(Received for publication, August 15, 1995; and in revised form, January 4, 1996)
From the
In mouse lacrimal acinar cells, microinjection of the
metabolically stable analog of inositol 1,4,5-trisphosphate, inositol
2,4,5-trisphosphate ((2,4,5)IP), stimulated both
intracellular Ca
mobilization and Ca
entry. Microinjection of inositol 1,3,4,5-tetrakisphosphate
((1,3,4,5)IP
), the inositol 1,4,5-trisphosphate-3-kinase
product, was ineffective at mobilizing intracellular Ca
or activating Ca
entry. In lacrimal cells
previously microinjected with submaximal levels
of(2, 4, 5) IP
, the subsequent
microinjection of low to moderate concentrations of (1, 3, 4, 5) IP
did not
result in additional release of intracellular Ca
, nor
did it potentiate the Ca
entry phase attributable
to(2, 4, 5) IP
. However, as
previously demonstrated (Bird, G. S. J., Rossier, M. F., Hughes, A. R.,
Shears, S. B., Armstrong, D. L., and Putney, J. W., Jr. (1991) Nature 352, 162-165), additional injections of (2, 4, 5) IP
induced further
mobilization of intracellular Ca
and increased the
elevated and sustained Ca
entry phase. Introduction
of high concentrations of (1, 3, 4, 5) IP
appeared
to inhibit or block the (2, 4, 5) IP
-induced
Ca
entry phase. These results were consistent with
the observed effect of (1, 3, 4, 5) IP
in
permeabilized lacrimal cells, where (1, 3, 4, 5) IP
did not
release cellular
Ca
but at high
concentrations inhibited the ability of submaximal concentrations
of(2, 4, 5) IP
to release
Ca
. Likewise, injection of a high
concentration
of(1, 3, 4, 5) IP
prior
to injection of (2, 4, 5) IP
blocked both release and influx of Ca
. The
inhibitory action
of(1, 3, 4, 5) IP
on
Ca
signaling observed in intact cells occurred at
concentrations that might be obtained in agonist-stimulated cells.
However, in permeabilized
cells,(1, 3, 4, 5) IP
inhibited Ca
mobilization at concentrations
exceeding those likely to occur in agonist-stimulated cells. These
results suggest that physiologically relevant levels
of(1, 3, 4, 5) IP
in the
cell cytoplasm do not release Ca
, nor do they
potentiate inositol trisphosphate-induced Ca
entry
across the plasma membrane. Rather, the possibility is raised that (1, 3, 4, 5) IP
or one
of its metabolites could function as a negative feedback on
Ca
mobilization by inhibiting inositol
1,4,5-trisphosphate-induced Ca
release.
In many cell types, surface receptor activation results in a
complex, biphasic Ca response composed of an initial
mobilization of internally stored Ca
, followed by
entry of extracellular Ca
. An early event following
activation of the muscarinic receptor is the breakdown of
phosphatidylinositol 4,5-bisphosphate generating the putative second
messenger, inositol 1,4,5-trisphosphate
((1,4,5)IP
)(
)(1) , which can
subsequently undergo complex metabolic processing(2) . The role
played by the inositol phosphates in Ca
homeostasis
has been the subject of much study, and it is widely believed that (1, 4, 5) IP
is responsible for
the first phase of Ca
mobilization from intracellular
pools(1) . However, the mechanism underlying the second phase
of Ca
entry is poorly understood(3) , and
controversy particularly surrounds the role of
the(1, 4, 5) IP
metabolite,(1, 3, 4, 5) IP
,
in this process(3, 4, 5, 6) .
In
previous studies, it was reported that intracellular application of (1, 4, 5) IP into lacrimal acinar
cells, by means of perfusion of patch clamp pipettes in the whole cell
configuration, resulted in transient activation of a
Ca
-activated K
conductance.
Sustained activation could only be achieved
when(1, 3, 4, 5) IP
was
applied together with (1, 4, 5) IP
(7, 8, 9, 10) .
