarified. However, a
possible working hypothesis to explain the effect of PTK inhibitors on
Ca
channel function could be that phosphorylation by
PTKs exerts a permissive role on the activation of Ca
channels elicited by both the dihydropyridine agonist Bay K 8644
and depolarizing stimuli. Such a model has already been proposed by
Armstrong et al.(2) to explain the effect of PKA on
Ca
channel activation.