These studies suggested that both (1, 4, 5) IP
and(1, 3, 4, 5) IP
were necessary for the activation of Ca
entry
in these cells. Subsequently, we measured
[Ca
]
changes in
lacrimal cells with the Ca
-sensitive fluorescent dye,
fura-2, and introduced(1, 4, 5) IP
or its poorly metabolized analog, (2, 4, 5) IP
into the cells by
microinjection(11) . These studies demonstrated
that(1, 4, 5) IP
alone is both a
necessary and sufficient signal for intracellular Ca
([Ca
]
)
mobilization as well as Ca
entry across the plasma
membrane. However, as pointed out by Irvine(12) , possible
augmenting actions of (1, 3, 4, 5) IP
on
calcium signaling were not directly examined. In the present study, we
have used fura-2-loaded mouse lacrimal acinar cells to examine effects
of(1, 3, 4, 5) IP
on
Ca
entry induced
by(2, 4, 5) IP
. Our results
indicate that physiological concentrations
of(1, 3, 4, 5) IP
clearly do not augment but rather may inhibit (1, 4, 5) IP
-induced
Ca
signaling.
During the microinjection of inositol phosphates, the mouse
lacrimal cells were maintained in nominally Ca-free
medium. Under these conditions, microinjection of a (1, 3, 4, 5) IP
(10
mM pipette concentration, final cellular concentration,
100-200 µM(11) ), did not mobilize
intracellular Ca
([Ca
]
), nor did it
promote Ca
entry into the cell on restoring the
extracellular Ca
(Fig. 1). Further, the
injection of (1, 3, 4, 5) IP
did not prevent the ability of thapsigargin to activate
Ca
entry in these cells (Fig. 1). As shown
previously in lacrimal cells(11) , microinjection of submaximal
concentrations of the metabolically
stable(1, 4, 5) IP
analog,(2, 4, 5) IP
(1
mM pipette concentration, final cellular concentration
10-20 µM), resulted in a submaximal Ca
release and a submaximal level of Ca
entry (Fig. 2a). Subsequent microinjection of
additional(2, 4, 5) IP
released
additional Ca
and further increased the level of
Ca
entry (Fig. 2a). Control
injections not containing an inositol phosphate did not modify the
second Ca
entry phase as compared with the first (Fig. 2b).
Figure 1:
The effect of microinjected (1, 3, 4, 5) IP on
Ca
signaling in a single mouse lacrimal acinar cell.
Lacrimal cells were incubated in a nominally Ca
-free
medium, microinjected with 10
mM(1, 3, 4, 5) IP
(indicated by the arrow on the left), and the horizontal bar indicates when extracellular Ca
was restored to 1.8 mM. This was followed, where
indicated, by 2 µM thapsigargin.
Figure 2:
The effect of inositol polyphosphate
microinjection on Ca mobilization and entry in a
mouse lacrimal acinar cell previously microinjected with a submaximal
concentration of(2, 4, 5) IP
. As
in Fig. 1, lacrimal cells were incubated in a nominally
Ca
-free medium during the microinjections (indicated
with arrows), while the horizontal bars indicate when
extracellular Ca
was restored to 1.8 mM. a, (2, 4, 5) IP
was
first injected as indicated by the first arrow. The pipette
solution contained 1 mM(2, 4, 5) IP
, which gave
sufficient intracellular (2, 4, 5) IP
to induce a submaximal release of Ca
. The
subsequent injection of submaximal (2, 4, 5) IP
(second
arrow) caused a second, additional release of intracellular
Ca
, and the cumulative effects of these injections
resulted in an increased level of Ca
entry. b, as a control for any possible effect that the injection
procedure itself may have on the Ca
entry phase
itself, a second injection was made in the absence of any inositol
phosphate. In c and d, the protocol was similar to
that described for a, except that the second injection was
either 100 µM(1, 3, 4, 5) IP
(c) or 10 mM(1, 3, 4, 5) IP
(d). In all cases, the injection did not induce
intracellular Ca
mobilization, nor did it potentiate
Ca
entry. However, with high concentrations of (1, 3, 4, 5) IP
, the
subsequent Ca
entry phase was reduced, and in some
cases it was blocked. Each experiment is representative of three to
seven observations.
Although it is apparent from these
results and our earlier report (11) that(1, 4, 5) IP provides both a necessary and sufficient signal for both
intracellular Ca
release and Ca
entry, our previous study did not directly address the
possibility
that(1, 3, 4, 5) IP
might modulate or augment IP
-induced Ca
signaling in intact lacrimal cells. In Fig. 1, c and d, the effect of (1, 3, 4, 5) IP
on cells
previously microinjected with submaximal concentrations
of(2, 4, 5) IP
was examined to
determine if(1, 3, 4, 5) IP
could either mobilize additional intracellular Ca
or potentiate the Ca
entry phase induced
by(2, 4, 5) IP
alone.
After
establishing the response of a single lacrimal cell to a submaximal
concentration of(2, 4, 5) IP, the
cells were returned to a nominally Ca
-free medium and
microinjected a second time with different concentrations
of(1, 3, 4, 5) IP
(Fig. 2, c and d). In all cases, (1, 3, 4, 5) IP
(pipette
concentrations from 100 µM to 10 mM) neither
released additional intracellular Ca
nor potentiated
the Ca
entry phase seen with
the(2, 4, 5) IP
alone (n = 15/15). Rather, high concentrations of microinjected (1, 3, 4, 5) IP
appeared
to reduce the subsequent Ca
entry phase, and the
highest concentrations used almost completely blocked the entry phase (Fig. 2d, 10
mM(1, 3, 4, 5) IP
in the pipette, cellular concentration 100-200
µM, n = 6/6). Note that although the
Ca
entry phase appears blocked, Ca
entry can still be activated by treatment with thapsigargin (Fig. 1). Fig. 3summarizes results of experiments
showing that the inhibition of calcium entry by injected (1, 3, 4, 5) IP
was
dependent on the concentration of (1, 3, 4, 5) IP
in the
injection pipette.
Figure 3:
Concentration effect curve for
microinjected (1, 3, 4, 5) IP on the Ca
influx response to 10
µM(2, 4, 5) IP
in
single lacrimal cells. The effects
of(1, 3, 4, 5) IP
injection on the (2, 4, 5) IP
-induced
Ca
entry described in Fig. 2are summarized.
In each case, a single cell was initially injected
with(2, 4, 5) IP
(1 mM in the pipette), and the Ca
entry level was
established. Subsequently, the same cell was injected with additional
inositol phosphates, and the Ca
entry level
reexamined. Thus, 100% would indicate that there was no change in the
level of the second sustained Ca
entry phase when
compared with the sustained Ca
-entry phase induced by
the initial injection of (2, 4, 5) IP
. As can be seen, a
second injection of 1
mM(2, 4, 5) IP
results
in an approximate doubling of the Ca
entry; no such
effect is seen when the second injection is carried out in the absence
of inositol phosphates in the injection solution (control). In
contrast, increasing the concentration of
injected(1, 3, 4, 5) IP
decreases the second Ca
entry phase (to 11%
with 10 mM(1, 3, 4, 5) IP
).
Each data point represents three to five
experiments.
We next considered that the inhibitory effect of (1, 3, 4, 5) IP might
result from an inhibition of the action
of(2, 4, 5) IP
at the IP
receptor. Thus, we examined the effect
of(1, 3, 4, 5) IP
on
Ca
release from saponin-permeabilized
mouse lacrimal cells, particularly to see
if(1, 3, 4, 5) IP
could
modulate the effect of a submaximal concentration of (2, 4, 5) IP
on
Ca
release (Fig. 4). In
permeabilized lacrimal cells, (2, 4, 5) IP
released
Ca
in a dose-dependent fashion
(EC
= 6.5 ± 0.8 µM; maximal
release was 46.2 ± 4.8% of the ATP-dependent
Ca
pool(16) ). As shown in Fig. 4, a submaximal concentration (10 µM) of (2, 4, 5) IP
released 31.8
± 5.4% (n = 4) of the ATP-dependent
Ca
-pool (total ATP-dependent
Ca
pool = 6.40 ± 0.98 nmol
of Ca
/mg of protein; n = 4). Under
these
conditions,(1, 3, 4, 5) IP
(500 µM) did not cause significant
Ca
release (4.0 ± 4.9%; n = 4). When 10 µM(2, 4, 5) IP
and various
concentrations of (1, 3, 4, 5) IP
were
added together to the permeabilized
cells,(1, 3, 4, 5) IP
did not augment Ca
release due
to(2, 4, 5) IP
. Rather,
consistent with the observations in single cells, concentrations of (1, 3, 4, 5) IP
greater
than 10 µM apparently inhibited the ability of 10
µM(2, 4, 5) IP
to
release
Ca
.
Figure 4:
Concentration effect curve for the effect
of (1, 3, 4, 5) IP on
the ability of 10 µM(2, 4, 5) IP
to induce
Ca
release from permeable lacrimal
cells. Release of
Ca
from permeable
lacrimal cells was determined as described under ``Materials and
Methods.'' The data are expressed as the percentage released of
the ATP-dependent
Ca
pool (6.40 ±
0.98 nmol of Ca
/mg of protein). 10 µM(2, 4, 5) IP
maximally
released 31.8 ± 5.4% of the pool,
whereas(1, 3, 4, 5) IP
alone was ineffective up to 500 µM (4.0 ±
4.9%). The effect of increasing concentrations
of(1, 3, 4, 5) IP
on the
ability of 10
µM(2, 4, 5) IP
to
release
Ca
are shown. At the highest
concentration
of(1, 3, 4, 5) IP
tested, the effect of 10 µM(2, 4, 5) IP
was reduced to
11.7 ± 2.1%. Results are means ± S.E. of four independent
experiments.
We also confirmed
that(1, 3, 4, 5) IP was
capable of inhibiting the Ca
-mobilizing action of (2, 4, 5) IP
in intact cells. In
experiments shown in Fig. 5, 10
mM(1, 3, 4, 5) IP
was injected into a single lacrimal cells prior to injection of 1
mM(2, 4, 5) IP
. This
resulted in a complete blockade of both the Ca
release and Ca
entry phases of the response
to(2, 4, 5) IP
.
Figure 5:
Prior injection
of(1, 3, 4, 5) IP blocks
both Ca
release and Ca
entry due to (2, 4, 5) IP
injection. Top, immediately prior to data collection, a single lacrimal
acinar cell was injected with (1, 3, 4, 5) IP
(pipette
concentration 10 mM); this blocked both Ca
release and Ca
entry due
to(2, 4, 5) IP
injection (pipette
concentration 1 mM). The response to thapsigargin was
unaffected. Bottom, In a control experiment, a cell was
mock-injected (injected with fura-2-containing diluent
only);(2, 4, 5) IP
injection
induced both release of intracellular Ca
as well as
Ca
entry. The results illustrate findings from three
independent experiments.
(1,3,4,5)IP did not release intracellular
Ca
in intact or permeabilized cells, nor did it
induce or facilitate Ca
entry in intact cells.
Rather, and
surprisingly,(1, 3, 4, 5) IP
appeared to block the Ca
entry phase induced
by(2, 4, 5) IP
microinjection in
intact cells. Results from experiments in permeabilized and intact
cells would suggest that the inhibitory effect on the Ca
entry phase may be due to (1, 3, 4, 5) IP
interfering with the ability of (2, 4, 5) IP
to maintain
depletion of the intracellular Ca
pool. It is now
well established that depletion of the intracellular Ca
pool by(1, 4, 5) IP
proportionally activates Ca
entry(17) .
Heparin, an antagonist of
the(1, 4, 5) IP
receptor, blocks
agonist-activated calcium entry presumably by virtue of its ability to
prevent(1, 4, 5) IP
-induced
depletion of intracellular stores(11) ; heparin does not block
thapsigargin-activated calcium entry that does not involve interaction
of(1, 4, 5) IP
with its receptor (11) (but see (18) ).(1, 3, 4, 5) IP
similarly blocked calcium entry as well as calcium release by (2, 4, 5) IP
but not responses to
thapsigargin. Thus the antagonistic effect of high concentrations
of(1, 3, 4, 5) IP
on(2, 4, 5) IP
-induced
Ca
entry in intact lacrimal cells most likely is a
result of the inhibition of Ca
release
by(2, 4, 5) IP
.
(1,3,4,5)IP is metabolized in the cytoplasm of cells by
a 5-phosphatase, and thus the question arises as to whether the
injected material would persist long enough to exert any physiological
action. The half-time
for(1, 3, 4, 5) IP
in
exocrine gland cells has been estimated to be on the order of 45 s to a
minute(19) . Thus, since Ca
entry was
examined within 1 min after injection, and with a wide variety of
concentrations, one would expect to have detected some effect of the
injected(1, 3, 4, 5) IP
if in fact such could occur. Indeed, an inhibitory effect was
seen at the higher concentrations
of(1, 3, 4, 5) IP
, an
effect also seen with the addition
of(1, 3, 4, 5) IP
to
permeabilized acinar cells.
Because(1, 3, 4, 5) IP
is metabolized to (1, 3, 4) IP
and subsequent metabolites, we cannot at present determine if
this is a direct effect
of(1, 3, 4, 5) IP
or an
effect of a metabolic product. In fact, we note that the concentrations
of(1, 3, 4, 5) IP
that
inhibited Ca
entry in intact cells appear to be
considerably less than those that inhibited Ca
mobilization in permeable cells, based on the estimate (11) that the injected material is diluted 50-100-fold.
Such a discrepancy might result if a metabolite
of(1, 3, 4, 5) IP
inhibits the(1, 4, 5) IP
response since the concentration of such a metabolite would be
considerably diluted in the permeable cell experiments. Alternatively,
if the effect is due to a more direct action
of(1, 3, 4, 5) IP
, then
the discrepancy may reflect factors that influence IP
receptor sensitivity and accessibility in intact cells that are
absent or changed in permeabilized cells.
In conclusion, these
findings confirm and extend our previous conclusion (11) that
IP provides a necessary and sufficient signal for both the
intracellular release of Ca
and entry of
Ca
across the plasma membrane. This action of
IP
is presumably due to its ability to release
Ca
from intracellular stores, resulting in the
transmission of an as yet unknown message to the plasma
membrane(3, 20, 21) . In the current studies, (1, 3, 4, 5) IP
neither
mobilized Ca
nor potentiated the effects
of(2, 4, 5) IP
. However, (1, 3, 4, 5) IP
was able
to antagonize the effects of (2, 4, 5) IP
and reduce
Ca
entry, perhaps by interfering with the ability
of(2, 4, 5) IP
to bind to its
receptor, although this was not directly determined in the present
study. Interestingly, Wilcox et al.(22) have reported
that in neuronal
cells,(1, 3, 4, 5) IP
can act as an agonist at
the(1, 4, 5) IP
receptor. The
reason for the different effects in these two different preparations is
not known, but a possibility is that the two cell types express
distinct forms of the (1, 4, 5) IP
receptor, both of which can bind (1, 3, 4, 5) IP
but with
different effects on activation of the Ca
channel.
These data raise the possibility
that(1, 3, 4, 5) IP could inhibit the effects
of(1, 4, 5) IP
under
physiological stimulation. Co-injection of 100 µM(1, 3, 4, 5) IP
together with 1 mM(2, 4, 5) IP
resulted in
significant inhibition. Since (1, 4, 5) IP
is roughly 10-fold
more potent than (2, 4, 5) IP
,
this would indicate that significant inhibition would occur
when(1, 4, 5) IP
and(1, 3, 4, 5) IP
are present in approximately equal concentrations, a situation
that occurs in most cases of sustained stimulation. On the other hand,
from the data in permeable cells an approximately 30-100-fold
excess of (1, 3, 4, 5) IP
over(1, 4, 5) IP
would be
required for inhibition. The levels
of(1, 3, 4, 5) IP
in
maximally stimulated lacrimal acinar cells range from approximately
equal to, to 2-3 times the level
of(1, 4, 5) IP
(23) and
do not to our knowledge reach the 30-100-fold excess level in any
cell type. However, as argued above, the sensitivity of
the(1, 4, 5) IP
receptor
to(1, 4, 5) IP
and/or
to(1, 3, 4, 5) IP
in
intact cells may be markedly different than in permeable cells. Errors
in estimates of cellular dilution of inositol phosphates are not an
issue, because these would apply equally to the injected (2, 4, 5) IP
and the
injected(1, 3, 4, 5) IP
.
Thus, although additional work is clearly needed, we suggest that
inhibition
by(1, 3, 4, 5) IP
, or
possibly one of its metabolites, of the (1, 4, 5) IP
response may occur
under conditions of physiological stimulation, and this could represent
yet another important negative feedback on Ca
signaling, at least in this cell type.